in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (6), pp. 201-205, june, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper an assay for detecting foodborn salmonella using loop-mediated isothermal amplification wang deguo 1 , huo guicheng 1 *, wang fugui 2 , li yonggang 1 and ren daxi 1 1 key laboratory of dairy science, ministry of education of china, northeast agricultural university, harbin150030, china. 2 gansu animal husbandry engineering vocational technical college, china. accepted 07 april, 2017 loop-mediated isothermal amplification (lamp) was a novel nucleic acid amplification method that amplified dna with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a dna polymerase with strand displacement activity. in our research, lamp was developed to detect foodborn salmonella in raw milk, a set of six primers, two outer, two inner and two loop primers, were designed from salmonella genomic dna targeting inva. temperature condition for detection of salmonella was optimized to be 61°c. at the same time, lamp was found to be effective when the template was pure; however, the usefulness of lamp methods can be limited by the presence of inhibitors in the analysis of raw milk, unlike all the reports about lamp, the sensitivity of lamp was lower than that of pcr methods. key words: loop-mediated isothermal amplification, lamp, foodborn salmonella, raw milk, sensitivity. introduction recently, a new technique called loop-mediated isothermal amplification (lamp) had been developed which can amplify nucleic acids with high specificity, sensitivity and rapidity under isothermal conditions (notomi et al., 2000). the method was easy to perform (mori et al., 2001; nagamine et al., 2002a), which was based on the principle of the reaction performed by a dna polymerase with strand displacement activity and a set of two specially designed inner primers (fip and bip) and two outer primers (f3 and b3). lamp had high specificity for the target sequence because the target sequence was recognized by six independent sequences (f1c, f2, f3, b1c, b2 and b3) in the initial stage and by four independent sequences (f1c, f2, b1c, and b2) in the later stages of the lamp reaction. the amplification efficiency of the lamp method was extremely high because of its no time loss for thermal change, based on its isothermal reaction. therefore, the lamp assay had the advantage in specificity, selectivity and rapidity over other nucleic acid amplification methods (mori et al., 2001). moreover, nagamine et al. (2002) had advanced the method by putting forward loop primers (lf and lb) that accelerated the lamp reaction. *corresponding author. e-mail: gchuo@vip.0451.com. salmonella was an aetiologic agent of diarrhoea and a source of many food-related outbreaks (trafny et al., 2006), detection of salmonella was an important parameter in microbiological analysis to control food safety, many methods had been developed, among which standard culture method was commonly used, but culture method was laborious and time-consuming, requiring nonselective pre-enrichment, selective enrichment, plating on differential agar media, presumptive biochemical identification, and serological confirmation (španová et al., 2001). lamp may be a rapid and sensitive method to replace culture method. in the present study, an assay was developed for detecting foodborn salmonella with inva in raw milk using lamp, unfortunately, unlike all the reports about lamp, lamp method was not superior to pcr method, hereby, and the drawback of lamp was reported for the first time. materials and methods bacterial strain and artificially polluted raw milk salmonella enterica 13076 (key laboratory of dairy science, ministry of education of china) was stored at –80°c until use. raw milk was subjected to treatment of pasteurization and spread on bs agar to determine that there was no salmonella. samples of 200 ml raw milk, free of salmonella, were polluted with salmonella, and the concentrations of salmonella were 10 8 , 10 7 , 10 6 and 10 5 cfu ml -1 , respectively. deguo et al. 202 table 1. purity of dna templates extracted with different methods. read(260)/read(280) read(230)/read(260) abs(260) concentration method i) 1.8048 0. 5483 1.0169 — method ii) 1.5197 1.0986 0.9214 — dna preparation i) dna from a pure culture of s. enterica 13076 was extracted according to the manufacturer’s instructions of uniq10 column dna extraction kit (shanghai bioengineering co., ltd), the dna template was used for determining the effect of temperature and the effect of dna purity on lamp. ii) bacteria were split by sds and protease k, cell debris and polysaccharides were precipitated by cetrimonium bromide (ctab), total dna then was precipitated and extracted by isopropyl alcohol (li et al., 2005), the dna template was used for determining the effect of dna purity on lamp. iii) artificially polluted milk was centrifuged at 10 000 rpm for 10 min to obtain bacterial pellet, and then bacterial genomic dna was prepared by lysis of the bacterial pellet in 100 al of lysis buffer (20 mm tris–hcl, ph 8.0, 2 mm edta, ph 8.0 and 1.2% triton x-100) that was boiled for 10 min (enosawa et al., 2003; yano et al., 2007). the dna template was used for comparing sensitivities of lamp and pcr. design of primers based on the inva (genbank accession no: nc_006905) of salmonella enterica subsp. enterica serovar choleraesuis str. scb67, six primers were designed using primerexplorer3, the forward inner primer of inva (fip) consisted of the complementary sequence (20 nt) of f1, a tttt linker and f2 (19 nt): 5’gcgaggtttggcggctgatatttttcgcgacgacaaaatctgg 3’; the backward inner primer of inva (bip) consisted of b2 (21 nt), a tttt linker and the complementary sequence (21 nt) of b1: 5’ aactttagcgcaaggtgcctctttttgccggt aaactacacgatgacatg-3’; primers f3 (20 nt) and b3 (20 nt) for inva were 5’ ctgggcgtttttttgtcctg -3’ and 5’gggaaggttaaggagggtga -3’; loop primers lf (18 nt) and lb (19 nt) for the set of primers were 5’aggcttcgcgtacagagg 3’ and 5’cacgtcagcaaagcgtacc -3’. temperature determination of amplification lamp was performed in a total 25-ml reaction mixture containing 0.8 mm each of fip and bip, 0.2 mm each of the outer primers, 0.4 mm each of loop primers f or b or f and b, 1.6 mm dntps, 1m betaine (sigma), 20 mm tris-hcl (ph 8.8), 10 mm kcl, 10 mm (nh4)2so4, 4 mm mgso4, 0.1% triton x-100, 8 units of the bst dna polymerase large fragment (new england biolabs), and the specified amounts of double stranded target dna (nagamine et al., 2001; nagamine et al., 2002; fukuta et al., 2003; gunimaladevi et al., 2004). the reaction temperature (57°, 59°, 61°, 63° and 65°c) was optimized, and lamp was carried out for 60 min and terminated at 80°c for 10 min (yeh et al., 2005; itano et al., 2006). lamp products were subjected to electrophoresis on a 2.0% agarose gel and visualized under uv light of chemidocxrs after ethidium bromide staining (yano et al., 2007; shoemaker and klesius, 2006). effect of dna purity on lamp the purity of dna extracted with methods i) and ii) were determined by uv-visible spectrophotometer 100conc and then the templates were subjected to lamp reaction. the reaction system and conditions were the same as above. detection limit of lamp and pcr detection limit of salmonella with inva by lamp the dna extracted from artificially polluted raw milk samples by method iii) were used as templates for lamp following the conditions described above. the products were subjected to electrophoresis on a 2.0% agarose gel and visualized under uv light of chemidocxrs after ethidium bromide staining. detection limit of salmonella with inva by pcr in order to make a comparative analysis of both the protocols (lamp and pcr), dna obtained from artificially polluted raw milk samples were simultaneously subjected to lamp and pcr. the pcr reaction was carried out according to the method described by malorny et al (2003). results dna template purity the purity of dna template extracted by the method i) was higher than that extracted by the method ii), as indicated in table 1. there were proteins and sugars in the dna template extracted by the method ii). effect of temperature on lamp the lamp was carried out using the dna extracted by method i) as template to determine the optimal temperature. from figure 1, it can be seen that lamp products were formed at all temperatures. however, 61°c was considered as the optimal temperature as specificity of the reaction increased at this temperature. effect of dna purity on lamp dna templates extracted by method i) and method ii) were used to determine the effect of dna purity on lamp. as indicated in figure 2, there was no product when using the dna template extracted by method ii); however, when figure 1: effect of temperature on amount of lamp products m: marker iii; lanes 1 – 5: lamp carried out at 57°c, 59°c, 61°c, 63°c and 65°c, respectively. all the products were electrophoresed on 2% agarose gels and stained with ethidium bromide. using the dna template extracted by method i), the result was reverse. detection limit of lamp and pcr comparative analysis of sensitivity of salmonella detection by lamp and pcr was carried out using 10-fold serial dilutions of salmonella dna. the test was repeated for 20 times by our 5 researchers. figure 3 shows that lamp could not detect salmonella even when the dna template was extracted from the artificially polluted raw milk with 10 8 cfu ml -1 salmonella, while pcr could amplify up to 10 6 cfu ml -1 . hence, the detection limit or sensitivity of lamp was lower than pcr. 203 afr. j. food sci. res. figure 2. effect of dna purity on lamp m: marker dl2000; lane1: lamp carried out with the dna template extracted with method i); lane2: lamp carried out with the dna template extracted with method ii); nc: negative control. deguo et al. 204 m: figure 3. detection of salmonella dna by lamp and pcr from artificially polluted raw milk. marker dl2000 (2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp); p10 8 , p10 7 , p10 6 and p10 5 were the lanes detected by pcr; l10 8 , l10 7 , l10 6 and l10 5 were the lanes detected by lamp; nc: negative control. discussion since the development of lamp, many applications and modifications had been reported, including using the method to detect foodborn pathogens in food (wang et al., 205 afr. j. food sci. res. 2007). almost in all the reports, the detection limit of lamp had been found to be higher than that of pcr. furthermore, in the research of kaneko et al. (2007), the toleration of lamp for biological substances was found to be superior to pcr, so they suggested that the dna extraction step can be omitted in lamp assay. reported dna preparation methods (enosawa et al., 2003; yano et al., 2007) were used to detect foodborn pathogens in raw milk with lamp in our research. for most foodborn pathogens such as staphylococcus aureus, listeria monocytogenes, shigella and etc, the dna extraction step can be omitted as suggested by kaneko et al (2007). however, when detecting salmonella with lamp and primers as described above, there were plentiful products if the dna template was pure; in other hand, there was no amplified product if the dna template was not pure or dna extraction step was omitted, and the detection limit of salmonella by lamp was stupendously lower than that of pcr. the situation may be caused by two reasons: firstly, as reported by kaneko et al. (2007), a wide range of inhibitors including organic and inorganic substances such as detergents, antibiotics, phenolic compounds, enzymes, polysaccharides, fats, proteins, and salts might inhibit lamp and pcr reaction; secondly, the primers may have indirect influence on lamp reaction, because dna purity did not affect the detection of other foodborn pathogens in raw milk with lamp. the viewpoints speculated by us need to be further verified. in a word, further research on stability and sensitivity of lamp is still in need for generalization, popularization and application of lamp in clinical diagnosis and food detection fields. acknowledgement we were indebted to researcher li shulong of heilongjiang import and export inspection and quarantine bureau for advices on the experiment. references notomi t, okayama h, masubuchi h, yonekawa t, watanabe k, amino n, hase t(2000). loop-mediated isothermal amplification of dna. nucleic acid res. pp. 28-63. mori y, nagamine k, tomita n, notomi t (2001). detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. biochem. biophys. res. commun. 289:150–154. nagamine k, kuzuhara y, notomi t (2002). isolation of singles tranded dna from loop-mediated isothermal amplification products. biochem. biophys. res. commun. 290: 1195-1198. nagamine k, hase t, notomi t (2002). accelerated reaction by loopmediated isothermal amplification using loop primers. molecular and cellular probes. 16: 223-229. trafny ea, kozłowska k, szpakowska m (2006). a novel multiplex pcr assay for the detection of salmonella enterica serovar enteritidis in human faeces. lett. appl. microbiol. 43: 673679. alena španová, bohuslav rittich, renata karpíšková, leona echová, denisa škapova (2001). pcr identification of salmonella cells in food and stool samples after immunomagnetic separation. bioseparation 9: 379-384. enosawa m, kageyama s, sawai k, watanabe k, notomi t, onoe s, mori y, yokomizo y (2003). use of loop-mediated isothermal amplification of the is900 sequence for rapid detection of cultured mycobacterium avium subsp. paratuberculosis. j. clin. microbiol. 41, 4359-4365. akemi yano, rika ishimaru, rie hujikata (2007). rapid and sensitive detection of heat-labile i and heat-stable i enterotoxin genes of enterotoxigenic escherichia coli by loop-mediated isothermal amplification. j. microbiol. met. 68: 414-420. wenbin li, ronald h, brlansky, john hartung s (2005). amplification of dna of xanthomonas axonopodis pv. citrifrom historic citrus canker herbarium specimens. j. microbiol. meth. 7: 014023. nagamine k, watanabe k, ohtsuka k, hase t, notomi t (2001). loopmediated isothermal amplification reaction using a nondenatured template. clin. chem. 47: 1742-1743. kentaro nagamine, yoko kuzuhara, tsugunori notomi (2002). isolation of single-stranded dna from loop-mediated isothermal amplification products. biochem. biophysi. res. comm. 290: 1195-1198. shiro fukuta, shinro kato, keiko yoshida ( 2003). detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction. j. virolog. meth. 112: 35-40. gunimaladevi, t kono, venugopal mn, sakai m (2004). detection of koi herpesvirus in common carp, cyprinus carpio l., by loop-mediated isothermal amplification. j. fish dis. 27: 583-589. hung-yueh yeh, craig a, shoemaker, phillip h, klesius (2005). evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish ictalurus punctatus important bacterial pathogen edwardsiella ictaluri. j. microbiol. meth. 63: 36-44. itano t, kawakami h, kono t, sakai m (2006). detection of fish nocardiosis by loop-mediated isothermal amplification. j. appl. microbiol. 100: 1381-1387. yeh h-y, shoemaker ca, klesius ph (2006). sensitive and rapid detection of flavobacterium columnare in channel catfish ictalurus punctatus by a loop-mediated isothermal amplification method. j. appl. microbiol. 100: 919-925. malorny burkhard, hoorfar jeffrey, bunge cornelia, helmuth, reiner jan (2003). multicenter validation of the analytical accuracy of salmonella pcr: towards an international standard. appl. environ. microbiol. 69(1): 290-296. wang l, shi l, alam mj, geng y, li l (2007). specific and rapid detection of foodborne salmonella by loop-mediated isothermal amplification method, food res. inte. 41(1): 69-74. hisatoshi kaneko, takashi kawana, eiko fukushima, tatsuo suzutani (2007). tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. j. biochem. biophysical meth 70: 499-501. african journal of food science research vol. 1 (2), pp. 018-020, october, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper incorporation of turmeric-lime mixture during the preparation of tomato puree chaitali dutta, banani ray chowdhury*, runu chakraborty and utpal raychaudhuri department of food technology and biochemical engineering department, jadavpur university, kolkata, india. accepted 19 september, 2013 new types of tomato puree products were developed by blanching matured tomatoes (lycopersicon esculentum) for 1 min, 2 min and 3 min individually with or without addition of the mixture of turmeric and lime during the blanching time. soluble solid content and ph of the puree products were in the range of 11 12.6 brix and 4.32 4.68 respectively. total hunter lab colour difference (e) of treated sample following 2 min and 3 min blanching significantly (p < 0.05) higher than corresponding control samples. again, 2 minand 3 min-blanched treated samples did not have significantly different (p > 0.05) lab values (l, brightness; a, redness and b, yellowness). also, yield stress (measure of flow behaviour) of 2 minblanched samples (both treated and control) were the maximum among other corresponding puree samples. thus, 2 min blanching time may be preferred for the preparation of this new type of turmeric-lime treated tomato puree product. key words: turmeric-lime, lycopersicon esculentum, tomato puree. introduction tomato (lycopersicon esculentum) is cultivated in different countries for its edible red fruit. lycopene is a phytochemical nutrient element found in many fruits and vegetables, but excessively found in tomato that imparts natural red colour (holden et al., 1999). gertner et al. (1997) reported that lycopene is the predominant carotenoid in tomatoes. supplementation of tomato products, containing lycopene, has been shown to lower biomarkers of oxidative stress and carcinogenesis (basu and imrhan, 2006). many factors affect the lycopene concentration in raw tomatoes, such as genetics, soil, and plant nutrition, handling, maturity and seasonal variations. the green raw tomato turns into red when it ripens, as the cisform of lycopene present in tomato gradually changes into transform of lycopene with the maturity (xianquan et al., 2005). the redness of tomato depends upon the majority concentration of trans-form. retention of natural pigment is one of the symbols of livelihood. thermal treatment is one of the most important methods of preservation of vegetables (lund, 1975). thermal processing inactivates pathogens and other *corresponding author. e-mail: brchowdhury1@rediffmail.com. tele-fax: 9133-24146822 microorganisms and also improves the bioavailability of lycopenes since it breaks down the cellulose structure and plant cell. however, unfortunately thermal processing is also responsible for the degradation of red coloured lycopene pigment present in tomatoes. therefore discolouration during thermal processing (blanching) renders tomato puree unmarketable and leads to poor consumption. to compensate the reduction of red colour of tomato puree during its preparation, an attempt is made by adding equal proportion of turmeric and lime at the time of blanching of tomatoes. the objective of this paper is to intensify the colour of tomato puree for its better use and consumption. another objective of this research work is to measure the rheological characteristics of fortified tomato puree as fortification of turmeric-lime mixture might be responsible for the appetizing characteristics of tomato puree such as colour and consistency (rheology). materials and methods preparation of tomato puree matured red coloured fresh tomatoes were purchased from the local market in kolkata. the tomatoes were sorted and washed with clean water. then it was blanched in hot water at 100 c for 1, 2, and 3 min, separately. in case of pretreated tomato puree prepara dutta et al. 018 table 1. soluble solid content of different tomato puree samples. sample blanching time 1 min 2 min 3 min control 11ax* 11.2 ax 11.4 ax treated 12.6 by** 12 bx 12 ax a – b different letters corresponds to the samples of a particular blanching time differ significantly (p < 0.05). x – y different letters corresponds to the same type of samples differ significantly (p < 0.05). table 2. ph of different tomato puree samples. sample blanching time 1 min 2 min 3 min control 4.32 ax 4.46 ax 4.38 ax treated 4.82 by 4.57 ax 4.68 ax a – b different letters corresponds to the samples of a particular blanching time differ significantly (p < 0.05). x – y different letters corresponds to the same type of samples differ significantly (p < 0.05). 27 control sample b treated sample u n it ) 26 l a b c o lo u r b a a l ( h u n te r 25 a a 24 0 1 2 3 4 blanching time (min) figure 1. l-value colour parameter of tomato puree samples. a – b different letters corresponds to the samples of a particular blanching time differ significantly (p < 0.05). tion, 4 mg of each of turmeric and lime were added in the blanching water during blanching. after blanching, the tomatoes were taken out of cooking water and deskined by keeping it under running water. then it was cut into two halves, with the seeds kept out, and ground the pulp in a food processor (inalsa appliances ltd., india). the excess water was discarded by a strainer to obtain tomato puree. the puree was stored in a sterile container at 0 o c. puree prepared by adding no turmeric and lime was considered to be as control sample. soluble solid content soluble solid content of the samples were determined by using a bellingham-stanley digital refractometer (model rfm 110) and were expressed in terms of o brix. ph ph of both treated and control tomato puree samples were determined by using a ph-meter (model no. 355, systronics, india). determination of colour visual colour was measured by using a hunter lab colour measurement system model color flex 45/0 (hunter associates laboratory, inc., usa) in terms of universally accepted hunter lab colour scale. the intensity of the colour of both the treated and control tomato puree samples were expressed in l, a and b (brightness, redness and yellowness, respectively). the instrument (10 observer, illuminant d-65) was calibrated against a standard white reference tile. determination of rheology approximately 17 ml of tomato puree was placed in a concentric cylindrical cup in a rotational rheometer (anton paar, model physical mcr 51, germany). rheological measurements (shear stress and shear rate) of the samples were done and the yield stress (in pascal unit) readings were obtained directly from the instrument. flow model the power law model with or without yield term describes the flow behaviour of viscous food over wide ranges of shear rate (vitali and rao, 1984). the rheological model that has been generally used for non-newtonian fluids, especially puree/paste is the herschelbulkley model, as shown in equation 1 below: y p = a + b.x ………… (1) a, b and p are regression parameters; p is the flow behaviour index. statistical analysis all the tests were done in triplicate and the samples were subjected to f-test for significant difference (p < 0.05) using microsoft excel software. results and discussions soluble solid content of tomato puree samples were observed to be in the range of 11 – 12.6 (table 1), which followed the conventional rule of tomato puree preparation (crandall and nelson, 1975). ph of the treated and control puree were in the range of 4.32 – 4.68 (table 2). it was observed from table 2 that the treatment of lime (alkali) along with turmeric during tomato puree preparation did not change significantly (p < 0.05) the acidic ph of the puree sample compared to the control sample without any turmeric-lime treatment. this observation indicated nonsignificant (p < 0.05) change in sourness of the puree product from the control sample and the acceptability of the treated puree product was comparable to the traditional control product. colour and rheology of tomato puree are important appetizing properties of tomato puree. it was observed from figures 1 and 2 that the colour parameters; l-and a 019 afr. j. food sci. res. 25 control sample b b treated sample u n i t ) 24 l a b c o l o u r b 23 a a ( h u n t e r a a 22 0 1 2 3 4 blanching time (min) figure 2. a-value colour parameters of tomato puree samples. a – b different letters corresponds to the samples of a particular blanching time differ significantly (p < 0.05). 13 control sample treated sample b u n it) 12 co lo u r b a a a l a b b ( h u n te r 11 a 10 0 1 2 3 4 blanching time (min) figure 3. b-value colour parameter of tomato puree samples. a – b different letters corresponds to the samples of a particular blanching time differ significantly (p < 0.05). 7 control sample b d if fe re n c e 6 treated sample 5 b c o lo u r 4 a a l a b 3 a a t o ta l h u n te r 2 1 0 1 min 2 min 3 min blanching time (min) figure 4. changes in total colour difference of the tomato puree samples. a – b different letters corresponds to the samples of a particular blanching time differ significantly (p < 0.05). values of both the control and treated samples increased with the time of blanching and a-value of the treated samples were significantly (p < 0.05) higher than the corresponding control samples. however, b (i.e. yellowness of the samples) of the treated samples decreased with increasing of blanching time and was significantly (p < 0.05) lower than the corresponding control samples (figure 3). this finding indicated that the treatment with turmeric and lime increased the redness of the tomato puree. in alkaline medium (due to presence of lime), the yellow coloured curcumin pigment of turmeric turns into red and the addition of turmeric-lime mixture in the blanching water of tomato puree preparation minimizes the conversion of trans-form of lycopene (red coloured 56 control sample 55 treated sample by by 54 u n it) 53 co lo u r 52 ax l a b 51 (h u n te r 50 49 ay l a /b 48 ax 47 ax 46 0 1 2 3 4 blanching time (min) figure 5. lab value colour parameter of tomato puree samples. a – b different letters corresponds to the samples of a particular blanching time differ significantly (p < 0.05). x – y different letters corresponds to the same type of samples differ significantly (p < 0.05). tomato pigment) into cisform of lycopene (yellow coloured pigment in tomato) and minimizes the cause of discolouration. the total colour difference ( e) of the tomato puree a product (as shown in equation 2) were determined and was shown in figure 4: where e = ( (a) 2 + (b) 2 + (l) 2 ) 1/2 (2) it was observed from figure 4 that the total colour difference e of treated sample following 2 min and 3 min blanching significantly (p < 0.05) higher than corresponding control samples. the colour differences were measured with respect to the colour parameters of 0 min blanched tomatoes (a, b and l values were 21.63, 11.62 and 22.31 hunter lab colour unit, respectively). however, as b-value of purees increased with blanching time, combination of hunter lab parameters (la/b) was found to be increased exponentially with increasing of blanching time up to 2 min and then decreased when dutta et al. 020 table 3. yield stress values and corresponding model-fitting parameters of different tomato puree products. sample blanching time regression coefficients regression (%) yield stress (pa) (min) a b p 1 102.27 -0.09 0.92 0.93 102.27 b control 2 90.25 -18.18 0.29 0.94 90.25 ab 3 86.73 -2.36 0.61 0.91 86.73 a 1 132.94 -12.22 0.43 0.95 132.94 b treated 2 126.84 -0.41 0.91 0.94 126.84 b 3 112.45 -2.68 0.68 0.93 112.45 a a – b different letters corresponds to the same type of samples differ significantly (p < 0.05). the tomatoes were blanched for 3 min (figure 5). la/b has also been expressed to colour change of puree products (avila and silva, 1999; shin and bhowmick, 1995). 2 min-and 3 min-blanched treated samples showed insignificant (p < 0.05) la/b values. rheology of tomato puree products can be measured in terms of yield stress (in pascal unit) and data were fitted by herschel bulkley model. the yield stress, regres-sion coefficients and corresponding r (percentage of model fitness) of different tomato puree samples were shown in table 3. it was observed that the yield stress of both the treated and control samples decreased with blanching time. however, of 2 min-blanched puree sho-wed no significant (p < 0.05) difference of yield stress value from 1 min-blanched tomato puree products. toma-to puree exhibited yield stress, which decreased with the blanching time, due to the reason that the rapture of tomato skin occurred and the food structure becomes weak resulting in the lowering of yield stress (steffe, 1992). the flow behaviour index (p) was less than unity. this indicated that the puree behaved its pseudoplastic (shear thinning) nature. treated samples had significantly (p < 0.05) higher yield stress value than corresponding control sample irrespective of blanching time. conclusion degradation of lycopene, that is, deterioration of red colour of tomato in tomato puree preparation can be best compensated by using turmeric and lime mixture during the blanching of tomatoes. also, yield stress of 2 minblanched samples (both treated and control) were the maximum compared to the corresponding samples of different blanching time, indicating their most acceptable flow behaviours. thus, 2 min blanching time may be preferred for the preparation of this new type of turmericlime treated tomato puree product. references avila imlb, silva clm (1999). modeling kinetics of thermal degradation of color in peach puree. j. food eng. 39:161–166. basu a, imrhan v. 2006. tomatoes versus lycopene in oxidative stress and carcinogenesis: conclusions from clinical trials. eur. j. clin. nutri. adv. online. 16 th aug. crandall pg, nelson pe (1975). effects of preparation and milling on consistency of tomato juice and puree. j. food sci. 40:710 gartner c, stahl w, sies h (1997). lycopene is more bioavailable from tomato paste than fresh tomatoes. am. j. clin. nutri. 66: 116-122. holden jm, eldridge al, beecher gr, buzzard i, marilyn b, seema davis cs, doughlass lw, gebbardt shd, schakel s (1999). carotenoid content of u.s. foods: an update of the database. j. food compos. anal. 12: 169-196. lund db (1975). effects of blanching, pasteurization and sterilization on nutrient. in harris rs and karmas (eds.). nutritional evaluation of food processing (pp. 205 –240). new york: avi publishing. shin s, bhowmick sr (1995). thermal kinetics of color changes in pea puree. j. food eng. 27: 77–86. steffe jf (1992). rheological methods in food process engineering. michigan, usa: freeman press. pp. 9-30. vitali aa, rao ma (1984). flow properties of orange juice-serum viscosity and effect of pulp content. j. food sci. 49: 876-881. xianquan s, shi j, kakuda y, yueming j (2005). stability of lycopene during food processing and storage. j. med. food. pp. 413-422. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 4 (5), pp. 059-062, december, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of steeping time on the physico-chemical, nutritional quality and consumer acceptability of kunnun-zaki obadina, a.o 1 , oyewole, o.b 2 , and awojobi, t.m 2 1 department of food science and technology, bells university of technology, p.m.b. 1015, ota, ogun state, nigeria. 2 department of food science and technology, university of agriculture, abeokuta, p.m.b. 2240, ogun state, nigeria. accepted 22 july, 2015 millet grains were steeped in water for varying period of time during “kunun zaki” production in order to study the effect of the duration of steeping on the quality of “kunu zaki”. other processing factors were kept constant in the course of this study. kunun zaki produced from millet grains steeped for 36 h was rated best in terms of sensory characteristics. the steeping period had no significant effect on the specific gravity of the produced “kunun zaki”. as expected the titratable acidity and ph were inversely proportional, with the latter decreasing and the former increasing during the fermentation-steeping period. the protein content increased between 12 and 48 h steeping time. during the steeping period, the carbohydrates decreased rapidly in the first 12 h. however, the rate of carbohydrates decrease reduced beyond the first 12 h. this may be due to the decrease in the rate of fermentable sugars. key words: kunun-zaki, millet, steeping, quality, ph, titratable acidity, sensory. introduction the soft drink segment of the nigerian beverage industry is heavily dependent on imported raw material and nige-ria is presently passing through a developmental stage in which there is a strong emphasis on local sourcing of raw materials. this awareness has transformed into a general interest in commercial processing of indigenous foods in order to conserve the scarce foreign exchange by limiting importation of raw materials. cereals are the major local raw materials in the production of beverages in nigeria which include pito, burukutu, local gin and kunnu-zaki. cereals are eaten in large quantities and are the main sources of both major and minor nutrients (adeyemi and umar, 1994). due to their high viscosity on cooking when made into gruel, large amount of water is used during preparation to obtain the right consistency; this obviously decreases the nutrient density (mosha and svanberg, 1983). kunnu-zaki is a traditional non-alcoholic fermented beverage widely consumed in the northern part of nigeria. it is however becoming widely consumed in the southern parts among low and middle income workers who cannot afford industrially produced beverages like coca-cola, pepsi etc. its popularity is due to its charac *corresponding author. e-mail: obadinaw@gmail.com. teristic sweet-sour taste, its refreshing quality as well as the creamy or milky appearance and also its flowing consistency. adeyemi and umar (1994) described the traditional process for the manufacture of kunnu-zaki which involves steeping of millet grains, wet milling with spices (ginger, cloves, and pepper), wet sieving and partial gelatinization of the slurry, followed by addition of sugar to taste and bottling. as a cereal product kunnu-zaki can supply carbohydrates, proteins, fats, minerals and vitamins if the nutrients are not destroyed by over processing (chapman and cater, 1976). according to hamad and fields (1979) there is significant increase in the relative nutritive value of several cereals after natural fermentation. fermen-tation in the case of kunnu-zaki is by lactic acid bacteria and yeast (adeyemi and umar, 1994). unlike other traditional cereal-based alcoholic beverages, information is scanty on the manufacturing process, quality and rheological characteristics of kunnu-zaki. in its traditional manufacture, the basic processes are not standardized and levels of ingredients such as spices and sweetener are not quantified. one of the unit operations that vary from one processor to the other is the steeping period. the effect of these variations on the quality of kunnu-zaki is yet to be inves obadina et. al. 060 tigated. the objective of this work was to study the effect of steeping time on the physico-chemical, nutritional quality and consumer acceptability of the kunnun-zaki materials and methods the materials used are millet ( pennisetum typhoides ), spices-ginger (zingiber officinale), black pepper (piper niger), red pepper (capsian annum) and cloves (syzygium aromaticum), and granulated sugar. these materials were obtained from local markets in abeokuta, they were cleaned and sorted. preparation of kunnu-zaki 500 g of dehulled millet grains were cleaned and sorted, weighed and steeped in 1000 ml of water at room temperature for 48 h. the same procedure was repeated four times after steeping each sample was milled with 3.25 g ginger, 0.25 g cloves, 1.25 g red pepper and 0.25 g black pepper .the resulting slurry was sieved using a sterile sieve until all the starch is extracted, the shaft were then discarded. the filtrate was allowed to settle, the supernatant decanted and the sediment was divided into 2 parts (ratio 3:2). the larger portion was cooked by the addition of 500 ml boiling water for 5 min while 500 ml cold water was added to the second part. the two slurries were thoroughly mixed and sweetened with 50 g granulated sugar (i.e. 10% sugar w/w millet used). proximate analysis moisture content the moisture content was determined according to (aoac, 1990) method. ash content determination the ash content was determined according to (aoac, 1990) method. crude fibre determination 2 g of the sample was accurately weighed into a fibre glass and 100 ml of 0.255 n h2so4 was added. the mixture was heated under reflux for 1 h with the heating mantle. the hot mixture was filtered through a fibre sieve cloth. the filterate obtained was thrown off and the residue was returned to the fibre glass to which 100 ml of 0.31 n naoh was added and heated under reflux for another 2 h. the mixture was again filtered through a fibre sieve cloth and 10 ml of acetone was added to dissolve any organic constituent. the residue was washed with about 50 ml hot water twice on the sieve cloth before it was finally transferred into the crucible. the crucible and the residue were oven dried at 105ºc overnight to drive off moisture. fat determination gravimetric rose-gottlieb method as described by pearson (1981) was used. protein determination the protein content of the sample was determined by using formal titration method as described by pearson (1981). ph determination the ph was measure using electrometric ph meter (genway, 4590). this was calibrated by the use of prepared buffer solutions of accurately known ph (ph 4 and ph 9). 30 ml of the sample was measured into a curvette and the glass electrode of the ph metre was dipped into the sample. total titratable acidity the titratable acidity of the product was measure by direct titration as described by pearson (1981).10 ml of the sample was pipetted into each of two beakers labeled c and s. to the colour control beaker, 1 ml of rosanilline solution was added and stirred. to sample beaker s, 1 ml of phenolphthalein indicator was added and titrated with 0.1 m naoh, with continuous stirring until the colour matched the pink colour of c. the acidity was calculated as the lactic acid (percent m/v). specific gravity determination the specific gravity of each sample was determined using the picnometer specific gravity bottle. the bottle was washed, rinsed and dried. the empty bottle was weighed and mass recorded as m. it was filled with sample and weighed, mass recorded as m1. the bottle was emptied, rinsed and filled with water and weighed, mass recorded as m2. the specific gravity was calculated sensory evaluation the four different kunun zaki steeped for varying period of time were subjected to sensory evaluation. a total of twenty untrained panelist drawn from the university of agriculture, abeokuta based on their familiarity with the product were used for the evaluation. the parameter evaluated includes taste, colour, flavour, sweetness and overall acceptability. the coded samples were served in clean transparent plastic bottles at room temperature (25 o c) in individual booths with adequate florescent lights. sample presented to the panelists was at random and one at a time. the panelists were given enough water to rinse their mouths between each sample. the nine-point hedonic scale (larmond, 1977) was used for the evaluation and the resulting data were analyzed using analysis of variance (anova) to establish significant differences among treatment. duncan multiple range test was used to separate means where significant differences existed (duncan, 1955). results and discussion sensory evaluation the mean sensory scores for mouth feel, colour, taste, flavour, sweetness and over all acceptability of kunun zaki samples prepared from millet grains steeped for varying period of time are given in table 1. the highest mean values of preference were obtained from 36 h steeping. the analysis of variance revealed no significant difference in all the samples in terms of mouth feel, flavour and sweetness at 5% probability level. however, sample steeped for 36 h was rated highest in mouth feel and flavour. relatively equal rating of the samples in terms of sweetness and flavour were probably as a result table 1. mean of sensory scores. duration of steeping parameters 12 hours 24 hours 36 hours 48 hours mouth feel 5.45 a 1.9 6.20 a 1.3 6.20 a 1.29 5.45 a 1.76 overall acceptability 5.65 b 2.0 6.65 ab 1.7 6.80 a 0.9 6.35 ab 1.3 colour 6.00 b 2.0 6.65 ab 2.0 7.40 a 1.1 7.25 a 1.2 taste 5.45 b 1.6 6.40 ab 1.6 6.70 a 0.9 5.95 ab 1.7 flavour 5.75 a 1.6 6.40 a 1.2 6.20 a 1.7 5.70 a 1.7 sweetness 5.90 a 1.9 6.05 a 1.9 6.85 a 1.1 5.90 a 1.4 1 values are mean standard deviation of 20 participants 2 mean along the same row with different superscript are significantly different from each other at 1% probability level. table 2. proximate composition of kunun-zaki on dry basis. duration of steeping parameters 12 hours 24 hours 36 hours 48 hours crude protein 5.16d 0.003 5.78c 0.005 5.86b 0.007 6.35a 0.002 fats content 19.02c 0.009 22.67b 0.002 24.08a 0.001 24.13a 0.005 ash content 2.20a 0.002 2.19a 0.007 2.17b 0.007 2.13c 0.007 carbohydrates 73.59a 0.007 69.38b 0.003 67.84c 0.009 67.44d 0.003 mean along the same row with different superscript are significantly different from each other at 1% probability level. figure 1. variation in protein, fats, carbohydrates and ash contents of kunun-zaki with different steeping time. of equal amount of sugar and spices added to each sample after fermentation, during the production to ensure that there is no variation in the factors of production other than the steeping time. there were significant differences in the colour of the samples at probability level of 5%, with samples steeped for 36 and 48 h rated relatively equal and better. there were no significant differences in the taste and over all acceptability; however from the mean separation, 2 homogenous sub-sets were obtained with sample steeped for 36 h rated highest (best) in taste and overall acceptability. proximate composition table 2 shows the proximate composition of kunun zaki on dry basis. the result of the analysis of variance revealed significant difference between the values of crude protein, fats, ash and carbohydrates contents obtained for each steeping time. a plot of the variation in crude protein, carbohydrates, fats and ash contents with steeping time was made (figure 1). kunun zaki steeped for 12 h contains 5.2% protein, while that of 36 h has 6.4% protein. in general, a gradual increase was observed in trend of protein with steeping time. this agrees with the report of hamad and fields (1979), au and fields (1981), kazanas and fields (1981), that there was significant increase in the relative nutritive value of proteins after natural fermentation (occurring during steeping). kazanas and fields (1981) also reported increase in average nutritive value of proteins by 10.67% lysine and iso leucine increase from 8.60 to 10.50 mg/g and nitrogen from 8.33 to 109.58 mg/g after fermentation. suberu (2001) also reported that a gradual increase in total soluble proteins of kunun zaki accompanied natural fermentation. figure 1 also shows the variation in carbohydrates with steeping time. 73.6% was reported in the product steeped from 12 h while 69.4% was recorded for 24 h. the rapid decrease was probably as a result of decrease in starch content during fermentation according to usha et al. (1996) and high consumption of sugar by microbes 061 afr. j. food sci. res. obadina et. al. 062 7 6 5 p h 4 ph 3 2 1 0 12 18 24 30 36 42 48 54 time (h) figure 2. variation in ph of kunun-zaki with different steeping time. 0.04 0.035 a c id it y 0.03 0.025 t it ra ta b l e 0.02 tta 0.015 0.01 0.005 0 12 18 24 30 36 42 48 54 time (h) figure 3. variation in total titratable acidity kunun-zaki with different steeping time. which are mainly active at this stage. carbohydrates recorded for the subsequent steeping time decrease at a lower rate. consumption of sugar in the production of organic acids was probably slower due to the feedback inhibition of the microbes by accumulated acidity. the variation in fats content with steeping time was also demonstrated in figure 1. from 12 h of steeping time there was a gradual increase in fats content of the products. the ash content with corresponding steeping time was also recorded in figure 1. the ash content of product steeped for 12 h was 2.2%, for 24, 36 and 48 h lower value were obtained. figure 2 shows the value for ph, titratable acidity and specific gravity of the kunun zaki steeped for different period. the result of analysis of variance indicates that there was a significant difference between the values. decrease in ph was a result of increasing hydrogen ion content probably due to the microbial activity on the carbohydrates and other food nutrient to produce organic acids. the range agrees with the report of adeyemi and umar (1994). analysis of variance of the results of the titratable acidity shows that there was a significant difference at (p<0.05) between the values in general; there was increase in titratable acidity with increasing steeping time. 0.0154% was obtained at 12 h steeping, 0.0262% at 24 h and 0.0340% at 48 h (figure 3). conclusion the relationship between the physical, chemical properties and nutritional parameters of kunun zaki and the steeping time of the millet grains was described. there were significant differences in the colour of the samples at probability level of 5%, with samples steeped for 36 and 48 h rated relatively equal and better. it can be concluded that in conclusion section, the relationship between the physical, chemical properties and nutritional parameters of kunun zaki and the steeping time of the millet grains was described. it can be concluded that kunun zaki from millet grains steeped for 36 h produced the best sensory and nutritional characteristics. references adeyemi la, umar s (1994). effect of method of manufacture on quality characteristics of kunu-zaki, a millet based beverage. niger. food j. 12: 34-42. au pm, fields ml (1981). nutritive quality of fermented sorghum. j. food sci. 46: 652. chapman rs, carter pl (1976). crop production principles and practices. freeman and company, san francis and co., pp. 67-69. hamad a, fields ml (1979). evaluation of protein quality and available lysine of germinated and fermented cereals. j. food sci. 44: 456. mosha ac, svanberg ed (1983). preparation of weaning foods with high nutrient density using flour of germinated cereals foods. nutr. bull. 5: 10 -14. suberu pa, kassum a (1992). rheological characterization of nigerian liquid and semi-liquid foods, kunu-zaki and kunungyada. niger. food j. 10: 23-33. usha as, chandra ts (1996). effect of fermentation on the primary nutrients in finger millet (eleusine coracana). j. agric. food chem. 44: 2616-2618. african journal of food science research vol. 2 (7), pp. 115-118, july, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper the effect of soil chemical properties on basic gum composition kabatila bedan*, kavat brayant and mishflo e. botany department, faculty of science, africa nazarene university, ongata rongai, kenya. accepted 24 june, 2014 geographical positioning system was used to mark the sites of acacia senegal variety kerensis in marsabit and samburu districts. soil and gum samples were collected for analysis of ph, carbon, nitrogen and phosphorus. soil ph (6.0 6.7) varied significantly (p < 0.05) with ph of gum (4.54, 4.50, 4.51 and 4.52) in all the sites. in merrile, organic carbon in gum (0.15%) was significantly higher than 0.073, 0.055 and 0.027% in logologo, laisamis and sereolipi, respectively. soil nitrogen (0.30, 0.4 and 0.8%) in merrile, laisamis and logologo were significantly correlated (p < 0.05) to the nitrogen (0.31 0.32%) in gum, while soil n (0.3%) in sereolipi was not significantly correlated with gum nitrogen (0.23%) and was significantly lower than those of merrile, laisamis and logologo (0. 31, 0.32 and 0.32%). phosphorus (700.2 and 705.2 ppm) in gums from sereolipi and merrile were significantly higher than 412.2 and 412.2 ppm in laisamis and logologo. ph (4.5 4.54) and nitrogen content (0.31 0.32%) in gum from merrile, laisamis and logologo were within the international standards (ph 4.2 4.8) and (0.24 0.41%). chemical properties of soils were major factors that influenced the gum quality. key words: acacia senegal, soil, gum arabic quality, sites. background and justification acacia senegal varieties are thorny and spiny trees, which grow abundantly in areas with annual rainfall of 200 800 mm and high temperatures in the arid and semi-arid lands of marsabit and samburu districts in northern kenya. the tree plays an important role in enriching the fertility of the soil through biological nitrogen fixation. it has remarkable adaptability to drought. the tree produces gum arabic in form of nodules or tears, under internally controlled physiological process during the dry season and in poor soil conditions. gum arabic exudate is produced through insect attack, mechanical injury or by making incisions in the stems and branches to strip away the bark to accelerate exudation. gum arabic is used as an emulsifier and stabilizer in the food and pharmaceutical industries (mocak et al.1998).other industrial products that use technical grades of gum arabic include adhesives, textiles, printing, lithography, paints, paper sizing and pottery glazing. gum arabic is harvested by herdsmen and women groups *corresponding author. e-mail: kaba_bedan@yahoo.com. from the natural stands of acacia senegal varieties from different botanical sources, locations, and soil types, age of trees, seasons, topographical conditions and temperature (chikamai and gachathi, 1994). it has been reported that the quality of gum may be influenced by geographical origin and age of the trees, climatic conditions, soil environment, and even on the place of exudation on the tree (chikamai, 1993; idris et al., 1998; islam et al., 1997; karamalla et al., 1998). the quality of gum arabic must conform to international specifications (fao 1995). most concerned in this regard are the us food and drug administration, the british pharmacopoeia, and the fao/who joint expert committee on food additives (jecfa), all of which aim to protect the consumer of processed foods containing additives, and thus ensusre the safety of gum arabic from toxicological hazards. to achieve this end, gum arabic must conform to certain chemical specifications, and these must be adhered to, by both the producers and the processing enterprises. among these requirements are that the gum arabic has to have a specific optical rotation of -26 to -34 degrees and nitrogen content of 0.24 0.41% (anderson et al., 1990, 1991). these characteristics are met only by http://www.internationalscholarsjournals.org/ kabatila et al. 115 gum arabic from a. senegal (l.) willd. var. senegal and not by gum talha from acacia seyal. the kenyan gum faces major problems relating to quality in the world market. according to a private sector player, kenya’s gum arabic obtained from acacia senegal variety kerensis is not able to attract premium prices because of problems relating to inherent high viscosity. some studies have shown differences between kenyan gum arabic and gum from other countries such as the sudan and nigeria in terms of specific rotation, nitrogen and viscosity. these differences may have evolved as a result of the differences in edaphic conditions and climatic factors. in addition, quality problems (as a function of site and location of the tree) may also contribute towards the quality of gum (chikamai and odera, 2002) the overall objective of this study was to establish the effect of chemical properties of soils on quality of gum arabic obtained from the natural stands of a. senegal (l.) willd. var. kerensis schweinf. trees. the specific objectives were to identify the natural stands of acacia senegal variety kerensis and to collect soil and gum samples for analysis of gum quality in logologo, laisamis, merille and sereolipi in marsabit and samburu districts. materials and methods study areas were logologo, seepi and turung’ung in koya location of laisamis, santait and kisapatai in merille, and kauro, nenyirao in sereolipi. the criteria used to select the study sites were high populations of naturally occurring stands of acacia senegal variety kerensis trees. the sites were surveyed, selected and marked using geographical positioning system (gps). twenty soil samples of 500g were collected randomly at depth of 0 25 cm in polythene bags and nine gum samples of 100 g collected in paper bags from the stems and branches of acacia senegal trees in each site. the samples were packed and taken for further pre-treatment and analysis. ph, organic carbon, nitrogen and phosphorus in soil and gum samples were analyzed respectively according to methods of anderson and ingram (1993) and okalebo et al. (2002). analysis of variance (anova) was used to analyse data on chemical properties of soils and gum arabic from the study sites. results and discussion the results of chemical properties of soils and gum arabic are given in table 1. the soil ph in laisamis, lo-gologo, sereolipi and merille (6.6, 6.7, 6.3 and 6.0) were higher (p < 0.05) than those of gum arabic (4.54, 4.51, 4.54 and 4.52) from each particular site, respectively. the soils of laisamis and logologo were less acidic than those of sereolipi and merrile, which were more acidic (table 1). ph of gum arabic was strongly acidic and varied significantly with soil ph in all the four sites. these ph values are similar to ph of gum arabic (4.5) from kargi and different from those of isiolo and ngurunit (4.4 and 4.3) given by chikamai and banks (1993). the ph values (4.50 4.54) in gum arabic from laisamis, logologo sereolipi and merrile were within the range of internatio nal specifications (ph: 4.2 4.8) fao (1996,1999). soil organic carbon in logologo and laisamis (1.4 and 0.7 %) were significantly higher (p < 0.05) than those of merrile and sereolipi (0.3 and 0.2%), respectively. there were no significant difference (p > 0.05) in soil carbon from each other in merrile and sereolipi. the levels of soil carbon in logologo and laisamis were ascribed to soil ph (6.7 and 6.6) which influences the activity of microorganisms in soil to increase uptake of carbon (h2co3) by the tree leaves to form carbohydrates through the process of photosynthesis. carbohydrates were transferred to stems and branches, which produced gum exudates with high carbon content. soil organic carbon in logologo and laisamis (1.4 and 0.7%) were significantly higher (p < 0.05) than the carbon content in gum arabic (0.07 and 0.06) from the respective sites. the carbon content in soils of merrile and sereolipi (0.3 and 0.2%) were also significantly higher (p < 0.05) than those of gum arabic (0.15 and 0.03%) from same sites (table 1). the carbon in gum arabic of merrile (0.15%) was significantly higher than those of gum arabic found in logologo, laisamis and sereolipi (0.073, 0.055 and 0.027%) respectively. the levels of soil nitrogen in logologo and laisamis (0.8 and 0.4%) were significantly higher than those of merrile and sereolipi (0.3 and 0.3%), respectively (table 1). nitrogen content in the soils of merrile and sereolipi (0.3 and 0.3%) was significantly higher (p < 0.05) than those of gum arabic (0. 31 and 0.23%) found in each respective site. the levels of nitrogen in gum arabic from acacia senegal variety kerensis in sereolipi, merrile, laisamis and logologo (0.31, 0.32 and 0.32%) were similar to nitrogen contents in gum arabic (0.28% 0.34%) from acacia senegal variety kerensis and acacia senegal variety senegal in marigat, baringo district (lelon, 2008). these results are different from the work by chikamai and banks (1993) on levels of nitrogen in gum arabic from acacia senegal variety kerensis found in kargi, isiolo and ngurunit (0.5, 0.4 and 0.43%). nitrogen content in gum arabic from laisamis, logologo and merrile (0.31, 0.32 and 0.32%) were within the range of international specifications (phillips and williams, 2001). nitrogen content in gum arabic from sereolipi (0.23%) fell outside the specifications (0.24 0.41%) (fao, 1996); (osman et al. 1993a, b). the level of soil phosphorus in laisamis (21.3 ppm) was significantly higher (p < 0.05) than those of logologo, sereolipi and merille were (4.2, 6.4 and 4.5 ppm), respectively. this was attributed to soil ph (6.0 and 6.3) a dominant factor affecting the availability of phosphorus. phosphorus becomes more available the lower the ph, since the solubility of phosphorus is highly dependent upon soil ph (woolhouse, 1983). phosphorus levels in gums from sereolipi and merrile (700.2 and 705.2 ppm) were significantly higher than those of laisamis and logologo (412.2 and 412.2 ppm), respectively. the levels of phosphorus in gums (700.2, 705.2, 412.2 and 412.2 ppm) were very high compared with those of soil phos 116 afr. j. food sci. res. table 1. chemical properties of soils and gum arabic in study sites. sites soil chemical properties chemical properties of gum arabic ph organic nitrogen phosphorus ph organic nitrogen phosphorus carbon (%) (%) (ppm) carbon (%) (%) (ppm) sereolipi 6.3 0.2 0.3 21.3 4.54 0.03 0.23 700.2 merrile 6.0 0.3 0.3 6.4 4.52 0.15 0.31 705.2 laisamis 6.6 0.7 0.4 4.5 4.50 0.06 0.32 412.2 logologo 6.7 1.4 0.8 4.2 4.51 0.07 0.32 412.2 % c soil ph gum % c 3 2.5 2 1.5 1 0.5 0 sereolipi merrile laisamis logologo sites figure 1. comparison of soil ph with % carbon in gum arabic in sites. phorus (21.3, 6.4, 4.5 and 4.2 ppm), respectively in all the sites (table 1). effect of chemical properties of soils on gum arabic quality comparison of soil ph with percent carbon in gum arabic is given in figure 1. soil ph in logologo merrile, laisamis and sereolipi was not significantly different (p > 0.05) while percent carbon in gum arabic was significantly higher (p < 0.05) in merrile than those of sereolipi, laisamis and logologo (figure 1). the soil ph had effect on the level of soil organic carbon in the soils of laisamis and logologo which had direct relation to level of carbon in gum arabic obtained from the natural stands of a. senegal variety kerensis. in addition, increase in soil organic carbon resulted in low level of carbon in gums. in merrile, soil ph influenced the level of carbon in gum arabic as shown in figure 1. soil nitrogen had direct effect on the amount of nitrogen in gum arabic obtained from the natural stands of a. senegal in merrile, laisamis and logologo which had direct relation to level of nitrogen. however, the impact of soil nitrogen was more pronounced in merrile than in laisamis and logologo. in sereolipi, soil nitrogen had no direct impact on the amount of nitrogen in gum arabic; this may be attributed to high level of soil phosphorus, which probably influences strong acidic nature of gum arabic (4.54) as shown in table 1. in addition, increase in soil organic carbon resulted in high level of nitrogen in gum arabic obtained from the natural stands of a. 1 soil n gum % n 0.8 n0.6 % 0.4 0.2 0 sereolipi merrile laisamis logologo sites figure 2. comparison of soil n with % nitrogen in gum arabic in sites. senegal in merrile, laisamis and logologo. comparison of soil nitrogen with percent nitrogen in gum arabic is given in figure 2. soil nitrogen in logologo was significantly higher (p < 0.05) than those of merrile, laisamis and sereolipi, while percent nitrogen in gum arabic was not significantly different in all the sites (figure 2). the levels of soil nitrogen in merrile, laisamis and logologo were correlated (p > 0.05) to the nitrogen content (0.31 0.32%) in gum arabic, respectively, while soil n (0.3%) in sereolipi was not significantly correlated to the nitrogen content (0.23%) in gum arabic harvested from natural stands of a. senegal variety kerensis(figure 2). nitrogen content in gum arabic from merrile, laisamis kabatila et al. 117 % c a n d n 1.6 1.4 1.2 1 sereolipi 0.8 merrile laisamis 0.6 logologo 0.4 0.2 0 gum % gum % soil % soil % n c n c figure 3. comparison of gum n with % soil nitrogen in the study sites and logologo were similar to those of international standards (0.24 0.41%) (anderson et al., 1990, 1991; biswas et al. 2000; buffo et al., 2001). soil carbon and soil nitrogen in logologo and laisamis were significantly higher (p< 0.05) than those of merille and seereolipi (figure 3). logologo and laisamis had similar trend in soil carbon, soil nitrogen and gum nitrogen, while merille and seereolipi varied in soil carbon, soil nitrogen, gum carbon and gum nitrogen (figure 3). conclusions and recommendations the soil characteristics are major factors that influence the gum quality and production, particularly soil reaction (soil ph), organic carbon, nitrogen and phosphorus. nitrogen content in gum arabic from merrile, laisamis and logologo were similar to those of international standards (0.24 0.41%). gums from the natural stands of acacia senegal variety kerensis in merrile, laisamis, logologo and marigat, baringo district can be mixed because of similar levels of nitrogen. the ph of gum arabic from the natural stands of acacia senegal variety kerensis in sereolipi, merrile, laisamis and logologo were 4.54, 4.5, 4.51 and 4.52 similar to the required international standards (ph: 4.2 4.8). further research is required to determine factors influencing the inherent high viscosity, nitrogen and specific rotation in relation to soil characteristics and varieties. acknowledgements the authors are grateful to chairpersons of project management committees, acacia operation project (aop) and their local communities for guidance and selection of sites in samburu and marsabit districts. the director, kenya forestry research institute (kefri) and aop fao -italian corporation for funding this work through dry land programme. references anderson dmw, brown ddm, morrison na, wang w (1990). specifications for gum arabic (acacia senegal) analytical data for samples collected between 1904 and 1989. food addit. contam. 7 (3): 303-321. anderson dmw, millar jra, wang w (1991). gum arabic (acacia senegal): unambiguous identification by 13c-nmr spectrograph as an adjunct to the revised jecfa specification and the application of 13c-nmr spectra for regulatory legislative purposes. food addit. contam. 8(4): 405-421. anderson dmw, weiping w (1990). composition of the gum from combretum paniculatum and four other gums which are not permitted food additives. phytochem. 29: 119-1195. anderson jm, ingram jsi. (1993). in tropical soil biology and fertility: a handbook of methods. cab international, wallingford, u. k. pp. 68 71. biswas b, biswas s, phillips go (2000). the relationship of specific optical rotation to structural composition for acacia and related gums. food hydrocolloids 14: 601-608. buffo ra, reineccius ga, oehlert gw (2001) factors affecting the emulsifying and rheological properties of gum acacia in beverage emulsions. food hydrocolloids 15: 53-66 chikamai bn, banks wb (1993). “gum arabic from acacia senegal (l) wild. in kenya. food hdrocolloids 7(6): 521-534. chikamai bn, gachathi n (1994). “gum and resin resources in isiolo district, kenya. ethnobotanical and reconnaissance survey”. east afr. for. j. 59(4): 345-351. chikamai bn, odera ja (2002). commercial plant gums and gum resins in kenya. sources of alternative livelihood and economic development in the drylands of kenya. executive printers. nairobikenya. fao (1995) gums, resins and latexes of plant origin. (non-wood forest products 6) fao, rome fao (1996). a review of production, markets and quality control of gum arabic in africa. fao, rome. fao (1999) gum arabic. (food and nutrition paper 52, addendum 7) fao, rome idris ohm, williams pa, phillips go (1998). characterization of gum from acacia senegal trees of different age and location using multidetection gel permeation chromatography. food hydrocolloids 12: 379-388 islam am, phillips go, sljivo mj, williams pa (1997). a review of recent developments on the regulatory, structural and functional aspects of gum arabic. food hydrocolloids 11: 493-505. karamalla ka, siddig ne, osman me (1998). analytical data for acacia senegal var. senegal gum samples collected between 1993 and 1995 from sudan. food hydrocolloids 12: 373-378. lelon jk (2008).uptake of micronutrients by acacia senegal varieties and it possible effect on gum arabic quality. phd thesis, university of nairobi. mocak j, jurasek p, phillips go, vargas, casadei e, chikamai bn (1998). the classification of natural gums. x. chemometric characterization of exudates gums that conform to the revised specification of the gum arabic for food use, and the identification of adulterants. food hydrocolloids 12: 141-150. okalebo jr, gathua kw, woomer pl (2002). laboratory methods of soil and plant analysis: a working manual. second edition. tsbfciat and sacred africa, nairobi, kenya. 118 afr. j. food sci. res. osman me, menzies ar, williams pa, phillips go, baldwin tc (1993a). the molecular characterisation of the polysaccharide gum from acacia senegal. carbohydr. res. 246: 303-318. osman me, williams pa, menzies ar, phillips go (1993b). characterization of commercial samples of gum arabic. j. agric. food chem. 41: 71-77. phillips go, williams pa (2001) tree exudate gums: natural and versatile food additives and ingredients. food ingred. anal. int. 23: 2628. sanderson gr (1996). gums and their use in food systems. food technol. 50: 81-84. woolhouse hw (1983). toxicity and tolerance in the responses of plants to metals. in: lange oc, nobel ps, osmond cb, ziegler h, (eds) encyclopedia of plant physiology ii. 12b. new york, ny, springer-verlag. pp. 245-300. african journal of food science research vol. 1 (4), pp. 033-036, december, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper effect of storage on the brewing properties of tropical hop substitutes okoro, casmir chukwuemeka 1* and aina, j. o. 2 1 department of food technology, yaba college of technology, lagos, nigeria. 2 university of ibadan, ibadan, nigeria. accepted 30 september, 2013 tropical hop substitute from utazi (utz) gongronema latifolium, bitter cola (btc), garcinia kola, bitter leaf (btl), vernonia amygdalina and a blend (1:1.41:2.89) of the three (hsb) respectively, were produced. stability studies were carried out to predict their suitability for brewing after one to six months storage at 5  1 o c and 27  1 o c, respectively. the level of reduction in their -acid, iso--acid, soft resin, analytical bitterness and degree of utilization levels were determined. result showed that there was a general reduction of between 10 to 30% in these parameters. however, the (hsb) recorded lower losses than btc, blf, and utz. also the samples were more stable at 5  1 o c than at 27  1 o c. samples treated with ca(oh)2 had lower rate of decrease instability with percentage loses of between 5 to 15% recorded in all the samples. pertinently, these levels of reduction were comparable to the level of losses reported in conventional temperate hops (humulus lupulus) stored under similar conditions. conclusively, tropical hop substitutes stored at 5  1 o c to 27  1 o c can still be used for brewing even after three to six months storage. key words: hop substitutes, hops, -acid, iso--acid analytical bitterness, brewing. introduction the conventional hops are produced from the flowers of the plant, humulus lupulus, and are a major raw material used in beer brewing for imparting flavour, colour, bitterness, foam head stability and antiseptic properties (hough, 1980). however, hop plant is a temperate crop and cannot be successfully grown in tropical countries like nigeria: hence its importation for beer brewing is imperative. according to the federal office of statistics (1986) report, it cost nigeria about 5.5 million dollars to import hops in 1985. this high cost trend could be reduced if hops substitutes can be sourced locally. since the hops of commerce are bitter, some edible tropical vegetables with bittering principles have been researched into as potential hops substitutes. gentalium (1975) reported the use of bitter leaf (gongronema latifolium) in brewing the popular telabeer in ethiopia. okafor and anichie (1983) brewed an acceptable lager beer with utazi leaf (vernonia amygdalina). bitter cola *corresponding author. e-mail: emoko102003@yahoo.com. (garcinia kola), according to hutchinson and dalziel (1985), enhances flavour of local drinks when chewed while drinking them. the work of okoro, (1990, 1993) showed the successful development of a tropical hops substitute from a blend of utazi, bitter cola and bitter leaf combined in the ratio of 1: 1.41:2:89, respectively. the lager beer produced using this tropical hop substitute blend (hs-blend) was reported to be comparable and significantly not different from beers brewed with the conventional temperate hops. the use of these tropical hop substitutes were due to their high content of -acids, iso-acid and essential oils at levels comparable to those of the temperate hop substitutes (okafor and anichie, 1983; okoro, 1993). however, for the successful use of these developed tropic hops substitute or their blends, the shelf stability of these products with storage has to be determined to obtain best storage conditions or duration or treatment that will improve their shelf stability in terms of retaining their brewing potentials. this is necessary because the -acids, iso--acids and essential oils found in these tropical hop okoro and aina 033 substitutes may be unstable with storage. the aim of this work is therefore to determine the level of retention of bitterness and flavour principles in these tropical hop substitutes at different storage conditions and periods, as well as to determine the influence of different preparations or treatments on the shelf life of these hop substitutes. materials and methods raw materials procurement the bitter leaf, bitter cola and utazi were procured fresh from mile 12 market, lagos. they were washed, destalked or decorticated (for bitter cola) sorted and dried at 50  2 o c to moisture content of 10  2% in drought air oven. after which they were milled into powder, using hammer mill (chrysty – lab mill model 8) to 0.1 m diameter particle size. the powders were blended in the ratio of 1:1.41:2.89, utazi : bitter cola : bitter leaf, respectively, as established using linear programming (okoro, 1990, 1993) . the blend was compounded into 1 g pellets using a laboratory hand screw press locally designed and fabricated. preparation of samples for shelf-stability studies reports on the trial brewing with these samples were reported by okoro (1993). the four hop substitutes were utazi pellet (utz), bitter leaf pellet (blt), bitter cola pellet (btc) and hop substitutes blend (hsb). to further improve the stability before storage, another set of the hsb was blended with 1% ca(oh)2 before palletizing it. all the samples were vacuum packed respectively in high density polyethylene bags and stored at 27  1 o c and 5  1 o c for period ranging from 1 to 6 months. the stability and quality changes of the samples over this storage period were monitored every two months by determining their levels of soft resin retention, acid retention, iso--acid retention, bitterness level retention and the degree of hop utilization. soft resin determination 10 g of each sample was dissolved in 10 ml of hexane, thoroughly stirred and filtered (using watman no 14 filter paper) . filtrate was dried to a constant weight at 50 o c. the soft resin was calculated as the percentage of the original weight of sample dissolved in the hexane. -acid determination to a 0.15 g of the samples was added 100 ml cold methanol in a (gallenkamp) flask shaker. the solution was then centrifuged at 2500 pm for 20 min and the decanted supernatant was acidified with 0.002 n hcl and its absorbance at 355, 325 and 275 nm was determined using spectrophotometer (pye-unicam sp6-550 uv/vis. model) and the -acid calculated using aoac (2000) and asbc 1976 methods: -acid (mg/l) = 73.79 (a325) – 51.56 (a355) – 19.07 (a275) where a is absorbance reading at the specified wave length. iso--acid determination 15 ml sample extract was acidified with 0.5 ml 6 n hcl and mixed with 15 ml of pure iso-octane in a shaker (gallenkamp flask shaker) , 10 ml of the isoactane extract was washed with 10 ml of a mixture of methanol and 4 n hcl (68:32, v/v). after which 5 ml, of the washed iso-octane layer was diluted with 5 ml of alkaline methanol (60:40, v/v methanol : 0.5 n naoh) and its absorbance read at 255 nm. the iso--acid (mg/l) was calculated according aoac (2000) method of analysis. iso--acid (mg/l) = a255 (96.15) + 0.4 preparation of the vegetable water extract for analytical bitterness determination an 0.15% (w/v) solution of the respective samples was made using distilled water. the solution was boiled for 90 min cooled and filtered using watman no 14 filter paper. 10 ml of the water extract of each sample were acidified with 0.5 ml 6 n hcl and subsequently extracted with 20 ml of isooctane in a shaker (gallenkamp flask shaker). the absorbance of the iso-octane extract was determined at 275 nm using a spectrophotometer (pye-unicam sp 6-550 uv/vis model) . the analytical bitterness was calculated according to ebc (1975) method and reported as analytical bitterness unit ( 0 ebu). a275 = 0 ebu, where a is absorbance at 275 nm. degree of utilization determination the degrees of utilization of the bitterness potentials in the hop substitute were calculated as: % utilization = [iso--acid (mg/l) x 100]/-acid (mg/l) results and discussion results in table 1 show that the soft resin content of all the tropical hop substitutes (ths) decreased with storage; hsb (1015%), utz (15-30%) blf (12-19%) and btc (10-23%) over 6 months storage. these results compares well with losses in resins reported for the conventional hops stored at 25 o c for 30 weeks (12-17%) by marr (1985) and laws (1983). the reduction in the soft resin content of hops is a common phenomenon which is associated with the oxidative depreciation of the soft resins to hard resins with storage, hough (1980). however, the low percentage reduction especially, with storage at 5 o c show that the ths can still retain up to 7085% of their bitterness properties, and could still function well as hop substitute for brewing after 6 months of storage. the stability of the -acid component of the soft resin of any given hop is very important in determining the suita bility of the hop for brewing. it is the -acid that impacts the bitterness in the beer. results in table 2 show that the -acid content of the tropical hop substitutes (ths) were more stable at 5  1 o c than at 27  1 o c storage with reduction of 15.0% for hsb, 21% for utz, 15.41% for blf and 31% for btc. however, the -acid content of the hop substitutes blend was more stable than those present in the individual substitutes. generally, the instability of the -acid is associated with that of the soft table 1. changes in the soft resin levels of hop substitutes with storage. samples soft resin levels (%) fresh samples (%) 1 month 3 months 5 months 6 months 51 0 c 271 0 c 51 0 c 271 0 c 51 0 c 271 0 c 51 0 c 271 0 c hsb 15.70 15.65 14.98 15.40 14.10 15.10 13.63 14.77 15.03 (1.00)* (4.59) (2.55) (9.87) (3.84) (13.18) (4.77) (15.03) utz 16.10 15.88 14.32 15.86 12.86 15.25 12.03 14.21 11.22 (1.37) blf 12.84 12.70 12.20 12.01 11.60 11.92 10.68 11.70 10.46 (1.09) (4.98) (6.46) (9.66) (7.17) (16.82) (8.91) (18.54) btc 9.74 9.22 8.85 9.10 8.13 8.25 7.82 9.97 7.54 (5.33) (9.14) (6.75) (16.54) (15.29) (19.71) (18.17) (22.59) hsb = hops substitutes blend, utz = utazi, blf = bitter leaf, btc = bitter cola. *values in parenthesis indicate % reduction. table 2. -acid stability of hop substitutes with storage. samples -acid stability (mg/l) fresh 1 month 3 month 5 month 6 month samples 0 271 0 c 0 271 0 c 0 271 0 c 0 271 0 c 51 c 51 c 51 c 51 c hsb 10.71 10.68 10.26 10.27 9.63 10.11 9.46 9.48 9.11 (0.28) (4.20) (4.11) (10.08) (6.14) (11.6) (11.48) (8.25) utz 12.81 12.42 12.12 11.81 10.73 11.51 10.05 11.20 9.25 (3.04) (5.39) (7.81) (16.24) (10.15) (22.10) (12.57) (27.80) blf 8.98 8.87 8.58 8.53 8.00 8.31 7.73 8.01 7.04 (1.22) (4.45) (5.01) (10.91) (7.46) (13.92) (10.80) (21.60) blc 4.94 4.84 4.70 4.61 4.23 4.20 4.00 4.20 3.41 (2.02) (4.46) (6.68) (14.57) (14.98) (19.43) (14.98) (30.97) hsb = hops substitutes blend, utz = utazi, blf = bitter leaf, btc = bitter cola. *values in parenthesis indicate % reduction. table 3. analytical bitterness of hop substitutes. samples 0 month 1 month 3 month 5 month 6 month 0 ebu 51 0 c 271 0 c 51 0 c 271 0 c 51 0 c 271 0 c 51 0 c 271 0 c hsb(% 24.51 24.29 24.13 24.15 23.62 24.14 23.28 23.33 22.55 reduction) (0.89) (1.55) (1.42) (3.63) (1.51) (5.01) (4.81) (8.00) utz 26.50 26.17 25.91 25.43 24.50 25.69 25.69 24.41 22.90 (% reduction) (1.32) (1.92) (4.04) (7.55) (6.84) (6.84) (7.89) (13.58) blf 24.00 23.70 23.24 23.43 22.94 23.50 23.29 22.62 21.97 (% reduction) (1.25) (3.16) (2.38) (4.42) (2.98) (7.12) (5.75) (8.45) blc 15.00 14.90 14.78 14.74 14.50 13.70 14.36 14.48 14.06 (% reduction) (0.67) (0.67) (0.67) (3.33) (2.00) (4.27) (3.47) (6.27) resins. this, according to hough (1986), is due to oxida tion of -acid with storage. the bitterness levels of the hop substitutes samples (table 3) reduced, with storage at both storage tempera tures. however, the percentage reductions in bitterness units were observed to be lower (between 0.5 to 8%) than percentage losses in -acid of the samples. this is consistent with the report of gill et al. (1979) that the loss 034 afr. j. food sci. res. okoro and aina 035 table 4. percentage utilization of hop substitute with storage. samples 0 month 1 month 3 months 5 months 6 months hsb acid iso-acid -acid iso--acid -acid iso--acid -acid iso--acid -aid iso--acid (% utilization) mg/l (mg/l) mg/l (mg/l) mg/l (mg/l) mg/l (mg/l) mg/l (mg/l) hsb 10.71 5.17 10.26 4.91 9.63 4.02 9.36 3.44 8.91 3.03 (% utilization) (48.9%) (47.90%) (41.74%) (36.75%) (34.01) utz 12.82 4.98 12.12 4.46 10.73 3.73 10.05 3.04 9.25 2.57 (% utilization) (38.85%) (36.81%) (34.97%) (30.23%) (27.80) blf 8.48 4.17 8.58 3.83 8.00 3.24 7.73 2.81 7.04 2.31 (47.12%) (44.64%) (40.45%) (36.32%) (32.82) btc 4.94 1.98 4.84 1.83 4.24 1.48 4.00 1.27 3.41 0.99 (% utilization) (40.00%) (37.89%) (35.10%) (32.73%) (26.91) table 5. stability effect of ca(oh)2 treatment on hop substitutes. samples 0 month 1 month 3 month 5 month 6 month 51 0 c 271 0 c 51 0 c 271 0 c 51 0 c 271 0 c 51 0 c 271 0 c -acid acid -acid -acid -acid -acid -acid -acid -acid mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l hsb % red. 10.71 9.58 10.26 8.58 9.63 8.72 9.36 8.91 8.91 (% reduction) (10.05%) (4.20%) (17.10%) (18.50%) (19.50%) (12.50%) (20.54%) (12.81%) utz 12.82 10.88 10.26 10.21 10.73 10.13 10.05 8.75 8.91 (% reduction) (15.12%) (5.45%) (23.0%) (16.32%) (28.78%) (4.75%) (20.54%) (12.81%) blf 8.98 7.22 8.58 7.15 8.00 7.19 7.73 7.08 7.04 (% reduction) (11.50%) (4.57%) (18.8%) (11.08%) (19.90%) (13.90%) (21.20%) (14.41%) btc 4.94 4.20 4.84 1.86 4.24 3.78 4.00 3.53 3.41 (% reduction) (14.98%) (4.85%) (21.10%) (14.46%) (26.48%) (19.25%) (28.50%) (21.25) in bitterness potentials of stored hops was usually less that 50% of the reduction in its -acid and soft resin values. this, according to hough (1986), is because some oxidation products of -acid and -acids are themselves bitter and that contributes to the bitterness values of hops. the reduction in the percentage utilization of the bitterness principles in the hop substitutes with storage (table 4), were also not as high as recorded for -acid reduction with storage (table 2). a net reduction in utilization of 14.89% for hsb, 11.05% for utz, 14.30% for blf and 11.09% for btc were observed. this is because the percentage utilization, like the bitterness level (table 4) is not only caused by the -acid level but also by its iso--acid level. according to hough (1986), the percentage utilization is measure of the extent of extrac tion of -acids and its isomerization and bitterness potentials in water, wort or beer. the utilization level obtained from the hsb (34%), btl (32%), btc (30%) and utz (28%) after 6 month of storage, compares well with those reported by laws (1983) for the conventional hops (34 37%). there was a marked increase in the -acid stability of tropical hop substitutes treated with ca(oh)2 before pal letizing and those not treated (table 5). hsb treated with ca (oh)2 and stored for six-months at 27  1 o c had a 12.81% reduction in -acid level compared to the untreated hsb with 20.54% reduction in -acid values. the same trend in reduction was observed in utz (26.51%), blf (14.11%) and btc (21.25%). this is consistent with the use of ca(oh)2 as hop stabilizer in the conventional hop pellet production. the observed improvement in the stability of -acids in ca(oh)2 treated pellets may be due to the formation of calcium salts of the -acid. the caacid salts, according to grant (1979) are more stable to oxidation than -acid. expectedly, all samples stored at 5  1 o c recorded more stability in all parameters than those stored at 27  1 o c which is consistent with the stabilization effect of cold temperature storage against oxidation changes. conclusion the observed reduction in the soft resin levels, -acid levels, bitterness levels and utilization levels with storage of the tropical hop substitutes are consistent with storage changes, but their levels of reduction are similar to those 036 afr. j. food sci. res. recorded for the stored conventional hops especially, if treated with ca(oh)2 before palletizing and storing at 5  1 o c. essentially, tropical hop substitutes, if produced and utilized within three to six-month can yield sufficient bitterness principles when used in beer brewing. to obtain a shelf stable hop substitute from the tropics, a blend of the three identified substitutes (utz, bfl and btc) treated with 1% ca(oh)2, palletized, vacuum packed and stored at 5  1 o c and used within 6 months of storage is recommended. references american society of brewing chemist (asbc) (1976). methods of analysis. 7 th edition. washington d.c. pp 560-562. aoac (2000) official methods of analysis, 17th edition vol ii chapter 27, p. 16. european brewing convention. analytical (ebc) (1975). issued by the analysis committee of the european convention. 3 rd edition. london. federal office of statistics (fos) (1986). the nigerian trade summary. federal ministry of commerce and industry abuja nigeria. gentalium a (1975). some common medicinal and poisonous plants used in ethiopian folk medicine in: useful plants of west tropical africa. vol. 1, 2 nd edition (edited by hutchinson kj, dalziel mj) royal botanical gardens, kew, london pp. 503-508. gill r, laws drj, goldfinch p, henley hp (1979). storage characterristics of some hop varieties. j. inst. brew. 85(2): 243-247. grant hl (1979). developments in the science of hops. master brewers association of the americas 15(14): 224-228. hough js (1986). hops and wort treatment in: introduction to brewing science and technology part 1 (eds rainbow c, fleat ges). the inst. brew. london pp. 28-41. hutchinson kj, dalziel mj (1985). the useful plants of west tropical africa vol. 1, 2 nd edition. royal botanic gardens, kew, london pp 501-508. laws drj (1983). commercial brewing and storage trials with yeamen hop. j. inst. brew. 89(2): 92-95. marr jf (1985). hop industry committee analysis of hop of 1984 crop: rep. j. inst. brew. 91(1): 184-191. okafor n, anichie gn (1983). west african hop substitutes for sorghum lager beer. brew. dist. int. 13(1): 20-23, 31. okoro cc (1990). development of tropical hops substitute for the nigerian brewing industry. msc thesis, submitted at university ibadan, nigeria. okoro ce (1993). development of hop substitutes from tropical plants. nbte j. agric. technolo. maiden ed. p. 30-35. author(s) retain the copyright of this article. full length research paper vitamin a losses in a supply chain of fortified food vehicles which comprised two brands of vegetable cooking oil and maize flour tomika orton mponda*, patrick fisher khumbo and peter robert mabedi department of physics and biochemical sciences, the polytechnic, university of malawi, private bag 303, blantyre 3, malawi. accepted 10 november, 2016 vitamin a levels were analyzed in fortified vegetable cooking oil and maize flour through a commercial food supply chain, from the production line to selected retail outlets using standard procedures over different times of exposure to sunlight. samples from the production line acted as controls. in all the cases, vitamin a levels decreased at various stages of the supply chain with the least retention in products sold by street vendors. statistical analysis showed significant losses (p<0.05) in vitamin a after the samples were exposed to sunlight. these results indicate that although food fortification is crucial in making micronutrients available to poor households, especially in developing countries like malawi, there is need to sensitize retailers on proper handling and storage of these products to minimize losses in the supply chain. key words: fortification, vitamin a, supply chain, sunlight, vegetable cooking oil, maize flour. introduction food fortification has recently been highlighted as one way of ensuring the supply of micronutrients to most of the population in the developing world (heikens, 2007). for example, there have been mass fortification programmes in zambia, central america and egypt (who, 2009) with the aim of decreasing micronutrient deficiencies among poor communities, especially children of 6-59 months and lactating women within 8 weeks of *corresponding author. e-mail: tomika.orton@yahoo.com child birth (gom, 2009). a national micronutrient survey conducted in malawi in 2001 found that 60% of children under the age of five, 57% of women of child bearing age, and 38% of school children were suffering from subclinical vitamin a deficiency (vad) (gom, 2009). such high levels of micronutrient malnutrition have been linked to ailments which include low immunity, impaired physical, mental and psychomotor development and severe cases, night blindness. such effects may affect child’s mental development and in the long term national economic development may suffer. vitamin a deficiency has been reported to cause childhood blindness to in ternationa l scholars journa ls african journal of food science research issn: issn 2375-0723 vol. 4 (5), pp. 033-037, december, 2016. available online at www.internationalscholarsjournals.org © international scholars journals tomika et al. 034 estimated 140 million children worldwide (combs, 2012). severe vitamin a deficiency has also been reported in several sub-saharan countries including nigeria, egypt, south africa, kenya, namibia and tanzania (klemm et al., 2010). in south africa, a country with a relatively good economic standing in africa, it was recently found that 49% of preschool aged children and up to 68% among women of reproductive age had vad (mostert et al., 2005). these data are worrisome and certainly suggest an urgent need for interventions. despite the severe consequences that result from vitamin a deficiency, the good news is that a diverse diet, which includes foods of animal origin that are rich in preformed vitamin a (esters of retinol), might be sufficient to satisfy the daily requirements of vitamin a. however, in most developing countries, diets are monotonous (ruel, 2001) and mainly based on cereals and legumes that are poor sources of vitamin a (west et al., 2002; who, 1998). vitamin a is virtually absent in whole-grain cereals and flours. because vitamin a deficiency results mainly from chronic dietary insufficiency, food fortification has been identified as an effective approach to abate the problem (klemm et al., 2010). for example, it is possible to fortify flour from cereal grains using a powdered form of vitamin a, retinyl palmitate which has been found to be more stable than retinyl acetate (combs, 2012). the fortification of vitamin a, which is fat soluble, in cooking oil is even much simpler and cheaper and can be done either with retinyl acetate or retinyl palmitate in oil base (johnson, 1997). in a drive to promote the reduction in micronutrient deficiency, the government of malawi has been advocating for micronutrient supplementation of foods. in addition to iron and iodine, one of the target micronutrient for this exercise is vitamin a and the food vehicles chosen are sugar, vegetable cooking oil and maize flour (yeudall et al., 2005). fortifying a widely consumed food product or additive makes it easy to deliver low doses of vitamin a daily to a large number of people (dary and mora, 2002). this was the rationale behind the choice of these foods that are consumed by the majority of malawians most of who live on less than 1 us$ per day. actually, the current human development index for malawi is 0.400 and not only ranks malawi at 141 out of 187 countries with comparable data but also puts it below the average for sub-saharan africa which is at 0.463 (undp, 2012). regardless of whether the vitamin a occurs naturally or has been added to a food product through fortification or other means, the potential exists for losses by chemical or physical means. these losses may also occur due to exposure to light and heat exposure resulting into oxidation (butt et al., 2007). vitamin losses are to some extent inevitable in the manufacturing, distribution, storage and preparation of processed foods (sustain, 1999). retention of vitamin a in fortified foods is important for determination of the efficacy of the fortification programs. it is therefore important to understand the degree of loss in the supply chain so that proper strategies can be put in place to ensure that the consumer is getting the intended dosage of the micronutrient (butt et al., 2007). the malawi bureau of standards (mbs) recommends a vitamin a content range of 30 to 60 mg/l for vegetable cooking oil and 10 to 40 mg/l for maize flour (sustain, 1999). in malawi, however, vitamin a losses in the food supply chain have not been evaluated. this study therefore assessed the extent of vitamin a losses in a supply chain of fortified food vehicles which comprised two brands of vegetable cooking oil and maize flour. materials and methods the study was done in blantyre city, malawi, targeting one major flour producing company ,and two major oil manufacturing factories in malawi´s commercial capital. one of the oil factories produces vitamin a fortified soybean cooking oil while the other produces vitamin a fortified sunflower cooking oil. sample collection from production line triplicate samples (1 l each) of freshly produced fortified vegetable cooking oil (sunflower and soybean) were collected from the two sampling points. triplicate (1 kg) samples of fortified maize flour were also collected from the production line of the flour company. the fortified cooking oil and fortified flour bought form the manufacturing companies acted as a control. fresh samples from production line were analyzed immediately after sampling and the remaining portions were analyzed after exposure to sunlight at intervals of seven, fourteen and thirty five days. sample collection from retail markets sampling from the retail markets was done by gathering information on the cooking oil bottles (batch coding and manufacturing date) to determine the most recent batch for use in the study. this was done because it was difficult to trace the same batches that had been sampled from the production line to the targeted retail markets. the most recent batches of soybean cooking oil sampled from usave supermarket were the ones that had been stored in the shop for fifteen days from the manufacturing date. ndirande market soybean cooking oil samples were analyzed after being retained by the retailer for six days while zingwangwa market soybean cooking oil samples were analyzed after being retained by the retailer for seven days. recent batches for sunflower oil had been in the supermarket for thirteen days on the day of sampling and the sunflower oil from ndirande and zingwangwa market were three and five days old on the day of sampling. flour samples collected from usave supermarket had been stored for ten days from the manufacturing date while ndirande and zingwangwa samples were five and six days old respectively on sampling day. triplicate 1 l samples of the same brands of fortified vegetable cooking oil were bought from a supermarket (usave) and street vendors in two open markets (ndirande and zingwangwa) in blantyre city. samples of cooking oil and maize flour from the retail markets were analyzed immediately after collection. the percent loss in vitamin a was calculated using the following formula: loss (%) = [(initial vitamin a content new vitamin a content) / initial vitamin a content] x 100. vitamin a analysis vitamin a analysis in oil analytical procedures used in vitamin a analysis of the oil samples are those from the manual for internal monitoring of oil fortified with vitamin a (east, central and southern african health community, 2007). approximately two grams of oil was weighed into a twenty 5 ml 25 ml volumetric amber flask and mass was recorded to four decimal places. dichloromethane was added to the flask to dissolve the oil and mixed thoroughly. the same process was repeated using unfortified (blank) oil. absorbance reading of samples and unfortified control was read on a spectrophotometer at 325 nm. retinyl palmitate concentration of the oil sample was estimated using the following equation: abs corrected x vf x cf spec retinyl palmitate (mg/kg ) = a x w where abs corrected = abs sample – abs unfortified oil; vf = final volume; cf=correction factor of the spectrophotometer, ideally; a= retinyl palmitate absorption coefficient in dichloromethane (mg-1 cm-1 l) 0.094; w= weight of sample. vitamin a analysis in flour vitamin a in flour was determined using spectrophotometric method (aoac, 2002). about 0.05-1 g of flour and thick porridge samples were weighed using analytical scale (model: adam pw 124) into 50 ml conical centrifuge tube. six milliliters of dichloromethane was added to samples. the mixture was vortex for 2 min, and then 1.0 ml of methanol was added to the mixture and vortexed for 2 min. ten milliliters of distilled water was added to the mixture, the vortex for 1 min. the mixtures were centrifuged for 2 min to separate the two phases. the dichloromethane phase went to the bottom. using the pasteur pipette, the flour pellet that were formed between the two liquid phase was set aside, and then the organic phase was transferred in to 10 ml measuring cylinder. the mixture was left to stand and the remaining water was removed with a pasteur pipette, then the volume of the extract was recorded. the volume of extract was used to calculate the concentration. the extract was used for the determination of vitamin by directly recording the absorbance at 325 nm. preparation of standard vitamin a solution one hundred milliliters amber was tare on an analytical balance. using the pipette, 75.6 mg of standard retinyl palmitate (usp) was transferred into volumetric flask. then dichloromethane was added to the flask to dissolve the vitamin a and make up the volume. taking into account the actual concentration of the stock solution, appropriate aliquots was pippeted into a set of 50 ml amber volumetric flasks to give a set of standard solutions. a standard plot was prepared by reading the absorbance of the standard solutions of vitamin a. to 50 ml of volumetric flask, 0.1, 0.2, 0.3, 0.4 and 0.5 ml of standard solution was pippeted, then made up to the volume. the absorbance of these solutions was read at 325 nm and a standard plot was made. the absorbance of sample extract was measured and compared against the standard plot to determine the concentration. the following formula was used to calculate vitamin a concentration in samples. the concentration of vitamin a in extract of samples, treated in a similar manner to the standard solution was calculated using slope and constant of the standard plot. absorbance = slope x concentration + constant 035 afr. j. food sci. therefore concentration of vitamin a = (absorbance – constant) / slope of plot concentration of vitamin a (mg retinol/kg flour ) would therefore be according the following: conc. of vit.a in extract (m) x vol ( ml) of extract solution x 5.249 x 105 mass of flour weighed ( g ) data analysis the mean, standard deviation and 95% confidence intervals (ci) of the mean were calculated using spss version 20. single factor analysis of variance (anova) and turkey honest significant difference (hsd) post hoc tests were also done using spss. results and discussion the malawi bureau of standards recommends 30-60 mg/l (malawi standard 51, 1988), in vegetable cooking oil and 10-40 mg/l in maize flour. this study results generally indicated varied vitamin a losses in all samples along the supply chain and during exposure to sunlight but drastic changes were observed in maize flour which lost up to 61% after 35 days of exposure to sunlight and an average of 55% in samples from street markets (table 1). actually, after two weeks of exposure to sunlight, maize flour had already lost most of the vitamin a to levels below the malawi bureau of standards minimum value of 10 mg/l. the vitamin a loss in maize flour could be attributed to storage conditions (left in open basins at the market) of the flour which led to direct exposure to light. one way of analysis of variance ( anova) showed that there was significant loss of vitamin a levels in fortified sunflower oil after 35 days of sunlight exposure as compared to fortified soybean oil; 40% versus 27% (p < 0.05) respectively. the results obtained in this study for vitamin a loss in fortified soybean oil are in agreement with the finding of puysuwan et al. (2007) who noted that fortified soybeans oil in losed pet bottles exposed to sunlight for 4 weeks at room temperature reduced thr initial vitamin a concentration by 27.1 ± 12.1% without accounting for the oxygen exposure upon opening.the vitamin a losses in oils and maize flour from samples from street vendors (table 2) could be attributed to prolonged exposure to sunlight and also the nature of the packaging material used (sachets for oils and basins for flour) by the vendors (chakravarty,2000). this is so because packaging is also a contributing factor to the losses in vitamin a (oluwalana et al., 2015). loss of vitamin a in vegetable cooking oils was lower than in maze flour and this could be attributed to the fact that oil stabilizes retinol and delays oxidation of the vitamin (dutrade – oliveira, 1994, pignitter et al., 2012). though the samples used were produced by large tomika et al. 036 table 1. vitamin a levels (mg/l) in the various samples analyzed from production line. sample source and treatment (soybean oil) (sunflower oil) (maize flour) production line – fresh 41.37±0.09a 36.76±0.13 a 21.53±0.93 a production line – 7 day sunlight exposure 33.89±0.64 b 27.35±0.56 b 10.92±0.46 b production line – 14 day sunlight exposure 31.98±0.52 c 24.06±0.29 c 9.22±0.35 c production line – 35 day sunlight exposure 30.11±0.04 d 22.83±1.69 c 8.49±0.06 c values are mean ± standard deviation of triplicate samples. values within the same column with the same superscript are not significantly different (p < 0.05). table 2. vitamin a levels (mg/l) in the various samples analyzed from retail markets. sample source and treatment (soybean oil) (sunflower oil) (maize flour) supermarket 37.51±0.86 36.12±0.17 17.68±0.53 ndirande market 31.91±0.18 30.15±0.44 9.25±0.91 zingwangwa market 31.57±0.80 29.78±0.89 10.04±0.34 values are mean ± standard deviation of triplicate samples. manufacturing industries in malawi, the authors acknowledge the limitation that the sampling points were only in one location (blantyre). other retailers who also serve a large portion of the population were left out, making the findings suggestive of the likely trend to be observed nationwide rather than being conclusive. the study did not measure the peroxide values of the oils which are also influenced by exposure to sunlight. for this reason, it is recommended that further studies should include a measure of the peroxide values of the oils which would give an indication of the quality (chabiri et al., 2009) and storage stability of the product. conclusions this study shows loss of vitamin a in the commercial food supply chain of vegetable cooking oil and maize flour upon exposure to sunlight. handling of vitamin a fortified foods should therefore be away from sunlight.the study also revealed higher vitamin a lose in maize flour than vegetable cooking oil when exposed to sunlight for the same duration. sunflower cooking oil also exhibited higher vitamin a losses than soybeans oil.it is therefore recommended that more effort in future interventions of abating vitamin a deficiency should focus on soybean cooking oil as a vehicle. retailers should also be sensitized on the importance of proper handling and storage of fortified cooking oil and maize flour to avoid loss of vitamin a. conflict of interests the authors have not declared any conflict of interests. acknowledgement the authors thank the university of malawi through the department of physics and biochemical sciences for financially supporting this study. references aoac (2002). official methods of analysis of association of official analytical chemist, 17th ed., arlington virginia: association of official analytical chemist. chapter 32, pp. 1, 2, 23 and 43. butt ms, arshad mu, alam ms, nadeem mt (2007). bioavailability and storage stability of vitamin a fortificant (retinyl acetate) in fortified cookies. food res. int. 40:1212-1219. chabiri, sa, hati ss, dimari ga,oguguaja vo (2009). comparative quality assessment of branded and unbranded edible vegetable oils in nigeria. pac. j. sci. technol. 1(2):927-934. chakravarty i (2000). food-based strategies to control vitamin a deficiency. food nutr bull 21:135-43. combs gf (2012). vitamin a. the vitamins (fourth edition) 2012. pp. 93-138. dary o, mora jo (2002). international vitamin a consultative group. food fortification to reduce vitamin a deficiency: international vitamin a consultative group recommendations. j. nutr. 132:2927-2933. dutra-de-oliveira je (1994). effect of heat treatment during cooking on the biological value of vitamin a fortified soybean oil in human. int. j. food sci. nutr. 45:2003-2017. east, central and southern health community (2007). manual for internal monitoring of oil fortified with vitamin a (quality assurance and quality control, qa/qc). arusha, tanzania. gom-malawi government (2009) national nutrition policy and strategic plan (2007-2011). office of president and cabinet, department of nutrition hiv and aids. lilongwe, malawi. heikens gt (2007). how can we improve the care of severely malnourished children in africa? plos med; 4(2):45. doi: 10.1371/journal.pmed.0040045. johnson le (1997). oils, fats and margarine: overview of technology. food fortification to end micronutrient malnutrition. state of the art :22-26 the micronutrient initiative, international development research centre ottawa, canada. klemm rdw, keith p, west jr., amanda cp, johnson q, randall p, ranum p, northrop-clewes c (2010). vitamin a fortification of wheat flour: considerations and current recommendations. food and nutrition bulletin, supplement no. 1.the united nations university. vol. 31. malawi standard 51 (1988). edible oil – general standard, malawi bureau of standards, blantyre, malawi. mostert d, steyn np, temple nj, olwagen r (2005). dietary intake of pregnant women and their infants in a poor black south african community. curationis 28:12-19. oluwalana ib, oluwamukomi mo, toriola bo, karim or (2015). influence of packaging materials and storage conditions on the vitamins a and e storage stability of palm oil in nigeria. adv. res. 4(3):191-202. pignitter m, somoza v (2012). are vegetable oils always a reliable source of vitamin a? a critical evaluation of analytical methods for the measurement of oxidative rancidity. sight and life, 26:18–27 puysuwan l, chavasit v, sungpuag p, hediger d, punvichai t (2007). feasibility and use of vitamin a fortified vegetable oilsamong consumers of different socioeconomic status in thailand. food nutr. bull. 28:181−188. ruel mt (2001). can food-based strategies help reduce vitamin a and iron deficiencies? a review of recent evidence. international food policy research institute; washington, dc, usa. solon fs, sancex-fermin le, wambangco ls, solon mam (1999). final report-iron and vitamin a stability in flour and products. manila: nutrition center of the philippines. sustain (1999). the progress of wheat flour fortification with vitamin a in the philippines. final report of the micronutrient assessment project u.s. agency for international development, sustain washington, dc. undp-united nations development programme (2012). human development report 2011/2012-country fact sheets –malawi. 037 afr. j. food sci. west ce, eilander a, van lieshout m (2002). consequences of revised estimates of carotenoid bioefficacy for dietary control of vitamin a deficiency in developing countries. j. nutr. 132(9):2920s-2926s. who-world health organization (1998). safe vitamin a dosage during pregnancy and lactation. document who/nut/98 4 world health organization geneva, switzerland 2. who-world health organization (2009). global prevalence of vitamin a deficiency in populations at risk 1995–2005: who global database on vitamin a deficiency. who, geneva. yeudall f, gibson rs, cullinan tr, mtimuni b (2005).efficacy of a community-based dietary intervention to enhance micronutrient adequacy of high-phytate maize-based diets of rural malawian children. public health nutr. 8:826-836. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (1), pp. 155-159, january, 2015. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper proximate composition and sensory properties of moringa fortified yellow maize-ogi abioye, v.f department of food science and engineering, lautech, ogbomoso, oyo state, nigeria. e-mail: vf_abioye@yahoo.com accepted 11 january, 2015 ‘ogi’ was produced from yellow variety of maize to obtain a fermented gruel from ogi and the effect of moringa leaves fortification on the nutritional value and the sensory properties of yellow maize-ogi was investigated. the moringa leaf powder was substituted into yellow maize-ogi in these formulations; 100:0, 90:10 and 85:15. the effects of the moringa leaves powder substitution on the proximate content, mineral content, betacarotene, swelling capacity and the sensory properties were determined. the sample with 15% moringa leaf substitution had about 95% increases in protein content. the crude fibre and ash contents increased from 2.33 and 1.75% to 3.57 and 2.33% with sample substituted with 15% moringa leaf having the highest. there was increase in the values of the mineral contents of the ogi samples with increase in the level of moringa leaf substitution; calcium content increased from 136.0 to 466.0 mg/100 g; magnesium from 31.67 to 123.00 mg/100 g; iron increased from 5.23 to 14.77 mg/100 g; potassium from 33.33 to 215.00 mg/100g; zinc from 0.20 to 0.73 mg/100 g and copper from 0.27 to 0.60 mg/100 g. beta – carotene of 1126.67 µg/100 g was obtained with 15% moringa leaf substitution. the swelling capacity decreased with increase in the level of moringa leaf substitution. this study revealed that fortification of ogi with moringa leaves at 10% improved the nutritional quality of ogi and was still generally acceptable. key words: maize, yellow maize-ogi, substitution, moringa leaves, fortification. introduction „ogi‟, a fermented gruel or porridge made from maize (zea mays), sorghum (sorghum bicolor) or millet (pennisetum glaucum) is a common food in west africa. it serves as a major weaning food for infants, a breakfast meal for both children and adults and sometimes it is chosen as food for the sick (oyewole, 1997). the colour of ogi depends on the colour of the cereals from which the ogi is made, white to cream for maize, reddish brown for sorghum and dirty grey for millet (ohenhen and ikenebomeh, 2007). the nutritional value of ogi is deficient in some nutrients especially lysine because of the poor processing techniques involved in its traditional production (adeyemi and beckly, 1986; adeniyi and porter, 1978; adeyemi et al., 1987). maize is the third most important cereal food in the world and it is a staple food for more than one billion people in sub-saharan african and latin america (akinbode and bamire, 2015). it‟s high in carbohydrates but lacks essential micronutrients such as vitamin a. new varieties of maize called yellow maize which is rich in vitamin a have been developed in nigeria to alleviate the problem of vitamin a deficiency (uchendu, 2013). moringa oleifera is an underutilized perennial tree with various uses. the young leaves are edible and can be consumed fresh, cooked and eaten like spinach or used for soups and salads. the leaves can also be stored as dried powder for many months without refrigeration and without loss of nutritional value. moringa leaf is an inexpensive source of cheap and abundant source of proteins, carbohydrate, minerals, vitamins and fibres to most vulnerable groups. edible leaves from vegetable plants are rich source of beta-carotene a precursor for abioye 155 vitamin a (eroarome, 2004). the powder has the highest protein content than any other vegetable. fresh leaves of moringa oleifera contain at least twice more proteins than the protein of milk and of eggs (broin, 2006). several authors have reported improvement in the nutritional quality of ogi by fortifying it with other food substances such as soybean (nnam, 2000, oluwamukomi et al., 2005; adelekan and oyewole, 2010); cowpea (sanni et al., 2001, egounlety et al., 2002); mellon (osundahunsi and aworh, 2003), okra (akingbala et al., 2005, aminigo and akingbala, 2004), baobab fruit (adejuyitan et al., 2012) and groundnut seed (ajanaku et al., 2012). this work is therefore aimed at investigating the effect of moringa leaves powder fortification on the nutritional composition and sensory properties of ogi produced from yellow variety of maize thus producing nutritionally and affordable weaning food or breakfast food for the populace. materials and methods the yellow variety of maize used was obtained from a local market in ogbomoso, oyo state, nigeria while the moringa leaves were obtained from the research farm of ladoke akintola university of technology, ogbomoso, oyo state, nigeria. production of ogi ogi was prepared using a method described by akingbala et al. (1981) with a slight modification. the yellow maize was thoroughly cleaned by picking out all broken kernels together with other foreign particles and then sorted to obtain the good ones. then the maize grains were washed, soaked in a bucket and allowed to steep for 72 h at room temperature (27 ˚c). the steep water was changed each day for the three days. after the third day, the steep water was discarded and the grains were wet milled using a grinding machine/grinder. the milled slurry was then wet sieved using a muslin cloth to remove bran, hull and germ. the ogi slurry was collected in a muslin cloth and hand squeezed to remove excess water leaving behind a semi wet ogi which was then dried at 50 ºc for 48 h in the cabinet drier to obtain dry ogi powder. preparation of moringa powder freshly plucked moringa leaves were weighed, cleaned (rinsed) and dried at 45 ºc in the cabinet dryer. it was then blended, allowed to cool and packaged in cellophane bags until it was needed for further use. preparation of moringa-ogi powder moringa-ogi powder was produced by blending ogi powder and moringa powder in the different proportions needed for the formulations i.e. 100:0, 90:10 and 85:15 and then thoroughly mixed to obtain homogenous ogi samples. determination of proximate composition the different formulations of moringafortified ogi samples were analyzed for moisture, ash, crude fibre, protein (n*6.25), crude fat and the carbohydrate determined by difference according to the method described by aoac (2005). determination of minerals selected minerals including calcium, magnesium, iron, potassium, zinc, and copper were extracted from dry ashed samples and determined by atomic absorption spectrophotometer (aoac, 2005). swelling capacity the swelling power of the ogi sample was determined by the method described by tester and morrison (1990). about 0.2 g ground samples (< 60 mesh) was suspended in 10 ml of water and incubated in a thermostatically controlled water bath at 95 ºc in a tarred screw cap tube of 15 ml. the suspension was stirred intermittently over 30 min periods to keep the starch granules suspended. the tubes were then rapidly cooled to room temperature (27 ºc). the cool paste was centrifuged, at 2200 x g for 15 min to separate the gel and the supernatant. then, the aqueous supernatant was removed and the weight of the swollen sediment was determined. sensory evaluation ogi was prepared by making the flour into slurry by heating on fire with constant stirring until a thick paste was formed. the prepared ogi was then dished into sample plates for the panelist. sensory evaluation of the composite ogi samples was carried out by a panel of ten people comprising of the students of the ladoke akintola university of technology, ogbomoso who are familiar with the product. the parameters tested for are appeal, colour, mouthfeel, taste and flavour using a nine point hedonic scale ranging from 9 = like extremely to 1= dislike extremely. statistical analysis statistical analysis of all data was done with the statistical analysis systems (sas) package (version 9.2 of sas institute inc, 2003). statistically significant differences (p<0.05) in all data were determined by general linear model procedure (glm) while least http://www.scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=vitamin+a http://www.scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=moringa+oleifera http://scialert.net/fulltext/?doi=jfrs.2012.1.14&org=11#47011_an 156 afr. j. food sci. res. maize sorting washing soaking in clean water for 3days decanting wet milling sieving and discarding of pomace ogi slurry drying yellow maizeogi powder figure 1. flow diagram of production of ogi powder source: akingbala et al. (1981). table 1. proximate composition of moringa fortified yellow-maize-ogi. samples moisture content crude protein crude fat crude fibre ash cho a 9.13a 9.23a 3.56a 2.33c 1.70c 74.04a b 8.63b 13.03b 3.70ab 2.93b 2.50b 69.20b c 8.77b 18.00c 3.87a 3.57a 3.30a 62.50c means with the same alphabet in the same column are not significantly (p<0.05) different from each other. a – 100% yellow maize-ogi, b – 90% yellow maize-ogi and 10% moringa leaves powder c 85% yellow maize-ogi and 15% moringa leaves powder significant difference (lsd) was used to separate the means. result the proximate composition of the powdered ogi samples is as presented in table 1. the sample with 15% moringa leave substitution had the highest protein content (18.00 %) while the sample with no moringa substitution had the lowest (9.13 %). the proximate composition of the fortified ogi samples revealed an increase in the nutritional content. this is similar to the reports from studies in which ogi was supplemented with other substances such as okra seed meal, soybean (aminigo abioye 157 table 2. mineral composition of moringa fortified yellow maize -ogi samples. sample calcium magnessium iron potassium zinc copper a 136.6c 31.67c 5.23c 33.33c 0.20c 0.27c b 3.85b 80.00b 8.37b 173.33b 0.50b 0.50b c 466.0a 123.00a 14.77a 215.00a 0.73a 0.60a means with the same alphabet in the same column are not significantly (p<0.05) different from each other. a – 100% yellow maize-ogi, b – 90% yellow maize-ogi and 10% moringa leaves powder c 85% yellow maize-ogi and 15% moringa leaves powder. table 3. beta-carotene content of moringa fortified yellow maize-ogi samples. sample beta-carotene content (µg/100g) a 141.67a b 858.33b c 1126.67c means with the same alphabet in the same column are not significantly (p<0.05) different from each other a – 100% yellow maize-ogi, b – 90% yellow maize-ogi and 10% moringa leaves powder c 85% yellow maize-ogi and 15% moringa leaves powder. table 4. swelling capacity of moringa fortified yellow maizeogi samples. sample swelling capacity a 33.33a b 24.00b c 10.00c means with the same alphabet in the same column are not significantly (p<0.05) different from each other a – 100% yellow maize-ogi, b – 90% yellow maize-ogi and 10% moringa leaves powder. c 85% yellow maize-ogi and 15% moringa leaves powder. and akingbala, 2004; akinrele et al., 1971; adesokan et al., 2011). the increase is higher compared to other researchers; ajanaku et al. (2012) who obtained about 258% percentage increase in sorghum-ogi sample but with 100% level of substitution with groundnut seed. aminigo and akingbala (2004) also obtained about 122 and 106% increase in protein content with 20% substitution with defatted and roasted okra seed meals. there was increase in the crude fibre and ash content with increase in the level of moringa substitution. this is a reflection of the composition of moringa leaves, it is reported to have about 13.2% ash content and 8.51% crude fibre (melesse, 2011). the percentage carbohydrate for the samples ranged between 62.50% and 74.04%. the result of the analysis of selected minerals is as presented in table 2. an increase in mineral content with increase in moringa leaves powder substitution addition was recorded in all the mineral content tested. there was increase in the calcium content from 136 mg/100 g to 466 mg/100 g, iron content from 5.23 mg/100 g to 14.77 mg/100 g, the magnesium increased from 31.67 to 123.00 mg/100 g, the potassium 33.33 to 215.00 mg/100 g, zinc from 0.23 to 0.73 mg/100 g and copper from 0.27 to 0.60 mg/100 g. there was increase in the betacarotene content with increase in the level of moringa leaf substitution as shown in table 3. the beta-carotene is very important for sight and general immunity of the body. the swelling capacity of the ogi samples is as presented in table 4. there was a decrease in swelling capacity with increase in moringa leaves powder substitution. the values for the swelling capacity ranged from 10.00 % 33.33 %. the results from this study showed that there was a reduction in swelling capacity with increase in mor 158 afr. j. food sci. res. table 5. evaluation of the sensory properties of moringa fortified yellow maizeogi samples. samples colour taste mouth feel appeal flavour general acceptability a 8.40a 8.10a 7.60a 8.00a 6.80a 8.40a b 6.40b 5.60a 5.80b 5.30b 5.40ab 6.20b c 5.50c 5.20b 5.70b 4.50b 5.70ab 5.10b means with the same alphabet in the same column are not significantly (p<0.05) different from each other a – 100% yellow maize-ogi, b – 90% yellow maize-ogi and 10% moringa leaves powder c 85% yellow maize-ogi and 15% moringa leaves powder moringa leaves powder addition and this means that more of the ogi can be consumed in terms of quantity. the result of the sensory evaluation of the ogi samples is as shown in table 5. the result showed that the sample substituted with 10% moringa leaf did not have any significant (p<0.05) difference in taste with the unfortified ogi samples. this indicates that the sample with 10% substitution had a close rating with the unfortified samples. conclusion this research work reflected the effects of moringa leaf substitution on the nutritional value of the yellow-ogi samples. there was increase in the protein content, ash content and crude fibre the samples substituted with 15% moringa leaf powder recorded a good nutritional content while the yellow-ogi sample substituted with 10% moringa leaf did not have a significant(p<0.05) difference in taste. this reveals that fortification of yellow maize ogi with moringa oleifera leaf is possible and this will also enhance the nutritional status of the populace especially the children and adult who consume ogi as breakfast foods. references adejuyitan ja, abioye ao, otunola et, oyewole yn (2012). an evaluation of some properties of baobab fruit powder and ogi mixes. transnat. j. sci. technol. vol 2(7): 91-102. adelekan ao, oyewole ob (2010). production of ogi from germinated sorghum supplemented with soybeans. afr. j. biotechnol. vol. 9(42): 7114-7121. adeniyi ao, porter nn (1978). properties of ogi powders made from normal fortified and opaque-2 corn. j. food sci., 43:1571. adesokan ia, fawole ao, ekanola ya, odejayi do, olanipekun ok (2011). nutritional and sensory properties of soybean fortified composite ogia nigerian fermented cereal gruel. afr. j. microbiol. res. vol. 5(20), pp. 3144-3149. adeyemi ia, beckley o (1986). effect of period of maize fermentation and souring on chemical properties and amylograph viscosity of ogi. j. cereal sc., 4: 353-360. adeyemi ia, ogunsanmi at, fakorede mab (1987). effect of corn varieties on ogi quality. j. food sci., 52:322-324. ajanaku ko, ajanaku co, edobor-osoh a, nwinyi oc (2012). nutritive value of sorghum -ogi fortified with groundnut seed (arachis hypogaea l.). am. j. food technol. 7(2): 82-88. akinbode wo, bamire as (2015). discontinued use decision of improved maize varieties in osun state, nigeria. j. dev. agric. econ. vol. 7(3):85-91. akingbala jo, rooney lw, faubion jm (1981). a laboratory procedure for the preparation of ogi, a nigerian fermented food. j. food sci., 46, 1523-1526. akingbala jo, akinwande ba, uzo-peters, pi (2005). effects of color and flavor changes on acceptability of ogi supplemented with okra seed meals. plant food. hum. nutr., 58(3): 1-9 akinrele ia, edwards cca (1971). an assessment of the nutritive value of a maize-soya mixture, “soy-ogi” , as a weaning food in nigeria. brit. j. nutr., 26, 177. aminigo er, akingbala jo (2004). nutritive composition of ogi fortified with okra seed meal. j. appl. sci., environ. mgt. 8(2):23-28. aoac (2005). offical methods of analysis, association of official analytical chemists. food compositition, additives natural contaminants. adrich rc (ed.) vol 2, abioye 159 15 th ed association of official analytical chemist inc. usa. broin m (2006). the nutritional value of moringa oleifera lam. leaves: what can we learn from figures. moringanews network. egounlety m, aworh oc, akingbala jo, houben jh, nago mc (2002). nutritional and sensory evaluation of tempefortified maize-based weaning foods. int. j. food sci. nutr., 53 (1):15-27. eroarome ma (2004). nutritive value and inherent antinutritive factors in four indigenous edible leafy vegetables in human nutrition in nigeria: a review. j. food res. sci. 1: 1-14. leaves of moringa stenopetala and moringa oleifera using in vitro gas production method. ethiop .j. appl. sci. technol., 2(2): 31 – 41. melesse a (2011). comparative assessment on chemical compositions and feeding values of nnam nm (2000). chemical evaluation of multimixes formulated from some local staples for use as complementary foods in nigeria. plant food. hum. nutr., 55(3):255-263. ohenhen re, ikenebomeh mj (2007). shelf stability and enzyme activity studies of ogi: a corn meal fermented product, j. am. sci. 3(1): 38-42. oluwamukomi mo, eleyinmi af, enujiugha vn (2005). effect of soy supplementation and its stage of inclusion on the quality of ogi-a fermented maize meal. food chem., 91:651-657. osundahunsi of, aworh oc (2003). nutritional evaluation, with emphasis on protein quality, of maizebased complementary foods enriched with soya bean and cowpea tempe. int. j. food sci.technol., 38: 809813. sanni ai, asiedu m, ayernor gs (2001). influence of processing conditions on the nutritive value of ogibaba, a nigerian fermented sorghum gruel. plant foods hum. nutr.56:217-223. uchendu fn (2013). the role of biofortification in the reduction of micronutrient food insecurity in developing countries. afr. j. biotechnol. vol.12 (37): 5559-5566. http://scialert.net/fulltext/?doi=jfrs.2012.1.14&org=11#47011_an african journal of food science research vol. 2 (2), pp. 044-050, february, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper effects of food security and agricultural land access in nigeria oseni abiodun1*, uzezi utunedi and ome ayodeji2 1 department of agricultural extension and rural development, obafemi awolowo university, ile ife, osun state, nigeria. 2 department of agriculture, faculty of food science and technology, obafemi awolowo university, ile ife, osun state, nigeria. accepted 02 january, 2014 this study assessed gender discrimination in agricultural land access: implications for food security in ondo state nigeria. specifically, it analysed men and female accessibility to forms of land holding and the factors affecting agricultural land accessibility in the study area. multistage sampling technique was used in selecting 240 respondents used for this study. data collected were summarized using descriptive statistics such as frequency counts and percentages and correlation analysis was used to test the hypothesis stated. the results revealed that the mean age of the male respndents was 48.3 while that of female was 43.7 with the standard deviation of 14.9 and 11.3, respectively. also, at p ≤ 0.05, there was significant relationship between accessibility to agricultural land and male and female socio-economic characteristics such as age (r = 0.484), marital status (r = 0.568), farm size (r = 0.504), farming experience (r = 0.479), household (r = -0.668), access to credit facility (r = 0.476), and membership of social organization (r = 0.593). this study therefore concluded that gender differentials, especially with regards to land favour the males. it is therefore recommended that redesigning and redeveloping the structure of land policies to be more gender sensitive and inclusive. key words: gender, land acess, food security, correlation, discrimination. introduction in a rapidly changing world, food and agricultural land holding systems in developing countries are facing new and increasingly complex challenges (derman et al., 2007). fighting poverty, ensuring food and nutrition security while protecting the environment still remains a major challenge facing global development practitioners today. in discussing agriculture, two key factors that cannot be ignored are land and labour. land ownership, accessibility, and the sustainability of this access are very crucial for any meaningful agricultural development. either by design or circumstance, women constitute a large proportion of farm labour and agricultural workers. these women usually control very little or no amount of land, which is very important in determining farm productivity *corresponding author. e-mail: osenimd@yahoo.com and labour welfare (afonja et al., 2002). land is the most valuable form of property in agrarian societies because of its economic, political symbolic and ritual importance (bioye et al., 2006). it is the basis of political power and social status in most societies of the world. it is a productive wealth-creating and life sustaining asset which every human being craves for and provides a sense of identity and rootedness within a community (argwal, 1994). land is used for production of biomass, ensuring food, fodder, renewable energy and raw materials for existence of human and animal life. it is a base for settlement and industrial use and a store of our cultural heritage and is actually a source of raw materials like minerals, clay, energy and water (blum, 1998). land stands for continuity of ownership since it is a burial ground where all clansmen are buried and consequently a central place for the spirits of their ancestors for example in african countries. http://www.internationalscholarsjournals.org/ oseni et al. 044 in most developing countries, land is not only the primary means for generating livelihoods but often the main vehicle for investing, accumulating wealth, and transferring it between generations. thus, the ways in which access to land is regulated, property rights are defined, and ownership conflicts are resolved has broad implications for food security (deininger and binswanger, 1999, fao, 2006, 2008, 2009). access to land can be through right of ownership, through informal concessions granted by individuals to kin or friends. legal ownership is normally accompanied by legal restrictions on disposal, that is, there is no effective control here. in most african societies, women have land use priorities from husbands but have no independent rights which allow them control or produce from the land. the direct advantage of land rights are that a woman can use it to grow food crops, fodder for animals, keeping livestock, practicing sericulture, growing trees and vegetable gardening (cousins and claassens, 2006). land rights facilitate access to credit and strengthen support that the women receive from relatives. access to land means reliable food supply, better healthcare, better housing and reliable income in most cases. the percentage of men and women employed in agricultural sector decreased between 1997 and 2008 (due to the increasing industrialization of the considered countries), the percentage of women employed in agriculture is still higher in almost all developing regions as shown in apendix 1. in the last years, migration of men towards the cities led to a gradual feminization of smallscale agriculture, with an increasing percentage of womenheaded households in rural areas (fao, 2008). the relevance of women’s agricultural labour can be appreciated if we consider that, for instance, the agricultural sector in sub-saharan africa contributes about 30% of the gnp of the continent, employing from 60 to 90% of the population and producing from 25 to 90% of the income deriving from exports (fao, 2009) and fao (2002) lawanson (2010) opined that nigeria is a typical patriarchic society where male superiority and dominance originated from historically rooted culture and religion. in pre-colonial times, females generally were accorded less value and lower social status. western culture reinforced this anomaly. however, bruce and lloyd (1991) stated that the western culture has failed to address gender inequality in access to land, in spite of the role that land plays in the lives of the women who are increasingly being saddled with the responsibility of heading and maintaining households, especially in developing countries. women constitute over 50% of the nigerian population, make up about 37% of the formal sector (world bank, 2001), and dominate the informal economic sector which is principally made up of home-based enterprises (soetan, 2002); moreso, lawanson (2010) stated that their relative powerlessness both economically and politically, are unable to exercise control over resources, particularly land. some cultures in nigeria, through marriage and inheritance practices, prohibit women from owning land. however, soetan (2002) posited that marital status increases the women ability to own land. at rural level, women work mainly on their own, linking their activities to the family needs, and just a small percentage of them, everywhere lower than men’s receives a wage. in latin america, for instance, only 2.3% of women in agriculture get a wage against 20.9% of men; in southern asia salaried rural women are 11.9%, while men are 21.8%7 (alice, 2008). with the increasing roles of rural women in agriculture and contributions to food security and the consequence inequality on land access across gender, there is therefore the need to access the gender discrimination in land accessibility and implications to food security in nigeria the specific objectives for the study were to: 1. examine the socio-economic characteristics of the respondents 2. analyse male and female accessibility to forms of land holdings in the study area 3. assess the factors affecting male and female accessibility to land. 4. profile the security of tenue over land across gender. hypothesis for this study was stated in the null form as follows: there is no significant relationship between selected socio-economic characteristic of male and female respondents and their accessibility to land in the study area. the study area the study was conducted in ondo state of nigeria. the state was carved out of the old oyo state on the 3 rd february, 1976 with the capital in akure. the state covers an area of approximately 15,500 km 2 and it is bounded in the south by the bight of benin and atlantic ocean; north by ekiti and kogi states; east by edo and delta states and west by osun and ogun states (figure 1). the state lies between longitude 5°45′ and 7°52′ on the north – south pole, and longitude 4°20′ and 6°5′ on the east – west pole. according to analytical report of the national population commission (npc) (2006), ondo state has 3,441,024 million people with eighteen (18) local government areas. the tropical climate of the state is broadly of two seasons: rainy season (april to october) and dry season (november to march). a temperature throughout the year ranges between 21 to 29°c and humidity is relatively high. the annual rainfall varies from 2,000 mm in the southern areas to 1,150 mm in the northern areas. the soils derived from the basement complex rocks are mostly well drained, with a medium to fine texture. the state enjoys luxuriant vegetation with high forest zone (rain forest) in the south and sub-savannah forest in the northern fringe. the indigenes of the state belong to the yoruba ethnic group 045 afr. j. food sci.res. figure 1. map of the study area. and are composed of the akokos, the ondos, the ikales/ilajes and the apoi/ijaw arogbos. however, non – indigenes from every part of the country and outside reside in the state. yoruba and english are the languages of the people for official and business transactions. the state is basically agrarian with large scale production of cocoa, palm produce and rubber. other crops like maize, yam and cassava are produced in large quantities. sixty-five percent of the state labour force is in the agriculture sub-sector (coastalnews, 2012). the state is also blessed with very rich forest resources where some of the most exotic timber in nigeria abounds. ondo state is equally blessed with extensive deposits of crude oil, bitumen, glass sand, kaolin, granites and limestone. therefore, the state has great potentials for rapid industrial growth in view of its raw materials base. reasonable segment of the populace are also traders and artisans. other occupations of the people include weaving, mat – making, dying, soap making, wood oseni et al. 046 table 1. distribution of respondent’s demographic characteristics. variable male (n= 160) female (n=160) frequency percentage frequency percentage age >30 42 27.2 34 21.3 31-60 90 56.3 102 63.8 60 years and above 28 16.5 24 14.9 level of education no formal education 19 11.9 31 19.4 primary school 56 35.0 61 38.1 secondary education 63 39.3 50 31.2 tertiary education 22 13.8 18 11.3 farming experience < 5 10 6.3 5 3.1 6-10 32 20.0 40 25.0 11-20 68 42.5 79 49.4 >20 50 31.2 36 22.5 household size >2 15 9.4 12 7.5 2-5 35 21.9 89 55.6 6-9 84 52.5 40 25.0 >10 26 16.2 19 11.9 farm size <1 12 7.5 62 38.8 1-3 91 56.8 83 51.9 >3 51 35.7 15 9.3 source: field survey, 2010. carving, among many others. the state lies entirely within the tropics of 162 mm per annum. sampling procedure and percentage while the inferential statistics were correlation analysis and chi-square. data were analysed with the aid of statistical package for social sciences (spss) version 14.0 and costat analytical software. a multi-stage sampling technique was adopted to select respondents for this study. the first stage involved purposive sampling of six local government areas based on their degree of involvement in farming. the local government areas were irele, okitipupa, odigbo, owo and akoko, ose and akoko south-west. in the second stage, the local government areas were grouped into communities. in the third stage, two communities were randomly selected from each of the local government areas. in the final stage, twenty respondents with equal number of male and female were sampled from each of the communities primary data were used for this study. data were collected using structured questionnaire and interview schedule for both the literates and illiterates respondents respectively. a total of 240 respondents were used for this study. analytical technique data were analyzed using descriptive statistics and inferential statistics. the descriptive statistics employed were frequency counts results and discussion the result of the socio-economic characteristics as shown in table 1 revealed that 27.2% of male respondents were less than 30 years, 56.3% were found within the age bracket of 31 to 60 years while only 16.5% were 61 year sand above. in the female category, 21.3% of the respondents were less than 30 years, 63.8% were within the age group of 31 to 60 years while just 14.9% of the respondents were 60 years and above. the mean ages of male and female respondents were 48.3 and 43.7 years respectively. the findings revealed that majority (56.3 and 63.8%) of respondents were in their middle active ages, an indication that they will still be active to access land. furthermore, 35.0% of male respondents had primary education, 39.3% had secondary education and only 13.8% had tertiary education while 11.9% did not have 047 afr. j. food sci.res. table 2. distribution of respondents’ accessibility to forms of land holdings. gender male (n=160) female (n=160) variable frequency percentage frequency percentage access to community land 152 95.0 38 23.8 access to inherited land 138 86.3 52 32.5 access to land by purchase 160 100.0 72 45.0 access to land by lease 160 100.0 61 38.1 access to land by gift 115 71.9 29 18.1 source: field survey, 2010. multiple responses were given. table 3. chi-square analysis showing the difference between gender and land accessibility. variable x 2 c x 2 t df c gender 5.83* 3.84 1 0.57 p-values at 0.05 level of significance; c= contingency coefficient; x2c = chisquare calculated; x2t = chi-square formal education. in the female group, 38.1% had primary education and only few (11.3%) had tertiary education. this implied that male respondents were more educated than their female counterparts in the study area. for farming experiences in years, majority (42.5%) of male respondents had between 11 to 20 years and 31.2% had greater than 20 years. more so, 49.4% of female respondents had 11 to 20 years of farming experience while only 22.5% were found to have more than 20 years of farming experience as shown in table 1. in addition, majority (52.5%) of male respondents had between 6 to 9 household sizes while majority (55.6%) of female respondents had between 2 to 5 household sizes. this implied that male respondents had higher household sizes than the female. this could be as a result of male having more than one wives. also, 56.8% of male respondents had between 1 to 3 hectares farm size, 35.7% had more than 3 hectares farm size while 51.9% of female respondents had between 1 to 3 hectares and a few (9.3%) cultivated more than 3 hectares of land in the study area. this analysis shows that male respondents had higher farm size than their female counterparts in the study area. results in table 2 revealed that majority (95.0%) of male respondents had access to communal land while few (23.8%) of female respondents had access to community land. also, 86.3% of male had access to inherited land while only 32.5% of female respondents had access to inherited land. more so, 100.0% of male respondents were found to have access to land by purchase while only 45.0% of female could access land by purchase. in addition, majority (71.9%) of male could access land through gift. the above analyses indicated that male had more access to any form of land than their female counterparts. also, the result of chi-square analysis revealed that there was a significant difference between gender and accessibility to land at 0.05 level of significance as shown in table 3. gender was found to exert a little above avarage (57%) strenght of association on land accessibilty. this significant difference further revealed that male have more access to tabulated; df = degree of freedom; source: field survey, 2010. land than female in the study area. this finding was in conformity with lawanson (2010), bruce and lloyd (1991) and afonja et al. (2002). the low accessibility of female to agricultural land could be as result of sociocultural factors that could hinder female from owning land (lawanson, 2010). results in table 4 revealed that 70.0% of male respondent indicated that income level affects land holding, while majority (100.0%) of female indicated that marital status is the major determinant to hold land in the study area. more so, about 84.5 percent of the female respondents viewed cultural belief as a factor that affects land holding while a few (38.8%) of the male respondents indicated that cultural belief is a factor that affects land holding among men in the study area. this finding revealed that men were less affected by the various factors indicated, an indication that female were not having equal access to agricultural land despite their contributions to the food production in sub-saharan africa. this affirms the position of alice (2008) in the study ‘’effect of land tenure system on women’s knowledge-base and resource management in manjiya county, uganda’’. the study indicated that a very low percent of women in agriculture gets a wage as aginst the percentage of men. the pearson correlation analysis revealed that marital status was positively correlated with land accessibility. this conforms with soetan (2002). multiple responses were given on the security of tenure over land, results in table 5 indicated that about 83.1 percent of male owned land while only few (17.5%) of women owned land in the study area. more so, about 74.4 percent of male had been in possession of their land for more than 5 years while about 1.3% of women had held land for the same duration with men. in addition, about 88.1% of male had the rights to transfer land while none of the female respondents had the same priviledge of transferring land like their male counterparts. this analysis reveals that gender differential oseni et al. 048 table 4. factors affecting land ownership. variable male (n=160) female (n=160) frequency percentage frequency percentage income 112 70.0 129 80.6 marital status 70 43.8 160 100.0 access to credit facility 132 82.5 108 67.5 age 125 78.1 115 71.9 cultural belief 62 38.8 135 84.5 source: field survey, 2010. table 5. security of tenure over land. variable male female frequency percentage frequency percentage land ownership owned 133 83.1 28 17.5 otherwise 27 16.9 132 82.5 duration of land use <2 years 39 24.3 3-5 years 41 25.6 118 73.8 >5 years 119 74.4 3 1.9 rights to land use only 19 11.9 160 100 transfer 141 88.1 source: field survey, 2010. table 6. results of correlation analysis showing the relationship between male and female socio-economic characteristics and accessibility to agricultural land. variable correlation coefficient (r) coefficient of determination (r 2 ) age 0.484* 0.064 marital status 0.568* 0.059 level of education 0.115 0.013 farm size 0.504* 0.039 farming experience 0.479* 0.121 household size -0.668* 0.072 access to credit facility 0.476* 0.141 membership of social organization 0.593* 0.154 source: field survey, 2010. critical value of r = 0.427, level of significance= 0.05. exists in security of tenure over land in the study area. this findins conform with lawanson (2010), who stated that women relative powerlessness both economically and politically in a typical african setting limits their control over resources, particularly land and also that male superiority and dominance over resources in nigeria originated from historically rooted culture and religion. the results of the hypothesis stated and tested on table 6 revealed that there were positive and significant correlation between accessibility to agricultural land and age (r = 0.484); marital status (r = 0.568); farm size (r = 0.504); farming experience (r = 0.479); access to credit (r = 0.476) and membership of social organization (r = 0.593). the r values were compared with the critical value of r = 0.427. household size had negative but significant relationship with respondents’ socio-economic characteris tics and accessibility to agricultural land. only level of education was found to have non significant relationship. the above information established that an increase in the value of the independent variables would result in corresponding relationship between respondents’ socioeconomic characteristics and accessibility to agricultural land. the values of coefficient of determination (r 2 ) further revealed the percentage contribution of the independent variables to agricultural land accessibility. the higher the values of r 2 , the stronger the influence as reflected in the percentage contribution of the significant variables. this information therefore justified these variables as highly significant between respondents’ socio-economic characteristics and accessibility to agricultural land. conclusion and recommendations based on the findings of this study, most of the respondents were within their productive age of 31 to 60 years. there was gender differences in land accessibility in the study area as male were found to have more access to agricultural land than their female counterparts. female were more affected by the factors affecting land holding in ondo state. age, marital status, farm size, farming experience; access to credit facility and cultural beliefs were found to determine accessibility to agricultural land in the study area. there is therefore the need for an intensive effort and emphasis on mainstreaming gender in agricultural programmes. this will facilitate the entry of women as active decision makers on issues that relate to food security and income generation. women are important links to development: they maintain food security and the general well-being of their families/households. therefore, improving women's status and control over land should be considered as strategically important efforts at all levels in combating the state of food security in the country. it is therefore recommended that society and government should consider gender in agriculture by restructuring the system of land holding in order to include the vulnerable group as this will have a significant effect on food production in ondo state and in nigeria at large. also, land tenure policies should be restructured to ensure that farmers (male and female) have equal access to agricultural land especially for perennial crops and are able to obtain land on a more permanent basis would be helpful in combating the state of food insecurity in the country. 049 afr. j. food sci.res. references afonja s, mills-tettey r, amole d (2002). gender differentials in access to land and housing in a nigerian city, being monograph of the center for girder and social policy studies, obafemi awolowo university, ile-ife. alice mk (2008). the effect of land tenure system on women’s knowledge-base and resource management in manjiya county, uganda. argwal b (1994). a field of one’s own: cambridge university press. fao corporate document repository: current and emerging issues for economic analysis and research, 2010 blum cl (1998). land tenure and administration in africa: land tenure and resource access in africa, iied/fao, london. bioye ta, abdul ra, joseph bo (2006). women and land rights reforms in nigeria: promoting land administration and good governance 5th fig regional conference. bruce j, lloyd c (1991). family research and policy issues for the 1990's’, in understanding how resources are allocated within households. cousins b, claassens a (2006). ‘more than simply ‘socially embedded’: recognizing the distinctiveness of african land rights’. keynote address at the international symposium on the frontier of land issues: social embeddedness of rights and public policy’ montpellier, may 17-19. coastalnews (2012). profile of ondo state, nigeria. deininger k, binswanger h (1999). the evolution of the world bank’s land policy: principles, experience and future challenges’, the world bank research observer 14:247-276. derman br odgaard o, sjaastad e (2007). conflicts over land and water in africa. oxford: james currey. the world bank research observer 14:247-270. food and agricultural organization (2002). assessment of the world food security and nutrition situation. background document for the 38th session of the fao, rome, pp. 34-39. food and agricultural organization, (2006). the state of food insecurity in the world rome. food and agricultural organization (2008). gender, property rights and livelihoods in the era of aids. food and agricultural organization (2009). gender equity in agriculture and rural development. a quick guide to gender mainstreaming in fao’s new strategic framework. lawanson to (2010). gender differentials in nigeria: implications for sustainable development. j. extension. syst. 21(1):46-57. soetan r (2002). ‘women, small scale enterprises and social change: implications for changes in industrialization strategy”, in afonja.s and aina o (eds) gender and social policy studies. obafemi awolowo university, ile ife the world bank (2001). integrating gender into the world bank's work: a strategy for action. www.worldbank.org oseni et al. 050 appendix appendix 1. percentage of women and men employed in the primary sector. region 1998 2007 male women male women world 39.4 42.9 33.1 36.4 eastern asia, south-eastern asia and pacific 44.3 51.6 36.4 41.2 latin america and caribbean 26.4 12.6 22.1 9.7 southern asia 53.7 74.4 41.5 65.1 sub-saharan africa 65.1 71 60.3 65.1 source: global employment trends for women, ilo, 2009-data for 1998 and 2007 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (5), pp. 177-181, may, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the risks and microbial quality of kunun-zaki beverage for the consuming public elmahmood, a. m.* and doughari, j. h. department of microbiology, school of pure and applied sciences, federal university of technology, pmb 2076 yola 640002, adamawa state, nigeria. accepted 04 january, 2015 thirty kununzaki samples were obtained as freshly formulated beverages from 10 different local hawkers in girei town, adamawa state, nigeria and screened for microbial contamination. the ph of the samples ranged between 3.44 4.34 and total bacterial count ranged between 1.0 x 10 3 1.8 x 10 4 cells/ml. the presence of high microbial loads was indication of poor hygiene and/or poor quality cereals and water used in the preparations. the microorganisms recovered were escherichia coli, staphylococcus aureus, streptococuus pyogenes, rhizopus nigricans, penicillium digitatum, asper-gillus fumigatus and monilia sitophila. the types and density of microorganisms recovered from calls for urgent measures to be taken by regulatory authorities in the processing and handling of the product before being sold to the unsuspecting general public. key words: microbial quality, kunun-zaki, microbial load, microbial contamination. introduction kunun-zaki is an indigenous fermented non-alcoholic beverage that is widely consumed for its thirst quenching properties. though consumed throughout the year, it is extensively consumed during the dry season. the drink is produced from fermented millet, sorghum, guinea-corn and maize in decreasing order of preference. in some cultures, the grains are used in a composite from espe-cially millet, guinea-corn and sorghum in a ratio of 1:2 w/w (abegaz, 2007). it is sweetened with honey and sug-ar together with small quantities of sweet potatoes and spices (ginger, black pepper or clove). the method of production is crude, not standardized with levels of ingre-dients not quantified and largely a family art. the proce-dure involves steeping the cereals in local household utensils such as buckets, calabashes and earthenware vessels. this is then followed by grinding of the steeped grains into a mush which is then mixed with spices (clo-ve, red or black pepper and ginger). this is then divided into two unequal portions, one portion is gelatinized with hot water and the other portion mixed with liquefying agents (sweet potato paste, malted rice and extracts of cadaba farinose stem). the two portions are then mixed together at 70 75 o c and the mixture left at room tempe-rature for chance fermentation for 18 24 h. this is then *corresponding author. e-mail: elmahmu@yahoo.com. filtered first using a piece of muslin cloth and then a sie-ve. after filtration, honey or sugar is then added to the filtrate to taste and is now ready for consumption (onuo-rah et al., 1987; akoma et al., 2006). significant varia-tions exist in the procedures depending on taste and cul-tural habits leading to differences in quality and stability. while some cultures prefer kunun-zaki with much pep-per or sweet taste, others prefer it with no pepper or sug-ar (adeyemi and umar, 1994) .it is usually packaged and sold in 1litre and 500 ml plastic bottles or even tied in small polyethene bags. kunun-zaki must be consumed within 18 36 h of production due to its poor keeping qua-lity. the drink is very cheap because the cereals and ad-ditives used in its production are locally sourced as they are grown throughout the savannah belt of west africa. packaging materials are also cheap and easily available. ayo and okaka (1998) have reported that kununzaki is rich in carbohydrates, vitamins and minerals but low in proteins. furthermore, the methods of production are simple and cheap as no elaborate equipment and exper-tise required (agboola, 1987). the high water content coupled with crude methods of production and packaging under improper sanitary conditions predisposes kununzaki to microbial contamination. this study was designed to assess the microbial quality of this immensely popular beverage and possibly high-light the risks involved for the consuming general public. elmahmood and doughari 178 table 1. mean ph values and total bacteria counts (cfu/ml) for fresh kunun—zaki. vendor ph samples nutrient agar macconkey agar mannitol salt agar a 4.34 a1 4.0 x 10 4 1.8 x 10 4 a2 3.1 x 10 4 0.2 x 10 4 a3 3.6 x 10 4 0.8 x 10 4 b1 2.5 x 10 4 2.0 x 10 4 0.4 x 10 4 b 3.75 b2 4.6 x 10 4 2.6 x 10 4 1.0 x 10 4 b3 3.1 x 10 4 1.8 x 10 4 0.6 x 10 4 c1 1.9 x 10 4 1.3 x 10 4 0.1 x 10 4 c 3.51 c2 3.2 x 10 4 0.9 x 10 4 0.3 x 10 4 c3 3.4 x 10 4 0.5 x 10 4 0.5 x 10 4 d 4.15 d1 1.56 x 10 4 8.9 x 10 4 2.8 x 10 4 d2 1.26 x 10 4 7.4 x 10 4 4.0 x 10 4 d3 8.6 x 10 4 5.8 x 10 4 3.6 x 10 4 e 3.44 e1 5.0 x 10 4 2.1 x 10 4 4.6 x 10 4 e2 9.2 x 10 4 1.7 x 10 4 5.1 x 10 4 e3 7.5 x 10 4 1.3 x 10 4 3.7 x 10 4 g 4.10 g1 8.0 x 10 4 0.5 x 10 4 1.76 x 10 4 g2 5.9 x 10 4 2.0 x 10 4 8.8 x 10 4 g3 1.27 x 10 4 1.3 x 10 4 1.19 x 10 4 h 3.48 h1 1.84 x 10 4 5.0 x 10 4 1.03 x 10 4 h2 1.15 x 10 4 1.3 x 10 4 9.6 x 10 4 h3 1.46 x 10 4 2.0 x 10 4 1.23 x 10 4 i 3.49 i1 1.60 x 10 4 1.0 x 10 4 2.6 x 10 4 i2 1.48 x 10 4 2.3 x 10 4 4.2 x 10 4 i3 1.75 x 10 4 0.3 x 10 4 6.5 x 10 4 j 3.69 j1 1.23 x 10 4 1.6 x 10 4 3.9 x 10 4 j2 1.76 x 10 4 0.7 x 10 4 5.3 x 10 4 j3 1.08 x 10 4 0.2 x 10 4 2.6 x 10 4 key: no growth materials and methods collection of samples three samples of freshly prepared kunun-zaki were collected from each of 10 different hawkers between the months of february and july, 2005 in girei town, girei local government of adamawa state, nigeria. the samples were packaged in 500 ml sterile plastic bottles and immediately transferred to the microbiology laboratory for isolation of microorganisms and enumeration of bacteria. determination of ph of the samples the ph of the various samples was immediately determined using sterile probes of the ph meter (corning 35). determination of total count of bacteria this was carried out on agar plates of nutrient agar (na), mac-conkey agar (mca) and mannitol salt agar (msa) all of oxoid gra-de, using the pour plate method. the samples were serially diluted and 1ml of appropriate dilution was used to inoculate each of the plates in triplicates. the culture plates were then incubated at 37ºc for 24 48 h and colonies counted on a gallenkamp colony counter. the mean of triplicate results were then recorded as the colony count (lateef et al., 2004). isolation and identification discrete colonies of the organisms (for bacteria) were selected and sub cultured from the mixed cultures of the plates to respective na plates and incubated at 37ºc for 24 h. the bacterial isolates were then identified following standard microbiological procedures as described by buchanan and gibbons (1974) and cheesbrough (2002). for the filamentous fungi, appropriate spore dilutions (1.0 x 10 7 spores/ml) of the kununzaki samples were surface-spread in triplicates on potato dextrose agar (pda, oxoid) plates and incubated at room temperature (30 32ºc) for 48 72 h. after incubation, the colonies were screened and identified based on the taxonomic schemes and descriptions by ainsworth et al. (1973) and mislivec et al. (1992). results the mean ph values of the kunun-zaki and the extent of microbial contamination are shown in table 1. results of table 2. microorganisms isolated from kunun-zaki samples. organisms characteristics samples from vendors a b c d e f g h i j 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 s. aureus slightly raised golden yellow x x x x x x x x x x x x x x x x x x x x x x x x x x x x x g+ colonies that ferments manitol e. coli large, circular, low cobnvex x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x colorless opaqueg+, lf colonies on mc str. pyogenes small dry shiny mucoid x x colonies on ba, g+e cocci in chains p. digitatum grren/black mycelia, spores x x x x x x x x on flask-shaped sterigmata m. sitophila red mycelia, floccose and x x x x x x x x salmon-colored spores r. nigricans white and cottony mycelia, x x x x floccose white to gray spores a. fumigatus bluish green floccose matted x x x x mycelia, conidiophore-bearing phialides (flask-shaped) that produce spores note: x = presence; = absence; g+ = gram positive; lf = lactors fermenting; mc = macckonkey agar; ba = blood agar; a-j = vendors; 1-3 = samples 179 afr. j. food sci. res. elmahmood and doughari 180 ph determination showed that all the samples were acidic in nature. samples collected from vendor e had the lowest ph of 3.44 and those collected from vendor a had the highest ph of 4.34. samples a1 a3 (from vendor a) had bacterial counts (cfu/ml) range of 3.1 4.0 x 10 4 cfu/ml on na, 2.0 x 10 2 1.8 x 10 4 cfu/ml in msa but none on mca. samples b1 b3 (from vendor b) contained bacteria ranging from 2.5 4.6 x 10 4 cfu/ml on na, 1.8 2.6 x 10 4 cfu/ml on mca and 4.0 x 10 2 1.0 x 10 4 cfu/ml on msa. these results follow similar trend for all the other samples. sample e (e1 e3) had highest number of counts on na (5.0 9.2 x 10 4 cfu/ml ), closely followed by sample f (f1 f3) with a cell density of 4.4 6.2 x 10 4 cfu/ml and sample j (j1 j3) with the lowest number of cells ranging from 1.08 1.74 x 10 4 cfu/ml. on mca, sample d (d1 d3) had highest number of cells ranging from 5.8 8.9 x 10 4 cfu/ml and sample j (j1 j3) with lowest bacterial population of 2.0 x 10 3 1.6 x 10 4 cfu/ml. on msa, sample from vendor i (i1 i3) had the highest bacterial cell density of 2.6 6.5 x 10 4 cfu/ml, while sample c (c1 c3) had the lowest cell density of 1.0 x 10 3 -5.0 x 10 3 cfu/ml. table 2 shows the microbial isolates obtained from the various kunun-zaki samples collected. staphylococcus aureus and escherichia coli were isolated from samples a1 j3, while samples a1 and d1 contained streptococcus pyogenes and penicillium digitatum was isolated from each of samples a1 a3, b2 b3, d1, e1, g2, i1 and j1 j3. also monilia sitophila was isolated from samples b1, d2 d3, f1 f2, and i1 13; r. nigricans was isolated from samples c1-c3 and g1; while aspergillus fumigatus was isolated from samples e1 e2, f1, and h1. discussion being a thirst quenching beverage, kunun-zaki has high moisture content. the proportion of water varies from 55 98%, the remainder being mostly additives (giese, 1995). all the samples were acidic in nature (ph 3.34 4.42). this level of acidity of kunun-zaki have been described by several researchers including efiuvwevwere and akoma (1995) and akoma et al. (2006) who attributed these to the presence of certain species of lactic acid bacteria, namely lactobacillus leichmannii and lactobacillus fermentum during the fermentation process. in this study however, attention was directed at isolating pathogenic bacteria. similar local drinks with acidic ph values have been reported for zobo and for orange juice products (lateef et al., 2004) as well as burukutu and pito (kolawole et al., 2007). although these classes of beverages are acidic in nature, the acidity tends to increase with increase in fermentation period resulting into spoilage. consequently, the low ph values may have encouraged the growth of fungi and this could be responsible for the species of microorganisms isolated. the main components of cereals from which kunun-zaki is made are carbohydrates, proteins, vitamins and minerals, and the chief product of fermentation is lactic acid and this leads to a decrease in ph values and an increase in acidity (nkama, 1993). the acidic nature of the samples may also be due to the fact that the kunun-zaki might have started undergoing spoilage even before the time of purchase, and such may lead to production of certain metabolites that could bring about reduction in ph of the product. kunun-zaki and other indigenous nigerian nonalcoholic beverages, such as kunun-aya and simi have been reported to contain high nutritional values because of the raw materials from which they are made. spices are usu-ally added in small quantities to improve taste and flavour and as these are agricultural commodities, which may contain a high level of microbial impurities (adeyemi and umar, 1994). these can be sources of spoilage and pat-hogenic microorganisms (bibek, 2001). the ph of kunun-zaki is usually too low to allow the growth of pathogenic microorganisms, but the presence of e. coli, s. aureus and streptococcus spp. could be a matter of serious concern. s. aureus is a normal flora of the skin, nose, throat, palms, hairs and mucus membrane and a common etiological agent of septic arthritis (alice, 1976). e. coli is an important member of the coliform group it is part of the normal flora of the intestine of human and vertebrates. some strains of e. coli can cause gastroenteritis, diarrhea and urinary tract infections (pelczar et al., 1993). the streptococci are normal flora of the throat and the buccal cavity. in their own study on two hundred and forty samples of kunun-zaki, umar et al (2004) reported the presence of organisms like bacillus cereus, s. aureus and e. coli. the presence of these pathogens even in small numbers could render a beverage unsuitable for human consumption (phls, 2000). it is possible that contamination by these pathogens could have occurred during sieving and packaging, as most of the people involved in the production, packaging and hawking do not take necessary precautions, and as such contamination could be very prominent. contamination of food items by specific species of microorganisms is largely due to the presence of these organisms and their entrance into the food or beverage as a result of poor hygiene and sanitation (bibek, 2001). the dominance of lactobacillus leimannii and l. fermentum in their own samples led akoma et al. (2006) to conclude that kunun-zaki is a lactic acid bacteria fermented beverage. fungi isolated are p. digitatum, a. fumigatus, r. nigricans and m. sitophila. the presence of these fungal species is associated with spoilage of the beverages (kolawole et al., 2007). the total bacterial counts in the various kunun-zaki samples ranged from 1.0 x 10 2 -8.9 x 10 4 cfu/ml as shown in table 1. sample a1-a3 obtained from vendor a had a bacterial count of 3.1 x 10 4 cfu/ml on na, a range of 2.0 x 10 2 1.8 x 10 4 cfu/ml on msa corresponding with ph 4.34. there was no growth on mca which may be 181 afr. j. food sci. res. due to relative absence of coliforms in this sample a .the total bacterial counts obtained in this study fall within then ranged between 1.0 x 10 2 1.0 x 10 5 cfu/ml. earlier works by hatcher et al. (1992) also reported similar abnormally high bacterial populations in orange juices (hatcher et al., 1992). this high colony counts is an indication of spoilage as a consequent of either poor hygiene or poor quality of cereals and water used. many native african beverages are little known outside the parent continent. a concerted effort should therefore be made to improve in the quality and production techniques of these indigenous exotic beverages so that large scale production for export outside the continent can be carried out. many people now prefer imported and exotic beverages because of their attractive forms, long shelf life, ease of transportation and other forms of utility which consumers associate with them (achi, 2005). as of now, there are no industries involved in production of kunun-zaki. kunun-zaki is widely believed to be of immense social, economic and medicinal importance to its numerous consumers (akoma et al., 2006). to safeguard public health, governments and regulatory authorities should intervene by setting standards in acquisition of raw materials, production procedures and techniques as well as health status of personnel. producers and vendors of kunun-zaki should be encouraged to utilize the technical assistance of national agency of food, drugs administration and control (nafdac) towards attaining quality standards. reports have indicated that most nafdac approved sachet water produced by small scale industries in nigeria have attained acceptable microbiological standards (lateef and yusuf, 2002). the extent of organic and inorganic contaminants of kunun-zaki beverage has not been evaluated. further studies are therefore recommended as these too equally affect the health and well being of the general public. reference abegaz k (2007). isolation, characterization and identification of lactic acid bacteria involved in traditional fermentation of borde, an ethiopian cereal beverage. afr. j. biotechnol. 6 (12):1469-1478. achi ok (2005). the potential for upgrading traditional fermented foods through biotechnology. afr. j. biotechnol. 4(5): 375-380. adeyemi t, umar s (1994). effect of manufacture on the quality characteristics of kunun-zaki , a millet based beverage. niger. food j. 12:34-40. akoma o, jiya ea, akumka dd, mshelia e (2006). influence of malting on the nutritional characteristics of kunun-zaki. afr. j. biotechnol. 5(10):996-1000. agboola sd (1987). storage and preservation of agricultural products in nigeria, current status of methods; seminar on integrated rural development in kwara state. ajani aa, bello o (eds). ilorin. directorate of foods, roads and rural infructure. pp.10-15. ainsworth gc, sparrow fk, sussman as (1973). the fungi vol.1 va:a taxonomic review with keys; ascomycetes and fungi imperfecti, london: academic press. pp.13-67 . alice ls (1976). microbiology and pathology 11 th edn, cv mosby company. pp. 202-203. ayo ja, okaka jc (1998). interaction effect of cadaba farinose extract and ph levels on some physiochemical properties of kunun-zaki . proceedings of the 22 nd annual nifst conference. 23 rd -26 th , november, abeokuta , nigeria. pp. 31-33. bibek r (2001). fundamental food microbiology 2 nd edn. the crc press ltd washington, dc. pp 56-90. buchanan re and gibbons ne (1974). bergey’s manual of determinative bacteriology, baltimore. williams and wilkins co. 8 th edn. pp. 34-89. cheesbrough m (2002). biochemical tests to identify bacteria. in: laboratory practice in tropical countries, cheesbrough m (eds). cambridge edn. pp. 63-70 . efiuvwevwere bjo, akoma o (1995). the microbiology of kunun-zaki, a cereal beverage from nothern nigeria during the fermentation (production) process. world j. microbiol. biotechnol .11: 491-493. giese g (1995). measuring physical properties of foods. j. food technol. 2: 49 hatcher ws jr, weihe jl, splittstoesser df, hill ec, parish me (1992). fruit beverages. in: compendium of methods for the microbiological examination of foods. vanderzant c, splittstoesser d.f (eds). american public health association, washington, d.c. kolawole om, kayode rmo, akinuyo b (2007). proximate and microbial analysis of burukutu and pito produced in ilorin , nigeria. afr. j. biotechnol. 6 (5): 587-590. lateef a, oloke jk, gueguim-kana eb (2004). antimicrobial resistance of bacterial strains isolated from orange juice products. afr. j. biotechnol. 3 (6): 334-338. mislivec pb, beuchat lr, cousin ma (1992). yeasts and molds in: compendium of methods for the microbiolocal examination of foods. vanderzant c, splittstoesser df (eds). american public health association, washington, dc. nkama i (1993). studies on improving the nutritional quality of masa, a traditional nigerian fermented cereal base food. a report for uni, cftri, mysore , india. pp. 30-32. oloke jk (2000). activity pattern of natural and synthetic antibacterial agents among hospital isolates. microbiol. 102: 175-181. onuorah si, adesiyun aa, adekeye jo (1987). survival and multiplication of staphylococcus aureus and escherichia coli in a nigerian cereal drink (kunun-zaki): effects of spices, ph and temperature. j. food agric. 1: 31-34. pelczar mj, chane cs, noel rk (1993). microbiology 5 th edn., mcgraw-hill publishing company , new delhi. p. 272. phls advisory committee for food and dairy products (2000). guidelines for the microbiological quality of some ready –toeat foods sampled at the point of sale. comm. dis. pub. health. 3: 163167. african journal of food science research vol. 2 (6), pp. 099-104, june, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper extractive performance of solvents on the indices of african breadfruit (treculia africana) seed oil okocha nwabueze * and chinedu nwafor department of food science and technology, university of lagos, akoka, lagos state, nigeria. accepted 05 may, 2014 the physical and chemical indices of african breadfruit (treculia africana) seed oil extracted with polar (isopropanol, hexane and butanol) and non polar (acetone) solvents were investigated. the oil (19.85%) and energy contents 452.35 (kcal) suggest that african breadfruit seeds are a high -energy food. the yields were significantly (p 0.05) different with hexane extracted (non-polar solvent) oil having 19.85 % whilst oil extracted with polar solvents ranged from 15.58 -19.30 %. melting points were 34, 27, 26, 21 0 c for oil extracted with hexane, isopropanol, butanol and acetone respectively. smoke points were within the limits of 170 – 255 0 c for the four oil samples. iodine values ranged from 14.50 (hexane extracted) t0 25.17 (acetone extracted) . saponification values ranged from 125.89-267.85 while peroxide values were 3.20 mg/kg (hexane) and 3.60-3.83 mg/kg for polar solvents. free fatty (oleic) acid of oil extracted with hexane was 1.71% and polar solvent extraction ranged from 1.651.78%. hexane had the least peroxide value (8.74 mg/kg) compared to higher values for oil extracted with the polar solvents (9.10 9.83 mg/kg). keywords: african breadfruit, treculia africana, oil extraction, saponification, fatty acids, peroxide value, thiobarbituric acid introduction african breadfruit (treculia africana) constitutes a strategic reserve of essential food nutrients that are available at certain critical periods of the year when reliable sources of these nutrients are under cultivation and are very scarce. diverse food forms could be produced from the seeds on the basis of custom, tradition, ethnic background. it is boiled and consumed as white porridge and sauce with or without fresh corn, roasted and consumed with coconut or palm kernel as snack, made into refreshing milk drink, prepared into flour as soup condiment or thickener and for bakery and confectionaries. in the past the consumption was limited to poor village dwellers for whom it supplemented their diets during times of food scarcity and substituted the more expensive rice during festivals and other ceremonies on the basis of tradition and cost (nwabueze *corresponding author e-mail: nwa_okocha@yahoo.com and nwokenna, 2006). but today, african breadfruit has become a delicacy and a specialized meal not only for the rich and the urban dwellers in nigeria but has also become a foreign exchange earner. dehulled kernels are sun-dried and exported to cater for the african consumer interests overseas. african breadfruit seed is of high nutritional value. each seed contains about 14-17% crude protein, 2.5 % crude fibre, 35-60 % carbohydrate and a good supply of vitamins and minerals (akubor, 2000). the amino acid composition has also been highlighted to further buttress its nutritional potentials (nwabueze, 2007) . the oil potential of the seed has been reported (ajiwe et al., 1995). generally, plant oil serves as rich source of essential fatty acids and energy yielding approximately 9 kilocalories per gram as well as being a carrier of fat-soluble vitamins (a, d, e, and k) which help the body to absorb most of its nutrients (ajiwe et al., 1995). plant oils play other distinctive role in the natural flavour and palatability of a wide of food commodities (ihekoronye and ngoddy, 1985) http://www.internationalscholarsjournals.org/ okocha and chinedu 099 while certain of its components (linoleic, linolenic and arachidonic acid) are essential in all diets for body growth and normal skin condition (ebuehi et al., 2006). it has been reported (bjorck and asp, 1983) that only 45-55 % of the oil present in raw materials could be extracted with ethyl ether after extrusion. but nierle et al. (1980) report an average oil recovery in extruded wheat to be 40 and 20 % for maize. a number of reasons were advanced for this reduction of oil extraction in extrudates. monoglycerides and free fatty acids have been reported to form complexes with amylose during extrusion processing (mercier, 1980; badrie and mellowes, 1992). such complexes are likely to cause difficulty in plant oil extraction with petroleum ether. a literature search did not reveal much information deal ing with selection of organic solvents for plants and in particular african breadfruit seed oil extraction. the objective of this research was to evaluate the effectiveness of polar and non polar solvents in african breadfruit seed oil extraction and to study their effects on the physical and chemical properties of the oil. this study is necessary to assess african breadfruit seed oil quality indices for proper application in food and industrial system. materials and methods raw materials and preparation about 10 kg of african breadfruit (treculia africana) seeds were purchased from umuahia main market, abia state, nigeria. they were cleaned by hand picking to rid them of any contaminating stones or extraneous and organic materials before being parboiled at 100 o c for 15min. parboiled and drained seeds were then threshed in a commercial attrition mill and manually dehulled to recover the kernels. the kernels were sun dried for about 17 h and then milled in a blender (moulinex 276, france, speed 1) to fine flour (2 mm particle size). the flour was preserved in a tight polyethylene bag at room temperature (28± 2 o c) from which samples were collected for different analyses. extraction and yield of african breadfruit seed oil african breadfruit seed oil was extracted from the resulting flour using four different food grade organic oil extraction solvents (boiling point of 60 80 o c). these included iso-propanol, butanol, and acetone (polar solvents) and hexane (non polar solvent). the extraction involved the use of soxhlet extraction method (aoac, 1995). about 100g of the flour were weighed into a thimble in the soxhlet extractor fitted to conical flask. african breadfruit seed oil was extracted with 250 ml of each solvent. the solvent was boiled under reflux for about 6 h. african breadfruit seed oil yield was calculated for each extraction solvent by weight difference of the sample before and after extraction and reported as mean of duplicate determinations. proximate composition of dehulled full fat african breadfruit seed flour proximate composition of dehulled-sun dried african breadfruit seed was determined in triplicate for moisture, crude protein (micro kjeldhal method), fat (soxhlet method), crude fibre, and ash according to aoac (1995) methods. total carbohydrate was determined by difference. energy was calculated using the at water factors of 4 x protein, 4 x carbohydrate and 9 x fat (nwabueze, 2006). determination of vitamins in african breadfruit seed oil vitamin a was determined by the method described by delia and meiko (2003) while the spectrophotometeric method described by pearson (1976) was used for the determination of vitamin e. physical properties of african breadfruit seed oil the physical characteristics of african breadfruit seed oil determined included yield, colour (photometric system), specific gravity, melting and smoke points were determined by the standard methods as described by aoac (1995). specific gravity was determined by use of specific gravity bottles at a temperature of 28±2 o c. photometric colour index (pci) of african breadfruit seed oil was determined on 1g sample according to the method described by pike (2003). the sample was weighed and dissolved in 20 ml water/ethanol mixture. the mixture was filtered after standing for 30 min. the absorbance of the filtrate was measured at 400, 550, 620 and 670 nm using spectrophotometer (unican he 105y, england). the solvent was used as blank. photometric colour index was calculated as pci = 1.29 (a400) + 69.70 (a500) + 41.20 (a620) –56.41 (a670) (2) where a = absorbance. chemical properties and acid concentrations the acid values, iodine value, saponification value, peroxide and thiobarbituric acid values were determined by the standard methods of aoac (1995). fatty acid composition of the oil was determined by the method described by christie (1980) using a flow-mac flame ionization gas liquid chromatography. the methylation of the fatty acids was prepared according to standard methods of aoac (1995) . the equipment was set at oven temperature of 200 o c with a 1.8 m by 0.32 cm stainless column packed with 10 % degs on material support of silicon mesh size. nitrogen was used as a carrier gas at a flow rate of 25 ml/min and the quantification of the fatty acid was obtained by automatic integration with linear response attached to the system. effect of ambient storage on african breadfruit seed oil extracted oil samples from different extraction solvents were stored in plastic bottles tightly corked to practically prevent entrance of air at (ambient) room temperature (28± 2 o c) for 25 days. during this period the thiobarbituric acid and peroxide values were determined at 0, 8, 19 and 25th days for each extract and reported as mean duplicate determinations. statistical analysis differences between means were assessed by students t – test, while the levels of significance of the data were calculated by 100 afr. j. food sci. res. table 1. proximate composition and energy values of dehulled full fat african breadfruit seed flour. moisture (%) protein (%) fat (%) ash (%) carbohydrate (%) energy (kcal) 9.425±0.002 8.76±0.002 19.85 ±0.025 2.30±0.001 59.67 ±0.006 452.35±0.041 table 2. oil yield and vitamin content of african breadfruit seed oil extracted with different solvents. extraction solvent yield (%) vitamin a ( g/g) vitamin e (mg/100g) isopropanol 18.10 c ± 0.01 21.32 b ± 0.01 3.11 b ± 0.01 butanol 19.30 b ± 0.01 16.09 c ± 0.03 2.05 d ± 0.01 hexane 19.85 a ± 0.02 27.39 b ± 0.01 5.67 a ± 0.04 acetone 15.58 d ± 0.06 12.78 d ± 0.03 2.56 c ± 0.03 means not followed by the same superscripts along the same column are significantly different (p 0.05). analysis of variance at 0.05 % level of significance according to steels and torrie (1981). results and discussion proximate composition of dehulled full fat african breadfruit seeds flour the proximate composition of dehulled sun dried full fat breadfruit seeds flour is presented in table 1. with a moisture content of 9.43 %, the african breadfruit seed full fat flour will keep well. protein content was 8.70 % which was lower than the range (15.76 -17.52 %) reported by nwabueze (2006) probably due to variety used. since african breadfruit seeds have not been classified or graded into varieties or breeds or cooking types, they are marketed as mixtures of varieties, a problem that also affects its cooking time (nwabueze and nwokenna, 2006). the ash content of 2.30 % suggested a good source of minerals (nwokolo, 1987; akubor, 2000) while the oil (19.85%) and energy contents suggest the seed to be a high-energy food. the carbohydrate content of 59.66 % could pass african breadfruit seed for composite flour in baked and confectionary products. african breadfruit seed oil yield and vitamins oil yield and vitamin content (a as carotenoid and e) of african breadfruit seed oil extracted with different solvents are shown in table 2. generally, all the african breadfruit seed oil extracts was yellow in colour and remained liquid at room temperature (28±2 o c) . the photometric colour index shows significant (p 0.05) differrences in their values. this could be attributed to the selective permeability of the different solvents used. the yields of oil extracted with the hexane (non-polar solvent) were significantly (p 0.05) different from that of the oil extracted with polar solvents (isopropanol, butanol and acetone), having a yield of 19.85% while the amount of oil extracted by the polar solvents ranged from 15.58% (acetone) to 19.30% (butanol). various authors (nierle et al., 1980; ajiwe, et al., 1995; ebuehi and avwobobe, 2006) have reported varying extraction values with differrent solvents or seeds. ajiwe, et al. (1995) reported an extraction yield of 20.83% oil from african breadfruit seed by soxhlet using petroleum ether as extraction solvent, nierle et al. (1980) reported an average oil reco-very of 20% in extruded maize while ebuehi and avwobobe (2006) reported a yield of 17.36% for water melon. in this study, hexane gave highest oil yield but the use of such oil has been questioned in terms of safety particularly if not purified (bera, et al., 2004). ethanol, methanol and acetone have been recommended as solvents for extraction of vegetable oils. thus, oil extraction yield from african breadfruit seed may correlate closely with polarity of the extraction solvent. non-polar solvents have both sides charged and are able to penetrate into the matrix of a feed. this is so, because they lack an o-h end which otherwise would interfere with the extraction process. the low extraction yield of african breadfruit seed oil reported in this study and in literature could be attributed partly to extraction solvents used, state of the food material and partly due to complex formation between fatty acids and carbohydrate breakdown components (mercier, 1980). such complexes will definitely cause difficulty in plant oil extraction with petroleum ether. following the minimum fat requirement of 6% in complementary formulation (obatolu, 2002), the oil yields by all the solvents meet this requirement. vitamin a ( – carotene) varied significantly (p 0.05) with a range of 12.78 g/g in oil extracted with hexane to 37.39 g/g in oil extracted with acetone. vita-min a deficiency has been recognized as the second okocha and chinedu 101 table 3. physical properties of african breadfruit seed oil extracted with different solvents extraction solvent specific gravity (g/ml) melting point ( 0 c) smoke point( 0 c) photometric colour index isopropanol 0.8627 c ± 0.001 27.15 b ± 0.002 149.67 d ± 0.03 148.08 c ± 0.03 butanol 0.8756 b ± .0001 26.00 c ± 0.004 255.00 a ± 0.06 367.11 b ± 0.05 hexane 0.8656 c ± .0001 34.00 a ± 0.001 250.00 b ± 0.02 476.55 a ± 0.02 acetone 0.9760 a ± .0003 21.01 d ± 0.002 180.27 c ± 0.08 140.77 d ± 0.03 means not followed by the same superscripts along the same column are significantly different (p 0.05). table 4. chemical properties of african breadfruit seed oil extracted with different solvents extraction solvent iodine value saponification value peroxide value (mg/kg) isopropanol 15.72 c ± 0.06 125.89 d ± 0.3 3.83a±0.1 butanol 21.51 b ± 0.01 238.43 c ± 0.01 3.60 a ± 0.01 hexane 14.50 d ± 0.01 259.46 b ± 0.01 3.20 b ± 0.01 acetone 25.17 a ± 0.1 267.85 a ± 0.03 3.78 a ± 0.03 means not followed by the same superscripts along the same column are significantly different (p 0.05). most common form of malnutrition. the difference in vitamin a content of the oil samples could be attributed to a stability factor since it is readily stable in non polar solvents. thus the amount extracted could be as a result of the solvent used as well as the amount of oil extracted (yield) and the concentration of the vitamin in the extracted oil. the vitamin e content varied from 2.05 mg/100g in oil extracted with hexane to 56.69 mg/100g in oil extracted with butanol. all the oil samples extracted with polar solvents (isopropanol, butanol and acetone) had low values (2.05 – 3.11 mg/100g) of vitamin e. these vitamin e values are higher than those reported (baurnfeind, 1980) for soybean oil (1.2 mg/100g) and corn oil (2.0 mg/100g). with the working solvents in this study, african breadfruit seed oil will make a good source of vitamin e and hence have an antioxidant stability effect on the oil. this is because vitamin e has been recognized as the most biological oxidant in humans. physical properties of african breadfruit seed oil table 3 shows physical properties of african breadfruit seed oil extracted with different solvents. specific gravity was significantly (p 0.05) different varying from 0.8627 g/ml isopropanol to 0.970 g/ml (acetone). this range of specific gravity of african breadfruit seed oil compares with the ranges reported for palm oil (0.91) and groundnut oil (0.84) (ebuehi, et al., 2006) and 0.91 for pumpkin (curcubita pepo) seed oil (ihediohanma et al., 2006). higher deviations from these values point to hydrolytic and oxidative changes. melting and smoke points of the oil samples were significantly (p 0.05) different with extraction solvent used. the smoke points showed that african breadfruit seed oil could be used in deep frying food systems as oils with high smoke points resist ignition. chemical properties of african breadfruit seed oil chemical properties of african breadfruit seed oil extracted with different solvents are presented in table 4. iodine values were generally low when compared to other oils of plant origin. it ranged from 14.50 (hexane extracted) t0 25.17 (acetone extracted) which functionally may not qualify the oil to be used as a drying oil. the degree of unsaturation of oil is determined by measuring its iodine value. iodine value is a measure of fat or oil stability and resistance to oxidation. oresanya et al. (2000) stated that fat oxidation which occurs during fat extraction or storage and transportation leads to hydrolysis of triacylglycerols to free fatty acids. lee (1983) strongly suggested that iodine value of oil be determined by the temperature at which the oil seeds are produced, thus the higher the temperature, the lower the iodine value and vice-versa. african breadfruit seed oil from the four different solvents showed significantly (p 0.05) different values with respect to their saponification values. they ranged from 125.89-267.85. oil extracted with isopropanol had the least value while that of acetone was the highest. high saponification value indicates high molecular weight oil good mainly for soap making and for hair shampoo (ajiwe, et al., 1995). according to champe and harvey (1994), saponification values measure the amount of alkali required to combine with the fatty acids liberated by the hydrolysis of fats and oils from which weights of fatty acids could be determined. 102 afr. j. food sci. res. table 5. acid concentrations of african breadfruit seed oil extracted with different solvents extraction solvent ph acid value (%) thiobarbituric acid (mg/kg) oleic acid (%) isopropanol 3.38 d ± 0.1 3.21 c ± 0.06 18.42 a ±0.3 1.65 b ± 0.06 butanol 4.45 c ± 0.0001 2.48 d ± 0.0001 16.63 b ± 0.0001 1.25 c ± 0.0001 hexane 5.70 a ± 0.0001 3.40 b ± 0.0001 15.69 c ± 0.0001 1.71 ab ± 0.0001 acetone 5.16 b ± 0.03 3.55 d ± 0.03 14.83 d ± 0.03 1.78 a ± 0.03 means not followed by the same superscripts along the same column are significantly different (p 0.05). t h io b a rb it u ri c a c id ( m g m a lo n a ld e h y d e /k g o il) 70 60 50 40 30 20 10 0 day 0 day 8 day 19 day 25 isopropanol butanol hexane acetone extraction solvents figure 1. effect of storage on thiobarbituric acid of african breadfruit seed oil extracted with different sovents. peroxide value of african breadfruit seed oil varied with solvent used. peroxide value is used as an indicator of deterioration of fats and oils. as oxidation takes place, the double bonds in the unsaturated fatty acids are attacked producing peroxides. this in turn decomposes releasing secondary products which cause rancidity (champe and harvey, 1994; ebuehi, et al., 2006). in this study, peroxide value of oil extracted with hexane (3.20 mg/kg) was significantly (p 0.05) lower than oil samples extracted with polar solvents (3.60-3.83 mg/kg). acid concentrations of african breadfruit seed oil table 5 shows acid concentrations of african breadfruit seed oil extracted with different solvents. mean ph values of the oils varied significantly (p 0.05) with different solvents. it ranged from 3.38 (isopropanol extraction) to 5.70 (hexane extraction). this variation could have affected the values of vitamins observed in this study as ph < 7 has been reported (ihekoronye and ngoddy, 1985) to affect the stability of vitamin a and its precursors. the table showed that there was no significant (p 0.05) difference between the free fatty acid (oleic acid) of oil extracted with hexane (1.71%) and that extracted with either acetone (1.78%) or with isopropanol (1.65%). oil samples extracted with butanol had the least free fatty acid as oleic acid and so may not be easily prone to oxidation. champe and harvey (1994) stated that several cardiovascular diseases have been implicated in human population consuming diets rich in polyunsaturated fatty acids. the acid value for the four different crude oils were high thus, the oil extracts would need some form of purifi okocha and chinedu 103 12 10 /k g o i) 8 (m e g .o 2 6 v a lu e p e ro x id e 4 2 0 day 0 day 8 day 19 day 25 isopropanol butanol hexane acetone extraction solvents figure 2. effect of storage on peroxide valves of african breadfruit seed oil extracted with different sovents. cation (refining) to enhance stability and storage. low acid values suggest stability of oil. oscar (2002) reported that acid value or free fatty acid (ffa) in crude oils, estimates the amount of oil that will be lost during refining. thus the higher the acid as ffa values the higher the amount of oil that could be lost during processing. effect of storage on thiobarbituric acid and peroxide values of african breadfruit seed oil effect of storage of african breadfruit seed oil extracted with different solvents on thiobarbituric acid and peroxide value are shown in figure 1 and 2, respectively. thiobarbituric acid and peroxide values are two important rancidity indices which were found to vary with storage in this study. at the end of the 25 day storage period at room temperature (28 o c), the oil extracted with hexane had the least peroxide value of 8.74 mg/kg compared to higher values for oil extracted with the polar solvents which ranged from 9.10 mg/kg (butanol) to 9.83 mg/kg (isopropanol). this observation indicates that rate of autocatalytic degradation of oil extracts, measures rancidity, increased with storage. the relatively higher peroxide values of oil samples extracted with polar solvents with storage were still lower than 20 mg/kg allowed for use olive oil (fao/who, 1993). lower values imply that the oil has lower degree of rancidity. the rate of increase which was also observed in thiobarbituric acid of oils related to extraction solvents and perhaps to vitamin e content of the oil as antioxidant. this trend has been attributed to the fact that products formed during the reaction tend to catalyze the rate of autoxidation reaction. lee (1983) reported that the reaction increases exponentially. conclusion most of the studies reported in this favoured the use of non polar (hexane) solvent as an extraction solvent. the extraction of oil with hexane gave highest oil yield but the use of such oil has been questioned in terms safety. it is recommended above other physical and chemical properties for use in industries other than food. however the oil extraction with acetone a polar solvent is recommended for food use on safety grounds. references ajiwe vie, okeke ca, agbo hu (1995). extraction and utilization of breadfruit seeds oil (treculia africana). bioresources technology, 53: 183-184. akubor pi (2000). studies on food potentials and storage properties of african breadfruit kernel flour. an unpublished msc thesis, department of food science and technology, university of agriculture, makurdi, nigeria. aoac (1995). official methods of analysis . 15th edition. association of official analytical chemists, arlington, va: association of analytical chemists. badrie n, mellowes wa (1992). cassava starch or amylose effects on characteristics of cassava (manihot esculenta crantz) extrudate. j.food science. 57: 103-107. baurnfeind j(1980). in: machlin, l.j. 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(edition). cbs publishers & distributors. new delhi. pp. 195-204. pearson d (1976). the chemical analysis of foods: 572 churchill livingstone london. 7th edn. pike ao (2003).fat characterization. in: food analysis, 3rd edition. aluwer academic /planum. publishers new york. pp. 227 –235. steels rgd,torrie jh (1981). principles and procedures of statistics.a biomedical approach. 2nd edn. mc graw –hill intern auckland. pp.50102. african journal of food science research vol. 2 (2), pp. 051-058, february, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper the impact of rural agricultural development projects on agricultural products in preferred area of benin kashez asiamah, awuah nulty and kubby j. department of agricultural and resource economics, university for development studies, tamale, ghana. accepted 22 january, 2014 in this study, data collected from 120 rural households located in two distinct socio-cultural locales of benin was used to assess the impact of 20 development projects on agricultural productivity. a ‘withwithout’ approach of impact evaluation is followed using anova and econometric regressions. results reveal no significant differences of projects on agricultural productivity between participants in the two study zones. econometric regression estimates show significantly positive impacts on agricultural productivity for two selected project indicators in the two study zones. however, the goal achievement index was more remarked in the adja area, where the projects were found to have better addressed development problems and provided higher impact. the results suggest the need to improve management of agricultural projects to enhance their impact. likewise, objectives and activities of the projects should be oriented to deal better with development problems of rural people, in particular those of the poorest and marginalized communities. key words: productivity, rural projects, impact, benin. introduction most less developed countries depend on rural areas for most of their survival and development resources. agriculture, which is the main activity of these areas, employs between 70% and 80% of the working population. besides, local rural populations ensure food consumption from agricultural production, thereby guaranteeing household food security (world bank, 2008). in spite of this key role of rural areas in development, they are confronted more and more with severe problems, which retard their progress with consequent problems for livelihood survival of the local people. natural resources such as land, forest and water do not stop degrading gradually. the decline in land fertility has resulted in decreased agricultural productivity. for decades, developed countries, international corresponding author. e-mail: kashez360@yahoo.com institutions such as the world bank, fao, undp, as well as non-governmental organizations ngos) have been fighting ceaselessly to contribute to the development of rural areas via actions and interventions implemented through rural development projects. such projects have often been designed, planned and implemented to help the rural people to develop their agriculture and to have better access to farm inputs to increase their income. in addition, most projects tend to strengthen the capacities of rural farmers through education, training and institutional support. given the key role that project interventions play in the rural development process, it is imperative to assess both the specific and overall impacts of implemented projects. several approaches to evaluate rural development projects have evolved over time. according to kirkpatrick (1994), various appraisals of most projects have focused on cost-benefit or cost-effectiveness approaches by assessing project costs (monetary or non-monetary), in particular, their relation to alternative uses of the same http://www.internationalscholarsjournals.org/ kashez et al. 051 resources and to the benefits being produced by the projects. however, some outputs of rural development projects such as capacity building and improvement of food security are sometimes difficult to measure and/or provide unsatisfactory results via the cost-benefit approach. indeed, the decisive issue of a measure of project success is not whether the planned results have been achieved, but what impact the activities of the project have provided and whether they satisfy all the stakeholders. consequently, project evaluations in recent times have focused on the impact evaluation approach, whereby project success emphasizes more broadly on whether the project had the desired effects on individuals, households and institutions and whether those effects are attributable to the project intervention. accordingly, evaluating the impact of rural development projects on agricultural productivity becomes a challenge to deal with (gtz, 2008). in evaluating projects, the central problem is how to isolate and to estimate their impacts on target groups. since many other exogenous factors that are not related to project execution (government policy, market conditions, former experiences, etc.) also have an influence on target groups’ evolution, appraisal approaches of projects seem to be difficult. the literature proposes two main approaches with different concepts of measurement: the ‘before-after’ and ‘with-without’ approaches as illustrated in figure 1 (bauer, 2000) . according to kerr and kolavalli (1999) and adekambi (2005), if the ‘with-without’ approach is designed in a consequent way to isolate the exogenous influences and to carry out the project impact only; it may provide more reliable results. this paper therefore focuses on the use of the ‘with-without’ approach to estimate the impact of rural development projects on agricultural productivity of farmers in two regions of benin. review of impact of projects on household livelihood various authors have in the past focused on impact evaluation of development projects on sustainability. in central america for example, a number of projects have promoted soil conservation or soil recuperation technologies that benefitted farmers through increase in productivity (brunch, 2001). likewise, doppler and bothe (1999) showed that the adoption of cassia siamea in rural benin improved soil fertility and agricultural productivity and led to an increase in the overall family income. this helped to reduce poverty of many rural farming households. as a result of increase in productivity and income due to the adoption of new technology for bean growing in south-benin, allogni et al. (2008) found that food expenditure increased and food security in households showed some improvement. data from (2006) also show that, increases in agricul agricultural productivity and income over the years due to rural development projects have undoubtedly raised food availability and kept food prices low, providing critically important benefits for extremely poor households that spend more than half their income on food (kerr and kollavali, 1999). arguing in the same way, ifpri (2001) reported that the project “improving food security in bangladesh” implemented since the 1980s resulted in a significantly increased availability of and access to food in rural areas of bangladesh. in countries where starvation is disastrous for rural people, various projects are implemented to avoid malnutrition diseases and death, mainly among children. for example, (idrc, 2003) found that 30 projects implemented in ethiopia that focused on agriculture and water management saved more than 25% of rural communities from starvation, malnutrition diseases and death. the foregoing discussion shows an optimistic view of the adoption of technology leading to poverty alleviation through positive effects on consumer food prices, producer incomes and labourer waged incomes. in this scenario, higher productivity, better natural resource management and poverty alleviation are mutually reinforced and may lead to achievement of a sustainable food system (winkleman, 1998). in contrast to this optimistic point of view, the pessimist sees the overall process of project implementation and technology adoption in agriculture biased towards wealthy people so that the poor are made worse off. the rich get richer while the poor get poorer resulting in social unrest and a decidedly unsustainable food system. the key relationship according to this framework is that technologies, policies and institutions are biased in favor of wealthy farmers who have unequal access to assets to begin with. their income rises when they adopt the improved technologies while the income of non-adopting farmers fall, many agricultural workers are displaced and some of those who remain, suffer from overexposure to poisonous chemicals (winkleman, 1998; kerr and kolavalli, 1999). finally, in impact, evaluation of a rural development project, another decisive discourse regarding success is whether the impacts are maintained after the project is completed, or in short, whether the project is sustainable (gtz, 2008). sustainability is seen as a result of the impact on sustainability of the production system where the project is implemented and of a long-term duration of the impact, even after the termination of the projects. typically, sustainable projects are those designed and financed to build local capacities and to develop the ability of local people to manage and utilize the development activities themselves, that is institutional and empowerment supports (clayton et al., 1998; uphoff, 1989; mcallister, 1999) . the capacity building is particularly viewed as very important for sustainability and many institutions such as gtz, world bank, undp, etc. c indicator b impact(income) a beginning of the project figure 1. illustration of project impact (bauer, 2000). have directed their supports towards more technical assistance to achieve better capacity building of local people (rudovist and woodford-berger, 1996; gtz, 2008). methods and analysis theoretical and empirical modelling methods for impact evaluation provided in the literature include systematic comparison, indicator trend function, econometric models and more complex system modelling (bauer, 2000; yabi, 2004). to estimate direct changes in agricultural productivity when the participation in project changes, the study focussed mainly on econometric models to evaluate the impacts. supposing that ip is an index of projects and y represents agricultural productivity, then if a unit change in index ip induces  unit change in y, then:  y /  ip   (1) by taking an integral of both sides of the equation (1), we obtain:  y dip   dip (2)  ip it is evident from equation (2) that y can be a function of ip and the general mathematical form of the regression model is expressed as: y= f(x1, …, xn, ip, z1, …zm, ) (3) where,  is the error terms supposed to be a n(0, 2 ), the x1, …, xn are production inputs and z1, …, zm other explanatory variables such as economic, social or human capital variables, etc. considering its computational ease and extensive use in many studies, the cobb-douglas functional form is applied to equation (3) to estimate the impacts of participation in projects on agricultural productivity. the empirical model estimated in this paper can be 052 afr. j. food sci.res. without the project with the project status quo time evaluation specified as: ln( yi )    1 ln(landi )  2 ln(labori )  3 ln(capi i )  ipi  1proi 2 agei  3sex i  4 edu i  5 alph i  6ten i  i (4) where: 1n(.) = the natural logarithm; i = the i th farmer; yi = the value of agricultural productivity expressed in fcfa/ha(fcfa is the local currency for benin. 1 =655 fcfa ). the productivity is computed for major cultivated crops such as maize, cotton, cassava, nuts, beans and yams. land is the overall cultivated area in hectare (ha) while labour, the total family labour used is expressed in man-days per ha and capital, the total capital used in fcfa per ha. the total capital is calculated as the total amount of input expenditures (seed, fertilizer, pesticide, hired labour, etc.). pro is a dummy variable representing the project type; pro = 1 if the project is integrated and 0 if it is single activity project; age is the age of the farmer (year); sex is a dummy variable expressing the sex of the farmer; sex = 1 for a man and 0 for a woman; edu is a dummy education variable; edu =1 if the farmer is formally educated and 0 if not. alph is a dummy informal education variable; alph = 1 if the farmer had received informal education and 0 if not; ten is a dummy variable of land tenure; ten = 1 if the cultivated land is secured and 0 if not. socioeconomic and demographic variables that were essentially measured in this study as dummy variables can accordingly not be logged. the ip are indicators of agricultural projects at a beneficiary level. by the nature of the approaches employed during the project implementation in the study zone, most local people had the opportunity to participate in several projects at the same time without any restrictions. in order to appreciate the presence of the projects at a beneficiary level, two indicators, relative to the projects in which each stakeholder was involved, were computed, namely: contact index (ic) and goal achievement index (is). the ic is expressed as the sum of contact frequency at stakeholder level and kashez et al. 053 mathematically defined as: if the farmer i was involved in no project ici = 0 n if farmer was involved in n f ki projects; n = 1, 2, …, p k 1 where; ici represents the contact index of stakeholder i, fki the frequency of contact this stakeholder i made per week with the team of the k th project, n the number of projects in which he participated. as defined, the contact index considers only the frequency of contacts with the beneficiaries. it fails to take into account success in achievement of activities that were implemented through projects. conversely, the goal achievement includes the overall success in achievement of objectives and activities of the projects, and thus computing its index could help in appreciating these aspects at the beneficiary level. as suggested by sarbeck (1994), the utility value of the projects can be defined as: 1 (6) ua  gi * gai g i where; ua is the utility value with 0<ua<1, gi the grading or weight and gai the achievement of goal i. by introducing in equation (5) the utility value computed in equation (6), it is possible to define the goal achievement index (is) as: y / y y  ic   ic ic ic ic y ic ic / ic y ic y (8) y / y y is is  is  (5)   is y   is is is / is y y is (9) where; ic and is represent respectively the parameters of ic and is in equation (4), ic and is their means computed from the study sample. the elasticities ic and is gives the percentage of variation in the impact due to 1% variation in the project indicator. study area and data datas were collected from two distinct socio-cultural locales of benin (adja and nagot), where 20 selected agricultural projects were implemented. for the ‘with-without’ approach, kerr and kolavalli (1999) and pitt and khandker (1996) suggested the randomisation of the “with” and “without” groups selected for the study. according to the authors, if the sample is not drawn randomly, or if there are hidden relationships determining between relationships of interest, the findings will be biased, that is, the statistics estimated for the sample will not represent those for the entire population. therefore, representative samples of agricultural households were chosen by randomisation according to the number of projects in which they were involved. however, three stages of stratification were distinguished for prior to the final random sampling. in each socio-cultural area of the study zone, three villages were selected: one without a project, one with a single project and one with 2 or more projects. on the basis of this typology, three categories of households were identified for the study. the first concerned the “without project” group of households which had not participated in any development project. the second category involved “with one project” group of households that had participated in a single project. the third category of households was the “with two or more projects” group of households who had 0 isi = n ua k . f ki k 1 if the farmer i was involved in no project (7) if farmer was involved in n projects; n = 1, 2, …, p participated in two or more projects. in each of the identified villages, twenty (20) households were selected at random from each group. in total, a sample size of one hundred and twenty (20 × 3 × 2 = 120) was drawn for the study. as much as possible, the intensity of participation in agricultural projects remained the key criteria for selecting respondents for the randomisation between the two groups in each socio-cultural area. data collected related to outputs, inputs such as labour, fertilizer, pesticides, etc. and socioeconomic and demographic characteristics of producers (sex, age, education level and frequency of participating in projects). where; isi represents the goal achievement index of stakeholder i, uak the utility value of the kth project in which he was involved as defined in equations (6). the fki and n are defined as in equation (5). the i are the error terms and the , ,  and  are parameters to be estimated.  is the origin ordinate, the  gives the elasticities of the productivity with respect to the corresponding farmsupplied factors {(excluding land(since the productivity is the output per unit of land, the parameter of land represents the return to scale)} and the  the percentage increases in productivity in response to a unit increase in the related variables. the  are estimates of project indicators. according to a hypothesis of positive impact of agricultural projects on agricultural productivity, they are supposed to be positive and significant. from equation (4), the elasticity of productivity ic and is with respect to ic and is can be thereby computed as: results descriptive statistics of variables used in the models the descriptive statistics showed on one side low indexes of contact (ic) with no significant difference for the two areas of the study zone. the fact is that the projects’ teams did not work with the stakeholders as frequently as supposed. at the same time, the farmers did not also respect the appointments that they got from the projects’ teams to work together. as a consequence, the effective frequency of meeting with the project teams 054 afr. j. food sci.res. table 1. descriptive statistics of variables used in the models. qualitative variables adja area (n = 60) nagot area (n = 60) count (%) count (%) type of project not integrated 23 57.5 14 35.5 integrated 17 42.5 26 64.5 total 40 100 40 100 sex woman 1 1.67 13 21.67 man 59 98.33 47 78.33 total 60 100 60 100 education no 37 61.67 26 43.33 yes 23 38.33 34 56.67 total 60 100 60 100 alphabetisation no 38 63.33 34 56.67 yes 22 36.67 26 43.33 total 60 100 60 100 land security no 16 26.67 4 6.67 yes 44 73.33 56 93.33 total 60 100 60 100 ratio variables means variation coefficients (%) means variation coefficients (%) age (year) 39.77 26.54 41.63 33.13 contact index (ic) 1.6 8.7 1.6 5.6 goal achievement index (is) 0.54 20.4 0.49 20.41 cultivated areas (ha) 0.65 54.00 2.81 67.45 family labour (man-day/ha) 86.71 54.56 63.88 51.68 capital used (fcfa/ha) 44,328.64 45.81 71,914.5 38.82 productivity (fcfa/ha) 178,925.29 39.84 162,578.63 29.00 was lower than expected during the project design and the planning phase such that the indexes were as well low. besides, the indexes of goal achievement (is) were lower than those of contact. this result may be explained by a very poor goal achievement of these projects. conversely, the percentage of secured land remained very high in the nagot area where lands were more available and fertile. consequently, access to and conservation of land did not seem to have been a previous (prior to project implementation of projects considered in this study) problem of farmers in this area. this situation might explain the lower agricultural productivity in the nagot area as compared to the adja (table 1). since nagot farmers found their soil to be still relatively fertile, they did not care much about modern cultural techniques diffused by the projects and continued to employ local methods of soil conservation. with regards the use of inputs, land pressure was higher in the adja area and agricultural households had an average of 0.65 ha of endowed land as compared to 2.81 ha in the nagot area. in order to facilitate the adoption of modern technologies, adja farmers utilized more family labour. however, they used less mineral fertilizers, pesticides and hired labour than farmers of the nagot area, though, they produced more intensively. the situation could be explained by improved availability of and access to inputs in the nagot area. indeed, by producing more cotton, the existing agricultural policy that guarantees availability of and access to agricultural inputs to cotton producers favoured them. however, pro t h o u sa n d s o f fc fa p er h a 240 180 outputs 120 variable costs gross margins 60 0 without project with 1 project with 2 or more (n = 20) (n = 20) projects (n=20) groups of participation figure 2. outputs, variable costs and gross margins (in thousands of fcfa per ha) of agricultural production according to groups of participation in projects in the adja area. producers of the nagot area had lower agricultural output than those of the adja area (table 1). groups of participation in projects and agricultural productivity the analysis (anova) of economic indicators of agricul-tural production showed no significant difference between their averages according to the groups of participation in projects in the two sociocultural areas. nevertheless, the farmers that participated in the projects had agricultural outputs higher than non-project participants. even though, the variable costs of production were higher for project participants, their gross margins were also better in comparison with farmers without project. additionally, the results demonstrated that farmers with a single project had higher gross margins than those with 2 or more projects (figures 2 and 3). this could be due to the fact that those with a single project were more efficiently involved in the project than those with 2 or more projects. in addition, the effects of projects would be counter-productive to each other instead of being complementary. these results challenge the positive effects of simulta-neously implementing several agricultural projects at the same place and as well the necessity for farmers to con-currently participate in such multiple projects at the same time. but, an absence of significant difference between the groups of a participa-tion in projects does not expli-citly imply similar conclusions on impacts of project. for example, the absence of observed differences noted in this study may suggest that there is no correlation between groups of participation and the intensity of participation in projects. the results also reveal statistically significant differences between the selected economic indicators in the two distinct locales investigated in this paper. specifically, agricultural outputs and gross margins were significantly higher in the adja area than in the nagot area (178,925.29 fcfa/ha against 162,578.63 fcfa/ha and 134,596.64 fcfa and 90,664.15 fcfa; figures 2 and 3). nevertheless, a comparison of economic indicators with respect to areas of implementation fails to give the desired level of the impact of the projects on agricultural productivity in each area. impact of the projects on agricultural productivity the results of the econometric models are generally statistically significant and overall satisfactory for the two socio-cultural areas (tables 2 and 3). the parameters of indicators ic and is are found in all cases to be significant and positive. the results from this study show that the projects had positive impact on productivity. indeed, they popularized and diffused modern production techniques, which allowed farmer beneficiaries involved in the project to significantly improve their productivity. enhanced capacity building resulting from training and group formation did strengthen the managerial capacities of producers. however, the regression results call for two types of analyses and interpretations. kashez et al. 055 056 afr. j. food sci.res. 240 180 fa p er h a 120 o f fc t h o u sa n d s 60 0 without project with 1 project with 2 or more (n=20) (n=20) projects (n=20) outputs variable costs gross margins groups of participation figure 3. outputs, variable costs and gross margins (in thousands of fcfa per ha) of agricultural production according to groups of participation in projects in the nagot area. table 2. estimated parameters of factors affecting agricultural productivity in adja socio-cultural area. independent variables model with ic model with is notations b statistic t b statistic t constant 9.402*** 14.882 6.112*** 3.799 family labour (man-day/ha) labour 0.120** 2.043 0.526*** 4.057 capital (fcfa.ha -1 ) capi 0.145*** 2.837 0.235* 1.802 cultivated area (ha) land 0.018 0.420 0.048 0.460 type of project pro 0.08 0.976 0.187 0.836 formal education edu 0.135*** 2.979 0.034* 1.968 sex sex -0.011 -0.163 -0.072 -0.424 age (yr) age 0.001 0.533 0.004 0.809 informal education alpha -0.081 -1.550 -0.071 -0.548 land security ten 0.251** 2.031 0.325 1.131 contact index ic 0.639** 2.314 goal achievement index is 3.616*** 5.648 adjusted r2 0.55 0.80 f statistic 9.17*** 23.96*** observations number 60 60 dependent variable y output (fcfa.ha -1 ) elasticity of productivity with respect to project indicators  1.02 1.95 *** significant at 1%; ** significant at 5%; * significant at 10%. kashez et al. 057 table 3. estimated parameters of factors affecting agricultural productivity in nagot socio-cultural area. independent variables model with ic model with is notations b statistic t b statistic t constant 7.230*** 14.420 5.519*** 8.170 family labour (man-day/ha) labour 0.080* 1.691 0.439*** 4.500 capital (fcfa.ha -1 ) capi 0.364*** 8.290 0.274*** 4.149 cultivated area (ha) land -0.026 -0.870 0.131* 1.909 type of project pro 0.119* 1.823 -0.146 -1.105 formal education edu 0.111*** 2.713 0.166* 2.166 sex sex 0.179 1.224 0.323 0.903 age (yr) age 0.003 1.331 0.009* 1.886 informal education alpha -0.017 -0.383 -0.054 -0.463 land security ten 0.051 1.060 -0.023 -0.195 contact index ic 0.448*** 3.247 goal achievement index is 2.181*** 4.455 adjusted r2 0.84 0.9 f statistic 36.15*** 54.9*** observations number 60 60 dependent variable y output (fcfa.ha -1 ) elasticity of productivity with respect to project indicators  0.72 1.07 *** significant at 1%; ** significant at 5%; * significant at 10%. firstly, the impact level varied according to indicator considered. the computed elasticities from regression coefficients reveal that a variation of 1% of ic induced a variation of 1.02 and 0.72% in productivity in the adja and the nagot areas, respectively. when considering only the goal is of the projects, a variation of 1% of ic induced a variation of 1.95 and 1.07% in productivity in the adja and the nagot areas, respectively (tables 2 and 3). these results show that is, which includes overall aspects of management and the achievement of objectives, provides the highest impact on productivity. in fact, besides the direct contact and working with beneficiaries, the projects helped their organizations to acquire organizational skills of inputs distribution and product commercialization. they also built rural infrastructures such as rural roads, informal education centres, hospitals, etc. these additional activities, which were considered in the goal achievement evaluation, also affected productivity indirectly. using the is in the regressions has helped to estimate full impacts of the projects on productivity of beneficiaries. secondly, the impacts were more pronounced in the adja area than in the nagot area (ic =1.02 for adja against ic =0.72 for nagot and is = 1.95 for adja against is = 1.07 for nagot; tables 2 and 3). these results confirm the findings of our descriptive statistics, which showed that agricultural outputs and gross margins gained by producers in the adja area are significantly higher than those of nagot area producers. as previously explained, lands in adja area had already reached levels of degradation with decline in fertility to such an extent that the adoption of modern production practices and techniques resulted in considerable positive effects on productivity. in contrast, since lands in the nagot area remained relatively fertile and more available soil nutrients for crop growth, the impacts of modern production practices popularized by rural development projects, although positive, stay even lesser. indeed, producers of the nagot area still extended their cultivated areas, instead of intensifying their agricultural production to sustain the management of natural resources. conclusion the positive impacts provided by is of the projects at the beneficiaries’ level suggest that a scenario of improvement on design, management and monitoring, which leads to better goal achievement of the projects may improve the impact. therefore, agricultural policy in benin should focus more on efficiency in designing, managing and monitoring rural development projects. in addition, the results show that the impact was strongly related to the locale where the projects were implemented. consequently, policy makers in benin sholud place emphasis on implementing projects at the level of small scale, in order to tackle effectively development problems encountered by target beneficiaries, in particular the poorest. likewise, as suggested by various authors cited in the literature review section, it is important to support the sustainability of the impacts by strengthening capacity of stakeholders and their organizations. thus policy implication, conception, design, implementation and monitoring of rural development projects should also include aspects related to capacity building of stakeholders such as: training, reinforcement of organisational and management skills, etc. these inevitably call for a combination of financial and technical support. acknowledgments the authors are much grateful to the german academic exchange service (daad) for its financial support without which this study could not have been undertaken. we also thank prof. dr. n. sokpon for revising this paper. references adekambi s a (2005). ‘impact de l’adoption des variétés améliorées de riz sur la scolarisation et la santé des enfants au bénin : cas du département des collines’, fsa, uac, bénin, 100p. allogni wn, coulibaly o, honloukou an (2008). ‘impact des nouvelles technologies de la culture de niébé sur le revenu et les dépenses des ménages agricoles au bénin’. annales des sci. agron. du bénin 10 (2) :179-192. baeur s (2000). ‘konzeptstudie : evaluierung zukunftsinitiative rheinland-pfalz’, dept. of project and regional planning. justusliebig university giessen, giessen, germany, 40 p. brunch r (2001). ‘enabling long-term impact of soil conservation through farmer-driven extension’. 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(eds.). ‘farming and rural system economics’. margraf publishers, weikersheim, germany 135p. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (8), pp. 196-202, august, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of live prey’s enrichment with three different diets and their effects on survival and growth of common dentex larvae ali ait ali 1* , nadia rharbi 2 , houda akharbache 1 and said sedki 1 1 centre spécialisé en aquaculture, bp. 31-inrh. m’diq. morocco. 2 université hassan ii. faculté des sciences ain chock, casablanca, morocco. accepted 11 april, 2015 the rotifer brachionus plicatilis (s-type) and artemia were fed with three enrichment types (nanno: nannochloropsis occulata, cep: commercial enrichment products: selco ® from inve aquaculture nv and clo: cod liver oil emulsion). the polyunsaturated fatty acid composition in the regimes was tested to estimate survival and growth of common dentex larvae. during rotifers feeding phase, epa (20:5n-3) contents were of 4.6 ± 1.46 mg g -1 dw for nanno regime, 5.1 ± 0.22 for the cep regime and 6.1 ± 0.87 mg g -1 dw for the clo regime; for dha (22:6n-3), clo regime contains the highest level (7.2 ± 1.26 mg g -1 dw) than the two other regimes. during artemia feeding phase, epa contents were 2.4 ± 0.05, 3.2 ± 0.03 and 8.3 ± 0.41 mg g -1 dw and in dha 0.4 ± 0.00, 4.4 ± 0.13 and 8.6 ± 0.69 mg g -1 dw respectively for nanno, cep and clo enrichments. the weight growth of larvae showed a significant difference (p < 0.05) for the rotifers and artemia feeding phases. the respective sgr for the three enrichments are 8.8, 8.9 and 9.2 and survival rates were of 3.15, 4.03 and 8.35% respectively for nanno, cep and clo regimes. key words: dentex dentex, live-preys, n-3 hufa, larvae, growth, survival. introduction the major problem for mass production of common dentex is the high mortalities during the larval phase. this mortality can be associated to environmental factors (temperature, salinity, water renewal rate, lightening intensity, and handling). the priority of investigations should be focused up on the nutritional aspects, espe-cially the nutritional value of preys during the first stages of feeding. their nutritional value depends on the nature of the food source, particularly contents in (n3) highly unsaturated fatty acids ((n-3) hufa), such as docosa-hexaenoic acid (dha, 22:6n3) and eicosapentaenoic acid (epa, 20:5n3). the lipid sources in the enrichment diets differ in lipid class composition (mcevoy et al., 1996; tocher et al., 1995), (n-3) hufa content (dhert et al., 1993; evjemo et al., 1997) and dha/epa ratio (naess et al., 1995; evjemo et al., 1997). the low diets in fatty acids can be at the origin of massive mortality in larval phase for aquacultured marine fish species. the enrich *corresponding author. e-mail: a.aitali2@caramail.com. ment protocols improve the hufa content rotifers and artemia nauplii (léger et al., 1987; takeuchi et al., 1992; watanabe, 1993) . sorgeloos et al. (1988) reported a strong correlation between the epa content and larvae survival and between dha content and the growth of asiatic sea bass larvae. watanabe (1993) conclude that epa and dha increase survival and growth of larvae of several marine fishes. koven et al. (1990) suggested that such acids function as essential component of biomem-branes, and their level in the tissue phospholipid fraction are associated with larval growth. the purpose of this study was to determine the effectiveness of live prey’s enrichment with three different diets and their effects on survival and growth of common dentex larvae. material and methods brood stock and origin of dentex’s larvae the brood stock was acquired in march 2001 and acclimatized to captivity conditions. parents were maintained in a polyester rect ali et al. 197 table 1. fatty acid composition (mg g -1 dw) of dentex’s eggs and larvae. data represent means ± sem egg newly hatched larvae total lipids(%dw) 0.38±0.00 a 1.97±0.01 b 14.0 4.6±0.01 4.8±0.23 15.0 1.8±0.01 1.7±0.04 16.0 20.4±0.39 a 16.5±0.25 b 17.0 2.6±0.01 2.5±0.03 18.0 6.2±0.08 a 4.5±0.03 b 16:1 8.3±0.13 a 9.1±0.14 b 17:1 1.5±0.01 a 1.2±0.01 b 18:1n-9c 16.9±0.22 a 18.0±0.08 b 18:1n-7 4.0±0.06 3.7±0.00 20:1n-9 1.9±0.04 1.8±0.01 22:1n-9 1.2±0.00 1.2±0.02 24:1n-9 1.3±0.00 1.2±0.00 18:2n-6c 2.6±0.04 2.7±0.04 20:2n-6 5.3±0.01 5.3±0.01 18:3n-6 4.1±0.00 4.2±0.00 20:3n-6 nd 0.1±0.01 20:4n-6 1.2±0.02 a 0.9±0.01 b 18:3n-3 0.9±0.21 0.9±0.01 20:3n-3 nd nd 20:5n-3 3.6±0.08 a 4.7±0.04 b 22:5n-3 1.0±0.01 a 1.5±0.04 b 22:6n-3 16.9±1.10 a 22.1±0.69 b saturated 35.3±0.50 a 30.2±0.57 b mono saturated 35.1±1.05 36.2±0.09 dha/epa 4.7±0.19 4.7±0.10 n-3 hufa 21.5±1.39 a 28.3±0.78 b n-3 hufa = > 20:3n-3. nd= not detected means ± sem having different letters indicate that treatments are significantly different (p<0.05) according to kruscal wallis test. (n = 2) rectangular tank, of volume 10 m 3 . the renewal sea water rate was maintained about 300 500%d -1 and oxygenation was provided by two air inflows. the temperature (14 22°c) and salinity (36 38 ppm) conditions were natural. the spawning was spontaneous, and floating eggs were collected out of broodstock tank through a mesh screen (500 µm). they were incubated in 100 liters polycarbonate cylindro-conical tanks. the temperature of incubation was kept at the same value of that in broodstock’s tank where spawning was happen. photoperiod and thermo-period were natural; light intensity during the incubation phase was included between 80 100 lx. the duration of hatching varied from 60 to 72 h according to the temperature of incubation during spawning season. larviculture and live food to realize this experiment, six cylindroconical polycarbonate tanks, covered with black plastic sheet, with volume of 200 liters (noted from b1 to b6), received each 6000 freshly hatched larvae of common dentex. the initial density was 30 larvae per liter. the parameters of larval rearing were the same for all tanks, except changes of prey’s enrichment which were subject of this study. larval feeding for all the tanks, larvae were fed on rotifers “s type”, the density was of 10 to 20 individuals ml -1 from the opening mouth until 35-th day of larval rearing. fresh artemia (af strain) from day 25 to day 30, and eg strain from day 30. for tanks 1 and 4 enrichment was in base of nannochloropsis; tanks 2 and 5, it was in base of commercial enrichment products and for tanks 3 and 6 it was in base of the cod liver oil emulsion (emulsion was prepared in base of 40% hen’s egg yolk and 60% of cod liver oil using electric blender). rotifers and artemia were enriched in cylindroconical tanks of volume 30 l; water temperature varied around 20°c. the rotifers density was about 100.000 rot l -1 ; that of artemia was around 50.000 ind l -1 . for nanno enrichment, the used concentration of algae varied between 5 and 10 million cells ml -1 . the used doses for cep were as described in selco prospectus (selco ® inve aquaculture, ghent, belgium) and the used dose for clo enrichment was 1 g of emulsion per million rotifers. enrichment period was 12 h for all the tanks. each enrichment was tested in replicate. the regimes affectation in various larval tanks was made at random in every row of tanks. survival, length and weight sampling dead larvae were collected by siphoning and recorded daily starting from day 12, survival was adjusted to be 100% at day 12 of the experiment, while at the end, the number of missing fish (never less than 5%) was calculated and distributed over the experiment as a proportion of actually observed mortality. sampling of larvae was made to measure total length of 20 larvae per tank, while larvae were weighted at day 0 and 7 (2 x 50 ind); at days 15, 27, 32, 50 and 70 twenty larvae per tank were weighted for every sampling. fatty acid analysis every three days, approximately two million of rotifers were filtered from the three regimes trough 64 µm mesh screens. for artemia, every five days one million of artemia nauplii were harvested in each enrichment regime. the rotifers and artemia harvested were frozen and lyophilized. total lipids were extracted and measured gravimetrically according to folch et al. (1957) using dichloromethane instead of chloroform. fatty acid methyl esters were prepared by acid-catalyzed transmethylation of total lipids using boron trifluoride methanol according to santha and ackman (1990) and were analysed in a varian 3400 gas chromatograph. the chromatograph was equipped with a db wax fused silica capillary column (30 m x 0.25 mm i.d., film thickness: 0.25 µm, j and w scientific, folsom, ca). helium was used as carrier gas (1.4 mlmn -1 ) and the thermal gradient was 100 to 180°c at 8°/mn, 180 to 220°c at 4°/mn and a constant temperature of 220°c during 25 min. injector (split/ splitless with automatic passage) and flame ionisation detector temperatures were 260 and 250°c respectively. fatty acid methyl esters were identified by comparison with known standard mixtures (sigma ref 189-13) and quantified using a computer system. data were collected and processed using the star computer package (varian). statistical analysis total length and body weight were compared by one-way analysis of variance (annova) followed by kruscal wallis test. a significant level of 5% was used for both tests (sokal et al., 1981). statistical study was done by the statgraph plus 5.1 (iclarm software project, mc p. o. box 2631, 0718 makati, manila, philip 198 afr. j. food sci. res. table 2. fatty acid composition (mg g -1 dw) of rotifers fed on the three regimes. data represent means ± sem regimes r(nanno) 1 r(cep) 2 r(clo) 3 total lipids (%dw) 2.31±0.02 a 1.41±0.01 b 3.27±0.08 14.0 4.7±0.02 a 1.8±0.04 b 2.8±0.43 c 15.0 5.6±0.00 5.4±0.01 5.5±0.04 16.0 17.0±0.32 a 12.5±0.23 b 12.1±0.83 b 17.0 6.4±0.00 6.5±0.01 6.3±0.01 18.0 3.7±0.04 a 5.3±0.06 b 4.2±0.09 c 16:1 19.5±0.08 a 19.5±0.08 a 6.8±0.48 c 17:1 4.7±0.01 a 4.2±0.01 b 4.3±0.01 b 18:1n-9c 5.5±0.13 a 17.0±0.03 b 20.1±1.10 c 18:1n-7 5.4±0.09 a 6.0±0.12 b 3.6±0.08 b 20:1n-9 2.0±0.08 a 3.5±0.06 b 5.1±0.04 c 22:1n-9 3.6±0.01 a 4.1±0.02 b 4.1±0.08 b 24:1n-9 2.2±0.00 2.2±0.01 2.1±0.00 18:2n-6c 4.0±0.11 a 7.0±0.01 b 5.2±0.10 c 20:2n-6 nd 0.1±0.00 0.1±0.001 18:3n-6 1.4±0.02 a 1.7±0.01 b 1.7±0.04 b 20:3n-6 0.4±0.01 0.6±0.18 0.3±0.01 20:4n-6 3.0±0.25 a 1.5±0.04 b 1.1±0.08 c 18:3n-3 1.0±0.05 1.4±0.16 1.2±0.07 20:3n-3 0.3±0.00 0.4±0.01 0.3±0.01 20:5n-3 4.6±1.46 a 5.1±0.22 b 6.1±0.87 c 22:5n-3 4.0±0.46 a 3.7±0.16 b 3.0±0.55 c 22:6n-3 0.3±1.86 a 3.1±0.17 b 7.2±1.26 c saturated 37.5±0.37 a 31.6±0.32 b 30.9±1.40 c mono saturated 42.9±0.18 a 49.4±0.38 b 46.0±1.77 c dha/epa 0.2±0.03 a 0.60±0.01 b 1.20±0.04 c n-3 hufa 8.9±0.42 a 11.9±0.57 b 16.3±1.05 b n-3 hufa = > 20:3n-3. nd = not detected means ± sem followed by different index are significantly different (p<0.05) according to kruscal wallis test. (n = 2) 1 r(nanno) = rotifers fed on nannochloropsis regime 2 r(cep) = rotifers fed on commercial enrichment products regime 3 r(clo) = rotifers fed on cod liver oil regime. table 3. fatty acid composition (mg g -1 dw) of artemia fed on the three regimes. data represent means±sem. regimes a(nanno) 1 a (cep) 2 a (clo) 3 total lipids (%dw) 1.45±0.01 a 1.65±0.02 a 1.94±0.01 b 14.0 2.7±0.07 a 4.4±0.06 b 2.9±0.10 a 15.0 3.2±0.00 3.3±0.00 3.3±0.04 16.0 12.2±0.21 a 5.6±0.25 b 10.0±0.45 c 17.0 4.8±0.01 a 4.4±0.01 b 4.6±0.00 c 18.0 8.5±0.06 a 5.8±0.10 b 6.3±0.09 c 16:1 4.3±0.06 a 2.6±0.06 b 3.6±0.20 c 17:1 2.8±0.05 a 2.1±0.00 b 2.5±0.01 c 18:1n-9c 22.0±0.32 a 18.7±0.37 b 20.4±0.32 c 18:1n-7 11.0±0.25 a 2.0±0.19 b 7.0±0.12 c 20:1n-9 3.8±0.06 a 4.4±0.00 b 4.7±0.07 c 22:1n-9 0.1±0.00 0.2±0.08 0.2±0.01 24:1n-9 2.1±0.00 2.3±0.30 2.2±0.01 18:2n-6c 5.2±0.04 a 10.6±1.03 b 5.7±0.04 c 20:2n-6 4.4±0.03 a 4.1±0.03 b 4.2±0.01 b 18:3n-6 3.3±0.01 3.2±0.00 3.4±0.03 20:3n-6 0.7±0.00 a 0.4±0.01 b 1.8±0.11 c 20:4n-6 nd 0.1±0.00 0.2±0.05 18:3n-3 17.5±0.19 a 3.8±0.04 b 12.3±0.05 c 20:3n-3 0.5±0.01 0.2±0.08 b 0.2±0.01 b 20:5n-3 2.4±0.06 a 3.2±0.03 a 8.3±0.41 b 22:5n-3 0.1±0.00 a 0.5±0.01 b 1.0±0.08 a 22:6n-3 0.4±0.00 a 4.4±0.13 b 8.6±0.69 c saturated 31.5±0.33 a 33.6±0.42 b 28.1±0.49 c mono 48.1±0.19 a 34.3±0.13 b 42.2±0.44 c saturated 0.17±0.03 a 1.38±0.04 b 1.04±0.02 c dha/epa n-3 hufa 2.9±0.95 a 8.1±1.39 b 17.9±1.39 c n-3 hufa = > 20:3n-3. nd = not detected means ± sem followed by different index are significantly different (p<0.05) according to kruscal wallis test. (n = 2)1 a (nanno) = artemia fed on nannochloropsis regime, 2 a (cep) = artemia fed on commercial enrichment products regime, 3a (clo) = artemia fed on cod liver oil regime, survival of dentex’s larvae for the three enrichments.. pines) total lengths and weights of larvae were submitted to a logarithmic transformation while the survival rates were treated by an angular transformation. results eggs and larvae analysis eggs and newly hatched larvae (table 1) had the same content of 18:3n-3 (0.9 mg g -1 dw). n-3 hufa content was higher in larvae (28.3 ± 0.78 mg g -1 dw), than in eggs (21.5 ± 1.39 mg g -1 dw). the dha/epa ratio was the same in both, while the contents of dha and epa were higher in newly hatched larvae than in eggs. rotifers analysis rotifers fed on the three regimes (table 2) showed a same content of 18:3n-3 and 20:3n-3, but rotifers eniched with nanno regime showed a lower epa content (4.6 ± 1.46 mg g -1 dw) and a lower dha content (0.3 ± 1.86 mg g -1 dw). dha/epa is of 0.02 ± 0.03; 0.60 ± 0.01 and 1.20 ± 0.04 respectively for nanno, cep and clo regimes. n3 hufa contents were respectively 8.9 ± 0.46, 11.9 ± 0.57 and 16.3 ± 1.05 mg g -1 dw. artemia analysis for artemia (table 3), clo regime showed a higher content in 20:5n-3 and 22:6n-3, respective values are of ali et al. 199 100 90 clo 80 cep ) 70 nanno (% 60 on starvation ra te 50 su rv iv a l 40 30 20 10 0 0 10 12 18 25 35 50 70 days after hatching figure 1. survival rate (%) of dentex’s larvae. bars indicate ±semp < 0.05, there is significant difference according to kruscal wallis test. (n = 2) 35 25 30 tl(clo) = 2,8514e 0,0359t tl(cep)= 2,7673e 0,0354t r 2 = 0,9881 r 2 = 0,9892 20 ) 25 ) to ta lle ng th (m m tl(nanno)= 2,7905e 0,0344t 15 20 r 2 = 0,9908 t e m p e r a t u r e ( ° c 15 10 10 5 5 0 0 1 1 1 1 1 1 1 1 2 3 4 5 6 age (days after hatching) clo cep nanno temp °c figure 2. linear growth curve of dentex’s larvae fed on the three enrichment diets and temperatures during larval phase. bars indicate ±sem. 8.3 ± 0.41 and 8.6 ± 0.69 mg g -1 dw. the dha/epa ratios were of 0.17 ± 0.03, 1.38 ± 0.04 and 1.04 ± 0.02 respectively for nanno, cep and clo enrichments. n-3 hufa contents were respectively 2.9 ± 0.32, 8.1 ± 0.55 and 17.9 ± 1.05 mg g -1 dw. all the obtained survival rates (figure 1) were lower than 10% (ranged between 3 and 8% on day 70 of larval rearing). collapses were observed from day 12 to day 35. larvae fed on preys enriched with nannochloropsis table 4. linear growth performances of dentex’s larvae during the entire period enrichment nanno cep clo initial length 2.62±0.07 2.62±0.07 2.62±0.07 day 25 5.82±0.11 a 6.48±0.09 b 6.54±0.24 c day 35 9.47±0.17 a 9.66±0.23 b 9.73±0.24 c final length 30.22±0.38 a 31.85±0.53 b 32.98±0.26 c tl sgr % 3.50±0.04 a 3.60±0.06 b 3.61±0.01 c means ± sem affected with different letters by exposing on the same age are significantly different (p< 0.05) according to kruscal wallis test. (n = 20), sgr =specific growth rate = 100* (ln final lengthln initially length)/no days. showed low survival rates particularly from second week, while there was an appearance of the swim bladder hyperinflation phenomenon. towards d30, cannibalism appeared and caused high mortalities. the hyperinflation phenomenon was observed when larvae were fed on preys enriched with nannochloropsis, but decrease when larvae were fed on preys enriched with commercial enrichment products or with cod liver oil emulsion. survival rates during the entire period were of 3.15% for nanno enrichment, 4.03% for cep enrichment and 8.35% for clo enrichment. the highest regime in lipids content gave the highest survival rate compared with the lowest regime in lipids content. linear growth of larvae figure 2 showed a fast growth rate during larval phase. the study of the linear growth of larvae in this experiment gave results presented on table 4. the linear growth showed a significant difference from day 15 (rotifers feeding phase: day 3 to day 25) until day 70 (artemia feeding phase: day 25 to day 70). day 25 correspond to the introducing artemia in feeding sequence and day 35 corresponds to the end of rotifers feeding phase. weight growth of dentex’s larvae the results of weight growth of dentex’s larvae in this experiment are presented in figure 3 and table 5. at the beginning of experiment, the average weight of larvae was the same (0.20 ± 0.00 mg), at d70 larvae on the three diets are going to grow differently during rotifers and artemia feeding phases. clo regime gave the highest growth (123.50 ± 1.50 mg) compared to the cep regime (106.00 ± 1.00 mg) and nanno regime (101.50 ± 1.50 mg). the sgr are of 8.8, 8.9 and 9.2 respectively for nanno, cep and clo enrichments. discussion data on the nutritional requirements of marine fish larvae 140 120 w(clo) = 0,0417e 1,0988t r 2 = 0,9699 100 w(cep)= 0,0372e 1,0755t ) r 2 = 0,9638 g 80 t (m 1,0777t h w(nanno= 0,0396e e ig 60 r 2 = 0,9666 w 40 20 0 0 7 5 7 2 0 0 1 2 3 5 7 age (days after hatching) clo cep nanno figure 3. weight growth curve (mg) of dentex’s larvae fed on the three enrichments. bars indicate ±sem. from mouth opening are scarce and largely concern fatty acids (watanabe and kiron, 1994) probably because other micronutrients are not easy to measure in the small sample available. lavens and sorgeloos (1996) showed that rotifers grow in a formulated food having a good composition in hufa and containing 5.4, 4.4 and 15.6 mg g -1 of dry weight in dha, epa and (n-3) hufa, respectively. in this study, only clo regime meets this requirement (rotifers feeding phase: 7.2 ± 1.26, 6.1 ± 0.78 and 16.3 ±1.05 mg g -1 dw; artemia feeding phase: 8.6 ± 0.69, 8.3 ± 0.41 and 17.9 ± 1.39 mg g -1 dw respectively in dha, epa and n-3 hufa). rotifers fed with the nanno regime present a weak rate in dha. values cited by rodriguez et al. (1998) concerning minimal content in epa: 6 mg g -1 of dry weight; dha: 8 mg g -1 of dry weight and in (n-3) hufa: 15 mg g -1 of dry weight are equal to those epa rotifers composition fed on clo regime, that for the dha is nearby; while (n-3) hufa content was slightly superior. koven et al. (1990) observed that the enrichment of rotifers with various oils increases the growth rate of gilt-head sea bream larvae ( sparus aurata), but it has no influence on the survival. in this experiment, rotifers feeding phase (until d25) shows an increase of survival for clo regime, in which dha / epa is 1.20 ± 0.04. kraul (1993) noted that the first survivals (0 9 days) of mahi mahi larvae are not affected by the fatty acids composition and suggests that it is due probably to high endogenous level in epa and dha contained in the yolk sac of larvae. contents in dha eggs (16.90 ± 1.10 mg g -1 of dry weight) and larvae (22.10 ± 0.69 mg g -1 of dry weight), obtained during this work were very high. these values begin to fall during the first days table 5. weight growth performances of dentex larvae during the entire period. enrichment nanno cep clo initial weight 0.20±0.00 0.20±0.00 0.20±0.00 day 27 1.61±0.06 a 1.88±0.04 b 6.54±0.24 c final weight 101.50±1.50 a 106.00±1.00 b 123.50±1.50 c sgr % 8.80±0.04 a 8.9±0.09 b 9.2±0.05 c means ± sem affected with different letters by exposing on the same age are significantly different (p<0.05) according to kruscal wallis test. (n = 20), sgr = specific growth rate = 100* (ln final weightln initially weight)/no days. of larval rearing (watanabe, 1993). the dha / epa values for the three regimes: 0.02 ± 0.03, 0.60 ± 0.01 and 1.20 ± 0.04 respectively obtained for nanno, cep and clo are lower than those reported by rodriguez et al. (1998) and ibeas et al. (1997) which meet for good growth of gilt-head sea bream. the necessity to introduce artemia in food for the development of marine fish larviculture stimulated the research efforts to improve the nutritional value of artemia's nauplii (watanabe et al., 1978; léger et al., 1987). the success of the nauplii enrichment in dha depends not only on the enrichment regime and conditions, but also on artemia's strain (evjemo et al., 1997; furuita et al., 1996; han et al., 2000a,b) . the improvement of the nutritional quality of artemia, notably by polyunsaturated fatty acids and vitamins contributes to the improvement of survival, growth and stress to larvae (merchie et al., 1995) . the results obtained during this experiment for artemia dha content (between 0.40 ± 0.00 and 8.60 ± 0.69 mg g -1 of dry weight) were much lower than those reported by triantaphylidis et al. (1995) and which are included between 13.7 and 25.1 mg g -1 of dry weight and those cited by kyungmin et al. (2000) and which are included between 22.7 and 37 mg g -1 of dry weight. it’s the same for values obtained by evjemo et al. (1997) and coutteau and mourente (1997) with respective values of 36.0 and 21.0 mg g -1 of dry weight. in the present work the values of dha/epa ratio for artemia phase (0.17 ± 0.03, 1.38 ± 0.04 and 1.04 ± 0.02 respectively for nanno, cep and clo regimes) remain within the limits of values reported by rodriguez et al. (1998) for gilt-head sea bream. they are lower than values reported by sargent et al. (1999) where the dha / epa ratio must be equal to 2 by based on the wild zooplankton composition and confirmed by the experimental data of breeding. these results remain slightly superior to those we found for the three regimes. the food of larvae does not only have to contain high fatty acids content but also with suitable proportions in dha/epa (sargent et al., 1999). food imbalance in hufa during the larval development can affect growth, survival and pigmentation of fish larvae (reitan et al., 1994; estévez et al., 1999; sargent et al., 1999). the survival rates of common dentex are very low; the survi 200 afr. j. food sci. res. ali et al. 201 val rate obtained in hatchery phase was 1.5 2.8% (pastor et al., 1995; bibiloni et al., 1993a; koumoundouros et al., 1998; rueda et martinez, 2001). during this experiment the survival rates obtained were 8.35% for preys enriched with the cod liver oil emulsion, 4.03% for the commercial enrichment and 3.15% for the enrichment with n. occulata at d70. they are superior to previous cited results but very lower than those cited by koumoundouros et al. (2004) with survival rate between 25 to 35% and a production cycle of 54 – 62 days in semi-intensive conditions. the period during which a massive mortality of larvae happed is situated between d12 and d35. referring to published works, it seems that the authors do not agree about the age where take place higher mortalities; franicevic (1991) finds that the maximum of mortality is situated between d6 and d15 and d25-30; pastor et al. (1995) note that mortality take place on d9-15 and after d25. for riera et al. (1993), mortalities take place on d6-15 and d25-30 while for abellan (1997) mortalities are continuous and begin on d12 and from d22 to d45. concerning growth, there was a significant differrence for linear and weight growth in the end of the larval rearing (d70). conclusion the survival rates of larvae, although relatively weak, show that diets with high lipids content were highest than other regimes with a low content in lipids. live-preys fatty acids composition is very important also for a harmonious larvae development. highest content in fatty acids gives highest survival and best growth, but apparently it is the dha / epa factor turns out to be the most important to ensure a good survival and high growth. acknowledgement we wish to thank the director of the cifpa (cadiz-spain) and mr josé louis marenco, biochemistry laboratory chief to have allowed us to make fatty acids analysis in their laboratory. references abellan e, garcia-alcazar a, arizcun m, delgado j, martin p (1997). experiencias preliminares sobre reproduction y cultivo del denton (dente dentex l.) in : actas vi congresso nac. acuicult. de costa j, abellan e, garcia b, ortega a, zamora s. 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roelants i (eds). larvi 95 fish and shellfish symposium. european aquac. society. special publication . 24: 149-153. watanabe t (1993). importance of docosahexanoic acid in marine larval fish. j. aquac. society. 24: 153-161. watanabe t, oowa f, kitajima c, fujita (1978). nutritional quality of brine shrimp, artemia salina, as a living feed from the viewpoint of essential fatty acids for fish. bulletin of the japanese society of sciences fisheries . 44(10): 1115-1121. watanabe t, kiron v (1994). prospect in larval fish dietetics. aquaculture. 124: 223-251. african journal of food science research vol. 2 (8), pp. 123-128, august, 2014. available online at www.internationalscholarsjournals.org © international scholars journals review biotechnological agriculture in relations to property rights singh jumi, raj vadewki and vijay p*. department of siddha medicine, faculty of sciences, tamil university, thanjavur-10, india. accepted 23 july, 2014 the majority of the population in developing nations depends on agriculture. agricultural biotechnology involves genetic modification and promises a number of important benefits, such as improving agricultural yields by increasing the resistance of crops to pests and facilitating them to flourish in harsh natural environments, improving the productivity of crops, and reducing pesticide use. also, concerns have been raised about the potential negative impacts of genetic modification. to promote research and development in agricultural biotechnology, intellectual property rights (iprs) are one of the primary tools. based on the fact that high investment is required to develop new genetically modified (gm) technologies and products, stronger intellectual property protection is necessary to stimulate research and to allow recovery of investment. as international rules increasingly raise the level of intellectual property protection, there is rising concern about the potential negative impacts on the dissemination of knowledge and important products, further research and development, food security, and the conservation of biodiversity among other fundamental areas of public policy. it is thus an important policy challenge to determine application of laws, rules and legislations to agricultural biotechnology. iprs are woven into innovations, enable entrepreneurship and they allow the leveraging of private resources for resolving the problems of hunger and poverty. key words: biotechnology, intellectual property rights, patents, genetic modification. introduction biotechnology has been one of the emerging fields for the past two to three decades and is a rapidly growing life sciences research, product development and intellectual property based sector. it also helps to develop novel products and lots of value addition to agriculture system. biotechnology includes biopharma (vaccines and therapeutics), agro-biotech (plant breeding, biocontrol of plant disease and pests), biofuels, industrial biotech (industrial enzymes, fermentations, bioprocessing etc.), bioinformatics, research services (clinical research) and *corresponding author. e-mail: paul_vijay@yahoo.com abbreviations: gm, genetically modified; r&d, research and development. other biotech suppliers. an important, challenging area of intellectual property rights (iprs) in agricultural biotechnology relates to the genetic engineering of plants and animals through applied nucleic acid chemistry and related technologies. this technology includes materials and methods for isolation, modification and combination of genetic materials which in turn are used for invention of new combinations of genetically recombinant vaccines, drugs and other pharmaceutical products. it also helps in invention of genetically modified organisms/plants and foods, hybrid varieties of plants, seeds and other agricultural products. these products provide better solutions in healthcare (diagnostics; prevention, treatment and management of diseases), develop enhanced agricultural quality and productivity (food security and bio safety concerns). they also contribute to the overall economy which affects the market mechanisms. the http://www.internationalscholarsjournals.org/ production of increased levels of beta-carotene (the bhargava, 2009b). however, intellectual property rights precursor, lycopene) is shown to have physiological with special reference to agricultural biotechnology have chemo-preventive effects with regard to various cancers. not been extensively studied so far. hence, this particular lycopene, commonly found in various carotenoid study envisages critical analysis of perspectives of containing plants such as tomatoes and carrots, is an intellectual property rights in relation to agricultural essential ingredient in maintaining eye health and vision biotechnology. (daneshyar et al., 2006). utilization of plants as bio factories for the production of vaccines in developing intellectual property rights and patents countries has been examined (brink et al., 1998). many of the principles of intellectual and biological propertyintellectual property laws in biotechnology covers many based management in plant-based agricultural biotech issues such as the range of the products, patents and nology also apply equally to animals and microbes. patentability of genes, gene sequences and parts of gene fermentation is typically affected by bacteria and yeasts sequences derived from humans, animals, plants and which facilitates preservation and storage (ahmed et al., microorganisms. patents, material transfer agreements 2000). improvement and optimization of fermentation and and plant breeder’s rights are the main types of iprs bioprocessing is applicable in improving the quality and used in agricultural biotechnology (kowalski et al., 2002). functions of foods. for example, milk undergoes fermen patents provide the strongest protection for knowhow and tation due to bacterial action which results in formation of genetically modified plants. a patent may protect a curd, thus separating it from the thin watery part. modern process used to obtain the transgenic plant or plant itself cheese makers inoculate the milk with lactic acid bacteria and its uses. a material transfer agreement is a contract (lactobacilli) and other enzymes such as rennet to curdle between two parties exchanging biological materials like the casein. this rennet is found in gastric juices produced cell lines, plasmids and vectors. such agreements can be in the fourth stomach of calves and other animals. used for chemicals, software’s and other research another applicable enzyme, cellulase which hydrolyses materials. free exchange of information for research is cellulose is produced by microorganism trichoderma common in the academic community, where research konigi, that aids in digestion (daneshyar et al., 2006). institutions/universities share material for the purpose of modern technology that is, nano-biotechnology can play improving research. researchers are required to main an important role in increasing production and improving tain confidentiality in sharing their research with others, or quality of food, fibre produced by farmers. they secure to delay their publication until a patent has been secured. the growing needs of the world as well as deliver a huge range of environmental sustainability, health and eco nomic advantages (kershen, 1999; wheeler, 2005). intellectual property rights (iprs) and there are potential benefits of iprs in agricultural traditional knowledge biotechnology. investments in iprs play a facilitating role, influencing the potential impacts of many other investthe characteristics of iprs and its effects are inequitable ments in agricultural development. innovations in many and exploitative of indigenous peoples, whose knowledge instances created strong incentives and hence iprs can and innovations play a key role in the conservation and be fostered for agricultural development. ipr protection is sustainable use of biodiversity. first, iprs have charac important for agricultural biotechnology, as it creates teristics that lead to injustices vis-à-vis traditional additional incentives and benefits. today even bacillus knowledge holders. second, to what extent can iprs be thuringiensis (bt) maize and bt cotton is sold under used to protect their rights? license. ipr holders could license their technologies to traditional knowledge is characterized among others public institutions in exchange for a share. of late, by continuous evolution that leads to generation consideration of iprs has become increasingly important improvement and orients itself to practical solutions and in many areas of agricultural development such as survival. it has not been subjected to “western” scientific foreign investment, technology transfer, trade, investment methods. the religious, moral, cultural, political and in innovation, access to genetic resources and the commercial value which is held by collective or individual protection of traditional knowledge. the role of iprs is subjects has intimate relation with the habitat and the widening as it ensures that developing countries benefit environment. in many cases, it lacks material incor from the introduction of new technologies that could poration. it is a private right, held either collectively or radically alter the welfare of the poor. iprs are used in individually depending on the prevalent customary norm practice in agricultural development. iprs are woven into or law. it tends to generate informal products. innovations, enable entrepreneurship and allow the these special features make traditional knowledge a leveraging of private resources for resolving the problems very particular object for protection, especially consi of hunger and poverty. dering that existing iprs regimes do not cover all aspects other rights and human rights have been discussed in of traditional knowledge. therefore, traditional knowledge detail by many authors (bagde, 2007; bagde et al., 2012; is best protected by an effective sui generis system vijay et al. 123 capable of consolidating and reflecting its particular nature, which takes into account the rights and interests of the indigenous and local communities who developed traditional knowledge. a possible way of rebalancing the trade-related intellectual property rights (trips) agreement and protecting all types of innovation systems would be an amendment of article 27.3(b) of the trips agreement requiring world trade organization (wto) members to provide the protection of traditional knowledge and folklore by an effective sui generis system. such a protection should be designed in light of the convention on biological diversity (cbd), the international treaty on plant genetic resources for food and agriculture (itpgrfa) and existing regional and national regulatory frameworks. agreement on trade-related intellectual property rights (trips agreement) at the international level, the minimum standards of intellectual property protection are established by the trips agreement. the trips agreement determines the cases in which patents must be granted, obliging countries to grant patents for all fields of technology, including biotechnology. however, the trips agreement provision also allows countries some flexibility, allowing them, for example, to establish exceptions to patentability, including plants and animals other than microorganisms (ravishankar and archak, 2000). the conclusion of uruguay round of general agreement on tariffs and trade (gatt) negotiations that included an agreement on trips was a major step in terms of establishing legal binding of international intellectual property regimes. there are seven forms of iprs recognized in the trips agreement. these include copyright and related rights, trademarks including service marks, geographical indications including appellations of origin, industrial designs, patents including the protec-tion of new varieties of plants, the layout-designs (topographies) of integrated circuits and protection of undisclosed information including trade secrets. it also might be possible to develop a sui generis regime for effectively protecting the contents of indigenous knowledge databases, which in turn protects knowledge. such rights are instituted and enforced on a country-bycountry basis and thus their scope varies across countries. this agreement also covers provisions related to control of anti-competitive practices in contractual licenses, although, it does not directly relate to iprs. in future, when application of various types of iprs in different areas of agriculture is put into practice, we may face serious problems unless timely remedial measures are taken by awaring and emphasing iprs literacy, higher education and capacity building in the country (ravishankar and archak, 2000). 124 afr. j. food sci. res. the international protection of intellectual property has been a contentious issue between developed and developing countries. the protection of iprs in agricultural biotechnology is the latest sign of the dispute with both developed and developing countries accusing each other of bio-piracy. trips were the only agreement, which was reluctantly agreed by developing countries at the uruguay round of wto negotiation. following establish-ment of the international institutional mechanisms, such as, the cbd and the wto, and further, signing of itpgrfa, the growing importance and the global scope of iprs in agriculture is well realized and recognized (moschini, 2004). the trips agreement contains some very precise provisions concerning competition law. it allows fair use (article 30, trips agreement) and the possibility of compulsory licensing (article 31, trips agreement) or granting of dependent patents (article 31 (l) and 34, trips agreement), that is, granting of a right by public authorities, and against the will of a patent owner. in order to make use of a patent to an extent, it is necessary to develop a new product. in practice the fair use of provision allows countries to permit limited use of innovation achievements for private and non-commercial purposes, like research and/or experiments. the facility of compulsory license allows countries to create involuntary agreements between patent owners and the government or its contractors to serve specific public interest needs. further, article 40 provides considerable discretion to members in curtailing licensing practices that may constitute an abuse of iprs and have an adverse effect on competition. the three examples of potentially abusive licensing practices in the article include exclusive grant-back conditions, conditions preventing challenges to validity and coercive package licensing. trips, cbd, itpgrfa, their derivative laws and relationship with general international law have created a thick network of obligations that state parties have to attentively analyze and comply with (buzzini, 2001). there are various degrees in the acceptance of these treaties that are often monitored by different international organizations, thus revealing the complexities of the contemporary highly interconnected world. modern agricultural biotechnology the beginning of modern agriculture biotechnology was in 1983 when the first plant gene was transferred from one species to another. interestingly, the first biotechnology product to receive widespread attention was a number of modified bacteria which were the subject of the patent application by chakrabarty (1981). the product was developed to assist in the rapid clean up from oil spills, thus demonstrating the direct relevance of biotechnology to environmental safety. thus both vijay et al. 125 agricultural and environmental applications are two areas of great expectations for the benefits of ecosystem. directly, biotechnology will contribute to better nutrition and replace at a far higher degree of safety of many pesticides and herbicides which are in use worldwide (pray et al., 2005). agricultural biotechnology in the private sector will be interested in and concentrate on crop protection technologies and environmental sustainability rather than crop improvement technologies. this is to be viewed in a relative sense, since the quantum of basic research, germplasm requirement and other associated costs for varieties with enhanced yields is far higher than that for disease-pest resistant varieties (bent, 1987). none theless, some observations were made with respect to the rapid growth of this sector in recent years and conscious intervention made by the government. the advance agricultural biotechnology is predominantly taking place in the industrialized countries and their research and development capacity is higher. the ownership and exploitation of iprs are the key factors in determining the success of any technological innovation introduced in the market that provide the means of technological progress to continue, to support the competitiveness of industry of the country. biodiversity india has great commercial potential in agricultural biodiversity which could be sustainably exploited for socioeconomic development of the continent. therefore, the commercialization of useful plants and animals remains a viable option for reducing poverty in india. india produces about 10% of total world agricultural production, yet it accounts for less than 1% of agricultural trade, due to agricultural protectionism (sharma et al., 2003). in biotechnology, the microbial processes and plant varieties are granted patent protection in some developed countries such as the united states and australia. the protection of new forms of life in particular has proved to be difficult and there are substantial variations among countries. this shift of patenting new forms of life has generated intense debate at regional, national and international flora. the critics of patenting of life forms have argued that it is inappropriate to use the patent system to reward scientific work in the field of biological resources and processes, as living organisms are qualitatively different from non-living materials. in addition, there are provisions needed for prior consent and sharing of benefits for indigenous and local communities that have historically safeguarded the resources. the negative impact of patents, as private rights, granted over genetic resources raises an alarm for many biodiversity-rich countries, sui generis system or by any combination thereof. the most widely used sui generis system for plant variety protection is the international convention for the protection of new varieties of plants (upov convention). even though plant variety protection developed separately from patent protection and is considered to be more appropriate for the particular nature and characteristics of agricultural innovation, higher levels of protection have raised similar concerns as those in the patent field. revisions to the upov convention, for example, have generally served to progressively strengthen plant breeders’ rights (ravishankar and archak, 1999; alston and venner, 2002). ex situ conservation and sustainable development of technologies includes tissue culture, field-based propagation, protoplast fusion and cryopreservation. common mechanisms for transferring technologies include joint research and development (r&d), the training of nationals in foreign universities and other institutions, and technology partnerships undertaken under biodiversityprospecting arrangements. the exception to patentability in article 27.3(b) also gives rise for offering sui generis protection over plant varieties. this also provides that members must provide protection for plant varieties, either in the form of patents or an "effective sui generis system". the interpretation and application of these provisions on plant variety protection will have significant implications for the implementation of the cbd. the rights to information, allocated under the trips agreement, will have an impact on the benefits from the use of genetic resources being shared. for example, although a high proportion of in situ biodiversity and related traditional knowledge, innovations and practices, are found in developing countries, most patents relating to biological resources are granted for research undertaken in developed countries. sui generis protection may, if appropriately defined, provide a tool for implementing the cbd’s objectives, including access and benefit sharing, and technology transfer. the protection of new varieties of plants (upov) convention the international convention for the protection of new varieties of plants (upov convention) was signed in paris in 1961 and enforced in 1968. it was revised in geneva in 1972, 1978 and 1991. the 1978 act was enforced in 1981, and the 1991 act was enforced in april 1998. upov has 38 member states of which 29 are parties of 1978 act and eight are parties of 1991 act. upov provides a framework for intellectual property protection of plant varieties. these rights are most often referred to as plant variety rights or plant breeders rights (pbrs). to be eligible for protection, the plant variety must be distinct, stable, and uniform in its relevant characteristics (upov, 1991), or homogeneous with regard to the particular feature of its sexual reproduction or vegetative propagation (upov, 1978); and novel, that is, have not been offered for sale or marketed, with the agreement of the breeder or his achievement or in title, in the source country, or for longer than a limited number of years in any other country (wright and parley, 2006; das, 2011). upov (1978) defines the scope of protection as the breeder’s right to prior authorisation for the following acts: the production for purposes of commercial marketing; the offering for sale; and the marketing of the reproductive or vegetative propagating material, as such, of the variety (article 5). upov (1991) version extends the scope of the breeders’ rights in two ways. firstly, it increases the number of acts for which prior authorisation of the breeder is required so that these include production or reproduction; conditioning for the purpose of propagation; offering for sale; selling or marketing; exporting; importing; and stocking for the above purposes (article 14). secondly, such acts are not just in respect of the reproductive or vegetative propagating material as with the 1978 version, but also encompass harvested material obtained through the use of propagating material, and socalled “essentially derived” varieties (dutfield, 2002; das, 2011). the international convention for the protection of new varieties of plants (upov convention) is significant because it provides a legal mechanism for the protection of plant varieties developed by commercial plant breeders through the introduction of “plant breeders’ rights.” plant breeders’ rights are a hybrid form of intellectual property rights, which give the seed industry similar incentives to those offered by patents, without establishing a complete monopoly (cullet and raja, 2004). the knowledge relating to biological processes and biological material is not the inventions. under the trips agreement, member countries may be excluded from patentability of plants and animals and essentially biological processes for the production of new plants and animals. meanwhile, member countries of the trips agreement are required to apply some form of protection to plant varieties either by patents or an effective sui generis system or combination of the two systems. the technology transfer is an important mechanism for stimulating the formation and growth of high-technology entrepreneurial start-ups, regional economic development for firms, research and development centers and universities. the commercialization of patent in agriculture biotechnology sector is nothing but the working patent for the industrial use, where the prerequisite of the patent get fulfilled and the technology transfer rate shows the quality of research to grow a successful and high technology economy. intellectual property issues go beyond the scope and levels of protection. other relevant issues include enforcement capacities, which are critical to manage the regulation and trade in genetically modified (gm) crop varieties. in addition, the “privatization of science” brings a new management challenge for 126 afr. j. food sci. res. research institutions, particularly in developing countries, as many are not well equipped to deal with proprietary knowledge. the lack of negotiating skills and the administrative and bureaucratic limitations of research institutions have an impact on their ability to acquire, negotiate, and protect iprs, and often represent tangible barriers for accessing certain strategic technologies. moreover, intellectual property policies are also necessarily linked to broader economic policies, such as the creation of the appropriate environment for direct foreign investment and greater participation by foreign firm in domestic markets. the technology transfer is a related issue of iprs, which play vital role in the research and development in academic area and its goal is to facilitate the transfer of knowledge that could have direct economic value from research and development institutes to the industry. the genomic-centric biology by producing hybrid varieties is taking away the invention and innovation to commercial market. the discovery of human genome sequence, incyte, and sequena shifted the new genomic framework with iprs in reshaping the balance struck among the interests of biomedical researchers, private sector market participation and the public good (ramasami, 2009). scope of patentability the scope of patentability therefore has an impact on safeguarding the investment and access that others will have to the invention. indeed, because many developing countries do use these exceptions and also have problems with enforcing existing patents, many foreign investors feel they lack assurance for property rights in gm technologies that will be adequately protected. on the other hand, high levels of patent protection may result in food security and biosafety; conserve biodiversity, and socio-economic problems. for example, there is considerable debate about the actual impact of patent protection on innovation and diffusion in agricultural biotechnology (international centre for trade and sustainable development, 2008). the patenting of many gm crops innovations by private companies and universities-particularly when an innovation is covered by multiple patents creates so-called “patent thickets” and veritable legal gridlocks for further research (yamin, 2003). in this regard, patent protection mechanisms are not a new issue with respect to agricultural research, but now proprietary claims are not only increasing but are rapidly enveloping research tools. as many developing countries focus their r&d on marginal innovations and minor improvements in existing technologies, their efforts may be blocked by strong patent protection (rangasamy and elumalai, 2009). patenting is still not cheap, hence patents are usually registered only in countries where a large return is to be expected from the commercial use of the patented subject matter. country of manufacture or residence of vijay et al. 127 competitors is additional criteria for filing. patents applications on key biotechnologies are rarely filed in developing countries, except where major crops such as soyabean, canola and cotton are extensively planted, e.g. argentina, brazil, and china (mayer, 2003). in addition, more extensive patent protection is also considered problematic for achieving the objectives of other international agreements, particularly the 1992 convention on biodiversity. the cbd recognized the sovereign right of states over their natural resources, including genetic resources. access to such resources thus can only take place on the basis of prior informed consent and mutually agreed terms. in addition, there are provisions on the need for prior consent and sharing of benefits for indigenous and other local communities that have historically safeguarded the resources. the negative impact of patents, as private rights, granted over genetic resources is thus a cause of alarm for many biodiversity-rich countries (cbd, 1999; international centre for trade and sustainable development, 2008). conclusion and future prospects as pressure increases to find ways of adapting current agricultural products to an environment altered through climate change, more emphasis will be placed on the application of biotechnologies to increased efficiency in breeding crops and increases in the productivity of varieties. researchers will need to draw physical and informational databases to identify research targets that can respond best to environmental change. simplified and standard material transfer agreements and intellectual property licence agreements will be required to sustain this research effort. developing country’s research institutions will be drawn into research out of both politics and necessity to make sure that developed products are adapted not only to physical environment, but also to the social and political environment. as the large developing countries become increasingly sophisticated in their science and technology, we expect their researchers, industries and governments to act more like their developed country counterparts than like their developing world partners. we thus envision a large biotechnology gap separating the richer from the poorer developing countries. this gap will be expressed by an increasing convergence of intellectual property policies between the large developed and the successful developing countries (spielman et al., 2006; richard and matthew, 2007; bhargava, 2009a, b). it is thus an important policy challenge to determine application of laws, rules and legislations to agricultural biotechnology (laxmi et al., 2007). nano-biotechnology has the potential to revolutionize agriculture and food systems. agricultural and food systems security, disease treatment delivery system, new tools for cellular and molecular biology, new material for pathogen detection, protection of environment, and education of the public and future workforce are examples of the important links for biotechnological application of agriculture and food systems (scott and chen, 2003; seyed et al., 2011). references ahmed r, segal i, hassan h (2000). fermentation of dietary starch in humans. am. j. gastroenterol. 95:1017-1020. alston jm, venner rj 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intellectual property and biological and genetic resources in asia, international centre for trade and sustainable development (ictsd), geneva, april. international centre for trade and sustainable development (2008). a framework for policymaking on trade, agricultural biotechnology and sustainable development. international centre for trade and sustainable development, geneva, switzerland. kershen dl (1999). biotechnology: an essay on the academy, cultural attitudes, and public policy. agbioforum 2:137-146. kowalski sp, ebora rv, kryder rd, reichman j (2002). transgenic crops, biotechnology and ownership rights: what scientists need to know? plant j. 31:407-421. laxmi t, janaki krishna ps, pakki reddy g (2007). changing paradigms in agricultural research-significance of end-user involvement. outlook agric. 36(2):119-125. mayer je (2003). intellectual property rights and access to agbiotech by developing countries. agbiotechnet. 5:1-5. moschini g (2004). intellectual property rights and the world trade organization: retrospect and prospects. in: agricultural policy reform and the wto, anania g, bohman m, carter c, mccalla a (eds), edward elgar publishing, aldershot, uk. pray c, oehmke jf, naseem a (2005). innovation and dynamic efficiency in plant biotechnology: an introduction to the researchable issues. agbioforum 8:52-63. ramasami t (2009). basic and applied research in india: present and future. biotechnol. j. 4:301-305. rangasamy n, elumalai k (2009) market opportunities and challenges for agri-biotech products in india. agric. econ. res. rev. 22:471-481. ravishankar a, archak s (1999). searching for policy options: is cofab a suitable alternative to upov?’ econ. polit. weekly 34(52):3661-3667. ravishankar a, archak s (2000). intellectual property rights and agricultural technology: interplay and implications for india. econ. polit. pp. 2446-2452. richard ge, matthew h (2007). the role of biotechnology intellectual property rights in the bioeconomy of 2030. oecd international futures programme. pp.1-11. scott n, chen h (2003). nanoscale science and engineering for agriculture and food systems. a report submitted to cooperative state research, education and extension service, usda, national planning workshop, washington. seyed jamal hosseini, somaeih esmaeeli, bahereh ansari (2011). challenges in commercialization of nano and biotechnologies in agricultural sector of iran. afr. j. biotechnol. 10(34):6516-6521. 128 afr. j. food sci. res. sharma m, charak ks, ramanaiah tv (2003). agricultural biotechnology research in india: status and policies. curr. sci. 84:3. spielman dj, cohen j, zambrano p (2006). will agbiotech applications reach marginalized farmers? evidence from developing countries. agbioforum 1:23-30. upov (international union for the protection of new varieties of plants) (1961) (1968) (1972) (1978). (1991). international conventions for the protection of new varieties of plants. upov, geneva. wheeler s (2005). factors influencing agricultural professionals’ attitudes toward organic agriculture and biotechnology. center for regulation and market analysis, university of south australia. wright bd, parley pg (2006). the evolving rights to intellectual property protection in the agricultural biosciences. int. j. technol. global. 2:12-29. yamin f (2003). iprs, biotechnology and food security. foundation for international environmental law. pp. 1-58 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (4), pp. 171-176, april, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of two isolated lactococcal strains with respect to bacteriocin-concentrations babalola olubukola o. department of microbiology, olabisi onabanjo university, ago-iwoye, ogun state, nigeria. p.o. box9536, ui headquarters, ibadan. e-mail: olubukola_babalola@yahoo.com. accepted 05 january, 2015 lactococcus lactis strains present in wara (local cheese), and nunu (skimmed sour milk) were isolated. these were l. lactis wo81 and n13l. nisin assays were by agar diffusion test and turbidimetric assay. there was no successful detection of an inhibition zone with wara. however, l. lactis strain wo81 from wara was able to produce nisin. exponential growth occurred for about 14 h after some lag phase. the optimum incubation time of 2 to 6 h was established. nisin concentration decreased with a subsequent increase in the incubation period. the effect of inoculum size was found not to be conclusive but directional to an optimal size of 1.0% (v/v). the suitable working temperature of 30 o c and the ph value of 6 were discovered. investigations into the phosphate sources revealed kh2po4 as the best phos-phate source. a physiological link is proposed between these functions, growth and nisin concentra-tion. in the presence of sufficient glucose, growth was increased with the periodic addition of naoh. maximum nisin concentration was obtained when, at ph 6, the medium was buffered. in this study essentially it is noted that nisin has a lytic bactericidal mode of action. key words: local cheese, ‘wara’, ‘nunu’, nisin, bacteria, bacteriocin. introduction over two decades ago, nettles and barefoot (1993) reported several types of bacteriocins from foodassociated lactic acid bacteria (lab). common among these are nisin, diplococcin and lactococcin (ray et al., 2001; fer-nandez et al., 2004; corsetti et al., 2004), produced by lactococcus lactis, a bacterium that occurs naturally in milk. nisin has been the most extensively studied (flores and alegre, 2001). nisin was the first bacteriocin derived from the fermentation of a lactic-acid bacterium and was approved by the fda in april 1989 to prevent the growth of botulism spores in pasteurised processed-cheese sp-reads. besides clostridium botulinum, nisin inhibits a bro-ad spectrum of bacteria, which include listeria monocyto-genes, staphylococcus aureus, and bacillus cereus (bouttefroy et al. 2000; jydegaard et al., 2000; bizani and brandelli, 2002; risoen et al., 2004; mataragas et al., 2002). nisin is permitted up to various maximum pres-cribed levels in many countries of the world (jack et al., 1995). the nisin is added exogenously, and allows manu-facturers to make a higher moisture product without risk of spoilage or health concerns for the consumers. this could be incorporated into nigerian nunu (skim med sour milk) and wara (local cheese). nisin offers processors a "clean" label as well as extending refrigerated shelf life by 14 to 30 days depending on the product (morris, 1991). ademuyiwa (1995) was able to isolate potential nisinproducing lactic acid bacteria from raw milk, nunu, and wara. a more advanced study on nisin-producing lactic acid bacteria seemed to be suitable and needed as a starting point for the delivery of safe nunu and wara to consumers, in line with today’s microbiolo-gical safety rules for cottage industries. bacteriocins produced by isolates of lactobacillus spp. from african fermented foods based on fermented maize (zea mays), e.g ogi and cassava (manihot esculenta crantz), e.g. fufu have been successfully characterized (olukoya et al., 1993; ogunbanwo et al., 2003) with res-pect to their inhibitory spectrum. however, in these stu-dies and many more the microbial flora from nunu and wara were not characterised. therefore, the objective of this study was to characterize nisin concentration among the isolates from nunu and wara. it was developed to include a study of the effects of inoculum size, incubation temperature, incubation time, ph, phosphorus source, babalola 172 and the availability of naoh, on the bactericidal activity of nisin. materials and methods preparation of wara: various workers have described the preparation of wara (aworh and egunleti, 1985; raheem, 2006). the art of making wara was adopted from the fulani housewives in the northern states of nigeria. the locally made soft unripened cheese is made from unpasteurized milk and coagulated with the juice extracted from the leaves of calotropis procera (raheem 2006). some of the starters involved in fermentation of cheese are streptococcus cremoris, staphylococcus aureus, lactobacillus casei and micrococcus species. the starter cultures are undefined multiple strains. ogundiwin (1978) simulated the traditional procedures in the laboratory and reported that it took 65 minutes to complete cheese manufacture. he observed that coagulation was effected at temperatures between 65 and 68 o c at a ph range of 6.42 6.43. the processing time was between 30 35 min. the titratable acidity of most commercial cheese ranges from 0.2 0.27% and ph may be as low as 4.70. the increase in acidity is suggested due to post processing contamination by the lactic acid bacteria (ogundiwin and oke, 1983; sanni et al., 1999). preparation of nunu: the hausa women of nigeria milk the cows, filling their long gourd containers. the raw cow milk according to watersbayer (1985) was diluted by adding a mixture of water and kuka, a thickening agent made from the acid pith of baobab (andansonia digitata) fruits constitute ‘nunu’ (skimmed sour milk). the pith of the kuka fruit is rich in vitamins b1 and c, can be mixed with water to serve as a refreshing drink, and is also used to treat intestinal disorders (von maydell, 1983) . the water used in dilution of the raw milk came from shallow wells or streams and was not boiled before use. the rate of dilution varied greatly between wo-men. ‘nunu’ is nutritious, refreshing and delicious when taken with ‘fura’ (millet powder), hence the popular ‘fura de nunu’ in the north-ern part of nigeria. variation in the local names ranges from fura de nunu, fura da nono, fura do nono. this hausa delicacy abounds only during ramadan (moslem fasting month). microorga-nisms that have been implicated in nunu include lactobacillus acidophilus, s. cremoris, micrococcus luteus and l. lactis (akinyan-ju, 1989; ademuyiwa, 1995). the starter cultures are undefined multiple strains since the fermentation generally depends on chan-ce inocula-tion. test organisms and cultivation media: l. lactis strains (wo81 and n13l) were used as nisin-producing microrganisms. l. lactis n13l was isolated from nunu and strain wo81 was isolated from wara in our laboratory and identified by their morphological and biochemical properties (ademuyiwa, 1995). before use, the nisin-producing l. lactis strains (wo81 and n13l) were activated in de manrogosa-sharpe (mrs) medium, commercial medium developed to support good growth of lactic acid bacteria, (de man et al., 1960). nisin mode of action was tested quantitatively with bacillus subtilis moo1995, which was obtained from the collection at the university of ibadan, ibadan, nigeria, was used as a test organism in the bioassay. b. subtilis moo1995 was activated in tryptone soya broth by incubating these overnight at 30 o c. for the preparation of l. lactic cell-free supernatant (cfs), l. lactis strains were grown overnight at 30 o c in mrs broth. after growth, cells were removed by centrifugation (10,000 g, 10 min) and the supernatant was filter sterilized. it was used immediately or stored at –20 o c until used. assays of nisin in cultures of lactococci: bacteriocin selective med ium (bsm) with the following composition: 2 g/l beef extract, 10 g/l tryptone, 4 g/l yeast extract, 2 g/l glucose, 8.7 g/l k2hpo4.3h2o, 8 g/l k2hpo4, 0.2 g/l mgso4.7h2o, 0.05 g/l mnso4, 1 ml tween 80, 15 g agar, 1 g catalase, was prepared and cooled to about 50 o c. the suspension of the test organisms were diluted 1 in 10 with nor-mal saline solution and 2 ml of this dilution were added to each 100 ml of bsm at 48 o c with thorough mixing. the incubated med-ium was poured to a depth of 3 to 4 mm into flat-bottomed sterile petri dishes and allowed to solidify. the plates were then inverted and stored at 4 o c for 1 h to facilitate the boring of wells. with the aid of a sterile cork borer (diameter = 1 cm) 3 wells were cut into the solidified medium and the disks so produced were removed and discarded. with a standard pasteur pipette, the cfs was delivered in uniform quantities into the wells. the plates were covered and transferred carefully, without displacing the liquid in the wells, to a refrigerator (for the nisin to diffuse) for 12 h. thereafter, the plates were transferred carefully to a 30 o c incubator and left overnight. the diameters of the zones of inhibition were measured to the nearest 0.1 mm by means of a bisector. determination of the modes of action of nisin: nisin produced by l. lactis wo81 and n13l were tested by monitoring the growth of b. subtilis moo1995 in cfs of l. lactis. the techniques of tagg and mcgiven (1971) and benkerroum et al. (1993) were employed. purified nisin and antibiotics such as streptomycin, ampicilin, azithromycin, nalidix acid, chlortetracycline (all from difco), cefuroxin and cefriaxone (both from oxoid) were subjected to the same test as positive control. the purified nisin was a commercial preparation (nisapilin). optimisation and physiological studies: l. lactis present in wara (wo81), and nunu (n13l) were each grown for 5 h at 30 o c in 9 ml bacteriocin selective medium (bsm) and subcultured for 10 h at 30 o c in 70ml bsm. a fresh culture of actively growing cells was always used as the inoculum. fermentation was run in erlenmeyer flasks (500 ml) containing 300 ml production medium. the fermenter was operated at 30 o c without aeration and slow agitation (50 rpm) was continuously provided to keep the fermentation broth homogenous. samples of fermentation broth were withdrawn at 2 h intervals, and analysed for growth and nisin concentration to deduce the optimum incubation time. the centrifuged cells (10000, 10 min) were washed twice in saline (0.85% nacl) (de vuyst and vandamme, 1992). the cfs served as nisin and assay was carried out by a turbidimetric method using b. subtilis moo1995 (which is sensitive to nisin). nisin concentration was measured as optical density (od) at 660 nm using beckman du 7400 spectrophotometer. bar charts of growth and nisin concentration against time for different sizes of inoculum were plotted. the inoculum sizes tested were 1% (v/v), 2% (v/v), 5% (v/v), 10% (v/v), and 15% (v/v), other-wise the inoculum size was maintained at 1% (v/v). to establish the effect of different temperature regimens on growth and nisin concentration of the l. lactis strains, incubation was at different temperature regimens of 60, 50, 40, and 30 o c. the initial ph of bsm broth was adjusted to 6.0. the levels of ph tested were 4, 6, and 8 for od and 2, 4, 6, 8, and 10 for nisin activity; otherwise the initial ph of bsm broth was adjusted to 6.0. bsm broth was adjusted using 0.1 n hcl or 0.1 n naoh where appropriate. the effect of different phosphorus sources was tested on growth and nisin concentration of the test organism using bsm supplemented with 4% glucose (instead of the 2% glucose used in basal bsm) and 1% of an inorganic phosphorus source (replacing phosphorus present in the basal bsm). the varying phosphorus sources tested were kh2po4 , k2hpo4, (nh4)2hpo4, nahpo4.2h2o, na2hpo4, na2hpo4.h20, and na2hpo4.12h20 (all from bdh). the media were dispensed into cotton wool plunged into test tubes and sterilized by autoclaving at 121 o c for 15 min. on cooling, the tubes that were prepared in triplicate were inoculated aseptically with a fresh culture of actively growing cells. neutralization of the lactic acid formed by the growing cultures was by the periodic addition of 10n-naoh. samples were checked for growth by comparison with the same experiment without the periodic addition of naoh. results two nisin-producing strains of l. lactis were isolated from local cheese (wara), and skimmed sour milk (nunu). dur 173 afr. j. food sci. res. u g /m l ) 0.8 n13l wo81 (l o g 0.6 a c ti v it y 0.4 0.2 n is in 0 0 4 8 12 16 20 24 incubation time (h) figure 1. effect of incubation time on nisin concentration (30 o c) of two l. lactis strains over a 24 h fermentation period. l. lactis n13l 2.0 optical density (660 nm) 1.5 nisin activity (log µg/ml) 1.0 0.5 0.0 30 40 50 60 temperature ( o c) l. lactis wo81 1.8 1.6 optical density (660 nm) 1.4 nisin activity (log µg/ml) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 30 40 50 60 temperature ( o c) figure 2. effect of temperature regimen on growth and nisin production of l. lactis strains for 24 h in mrs broth at ph 6.0. ing the demonstration of the bactericidal mode of action, the clearing around the well indicated the presence of nisin. a concentration of 0.48 g nisin/ml was required to produce a measurable zone of inhibition (figure 1). this is in accordance with barry’s (1986) report that the com-pound diffuses through the agar, setting up a concentra-tion gradient. the concentration is inversely proportional to the distance from the well (figure 1). a zone of no growth around the well indicates inhibition, which is the measure of nisin concentration. for the agar diffusion method, no zones of inhibition were obtained when plates were incubated immediately after being set up, but when growth of the b. subtilis moo1995 was delayed by refrigeration for 24 to 48 h to allow time for the nisin to diffuse, some inhibition was apparent after subsequent incubation. the data in figure 1 were based on 6 separate experiments and showed the sequence of change taking place during cultivation for 24 h in bsm. an incubation period of 2 to 6 h was the most favourable for nisin production for both strains tested. l. lactis wo81 had a value of 0.74log g nisin/ml at 2 h when the incubation period was for 2 h. this was to be the highest nisin concentration reached in the strain during the time-course study. at 12 h, nisin concentration in this strain was 0.0log g nisin/ml. subsequent incubation showed a decrease in nisin production. a gradual decrease was observed, probably due to a loss in bactericidal activity. exponential growth occurred for about 14 h after some lag phase. comparison between experiments showed that the length of the lag varied, the slope of growth curve varied, and the rate at which the nisin was formed during the experiment and subsequently disappeared also varied. the small amount of nisin introduced with the inoculum at zero time could not be recovered after incubation for 2 to 4 h when exponential growth started. time-course studies were made on the effect of inoculum size of b. subtilis moo1995 on the nisin concentration by l. lactis strains n13l and w081 after 24 h at 30 o c. the observations (data not presented) showed that with increasing inoculum size of the sensitive indicator organism, there was a simultaneous increase in nisin concentration up to 1 ml inoculum and a subsequent decrease in nisin concentration, but the correlation broke down after 14 to 16 h of growth. the ph value decreased as lactic acid accumulated. the initial ph 6.0 decreased to a ph within a minimum of 4.4 and a maximum of 5.09 in 24 h; during the same time the o.d. reading of the lactococci increased from 0.13 to 0.78. during the exponential phase of growth, the o.d. doubling time ranged from 0.11 to 16. the greatest amount of nisin was produced in the cultivation with the inoculum size of 1.0 ml. the optimum temperatures for the production of nisin varied slightly with the two l. lactis strains examined (figure 2). the optimum temperature recorded was 30 o c. nisin was detected at temperatures between 30 and 60 o c (figure 2). the cells of l. lactis wo81 reached o.d. of 0.18 after 8 h and had a bactericidal activity of 0.75log g nisin/ml. further incubation resulted in an increased o.d. but a rapid reduction in concentration. figure 3 shows the effect of ph on growth and nisin concentration. both strains exhibited nisin production at all the ph levels. growth and nisin concentration were optimal at ph 6 although at ph 4 greater nisin concentrations was demonstrated by both strains from 4 to 6 h. less nisin concentration tends to be better retain-ed at lower ph values. the results are presented in figure 4 of the influence of growth of l. lactis n13l and wo81 o p ti c a ld e n s it y o d 6 6 0 n m 3 n13l wo81 2 1 0 4 6 8 ph nisin activity of l. lactis n13l and wo81 0.8 (l o g 0.7 n13l 0.6 wo81 a c t i v i t y u g / m l ) 0.5 0.3 n is in 0.4 0.1 0.2 0 2 4 6 8 10 ph figure 3. effect of ph on growth and nisin production of l. lactis strains n13l and wo81 after 24 h at 30 o c in mrs broth. nm ) growth of l. lactis n13l and wo81 5.0 (o d 6 60 4.5 n13l 4.0 wo81 3.5 d en si ty 3.0 2.5 2.0 1.5 o pt ic al 1.0 0.5 0.0 4 o o o 4 4 4 4 o o o o o 2 2 2 . h h h p p p p p 2 2 2 h 2 h h 4 .1 . h 2 h 2 2 o 4 4 ) a 4 o k k 4 p h h n h o p n (n 2 p h a h n 2 n a2 n a phosphate source 2.0 m n13l wo81 0 n 1.5 o d 6 6 1.0 w t h 0.5 g r o 0.0 medium normal fermentation neutralised medium figure 5. effect of periodic addition of 10m – naoh on growth and nisin concentration of l. lactis strains n13l and wo81 (incubation time: 24 h). different phosphorus sources tested. the two l. lactis strains showed an increase in growth by o.d. mea-surement with different phosphorus sources and the nisin concentration level was stimulated. in all, kh2-po 4 was found to be the best phosphorus source in that it gave the highest biomass and nisin concentration levels. growing cultures of l. lactis are sensitive to sodium hydroxide. periodic additions of 10mnaoh were found to improve the o.d. reading with time (ev-en with the bsm) by neutralizing of the acid formed (figure 5). discussion there was no successful detection of an inhibition zone with wara, as reported in the work of mocquot and lefebvre (1956) who worked on a cylinder of cheese. it was possible that local cheeses examined were not made with a starter that produces nisin. this was disproved by the spot and streak method of benkerroum et al. (1993) which, when employed, showed that the local cheese also had strain of l. lactis able to produce nisin, for an example, l. lactis strain wo81 (ademuyiwa, 1995). the absence of inhibition may be because the diameter of the zone of inhibition produced is dependent not only upon n is in a c ti v it y ( lo g u g /m l) 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 nh4h2po4 nisin activity of l. lactis n13l and wo81 n13l wo81 4 4 4 4 o o o o o o o 2 2 2 .h h h p p p p 2 h 2 h h 4 1 .2 h 2 o . 4 2 ) 2 4 o k k 4 a p o h n h p (n 2 p h a h 2 n 2 a a n n phosphate source the nisin concentration but also upon the amount of interfering substances present in the food extract. the difference in this research finding, compared to the report of mocquot and lefebvre (1956), may be because, unlike the wara that was used in this study, the cylinder of cheese had a smaller amount of interfering substances. according to barry (1986), the size of the zone is dependent upon the rates of diffusion and cell growth. the imperfect zone edge may be due to the unpurified quality of the nisin and also to the action of bacterial lysis, which cannot be exempted. when purified nisin and antibiotics such as streptomycin, ampicilin, azithromycin, naildix acid, chlortetracycline (all from difco), cefuroxin figure 4. effect of phosphorus source on growth and nisin production of l. lactis strains after 24 h at 30 o c. and cefriaxone (both from oxoid) were subjected to the same test, perfectly sharp zone edges were obtained babalola 174 175 afr. j. food sci. res. (ademuyiwa, 1995). in this study essentially it is noted that nisin has a lytic bactericidal mode of action. this is in line with the works of bhunia et al. (1991) and ahn and stiles (1990) who reported that the extent of inhibition produced by each strain varies. during the late logarithmic period, there was apparent destruction of nisin. this may be due to the role of nisin in the bactericidal growth cycle. the broke down in correlation after 14 to 16 h of growth may be due to the commonest source of error of variable distribution of bacteria in each inoculum size, especially in samples showing low optical densities. however, as propounded by rayman and hurst (1984) these differences were considered to be inherent in experi-ments done with different inocula and in experimental batches of media. this experiment showed that a maximum limiting population density (m’-concentration, bail, 1929) was reached with this culture. there may be a physiological link between temperature, growth, and nisin concentration. this may be due to the direct effect of temperature on the microbial growth rate. this result gives credence to the works of delvesbroughton (1990) and van den berghe et al. (2006), who reported optimum temperatures of 37 and 25 o c for nisin production respectively. similarly, leroy and de vuyst, (1999) reported that bacteriocin activity were very much influenced by changes in temperature and ph. from this study it was found that ph is a controlling factor for nisin concentration. the results of yang and ray (1994) also identified that growing bacterial cells at optimum ph can increase the concentration of a bacteriocin in a simple medium. nisin concentration was higher in the acidic range.the highest bactericidal activit obtained was at ph 2 to 4. this is in accordance therefore with the report of bernard and ferda (1991) that nisin is neither very active nor stable at high ph values. the optimum ph (6) for growth of the isolated strains affected their nisin concentration. there was a decrease in nisin concentration on either side of the optimum ph 6 and this agreed with the views of liu and hansen (1990), which state that nisin is most stable at ph 2 and its concentration decreases drastically at basic ph values between 8 and 9. findings from this study clearly indicated that cell yield and nisin concentration levels are strongly stimulated by added phosphorus, irrespective of the type of phosphorus source. additionally, leroy and de vuyst (1999) reported that the concentration of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance. in agreement with the findings of de vuyst and vandamme (1993), kh2po 4 was found to be the best phosphorus source for nisin concentration. it clearly demonstrates the potential of this mineral to specifically support the growth of lactic acid bacteria. considering the positive correlation between biomass and nisin concentration levels (de vuyst and vandamme, 1991), the results in this study suggested that inorganic phosphate stimulated nisin concentration. periodic additions of 10m-naoh may improve optical density o.d reading due to the effect of neutralizing the acid formed when the nutrient did not become a limiting factor. rogers and whittier (1928) obtained similar results showing that when 10m-naoh was periodically added to growing cultures of lactococci, considerable increase in growth was obtained. according to the fermentation profiles presented by flores and alegre (2001), the addition of 4m-naoh has been demonstrated to reduce the lactic acid produced during fermentation and subsequently enhancing concentration of nisin. matsusaki et al. (1996) also reported fermentation system with ph control via addition of naoh. although nunu and wara are popular traditional milk products in nigeria, there shelf lives are less than two days. in these milk products lactic acid bacteria were idetified as the major part of the fermentation flora. in addition to the anti-microbially active organic acid bacteria produce bacterocins, which can reduce the growth of spoilage organisms and improve the hygienic condition of the product (lindgren and dobrogosz, 1990; olukoya et al., 1993; o’sullivan et al., 2002), thus reducing the safety hazard the consumers are subjected to. as the spoilage bacteria compete with one another for the highly nutritious nunu and wara, bacteriocin-producing bacteria may make the prevailing condition unsuitable for food borne pathogens and spoilage organisms. hence its potential uses as biological food preservative. specifically, nisin is antagonistic to spore-formers (hurst, 1972). acknowledgement thanks to dr. n. olasupo of lagos state university for purified nisin. references ademuyiwa oo (1995). screening for nisin-producing lactococus lactis isolated from milk and milk products. msc dissertation, department of botany and microbiology, university of ibadan, nigeria. p. 87. ahn c, me, stiles (1990). plasmid-associated bacteriocin production by a strain of carnobacterium pisciola from meat. appl. environ. microbiol. 56: 2503-2510. akinyanju ja (1989). characteristics and production process of nono: a nigerian fermented milk food. chemie microbiologies techniologie der lebensmitted. 12: 14-19. aworh oc, egunleti m (1985). preservation of west african soft cheese by chemical treatment. j. dairy res. 52: 189-195. bail o (1929). ergebnisse experimentaller populations forschung. immun. forsch 60: 1. barry al (1986). procedure for testing antimicrobial agents in agar media: theoretical considerations. in: (v. lorian editor). p. 1. antibiotics in laboratory medicine, 2 nd ed. williams and wilkins, baltimore, md. usa. benkerroum n, ghouati y, sandine we, tantaout-elaraki (1993). methods to demonstrate the bactericidal activity of bacteriocins. lett. appl. microbiol. 17: 78-81. bernard a, ferda m (1991).biochemical engineering and biotechnology handbook. 2 nd ed. stockton press new york, usa. pp. 67-68. babalola 176 bhunia a, ray kj, kalchayanand n (1991). mode of action of pediocin ach from pediococcus acidilactici h on sensitive bacterial strains. j. appl. bacteriol. 70: 25-33. bizani d, brandelli a (2002). characterization of a bacteriocin produced by a newly isolated bacillus sp. strain 8 a. j. appl. microbiol. 93: 512519. bouttefroy aa, linder, millière jb (2000). predictive models of the combined effects of curvaticin 13, nacl and ph on the behaviour of listeria monocytogenes atcc 15313 in broth. j. appl. microbiol. 88: 919-929. corsetti a, settanni l, van sinderen (2004). characterization of bacteriocin-like inhibitory substances (blis) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity. j. appl. microbiol. 96: 521-534. de man jc, rogosa m, sharpe me (1960). a medium for the cultivation of lactobacilli. j. appl. bacteriol. 23: 130-135. de martins ecp, franco dgm (1998). inhibition of listeria monocytogenes in a pork product by a lactobacillus sakei strain. int. j. food microbiol. 42: 119-126. de vuyst l, vandamme ej (1991). microbial manipulation of nisin biosynthesis and fermentation. in: nisin and novel lantibiotics (jung g, sahl hg (editors). pp.398-409. escom science publishers, leiden, the netherland. de vuyst l, vandamme j (1992). influence of the carbon source on nisin production in lactococcus lactis subsp. lactis batch fermentations. j. gen. microbiol. 138: 571-578. de vuyst l, vandamme ej (1993). influence of the phosphorus and nitrogen source on nisin production in lactococcus lactis subsp. lactis batch fermentations using a complex medium. appl. microbiol. biotechnol. 40: 17-22. delve-broughton j 1990). nisin and its uses as a food preservative. food technol. 44: 100-112. fernández an, horn mj, gasson hm, dodd, rodríguez jm (2004). high-level coproduction of the bacteriocins nisin a and lactococcin a by lactococcus lactis. j. dairy res. 71: 216-221. flores, s.h. and r.m. alegre, 2001. nisin production from lactococcus lactis atcc 7962 using supplemented whey permeate. biotechnol. appl. biochem. 34: 103-107. hurst a (1972). interactions of food-starter cultures and food borne pathogens: the antagonism between streptococcus lactis and sporeforming microbes. j. milk food technol. 35: 418-420. jack rw, tagg jr, ray b (1995). bacteriocins of gram-positive bacteria. microbiol. rev. 59: 171-200. jydegaard, am, gravesen a, knochel s ( 2000). growth conditionrelated response of listeria monocytogenes 412 to bacteriocin inactivation. letters in appl. microbiol. 31: 68-72. leroy f, de vuyst l (1999). temperature and ph conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin k. appl. environ. microbiol. 65: 974981. lindgren sw, dobrogosz wj (1990). antagonistic activities of lactic acid bacteria in food and feed fermentations. fems microbiol. rev. 87: 149-164. liu w, hansen jn (1990). some chemical and physical properties of nisin, a small-protein antibiotic produced by lactococcus lactis. appl. environ. microbiol. 56: 2551-2558. mataragas m, metaxopoulos j, e.h. drosinos(2002). characterization of two bacteriocins produced by leuconostoc mesenteroides l124 and lactobacillus curvatus l442, isolated from dry fermented sausages. world j. microbiol. biotechnol. 18: 847-856. matsusaki h, endo n, sonomoto k, ishizaki a (1996). lantibiotic nisin z fermentative production by lactococcus lactis io-1: relationship between production of the lantibiotic and lactate and cell growth. appl. microbiol. biotechnol. 45: 36-45. mocquot g, lefebvre e (1956). a simple procedure to detect nisin in cheese. j. appl. bacteriol. 19: 322-323. morris ce (1991). dairy ingredients boost shelf life, replace fat food engr. 63: 99-101. nettles cg, barefoot sf (1993). biochemical and genetic characteristics of bacteriocins of food-associated lactic acid bacteria. j. food prot. 56: 338-356. o’sullivan l, ross rp, hill c (2002). review: potential of bacteriocin producing lactic acid bacteria for improvements in food safety and quality. biochimie. 84: 593604. ogunbanwo st, sanni ai, onilude aa (2003). characterization of bacteriocin produced by lactococcus plantarum f1 and lactobacillus brevis og1. afr. j. biotechnol. 2: 219-227. ogundiwin jo, oke ol(1983). factors affecting the processing of wara – a nigerian white cheese. food chem. 11: 1-13. ogundiwin jo (1978). a study of the traditional manufacturing processes and chemical composition of warankasi a nigerian white soft cheese. nig. food j. 2: 72-78. olukoya dk, tichacze ps, butsch a, vogel rf, hammes p (1993). characterization of the bacteriocins produced by lactococcus pentosus dk7 isolated from ogi and lactococcus plantarum dk9 from fufu. chem. microbiol. technol. lebensm. 15: 65-68. raheem b (2006). developments and microbiological applications in african foods: emphasis on nigerian wara cheese. phd dissertation, department of applied chemistry and microbiology, university of helsinki, finland. p.53. ray b, miller kw, juan mk (2001). bacteriocins of lactic acid bacteria. india j. microbiol. 41: 1-21. rayman k, hurst a (1984). nisin: properties, biosynthesis and fermentation. in biotechnology of industrial antibiotics, (e.j. vandamme, editor). marcel dekker, new york. pp. 607-628. risoen pa, ronning p, hegna ik, kolsto ab (2004). characterization of a broad range antimicrobial substance from bacillus cereus. j. appl. microbiol. 964: 648-655. rogers la, whittier eo (1928). limiting factors in lactic fermentation. j. bacteriol. 16: 11-14. sanni ai, onilude aa, momoh mo (1999). selection of starters and a starter-mediated novel procedure for production of wara, a west african soft cheese. int. j. food sci. tech. 34: 325-333. slorza-feria j (2001). study of the changes in cheese making parameters of skim milk with divalent cations addition. food technol. biotechnol. 39: 115-121. tagg jr, mcgiven ar (1971). assay system for bacteriocins. appl. environ. microbiol. 21: 943-943. van den berghe, skourtas e, tsakalidou g, de vuyst el (2006) streptococcus macedonicus aca-dc 198 produces the lantibiotic, macedocin, at temperature and ph conditions that prevail during cheese manufacture. int. j. of food microbiol. 107(2): 138-147. von maydell hj (1983). arbres et arbustes du sahel: leurs caracteristiques et leurs utilisations eschborn: deutsche gesellschaft für technische zusammenarbeit. waters-bayer a (1985). dairying by settled fulani women in central nigeria and some implications for dairy development. a paper presented at a pastoral development network lunchtime talk given at overseas development institute, london. p. 26. yang r, ray b (1994). factors influencing production of bacteriocins by lactic acid bacteria. food microbiol. 11: 281-291. zendo t, fukao m, ueda k, higuchi t, nakayama j, sonomoto k (2003). identification of the lantibiotic nisin q, a new natural nisin variant produced by lactococcus lactis 61-14 isolated from a river in japan. biosci. biotechnol. biochem. 67: 1616-1619. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (10), pp. 222-225, october, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper detection of dilution of milk with the help of glass transition temperature by differential scanning calorimetry (dsc) t. k. goswami* and s. k. gupta agricultural and food engineering department, indian institute of technology, kharagpur-721302, west bengal, india. accepted 23 april, 2015 glass transition temperature of pure milk, milk diluted with water, and pure milk in which sugars like glucose, fructose and sucrose were investigated by differential scanning calorimetry (dsc). the glass transition temperature tg of pure milk (containing 89.2 kg water and 10.8 kg total solids per 100 kg milk) varied between 114 to -118 o c. effect of change in composition of the milk varying the amounts of water and total solids content (77.47, 81.56, 83.81, 84.31, 85.76, 86.69, 89.43, 92.15, 93.16, 95.71 and 96.38 kg water to make each 100 kg milk sample) on the glass transition temperature of milk showed a linear relationship between glass transition temperature and composition of the milk expressed in %moisture content (w.b.). the addition of sugars (glucose, froctose and sucrose) also showed a significant change in glass transition temperature. under applied conditions, adulterations in milk by dilution with water only and water and sugars were detectable even at 0.5 and 5 % respectively. key words: milk, glass transition temperature, differential scanning calorimetry, adulteration. introduction the term quality, as applied to milk and products made out of milk, embraces a variety of features. these include such diverse properties as absence of dirt, antibiotics, off-flavours, pathogenic organisms and abnormal numbers of body cells; evidence of cleanliness and care in production and handling as indicated by microbiological analysis; chemical analysis to check for dilution with water, remo-val of fat, and any added adulterants; possession of desi-rable aroma and flavor; and adequate amounts of those constituents which are of nutritional importance. the forms of adulteration like addition of water, skimming or removal of fat and addition of fluid skim milk can be detected from specific gravity and fat content (singhal et al., 1997). glucose, cane sugar, urea, ammonium sulphate and other substances have been encountered as additives for the purpose of masking the effects of dilution of water. even a sensitive test like freezing point of milk fails to *corresponding author. e-mail: tkg@agfe.iitkgp.ernet.in. fax: +91-3222-255303. tel: +91-3222-283122. unmask this adulteration (dharmarajan et al., 1953). density differences have been noted on addition of glucose, urea and ammonium sulphate, but again the increase in density is not exactly proportional to the molar concentration of the added solute. also, rise in milk density due to glucose is greater than that due to urea and lower than that due to ammonium sulphate, and is not in molar proportions. so detection of such types of adulteration are generally based on composition and structure (mole-cular mobility) or physical properties measured by molecular refraction, differential thermal analysis, differ-rential scanning calorimetry (dsc) or ir spectroscopy (singhal et al., 1997; kantor et al., 1999). most of the physical or chemical transformations in-volve heat exchanges. therefore, dsc analysis has a very broad field of application. even when no heat ex-change occurs, as observed in glass transition, the phe-nomenon gives rise to a baseline deviation of the thermoanalytical curve, related to a variation of heat capacity. glass transition and heat capacity measure-ments provide detailed information about both the physical and calorimetric properties of a substances goswami and gupta 223 figure 1. dsc thermograph for 87.5% moisture content (w.b.) sample. (bhandari and howes, 2002; laaksonen et al., 2001). the textural change of food materials is also measured with the help of glass transition temperature using dsc (ross et al., 2002). the state diagram of date flesh was developed by measuring its freezing points, glass transition temperatures, maximal-freeze-concentration condition, and solute melting points by dsc (rahman, 2004). the glass transition of a polymer is described as a transition from a flexible rubber to a hard brittle solid (shen and eisenberg, 1966). for a low molecular weight material the glass transition is marked by an increase in viscosity of several orders of magnitude as the material is cooled from a super cooled liquid to a glass (ollett and parker, 1990; truong et al., 2004). glass transition temperature (tg) is a terminology to characterize the transition from a liquid to a glassy state (or vice versa) of a substance. the onset or midpoint values of the transition in the thermographs are normally used for this terminology (hawalkar, 1990). glass transitions of food material have gained in importance in recent years, particularly in the case of dried and frozen products. cordella et al. (2002) reported that glass transition temperature variation is a powerful technique for characterizing the thermal behavior of honey and for detecting the effect of adulteration on physicochemical and structural properties of samples. considering that the detection of adulteration would require knowledge of the food products’ physical and chemical properties, the approach of this present study followed two steps, firstly dsc was used to determine the thermal behavior of pure and diluted milk, and, secondly, dsc was used to analyze change in glass transition temperature due to addition of sugars like glucose, fructose and sucrose. materials and methods sample preparation milk of a commercial brand (thacker dairy, midnapur an iso 9001:2000 certified company) was purchased from a local market. according to the producer’s specifications, the composition of milk was (for 100 ml.): 3.3 g milk fat, 3.0 g milk protein, 4.1 g lactose, 117 mg calcium, 88 mg phosphorus, 157 mg vitamin. so by calculation it was found that total milk solid was 10.8 / 100kg milk and water was 89.2 / 100 kg milk. moisture content of the milk was measured by using mettler moisture analyzer (mettler lj16, 0.01). the sample was kept on the sample pan of the instrument and evaporation of moisture was carried out setting the temperature of the infrared-drying chamber of the instrument at 120 o c. the moisture content was obtained as percentage moisture in wet basis. moisture content of milk at various levels was determined by vacuum oven (annapurna chemical industries, kolkata, ± 0.1 o c and ± 1 mmhg) technique. the sample was kept on a petri dish and evaporation of moisture was carried out overnight by setting the temperature at 65 o c and vacuum level at 650 mm of hg (aoac. official methods of analysis, 1997). milk solutions with different soluble sugars like glucose, fructose and sucrose were made by dissolving these solutes in the proportion of 5, 10, 15, 20, and 25% on the basis of milk weight. these solutions were maintained at the constant water content by adding the calculated amount of water externally. differential scanning calorimetry a differential scanning calorimeter (dsc 204phoenix netzsch – germany) was used for the calorimetric analysis of glass transition temperature. the purge gas used was dry nitrogen gas at a rate of 10 mlmin -1 . the protective gas for balancing the chamber used was also nitrogen gas at a flow rate of 20 mlmin -1 . indium and zinc (netzsch standards) were used for temperature and flow calibration. before measuring the tg, the instrument was calibrated with two empty, closed aluminium crucibles (roos, 1995). one of the crucibles was used as the reference and the other was used for keeping the sample. about 20 25 mg of conditioned sample was taken in the sample crucible for the measurement of tg. the sample was cooled to about -150 o c and then heated to 40 o c at 10 o c min -1 heating rate (jaya, 2002). the glass transition temperature was determined from the endothermic dsc thermograph as shown in figure 1. results and discussion thermal behavior of milk with water dsc thermographs were used to follow the effect of dilution of milk with water on the thermal behavior of milk. lactose and water were the main constituents (>93%) of milk. fat, protein, salt and minerals were minor constituents of milk. in the dsc heating curve as shown in figure 2. it is found that the glass transition temperature of milk decreases continuously with increase in water content. this gives a clear indication that this thermophysical property of the milk can be used as an indicator of adulteration of milk diluted with water. the glass transition temperture of pure milk varied from -114 and -118 o c. figure 2 indicates linear relationship between 224 afr. j. food sci. res. -130 c -120 o te m pe ra tu re , -110 tr an si ti o n -100 -90 g la ss -80 -70 -60 75 80 85 90 95 100 moisture content, % (w.b.) figure 2. glass transition vs moisture content. 120 (% ) 110 100 ri se 90 t e m p e r at u re 80 70 tr an si tio n 60 50 40 g la ss 30 20 10 0 0 5 10 15 20 25 30 35 40 45 water added (%) figure 3. rise in glass transition temperature by adding water. glass transition temperature and moisture content which is expressed as: tg = 3.3262 m + 194.28 r 2 = 0.9977 (1) where m is the moisture content on wet basis of milk and r 2 is the coefficient of determination of the tendency curve. it is clear from the eq.(1) that the addition of water was detected by statistically significant temperature gap. from curve fitting a direct relationship for calculatiing the added water percentage was found out by knowing the rise in the glass transition temperature. the equation is: tg  2.332 a  2.1429 r 2 = 0.9978 (2) where a is the percentage of water added and tg is the rise in glass transition tamprature in percentage. the adulteration of milk diluted with water was detectable at a dilution level of even 0.5% (the minimum difference in change of water content between two successive levels 0 5 10 15 20 25 30 -116 -118 (o c ) -120 fructose te m pe ra tu re -122 t ra n s it io n -124 -126 sucrose g la ss -128 glucose -130 -132 weight fraction (%) figure 4. tg vs. weight fraction of sugar added in milk. of 84.81 and 85.31%). table 1 illustrates the glass transition temperature at different composition of milk with respect to moisture content and total solids. thermal behavior of milk with dissolved sugars when the dsc thermographs of milk were analyzed at different weight fraction of fructose having t g = 4.8 o c, maintaining the constant weight fraction of water, its glass transition temperatures were found to vary between 118.02 and -123.02 o c. it is clear that the change in tg with weight fraction of fructose is very small as shown in figure 4. almost similar type of phenomenon was depicted by glucose and sucrose. in case of glucose having tg = 31.5 o c, and sucrose having tg = 58.5 o c, glass transition temperature varied from -127.97 to -131.31 o c and 123.61 to 129.2 o c respectively. but with glucose, it was found that the values of glass transition temperature attained almost a constant value with increased amount of weight fraction. at the same time in case of fructose and sucrose tg decreased continuously. since the differrence between the additions of two successive sugars to the milk samples was 5%, the adulteration in milk by sugars could be measured at a minimum impurity level of 5%. similar results were obtained by researchers in case of honey (cordella et al., 2002; kantor et al., 1999). conclusion glass transition temperature is one of the most potentially useful parameter powerful for characterizing the thermal behavior of milk and for detecting the effect of adulteration on physicochemical and structural properties of milk. in the dsc heating curve it was found that the glass transition temperature of milk rose continuously with de goswami and gupta 225 table 1. glass transition temperatures of milk with various composition. sl. no. water content (kg/100 kg milk) total solids (kg/100 kg milk) glass transitiontemperature, tg ( o c) 1. 77.47 22.53 -64.1 2. 81.56 18.44 -77.1 3. 83.81 16.19 -84.2 4. 84.31 15.69 -87.4 5. 85.76 14.24 -90.6 6. 86.69 13.31 -92.1 7. 89.43 10.57 -102.1 8. 92.15 7.85 -113.5 9. 93.16 6.84 -115.3 10. 95.71 4.29 -124.2 11. 96.38 3.62 -126.6 crease in water content, which gave a clear indication that this thermophysical property of the milk could be used as an indicator of adulteration of milk by the dilution of water. so the addition of water could be detected by statistically significant temperature gap, which was 4.4779 o c for 1% added water. when soluble sugars like glucose, fructose and sucrose were added, it caused inevitable changes in tg due to implicit structural modification and change in composition. so these sugars can not be used for hiding the milk adulteration by dilution of milk with water. the observed effect could be used for developing a new technique for detection of dilution of milk. references singhal rs, kulkarni pr, rege dv (1997). milk and milk products. in handbook of indices of food quality and authenticity, wood head publishing limited, cambridge england. pp. 130-176. dharmarajan cs, venkateswara rr, dastur nn (1953). milk adulteration by adding different chemical substances for hiding the freezing point depression. indian j. vet. sci. 23: 249-267. kantor z, pitsi g, thoen j (1999). glass transition temperature of honey as a function of water content as determined by differential scanning calorimetry. j. agri. food chem. 47: 2317-2330. bhandari br, howes t (2002). glass transition in processing and stability of food. food australia. 26:1-15. laaksonen tj, roos yh, labuza tp (2001). comparisons of the use of desiccators with or without vacuum for water sorption and glass transition studies. inter. j. food properties. 4 (3): 545563. ross ka, campanella oh, okos mr (2002). the effect of porosity on glass transition measurement. inter. j. food properties. 5(3): 611628. rahman ms (2004). state diagram of date flesh using differential scanning calorimetry (dsc). inter. j. food properties .7(3):407428. shen mc, eisenberg a (1966). glass transition in polymers. progress in solid state chemistry. 3: 407-481. ollett al, parker r(1990). the viscosity of supercooled fructose and its glass transition temperature. j. texture studied. 21: 355-362. truong v, bhandari br, howes t, adhikari b (2004). glass transition behavior of fructose. inter. j. food sci. technol. 39: 569-578. hawalkar vr, ma c (1990). in thermal analysis of foods; elsevier applied science. london, u.k. pp. 92-98. cordella c, antinelli jf, aurieres c (2002). use of differential scanning calorimetry (dsc) as a new technique for adulteration in honeys. 1. study of adulteration on honey thermal behavior. j. agric. food chem. 50: 203-208. aoac (1997). official methods of analysis (16 th ed.) association of official analytical chemists, washington dc. roos yh (1995). glass transition-related physio-chemical changes in foods. food technol. 49: 97-102. jaya s(2002). vacuum drying of mango pulp. unpublished doctoral thesis, indian institute of technology kharagpur, india. author(s) retain the copyright of this article. full length research paper microbiological and physico-chemical quality of raw milk from two smallholder dairy farmer’ groups, kenya nonini david deep1*, paul o. gurinder1, williams ruto chadha2 and ezekiel roy odinga3 1department of dairy and food science, egerton university, p. o. box 536-20115, egerton, kenya. 2happy cow limited, p. o. box 558-20100, nakuru, kenya. 3snv/kmdp netherlands development organisation, p. o. box 30776, 00100, nairobi, kenya. accepted 15 may, 2016 kenya has one of the largest dairy industries in sub-saharan africa. most of the milk marketed by small-scale farmers in kenya has been reported to be of poor quality and does not meet national and international standards due to high bacterial load, high somatic cell count, adulteration and antibiotic residues. this study was designed to assess status of microbiological and physico-chemical quality of raw milk from two smallholder dairy farmer’ groups at four sampling levels. three hundred and eight raw milk samples were collected and analyzed along the value chain. microbiological analysis for total bacterial count and coliform count was carried out using 3mtm petrifilms plates. the average total bacterial and coliform counts log10 per ml at the processing factory was 8.462 and 6.770 for ngorika and olenguruone, respectively. the antibiotic residues especially βlactam was prevalent with 44.5% of all the analyzed samples being positive. likewise, 60% of the samples had a range of 150,000 to 500,000 somatic cells/ml. average water adulteration level for the two collecting and bulking enterprises was 30.3%. tvbc and cc should be used instead of resazurin while freezing point determination should be used for adulteration. key words: raw milk safety, adulteration, antibiotic residues resazurin test. introduction kenya produces an estimated volume of 5 billion liters of milk annually and is therefore the leading milk producer in the east africa region (muia et al., 2011). kenya’s dairy *corresponding author. e-mail: david.deep@yahoo.com industry, the single largest livestock production subsector contributes 14% of the agricultural gross domestic product (gdp) and 3.5% of the total gdp (muriuki et al., 2003). kenya’s dairy industry is a dynamic and plays an important economic and nutrition role in the lives of many people ranging from farmers to milk hawkers, processors, in ternationa l scholars journa ls african journal of food science research issn: issn 2375-0723 vol. 4 (3), pp. 018-026, october, 2016. available online at www.internationalscholarsjournals.org © international scholars journals nonini et al. 019 and consumers. kenya is generally self-sufficient in milk and dairy products. however, the demand for milk and dairy products in developing countries is estimated to increase by 25% by 2025 (delgado et al., 1999), mainly due to human population growth, further urbanization, increased disposable income, greater diversity of food products to meet nutritional needs, and increased opportunities for domestic and external trade. indeed, dairy imports in developing countries may reach 38.9 billion litres of milk equivalent by 2030 (food and agriculture organization (fao) and international dairy federation (idf), 2004). fortunately, kenya has the potential to increase milk production from the current 4.2 billion litres in 2009 to over 5.0 billion litres in 2014 (cherono, 2005). kenyan milk production systems can be divided into two general categories: large-scale and small-scale. the small-scale or smallholder dairy production system dominates. the differences between the two dairy systems are in their sizes of operation, level of management and use of inputs. dairy cattle in smallholdings feed mainly from forage and very small quantities of concentrate, but some small-holder dairy farmers are highly commercial and well versed in dairy production, with high-quality management. dairy production is dominated by smallholders who own about 98% of the total dairy herd (peeler and omore 1997). smallholder dairying households estimated to number over 1.5 million households, account for more than 85% of the annual total milk production and 80% of the 1.8 billion litres of milk marketed annually (mol & fd, 2003; staal et al., 2001). over the years, significant changes in the traditional dairying have occurred resulting in a major shift towards market-oriented smallholder production. farmers’ groups handle only about 40 percent of marketed milk production and about 20 percent of total milk (muriuki, 2003). over 800,000 smallholder farmers in kenya depend on dairy farming for their livelihoods. small-scale farmers account for 80% of the total milk production and 70% of the total marketed milk in the country. this has positive implications on food security and nutrition and has the potential to reduce poverty, particularly in the rural areas (chesterman and neely, 2015). milk is synthesized by cells within the mammary gland and is virtually sterile when secreted into the alveoli of the udder. beyond this stage of milk production, bacterial contamination can generally occur from within the udder, outside the udder, and from the surface of equipment used for milk handling and storage. cow health, environment, milking procedures and equipment sanitation can influence the level of microbial contamination of raw milk. equally important is the holding temperature of milk and the length of time milk is stored before testing and processing that allow bacterial growth. all these factors will influence the total bacteria count and the types of bacteria present in raw milk bulk tank (murphy and boor, 2000). raw milk safety in kenya has been disputed over a decade but no measurable data exists despite the fact that it requires monitoring from production to consumption. the regulatory institutions are constrained by lack of resources in terms of personnel and equipment (muriuki et al., 2003) even though, the kenya bureau of standards developed a hygienic code of practice for milk production to assist farmers in producing hygienic milk. rejection at market is a result of poor handling and the time taken to reach markets (long distances and bad roads). rejections are higher during the wet season, when production is high and roads are impassable. losses at the farm level can be more than 6 percent of total production, which means that at current production levels, national annual losses may reach 60 million kg (muriuki, 2011). consequently, most of the milk marketed by small-scale farmers in kenya has been reported to be of low quality and does not always meet national and international standards due to high bacterial load (mwangi et al., 2000), antibiotic residues (omore et al., 2005; shitandi and sternesjo, 2004) and water adulteration. the greatest limitations in the whole raw milk collection chain are proper ways to maintain cold collection due to the high investment costs demanded. this particularly affects the informal sector but also relatively in the formal sector (orregård, 2013). the formal milk trade is the market segment licensed by kdb. the informal markets controls an estimated 70 percent of the total milk marketed in kenya (kenya dairy board (kdb), 2009; government of kenya, 2006). this sector is important and is driven by among other factors the traditional preferences for fresh raw milk and its relatively lower cost. raw milk markets offers both higher prices to producers and lower prices to consumers but with several challenges relating to quality control and standards, and the associated health and safety concerns. other players in milk marketing include informal traders, distributors and retailers. the existence of informal trade results from a combination of the formal system’s failure or inefficiency, consumer habits/preferences, and price differences between raw and processed milk (muriuki, 2011). additional factors like unhygienic milking and handling practices, results in poor raw milk quality. according to orregård, (2013), plastic jerry cans are impossible to clean and are often used for transporting milk by most motor bike transporters. this result in a less hygienic handling compared with the use of aluminum cans whose only limitation is the acquisition cost. plastic jerry cans which could contribute to milk quality deterioration. this is in line with gemechu, (2015), who found out that milk producers use plastic containers which are difficult to clean and disinfect and thus it might contribute to poor quality of the milk. the collection and bulking enterprises (cbe’s) critical quality control challenges in line with milk bulking are; adulteration (both water and preservatives), table 1. the number of samples collected and analyzed from the four sampling levels in each cbe per replicate. cbe ngorika olenguruone can 44 24 collection routes 6 6 cooling tank 1 1 tanker 1 1 high bacterial load due to warm collection, potential for contamination with coliforms due to handling, presence of anti-microbial residues and zoonotic diseases like tuberculosis and brucellosis (muriuki, 2011). owing to the large amount of milk that is marketed unprocessed, and to weak monitoring of the market, public health risks are a concern. the main public health concern is the potential risk of diseases such as brucellosis and tuberculosis (tb). drug residues are also of concern, even in the processed milk channel. nyandarua county produces the highest amount of milk due to its higher population of dairy cows as compared to the other regions in central kenya (muia et al., 2011). however, reports for central kenya indicates that dairy production potential for nyandarua county is the least exploited (romney, 2004; staal et al., 2001; schreiber, 2000; baltenweck et al., 1998). nakuru county had many districts; it has an area of 166 square km and human population of 25,800 people (gok, 2009). the area has now settled down as a productive area with a high potential for dairy farming. the division has a total of 8925 cattle producing 7.5 million litres annually (district livestock production annual report, 2012). this study was designed to monitor microbiological and physico-chemical quality of raw milk from two smallholder dairy farmer’ groups at four sampling levels according to the requirements of the kenya bureau of standards (kebs). materials and methods study site the study was carried out in new ngorika milk producers limited in nyandarua county and olenguruone dairy farmer’s cooperative society in nakuru county. for both cbe’s, milk from individual farmers was collected and bulked into milk-cans while warm and transported in the same condition to the cbe cooler. the mode of transport per cbe varied from truck, tractor with trailer, tricycles, donkeys, individual farmer’s delivery and motor bikes. milk collection was only done once in the morning with some few farmers offering their evening milk separately along the routes. laboratory tests were carried out at the happy cow ltd laboratory and the food chemistry laboratory at the egerton university department of dairy and food science. the table 1 shows the number of samples carried out in every cbe during the analysis. 020 afr. j. food sci. milk sampling milk was initially stirred using a plunger to obtain a homogenous sample. the samples were obtained aseptically per the farmers group cans after acceptance at the reception platform, composite sample per route, cbe cooler and processor tanker. additional samples were collected from the milk delivered late in the afternoon in olenguruone, bulk samples from the rejected milk at the cbe platform, composite samples at the dump tank for the farmers that delivered individually to the cooling plant and extra samples for the freezing point determination. antibiotic residues analysis involved only the bulk samples at the routes and cooler/ tanker levels due to the cost of the analytical method applied. microbial analysis total viable bacterial count and coliform count were done according to aoac (2005) methods 991.14 and 990.12, respectively using 3mtm petrifilms plates. serial dilution was done up to 10-5 and 10-6 for coliform count and total viable bacterial count respectively. incubation was done at 370c for 24 hr and at 320c 48 hr for coliform count and total bacterial count, respectively. after the growth, the colony counting was done using the 3m plate reader (6499, 3m health care, germany) and the total colony forming units (cfu) /ml recorded in an excel sheet. adulteration the freezing point determination was carried out to assess adulteration according to draaiyer et al. (2009). when it is adulterated with water or other materials are added the density and freezing point of milk change from its normal value causing a detectable elevation of the freezing point of milk from its normal values of -0.54°c. this was done using a cryoscope according to the manufacturers operating instructions. after calibrations, 2.5 ml of the sample were put in the sample vial and placed at the measuring point of the cryoscope. the start measure of the machine was selected and the results presented as a percentage on the display of the machine and as well as printed. antibiotic residues the presence of the antibiotic residues was detected using the delvo test (sp nt and blf) according to delvotest technical bulletin (2011). delvo test is easy to use and covers the broadest spectrum of antibiotic residues in the industry. moreover, it is reliable and accurate with detection levels closest to maximum residue levels and safe tolerance levels (hillerton et al., 1999). the milk sample (0.15 ml) was added to the ampule and incubated at 64°c in a delvo incubator for 3 h to observe colour changes (sp nt). the other delvo test (blf) that involved use of ampules together with strips, was carried out. the incubator was set and the ampules with 0.15 ml milk sample inserted in it for 2 min. the milk sample was swirled again before inserting the strips in the ampules for 3 min and the results recorded. somatic cell count the somatic cell count was done on all the samples using california mastitis test (cmt) according to mellenberger and roth (2000). an equal amount of commercial cmt reagent was added to each cup and a gentle circular motion applied to the mixture in a horizontal plane. a positive gelling reaction and colour change occurred in 10 s with the positive samples. the gel formation and colour changes nonini et al. 021 table 2. means for log10 tvbc and cc (cfu /ml), lactic acid (la, %) and resazurin test (rt) for ngorika samples. sampling levels test can route cooler tanker rejected tvbc 8.396b 8.818a 8.708a 8.828a 8.889a cc 6.785b 7.240a 6.953a 7.150a 7.590a la 0.151c 0.156b 0.150c 0.150c 0.178a rt 3.971a 3.647a 3.846a 4.111a 1.778b means within a row marked with different letters are significantly different at (p< 0.05). table 3. means for log10 tvbc and cc (cfu/ml), lactic acid (la, %) and resazurin test (rt) for olenguruone samples. sampling levels test can route cooler tanker rejected tvbc 6.455c 6.276c 6.369c 7.138b 8.222a cc 4.137c 4.683bc 5.322ab 5.390ab 6.422a la 0.148b 0.152b 0.151b 0.153b 0.167a rt 4.817a 5.000a 5.000a 4.889a 3.222b means within a row marked with different letters are significantly different at (p< 0.05). were observed and compared using the colour and viscosity comparison table. ten minute resazurin test the resazurin test was done as per draaiyer et al. (2009) where a resazurin tablet was completely dissolved in 50 ml of sterile distilled water according to the manufacturer’s instructions. one milliliter of the resulting solution was added into 10 ml of the milk sample in a test tube, mixed and then incubated at 37°c in a water bath for 10min in a water bath. the samples were then read using a lovibond comparator (14 20 00, tintometer ltd, england) from a good source of light for colour change and numerical score value ranging from 1 to 6, assigned. a milk sample without the resazurin dye was similarly treated and used as the blank in the comparator. samples with comparator readings ranging from 4 to 6 were acceptable based on the kenya bureau of standards (kebs) on milk quality. titratable acidity the titratable acidity test was done as per draaiyer et al. (2009). this measured by titration whereby 3 to 4 drops of 0.5% phenolphthalein were added in 9 ml of milk sample in a beaker on a white tile and titrated against 0.1 equivalents/litre naoh with constant shaking of the milk until a permanent colour change (pink) was observed. by recording the volume of base used and the volume of the milk sample, the amount of developed lactic acid was calculated and expressed as a percentage lactic acid. statistical analysis the laboratory experimentation employed a randomized complete block design with a 2×5 factorial arrangement having three replications. the blocks of the experiment were the two cbe while the treatments were the four different sampling levels. analysis of variance (anova) was used to analyze the results obtained . this was done using proc general linear model (glm) procedure of the statistical analysis system (sas) version 9.0 (sas, 1999). means were separated using least significant difference (lsd). results and discussion total viable bacterial count (tvbc) and coliform count (cc) according to ks 05-1552, bacteriological grades for raw milk grade iii for total bacterial count and coliform count are 2,000,000 and 1,000 (cfu/ml), respectively. this study realized results that were generally higher compared to the standards. mean log10 for total bacterial count and coliform count per ml (cfu/ml) in ngorika (table 2) was not significantly different (p ≤ 0.05) among the milk samples collected from the route composite, cooler and tanker but it was significantly different (p ≤ 0.05) for the composite cans. this could have been contributed by the warm collection and the much time spent during transportation favouring the bacterial multiplication. warm collection in the dairy value supply chain creates an optimum environment for microbial growth consequently causing milk quality deterioration (mwangi et al., 2000; orregard, 2013). the rejected milk analysis results represent the plates that had countable colonies and it had no significant difference (p ≤ 0.05) from the route, cooler and tanker samples. moreover, plates with too many to count (tmtc) microbial colony growth were 44.4 and 33.3% for tvbc and cc, respectively for both locations. these uncountable results were not included in the statistical analysis of the data. in olenguruone (table 3), the means log10 for tvbc were significantly different (p ≤ 0.05) at the tanker level table 4. means for log10tvbc and cc cfu/ml for all sampling points in the two locations. sampling point tvbc cc rejected 8.889a 7.590a tanker 8.462c 6.770ab cooler 8.305bc 6.694ab route 8.187bc 6.474b can 8.078b 6.226b means within a column marked with different letters are significantly different at (p< 0.05). compared to the can route and cooler levels. additionally, significant difference (p ≤ 0.05) was observed between the tanker and the rejected milk. the means log10 cc count cfu/ml for the can and route sample were not significantly different (p ≤ 0.05) similarly to the means for the route, cooler and tanker. nonetheless, significant difference (p ≤ 0.05) for the rejected milk was observed for both tvbc compared to all the sampling levels (table 3). these samples were collected from the longest routes with poor infrastructure and took longer time for milk collection in olenguruone. the microbial quality of the rejected milk samples in olenguruone was similar to the samples at the tanker level in ngorika. in table 4, the overall mean log10 for total bacterial count in both locations was 8.078, 8.187, 8.305 and 8.462 tvbc log10 cfu/ ml for milk samples collected directly from the can composite, route composite, cooler (bulk) and upon arrival at the processing plant (tanker), respectively. an increasing trend of tvbc was observed at the different points considered from the can to the tanker and consequently the rejected milk samples. however, the means log10tvbc for the two locations indicated that the can level, route level and cooler level were not significantly different (p ≤ 0.05) unlike the tanker level, but significant difference (p ≤ 0.05) for tvbc between all the levels and the rejected milk samples was observed. coliform count indicated no significant difference (p ≤ 0.05) between can, route composite, cooler and tanker levels. furthermore, no significant difference (p ≤ 0.05) was observed between the rejected milk, cooler and the tanker levels. accordingly, the tvbc increased by 4.75% (0.384 log10 cfu/ml) from the can to the tanker milk sampling points. increase in tvbc observed along the value chain may be due to several factors like contamination at the farm, storage and transport using improperly cleaned milk cans, and lack of controlled temperature during transportation (doyle et al., 2015). the difference between the two cbes might be due to the fact that the olenguruone was sampled from the smallest radius to the cooling plant and the milk was transported using motor bikes that are very fast in terms 022 afr. j. food sci. of delivery time. an increase in microbial growth was observed between the cooler and the tanker which could have been majorly contributed by the cooler efficiency that is taking over three hours to cool the milk from 18 to 4°c (which was the required temperature for the processor), the use of the plastic containers in the collection of milk, the milking practices and the handling hygiene through the chain. additionally, the sample was taken immediately after the cooler filled up. in ngorika, the sampling was done in all the routes, and collection the raw milk microbial quality was very poor at all levels for this cbe. for both cbe, this may be due the contribution of insufficient pre-milking udder preparation, insufficient cleaning of milk handling equipment, use of poor quality water for cleaning, the storage time and lack of cold chain facility starting from the production site (doyle et al., 2015). as reported by van kessel et al. (2004), the use of insufficient and poor quality water for cleaning of milk handling equipment can result in milk residues on equipment surfaces that provide nutrients for the growth and multiplication of bacteria that can then contaminate the milk. murphy and boor (2000) noted that ineffective cleaning, use of water without heat treatment and the absence of sanitizers tend to fasten growth of less heat resistant organisms. similarly, mastitis infected cows can also contribute to high tvbc. generally, the presence of coliforms in milk confirms that the milk has been contaminated with fecal materials and it is an indicator of the sanitary conditions in the production and handling of the milk starting from production (orregard, 2013) accordingly, poor herd/farm hygiene, use of contaminated water, unsanitary milking practices, and use of improperly washed equipment for storage and distribution can all lead to elevated coliform count (cc) in raw milk (gemechu et al., 2015). the fact that high proportion (90%) of the milk samples taken from all levels had coliform counts more than the upper limit of kebs standards accepted for cc in raw milk, provides irrefutable evidence that the udder of the cows have been soiled with fecal materials and/or the udder is improperly washed; that is, milk contamination in the study area happened starting from milking of the cows. in addition, the presence of coliform in an aseptically collected sample of raw milk shows the use of bacteriological low quality water, either for washing utensils or mixing in raw milk (farhan and salik, 2007). apart from safety and public health concerns, high contaminations by coliforms results in off-flavours in milk and reduced shelf life of dairy products (reta and addis, 2015: kaindi et al., 2011). generally, the bacterial generation (doubling) time is between 10 to 15 min depending on the conditions. according to orregard (2013), aluminum cans allows better hygienic handling unlike plastic jerry cans. in this study, milk was transported while warm and in plastic jerry cans which could contribute to milk quality deterioration, unlike the recommended containers that do not have adhesive properties and are easy to clean when nonini et al. 023 40 35 fi gu re s 30 20 25 % 15 10 5 0 ngorika olenguruone locations figure 1. the average percent water adulteration in both cbe's. compared with plastic containers. moreover, more than 3 h, where natural lactoperoxidase enzyme could sustain the milk quality, were surpassed in some routes before refrigeration could take place. cooling of milk is advocated to help in significantly reducing the multiplication of bacteria and in turn reduce spoilage (kurwijila, 2006). adulteration freezing point of milk is its most constant property. according the kenya standard (ks 05-1552), approximately 0.545°c; but not less than 0.525°c is the freezing point of milk. results indicated that adulteration incidences in ngorika and olenguruone were 23.8% and 36.8%, respectively (figure 1). adulteration in ngorika was lower than in olenguruone. this could have been contributed by the effect of the penalty attached to a farmer when caught in this act. added water can occur in milk due to both unintentional (e.g., poor system drainage) and intentional addition (kurwijila, 2006). agents in some areas add water to increase the volume to make a larger profit, especially during the dry period when milk supplies are low and prices are high (orregård, 2013). this can be detected using a cryoscope by measuring its freezing point. normally, the freezing point of milk is slightly less than that of pure water and is relatively constant. typical milk generally has a freezing point below minus 0.542 degrees hortvett but when water is added to milk, the freezing point increases approximately 0.005°h for every 1% water addition. the lactometer test could not detect water adulteration especially when done at minimal levels. the freezing point determination as a standard gauge for water adulteration (draaiyer et al., 2009) was applied to compliment the lactometer test. this study corresponds a study carried out by orregård, (2013), where the results showed that about 33% of the samples from farmers were not within the acceptable density limit. according to (ks 05-1552), density of milk of 20°c shall be within the following range: 1.026-1.032 g/ml. antibiotics antibiotics in milk are a major concern due to the risk of allergic reactions and the development of antibiotic resistant pathogen and inhibition of dairy starter cultures used to develop acid (e.g., lactic acid bacteria), which can result in the loss of significant amounts of product and milk (popelka et al., 2004). the delvo test was carried out for the composite samples at the route level and the cooler. in total, 74 samples were analyzed in both cbe and out of these, 54 and 35% from olenguruone and ngorika, respectively were positive (figure 2). this study corresponds to another study carried out by aboge et al. (2000) on antimicrobial residues detected on marketed milk in kenya. according to shitandi, (2004), eighteen percent (18%) of the samples from small-scale producers in his study area were β-lactam positive significant (p<0.001) while other studies carried out within kenya showed that many animal products in kenyan market have high level of drug residues which is unacceptable (muriuki, 2001). according to gallagher (2015), consumption of food with antibiotics residues can lead to bacteria becoming completely resistant to treatment in human beings, a situation reffered to as antibiotic apocalypse. somatic cell count somatic cell count should not exceed 300 000 per ml when tested in accordance with iso 13366. the california mastitis test (cmt) has been used for more than 50-years and continues to be the most accurate farm screening test for subclinical mastitis (ruegg and reinemann, 2002). the heavier the gel the higher the somatic cells in the milk and vice versa, indicative of the leukocyte count (quinn et al., 1994). most of the samples were in the range of 150,000 to 500,000 cells/ml which is an indicator of presence of clinical mastitis in the farms. in ngorika, no sample was within the 0 to 200,000 cells/ml which reflects absence of somatic cells in milk suggesting that it has higher incidences of mastitis compared to olenguruone which had 5% samples being mastitis negative. additionally, 65% of the samples collected ngorika were lying under the range150,000 to 500,000 cells/ml which is higher than olenguruone (55%) (figure 3). the farmers in olenguruone have a low average production per cow compared to ngorika which could be a contributing factor to the high incidences of mastitis observed. s . agalactiae is known to be an occasional 024 afr. j. food sci. ngorika olenguruone positive 35% negative 65% positive 54% negative 46% figure 2. detection percent of the antibiotic residues in both cbe’s. olenguruone ngorika 0-200 0% 0-200 5% 800-5000 800-5000 15% 400-1000 13% 22% 400-1000 25% 150-500 150-500 55% 65% figure 3. california mastitis test somatic cell count per 1000 mls per cbe. cause of high bacterial counts and subclinical mastitis problems, hence it should be considered when both the scc and tvbc are high (ruegg and reinemann, 2002). for that reason, the higher somatic cell counts detected could have contributed to the high levels of the total bacterial counts observed in both locations. increased somatic cell numbers are positively correlated with concentrations of plasmin, a heat-stable protease, and of lipoprotein lipase in freshly produced milk (barbano et al., 2005). activities of these enzymes can supplement those of bacterial hydrolases, hence shortening the time to spoilage. the major determinants of quantities of these enzymes in the milk supply are the initial cell numbers of psychrotrophic bacteria, their generation times, their abilities to produce specific enzymes, and the time and temperature at which the milk is stored before processing. several conditions must exist for lipolyzed flavor to develop from residual lipases in processed dairy foods, that is, large numbers (>106 cfu/ml) of lipase producers (stead, 1986), stability of the enzyme to the thermal process, long-term storage and favorable conditions of temperature, ph, and water activity. resazurin test it uses the indicator resazurin to measure the bacteriological quality of milk. the majority of the organisms in milk are capable of reducing and decolorizing the resazurin dye. when bacteria grow in the milk they utilize oxygen, the rate of removal or reduction is proportional to the keeping quality (draaiyer et al., 2009). the resazurin dye is more sensitive than the methylene blue and for this reason, this test provides a rapid measure of the keeping quality of milk. there was no significant difference (p ≤ 0.05) for 10 min resazurin test at the four sampling levels except for the rejected milk for both cbe’s (table 2 and 3). the correlation of the 10 min resazurin test a nd tvbc was not nonini et al. 025 table 5. correlation of general quality results evaluated at four sampling levels in smallholder dairy farmers supply systems in kenya. time route tvbc cc la rt time 1.00000 -0.74248*** 0.42845** 0.53512*** 0.22400 -0.47639** route 1.00000 -0.46522*** -0.47591*** -0.17855 0.64902*** tvbc 1.00000 0.67682*** -0.06786 -0.23962 cfu 1.00000 0.02256 -0.21445 la 1.00000 -0.34558* rt 1.00000 *,**,***, indicates significance at p ≤0.05, p ≤0.01 and p ≤0.001, respectively. significantly different (p ≤ 0.05) at the route level (table 5). according to kebs, (2007) raw milk specification, only 20.6% of samples were unacceptable based on this test contrary to the tvbc where all the samples were way above grade iii raw milk requirements (2,000,000 cfu/ml) which is unacceptable. the study results agrees with the study carried out by muliro et al. (2013) on quality assessment of raw camel milk using dye reduction tests that the resazurin test is not reliable as a measure of total viable bacterial count. according to murphy and boor (2000), a significant correlation (p ≤ 0.01) between the total bacterial count and time used to deliver milk for cooling was observed. the plate count test has been reported to be generally accepted as the most accurate and informative method of testing the bacteriological quality of milk (kurwijila, 2006: muliro et al., 2013). titratable acidity test the means separation indicated no significance difference (p ≤ 0.05) at the can, cooler and tanker level unlike the route level and the rejected milk in ngorika (table 2). this could have been contributed to the dilution factor as milk is being bulked together in the cooler. a different scenario was observed in olenguruone where no significance difference (p ≤ 0.05) at the can, route, cooler and tanker level but significant difference (p ≤ 0.05) was observed with the rejected milk (table 3). all the samples at the can, route, cooler and tanker levels were found to have acidity levels within the range of 0.16±0.02 and therefore judged to be of good quality for the titratable acidity test. this was contrary to all the milk samples rejected at the reception platform that were above 0.18% lactic acid, hence rejected as per the kebs standards. conclusion and recomendations the study showed that total bacterial count and coliform count were way above the kebs standards although along the dairy value chain. factors like famers training on the clean milk production, milk handling hygiene, use of appropriate containers and reduced delays in milk collection could assist in reducing the microbial load in raw milk. milk quality tracking and tracing was lacking hence the concept of quality based milk payment system (penalties and premium) would be difficult to introduce. can labeling and subsequent ownership which was lacking should be enhanced for tracking and tracing. the cooling plants were taking more than 3.5 h to cool the milk to below 5°c which justifies use of plate heat exchangers as an alternative for speedy cooling. the study also revealed higher figures for antibiotic residues which suggests that withdrawal period was not observed. to overcome this, inclusion of the test in quality based payment system and vigorous training to farmers will ensure that the antibiotic residues are within the maximum allowable limit. adulteration of milk with water was evident which could be due to the fact that the farmers are paid according to supplied quantities. milk policies should be established and punitive penalties introduced to farmers found to have adulterated the milk unlike the current situation where no action is taken. for raw milk quality based payment system, resazurin test and lactometer test are not sufficient. tvbc and cc should be used instead of resazurin while freezing point determination should be used for adulteration. conflict of interests the authors have not declared any conflict of interests. references aboge go, kang’ethe ek, arimi sm, omore ao, mcdermott jj, kanja lw, macharia jk, nduhiu jg, githua a (2000). antimicrobial agents detected in marketed milk in kenya. 3rd all africa conference on animal agriculture. 6-9 november 2000, alexandria, egypt. nairobi (kenya): smallholder dairy project (sdp). aoac (2005). official methods of analysis of aoac international. 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(1999). sas/stat users’ guide, version 8 release 6.03. statistical analysis systems (sas), carry, nc. 549-640. schreiber c (2000). sources of innovation in dairy production in kenya. international services for national agricultural research (isnar) briefing paper no. 58. ministry of agriculture and rural development (moard), kenya agricultural 53 research institute (kari) and international livestock research institute (ilri)/isnar. the hague, the netherlands. shitandi a (2004). risk factors and control strategies for antibiotic residues in milk at farm level in kenya. doctoral dissertation. department of food science. swedish university of agricultural sciences. sweden. 91(3):576-6477. shitandi a, sternesjo a (2004). factors contributing to the occurrence of antimicrobial drug residues in kenyan milk. j. food prot. 67:399-402. staal sj, owango m, muriuki h, lukuyu b, njoroge l, njubi d, baltenweck i, musembi f, bwana o, muriuki k, gichugu g, omore a, thorpe w (2001). dairy systems characterization of the greater nairobi milk shed. smallholder dairy (research & development) project. kenya agricultural research institute (kari), ministry of agriculture (moa) and international livestock research institute (ilri) collaborative dairy research program, nairobi, kenya. 73. http://www.smallholderdairy.org/collaborative res reports.htm van kessel sj, karns sj, gorski l (2004). prevalence of salmonellae, listeria monocytogenes, and fecal coliforms in bulk tank milk on us dairies. j. dairy sci. 87:2822-2830. african journal of food science research vol. 2 (9), pp. 139-141, september, 2014. available online at www.internationalscholarsjournals.org © international scholars journals review agroforestry concepts and practices in maintainable land use systems in nigeria bello ibrahim and mohammed dave department of forestry and wildlife management, faculty of agriculture and agricultural technology, ahmadu bello university, zaria, kaduna state, nigeria. accepted 23 august, 2014 agroforestry has been defined as a dynamic ecologically based natural resources management system that through the integration of trees on farms and in the agricultural landscape, diversifies and sustains production for increased social, economic and environmental benefits for land users at all levels. this paper highlighted agroforestry practices and concepts in sustainable land use systems. the benefit derivable from the interface between forest trees and agricultural crops are enormous. they include the optimal use of land for both agricultural and forestry production on a sustainable basis including the improvement of the quality of soil. this is in addition to the socio-economic benefits that are accruable from agroforestry. indeed the advantage of agroforestry is all encompassing and germane to a sustainable production system and livelihood. key words: agroforestry, practices, concepts, sustainable, land use systems. introduction agroforestry practices offer practical ways of applying various specialized knowledge and skills to the development of sustainable rural production systems. agro-forestry is recognized as a land use option in which trees provide both products and environmental services. in agroforestry systems, the trees grown on different farmlands in the same locality when aggregated can bring about improved wooded situation thereby enhancing environmental protection (otegbeye, 2002). in most agroforestry systems, the trees grown do not have the usual silivicultural recommendations in terms of spacing (owonubi, 2002). given the reality of awareness among the farmers of multiple land use management, the need to improve on the existing agroforestry practices becomes necessary in the face of increasing population and limited nature of land. rural people have been discovered to have a wealth of indigenous knowledge and have incorporated trees in production systems in areas where they lived for a very long period of time (evans and alexander, 2004). agroforestry has both protective and social-economic benefit. kang (1993) reported that besides direct agricultural benefit, trees exhibit social economic values. the benefit of the tree components derived by farmers from agroforestry was evaluated from a social-economic and ecological perspective (anderson and sinclair, 1993). the social economic benefits of agroforestry can be evaluated in terms of productivity, stability and sustainability. the objective of this paper is to highlight the importance and concepts of agroforestry as a veritable tool in sustainable land use systems. *corresponding author. e-mail: ib_bello@yahoo.co.uk http://www.internationalscholarsjournals.org/ ibrahim and mohammed 139 table 1. agroforestry practices among farmers in lafia local government area, nasarawa state, nigeria. types of agroforestry practices lafia north lafia south lafia west lafia east respondents percentage row/hedges 11 8 6 5 30 25 trees on farm land 2 4 6 7 19 15.8 scattered trees on farmland 13 10 16 14 53 44.2 wind break 4 8 2 4 18 15 total 30 30 30 30 120 100 source. alao and shuaibu (2011). types of agroforestry systems there are various types of agroforestry systems, some of which are listed as follows: 1. trees on farmland: the farmers plant or retain trees on their farmland, both for food, income, soil improvement and environmental amelioration and for shade during the harsh weather period. 2. parkland also known as scattered trees: parklands are characterized by well grown scattered trees on cultivated and recently fallowed land (cta, 2003). these parklands develop when crop cultivation on a piece of land becomes more permanent. the trees are scattered far apart so that they do not compete with their neighbours. parklands consist of indigenous trees like parkia biglobosa, vitellaria paradoxa, tamarindus indica, azadirachta indica, etc. parkland trees have the following characteristics:they are deep rooting, preferably reaching ground water table. they have capacity to fix nitrogen produce litter that decomposes well and add as much as possible to soil organic matter. 3. alley cropping as described by (cta, 2003) is a system in which strips of annual crops are grown between rows of trees or shrubs. lining up the woody plants in hedges should ensure that there is little interference with cultivation of the field. the extension of alley cropping to include animal husbandry by the international livestock research institute (ilri) has led to the concept of alley farming (okali and submerge, 1985). 4. wind breakers and shelter belts. their major purpose is primarily to control wind erosion. the species used include, azadirachta indica, anacardium occidentale, mangifera indica, musa species, khaya senegalenses, etc. alao and shuaibu (2011) have shown that these practices are commonly practiced by farmers as shown in table 1. based natural resources management system that through the integration of trees on farms and in the agricultural landscape, diversifies and sustains production for increased social, economic and environmental benefits for land users at all levels (leakey, 1996). agroforestry, generally refers to land used system or farming system in which trees or shrubs are grown in association with agricultural crops, pastures or livestock and in which there is ecological and economic interaction between the trees and other components. agroforestry practice is a distinctive arrangement of components in space and time. it is a specific local example of a practice, e.g taungya. it is characterized by environment, plant species, and arrangement, management, social and economic functions. range and classification of agroforestry the range and classification of agroforestry practices are the following. agro silvicultural agrosilviculture was aptly regarded as a variant of taungya to be practiced outside of forest reserve (nwoboshi, 1982). it envisages multiple land use involving arable and tree crops, but the emphasis here is shifted to the agricultural crops which are of dominant interest. indeed, agrosilviculture could be likened to shifting cultivation (nwoboshi, 1982) except that the fallow vegetation is planted with economic trees whose gestation period is equivalent to the fallow period. silvo – pastoral these are mostly trees with pastures and livestock. it is essentially the practice of animal production along with trees and pastures. concepts of agroforestry shifting cultivation agroforestry has been defined as a dynamic, ecologically shifting cultivation was the farming system widely table 2. uses and importance of agroforestry to farmers in lafia local government area, nasarawa state, nigeria. uses of trees respondents percentage additional income 23 19.2 human nutrition 8 6.7 reduce weeding 6 5 fuel, stakes and timber 34 28.3 shade for livestock 9 7.5 medicinal herbs 4 3.3 wind break 9 7.5 soil improvement 27 22.5 total 120 100 source. alao and shuaibu, 2011. embraced by peasants in the tropics in the past (greenland, 1974). this form of farming is no longer common, because rapid population growth has increased food demand tremendously to the level that fallow periods had to be reduced and the forestry sector had to give way gradually to agricultural needs. this has led to unprecedented deforestation, lowering of soil productivity, loss of biodiversity, increased soil erosion and weed infestation, and consequently lowered crop yield (okigbo, 1984a). unfortunately, the continued relying on expanding cultivated areas has not been able to contribute substantially to resolving the food crisis, because not all the available land is equally productive (okigbo, 1984b). it is not even economical on the long run. utilization levels of land and water resources are close to maximum potentials and future growth will be possible only through better management of a fixed resource base (banuri and holmberg, 1992). intensive rather than extensive use would be the way out of the log jam (fagbemi, 1997). in order to achieve the twin goal of satisfying increasing demands for food as well as retain the biologically beneficial effects of shifting cultivation, many workers have in the last two decades advocated the development of land use systems based on age-old practices of intentionally mixing trees in crop animal production fields (nwoboshi, 1980). mixed farming mixed farming system practiced by majority of the farming communities indicated the existence of traditional agroforestry system common in the semi-arid zones of nigeria (oboho, 1989). integration of trees into farming system and subsequent modification of the system could be easy with earlier understanding of the importance of trees in the farming system. similarly, the practice of animal production could make the intensification of fodder bank system an easily acceptable agroforestry model. with soil fertility, maintenance as the major problem of 140 afr. j. food sci. res. the farming system, adoption of appropriate agroforestry system will be easy. hayashi et al. (1995) opined that since trees have traditional value and importance among the farmers in the agro-ecological zone the modification of their integration within the farming system could be readily adapted and accepted by the farmers. fruits, fodder and fire wood species are common on the farmlands. improvement on these species will make it readily acceptable. advantages of agroforestry it should be noted that the attempts being made under agroforestry are to optimize the use of land for agricultural production on a sustainable basis at the same time meeting other needs from forestry (fagbemi, 2002). nitrogen-fixing and non-nitrogen-fixing trees thrive adequately in agroforestry with annual crops, presents a farming system in which arable crop yields can be enhanced. the tree rooting system brings about stability that can lead to soil conservation. what is needed would be mutual interaction and proper management techniques that would reduce the adverse effects that may result when trees are integrated into agro-ecosystem (connor, 1983). various authors (kang et al., 1990; young, 1986; rocheleau and dianne, 1987) were of the view that successful agroforestry practices benefits the farmers in the following ways: a. consistent restoration of the fertility status of the soil through the recycled litter deposition and nitrogen fixing mechanism of trees. b. a variety of products, firewood, poles, woodcraft, fodder, medicinal herbs and food for livestock and man respectively. c. prevention of wind and water erosion by trees acting as wind break and intercepting the raindrop impact on the soil respectively. d. improving the micro-climate effect of the immediate and adjourning environment. e. restoration of water table to an absorbable level for crops use. f. increased income opportunities. g. increased economic stability h. reduce cost for establishing plantation i. increased ability to manage for sustained yield. also, alao and shuaibu (2011) from their studies have shown in table 2, the inherent advantages in agroforestry accruable to farmers conclusion the role of agroforestry in sustainable land use system cannot be over emphasized. agroforestry practices offer ibrahim and mohammed 141 practical ways of applying various specialized knowledge and skills to the development of rural production systems. it evolves a synergy between agricultural production and forestry that is beneficial for increased food production, sustainable wood production and improvement of the quality of the soil. this is a win-win situation. the advantages of agroforestry are quite quantum. agroforestry, among other benefits strive to optimize the use of land for agricultural production on a sustainable basis and at the same time meeting other needs from forestry. references alao js, shuaibu rb (2011). agroforestry practices and preferential agroforestry trees among farmers in lafia local government area, nasarawa state, nigeria. waste management and bioresource technology 1(2)12-20. www.waoj.com anderson is, sinclair fl (1993). ecological interactions in agroforestry systems. agroforestry systems 6(2);58-61. banuri i, holmberg j (1992). governance for sustainable development: a southern perspective. islamabad and london. p. 50. connor bj (1983). plant stress factors and their influence on production of agroforestry plant association. in: plant research and agroforestry (p.a. huxley: ed) icraf, nairobi, pp. 401-426. cta (2003). agroforestry. arbrus et agricultures multietagees d’ afrique. cta, wageningan, the netherlands, p. 280. evans a, alexander i (2004). organic farming supports biodiversity in farming with nature, low external input and sustainable agriculture amersfoort, the netherlands. p. 36. fagbemi t (1997): agroforestry for sustaining agricultural production in the tropics. in: strategies and tactics of sustainable agriculture in the tropics (m.a. badejo and a.o. togon, eds) stasat 1:45-68. fagbemi t (2002). investment opportunities in renewable resources industry-forestry. 1st edn., belodan press, nigeria. greenland ds (1974). evaluation and development of different types of shifting cultivation: in shifting cultivation and soil conservation in africa. sida/fao soils bull. 24:5-13. hayashi x, carsky rj, ladipo do (1995): uses of indigenous tree species in a selected area in the northern guinea savanna of nigeria. nig. j. forest. 26(1):14-21. kang bt, reynolds l, atta-krah an (1990). alley farming advances in agro-forestry. agroforestry sys. 43:315-359. leakey r (1996). definition of agroforestry revisited. agroforestry today 8:1. nwoboshi lc (1980). agroforestry and the nigeria food production target. in: food and nutrition policy for nigeria eds: atinmo and akinyele. ibadan university press. pp. 117-124. nwoboshi lc (1982). tropical silviculture-principles and techniques. ibadan university press. p. 333. oboho eg (1989). important forest products in the semi-arid area of sokoto state. in forest resources development and the sourcing of local raw materials in nigeria. proceedings of the 19 th annual conference of the forestry association of nigerian. eds: j.a okojie and p.c obiaga. pp. 33-38. okali c, submerge je (1985). sheep and goats, men and women: household relations and small ruminant development in southwest nig. agric. sys. 18:39-45. okigbo bn (1984a) improved permanent production systems as an alternative to shifting cultivation. in: improved permanent production systems as an alternative to shifting cultivation. fao, rome, italy 53:1-100. okigbo bn (1984b). cropping systems and rotations development from improving shifting cultivation and related intermittant production systems in tropical africa. in: improved permanent production systems as an alternative to shifting cultivation. fao, rome, italy 50:121-140. otegbeye go (2002). report on agroforestry and land management practices, diagnostics survey of katsina state of nigeria. may 2000, katsina state agricultural and rural development authority.katsina. p. 89. owonubi jj (2002). disappearing forests: a review of the challenges for conservation of genetic resources and environmental management. j. forest. res. manage. 1:11-20. young a (1986). effects of trees on soils. in amelioration of soil by trees. commonwealth science council, longon. technical publication 190:28-41. african journal of food science research vol. 1 (3), pp. 024-032, november, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper isolation, characterization and identification of lactic acid bacteria involved in traditional fermentation of borde, an ethiopian cereal beverage kebede abegaz department of food science and post-harvest technology, awassa college of agriculture, debub university, p. o. box 5, awassa, ethiopia. e-mail: abegaz2005@yahoo.co.uk. accepted 13 november, 2013 changes in ph, titratable acidity, enterobacteriaceae, aerobic mesophiles, lactic acid bacteria (lab) and yeast counts were investigated during borde fermentation. a rapid decrease in ph was associated with accelerated growth rate of lab and inhibition of enterobacteriaceae. wide diversities of lab strains were present at early stage of borde fermentation. the number of species reduced as the fermentation progressed. representatives of lab involved in borde fermentation were isolated at 6 h intervals, characterized and identified using physiological and biochemical methods including api 50ch kit. the lab strains were grouped into 3 major and 2 minor clusters. most of the lab strains were heterofermentative lactobacillus (79.4%), while pediococcus species dominating the cocci (20.6%). weissella confusa, lactobacillus brevis, lactobacillus viridescens, pediococcus pentosaceus and p. pentosaceus subsp. intermedius were identified throughout borde fermentation as dominant lab. the frequency of dominating strains were: w. confusa (30.9%), lb. viridescens (26.5%), lb. brevis (10.3%) and p. pentosaceus (7.4%) and p. pentosaceus subsp. intermedius (8.8%), while sporadically isolated species of lactobacillus curvatus, p. acidilactici, lactobacillus collinoides, lactobacillus sanfrancisco, lactobacillus pontis and lactobacillus delbrueckii subsp. delbrueckii were 16.3%. the dominating lab strains can be potential starter cultures for borde fermentation. key words: beverage, cereal fermentation, ethiopian borde, identification, lactic acid bacteria. introduction fermentation has been used as a means of improving the keeping quality of food for more than 6000 years (holzapfel, 1997). beer brewed by the babylonians and exported to egypt around 3000 b. c. was most likely the product of both alcoholic and lactic fermentations. present day sorghum, maize and millet beers in africa possess similar features in which the lactic fermentation plays a key role in safety and acceptability of these products in tropical climates (haggblade and holzapfel, 1989). many african foods that are prepared by the action of diverse species of fungi, bacteria and yeasts on plant materials are little known outside their native countries. otherwise, foods are considered as habitats for microbial growth. the origin, development and succession of a particular microbial community in any food are governed by its ecological factors (deak and beuchat, 1996), which influence the physiological expression of microbial cells. thus, strategies for food processing and preservation can be developed on the basis of ecological factors associated with specific foods and beverages (gould, 1992). ethiopian borde is a spontaneously fermented, low or non-alcoholic cereal beverage. it is produced from a variety of locally available cereal ingredients using traditional techniques. the adjunct (all unmalted cereal ingredients) and malt could each be from one type or a mixture of cereals. borde is an opaque, effervescent, whitish-grey to brown colour and has a thick consistency and sweet-sour taste. it must be consumed while actively fermenting within a day due to its poor keeping quality. it is widely consumed by adults and children mainly in southern and western parts of ethiopia. borde is often consumed as a low-cost meal replacement for many poor people. kebede 024 the traditional technology of borde preparation is timeconsuming, unhygienic and complex, which involves grinding, souring, roasting, steam cooking, boiling, cooling, mashing, wet-milling and wet-sieving operations (abegaz et al., 2002a). this may create a variable bio-physicochemical environment which select for involved microflora that overcome technological hurdles in the natural fermentation of borde. although cooking of the adjuncts at about 90c killed vegetative microbial cells (abegaz et al., 2002b), addition of malt serves as a source of microorganisms (sahle and gashe, 1991; ketema et al., 1998) and endogenous amylolytic enzymes. this would result in the hydrolysis of starch and thus create an environment suitable for proliferation of microorganisms during traditional (“four phases”) fermentation of borde. however, the spontaneous fermentation resulted in unpredictable quality of borde with a low ph, high counts of aerobic mesophiles, lactic acid bacteria (lab) and yeast (abegaz et al. 2002b). in general, a wide spectrum of microorganisms is involved during fermentation processes but a few types usually determine quality of the end product. given adequate environmental conditions, a particular microbial community will determine the quality of a specific food. therefore, isolation, characterization and identification of the involved microorganisms with a prospective selection of starter cultures that are adapted to borde production would be important to support the technical process and to obtain a predictable end product with a desired quality. this may help in development of starter culture and devising appropriate and affordable technology that could modernize borde production. thus, there is a need of information on systematic isolation and identification of lab involved in borde fermentation. the objective of this work is, therefore, to isolate lab during the course of traditional borde fermentation, to characterize and identify them using biochemical, morphological and physiological techniques with the aim of starter culture development. materials and methods borde was prepared by an experienced brewer from a mixture of cereal ingredients following the traditional recipe and four phases of its fermentation (abegaz et al., 2002a). appropriate equipment and raw materials for borde production were purchased locally. for convenience of sample analysis and isolation of lab at 6 h intervals during the course of traditional borde fermentation, the experiment was carried out at awassa college of agriculture, awassa (272 km south from ethiopian capital addis ababa). equipment and preparation of ingredients the main equipments used in this study were plastic jars, plate and pan made from metal, grinding stones for wet-milling, bowls, grass baskets and sieve (1 mm pore). the adjuncts used were whole flour (abegaz et al., 2004) from maize (zea mays), sorghum (sorghum bicolor) and from a mixture of wheat (triticum sativum), finger millet (eleusine coracana) and tef (eragrostis tef). barley (hordeum vulgare) malt (bikil) was prepared following the traditional malting technique and 3% malt flour on weight basis of maize flour fermented at phase i was used. the adjuncts were cooked at about 90c (abegaz et al., 2002b) into various forms (flat bread, steamed dough balls and thick porridge) and cooled to 23 25c. the cooled flat bread, steamed dough balls and thick porridge were separately added into the plastic jar at phase ii, iii and iv of borde fermentation, respectively; and blended with appropriate malt flour and/or the fermenting mash. the proportions of adjuncts (on weight basis) were 3 kg maize flour for phase i fermentation, 3 kg mixed flour (1 kg wheat, 1 kg finger millet, 1 kg tef) for dough balls, and 1.5 kg sorghum flour for porridge. the maize flour was fermented with equal amount (3 l) of water (w/v) for 24 h before baking a portion of it (40%) into flat bread on a hot plate. the remaining portion of dough (60%) was fermented for a further 24 h before steam cooking 40% of it with 3 kg mixed flour into dough balls or boiling 20% of it with 1.5 kg sorghum flour into a thick porridge. on the other hand, the proportion of total adjunct flour (on weight basis) before cooking was 14, 56 and 28% according to the adding time of cooked adjuncts at phase ii (flat bread), iii (steamed dough balls) and iv (thick porridge) into the main fermentation jar, respectively (abegaz et al., 2002b). malt was added at phase ii (80% of the required malt) and phase iv (the remaining 20%). steps in traditional fermentation of borde the traditional fermentation of borde has four major phases marked by the addition of fresh ingredients into the fermentation jar at different time. the full details of the process and ingredients used in each phase are described by abegaz et al. (2002a, b). however, the four phases are described briefly as follow: phase i: fermentation of maize flour for 24 to 48 h before various types of cooking. phase ii: blending 80% of the required malt with pancakelike flat bread (kita) prepared from 24 h fermented maize dough into a thick mash (tinsis) and the tinsis left for 24 h fermentation/ phase iii: addition of steamed dough balls (gafuma), prepared from 48 h fermented maize dough and mixed flour, into the 24 h fermented tinsis, blending the mix to a thick mash (difdif) and the difdif left for a further 18 h fermentation. phase iv: addition of thick porridge from 48 h fermented maize dough and sorghum flour along with 20% of the required malt into the 18 h fermented difdif. after homogenization of the mix to thick slurry (mulet), it was wet-sieved using a sieve (wonfit) of 1 mm pore. the filtrate was poured back into the jar and left for 4 h final fermentation. the traditional fermentation of borde was done in duplicate and repeated three times at room temperature (2023 o c). during fermentation, appropriate samples were taken at 6 h intervals for determination of ph, titratable acidity (ta), microbial counts and isolation of lab. microbiological enumeration and isolation samples (25 g or ml) were taken aseptically at 6 h intervals during traditional fermentation of borde and transferred separately to a stomacher (lab blender 400, seward medical, london, england) 025 afr. j. food sci. res. with 225 ml sterile 0.1% peptone water and then homogenized for 30 s, ‘normal’ speed. the homogenate was then serially diluted and aliquots of 0.1 ml from appropriate dilutions were spread-plated in duplicate on pre-dried agar plates of violet red bile dextrose (vrbd), plate count agar (pca), mrs, and yeast extract glucose chloramphenicol bromophenol blue (ygc). all the culture media are from merck (kgaa, 64271 darmstadt, germany) except ygc, which consisted of (gram l 1 ): yeast extract, 5.0; glucose, 20.0; chloramphenicol, 0.1; bromophenol blue, 0.01; agar, 15; ph, 6.0 to 6.2. after incubation at 30 o c for 24 h, purple-red colonies on vrbd agar plates were counted as enterobacteriaceae (eb). the total aerobic mesophilic count (amc) was enumerated on pca plates after incubation at 30 o c for 48 h. colonies of lab were counted on mrs agar plates after anaerobic incubation in gaspak jars (gaspak system, bbl) at 30 o c for 72 h. yeast and mould colonies were counted on ygc plates after incubation at 28 o c for 3 to 5 days. the numbers of eb, amc, lab or yeasts from their respective duplicate countable plates are reported as log cfu g -1 calculated from the arithmetic mean of three replicates of borde preparation. three hundred fifty eight (358) representative colonies with distinct morphological differences such as colour, size and shape were randomly picked from countable mrs agar plates as presumably lab isolates. each lab isolates were purified by repeated streak-plating on mrs agar for three times; maintained on appropriate slants at 4 o c and subcultured every four weeks until required for characterization. characterization and identification all the lab isolates were characterized using phase-contrast microscopy and conventional biochemical and physiological tests. all isolates under examination were separately propagated twice in appropr-iate mrs broth and an overnight culture (inoculum) was used for all tests and incubated anaerobically (gaspak system, bbl) at 30 o c. each strain was initially examined for colony and cell morphologies, cell grouping, presence or absence of spores and motility using microscopy; gram-reaction using the koh test of gregersen (1978); catalase production with 3% h2o2 (v/v); and separation into phenotypic groups was undertaken. the vigor of colony and broth culture in the growing media was also examined. only the gram-positive, catalasenegative and non-spore former isolates were characterized. growth at different temperatures was observed in mrs broth after incubation at 15, 37 and 45 o c for 5 days, and at 4 o c and 10 o c for 12 days. growth in the presence of 4, 6, 8 and 10% nacl was performed in mrs broth and incubated at 30 o c for 5 days. gas (co2) production from glucose was determined in modified mrs broth containing inverted durham tubes, with diammonium citrate replaced by ammonium sulphate (samelis et al., 1994). growth of isolates was also assessed after inoculating 1% of 6 log cfu ml -1 in sterilized tinsis (100 g) for borde production. eighty seven (87) representatives of the isolates from the best borde that showed vigorous growth (increased by 3 to 4 log cycles in 24 h) in the sterilized tinsis were selected for further charac-terization as potential starter culture. gas (co2) production was determined after vigorously shaking of sealed headspace vials that contained 10.0 ml modified mrs broth culture (24 h) for 2 min and injecting 0.5 ml headspace gas twice using a sterile 1ml disposable syringe (terumo europe n.v., leuven, belgium) directly into the rubber tubing supplying nitrogen to the infrared carbon dioxide analyzer (adc 225 mk3, analytical development co. ltd., hoddesdon, hertfordshire, england) conn-ected to a chromatopac (c-r3a, shimadzu corporation analytical instruments, kyoto, japan). growth in ph 3.9, adjusted with hcl, was tested in mrs broth. production of ammonia from arginine was tested according to the method described by harrigan and mccance (1976) and also in modified mrs broth (samelis et al., 1994). the lab isolates were tested for fermentation of the following 14 sugars (merck): l-arabinose, ribose, d-xylose, galactose, fructose, maltose, lactose, saccharose, trehalose, raffinose, mannltol, sorbitol, salicin and esculin. in this test, phenol-red broth base was used as basal medium. a 1% filter-sterilized sugar solution, using 0.2 m filter (minisart-plus, sartorius, germany), was added aseptically into autoclaved phenol-red broth base before inoculation with overnight culture of each lab strain. the results were assessed with reference to the control after anaerobic incubation at 30 o c for 5 days. further-more, to determine the biochemical profiles of lab strains, 68 isolates were tested for fermentation of 50 carbohydrates, using api 50ch lactobacillus identification system (bio merieux, 69280 marcy i’etoile, france). the api 50 chl medium was used according to the manufacturer’s instructions. anaerobiosis in the inoculated strips was obtained by overlaying with sterile paraffin oil and incubated at 30 o c and then observations were made after 24 and 48 h. results from conventional method of sugar test and the api 50 chl system were compared for curiosity. after identification, 24 h broth cultures at 30 o c were centrifuged (kubota 2010, kubota corporation, tokyo, japan) at 3000 rpm for 15 min and the pellets were re-suspended in mrs broth containing 10% (v/v) glycerol (merck). the suspension was aseptically transferred into sterile cryo-tubes containing acid-washed glass beads and stored at 80 o c for further investigation aimed at development of starter culture for borde fermentation. statistical analysis the data from biochemical, morphological and physiological tests were analyzed with principal component analysis using the unscrambler ® programme (unscrambler ® v7.5, camo asa, n0115 oslo, norway). the positive and negative responses (table 2) that are made by all the lab strains were not included to this statistical analysis. interpretation of the data was made by inspection of the scores and loadings plots. identification of lab strains was facilitated by the use of a computer programme, apilab plus, version 3.2.2 (bio merieux), bergey’s manual of systemic bacteriology (sneath et al., 1986) and the genera of lactic acid bacteria (wood and holzapfel, 1995). results the initial counts of lab at phase i were low (10 4 cfu ml -1 ) but a rapid increase was observed within 12 h and then reached to about 10 9 cfu ml -1 after 24 h fermentation. the changes in ph, ta and microbial load during fermentation of borde are shown at figure 1. the growth of amc was comparable to that of lab though the amc were somewhat higher until 12 h from the initiation of fermentation. a rapid decrease in ph was in agreement with accelerated growth rate of lab and then followed by reduction of enterobacteriaceae. despite the amc and lab enter stationary growth phase, the yeasts were constantly increasing. moulds were declined as the fermentation progressed and reached at non-detectable level after 12 h at phase i and after 6 h at phase ii (data not shown). there were no moulds at phase iii and iv fermentation. representatives of lab from each phase of borde fermentation were isolated, characterized and identified using physiological and biochemical methods kebede 026 table 1. morphological and physiological properties of lab isolated during borde fermentation. percentage of strains with positive reactions characteristics cluster no. a i ii iii iv v no. of strains 31 21 5 10 1 cell morphology b r r 4c/1r c r co2 from glucose 100 100 0 0 0 growth at 4c 39 (48) c 81 (19) 20 (80) 90 (10) 100 10c 100 100 100 100 100 15c 100 100 100 100 100 37c 100 100 100 100 100 45c (39) 48 (43) 60 90 0 growth in 4% 100 100 100 100 100 6% 100 100 100 100 100 8% nacl 84 (13) 86 (5) 100 100 100 10% nacl 0 (3) 0 0 30 0 growth at ph 3.9 42 (58) 100 60 (40) 100 100 a cluster no. i = lb. viridescens and lb. brevis; cluster no. ii = w. confusa; cluster no. iii = p. acidilactici, and ent. flavenscens; cluster no. iv = p. pentosaceus and p. pentosaceus subsp. intermedius; cluster no. v = lb. delbrueckii subsp. delbrueckii. b r, rod shape; c, cocci (spherical shape); c the numbers in parenthesis indicate percent of strains that showed weak or delayed reactions. a1 a2 a3 a4 6 0.8 4.5 0.8 5 0.8 0.8 4.3 5.5 0.7 4.3 0.7 0.7 ph ta(%) 4.5 4.1 0.7 5 0.6 4.1 0.6 0.6 p h 0.6 3.9 t a 0.5 3.9 0.5 0.5 0.5 4.5 4 3.7 4 0.4 3.7 0.4 0.4 0.4 3.5 0.3 3.5 0.3 3.5 0.3 3.5 0.3 0 6 12 18 24 30 36 48 0 6 12 18 24 0 6 12 18 0 4 b1 b2 eb b3 b4 10 10 amc 10 10 c f u /m l lab 8 8 yeast 8 8 6 6 6 6 l o g 4 4 4 4 2 2 2 2 0 6 12 18 24 0 6 12 18 0 4 0 6 12 18 24 30 36 48 phase ii: fermentation (h) phase iii: fermentation (h) phase iv: fermentation (h) phase i: souring (h) figure 1. changes in ph and ta and microbial growth during fermentation of borde. ta, titratable acidity expressed as lactic acid (%); eb, enterobacteriaceae; amc, aerobic mesophilic count; lab, lactic acid bacteria; a1-a4, changes in ph and ta; b1-b4, changes in microbial growth. aiming at selection and development of starter culture. all the isolates were gram-positive and catalase negative rods or cocci except two of them. basic data including gram-reaction; morphology; gas production from glucose; growth at 4, 10, 15, 37 and 45c; and growth in 2, 4, 6, 8 and 10% nacl were essential (table 1) in addition to the pattern of carbohydrate fermentation using api 50ch strips (table 2). all the strains showed kebede 1473 027 afr. j. food sci. res. table 2. differences in fermentation of carbohydrates by lab strains isolated from borde. carbohydrates a percentage of strains with positive reactions to api 50 ch cluster no. b i ii iii iv v no. of strains 31 21 5 10 1 arabinose 65 (13) c 57 80 40 0 ribose 42 52 (5) 60 100 100 xylose 3 100 100 10 0 galactose 3 (65) 76 (19) 100 100 100 fructose 94 100 100 100 100 mannose 94 100 100 100 100 rhamnose 0 0 40 (20) 50 0 mannitol 0 0 (5) 40 0 0 methyl-d-mannoside 0 0 0 (20) 0 (10) 0 methyl-d-glucoside 3 0 0 0 0 amygdalin 3 86 (10) 100 100 0 arbutin 3 (6) 76 (14) 100 100 0 esculin 3 (3) 100 100 100 0 salicin 3 (3) 81 (14) 100 100 0 cellobiose 0 95 (5) 100 100 0 maltose 100 100 80 100 100 lactose 0 14 (10) 40 (20) 80 (20) 0 melibiose 3 0 0 (20) 40 (10) 0 sucrose 94 90 0 0 100 trehalose 0 0 80 90 100 starch 0 0 40 0 0 gentiobiose 0 95 (5) 100 100 0 d-togatose 0 5 40 90 0 gluconate 55 (10) 81 (5) 20 0 (30) 0 5-keto-gluconate 3 0 0 0 0 a all strains fermented glucose and n-acetyl glucosamine, but none of the strains fermented glycerol, erythritol, d-arabinose, lxylose, adonitol, -methyl xyloside, sorbose, dulcitol, inositol, sorbitol, inulin, melezitose, raffinose, glycogen, xylitol, d-turanose, d-lyxose, d-fucose, l-fucose, d-arabitol, l-arabitol, and 2-keto-gluconate. b,c see a and c under table 1, respectively. growth at 10, 15 and 37c incubations and in 4 and 6% nacl; but growth at 45c and growth in 8 and 10% nacl were important for differentiation. when the strains inoculated into api 50chl medium were further incubated for 24 to 72 h, their responses were similar to that of the conventional method of sugar test carried out in test tubes for 5 days. although the data on response of lab strains in api 50ch strips was taken after 24 and 48 h incubation (according to manufacturer’s instruction), some of the strains showed slow positive reaction after 24 to 48 h further incubation. however, except for curiosity on the response of wild-type lab strains on api 50chl compared to the conventional method, the data after 48 h incubation of api 50chl were not utilized for identification of the isolates. results from arginine test were not included for identification. because, some of the strains were repeatedly showed positive responses both on treatment and control tests for ammonia production that could be from hydrolysis of arginine or other compounds. most of the strains appeared to be lactobacillus species (79.4%), while pediococcus species were dominating the cocci (20.6%). however, the strains of lactobacillus were found to be heterofermentative, which is in agreement with reports elsewhere (hounhouigan et al., 1993; ketema et al., 1998), except one incidence of homofermentative species identified from the end product. as a result of principal component analysis, the lab strains were grouped into 3 major and 2 minor clusters (figure 2) based on similarities in biochemical, morphological and physiological characteristics. a total of 68 strains of lab isolates were identified. cluster i consisted of lb. viridescens, lb. brevis, lb. curvatus, lb. collinoides (fructose negative and grew at kebede 028 1.0 pc2 bi-plot d-xyl 1923667 cluster ii gnt 33 26 3068 w. confusa gas 49 295 7066 shape 6 25 sac 50 59 44372 0.5 47 45c escgceln amy l-ara sal cluster i arb 7240351 64 dmne-fru gal 0 63 mal4c 613428 5md-cg 5420 583821 amdman 1 sc8 39 4862 52 9 43 8 lb. brevis sc10 7 18 cluster iii 4 571 22 12 mel lb. viridescens 46 rha 1145 69 lac -0.5 24360 rib 15 tag pediococcus cluster v tre 13 65 cluster iv 56 55 743 42101 lb. delbrueckii -1.0 14 pc1 -1.0 -0.5 0 0.5 1.0 clusters of labx…-expl:, 46%,15% figure 2. principal component analysis of characterization tests on lab isolated during traditional fermentation of borde. number 1 to 74 are reference no. of strains; cluster i includes also sporadic strains: lb. collinoides (1), lb. carvatus (45, 46 and 60), lb. sanfrancisco (18), lb. pontis (22); cluster iii includes p. acidilactici (4), e. flavescens (7 and 9); p. pentosaceus (8), unidentified (43); gas, co2 production from glucose; 15c or 45c, growth at 15 or 45c; sc8 or sc10, growth in 8 or 10% salt concentration; carbohydrates, three letter abbreviations from api 50 chl medium. 45c), lb. pontis (l-arabinose positive) and lb. sanfrancisco (mannose positive) . the latter three strains were isolated only at phase i, while lb. curvatus (cellobiose negative) was isolated from phase ii and iii fermentation. in contrast, lb. viridescens and lb. brevis were found throughout the four phases of borde fermentation. however, differentiation between some of the lb. viridescens (larabinose, ribose and gluconate positives)and lb. brevis (ribose and gluconate negatives but grown at 45c) strains may require further studies using techniques of molecular genetics. there are also one salicin positive lb. brevis and another amygdalin positive lb. viridescens strains. otherwise, all the lb. brevis and lb. viridescens strains in this cluster are showed positive (fructose, mannose and sucrose) and negative (xylose and melibiose) responses on these tests. in general, lb. viridescens strains showed weak growth both in ph 3.9 and at 4c incubation tests. cluster ii contained w. confusa where some of the 21 strains were arabinose positive and ribose negative (8), arabinose positive (2), lactose positive (2), amygdalin and salicin negative (1). four of the above 8 strains were not fermented gluconate and another one was sucrose nega tive. cluster iii consisted of enterococcus flavenscens, p. acidilactici, p. pentosaceus that were isolated during fermentation of maize dough at phase i. this cluster also included an unidentified short rod lab strain isolated from phase iii fermentation. in this study, ent. flavenscens strains were the only isolates that fermented mannitol and starch. the ent. flavenscens grew weakly at ph 3.9 but not grew at 45c. cluster iv included p. pentosaceus and p. pentosaceus subsp. intermedius (wood and holzapfel, 1995), which are isolated throughout the fermentation. fermentation of arabinose and rhamnose by p. pentosaceus was important to discriminate the subspecies. but one of the p. pentosaceus subsp. intermedius strains is rhamnose positive and d -togatose negative. in addition, one p. pentosaceus strain is xylose positive and trehalose negative. cluster v contained lb. delbrueckii subsp. delbrueckii, the only homofermentative lactobacillus strain in this report. it is galactose and ribose positive and also does not grow at 45c. the frequencies of dominating lab strains were as follows: w. confusa (30.9%), lb. viridescens (26.5%), lb. brevis (10.3%) and p. pentosaceus (7.4%) and p. pentosaceus subsp. intermedius (8.8%). in addition, p. acidila kebede 1475 029 afr. j. food sci. res. table 3. distribution of the dominating lab strains in the four phases of borde fermentation. phases and major activities lab a counts reference no. dominating (log cfu/g) of lab strains strains of lab phase i fermentation (souring) of maize 0 h (4.3) 1, 4, 5, 6, 7, 8, 9, 10 11, w. confusa, l. brevis, dough before cooking 24 h (9.4) 12, 13, 14, 15 l. viridescens and 48 h (9.3) p. pentosaceus phase ii blending of flat bread and malt flour 0 h (3.8) 18, 19, 20, 21, 22, 23, 24, w. confusa, l. brevis, into tinsis 24 h (8.7) 25, 26, 27, 28, 29, 30 l. viridescens and p. pentosaceus phase iii blending of steamed dough balls and 0 h (8.0) 32, 33, 34, 35, 36, 37, 38, w. confusa, l. brevis, tinsis into difdif 18 h (9.9) 39, 40, 41, 42, 43, 44, 45, l. viridescens and 46, 47, 48, 49, 50, 51, 73 p. pentosaceus phase iv blending of porridge and malt with 0 h (9.4) 52, 54, 55, 56, 57, 58, 59, w. confusa, l. brevis, difdif, wet-sieving into mulet 4 h (10.2) 60, 61, 62, 63, 64, 65, 66, l. viridescens and 67, 68, 69, 70, 71, 72, 74 p. pentosaceus a lab (lactic acid bacteria) counts at the beginning (0 h) and the end of each phase. ctici (1.5%), ent. flavescens (2.9%) and lb. collinoides (1.5%) at phase i fermentation; lb. sanfrancisco (1.5%) and lb. pontis (1.5%) at phase ii fermentation; nonidentified isolate (1.5%) at phase iii fermentation; and lb. delbrueckii subsp. delbrueckii (1.5%) at phase iv fermentation; lb. curvatus (4.4%) both at phase iii and iv fermentation were sporadically found as part of the lab population during the fermentation of borde. succession of the dominating lab strains in borde fermentation is shown in table 3. a wider range of sporadic species of lab including e. flavescens was also present at early period of the fermentation. in general, heterofermentative lactobacillus strains and homofermentative pediococcus spp were found to be involved from the beginning to the end of borde fermentation. however, w. confusa, lb. viridescens, lb. brevis and p. pentosaceus and p. pentosaceus subsp. intermedius appeared to be the most important lab population in borde fermentation. discussion the natural fermentation of borde is unpredictable. spontaneous fermentation typically results from the competitive activities of different microorganisms whereby strains best adapted and with the highest growth rate will dominate during particular stages of the process. among the bacteria associated with food fermentation, lab are of predominant importance. the lab are known to be associated with many natural and man-made environments. it was reported elsewhere (damelin et al., 1995) that samples from plant material showed the greatest diversity of lab with lactobacillus strains being predominant in food-related ecosystems. it appeared that enterobacteriaceae pioneered borde fermentation at phase i as shown by their early leading rate of growth that was followed by the succession of lab (figure 1 b1). the initial high ph 6.0 of the dough fermentation at phase i (figure 1 a1) would explain the growth of enterobacteriaceae (figure 1 b1), including enterococcus spp which is indicated by the isolation of ent. flavescens, while the lower initial ph 4.4 at phase ii fermentation inhibited their growth (figure 1 a2). the high number of lab attained after 12 h dough fermentation in phase i was responsible for a marked reduction of ph and increase in ta (figure 1 a1) probably thereby the inhibition of enterobacteriaceae (nout et al., 1989; lorri, 1993). in phase i, dough fermentation for 24 h appeared to be a turning point for an accelerated reduction in number of enterobacteriaceae and stabilizing the maxi-mum numbers of aciduric bacteria that are involved in the fermentation. this would be the maximum aciduric bacterial population attainable in this dough fermentation. the cooking of fermented dough at about 90c would kill vegetative microbial cells in it. otherwise, the cooked cereal ingredients showed no lab and yeast except bacterial spores (abegaz et al., 2002b), which cannot grow in acidic condition. however, the addition of malt flour to unmalted and cooked ingredients at phase ii is the source of both starter microorganisms and endogenous enzymes that initiate the main fermentation of borde. the microflora on the malt would comprise the normal flora of kebede 030 the grains and also contaminants from threshing and equipment associated with storage and malting process (sahle and gashe, 1991; ketema et al., 1998). processing utensils, water and the brewing personnel constitute additional sources of microorganisms that could initiate the fermentation at phase ii. a balance between effects of low ph on selection of aciduric microorganisms and on activity of malt enzymes at phase ii would be vital for the subsequent stages of borde fermentation. the addition of pancake-like flat bread from 24 h fermented maize dough was responsible for a low initial ph at phase ii and also partially affected the ph at the start of phase iii and iv. in fact, the bulk of unmalted mixed cereal ingredients (56%, on weight basis) and high microbial inoculum that had been built up in phase ii (tinsis) were blended at the start of phase iii. thus, the fermentation at phase ii and phase iii can be considered as starter culture building up and bulk fermentation phases, respectively. the high microbial load observed in borde is in agreement with zimbabwean mangisi (zvauya et al., 1997). since the number of fermenting microorganisms at the start of phase iv is very high, the small amount of malt added in phase iv is mainly to hydrolyse the newly added unmal-ted adjuncts and sweeten the borde. a few hours fermen-tation at phase iv meant for attainment of optimal sens-ory attributes of a sweet-sour borde. finally, since further holding renders borde too sour, it should be consumed as a sweet-sour product while actively fermenting. despite unhygienic wetmilling and wet-sieving process (abegaz et al., 2002a, b), the low ph of borde would hopefully make it safe for consumption. the acidic fermentation and lab metabolites are also responsible for inactivation of enterobacteriaceae including toxin-producing and foodborne infectious pathogens (nout et al., 1989; lorri, 1993, dessie et al., 1997; byaruhanga et al., 1999; kunene et al., 1999; ana et al., 2006). although the possible sources of chance inocula for fermentation of dough at phase i from maize flour and main fermentation at phase ii from malt flour appeared to be different due to the effects of cooking and malting processes, the dominating lab were similar in all the four phases of borde fermentation. the lab were in the minority at start of phase ii fermentation with the addition of malt flour but they were the dominating flora as the fermentation progressed. this indicates the significant role of lab in borde fermentation. the growth of both lab and yeast populations during fermentation of borde would be indicative for their involvement. however, yeasts are known to facilitate alcoholic fermentation, while borde is supposed to be non-alcoholic beverage. hence the present work focused on characterization and identification of lab aimed at selection of potential starter culture for controlled fermentation of borde. the group of lab includes several genera of bacteria that differ considerably in morphological, physiological and functional properties. the api lab program distinguishes between species on the basis of pattern-matching principle and assesses the entire group of lab as a whole (figure 2). the differentiation efficiency of commercially available identification test kits such as api 50 chl system is based on testing the organisms’ ability to assimilate or ferment a fixed number of specific substrates. the resulting fermentation patterns of wild-type lab strains in the present work were compared with patterns obtained from recognized test organisms from known source. in a group as heterogeneous as lab, this approach may lead to results with wrong species and even wrong genus particularly for those wildtype strains isolated from peculiar niche. according to api 50 chl system, it was particularly difficult to distinguish between some of the lb. brevis and lb. virides-cens strains isolated from borde. however, this investi-gation is aimed at identification of wild-type lab strains to select suitable starter cultures that are adapted to the ecological niche of borde fermentation. accordingly, the identified w. confusa, lb. brevis, lb. viridescens, p. pentosaceus and p. pentosaceus subsp. intermedius are adapted to the ecological niche of borde fermentation and overcome its production technology hurdles. thus, they could be suitable candidate starter cultures for borde production in the near future. several of the lab isolated from borde have also been isolated from various fermented foods. hounhouigan et al. (1993) isolated w. confusa, lb. brevis, lb. curvatus, p. pentosaceus and p. acidilactici during spontaneous fermentation of mawe from maize. uchimura et al. (1991a) reported that the principal coccus in indonesian ragi was p. pentosaceus, which is in agreement with the present report. hancioglu and karapinar (1997) and getcheva et al. (2000) isolated w. confusa, lb. brevis and lb. sanfrancisco from boza, a thick beverage related to borde (the investigator’s personal observation). paludan-muller et al. (1999) isolated w. confusa, lb. brevis, p. pentosaceus and lb. curvatus during fermentation of som-fak prepared from minced fish fillet, ground boiled rice and garlic. many other investigators also isolated w. confusa, lb. brevis, lb. viridescens and pediococcus spp. (nout, 1980; sakai and caldo, 1985b; samelis et al., 1994; johansson et al., 1995; olasupo et al., 1997; sanchez et al., 2000; corsetti et al., 2001; leisner et al., 2001; gobbetti et al., 2005), p. pentosaceus and p. acidilactici (halem et al., 1993), lb. collinoides (leisner et al., 2001) and lb. delbrueckii subsp. delbrueckii (olasupo et al., 1997; corsetti et al., 2001) as part of the lab population in different fermented foods. ent. flavescens has been isolated from human clinical specimens and its habitat and pathogenic significance are unknown (wood and holzapfel, 1995). however, the ent. flavescens was not detected after 18 h of fermentation at phase i, even if, cooking of the dough could safeguard from further contamination of the product. the presence of ent. flavenscens strains that fermented starch at phase i may 031 afr. j. food sci. res. also be an advantage for hydrolysis of starch and thus create a suitable ecosystem for the proliferation of microbes, like that of the malt enzymes at phase ii. it was reported elsewhere (nout, 1980; johansson et al., 1995; olasupo et al., 1997; paludan-muller et al., 1999; getcheva et al., 2000; sanchez et al., 2000; leisner et al., 2001) that lb. plantarum in fermented plant materials was dominating the lab flora. however, there was no lb. plantarium observed during the acidic fermentation of borde in the present work. among the dominating strains, lb. viridescens may not be a potential starter culture for acidic fermentation of borde due to its weak growth in ph 3.9. moreover, there was no accessible publication on use of lb. viridescens as starter culture in fermentation of plant material. if any of the strains appeared to be important starter culture for borde fermentation, neces-sary investigation would be carried out for further details. otherwise, the evaluation of w. confusa, lb. brevis and p. pentosaceus as starter culture for borde fermentation is underway. in conclusion, the results of this study indicated an association of lab, composed of heterofermentative lactobacillus strains and homofermentative pediococcus spp, involved from the beginning to the end of borde fermentation. the least initial counts, at phase i and ii fermentation, and the highest final counts of lab indicate their important role in borde fermentation. a wider diversity of lab strains (cluster i and iii), both rods and cocci, were present at the early period of maize dough fermentation at phase i than any other phases of borde fermentation. the number of species reduced as the fermentation progressed and finally, w. confusa, lb. brevis, lb. viridescens and p. pentosaceous spp were the most significance species. despite these species of lab are adapted to the substrates and overcome the possible physiological, technological and biochemical hurdles during the four phases of borde fermentation, it will also be important to assess their performance on the actual ingredients of borde under controlled fermentation and then select for the best starter culture. this approach would be a paramount strategy (among others) to develop starter culture, to produce microbiologically stable and predictable end product aimed at upgrading and commercial production of borde. acknowledgements the author would like to thank the norwegian universities committee for development research and education (nufu), for sponsoring this work; 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length research paper nutritional composition of m. senegalense in order to increase the value of this macroalgal resource for economic development abdoulaye c. macky1, baaba g. diouf2,4*, patrick h. ousmane2,3, leopold abdou sembene3 and youssou keita cisse1 1département de biologie végétalelaboratoire de botanique et de biodiversité. faculté des sciences et techniques, université cheikh anta diop de dakar, sénégal. 2laboratoire de electrochimie et des procedes, école supérieure polytechnique, université cheikh anta diop, b.p 5085 dakar fann, sénégal. 3laboratoire des biotoxines et contrôle des aliments, institut de technologie alimentaire (ita) route des pères maristes, b.p 2765 dakar, sénégal. 4centre d’etudes sur la sécurité des aliments et le développement des molécules fonctionnelles (cesam-rescif) université cheikh anta diop de dakar, sénégal. accepted 01 october, 2016 macroalgae are used in diverse global regions. meristotheca senegalense j. feldmann, a macroalga species found in a senegalese bay, was the subject of this study focusing on the chemical and mineral composition. the present study was done in order to evaluate the potential of this macroalgal resource for biomass development and contribution to the economy in senegal. the results of this study showed that m. senegalense j. feldmann was relatively a good source of nutrients including fiber (6.67 ± 0.7%) and protein (6.37 ± 0.8%). mineral analysis also showed significant concentrations of magnesium (216.87 ±12.9 mg/100 g), calcium (81.6 ± 17.5 mg/100 g), iodine (31.16 ± 1.5 mg/kg) and iron (28.13 ± 2.15 mg/100 g). however, zinc and copper 3.31 ± 0.26 and 2.43 ± 0.13 mg/100 g respectively were found at relatively low concentrations. interestingly, the vitamin b12 content was significant with a content of 20 ± 1.0 mg/kg, potentially allowing for the use of the alga as a supplemental. the nutrient concentrations reported for m. senegalense j. feldmann suggests that its cultivation and harvest can be a source of diversification in the activities of fishermen. in terms of food consumption, the results also showed that the red alga used in this study can be added to human diets as supplementation and might also be f key words: meristotheca senegalense, nutrient composition, macroalgae, supplementation, food diversification. introduction there are 218270 known macro and microalgae varieties of which 36238 have been identified at the species level (john, 1994). however only about 665 of the known species of marine macroalgae are used for food or *corresponding author. e-mail:di.baba@gmail.com in ternationa l scholars journa ls african journal of food science research issn: issn 2375-0723 vol. 4 (4), pp. 027-032, november, 2016. available online at www.internationalscholarsjournals.org © international scholars journals baaba et al. 028 figure 1. map of cap-vert peninsula (dakar region) showing ngor bay position (modified from google earth 04/2015). animal feed, and extracted for individual chemical components (lobban and harrison, 1994; pereira, 2016). components from industrial extraction of macroalgae include: hydrocolloids, nutraceuticals, minerals and food color. according to mouritsen (2013) and pereira (2016), the primary mineral components in seaweeds were iodine, calcium, phosphorus, magnesium, iron, sodium, potassium, and chlorine with trace elements such as zinc, copper, manganese, selenium, molybdenum and chromium. the mineral composition varies significantly from one seaweed species to another depending on oceanic residence time, seasonal variations, environmental and physiological conditions, and the processing method of mineralization. meristotheca senegalense has been harvested for approximately three decades (pérez, 1997; mchugh, 2002; john et al., 2004). it was originally isolated in dakar between ngor and ouakam bays and initially characterized by faye et al. (2004) and named m. dakarensis and was subsequently renamed m. senegalense. previous studies on m. senegalense showed the presence of the phycocolloid iota carrageenan (fostier et al., 1992) containing a potentially promising pharmacological glycolipid biomolecule with anti hiv properties previously extracted from cyanobacterium (diop and samb, 2004). this species of algae was previously exported as raw material to japan (pérez, 1997; mchugh, 2002) where m. senegalense (mchugh, 2002), m. papulosa (faye et al., 2005; rao et al., 2007; pereira, 2016) and m. procumbens (rao et al., 2007; pereira, 2016) were used for human consumption (pérez, 1997; rao et al., 2007; mouritsen, 2013; pereira, 2016). unfortunately the mineral composition of m. senegalense was unknown at this time. the exploitation of this marine resource was recently restricted for exportation as a harvested biomass by the senegalese government. however due to malnutrition faced by some segments of the population of senegal and elsewhere, diversification of sources of essential micronutrients appears as one of the solutions next policy strategies and enhancement of certain fruit and vegetables neglected (ayessou et al., 2009, 2011, 2014; gueye et al., 2014). thus, strengthening nutritional balance requires the identification of new food resources. accordingly, the purpose of the present study was to determine the nutritional composition of m. senegalense in order to increase the value of this macroalgal resource for economic development through the local use of the biomass which is still underexploited in senegal. materials and methods plant materials m. senegalense samples were collected in dakar peninsula ngor bay located at n 14° 44’715; w017° 30’ 857 (figure 1). samples were collected early march (the beginning of the cold season) until late june (beginning of the warm season) from crops grown on the mariculture station nets.(figure 2) this time interval corresponded to the period during which the natural fields of the species were harvested for biomass traded by local coastal populations. for biochemical analysis, three batches of m. senegalense samples were targeted. to insure preservation, samples were first naturally dried in shade with adequate ventilation. prior to analysis samples were washed in distilled water and dried at 70°c in an oven according to afnor’s method nf v 03-707 (afnor, 1982) for 48 h before grinding with a mortar pestle. major macronutrient analysis analyses of lipid and protein content were carried out according to the procedure described according to afnor standards (afnor, 1982). samples were dried in an oven at 105°c for two hours, cooled and then weighed to determine moisture content. lipid extraction was performed using a soxhlet extractor with diethyl ether as the solvent (nfv 03-905 standard). nitrogen determination was performed using kjeldhal method (nf 03-050 standard) and the protein content was calculated by using a coefficient factor of 5.7. fiber content was determined in sample through afnor standards v76-101. samples were hydrolysed with acid solution and then with basic solutions. then it’s mineralizing during 3 h incineration at 550°c. ascorbic acid ascorbic acid content of the samples was determined according to the method of dhuique et al. (2007). ten grams of m. senegalense was homogenized in ice-cold metaphosphoric acid solution (4% in distilled water) to extract the ascorbic acid. the mixture was centrifuged at 1000 x g for 15 min with a second extraction repeated after removing supernatant. supernatants were pooled before determining the total ascorbic acid content. the analysis was performed using a thermo scientific hplc 1000 scm (thermo fisher scientific france, illkirch) with an rp 18 licrospher 100 column (4.6 × 250 mm; 5 m, merck, darmstadt, germany). the separation used a mobile phase consisting of a 0.01% isocratic sulfuric acid solution (sigma-aldrich, saint-quentin fallavier, france) with an injection volume of 10 µl. quantification was performed using a uv 3000 spectra (254 nm). quantification and identification of the peaks were done using an external calibration curve using a concentration range from 10 to 200 mg/l of l-ascorbic acid solution (a5960 bioxtra, ≥99.0%, crystalline (sigma) standards (200 to 10 mg.l-1) with a 0.01 % limit of detection. 029 afr. j. food sci. mineral determination samples were mineralized by incineration at 500°c and desiccated by adding fluorhydric acid at 40% (sigma-aldrich, saint-quentin fallavier, france) and then evaporated to dryness. sodium, potassium, calcium, magnesium, phosphorus, copper, zinc and iron were quantified using an icp-aes (inductively coupled plasma atomic emission spectrometry) varian vista spectrophotometer containing a charge coupled device for detection (agilent france, massy). mineral standards used in this assay (ca, k, mg, fe, zn, cu) were obtained from fisons scientific equipment (loughborough, england). vitamin b12 extraction of vitamin b12 was carried out in acetate buffer (ph 4.0) containing pepsine [50 mg.ml-1 (4500 u.ml-1)]. partial purification was achieved using amberlite xa d-2 followed by elution with 80% (v/v) methanol. activated charcoal facilitated removal of impurities in the extract and in the further purification of vitamin b12. the purified fraction containing methyl cobalamin was analyzed by hplc using a silica c18 column with water/methanol 50/50 (ph 4.5) as a mobile phase, and a uv detector. iodine iodine was assayed by “aquanal” (bordeaux, france) using the standard method en 15111/icp-ms with a varian spectrometer icp-vista coupled to a mass spectrometer. quality control and statistical analysis hplc limits of detection (lod) of vitamin c, β-carotene, and vitamin b12 were determined using a 5 point external calibration systematically performed before each series of analyses. the spectrophotometer limits of detection (lod) used for mineral determination was a 5 point calibration and analysis of control samples whose mineral contents were known. all analyses were carried out in duplicate and the data were analyzed using sas software (version 8.1 2000; sas institute, inc., cary, nc, usa). vitamin a to determine vitamin a content samples were extracted with a mixture of ethanol/hexane (4:3 v/v), with caco3 and 0.1% butylhydroxytoluene (added as antioxidant). m. senegalense (0.5 g) samples were mixed with 20 ml of the extraction buffer followed by centrifugation at 15000 x g for 15 min at 4°c. a second extraction was repeated using the same conditions after collecting the supernatant. all supernatants were pooled and evaporated to dryness under nitrogen. after evaporation to dryness, samples were solubilized in 1 ml of a dichloromethane/tert-butyl methyl ether/methanol mixture. separations were carried out by gradient elution using an agilent hplc 1100 with water (solution a)/methanol (solution b)/tert-butyl methyl ether (solution c) as follows: initial conditions 40 %a/60 % b; 0-5 min, 20% a/80% b; 510 min, 4%a/81% b/15% c; 10-60 min, 4% a/11% b/85% c; 60-71 min, 100% b 71-72 min, and back to the initial conditions for reequilibration. samples were quantified at 350, 400, 450 and 470 nm wavelengths. comparisons were carried out using authentic standards as reported by dhuique et al. (2007) with a limit of detection (lod) of 0.035 mg/100 g and a limit of quantification (loq) of 0.119 mg/100 g per sample. results and discussion the results from table 1 show that the algae m. senegalense is very low on lipid content (0.08 mg /100 g) and rich in carbohydrates especially with a dry weight fiber content of 6.67%. the protein content was 6.38% and the minerals were relatively abundant most notably magnesium (216.87 mg/100 g), calcium (81.6 mg/100 g) and iron (28.13 mg/100 g). moreover, m. senegalense also contained a significant concentration of iodine (31.16 mg/kg) zinc (3.31 mg/100g) and copper (2.43 mg/100g). vitamin b12, an essential hydrosoluble vitamin was found at a concentration of 20 mg/kg. however, the vitamin a and vitamin c content were relatively low 0.7 mg/kg of sample and < 10 mg/kg respectively. in this study, sample averages were obtained during a complete season and showed large variances for calcium (17.5 mg/kg) and magnesium (12.9 mg/kg) due to the minima and maxima values (64 and 99 for ca; 203 and 228.5 for mg). these variations could be related to baaba et al. 030 table 1. average chemical composition of meristotheca senegalense components average (n=3) components average (n=3) lipid (mg/100 g) 0.08 ± 0.1 zn (mg/100g) 3.31 ± 0.26 protein (%) 6.38 ± 0.8 cu (mg/100g) 2.43 ± 0.13 fibers (%) 6.67 ± 0.7 iodine (mg/kg) 31.16 ± 1.5 ca (mg/100 g) 81.6 ± 17.5 vitamin b12 (mg/kg) 20 ± 1.0 k (mg/100 g) 9.25 ± 2.58 vitamin c (mg/kg) ˂ 10 mg (mg/100 g) 216.87 ±12.9 vitamin a (mg/kg) 0.7± 0.06 fe (mg/100 g) 28.13 ± 2.15 table 2. comparative study between m. senegalense and other macroalgae species. species* protein lipid (%) fibers ca fe zn cu vit. b12 (%) (%) (mg/100 g) (mg/100 g) (mg/100 g) (mg/100 ng) (mg/kg) m. senegalense1 5.6 -7.2 2-8.10-5 6 -7.5 64-99 25.729.8 3.13.6 2.3-2.5 20 ulva lactuca2 10-25 0.6-1.6 29-55 840 66 600 laminaria digitata2,3 8-15 1 36-37 1005 3.2-9 1.77 < 0.5 0.05 undaria pinnatifida2 12-23 1.0-4.5 16-51 680-1380 1.54-30 0.94 0.18 0.36 chondrus crispus2 11-21 1-3 10-34 420-1120 1.54-30 7.14 < 0.5 60400 palmaria palmata2 8-35 0.7-3 29-46 560-1200 50 2.86 0.37 0.9 porphyra tenera2 28-47 0.7-1.3 12-35 390 10-11 2-3 < 0.63 p. umbilicalis2 29-39 0.3 29-35 330 23 2.9 p. yezoensi2;3 31-44 2.1 30-59 440 13 10 1.47 0.52 *results expressed according to the dry wet; 1this article; 2pereira (2011). 3pèrez (1997). seasonal aspects of the samples which are linked to the physiological state of m. senegalense during sample collection was in april, may (cold season) and june (beginning of the warm season). m. senegalense was compared to other edible seaweeds (table 2). the results show that m. senegalense contains less fiber and calcium compared to other species; whereas, the vitamin b12 contents of different edible seaweeds were highly variable. when considering seafood as an important source of iodine both red and brown algae are generally richer in iodine than green algae (fleurence and guéant, 1999). according to teas et al. (2004) rhodophyceae contained 10 to 100 mg/kg of iodine similar to the content of palmaria palmata, while species like porphyra umbilicalis and undaria pinnatifida had iodine contents of 17.3 and 22 to 30 mg/kg respectively. the results of this study show that m. senegalense is a good source of iodine with an higher average concentration (31.16 mg/kg) than the species mentioned above. in the past years important actions were taken by world organizations, such as world health organization, to fight against iodine deficiency. the recommended concentration of iodine in iodized salt was fixed at 30 mg/kg. therefore, m. senegalense could be a valuable dietary source of iodine. the specie used in this study could be used to fill the gap of iodine concentration in many foods which who and other organizations have suggested as an alternative source of iodine. in this context, m. senegalense should be promoted as a new local food. in fact edible seaweeds present many advantages such us the quality of their fatty acids content (khotimchenko and levchenko, 1997), traces metals such us fe, zn, cu (robledo and pelegrin, 1997) or iodine content (teas et al., 2004; zava and zava, 2011). in addition, some reports have shown quality protein content in some species including chlorophyta and rhodophyta (galland-irmouli et al., 1999; fleurence and guéant, 1999). although m. senegalense is considered as a rich source of iodine, the fda and other world dietary guidance programs warn that consumption of excess iodine rich seaweeds could be dangerous for health (brownlee et al., 2012; leung and braverman, 2014). an example of the potential health risk was reported by brownlee et al. (2012), when seaweed was used in a study composed of lactating mothers in japan and korea. they found that the high concentrations of iodine in seaweed were transmissible from mother to infant through breast milk which could result in neonatal iodine toxicity and subsequent hypothyroidism or hyperthyroidism. however, m. senegalense could be very valuable in new foods which are rich in iodione. 031 afr. j. food sci. figure 2. plant of meristotheca senegalense collected during may 2010 at ngor bay. scale bar = 5 cm. currently new types of foods are been developed using m. senegalense as a source of some important nutrients such calcium, magnesium and iron as well as iodine. the increase of the local consumption of this endemic seaweed in spite of exportation will also induce development of local markets. conclusion like other seaweed, m. senegalense could be an important source of nutrients, iodine, minerals and vitamin b12. these results also show that these red algae can be added to 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japanese iodine intake based on seaweed consumption in japan: a literature based analysis. thyroid res. 4:1-14. african journal of food science research vol. 1 (1), pp. 010-014, september, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper effects of thermal treatments and germination on physico-chemical properties of corn flour prakash bhattacharyya, uma ghosh, hiranmay gangopadhyay, utpal raychaudhuri * department of food technology and biochemical engineering, jadavpur university, kolkata 700 032, india. accepted 17 july, 2013 certain physicochemical properties including viscoelasticity, crystallinity and maltose content of corn depends on the gelatinization of starch under different treatments. three different treatments were performed; boiling in water, steam heating, and germination. the effects of gelatinization on viscoelastic property of corn starch were measured and the morphological changes of corn after different treatments were determined by scanning electron microscope (sem). various grooves and fissures on the exterior surface of the granules were noticed in the treated sample compared to control. the crystallinity of corn after different treatments was determined by x-ray diffraction studies (xrd). the control showed higher crystallinity than that of thermal treated samples. key words: crystallinity, viscoelastic property, instron texture analyzer, scanning electron microscope, and x-ray diffraction. introduction starch in abundant quantity is available in india. starchy foods are staple foods for the growing millions of indians. starch, is an extraordinarily versatile polymer used in a wide array of products from paper to prepared foods (mua and jackson, 1995). in addition, starch is one of the most economic carbohydrates in nature (hye young et al., 2001), biosynthesized in granular form by plants. whether modified or in its native state starches are used for thickening, stabilizing and gelling abilities in a wide variety of food and nonfood products (jennings et al., 2002). again in response to concerns about health implications of excess dietary fat and calories, a variety of fat substitutes were developed in recent years (duxbury, 1990; duxbury, 1990; chun et al., 1997). modified food starches partially replace fat to reduce calories and provide desirable textural qualities, mouth feeling appeal, etc. rapid advances continue in acquisition of new knowledge of starch and a vigorous expansion in the use of starch is proceeding in food applications. some remarkably significant and interesting practical developments have occurred. consequently it is important to explore *corresponding author. e-mail: utpal31@hotmail.com, urc@vsnl.in. again the accumulated knowledge of starch and its practical use (grist, 1975). availability makes dent corn the grain of choice for food starch manufacture, thus allowing corn starch to be an inexpensive food starch available in india. corn flour is extensively used to produce products such as corn tortillas, corn chips, and tortilla chips, etc (sahai et al., 2001; serna et al., 1990). it is generally recognized that physical characteristics of corn are important factors influencing product characteristics (sahai et al., 2001). the major component of the corn kernel is starch, and according to trejo-gonzales et al. (1982), starch is responsible for the mechanical properties of the dough (cory and bruce, 1997) and consequently plays an important role as a determinant of the food product quality. functional properties of starch have considerable effects on the quality of starch-based products. starch gelatinization is most important in a number of food modifications inclusive cooking, baking and extruding starch-based foods. the overall starch gelatinization process is generally supposed to be obeying a first order kinetics and depends on temperature. nonetheless the phenomenon is very complex and implies significant changes in physical, chemical and nutritional properties of starch as well as water and heat diffusivity, viscosity, rheological behavior, swelling and deformation of the prakash et al. 010 original shape of starchy products and susceptibility to enzymatic digestion. actually the crystalline order of starch granules is lost during gelatinization (bhattacharyya et al., 2004). cereal grains synthesize -amylase during germin-ation, which enhances the starch degradation process. this enzyme enhances the conversion of insoluble gra-nules to soluble starch and dextrin. hence it is important to study the effect of germination on quality of the grains (dona et al., 1991; beck and ziegler, 1989; hill and macgregor, 1988). the present paper has studied the effects of thermal treatments and germination on gelatinization of corn starch, which affect viscoelastic property. the viscoelastic properties of starch-based doughs were measured by instron texture analyser (london, u.k.). starch crystallinity was determined by x-ray diffractometer (xrd). scanning electron microscope (sem) was applied to observe the morphological changes of corn after different treatments. materials and methods materials corn seeds (zea mays l) were procured from local market. 3,5dinitrosalicylate (loba chemie, mumbai), potassium iodide (e. merck india pvt. ltd, mumbai), and iodine (qualigens fine chemicals, mumbai, india) were purchased. different treatments on corn kernel corn seeds were used throughout the experiments. the proportion of corn flour and water was kept constant (1:5, w/v) in each case. corn was treated in boiling water for 5, 10 and 15 min followed by drying at 60ºc in tray drier (international commercial traders, kolkata, india) for 56 h. another portion of corn was steam heated at 15 psi for 10, 20 and 30 min before drying at 60ºc for 5-6 h. beside steam heating another portion of corn was allowed to germinate in dark for 2,3 and 4days respectively at 37 0 cand then dried at 60 0 c for 5-6 h. developments of doughs all the dried samples were milled finely with a blender (bajaj mixer grinder, gm550) for 1 min and used for dough development. the proportion of corn flour and water was kept constant (1:1, weight basis) in each case. doughs were formed by mixing flour and water in a twin z arm mixer for 0.5 min. this produced a (just) cohesive very undeveloped dough that was finally shaped by hand into a 1cm cube. moisture content moisture content of control and treated corn seed powders were measured using standard air oven (model no-06104, s.c.dutta andco, kolkata, india) according to aoac no14.002 (aoac,1965). texture analysis texture analysis (viscoelasticity) of all the doughs were made using instron texture analyser 4301 (london, u.k.) of maximum load 100 n and operating at 25%. the dough was placed onto the flat platform under a stainless steel probe with rounded end. the probe traveled at 15 mm/min until the force detected 10 n and then traveled upward until the gap between dough and probe was 2 mm. finally the probe traveled downward until the force was 10n. the difference between the forces of these two bites was the viscoelasticity of that dough. determination of starch digestion effects of different treatments on corn starch damage were estimated by determining reducing sugar (maltose) content of each sample. reducing sugar content was determined by standard aoac no 14.023 method. enzyme assay alpha-amylase activity was assayed at 40 o c by measuring the reducing sugar released during the reaction, using starch as the substrate, according to somogyi-nelson method (nelson, 1994). the reaction mixture contained 0.5 ml of 1.1% soluble starch in 0.05 m imidazole-hcl buffers, ph 7.0 and 2.5 ml of enzyme solution. after incubating for 1 h at 60 o c the reaction was stopped by heating the reaction mixture in boiling water bath for 10 min. this reaction mixture was diluted with distilled water. 1 ml of dinitrosalicylic acid solution (miller, 1959) was added in each 3 ml of dilutions (samples) and heated in boiling water bath for 5 min. absorbance at 540 nm wavelength was measured (spectrophotometer, u2000, hitachi, japan) after cooling the dns-sample mixture at room temperature (shaw et al., 1995). one unit of alpha-amylase activity is defined as the amount of enzyme that releases 1 m (micromole) of reducing sugar per minute from soluble starch at ph 6.9 and 40 o c. x-ray diffraction patterns the x-ray patterns of the flours were obtained with copper, nickel foil filtered, k radiation using a diffractometer (rigaku, miniflex, japan). the diffractometer was operated at 10 ma and 30 kv. the scanning region of diffraction angle (2) was 4 40 o at a 0.05 o step size with a scanning speed 1 o per min (mcpherson et al., 2000; jane et al., 1997). scanning electron microscope (sem) scanning electron microscope (jeol, jsm5200, tokyo, japan) were taken at an accelerating voltage of 20 km to view the corn flour in three dimensions and to determine the shape and surface features of the granules. corn flour from all the treatments and the control sample were mounted stubs with adhesive tape and sputters coated gold approximately 190 a o thick for 2.5 min at 10 ma before observation with sem. one micrograph was taken for each starch sample at 1000 x magnification. all the images for each sample showed representative results. statistical analysis data were analyzed using single factor anova in excel (micro 011 afr. j. food sci. res. soft office 2000). significance level at p 0.05 was applied to the results to test the significant difference. results and discussion texture analysis figure 1 shows the viscoelasticity of doughs prepared from corn powder. it is evident from the figure that viscoelasticity of doughs prepared from corn under different treatments were influenced by starch damage. it was also found that in the case of boiling and germin-ation, the viscoelasticity of doughs decreased rapidly. in figure 1 two bars showed effect of each treatment and difference between the values of two bars is the elasticity of that sample. the stress values were: 2.5±0.11, 3.75±0.041, 3.27±0.15 n for 5, 10 and 15 min boiling and 4.86±0.33 n for 3-days germination, respectively, than that of steam treatment where the stress values were: 1.02±0.024, 1.25±0.071, 2.5±0.041 n for 10, 20 and 30 min at 15psi. again it was found that the viscoelasticity of doughs made by control corn was the highest, with corresponding stress value of 0. 99±0.061 n. 16 (n ) 14 12 l o a d 10 8 m a x im u m 6 4 2 0 1 2 3 4 5 6 7 8 pretreatments figure 1. texture analysis of doughs. pretreatments: control (p1), boiling in water for 5min (p2), boiling in water for 10 min (p3), boiling in water for 15 min (p4), 10 min team heating at 15psig (p5), 20 min steam heating at 15 psig (p6), 30 min steam heating at 15psig (p7), 3 rd day germination (p8). starch crystallinity the x-ray diffraction patterns of the treated starches showed that crystallinity decreased on thermal treatments (boiling and steam heating) . native corn starch displayed an a-type xray diffraction pattern (figure 5). the corn starch boiled in water for 10 min and steam heated for 10 min at 15 psi was gelatinized as indicated by the absence of crystalline peaks (figure 5). the native corn starch exhibited intensified peaks at 18.15 o and 23.1 o . jane and coworkers (1986) reported that the native double helical structure in starch was transformed into single helices during heating and this transformation occurred in both amylose and amylopectin molecules. the crystalline structure of native corn starch might be partially disrupted figure 2. scanning electron microscopy of corn flour. a: p1, b: p2, c: p3, d: p4. see figure 1 for the meaning of p1 to p4. figure 2. scanning electron microscopy of corn flour. e: p5, f: p6, g: p7, h: 2nd germination (p9). see figure 1 for the meaning of p5 to p7. figure 2. scanning electron microscopy of corn flour. i: p8, j: 4th day germination (p10). see figure 1 for the meaning of p8. during heat treatments (boiling in water and steam heating) and as a result representing vtype diffractions (figure 5). on the other hand the starch crystallinity was not significantly affected during germination. scanning electron micrographs sem pictures illustrate the effects of different treatments on the external structure of the individual starch granule compared to control (figure 2). sem showed that the corn starch granules under various treatments differed in shape from control one. native corn starch was polyhedric (mcpherson and jane, 2000) in shape and had axial diameters of 5-20 µm (figure 2a) as has been reported by jane et al. (1994). various grooves and fissures on the exterior surface of the granules are noticed in the treated samples compared to control. the control (figure 2a) showed compact crystalline starch granules while in case of boiling and steam treatments the starch crystal granules were ruptured in the order figures 2b to 2d and figures 2e to 2g. these results agreed with those of xray diffraction patterns as well as percent (%) maltose content. during germination corn starch granules were converted to small crystal fragments. germination (figure 2i) on 3 rd day exhibits highest fragmentation. prakash et al. 012 013 afr. j. food sci. res. m a lt o s e c o n te n t (% ) series1 series2 series3 series4 series5 series6 series7 series8 series9 series10 40 35 30 25 20 15 10 5 0 pretreatments figure 3. determination of maltose content (%). series1: p1, series2: p2, series3: p3, series4: p4. series5: p5, series6: p6, series7: p7, series8: p8, series9: p9, series10: p10. (µ m o le /m in ) 70 58.967 60 50 40 37.037 a c ti v it y 30 28.752 38.011 20 e n z y m e 9.746 10 0 1 2 3 4 5 germination time (days) figure 4. effect of germination on the activity of corn -amylase. effect of germination time on the activity of enzyme during germination the seeds undergo pronounced metabolic changes and the structural profile of the various organic components are altered. protein and carbohydrates are broken down during germination to simpler units mediated by hydrolytic enzymes (jaya and venkataraman, 1980). studies were made on the effects of germination time on the activity of -amylase produced. it was found (figure 4) that the enzyme produced showed maximum activity (58.967 unit) at 3days germination. this result supported the fact that germinated sample for third day showed highest maltose content (%). starch digestion during different treatments on starch, maltose (reducing sugar) free from corn starch to a great extent. thus the starch damage during different treatments was determined by measuring percentage maltose content of each sample, which is shown in figure 3. it is evident from the figure that in case of boiling in water and germination, percentage (%) maltose content of corn starch increased rapidly than that of steam-heated samples. the values of percentage (%) maltose content were: 11.88±0.071, 15±0.085, 21.75±0.057 percentages (%) for 5, 10, 15 min boiling and 16±0.092, 30.25±0.29, 18.95±0.092% for 2, 3 and 4-days germination respectively while in case of steam heated samples the values of maltose content were: 9.38±0.38, 10±0.37 and 15.11±0.32% for 10, 20 and 30 min at 15 psi. conclusion viscoelasticity (figure 1), percent maltose content (figure 3), surface structure of corn starch (figure 2) and crystallinity (figure 4) were influenced by the action of different pretreatments. treatments in boiling water and germination show great changes in maltose content and viscoelastic property of cornstarch compared to steam heated samples. the crystallinity of cornstarch was prakash et al. 014 figure 5: x-ray diffraction patterns (xrd) of treated samples. 10b:p3, 10s:p5, 3g:p8. decreased during the thermal treatments; boiling in water and steam heating. acknowledgement authors thank to departmental special assistance programme phase iii (ugc, india) and department of biotechnology (govt of india) for their financial support. references aoac methods of analysis (1965). 10 th ed, (aoac washington d c). beck f, ziegler p (1989). biosynthesis and degradation of starch in higher plants. annul rev. plant physiol. plant mol. biol. 40: 95. bhattacharyya p, ghosh u, roychowdhuri u, chattopadhyay p, gangopadhyay h (2004). effects of different treatments on physicochemical properties of rice starch. j. sci. industrial res. 63:826-829. chun j, lim s, takeda y, shoki m (1997). properties of highcrystalline rice amylodextrins prepared in acid –alcohol media as fat replacers. cereal foods world.42: 813-819. cory m. bryant, 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serna-saldirar so, gomez mh, rooney lw (1990). technology, chemistry and nutritional value of alkaline cooked corn products. advances in cereal science and technology, vol 10y pomeranz, ed, am assoc. cereal chem., st paul m n, 243-307. shaw jf, lin fp, chen sc, chen hc (1995). purification and properties of an extra cellular -amylase from thermus sp. bot bull acad sin. 36: 195-200. sahai d, buendia mo, jackson ds (2001). analytical techniques for understanding nixtamalized corn flour: particle size and functionality relationships in a masa flour sample. cereal chem. 78: 14-18. sahaid, mua jp, surjewan i, buendia m, rowe m, jackson ds (2001). alkaline processing (nixtamalization) of white mexican corn hybrids for tortilla production: significance of corn physicochemical characteristics and process conditions. cereal chem. 78: 116-120. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (2), pp. 072-077, february, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of canned fermented oil bean seed slices during ambient storage victor n. enujiugha 1 * and charles t. akanbi 2 1 department of food science and technology, federal university of technology, akure, nigeria. 2 department of food science and technology, obafemi awolowo university, ile-ife, nigeria. accepted 08 july, 2016 african oil bean (pentaclethra macrophylla benth) seed slices were prepared, fermented and canned in three different media using conventional batch retort procedures. the canned products were subjected to sensory evaluation using a 20 man panel of judges, and to six months’ ambient storage with monthly analysis being carried out for total viable counts, peroxide values, free fatty acids and acid values of the sample oils, as well as for hardness and appearance indices of the canned seed slices. the results show that after 6 months storage the total viable counts were 9.3 x 10 3 cfu/g, 1.7 x 10 4 cfu/g, and 6.0 x 10 3 cfu/g for the brine-canned, refined groundnut oil-canned and tomato sauce-canned samples, respectively. peroxide values and free fatty acids had lowest concentrations in the product canned in refined groundnut oil; whereas the same product was least accepted organoleptically in terms of aroma and overall acceptability. the hardness and appearance indices show increased softening and colour darkening with prolonged storage period, although the samples were still considered acceptable after 6 months tropical ambient storage. key words: fermented oil bean seeds, canning, quality attributes, storage. introduction many tropical underutilized crop plants have seeds that are recently attracting worldwide attention because of their high nutrient potentials, especially with regard to solving the prevalent protein-energy malnutrition (pem) that has ravaged populations in the less developed parts of the world. some of these seeds have been exploited as soup bases, such as gbegiri from vigna unguiculata (akanbi, 1992); others are fermented and used as condi-ments and seasonings, such as okpei from prosopis africana (achi, 1992), and ogiri from ricinus communis (odunfa, 1985). the most prominent use of the african oil bean (pentaclethra macrophylla benth) seeds is the fermentation into ugba, a nutritious condiment and local snack, which is consumed by more than 40 million people in west tropical africa (enujiugha et al., 2002). fermentation of african oil bean seed cotyledons plays many roles, including bringing about better nutrient availability and digestibility with significant softening of the cotyledons; formation of hydrolytic non-toxic products with flavour, texture and co *corresponding author. e-mail: venujiugha@yahoo.com. lour that appeal to the consumer; and reduction in antinutritional and toxic factors (enujiugha and akanbi, 2002). however, the fermented ugba has restricted avai-lability and exploitation because of its high rate of deterio-ration, as it is known to lose acceptability beyond 2 weeks ambient storage (enujiugha, 2000). softness increases progressively with storage duration at a very rapid rate, possibly due to the fact that the microbial and plant enzymes still remain active after fermentation and the major fermentative organisms still remain viable. accompanying the increasing softness is the production of offflavours and off-colours, as well as slimy dextri-neous appearance. the african oil bean seed contains more than 52% oil in its cotyledons (enujiugha and ayodele-oni, 2003), with polyunsaturated fatty acids (especially linoleic and oleic acids) constituting more than 88% of the fatty acids in the seed oil (enujiugha, 2003). this means that the seed and its products have potential predisposition to oxidative rancidity with attendant off-flavours, especially during tropical ambient (ca. 28 30 o c) storage. thermal pro-cessing has been applied in the preservation of the fer-mented oil bean seed slices with remarkable improve-ments in nutrient bioavailability, digestibility and function enujiugha and akanbi 073 nality (enujiugha and akanbi, 2005). however, the preservation of the quality and freshness of the fermented product is a critical factor in the choice of processing and preservation technique. since thermal processing is viewed as an achievable technique for the maintenance of the preferred form and quality of ugba while at the same time ensuring the elongation of its shelf life (enujiugha and akanbi, 2002), it would be worthwhile to carry out storage studies on the canned product with a view to evaluating the impacts on some spoilage parameters. as an oilseed, the major indicators of spoilage for the canned product are likely to be those that are useful in rancidity measurements, such as peroxide value and free fatty acids content. also, since the fermented product becomes progressively softer with storage period, the measurement of softness via thumb pressure will show the level of acceptability during the storage period. in the present study, the quality of the canned ugba slices was evaluated with a view to determine the optimum storage duration under tropical ambient conditions. materials and methods preparation of the fermented seed slices freshly harvested african oil bean seeds were obtained from local farmers at nri in eastern nigeria and fermented in the laboratory using a modification of the previously outlined procedure (enujiugha and olagundoye, 2001). in short, parboiling of whole seeds was done for 4 h using heating plate, and after dehulling and slicing (3.5 5.0 x 0.2 0.4 cm), cooking of sliced cotyledons was done for 6 8 h. slicing was done manually with a kitchen knife. the cooked oil bean seed slices were soaked in tap water overnight, washed in three changes of water, and spread out for 4 h natural inoculation on a matted surface, before being fermented at 30 o c for 4 days. canning of the fermented product the fermented oil bean seed slices were canned using 300 x 208 kk lac/lac cans with 300 kk lined ends (carnaud metal box nig. plc., ikeja – lagos, nigeria) in three different media (reflecting the use to which the fermented product is put); namely, tomato sauce, refined groundnut oil and brine solution. can size was not varied since taiwo et al. (1997) observed that variations of can size did not affect the d-value (decimal reduction time) in the thermal processing of cowpeas in tomato sauce. the tomato sauce used in the present study was prepared from the following ingredients: tomato puree (30% solids), 75%; flour, 5%; sugar, 14.5%; salt, 3%; vinegar, 1%; and spices, 1%; to give a total solids content of 14.0  0.5% (taiwo et al., 1997). the brine solution used in the study contained 1.5% sodium chloride and 3% sucrose (w/v) as described by ekanayake and nelson (1990) and lu et al. (1984). the refined groundnut oil used was a commercial brand purchased from a local market at ile-ife, nigeria. portions of the fermented ugba slices (about 100 g each) were placed in the cans and the appropriate medium added as described by aubourg et al. (1990), with a headspace of about 15 mm. the cans were covered with lids, exhausted in a steam chamber for 15 min, and sealed immediately by means of a semi-automatic sealer (dixie model 23-500, md-75, usa). thermal processing was done at 116 o c for 60 min in a still retort and the cans were cooled under running water at 38 o c and stored in a cool place (about 15 o c) for 1 week preceding quality evaluation and sensory analysis. sensory evaluation and storage studies a 20 man panel of judges based on familiarity with ugba flavour, colour and texture was constituted to assess the products on the characteristic sensory parameters of appearance, aroma, taste and texture, as well as overall acceptability. the samples for evaluation included commercially-available fermented ugba (control) and the canned product in tomato sauce, brine solution and refined groundnut oil after the 8 days post-canning stabilization period. table water was provided between samples to cleanse the palate; also the sample presentation order was randomized for each assessor (enujiugha, 2006). the products were assessed on a 7point hedonic scale, with 7 equaling like very much and 1 equaling dislike very much. for the storage studies, the canned products were stored under shelf (tropical ambient) conditions (ca. 28 30 o c) for a period of 6 months, and monthly analysis was carried out for physicochemical properties and total viable counts. the samples taken immediately after the 7 days stabilization period served as control (or day zero samples). microbiological analysis a 10 g sample of the canned product was taken and homogenized in 90 ml of 0.1% peptone water (difco, 0118 – 17) . from the 10 -1 dilution, other decimal dilutions (10 -2 , 10 -3 , 10 -4 , 10 -5 ) were prepared and 0.1 ml of inoculums from each dilution level was used in the evaluation (enujiugha and badejo, 2002). the total viable counts were determined by the pour plate method, using plate count agar (difco, 0479 – 17) as the medium. the plates were incubated at 30 o c for 24 48 h (kilinc and cakli, 2005), and examined for growth. separate colonies were identified using colony counter and the results were expressed as cfu/g. physico-chemical analysis the seed oils of the canned products were extracted using soxhlet apparatus and the rancidity indices (peroxide value, free fatty acids content and acid value) were determined using the methods of pearson (1976). the peroxide values were expressed as milliequivalents of peroxide oxygen per kg of sample (meq/kg) while the free fatty acids were expressed as g oleic acid per 100 g of sample (g/100g). the acid values were expressed as mg naoh per g of sample (mg naoh/g). hardness/softness index was determined using thumb pressure while the appearance/colour index was determined via visual observation. statistical analysis all experiments were carried out in triplicates, and except for the sensory evaluation data, all other data obtained were expressed as means  s.d. data obtained from sensory evaluation were subjected to analysis of variance (anova) as described by snedecor and cochran (1976). differences among means were separated using duncan’s multiple range test (duncan, 1955); significance was accepted at 5% level (p  0.05). results table 1 shows the results of sensory evaluation of the 074 afr. j. food sci. res. table 1. results of sensory evaluation of the ugba samples*. aroma taste colour texture overall acceptability brine 4.90 bc 5.15 a 5.20 b 5.50 a 5.40 a sauce 5.20 ab 5.05 a 4.55 b 5.45 a 5.10 ab g/oil 4.30 c 4.50 a 4.65 b 5.05 a 4.40 b retail** 5.75 a 5.15 a 6.25 a 5.60 a 5.55 a *means in column with the same following letters in superscript are not significantly different (p 0.05). also 7 = like very much; 1 = dislike very much. **commerciallyavailable fermented seed slices. table 2. total viable counts of the canned samples during ambient storage (cfu/g)*. storage total viable counts (cfu/g) duration (months) brine-canned g/oil-canned t/sauce-canned 0 0 0 0 1 1.3 x 10 2 2.9 x 10 2 2.2 x 10 2 2 1.8 x 10 2 3.0 x 10 3 2.5 x 10 2 3 2.0 x 10 2 3.2 x 10 3 8.1 x 10 2 4 3.5 x 10 3 5.4 x 10 3 2.0 x 10 3 5 4.0 x 10 3 9.5 x 10 3 3.7 x 10 3 6 9.2 x 10 3 1.7 x 10 4 6.0 x 10 3 *weighted means from replicated experiments (n = 3; p 0.05) canned ugba samples compared with the commerciallyavailable fermented ugba. the canned ugba in brine was comparable to the commercially-available ugba in terms of overall acceptability. the ugba samples canned in brine and sauce were found not to be significantly differ rent (p  0.05) from the commercial sample in terms of flavour (aroma and taste) and texture. most of the panelists remarked negatively on the darker brown colours of the canned samples compared to the comercial ugba. on a 7point hedonic scale, with 7 equaling like very much and 1 equaling dislike very much, the best rated sample in terms of aroma was the commercial ugba sample, closely followed by the fermented product canned in tomato sauce. however, in terms of colour and stickiness, the brinecanned sample was more preferred to the sauce-canned ugba. the product canned in refined groundnut oil was the least accepted by the panelists in terms of aroma and overall acceptability. table 2 gives the results of the total viable counts in the canned ugba samples during the six months’ ambient storage. at the end of the storage period the total bacterial counts were 9.2 x 10 3 cfu/g, 1.7 x 10 4 cfu/g and 6.0 x 10 3 cfu/g for the canned ugba samples in brine, refined groundnut oil and tomato sauce, respectively. the higher acidity of the tomato sauce and the higher salt concentration of the brine solution may have affected the luxuriant growth of the microorganisms in the samples. there were gradual increases in bacterial density in all the examined products, although the increases were not very significant (p 0.05) within the first three months of storage. the rancidity indices for the canned products (being oilseed-based) comprise peroxide and acid values as well as the free fatty acids content, as these are reflective of oxidative rancidity, total acidity and hydrolytic rancidity, respectively. the peroxide values of the seed oils of the canned ugba samples during 6 months of ambient storage are presented in table 3. at the end of the storage period, the peroxide values for the samples were 11.63 meq/kg for brine-canned sample, 9.54 meq/kg for the vegetable oil-canned sample, and 10.02 meq/kg for the tomato sauce-canned sample. the peroxide value of the seed oil of brine-canned samples after 5 months storage and that of the tomato sauce-canned samples after 6 months storage were slightly higher than the value (10 meq/kg) recommended for fresh oils (pearson, 1976). table 4 shows the free fatty acids (ffas) contents of the samples during the 6 months storage period. the free fatty acids increased from 0.16 ± 0.05 g to 3.12 ± 0.10 g in the brine-canned samples; from 0.12 ± 0.01 g to 2.54 ± 0.10 g in the groundnut oil-canned samples; and from 0.13 ± 0.02 g to 3.98 ± 0.05 g in the tomato saucecanned samples within the 6 months storage period. onyeike and acheru (2002) obtained a value of 0.37 ± 0.10 g/100g for the raw oil bean seed cotyledons. table 5 gives the acid values of the extracted oils which is an indication of the total acidity as contributed by the fatty acids in the sample. the values obtained after 1 month ambient storage agree with the value (1.57 mg/g) obtained for fermented oil bean seeds by okafor et al enujiugha and akanbi 075 table 3. peroxide values of samples during storage. storage peroxide value (meq/kg)* duration (months) brine-canned g/oil-canned t/sauce-canned 0 6.21 ± 0.40 6.30 ± 0.07 6.05 ± 0.12 1 7.15 ± 0.09 7.08 ± 0.51 7.00 ± 0.43 2 7.82 ± 0.20 7.20 ± 0.04 7.64 ± 0.22 3 7.97 ± 0.05 7.32 ± 0.06 7.90 ± 0.10 4 9.23 ± 0.57 8.02 ± 0.21 8.52 ± 0.04 5 10.04 ± 0.12 9.25 ± 0.20 9.07 ± 0.26 6 11.63 ± 0.80 9.54 ± 0.03 10.02 ± 0.70 *figures represent means of triplicate determinations (mean ± s.d.) at 5% level. table 4. free fatty acids (ffas) contents of the products-oils during storage. storage free fatty acids content (g oleic acid)* duration(months) brine-canned g/oil-canned t/sauce-canned 0 0.16 ± 0.05 0.12 ± 0.01 0.13 ± 0.02 1 0.92 ± 0.02 0.70 ± 0.05 0.65 ± 0.05 2 1.36 ± 0.05 1.20 ± 0.02 1.50 ± 0.10 3 2.01 ± 0.06 1.53 ± 0.10 2.48 ± 0.12 4 2.46 ± 0.10 1.63 ± 0.04 3.57 ± 0.03 5 2.97 ± 0.02 2.01 ± 0.07 3.70 ± 0.10 6 3.12 ± 0.10 2.54 ± 0.10 3.98 ± 0.05 *figures represent means of triplicate determinations (mean ± s.d.) at 5% level. table 5. acid values of oil samples from the canned products during storage. storage acid values (mg naoh/g oil)* duration(months) brine-canned g/oil-canned t/sauce-canned 0 0.30 ± 0.12 0.27 ± 0.07 0.22 ± 0.03 1 1.90 ± 0.06 1.43 ± 0.23 1.40 ± 0.09 2 2.73 ± 0.10 2.30 ± 0.05 2.98 ± 0.10 3 4.50 ± 0.03 3.15 ± 0.07 4.51 ± 0.12 4 4.47 ± 0.15 3.82 ± 0.04 6.98 ± 0.25 5 5.90 ± 0.08 4.55 ± 0.10 7.02 ± 0.13 6 6.43 ± 0.09 5.10 ± 0.07 7.92 ± 0.06 *figures represent means of triplicate determinations (mean ± s.d.) at 5% level. (1991) and enujiugha (2000) . after 6 months of tropical ambient storage, the acid values of the samples increased to 6.43, 5.10 and 7.02 mg/g for the brinecanned, groundnut oil-canned and tomato sauce-canned ugba, respectively. there were general gradual increases in acid values of the samples during the period of storage the results of the hardness (thumb pressure) and appearance (visual observation) indices of the canned oil bean samples during the 6 months storage period are presented in table 6. the hard oil bean slices gradually became soft and melting as the storage period prolonged. the groundnut oil was able to preserve to some extent the hardness of the canned seed slices more than both the brine solution and the tomato sauce. however, in all the cases, the texture changed with increasing storage duration. the canned oil bean slices were still acceptable after 5 months for the groundnut oil-canned product; after 4 months for the brine-canned product; and after 3 months for the tomato sauce-canned product. the results of the appearance index show that the desirable light brown colour of ugba was lost after 2 months storage in both brine-canned and tomato sauce-canned samples, and after 4 months in the groundnut oil-canned samples. the product became darker with increasing 076 afr. j. food sci. res. table 6. hardness and appearance indices of the canned products during storage. storage hardness index appearance index duration (months) brine g/oil t/sauce brine g/oil t/sauce 0 hard hard hard light brown light brown light brown 1 slightly hard hard slightly hard light brown light brown light brown 2 slightly soft slightly soft soft brown light brown brown 3 soft soft soft dark brown light brown brown 4 soft soft very soft dark brown brown dark brown 5 very soft soft very soft dark brown brown dark brown 6 melting very soft melting black grayish dark brown dark brown storage period. in the brine-canned sample the colour turned grayish black after 6 months ambient storage. discussion sensory evaluation has shown that fermented seed slices of african oil bean canned in brine and tomato sauce using conventional thermal processing procedures had fairly comparable consumer acceptability to retail samples of fermented ugba. product colour and appearance seemed to be the major problem in consumer acceptance of the sensory quality of the canned ugba. it has been observed that browning during storage and processing is a significant problem in the food industry and is believed to be one of the main causes of quality loss during postharvest handling of legumes (nagai and suzuki, 2003). the acceptable colour of ugba is light brown; darker brown colours are undesirable. the colour changes are believed to be the result of both enzymatic and nonenzymatic browning. polyphenol oxidase (a major enzyme contained in oil bean seed cotyledons) catalyzes the oxidation of phenolic substances to quinines, which spontaneously polymerize to form a brown pigment (enujiugha and akanbi, 2005a). in the present study, groundnut oil as a medium seemed to prevent the undesirable browning activities up to 5 months of storage. the total viable counts of the canned product in brine and sauce were still within safe levels even after 3 months of ambient storage, which is indicative of their edibility and stability. for the product canned in groundnut oil, the microbial load rose sharply after 1 month of storage, revealing its lower safety ratio. the increasing microbial load with longer storage periods could be attributed to the product’s low acidity level. ugba is known to be a low acid food (enujiugha and akanbi, 2002; 2005), and such foods, according to garbutt (1997), may need the addition of the antibiotic nisin to effectively prevent growth of microorganisms, especially during prolonged storage. however, the results of the present study show that products could safely be consumed after 6 months of tropical ambient storage. this is premised on the fact that the conventional thermal processing schedule applied was enough to destroy all spores of pathogenic organisms. the lower peroxide value recorded for the oil-canned product compared to the other samples is an indication of its relative stability to oxidative rancidity. peroxide value correlates to some extent with the off-flavour caused by aldehydes and other oxidative rancidity products. a rancid taste often begins to be noticeable when the peroxide value is between 20 and 40 meq/kg. the results of the present study indicate that the canned products could still be consumed after 6 months tropical ambient (ca. 28 o c) storage. free fatty acids (ffas) were generally low in the samples throughout the storage period. free fatty acids content is an indicator of the level of hydrolytic rancidity of an oil, and this is usually enhanced by the seed lipases in the presence of moisture. already a previous study (enujiugha et al., 2004) has demonstrated an active lipase in dormant seeds of the african oil bean. the results of the present study show that the canned ugba slices in tomato sauce is highly prone to hydrolytic rancidity beyond 3 months of tropical ambient storage compared to those canned in the other media. this could be attributed to more hydrophilic molecules in solution. nacl exerts a more drying effect so that the brine-canned samples are less prone to hydrolysis. acidity increased with storage, indicating continued hydrolysis of the triglycerides and other macromolecules in the seed oil. the high margin of increase recorded in the tomato saucecanned sample after 4 months of storage compared to those canned in the other media could be attributed to increased degradation of the macromolecules and subsequent accumulation of fatty acids in the oil, and other oilbased degradation products. the canned products were progressively getting softer with longer storage periods. rodrigo et al. (1992) and mallidis and katsaboxakis (2002) observed that thermal processing of foods to the level necessary to achieve commercial sterility causes considerable softening of the tissues. the results of the present study agree with this observation. the softening could also be attributed to internal enzyme hydrolysis. enujiugha et al. (2002) suggested that the generally reported softening of the seed slices could be a consequence of enzyme hydrolysis of enujiugha and akanbi 077 macromolecules such as polysaccharides, peptides and glycerides. conclusion fermented african oil bean seed slices (ugba) have been canned in three different media and stored for 6 months under ambient conditions, with remarkable results. the results of total viable counts (tvc) and peroxide values show clearly that the products kept longer in refined groundnut oil, followed by tomato sauce, and finally brine solution. the hardness and appearance indices show increased softening and colour darkening with prolonged storage period. the free fatty acids and acid values were lowest in the product canned in refined groundnut oil. the products were still acceptable after the storage period. references achi ok (1992). microorganisms associated with fermentation of prosopis africana seeds for production of okpiye. plant fds. hum. nutr. 42: 279-304. akanbi ct (1992). processing, physical and sensory characteristics of drum dried cowpea bean (gbegiri) soup. niger. food j. 10: 44-50. aubourg sp, sotelo cg, gallardo jm (1990). changes in flesh lipids and fill oils of albarcore (thunnus alalunga) during canning and storage. j. agric. food chem. 38(3): 809-812. duncan db (1955). multiple range and multiple f tests. biometrics. 11: 1-42. ekanayake a, nelson pe (1990). effect of thermal processing on lima bean vitamin b-6 availability. j. food sci. 55(1): 154-157. enujiugha vn (2000). development of a new food paste from pentaclethra species. appl. trop. agric. 5(2): 89-94 enujiugha vn (2003). nutrient changes during the fermentation of african oil bean (pentaclethra macrophylla benth) seeds. pak. j. nutr. 2(5): 320-323. enujiugha vn (2006). supplementation of ogi, a maize-based infant weaning food, with african oil bean seed (pentaclethra macrophylla benth). int. j. postharvest technol. innovation. 1(2): 202-211. enujiugha vn, akanbi ct (2002). adaptable techniques for the postharvest processing and preservation of african oil bean seeds. proceedings, regional workshop on promotion of appropriate agroprocessing technologies in west africa, ile-ife, nigeria (23 rd -26 th october, 2002). pp. 164-169. enujiugha vn, akanbi ct (2005). compositional changes in african oil bean (pentaclethra macrophylla benth) seeds during thermal processing. pak. j. nutr. 4(1): 27-31. enujiugha vn, akanbi ct (2005a). colour variations in oil bean seeds as influenced by processing steps in the canning of ugba. proceedings, 29 th annual nifst conference, abakaliki, nigeria. pp. 211-212. enujiugha vn, ayodele-oni o (2003). evaluation of nutrients and some anti-nutrients in lesser-known, underutilized oilseeds. int. j. food sci. technol. 38(5): 525-528. enujiugha vn, amadi co, sanni tm (2002). -amylases in raw and fermented african oil bean seeds (pentaclethra macrophylla benth). euro. food res. technol. 214(6): 497-500. enujiugha vn, olagundoye tv (2001). comparative nutritional characteristics of raw, fermented and roasted african oil bean (pentaclethra macrophylla benth) seeds. riv. ital. sostanze grasse. 78(4): 247-250. enujiugha vn, thani fa, sanni tm, abigor rd (2004). lipase activity in dormant seeds of the african oil bean (pentaclethra macrophylla benth). food chem. 88: 405-410. eriksson ce (1982). lipid oxidation catalysts and inhibitors in raw materials and processed foods. food chem. 9(1,2): 3-7. garbutt j (1997). essentials of food microbiology. arnold, (publishers), euston road, london, uk. pp. 87-94. kilinc b, cakli s (2005). the determination of the shelf-life of pasteurized and non-pasteurized sardine (sardina pilechardus) marinades stored at 4 o c. int. j. food sci. technol. 40: 265-271. lu cl, hsu kh, wilson la (1984). quality attributes and retention of selected b-vitamins of canned faba bean as affected by soaking treatments. j. food sci. 49(4): 1053-1056. mallidis cg, katsaboxakis c (2002). effect of thermal processing on the texture of canned apricots. int. j. food sci. technol. 37: 569-572. nagai t, suzuki n (2003). polyphenol oxidase from bean sprouts (glycine max l.). j. food sci. 68(1): 16-20. odunfa sa (1985). microbiological and toxicological aspects of fermentation of castor oil seeds for ogiri production. j. food sci. 50: 1758-1759. okafor jc, okolonkwo uc, ejiofor man (1991). evaluation of oils from fermented and unfermented seeds of the african oil bean tree pentaclethra macrophylla. int. tree crops j. 7(1,2): 95-101. onyeike en, acheru gn (2002). chemical composition of selected nigerian oilseeds and physicochemical properties of the oil extracts. food chem. 77: 431-437. pearson da (1976). chemical analysis of foods (7 th edition). churchill living stone, edinburgh. pp. 422-511 rodrigo m, garcia mg, ramirez l, martinez a, giner v, safon j (1992). physical texture as an indicator of processing conditions for canning low-acid artichoke hearts. int. j. food sci. technol. 27: 41-48. snedecor gw, cochram wg (1976). statistical methods (6 th edition). the iowa state university press, ames i.a. usa. pp. 258-299. taiwo ka, akanbi ct, ajibola oo (1997). establishing processing conditions for canning cowpea seeds in tomato sauce. int. j. food sci. technol. 32: 313-324. african journal of food science research vol. 2 (3), pp. 059-064, march, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper rheological categorization and quality of large-scale marketable mayonnaise using back extrusion tawongsa w*. and adran abdelmoula biochemical-pharmaceutical technology department, pharmaceutical sciences faculty, thaksin university, phatthalung 93110, thailand. accepted 08 february, 2014 when creating or redesigning food product researchers need to pay special attention to textural and rheological properties. texture is a sensory perception derived from the structure of food and is related to viscosity and elasticity. mayonnaise is semi-solid oil-in-water emulsion with starch in its formulations when fat-reduced. under shear forces it exhibits different types of macroscopic flow behavior, such as shear thinning, yield stress behavior, thixotropy, and viscoelasticity. one problem with the rheological characterization of mayonnaise is that it is particularly difficult to isolate and quantify individual effects. only sensory evaluation is capable to fully detect and describe them. hence the ability to measure and to characterize them accurately and quickly enables the food industry to set standards for quality and to monitor deterioration during storage and distribution. back extrusion is a method used to quantify the behavior of thixotropic fluids. the relatively short time and low cost required to conduct the test makes it a suitable technique for quality control in product development. this paper evaluates back extrusion as method to analyze mayonnaise. it is performed at 5, 25 and 40ºc in a stable micro systems ta-xt2 using twelve commercial mayonnaises. the method has provided qualitative and quantitative characteristics of samples in an accurate and quick way. key words: rheology, back extrusion, texture, mayonnaise. .. introduction texture is a difficult term to define since it means different things to different people. food texture is a sensory perception derived from the structure of food at molecular, microstructure, and macroscopic levels (chen, 2009; weenen et al., 2003; rosenthal, 1999). szczesniak (2002) pointed out the multidimensional nature of texture and its importance to the consumer saying that only a human being can perceive and describe it and the texture testing instruments can detect physical parameters which then must be interpreted in terms of sensory perception, the most important ones being the sense of touch and pressure. when creating or redesigning food product researchers need to pay special attention to textural and rheological properties. the science of rheology has many applications in the field of food acceptability, food processing and handling. a number of food processing operations depend heavily upon rheological properties of the product at an intermediate stage of manufacture because this has a profound effect upon the quality of the finished product. plasticity, pseudoplasticity, and the property of shear thinning are important quality factors in foods and the study of these properties is part of the science of rheology. some foods, like mayonnaise, are either plastic or pseudoplastic in nature. they are required to spread and flow easily under a small force but to hold their shape when not subjected to any external force other than gravity. all of these properties fall within *corresponding author. e-mail: tawongsalx@yahoo.com http://www.internationalscholarsjournals.org/ tawongsa and adran 059 the field of rheology (bourne, 2002). mayonnaise is a mixture of egg, vinegar, oil and spices, with 70 to 80% fat. it is a semi-solid oil-in-water emulsion formed by first mixing the eggs, vinegar and spices and then slowly blending in the oil resulting in closely packed ‘foam’ of oil droplets (depree and savage, 2001; szczesniak, 2002). mayonnaise rheology and texture can depend on the distribution of the oil, the interaction between oil droplets, and the egg yolk emulsifier. alteration of the mayonnaise’s microstructure leads to different rheological properties. this means that the rheology is sensitive to any change in the materials microstructure (stokes and telford, 2004). in emulsions, droplet size is perhaps the most important factor in determining properties like consistency, rheology, shelf life stability, color and taste. in general, emulsions with smaller droplet size result in great stability. current equipment used for emulsion preparation includes colloid mills, sonicators or high pressure homogenizers. the advantage of high pressure homogenizers over other technologies is that more uniform droplets size distribution are obtained since the product is subjected to strong shear and cavitation forces that efficiently decrease the diameter of the original droplets (martin-gonzalez et al., 2009) rheology of mayonnaise can provide valuable information that can be used in quality control of commercial production, storage stability, sensory assessments of consistency, knowledge and design of texture, design of unit operations, and knowledge of the effects of mechanical processing on the structure of the emulsions (gallegos and berjano, 1992). mayonnaise has been shown to be shear thinning, viscoelastic and thixotropic and it has a yield stress. the yield stress is the minimum stress required to enable flow or fracture due to the breakdown or alteration of a materials microstructure. one of the many difficulties encountered during the characterization of such a complex material is to isolate and quantify individual effects (goshawk and binding, 1998). ingredients used in mayonnaises interact with each other either physically or chemically and determine the quality of the final products. the necessity in reducedfat/cholesterol also in mayonnaises has made it necessary to identify appropriate ingredients for formulating these products as the removal of certain ingredients like fat may have a significant influence on the quality and taste of food emulsions (ma and boye, 2013). back extrusion is a method used to quantify the behavior of thixotropic fluids. the relatively short time and low cost required to conduct the test makes it a suitable technique for quality control in product development. yield stresses can be easily determined (brusewitz and yu, 1996). it involves two physical movements: (a) a cylindrical plunger is forced down into the fluid and (b) the fluid flows upward through a concentric annular space. usually, the cylindrical plunger is a solid rod and the fluid is contained in a cylindrical cup. the force required to extrude the fluid in the direction opposite to that of the rod is measured (gujral and sodhi, 2002; paoletti et al., 1995; kaneda and takahashi, 2011). since mayonnaise rheological properties are related to the formulation, process parameters, the storage temperature and commercial mayonnaises present differentiated formulations analysis performed in distinct temperatures demonstrates such differences in its rheology. moreover, as the complexity of texture can only be fully detected and described by sensory evaluation, the ability to measure it accurately and quickly enables the food industry to set standards for quality and to monitor deterioration that occurs during storage and distribution. this paper evaluates back extrusion as a method to analyze mayonnaise rheologically and texturally, discussing the results in different formulations. materials and methods twelve commercial mayonnaises were analyzed. eight of them were traditional type (codes i, ii, iii, iv, v, vi, vii and viii) and four of them were light type (codes iii light, iv light, v light and vii light) as shown in table 1. the exact composition of these commercial mayonnaises is not known. all of them were bought in local supermarket. tests were carried out in a ta-xt2 texturometer (stable micro systems, uk) using back extrusion cell (a/be) using 5 kg load cell. dimensions of the back extrusion probe are: inner cup diameter 55 mm; cup height 70 mm and compression plate diameter 45 mm (figure 1). the data were captured by the program "texture expert exceed” (stable micro systems, uk). the parameters used were tested in advance and are shown in table 2. back extrusion was performed at 5, 25 and 40ºc. mayonnaises were stored at each one of these temperatures in a controlled temperature refrigerator for 24 h prior to analysis. by the time of the analysis the samples were removed from the refrigerator, filled in the cup and tested immediately. at least two independent tests were made in each mayonnaise. results and discussion force as a function of time curves (also force-time curve) obtained using probe back extrusion for commercial mayonnaises at 25°c are in figure 2. since similar overall behavior was seen in all samples only a few curves are shown in order to make the understanding easier. the form of these curves is consistent with a typical force-distance curve obtained from back extrusion test described by bourne (2002), who also divided the curve in sections explaining what is happening in each one of them. the sections displayed in figure 2 are described in table 3 using bourne’s as a base. the reader should be warned that the use of forcetime graphic instead of force-distance is due to the fact that time and distance parameters are numerically equal (25 s and 25 mm, respectively) and the velocity parameter is 1 mm/s (table 2), therefore, the two http://www.jstage.jst.go.jp/search/?typej=on&typep=on&typer=on&d3=au&dp3=isamu+kaneda&ca=999999&alang=all&rev=all&pl=20&search=%8c%9f%8d%f5%8e%c0%8ds http://www.jstage.jst.go.jp/search/?typej=on&typep=on&typer=on&d3=au&dp3=isamu+kaneda&ca=999999&alang=all&rev=all&pl=20&search=%8c%9f%8d%f5%8e%c0%8ds 060 afr. j. food sci.res. table 1. sample codes for commercial mayonnaises used. commercial mayonnaise sample code traditional i, ii, iii, iv, v, vi, vii and viii light (same brand as iii) iii light light (same brand as iv) iv light light (same brand as v) v light light (same brand as vii) vii light (a) (b) figure 1. back extrusion probe set (a) cup and base disassembled and (b) cup and base assembled. letters a to e mean: (a) inner cup, (b) compression plate, (c) extension bar, (d) base and (e) screwnut. table 2. parameters settings used in the stable micro systems ta-xt2. parameter setting test mode and option measure force in compression/return to start pre-test speed 1.0 mm/s test speed 1.0 mm/s post-test speed 1.0 mm/s distance 25 mm time 25 s trigger type auto trigger force 10 g data acquisition rate 200 pps graphics produce curves with exactly same shape. from section a to b the mayonnaise is immediate compressed with an instantaneous elastic deformation, which means its original form can be recovered instantaneously and completely if the force is removed, theoretically speaking, because few foods are perfectly elastic possessing flow properties in addiction to elasticity, most frequently ‘plastic’ and ‘viscoelastic’ (bourne, 2002). in section b the yield value is reached. when expressed in terms of shear stress this represents the ‘yield stress’ the minimum force that must be exceeded before flow begins. this type of flow is often found in foods and mayonnaise is a typical example of them. permanent deformation starts in section b going through section c because now original form cannot be recovered neither instantaneously nor completely if the force is removed because the flow has started through the annular concentric space and the behavior starts to changes from solid to liquid. in section c maximum force is reached and a viscous deformation with a constant force from section c to d starts showing the viscoelatic property of mayonnaise. this section is a newtonian region since force is independent of distance or time (or shear stress), leading to a constant viscosity. tawongsa and adran 061 figure 2. force-time curve obtained using back extrusion method for commercial mayonnaises at 25°c. symbols represent samples (  )viii, (  )vi, (  )iii, (  )iii light and (  )vii light (table 1). sections a to g are explained in table 3. table 3. steps that can be indentified in a typical force-distance/time curve obtained from back extrusion test. s/n step section in figure 2 1 2 3 4 5 6 7 8 9 compression plate begins to plunge into the sample; force rises steeply over a short time of movement compressing the sample; yield value is reached and elastic deformation begins; extrusion starts through the annular concentric space behavior change region “from solid to liquid” maximum force is reached and constant stress region begins with viscous deformation; newtonian region, the shear stress is independent of shear rate, leading to constant viscosity (in point d, the compression plate starts to go up); compression plate reverses direction and starts to move upward and force falls to zero force falls steeply over a short time of movement decompressing the sample yield value is reached and elastic deformation begins a a to b b b to c c c to d d e f 10 extrusion starts through the annular concentric space – behavior change region “from solid g to liquid”; szczesniak (2002) defined ‘hardness’ as the force necessary to attain a given deformation, a resistance of the food to the applied compressive forces and bourne (2002) defined it as the peak force in a force-time curve resulting from a test that compresses a bite-size of food that imitates the action of the jaw, therefore, section c can be termed as ‘hardness’. after section d same sequence of phenomena is observed in a mirror form between sections e and g with negative values of force. these forces in module are smaller than the ones observed in respective a-d sections, which means mayonnaise exhibits shearthinning and thixotropy characteristics. by backing extrusion twice the same sample, one following immediately the other, mayonnaise thixotropy and dependency of shear stress and of shear historical became clear as can be seeing in figure 3. here two different samples from brand v, v1 and v2, are back extruded twice: v11 and v21 by the first time and v12 and v22 by the second time. force and viscosity 062 afr. j. food sci.res. figure 3. force-time curve obtained using back extrusion method for brand v at 25ºc in a repeated extrusion. (  )v11: first sample, first extrusion; (  ) v12: first sample, second extrusion; (  )v21: second sample, first extrusion; (○)v22: second sample, second extrusion. viii v iii iv light iv vi 5ºc v light 25ºc i 40ºc ii vii iii light vii light 0 200 400 600 800 1.000 1.200 hardness (gf) figure 4. hardness obtained from back extrusion test of mayonnaise samples at 5, 25 and 40°c. reduction presented in second extrusion also indicates that compression force affect mayonnaise structure irreversibly by breaking it down. the non-linear rheological properties of commercial mayonnaise were studied by kaneda and takahashi (2011). the stress growth behavior under constant shear flow was observed using a strain control-type rheometer. as the stress-strain curves of commercial mayonnaise showed non-linear behavior, the curves were analyzed with a polynomial model that is a power series of strain. two types of commercial mayonnaise were investigated traditional and low-fat mayonnaise. the second order coefficient of the polynomial model revealed that the structure fracture behavior of these types of mayonnaise was very different. because the fracture behavior of semisolid foodstuffs is considered to be an important mechanical property, the approach may be useful for quantitative estimation of the texture of such foods. mayonnaise rheological properties are related with formulation, process parameters and storage temperature. by performing rheological analysis in distinct temperatures it is possible to demonstrate such differences. figure 4 and table 4 present hardness values (medium) obtained at 5, 25 e 40°c using back tawongsa and adran 063 table 4. hardness obtained from back extrusion test of mayonnaise samples at 5, 25 and 40°c. sample/brand 5ºc 25ºc 40ºc i 407.22 a,b 430.03 a,b 229.99 a ii 396.48 a,b 419.20 a,b 314.88 a,b iii 544.04 b,c 590.25 b,c 427.06 b,c iii light 355.25 a,b 349.65 a,b 320.28 a,b iv 563.70 b,c 556.25 b,c 452.32 c iv light 447.59 a,b,c 561.35 b,c 479.80 c v 640.37 c 698.26 c 510.02 c v light 361.95 a,b 450.27 a,b 312.22 a,b vi 536.07 b,c 515.78 a,b,c 447.51 c vii 272.89 a 362.96 a,b 287.20 a vii light 243.91 a 287.67 a 209.32 a viii 894.75 d 1054.83 d 823.20 d a,b,c same letter shows no significative difference at 5% level at tukey-hsd test. extrusion method for all samples. it is possible to see the variation due to temperature and brand. laverse et al. (2012) studied the microstructure and quantification of fat in four types of mayonnaise. the dynamic-mechanical properties of the mayonnaise samples were also studied using a controlled-strain rotational rheometer. four types of commercially produced mayonnaises, chosen to exhibit variability in terms of visible structure of fat, were used for the experiments. appropriate quantitative three-dimensional parameters describing the fat structure were calculated. with regards to the microstructural and rheological relationship, results from the correlation carried out show that a correlation exists among some microstructural and rheological parameters of the mayonnaise samples. the results showed that x-ray microtomography technique was used by is a suitable technique for the microstructural analysis of fat as it does not only provide an accurate percentage volume of the fat present but can also determines its spatial distribution. marinescu et al. (2011) studied the application of betaglucan prepared from spent brewer's yeast as a fat replacer in mayonnaise. fat was partially substituted by p-glucan at levels of 25, 50 and 75%. the results indicated that all mayonnaises exhibited thixotropic shear thinning behavior under steady shear tests and were rheologically classified as more solid like gels. the mayonnaise with 50% beta-glucan showed higher storage stability than the other samples. it has been demonstrated that spent brewer's yeast beta-glucan can be used as a fat replacer in mayonnaise as well as an emulsion stabilizer. brand viii presents the highest hardness values in the three temperatures. brand vii light presents the smallest hardness in the three temperatures. brand viii is the harder and brand vii light is the softer in terms of texture. a large number of ingredients called “texturizing agents” (also described as texturizers, thickeners, viscosity modifiers, bodying agents, gelling agents and stiffening agents) are available to help bring the texture of foods in to the range preferred by consumers (bourne, 2002). a thickener is a chemical component or mixture of components that can impart long-term emulsion stability by thickening a food system, reducing the movement of the system and by forming viscous, ordered networks in the continuous phase to prevent oil separation (ma and boye, 2013). brand viii has higher hardness due to the starch (texturizing agents) in its formulation, possibly starch or modified starch that can be used combined with non-starch hydrocolloids to obtain products such as reduced-fat mayonnaise with desirable textural properties. textural change can be obtained due to specific interactions between starch and emulsifiers and non-starch hydrocolloids in these products, and the starch and hydrocolloids combination can reduce the long and slimy texture of reduced-fat products compared to when hydrocolloids are used alone (ma and boye, 2013). nikzade et al. (2012) optimized mixture proportions of low cholesterol-low fat mayonnaise contained soy milk as an egg yolk substitute (10%) with different composition of xanthan gum, guar gum and monoand diglycerides emulsifier (0 to 0.36% of each component) were determined by applying the simplex-centroid mixture design textural and rheological properties and sensory characteristics for effective formulation process. results revealed that the best mixture was the formulation contained 6.7% monodiglycerides, 36.7% guar gum and 56.7% xanthan gum. the xanthan gum was the component showing the highest effect on all the properties of mayonnaise samples. in addition, an increase of xanthan gum followed by guar gum caused greater values for the stability, heat stability, consistency coefficient, viscosity, firmness, adhesiveness, adhesive force and overall acceptance and lower value for flow behavior index. except for brands iii light, iv and vi all other brands exhibit a reduction in their hardness from 25 to 5°c. this can be justified by a small break down in emulsion with low temperatures. the bigger stability was showed at 25°c. brand iv do not show difference between its traditional and light form (vi and vi light, respectively) at 1% of significance level (tukey test, lsd). it is the bestselling brand which means it can be used as a standard for hardness comparison since its texture is already well accept by consumers. most brands show biggest hardness values at 25°c which demonstrate more stability since this is a normal storage temperature before selling. market offers mayonnaises with a big variety of textural characteristics. conclusion the back extrusion method to determine rheological properties of mayonnaises showed sensible and very efficient for providing quantitative and qualitative characteristics. however, it is necessary a standardization of samples to get repeatability of measurements. most results showed no differences between traditional and light mayonnaises texture, meaning the standardization and quality of the formulations. references bourne mc (2002) food texture and viscosity: concept and measurement. london: academic press. p. 427. brusewitz gh, yu h (1996) back extrusion for determining properties of mustard slurry. j. food eng. 27:259-265. chen j (2009). food oral processing-a review. food hydrocolloids 23(1):1-25. depree ja, savage gp (2001) physical and flavour stability of mayonnaise. trends. food. sci. technol. 12(5/6):157-163. gallegos c, berjano m (1992) linear viscoelastic behavior of commercial and model mayonnaise. j. rheol. 36(3):465-478. goshawk ja, binding dm. (1998). rheological phenomena occurring during the shearing flow of mayonnaise. j. rheol. 42(6):1537-1553. gujral hs, sodhi ns (2002) back extrusion properties of wheat porridge (dalia). j. food eng. 52:53–56. kaneda i, takahashi s (2011) stress growth behavior of commercial mayonnaise under constant shear flow. food sci. technol. res. 17(4):381-384. 064 afr. j. food sci.res. laverse j, mastromatteo m, frisullo pdell, nobile ma (2012). x-ray microtomography to study the microstructure of mayonnaise. j. food eng. 108:225-231. ma z. boye jj (2013). advances in the design and production of reduced-fat and reduced-cholesterol salad dressing and mayonnaise: a review. food bioprocess technol. 6:648-670. marinescu g, stoicescu a, patrascu l (2011) the preparation of mayonnaise containing spent brewer's yeast beta-glucan as a fat replacer. roman. biotechnol. letters, 16:6017-6025. martin-gonzalez mfs; roach a, harte f (2009) rheological properties of corn oil emulsions stabilized by commercial micellar casein and high pressure homogenization. lebensm.-wiss. technol. 42:307311. nikzade v, tehrani mm, saadatmand-tarzjan, m. (2012) optimization of low-cholesterol-low-fat mayonnaise formulation: effect of using soy milk and some stabilizer by a mixture design approach. food hydrocol. 28:344-352. paoletti f, nardo n, saleh a, quaglia gb (1995) back extrusion test on emulsions stabilized whey protein concentrates. lebensm.-wiss. technol., 28:616-619. rosenthal aj (1999). food texture measurement and perception. gaitherburg: aspen publishers,. p. 311. stokes jr, telford jh. (2004) measuring the yield behaviour of structured fluids. j. non-newtonian fluid mech. 124:137-146. szczesniak as (2002). texture is a sensory property. food qual. prefer. 13:215–225. weenen h, van gemert lj, van doorn jm, dijksterhuis gb, de wijk ra (2003) texture and mouthfeel of semisolids foods: commercial mayonnaises, dressings, custard dressings and warm sauces. j. texture. stud. 34:159-179. http://www.jstage.jst.go.jp/search/?typej=on&typep=on&typer=on&d3=au&dp3=isamu+kaneda&ca=999999&alang=all&rev=all&pl=20&search=%8c%9f%8d%f5%8e%c0%8ds http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=1cgmi@mkc2oflci3fg1&page=1&doc=9 http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=1cgmi@mkc2oflci3fg1&page=1&doc=9 http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=1cgmi@mkc2oflci3fg1&page=1&doc=9 http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=1cgmi@mkc2oflci3fg1&page=1&doc=9 http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=1cgmi@mkc2oflci3fg1&page=1&doc=9 http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=2b4a7j2feahlj42a4da&page=1&doc=1&cacheurlfromrightclick=no http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=2b4a7j2feahlj42a4da&page=1&doc=1&cacheurlfromrightclick=no http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=2b4a7j2feahlj42a4da&page=1&doc=1&cacheurlfromrightclick=no http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=2b4a7j2feahlj42a4da&page=1&doc=1&cacheurlfromrightclick=no http://apps.webofknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=1&sid=2b4a7j2feahlj42a4da&page=1&doc=1&cacheurlfromrightclick=no in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (2), pp. 160-166, february, 2015. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper comparison of different types of fertilizers on growth, yield and quality properties of watermelon (citrullus lanatus) mohammed massri1 and louay labban2 1 department of food sciences, faculty of agriculture, al-baath university, syria. 2 department of nutrition and food sciences, faculty of health science, university of kalamoon, syria. accepted 24 july, 2015 watermelon is a very important crop because it has many nutritional and economical values. this crop has been known to human for ages and has been cultivated for centuries in many middle eastern countries including syria. several types of fertilizers are being used in order to increase the productivity of this crop but some fertilizers have shown to have adverse effect on the environment. therefore, the aim of this study was to examine the effect of several types of organic fertilizers (cow’s, sheep, poultry and pigeon manure) and to compare their effect on growth, productivity and quality parameters with chemical fertilizers (npk) and control group. two local cultivars spherical and cylindrical (audrey and shapah) were used to examine the effect of cow’s manure (8m/donum), sheep manure (6 m/donum), poultry manure (3 m/donum) and pigeons (2 m/donum) and were compared with chemical fertilizer ( n 20: p 40: k 25). the number of fruits on each vine, length of each vine (cm), fruit weight (kg), and estimated yield (kg/donum) were measured and recorded as well as some quality parameters such as rind thickness (cm) and heart color. cows manure proved to be superior to other types of fertilizers (organic and inorganic) in many traits but chemical fertilizer gave the highest yield (kg/donum) in audrey cultivar but in shapah cultivar there was no significant difference in productivity between chemical fertilizer compared with control group. using organic fertilizers to cultivate watermelon does affect quality watermelon and the productivity was too close to chemical fertilizer. on the other hand, organic fertilizer can reduce the harmful effect of chemical fertilizers on environment and human health alike. key words: audrey, shapah, manure fertilizers, chemical fertilizers, productivity, watermelon. introduction human have known watermelon (citrullus lanatus) for centuries and have noticed the nutritional importance of its fruits. watermelon is a herbaceous creeping plant that belongs to the botanical family curcurbitaceous or gourd ________________________________ corresponding author. e-mail: drlouay@gmail.com family which thrive in the tropical region and has been cultivated for thousands of years in the middle east (syria, egypt, jordan, tunisia, lebanon….etc) and south east asia (koocheki et al., 2007). watermelon is a very rich source of nutrients such as photochemical and lycopene (perkins-veazie et al., 2001; perkins-veazie; collins 2004) and often used as an appetizer or healthy snack, depending on the way it is prepared (erukanure et massri & labban 160 al., 2010). there are many local names for watermelon in the middle east region such as bateekh in syria, habhab in saudi arabia, raki in iraq, jah in uae. watermelon is considered a summer crop and it has good economical importance because it is a highly marketed crop in the local markets and for export. watermelon is among the most widely cultivated crops in the world and the acreage of watermelon increased in the past years (fao, 2007). according to fao 2011, syria produces 670559 tons and the cultivated area was 31044 hectares. watermelon not only tolerates hot weather but for best growth requires more heat than any other vegetables. watermelon seeds germinate well and plants thrive at 25°c – 30°c. fruits mature best at 30°c. watermelon citrullius lanatus plant is a herbaceous creeping plant which produces from 3 to 5 fruits weighing from 3 to 10 kilogram. some cultivars such as the ‘florida giant’ may weigh up to 20 kilogram, (pamplona-roger, 2008). in order to improve the yield of watermelon, the soil content of nutrients should be increased to boost the fertility which can be achieve by either using organic fertilizer such as cattle manure, poultry manure, animal waste and use of compost (dauda et al., 2005) or by using chemical fertilizers mainly potassium and nitrogen compounds. scientists have been interested in studying organic materials especially for its benefits for the plant, soil and recently for the environment. modern farming system is heavily dependent on chemical fertilizers and reducing the number of farmers who use organic fertilizers which created a wide range of problems mainly depleting the soil fertility and thus reducing the yield year after year putting some farmers out of business not to mention the effect on the soil texture since organic fertilizers can improve the soil texture especially the clay and sandy soils (mahmoud et al., 2009). over-use of chemical fertilizer can be harmful to the environment by polluting water and increasing the volumes of farm crops which proved to be hazardous to human health. in organic farming, the soil becomes rich in nutrients therefore, crops grow healthy and resistant to pest and diseases making the quality of the products more nutritious, tastier and contain substances that are good for health (wakui, 2009). john et al. (2004) had advocated for an integral use of organic manure and inorganic fertilizers for the supply of adequate quantities of nutrients to improve crop productivity while minimizing environmental impact from fertilizer use. large quantities of animal wastes are produced each year in syria, and mismanagement of organic wastes, have impacted public health and environment. the aim of the study was to assess the effect of different types of organic fertilizers ( cow’s manure, sheep manure, poultry manure and pigeon manure) and one type of chemical fertilizer (npk) on the characteristics of watermelon (growth parameters, yield and quality) of two local cultivars in syria. materials and methods four different types of fermented (for one year) organic fertilizers (cows, sheep, poultry, and pigeons) were used in addition to one chemical fertilizer as shown in table (1). *1 donum= 1000 square meters the fertilizer is added to the soil prior to cultivation, and then seedlings are brought from nursery and planted in the soil after irrigation at a distance of 1.5 meter of each other. two local cultivars were used; audrey round (spherical shape) and shapah oblong (cylindrical shape). the experiment was replicated for three times and the average was calculated. physical properties of watermelon: the physical properties were determined for each cultivar including width and length circumference of the fruits (cm), thickness of the rind (cm) and weight of the fruits (kg), percentage of the seeds, vine length and number of the branches in each plant were measured. estimated productivity was calculated by multiplying the number of fruits in each plant by the average fruit weight. three fruits were left to grow in each plant. methods of analysis the following measurements were taken after 75 days of transplantation: -the number of branches in each plant, the length of each vine (cm), the number of fruits in each vine. the weight of fruit (kg), estimated yield (kg/donum) and the rind thickness (cm) were taken 75 days after plantation. 1. moisture determination: was determined by drying the flesh in an oven at 105°c until a constant weight according to (a.o.a.c. 2002). 2. ash content determination: was determined in muffle at 525 °c according to the (a.o.a.c. 2002). 3. ph measurement: by using jenway ph meter 3510 according to( aoac, 2002). 4. texture measurement: a stable micro system ta.xt computerized digital texture analyzer equipped with (p/10) cylindrical probe and cell (5kg) and speed 2.0 mm/sec to depth 10mm, were used. it was measured for the heart and rind (xisto, et al., 2012). 5. color determination: heart color (cie l*, a*, b* 161 afr. j. food sci. res. table 1. type of fertilizers and the amount used in the experiment. treatment type of organic fertilizer(amount used) 1 cow’s manure (8 m³/ donum)* 2 sheep manure (6 m³/ donum) 3 poultry manure (3 m³/donum) 4 pigeon’s manure (2 m³/ donum) 5 chemical fertilizer (npk) 20-40-25 kg/ donum 6 control (no fertilizers) units) was measured on the cut surface of heart tissue using a konica minolta (model cm-3500d, japan) calibrated with a white tile. l* degree of lightness (100=white, 0=black) , a* degree of redness or greenness (+red; -green), and b* degree of yellowness or blueness (+yellow; -blue). one reading was taken from each of six slices of each replicate sample. (saftner et al., 2007 & nadzirah et al., 2013). 6. total soluble solids content: from juice extracts (tissue purees) were analyzed using an abbe refractmeter, according to (aoac, 2002). 7. fruits weight: was done by using electric scale (jenaway), weighs up to 30 kg with accuracy of 0.01 kg. 8. dimensions: were measured by using regular measuring tape (100 cm). 9. rind thickness was measured by using vernier thickness caliper. 10. soil analysis data: the soil in the planting sites was analyzed and the results are shown in table (2). statistical analysis collected data were presented as mean ± sd and statistically analyzed using one way analysis of variance (anova). student t-test repeated measure was used for significance. differences were considered significant at p<0.05 according to artimage and berry (1987). results and discussion with regard to audrey cultivar, cow’s manure fertilizer was superior to other treatments regarding the number of fruits on each vine, number of vines and length of vine. the difference was statistically significant between cow’s manure, sheep manure and poultry manure and between chemical fertilizer and control sample (p<0.05) as shown in table 3. dauda et al., 2008 studied the efficacy of different levels of poultry manure at the rates of 0, 3.3, 6.6 and 9.9 t haˉ¹ on growth and yield of citrullus lanatus, in nigeria. they found that application of poultry manure significantly enhance growth parameter vigor and number of fruits during the two seasons. when looking at shapah cultivar, cow’s manure was the best in producing more fruits on each vine and other treatments: sheep manure, poultry manure, pigeons manure and chemical fertilizer. there was a significant difference among these treatments and control group but the difference was highly significant (p <0.05) between cow’s manure and control group. these results are demonstrated in table 4. data collected from this study have shown that in audrey cultivar, cow’s manure had the highest length circumference compared with control group (p =0.02) followed by chemical fertilizer (p =0.03). sheep and pigeon manure were similar in their effect on length circumference (85±20.8 and 87±19.3) and the difference was significant (p =0.04) but the difference was not significant between poultry manure and control group (p =0.05) as shown in table 5. with regard to width circumference in audrey cultivar, control group and chemical fertilizer had the highest width circumference but there was no significant difference between chemical fertilizer and control group regarding width circumference (p = 0.05) but the difference was significant between control group and all other treatments. sheep and poultry manure (85±14.8 and 80±13.9 cm respectively) and the difference was significant (p =0.02) followed by pigeon manure and cow’s manure which were similar in their width circumference (78±14.1 and 75±13.3 cm) and the difference was highly significant (p =0.01). rind thickness was the highest in sheep and cow’s manure (2.16±0.3 cm and 1.92±0.2 cm respectively) and the difference was significant compared with control group (p =0.02), followed by poultry manure 1.78±0.2 cm and pigeon manure 1.74±0.2 cm (p =0.04). there was no significant difference between control group and chemical fertilizer (p = 0.05). the weight of fruit was the highest in chemical fertilizer 18.37±4.9 kg (p =0.01) followed by cow’s manure 16.29±4.3 kg (p=0.02), sheep manure massri & labban 162 table 2. soil analysis data for planting sites. soil analysis data ppm g/100g of soil millimose ph calcium k p mineral n calcium carbonate organic matter ec 1/5 18% 213.4 11 9.42 42.66 1.4 0.29 5.9 table 3. number of fruits, vines and length of vine in audrey cultivar. treatment no. of fruits on each vine p value no. of branches p value length of vine (cm) p value 1 4±0.26 0.03* 15±4 0.03* 98±20.4 0.02* 2 3±0.12 0.04* 5±1 0.10 66±19.9 0.04* 3 3±0.12 0.04* 8±2 0.07 82±20.69 0.03* 4 2±0.09 0.06 8±2 0.07 68±20.4 0.04* 5 2±0.09 0.06 6±1 0.2 42±10.76 0.07 6 1±0.03 6±1 33±9.94 *significant difference p <0.05 table 4. number of fruits, vines and length of vine in shapah cultivar. treatment no. of fruits on each vine p value no. of branches p value length of vine (cm) p value 1 3±0.12 0.02* 17±4 0.007* 70±19.84 0.02* 2 2±0.09 0.04* 8±2 0.06 97±25.33 0.002* 3 2±0.09 0.04* 10±2 0.04* 100±25.11 0.001* 4 2±0.09 0.04* 12±3 0.02* 88±22.85 0.02* 5 2±0.09 0.04* 10±2 0.04 96±24.85 0.002* 6 1±0.02 8±2 44±11.23 *significant difference p<0.05 15.5±4.2 kg (p =0.03), poultry and pigeon manure 14.26±3.9 kg and 13.26±3.6 kg (p =0.04). the lowest fruit weight was in the control group 10.62±2.1 kg. this also applies to estimated yield (kg/donum). the highest yield was in the chemical fertilizer 11026.2±343.3 kg/donum and the difference was highly significant (p =0.007) when compared with the control group which had the lowest yield (6370.2±132.1 kg/donum). cow’s manure came in the second place with 9970.4±250.4 kg/donum (p =0.01) followed by sheep manure 9300±233.3 kg/donum (p =0.02) , poultry and pigeon manure (8558.6±243.5 and 7956.6±222.4 kg/donum respectively (p=0.04). control group had the lowest yield (6370.2±132.1 kg/donum). these data are shown in table 5. the results are in total agreement with those obtained by olson and simonne, 2010 who found that chemical fertilizers (npk) had positive impact on watermelon productivity. in their study, chemical fertilizer gave around 90 tons/ ha which are very close to our result. with regard to length circumference in shapah cultivar, there was no significant difference between control group and treatment 6 but there was a significant difference between control group and other treatment (p <0.05). treatments 2 and 3 came in the first and second place whereas treatment 1 and 4 were equal with regard to this trait. the difference was significant between control group and all treatments with regard to width circumference. treatments 4 and 5 were equal (p=0.03) whereas treatments 1, 2 and 3 were equal (p =0.04). 163 afr. j. food sci. res. table 5. fruit dimensions, rind thickness, fruit weight and estimated yield of audrey cultivar treat. length circumferen ce (cm) p value width circumferen ce (cm) p value rind thickness (cm) p value weight kg p value estimated yield (kg/donum) p value 1 97±23.3 0.02* 75±13.3 0.01* 1.92±0.2 0.02* 16.29±4.3 0.02* 9970.4±250.4 0.01* 2 85±20.8 0.04* 85±14.8 0.02* 2.16±0.3 0.02* 15.5±4.2 0.03* 9300±233.3 0.02* 3 81±18.9 0.05 80±13.9 0.02* 1.78±0.2 0.04* 14.26±3.9 0.04* 8558.6±243.5 0.04* 4 87±19.3 0.04* 78±14.1 0.01* 1.74±0.2 0.04* 13.26±3.5 0.04* 7956.6±222.4 0.04* 5 93±22.8 0.03* 91±15.3 0.05 1.62±0.1 0.05 18.37±4.9 0.01* 11026.2±343.3 0.007* 6 77±17.4 94±15.9 1.58±0.1 10.62±2.1 6370.2±132.1 significant difference p <0.05 table 6. fruit dimensions, rind thickness, fruit weight and estimated yield of shapah cultivar. treat. length circumference (cm) p value width circumference (cm) p value rind thickness (cm) p value weight kg p value estimated yield (kg/donum) p value 1 105±23.8 0.04* 64±15.3 0.03* 1.29±0.2 0.05 10.51±2.11 0.03* 6309.6±1521 0.04* 2 112±24.2 0.02* 65±15.7 0.03* 1.5±0.3 0.02* 11.39±2.53 0.02* 6836.4±1543 0.03* 3 111±21.6 0.03* 64±14.5 0.03* 1.26±0.2 0.04* 11.9±2.43 0.02* 7140±1642.4 0.02* 4 109±23.9 0.04* 70±15.9 0.02* 1.38±0.2 0.03* 10.6±2.12 0.03* 6360±1584.3 0.04* 5 99±20.8 0.05 66±13.5 0.02* 1.38±0.2 0.03* 9.79±1.78 0.06 5874.4±1320.3 0.05 6 103±23.4 64±13.8 1.18±0.1 9.47±1.33 5685.2±1273.2 significant difference p <0.05 rind thickness was the highest in sheep manure (1.5±0.3 cm) and the difference was significant (p =0.02), followed by pigeon manure and chemical fertilizer 1.38±0.2 cm (p =0.03). there was no significant difference between control group and cow’s manure (p = 0.05). the weight of fruit was the highest in poultry manure 11.9±2.43 kg (p =0.02) followed by sheep manure 11.39±2.53 kg (p =0.02), pigeon manure 10.6±2.12 kg (p =0.03), cow’s manure 10.51±2.11 kg (p =0.03). the lowest fruit weight was in chemical fertilizer and control group and 9.79±1.78 kg and 9.47±1.33 kg (p =0.06). the highest yield (kg/donum) was in poultry manure 7140±1642.4 kg/donum and the difference was significant (p =0.02) when compared with the control group which had the lowest yield (5685.2±1273.2 kg/donum). sheep manure came in the second place with 6836.4±1543 kg/donum (p =0.03) followed by pigeon manure 6360±1584.3 kg/donum and cow’s manure 6309.6±1521 (p=0.04), control group had the lowest yield (5685.2±1273.2kg/donum).there was no significant difference between control group and chemical fertilizer (p =0.05). the results are similar to those obtained by olson et al., 2010. poultry manure is relatively resistant to microbial degradation. however, it is essential for establishing and maintaining optimum soil physical condition and important for plant growth. pm is also very cheap and effective as a good source of n for sustainable crop production, but its availability remains an important issue due to its bulky nature, while inorganic fertilizer is no longer within the reach of poor-resource farmers due to its high cost (rahman, 2004). however, john et al. (2004) had advocated for an integral use of organic manure and inorganic fertilizers for the supply of adequate quantities of plant nutrients required for sustaining maximum crop productivity and profitability, while minimizing environmental impact from nutrient use. with regard to seed percentage, chemical fertilizer and poultry manure had the highest percentage of seed (1.16 and 1.12% respectively) followed by control group (0.98%), sheep manure (0.72%), pigeon manure (0.63%) and finally cow’s manure that produced 0.57% seeds. as for moisture percentage in audrey cultivar, control group had the highest moisture percentage (93 %), followed by chemical fertilizer (91.1%) and then sheep, pigeon and poultry manure (90.96, 90.95 and 90.13% respectively). cow’s manure gave the lowest moisture percentage 89.75%. chemical fertilizers had the highest ash percentage (0.44%) followed by poultry manure (0.37%), control group massri & labban 164 table 7. seeds, moisture, ash percentage, ph and tss of audrey cultivar. treatment seed % moisture % ash % ph tss 1 0.57ᵈ 89.75ᶜ 0.23ᶜ 5.5ᵇ 10.24ᵃᵇ 2 0.72ᶜ 90.96ᵇ 0.22ᶜ 5.7ᵃᵇ 7.85ᶜ 3 1.12ᵃ 90.13ᵇ 0.37ᵃᵇ 5.7ᵃᵇ 8.02ᵇ 4 0.63ᶜᵈ 90.95ᵇ 0.3ᵇ 5.8ᵃ 10.4ᵃ 5 1.16ᵃ 91.1ᵃᵇ 0.44ᵃ 5.7ᵃᵇ 10.35ᵃ 6 0.98ᵇ 93ᵃ 0.33ᵇ 5.5ᵇ 7.25ᵈ ᵃ ᵃᵇ ᵇ significant difference. ᶜ ᵈ insignificant difference. table 8. seeds, moisture, ash percentage, ph and tss of shapah cultivar. treatment seed % moisture % ash % ph tss 1 1.08ᵇ 89.39ᶜ 0.45ᵃ 5.4ᵇ 10.1ᵇ 2 0.87ᵈ 92.4ᵃ 0.36ᵇ 5.5ᵃᵇ 8.1ᵇᶜ 3 1.68ᵃ 91.43ᵃᵇ 0.35ᵇ 5.6ᵃ 7.1ᶜ 4 1.4ᵃᵇ 90.99ᵇ 0.36ᵇ 5.3ᵇ 10.6ᵃ 5 0.99ᶜ 92.16ᵃ 0.41ᵃᵇ 5.6ᵃ 10.4ᵃᵇ 6 1.06ᵇ 90.58ᵇ 0.41ᵃᵇ 5.4ᵇ 7.9ᵇᶜ ͣ ͣᵇ ᵇ significant difference. ᵇᶜ ᶜ ᵈ insignificant difference. and pigeon manure ( 0.33% and 0.3% respectively) and finally cow’s manure and sheep manure (0.23 and 0.22% respectively). ph values for audrey cultivar for all treatment were very close, but pigeon manure had the highest (ph=5.8) followed by sheep manure, poultry manure and chemical fertilizer (ph=5.7) then control group and cow’s fertilizer (ph=5.5) were the lowest. when it comes to total soluble solids (tss), pigeon manure and chemical fertilizer had the highest (10.4 and 10.35 respectively) followed by cow’s manure (10.24), poultry manure (8.02), sheep manure (7.85) and finally control group (7.25). the results are presented in table 7. shapah cultivar had different values for seeds, moisture and ash percentage, also had different values for ph and tss for all treatments. poultry manure had the highest seed percentage (1.68%), followed by pigeon manure (1.4%) then cow’s manure and control group (1.08 and 1.06 % respectively) and then chemical fertilizer ( 0.99%) and finally sheep manure (0.87%). moisture percentage was the highest in sheep manure and chemical fertilizer (92.4 and 92.16% respectively) followed by poultry manure (91.43%), pigeon manure and control group (90.99 and 90.58% respectively) whereas cow’s manure had the lowest moisture percentage for a value of 89.39%. in the contrary, the highest ash percentage was in cow’s manure (0.45%) followed by control group and chemical fertilizer (0.41%) whereas sheep manure, poultry manure and pigeon manure had almost the same ash percentage (0.36%). poultry manure and chemical fertilizer had the highest ph values (ph=5.6) followed by sheep (5.5) and then cow’s manure, control group and pigeon manure (ph= 5.3-5.4). pigeon manure had the highest tss (10.6) followed by chemical fertilizer (10.4), cow’s manure (10.1) followed by sheep manure and control group (8.1 and 7.9 respectively). poultry manure had the lowest tss in shapah cultivar for 7.1 as shown in table 8. the results obtained from this study especially ph values and poultry manure was similar to those obtained by duda et.al., 2005 in which he found that chemical fertilizer increased ph values of soils. the superficial appearance and color of food are the first parameters of quality evaluated by consumers, and are thus critical factors for acceptance of the food item by the consumer (leon et al., 2006). the aspect and color of the food surface is the first quality parameter evaluated by consumers and is critical in the acceptance of the product, even before it enters the mouth. the color of this surface is the first sensation that the consumer perceives and uses as a tool to accept or reject food ( du & sun, 2004). this study has also examined the degree of lightness (l), the degree of redness or greenness (a) and the degree of yellowness or blueness (b) in both cultivars. in audrey cultivar, chemical fertilizer had the highest lightness degree (l=41.44) followed by poultry manure and control group (32.08 and 32.07 respectively) whereas sheep manure and pigeon manure had 30.19 and 165 afr. j. food sci. res. table 9. heart color measurement (units) of both audrey and shapah cultivars. treatment audrey cultivar shapah cultivar l* a* b* l* a* b* 1 31.63ᵇ 28.5ᵇ 14.25ᵇᶜ 32.78ᵃ 30.29ᵇ 17.04ᵃ 2 30.19ᵇᶜ 28.15ᵇ 13.43ᶜ 29.47ᵇ 31.94ᵃᵇ 16.39ᵇ 3 32.08ᵃᵇ 29.15ᵃᵇ 15.58ᵇ 29.98ᵇ 32.53ᵃ 16.48ᵇ 4 30.18ᵇᶜ 30.95ᵃ 16.76ᵃ 30.81ᵃᵇ 31.14ᵃᵇ 16.75ᵃᵇ 5 41.44ᵃ 29.23ᵃᵇ 16.26ᵃ 30.36ᵃᵇ 30.62ᵇ 16.02ᵇᶜ 6 32.07ᵃᵇ 28.84ᵇ 14.44ᵇᶜ 30.36ᵃᵇ 30.63ᵇ 15.45ᶜ ͣ ͣᵇ ᵇ significant difference. ᵇᶜ ᶜ ᵈ insignificant difference. 30.18 respectively. pigeons manure had the highest score with regard to redness (30.95) followed by chemical fertilizer and poultry manure (29.23 and 29.13 respectively) and then control group (28.84), cow’s manure (28.5) and finally sheep manure for a value of 28.15. yellowness score was the highest pigeon manure and chemical fertilizer (16.76 and 16.26 respectively) followed by poultry manure (15.58), control group and cow’s manure (14.44 and 14.23 respectively) whereas the sheep manure was the lowest (13.43). these results are shown in table 9 and are similar to the results found by pedreschi et al., 2000. table 9 has shown that the highest lightness score in shapah cultivar was in cow’s manure (32.78) followed by pigeon manure, chemical fertilizer and control group (30.81 and 30.36 respectively). poultry manure and sheep manure had the lowest lightness score 29.98 and 29.47. redness score in shapah cultivar was the highest in poultry manure (32.53) maybe was due to increase lycopene content of the flesh (perkins-veazie et al., 2001) followed by sheep manure and pigeon manure (31.94 and 31.14) whereas control group, chemical fertilizer and cow’s manure scored the lowest in redness scores (30.63,30.62 and 30.29 respectively). yellowness score was the highest in cow’s manure (17.04) followed by pigeons manure (16.75). poultry manure and sheep manure had almost the same value (16.48 and 16.39 respectively) and then chemical fertilizer (16.02) whereas the lowest was control group (15.45). the results obtained from this study are parallel to those results reported by vimala et al., 2001 and abdullah et al., 2004 who found the chemical fertilizer has a great effect on the color of the flesh and heart of watermelon, this eas due by increasing lycopene content of the flesh. conclusion as a conclusion for this study, organic fertilizers mainly cows, sheep and poultry manure had the highest results with regard to quality parameters such as redness and rind thickness whereas chemical fertilizer had a slight advantage for the productivity (kg/donum) compared with control and organic fertilizer but quality parameters were lower than organic fertilizers especially in those cows, sheep and poultry manure. references abdullah mz, guan lc, lim kc, karim aa (2004). the applications of computer vision and tomographic radar imaging for assessing physical properties of food. j. food eng. 61: 125–135. aoac (2002). official methods of analysis of aoac, internstional 17 th addition, current through revision #1. dauda, s.n.; ajayi, f.a.; and ndor, e. 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(1). wakui y (2009). organic farming technology in japan. pilot project for better farm income by organic-based vegetable production. koibuchi college of agriculture and nutrition. japan. xisto alrp, vilas boas evb, nunes ee, vilas boas em, mário césar guerreiro mc (2012). volatile profile and physical, chemical, and biochemical changes in fresh cut watermelon during storage. ciênc. tecnol. aliment., campinas, 32(1): 173-178. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 4 (5), pp. 049-053, december, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper quality estimation of cashew gum in the production of chocolate pebbles e. gyeduakoto 1 , i. oduro 2 , f. m. amoah 1 , j. h. oldham 2 , w. o. ellis 2 , k. opokuameyaw 1 , f. asante 3 and s. bediako 3 1 cocoa research institute of ghana, akim-tafo, ghana 2 department of biochemistry, kwame nkrumah university of science and technology, kumasi, ghana. 3 cocoa processing company, tema, ghana. accepted 12 july, 2016 due to the limited supply and high cost of gum arabic, cashew gum (cg) was assessed as a quick coating agent in the production of chocolate pebbles, using gum from both young and mature cashew trees. acute toxicity tests on rats did not reveal any abnormal changes attributable to ingestion of cg. pebbles produced with cg samples were compared with those produced with gum arabic. the ash contents of the three products ranged from 2.37 to 2.63% and the moisture content from 2.33 to 2.76% whilst the sugar concentration ranged from 26.05 to 29.66%. the microbial status of the three products conformed to specifications of the cocoa processing company (cpc). physicochemical parameters determined showed significant differences among the three products (p < 0.05). sensory analysis showed no significant difference among products in terms of flavour, hardness and smoothness. the overall acceptability of the products were similar and the mean scores were 7.4, 6.8 and 7.1 for pebbles produced with gum arabic, that produced with cashew gum from young and mature trees respectively. on a 9-point hedonic scale, this range varies from “like moderately” to “like extremely”. pebbles produced with cashew gum compared favourably with that produced with gum arabic. key words: cashew gum, gum arabic, chocolate pebbles, acute toxicity. introduction gums consist of heterogeneous complex mixtures of closely related polysaccharides, which produce viscous solutions or dispersions when dissolved in hot or cold wa-ter (belitz et al., 2004). they are best known as powerful thickeners but perform an extraordinary number of other functions essential to food quality. in recent years, gums have been recognized as healthy sources of fiber as well (hundley, 2002). there are four different types of natural gums, which are plant/tree exudates, seed gums, micro-bial gums and seaweed gums (glicksman, 1969). plant exudates are gums from various plant species obtained as a result of tree bark injury. they are normally collected as air-dried droplets (fitwi, 2000). they have been found to have many industrial potentials. about one billion pounds of gum are consumed in the united states each year where the growth in demand exceeds 8% per year (fao, 2002). in ghana about 10 tonnes of gum is used annually by industries (frimpong-mensah, 2000). *corresponding author. e-mail: akua_akoto2004@yahoo.co.uk gum from the cashew tree ( anacardium occidentale l), which is a plant exudate, has physico-chemical and rheological properties similar to gum arabic, which is obtained from acacia senegal (smith and montgomery, 1959; owusu et al., 2005)). cashew gum is a complex polysac-charide of high molecular mass comprising 72 73% galactose, 4.6 5% arabinose, 3.2 4% rhamnose, 11 14% glucose and 4.7 -6.3% glucuronic acid (cunha et al., 2007). gum arabic serves as a coating agent and film-former in panned confections such as chocolate pebbles (tic gums, 2001). however, its high cost has led to the assessment of other tree gums such as cg in the pro-duction of chocolate pebbles. materials and methods materials used included milled cg samples, gum arabic from the forest research institute of ghana, kumasi and rats obtained from the department of pharmacy, knust. chocolate liquid and powder were obtained from cocoa processing company, tema. peanuts, icing sugar, cornstarch, titanium dioxide, bees wax, equipment, apparatus and reagents. gyedu-akoto et al. 050 acute toxicity of cg thirty mature male rats divided into groups of five were used for the toxicity test. they were fed with food and hygienic water and their initial weights measured. six doses of cg per kg body weight (b.w.) were selected and administered to the different groups of rats orally. a control group was given water. the doses were administered as follows: 3, 5, 10, 15, 20 and 30 g/kg b.w. the rats were then observed continuously for 14 days for changes in movement, appetite, water intake, salivation, diarrhea, and urination. microbial status of cg enumeration of total microbial growth serial dilutions of cg samples were made in ringer’s solution and allowed to stand for 15 min in a water bath at 40 o c. aliquots of 1 ml were surface spread on highly rich nutrient agar and incubated at 35 o c for 48 h. enumeration of yeasts and moulds serial dilutions of cg samples were made in ringer’s solution and allowed to stand for 15 min in a water bath at 40 o c. aliquots of 1 ml were surface spread on malt extract agar and incubated at 27 o c for 120 h. determination of coliforms serial dilutions of cg samples were made in ringer’s solution and allowed to stand for 15 min in a water bath at 40 o c. aliquots of 1 ml were put into test tubes containing 9 ml lauryl sulphate broth and inverted tubes. malt extract agar was then added to the contents of the test tubes and incubated at 35 o c for 24 h. absence of air bubbles in the inverted tubes indicates the absence of coliforms. preparation of beeswax, sugar syrup, starch and gum solutions gums from both mature and young cashew trees were sterilized in a gallenkamp autoclave (uk) at 121 o c for 15 min and used in the preparation of aqueous gum solutions in a water: gum ratio of 60:40 w/v (66.7%). an aqueous solution of gum arabic, which is the conventional gum used for pebbles production (tic gums, 2001) in a water:gum ratio of 50:50 w/v (100%) was also prepared. sugar syrup was prepared to 72% sugar solids. starch solution was made by mixing cornstarch with sugar syrup. powder starch was also produced by mixing cornstarch and icing sugar in the proportion of 1:3. titanium dioxide was added to the starch solution to give it a white colour. the beeswax was then melted in frytol oil in the proportion of 1 kg beeswax in 4 l frytol oil. production of chocolate pebbles production of chocolate pebbles was done adopting the method of panned goods of fabry (1992), which involved three steps. preparation and pre-coating of centres the peanuts, which are the centres of the pebbles were cleaned and calibrated into even sizes. the centres were then fried with cocoa butter and put into a mechanically operated elliptical pan, which rotates on an inclined shaft at an angle of about 40 o to the horizontal (plate 1). the pan has a fan which blows cold air for drying attached to it. the centres were then pre-coated with the gum solution and sugar syrup and then air-dried. chocolate coating this involved the application of chocolate liquid to the centres and dusting with chocolate powder. they were then air-dried. dusting with chocolate powder ensures proper drying. this was repeated five times to build up the chocolate layer. they were then left overnight for drying. the application of chocolate layer was repeated about 8 10 times to obtain a desired number of layers or about half of the total weight of the final product. they were again left overnight for drying starch coating a second gum coating was applied and dried to protect the chocolate layer. the starch solution was applied and dusted with starch powder and air-dried. this was also repeated five times. they were then coloured with a colour solution (colour and sugar syrup) and then polished with the beeswax. determination of microbial status and physico-chemical composition of pebbles total microbial growth, yeasts and moulds, and coliforms of the products were determined. the results were then compared to internal specification of cpc (tema, ghana) for chocolate products. the ash, sugar and moisture contents were determined using mm55 plus (infrared engineering) for all the products and the data analyzed using statgraphics plus for analysis of variance (anova). sensory analysis this was done using the triangle test where 40 judges were presented with sets of 3 coded samples of pebbles, two of which were the same. judges were asked to identify the odd sample in terms of flavour, hardness and smoothness (stone and sidel, 1992). data obtained was analyzed using binomial distribution. acceptability test was also conducted .by asking the panelists to determine their overall acceptability using a 9 point hedonic scale with 1 3 = dislike extremely, 4 6 = neither like nor dislike and 7 9 = like extremely (stone and sidel, 1992). data obtained was analyzed using binomial distribution. results and discussions results of the acute toxicity studies showed that the median lethal dose (ld50) for cg was more than 30 g/kg b.w. cashew gum had no adverse effect on the rats (table 1) indicating that cg is not acutely toxic according to world health organization (who) acute hazard rankings (who, 2001) (table 2). this compares favourably with fao (1974) report on the acute toxicity of gum arabic which says that groups of rats fed with gum arabic in their diet for 62 days showed normal weight gain and food efficiency. haematological findings and organ weights were also normal. the ld50 of gum arabic was in the range 8 – 18 g/kg b.w. (booth et al., 1963). further tests by determining the microbial load of cg also reveal 051 afr. j. food sci. res. table 1. visual observation of rats. movement appetite water intake salivation diarrhea urination control normal normal normal normal absent normal rat normal normal normal normal absent normal table 2. who acute hazard rankings (who, 2001). who toxicity classification rat ld50 (mg of chemical per kg body weight class description solids (oral) liquids (oral) ia extremely hazardous <5 <20 ib highly hazardous 5-50 20-200 ii moderately hazardous 50-500 200-2000 iii slightly hazardous >500 >2000 iv not acutely toxic >2000 >3000 table 3. microbial status of cg from young and mature cashew trees gum from young tree gum from mature tree total microbial load <3.0 x 10 3 cfu/ml <3.4 x 10 3 cfu/ml yeasts and moulds 660 cfu/ml 270 cfu/ml coliforms negativ e negative table 4. physico-chemical properties of pebbles produced from gum arabic, gum from young cashew trees and gum from mature cashew trees. parameter gum arabic gum from young tree gum from mature tree ash (%) 2.46 2.63 2.37 0.02 0.008 0.005 mc (%) 2.33 2.38 2.76 0.01 0.09 0.11 sugar (%) 26.05 29.66 27.55 0.03 0.7 0.8 *std dev in italics table 5. microbial status of pebbles produced from the 3 different gums and the standards for foods in general. gum from young cashew tree gum from mature cashew tree specifications of cpc tpc 3.0 x 10 2 cfu/ml 3.0 x 10 2 cfu/ml 5.0 x 10 3 cfu/ml y/m 0 0 <5 cfu/ml coliforms negative negative negative ed that cg contained an average of 3.2 x 10 3 cfu/ml of total microorganisms and 465 cfu/ml of yeasts and moulds (table 3) which can easily be destroyed by heating. cashew gum was also found to be free from coliforms, which is an indicator of the presence of disease-causing bacteria. at a concentration of 80% and higher cg will form a gel (gyedu-akoto et al., 2007) and this could not be used in coating the centres of the pebbles since it caused the sticking of centres to each other resulting in uneven surface of products. this led to the use of 66.7% cg solutions in the production of pebbles in stead of 100% used for gum arabic. physicochemical analysis conducted on the three products gave significant differences bet gyedu-akoto et al. 052 table 6. results on triangle test (binomial distribution) category n observed prop test prop assymp. sig.(2-tailed) hardness gp 1 1.00 25 0.63 0.50 0.155 gp 2 2.00 15 0.38 total 40 1.00 flavour gp 1 1.00 19 0.48 0.50 0.874 gp 2 2.00 21 0.53 total 40 1.00 smoothness gp 1 1.00 20 0.50 0.50 1.000 gp 2 2.00 20 0.50 total 40 1.00 (% ) 50 40 f re qu en cy 30 g arabic 20 young cg mature cg 10 0 1 2 3 4 5 6 7 8 9 hedonic score figure 1. frequency of hedonic scale scores of pebbles produced with gum arabic, cashew gum from both young and mature trees for overall acceptability (1 = dislike; 9 = like extremely) ween the products (p < 0.05). multiple comparison tests showed that pebbles produced with gum from both young and mature cashew trees differed significantly from that produced with gum arabic. the results are presented in table 4. the moisture and sugar contents of the three products fell within the acceptable levels for chocolate pebbles (fabry, 1992) which are 1 3 and 20 30% for moisture and sugar contents respectively. the microbial status of the three products conformed to the internal specification of cpc for chocolate products (table 5). sensory analysis showed no significant difference between the three products in terms of flavour, hardness and smoothness (table 6). overall acceptability of the products was similar. the results confirm the similarity of cg and gum arabic reported by smith and montgomery, (1959). mean scores observed for overall acceptability of the pebbles were 7.4, 6.8 and 7.1 for gum arabic, gum from young cashew trees and mature trees, respectively. on a hedonic scale, this range varies from "like moderately" to "like extremely” (mothé and correia, 2004). the deserved frequency percentage of hedonic scale scores of overall acceptability, obtained for products is presented in figure 1. the frequency of responses are more concentrated between scores of 7 and 9, meaning that pebbles produced with cg compared favourably with that produced with gum arabic conclusion cg was found to be nontoxic and free from diseasecausing bacteria. results from sensory analysis are indicative of consumers’ acceptance to pebbles produced with cg to be similar to that produced with gum arabic. the study therefore showed that cg can be used as a substitute for gum arabic in the production of chocolate pebbles. acknowledgement this paper is published by the kind permission of the executive director of the cocoa research institute of ghana. references belitz hd, grosch w, schieberle p (2004). food chemistry, 3 rd edition, springer, berlin, pp. 309-314. booth a n, hendrickson a p, de eds f (1963). toxicol. appl. pharmacol. 5: 478. cunha plr, maciel js, sierakowski ms, de paula rcm, feitosa jpa (2007). oxidation of cashew tree gum exudate polysaccharide with tempo reagent, j. braz. chem. soc. 18 (1): 85-92. cunha plr, maciel js, sierakowski ms, de paula rcm, feitosa jpa (2007). oxidation of cashew tree gum exudate polysaccharide with tempo reagent. j. braz. chem. soc. 18 (1): 85-92. fao (1974). arabic gum, in compendium of food additive specifications. fao food and nutrition paper 53 (joint fao/who expert committee on food additives. combined specifications from 1st through the 37th meetings,(1956-1990). fao (2002). int. trade on non-wood forest products: an overview – plant gums, fao corporate document repository. fabry i (1992). panned goods, drouven, fabry gmbh, confectionery technology centre, germany. fitwi g (2000). the status of gum arabic and resins in ethiopia, report of the meeting of the network for natural gums and resins in africa (nagra), kenya college of communications technology, nairobi, kenya. 29-31 may, 2000. fao/kepri. pp. 14-22. frimpong-mensah k (2000). characterization of gums from local acacia species for food and pharmaceutical uses, j. gh. sci. ass. 21: 1-79. glicksman m (1969). gum technology in food industry, academic press, usa. pp. 11-16. 053 afr. j. food sci. res. hundley k (2002). starches and gums: a thousand and one functions, in: natural products insider, virgo publshing. mothé cg, correia dz (2004). rheological properties of cashew/xanthan gums blends in cashew juice, 3rd international symposium on food rheology and structure. p. 491. owusu j, oldham jh, oduro i, ellis wo, barimah j (2005). viscosity studies of cashew gum. trop. sci. 45: 86-89. smith f, montgomery r (1959). the chemistry of plant gums and muclages, reinhold publishing corporation, new york. pp.1-30. stone h, sidel jl (1992). sensory evaluation practices. 2. ed. san diego: academic press. p. 336 tic gums inc. (2001). focusing on gum arabic – prepared foods: p. 49. african journal of food science research vol. 2 (5), pp. 084-093, may, 2014. available online at www.internationalscholarsjournals.org © international scholars journals review a review of the food security and poverty issues in south africa ncanywa jabulani department of agricultural economics and extension, faculty of business grahams town, eastern cape, south africa, cape town,south africa. email:jabu789@gmail.com accepted 15 april, 2014 poverty and high levels of food insecurity have been some of the biggest problems facing the nation of south africa since the early days of democracy. whilst the black majority hoped for a more improved economy after the country’s first democratic elections, the legacy of apartheid laws has made the reality to be a different story. yes, it is true that the nation has continued to grow over the last decade but only the minority has enjoyed this growth at the expense of the majority. this paper attempts to review the situation in south africa (sa) through discussing food insecurity and the different faces of poverty faced by the citizens of this rainbow nation. this paper covers a number of concepts, which include the concept of food security, poverty, their definitions and application in south africa’s context. poverty situation and their indicators in south africa were also discussed. the paper advocates for the development of smallscale agricultural system in rural areas, provision of financial support and commercialization of the farm produce as the means to reduce poverty, achieve food security and decimate the high rate of unemployment in south africa. key words: poverty, food security, smallholder agriculture, rural development. introduction food security food security is part of section 27 of the constitutional rights in south africa (sa) (department of agriculture, 2002). concerning these rights, the sa constitution states that, “every citizen has the right to have access to sufficient food and water, and that the state must, by legislation and other measures, within its available resources, avail to progressive realization of the right to sufficient food.” the reconstruction and development programme (rdp) in 1994 identified food security as a priority policy objective. as a result, the government reprioritized public spending to focus on the food security conditions of the historically disadvantaged people. in terms of definition, the concept of food security has many interpretations. for instance, at the 1974 world food summit, it was defined by the united nations (1975) as “the availability at all times of adequate world food supplies of basic foodstuffs to sustain a steady expansion of food consumption and to offset fluctuations in production and prices.” this definition has been refined and re-refined over the years in an attempt to come up with a more common and comprehensive one that encompasses all aspects of food security. for the purposes of this paper, the definition used was postulated by fao (2002). it regards food security at individual, household, national, regional and global levels as being achieved when all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life. this definition was partly constructed from the earlier works of campbell (1991) who insists that the acquisition of “sufficient food” in defining food security should be through socially accepted means such as purchasing without having to beg, rely on charity or steal. if anyone does not have access to such socially accepted norms with which to acquire the right quantity and quality of food at the right times, then, they are thus, considered as food insecure. in short, all these definitions encompass just three main aspects which latham (1997) documented as food availability, food access and food adequacy. the aspect of food availability means that the food required has to be available at the right time and in right quantities and quality. therefore, food availability, though crucial in ensuring food security, does not guarantee it as this food http://www.internationalscholarsjournals.org/ ncanywa 084 should also be adequate both in quantity and quality. one of the most vital objectives of being food secure is to lead a healthy life. with this in mind, latham (1997) holds the view that not only should food be available in correct quantities and quality when needed but its supply should be consistent in order to sustain a healthy life in terms of ensuring proper growth, resistance or recovery from disease, pregnancy, lactation and or physical work. this means that the means used to acquire this food should be always available so that the required quality and quantities of food can be accessed at any given time, from anywhere. the department of agriculture (2002) acknowledges the importance of these three aforementioned aspects of food security but stresses that these should exist within a certain food production system in order to achieve food security effectively and efficiently. this food system should have the capacity to produce, store, distribute and if necessary, import sufficient food to meet the basic food needs of the people. it should also be robust enough to reduce vulnerability to market fluctuations and political pressures, together with minimal seasonal, cyclical and other variations in access to food. poverty according to the studies in poverty and inequality institute (spii) (2007), poverty is a greatly contested concept as defining and measuring what is unacceptable in a society means a lot about the way we as humans would like things to be. in defining poverty though, consensus has been around including issues such as having access to a far wider range of resources than those necessary to ensure mere survival. these different resources are needed in order to promote social solidity and comprehensiveness which are inherent in the right to human dignity. unlike food security which is limited only to having the right type of food in correct quality and quantities, poverty encompasses other important basic human needs such as safe drinking water, sanitation facilities, health, shelter, education and information. shinns and lyne (2004) regard these goods and services as essential to human wellbeing. to define the concept of poverty formally, frye (2005) suggests this be done in two ways, namely, in absolute and relative terms. absolute poverty: to understand poverty in absolute terms, frye (2005) proposes that reference be made to a “certain quantitative measure” which is used to distinguish the poor from the non-poor. thus, the ability to purchase the minimal quantity of basic goods and services required for human survival should be used to distinguish the poverty status of different individuals in absolute terms (frye, 2005). the fact that the concept is defined by measuring individuals’ purchasing power means that income is a strong factor to be considered when ranking the absolute poverty status of people. anyone whose income is below a certain level or threshold is regarded as poor. the copenhagen declaration of 1995 regards absolute poverty as a characteristic that defines most poor countries (spii, 2007). its existence in a community can be identified through the severe deprivation of basic human needs such as food, safe drinking water, sanitation facilities, health, education and information. thus, it goes beyond lack of income to buy the minimum required food basket. from the social point of view, the united nations (un) (2009) says it manifests itself through hunger and malnutrition, ill-health, limited or lack of access to education and other basic services, increased morbidity and mortality from illness; homelessness and inadequate housing; unsafe environments; and social discrimination and exclusion. it is also characterized by a lack of participation in decisionmaking and in civil, social and cultural life. relative poverty: a more formal definition of poverty should embrace more than just financial issues. townsend (1979) elaborates and states that it should also emphasize the individuals’ involvement and participation in the societies they live in. with this in mind, townsend (1979) formally defines relative poverty as a situation that exists when individuals, families and groups in the population “lack the resources to obtain the types of diet, participate in the activities and have the living conditions and amenities which are customary or at least widely encouraged or approved, in the societies to which they belong. their resources are so seriously below those commanded by the average individual or family that they are, in effect, excluded from the ordinary living patterns, customs and activities”. put in a different way, relative poverty attempts to understand inequality in terms of distributions of resources in a society (frye, 2005). unlike absolute poverty that measures the number of households that are unable to afford certain basic goods and services, this concept seeks to measure the extent to which financial resources of these households fall below an average income threshold for the economy. thus, one’s poverty status is measured against other people within the same society hence; the concept argues that an individual is poor when he/she is very much worse off than other people in their society. chronic poverty: this type of poverty encompasses both relative and absolute poverty. instead of focusing on what actually results in poverty, chronic poverty is about the duration of that period of deprivation. aliber (2001) describes it as a situation whereby people continue to live under circumstances of deprivation for a very long period of time, without any means with which to support themselves. thus, under chronic poverty, the most important thing to consider is the duration of this state of “lack” more than what actually caused it. mathole ( 2005) agrees with aliber (2001) in that the household or individual’s inability to sustain themselves through difficult times due to factors like lack of opportunities shows the existence of chronic poverty. a review of the poverty situation in south africa according to the world bank (2001), the sub-saharan africa (ssa) region has about 250 million people living in dire poverty. this translates to around 45% of the region’s total population. moikowa (2004) writes that the continent of africa has the highest number of least developed countries (ldcs), with 32 of the world’s 48 least developed countries found in the continent. the majority of the poor in these ldcs live on less than $2 a day and another handful on less than $1 a day (world bank, 2002). as a consequence, eight out of every 100 infants do not live to see their fifth birthday. furthermore, nine of every 100 boys and 14 of every 100 girls who reach school age do not attend school (world bank, 2002). south africa’s poverty situation is not different in any way. whatever type of poverty is studied, the fact remains that despite south africa’s abundance of resources, the skewed distribution of these resources has meant that the majority of the citizens are still deprived of their fair share of them. for this reason, it is not surprising that the poverty levels in sa have continued to rise even as the country continues to develop. as may (2000) puts it, the experience of south africans is either that of outright poverty or of continued vulnerability to becoming poor. at the present moment, there are so many south africans in need of basic resources such as food, proper shelter and sanitation, clean water and health care facilities etc. the greatest wealth of the nation remains in the hands of the minority. even with the attainment of democracy, racial segregation is still not a thing of the past. literature from mathole (2005) also points out that the most disadvantaged racial group in south africa seems to be the black community, with 61% of its population classified as poor. this is followed by the coloureds (38%), asians (5%) and whites only contribute 1% towards the nation’s poor population. due to such a skewed distribution of the nation’s wealth, mathole (2005) thus, considers sa as a country with two countries, one being a third world and the other is a first world. the most common notion is that the prevailing socio-economic situation in the country is a direct result of the past, the fruit of a historical harvest (voster, 1991). be that as it may, mathole (2005) insists that the legacy of apartheid should not be taken to imply that the citizens of the country are nothing but just prisoners of their past. instead, they have a vision that they should work towards accomplishing but without forgetting their past. democracy has paved way for the previously 085 afr. j. food sci.res. disadvantaged to be recognized in their respective communities and live better lives than before. even though development policies have not accomplished a complete reversal of the legacy of apartheid, the sa government has tried to make “a better life for all” by making everyone equal and with equal resources regardless of skin colour. what should be noted though, is that poverty is not only based on race but other social characteristics such as gender. oettle et al. (1998) provides evidence that the most affected households are those headed by females, especially, in the deepest corners of rural areas. it is true that male-headed households also carry the burden of poverty but women-headed ones suffer it the most for various reasons. this means that development initiatives and policies should also be implemented with women in mind to have a greater impact particularly in the rural areas whose most households are female-headed. symptoms of poverty in the south african context in sa, poverty manifests itself in a number of ways. the first of these are the low income levels that poor people have. other symptoms include the poor housing facilities, high infant mortality rates, high unemployment rates and poor health facilities etc. low income levels leibbrandt et al. (2010) maintain that poverty levels in sa have remained very high despite increases in the real income levels of people within different races in the country. blacks have also benefited from these increases even though the increases have not been enough to get them out of poverty. to understand how income is distributed amongst different households in each of south africa’s provinces, statistics sa (2006) carried out a general household survey (ghs) in 2005. the results showed the existence of big provincial variations, with the more urbanized provinces having relatively low proportions of their populations living below the ultra poverty line. the poorest provinces were found to be those with large rural populations and little access to employment opportunities. limpopo exhibits the most poverty-stricken profiles, followed by the eastern cape (statistics sa, 2006). the western cape province has the highest number of households with formally employed heads in spite of having nearly one in every five children (18%) living in very poor households in terms of earned income. in the year 2002 alone, woolard (2002) claims that eight of the 42 million people in the country were surviving on less than one us dollar (us$1) per day. during the same period, another 18 million people survived on us$2 per day. this is consistent with the ncanywa 086 table 1. number of adult and child beneficiaries of social grants from 2004 july, 2006. grant type number of adult recipients number of child recipients old age pension 2 162,990 -- war veterans grant 2,624 -- disability grant 1 356,937 -- grant in aid 28,441 -- child support grant --7 410,760 foster child grant --351,702 care dependency grant --92,853 total 3 550,992 7 855,315 department of social development (2006). world bank’s (2002) estimates of the extent of poverty in ldcs. even though millions of people in sa are still poor twelve years into the new millennium, the government’s social security system should be applauded for its huge contribution towards ameliorating poverty. this system offers grants to those that are too old, too young, too disabled or busy caring for disabled dependants to work for an income. the grants are in the form of cash transfers to a substantial percentage of the population and they are meant to help the beneficiaries with poverty alleviation. as such, the grants have become a major non-employment source of income and livelihood for most poor south africans. according to the department of social development (2006), south africa’s social security system benefits at least 25% of the entire country’s population each month (table 1). it is therefore not surprising that the budget for the social security system provided for grant allocations of r57,7 billion in 2006/2007, r62,6 billion in 2007/2008 and r68,3 billion in 2008/2009 (department of social development, 2006). reports on the intergovernmental fiscal review by the national treasury (2005) further highlight that 88.5% of social development spending went to social assistance grants in 2004/2005. by the end of july 2006, the social grant beneficiaries were presented (table 1). table 1 show that children were the biggest beneficiaries of this grant system between 2004 and july 2006. however, other grant categories such as the old age pension, though not meant for children, are also used to assist the children by their recipients (department of social development, 2006). through these grants, the government is trying to get its people out of deeper poverty. even though this has been achieved to a certain extent, it should be realized that these grants do not help all the poor. according to the department of social development (2006), the reason behind this, is that only individuals with certain characteristics qualify. thus, the system excludes those that are physically well but too poor and unable to get employment. furthermore, the limited size of the government’s coffers means that the amount given to each beneficiary, especially for the child support grant, is too small to contribute much towards a better life. nevertheless, the social grants have become the greatest source of income for the majority of rural households in sa. their role has surpassed that of smallholder agriculture by far. this is according to the findings by fraser et al. (2003), monde et al. (2005) and van averbeke and hebinck (2007). white and killick (2001) blame this behaviour of turning away from agriculture and towards social grants for income on the poor natural resources and high transaction costs in remote areas where physical infrastructure and services are inadequate. consequently, these factors lead to low agricultural production which in turn promotes poverty in these rural areas. the majority of rural dwellers therefore tend to rely on social grants for their income and cultivate small pieces of land just to produce enough only for domestic consumption. low levels of health a nation’s health levels can be used to assess its poverty status. health indicators that can be used to assess poverty are (i) infant mortality rate and (ii) life expectancy. high infant mortality rates the prevailing low levels of health in most rural areas are another symptom of poverty. with the vast amount of resources in sa, one would expect the nation’s health system to be first class but the reality is a nightmare to most citizens. the scars of the poor health system, especially, the rural areas can be seen through analyzing the infant mortality rates (imr) and the under-five mortality rate (u5mr). nannan and hall (2010) define imr as the probability of babies dying within their first year of life while u5mr is an overall measure of child mortality that usually encompasses the probability of dying during infancy, between ages 1 to 4 years and overall before the 5 th birthday. these two measures of infant mortality are estimated per 1000 live births during 087 afr. j. food sci.res. table 2. imr and u5mr for all sa provinces between 1988 and 1997 (n/1000). province infant mortality rate – 1988under-five mortality – 1988 1997 (n/1000) 1997 (n/1000) eastern cape 61 81 free state 53 72 gauteng 36 45 kwa-zulu natal 52 75 limpopo 37 52 mpumalanga 47 64 north west 42 56 northern cape 42 56 western cape 30 39 national average 45 59 department of health (1998). the same year. nannan and hall (2010) recognized that both imr and u5mr are the most widely used indicators of health status and socio-economic development because they reflect not only the child mortality levels but also the health status of the broader population. for this reason, the department of health (1998) used these two approaches to review the health levels for each of the nine provinces of sa between 1988 and 1997 as shown in table 2. the highest imr (61) and u5mr (81) were observed in the eastern cape province during the period studied. this was followed by the free state with imr and u5mr of 53 and 72 per every 1000 births respectively (table 2). the lowest mortality rates in the country were in the western cape province. while who’s “health for all” has a target of a maximum of 50 deaths per 1000 births, south africa’s imr’s average stood at 45 deaths for every thousand children born whereas the death rate for kids below the age of five was 59. however, in spite of this result, this figure hides the variations between population groups according to socio-economic status or region (bradshaw et al., 2003). in fact, as stated by bradshow et al. (2003), this study does not give any details pertaining to the causes of mortality. despite an estimated decrease in the sa’s imr to 22.45 deaths/1000 population, the rate still remains very high especially, when compared with other developed countries like japan whose imr is 2.79 deaths/1000 live births. although, hiv/aids is blamed for the death of at least 40% of children under the age of five in sa, the role of poverty should be acknowledged too. murray and lopez (1996) rated the top twenty specific causes of death in south african children under the age of five years in 2000. poverty-related causes included in this list were low birth weight (11.2%), diarrheal diseases (10.2%), protein-energy malnutrition (4.3%) and neonatal infections (2.8%). using these findings by murray and lopez (1996), bradshow et al. (2003) concludes that apart from the hiv/aids pandemic, most of the other causes of death of infants are associated with poor socioeconomic conditions such as the lack of access to clean water and basic sanitation, a problem prevalent in the eastern cape. low life expectancy life expectancy is an average estimate of the number of years citizens of a certain country are expected to live, taking all their socio-economic conditions into consideration. the number of years lived by people in different countries tends to differ. in 2009, the cia world fact book ranked south africa as number 208 in the entire world in terms of the average life expectancy of its citizens that are expected to live an average of 48 years and 10 months. women live longer than their male counterparts by 12 months. this was lower than the estimated global average of 66.57 years at birth (64.52 years for males and 68.76 years for females) for 2009 (cia world fact book, 2009). it was also very low especially when compared to the life expectancy of other developed nations like japan, canada, singapore, france, sweden, just to mention but a few, whose life expectancy remains at least at an average of 80 years. south africa is not the only country with a life expectancy below 50 years. findings from the cia world fact book (2009) show that other african countries that are worse than sa include swaziland, angola, botswana, lesotho, zimbabwe, south africa, namibia, zambia, malawi, the central african republic, mozambique and guinea-bissau. hiv/aids has been labeled as the greatest threat to high life expectancy levels in all these ssa countries. of south africa's nine provinces, those with the highest hiv prevalence rates also had the lowest life expectancy free state and mpumalanga, both at 47 years followed by kwazulu-natal at 43 years. the leading causes of death were tuberculosis (tb), influenza and pneumonia, all common opportunistic infections ncanywa 088 associated with hiv/aids. poor standards of housing the housing crisis in south africa has reached extreme levels with statistics sa (2004) estimating that there were 1.376 million people living as squatters in 2004. one might say this is extreme but hemson and o’donovon (2005) are convinced that the worst is yet to come. one might say this is extreme but hemson and o’donovon (2005) argued that the worst is yet to come. these predictions by hemson and o’donovon (2005) have proven not to be so unrealistic after all as the number of slum buildings increased between 2004 and 2008 from 1.376 to 2.4 million. the city of cape town is one of the most affected areas in terms of housing problems as indicated by its 144 informal settlements. the cape times newspaper (2007) has evidence that the majority of the inhabitants of these informal settlements in the city are the young, unemployed and poor. indications are that this situation keeps getting worse by the day, with the housing backlog expected to reach 460 000 by 2020 (cape times newspaper, 2007). according to may et al. (1995) and who (1997), most of these informal settlements are not only over-populated but have neither hygienic sanitation nor safe drinking water. who (1997) further states that such dwellings are also deficient in electricity, ventilation, food preparation and storage. inadequate access to these resources brings rise to a range of health risks including diarrheal and respiratory diseases. woolard (2002) reveals that by year 1999, the population in sa without clean drinking water and adequate sanitation was 47 and 38% respectively. high illiteracy rates the level of education amongst a nation’s citizens is one way of determining that particular country’s poverty status. in sa, aitchison and harley (2004) observed during the 2001 census that 18% of people aged between 20 years and older had no formal education at all. during the same period, 16% had only gone as far as primary education (grade one to six). in real terms, this translates to 4 567 497 and 4 083 752 people with no education at all and those with only primary education, respectively (aitchison and harley, 2004). this tells that at least a third of the nation’s population could be regarded as illiterate, with limpopo and western cape being the provinces with the highest and lowest levels of adult illiteracy, respectively. high unemployment rates barker (1999) defines an unemployed person as an ablebodied person who is without work, is available to work and therefore is seeking work. factors such as lack of proper education and skills, low labour absorption rates by industries and change of seasons (seasonal unemployment) can be pointed as causes of high unemployment rates in most developing countries. however, this view goes against that of harris and todaro (1970) and harris and sabot (1982) who had earlier on argued that unemployment in most developing countries is voluntary. if this is the case, then its cure can be downgraded as a policy concern (kingdon and knight, 2001). to challenge harris and todaro (1970) and harris and sabot’s (1982) stance on ‘voluntary unemployment’, clark and oswald (1994) assessed the factors behind unemployment by analyzing the utility levels of the jobless. their findings were that unemployed persons in various countries have much lower levels of happiness or wellbeing than those in work. consequently, the conclusion drawn is that unemployment is involuntary. this suggests that the greatest levels of poverty are mostly amongst the unemployed in south africa. snower and de la dehesa (1997) thus remain convinced that large-scale unemployment in south africa is responsible for poverty and inequality as it erodes human capital and also creates social and economic tensions. for the period of 1998, kingdon and knight (2001) documented that south africa’s unemployment rate was officially measured at 39%. by year 2009, the rate had dropped to 24.9% due to economic growth which later resulted in an increase in employment opportunities particularly in the formal sector. major contributing industries were private households which comprised mainly of domestic workers (137 000 jobs) and the personal services (132 000 jobs). however, the recent global economic slump has made it difficult for industries to increase their labour absorption rate. as a result, there is still a great need for the unemployment problem to be addressed as a matter of urgency. perhaps, this could be the entry point that the government, agricultural economists and other stakeholders can utilize to push for smallholder agricultural development to create employment and alleviate rural poverty. furthermore, taking the agriculture route can also ease the pressure created in urban centres by the influx of rural migrants in search of formal employment as their source of livelihood. role of agriculture in addressing poverty and food insecurity south africa is self-sufficient in food production (machete, 2004; mclachlan and kuzwayo, 1997). however, this selfsufficiency is only at national and not household level as shown by statistics from the national treasury (2003) and confirmed by terreblanche (2002). these two sources maintain that about 14 million people in the country are vulnerable to food insecurity whilst another 43% of households suffer from poverty. the impact of this problem is the major characteristic of people living in 089 afr. j. food sci.res. rural areas, particularly, in the former homelands. baiphethi and jacobs (2009) and fao (2004) provid evidence that not only do the former homelands have the majority (65%) of south africa’s poor but also 78% of those likely to be chronically poor dwell in such corners of the nation. with such high numbers of the poor staying in the rural areas, development economists have debated a lot on how to tackle this problem but without any consensus being reached. eicher (1994) regards both middle and smallholder agriculture as a positive force in ‘getting agriculture moving’. the same sentiment is shared by the likes of lopez (2002) and machete (2004) who also advocate for the use of agriculture as the main tool with which to tackle rural poverty. mwaniki (2005) also argues that the key lies in increasing the agricultural profitability of smallholder farmers and creating rural off-farm employment opportunities. the inclusion of off-farm employment in addressing poverty is based on the notion that food insecurity in africa is directly correlated with poverty hence it is necessary to not only alleviate poverty but also create wealth for the target population (mwaniki, 2005). however, not every development economist is happy with this approach of using agriculture as the main avenue out of poverty. nonetheless, the importance of smallholder agriculture in developing countries has been acknowledged globally. machete (2004) writes that in an attempt to find how best to use smallholder agriculture to drive rural development, two schools of thought have emerged. the first school of thought based its argument on the statistics published by the national treasury (2003) that the majority of south african citizens, just like in many other developing countries, are poor and dwell in the rural areas. for this reason, this school of thought is of the opinion that since most of these rural poor are already involved in agricultural production or agriculture-related activities, investing in agriculture is the most effective way to reduce poverty. thus, primary focus and investment should be directed towards developing the smallholder agricultural sector than anything else. to lobby for the support of this approach to rural development, advocates of this school used kenya’s example, as cited in lele and agarwal (1989) where smallholder farmers with farms of less than two hectares increased their share of national agricultural production from four percent (4%) in 1965 to 49% in 1985 after getting proper government support. in zimbabwe, smallholder farmers tripled their maize production between 1980 and 1987 due to the launch of a government programme to boost maize and cotton production and development of hybrid maize varieties. according to eicher (1994), these farmers in zimbabwe also managed to increase their share of the national marketable maize surplus from ten percent (10%) in 1980 to 40% in 1987, an improvement that made them to be referred to as “africa’s green revolution success story”. whilst assistance is vital to developing the sector, aid should be geared towards the sustainability of the agricultural systems. in order to achieve this, farmers should be trained on commercialization of their farm produce in order to maximize profits. in this way, the agricultural sector will become attractive to the unemployed. once smallholder farmers become commercial, they tend to become more labour-productive there by creating enhanced chances of getting better financial returns for their efforts. however, even though fan and connie (2005) support this notion, they believed that in order to increase labour productivity, and therefore farmer’s income, there should be either an increase in the land productivity or an improvement in the land to labour ratio. dyer (1997) and havnevik and skarstein (1997) further uphold the view that this inverse relationship between farm size and labour productivity is only in the short term as further increases in farm size in the long run against a small labour force renders smallholders less efficient. as a result, for smallholders to become commercial and efficient in their operations, there should be a balance between the land resources available vis-àvis the quantity of labour available to work on that particular land. failure to strike this balance will result in the overor under-employment of resources which in turn shows inefficiency. other indirect benefits of this initiative of commercializing smallholders include a reduction in rural-urban migration, while the rate of unemployment reduces drastically. apart from the employment advantages expected to emanate from improving the sector, delgado (1998) holds the view that smallholder agriculture is simply too important to human welfare and political stability in sub-saharan africa (ssa). for this reason, it should neither be ignored nor treated as just another small adjusting sector of a market economy. the second school of thought on rural development focuses on the provision of social services and other nonagricultural activities (mwaniki, 2005; orr and orr, 2002; ellis, 1998, 1999). the school believes that it is important for the rural folks to have gardens and farms especially, for purposes of household food production but non-farm enterprises should also be established to help in income generation (machete, 2004; mwaniki, 2005). some of the non-farm income sources include old-age pensions, remittances, wages and family businesses. other non-farm enterprises identified by mahajan and gupta (2011) include trading, agro-processing, manufacturing, crafting and construction. according to dfid (2002), some of these rural non-farm sectors are crucial in economic growth, rural development and poverty alleviation in that they act as growth engines in their own right by supplying urban, rather than rural, demand. in other words, instead of competing with urban enterprises, they actually complement them and thrive on increased urban connectivity. the dfid (2002) further accepts that having such secondary sectors in the rural communities could also provide cheap and effective goods and services thereby adding value to farm commodities. a good ncanywa 090 example of value addition comes in the form of agroprocessing which gives more value to agricultural output whose prices have always been regarded as very low when compared to those of products from other sectors such as the manufacturing sector (fao, 2004). in other words, the second school recognizes and acknowledges the importance of smallholder agriculture in fighting poverty but goes further to advocate for the supporting of other sectors that could run parallel and complement the agricultural sector. according to haggblade et al. (1989), the growth of the small farms also stimulates the growth of business activities created through forward and backward linkages which in turn generates economic growth through production and consumption linkages. van zyl et al. (1996) share the same view as haggblade et al. (1989) that the demand for production inputs from other sectors could be stimulated if there are gains in output resulting from investments in any given sector of the economy. if this happens, then it creates what is known as backward linkages. backward linkages also exist if the farming households use the income they obtain from selling their produce to purchase more farming inputs (investment) or even spend it on other items that have nothing to do with agriculture at all (expenditure), such as television sets, private cars and furniture etc (estudillo and otsuka, 1999). by doing so, they support the manufacturing sector through their agricultural income. dorosh and haggblade (1993) highlight that the initial output gains also raise incomes and consequently, spur consumer demand for other goods and services (forward linkages). this means that there are some non-farm sectors that rely on agricultural produce for their survival (estudillo and otsuka, 1999). thus, the agricultural sector, smallholder sector included, provides other sectors with raw materials and a market. the two schools of thought seem to be both centered on agriculture. the first school is clear on its support for using the smallholder sector as the vehicle with which to drive rural south africans out of poverty. as for the second school, one could actually conclude that even though it advocates for alternative income sources that are nonagricultural, it is also partly dependent on the success of the smallholder agricultural sector for the backward and forward linkages. therefore, regardless of which school of thought one opts to support, the fact remains that agriculture will always have a critical role to play in poverty alleviation and rural development be it directly or indirectly. as part of interrogating the different ways through which agriculture could be used to ameliorate rural poverty, one topic that has been greatly debated amongst development and agricultural economists is the inverse relationship between farm size and efficiency. the big question this debate has been trying to answer is whether smaller farms are more efficient than commercial ones. if they are, then, perhaps agricultural investment should focus towards smallholders at the expense of their largescale counterparts because of the former’s efficient use of limited resources. general findings from the likes of byerlee and de janvry (2009) and ngqangweni et al. (2001) suggest that smallholders can produce efficiently even when they are subjected to opportunity cost scenarios similar to their better-resourced commercial counterparts. this is usually the case with smallholder farmers that have turned commercial in their operation by producing for marketing purposes and not entirely home consumption. the guardian (2012) attributes such efficiency to the fact that the opportunity costs for their land and labour are relatively low thereby making smallholders competitive food producers. in addition to producing enough food, smallholders also represent an emerging market opportunity for local and international agribusiness alike. all this suggests that smallholder agriculture could be as much of a solution to household food security as it is to providing efficient use of scarce resources. in a country like sa where land for large-scale farming is very scarce, small-scale farmers can play a vital role in providing food for the nation without demanding too much of this scarce resource. this is because they are very efficient in terms of production per hectare due to their small farm sizes (carter, 1994; berry and cline, 1979; bharadwaj, 1974). vietnam is a good example where smallholder agriculture has enhanced the food security situation since 1979. according to pinda (2008), the country of vietnam was a very food-deficit country and relied heavily on imports. however, through the development of its smallholder sector, the nation has since become a major food exporter and it is now the second largest rice exporter in the world. furthermore, by 2007 the poverty rate had dropped to below 15%, compared to 58% in 1979 (pinda, 2008). however, the sa case is different. baiphethi and jacobs (2009) estimate that in south africa, at least four million people engage in smallholder agriculture. over the past few years, the main reason for farming has emerged as the need to supplement food rather than use farming as the main source of food in rural areas (baiphethi and jacobs, 2009). this conclusion is consistent with the situation in the eastern cape province where monde (2003) reports agriculture’s contribution towards household income to be very unpretentious. according to aliber (2005), on the average, smallholder agriculture contributes 15% of the total household income in sa, but for the poorest quintile the contribution is estimated at 35%. all together, the high number of people trapped in the poverty circle in the rural areas of sub-saharan africa (ssa) has created an urgent need to find ways to improve smallholder agriculture in order to improve food security at household level. as a solution, peacock (2004) recommends more investment in the smallholder agricultural sector. however, he also recognizes that whilst paying much attention to liberalizing markets and creating macro-economic stability in order to stimulate demand for and prices of agricultural products has dominated the past few years, such policies are necessary but not sufficient. as such, peacock (2004) further proposes the introduction of farmer-led initiatives as another solution. with these initiatives having been introduced, african governments need to increase their financial contributions to help the resource-poor farmers cultivate their land. in the opinion of peacock (2004), governments also need to cooperate with ngos to encourage farmer-led initiatives, work with african other governments to make the case for investment in smallholder agriculture and to lobby for farmers to be involved in the policy-making process. however, if this is to work, there should be a clear policy framework laid down to guide the stakeholders on the proper ways to assist the smallholder sector. this policy framework should be specific to the agriculture sector and allow for a harmonious working relationship between the farmers and both the public and private sectors. andriessi et al. (2007) uphold the view that any form of intervention in the sector should focus on at least one or more of the following; (1) increasing agricultural productivity, (2) enhancing access to resources and services, (3) getting competitive markets and (4) creating support institutions for smallholder farmers, particularly those based in the rural areas. in achieving increased yields, effort must be made to ensure that such improvements are sustainable and not at the expense of other natural resources. therefore, andriessi et al. (2007) are in favour of intensive research and development (r and d) programmes with the results being used as a starting point to inform the intended intervention policies. in consequence, as much as interventions are needed, they should be based on r and d findings and not the personal ambitions or desires of the interveners. in carrying out these r and d programmes, andriessi et al. (2007) have suggested that priority should be given to integrated soil management practices, rain-water harvesting, high quality seeds, post-harvest manage-ment, mechanization, processing and value-chain management. since all these requires level of expertise rarely possessed by the rural smallholder farmers, capacity building is very important to make sure that they are introduced to the farmers in a way that will be understandable (andriessi et al., 2007). synopsis the paper has highlighted a number of critical issues pertaining to poverty and food security in sa and other developing countries. it reviewed the different definitions of the term food security as proposed by different individuals or organizations. a critical discussion of the food security and poverty levels in sa was made, 091 afr. j. food sci.res. including the symptoms of the latter. however, the most sensible conclusion that one can draw based on the discussion made above is that poverty (or food insecurity) is a phenomenon that is going to be around for years to come, especially, with the ever-increasing global population coupled with land degradation, deforestation and global warming. whilst efforts are being made by various governments or organizations to respond to the un’s mdg of halving poverty by year 2014, it is going to take a lot more than what is currently being done. at the same time, it is widely agreed that if proper interventions are put in place, smallholder farmers can contribute to a vibrant rural sector, where locally-produced products and services meet growing local demand. this, in turn, can spur sustainable off-farm employment growth in services, agro-processing and small-scale manufacturing. nonetheless, there is still a long way to go if food insecurity is to be tackled using smallholder agricultural approach. the type of intervention needed in the smallholder sector is not easy to isolate due to the existence of various factors that affect the sector such as the skills levels, illiteracy and financial institutions etc. as such, formulating the “best” piece of intervention that will allow the smallholder sector to deliver what large businesses require, and in turn to encourage businesses to adapt their models to be inclusive and supportive of small-scale agricultural producers will take a while and also a number of different attempts. with poverty having so many faces, enabling the rural population to produce their own food is a good strategy but on its own, it is not enough to completely eradicate this problem. as such, the sa government has already embarked on a number of other poverty alleviation strategies such as cash transfers, provision of health care, education, housing, and basic services (water, electricity, sanitation). regarding agriculture, it has also tried to stimulate the performance of the sector by introducing a few programmes like the massive food programme in the eastern cape, the revitalization of irrigation schemes, supporting of farmer cooperatives, farmer support programmes (fsps), etc. in conclusion, one might ask how then can improving the agricultural sector help ameliorate the different faces of poverty and also meet the different mdgs set by the un. the answer is in the revitalization of the smallholder agricultural sector to improve the lives of the rural poor thereby relieving some pressure from the government when it comes to issues such as the extent of social grants, high unemployment and poverty levels and malnutrition problems etc. the fact that most of the food insecurity and poverty problems are experienced mostly in the rural areas of sa where the majority of the country’s population is found means that agriculture, especially smallholder agriculture can be the correct remedy for these problems. this is because the sector promotes food security and enhances rural livelihoods at household level first where improvements are needed. ncanywa 092 once the household needs have been met, then the agricultural sector’s effects can trickle down to the rest of the country. one could therefore could by saying that the arguments raised by the two schools of thought regarding dealing with poverty are very valid. policy developers, developments economists, the government and all other role-players should find roles that suit their resources and abilities within the two schools. this is to say that as others concentrate on boosting the productivity of the smallholder farmers, others could perhaps focus on how to stimulate the forward and backward linkages so that there is synergy between the sector and other critical sectors. focus will be on agriculture together with all the complementing sectors to achieve fast and overall success. references aliber m (2001). chronic poverty and development number 1: study of the incidence and nature of chronic poverty and development policy in south africa: an overview. university of the western cape, cape town, south africa. aliber m (2005). 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in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (8), pp. 190-195, august, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of ractopamine detection in various tissues of swine wentao xu 1 , kunlun huang 1 , aike deng 1 , baiqiang zhai 1 , heng zhao 1,2 , yingcong li 1 , zhihong liang 1 and yunbo luo 1 * 1 college of food science and nutritional engineering, china agricultural university, beijing 100083, china. 2 current address: cell biology and immunology group, wageningen university, p.o. box 338, 6700 ah, the netherlands. accepted 09 april, 2015 ractopamine has been developed to be the main -agonist substance used illegally in meat producing animals. a simple and efficient extraction and purification procedure for ractopamine was developed by means of the immunoaffinity column (iac) as a cleanup tool. purified polyclonal antibodies against rct were produced and coupled covalently to cnbr-activated sepharose 4b. both the binding conditions and elution protocols were optimized and the capacity, reusability, precision and accuracy of iac were determined. the iac was successfully employed to isolate and purify the rct from the various tissues of swine. subsequently, enzyme linked immunosorbent assay (elisa) procedures were established further on to measure rct. the antibodies showed negligible cross-reactivity with other -agonists. iac-elisa allowed 0.2 ng/ml of rct to be detected in urine and 0.5 ng/ml to be detected in other various tissues of swine, which makes this method an acceptable screening tool to access rct. iac-elisa for the detection of rct was validated by lc-ms and the correlations between the results from lc-ms and those from iac-elisa were all high (above 0.89). key words: purification, ractopamine, swine, immunoaffinity column, enzyme linked immunosorbent assay. introduction -agonists have been misused as growth promoting agents in meat producing animals over 20 years. ractopamine (rct) has been developed to be the main -agonist substance used illegally for this purpose. ractopamine is a phenethanolamine leannessenhancing agent that has been approved by u.s. food and drug administration (fda, 2000; muirhead, 2000) and regula-tory agencies in brazil, venezuela, colombia, guatemala, dominican republic, and the philippines as a feed addi-tive for swine (shelver and smith, 2002). phenethanola-mine -agonists have a history of being used for unautho-rized purposes by livestock producers (kuiper et al., 1998; mitchell and dunnavan, 1998; shiu and chong, 2001). the positive influence of ractopamine hydrochlo-ride on these economically important traits should make the product attractive to swine growers, and to producers *corresponding author. e-mail: lyb@cau.edu.cn. of livestock species for which ractopamine is not approve the presence of drug residues in animal tissues is a potential food safety concern, especially when the compound has been used illegally or in a manner not prescribed by regulatory official. in an effort to combat the illicit use of -agonists compounds, regulatory organizations worldwide test animal tissues for the presence of illicit drugs through both screening and confirmatory assays. for residue analysis, the analyte is separated from the matrix by liquidliquid or solid-phase extraction (spe), followed by liquid or gas chromatography or immunological analysis. sample preparations steps are often time consuming and tedious, making an efficient cleanup method very desirable when high throughput is required. high sensitivities of immunoassays are practically desirable for off-label drug monioring because it may be desirable to detect the analyte even after extended withdrawal periods. -agonists immunoassays have been developed for clenbuterol (yamamoto and iwata, 1982), fenoterol (haasnoot et al., wentao et al. 191 1994) and ractopamine (elliott et al., 1998; ba, 1998). materials and methods reagents and instruments: cnbr-activated sepharose 4b was obtained from pharmacia biotech. protein a-sepharose 4b was purchased from sigma. ractopamine was provided by eli-lilly (usa). liquid scintillation cocktail (ecolitetm) was purchased from icn biomedicaal, inc. animal specimens including swine live and muscle samples were obtained from swine known to be free of ractopamine and were pooled (beijing laboratory animal research center, china). microplate reader mk3 was from finland labsystems. all other chemicals or solvents were obtained from sigma or amerscreso and were analytical grade or better. immunogen and enzyme conjugate preparation: rct (99.999%, daming company henan, china) was coupled to a carrier, bovine serum albumin (bsa), and to an enzyme, horseradish peroxidase (hrp), using the coupling agent, butane-1,4-diol diglycidyl ether (yamamoto and iwata, 1982; haasnoot et al., 1994). immunogen: a 30 mg amount of bsa was dissolved in 0.5 ml of de-ionized water and the ph was adjusted to 10.8 using 0.5 mol/l sodium hydroxide. a 6 ul volume (30 umol) of butane-1,4-diol diglycidyl ether was added and the mixture was incubated for 20 h at room temperature under a nitrogen atmosphere. a 15 mg (50 umol) amount of rct was dissolved in 0.5 ml of 0.1 mol/l sodium hydroxide, 200 ul dimethylformamide being added to help solubility. this solution was degassed using nitrogen then added to the epoxy-activated bsa solution. the reaction mixture was incubated for 20 h at room temperature under a nitrogen atmosphere. purification of the rct-bsa immunogen was achieved using extensive dialysis (10 000 molecular mass off) against 0.15 mol/l -1 sodium hydroxide. the immunogen was diluted with 0.15 mol/l sodium hydroxide to give a final protein concentration of 1 mg/ml and stored at –20 o c until required. enzyme conjugate: a 20 mg amount of hrp was dissolved in 0.5 ml of de-ionized water and the ph was adjusted to 10.8 using 0.5 mol/l sodium hydroxide and the solution was de-gassed with nitrogen. a 50 l volume (5 mol) of a solution of butane-1,4-diol diglycidyl ether was added and the mixture was incubated for 20 h at room temperature under a nitrogen atmosphere. unreacted butane1,4-diol diglycidyl ether was removed by gel filtration (sephadex g25-m, equilibrated with 1 mmol/l sodium acetate). a 20 mg amount of rct was added to the activated hrp solution, 200 ul dimethylformamide being added to increase solubility. this solution was degassed using nitrogen. the enzyme-labelled conjugant was then purified by gel filtration (sephadex g25-m, equilibrated with 1 mmol/l sodium acetate) and by use of dextran-coated charcoal. the hrp-rct was stored at –20 o c until required. production and purification of polyclonal antibodies. the polyclonal antibodies against rct-bsa were obtained from new zealand white rabbits (beijing laboratory research center, china). in brief, immunogen was emulsified with an equal volume of freund’s complete adjuvant to give a final concentration of 0.5 mg/ml. this mixture was given in four intradermic injections. boosting injections of freund’s incomplete adjuvant were made at 2-week intervals. blood samples were collected 1-week after the third booster injections and tested for titer determination. blood collected from rabbits was first allowed to stand overnight at 4 o c, then centrifuged at 200 × g for 15 min. the obtained antiserum was purified in three steps by firstly using a saturated ammonium sulfate method (elliott et al., 1998). the 20 ml antiserum was purified by the saturated ammonium sulfate method. at last the precipitates were dissolved in 2 ml of pbs and dialyzed against pbs until no sulfate ion could be detected in the dialysis solution with 0.5 m bacl2 acidified with hcl. the obtained immunoglobulin (igg) was purified in the second step by the way of protein a-sepharose 4b (zhao et al., 2003). the eluates (0.1 m glycine buffer ph 7.2, 0.5 ml) were collected in tubes containing 50 ml of 1 m tris (ph 8.0) and mixed gently to bring the ph back to neutral. at last, the antiserum was purified to remove the anti-bsa antibodies by adsorption of anti-bsa antibodies to a column containing bsa cross-linked to an affiprep 10 matrix (bio-rad) and the column could be regenerated by elution with 50 mm glycine (ph 2.3)-0.5 m nacl-0.02% triton x-100 (giraudi et al., 1998). the igg concentration in the obtained solution was calculated based on the uv absorption difference between 280 and 260 nm (chen et al., 1993). the formula used for the calculation was cprotein (mg/ml) = 1.45 × a280 nm 0.74 × a260 nm. the purity of the purified igg was checked on sds-page gel. enzyme immunoassay procedure: a direct competition elisa for-mat was utilized to measure ractopamine binding and cross reac-tivity to related compounds. the checker board procedure was used to optimize the enzyme tracer and the anti-rct antibody concen-trations. after optimization, the elisas were processed as follows: microtiter plates were coated with 200 ul diluted antiserum in bicarbonate buffer (0.05 m, ph 9.6) overnight, 4 o c or 2 h at room temperature. the plate was washed with pbst (10 mm pbs containing 0.05% tween 20, ph 7.4) three times. serial dilutions (50 ul) of the analyte or sample solution in pbs-organic solvent, together with 50 ul of the tracer, was added to the wells and incubated for an appropriate time. after another washing step, 100 ul per well of tmb solution (400 ul of 0.6% tmb-dmso and 100 ul of 1% h2o2 diluted with 25 ml of citrate-acetate buffer, ph 5.5) was added. followed by incubation at 37 o c for 30 min. color development was stopped by adding 50 ul/well of 2 m sulfuric acid. the plates were read at 450 nm with microplate reader mk3 (thermo labsystems, vantaa, finland) and the resulting curves fitted with a four-parameter logistic equation to determine the ic50. the ic50 was defined as the concentration of inhibitor required to inhibit color development by 50% compared to control wells containing no competitors. antiserum cross-reactivities: competitors used for the competetion studies are fenoterol, isoxsuprine, salmeterol, fomoterol and clenbuterol, which have comparable structure to ractopamine. the sensitivity of the purified rabbit antiserum was determined by constructing calibration curves and calculating the 50% inhibition of control index (ic50), that is, the mid-point of the calibration curve. competitive elisa was performed using direct assay format with competitor concentration of 1 ug/ml. compounds that produced sigmoidal like curves were fitted to the four parameter logistic equation using sas; y (%b/b0) = (a d)/ [1 + (x/b) c ] + d (raab, 1983). coupling antibody to cnbr-activated sepharose (primot et al., 2000): purified antibodies against rct were coupled to cnbractivated sepharose to generate the immunoaffinity column. the igg solution was dialyzed against 0.1 m nahco3, 0.1 m na2co3, 0.2 m nacl, ph 8.3, and was added to the sepharose beads activeted with 1 liter of 1 mm hcl under constant rotation at 4 o c overnight at a concentration of 2 mg/ml (igg/sepharose beads). then the supernatant was removed and the beads were incubated with 1 m ethanolamine ph 9.0 to deactivate the remaining active sites for 2 h under constant rotation at 4 o c. beads were washed with 0.1 m acetate, ph 4.0, 0.5 m nacl, then with bead buffer (pbs ph 7.4, 0.02 nan3) and stored in 20% suspension in bead buffer at 4 o c until further use. negative rabbit antiserum containing no antibody against rct was applied to generate the blank column as a negative control (xu et al., 2005). determination of the optimal conditions of ractopamine on the immunoaffinity column (xu et al., 2005): iacs were subjected to multiple loadings and elutions to determine column stability. col-umns were loaded with 5 ug ractopamine, washed with both 10 ml 10% meoh and 10 ml pbs (ph 7.4), and eluted with either 10 ml 100% meoh or 10 ml 0.2 m glycine buffe (ph 2.5). all eluants were measured by elisa and lc to determine recovery. between uses, 192 afr. j. food sci. res. columns were stored at 4 o c in pbs-0.02 nan3 (ph 7.4). to select the optimal ph condition for elution of rct. 1 ml solution of a mixture of antibody rct-sepharose beads were washed with 5 ml of bead buffer. the matrix was incubated with 2 ml of rct solution (0.25 mg/ml) and 2 m of ova (0.25 mg/ml) for 40 min under constant rotation at 4 o c. after 7 times washing, the column was eluted with 0.2 m glycine buffer (2 ml) at various ph conditions (ph 5.5, 5.0, 4.5, 4.0, 3.5, 3.0 and 2.5). the collected fractions were assayed for rct by elisa. column capacity and reusability of iac: column capacity for rct was determined by passing 10 ug rct in 10 ml pbs through an affinity column at 1 ml/min. to ensure maximum binding, the effluent was loaded to the column once more and washed with 10 ml of 10% meoh (v/v) and 10 ml pbs (ph 7.4). bound rct was eluted with 0.2 m glycine buffer (ph 2.5) at 0.5 ml/min. rct in the glycine eluate was determined by elisa and lc. to determine column reusability, 1 ml samples (100 ppb) were loaded onto 2 ml iac for ractopamine. after the column was washed and eluted as the above, the column regenerated with equilibrating buffer and allowed to stand for various periods, from 10 min to 24 h. the capacity of the regenerated column was then determined by reloading with standards. this process was repeated ten times. rct both in the washing eluate and in the glycine eluate was determined by elisa and lc. precision and accuracy of iac. samples of 100, 1000, 2500, and 5000 ng ractopamine mixture were fortified in 10 ml urine and loaded onto a 1 ml iac. these represent 10, 100, 250, and 500 ppb fortified ractopamine samples. columns were washed with 10 ml of 10% meoh (v/v) and 10 ml 0.1 m sodium phosphate buffer (ph 7.4), and then eluted with 0.2 m glycine buffer (ph 2.5). ractopamine in the glycine eluant was quantified by elisa and lc. the procedure was repeated 3 times for each fortification level. preparation of urine, muscle, kidney, and liver samples, and application of iac-elisa: a 10 g test portion of tissue was added to 10 ml pbs (ph 7.4) and the mixture was homogenized for 1 min with a tissumizer (tekmar company, cincinnati, usa) set a high torque. samples were then cooled in an ice bath. this was repeated until no large particles were visually observed (usually 2 4 homogenization cycles) . supernatants were separated from tissue debris by centrifugation at 12 000 × g for three times and stored at – 20 o c until used. ractopamine (10 and 50 ng) was dis-solved in pbs (ph 7.4) or various supernatants as mentioned above from various issues of swine. these samples were to be assayed by elisa and iac-elisa in order to determine the matrix effect and to calculate the detection limit of various issues. validation of ractopamine lc/ms (kootstra et al., 2005). all the samples were evaporated to dryness at 55 under a stream of nitrogen. the residue is dissolved in 100 ul 10:90 (v/v) methanol: water. calibration curve was set up with ten-fold dilution of ractopamine in 10:90 (v/v) methanol:water, from 100 to 0.1 ng/ml. these solutions were transferred to a lc vial and 20 ul is injected onto the lc-ms system. equipment the ms system is a agilent lcq classic-system equipped with an apci+ interface. the lc system consists of a p4000 spectra system quaternary pump and an as3000 spectrasystem autosampler (agileng, wilmington, de, usa). chromatographic separations are carried out on a phenomenex luna 5 m c18 250 mm × 4.6 mm column at an oven temperature of 50. the lc gradient uses two solvents: 95% methanol 10 mm ammonium acetate (a) and 5% methanol 10 mm ammonium ace-tate (b) . flow rate 0.20 ml/min, linear gradient from 6% a80% a in 20 min. after 20 min the system is reconditioned for 10 min at 6% a. ms detection and conditions the analytes are detected in mrm (ms2) mode. the mass fragments are used for the screening method. the instrument was tuned and calibrated according to manufacturer’s specifications. results and discussion production and purification of polyclonal antibodies the polyclonal antibody against rct was firstly purified through a saturated ammonium sulfate and a protein a sepharose 4b affinity column. this twostep purification using 20 ml of antiserum resulted in a 70 mg of pure antibody. the antibody purity was checked by sdspage, which showed one strong band (the heavy chain) and one weak band (the light chain) under the reducing conditions (data not shown). the purified antibodies were repeatedly flown through the column for over three times and the anti-bsa antibodies were efficiently separated from the anti-rct antibodies. both polyclonal antibody and monoclonal antibody were used in immunoaffinity chromatography. the most usable antibody was the polyclonal antibody (james et al., 2004). polyclonal antibodies, obtained by immunizing a rabbit or goat and purifying the immunoglobulin fraction from the resulting serum. one can use quite pure antigen to avoid raising unwanted antibodies to minor impurities in the protein preparation. the obtained antiserum can be purified to remove many impurities and other unwanted antibodies by methods such as saturated ammonium sulfate, protein a sepharose 4b affinity column or affiprep 10 matrix. control column when the blank column was loaded with rct spiked water using the modified immunoaffinity protocol as the above, rct was not specifically adsorbed with the blank column containing the control igg that not generated toward ractopamine. the amount of rct in the washing and elution fractions was the same as that of columns containing antirct antibodies. the above result demon-strated that the retention of rct was primarily due to the anti-rct antibody. the presence of rct in the blank column eluate indicated that the rct was only slightly re-tained by the blank column. this might possibly be due to insufficient blocking of active sites or might indicate the wash volume was not adequate. these results show that ractopamine was specifically retained by the ractopamine iac, but not by the control column. determination of the optimal conditions of ractopamine on the immunoaffinity column elution of ractopamine from the iac with glycine resulted in excellent recovery of 80 100% and offered two main advantages over elution with methanol. firstly, elution with glycine buffer resulted in cleaner chromatograms of parent ractopamine after fortification in samples. elution with methanol resulted in the co-elution of fluorescent compounds that interfered with chromatographic analysis wentao et al. 193 figure 1. ph reducing elution of antibody against rct-sepharose beads with 0.2 m glycine buffer. the eluting amount of rct increased with the decrease of eluting ph. figure 2. iac stability with multiple elution using 0.2 m glycine, ph 2.5 or 100% meho. results are expressed as percentage of loaded ractopamine in the elution fraction (n = 3). almost half of the error bars for the glycine elution are too small to be visible. ractopamine iacs quickly degraded when eluted with > 50% methanol and showed marked performance problems in the second use. however, when 0.2 m glycine, ph 2.5, was used to eluate the analytes, only minimal decreases in column capacity occurred after 10 uses. of ractopamine (shelver and smith, 2002). secondly, iacs were stable after glycine buffer was used, whereas columns eluted sequentially with solvents containing a high percentage of methanol rapidly degraded (discussed below). the major disadvantage of using glycine as the eluting buffer is the increased amount of time required to concentrate it (that is by evaporation) comparing to methanol. figure 1 showed that the antibody (anti-rct)-sepharose beads was specifically combined with rct, and the eluting amount of rct increased with the decrease of eluting ph. if the eluting ph was too low, the antibody (against rct)-sepharose beads were unstable which affected the combination between antibody and antigen, so the prime ph of elution buffer was selected as ph 2.5. in stability tests, ractopamine iacs quickly degraded when eluted with >50% methanol and showed marked performance problems in the second use. however, when 0.2 m glycine, ph 2.5, was used to eluate the analytes, only minimal decreases in column capacity occurred after 10 uses (figure 2). furthermore, ractopamine iacs stored at 4 o c in pbs-0.02% sodium azide were stable over 3 months. because of the apparent advantages of glycine elution, subsequent studies on the effects of matrix (urine, muscle, kidney, and liver) were performed with glycine as the elution solvent. spiked ractopamine urine samples were used to determine the precision and recovery at various ractopamine levels. the recoveries of ractopamine from the urine samples for 10, 50, 100, 250, 500 ng/ml were 81.6, 87.3, 90.8, 86.5 and 83.4%, respectively. the standard errors of the measurements were 6.5, 7.8, 10.3, 8.4, and 5.6 ng/ml respectively. the iac gave excellent recovery and reproducibility. the clean sample from iac allows the sensitivity to be easily controlled by concentrating the eluant to obtain greater sensitivity. the method was confirmed useful over a very wide range of concentrations with only minor alterations in the lc procedure. matrices such as urine, tissues, and serum have great impact on the results of elisa when these samples were to be detected directly by elisa. but after these samples were purified by iac, the effect can be eliminated. so iac-elisa enhances not only the sensitivity but also the accuracy of detection for ractopamine. antiserum cross-reactivity under these conditions, cross-reactivity to-wards other agonists was determined. a number of -agonists, which table 1. cross-reactivity profile toward -adrenergic agonists from antiserum raised to ractopamine. compound ractopamine fenoterol isoxsuprine salmeterol fomoterol clenbuterol ic50 3.3 650 >80.000 >80.000 >80.000 >80.000 % cr 100% 0.5 <0.05 <0.05 <0.05 <0.05 figure 3. representative standard curve for rct by iac-elisa. each point represents the mean of 5 determinants. vertical bars indicate + sd about the mean. figure 4a and 4b. the precursor ion and the daughter ions of ractopamine in all samples detected and qualified by ms-ms. the precursor ion of ractopamine is 302.1 and the daughter ions of ractopamine are 284 and 164. were fenoterol, isoxsuprine, salmeterol, fomoterol and clenbuterol, tested for their cross-reactivity because they have comparable structure to ractopamine. fenoterol showed a cross-activity of 0.5% and other -agonists re 194 afr. j. food sci. res. wentao et al. 195 reacted 0.05 % (table 1). thus, this elisa can only recognize ractopamine of the compounds tested. detection limit and standard curve: the results obtained from the analysis of residue-free urine and other samples were used to estimate the detection limit. figure 3 showed a representative standard curve for ractopamine. the detection limit was 0.2 ng/ml of ractopamine in urine, based on the mean response for the control sample extracts plus three times the standard deviation with ic50 of 3.3 + 0.25 ng/ml (figure 3). in addition, iac-elisa allowed 0.5 ng/ml of rct to be detected in other various tissues of swine. validation of iac-elisa by lc-ms the validation indicated that all the eluates contain the ractopamine (the precursor ion of ractopamine is 302.1 and the daughter ions of ractopamine are 284 and 164) (figure 4a and 4b) and the correlations between the results from lc-ms and those from iac-elisa were all high (above 0.8943). the amounts of ractopamine in the samples measured by iac-elisa agree with the results from lc-ms but the concentrations of rct found with the iac-elisa were more or less higher compared with those obtained using lc-ms. this might be owing to the pre-sence of metabolites of rct in the samples, which could be detected by iacelisa but could not be detected by lc-ms. the method of iac-elisa has been proven to be a simple, practical and reliable method for screening ractopamine. acknowledgements this work was funded by the ‘863’ project and “948” project of china and we express our gratitude to the ministry of science and technology and the ministry of agriculture of china for financial support respectively. references ba d (1998). contemporary immunological technology and application, joint press of beijing medical sciences university and china union medical college, beijing, china. p. 312. chen c, ratcliffe na, rowley af (1993). detection, isolation and characterization of multiple lectins from the haemolymph of the cockroach, blaberus discoidalis. biochem. j. 294: 181-191. elliott ct, thompson cs, arts cjm, 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shiu tc, chong yh (2001). a cluster of clenbuterol poisoning association with pork and pig offal in hongkong, pubic health epidemiol. bull. (hongkong). 10: 14-17. xu wt, huang kl, zhao h, luo yb (2005). application of immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of phosphinothricin-n-acetyltransferase detection in genetically modified maize and rape. j. agric. food chem. 53: 43154321. yamamoto i, iwata k (1982). enzyme immunoassays for clenbuterol, a adrenergic stimulant. j. immunoassay. 3: 155-171. zhao mp, liu y, li yz, zhang xx, chang wb (2003). development and characterization of an immunoaffinity column for the selective extraction of bisphenol a from serum samples. j. chromatography b. 783: 401-410. african journal of food science research vol. 2 (4), pp. 071-073, april, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper study on the angiotensin converting enzyme inhibiting properties of the terpenoid extract of (crataegus monogyna) habib naeem, hasnain haider, yaseen b. department of microbiology, school of medicine, infectious and tropical diseases research, ahvaz jundishapur university of medical sciences, ahvaz, iran. accepted 11 march, 2014 crataegus monogyna is mainly used in the treatment of cardiac and circulatory system disorders. in vitro and clinical studies are indicative of the fact that the hydroethanolic extract of c. monogyna has angiotensin converting enzyme (ace) inhibitory activity. this study sought to support these claims through the use of in silico modelling techniques. possible binding conformations for β-amyrin, oleanolic acid and ursolic acid were generated using captopril, as well as enalaprilat and lisinopril, as template ligands. the ligand binding affinity (lba) of each was calculated and the best binding conformation of each triterpene was established. results indicate that these naturally occuring terpenes possess in silico predicted ligand binding affinities that are superior to both the small molecule captopril and the larger molecules enalaprilat and lisinopril. key words: crataegus monogyna, hydroethanolic extract, angiotensin converting enzyme (ace) inhibition, in silico. introduction hypertension is recognised as being one of the most preventable causes of premature morbidity and mortality. the world health organization (who) statistics indicate that hypertension affects approximately 40% of all individuals aged 25 years and over. it is prevalent worldwide in both developed and developing countries (who, 2012). the majority of hypertensive patients currently rely on the use of angiotensin converting enzyme (ace) inhibitors for management of their condition (sweileh et al., 2009). in silico models are of value in that they allow virtual screening for desirable properties and further optimization *corresponding author. e-mail:habib_n@gmail.com optimization of identified lead candidate molecules, thus drastically lowering financial and time requirements for the chemical synthesis and biological testing of promising leads. they also minimise animal testing; a factor that should not be considered superficial in the highly regulated and ethically conscious scenario in which contemporary drug discovery operates (kapetanovic, 2008). in vitro and clinical studies have indicated that the triterpenic extract of crataegus monogyna is capable of exerting an inhibitory effect on the ace (attard and attard, 2006). subsequent to this work, we hereby report the results of a static in silico investigation of the ace and of the molecular and conformational bases for the ace inhibitory effect exerted by the terpenoid extract of c. monogyna, which to our knowledge has as yet not been described. http://www.internationalscholarsjournals.org/ habib et al 071 materials and methods protein data bank (pdb) deposition selection three protein data bank (bernstein et al., 1977) depositions were selected as templates for this study. these were pdb ids 1uzf (natesh et al., 2004), 1uze (natesh et al., 2004) and 1o86 (natesh et al., 2003), describing the bound co-ordinates of testicular ace and the small molecules captopril (resolution 2.00 ǻ), enalaprilat (resolution 1.82 ǻ) and lisinopril (resolution 2.00 ǻ). pdb deposition visualisation and modelling molecular visualisation and modelling were carried out using sybyl ® (sybyl 7.3, tripos international, 1699 south hanley rd., st. louis, missouri, 63144, usa.) (sybyl 7.3, tripos international, cartera™). the selected pdb depositions were treated identically during this phase of the study. specifically, each crystallographic deposition 1uzf, 1uze and 1o86 was read into sybyl ® (sybyl 7.3, tripos international, cartera™) with precautions being taken to preserve the bound co-ordinates and consequently the bioactive conformation of each. all moeities considered as superfluous to binding were edited. this means that in the case of 1uzf, two chlorine atoms, 2 n-acetylglucosamine molecules and all water molecules lying at a distance ≤ 5 å were removed. in the cases of 1uze and 1o86, two chlorine atoms and two glycine molecules were removed. the result of this editing process consequently was holo-ace bound to captopril, enalaprilat and lisinopril, respectively with water molecules at a radius ≤ 5 å being retained. retention of these water molecules was carried out on the premise that crystallographic data was suggestive of the fact that their proximity to the bound small molecules and the ligand binding pocket (lbp) could give rise to a situation in which bound ligands could be stabilised within the lbp through the formation of water bridges. this editing process was performed in preparation for molecular dynamics studies which will be carried out during subsequent stages of this study. the small molecules captopril, enalaprilat and lisinopril were subsequently extracted, using sybyl ® (sybyl 7.3, tripos international, cartera™), from their respective ace ligand binding pockets (ace-lbp). each small molecule was saved in pdb and mol2 formats while the apo-ace with the retained water molecules (n = 143, 455 and 569, respectively) were saved in pdb format. the choice of file format in which to save each moeity was a function of software requirements of subsequent phases of the study. specifically, ligand binding affinity (lba) and de novo design were carried out using the algorithms of wang and co-workers in score ® (wang et al., 1998) and ligbuilder® (wang et al., 2000), respectively. these require that small molecules be saved in mol2 format and that the protein receptor be saved in pdb format. vmd® (humphrey et al., 1996) was used for image generation and this required that all molecules be read in pdb format. analysis of the ligand binding pocket the extracted bioactive conformations of the small molecules captopril, enalaprilat and lisinopril were used as probes in order to generate ace-lbp maps of each bound conformation of the ace. this was done using the pocket module of ligbuilder ® (wang et al., 2000) which exploited the contacts forged between the small molecule and the ace-lbp in each case to generate a 3d bond type specific map of the ace-lbp as described in each deposition being studied. this process also generated proposed pharmacophores for each deposition. elucidation of ligand specific lbp maps and pharmacophores was carried out based on the fact that it is known that receptor tertiary structure and lbp conformation is specific ligand driven. this process consequently served to highlight the conformational differences that the 3 ligand probes induced within the ace-lbp. at the end of this process, consequently, 2 output files were generated for each deposition. these were: 1. the key_site_file depicting the key interaction sites between the amino acid side chains lining the ace-lbp and the resident small molecule. by convention, hydrogen bond donor sites were represented in blue, hydrogen acceptor sites were represented in red, while hydrophobic sites were represented in cyan. 2. the pharmacophore_file depicting the proposed pharmacophoric model. colour conventions were assigned in the same manner as described for the key_site_file. both files were saved in pdb format such that they could be later used in the visualization software vmd® (humphrey et al., 1996). estimation of the ligand binding affinity (lba) of the template ligands for the ace receptor the apo-ace files which were generated when the selected pdb depositions were edited together, with their respective extracted small molecules saved in mol2 format, which were read into and processed in score ® (wang et al., 1998). through its static algorithm, the in silico provided lba (pkd) of each of the small molecules captopril, enalaprilat and lisinopril and was calculated for the respective cognate receptor. superimposition of the triterpene molecules onto the bound coordinates and 3d volume of the template ligands in this part of the study, the bioactive conformations of the small molecules captopril, enalaprilat and lisinopril together with the 3d volumes which they occupy within their respective ace-lbp conformations were used to guide β-amyrin, oleanolic acid and ursolic acid, all of which are constituents of c. monogyna, into the 3 conformations of the ace-lbp. this was done in order to identify the highest affinity conformers for each molecule, and to utilise these during subsequent stages of the rational drug design process. identification of the optimally binding conformations of the triterpenic small molecules was carried out using the similarity suite algorithm in sybyl ® (sybyl 7.3, tripos international, cartera™). this algorithm facilitated initial positioning of each small molecule within the ace-lbp based on the conformation of its cognate ligand. subsequent to this process the triterpenic molecules were allowed conformational rotation within each ace-lbp conformation. the 21 conformers for each molecule which exhibited optimal binding characteristics were selected and their in silico lba (pkd) was then quantified in score ® (wang et al., 1998). results the in silico lba (pkd) of captopril, enalaprilat and lisinopril for their cognate receptor was predicted to be 5.36, 6.44 and 6.53, respectively. significant differences in ace-lbp occupation by captopril, enalaprilat and lisinopril could be seen when the lbp maps generated by 072 afr. j. food sci.res. table 1. the lba (pkd) of the 21 optimally binding conformers of each terpene ligand based on the three different template ligands employed in this study as calculated in score ® . captopril enalaprilat lisinopril β-amyrin oleanolic ursolic β-amyrin oleanolic ursolic β-amyrin oleanolic ursolic acid acid acid acid acid acid 6.56 6.95 6.74 7.03 7.04 7.26 6.98 6.78 7.37 6.56 6.95 6.74 7.00 7.13 7.23 7.05 6.66 7.34 6.30 7.04 6.82 7.24 7.14 7.35 6.81 7.19 7.41 6.55 6.62 6.68 7.10 7.21 7.21 7.09 6.93 6.95 6.56 6.70 6.69 7.04 7.17 7.15 7.11 6.94 6.85 6.62 6.64 7.18 7.15 7.13 7.59 7.00 7.00 6.96 6.74 6.68 7.14 7.18 7.20 7.29 6.64 7.04 7.02 6.63 6.94 6.53 7.03 7.20 7.19 6.89 6.99 7.30 6.60 6.99 6.94 6.94 7.19 7.36 6.62 6.89 7.34 7.25 6.53 7.27 7.30 7.41 7.25 6.53 7.35 6.85 6.57 6.94 7.30 7.25 7.05 7.31 7.25 6.59 7.27 6.67 6.89 7.24 7.20 7.42 6.93 6.88 6.87 7.08 6.61 6.64 7.29 7.20 7.43 7.42 6.89 6.90 7.05 6.64 6.69 7.69 6.69 7.44 7.43 6.94 6.90 7.17 6.56 6.98 7.84 6.67 7.46 7.26 6.94 6.52 7.07 6.47 6.36 7.11 6.68 7.25 7.08 6.55 6.52 6.62 6.47 6.94 7.79 6.77 7.29 7.21 6.74 6.37 6.81 6.47 6.47 7.15 7.05 7.11 7.20 6.39 6.73 6.63 6.82 6.61 7.41 6.80 7.02 6.67 6.68 6.74 6.80 6.86 6.17 6.76 6.77 7.05 6.58 6.74 6.11 6.55 6.56 6.95 6.83 7.03 7.04 7.26 6.98 6.78 7.37 figure 1. key interaction sites of captopril (in orange), enalaprilat (in green) and lisinopril (in blue) rendered in vmd ® . figure 2. proposed pharmacophores of captopril (in orange), enalaprilat (in green) and lisinopril (in blue) rendered in vmd ® . each molecule were compared (figure 1). similarly, the proposed pharmacophores in each case were also dissimilar (figure 2). the lbas of the 21 optimally binding conformers of β-amyrin, oleanolic acid and ursolic acid for the captopril-, enalaprilatand lisinoprilbound conformations of the ace were calculated and are shown in table 1. the most salient finding in this case was that the predicted in silico lbas of all 3 experimental triterpenes (pkd 7.25, 7.46 and 7.84) exceeded those calculated for captopril, enalaprilat and lisinopril (pkd 5.36, 6.44 and 6.53, respectively). discussion this in silico study further corroborates the hypothesis habib et al 073 of attard and attard (2006) that the triterpenic extract of c. monogyna has ace inhibitory activity. all three molecules β-amyrin, oleanolic acid and ursolic acid exhibit binding affinities (7.25, 7.46 and 7.84, respectively) that are superior to those of captopril, enalaprilat and lisinopril (5.36. 6.44 and 6.53, respectively), implying a superior inhibitory activity when compared to the ace inhibitors that are currently in widespread clinical use. although this study utilised algorithms that were static, it was still possible to infer the importance of understanding the dynamic nature of both the ace and its cognate small molecules. this is borne out by the fact that ace-lbp maps and proposed pharmacophores were obtained that differed according to resident ligand. this was indicative of the fact that lbp conformation is essentially ligand driven. the fact that for example, ursolic acid had a lba which was highest when captopril was used as a template may be taken to imply that ursolic acid adopts a captopril-like conformation within the ace-lbp. a similar conclusion may be drawn for oleanolic acid whose best lba was observed when its conformation within the ace-lbp was modelled on that of enalaprilat. it was also interesting to note that in the case of β-amyrin, the in silico calculated lba for its optimally binding conformation remained constant irrespective of which ace inhibitor it was modelled on. this conformation of β-amyrin is consequently very interesting from a rational drug design point of view. the results obtained from this study consequently open new avenues for the rational design of ace inhibitors based on the triterpenic scaffold. a molecular dynamics simulation through which the dynamic interactions between the ace and the ace inhibitors captopril, enalaprilat and lisinopril may be compared with those between the ace and the triterpenic molecules β-amyrin, oleanolic acid and ursolic acid represents the next step in this direction. references attard e, attard h (2006). the potential angiotensin-converting enzyme inhibitory activity of oleanolic acid in the hydroethanolic extract of crataegus monogyna jacq. nat. prod. comm. 1(5):381386. bernstein fc, koetzle tf, williams gj, meyer jr. ee, brice jf, rodgers jr, kennard o, shimanouchi t, tasumi m (1977). the protein data bank: a computer-based archival file for macromolecular structures. j. mol. biol. 112(3):535-42. humphrey w, dalke a, schulten k (1996). vmd visual molecular dynamics. j. mol. graph. 14(1):33-8, 27-8. kapetanovic im (2008). computer-aided drug discovery and development (caddd): in silico-chemico-biological approach. chem. biol. interact. 171(2):165-76. natesh r, schwager slu, evans hr, sturrock ed, acharya kr (2004). structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin i-converting enzyme. biochemistry 43:8718-8724. natesh r, schwager slu, sturrock ed, acharya kr (2003). crystal structure of the human angiotensin converting enzyme-lisinopril complex. nature 421(6922):551-4 sweileh wm, sawalha af, zyoud sh, al-jabil sw, tameem ej, shraim ny (2009). evaluation of antihypertensive therapy in diabetic hypertensive patients: impact of ischemic heart disease. j. pharm. pract. 7(1):40-46. wang r, gao y, lai l (2000). ligbuilder: a multi-purpose program for structure-based drug design. j. mol. model. 6(7-8):498-516. wang r, liu l, lai l, tang y (1998). score: a new empirical method for estimating the binding affinity of a protein-ligand complex. j. mol. model. 4:379-394. who (2012). world health statistics 2012. 1. health status indicators. 2. world health. 3. health services statistics. 4. mortality. 5. morbidity. 6.life expectancy. 7. demography. 8. millennium development goals – statistics. 9. statistics. world health organization. (http://www.who.int/gho/publications/world_health_statistics/en_wh s2012_full.pdf). african journal of food science research vol. 2 (7), pp. 105-114, july, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper reliable ways in advancing employee motivation in fast food outlets in cape town, south africa aotunex ihemanma and chigaemezu ebutex department of business administration, faculty of business, stellenbosch university, stellenbosch 7600, south africa. accepted 03 june, 2014 motivation increases the level of performances of employees and also increases their commitment in the workplace. this implies that motivating workers is very important. the fast food workers are unique and have their individual needs, potentials, values and goals. job satisfaction leads to job motivation. therefore, when workers are satisfied, they tend to be motivated to work. the study points out the strategies that could be used to improve the level of motivation of the fast food workers and also discusses the causes of low employee motivation within the organization. this will also assist the management of the fast food industries in improving the performances of their employees. a case study approach was used for the survey because only the fast food industries were involved. information was obtained from both the operational workers, administration, cashiers and the managers. a total of 200 closed-ended questionnaires and open-ended semi-interview questions were distributed and 123 employees responded which gave a response rate of 62.5%. research proved that the rate of personal growth of the employee’s in their workplace was not satisfactory with a rate of 56.1% respondents. also, the flexible time plan was not satisfactory to the workers because they were being given a flexible time sometimes especially when their workplace is very busy. in addition, the employee’s were not given the privilege of making decision in their workplace especially in the areas that concerns them. however, the researcher recommended that the fast food workers should be empowered in different ways, to give them opportunities to share their views. also, the skills should be developed through coaching and feedback system. the study includes only the fast food workers that have at least six (6) months experience. this is to obtain a reliable and valid result. keywords: motivation, work environment, working conditions, employee performance, organizational commitment, job satisfaction, recognition, promotion. introduction people management is an important aspect of any organization, and this includes the fast food industry. a well managed fast food industry will normally consider *corresponding author. e-mail: aotunex07@yahoo.co.uk abbreviations: qwl, qualities of work life; fasa, franchise association of south africa; nsdsfsa, national strategy for the development and support of franchising in south africa; bna, bureau of national affairs; hr, human resource. employees rather than financial capital as the core foundation of the business, which also contributes to the industry’s development (morato, 2008). therefore, in order to ensure achievement of the industry’s goals, the fast food industry should create an atmosphere of commitment and cooperation for its employees through policies that facilitate employee motivation and satisfaction. satisfaction of human resources is closely linked to highly motivated employees. motivated employees normally perform better, which result in greater productivity and lower labour turnover rates. moreover, highly motivated employees strive to produce at the highest http://www.internationalscholarsjournals.org/ aotunex and chigaemezu 105 self actualization esteem social safety physiological figure 1. maslow`s hierarchy of needs. possible level and exert greater effort than employees who are not motivated (schultz et al., 2003). when motivating factors such as challenging jobs, responsibility and so on are inherent in a job, satisfaction is perceived as possible and work-directed energy is aroused and this leads to motivation (bloisi et al., 2007). the importance of employee motivation should be emphasised within the fast food industry. some ways to improve motivation in the fast food industry include good remuneration, effective training and skills development, a proper recognition and reward system, and employee growth prospects (schultz et al., 2003). employees’ motivation is one aspect of a human resource management development strategy. championhughes (2001, cited in morato, 2008) states that a holistic approach should be used to improve certain qualities of work life (qwl) such as fringe benefits, better employment conditions, and career development to support facilitation of motivation, which is directed towards achieving the organization’s goal (ukandu and ukpere, 2011). literature review employee motivation is a process, which accounts for an individual’s intensity, direction and persistence of effort towards attaining a goal (robbins et al., 2007). therefore, intensity relates to how hard a person tries, while direction is the channel through, which a job is performed. persistence is a measure of how long persons can maintain their effort (robbins et al., 2007). through employee motivation, an organization can achieve a competitive advantage through higher productivity and improved customer service (stone, 2005). consequently, when employee motivation is downgraded, the organization will be at risk in relation to finances and other strategic objectives. hence, employee motivation regulates the behaviour of employees and enables them to achieve the desired goals of an organization. therefore, motivation is the psychological process that provides behaviour, purpose and direction. it is also defined as an internal force, which is based on an individual’s conscious and unconscious needs that drive him/her to achieve a goal (robbins, 1993) indeed; it is an internal drive to satisfy an unsatisfied need (higgins, 1994, cited in lindner, 1998). motivation theories when discussing motivation, some theories cannot be ignored, since they explain why people act the way that they do and why others refrain from doing certain things (swanepoel et al., 2003). regarding this research, theories of motivation will be explored in relation to work motivation in the fast food industry, namely maslow`s hierarchy (figure 1) of needs and herzberg’s theory. maslow`s theory maslow proposed a theory, which he called the needs hierarchy. these needs are physiological, safety, social fulfillment, satisfaction of the ego and self-actualization. people always have needs, and as soon as one need is satisfied, another need takes its place (nel et al., 2004). from figure 1, maslow stated that individuals move up the steps of a hierarchy and in order to be motivated, one should know, which level of hierarchy that person is currently on and focus as on satisfying those needs first (robbins et al., 2007). maslow distinguished between higher and lower order needs, hence, phy-siological and safety are grouped as lower order needs while social, esteem and self-actualization are referred to as higher order needs. according to maslow, when the lower order needs, namely physiological and safety, are substantially satisfied, the next need becomes dominant (robbins et al., 2007). therefore, in order to motivate someone, a person should understand what level of the hierarchy that particular person is currently on and focus on satisfying those needs within the particular level. accordingly, higher order needs can be satisfied internally while lower order needs are satisfied externally. incentives such as pay, union contracts and tenure are examples of lower order needs. hence, if basic needs or lower order needs are not met, efforts to satisfy higher order needs will be postponed, according to maslow’s perspective (prasad, 2003). herzberg`s theory herzberg investigated the question: “what do people want from their jobs?” (swanepoel et al., 2003). there are different variables that can make people feel either good or bad about their jobs. these factors were indicated by herzberg, which he called the two-factor theory of motivation, namely intrinsic and extrinsic factors. intrinsic factors include advancement, recognition, responsibility and achievement, while extrinsic factors comprises of status, security, company policy, administration, remuneration, supervision and interpersonal relations, which are referred to by herzberg as “hygiene” factors. these latter factors do not motivate employees, although they should be present in the workplace to placard employees. employees are motivated by internal values rather than external values. hence, motivation should be internally generated through those intrinsic factors, which are known as “motivators”. among many organizations that need to motivate their employees is the fast food industry (ukandu and ukpere, 2011). fast food industries in south africa fast food industries started as a franchise in south africa under the canopy of the franchise association of south africa (fasa). fasa was established in 1979 and operates as a non-profit, self-funding organisation (national strategy for the development and support of franchising in south africa) (nsdsfsa). its main aim is to promote and maintain ethical franchising in south africa. this organisation consists of two memberships, namely franchisors and affiliates, which are service providers to franchisors and franchisees (fasa, 2000). the fasa is universally accepted as one of the most successful business formats in south africa. they ensure that all members follow internationally accepted franchise 106 afr. j. food sci. res. business principles, and serve the needs of the public and the franchise community (fasa, 2009). fasa has about 209 members, which comprise 170 franchisors and 39 affiliates (sibeko and tambani, 2000). they also have a self-regulatory measure, which every member of the franchisor should meet. these include. i. fasa members are subject to fasa`s code of ethics and business practice; and ii. fasa assists its members with a dispute resolution by providing a mediation service. fast food and restaurant industries, under the canopy of the fasa, include the following: barcelos flamed chicken, chicken licken, kentucky fried chicken, mcdonald’s south africa, king pie and wimpy restaurants, to mention but a few. an observer declared that there is a need to enhance career development and job characteristics of the fast food employees in order to increase the level of motivation among employees (ukandu and ukpere, 2011). motivation and the work environment motivation of employees in the fast food industry is affected by the environment in which they work (smithers and walker, 2000). research conducted proved that workers’ environments do affect their level of motivation, for instance, long hours of work, non recognition for work done and colleagues’ aggressive management style. in order to motivate employees to perform their best, there is a need to provide a work environment that provides achievement, recognition, meaningful work, advancement and growth (musselwhite, 2007). some of these variables that affected employees’ level of motivation in the workplace are discussed subsequently. working conditions fast food workers need enough resources such as time, money and equipment to be able to do their work effectively. however, resources are scarce, which means that decisions should be made to distribute them fairly. the needs and goals of the industry should be prioritized. the physical layout of the work environment such as neatness, organization, convenience, attractiveness and stimulus (noise, air, hazards and so on) should not threaten the well-being of employees (nel et al., 2004). (ester, 1986; cited in royle and towers, 2002) states that public humiliation; unsafe working conditions and total lack of respect and dignity are amongst reasons why fast food employees are not happy. most jobs at fast food outlets have reflected abusive working relationships, poor working conditions, reduced wages, long working hours and less participation in the decision making of the aotunex and chigaemezu 107 organisation, especially in areas that concern workers (ukpere, 2007). almost all the aspects of fast food work are highly standardized and rigorously monitored (royle, 2004). their methods of work produce identical products and their standards and productivity are broken down into the smallest steps. work in the fast food industry is computerised, which reduces work movements and hence speed up production. employees’ skills are depleted and their work becomes intensive with machinery making decisions for them. lights and buzzers tell workers when to fry or bake. furthermore, workers learn a routinized job in a day with no previous experience and the minimum of training (royle, 2004), which results in monotony. furthermore, employees in the fast food industry tend to quit after a short term (thoms et al., 2004). this is because they make poor earnings and work in less than satisfactory conditions. he further stated that their hours of work are long, shifts unpredictable and promotions are as scarce as water in a desert. there are times when workers want to join a union to negotiate with the company in which they work, but are not allowed to do so because the fast food industry works under a policy of anti-union, which means that they do not allow their employees to join unions. if any employee joins the union he/she may be sacked and management will not attribute the sacking to joining of a trade union (harikripahai, 2007). another factor is the workforce, which consists of 75% of youths under the age of 21 years (steinberg and greenberger, 1986; harikripahai, 2007). this encourages the industry to have no legal basis for paying minimum wages to its workers. a witness mentioned that the workers were not given sick leave and if any staff member is sick, his/her schedule will be rewritten. working hours managements at fast food outlets have created an environment, which looks like a family, while on the other hand, it is an environment filled with intimidation and fear (reischman, 2003). employees are pushed to their physical limits with long hours of work, and are not given enough breaks. in addition, their wages barely meet the minimum requirement. a reason for this is that most employees have a low standard of education and some of them are immigrants and high school students (reischman, 2003). the working hours speculated by the federal fair labour standard act is at least forty hours a week and employees who work overtime should be paid additionally for this. fast food employees work long hours for low pay (schlosser, 1998). furthermore, fast food assistants who work sixty hours a week earn a lower hourly wage than some of their crew members (ibid). fast food managers receive promotions, while most assistant managers never receive promotions. employees in the fast food industry prefer work hours that are compatible with their activities outside of work, thus a huge popularity of flexi-time and part-time work (brown et al., 1992). their schedules should give them freedom to integrate their jobs with the rest of their lives; however, shift work is typically disliked because it usually interferes with their off-the-job routine (reiter, 1986). pay and benefits a low minimum wage has long been a critical issue of the fast food industry`s business plan (schlosser, 1998). in view of this, they also import workers from other countries to work for low wages (reischman, 2003). according to streeten (2001, cited in ukpere, 2007), outsourcing is a cause of the decline in the demand for wages of semiskilled and unskilled labour. their employees are treated poorly especially in the area of pay, which results in the fact that an average worker that wants to make a living by supporting his or her family, cannot do so with the average pay or minimum wage that is given to him or her (harikripahai, 2007). he further states that instead of the fast food industry increasing their employees’ wages, they will prefer to hire other people who are willing to work for that amount or even less. this mistreatment does not only include poor hourly wages, but the denial of the right to join a union (schlosser, 2003). workers assume that picking strawberries is far better than cooking french fries and jobs, which are filled with people who are young and unskilled. the extreme standardization of work in the fast food industry justifies low wages and benefits (leidner, 1993, cited in royle and towers, 2002). inexperienced people who are capable of doing the work easily accept low paid jobs because they do not have a choice. this also causes low motivation and high labour turnover, since workers’ needs cannot be met with low paid jobs in the fast food industry (leidner, 1993). benefits in the fast food industry are almost non-existent. other organizations provide benefits to their workers such as medical insurance and paid vacations. but the fast food industry does not. also, the work and life balance of the employee is not feasible, since they have a lowly, erratic and unpredictable work schedule that must be planned around, and if they do not comply with this unfair treatment, they will be asked to quit and their position will be filled by another person (williams and hazer, 1986, harikripahai, 2007). recognition an employee’s performance can be recognized through promotion. employee recognition consists of personal attention, expressing interest, promotion, pay, approval and appreciation for a job well done (robbins, 2003). generally, in the workplace, recognition is considered as the most powerful tool for employee motivation (robbins, 2003). fast food workers want to be recognized. they want to know that their superiors appreciate their work in the industry. most of the fast food industries value ‘people principles’ and ‘social responsibility’. people principles refer to the relationship between management, the workers and the treatment of workers (harikripahai, 2007). according to an observer, the fast food industry holds five basic ideas: respect and recognition, values and leadership, pay, learning and developmental growth, and resources. he adds that four of these five are questionable because management regards workers as passive people who can be employed to work for a certain period of time and duped with a false sense of employment security and individuality. promotion promotion is the reassignment of an employee to a higher-level job (grobler et al., 2006). promotion means more responsibility. employees want promotion to be given fairly. fairness means promotion for the most qualified workers, although few employees who are competent may not have good managerial skills, or training that will enable them manage people. in the first paragraph of working hours, it was stated that fast food managers receive promotion while most assistant managers never receive promotion. strober (1990) points out that race and gender affect employees’ promotion on the ground of human capital theory. this contends that variation in upward mobility is largely attributed to differences in the quantity and quality of the educational opportunities. the most obvious aspect is the discrimination that exists between race and gender in promotion decisions. this discrimination, he states manifests by slowing the promotion rates of the minority or by creating barriers to upward mobility. employee promotions in the fast food industry are considered as vital both to the industry and their employees. promotions provide direct economic and psychological reinforcement for employees and in determining, which employee should be selected for the promotion exercise; job performance is a key factor (sheridan et al., 1997). ‘promotion may be an employee’s reward for good performance, that is, positive appraisal, which leads to employee motivation’ (louis, 2009). la motta (1995) defines job performance as the result of motivation and ability. ability includes training, education, equipment and simplicity of task. despite all these factors, an employee may still not perform well on the job if there is no motivation. motivation not only influences performance, but performance, if followed by rewards, can influence motivation (la motta, 1995). in some south african fast food outlets, research has shown that management makes false promises of job promotion that never occurs. this could be as a result of the downsizing of the industry or the inability of management to pay workers when 108 afr. j. food sci. res. promoted (inglish, 2010). smart and talented employees tend to give up and look for employment elsewhere (inglish, 2011). management of fast food outlets, including the south african fast food outlets, sometimes promote their workers without proper training and experience to become supervisors. this demoralizes staff because these supervisors are then incompetent and cannot perform in the post given to them. employee performance it was stated earlier that jobs in the fast food industry are highly routinized and capital intensive, which results in employees living their job. reduced work output, increased accidents, absenteeism, employee turnover and poor employee performances are examples of detrimental employee behaviour that have a significant effect on profitability (spector, 2003, cited in lo and lamm, 2005). about sixty two percent of first-time employees in the fast food industry usually engage in theft and misconduct in their workplace such as giving away goods, falsely claiming to be sick, stealing, damaging the organizations` property, or work while intoxicated during their first nine months of employment (reischman, 2003). turnover is high for non-management fast food employees at three hundred percent per year, and vacancies are reported to be eighty one percent (bureau of national affairs (bna), 1985, cited in reischman, 2003). it is the amount of training that is provided to fast food employees that enhances their performance rather than the quality or quantity of education achieved prior to employment (sheridan et al., 1997). as mentioned previously, employee job performance is a result of motivation and ability. motivation influences performance, but performance, if followed by rewards, can influence motivation. since the subject of motivation has become increasingly important in the fast food industry recently, there is need for total quality management and a flatter management structure, which will initiate an employee reward management system. deeprose (1994) postulates that effective reward management can help the management of a fast food outlet to achieve their business objectives by attracting and retaining competent workers. management and supervision abuse by managements of fast food outlets have come into the public interest increasingly because of increased awareness and workers are no longer tolerating it. abusive scenarios such as unclear performance goals, strange human resource (hr) practices, deadly teams and unannounced relocation, have become the order-ofthe-day (inglish, 2010). managements at fast food outlets do not create an environment that will allow them to aotunex and chigaemezu 109 exchange ideas and participate with the workforce. they also do not implement co-determination and participation of workers in decision making, especially regarding issues concerning the fast food industry (butod, 2009). he further states that co-determination and participation can increase workers commitment, job satisfaction and motivation and reduce resistance to change. it will also improve communication and consultation between management and employees (butod, 2009). some fast food management training material suggests that failure to provide adequate communication, correct management styles, adequate praise and recognition, adequate staffing levels and correct handling of holiday entitlements and pay details, are the major reasons why workers are becoming resistant to management power (royle, 2000). regimentation and standardization of the fast food industry have given managers authority over their employees, which means that management determines the way every task should be done and imposes rules on pace, output, quality and techniques of production (leidner, 1993). management has not been taking the feelings and emotions of their workers to heart. the workers are not given responsibilities in their workplaces to make them feel as part of the company (ukpere, 2007). furthermore, some fast food employees have demonstrated dissatisfaction with their work assignments and the degree to which they are utilized within their workplace. in addition, managers change their shift patterns, while working night shifts is a big concern for female workers. this is assumed to be affecting their primary family responsibilities. furthermore, it can lead to stress, anxiety and depression, which affect their psychology (michailids and e-ali elwkai, 2003). this is one of the major dysfunctional aspects of the fast food industry. research has further been conducted to prove the reliability of this information. need for the study this research was done to enable the fast food industry management implement practices that will increase job satisfaction in order to enhance employee motivation. it is needful to help direct the fast food managers on how to achieve a high employee performance and higher levels of productivity. the study also guides the fast food managers on how to stimulate their workers and bring about change in their job performances. objectives of the study the objective of this research is to find out the various strategies on how employees can be motivated in their workplace especially in the fast food industry. this study was conducted using fast food outlets within cape town metropolis. this study also investigated the causes of low employee motivation such as poor working conditions, poor managerial services and supervision, poor pay and others. research methodology: sample and research process the aim of this study is to derive a motivation strategy for the fast food outlets within cape town. a case study approach was used for this research because it was dealing with a specific organization which is the fast food outlets in cape town. the research methods used were the qualitative and quantitative methods (triangulation). this was to know the opinion of the fast food employees in the area of motivation. a total of 200 questionnaires were distributed and only 123 employees responded and were used for the analysis. furthermore, a pilot study was conducted with few staff members and managers of the fast food industry from different outlets and corrections made before distributing it to the others. this is to allow reliability and validity. also, openended, semi-structured interview questions were administered face-to-face to the senior and store managers of the different fast food outlets to know their opinion in the area of employee motivation, while a closed-ended questionnaire was distributed to the other staff members. as mentioned previously, the total population sampled was 200. the selected sample composed of all the staff members that have at least six (6) months of work experience within the fast food industry. this is to enable the researcher obtain reliable information. a report was made on the level of motivation of the workers. a high ethical standard was maintained as far as the information is concerned. data analysis to ascertain the motivation level of the fast food workers, the researcher posed some questions and statements. in analysing the data obtained, content analysis was used. information from other sources was also incorporated into the analysis for validity and reliability. the responses received from the questionnaires are given as follows: the growth rate of the fast food workers and how it has affected their motivation level was assessed. emanating from the interviews, it was evident that some fast food workers work in poor working conditions and environment. many interviewees maintained that they were not satisfied with the layout of their workplace. it was clear that the employees’ personal growth was not a major concern to their managers; however, employees are expected to carry out their jobs effectively. few interviewees noted that they do not have enough personal growth in their workplace which is discouraging them from working with the fast food outlet. as indicated in table 1, the rate of personal growth among staff members was not impressive. a total of 56.1% (25.2% plus 30.9%) of respondents noted that they were not satisfied with the rate of their personal growth within the fast food industry. from the result, one can conclude that the personal growth of the fast food workers is not satisfying. the questionnaires revealed that the fast food employees were not having flexible time in their workplace. employees in the fast food industry prefer work hours that are compatible with their activities outside of work, thus a huge popularity of flexi-time and part-time work. the rationale for the question was to determine whether or not flexible time is convenient for the fast food workers, and to find out if they are satisfied with their flexible time plan (table 2). of the 123 respondents, 7.3% are highly satisfied, which means that they are highly satisfied with the flexible time plan at their workplace and 19.5% are satisfied, which totals 26.8% (7.3% plus 19.5%). a total of 5.7% are neutral. however, 31.7% are not very satisfied, while 33.3% are not 110 afr. j. food sci. res. table 1. the rate of my personal growth in my workplace is? variable frequency percent valid percent cumulative percent valid highly satisfying 10 8.1 8.3 8.3 satisfying 24 19.5 20.0 28.3 neutral 17 13.8 14.2 42.5 not very satisfying 31 25.2 25.8 68.3 not satisfying at all 38 30.9 31.7 100.0 total 120 97.6 100.0 missing system 3 2.4 total 123 100.0 (n=123). table 2. flexible time plan at my workplace is? variable frequency percent valid percent cumulative percent valid highly satisfying 9 7.3 7.5 7.5 satisfying 24 19.5 20.0 27.5 neutral 7 5.7 5.8 33.3 not very satisfying 39 31.7 32.5 65.8 not satisfying at all 41 33.3 34.2 100.0 total 120 97.6 100.0 missing system 3 2.4 total 123 100.0 (n=123). table 3. what is your level of satisfaction regarding decision making in your workplace? variable frequency percent valid percent cumulative percent valid highly satisfying 9 7.3 7.5 7.5 satisfying 21 17.1 17.5 25.0 neutral 24 19.5 20.0 45.0 not very satisfying 42 34.1 35.0 80.0 not satisfying at all 24 19.5 20.0 100.0 total 120 97.6 100.0 missing system 3 2.4 total 123 100.0 (n=123). satisfied at all, totalling 65% (31.7% plus 33.3%). also, 70.7% (34.1% plus 36.6%) of respondents stated that they were not satisfied with overtime duty at their workplace (table 2). the data that was analyzed confirmed that fast food employees are not satisfied with the flexi-time plan at their workplace. the rationale for this question is to ascertain whether the fast food employees are really satisfied with decision making in their organization and whether they are allowed to participate in the decision making of the organization. findings from the data analysis disclosed that workers are not encouraged to participate in the decision making of most outlets and are also not recognized for good performances. during the interviews, several respondents indicated that they were not satisfied with decision making in their workplace. they added that they were not given freedom to provide input with regard to the organization’s objectives and standards. however, the data collected are given subsequently. table 3 points out that 7.3% of respondents are highly satisfied with decision making in the organization, and 17.1% said that they are satisfied, totalling 24.4% (7.3% plus 17.1%). a total of 19.5% of respondents are neutral regarding the question, while 34.1% of respondents said that they were not very satisfied and 19.5% were not satisfied at all, totalling 53.6% (34.1% plus 19.5%). the rationale for this question is for the researcher to know if the fast food employees are given freedom to make input with regard to objectives and standards of the industry. the interviewees maintained that they were not given freedom to provide input with regard to the organization’s objectives and standards. again, some respondents maintained that their job aotunex and chigaemezu 111 table 4. do employees have freedom to make input with regard to objectives and standards? variable frequency percent valid percent cumulative percent valid yes 35 28.5 29.2 29.2 no 85 69.1 70.8 100.0 total 120 97.6 100.0 missing system 3 2.4 total 123 100.0 (n=123). execution was not made challenging by their supervisors. the data analysis, as illustrated in table 4, indicates that 69.1% of respondents have not been given the freedom to provide input with regard to the organization’s objectives and standards. table 4 showed that 61.8% of respondents stated that their job execution is not made challenging by their supervisors. as previously mentioned, some interview questions were posed to the store managers, senior managers, and ex-staff to obtain information on the motivation level of the fast food workers. content analysis was used in analyzing the interview questions. the following are some interview questions and responses obtained from the store managers and senior managers: do you think your employees are motivated enough in your workplace? two managers noted that employees are motivated because of the type of work that they produce and of course they do not have a choice because they need the job. in addition, one interviewee noted that workers are sometimes motivated if a goal is set for them or a reward is provided to them. other managers said that workers are not motivated because there is no training given to them, while promises of recognition are made, but not delivered, and hence not enough recognition is practiced. furthermore, most employees have no passion for their job because they feel that there is a lot of negativity and unfairness amongst them. what is your view about the performance of your staff members? the managers revealed that staff performances were poor because they did not receive any formal training. they also lack self confidence and self respect. an interviewee stated that sometimes staff performance is good and sometimes it is not. another two managers stated that staff performance is good with no reason or further explanation provided. do you think that employee motivation can improve the staff performance? all interviewees reported that employee motivation can improve staff performance because it will make them stress–free at work; the workers will feel more empowered, and have more confidence and self respect. they will also feel more proud about their job provided that they receive proper training in order to perform above expectation; employee motivation enables staff to improve their performance by yielding good results and by lifting their self esteem. furthermore, employee motivation will help them to achieve organizational goals in a better manner. the following are some interview questions for the ex-staff and their responses. this is to find out their reasons for their leaving the fast food industry and to know if their reasons were related to their level of motivation. were you motivated at the fast food outlets? four ex-staff members said that they were not motivated because there was nothing to motivate them, except the fact that it was a production company, which focused on business. moreso, the salary was too little and the shifts were long (24 h), while they also felt that they were treated poorly. in addition, the work was too much and managers were rude to their workers and unfriendly. no transportation was available to and from work for late shifts, and they were abused, because they worked as cleaners instead of what they applied for. lastly, they felt that they were just a number doing duties, and could hence not be motivated under such conditions. however, three (3) ex-staff members stated that they were motivated because one said that she was promoted even though it was after six (6) years, while another said that she enjoyed working with people, and learned new ideas. the last said that he was always given an opportunity to prove himself and to learn new skills. what was your reason for leaving the fast food industry? the ex-staff members had different reasons for leaving. the first two said that the business closed down so they had to leave; another said that the working hours were too long and travelling was too costly, hence she could not afford it any longer and resigned to look for a better opportunity. the next two said that they were going back to varsity and another noted that they had a better job opportunity elsewhere with more pay. different reasons were given, but this shows that they were not motivated enough to enable them to stay at the fast food outlet. in your opinion, do you think that being motivated at your workplace would have made you to stay longer? they all noted that if they had been motivated at their workplace they would have stayed longer for the following reasons: if their salary was increased and transportation was made available for them to and from work. they also said that a friendly attitude with less work would have made them stay longer as well. moreover, one said that she likes the fast food industry and so being motivated would have made her to stay longer and another said that if he could see potential that would challenge him, then he would have stayed longer. lastly, the other two said that there was no need for motivation, since the outlet had closed down. strategies that could improve the motivation level of the fast food employees emanating from the interviews conducted with the staff members, it was revealed that the employees do not have enough time for their families and other personal responsibilities. therefore, in an effort to improve the work life of the employees, the management needs to recognize this balance in order to help improve the employees work life. the following are factors that could contribute to the improvement of the employee’s work life. training and development it is appalling to note that the fast food employees are not given proper training. investigations revealed that employees have not been performing well because of improper training. from the data analysis, it was revealed that 56.1% (25.2% plus 30.9%) respondents were not satisfied with their personal growth with regard to their job descriptions at the fast food outlets. most respondents also noted that their job execution is not made challenging by their supervisors. three interviewees maintained that training workers will help them to improve their general performances and also motivate them to work harder and better. reduced workload it was deduced from the interviews with staff members that there is work stress in the fast food industry, and too much pressure and heavy workloads. it is clear that employees’ workloads should be reduced to prevent low employee motivation and to improve the work life of employees. results from the interviews showed that employees do not have flexible work time and working hours were too long, hence they do not have enough time to spend with their families. moreover, the pay is little, compared to the work that they do. it is important to know that workers are eligible for flexible time and less hours of work, which would allow them to have time with their families and their personal life. incentive programmes the study has revealed that workers were not properly remunerated. moreover, bonuses and fringe benefits were not given to them. employees stated that they were not recognized for good performances. results from table 4.15 revealed that 63.4% (30.9% plus 32.5%) of respondents disagreed that they had a bonus plan in their organization. table 4.19 also noted that 69.1% (32.5% plus 36.6%) of respondents reported that there is no health plan for employees. increasing workers’ pay, introducing a benefit plan and recognizing employees` performance, should be important aspects of improving employees` standard of living and work life. retention strategy it was derived from the interviews with store managers that there is a high staff turnover and absenteeism at fast food outlets, which participated in the survey. an interviewee mentioned that some workers that were absent complained of low motivation. another interviewee maintained that some staff members who resigned did so because they had not received what they had expected from the company. a store manager also said that some workers who left the organization complained of job dissatisfaction and a lack of transportation to work. the researcher was not able to obtain data from employees who had resigned. findings from the data analysis disclosed that the quality of supervision at fast food outlets were not 112 afr. j. food sci. res. satisfactory. additionally, the workers’ salary is not competitive. moreover, they are not involved in the decision making of the outlet and are also not recognized for good performances. most interviewees noted that their jobs are too stressful and that their workloads are too big, hence they do not have personal fulfillment in their jobs. results from table 4 disclosed that 86.2% of respondents agreed that they were experienced stress related to their work. it is necessary to note that a reduced workload and recognition of employees’ performance and contribution would keep them in the industry. more than that, promotion of staff members and flexible time of work will attract talented candidates, which will boost the profile of staff members. results and discussion it can be said that the fast food employees are not motivated to work from the analysis in the foregoing. in the first instance, the rate of their personal growth in their work-place is not satisfying. table 1 shows 56.1% respondents who were not highly satisfied or satisfied with their rate of personal growth in the organization. more so, it was stated from the respondents that their flexible time plan is not encouraging. in spite of this, their level of satisfaction with the decision making in their workplace is low. res-pondents also claimed that they do not have freedom to make inputs with regards to the objectives and standards of the organization. the interviews with the store managers and the ex-staff also revealed that the fast food workers are not motivated at work. this has been clearly stated from the interviews. some interviewees noted that one of the reasons why the workers are not motivated is that there is no training given to them; rather there are promises of recognition which is not always delivered. also, few managers noted that most of the employees have no passion in the job because they feel that there is a lot of negativity and unfairness amongst them. however, the interviewees view the performance of the staff as poor because of lack of formal training. again, they noted that some of the workers lack self confidence and self respect. ex-staff members revealed that they were not motivated at work because the salary was too little and the shifts were long (24 h) and they also felt that they were treated poorly. in addition, the work was too much and managers were rude to their workers and unfriendly. no transportation was available to and from work for late shifts, and they were being abused and so they resigned to look for a better opportunity. moreover, the ex-staff members revealed that being motivated in the fast food industry would have made them to stay back. lastly, they added that if there were potentials and challenges in the industry, they would have stayed back. limitations and future research this study was conducted only with the fast food outlets aotunex and chigaemezu 113 in cape town. the intention is to develop a motivation strategy that could improve the level of motivation of the workers. it has also highlighted the need for further research on job satisfaction of the fast food employees to measure the level of satisfaction of the workers. recommendations and conclusion despite the good qualities of food provided from the fast food outlets, there are weaknesses with the way the management deal with their employees, which have adversely affected the state of motivation of the workers. employee motivation is essential to achieve the business goals of the fast food outlets in cape town and south africa in general. if the workers are satisfied with their working conditions, they tend to perform well and customers are also happy. the researcher has recommended strategies that could improve the level of motivation of the workers within the fast food outlets in cape town as follows: fast food employees should be empowered and given some degree of autonomy in the execution of their job. it is evident that when employees are empowered, they tend to work harder and faster. they are found to be loyal to their employers and they also enjoy their jobs more. it is imperative to empower employees by making them feel that the company has a high regard for them, and that they are the reason for the organisation’s success. fast food workers should be empowered in different ways, namely they should be given opportunities to share their views in terms of making suggestions. moreover, their suggestions should not be taken for granted, but should be used to solve problems in the organisation. the management should be able to communicate with their workers in a clear and understandable manner. managers should be able to know the abilities of their employees and assign them tasks that will allow them to enjoy the freedom of doing their work. fast food employees’ skills should be developed through a coaching and feedback system. a good degree of autonomy should indeed be given to fast food workers. in other words, they should be permitted to give their opinions on how a particular job should be performed. substantial freedom, independence, and discretion to schedule work and determine procedures that should be taken to do the job will increase levels of motivation for fast food employees. greater autonomy will give workers a sense of accomplishment in the workplace, as they begin to take charge of directing their jobs. contrary to popular belief, workers also enjoy taking responsibilities, especially when they are asked to stand in for their managers, in cases of absenteeism. this has an accelerator effect on motivating workers to hope that one day they may as well become managers/leaders in the organisation, and hence their levels of motivation will positively be affected. workers should be involved in the organisation’s decision making it is important to involve both managers and employees in joint decision making on a regular basis. employees should be empowered through decision making for the organisation, especially in areas in which they excel. there is a need for managers to mark out how much decision making authority will be assigned to employees to prevent competition. workers should be empowered to compile schedules for their vacation because they may have a better idea of how it will work. managers should consult employees before any decision is reached. this will help to prevent overlooking solutions that may appear obvious to front-line employees but unfamiliar to higherlevel managers. this will also help managers to make decisions since they may not have enough information to make a quality decision without the employee’s input. moreover, managers should involve their employees in decision making in order to make an effective decision. despite this, employees should be allowed to participate in planning their personal career paths as this will render a sense of ownership, and thereby increase their levels of motivation at work. workers should be recognised for their contribution towards organisational success every employee wants to be recognized and appreciated for good performance. fast food managers should always recognize and appreciate the efforts of their workers. monetary reward is important, but recognition and appreciation is critical for industrial harmony. therefore, managers may recognize their workers by announcing their good performance in staff meetings or by mentioning the good performers within the workplace. it is also best practice to send emails to all staff or publish staff members’ contributions and achievements in the company’s newsletter or notice board. this will motivate employees to do more for the organisation. employees should be recognized by giving them time off. many workers like to spend more time with their friends and families and will appreciate if they are given time off at least for a day in recognition of their good performance. they will come back to work feeling refreshed and grateful for the recognition. recognizing employees for good performance and contributions in the organization should enhance employee motivation. fast food managers should also recognize their employees by providing monetary incentives. this will not only motivate the beneficiary, but will also motivate other workers to do their best. additionally, they 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(online) available www.fasa.co.za/content/ memberslist/ restaurants.aspx thoms p, wolper p, scott ks, jones d (2004). the relationship between immediate turnover and employee theft in the restaurant industry. human sciences press inc. erie, pa. j. bus. psychol., 15(4). ukandu ne, ukpere wi (2011). the causes of low employee motivation within cape town fast food industry. unpublished paper. cape town: cput. ukpere wi (2007). the functional relationship between globalization, internationalisation, human resources and industrial democracy. cape town: cape peninsula university of technology phd thesis. williams lj, hazer jt (1986). antecedents and consequences of satisfaction and commitment in turnover models: a reanalysis using latent variable structure equation methods. j. appl. psychol., 71(2): 219-231. 114 afr. j. food sci. res. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (10), pp. 217-221, october, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the effects of peroxides on allergic reactions t. toyosaki department of foods and nutrition, koran women’s junior college, fukuoka, 811-1311, japan. e-mail: toyosaki@koran.ac.jp. tel: +81925811538. fax: +81925812200. accepted 11 august, 2015 the anti-allergic effects on dough fermentation of hydroperoxides produced by lipid peroxidation through lipoxygenase induction were studied. the anti-allergic effect of hydroperoxides to dough fermentation on antigen-antibody reaction involving ige was examined. crude proteins extracted from the dough including hydroperoxides showed weaker antigen-antibody reactions on allergic tests such as precipitin ring test with human-specific ige, and the ige binding activity on elisa. this inhibitory effect on the antigen-antibody reaction increased with increased hydroperoxide and a parallel relationship was observed between hydroperoxide level and inhibitory effect. the crude proteins extracted from the dough with hydroperoxides were applied to an affinity choromatography column of immobilized-trypsin chitin. then proteins having affinity was recovered. the recovered proteins were separated by sdspage. each proteins was examined for the ige binding activity on elisa. therefore, ovomucoid, which is present in egg, was identified as the main allergen. crude proteins of baked bread made from dough including hydroperoxides showed weaker ige binding activity on elisa than proteins of baked bread made from dough without hydroperoxides. suppression of antigenantibody reactions with ige due to denaturation of allergen protein (ovomucoid in the present study) present in dough by the produced hydroperoxide may be identified as a major factor. the importance of hydroperoxides as an ingredient of baked bread was confirmed. key words: hydroperoxide, ige, allergy, dough, baked bread, elisa. introduction over the last several years, numerous researchers have studied food allergies from a variety of angles and clarified various facts (buck and hefle, 1996; crespo et al., 1995; fuchs and astwood, 1996; sampson and mccaskill, 1985; sampson, 1999). various foods show food allergies, and it is not easy to find safe foods for allergy sufferers. in addition, most of the allergens in food allergies are proteins, various proteins are known as allergens, but detection of specific allergens in foods is difficult. proteins in normal bread also cause allergies for allergy sufferes. allergens are primarily thought to be proteins and gluten of origin from wheat flour or additional ingredients such as hen eggs in the bread-making process. most allergy sufferers require commercially prepared allergen-free bread because of the allergy risk. to overcome this issue, development of allergyfree bread has been studied from various viewpoint: the interaction between the allergens in hen eggs (mainly ovomucoid) and gluten in the bread effects on allergy. herein, the details are described. the aim of this study was to determine the effects of peroxides on allergic reactions by closely investigating the relationship between peroxides produced from the induction of lipid peroxidation reactions in the process of dough fermentation and proteins, also egg proteins that act as allergens. materials and methods materials the ovomucoid (type iii), ovomacroglobulin (type ii), ovomucin (type i), ovalbumin (type vii), ovotransferrin (type i), lysozyme, linoleic acid (more than 99% pure) and lipoxygenase (from soybean, type i, lyophilized) were purchased from sigma chemical co. (st. louis, mo, usa). kieselgels 60g and pf254 were purchased from merck (darmstadt, germany). the 0.25 mm thin-layer chromatography (tlc) was done with a tlc apparatus from yazawa toyosaki 218 scientfic apparatus manufacturing company limited. (tokyo, japan). n’,n’-dimethylformamide, chondroitin sulfate sodium salt, hematin, 2’,7’-dichlorofluorescin diacetate (dcfda) were purchased from tokyo kasei kogyo company limited. (tokyo, japan). gluten and other reagents were purchased from nacalai tesque inc. (kyoto, japan). preparation of linoleic acid hydroperoxide linoleic acid hydroperoxide was prepared by the procedure of matsuda et al. (1978) by enzymatic peroxidation with lipoxygenase. the standard reaction mixture containing 32 mm linoleic acid, 0.1% tween 80, 50 mm na2 hpo4 /naoh buffer (ph 9.0), and 50 units of the lipoxygenase in a total volume of 20 ml was incubated in a conical flask to facilitate flushing with pure oxygen. the reaction mixture was stirred mechanically for 40 min at 30ºc under a stream of pure oxygen. after incubation, hydroperoxide was extracted with diethyl ether. the formation of linoleic acid hydroperoxide was monitored by measurement of the increase in the absorbance at 234 nm. the hydroperoxide was purified by tlc with nhexane/diethyl ether/ acetic acid (60:40:0.1, v/v/v) as the solvent system and was monitored under uv light. bread making and preparation of the model system of bread conditions were as follows: wheat flour (1.0 kg), fresh live yeast (40 g), nacl (15 g), saccharose (40 g), hen egg (120 g) were added to water (350 ml), the materials were mixed using a mixer. the lipid added was linoleic acid (more than 95% pure), which was added at a 3% level. other ingredients used to make the bread were all commercially available. lipoxygenase was added at the end of bread dough adjustment and underwent primary fermentation in an incubator at 37°c with 75 80% humidity. after fermentation, gas was released; after a bench time of 15 min, the dough underwent final fermentation for 35 min and was then baked for 20 min at 180°c. conditions of model system of bread were as follows. wheat flour (1.0 kg), fresh live yeast (40 g), nacl (15 g), saccharose (40 g), ovomucoid (1.0 g), ovomuchin (1.0 g), ovalbumin (1.0 g), ovotransferrin (1.0 g), ovomacroglobulin (1.0 g), and lysozyme (1.0 g) were added to water (350 ml), the materials were mixed using a mixer. the various concentration of hydroperoxide was added, which was added at a 100 mm level, which served as the test sample. a sample system was created. this sample underwent primary fermentation in an incubator at 37°c with 75 80% humidity. after fermentation, gas was released; after a bench time of 15 min, the dough underwent final fermentation for 35 min and was then baked for 20 min at 180°c. the dough and baked bread was used for each experiment. preparation of crude protein extracts from dough and baked bread extract of crude proteins from the dough and baked bread were as following by chung and champagne(2001). dough (30 g) and baked bread (50 g) were stirred in 300 ml of 0.1 m phosphate buffer ph 6.5, and 10 mm egta for 30 min at 4ºc, followed by centrifugation at 85,000 x g for 10 min. the supernatants (extracts crude proteins) were filtered at 0.22 mm millex-gx (millipore co., usa). the filtrate, crude proteins, was used for was added into experiiment.concentr ation of crude proteins in the extracts was determined with the folin phenol reagent bassed on lowry et al. (1951) measurement of linoleic acid hydroperoxides hydroperoxide was estimated by the method of cathcart et al. (1984). extracted linoleic acid (30 µi) was then measured for hydroperoxide. first, 1.0 ml of a 1.0 mm solution of dcfda in ethanol and 2.0 ml of 0.01 m naoh were mixed and stirred for 30 min before being neutralized with 10 ml of 25 mm phosphate buffer (ph 7.4). then, 2.0 ml neutralized dcfda solution were added to a solution of hematin in 25 mm phosphate buffer (ph 7.4; 0.01 mg 2’,7’-dichlorofuluorescein (dcf)/ml) and 2.9 ml hematin-dcfda solution and 10 µi supernatant containing linoleic acid were mixed and left at 50ºc for 60 min before fluorometry treatment (excitation, 400 nm; emission, 470 nm) to measure dcf. this method measures hydroperoxide with more sensitivity than the iron rhodanide method usually used. the formation of linoleic hydroperoxides and other products was checked by tlc with n-hexane/diethyl ether/acetic acid (80:20:1, v/v/v) as the solvent system. the tlc plate was sprayed with 50% sulfuric acid, heated at 110ºc for 15 min, and checked under ultraviolet light. determination of antibody-antigen reaction human blood was collected from ten females (20 years old) with their permission. the human serum was separated in accordance with method of mirella et al. (1995). human serum containing kedta (1.6 mg/ml) was separated with a centrifugal separator (2000 x g, 8 min) and objective serum fractions were obtained. singleradial immunodiffusion was performed for measurement of antigenantibody reaction of crude proteins obtained from the dough including 100 mm hydroperoxide and the dough without hydroperoxide against human-specific ige. human serum was added into 1% agar solution, the agar solution was coated to a thickness of 4 mm on a slide glass surface. holes (2.5 mm diameter) were formed in the agar. the proteins obtained from the dough were added into the hole. after the glass was maintained on overnight at 37ºc, the diameter of the precipitin ring obtained was measured. enzymelinked immunosorbent assay (elisa) was conducted according to a method of engvall and perlmann (1972). separation of proteins by affinity chromatography and sds−polyacrylamide gel electrophoresis (page) protein fractions extracted from the dough with hydroperoxide and dough without hydroperoxide were separated by immobilizedtrypsin affinity column chromatography (mine and zhang, 2001). the adsorbed substances were eluted by the linear gradient of ph 6. 5 to 7.5 in 1.0 m phosphate buffer. the non-affinity fraction eluted in the flow through void volume solution, whilst the bound fractions were eluted from the column by the buffer linear gradient elution. latter fraction was further purified by sds-polyacrylamide gel electrophoresis (page). electrophoresis was performed in accordance with a method of laemmli (1970) using ready gel j 18 ma/gel (gradient gel) using a mini-protean ii electrophoresis cell (bio-rad laboratories, inc, tokyo, japan). after electrophoresis, the gel was stained using coomassie brilliant blue r250. the hast system automated electrophoresis device cell was used as well. amounts of protein in each fraction obtained by affinity column chromatography were measured by the lowry et al. (1951) method. statistical analysis the data were expressed as mean ± standard deviation. statistical analysis was performed by one-way analysis of variance , and differences between means were tasted using duncan’s multiple range tests. p-values of <0.05 were considered to be significant. figure 1. evaluation of binding activity of human-specific ige with dough added various concentrations of hydroperoxide by single-radial immunodiffusion. values are the means of three replicates ± standard deviation. figure 3. relationship between the concentration of hydroperoxide and time-course of the binding activity of human-specific ige with dough during fermentation process. values are the means of three replicates ± standard deviation. figure 2. evaluation by elisa of human-specific ige binding to dough with various concentrations of hydroperoxide. values are the means of three replicates ± standard deviation. results and discussion effects of hydroperoxide on ige antibody figure 1 shows the measurement results obtained using a single radial immunodiffusion method for ige antibody in a solution from which crude proteins in dough were extracted using 50 mm phosphate buffer (ph 7.4) . crude protein solution extracted from dough without hydroperoxide showed a concentration dependent antigen-anti body reaction (diameter of precipitin ring: 12 mm) against ige in the single-radial immunodiffusion method. in contrast, crude protein solution extracted from dough added hydroperoxide decreased precipitin ring. the diameter of the precipitin ring of crude protein extracted from dough having 60 mm hydroperoxide was 2.5 mm. the diameter of the precipitin ring of dough having a higher concentration of hydroperoxide decreased drastically (figure 1). figure 2 shows the results of confirmation using an enzyme-linked immunosorbent assay (elisa). the reaction of dough extracts of a higher concentration of hydroperoxide against ige got weaker. crude protein extracted from dough having more than 20 mm hydroperoxide decreased antigen-antibody reaction against ige drasticcally. it was concentration-dependent (figure 2) and these results consisted with the results in figure 1. these facts strongly suggested that the addition of hydroperoxide affected antigen-antibody reaction of dough crude protein. relationship between hydroperoxide and antigenic activity investigation of changes in peroxides produced by lipid peroxidation reactions in the process of dough fermentat-ion, in addition to associated changes in antigenic activity, revealed decreases in antigenic activity with progress of fermentation (figure 3).this finding suggests that hydroperoxides produced during fermentation were highly likely to have caused denaturation of allergen proteins, which led to inhibition or loss of antigenic activity. 219 afr. j. food sci. res. toyosaki 220 figure 4. elution profiles of proteins extracted from dough by affinity chromatography using immobilized-trypsin chitin. figure 5. sds-page of 14-40 fractions numbers from the affinity chromatography studies. isolation and purification of allergen proteins using affinity chromatography as shown in figure 4, after the extraction of crude proteins from dough, proteins with antigenic activity were isolated and purified using affinity chromatography. figure 5 shows the results of sodium dodecyl sulfate figure 6. comparison of the binding activity of humanspecific ige to proteins from dough with hydroperoxide. values are the means of three replicates ± standard deviation. polyacrylamide gel electrophoresis for fractions (fraction numbers 14 40) collected using elution buffer. eluted proteins primarily included ovomucoid, ovalbumin, ovotransferrin, ovomacroglobulin, and lysozyme. measurements of antigenic activity for these protein fractions confirmed a particularly high antigenic activity for ovomucoid (figure 6). allergens are primarily thought to be proteins and gluten of origin from wheat flour or additional ingredients such as hen eggs in the bread-making process. it has already been reported that ovomucoid in the hen egg or in gluten of origin from wheat flour causes the allergy (mine and zhang, 2001; buck and helfe, 1995; fuchs and astwood, 1996). therefore, ovomucoid, which is present in hen eggs or wheat flour was may be identified as the main allergen. peroxides produced during dough fermentation were thought to cause denaturation of the conformation of ovomucoid and thereby prevent the induction of antigen-antibody reactions with ige. antigen-antibody reactions of baked bread bread using the dough with 100 mm hydroperoxide or without hydroperoxide was actually made. antigen-antibody reactions of crude proteins extracted from both bread samples were studied. bread in which lipid peroxidation reactions were induced had a clearly lower antigenic activity than bread in which these reactions were 221 afr. j. food sci. res. figure 7. comparison of the binding activity of humanspecific ige to crude proteins from baked bread made from dough with 100 mm hydroperoxide and baked bread made from dough without hydroperoxide. values are the means of three replicates ± standard deviation. were not induced (figure 7). these findings indicate that induction of lipid peroxidation reactions during dough fermentation results in production of hydroperoxides that cause changes in the conformation of allergen proteins (ovomucoid in the present study) and consequently prevent induction of antigen-antibody reactions. the mechanism by which hydroperoxides acts promote fermentation the various experimental results that were obtained were comprehensively analyzed to determine the mechanism of action by which hydroperoxide acts to promote fermentation. during gluten formation or additional ingredients such as hen eggs, gluten is formed when gliagin and glutenin, and ovomucoid form a network structure. when is allergen protein denaturation in the presence of hydroperoxide, the molecules themselves from macromolecules. as a result, antigen-antibody reaction involving ige was become unresponsive. conclusions the current research demonstrated that the effects of lipid peroxides produced during dough fermentation on allergic reactions. the mechanism of inhibition on allergic reaction of crude protein (ovomucoid in the present study) by hydroperoxide induction is discussed in detail. crude protein (ovomucoid in the study) was denatured by hydroperoxides, the structure of ovomucoid was destroyed which consequently prevent induction of antigenantibody reactions. this phenomenon is advantageous when baking bread and can be used to enhance the quality of baked bread. based on the results of these tests of chemical properties, further detailed study is needed of the effect of lipid peroxides on the flavor of baked bread. acknowledgments this work was supported by a the elizabeth arnold fuji foundation to which we are grateful. references buck ok, hefle sl (1996). food allergens. clin. rev. food sci. nutr. 36: 119-163. cathcart r, schwiers e, ames bn (1984). detection of picomole levels of lipid peroxides using a dichlorofluorescein fluorescent assay. “methods in enzymology” (ed. packer l.), academic press, new york. 105: 352. chung sy, champagne et (2001). association of end-product adducts with increased ige binding of roasted peanuts. j. agric. food chem. 49: 3911-3916. crespo jf, pascual c, burks va, helem rm, esteban mm (1995). frequency of food allergy in a pediatric population from spain. pedi. aller. immunol. 6: 39-43. engvall e, perlmann o (1972). enzyme-liked immunosolvent assay, elisa. quantization of specific antibodies by enzyme-labeled antiimmunoglobulin in antigen-coated tubes. j. immunol. 109: 129-135. fuchs rl, astwood jd (1996). allergencity assessment of foods derived from genetically modified plants. food technol. 50: 83-88. laemmli uk (1970). cleavage of structural proteins during the assembly of the head of bacteriphage t4. nature 227: 680-685. lowry oh, rosebrough nj, farr al, randall rj (1951). protein measurement with the folin phenol reagent. j. biol. chem. 193: 263275. matsuda y, beppu t, arima k (1978). crystallization and positional specificity of hydroperoxidation of fusarium lipoxygenase. biochim. biophys. acta 530: 439-450. mine y, zhang jw (2001). the allergenicity of ovomucoid and the effect of its elimination from hen’s egg white. j. sci. food agric. 81: 15401546. mirella n, massimo da, gianni t, vincenzo g, maurizio df, cristima s (1995). inhibition of human low-density lipoprotein oxidation by caffeic acid and other hydroxycinnamic acid derivates. free rad. biol. med. 19: 541-552. sampson ha (1999). food allergy. part i: ommunopathogenesis and clinical disorders. j. aller. clin. immunol. 103: 717-728. sampson ha, mccaskill cc (1985). food hypersensiticity and atopic dermatitis: evaluation of 113 patients. j. paediatr. 107: 669-675. african journal of food science research vol. 2 (4), pp. 074-083, april, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper a provincial study of food safety measures in bundelkhand region in india singh jumi, raj vadewki and vijay p*. department of siddha medicine, faculty of sciences, tamil university, thanjavur-10, india. accepted 27 march, 2012 in the present study the authors have attempted to analyse the condition of food security in the bundelkhand region of uttar pradesh at district level. bundelkhand region has been chosen as a unit of study because it is one of the poorest regions in comparison with western, central and eastern regions of the state and even after being in close proximity to heartland of green revolution (punjab, haryana and western uttar pradesh) the region is still backward in agricultural development in india in general. agriculture is the mainstay of the economy and mostly dependent on monsoonal rainfall. the bundelkhand is continuously facing drought since last few years and people are migrating for employment. thus, there is an urgent need to look after the conditions of food security of the people living in this region. the standard statistical techniques have been used for the analysis of data. the study concludes that the condition of food security is unfavourable. only one district of jalaun has shown high food security. majority of the districts are moderate and low food secure because of low food grain availability, low purchasing power of the people, etc. the irrigation facilities are also not well developed leading to low consumption of fertilizer and low productivity of crops. employment rate, rate of urbanization, storage capacity and number of fair prices shops are also not satisfactory leading to pitiable food security condition in the study area. key words: food availability, food stability, food accessibility, food security, bundelkhand. introduction food security is the most heated and debated topic and in this context policy makers, planners, researchers and decision makers all over the world have been exploring ways and means to ensure food security to the millions of citizen (tripathy et al., 2011). a family is said to be food secure when it has physical, economic and social access to the required amount of food grains in terms of quantity, quality, safety, (sharma and sharma, 2008), the food available should be culturally acceptable and when it is not at undue risk of losing such access (mishra et al., 2008). the problem of food insecurity is prevalent all over the world but it is more common in developing countries like india which produces surplus food on one extreme but at the other extreme one third of the population is extremely *corresponding author. e-mail: paul_vijay@yahoo.com poor and one half of the children are malnourished in one way or the other (kannan et al., 2000). india has achieved a fourth fold increase in food grains from 50 million tonnes in 1950 to 219.3 million tonnes in 2007 to 2008 against a threefold increase in population from 33 million to more than 100 million (kumar, 2010). in terms of food factors, the concept of food security basically stands on three pillars, food availability, which deals with the domestic production of food grains in the area; food stability, which deals with the constancy in the availability of food in an area or to make the food available either through import from the surplus areas or by maintaining buffer stock at micro level at the time of manmade or natural disaster and harmful seasonal stability, and finally accessibility; which depends on the physical and economic access to food, guided by the purchasing power and per capita income of the people. it is of utmost importance because availability of food will be of no use, until and unless people have means to buy the available food (chaturvedi, 1997; grish, 1997; http://www.internationalscholarsjournals.org/ vijay et al 074 jain, 1997; ghosh, 2000). 2001), kravdal (2001) and anderson (2009). literature review burke (1989) examined that, in case of eastern uttar pradesh the productivity is low because there is acute pressure of rural population on the agricultural land, low per capita income and use of old agricultural implements. she further opines that, uttar pradesh will face acute shortage of rice and pulses in future, which constitute main diet of majority of the people and to overcome this problem one has to increase the irrigation potential of the agricultural backward areas and to increase the yield by applying modern inputs. alexandratos (1991) in his study opines that food security in terms of per capita availability of food is increasing around the world. but this improvement is uneven and there are many countries which are unable to contribute in such progress. the food security condition in some of the countries today is worse than it was 20 years before. the malfunctioning of economic development is responsible for the poverty and food insecurity. swaminathan (2001) opines that india has achieved satisfactory progress on the production side but in spite of this food insecurity is very common at the household level and poverty is the root cause behind this insecurity. he opines that the availability is not the problem but the accessibility to the available food is the main problem so, the new agriculture should aim to provide not only more and more food but also some employment opportunities in the farm and non-farm sector so that people gain some income to access the food. gorton et al. (2009) has discussed about various economic, physical and socio-cultural factors related to food security. they opine that a low level of education is associated with reduced food security. people living in urban areas as compared to their rural counterparts are more likely to experience food security. further speaking about transport they say that lack of transport to undertake food shopping has been linked with food security in mane studies. ubong et al. (2009) in their article says that, nigeria is the 10 th most populous state, 6 th largest petroleum exporting country with 50% of its land area cultivable in spite of this nigeria is unable to feed its population. a new approach of agricultural education has been canvassed and is therefore suggested that agricultural science should be made one of the core courses at the junior secondary level so as to give young people an early orientation on the role of agriculture not only in food security but in national, economic and social development. the problem of food security has attracted many scientists and has given voluminous and diverse literature in the problem from different perspectives. some of the important contributors are acharya (1983), mohammad (1978 and 1995), swaminathan (1996 and study area bundelkhand has been in the news for all unfortunate reasons. since many years, this region has been facing drought. due to the moneylender’s trap, the farmers are committing suicides, selling their land, cattle and even their wives (singh, 2010). the annual precipitation is low (600 to 1400 mm) and often erratic coupled with shorter duration which eventually makes the larger part of the region droughtprone with limited agricultural production. soils exhibit great variation ranging from highly impoverished, shallow and low water retentive red soils to highly productive, deep and good water retentive black soil, unlike the soil in other regions of uttar pradesh. if it does not rain at the right time with consistency, the drought-related distress affects the masses. the irrigation facilities are a total disaster and no one seems to care. unemployment is common and people are migrating in search of job. keeping all these points in view, the authors have selected bundelkhand region of uttar pradesh to provide an in-depth and detailed assessment about the condition of food security in the area. administratively, bundelkhand region of uttar pradesh (herein called as up-bundelkhand) comprises 48 blocks under the jurisdiction of 7 districts (figure 1). the geographical area of the up-bundelkhand is 2.94 million hectare which is about 12.21% of that state. depending upon the economic considerations and infrastructure development, up-bundelkhand is the poorest region in comparison with western, central and eastern regions of the state. up-bundelkhand has only 4.96% of the state’s population. objectives the objectives of the present study are to find out the spatial pattern of food availability, food stability and food accessibility and henceforth determine the food security in up bundelkhand and also to examine the interrelationship between the indicators of food security. finally, some measures and suggestions have been provided to improvement of food security in the study area. database and methodology the present study is based on the secondary sources of data to assess food security (table 1) at district level obtained from the bulletin of agricultural statistics of uttar pradesh for various years, published by the directorate, ministry of agriculture, department of statistics and economics, lucknow, uttar pradesh. food availability has been worked out at district level by taking three years moving average of agricultural produce to remove the weather abnormality and fluctuation in the data from 2006 to 2007 075 afr. j. food sci.res. figure 1. location of uttar pradesh bundelkhand region in india. vijay et al 076 table 1. variables of food security in bundelkhand region. s/ n description of indicators food availability 1 production of food grains (kg./head/annum) 2 calorie availability per head per day 3 population per livestock food stability 4 fertilizer consumption (kg per hectare) 5 percentage of gross irrigated area to net sown area 6 storage capacity per 1000 population (in quintals) 7 ration shop per lakh population 8 yield of food grains (kg per hectare) food accessibility 9 employment rate (percentage of main workers to total population) 10 literacy rate 11 length of roads per lakh population (in kms) 12 urbanization (percentage of urban population to total population) 13 per capita income (in rs) and 2008 to 2009. the food availability, the variables that have been included are cereals and pulses (food grains) but for the calories per head per annum along with the food grains, the production of sugar, oilseeds, potato and onion for each district have also been incorporated. there are many factors which affect the amount of food consumed, for example age, sex, occupation, income level, rural versus urban population, vegetarian and non vegetarian diet, body size, climate and for most of these the usable data is not available. the food consumed by a child and an adult is different. similarly food consumed by a male and a female, normal and pregnant women, person working in fields and office is not similar. according to singh’s scale each unit of population is equal to 0.773 consumption units or 1000 person equals to 773 consumption units (chakravarty, 1970). all the food grains produced are not available in the same amount for consumption. there are many sources of leakage between the production and consumption of food grains e.g. losses in storage and transport, destruction by insects and pests, kitchen waste, use of seeds etc but the exact data is not available for all these losses. after examining various estimates of losses given by different scholars, chakravarty (1970) has suggested a total deduction of about 16.8% of the total gross production for obtaining net food available for consumption. the coefficient of production is therefore, 0.832 (100-16.8=83.2). after calculating the net availability of food grains it has been converted into calorific values. as the nature of all the crops vary, so the calorie content per unit of crop is different. therefore all the crops taken under study have been converted into calorific value separately with the help of the conversion table and have been added together to get the food production in calories for each district. then the calories available for consumption are divided by total consumption unit to get calorie availability per head per annum and then further divide by 365 days to get calorie availability per head per day. the data regarding the variables of accessibility and district wise population for the year 2008 has been projected using the analytic method of population estimation (khan, 1998). the formula assumes, that the population has been increasing at a constant rate, observed during the census period that is 1991 to 2001, thus; pp = p1 + n/n * (p2 – p1) where pp is the projected population; p1 is the population of the previous census; p2 is the population of succeeding census; n is the number of years between the censuses; n is the number of years between the previous census and the year for which population would be projected. in order to standardize the raw data of all the variables, the method of z-score has been used (smith, 1973). z-score quantify the departure of individual observations, expressed in a comparable form. the formula is thus; where, zi is the standard score of the ith variable; xi is the original value of individual observation; i is the mean of variable and sd stands for the standard deviation. in first step district wise z-score of each indicator is calculated. the values so obtained are added district wise and average is taken to calculate the composite zscore as the index of development, by the formula; where, cs refers to composite z-score; zij stands for the sum of zscore of variable j in observation i; and n symbolize the number of variables. correlation matrix based on karl pearson’s technique has been used to examine the relationship between the variables of food availability, stability and accessibility. student’s ‘t’ test 077 afr. j. food sci.res. figure 2. uttar pradesh bundelkhand food availability regions, 2008. technique has been chosen to identify the level of significance at 0.01 and 0.05 of their correlation. results and discussion food availability regions food security of any region mainly depends on food availability which is a function of domestic agricultural production or through imports from surplus areas. to examine the overall scenario of food availability in up bundelkhand for the year 2008 the variables taken are; food grain availability (kg per head per annum), calorie availability (calorie per head per day) and livestock availability (population per livestock) (table 1). the standard z-scores have been obtained for the all the variables of food availability separately. with the help of z-scores, the composite z-scores have been calculated in order to delineate the food availability region at district level. the districts of the study area have been divided into three categories of high, medium and low food availability on the basis of mean and standard deviation method. figure 2 highlight that three districts of jalaun, lalitpur and hamirpur are having high availability of food. due to well coverage of canals, high consumption of fertilizer and high yield, the food grain availability and calorie availability is higher in these districts. availability of livestock is also high which has made these districts a high food available region. in spite of drought condition the availability of live stock is high because here animal husbandry is an integral part of the rural economy. considerable progress has been made in recent years in improving the live stock through breeding, culling undesirable animal and upgrading indigenous cattle with improved bulls of well known tried india breeds and distribution of seeds of improved varieties of fodder. district of jhansi and banda have moderate availability of food. though the food grain and calorie availability is low but livestock availability is high because of drought and migration. it is often said that there is more livestock than human population in up bundelkhand. people from jhansi and banda are migrating because of drought after selling their livestock to people who are still residing and this is the reason why the availability of livestock per head is greater. it is also possible that, due to hindu reverence for cows and buffalos up bundelkhand has high livestock population. the agricultural development of the district banda is not up to the standard and the hilly topography of the vijay et al 078 table 2. food availability, food stability and food accessibility with indicators in bundelkhand region, 2008. food food stability food accessibility availability storage number of employment urbanization production calorie consump % of gross capacity yield of rate road per s/n districts population fair prices (percentage of per capita of food availability tion of irrigated per 1000 food (percentage of literacy lakh per shops (per urban income grains in per head fertilizers area to net populatio grains main workers rate population livestock lakh population to (in rs) kg/h/annum per day (kg/ha) sown area n (in (kg/ha) to total (in km) population) total population) quintal) population) 1 jhansi 219.96 2324 2.54 62.52 64.11 298 38 1305.78 25.03 61.67 99.36 41.39 27602 2 lalitpur 374.99 3863 1.50 34.22 65.48 262 61 1162.34 29.90 45.86 118.60 14.74 22899 3 jalaun 338.39 3849 2.04 61.87 80.47 727 58 1612.55 60.57 60.57 134.09 24.12 28461 4 hamirpur 270.50 3730 1.98 29.29 45.04 196 43 1040.78 20.31 51.23 134.61 17.28 47043 5 mahoba 203.64 3171 1.57 31.60 24.04 329 55 712.49 31.09 54.64 161.49 22.70 27195 6 banda 225.58 2372 2.06 31.49 54.07 415 38 1004.86 21.83 49.35 96.57 16.69 23135 7 chitrakut 173.83 1836 1.42 43.76 25.52 14 64 848.21 26.70 66.21 108.88 10.07 19492 source: directorate, ministry of agriculture, department of statistics and economics, lucknow, u.p., census of india, 1991 and census of india, 2001. district jhansi does not permit large scale farming and cultivation of land under improved agricultural practices. this is the reason why the availability of food is moderate. mahoba and chitrakoot have low food availability. though the calories available for consumption are higher (because of inclusion of onion, potato, oilseeds and sugar) but low availability of food grains and livestock has made these districts to fall in the low category of food availability. these districts are also under the curse of droughts for past few years and this is the main reason for low food availability in these districts of study area. but at aggregate level the data shows that the food availability condition largely of food grains availability of up bundelkhand is good. this is because of nfsm (national food security mission) which aimed to increase production (especially for pulses which is suitable for dry land farming) through area expansion and productivity also creating employment opportunities and enhancing th farmlevel economy to restore confidence of farmers food stability regions the dimension of stability is influenced by the sustainability of the food system which minimizes the chances of risks related to food insecurity in normal and as well as in difficult times by ensuring the food consumption at a required level (tripathy, 2011). food stability calls for undertaking appropriate pre-emptive steps through which harmful, suspended and inter-annual instability of supplies of food can be reduced. the regional pattern of food stability has been examined on the basis of variables of yield per hectare, percentage of gross irrigated area to net sown area, storage capacity per thousand of population in quintals and ration shops per lakh population, and then composite zscore has been calculated out as shown in (table 3). on the basis of composite z-scores of the indicators of food stability (table 4), it can be viewed that, very high level of food stability is found only in one district of study area that is, jalaun (figure 3). due to the development in the infrastructure of agriculture that is increased consumption of fertilizer and area under irrigation the yield per unit of land in these districts has increased. storage capacity is highest in district of jalaun. in other words we can say that the concentration of all variables of food stability was high except ration shop per lakh of population. districts of jhansi, lalitpur and banda are having moderate food stability. though irrigational facilities, consumption of fertilizer and yield of food grain is adequate on one extreme but storage capacity and ration shops for the distribution of food grain is low, keeping these districts into moderate category of food stability. chitrakoot, hamirpur and mahoba have low food stability because concentration of almost all the variables of food stability was low in these districts. the storage capacity of these districts is low thus; they are unable to maintain stability in food supply even at the time of shortage. the farmers 079 afr. j. food sci.res. table 3. food availability, food stability and food accessibility with indicators (in z-score) in bundelkhand region (2008). food availability food stability food accessibility urbanization production calorie population consumption % of gross storage capacity no. of fair yield of employment rate road per (percentage per s/n districts of food availability irrigated area per 1000 prices shops food (percentage of literacy lakh of urban per of fertilizers capita grains in per head per to net sown population (in (per lakh grains main workers to rate populatio population livestock (kg/ha) income kg/h/annum day area quintal) population) (kg/ha) total population) n (in km) to total population) 1 jhansi -0.516 -0.830 1.675 1.409 0.609 -0.101 -1.202 0.695 -0.419 0.815 -0.977 2.007 -0.041 2 lalitpur 1.580 1.003 -0.925 -0.544 0.675 -0.269 0.922 0.215 -0.064 -1.325 -0.144 -0.616 -0.564 3 jalaun 1.085 0.986 0.425 1.364 1.385 1.858 0.607 1.723 2.173 0.666 0.526 0.307 0.054 4 hamirpur 0.167 0.844 0.275 -0.885 -0.294 -0.567 -0.700 -0.192 -0.764 -0.598 0.548 -0.366 2.119 5 mahoba -0.737 0.179 -0.750 -0.725 -1.290 0.042 0.378 -1.291 0.023 -0.137 1.710 0.168 -0.087 6 banda -0.440 -0.772 0.475 -0.733 0.134 0.435 -1.165 -0.312 -0.653 -0.853 -1.097 -0.424 -0.538 7 chitrakut -1.139 -1.411 -1.125 0.114 -1.219 -1.397 1.162 -0.837 -0.297 1.429 -0.565 -1.076 -0.943 table 4. districtwise food availability, food stability, food accessibility and food security by composite z-score, 2008 s/n districts food availability food stability food accessibility food security (composite z-score) (composite z-score) (composite z-score) (composite z-score) 1 jhansi 0.110 0.282 0.277 0.223 2 lalitpur 0.552 0.200 -0.543 0.070 3 jalaun 0.832 1.387 0.745 0.988 4 hamirpur 0.429 -0.528 0.188 0.030 5 mahoba -0.436 -0.577 0.335 -0.226 6 banda -0.246 -0.328 -0.713 -0.429 7 chitrakut -1.225 -0.436 -0.290 -0.650 ] calculation based on table 3. in these districts are still backward and have inappropriate knowledge about the modern agricultural technology like modern means of irrigation. the most recent data available from government of india shows that, in the study area the percentage of total irrigated area under food grains is 48.72 %, which is far below than that of state average of 74.81%. if we look district wise then it becomes evident that the percentage of total irrigated area in the districts of chitrakoot, hamirpur and mahoba is lowest at 25.6, 33.6 and 38.4%, respectively. agriculture in study area is dependent on rainfall but due to its erratic nature, the irrigation and productivity of crops has reduced to minimum. the adequate amount of rainfall required for districts chitrakoot, hamirpur and mahoba is 945, 851 and 940 mm, respectively but rainfall has deviated from the normal by -60, -50 and -66%, respectively. consumption of chemical fertilizer is lowest and area under food grains has continuously declined during last decade. thus, they are unable to increase the production and productivity of food vijay et al 080 figure 3. uttar pradesh bundelkhand food stability regions, 2008. figure 4. uttar pradesh bundelkhand food accessibility regions, 2008. grains on the existing agricultural land. on the other hand, public distribution system (pds), which was launched with the objective to provide food security to the needy and poor in india, does not have a growing curve in bundelkhand and the number of fair prices shops are still low per unit of population. food accessibility regions the availability and stability of food would loose its meaning until the people have access to the available food. the food accessibility in up bundelkhand has been assessed on the basis of employment rate, literacy rate, length of pukka/metalled road per lakh population in kilometres, percentage of urban population and per capita income. the district wise distributional pattern shows that two districts of jalaun and jhansi have high food accessibility (figure 4). the proportion of urban population and literacy rate is much higher in these districts and literacy is usually seen to have a high correlation with employment, providing better access to food. also, the good network of the roads in these districts enable the 081 afr. j. food sci.res. figure 5. uttar pradesh bundelkhand food security regions 2008. farmers to have an easy access to the agricultural inputs and better delivery and marketing of the agricultural produce to make the food available to the needy at right time. the per capita income of people in these two districts is also high leading to better purchasing power of the people. district hamirpur is the only district having moderate food accessibility. district of chitrakoot lalitpur and banda are showing low food accessibility. employment rate and urbanization is low leading to low purchasing power of the people which leads to low accessibility of food. though, the people are literate but the employment facilities and job opportunities are short and unemployment is common. despite the hype over nrega (national rural employment guarantee act) scheme, it has been found that 52% of the poor and needy households in the backward region of these districts did not get a single day of nrega employment. the majority of households in most parts of bundelkhand rely on income from local or inter-state, annual or seasonal migration for employment. till the end of year 2008 there were only two large manufacturing units in the entire region. no small or cottage industry in the region commanded a large market outside. the potential of tourism has not been well realized yet and services industry is limited to few pockets. urbanization is also low in these districts leading to reduced amount of urban facilities related to health and other infra structure facilities which are essential for improving the accessibility of food for population. bundelkhand is still backward in urban facilities and infrastructure. poverty and illiteracy is acute and also the main cause of food insecurity. for making the accessibility of food easier for the population, educational facilities should be provided. higher literacy will provide better employment opportunities, which in turn will lead to the better purchasing power and access to food. food security regions the study clearly shows that there are differences in the level of food availability, food stability and food accessibility among the districts of the study area. in order to bring out the whole picture of food security the zscores of all thirteen variables, three of food availability, five of food stability and five of food accessibility were taken district wise (table 3) and composite z-score for food security has been calculated (table 4). it becomes evident that the districts wise variation of zscore varies from maximum value of 0.988 in jalaun to minimum value of -0.650 in chitrakoot district of the study area. only one district of jalaun recorded high food security (figure 5) because out of thirteen variables all vijay et al 082 table 5. correlation matrix of indicators of food security. variables food availability food stability food accessibility food security food availability 1 0 0 0 food stability 0.656 1 0 0 food accessibility 0.368 0.483 1 0 food security 0.851* 0.889** 0.706 1 *correlation significant at 5% level, **correlation significant at 1% level. have contributed positively to food security. availability of food and livestock and higher consumption of fertilizer with adequate irrigational facilities accompanied by higher productivity of the crops are responsible for high food security of this district. purchasing power of the people and high employment are the main factors for the high level of food security in this district. jhansi, lalitpur, hamirpur and mahoba are showing moderate level of food security. the z-score also reveals that out of thirteen variables seven has contributed positively in district of jhansi, five in districts of lalitpur and hamirpur and six in mahoba district. the development in the infrastructure of agriculture is highly required because the consumption of fertilizer and yield per hectare is low. storage capacity and the number of fair prices shops should be increased to secure better access towards food. remaining two districts of the study area namely, banda and chitrakoot have shown low food security and out of thirteen only three variables are recorded as positive. relationship between the variables of food security the inter correlation matrix (table 5) exposes the causal relationship, among the variables of food security. the level of significance has also been tested on the basis of student’s‘t’ test technique (table 2). it can be inferred from table 2 that food security is positively correlated with food availability (r=0.851) and food stability (r=0.889) at five and one percent level respectively. however, it is positively correlated with food accessibility (r=0.706) but it is not significant. it may be concluded from the analysis that food security of the region depends upon the food availability and stability. food availability though insignificant, but positively correlates with food stability and accessibility. this shows that food stability and accessibility increases as much as food availability increases. conclusion and suggestions the study shows that, the condition of food security in up bundelkhand is very critical. the trio of food security that is, food availability, food stability and foods accessibility is responsible for food insecurity condition in one way or other. food security in the up bundelkhand is found to be positively correlated with food availability and food stability. though not significant, but also positively correlate with food accessibility. irrigation and drainage (i and d) sector in uttar pradesh had been in the forefront for more than a century and had been the vigour in transforming the ‘india of famines’ to ‘india with food security’. as is it also witnessed, that the success of green revolution in india has dependent on the area under assured irrigation facilities. unfortunately, this sector has not been getting the kind of growth attention and budgetary support which other sectors and other regions are getting for the development of irrigation. in bundelkhand region about 46% of the net sown area is still unirrigated and depends on the impulses of monsoon which is unsure and inconsistent. a large part of this region is badly affected by the menace of distressing drought thus drought mitigation measures should also be taken on large scale and diesel subsidy should be provided to the farmers in drought or limited rainfall areas so that the farmers could save the standing crops in the field. the storage capacity at community and household level should be developed to provide food to the needy at right time and also to avoid the exploitation of rural folks by middleman and traders. the availability of food grains at the government ration shops should be provided at subsidised rate and pds (public distribution system) should be strengthened because an effective pds can be the backbone of food availability to the consumers particularly the vulnerable sections of the society. the purchasing power of the people should be increased by providing them employment through various schemes under government, related to construction of roads and development of irrigation facilities through nrega (national rural employment generation act) and pradhan mantri gram sadak yojna. providing employment through food for work programme (fwp), integrated rural development programme (irdp) and through droughts control programmes like drought prone area programme (dpap). mass illiteracy prevails in the district of the study area, thus educational facilities should be provided to the people leading to employment and better access to food. references acharya kcs (1983). food 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swaminathan ms (1996). science and technology for sustainable food security, indian j. agric. econ., 51: 60-75. swaminathan ms (2001). food security and sustainable development. curr. sci., 81(8): 948-954. swaminathan ms (2001). science and sustainable food security, indian farming, 50(10): 4. tripathy kk, mishra kc (2011). food security in india: policy issues and challenges, 59(5): 14-20. ubong b, oguzor ns. wokocha am (2009). bulgarian j. sci. edu. policy (bjsep), 3(1): 96-108. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 4 (5), pp. 054-058, december, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of atherogenic property of oils extracted from cucumeropsis mannii mercy bih achu 1* , elie fokou 1 , clergé tchiégang 2 , martin fotso 3 and felicité mbiapo tchouanguep 4 1 department of biochemistry, faculty of science, p. o. box 812, university of yaoundé i, cameroon. 2 department of food science and nutrition, ensai/iut, ngaoundéré, cameroon. 3 centre for research in food and nutrition, impm, yaoundé, cameroon. 4 department of biochemistry, faculty of science, university of dschang, cameroon. accepted 03 february, 2016 the atherogenicity of cucumeropsis mannii and cucumis sativus oils was compared to that of corn oil and palm oil. female wistar albino rats of 6 weeks old weighing 58 – 65 g were randomly assigned to one of four diet groups: c. mannii and c. sativus oils (test diets) and corn and palm oil (control diets). there was weight gain in all the groups amounting to 128.65% (palm oil), 132.75% (c. mannii), 140.8% (c. sativus) and 153.45 (corn oil) with no significant difference. the weights of the livers ranged from 4.22 (palm oil) to 5.17 g (corn oil), ratio of weights of liver to that of rat, from 0.029 0.034 and percentage weight gain from 128.65 (palm oil) to 153.45% (corn oil). there was no significant difference in the values. for the atherogenic parameters measured, the triglyceride level ranged from 73 (palm oil) to 79.4 (c. sativus oil) with no significant difference. total cholesterol levels ranged from 49.6 (corn oil) to 64.2 mg/dl ( c. sativus oil) with significantly lower values in the corn oil group but similar values in the rest of the groups. hdl ranged from 18.94 (c. mannii oil) to 32.8 (palm oil) which was significantly high, ldl from 6.2 (palm oil) to 25.06 mg/dl ( c. mannii oil) and atherogenic ratio (ar) from 0.2 (palm oil) to 1.61 (c. mannii) which was significantly high. the levels of these atherogenic parameters are far below the borderline level for oils to cause atherosclerosis, indicating that they could be potential good edible oils for reducing cardiovascular illnesses. keywords: atherogenicity, oils, cucumeropsis mannii, cucumis sativus. introduction in africa, obesity has become a major problem in line with hiv. though obesity is traditionally seen as a sign of wealth, it is becoming a very serious health issue due to its complications (bbc news, 2004). in cameroon, according to information from an obesity clinic in the yaounde central hospital, 35% of the adult population is either overweight or obese and this often leads to diabetes and cardiovascular diseases. in addition to exercises and drugs (which usually have undesirable side effects), one of the ways of overcoming this health problem can be through the use of diets and foodstuffs, especially those that are locally available. cucurbitaceae (egusi) seeds are one of such foodstuffs. these seeds have been shown to be rich in proteins and oils and the *corresponding author. e-mail: lohmercy@yahoo.fr. oils contain mostly linoleic (an essential fatty acid which can only be got from the diet) followed by oleic acid (silou et al., 1999; murkovic et al., 1996; younis et al., 2000; achu et al., 2005; achu, 2006). essential fatty acids are important for normal foetal and infant growth and development, brain development and visual acuity (fao, 1994). the other essential fatty acids; linolenic acid, can be got from linoleic acid, and arachidonic acid from linolenic acid. arachidonic acid is a component of the liver, plasma phospholipids and cholesterol esters. it is an important precursor of prostaglandins, thrombosanes and prostacyclins. prostaglandins stimulate contraction of smooth muscles. prostacyclins have an antagonistic effect to that of thrombosanes by inhibiting platelet aggregation, relaxing coronary arteries and lowering blood pressure (ottaway and apps, 1984). linoleic acid (polyunsaturated) moderately reduces serum cholesterol and ldl levels. oleic acid (monounsaturated) appears to be achu et al. 055 neutral in regard to ldl but modestly raises hdl (fao, 1994). there is little data on the atherogenicity of oils from cucurbit seeds cultivated in cameroon. the main objective of the study was to investigate the atherogenic property of oils extracted from cucumeropsis mannii and cucumis sativus seeds from cameroon which can be exploited at the alimentary levels. this was in order to determine the levels of serum triglycerides, total cholesterol (tc), high density (hdl) and low density (ldl) lipoprotein cholesterol, atherogenic ratio (ar) and proteins of rats fed with oils extracted from the seeds of c. mannii (egusi melon) and c. sativus (“ibo” egusi) seeds compared to rats fed with palm and corn oils diets. materials and methods sample collection and treatment c. mannii seeds were collected from ebolowa (in the south province) and c. sativus seeds from bafia (in the centre province), which are amongst the regions of great cultivation of these seeds in cameroon. the seeds were bought already sun-dried by the farmers, transported in polyethylene bags to the laboratory, wiped with filter paper and dried in an oven at 70°c to constant weight. they were ground in an electric grinder, put in airtight bottles and stored in the desiccator for analyses. extraction of the oils oils were extracted from the ground seeds by continuous extraction in a soxhlet apparatus for 8 h using hexane as solvent (aoac, 1980). the hexane was evaporated on a rotary evaporator and the oil obtained was dried in an oven at 60°c for 24 h to remove all traces of solvent. the experimental design the experimental design was 4 types of oils tested x 4 groups of rats x 5 rats per group. that is: 4 oils x 4 groups = 4 x 4 groups. formulation of the diets given to rats the diet was formulated by weighing the various components according to the american institute of nutrition-76 (ain-76) composition, modified for casein level as follows: casein 10%, starch 36%, -cellulose 5%, vitamin mixture 1%, choline chloride 0.1%, dlmethionine 0,3%, sucrose 27.6%, salt mixture 5%, oil 5% (oil = corn oil, palm oil, oil extracted from c. mannii and c. sativus seeds). this diet was mixed with 10% of water to moisten the food for easy consumption by the rats. the food was given to the rats in the form of a paste of 20 g of food per rat. treatment of the rats 20 wistar albino rats of 6 weeks old weighing 58 – 65 g were used. one rat was put in a metabolic cage for an adaptation period of 5 days. during this time, they received the same food (that is, the food that was consumed by rats of the corn oil group which is the corn oil diet) and water ad libitum. they were then weighed and randomly distributed into 4 groups of 5 rats each, receiving 4 types of food differing only by the type of oil (corn oil and palm oil for the control groups and c. mannii and c. sativus oils for the experimental or test groups). the feeding took 3 months. this was to allow enough time to follow up the effect of the oils tested on the metabolism of the rats. the rats were weighed every 3 days. 12 h before sacrifice, these rats were left to fast. they only took water. on the morning of the sacrifice, they were weighed and sacrificed by decapitation. the blood of each rat was collected into dry 10 ml test tubes and centrifuged at 3000 rpm/min for 5 min. the separated serum was collected into 2 ml ependoff tubes. the serum was divided into 2 parts. the first part was preserved in aliquots at 4°c for hdl cholesterol analysis and the second part at -28°c for the rest of the analysis. the liver of each rat was equally collected and weighed in order to see the effect of the oils tested on the size of the liver. analysis of serum samples triglycerides, total and hdl cholesterol were assayed enzymatically according to the method on the randox kits used, while ldl levels were calculated using the formula of friedewald et al. (1972). the ar ratio was also calculated and the serum protein levels were assayed according to the method of gornall et al. (1949). statistical analysis the kruskal-wallis test (for the distribution of the sample as shown by the kolmogorov test was non normal) was used to find differences between the parameters measured in the rats that received diets prepared with the different oils. the studentnewmankeuls (s-n-k) test was used to locate these differences. the tests were done at 5% level of significance, using spss 10.1. results table 1 shows the amounts of food ingested, the evolution of the body weights of the rats and the ratio, weight of liver/weight of rat. the amounts of food ingested per rat per day ranges from 9.68 (corn oil) to 11.06g ( c. mannii oil). at the start of the experiment, the weights of the rats ranged from 58.28 (c. sativus oil) to 64.91 g (c. mannii). at the end of the experiment, they ranged from 139 (c. sativus) to 151.07g (c. mannii). there is an increase in weight in all the groups with a gain of 80 (palm oil) to 90.52 g (corn oil) amounting to 128.65% (palm oil), 132.75% (c. mannii), 140.8% (c. sativus) and 153. 45% (corn oil), showing no significant difference. the weights of the livers range from 4.22 (palm oil) to 5.17g (corn oil), ratio of weights of liver to that of rat, from 0.029 (palm and c. mannii oils) to 0.034 (corn oil). there is no significant difference between the food ingested and the weights of these rats that received diets prepared with the different types of oils. table 2 shows the serum levels of triglycerides (tg), total cholesterol (tc), hdl and ldl cholesterol, atherogenic ratio (ar) and serum proteins. no significant differrences were observed in the total tg levels among the four groups. however, total serum cholesterol was significantly higher in the rats on c. sativus compared to the rats on corn oil diet. there was no significant differrence in serum hdl cholesterol among palm oil and c. 056 afr. j. food sci. res. table 1. evolution of body weights of rats and the ratio, weight of liver/weight of rat. food initial weight final weight weight gain weight of liver/rat groups ingested % weight gain of rats (g) of rats (g) (g) liver (g) weight (g/rat/day) corn oil 9.68 ± 2.58 58.89± 3.82 149.40± 19.03 90.52± 16.67 153.45± 24.68 5.17± 1.26 0.034± 0.005 palm oil 9.77 ± 1.93 63.06 ± 3.36 143.73 ± 6.11 80.66 ± 8.36 128.65 ± 19.09 4.22 ± 0.37 0.029± 0.002 c. sativus oil 10± 1.67 58.28 ± 5.23 139.85 ± 6.37 81.57± 2.83 140.80± 12.58 4.42 ± 0.26 0.032 ± 0.001 c. mannii oil 11.06 ± 1.72 64.91 ± 3.17 151.07± 12.86 86.16 ± 11.49 132.75 ± 16.76 4.32 ± 0.28 0.029± 0.002 no significant differences were observed between groups in the amount of food ingested and in the total weight gain. table 2. serum levels of triglycerides, total cholesterol, hdl and ldl cholesterol, atherogenic ratio and serum proteins. 0 triglycerides total hdl ldl atherogenic 0 total groups (mg/dl) cholesterol cholesterol cholesterol ratio, proteins (g/l) (mg/dl) (mg/dl) (mg/dl) (ldl/hdl) corn oil 76.2± 12.66 49.6± 4.93 b 25.92± 2.52 ab 8.44± 1.14 b 0.33± 0.05 b 79.04 ± 13.4 palm oil 73 ± 10.2 53.6± 7.99 ab 32.8± 6.53 a 6.2 ± 4.57 b 0.20± 0.13 b 81.57± 8.01 c. sativus oil 79.4 ± 10.06 64.2± 8.64 a 27.72± 4.18 ab 20.6± 8.65 a 0.77± 0.38 a 78.17± 11.83 c. mannii oil 75 ± 6.96 59 ± 6.82 ab 18.94± 7.14 b 25.06± 7.25 a 1.61 ± 1.05 a 84.71±15.37 kruskal-wallis test: 0 = there is no significant difference (p>0.05) between values in the same column. student-newman-keuls test: values in the same column with different letter superscripts are significantly different (p<0.05). sativus oil. however, hdl cholesterol was significantly higher in the rats on palm oil compared to those on c. mannii oil (p<0.05). ldl cholesterol was significantly higher in the rats on c. mannii and c. sativus oils compared to the control diets, corn oil and palm oil. similarly the atherogenic ratio (ar) was significantly higher in the rats on c. mannii and c. sativus oils compared to corn and palm oil diets. total serum protein was not signifycantly different among groups. discussions the triglyceride (tg) levels were 75 in c. mannii and 79.4 in c. sativus oil rats compared to 73 in palm oil and 76.2mg/dl in corn oil rats, but with no significant differrence. however, from the results of analysis of the quality of c. mannii and c. sativus oils, c. sativus oil shows lower levels of free fatty acids (acid index: c. sativus oil = 1.76 and c. mannii oil = 4.56), higher degree of unsaturation (iodine index: c. sativus oil = 114.4 and c. mannii oil = 106.78; unsaturated fatty acids: c. sativus oil = 78.4% and c. mannii oil = 54.7%; saturated fatty acids or sfa: c. sativus oil = 21.7% and c. mannii oil = 42%), lower level of peroxides (peroxide index: c. sativus oil = 3.31 and c. mannii oil = 13.68), lower r1 value (r1 is the ratio of the sum of saturated to that of unsaturated fatty acids. the r1 of c. sativus oil = 0.28 and c. mannii oil = 0.77) and lower linolenic acid level (linolenic acid: c. sativus oil = 0.1% and c. mannii oil = 0.3%) (achu, 2006). these suggest that the quality of c. sativus oil is better than that of c. mannii oil and that c. sativus oil contains more unsaturated fatty acids than c. mannii oil. c. sativus and c. mannii oils have high levels of polyunsaturated fatty acids, pufa (39.3% in c. mannii oil and 62.2% in c. sativus oil) as in corn oil (58.5%) while palm oil has a low level (8.5%) (table 3). these results are similar to those of noubissi in djamen (1998), who after 2 months of experiment, found a non significant decrease in the tg level of rats fed with palm oil diet (rich in sfa) compared to those fed with cotton oil diet (poor in sfa). however, these results show that these egusi oils can be good edible oils (do not cause hypertriglyceridaemia) because their tg levels which are 75 in c. mannii oil and 79.4 mg/dl in c. sativus oil rats, are far below 150 mg/dl, the borderline level of tg, above which the person has hypertriglyceridaemia (randox assayed multisera, 2004b). the total cholesterol (tc) level of c. sativus oil (64.2) is similar to those of c. mannii oil (59) and palm oil (53.6) but significantly higher than that of corn oil rats (49.6mg/dl). c. sativus and corn oils all have high levels of pufa which are 62.2 and 58.5% respectively, due to their high linoleic acid levels. but c. sativus oil has higher levels of sfa (21.7%) than corn oil (16.5%) (table 3). hence, the high tc level in c. sativus oil compared to corn oil rats may be due to its higher sfa levels. however, the tc levels of all these four groups of rats were low when compared to levels that cause hypercholesterolaemia. secondly, other studies have shown that the hypercholesterolaemic effect of sfa is mostly due to c14:0 and c12:0 fatty acids rather than c16:0 (ng, 1994). c. sativus and c. mannii oils contain little or no achu et al. 057 table 3. fatty acid composition of oils used. corn oil palm oil c. sativus oil c. mannii oil (achu, fatty acid symbol (ngogang et al., (fao, 1981) (achu, 2006) 2006) 1996) caprylic acid c8:0 capric acid c10:0 lauric acid c12:0 1.5 myristic acid c14:0 1 3.5 4.7 palmitic acid c16:0 12 40.6 10.7 24.4 palmitoleic acid c16:1 0.5 0.8 stearic acid c18:0 2 4 10.6 11.2 oleic acid c18:1 24 43 16.2 14.6 linoleic acid c18:2 56.5 8.5 61.8 38.7 linolenic acid c18:3 2 0.1 0.3 arachidonic acid c20:0 1 0.4 0.2 gadoleic acid c20:1 0.5 arachidonic acid c20:2 0.3 0.3 behenic acid c22:0 lignoceric acid c24:0 0.5 total saturated fatty acids 16.5 48.1 21.7 42 total mufa 25 43.4 16.2 15.4 total pufa 58.5 8.5 62.2 39.3 total unsaturated fatty acids 83.5 51.9 78.4 54.7 pufa/sfa (r2) 3.55 0.17 2.87 0.93 = not found, mufa = monounsaturated fatty acids, pufa = poly unsaturated fatty acids. c14:0 and c12:0 fatty acids. their sfa are mostly c16:0 as in corn and palm oils (table 3) which do not lead to much rise in cholesterol levels. also, the non-hypercholesterolamic action of these oils is possibly due to their presence of tocotrienols, the unsaturated analogue of tocopherols. tocotrienols inhibit cholesterol synthesis in vivo, exerting a hypocholesterolamic action in humans and animals. this has been shown in palm oil by rukmini (1994). however, these results show that these oils are good edible oils (do not cause hypercholesterolaemia) because their tc levels (59 in c. mannii oil and 64.2 mg/dl in c. sativus ) are far below 200 mg/dl, the desirable blood cholesterol level, above which the person has hypercholesterolaemia (randox, 2004a). the hdl level of c. sativus (27.72) is similar to those of c. mannii (18.94), corn oil (25.92) and palm oil (32.8 mg/dl) rats. the hdl level of c. mannii oil is similar to those of c. sativus and corn oil rats but significantly lower (p<0.05) than that of palm oil rats (table 2) . this lower hdl value in c. mannii compared to palm oil rats may be due to the lower level of mufa in c. mannii (15.4%) than palm oil (43.4%) and higher pufa in c. mannii (39.3%) than palm oil (8.5%) (table 3) . this is in line with the results of a study carried out by o’callaghan et al. in during et al. (2000) who found that patients fed pufa dairy products showed lower plasma hdl levels than patients fed mufa dairy products. this might be the case with these oils where c. mannii had higher pufa, lower mufa and showed lower hdl levels than palm oil with lower pufa, higher mufa and high hdl levels. the ldl level of c. mannii oil rats (25.06) is similar to that of c. sativus (20.6) and these are significantly higher than those of corn (8.44) and palm (6.2) oil rats, with similar ldl levels. apart from increasing hdl levels, high mufa also reduce ldl levels. this high ldl level and the eventual higher atherogenic ratio in c. mannii and c. sativus oils may be due to their lower mufa and higher pufa levels as opposed to corn and palm oil rats. this is seen in the study carried out by during et al. (2000) where rats fed with experimental cheeses containing mostly mufa resulted in a significant increase of hdlcholesterol (11%) and a significant reduction of ldlcholesterol (31%). other findings have shown that, in addition to unsaturated tg, dietary phytosterols in vegetable oils can also reduce plasma ldl levels (jones et al. in during et al., 2000). hence, high hdl and low ldl levels are due to high mufa (low pufa) and dietary phytosterols (as in palm oil) and low hdl and high ldl levels are due to low mufa, high pufa and possibly low phytosterols (as in c. mannii and c. sativus oils). however, the hdl levels of c. mannii (18.94) and c. sativus oil rats (27.72mg/dl) are below the desirable hdl level (>40 mg/dl) and their ldl levels 25.06 and 20.6 mg/dl respectively are far below the borderline (<130 058 afr. j. food sci. res. mg/dl) (american academy of family physicians, 2005). this means that these oils could be very good in reducing ldl and to a lesser extent, raise hdl levels. however, these oils need to be tested on humans in order to better confirm these findings that were carried out on wistar albino rats. the atherogenic ratio, ar (ldl/hdl) in c. mannii oil rats (1.61) is similar to that of c. sativus (0.77) and these are significantly higher (p<0.05) than those of corn (0.33) and palm (0.2) oil rats with similar ar levels. these high ar levels in c. mannii and c. sativus oil rats are due to their high ldl levels, caused by their low mufa levels. although the atherogenic ratio is higher in the test groups (than the palm oil and corn oil groups), these ratios are good, for they are below 3.55 in men and 3.22 in women, the borderline levels for lipids to cause atherosclerosis. above these values, the risk of appearance of cardiovascular illness is high (laboratoires fournier, 1981). the serum protein levels are 84.71 g/l in c. mannii and 78.17 in c. sativus oil rats compared to 79.04 in corn and 81.57 g/l in palm oil rats but with no significant difference in all these values. conclusion this study which was aimed at investigating the atherogenic property of oils extracted from c. mannii and c. sativus seeds from cameroon showed that these oils are good in reducing ldl cholesterol levels thereby reducing the atherogenic ratio, for the ar levels were far below the threshold values expected to cause athero-sclerosis. c. mannii and c. sativus oils could be potential good edible oils for use in the reduction of cardiovascular illnesses. there is the need to carry out this study on humans to better confirm these findings that were carried out on wistar albino rats. acknowledgement this research was supported by the international foundation for science (ifs), stockholm, sweden and the organization for the prohibition of chemical weapons (opcw), the hague, netherlands, through a grant to mrs achu mercy bih. references achu mb, fokou e, tchiégang c, fotso m, tchouanguep fm (2005). nutritive value of some cucurbitaceae oilseeds from different regions in cameroon. afr. j. biotechnol. 4(10): 1329-1334. achu mb (2006). a study of some physicochemical characteristics and nutritional properties of five cucurbitaceae oilseeds from cameroon. thesis presented and defended for the award of a doctorate phd in biochemistry. faculty of science, university of yaounde i. p. 180 american academy of family physicians (2005). cholesterol: what your level means. heart disease and stroke. http://familydoctor.org/029.xml.htm. access date 05/12/2005. aoac (1980). official methods of analysis of the association of official analytical chemists. 13 th ed. william horwitz ed. washington d.c. 7: 56-132 bbc news (2004). obesity epidemic out of control. bbc news world africa. sunday, 31 october, 2004, 15:22 gmt. http://news.bbc.co.uk/2/low/africa/3969693.stm access date 15/03/2007. during a, combe n, mazette s, entressangles b (2000). effects on cholesterol balance and ldl cholesterol in the rat of a soft-ripened cheese containing vegetable oils. j. ame. college of nutr. 19 (4): 458-466 fao (1981). le rôle des graisses et huiles alimentaires en nutrition humaine. alimentation et nutrition; 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on cholesterol balance and ldl cholesterol in the rat of a soft-ripened cheese containing vegetable oils. j. ame. college of nutr. 19 (4): 458-466. ottaway jh, apps dk (1984). concise medical textbooks. biochemistry. 4 th edition. london. pp. 6100. randox (2004a). cholesterol, enzymatic endpoint method, manual. ch 200, randox laboratories ltd, ardmore, diamond road, crumlin, co. antrim, united kingdom, bt29 4qy. p. 2. randox (2004b). triglycerides, gpo-pap method, manual. randox laboratories ltd, ardmore, diamond road, crumlin, co. antrim, united kingdom, bt29 4qy. p. 2. rukmini c (1994). red palm oil to combat vitamin a deficiency in developing countries. food and nutrition bulletin. 15: 24. silou t, mampouya d, loka lonyange wd, saadou m (1999). composition globale et caractéristiques des huiles extraits de 5 espèces de cucurbitacées du niger. la rivista italiana delle sostanze grasse. lxxvi: 141-144. younis ym, ghirmay s, al -shihry ss (2000). african cucurbita pepo l. properties of seed and variability in fatty acid composition of seed oil. phytochem. 54 (1): 71-75 1 african journal of food science research issn 2375-0723 vol. 2 (11), pp. 149-154, december, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper prediction of drying model and determination of effects of drying temperature on mucilage and vitamin-c contents of fluted jute (corchorus capsularis) leaves famurewa j. a. v and akinmuyisitan f a department of food science and technology, federal university of technology, akure. accepted 9 december, 2014 this study investigated the suitable drying model and the effects of different drying temperatures on the mucilage property of fluted jute leaves (corchorus capsularis , . a cabinet dryer was employed to dry a well cleaned leaves in a thin layer, differently at air temperatures of 40, 50, 60 and 70 ℃ respectively. five thin layer-drying models (lewis, page, modified page, henderson and pabis, and logarithmic) were fitted to the experimental moisture ratio data. the dried samples were analyzed for mucilage and vitamin c contents. drying of corchorus leaves prominently occurred in falling rate period. among the mathematical models investigated, the logarithmic model satisfactorily described the drying behaviour of corchorus leaves with highest r 2 and lowest rmse , mbe, and χ 2 at 40, 50, 60 and 70 0 c respectively. viscosity was at its highest at 70 0 c and lowest values at 40 ℃ while vitamin c was highest at 50 0 c and lowest at 40 ℃. based on the result of this study, drying temperature of 50 ℃ and logarithmic model were recommended for drying of corchorus for better retention of vitamin c, though 70 0 c retained higher quantity of mucilage, but not significantly different to that of 50 0 c. key words: fluted jute leaves, mucilage, vitamin c, modelling, drying. introduction green leafy vegetables constitute an indispensable constituent of human diet. in africa, they are consumed as cooked complements to the major staples, like cassava, cocoyam, guinea corn, maize, millet, rice and plantains. many of these leaf vegetables are common in all parts of nigeria, but some are restricted in their natural distribution because of climatic factors. corchorus spp. is a member of the family tilaceae, common names include: long fluted jute, jew’s marrow, african sorrel, bush okra. local names include: ‘ewedu’, ‘lalo’, ‘krin krin’. two main types are common, one with finely serrate leaves, called ‘ amugbadu’ and the other is shorter with coarsely serrated short leaves called oniyanya’ *corresponding author. e-mail: jav_murewa@yahoo.com oniyanya’ (oguntona, 1998). it is cultivated as a vegetable and has the advantage of tolerance to many soil conditions. among any communities, the leaves are valued as a cooked vegetable because of its high proportion of mucilage. the renewed interest in non-orthodox medicine in the world has dramatically increased the demand for plantbased medicine in global market (natesh 2000). according to chen and saad (1981), corchorus species contain important bioactive compounds such as cardiac glycosides, stropanthidin, b-sitosterol, terpenoid-corosin, flavone glycoside, urasolic acid, vitamin c, b-carotene, mucilage, and others which are potential ingredients for developing plant-based drugs. the leaves are rich in protein, b-carotene, iron, calcium, vitamin b, and vitamin c. they also contain oxydase and chlorogenic acid. according to awogbemi and ogunleye (2009), the folic acid content is substantially higher than that of other folacin-rich vegetables. 2 famurewa and akinmuyisitan 149 table 1.results for vitamin c and viscosity contents of dried corchorus leaves. treatment ℃ vitamin c (mg/100g) viscosity (pascal/ses) 40 359.4 d 2.692 50 1078.75 a 2.693 60 890.03 b 2.694 70 531.25 c 2.969 values are means of three replicates; values in a column denoted by different letters differ significantly at p <0.05. drying is one of the oldest methods known for the preservation of agricultural products such as fruits and vegetables, and is the most common in food engineering unit operations. drying of agricultural products enhances their storage life, minimizes losses during storage, and save shipping and transportation costs (doymaz 2005). according to gogus (1994) and sokhansanj and jayas (2006), when carried out correctly, the nutritional quality, colour, flavour and texture of rehydrated foods are slightly less than fresh foods. however, if drying is carried out incorrectly there is a greater loss of nutritional and eating qualities and possibly even food poisoning. drying kinetics of food is a complex phenomenon and requires dependable models to predict drying behaviour (akpınar and bicer, 2003). there are several studies describing the drying behaviour of various fruits, vegetables and medicinal plants such as onions (singh and sodhi, 2000), garlic cloves (sharma and prasad 2001), black tea (panchariya et al., 2002), grapes (el-ghetany. 2006), apple (kaya et al., 2007), mint leaves, (kadam et.al 2011b), tomato slices (abano et al., 2011) and other various vegetables (awogbemi and ogunleye, 2009) the objectives of this study therefore are to investigate the effect of drying temperatures on the viscosity of corchorus capsularis and to predict the mathematical drying models, at four different temperatures, that best describe the drying phenomenon of ‘ewedu’. materials and methods materials a cabinet dryer was used. other materials used include a digital weighing balance (accuracy of ± 0.01g), and a bowl. methods drying experiment the corchorus leaves used for this experiment were all sourced from a farm located at alaba layout, south gate of the federal university of technology, akure. the leaves were separated from the stem and placed in a stainless steel tray. fifty (50) grams of the leaves were spread in thin layer inside a cabinet dryer. drying was performed at four different temperatures, 40 ⁰c, 50 ⁰c, 60 ⁰c and 70 ⁰c. drying continued until two successive constant weights were obtained. modeling of drying curves for this study, five (5) mathematical models were reported to predict drying conditions; other models showed a very wide variation. these models and their equations are lewis,. henderson and pabis, page, modified page and logarithmic. sigma plot software (windows version, 10.0, synstat software inc) was used for the modeling of equations, and the generation of the r 2 values. windows excel (microsoft office suite, microsoft inc.) was used in the calculation of the root mean square error (rmse), mean bias error (mbe) and χ 2 values and plotting of the drying curves and the model validation curves for each temperature. vitamin c and viscosity determination both vitamin c (ascorbic acid) and viscosity (mucilage) were carried out using a method described by aoac (1990). results and discussion effect of drying temperature on viscosity and vitamin c of corchorus the result in table (1) showed the effect of drying temperature on the vitamin c content and the viscosity of the leaves. the sample dried at 50 ℃ has the highest value of 1078 mg/100 g, while the sample dried at 40 ℃ had the lowest value at 359 mg/100 g. according to (ihenkoronye and ngoddy 1985), vitamin c is heat labile; the low value may be due to the elongated drying period as compared to other samples. this result also agreed with the one reported by famurewa (2011), when effect 3 150 afr. j. food sci. res. figure 1. drying curves of corchorus at different temperatures. table 2. values of model constants for corchorus dried at 70℃. model model constants r 2 rmse mbe χ2 lewis b=0.0344 0.8906 0.107177 -0.04486 0.014359 page n=0.1592, b=0.8468 0.9988 0.107177 -0.04486 0.074767 modified page n=0.1592, b=0.3519 0.9988 0.107176611 -0.04486 0.01914471 henderson and pabis a=0.9725, b=0.0332 0.8920 0.107176611 -0.04486 0.01914471 logarithmic c=0.1737, a=0.8263, b=0.0828 1.0000 0.107176611 -0.04486 0.028717065 of drying on worowo (senecio biafrae) was investigated. for the viscosity, the sample dried at 70 ℃ had the highest value, while the sample dried at 40 ℃ s still had the lowest values. observed drying rate during drying of corchorus leaves the drying times needed to reach equilibrium moisture content were 420, 390, 160, and 120 min respectively for 40℃, 50℃, 60℃ and 70℃. the results showed that as the temperature was increasing successively by a difference of 10 ℃, drying time was also decreasing. the drying curves showed that the constant rate period was absent; and drying took place in the falling rate period and the moisture loss was faster at the beginning than towards the end of the drying period (figure 1). these observations are in good agreement with the reports of sharma and prassad (2001), demir et al., (2004) and premi et al., (2010). mathematical models for fitting of the drying curves moisture ratio drying data for corchorus were fitted into five mathematical models (lewis, page, modified page, henderson and pabis, and logarithmic). the co-efficient of correlation and statistical analyses for the samples are listed in tables 2, 3, 4, and 5. in all the models, r 2 values were greater than 0.90 except the henderson and pabis model indicating, the fitness of the models in predicting the data and drying behaviour. results show that the highest value for r 2, , and lowest value for χ 2 , mbe and rmse were obtained using the logarithmic model. this model can therefore be considered as to represent the drying behaviour of corchorus leaf the results obtained are similar to those reported by kadam et al., (2011a) for 4 famurewa and akinmuyisitan 151 table 3. values of model constants for corchorus leaf dried at 60℃. model model constants r 2 rmse mbe χ2 lewis b=0.0192 0.9163 0.082938 -0.0254 0.008025 page n=0.5178, b=0.1442 0.9821 0.038389 -0.00226 0.002063 modified page n=0.5178, b=0.0237 0.9821 0.038389 -0.00226 0.002063 henderson and pabis a=0.9516, b=0.0181 0.9204 0.038389 -0.00226 0.009158904 logarithmic c=0.1565, a=0.8470, b=0.0340 0.9981 0.012446285 1.42857e05 0.000271093 table 4. values of model constants for corchorus leaf dried at 50℃. model model constants r 2 rmse mbe χ2 lewis b= 0.0092 0.8628 0.088378 0.02447 0.102258 page n= 0.5712, b= 0.0739 0.9672 0.043194 -0.00409 0.110779 modified page n= 0.5712, b= 0.0105 0.9672 0.043194 -0.00409 0.110779 henderson and pabis a= 0.8862, b= 0.0079 0.8836 0.081406463 -0.024021429 0.110779025 logarithmic c= 0.1620, a= 0.8423, b= 0.0168 0.9963 0.014506624 -3.96508e-18 0.120849845 table 5. values of model constants for corchorus leaf dried at 40℃. model model constants r 2 rmse mbe χ2 lewis b= 0.0080 0.8712 0.083912 -0.01865 0.007544 page n= 0.5872, b= 0.0627 0.9763 0.036 -0.00355 0.008124 modified page n= 0.5872, b= 0.0089 0.9763 0.036 -0.00355 0.008124 henderson and pabis a= 0.8766, b= 0.0067 0.8977 0.074780465 -0.01938 0.008124497 logarithmic c= 0.1664, a= 0.8307, b= 0.0145 0.9989 0.007607146 6.66667e-06 0.008801538 figure 2. validation curve for various models at 40℃. 0 0.2 0.4 0.6 0.8 1 1.2 0 100 200 300 400 500 m o is tu re r at o , ( p d ) drying time( min validation curve at 40⁰c for the models. exp lewis page m.page h and p logarithnic 5 152 afr. j. food sci. res. figure 3. validation curve for various models at 50℃. figure 4. validation curve for various models at 60℃. figure 5. validation curve for various models at 70℃. 0 0.5 1 1.5 0 200 400 600 m o is tu re r at io , ( m r ,( p re d ) drying time, (min) validation curve at 50⁰c for the models exp lewis page m. page h and p logarithmic 0 0.5 1 1.5 0 100 200 m o is tu re r at io , m r (p re d ) drying time, (min) validation curve at 60⁰c for the models exp lewis page m. page h and p logarithmic 0 0.5 1 1.5 0 50 100 150m o is tu re r at io , m r ( p re d ) drying time(min) validation curve at 70⁰c for the models exp lewis page m. page h and p 6 famurewa and akinmuyisitan 153 basil leaves, kadam et al., (2011b) for mint leaves, togrul and pehlivan, (2002) for apricots; erenturk et al., (2004) for rosehip and goyal et al.,(2007) for plum. the mathematical models were subjected to validation, by plotting both the experimented and predicted moisture ratio values against the drying time. (figures 2-5). the figures showed that logarithmic model established by mathematical model gave the best fit between experimental and predicted moisture ratios for the four levels of drying temperatures. conclusion corchorus leaves were dried at 40, 50, 60 and 70°c temperature, the drying rate decreased continuously throughout the drying period. constant rate period was absent and the drying process of the leaves took place in falling rate period. drying time decreased considerably with increased temperature. logarithmic model was the best among the selected models for describing the drying behaviour of corchorus leaves. the sample dried at 50 ℃ showed the highest retention of vitamin c, while the sample dried at 70 ℃ showed the highest retention of viscosity though not significant to that at 50 0 c. it can then be concluded from the results of this study that logarithmic model is the most suitable to describe the drying phenomenon of corchorus leaves and 50 0 c is the best drying temperature to retain its vitamin c and mucilage contents. references abano ee, ma h, qu w (2011). influence of air temperature on the drying kinetics and quality of tomato slices. j. food process technol. 2(5):3-9 aoac (1990). association of analytical chemists. official methods of analysis,11th edn. association of analytical chemists, washington dc. akpınar ek, bicer y (2003). modeling and experimental study on drying of apple slices in a convective cyclone dryer. j. food process eng. 26:515-541. awogbemi o, ogunleye io (2009). effects of drying on the quality of some selected nigerian vegetables, acsit int. j. eng. technol. 1(5):409-411 chen ts, saad s(1981). folic acid in egyptian vegetables: the effect of drying method and storage on the folacin content of mulukhiyah (corchorus olitorius). ecol. food nutr .10:249–255. demir v, gunhan t, yagcioglu ak, degirmencioglu a (2004). mathematical modelling and determination of some quality parameters of air-dried bay leaves. biosyst. eng.88(3): 325-335. doymaz i (2004). convective air drying characteristics of thin layer carrots. j. food eng.61: 359-364. doymaz i̇ (2005). drying kinetics of black grapes treated with different solutions. j. food eng. 76(2): 212-217. el-ghetany hh (2006). experimental investigation and empirical correlations of thin layer drying characteristics of seedless grapes. energy conversion and management 47:1610-1620. erenturk s, gulaboglu ms, gultekin s (2004). the thin layer drying characteristics of rosehip. biosyst. eng., 89 (2): 159 – 166. famurewa jav (2011). an assessment of physicochemical properties of worowo (senecio biafrae); emphasis on common drying methods. journal of food processing and preservation 35:327-320 gogus f (1994). the effect of movement of solutes on maillard reaction during drying. ph.d. thesis. leeds university, leeds goyal r k, kingsley arp, manikanthan mr, ilyas sm (2007).mathematical modeling of thin layer drying kinetics of plum in a tunnel dryer. j. food eng. 79: 176180. ihenkoronye ai, ngoddy po (1985).integrated food science and technology for the tropics, macmillian publishers, first ed. 168-170. kadam dm, goyal rk, gupta mk (2011a). mathematical modeling of convective thin layer drying of basil leaves j. med. plant. res. 5(19): 4722-4730 kadam dm, goyal rk, singh kk, gupta mk (2011b). thin layer convective drying of mint leaves. j. med plant. res. 5(2): 164-170 kaya a, aydın o, demirtaş c (2007). drying kinetics of red delicious apple. biosystems engineering 96(4):5175 natesh s (2001). the changing global scenario of herbal drugs: role of botanist. phytomorphology 51(3–4):75– 96 oguntona t (1998). proximate composition of three leafy vegetables common only in north-eastern nigeria. nigerian food journal, 3(6): 234-339 panchariya pc, popovic d, sharma a l (2002). thin layer modeling of black tea drying process.j. food eng., 52: 349-357. sharma gp, prasad s (2001). drying of garlic (allium sativum) cloves by microwave – hot air combination. j. food eng., 55: 99-105 singh h, sodhi ns (2000). dehydration kinetics of onions. j. food sci.technol.37(5): 520-522. premi m, sharma hk, sarkar bc, singh c(2010). kinetics of drumstick leaves (moringa oleifera) during convective drying. afri. j. plant. sci. 4(10): 391-400 sokhansanj s and jayas ds (2006).drying of foodstuffs. in handbook of industrial drying, ed arun s. mujumdar, 522-545.crc press. sharma gp, prasad s (2001). drying of garlic (allium sati 7 154 afr. j. food sci. res. vum) cloves by microwave – hot air combination. j. food eng.55: 99-105. togrul it, pehlivan d(2002). mathematical modelling of solar drying of apricots in thin layers. j. food eng., 55: 209-216. african journal of food science research vol. 2 (1), pp. 037-043, january, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper optimization of water-extract of phenolic and antioxidant compounds from kinkéliba (combretum micranthum) leaves beda marcel yapo1*, veronique besson2, sylvain beourou3 and kouassi koffi2 1 subunit of pedagogy in biochemistry and microbiology, unit of training and research in agroforestry, university of jean lorougnon guédé (ujlog), bp 150 daloa, côte d’ivoire. 2 food research and technology division, cargill west africa, abidjan, côte d’ivoire. 3 department of parasitology-mycology, pasteur institute, 01 bp 490 abidjan 01, côte d’ivoire. accepted 22 november, 2013 the hot water extract of dry kinkéliba leaves (dkl) is a traditionally widely delighted and consumed beverage in west africa, especially during the fasting periods. it is believed to possess various health benefit effects such as diuretic, purgative, digestive, anti-viral and anti-malarial properties. however, the preparation of this beverage has not been optimized so far, which may underestimate its health benefit potential. in this study, the traditional preparation of the beverage, by decoction, was optimized in order to maximize the amounts of health benefit polyphenolic and other antioxidant compounds. the results showed that an optimal preparation with maximum solubilization of polyphenols required the brewing of approximately 20-25 g of dkl in boiling water for about 15-20 min. these conditions afforded an optimized extract (oedkl) with a total phenolic content of 215.0 mg gae/g and arabinogalactan-rich pectic polysaccharide content of 553.0 mg/g. moreover, the oedkl exhibited a high antioxidant capacity, as evaluated by dpph [93.0% ip and ec50 = 15.0 (g/g)], abts (112.0 µmol te/ g), or frap (376.0 µmol te/g) assay, which was correlated with the total phenolic content (r 2 = 0.897-0.991). nevertheless, the pectic material detected within the beverage was likely to contribute to its interesting antioxidant properties. therefore, it was suggested that the total antioxidant capacity of the oedkl could result from synergistic interactions among the various antioxidant compounds present, including polyphenols and fractionated pectic materials. key words: combretum micranthum, beverages, polyphenols, pectic polysaccharides, antioxidant properties. introduction free radicals, of which reactive oxygen species (ros) are the most threatening, play a crucial role in the pathogenesis of various human disorders, such as colonic cancer, rheumatoid arthritis, neurodegenerative, coronary-heart, and pulmonary diseases (soobrattee et al., 2005). in vivo, some of the free radicals play positive roles in cell physiology; however, the balance between the production of ros and their neutralization by radical scavenging antioxidants is very delicate, and if this balance turns to overproduction of ros relative to the *corresponding author: bedamarcel@yahoo.fr tel.: +225 48 73 61 30/32 78 10 88; fax: +225 32 78 75 70 antioxidant stores, normal cells start to suffer the consequences of oxidative stress and related disorders (carocho and ferreira, 2013). phenolic compounds are usually associated with the health benefits, which stem from the consumption of polyphenol-rich food stuffs such as fruits, vegetables, teas, coffees, and cocoa-derived products (balasundram et al., 2006; martin et al., 2013). a direct relationship between the total antioxidant activity (taoc) and total phenolic content (tpc) of diverse (food) plant materials has widely been reported (soobrattee et al., 2005; carocho and ferreira, 2013). epidemiological studies have also shown that intake of polyphenols can effectively reduce the risk of development of various free radical-induced pathologies (maskarinec, 2009). besides, http://www.internationalscholarsjournals.org/ yapo et al. 037 it has recently been reported that biologically active pectin-derived polysaccharides from various plant fruits,stems, roots, and leaves, with commonly known immunological and anti-tumor properties (inngjerdingen et al., 2005; yapo, 2011), may also be endowed with antioxidant properties (gan and latiff, 2011). thus, there is a growing interest for seeking for food herbals and plants with high amounts of natural antioxidant compounds (aoc) to compensate for their shortage in refined modern diets. a diet with low aoc is indeed considered a risk factor for the development various free radical-induced disorders (ching et al., 2006). the intake of aoc-rich foodstuffs are therefore of primary importance for maintaining optimum health balance and welfare. kinkéliba (combretum micranthum g. don), also known as kinkiliba (or quinquéliba or quinquiliba), is a dicotyledonous plant which belongs to the family of combretaceae. it is generally an undomesticated shrub (4-5 m height) species which develops in the savannah regions of most west african countries (burkina faso, côte d’ivoire, guinea, mali, niger, and senegal). the fresh leaves are usually used to treat malarial fever (benoit et al., 1996; welch, 2010). the water extract of the leaf, the most biologically active plant tissue relative to stem, bark and roots, is one the most popular beverages in west africa, especially in senegal (welch, 2010). the dark green oval leaves are usually harvested and sundried, before being packaged and sold as dried kinkeliba leaves (dkl) in local stores for tisane preparation, most often with a specified direction for use. moreover, the health benefits, especially the pharmacological properties, of kinkéliba leaves are being recognized worldwide (welch, 2010). the beverage is commonly prepared by decoction by brewing dkl in boiling water. in traditional preparation of the so-called “long life tisane” of dkl, as recommended by producers and sellers in côte d’ivoire, 20 g of dlk are usually boiled in 1.5 l of water for 3-5 min, cooled to room temperature and filtered, after which the filtrate is consumed as a hot beverage or a cold drink within 24 h after being cooled to 5-7 °c. the beverage has good taste and is nearly substituted for commercially available teas. in addition, it may possess various health benefit properties, including diuretic, purgative, digestive, antiinflammatory, anti-viral, anti-nausea and anti-malarial potentials (ferrea et al., 1993; benoit et al., 1996; olajide et al., 2003; ancolio et al., 2002; welch, 2010). it is may also be effective against spleen fever and liver disorders, and type 2 diabetes (welch, 2010). these functional properties are believed to stem from various biologically active components present within dkl, namely high amounts of alkaloids, betains, heterosides, polyphenols, tannins, and potassium nitrate (ferrea et al., 1993; karou et al., 2005; welch, 2010; udoh et al., 2012). however, all these previous studies have used either (aqueous) organic solvent (acetone, alcohols, etc.) extractions or a one-point pure water extraction without any optimization of the extraction process. on the one hand, the use of aqueous-organic solvents, as commonly done, in the evaluation of tpc and taoc of plant extracts does not reflect the traditional preparation of this beverage by decoction. on the other hand, the utilization of a onepoint pure water extraction, without any optimization may solubilize low quantities of these bioactive compounds. the scope of this study was then to optimize the traditional preparation of dkl beverage in order to enhance its tpc and taoc. material and methods reagents, enzyme solutions, and analytical apparatus 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (abts), 2,2-diphenyl-1-picrylhydrazyl (dpph), 6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (trolox, a water-soluble vitamin e analogue), folin-ciocalteu reagent (fcr), gallic acid, 2,4,6-tri(2-pyridyl)-s-triazine (tptz), and glucose oxidase (god; ec 1.1.3.4) were from sigma-aldrich co. (st. louis, mo). all the uv-vis spectrophotometric analyses were performed with a perkin-elmer lambda 3 double beam spectrophotometer (perkinelmer corp., norwalk, ct). raw plant materials dry kinkéliba (c. micranthum g. don) leaves (dkl) were purchased from a local distributor of african medicinal plant products (cipradel, abidjan, côte d’ivoire). the plant materials were authenticated at the department of botany, university of felix houphouet boigny (ex. university of abidjan-cocody). optimization of the water extraction of bioactive compounds from dkl in a first experimental procedure, dkl (20 g) were treated in boiling water (1.5 l) for 3-60 min, cooled to room temperature, and filtered through g-3 sintered glass, and then on nylon cloth (0.45 µm). the different filtrates were concentrated, by rotary evaporation (40 °c), to the desired quantity of solutions, freeze-dried, and kept at room temperature under moisture-free conditions pending analysis. in a second experimental procedure, 555 g of dkl were treated in 1.5 l boiling water for 15 min and processed as above to obtain filtrates, which were concentrated to the desired quantity of solutions, freezedried, and stored under moisture-free conditions until analysis. according to the traditional preparation, as 038 afr. j. food sci.res. specified by producers and sellers, 20 g of dkl are boiled in 1.5 l of water for 5 min, cooled to room temperature and filtered and processed as above. the product obtained was referred to as the control extract. all the experiments were performed in three independent runs. determination of the total phenolic content of dkl the total phenolic content (tpc) of the extracts was determined by a colorimetric fcr assay (singleton et al., 1999). briefly, samples (0.1 ml) were added to test tubes, followed by addition of 6 ml of distilled water and 0.5 ml of fcr. after mixing and resting for 3 min, 1.5 ml of 20% (w/v) sodium carbonate were added to test tubes and adjusted to 10 ml. the mixtures were thoroughly vortex-mixed, and the increase in absorbance of the colour generated, after 2 h at room temperature, was spectrophotometrically read at 760 nm against an appropriate control (or “blank”). gallic acid (25-500 µm) was used as standard and the results obtained were expressed as gallic acid equivalents (mg gae)/g dried weight of sample. analyses were carried out in triplicates. determination of the total antioxidant capacity (taoc) dpph scavenging antioxidant assay the total antioxidant capacity (taoc) of the extracts was first evaluated for its ability to scavenge the dpph free radical by two ways. for the fixed duration reaction method (mishra et al., 2012), 0.2 ml ethanolic solution of antioxidant-containing sample (0.5 mg/ml) was added to 0.8 ml of a 50 mm tris-hcl buffer (ph 7.5) and vortex-mixed, followed by addition of 1 ml of a 0.1 mm ethanolic solution of dpph • . the reaction mixture was thoroughly vortex-mixed and left in the dark at 23 °c for 30 min. the decrease in absorbance of the mixture, at room temperature, was spectrophotometrically measured at 515 nm. trolox (0.11.0 mm) was used as antioxidant standard. antioxidant activity or inhibition percentage (ip), defined as the ability of the sample to scavenge the dpph free radical, was calculated as follows: ip (%) = [(abscontrol abssample)]/(abscontrol)] × 100 where abscontrol and abssample are the absorbances of the free radical dpph • in the absence and presence of the antioxidant-containing sample, respectively. for the steady sate saturation method (brand-williams et al., 1995), 0.1 ml of an ethanolic solution of sample was added to 3.9 ml of a 24 mg/l of ethanolic solution of dpph. the decrease in absorbance was determined at 515 nm at different time intervals until the reaction reached a steady state (a plateau-like curve). the initial concentration of dpph • in the reaction medium was calculated from a calibration curve, as obtained by linear regression (r 2 = 0.989), and the percentage of remaining dpph • at steady state (%dpph • rem) was calculated and plotted against the sample concentration. the antiradical activity was determined in terms of the efficient (or inhibition) concentration (ec50 (g dry weight sample/g dpph • )), viz. the amount of antioxidant-containing sample required for 50% decrease in the initial dpph • concentration. abts scavenging antioxidant assay the taoc of the extracts was also evaluated by its capability to scavenge abts radical (re et al., 1999). briefly, 7 mm abts stock solution was prepared in water. abts radical cation (abts •+ ) was produced by reacting equal quantities of abts stock solution with 2.5 mm potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 15 h before use. the generated abts •+ solution (1 ml) was then diluted with ethanol (60 ml) to an initial absorbance of 0.802  0.01, read at 734 nm, and the reagent obtained was equilibrated at 30 °c. after adding 0.9 ml of the latter abts •+ solution to 0.1 ml of antioxidant-containing sample in test tube and vortexmixing, the decrease in absorbance of the mixture, at 30 °c, was spectrophometrically measured at 734 nm for 1-7 min against an appropriate control (or “blank”). trolox (0.1-1.0 mm) was used as standard and the results were expressed as micromole of trolox equivalent (µmol te)/g dried weight of sample. analyses were carried out in triplicates for each sample used. ferric reducing antioxidant power (frap) assay the taoc of the extracts was finally evaluated through its ability to reduce ferric (fe 3+ ) cation to ferrous (fe 2+ ) cation (benzie and strain, 1996). the stock solutions included 300 mm acetate buffer (ph 3.6), 10 mm tptz solution in 40 mm hcl, and 20 mm fecl3.6h2o solution. the fresh working frap reagent was prepared by mixing acetate buffer, tptz, and fecl3.6h2o solutions in a ratio of 10:1:1 (v/v/v), respectively; and was warmed up to 37 °c before use. antioxidant-containing sample (0.1 ml) was added to 0.9 ml of the frap solution, and the mixture was vortex-mixed and allowed to react for 30 min in the dark. the increase in absorbance of the mixture, at 37 °c, was spectrophotometrically monitored at 593 nm for 4-30 min against an appropriate control (or “blank”). trolox (0.1-1.0 mm) was used as standard and the results were expressed as µmol of te/g dried weight of sample. sugar composition analyses aliquots were taken from the extracts with the highest yapo et al. 039 figure 1. effect of extraction time on the total phenolic content (tpc) of hot water-solubilized antioxidant fractions from dry kinkeliba leaves. taoc and mixed with 3 volumes of 70% (v/v) aqueous ethanol. the mixtures were then allowed to rest at 5 °c overnight and centrifuged (3000g, 20 min) to recover precipitates, which were dispersed in distilled water and extensively dialyzed in 3500 molecular weight cut off tubing against distilled water. the retentates were concentrated by rotary evaporation (40 °c) to desired quantity of solutions and were analyzed for glycosyl residue composition. they were therefore treated with 1 mol.l -1 h2so4 (100 °c, 3 h) to release the monosaccharide constituents of the solubilized polysaccharide material as described previously (yapo and koffi, 2008). galactose, glucose, mannose, and fucose were analyzed after finch et al. (1969). glucose was also analyzed by the rapid god-fenton’s reaction method (woodward et al., 1985). arabinose and rhamnose were determined using megazyme assay kits (megazyme international ireland ldt., bray, co. wicklow, ireland). xylose was analyzed according to pham et al. (2011) and finally uronic acid was determined as galacturonic acid as previously published (yapo, 2010). all the analyses were carried out in triplicates for each sample used. statistical analysis all the data were statistically appraised by a single-factor analysis of variance (anova), followed by the bonferroni’s posthoc test for multiple comparisons, whenever applicable, using a graphpad prism v.3 software (graphpad software inc., san diego, ca). the means of different treatments were considered to be significantly different at p-value <0.05. correlation between the tpc and taoc of extracts were determined by the pearson’s test. results and discussion optimization of the water extraction of total phenolic compounds the result of effect of the duration of extraction on the amount of water-solubilized phenolics from dkl is shown in figure 1. as can be seen, the tpc of the beverage increased with increasing time of extraction up to 20 min, after which it decreased substantially as the extraction time was further increased to 60 min. longer heating times have also been found to decrease the tpc and taoc of soluble phenolics from grape seed flour (ross et al., 2011). these results indicated that the heating duration considerably influenced the tpc of the beverage, thereby showing the importance of the optimization of the extraction process. the highest tpc was obtained after 15-20 min extraction. in comparison with the latter result, the tpc of the control extract was significantly lower (p <0.05), representing approximately 20-40% of the maximum yield obtained at 15-20 min extraction. this confirmed that a longer time of extraction (than 5 min) was required for maximizing the solubilisation of phenolics from dkl. furthermore, the variation in the initial quantity of dkl used for the preparation of the beverage showed that the best yield of phenolics was obtained when 20-25 g of dkl were boiled in 1.5 l of water for 15 min (figure 2). this solid to liquid ratio is in good accord with the one traditionally recommended. the optimized extract from dkl (oedkl), obtained by boiling 20 g of dkl in 1.5 l 040 afr. j. food sci. res. figure 2. effect of solid to liquid (s/l) ratio on the total phenolic content (tpc) of hot watersolubilized antioxidant fractions from dry kinkeliba leaves. for 15 min and the control extract were further analyzed for tpc and taoc. total phenolic content (tpc) and antioxidant capacity (taoc) of extracts the results of the analysis of the tpc and taoc of oedkl and control extract are presented in table 1. the tpc of oedkl and control extract were 214.8 and 79.6 mg gae/g, respectively; showing that there were 2.7 times more phenolics in oedkl than in the control extract. the tpc of oedkl was lower than the one (ca. 378 mg gae/g) reported by karou et al. (2005). this difference is likely due to difference in the methods used for the extraction of polyphenols from kinkéliba leaves, 70% (v/v) aqueous acetone being used, as commonly done, by the latter workers. 60-70% aqueous organic solvent (acetone and/or alcohol) extractions are known to be more efficient that polar solvent (water) extraction (cheynier et al., 1999). however, it should be underlined that we performed only pure water extraction, and not aqueous organic-solvent extraction, to be in line with the traditional preparation of the beverage from dkl. the differing origin of kinkéliba leaves (burkina faso in their study) may also contribute to discrepancies in data to some extent. however, the tpc of oedkl was higher than those reported in other hot beverages such as instant and ground coffees (52.5-151.0 mg/g) and black and green teas (61.0-200.0 mg gae/g) as summarized elsewhere (balasundram et al., 2006). the taoc of oedkl and control extract was evaluated by dpph, abts, and frap methods, because the taoc of a (food) plant material generally results from a mixture of various antioxidant compounds with different mechanisms of action, among which there may be synergistic (or antagonistic) interactions (aruoma, 2003; pérez-jiménez et al., 2008). moreover, there is not one analytical method that can provide unequivocal results (carocho and ferreira, 2013). therefore, a better practice nowadays recommends the combination of (at least two) different methods for the in vitro determination of the taoc of food products. the dpph radical measures a sample’s free radical scavenging capacity, which is based on the premise that a single electron transfer (set) or a hydrogen donor is an antioxidant. the abts radical also measures a sample’s free radical scavenging capacity. it is commonly referred to as a decolorization assay in which there is only a set reaction. frap measures the ability of a sample to reduce metal cations and is also based on a set reaction (pérez-jiménez et al., 2008; carocho and ferreira, 2013). the taoc of oedkl, as determined by the colorimetric dpph, abts, and frap assays, were 92.8% ip of dpph (ec50 = 14.5 g/g), 112.4 µmol te/g, and 375.9 µmol te/g, respectively. the results of the taoc of control extract, as determined by dpph, abts, and frap assays, were 52.6% ip of dpph (ec50 = 31.2 g/g), 49.7 µmol te/g, and 104.8 µmol te/g, respectively (table 1). it can be seen, in each case, that the taoc of oedkl was significantly greater than that of the control extract (p <0.05), thereby demonstrating the necessity for the optimization of the preparation of this beverage. in comparison with other workers, the taoc of oedkl, as appraised by the dpph assay (ec50 = 14.5 g/g), is rather comparable to the one (9.1 ± 0.28) found for a water extract of kinkéliba leaves from guinea (touré yapo et al. 041 table 1. total phenolic content, total antioxidant capacity, and glycosyl residue composition of the optimized extract (oedkl) and control extract from dry kinkeliba leaves. antioxidant properties oedkl control extract soluble phenolics (mg gae/g) 214.8 ± 5.7a 79.6 ± 2.2b dpph (%ip) 92.8 ± 3.6a 52.6 ± 1.7b dpph [ec50 (g/g)] 14.5 ± 0.2a 31.2 ± 1.4b abts (µmol te/g) 112.4 ± 5.7a 49.7 ± 2.3b frap (µmol te/g) 375.9 ± 3.4a 104.8 ± 2.7b sugar composition (mol%) rhamnose 14.8 ± 1.4 nd fucose nd nd arabinose 12.6 ± 1.1 nd xylose 3.2 ± 0.4 nd mannose nd nd galactose 34.7 ± 4.3 nd glucose 8.2 ± 0.9 nd galacturonic acid 26.4 ± 2.7 nd total sugar (mg/g) 553.1 ± 6.5 nd data are expressed on a dry weight basis as mean  sd (n =3). values in the same lines with different letters are significantly different (p <0.05). nd: not detected nd: not determined et al., 2011). the taoc of oedkl, as evaluated by the abts assay, is much lower than that (16.37 µmol te/µg) reported by karou et al. (2005). this discrepancy might be explained by a lower efficient of pure water extraction, compared with 70% aqueous acetone extraction (in their study) which is known to solubilize high molecular weight polyphenols, especially proanthocyanidins (cheynier et al., 1999). touré et al. (2011) have also observed that the ec50 (or ic50) values of the ethanol extract of guinean kinkéliba leaves in abts and dpph tests were significantly lower than those from the water extract. a strong correlation was found to exist between the tpc and taoc of oedkl for either frap (r 2 = 99.1%) or abts (r 2 = 97.8%). by contrast, the correlation was moderately high for dpph (r 2 = 89.7%). karou et al. (2005) have also found that the tpc of 70% aqueous acetone extract of kinkéliba leaves, from burkina faso, were highly correlated (r = 0.91) with the antioxidant activities using the abts assay. in order to know if polysaccharide components were also present in the hot water extract (oedkl), since biologically active (pectic) polysaccharides, which are generally present in all higher plant tissues, are usually extracted by this method (inngjerdingen et al., 2005; yapo, 2011), 70% aqueous alcohol-precipitation was carried out, thereby partitioning the hot water-extract into alcohol-soluble and alcohol-insoluble (precipitate) fractions. the latter fraction was then analyzed for different typical monosaccharide constituents of polysaccharides. the results are also shown in table 1. the quantitatively detected monosaccharides, in order of abundance, were galactose (34.7 mol%), galacturonic acid (26.4 mol%), rhamnose (14.8 mol%), arabinose (12.6 mol%), glucose (8.2 mol%), and xylose (3.2 mol%). this indicated that polysaccharides (553.1 mg/g), mainly pectic substances with possibly some xyloglucan moieties, were co-extracted with polyphenols by the hot water treatment of dkl. to substantiate the polysaccharide contribution or not to the taoc of the overall water extract (oedkl), the derived alcohol-soluble and alcohol-insoluble fractions were analyzed by dpph, abts, and frap assays. it was observed that each of the two fractions exhibited substantially lower taoc, compared with that of the whole extract (data not shown). it was therefore suggested that the interestingly high taoc of oedkl resulted from synergistic interactions among various biologically active compounds, including at least arabinogalactan-rich pectic polysaccharides and polyphenols, of which gallic acid might be the major components (touré et al., 2011). this observation is in agreement with the recent findings that pectic polysaccharides from mangosteen (gan and latiff, 2011) are effective free radical (dpph) scavengers. the free radical scavenging activity and metal-reducing power of pectic polysaccharides could be explained by the 042 afr. j. food sci.res. presence of several protonated carboxyl (-cooh) groups and secondary alcohol (-oh) groups, which render them potential hydrogen donors and contributors to the single electron transfer mechanism. thus, in addition to polyphenols, pectic polysaccharides may be the other effective antioxidant compounds present within the oedkl. conclusion investigation on the bioactive compounds content of the beverage, obtained by decoction, from dry kinkeliba leaves showed the presence of high amounts of soluble phenolics, together with antioxidant pectic polysaccharides. the product obtained (oedkl) under optimized conditions exhibited interesting antioxidant properties, which is likely to result from synergistic interactions among various compounds, including polyphenols and fractionated pectic materials. the wide interest for daily consumption of this beverage is therefore fully accounted for. however, as demonstrated in this study, the traditional preparation of the beverage should be optimized by brewing approximately 20-25 g of the dried raw material in 1.5 l of boiling water for about 15-20 min in order to enhance its health benefit potentials. our future study should deal with identification of the specific classes of the antioxidant compounds present in oedkl and in vivo analyses. acknowledgements we are grateful to cargill west africa and cosmivoire for some financial support. a list of abbreviations used abts: 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) aoc: antioxidant compounds dkl: dry kinkeliba leaves dpph: 2,2-diphenyl-1-picrylhydrazyl fcr: folin-ciocalteu reagent frap: ferric reducing antioxidant power gae: gallic acid equivalents god: glucose oxidase oedkl: optimized extract of dry kinkliba leaves taoc: total antioxidant capacity tpc: total phenolic content tptz: 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polysaccharide composition of yellow passion fruit rind cell wall: chemical and macromolecular features of extracted pectins and hemicellulosic polysaccharides. j. sci. food agric. 88(12): 2125-2133. african journal of food science research vol. 2 (5), pp. 094-098, may, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper effects of types of treatments of lead and cadmium on soil bioavailability: a potential human risk chimony mhembere*, stewart salau and tatender mapeto department of biochemistry, university of zimbabwe, box mp167, mt. pleasant, harare, zimbabwe. accepted 25 april, 2014 the effect of single and mixed treatments of lead (pb) and cadmium (cb) on soil bioavailability and uptake by brassica napus was investigated in green house using soil from golden ridge estate and wastewater from pakamisa sewage treatment plant, gweru, zimbabwe. pb and cd had significant effects on metal soil bioavailability and yield in single treatments. soil bioavailable pb in mixed treatments significantly correlated positively with uptake by b. napus. yield was significantly correlated negatively with soil bioavailable pb and cd as well as metal uptake. soil bioavailable pb accounted for 49.2% (single treatment) and 6.1 % (mixed treatment) of the variation in b. napus yield. pb uptake accounted for 4.63% (single treatments) and 88.77% (mixed treatments) of the variation in yield. cd accounted for 0.38% (single treatment) and 7.98% (mixed treatments) of the variation in yield. findings of this study indicate that b. napus grown in golden ridge estate soil and irrigated with wastewater from pakamisa sewage treatment plant accumulates pb and cd to above recommended maximum limits for human consumption and is not fit for human consumption. key words: lead, cadmium, soil bioavailability, brassica napus. introduction in zimbabwe, urban authorities and water authorities are collaborating with peri-urban farmers to strengthen the role of peri-urban horticulture in wastewater recycling and creating sustainable food systems. a major challenge of peri-urban horticulture using treated wastewater for irrigation is the supply of safe products in this often polluted environment. pollution by heavy metals presents a risk to the consumers as well as producers of these horticultural products (tandi et al., 2005). mapanda et al. (2005) indicated that vegetable gardens of the city of harare, zimbabwe, irrigated with wastewater cause significant heavy metal enrichment in soils. the city of gweru started irrigating its croplands that includes vegetables at golden ridge estate with wastewater in the early 1990s. the potential of lead (pb) and cadmium (cd) to accumulate in golden ridge estate soils is not known. no studies have *corresponding author. e-mail: chi_mhebere@yahoo.com been done to monitor heavy metals in wastewater and to quantify their uptake by crops at golden ridge estate. pb and cd are cumulative and indestructible toxins that can only be eliminated through excretion (moolenar and lexmand, 1999). pb and cd accumulate in the body causing health problems that include damage to the nervous system, reduced intellectual capacity and hypertension (staeson, 2002). humans acquire these metals from contaminated water and food after the addition of pb and cd to agricultural soils and uptake by food and fodder crops (johnson and jones, 1995). information on the uptake of pb and cd is therefore important in designing strategies for predicting uptake of the metals into the food chain. in zimbabwe, brassica napus (rape) is the most common green leafy vegetable that is consumed in most households. it is relatively cheaper than other vegetables and is available from the vegetable markets throughout the year. the objective of this study was to investigate the effects of single and mixed pb and cd treatments on soil bioavailability and uptake by b. napus, grown using soil http://www.internationalscholarsjournals.org/ chimony et al. 094 from golden ridge estate and irrigated with wastewater from pakamisa sewage treatment plant. materials and methods golden ridge estate golden ridge estate is located 10 km north of the city of gweru, zimbabwe. the estate is under irrigation with treated sewage effluent from pakamisa sewage treatment plant. major crops cultivated are vegetables such as b. napus (rape) that is sold to vendors in the city of gweru, maize and wheat. soil and wastewater characterization soil from golden ridge estate was collected in 10 l buckets and transported to the laboratory for analysis and use in greenhouse experiments. the soil was tested for pb and cd concentrations according to the method of mcgrath and ceggara (1992). soil texture was determined by the hydrometer method of gee and bauder, (1986). soil ph was determined using a 1:5 soil suspension of 0.01 m cacl2 after calibrating ph meter session 1 with ph buffers 7 and 10. cationic exchange capacity (cec) was determined by saturating the soil with 1m ch3coonh4 buffered at ph 5.2. total organic carbon was determined using the method described by houba et al. (1989). treated waste water from irrigation canals at golden ridge estate coming from pakamisa sewage treatment plant was collected and analyzed for pb and cd concentrations using the method of mcgrath and ceggara, (1992). pakamisa sewage treatment plant pakamisa sewage treatment plant processes both industrial and domestic effluent from the city of gweru. the industries include a radiator clinic, panel beaters and spray painters, alloy processing plants, motor vehicle garages and battery manufacturers. the plant uses the conventional wastewater biological trickling filtration system and ponds. the system produces low quality water that is used to irrigate crops at golden ridge estate. green house experiments the experiments were carried out in a green house at midlands state university from september to november 2007. soil from golden ridge estate and treated wastewater from pakamisa sewage treatment plant were used in the metal treatment experiments. single metal treatment five treatment levels each of pb and cd were used. the first level was a control without inorganic pb and cd and used drinking tap water from the university. the second level only received treated wastewater and no inorganic metals. the third level used the maximum permissible pb concentration of 100 mg/kg of soil or maximum acceptable cd of 3 mg/kg of soil (pescod, 1992). the fourth level doubled the maximum acceptable concentration of the metals while the fifth level received four times the acceptable metal concentration in the soil. each level was replicated three times. mixed metal treatments five treatment levels of pb and cd were used. the first and second levels were the same as those of single treatments. the third level had inorganic pb and cd mixed in proportions of 100 mg/kg pb and 3 mg/kg cd. the fourth level was a combination of 200 mg/kg pb and 6 mg/kg cd. the fifth level was a combination of 400 mg /kg pb and 12 mg/kg cd. b. napus growing in each 5 l soil packed pot (5 kg), three shallow holes were drilled and b. napus seeds were added and covered with a thin layer of soil. in order to eliminate nutrient deficiency compound d fertilizer (n8%, p14%, and k7%) was added at a rate 700 kg/hectare on planting. the soils in the pots were then enriched with lead nitrate [pb(no3)2] and cd(no3)2.. 4h2 o the inorganic sources of pb and cd respectively according to the relevant treatment level. ammonium nitrate (n34.5%) fertilizer was added to all the pots irrespective of treatment level at a rate of 100 kg/hectare at one month intervals (agritex, 1993). after the vegetables were established the rest of the seedlings were thinned to remain with five plants per pot that were almost the same size. two litres of treated wastewater were applied per pot for levels 2 to 5 while level 1 received tap water three days per week. plants were allowed to grow for a period of two and a half months. pots soil sampling and testing soil samples were taken from pots to a depth of 20 cm using a soil auger after harvesting b. napus. plant debris was removed from the soil by sieving with a 2 mm sieve. bioavailable pb and cd concentrations were determined using the procedure of mcgrath and ceggara (1992). pot b. napus sampling and testing harvesting of b. napus was made from each pot two and a half months after soil enrichment with inorganic salts of pb and cd. all above ground consumable leafy parts of b. napus were harvested to constitute a sample per harvest. leaves were washed with deionized water and oven dried at 65°c to a constant weight. dried b. napus was weighed to determine yield before analysis for pb and cd concentrations. the samples were ground, air dried and placed in conical flasks. they were wetted with water and 25 ml of nitric acid was added and gently heated to start the reaction and then cooled. ten milliliters of perchloric acid was added to each sample and gently heated to concentrate it. if the mixture became too dark, 2-3 ml of nitric acid was added with continuous heating. after the mixture had turned yellow or colourless, decomposition was complete. the mixture was left to cool and 2 ml of hydrochloric acid was added. de-ionized water was used to prepare fixed volumes of measurement solution. processed blank test solutions were prepared in the same way as the samples. samples were analyzed for pb and cd using atomic absorption spectrometry (shumadzu, 2007). data analysis one-sample t-tests were used to compare mean levels of pb and cd in the soils and treated sewage effluent to the standard permissible levels. data on bioavailable metal concentration in soil, metal uptake by b. napus and yield were tested for normality and then transformed using the formula y=in (x) anova was used to test the significance of treatment on biovailable pb and cd, their uptake by b. napus and yield. regression analysis was done to determine the correlation between soil bioavailable metal concentration and b. napus uptake and yield. all statistical analysis was performed using genstat version3. table 1. levels of pb and cd in golden ridge estate soil and wastewater. metal pb cd soil metal concentration (mg/kg) 0.83 nd recommended maximum level (mg/kg)* 100 3 treated wastewater (mg/l) 1.29 nd recommended wastewater maximum 5 0.01 level for irrigation (mg/l)** *source: who (1993) ** source: usepa (1992) nd – not detectable. results soil characterization golden ridge estate soil had clay content of 78%, soil ph 5.52, organic carbon 1.7% and cationic exchange capacity of 39.8 molkg -1 . pb and cd concentrations in soil and wastewater data on pb and cd concentrations in soil and wastewater are given in table 1. pb and cd levels were below the maximum permissible limits. bioavailable pb and cd in pot soils, metal uptake and b. napus yield data on soil bioavailable pb and cd, b. napus metal uptake and corresponding yield for single and mixed treatments are given in tables 2 and 3. from level 3 to 5 soil bioavailable pb and cd increased with increase in level in single treatments (p>0.05). the same trend was observed in mixed treatments except in level 5 where soil bioavailable pb and cd was less than level 4 (table 4). in levels 1 and 2 little pb in soil was accumulated in higher proportion in plants than in level 3. the uptake of pb and cd by b. napus in levels 1 and 2 was significantly higher than the recommended human maximum consumption limits [pb 3.5 g/kg and cd 1 g/kg (who, 1993) (p < 0.05)]. pb uptake by b. napus in level 4 and 5 was significantly lower than that in single treatments. treatment had significant effects on yield and soil bioavailable pb and cd in single treatments as well as soil bioavailable pb in mixed treatments (table 4). relationship between soil bioavailable pb and cd, and their uptake and yield by b. napus the regression coefficients for the relationship between soil bioavailable pb and cd, their uptake and b. napus yield in single and mixed treatments are given in tables 5 095 afr. j. food sci. res. and 6. particularly soil bioavailable pb in mixed treatments was significantly correlated positively with uptake by b. napus than in single treatments. weaker negative relations were found between b. napus pb and cd uptake and yield; soil bioavailable pb and cd and corresponding yield. strong negative relations were only found between soil bioavailable pb and b. napus yield (r=-0.701) and between b. napus pb uptake and yield (r=-0.942). the regression models were linear and the residuals were normally distributed. soil bioavailable pb accounted for 49.2% of the variation in yield in single treatments (table 5) whereas pb uptake accounted for 88.7% of the variation in yield in mixed treatments (table 6). discussion b. napus irrigated with sewage effluent as well as the control took up pb and cd concentrations that are above the maximum permissible limits for human consumption. therefore the soil in golden ridge estate and the wastewater from pakamisa sewage treatment plant are not fit for irrigating b. napus for human consumption. despite failure to detect cd in soil, its uptake by b. napus was high. cd might be detected in future studies corroborating tandi et al. (2005 ) who reported that repeated application of sewage effluent resulted in accumulation of heavy metals in the soil to detectable levels. selvam and wong (2008) found that cd tends to accumulate higher concentrations in the roots than the shoot of b. napus. it is possible that in our experiments higher cd levels were accumulated in the roots of b. napus than in the shoot. our study did not determine cd levels in roots of b. napus. soil bioavailability data indicated that golden ridge estate soils have the potential to accumulate high levels of pb and cd over a long period of time. the increase in soil bioavailable pb concentrations in mixed than single treatments tallies with the findings of madyiwa et al., (2002). b. napus accumulated pb and cd in clayey soils and the results were consistent with the findings of mathegasper and anton, (2005) and licinia et al., (2007). cd accumulation positively correlated with cd bioavailablity in the soil corroborating findings of selvam and wong, (2008). kachenko and singh (2006) reported that broad leaf vegetables are hyper accumulators of heavy metals. our findings suggest that the interaction of pb and cd in soils and possibly other factors reduce pb uptake by b. napus. the observed weak positive correlations between soil metal bioavailability and corresponding metal uptake by b. napus was similar to the findings of moolenar and lexmond, (1999). the negative correlations observed for pb uptake and yield is in agreement with the findings of miller (1997) who reported that cd in soil reduced uptake of pb in zea mays l (con). however, our results contradicted the findings of carlson and bazzaz (1997) who reported that uptake of pb by plants increased as cd chimony et al. 096 table 2. soil bioavailable pb and cd, vegetable metal uptake and corresponding yield by b. napus (rape) single treatments. treatment mean soil bioavailable mean metal uptake mean rape yield metal (mg/kg) (mg/kg) (g/pot) pb single treatments control 0.83 a 3.3 a 2.0 a wastewater 1.23 a 3.85 a 2.05 a pb100 176.9 b 7.27 b 3.51 a pb200 219.5 b 271.5 b 2.83 a pb400 422.1 b 123.0 b 0.29 b cd single treatments control 0a 0.025 a 2.0 a wastewater 0 a 0.03 a 2.05 a cd3 0.37 a 0.06 a 2.05 a cd6 0.54a 0.32 b 3.1 a cd12 1.84 b 0.79 b 3.0 a columns with different superscripts are significantly different (p<0.05). table 3. soil bioavailable pb and cd, vegetable metal uptake and corresponding yield by rape mixed treatments. treatment mean soil bioavailable mean metal uptake mean rape yield metal (mg/kg) (mg/kg) (g/pot) pb mixed treatments control 0.83 a 3.3 a 2.0 a wastewater 1.23 a 3.85 a 2.05 a cd3+pb100 223.15 b 8.6 b 3.0 a cd6+pb200 465.25 c 15.27 b 2.5 a cd12+pb400 417.25 c 41.53 c 1.4 b cd mixed treatments control 0 a 0.025 a 2.0 a wastewater 0 a 0.03 a 2.05 a cd3+pb100 0.21 b 0.13 b 3.0 a cd6+pb200 0.61 b 2.1 c 2.5 a cd12+pb400 0.27 b 0.17 b 1.4 b columns with different superscripts are significantly different (p<0.05). table 4. analysis of variance results of effect of single and mixed treatments on soil bioavailability, uptake and yield of b. napus using pb and cd inorganic salts. source of variation single mixed f p f p pb yield 4.31 p<0.069 2.40 p>0.186 soil bioavailability 4.79 p<0.069 7.82 p<0.065 cd yield 30.47 p<0.002 0.78 p>0.508 soil bioavailability 10.40 p<0.01 1.15 p>0.377 concentrations were raised in the soil. the observed high negative correlations between b. napus yield response to pb uptake in mixed treatments compared to single treatments is similar to the observations of molgorzata and hakan (2001). the observed decline in yield as pb concentrations increased was due to the toxicity of the metal to b.napus. this suggests that repeated irrigation of golden ridge estate crops with effluent over a long period of time could result in low crop yield. the absence of decline in yield in cd single treatments (table 2) could be attributed to the fact that cd accumulates in roots than in the shoot and the reduction in dry weight is seen in the root but not the shoot (selvam and wong, 2008). 097 afr. j. food sci. res. table 5. the regression coefficients of the relationship between soil bioavailable pb and cd, and their uptake and yield by b. napus in single and mixed treatments. b (mg/kg) r 2 (%) n significance pb single treatments soil bioavailability and uptake 0.231 0.69 7 ns soil bioavailability and yield -5.848 49.20 7 p<0.05 pb mixed treatments soil bioavailability and uptake 2.019 32.89 7 p<0.05 soil bioavailability and yield -1.177 6.10 7 ns cd single treatments soil bioavailability and uptake 1.559 78.39 7 p<0.01 soil bioavailability and yield -0.034 9.82 7 ns cd mixed treatments soil bioavailability and uptake -0.411 13.48 7 p<0.05 soil bioavailability and yield 0.351 10.15 7 ns table 6. relationship between pb and cd uptake by b. napus and corresponding yield in single and mixed treatments. b g/pot r 2 (%) n significance pb single 0.140 4.63 7 ns treatments mixed -0.798 88.77 7 p<0.01 treatments cd single 0.035 0.38 7 ns treatments mixed 0.088 7.98 7 ns treatments conclusion b. napus accumulates high levels of pb and cd to above recommended limits and poses a hazard to humans after consumption. contaminated golden ridge estate soil and wastewater from pakamisa sewage treatment plant are not fit for irrigating b. napus for human consumption. golden ridge estate should take action to reduce levels of pb and cd in the wastewater or stop selling rape to residents of the city of gweru as this is likely to result in health problems due to the cumulative nature of pb and cd. acknowledgements the authors are grateful to golden ridge estate and pakamisa sewage treatment plant for provision of soil and wastewater respectively. the midlands state university research board provided funds for the research. references agritex (1993). farm management handbook department of agricultural technical and extension services. harare. zimbabwe. carlson rw, bazzaz fa (1977). growth of rye grass and fescue as affected by lead-cadmium-fertilizer interaction. j. environ. quality. 8: 348-352. gee gw, bauder jw (1986). particle size analysis. methods of soil analysis. ed. klute, a. american society of agronomy, madison w. i. usa. johnson ae, jones kc (1995). the origin and fate of cadmium in soil. proceedings of the fertilizer society. london. pp.1-39. kachenko ag, singh b (2006). heavy metals contamination in vegetables grown in urban and metal smelter contaminated sites in australia. water, air and soil pollution. 169: 101-123 licinia v, antic-meladenovic s, kresovic m (2007). the accumulation of heavy metals in plants (lactuca sativa l, fragaria vesca l) after the amelioration of coalmine tailing soils with different organo-mineral amendments. arch. agron. soil sci. 53: 39-48 houba vjg, van der lee jj, novozamsky i, waling i (1989). soil and plant analysis, part 5. waginingen agricultural universuty. netherlands. pp. 4-10 madyiwa s, chimbari m, nyamangara j, bangira c (2002). phytoextraction capacity of cynodon nlemfuensis (star grass) at artificially elevated concentrations of pb and cd in sandy soils under greenhouse conditions. 3 rd waternet/warfsa symposium on water demand management for sustainable development: dar es salaam. tanzania. 30 – 31 october. molgorzata mm, hakan a (2001). influence of lead and cadmium on growth, heavy metal uptake, and nutrient concentration of three lettuce cultivars grown in hydroponics culture. commun. soil sci. plant anal. 32: 571-583 mapanda f, mangwayana e, nyamangara j, giller k (2005). the effect of long term irrigation using wastewater on heavy metal contents of soils under vegetables in harare, zimbabwe. agriculture, ecosystems and environment (in press) mathe-gasper g, anton a (2005). phytoremediation study: factors influencing heavy metal uptake by plants. acta biologica szegediensis. 49: 69-70. mcgrath sp, cegarra j (1992). chemical extractability of heavy metals during and after long-term application of sewage sludge to soil. j. soil sci. 43: 313-321. miller je, hasset jj, koppe de (1997). interaction of lead and cadmium on metal uptake and growth of corn plants. j. environ. quality. 6:1820. moolenar sw, lexmond tm (1999). general aspects of cadmium, copper, zinc, and lead balance studies in agro-ecosystems. heavy metal balances, part 1. j. indust. ecol. 2: 4-8. chimony et al. 098 pescod md (1992). wastewater treatment and use in agriculture. food and agricultural organisation (fao) irrigation and drainage paper 47. rome italy: fao. selvam a, wong jwc (2008). phytochelation and synthesis and cadmium uptake by brassica napus. environ. technol. 29 (7): 765773. shimadzu cook handbook (2007). atomic absorption spectrophotometry cookbook. shimadzu corporation. kyoto. japan. staeson j (2002). b4, a study on how to prevent toxic effect of cadmium in the population at large. kul, uz gasthuisberg, klinisch lab. hypertensie, inwendige., geneeskunde-cardio, herestratt 49, 3000 leuven. tandi nk, nyamangara j, bangira c (2005). enivironmental and potential effects of growing leafy vegetables on soil irrigated using sewage sludge and effluent: a case of zn and cu. j. environ. sci. health. 39: 461 – 471. usepa (united states environmental protection agency) (1992). soil screening guidance. technical background document. epa/540/r95/128. world health organisation. (1993). guidelines for drinking water quality. 2 nd ed. vol. 1 recommendations. who. geneva. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (3), pp. 167-170, march, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the variation in the composition of ‘chin chin’ and its acceptability among consumers evelyn n. bede department of food science and technology, federal university of technology, owerri, nigeria. email: abedec@yahoo.com. accepted 03 january, 2015 the composition and acceptability of a pastry product ‘chin chin’ prepared by substituting egg with peanut in the master mix has been studied. six different samples of the pastry product were produced. one sample was produced with neither egg nor peanut incorporated. another sample had only egg incorporated while four samples had ground raw peanut incorporated in them at 5, 10, 15 and 20% w/w relative to the flour content. sensory studies showed that all the ‘chin chin’ samples incorporated with peanut showed higher acceptability than the samples produced with egg. they also showed higher protein content which varied from 20.52 to 26.25% as against the protein content of the sample pre-pared with egg which was 18.51%. the sample containing 10% w/w peanut was the most preferred of all the samples. the use of 10% w/w peanut in the production of ‘chin chin’ as a substitute for egg will reduce the cost of the protein source in the pastry product by about 85%. key words: pastry, chin-chin, incorporation, peanut. introduction ‘chin chin’ a pastry product is usually eaten as snacks by all classes of individuals in nigeria. after a thorough mix-ing of the master mix which usually contains egg as pro-tein source, it is cut into desired shapes and sizes which is either baked or fried in oil. however, due to the relative high cost of eggs in the country (nigeria), most ‘chin chin’ produced commercially do not contain egg or any other alternative source of dietary protein, thus leaving the con-sumers with a very poor product on the bid to maximize profit by the producers. the need therefore arises to obt-ain a cheap and acceptable substitute for egg in the prod-uction of ‘chin chin’. however, the consumption of peanut and peanut products have been indicated as an alterna-tive source of dietary protein, especially in countries whe-re diary and poultry products are expensive or are in limi-ted supply (adair et al., 2001; jiang et al., 2003; mate et al., 1996; swanson and munsayac, 1999; hinds et al., 1997). a study to evaluate the diet quality of free-living men, women and children given to peanut consumption (griel et al., 2004) demonstrated improved diet quality of peanut users, indicated by the higher intake of the micro-nutrients, vitamin a, vitamin e, folate, calcium, magne-sium, zinc, iron and dietary fibre, and by the lower intake of saturated fat and cholesterol. peanut consumption was not associated with higher body mass index(bmi), how-ever it serves as a complementary food to reduce post-prandial glycemia (johnston and buller, 2005; coelho et al., 2006). in addition, it has been suggested that frequ-ent intake of peanut and its products may reduce colorec-tal cancer risk in women (yeh et al., 2006), improve indices of cardiovascular disease risk (alper and mattes, 2003) and maintain metabolic order and energy balance in adults (alper and mattes, 2002). consequently accep-tability studies of products rich in peanuts have been car table 1. formula for master mix of ‘chin chin’. ingredient % w/w flour basis wheat flour 100 water 160 ml / 500 g flour sugar 20 salt 0.1 shortening 10 nutmeg 2 baking powder 0.6 eggs 2 eggs/ 500 g flour table 2. basic chemical composition of ‘chin chin’ samples. samples a b c d e f content variation with egg without with 5% with 10% with 15% with 20% egg/peanut peanut peanut peanut peanut moisture content (%) 1.82 7.07 4.07 4.00 3.66 2.26 protein content (%) 18.51 13.51 20.52 21.62 25.20 26.25 fat content (%) 17.00 14.60 19.45 20.75 21.05 25.90 ried out by some authors (adair et al., 2001 swanson and munsayac, 1999).these studies showed increase in acceptability of products incorporated with peanuts and other fruits or nuts. with the above dietary and medical advantages of consumption of peanut and its products, peanut becomes a ready and cheap alternative to egg in the master mix of ‘chin chin’ production. in this study therefore, investigation of the variation in the basic composition of ‘chin chin’ and its acceptability among consumer with increasing percentage of peanut incorporation was carried out. the composition and acceptability of this ‘peanut chin chin’ compared with that of the ‘chin chin’ produced with egg and without egg were also investigated. materials and methods wheat flour produced by the nigeria flour mills plc was used for this work. the ‘red skin spp.’ of peanut ( arachis hypogea) commonly known as ‘gwongworo’ in the south eastern part of nigeria was used. other materials including sugar, salt, butter, nutmeg, baking powder and eggs were all sourced from a local market in owerri, nigeria. the purchased peanut was sorted to remove immature nuts, damaged nuts, stones and other dirt. the sorted peanuts were ground using an attrition mill. the formulation for the master mix of ‘chin chin’ used for this work is given in table 1. the formulation used the weight of the wheat flour as basis. 500 g of wheat flour were weighed each into six different cleaned plastic bowls. 0.5 g of salt, 100 g of granulated sugar, 10 g of nutmeg, 3 g of baking powder and 50 g of butter were added into each of the six bowls containing the wheat flour. the mixtures were thoroughly mixed using finger tips to obtain a uniform mixture. 2 eggs were added to only one of the bowls of flour mixtures and followed by addition of 160 ml of water to form dough. the dough was flattened to a thickness of about 10 mm using a rolling pin and was cut into strips of length 30 mm and width 10 mm. these strips were fried in deep hot vegetable oil at 155 o c for 20 min to produce the product sample a. in the second bowl of flour mixture, 160 ml of water was added with neither egg nor peanut addition. the product produced from this mixture gave the sample b, which represents the ‘chin chin’ produced commercially in nigeria. different quantities of raw ground peanut were incorporated into the remaining four bowls containing flour mixtures with no eggs added. 160 ml of water were added into each of the bowls. after a thorough mixture of the dough, the same sizes of ‘chin chin’ mentioned above were produced by deep frying in hot vegetable oil at 155 o c for 20 min. these sets of ‘chin chin’ produced were labeled samples c, d, e, and f. the variation in the content of the ‘chin chin’ mixtures is as given below; sample a with egg added sample b without egg or peanut added sample c with 5% (25 g) of peanut added sample d with 10% (50 g) of peanut added sample e with 15% (75 g) of peanut added sample f with 20% (100 g) of peanut added. the samples were allowed to cool to ambient temperatures and determination of the basic chemical composition of the samples was carried out immediately after cooling. the chemical composition determined include, moisture, fat and protein contents. the procedure followed was as outlined in literature (aoac, 1990). the samples were subjected to sensory evaluation to determine consumer preferences among the different samples, for colour, taste, crispiness, flavour, mouth-feel, aftertaste perception and overall acceptability. a panel of twenty five undergraduate students very familiar with the snacks ‘chin chin’ was used in the sensory evaluation exercise. the samples were presented in coded identical transparent polythene bags and the samples were tested individually. the order of presentation of the six samples wascompletely randomized. the panelists rinsed their mouth thoroughly with water after testing each sample and waited for a minute before proceeding to test the next sample. the sensory evaluation was based on a seven point hedonic scale as point (7) represented ‘liked extremely’ down to point (1) representing ‘disliked extremely’. the samples were packaged in transparent polythene bags of the same size similar to the packaging of ‘chin chin’ sold in the mar-ket and stored. the packaged samples were kept on the shelf for eight weeks. free fatty acid (ffa) measurements were taken on weekly basis for all the samples. the procedure was also as previously given (aoac, 1990). results the results of this work arepresented below. table 2 shows the basic chemical compositions of the ‘chin chin’ samples. in table 3, the results of the sensory scores of the samples are presented. the quality attributes considered were colour, taste, crispness, flavour, mouth feel, after taste perception and overall acceptability. table 4 gives the comparison of mean percentage ffa values of the samples over the weeks of storage, while figure 1 illustrates the trend of the percentage ffa values for the samples over the weeks of storage. discussion table 2 showed that sample a had the lowest percentage moisture of 1.82% while sample b had the highest bede 168 table 3. sensory scores for the ‘chin chin’ samples. samples a b c d e f content variation with without with 5% with 10% with 15% with 20% egg egg/peanut peanut peanut peanut peanut colour 5.58 c 5.88 ab 5.92 ab 6.19 a 5.88 ab 5.81 bc taste 5.12b ed 4.18 def 5.31 bcd 6.04 a 5.58 abc 5.50 abc crispness 4.27 f 5.31 f 5.69 ab 5.96 a 5.59 abc 5.19bcde flavour 4.77 cde 4.69 def 4.85 d 5.65 a 5.54 abc 5.31 abc mouth feel 4.73 cde 4.88bcde 5.04 bcd 5.92 a 5.35 abc 5.46 ab after taste perception 4.62cdef 4.67 cde 5.12abcd 5.65 a 5.38 ab 5.15 abc overall acceptability 4.63 cd 4.39 d 5.22 b 6.33 a 5.48 b 5.12 bc values within a row for each attribute with different superscript are significantly different (p < 0.05). figure 1. variation in percentage ffa values with storage time for the ‘chin chin’ samples. value of 7.07%. the moisture content for the samples c to f decreased with increase in peanut incorporation. the variations in moisture content of the samples could be as a result of the nature and quantity of protein contained in them. sample a which contained animal protein in form of egg bound up much moisture during its protein denaturation unlike the other samples (c to f) that contain plant protein in form of peanut. the sample b with no other protein save that contained in the wheat was left with the highest moisture content. the table also showed that with the addition of only 5% w/w peanut (sample c), the percentage of protein in the ‘chin chin’ product was higher than that in sample a (produced with egg). as the percentage of peanut increased, the percentage protein content also increased. however, incorporation of peanut up to 5% w/w showed a higher percentage of fat compared with sample a with egg incorporated. this increased fat content in samples c to f could be due to the high percentage of oil of about 40 56.2% in peanut (ihekoronye and ngoddy, 1988). in table 3, the sensory evaluation showed that the samples with peanut incorporated were more acceptable than the chin-chin samples made without peanut (samples a and b). sample d (sample with 10% w/w groundnut) was the most preferred in all the organoleptic quailties. this suggests that 10% w/w incorporation of peanut in ‘chin-chin’ will be the best mixing ratio of incorporation of peanut. at this 10% w/w incorporation of peanut, there was 16.8% increase in protein over the ‘chin-chin’ produced with egg. when the cost of 2 eggs were compared with the cost of 50 g of peanut in nigeria, a reduction cost for the protein source of about 85% was obtained (taking average retail cost of 2 eggs to be n40.00 as compared to the retail cost of 50 g of peanut which was calculated to be n6.00), hence suggesting that incorporation of groundnut as a substitute for egg in ‘chin chin’ will make the product more acceptable and cheap. in addition, consumption of peanut and its products showed a lower intake of saturated fat and cholesterol (griel et al., 2004) hence making it more preferred to egg which has a high cholesterol content (pesti and bakali, 1998; froning et al., 1998). the trend for all samples in figure 1 wassimilar as the percentage ffa values increased with weeks of storage. the ffa values for sample b showed least deviation, followed by sample a. the increase of ffa values with weeks of storage varied greatly with higher percentage of peanut incorporation. it could be observed that the difference in ffa values for sample a and sample c were very small until after the second week. this observation was also confirmed in the table for the statistical comparison of ffa values for the samples (table 4) . the per-centage ffa values for samples a and c were not signi-ficantly different (p < 0.05) till after the second week of storage. at the end of the second week of storage, the percentage ffa values for sample d had doubled. considering the trend of the percentage ffa values of the samples, it could be inferred that organoleptic qualities of the ‘chin chin’ samples incorporated with groundnut (sample c f) will deteriorate faster than the others (samples a and b). however, most ‘chin chin’ products 169 afr. j. food sci. res. bede 170 table 4. comparison of mean percentage ffa values for samples over the storage period. samples a b c d e f content with without with 5% with 10% with 15% with 20% variation egg egg/peanut peanut peanut peanut peanut week 0 0.65 a 0.43 b 0.71 a 0.85 c 1.05 d 1.15 e week 1 0.97 a 0.56 b 0.99 a 1.35 c 1.69 d 1.83 e week 2 1.32 a 0.71 b 1.33 a 1.63 c 1.97 d 2.40 e week 3 1.53 a 0.97 b 1.69 c 1.83 d 2.12 e 2.68 f week 4 1.62 a 1.05 b 1.97 c 2.05 d 2.40 e 3.12 f week 5 1.69 a 1.41 b 1.99 c 2.40 d 2.63 e 3.22 f week 6 1.83 a 1.55 b 2.00 c 2.63 d 2.93 e 3.40 f week 7 1.93 a 1.62 b 2.33 c 2.97 d 2.99 d 3.61 e week 8 2.12 a 1.69 b 2.67 c 3.06 d 3.37 e 3.82 f values within a row for each attribute with different superscript are significantly different (p < 0.05). sold in stores rarely exceed 2 weeks on the shelf. the comparison of percentage ffa values over the storage period (table 4) showed that the ffa values for the samples were significantly different from one another (p < 0.05) throughout the storage period except for sam-ples a and c with ffa values not significantly different in weeks 0 – 2. also ffa values for samples d and e were not significantly different in week 7. conclusion ‘chin chin’ produced with the incorporation of plant protein as an alternative to egg has shown acceptable quality characteristics. this substitution of peanut for egg will not only increase the protein content of the chin-chin but will highly reduce the cost of producing a protein rich ‘chin chin’ by about 85%. the most acceptable mixture was the chin-chin with 10% w/w peanut incorporation. as a result of the improved organoleptic quality of chin-chin due to the incorporation of peanut, the problem of produ-cing poor quality pastries in developing countries such as nigeria due to high cost of animal protein could be abated. acknowledgement the author acknowledges b. n. odili for her assistance in carrying out the laboratory and sensory aspect of this work. references adair m, knight s, gates g (2001). acceptability of peanut butter cookies prepared using mungbean paste as fat ingredient substitute. j. am. dietetic assoc. 101(4): 467469. alper c m, mattes rd (2003). peanut consumption improves indices of cardiovascular disease risk in healthy adults. j. am. col nutr. 22(2): 133-141 alper cm, mattes rd (2002). effects of chronic peanut consumption on energy balance and hedonics. inter. j. obesity and related metabolic disorders 26(8): 1129-1137. aoac (1990). official methods of analysis. association of official analytical chemists. 15th ed. virgnia usa. coelho sb, de sales rl, iyer ss, bressan j, costa nmb, lokko p, mattes r (2006). effects of peanut oil load on energy expenditure, body composition, lipid profile and appetite in lean and overweight adults. nutrition 22: 585-592. froning gw, wehling rl, cuppett s, niemann l (1998). moisture content and particle size of dehydrated egg yolk affect lipid and cholesterol extraction using supercritical carbon dioxide. poult. sci. 77: 1718-1722. griel ae, eissenstat b, juturu v, hsieh g, kris-etherton pm (2004).improved diet quality with peanut consumption. j. am. coll. nutr. 23(6): 660-668. hinds mj, chinnan ms, beuchat lr (1997). particle size distribution in a heat-processed beverage prepared from roasted peanuts. food res. int. 30(1): 59-64. ihekoronye ai, ngoddy po (1988). integrated food science and technology for the tropics. macmilian, london. jiang s, mansson je, stampfar mj (2003). nut and peanut butter con-sumption and risk of type 2 diabetes in women. acc curr. j. rev. 12(2): 41-42. johnston cs, buller aj (2005). vinegar and peanut products as complementary foods to reduce postprandial glycemia. j. am. diet. assoc. 105: 1939 -1942. mate ji, saltveit me, krochta jm (1996). peanut and walnut rancidity: effects of oxygen concentration and relative humidity. j. food sci. 61 (2): 465468. pesti gm, bakali ri (1998). studies on the effect of feeding cupric sulphate pentahydrate to laying hens on egg cholesterol. poultry sci. 77: 15401545. swanson rb, munsayac lj (1999). acceptability of fruit purees in peanut butter, oatmeal and chocolate chip reduced fat cookies . j am. dietetic. assoc. 99(3): 203-207. yeh cc, you sl, chen cj, sung fc (2006). peanut consumption and reduced risk of colorectal cancer in women: a prospective study in taiwan. world j. gastroenterol. 12(2): 222-227. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (4), pp. 297-299, april, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. short communication promoting the use of coconut in the production of yoghurt rita e. sanful department of hotel, catering and institutional management, cape coast polytechnic, p. o. box ad 50, cape coast, ghana. e-mail: resanful@cpoly.edu.gh or sanrita74@hotmail.com. accepted 03 march, 2017 yoghurt was produced from milk obtained from coconut milk and skimmed cow milk, by fermentation using starter cultures. the results obtained show that the ph of the various products ranged from 4.2 4.4. the acceptability of the yoghurt produced was investigated in a sensory evaluation study. the sensory evaluation study of the samples indicated that yogurt produced from skimmed cow milk did not differ from those produced from coconut and cow milk composites in all sensory quality attributes. thus coconut milk can be used with cow milk to produce acceptable and affordable yoghurt since coconut milk is cheaper. the results also showed that middle aged coconut (7 8 months old) could be used for the production of acceptable yoghurt. key words: sensory evaluation, starter culture, coconut, substrate. introduction yoghurt is a fermented product obtained through an anaerobic fermentation of lactose in milk by relevant microorganisms most of which are classified as probiotic (tull, 1996). most ghanaians regularly take yoghurt either as a dessert, snack or as a pro-biotic food drink to aid diges-tion and to re-establish a balance within the intestinal micro-flora. the most popular yoghurts known on the ghanaian market are those obtained from cow milk (sackey-addaquay, 2008). the substrate that is usually employed in this type of yoghurt is evaporated whole milk/skimmed solids or fresh milk from cow. although this substrate produces good quality yoghurt, there are certain limitations that make it difficult for the ordinary ghanaian middle income earner to patronize. this is because the substrate is relatively expensive compared with other possible substrates which have the potential to produce a comparable effect as seen with cow milk. it is realized that strict vegetarians are also limited in their quest for probiotic yoghurts when there is the confinement to only animal base yoghurt. it is therefore of great importance to find out the feasibility of using the coconut milk as substrate for yoghurt production as are used in other experimental substrates such as, soy bean milk and tiger nuts milk (belewu and belewu, 2007). yoghurt obtained by using coconut milk has been found to be delicious and a nutritional product (imele and atemnkeng, 2001). belewu et al. (2005) have also documented the combination of soymilk (50%) and coconut milk (50%) in the preparation of soy-coconut yoghurt. coconut (cocos nucifera) milk is being used by confectionaries, bakeries, biscuits and ice cream indus-tries worldwide to enhance flavor and taste of various products (persley, 1992). coconut milk was found to be rich in calcium. the milk was reported to be high in mine-rals and vitamin content (nieuwentus and nieuwelink, 2002) while total saturated fat was 10% of the total energy (thai food composition, 2004). in the light of the above, this research was taken to promote the use of coconut in the production of yoghurt. this is done by producing yoghurt from coconut milk extract, cow milk and their composites. sensory evaluation is conducted on the various yoghurts produced and the results analyzed using the statistical package for social sciences (spss) version ii for windows. materials and methods extraction and preparation of coconut milk the fresh middle aged coconut (7 8 months old) used in this work was purchased from the local market. the coconut was crushed open and the juice poured and stored in a refrigerator. 2 kg of coconut flesh was then removed from the shell, grated and homogenized in a blender together with the coconut juice for 2 min. it was then passed through a fine sieve twice, with the volume adjustted to 1.5 l and stored in a bottle in a refrigerator. the extracted coconut milk was transferred into a pot and pasteurized or heated at 90°c for 30 min and allowed to cool gradually to a temperature of 43°c. it was kept at this temperature for 12 h before it was finally cooled to room temperature of about 27°c. sanful 298 table 1. the physico-chemical properties of yoghurts. sample fat relative density ph a 1.4 1.017 4.20 b 1.4 1.021 4.22 c 1.8 1.026 4.27 d 2.0 1.034 4.40 reparation of cow milk half kg of skimmed milk powder was weighed into a measuring cylinder. 1.5 l of warm water was added to the skimmed milk and thoroughly stirred to give a homogenous mixture. the mixture was heated to 90°c and held at this temperature for 30 min and cooled to a temperature of 43°c for 4 h and then allowed to cool to room temperature of about 27°c. preparation of yoghurts the yoghurt was produced from the mixture of cow milk and coconut milk. the different mixtures were composed according to the following % presented below: sample a: 100% cow milk sample b: 75% cow milk and 25% coconut milk sample c: 50% cow milk and 50% coconut milk sample d: 100% coconut milk the mixtures were incubated with a starter culture, stirred properly and kept at a temperature of 43°c for 5 h. 0.02 kg of adamly starch and 0.01 kg of recodan fs were added to the samples. these act as stabilizers and emulsifiers to add up to the nutritional value of the main ingredients and also to bind all the ingredients together without any separation. 0.45 kg of sugar was added to the mixture to sweeten and the samples were stored at a temperature between 1°c and 6°c. determination of ph a hanna hi98128 stick ph meter was used to determine the ph of the yoghurts produced. the ph meter was switched on and allowed to warm up for about 15 min. the ph meter was adjusted to neutral value by using distilled water at ambient temperature. the electrodes of the meter were cleaned, dried and dipped into the different samples and the reading was noted. determination of fat a 5 g sample of yoghurt was weighed with 0.01 g accuracy into a cowbell milky butyrometer (0 6%). 16 ml of sulphuric acid (h2 so4) and 1 ml of isoamyl alcohol were added. the butyrometer was capped tightly and turned up and down repeatedly, to have a homogeneous mixture and to have the proteins dissolved entirely. the butyrometer then was immersed in a 65 ± 2°c water bath for 5 min. following heating, the butyrometer was centrifuged for 5 min at 1000 rpm. after removing from the centrifuge, the lower level of the fat column in the butyrometer was set at the start point of the scale by means of the cap. the reading was taken by the lower meniscus of the scale. determination of relative density (specific gravity) with the aid of a lactometer (zeal type), the specific gravity (or relative density) of the yoghurts prepared was measured. a lacto meter works on the principle of specific gravity of milk. the instrument was washed in distilled water and put in some amount of the yoghurt in a test tube, with the bulb of the meter dipped in the yoghurt. the meter sank until it floated. the level to which the meter sank in the yoghurt was read. sensory evaluation untrained panel of 40 made up of staff members of the maintenance department and 20 members of the catering department of the ankaful psychiatric hospital were selected. they ranked some sensory attributes, such as appearance, mouth feel, sourness, consistency, general acceptance and aroma of the samples using the hedonic descriptive scale 1 5. the identities of the samples were concealed. results and discussion physico-chemical properties the ph of the yoghurt produced ranged from 4.2 to 4.4 which agrees quite well with the results of other workers (akpan et al., 2007) . the fat content ranged between 1.4 to 2.0% which is in agreement with the results obtained by imele and atemnkeng (2001). the relative density of the yoghurt samples was found to range between 1.017 to 1.034, with the skimmed cow milk yoghurt having the least and the pure coconut yoghurt having the maximum. this indicated that the skimmed cow milk yoghurt contained more water than the pure coconut yoghurt. the physicochemical properties of the yoghurts are given in table 1. table 2 gives the score of comparative sensory evaluation of the coconut-cow milk yoghurt. appearance the appearance is the look and texture of the yoghurt as it is poured into a glass. the yoghurt was poured into a glass without labels for the panel to see and comment on. the panelists accepted the appearance of all the samples as good. there was insignificant difference between the mean of the values of the samples. sample c produced a clean natural colour with a smooth velvety appearance which agrees with acceptable standard described by tull (1996). sourness sourness of yoghurt is derived from the various acids present in the yoghurt during fermentation. the panelists gave their comments based on the tartness they felt in their mouth after tasting the yoghurt. the analysis of the sourness revealed that samples a and d were very good. the composite samples (b and c) relatively had lower sourness. consistency the panelists assessed the consistency of the stirred yoghurt. the stirred yoghurt should neither be watery nor 299 afr. j. food sci. res. table 2. percentage score on comparative sensory evaluation of the coconut-cowmilk yoghurt. sensory qualities sample appearance sourness consistency aroma mouth feel overall acceptance a 100 95 100 100 98 98 b 100 90 98 97 96 94 c 100 85 100 98 95 92 d 100 95 97 98 98 95 too thick. the panelists found the consistency of the samples was almost the same, with samples a and c being slightly preferred. the mean of the values showed no significant difference. aroma the aroma of yoghurt is important to consumers. the panelists were asked to compare the aroma of the 4 yoghurt samples. the panelists accepted the aroma of all the samples with a slight preference for sample a. mouth feel the panelists commented on the mouth feel of the four samples. 98% of the panelists accepted the mouth feel of samples a and b better than samples b and c. however, the difference between the samples was insignificant. overall acceptance the panelists tasted and rated the samples on the 5 point hedonic scale on their degree of liking for the 4 samples of yoghurt presented. the overall acceptance was determined by the assessment of appearance, sourness, consistency, aroma and mouth feel. 98% of the panelists accepted sample a while 95% of them accepted sample d. sample b was also preferred to sample c. thus there seem to be a slight preference for the pure cow milk yoghurt and the pure coconut yog-hurt. this study has shown that yoghurt produced from skimmed cow milk did not differ organoleptically from those produced from coconut and cow milk composites and pure coconut milk in all the sensory quality attributes. the results of this work agree quite well with the results of akoma et al. (2000). conclusion yogurt was produced from milk obtained from coconut milk and skimmed cow milk, by fermentation using starter cultures. the results obtained show that the ph of the various products ranged from 4.2 4.4. also the measurement of the relative density of the yoghurts indicates that the skimmed cow milk yoghurt contained more water than the pure coconut yoghurt. the sensory evaluation study of the samples indicated that yogurt produced from skimmed cow milk did not differ from those produced from coconut and cow milk composites in all sensory quality attributes. thus coconut milk can be used with cow milk to produce acceptable and affordable yoghurt since coconut milk is cheaper. acknowledgement the author is grateful to professor r. k. nkum, the rector of this polytechnic, for offering very useful suggestions during the course of this work and for reading through the manuscript. references akoma o, elekwa uo, afodunrinbi at, onyeukwu gc (2000). yoghurt from coconut and tigernuts, j. food technol. afr. 5: 132-134. akpan ug, mohammed ad, aminu i (2007). effect of preservative on the shelf life of yoghurt produced from soya beans milk, leornardo elect. j. pract. technol. 11: 131-142. belewu ma, bdelewu ky, olatunji sa (2005). soy-coconut yoghurt: preparation, composition and organoleptic properties, bio. sci. res. bull. 21: 129-37. belewu ma, belewu ky (2007). comparative physicoevaluation of tiger-nut, soybean and coconut milk sources. int. j. agric. biol. 9: 785-787. imele h, atemnkeng a (2001). preliminary study of the utilisation of coconut in yoghurt production. j. food. technol. 6: 121-125. nieuwentus r, nieuwelink j (2002). agrodok series no. 10 cta: the netherlands pp. 2830. persley jg (1992). replanting of the tree of life, cab international wallingford oxon pp. 38-40. sackey-addaquay s (2008). promoting the use of coconut in the production of yoghurt. hnd dissertation, cape coast polytechnic, cape coast, ghana pp. 128. tetteh e (2007). promoting the use of coconut in yoghurt production. hnd dissertation, cape coast polytechnic, cape coast, ghana pp. 5-21. thai food composition (2004). institute of nutrition mahidol university (inmu), thailand p.150. tull a (1996). food and nutrition, oxford university press pp. 109-111. african journal of food science research vol. 1 (1), pp. 001-009, september, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper optimization of process conditions for cassava (manihot esculenta) lafun production nwabueze, t. u.* and odunsi, f. o. department of food science and technology, michael okpara university of agriculture, umudike, p.m.b. 7267, umuahia, abia state, nigeria. accepted 28 th september, 2013 optimization of cassava (manihot esculenta, nr8082 clone) lafun production and evaluation was carried out using response surface methodology. the selected experimental design had three variables and five levels referred to as central composite design. process variables were inoculum volume, fermentation time and drying temperature. inoculum volumes were 1.50, 2.00, 2.50, 3.00 and 3.50 l. fermentation times were 24, 36, 48, 60 and 72 h while drying temperatures were 35, 41, 47, 53 and 59 o c. response surface data on lafun yields, colour index and cyanogenic potentials were analyzed in a regression model while threedimensional response surface plots were made. effects of process variables on lafun parameters varied depending on which response was analyzed. maximum and minimum yields were 82.68 and 66.68% for lafun flour and 68.24 and 56.20% for lafun starch, respectively. photometric colour index ranged from 10.03 to 30.51 while cyanogenic potentials of the flour ranged from 1.88 to 5.31 mg hcn/kg and that of the paste from 0.93 to 3.11 mg hcn/kg. models developed by response surface analysis for these responses were significant at low (1 to 5%) levels of probabilities and could be explained by regression coefficients in the models. variabilities explained by the models were low for lafun flour yield (r 2 = 0.3188) but high for photometric colour index (r 2 = 0.8098). optimum process conditions obtained for starch yield, photometric colour index and cyanide reduction were 2.0 l, 36 h and 41 o c; 2.50 l, 48 h and 35 o c; and 2.50 l, 72 h and 41 o c, respectively. key words: cassava, lafun, fufu, hydrogen cyanide, optimization, response surface methodology. introduction cassava (manihot esculenta) is an important food in the tropical areas of african, asia and latin america. it is estimated (iita, 1990) that the crop provides about 40% of all the calories consumed in africa and ranks second only to cereal grains as chief source of energy in nigerian diet (ngoddy, 1989). by this, cassava plays important role in alleviating african food crisis though poor in protein (1.20%) and rich in cyanide (> 10 mg/100g fresh weight) in some varieties such as tms 50395 (iita, 1990; janssens, 2001). the variety/clone nr8082 is low cyanide high-yielding cassava developed by the national root crops research institute, nigeria and commonly distributed to nigerian farmers. *corresponding author. e-mail : teeubueze@yahoo.co.uk. raw cassava has been reported (onabolu, 1989; cereda and mattos, 1996) to contain two cyanogenic glycosides known as linamarin and lotaustralin with the former being the most representative glucoside, accounting for about 80% of the total cassava glucoside (dicar, 1993) . linamarin and lotaustralin are -glucosides of acetone cyanohydrin and ethyl-methylketonecyanohydrin, respectively (cereda and mattos, 1996). linamarin produces the toxic compound (hydrogen cyanide, hcn), which can be hazardous to the consumer. cassava processing by fermentation is one of the most widespread techniques used in africa and is considered an efficient means of reducing cyanogenic potential in the resulting food (brainbridge, 1994). lafun is fermented cassava flour popular among the people in the southwestern states of nigeria (cereda and mattos, 1996) . the traditional method of processing cassava into lafun though unique for its ability to reduce nwabueze and odunsi 002 the toxic cyanogenic compound to a least possible level (numfor, 1983) imparts a strong smell to the product (cereda and mattos, 1996). lafun, like gari, another fermented product from cassava, is a dry product, which can be preserved for a long time under the prevailing local environment. it is a cheap and popular meal in the rural areas of nigeria and can be prepared into ready food much more quickly than gari (latunde-dada, 1997). lafun is generally prepared into a thick paste in boiling water and eaten with vegetable soup or stew. oyewole and ogundele (2001) reported that lafun quality varies with processing methods and with rocesssors. in spite of various reports in literature (oyewole and odunfa, 1990; oyewole and afolami, 2000; oyewole and ogundele, 2001) on lafun production from different cassava varieties, there is scarcity of information on efforts to optimize known process conditions for maximum product quality in mind. the need to optimize process conditions to enhance product quality in terms of yield, cyanogenic potentials and sensory acceptability through response surface methodology, becomes relevant given the present need for small scale food processing and spread of consumers. in many applications, the system to be optimized can be formulated as a mathematical model. with the advent of high-speed computers, very large and complex systems can be modeled and optimization can yield substantially improved benefits. although optimization has become popular in many sectors of the food industry, it is a procedure for developing the best possible product in its class (ruguo, 1999). the intention of optimization is to provide a more precise map of the path that has the highest probability of leading to a successful food product (ruguo, 1999; crapiste, 2000). the objective of this study is to use the response surface application to optimize process variables to maximize cassava lafun yields, colour and sensory attributes or reduce cyanogenic potentials without large changes in the operational parameters. these process variable conditions investigated included inoculum volume (l), fermentation time (h) and flour dry ing temperature (c) . the process variable combination that gave maximum cassava lafun response is recommended for scale up production in that response. materials and methods source of cassava roots cassava roots of the nr8082 clone were obtained from the national root crops research institute, umudike, abia state, nigeria. the cassava roots were freshly harvested at 10 – 12 months of age. cassava roots preparation freshly harvested cassava roots of the nr8082 clone were peeled, washed and fermented according to the method described in literature (oyewole, 1994; oyewole and odunfa, 1990) . the cassava roots were then grated with a local grating machine and fermented with appropriate inoculums from a 4-day-old starter culture containing about 10 cfu/g of microorganisms comprising mainly corynebacterum manihot and geotricum candida. processing variables process variable conditions investigated for optimization in lafun production included inoculum volume (l), fermentation time (h) and flour drying temperature (c). inoculums the methods described by mathew (2002) and numfor (1983) were used with minor modifications. inoculums from the starter culture were introduced into the cassava pulp at various volumes of 1.5, 2.0, 2.5, 3.0 and 3.5 l (x1 ). the medium was stirred to bring about uniform distribution of the microorganisms. drying temperature of lafun flour the fermenting liquor was decanted and the pulp processed using a local screw press. the dewatered pulp was dried in the oven (carbolite md 1430, england) at variable temperatures of 35, 41, 47, 53 and 59c (x2). fermentation time of the cassava pulp the cassava pulp was allowed to ferment at room temperature for variable time intervals of 24, 36, 38, 60 and 72 h (x3). lafun flour yield the percent yield of lafun flour was calculated from the weight of lafun obtained from known weight of the peeled cassava roots used (oyewole and ogundele, 2001). lafun starch yield a 50 g sample of lafun flour was thoroughly mixed with sufficient water and filtered through a 50 sieve. the mixture was allowed to stand overnight (oyewole and afolami, 2001). it was decanted and recovered starch was dried in oven to a constant weight. weight of dried starch starch (%) = x 100 (1) weight of sample photometric colour index photometric colour of various lafun flours was determined on 1 g sample according to the method described by pike (2003). the sample was weighed and dissolved in 20 ml water/ethanol mixture. the mixture was filtered after standing for 30min. the absorbance of the filterate was measured at 400, 550, 620 and 670 nm using 003 afr. j. food sci. res. table 1. central composite experimental design of 3 variable five level process conditions for production of lafun flour from cassava root. exptal a run inoculum volume (i) fermentation time (h) drying temperature(c) 1 2.0 (-1) 36 (-1) 41 (-1) 2 2.0 (-1) 36 (-1) 53 (+1) 3 2.0 (-1) 60 (+1) 41 (-1) 4 2.0 (1) 60 (+1) 53 (+1) 5 3.0 (+1) 36 (-1) 41 (-1) 6 3.0 (+1) 36 (-1) 53 (+1) 7 3.0 (+1) 60 (+1) 41 (-1) 8 3.0 (+1) 60 (+1) 53 (+1) 9 3.5 (+a) 36 (-1) 41 (-1) 10 1.5 (-a) 36 (-1) 53 (+1) 11 2.5 (0) 72 (+a) 41 (-1) 12 2.5 (0) 24 (-a) 53 (+1) 13 2.5 (0) 48 (0) 59 (+a) 14 2.5 (0) 48 (0) 35 (-a) 15 2.5 (0) 48 (0) 47 (0) 16 2.5 (0) 48 (0) 47 (0) 17 2.5 (0) 48 (0) 47 (0) 18 2.5 (0) 48 (0) 47 (0) 19 2.5 (0) 48 (0) 47 (0) 20 2.5 (0) 48 (0) 47 (0) 21 2.5 (0) 48 (0) 47 (0) 22 2.5 (0) 48 (0) 47 (0) 23 2.5 (0) 48 (0) 47 (0) numbers in brackets are the coded values of the independent variables in the experimental design. exptal = experimental. spectrophotometer (unican he 105y, england). the solvent was a small consumer acceptance panel (stone and sidel, 1985) used as blank. photometric colour index was calculated as: familiar with lafun consumption and with the sensory scale and method of assessment were drawn from southwestern nigeria pci = 1.29 (a400) + 69.70 (a500) + 41.20 (a620) – 56.41 (a670) (2) students of michael okpara university of agriculture, umudike and used to evaluate sensory attributes of the cooked paste. where a = absorbance. the samples were arranged randomly in similar plates each with coded 3-digit non-misleading or biasing numbers. a 9-point hedonic scale was used to make panelists express their feelings of cyanogenic potentials of lafun flour and paste like and dislike for the flavour, colour, texture, taste and overall acceptability of the pastes. on the scale score, 9 represented like cyanogenic potentials of lafun flour and paste were determined extremely, 1 represented dislike extremely while 5 represented neither like nor dislike. data generated from scores were analyzed using picrate paper kits method described by bradburg (1999). for variance as described by larmond (1977). one-gram sample of lafun flour was homogenized in a 250 ml conical flask containing 25 ml water. a strip of spot paper soaked in an alkaline sodium picrate solution was fixed in the solution with the experimental design cork. the flask was kept for 18 h at room temperature. the strip was removed and eluted in 60 ml water and the absorbance was the experimental design of 3-factor factorial in completely read at 540 nm using a spectrophotometer (unican he 105y, randomized design (crd) as reported by meilgaard et al. (1999) england). as shown in eq. (3) was adopted. thus: sensory evaluation of lafun paste lafun flour was first sifted through a 50 µ sieve. it was then cooked (3) where y is the dependent variable, xi and xj the independent into a paste by turning the flour in boiled water at flour/water ratio of 1:4 (w/v). sensory evaluation was carried out within ten minutes of variables in the model, k the number of independent variables, ßo preparation (oyewole and afolami, 2001) following the method the intercept (constants and regression coefficients of the model), described by stone and sidel (1985) with minor modifications. and the random error term. nwabueze and odunsi 004 table 2. analysis for quality parameters of lafun flour and starch yields (%), photometric colour index and cyanogenic potentials (mg hcn/kg) for flour and paste a . exptal b run yields (%) photometric colour index cyanogenic potentials c flour starch flour paste 1 82.68 68.24 14.47 3.75 2.17 2 80.00 64.62 14.52 4.06 2.17 3 78.68 56.72 19.74 3.13 1.86 4 66.68 64.32 29.26 3.44 2.17 5 80.26 58.74 14.54 3.75 2.48 6 78.68 64.32 13.80 4.06 2.48 7 80.08 56.20 15.48 3.75 2.48 8 73.32 56.70 17.59 4.06 2.17 9 71.04 58.68 12.04 4.06 1.86 10 67.04 65.70 13.44 4.38 2.48 11 77.32 55.80 10.03 1.88 0.93 12 66.68 57.20 10.08 5.63 3.11 13 77.32 57.80 12.74 5.31 2.80 14 66.68 64.66 30.51 4.38 2.48 15 73.32 60.86 20.83 4.69 2.48 16 77.32 60.72 22.34 4.69 2.48 17 66.68 62.72 24.79 4.69 2.17 18 73.32 60.24 29.59 5.00 2.48 19 66.68 62.30 26.62 5.00 2.48 20 78.68 60.30 21.06 4.69 2.17 21 78.68 61.28 18.41 4.69 2.48 22 77.32 60.74 21.10 5.00 2.48 23 77.32 60.24 20.46 4.69 2.17 a lafun flour cooked into paste by stirring in boiled water ready for consumption, b experimental, c cyanogenic potentials (mg hcn/kg). the three variables; inoculum volume (l), fermentation time (h) and results and discussion flour drying temperature (c) and five levels coded -a, -1, 0, +1 and +a gave 15 variable combinations which when replicated 8 times at response surface analysis for lafun flour the center point (0) generated a total of 23 experimental runs. the experimental design had upper (+a), intermediate (0) and lower (-a) data on quality parameters of cassava lafun are presen values of process variable conditions (table 1). ted in table 2. optimum process condition for maximum yield (82.68%) was 2.0 l, 36 h and 41c. (inoculum statistical analysis volume, fermentation time and drying temperature, res pectively). lower or upper process conditions reduced data collected were analyzed using an appropriate statistics flour yields. the independent variable with most signi package (spss/pc +) in a regression model. three-dimensional ficant (p 0.1) effect on flour yield was the length of response surface plots were made using a statgraphic computer fermentation. short fermentation times impair root tissue package (statistica, statsoft, inc., tulsa, ok). softening while long fermentation times as in the tradi tional practice favour it and result in increased flour yield. tissue softening is a product of associative interaction of (4) several microbes and enzymes such as polygalactu ronase, pectinase and cellulase with tissue degrading where activities (meilgard et al., 1999; okolie and ugochukwu, 1988). y = dependent variable, 0 …. 3 =estimated regression the polynomial equation (eq.5) shows that only cross coefficient, x1….x3 = independent variables, and = random error. product effects of process variables had significant (p 0.05) effect on lafun flour yield. essentially, length of 005 afr. j. food sci. res. figure 1. effect of process conditions on lafun starch yield (in = inoculum volume (l) and dt = drying temperature ( o c)). table 3. estimated regression coefficients for starch content of lafun four. source coeficient std error df p–value regression on constant 33.576623 84.594281 x1 -258.193308 54.030059 1 0.004 x2 5.933780 3.073175 1 0.0756 x1.x1 10.087864 4.386095 1 0.0387 x2.x2 -0.102818 0.021418 1 0.0003 x3.x3 -0.042018 0.009088 1 0.0005 x1.x2 4.105445 1.210963 1 0.0048 x1.x3 4.553212 0.865245 1 0.0002 x2.x3 0.060312 0.054932 1 0.2921 r 2 0.85464 fermentation was central in this effect on lafun flour yield. fermentation time (x 3) had a cross product order effect when interacted with either the inoculum volume (x1) or the drying temperature (x2). inoculum volume increased microbial load in the medium and hence favoured fermentation while drying temperature limited their activities. since flour yield was relative to the intact cassava root, loss of moisture and volatile components of fermentation (bokanga, 1992) could affect the actual values obtained for lafun flour in table 2: yflour = 30.14444–0.52332x1.x3–0.03695x2.x3 (5) the model for this parameter explained only 31.89% of the total variations in flour yield showing a significant (p 0.05) lack of fit. response surface analysis for lafun starch lafun flour was essentially of starch material with maximum yield of 68.24% at optimum process condition (table 2) . response surface plot of lafun starch yield had a twisted shape (figure 1) and linear, quadratic and cross product order effects were significant at 1 – 5% levels of probability. the plot showed that starch yield increased with inoculum volume and drying temperature effect up to a maximum of 2.0 l and 41c, respectively, before a twist effect of these variables occurred at an increased fermentation time. at longer fermentation times, disintegrated tissue pulps have the chances of leaching into the fermenting water, there by reducing starch yield. corynebacterum manihot population in the fermenting medium which initiates cassava starch break down could have bond breaking effect thereby lowering water holding capacity of the pulp. this activity invariably culminates in loss in moisture and consequently starch as a soluble cassava constituent. the response surface equation developed from the regression table (table 3) after removal of non significant (p>0.05) terms becomes: ystarch = 33.57662 – 258.19331x1 + 10.08764x1 2 – 0.10282x2 2 – 0.04202x3 2 + 4.10545x1.x2 + 4.55321x1.x3 (6) the model accounted for 85.46% of the total variation in lafun starch yield and significantly (p 0.05) fitted. response surface analysis of lafun colour index all lafun flour samples were visually white in colour. however the photometric determinations presented in table 2 showed that flour whiteness reading varied from 10.03 to 30.51. the highest index was obtained at process variable condition of 2.5 l (inoculum volume), 48 h (fermentation time) and 35c (drying temperature). cassava pulps fermented with higher or lower ( 2.5 l ) inoculum volumes and for longer or shorter ( 48 h ) periods, produced lafun flours with lower photometric colour indices particularly when dried at higher temperature range of 47 to 59c (figure 2). the figure confirms that white flour is produceable with >1.5l (inoculum volume) and <47c (drying temperature). lafun flour from 72 h fermented pulp had the least colour index (10.03) indicating that long fermentation times could encourage microbial breakdown of colour pigments as well as increase availability of reactive groups which can go on to take part in reactions in low molecular weight carbohydrates during drying. this effect was stronger at higher drying temperatures (47-59c) where mild reactions such as caramelization of carbohydrates and millard browning involving amino acid compounds of natural proteins and nwabueze and odunsi 006 table 4. estimated regression coefficients for photometric colour index of lafun flour. source coeficient std error df p–value regression on constant 63.100223 80.738146 x1 -82.826304 51.567161 1 0.1322 x2 -5.308687 2.933088 1 0.0935 x1.x1 -6.880421 4.186160 1 0.1242 x2.x2 -0.008258 0.020442 1 0.6928 x3.x3 -0.050130 0.008674 1 0.0001 x1.x2 4.207685 1.155763 1 0.0030 x1.x3 3.866535 0.825804 1 0.0004 x2.x3 0.214220 0.052428 1 0.0013 r2 0.80980 figure 2. effect of process conditions on photometric colour index (pci) of lafun flour. in = inoculum volume (l) and dt = drying temperature ( o c) reducing sugars affect flour whiteness. badrie and mellowes (1992) who considered effect of cassava starch or amylose on characteristics of cassava extrudate, reported the potentials for interactions between lipids, proteins and carbohydrates and their breakdown products, which under the present processing conditions could affect flour colour. the response surface analysis of data on the photometric colour index (table 4) showed that the response depended more significantly (p 0.05) on cross product order effects of independent variable interactions than on linear order effects of individual variables. fermentation time had significant (p 0.05) quadratic and cross product effect on the flour colour. the interac tive cross product effects of inoculum volume with drying temperature was most significant (p 0.05) (eq. 7) followed by that with fermentation time, while drying temperature and fermentation time was the least. on removal of non significant (p > 0.05) terms and recomputing, the polynomial becomes: ycolour = – 63.10022 – 0.05013x3 2 + 4.20769x1.x2 + 3.86654x1.x3 + 0.21422x2.x3 (7) the model accounted for 80.98% of total variation in lafun colour and significantly (p 0.05) fitted. response surface analysis of cyanogenic potentials of lafun flour and paste the independent process variable most affecting cyanide reduction was the length of fermentation. fermentation times of 60 to 72 h reduced cyanogenic potentials from 79.80 mg hcn/kg originally present in the raw cassava root (nr8082 clone) to a range of 3.75 to 1.88 mg hcn/kg in the flour and 2.17 to 0.93 mg hcn/kg in the paste. shorter fermentation times (24 –48 h) reduced the cyanide potentials from 5.63 to 3.75 mg hcn/kg in flour and from 3.11 to 2.17 mg hcn/kg in the paste (table 2). optimum process variable condition of 2.5 l, 72 h and 41c produced lafun flours with least cyanogenic potentials in flour (1.88 mg hcn/kg) and paste (0.93 mg hcn/kg). long fermentation periods gave fermenting microorganisms ample opportunity to produce enzyme linamarase which when combined with retting and drying drastically reduced cyanide content in the flour. optimum independent process condition of 2.0 l, 36 h and 41c (inoculum volume, fermentation time and drying temperature), reduced cyanogenic potentials to 95.30% in the flour and 97.28% in the paste. cooking the flour into paste resulted in a loss of 50.53% and 98.83% of cyanide 007 afr. j. food sci. res. table 5. estimated regression coefficients for cyanogenic potentials of lafun paste a . source coeficient std error df p–value regression on constant 3.469850 6.468992 x1 5.184381 4.131722 1 0.2317 x2 -0.043815 0.235008 1 0.8550 x1.x1 -0.868747 0.335408 1 0.0224 x2.x2 0.001506 0.001638 1 0.3747 x3.x3 -0.000705 0.000695 1 0.3291 x1.x2 -0.026021 0.092603 1 0.7831 x1.x3 0.019166 0.066166 1 0.0466 x2.x3 0.000196 0.004201 1 0.0636 r2 0.68678 a lafun flour cooked into paste by stirring in boiled water ready for consumption. figure 3. effect of process conditions on cyanogenic potential (cocnp) of lafun paste. in = inoculum volume (l) and st = fermentation time (h). present in the flour and raw cassava root (nr8082), respectively. the high percentage loss is attributable to its solubility and heat labile nature particularly in the paste. this is one major nutritional benefit of traditional practice of combining long period of fermentation (about 96 h) and twice cooking as unit operations in local cassava processing and utilization in africa. this observation agrees with the report (brainbridge, 1994) that fermentation is the most widespread techniques used in africa as effective means of reducing cyanogens in cassava products. response surface analysis of the cyanogenic potentials of lafun paste (table 5) indicated that quadratic and cross product coefficients of the model were significant (p 0.05) while the plot showed a dome shape (figure 3). reduction in cyanide increased quadratically with inoculum volume corresponding to 48 h fermentation time before declining. the cross product order effect of fermentation time and inoculum volume was significant up to 55 h before linear effect of fermentation time took over, being maximum at 72 h. effect of inoculum volume on cycanide reduction was due to heavy microbial concentration, which favoured fermentation. on removing the non significant (p>0.05) terms and recomputing, the polynomial equation becomes: ypaste =– 3.46985 – 0.8687x1 2 – 0.01917x1.x3 (8) the model contributed 68.68% of the total variation in cyanide content of lafun paste. sensory evaluation table 6 shows sensory evaluation of the lafun paste. one significant observation in this study is that all the lafun paste samples were acceptable to the panelists for the sensory attributes evaluated. the samples visually looked alike but for the experience of the traditional lafun consumers. differences in sensory data were not statistically significant (p >0.05). panelists had been used to consuming traditionally processed cassava lafun pastes, which are odourous, stickier, and darker in colour than products of this experimentation. they describe lafun pastes with little or no odour, having a characteristic white colour and good texture as good quality lafun (oyewole and afolami, 2001). perhaps loss of volatile products of fermentation during decanting and subsequent cooking operations reduced the offensive smell of the product. conclusion optimization of process variables to maximize cassava lafun yields, colour and sensory attributes or reduce cyanogenic potentials without large changes in the operational parameters was achieved in this study. the process variable condition that gave maximum cassava lafun flour (82.68%) and starch (68.24%) yields were 2.0 l, 36 h and 41c (inoculum volume (l), fermentation time (h) and drying temperature (c)). under these variable condition, cyanogenic potentials reduced by 95.30% in the flour and 97.28% in the paste from its 79.80 mg hcn/kg in the raw cassava pulp (nr8082 clone). since all lafun flours were visually white and the corresponding pastes generally liked by sensory panelists, the optimum nwabueze and odunsi 008 table 6. sensory evaluation of lafun paste* run colour flavour texture taste overall acceptability 1 7.3±0.25 ab 7.3±0.37 a 7.5±0.44 a 7.6±0.22 a 7.6±0.23 a 2 6.9±0.38 ab 7.00±0.44 a 7.7±0.27 a 7.3±0.32 a 7.1±0.34 a 3 6.7±0.27 a 6.8±0.53 a 6.8±0.38 a 7.2±0.34 a 7.0±0.27 a 4 7.5±0.25 ab 7.3±0.42 a 7.9±0.24 a 7.8±0.22 a 7.7±0.24 a 5 7.0±0.24 ab 7.2±0.27 a 7.5±0.36 a 7.3±0.25 a 7.3±0.20 a 6 7.6±0.23 ab 7.8±0.13 a 7.2±0.23 ab 7.6±0.23 a 7.7±0.23 a 7 7.8±0.21 ab 7.5±0.13 a 7.3±0.46 a 7.7±0.23 a 7.9±0.23 a 8 7.3±0.31 ab 7.7±0.23 a 7.5±0.26 a 7.2±0.23 a 7.8±0.20 a 9 7.4±0.33 ab 7.3±0.31 a 7.7±0.28 a 7.6±0.30 a 7.4±0.28 a 10 7.0±0.30 ab 7.8±0.40 a 7.6±0.23 a 7.9±0.19 a 7.4±0.20 a 11 7.6±0.23 ab 7.7±0.22 a 7.7 ±0.31 a 7.7±0.31 a 7.7±0.24 a 12 7.8±0.21 ab 7.4±0.31 a 7.5±0.43 a 7.6±0.35 a 7.5±0.25 a 13 7.4±0.22 ab 7.6±0.20 a 7.1±0.51 a 7.7±0.28 a 7.3±0.33 a 14 7.3±0.32 ab 7.3±0.39 a 7.0±0.45 a 7.3±0.37 a 7.6±0.31 a 15 7.1±0.29 ab 7.4±0.32 a 7.5±0.41 a 7.4±0.28 a 7.4±0.23 a 16 7.6±0.16 ab 7.3±0.28 a 7.5±0.38 a 7.2±0.20 a 7.5±0.17 a 17 6.6±0.36 ab 7.2±0.33 a 7.6±0.23 a 7.4±0.43 a 7.6±0.32 a 18 7.3±0.26 ab 7.3±0.28 a 7.9±0.30 a 7.1±0.35 a 7.1±0.29 a 19 7.4±0.16 ab 7.3±0.36 a 7.4±0.32 a 7.2±0.26 a 7.3±0.34 a 20 7.3±0.10 ab 7.3±0.23 a 7.6±0.24 a 7.3±0.34 a 7.3±0.24 a 21 6.7±0.38 ab 6.7±0.32 a 7.1±0.32 a 7.0±0.36 a 6.9±0.42 a 22 7.6±0.19 ab 7.6±0.27 a 7.0±0.44 a 7.9±0.32a 7.8±0.20 a 23 7.9±0.17 a 7.0±0.16 a 7.9±0.45 a 7.7 ±0.32a 7.2±0.20 a * lafun flour cooked into paste by stirring in boiled water ready for consumption. process condition producing highest lafun yields in terms of flour and starch with low flour and paste cyanide concentration (2.0 l, 36 h and 41c), is recommended for scale up production when using cassava nr8082 clone, on the basis of yield, safety and acceptability. references badrie n, mellowes wa (1992). cassava starch or amylase effects on characteristics of cassava (manihot esculenta crantz) extrudate. j. food sci. 57: 103 –107. bokanga m (1992). mechanisms of the elimination of cyanogens from cassava during traditional processing. in: traditional african foods – quality and nutrition. a westby, pja reilly (eds.) international foundation for science, stockolm, sweden. bradburg gm, egan sv, bradburg jh (1999). determination of all forms of cyanogens in cassava roots and cassava products using picrate paper kits. j. sci. food agric. 79: 593 –601. brainbridge z (1994). case studies in cassava processing and cyanogens elimination. proc. fifth symposium. istrc–ab, 343 –347. cereda mp, mattos mcy (1996). linamarin: the toxic compound of cassava. j. venom. anim. toxins 2: 6 –12. crapiste gh (2000). simulation of drying rates and quality changes during the dehydration of foodstuffs. in: trends in food engineering. je lozano, c anon, e parada-arias, gv barbosa-canovas eds technomic publishing co, inc pennsyvania, usa. dicar ha (1993). the indigenous foods of the sudan: a study in african foods and nutrition. center for agriculture and biosciences international (cbs) publication, walling ford, uk. iita (1990). cassava in tropical agric: a reference manual. iita, ibadan, nig. pp. 87 –120. janssens m (2001). cassava (manihot esulenta crantz). in: crop production in tropical africa. rh raemaekers ed. dgic, brussels, belgium. larmond e (1977). laboratory method of sensory evaluation of foods. department of agriculture, canada publications, 1637:33. latunde–dada go (1997). fermented foods and cottage industries in nigeria. food nutr. bull. 18: 4. mathew g (2002).cell separation and softening of cassava after fermentation with a mixed culture inoculum. proc 12 th symp. istrc – japan, pp. 104 –105. meilgard m, civille gv, carr bt (1999). sensory evaluation techniques. 3 rd edition. crc. press, boca raton, florida. ngoddy po (1989). processing and storage of cassava. proceedings of a course held at university of nigeria nsukka, 10 –23 april. 1988. dotam publications ltd. pp. 51-57. numfor fa (1983). an improved technique of processing cassava fufu. proceedings of 2 nd symposium. istrc –ab, 11 –113. okolie pn, ugochukwu en (1988). changes in activities of cell wall degrading enzymes during fermentation of cassava (manihot esculenta crantz) with citrobacter freundii. j. sci. food agric. 44: 51 –61. onabolu ao (1989). cassava utilization and nutrition. proceedings of cassava held at the university of nigeria nsukka, 10 –13april, 1988. dotam publication limited. pp. 90 –95. oyewole ob (1994). upgrading traditional cassava fermentation through process improvement. proceedings of 9 th symposium. istrc – ab. pp.261 –266. oyewole ob, afolami oa (2001). quality and preference of different cassava varieties for lafun production. j. food technol. afr. 6: 27–29. oyewole ob, odunfa sa (1990). characterization and distribution of 009 afr. j. food sci. res. lactic acid bacteria in cassava fermentation for fufu production. j. appl. bact. 68: 45 –52. oyewole ob, ogundele sl (2001). effects of length of fermentation on the functional characteristics of fermented cassava fufu. j. food technol. afr. 6: 38-40. pike oa (2003). colour in food analysis. 3 rd edn. kluwer academic hoenin publisher, ny. ruguo hu (1999). food product design: a computer aided statistical approach. crc press, boca raton, florida. stone h, sidel jl (1985). sensory evaluation practices. san diego. califonia: academic press, inc. p. 311. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (6), pp. 182-185, june, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the energy value and nutrient content of waterfufu and eru” consumed by cameroonians abia w. a. 1* , numfor f. a 2 , wanji s. 1 and tcheuntue f. 2 1 biochemistry programme, department of biochemistry and microbiology, faculty of science, university of buea. cameroon. 2 institute of agricultural research for development (irad) ekona, bp 25, swp cameroon. accepted 09 january, 2015 because of persistent reports of malnutrition in some parts of cameroon, a popular cameroonian food, “waterfufu and eru” was analyzed for its energy value and nutrient content in order to determine to what extent it satisfies recommended dietary allowances (rda). results showed that average serving size of the dish was 409 g. this amount provided 601 kcal of energy, 281 g of moisture, 82 g of carbohydrate, 30 g of lipids, 11 g of protein and 5 g of ash on fresh weight basis. micronutrients were found to be 1402 mg iron and 123 mg zinc. after due adjustment for absorption and bioavailability and considering the one serving size, constituted one of a three meal/day regime, although there was no significant difference (p > 0.05), lipid, zinc and iron contents of the dish satisfied rdas of all groups of consumers while energy value, carbohydrate and protein contents did not. this suggested that dependence of communities on this diet for supply of their daily needs may partly be responsible for the reported prevalence of malnutrition in these areas. it was suggested the food be improved upon through fortification and/or supplementation with the necessary elements. key words: malnutrition, cameroon, “waterfufu and eru”, rdas. introduction “waterfufu and eru” is a traditional dish popularly eaten in cameroon and neighbouring nigeria. it is made up essentially of a cooked cassava (manihot esculenta crantz) dough (“waterfufu”) and a vegetable soup (“eru”) whose major component is “eru” (gnetum africanum welw) leaves. the two components of the dish are prepared separately but served together (abia, 2003). previously the dish was associated with a particular tribe (the banyangs) as it was their staple meal. but in recent years, almost all ethnic groupings, especially those from the south west and north west provinces have learned to cook and eat “waterfufu and eru”. its popularity has spread to attain national dimensions (ewane, 2001). the dish forms a regular part of the menu in many households, parties, occassions and local restaurants, particularly in the forest zones of cameroon. the social, cultural and economic value of eru cannot be overstated (ewane, 2001; abia, 2003). in addition, ‘eru’ is one of the commonest vegetables eaten in cameroon today (ewa-ne, 2001). in some communities, it is not uncommon that the dish constitutes the three meals of the day in some *corresponding author. e-mail: wilfabia@yahoo.com. households (abia, 2003). because of the popularity and high frequency of consumption of the dish in some parts of the country, there is a need to know what contribution it makes to the well being of its consumers especially as malnutrition continues to be reported in these areas (numfor and noubi, 1995; horemans and jallow, 1997; ifrc, 2007). malnutrition affects every 1 in 5 children in cameroon, and was reported to have increased by 5.9% from 1991 (15.1%) to 1998 (21.0%), contrary to the country’s 1991 goal; to reduce malnutrition by 50% by the year 2000 (unicef, 2002; anbbia, 2002). more recent-ly, according to ifrc, (2007), the prevalence of malnutri-tion for the under fives stands at 27% and is most likely to increase. horemans and jallow (1997), documented that there are nutritional imbalances or deficiencies (in ener-gy, protein and iron) particularly in the forest regions of cameroon. similarly, ifeyironwa (2000), reported high level of anemia due to iron deficiency in cameroon. des-pite the apparent and reported malnutrition in the south west province of cameroon (horemans and jallow, 1997), no study has been documented on the impact of the popular diet “waterfufu and eru” on the nutrition and health of its consumer population. therefore, this paper reports the nutritive value of the dish and within estimated limits of absorption, the extent to which it satisfy the recommended daily energy and nutrient requirements of abia et al. 183 table 1a. percentage contribution of “waterfufu and eru” to rdas of children (7-10 years) and pvalues. issue lipid (g) protein(g) carbohydrates (g) zinc (mg) iron (mg) energy value (kcal) diet 25.8 9.6 69.8 6.2 70.1 511.4 rda 53.0 34.0 309.0 10.0 23.0 1900.0 %rda 48.7 28.2 22.6 62.0 304.8 26.9 p-value 0.4400 0.7863 0.5363 0.1995 0.7275 three categories of its consumers namely children 7 10 years, adult females 19 50 years and adult males 19 50 years. materials and methods the research was carried out in the south west province of cameroon based not only on the apparent and the reported malnutrition in the province (horemans and jallow, 1997) but also due to the high dependence on the diet, “waterfufu and eru” (abia, 2003). food samples were purchased randomly from amongst some previously surveyed restaurants in 5 communities (limbe, tiko, buea, muyuka and kumba) in the province, labeled and carried to the chemistry analytical laboratory of the institute of agricultural research for development (irad), ekona regional centre, buea, south west province cameroon where the average normal serving seize was determined and the samples processed for subsequent analyses. the analyses included determination of moisture content by drying samples in a draught oven at 105 o c until a constant weight was obtained. on dry weight basis, lipid content was obtained by hexane extraction using soxhlet apparatus and later concentrated using rotavapour and allowed overnight for complete evaporation of the solvent, after which weight was taken. total nitrogen content was determined using an adapted colorimetric method of the analytical chemistry laboratory of irad, ekona, buea (modified aoac, 1990): 0.03 g of re-dried samples were placed in to separate 100 ml tecator tubes, followed by addition of a quarter fraction of kjeldahl tablet, 6 drops of distilled water, and 2 ml of concentrated sulphuric acid. the contents of the tubes were swirled gently for a few seconds to mix. the tubes were then arranged in a rack and the rack was placed in a tecator block digestor (maintained in a fume cupboard) and its temperature raised gradually to 370 o c and let to digest for 70 min after which the digest was let to cool in the fume hood. thereafter, 50 ml of distilled water was added into each tube, swirled gently, its content made up to the 100 ml mark with distilled water, re-swirled gently and allow-ed to stand overnight for colorimetric determination of nitrogen. for colorimetric determination of nitrogen, 1 ml of each sample digest was pipetted into a fresh test tube and added 6 ml of buffer solution (50 g of sodium potassium tartrate and 26.8 g of disodium hydro-gen phosphate was dissolved in distilled water. 54 g of sodium hydroxide was dissolved in the above and made up to the 1000 ml mark of the volumetric flask with distilled water), 4 ml of sodium nitroprusside-sodium salicylate (150 g of sodium salicylate and 0.3 g of sodium nitroprusside were dissolved in distilled water and made up to mark of a 1000 ml volumetric flask), and 1 ml of sodium hypochloride solution or bleach solution (3 ml of commercial hypo-chlorite solution (10 12% cl) was diluted in distilled water to 100 ml). these tubes were shaken to mix and allowed to stand at ambient temperature for 45 min for complete colour development to occur. thereafter absorbance was read at 650 nm and the percenttage nitrogen calculated as: nitrogen (%) = standard factor (f) x a650nm. the standard factor (f = 12.081) is a value calculated from the amount of dry sample used and the volumes of the standards used for colorimetric determination. hence the total protein (%) was obtained by multiplying total nitrogen (%) by 6.25 (standard converting factor from nitrogen (%) to protein (%), total carbohydrate (and fibre) content of the dish was determined by difference (subtracting the sum total of lipid, protein and ash from total dry matter content), meanwhile ash content was determined by incineration in a muffle furnace at 550 ° c for 4 h, allowed to cool and weight taken. the micronutrient contents analyzed included zinc, and iron, which are currently a major concern in cameroon and the developing world in general (barbara, 2000; mi and unicef, 2004; hotz and brown, 2004), both using atomic absorption spectrophotometer (aas). considering that due to anti-nutritional factors as well as body stores of each of the nutrient, not all the amount of the food consumed may be assimilated, assimilability of each nutrient in the diet, “waterfufu and eru” was estimated based on the dietary components (latham, 1997). they were then compared with the food and agriculture organization (fao)’s recommended dietary allowances (rdas) (latham, 1997) of children 7 10 years, adult females 19 50 years and adult males 19 50 years (by multiplying the ratio of each nutrient content to its corresponding rda by 100, so as to express the adequacy of each nutrient as a percentage of rda it provides, and further statistically concluding whether the contribution made to rda is significantly different from the requirement or not), after which some necessary recommendations were made. results the “waterfufu” component of the dish which is a product of a starchy staple cassava tubers, is undoubtably the major source of carbohydrates, while the “eru” soup which is a combination of shredded dark green eru leaves and other ingredients (pepper, red palm oil, crayfish, magi and cowskin mainly), is the ultimate source of lipid, protein and the minerals zinc and iron. considering that eru (gnetum) leaves, a dark green leafy vegetable, forms about 85% of the quantity of “eru” soup, it is most probably responsible for the rich micronutrient and even protein contents of the soup (grivetti and ogle, 2000). table 1a, 1b and 1c respectively), present -within the estimated limits of assimilabilitythe extent (percentage) to which nutrient contents in the whole diet contribute to to which nutrient contents in the whole diet contribute to the recommended daily energy and nutrient requirements of major consumer categories (children 7 10 years, adult females and males 19 50 years), as well as their p values. on the basis that “waterfufu and eru” form one of three meals per day in the forest regions of cameroon a one third contribution or more ( 33.33%) of a nutrient a table 1b. percentage contribution of “waterfufu and eru” to rdas of adult females (19-50 years) and p-values. issue lipid (g) protein (g) carbohydrates (g) zinc (mg) iron (mg) energy value (kcal) diet 25.8 9.6 69.8 6.2 70.1 511.4 rda 62.0 49.0 359.0 12.0 48.0 2210.0 %rda 41.6 19.6 19.4 51.7 146.0 23.1 p-value 0.6596 0.4109 0.4030 0.3650 0.5960 table 1c. percentage contribution of “waterfufu and eru” to rdas of adult males (19 50 years) and p-values issue lipid (g) protein (g) carbohydrates (g) zinc (mg) iron (mg) energy value (kcal) diet 25.8 9.6 69.8 6.2 70.1 511.4 rda 81.0 55.0 470.0 15.0 23.0 2895.0 %rda 31.9 17.5 14.9 41.3 304.8 17.7 p-value 0.9519 0.3301 0.2405 0.6702 0.3375 to its recommended daily allowances was considered satisfactory or adequate. hence lipid, zinc and iron contents of the diet were observed to be in adequate amounts as each satisfies more than one third of their respective nutritional needs of the major consumer groups, while protein, energy and carbohydrate did not provide up to one-third of the rdas of any of the considered population. however, no statistical difference (p > 0.05) was observed in either case. although failure to satisfy the recommended dietary allowances does not in every case mean deficiency, however it is an indication of the likely hood and that the further contributed amount of any nutrient is below the rda of a target category, thus deficiency is most likely. discussion a fundamental strategy to ensure the well-being of a community with frequently reported malnutrition is the food-based approach which involve finding out the role of popular foods on the nutrition and health of its consumers. this was done by assessing the energy value and nutrient contents of a popular food (“waterfufu and eru”) in cameroon, whose communities in the forest zones in particular, highly depend upon. thereafter, estimated assailable nutrient amounts were compared with the rdas of major categories of consumers, and necessary conclusions and recommendations made. the observed energy value and nutrient contents of the “waterfufu and eru” were obvious based on earlier reports on their respective raw material contents: cassava and green leafy vegetables (bokanga et al., 1990; oshidi, 1992; mialoundama, 1993; numfor and noubi, 1995; ejoh et al., 1996; okafor et al., 1996; chavez et al., 2000; grivetti and ogle, 2000; danso et al., 2001, sango, 2007) . furthermore, it corroborates with the report of abia, (2003), who reported that eru leaves in eru soup generally contributes at least 60% of each of the nutrients in the soup. thus the whole diet, “waterfufu and eru”, is balanced in terms of the various nutrients. its energy value and nutrient content can be constituted into a food value table amongst others and made to the awareness of the public to probably appropriate diets of the population. however, with the knowledge that not all that is eaten is bio-available, the nutrient contents were adjusted for assimilability based on the dietary components: starchy roots, vegetables and little -or in most cases noanimal protein. thus, assimilability factor of each macronutrient (lipid, protein and carbohydrate) was similar to the factors of latham (1997), and that of the micronutrients (zinc and iron) correlated with those of who, (1996) and latham (1997) respectively. the comparison on table 1 provides relevant information which probably serves as an indicator of the inadequacy of the diet and the risk of nutritional pathology amongst individuals who depend predominantly on the food. it reveals that, considering the diet as one of three meals of a day –although it is not uncommon that it is consumed as the three meals of a day in some parts of the forest zones in cameroon (abia, 2003)-, despite its seemingly balanced nature it does not in all cases satisfy the rdas of the major consumer populations. these corroborates with the findings of horemans and jallow (1997). similarly, protein-energy malnutrition is very like probably due to dependence on the diet which is predominantly made up of starchy staple and very little animal protein (numfor and noubi, 1995). nevertheless, giving that the contributions made by the diet to rdas (whether they satisfactory or not) are not significantly different from the assumed one third part (33.33 %) of rda of each nutrient content, and also due to the fact that rdas are a range of safe values and not requirements, failure to provide recommended amounts of some nutrient contents is not conclusive of deficiency. that not withstanding, failure of a diet to provide the rda 184 afr. j. food sci. res. abia et al. 185 of its consumers is a vital indication of risk of nutritional deficiency and synergistically infections. thus, the further the nutrient contents are below the rda, the higher the risk of malnutrition and associated infections. therefore, the apparent and reported malnutrition in the province may be associated with the predominant dependence on this popular food, “waterfufu and eru” for proper nutrition and health. conclusion “waterfufu and eru” contain the necessary nutrients. energy and nutrient contents of a typical serving of the dish in restaurants in the forest zones of cameroon does not in all cases satisfy the recommended daily energy and nutrient allowances of major categories of consumers. though, there is no significant difference (p > 0.05) between the amount of nutrients provided and the rda for these nutrients, nutritional imbalances or deficiencies are still very likely to occur in communities which depend predominantly on “waterfufu and eru”. recommendation further studies on the absorption and bioavailability of the nutrients, including vitamin a of “waterfufu and eru” are necessary. to meet protein allowances, inter-alia, more meat and less cow skin (“nkanda”) should be used in the “eru” soup, and/or diet fortification and supplementation, measures to conserve nutrients during processing and preparation, as well as the findings of this study should be made to the awareness and understanding of the population. references abia wa (2003). energy value and nutrient content of “waterfufu and eru” (a popular cameroonian food). m.sc. research thesis, faculty of science, university of buea, cameroon. anb-bia (african news bulletin) (2002). cameroon: children under threat from malnutrition, anb-bia supplement issue/edition nr p. 442. aoac (association of official analytical chemists) (1980). officail methods of analysis. washington d.c. 13 th edition. barbara ua (2000). overcoming micronutrient deficiencies in developing countries: is there a role for agriculture? food and nutrition bulletin. the 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online at www.internationalscholarsjournals.org © international scholars journals full length research paper the impact of rural agricultural development projects on agricultural products in preferred area of benin kashez asiamah, awuah nulty and kubby j. department of agricultural and resource economics, university for development studies, tamale, ghana. accepted 22 january, 2014 in this study, data collected from 120 rural households located in two distinct socio-cultural locales of benin was used to assess the impact of 20 development projects on agricultural productivity. a ‘withwithout’ approach of impact evaluation is followed using anova and econometric regressions. results reveal no significant differences of projects on agricultural productivity between participants in the two study zones. econometric regression estimates show significantly positive impacts on agricultural productivity for two selected project indicators in the two study zones. however, the goal achievement index was more remarked in the adja area, where the projects were found to have better addressed development problems and provided higher impact. the results suggest the need to improve management of agricultural projects to enhance their impact. likewise, objectives and activities of the projects should be oriented to deal better with development problems of rural people, in particular those of the poorest and marginalized communities. key words: productivity, rural projects, impact, benin. introduction most less developed countries depend on rural areas for most of their survival and development resources. agriculture, which is the main activity of these areas, employs between 70% and 80% of the working population. besides, local rural populations ensure food consumption from agricultural production, thereby guaranteeing household food security (world bank, 2008). in spite of this key role of rural areas in development, they are confronted more and more with severe problems, which retard their progress with consequent problems for livelihood survival of the local people. natural resources such as land, forest and water do not stop degrading gradually. the decline in land fertility has resulted in decreased agricultural productivity. for decades, developed countries, international corresponding author. e-mail: kashez360@yahoo.com institutions such as the world bank, fao, undp, as well as non-governmental organizations ngos) have been fighting ceaselessly to contribute to the development of rural areas via actions and interventions implemented through rural development projects. such projects have often been designed, planned and implemented to help the rural people to develop their agriculture and to have better access to farm inputs to increase their income. in addition, most projects tend to strengthen the capacities of rural farmers through education, training and institutional support. given the key role that project interventions play in the rural development process, it is imperative to assess both the specific and overall impacts of implemented projects. several approaches to evaluate rural development projects have evolved over time. according to kirkpatrick (1994), various appraisals of most projects have focused on cost-benefit or cost-effectiveness approaches by assessing project costs (monetary or non-monetary), in particular, their relation to alternative uses of the same http://www.internationalscholarsjournals.org/ kashez et al. 051 resources and to the benefits being produced by the projects. however, some outputs of rural development projects such as capacity building and improvement of food security are sometimes difficult to measure and/or provide unsatisfactory results via the cost-benefit approach. indeed, the decisive issue of a measure of project success is not whether the planned results have been achieved, but what impact the activities of the project have provided and whether they satisfy all the stakeholders. consequently, project evaluations in recent times have focused on the impact evaluation approach, whereby project success emphasizes more broadly on whether the project had the desired effects on individuals, households and institutions and whether those effects are attributable to the project intervention. accordingly, evaluating the impact of rural development projects on agricultural productivity becomes a challenge to deal with (gtz, 2008). in evaluating projects, the central problem is how to isolate and to estimate their impacts on target groups. since many other exogenous factors that are not related to project execution (government policy, market conditions, former experiences, etc.) also have an influence on target groups’ evolution, appraisal approaches of projects seem to be difficult. the literature proposes two main approaches with different concepts of measurement: the ‘before-after’ and ‘with-without’ approaches as illustrated in figure 1 (bauer, 2000) . according to kerr and kolavalli (1999) and adekambi (2005), if the ‘with-without’ approach is designed in a consequent way to isolate the exogenous influences and to carry out the project impact only; it may provide more reliable results. this paper therefore focuses on the use of the ‘with-without’ approach to estimate the impact of rural development projects on agricultural productivity of farmers in two regions of benin. review of impact of projects on household livelihood various authors have in the past focused on impact evaluation of development projects on sustainability. in central america for example, a number of projects have promoted soil conservation or soil recuperation technologies that benefitted farmers through increase in productivity (brunch, 2001). likewise, doppler and bothe (1999) showed that the adoption of cassia siamea in rural benin improved soil fertility and agricultural productivity and led to an increase in the overall family income. this helped to reduce poverty of many rural farming households. as a result of increase in productivity and income due to the adoption of new technology for bean growing in south-benin, allogni et al. (2008) found that food expenditure increased and food security in households showed some improvement. data from (2006) also show that, increases in agricul agricultural productivity and income over the years due to rural development projects have undoubtedly raised food availability and kept food prices low, providing critically important benefits for extremely poor households that spend more than half their income on food (kerr and kollavali, 1999). arguing in the same way, ifpri (2001) reported that the project “improving food security in bangladesh” implemented since the 1980s resulted in a significantly increased availability of and access to food in rural areas of bangladesh. in countries where starvation is disastrous for rural people, various projects are implemented to avoid malnutrition diseases and death, mainly among children. for example, (idrc, 2003) found that 30 projects implemented in ethiopia that focused on agriculture and water management saved more than 25% of rural communities from starvation, malnutrition diseases and death. the foregoing discussion shows an optimistic view of the adoption of technology leading to poverty alleviation through positive effects on consumer food prices, producer incomes and labourer waged incomes. in this scenario, higher productivity, better natural resource management and poverty alleviation are mutually reinforced and may lead to achievement of a sustainable food system (winkleman, 1998). in contrast to this optimistic point of view, the pessimist sees the overall process of project implementation and technology adoption in agriculture biased towards wealthy people so that the poor are made worse off. the rich get richer while the poor get poorer resulting in social unrest and a decidedly unsustainable food system. the key relationship according to this framework is that technologies, policies and institutions are biased in favor of wealthy farmers who have unequal access to assets to begin with. their income rises when they adopt the improved technologies while the income of non-adopting farmers fall, many agricultural workers are displaced and some of those who remain, suffer from overexposure to poisonous chemicals (winkleman, 1998; kerr and kolavalli, 1999). finally, in impact, evaluation of a rural development project, another decisive discourse regarding success is whether the impacts are maintained after the project is completed, or in short, whether the project is sustainable (gtz, 2008). sustainability is seen as a result of the impact on sustainability of the production system where the project is implemented and of a long-term duration of the impact, even after the termination of the projects. typically, sustainable projects are those designed and financed to build local capacities and to develop the ability of local people to manage and utilize the development activities themselves, that is institutional and empowerment supports (clayton et al., 1998; uphoff, 1989; mcallister, 1999) . the capacity building is particularly viewed as very important for sustainability and many institutions such as gtz, world bank, undp, etc. c indicator b impact(income) a beginning of the project figure 1. illustration of project impact (bauer, 2000). have directed their supports towards more technical assistance to achieve better capacity building of local people (rudovist and woodford-berger, 1996; gtz, 2008). methods and analysis theoretical and empirical modelling methods for impact evaluation provided in the literature include systematic comparison, indicator trend function, econometric models and more complex system modelling (bauer, 2000; yabi, 2004). to estimate direct changes in agricultural productivity when the participation in project changes, the study focussed mainly on econometric models to evaluate the impacts. supposing that ip is an index of projects and y represents agricultural productivity, then if a unit change in index ip induces  unit change in y, then:  y /  ip   (1) by taking an integral of both sides of the equation (1), we obtain:  y dip   dip (2)  ip it is evident from equation (2) that y can be a function of ip and the general mathematical form of the regression model is expressed as: y= f(x1, …, xn, ip, z1, …zm, ) (3) where,  is the error terms supposed to be a n(0, 2 ), the x1, …, xn are production inputs and z1, …, zm other explanatory variables such as economic, social or human capital variables, etc. considering its computational ease and extensive use in many studies, the cobb-douglas functional form is applied to equation (3) to estimate the impacts of participation in projects on agricultural productivity. the empirical model estimated in this paper can be 052 afr. j. food sci.res. without the project with the project status quo time evaluation specified as: ln( yi )    1 ln(landi )  2 ln(labori )  3 ln(capi i )  ipi  1proi 2 agei  3sex i  4 edu i  5 alph i  6ten i  i (4) where: 1n(.) = the natural logarithm; i = the i th farmer; yi = the value of agricultural productivity expressed in fcfa/ha(fcfa is the local currency for benin. 1 =655 fcfa ). the productivity is computed for major cultivated crops such as maize, cotton, cassava, nuts, beans and yams. land is the overall cultivated area in hectare (ha) while labour, the total family labour used is expressed in man-days per ha and capital, the total capital used in fcfa per ha. the total capital is calculated as the total amount of input expenditures (seed, fertilizer, pesticide, hired labour, etc.). pro is a dummy variable representing the project type; pro = 1 if the project is integrated and 0 if it is single activity project; age is the age of the farmer (year); sex is a dummy variable expressing the sex of the farmer; sex = 1 for a man and 0 for a woman; edu is a dummy education variable; edu =1 if the farmer is formally educated and 0 if not. alph is a dummy informal education variable; alph = 1 if the farmer had received informal education and 0 if not; ten is a dummy variable of land tenure; ten = 1 if the cultivated land is secured and 0 if not. socioeconomic and demographic variables that were essentially measured in this study as dummy variables can accordingly not be logged. the ip are indicators of agricultural projects at a beneficiary level. by the nature of the approaches employed during the project implementation in the study zone, most local people had the opportunity to participate in several projects at the same time without any restrictions. in order to appreciate the presence of the projects at a beneficiary level, two indicators, relative to the projects in which each stakeholder was involved, were computed, namely: contact index (ic) and goal achievement index (is). the ic is expressed as the sum of contact frequency at stakeholder level and kashez et al. 053 mathematically defined as: if the farmer i was involved in no project ici = 0 n if farmer was involved in n f ki projects; n = 1, 2, …, p k 1 where; ici represents the contact index of stakeholder i, fki the frequency of contact this stakeholder i made per week with the team of the k th project, n the number of projects in which he participated. as defined, the contact index considers only the frequency of contacts with the beneficiaries. it fails to take into account success in achievement of activities that were implemented through projects. conversely, the goal achievement includes the overall success in achievement of objectives and activities of the projects, and thus computing its index could help in appreciating these aspects at the beneficiary level. as suggested by sarbeck (1994), the utility value of the projects can be defined as: 1 (6) ua  gi * gai g i where; ua is the utility value with 0<ua<1, gi the grading or weight and gai the achievement of goal i. by introducing in equation (5) the utility value computed in equation (6), it is possible to define the goal achievement index (is) as: y / y y  ic   ic ic ic ic y ic ic / ic y ic y (8) y / y y is is  is  (5)   is y   is is is / is y y is (9) where; ic and is represent respectively the parameters of ic and is in equation (4), ic and is their means computed from the study sample. the elasticities ic and is gives the percentage of variation in the impact due to 1% variation in the project indicator. study area and data datas were collected from two distinct socio-cultural locales of benin (adja and nagot), where 20 selected agricultural projects were implemented. for the ‘with-without’ approach, kerr and kolavalli (1999) and pitt and khandker (1996) suggested the randomisation of the “with” and “without” groups selected for the study. according to the authors, if the sample is not drawn randomly, or if there are hidden relationships determining between relationships of interest, the findings will be biased, that is, the statistics estimated for the sample will not represent those for the entire population. therefore, representative samples of agricultural households were chosen by randomisation according to the number of projects in which they were involved. however, three stages of stratification were distinguished for prior to the final random sampling. in each socio-cultural area of the study zone, three villages were selected: one without a project, one with a single project and one with 2 or more projects. on the basis of this typology, three categories of households were identified for the study. the first concerned the “without project” group of households which had not participated in any development project. the second category involved “with one project” group of households that had participated in a single project. the third category of households was the “with two or more projects” group of households who had 0 isi = n ua k . f ki k 1 if the farmer i was involved in no project (7) if farmer was involved in n projects; n = 1, 2, …, p participated in two or more projects. in each of the identified villages, twenty (20) households were selected at random from each group. in total, a sample size of one hundred and twenty (20 × 3 × 2 = 120) was drawn for the study. as much as possible, the intensity of participation in agricultural projects remained the key criteria for selecting respondents for the randomisation between the two groups in each socio-cultural area. data collected related to outputs, inputs such as labour, fertilizer, pesticides, etc. and socioeconomic and demographic characteristics of producers (sex, age, education level and frequency of participating in projects). where; isi represents the goal achievement index of stakeholder i, uak the utility value of the kth project in which he was involved as defined in equations (6). the fki and n are defined as in equation (5). the i are the error terms and the , ,  and  are parameters to be estimated.  is the origin ordinate, the  gives the elasticities of the productivity with respect to the corresponding farmsupplied factors {(excluding land(since the productivity is the output per unit of land, the parameter of land represents the return to scale)} and the  the percentage increases in productivity in response to a unit increase in the related variables. the  are estimates of project indicators. according to a hypothesis of positive impact of agricultural projects on agricultural productivity, they are supposed to be positive and significant. from equation (4), the elasticity of productivity ic and is with respect to ic and is can be thereby computed as: results descriptive statistics of variables used in the models the descriptive statistics showed on one side low indexes of contact (ic) with no significant difference for the two areas of the study zone. the fact is that the projects’ teams did not work with the stakeholders as frequently as supposed. at the same time, the farmers did not also respect the appointments that they got from the projects’ teams to work together. as a consequence, the effective frequency of meeting with the project teams 054 afr. j. food sci.res. table 1. descriptive statistics of variables used in the models. qualitative variables adja area (n = 60) nagot area (n = 60) count (%) count (%) type of project not integrated 23 57.5 14 35.5 integrated 17 42.5 26 64.5 total 40 100 40 100 sex woman 1 1.67 13 21.67 man 59 98.33 47 78.33 total 60 100 60 100 education no 37 61.67 26 43.33 yes 23 38.33 34 56.67 total 60 100 60 100 alphabetisation no 38 63.33 34 56.67 yes 22 36.67 26 43.33 total 60 100 60 100 land security no 16 26.67 4 6.67 yes 44 73.33 56 93.33 total 60 100 60 100 ratio variables means variation coefficients (%) means variation coefficients (%) age (year) 39.77 26.54 41.63 33.13 contact index (ic) 1.6 8.7 1.6 5.6 goal achievement index (is) 0.54 20.4 0.49 20.41 cultivated areas (ha) 0.65 54.00 2.81 67.45 family labour (man-day/ha) 86.71 54.56 63.88 51.68 capital used (fcfa/ha) 44,328.64 45.81 71,914.5 38.82 productivity (fcfa/ha) 178,925.29 39.84 162,578.63 29.00 was lower than expected during the project design and the planning phase such that the indexes were as well low. besides, the indexes of goal achievement (is) were lower than those of contact. this result may be explained by a very poor goal achievement of these projects. conversely, the percentage of secured land remained very high in the nagot area where lands were more available and fertile. consequently, access to and conservation of land did not seem to have been a previous (prior to project implementation of projects considered in this study) problem of farmers in this area. this situation might explain the lower agricultural productivity in the nagot area as compared to the adja (table 1). since nagot farmers found their soil to be still relatively fertile, they did not care much about modern cultural techniques diffused by the projects and continued to employ local methods of soil conservation. with regards the use of inputs, land pressure was higher in the adja area and agricultural households had an average of 0.65 ha of endowed land as compared to 2.81 ha in the nagot area. in order to facilitate the adoption of modern technologies, adja farmers utilized more family labour. however, they used less mineral fertilizers, pesticides and hired labour than farmers of the nagot area, though, they produced more intensively. the situation could be explained by improved availability of and access to inputs in the nagot area. indeed, by producing more cotton, the existing agricultural policy that guarantees availability of and access to agricultural inputs to cotton producers favoured them. however, pro t h o u sa n d s o f fc fa p er h a 240 180 outputs 120 variable costs gross margins 60 0 without project with 1 project with 2 or more (n = 20) (n = 20) projects (n=20) groups of participation figure 2. outputs, variable costs and gross margins (in thousands of fcfa per ha) of agricultural production according to groups of participation in projects in the adja area. producers of the nagot area had lower agricultural output than those of the adja area (table 1). groups of participation in projects and agricultural productivity the analysis (anova) of economic indicators of agricul-tural production showed no significant difference between their averages according to the groups of participation in projects in the two sociocultural areas. nevertheless, the farmers that participated in the projects had agricultural outputs higher than non-project participants. even though, the variable costs of production were higher for project participants, their gross margins were also better in comparison with farmers without project. additionally, the results demonstrated that farmers with a single project had higher gross margins than those with 2 or more projects (figures 2 and 3). this could be due to the fact that those with a single project were more efficiently involved in the project than those with 2 or more projects. in addition, the effects of projects would be counter-productive to each other instead of being complementary. these results challenge the positive effects of simulta-neously implementing several agricultural projects at the same place and as well the necessity for farmers to con-currently participate in such multiple projects at the same time. but, an absence of significant difference between the groups of a participa-tion in projects does not expli-citly imply similar conclusions on impacts of project. for example, the absence of observed differences noted in this study may suggest that there is no correlation between groups of participation and the intensity of participation in projects. the results also reveal statistically significant differences between the selected economic indicators in the two distinct locales investigated in this paper. specifically, agricultural outputs and gross margins were significantly higher in the adja area than in the nagot area (178,925.29 fcfa/ha against 162,578.63 fcfa/ha and 134,596.64 fcfa and 90,664.15 fcfa; figures 2 and 3). nevertheless, a comparison of economic indicators with respect to areas of implementation fails to give the desired level of the impact of the projects on agricultural productivity in each area. impact of the projects on agricultural productivity the results of the econometric models are generally statistically significant and overall satisfactory for the two socio-cultural areas (tables 2 and 3). the parameters of indicators ic and is are found in all cases to be significant and positive. the results from this study show that the projects had positive impact on productivity. indeed, they popularized and diffused modern production techniques, which allowed farmer beneficiaries involved in the project to significantly improve their productivity. enhanced capacity building resulting from training and group formation did strengthen the managerial capacities of producers. however, the regression results call for two types of analyses and interpretations. kashez et al. 055 056 afr. j. food sci.res. 240 180 fa p er h a 120 o f fc t h o u sa n d s 60 0 without project with 1 project with 2 or more (n=20) (n=20) projects (n=20) outputs variable costs gross margins groups of participation figure 3. outputs, variable costs and gross margins (in thousands of fcfa per ha) of agricultural production according to groups of participation in projects in the nagot area. table 2. estimated parameters of factors affecting agricultural productivity in adja socio-cultural area. independent variables model with ic model with is notations b statistic t b statistic t constant 9.402*** 14.882 6.112*** 3.799 family labour (man-day/ha) labour 0.120** 2.043 0.526*** 4.057 capital (fcfa.ha -1 ) capi 0.145*** 2.837 0.235* 1.802 cultivated area (ha) land 0.018 0.420 0.048 0.460 type of project pro 0.08 0.976 0.187 0.836 formal education edu 0.135*** 2.979 0.034* 1.968 sex sex -0.011 -0.163 -0.072 -0.424 age (yr) age 0.001 0.533 0.004 0.809 informal education alpha -0.081 -1.550 -0.071 -0.548 land security ten 0.251** 2.031 0.325 1.131 contact index ic 0.639** 2.314 goal achievement index is 3.616*** 5.648 adjusted r2 0.55 0.80 f statistic 9.17*** 23.96*** observations number 60 60 dependent variable y output (fcfa.ha -1 ) elasticity of productivity with respect to project indicators  1.02 1.95 *** significant at 1%; ** significant at 5%; * significant at 10%. kashez et al. 057 table 3. estimated parameters of factors affecting agricultural productivity in nagot socio-cultural area. independent variables model with ic model with is notations b statistic t b statistic t constant 7.230*** 14.420 5.519*** 8.170 family labour (man-day/ha) labour 0.080* 1.691 0.439*** 4.500 capital (fcfa.ha -1 ) capi 0.364*** 8.290 0.274*** 4.149 cultivated area (ha) land -0.026 -0.870 0.131* 1.909 type of project pro 0.119* 1.823 -0.146 -1.105 formal education edu 0.111*** 2.713 0.166* 2.166 sex sex 0.179 1.224 0.323 0.903 age (yr) age 0.003 1.331 0.009* 1.886 informal education alpha -0.017 -0.383 -0.054 -0.463 land security ten 0.051 1.060 -0.023 -0.195 contact index ic 0.448*** 3.247 goal achievement index is 2.181*** 4.455 adjusted r2 0.84 0.9 f statistic 36.15*** 54.9*** observations number 60 60 dependent variable y output (fcfa.ha -1 ) elasticity of productivity with respect to project indicators  0.72 1.07 *** significant at 1%; ** significant at 5%; * significant at 10%. firstly, the impact level varied according to indicator considered. the computed elasticities from regression coefficients reveal that a variation of 1% of ic induced a variation of 1.02 and 0.72% in productivity in the adja and the nagot areas, respectively. when considering only the goal is of the projects, a variation of 1% of ic induced a variation of 1.95 and 1.07% in productivity in the adja and the nagot areas, respectively (tables 2 and 3). these results show that is, which includes overall aspects of management and the achievement of objectives, provides the highest impact on productivity. in fact, besides the direct contact and working with beneficiaries, the projects helped their organizations to acquire organizational skills of inputs distribution and product commercialization. they also built rural infrastructures such as rural roads, informal education centres, hospitals, etc. these additional activities, which were considered in the goal achievement evaluation, also affected productivity indirectly. using the is in the regressions has helped to estimate full impacts of the projects on productivity of beneficiaries. secondly, the impacts were more pronounced in the adja area than in the nagot area (ic =1.02 for adja against ic =0.72 for nagot and is = 1.95 for adja against is = 1.07 for nagot; tables 2 and 3). these results confirm the findings of our descriptive statistics, which showed that agricultural outputs and gross margins gained by producers in the adja area are significantly higher than those of nagot area producers. as previously explained, lands in adja area had already reached levels of degradation with decline in fertility to such an extent that the adoption of modern production practices and techniques resulted in considerable positive effects on productivity. in contrast, since lands in the nagot area remained relatively fertile and more available soil nutrients for crop growth, the impacts of modern production practices popularized by rural development projects, although positive, stay even lesser. indeed, producers of the nagot area still extended their cultivated areas, instead of intensifying their agricultural production to sustain the management of natural resources. conclusion the positive impacts provided by is of the projects at the beneficiaries’ level suggest that a scenario of improvement on design, management and monitoring, which leads to better goal achievement of the projects may improve the impact. therefore, agricultural policy in benin should focus more on efficiency in designing, managing and monitoring rural development projects. in addition, the results show that the impact was strongly related to the locale where the projects were implemented. consequently, policy makers in benin sholud place emphasis on implementing projects at the level of small scale, in order to tackle effectively development problems encountered by target beneficiaries, in particular the poorest. likewise, as suggested by various authors cited in the literature review section, it is important to support the sustainability of the impacts by strengthening capacity of stakeholders and their organizations. thus policy implication, conception, design, implementation and monitoring of rural development projects should also include aspects related to capacity building of stakeholders such as: training, reinforcement of organisational and management skills, etc. these 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(eds.). ‘farming and rural system economics’. margraf publishers, weikersheim, germany 135p. african journal of food science research vol. 2 (3), pp. 065-070, march, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper a kinetic study in the changes of sds-page profile of whey proteins during storage at different temperatures tahmasebi e., moein bozorgi and keyvan khukhan school of health, environmental health research center, islamic azad university, tehran, iran. accepted 28 february, 2014 whey is the soluble fraction of milk separated from the caseins during manufacture of rennetcoagulated cheese, (sweet whey) or use of acids (acid whey). whey proteins are widely used in a variety of foods, primarily for their superior functional properties such as gelling and emulsification. solubility is the major protein attribute affecting all functional properties. in this study, the effect of storage at different temperatures on sweet and acid whey proteins solubility and gel electrophoresis pattern was investigated. acid whey was produced by adjusting the ph of pasteurized milk to 4.6 and sweet whey was produced by adding microbial rennet to the pasteurized milk held at 37°c for 1 h. whey samples were stored at -18, 4, 25, 40 and 60°c. at different time intervals, samples were removed and used for protein solubility determination and sds-page. results showed that the solubility of proteins of both whey types stored at -18 and 60°c significantly decreased, such that at -18 o c, 50 and 61% decrease for sweet and acid whey protein solubility, respectively, occurred after 6 weeks. these decreases were 44 and 55% for samples stored at 60°c for 5 days. the decrease in solubility at 4, 25 and 40°c was less drastic. at all temperatures, decrease in protein solubility followed a first order kinetic. the activation energies for loss of protein solubility were 6.3 to 6.4 kcal/mol for sweet and acid whey proteins. the sds-page patterns indicated significant denaturation and aggregation of major whey proteins during storage. key words: whey protein, solubility, kinetics, sds-page, activation energy. introduction whey is the soluble fraction of milk, rich in proteins, minerals and lactose, that is separated from the casein during manufacture of rennet-coagulated cheese or by adding acid to milk. the whey produced from rennetcoagulated casein or cheese is referred to as sweet whey whereas that from mineral or lactic acid-coagulated casein is called acid whey (corredig and dalgleish, 1996). whey proteins are globular proteins with molar mass ranging from 14 to 1000 kda and are composed of 60% β-lactoglobulin β-lg, 22% α-lactoalbumin α-la, 5.5% bovine serum albumin (bsa) and 9% immunoglobulins ((ig's) (cavallieri et al., 2007). the isoelectric points of these proteins are 5.2 for β-lg, 4.2 to 4.5 for α-la, 4.7 to 4.9 for bsa and 5.5 to 6.8 for ig's (bryant *corresponding author.e mail: mesabi_ahmed@yahoo.com and mcclements, 1998). heat-induced aggregation and gelation of whey proteins have been extensively studied (corredig and dalgleish, 1996; bryant and mcclements, 1999; livney et al., 2003). it has long been known that when temperatures are elevated above room temperature, β-lg dissociates from a dimer to a monomer, exposing its thiol group and interior hydrophobic residues, enabling thiol/disulfide exchange reactions. the hydrophobic, thiol-disulfide and electrostatic interactions induced by thermal aggregation have since been examined under various conditions for β-lg and α-la (alting et al., 2003; havea et al., 2004). it has been reported that the molecular masses of aggregates are dependent on concentration, ph, ionic strength and temperature of heating (kazmerski et al., 2003). however, the complete mechanism of heat denaturation and aggregation of the isolated proteins is not entirely understood (kazmierski and corredig, http://www.internationalscholarsjournals.org/ tahmasebi et al 065 2003). whey proteins are valuable by-products from the cheese industry. they are used widely in a variety of foods primarily for their superior gelling and emulsification properties (solar and gunasekaran, 2010). in addition, whey proteins have the advantage of their high nutritive value and are generally recognized as safe (gras) (kresic et al., 2006; herceg et al., 2007). the physicochemical properties of whey proteins suggest that they may be suitable for other novel food and non-food applications (sun and gunasekaran, 2010). whey proteins are used as functional ingredients in a wide variety of food products because of their ability to modify the texture and stability of emulsions, foams and gels ((bryant and mcclements, 1998, 1999; cavallieri et al., 2007; solar and gunasekaran, 2010; herceg et al., 2007). the development of foods with improved quality depends on improving our understanding of the molecular basis of protein functionality. one important functional property of proteins is their water solubility. the protein solubility is a function of many factors, such as the native or denatured state and environmental factors (that is ph, temperature) (lieske and konrad, 1996; guyomarc et al., 2003; floris et al., 2008; damodaran, 2008). the ph of the solution affects the nature and the distribution of the protein's net charge. generally, the proteins are more soluble at low (acids) or high (alkaline) ph values because of the excess of charges of the same sign, causing repulsion among the molecules and consequently, contributing to its largest solubility. temperature is also a factor that influences protein solubility. protein solubility increases with temperature between 40 and 50°c. when the temperature of the solution is raised high enough for a given time, the protein is denatured (pelegrine and gasparetto, 2005; damodaran, 2008). majority of studies on whey protein solubility and functionality are focused on elevated temperatures. little data is available on the behavior of whey proteins at low temperatures. in this study, the changes in solubility and sds-page patterns of sweet and acid whey proteins during storage at low and moderately high temperatures was investigated. the kinetics of solubility loss was also studied. materials and methods preparation of sweet and acid whey fresh milk was obtained from a local animal husbandry center and pasteurized at 63°c for 30 min using a batch pasteurizer. acid whey was produced as follows: the ph of pasteurized milk was adjusted to 4.6 with lactic acid at 25°c. after 1 h the whey was pressed in a cheese cloth. whey samples were aseptically transferred to 45 ml plastic tubes and stored at -18, 4, 25, 40 and 60°c. at different time intervals, samples were analyzed for protein solubility and sdspage. sweet whey was manufactured using rennet (microbial metio rennet, metio sangyo co., ltd, japan), according to the manufacturer’s recommendations. the enzyme was added to pasteurized milk at 37°c and the milk was incubated at 37°c for 1 h. the coagulated casein was cut into 1 cm 3 cubes and the whey was treated as before. samples were stored at different temperatures, as described earlier. time of sampling was, for both acid and sweet whey, every week for samples incubated at -18°c, every 4 days for samples at 4°c, and every day for samples stored at 25, 40 and 60°c. solubility measurement protein solubility was determined by the method of inklaar and fortin (1969) with some modification. total nitrogen in each sample was determined by the standard micro–kjeldahl procedure (aoac, 2002). each sample was centrifuged using international centrifuge universal, model uv, usa, at 2700g for 10 min. the nitrogen content of the supernatant was measured and the solubility was calculated as: % protein solubility = nitrogen content of the supernatant х 100 total nitrogen in the whey sample sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds– page) the supernatants which were used for solubility measurement were also used for sds–page studies. slabs for a 10% sds-page were formed according to the discontinuous buffer system of laemmli (1970). each sample was diluted with sample buffer to give a final concentration of 1 mg ml -1 protein, 0.01 mol l -1 trishcl, ph 6.8, 0.4% sds, 10% glycerol, and 0.004% bromophenol blue. the running gel was made of 5 to 20% gradient acrylamide in 1.2 mol l -1 tris-hcl, ph 8.8 and 0.3% sds. the stacking gel contained 3% acrylamide in 0.25 mol l -1 tris-hcl, ph 6.8 and 0.2% sds. samples were heated in boiling water for 10 min and 50 µl was applied to each well. the electrode buffer comprised 0.025 mol l -1 tris-hcl, 0.192 mol l -1 glycine and 0.1% sds at ph 8.16. electrophoresis was performed at constant 25 ma. gels were stained with 0.25% coomassie brilliant blue in 50% acetic acid/25% methanol and destained with a 10% acetic acid/7.0% methanol solution. statistical analysis the protein solubility data was statistically analyzed by spss/pc software (version 17). one–way anova, independent–sample and paired student’s t tests were used for comparison of the means. the significant differences were set at p=0.05. results and discussion the functional properties of proteins are often affected by protein solubility, and those most affected are thickening, foaming, emulsifying, and gelling. insoluble proteins have very limited use in food. the solubility of a protein is the thermodynamic manifestation of the equilibrium between protein-protein and protein-solvent interactions (damodaran, 2008). the changes in the solubility of acid and sweet whey samples are shown in figure 1. the significant (p<0.05) decreases in the protein solubility values of enzymatically-prepared whey samples stored 066 afr. j. food sci.res. 100 100 80 a b s o lu b il it y ( % ) 80 s o lu b il it y ( % ) a 60 60 acidic 40 40 enzymatic 20 20 0 0 0 1 2 3 4 5 0 1 2 3 4 5 days days 100 100 c 80 d 80 acidic (% ) 60 (% ) enzymatic 60 s o lu b il it y s o lu b il it y 40 40 acidic 20 enzymatic 20 0 0 0 1 2 3 4 5 0 4 8 12 16 20 days days 100 80 e s o lu b ili ty (% ) 60 40 20 0 0 7 14 21 28 35 42 days figure 1. changes in the solubility of whey proteins stored at a) 60°c, b) 40°c, c) 25°c, d) 4°c and e) -18°c. acid whey sweet whey . for 5 days at 25, 40 and 60°c were 11, 28 and 44%, respectively. corresponding values for acid-prepared whey samples were 22, 39 and 55% (figure 1), indicating higher solubility of rennet whey proteins than those of acid whey samples. for samples stored under refrigeration condition (4°c) after 20 days storage, solubility significantly (p<0.05) dropped by 11.5 and 22.3% for sweet and acid whey proteins, respectively, (figure 1). a drastic decrease in solubility occurred at 18°c such that at the end of the study (6 weeks) a 50 and 61% reduction in solubility was determined for sweet and acid whey proteins, respectively (figure 1). examination of the data of figure 1, indicates that loss of solubility (and consequently) aggregation of whey proteins, followed a first order kinetics which is described by conventional first order kinetics according to the following equation (tsoublli and labuza, 1991): ln (rsol) = kt where rsol is the relative residual solubility (solubility at any time relative to time zero of storage) and k is the first order insolubilization rate constant which can be obtained from the slope of the regression of line of ln (rsol) tahmasebi et al 067 2 1.9 a s o lu b il it y 1.8 p e rc e n ta g e 1.7 l o g 1.6 1.5 0 1 2 3 4 5 days 1 s o lu b il it y b 0.8 0.6 p e rc e n ta g e 0.4 l o g 0.2 0 0 7 14 21 28 35 42 days figure 2. semi-log plot of changes in the solubility of whey proteins stored at a) 60°c and b) -18°c. acid whey , sweet whey . versus time. figure 2 depicts semilog plots of data of figure 1 (shown only for data at 60 and -18°c). from the slope of these curves, the rate constants at different temperatures can be calculated (table 1). the temperature dependence of the rate of insolubilization can be best represented by the arrhenius plot (tsoublli and labuza, 1991). activation energy was determined using the slope of straight line obtained by plotting log k against 1/t (figure 4). from this plot, ea for 25 to 60°c was calculated to be about 6.3 kcalmol -1 , for the sweet whey proteins and 6.4 for acid whey proteins in the temperature range studied. these values are significantly lower than ~ 60 kcalmol -1 reported earlier for β-lb in the temperature range of 75 to 120°c (galani and apenten, 1999). it can be seen in figure 4 that a biphasic situation exists and the ea at 4 and -18°c is very small. several authors have reported such biphasic arrhenius plot for βlg aggregation rates, and is believed to be the consequence of there being two distinct rate determining steps (anema and mckenna, 1996; galani and apenten, 1997, 1999). these ea low values obtained in our study indicate low sensitivity of the rate of insolubilization and aggregation of whey proteins to temperature under the conditions of this study. the reason for this difference is not known at present, but might be related to the difference in the temperature ranges used or possible 068 afr. j. food sci.res. 2.4 2.2 ea enz = 6339 cal 2 k 1.8 l o g 1.6 1.4 ea acidic = 6402 cal 1.2 1 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 (1/t)×10 3 figure 4. the arrhenius plot for the insolubilization of sweet and acid whey proteins, acid whey , sweet whey . 1 2 3 4 5 6 7 8 9 10 figure 3. sds-page profile of whey proteins at 25°c (10%). 1-5: enzymaticly prepared whey: 1 day 1; 2 day 2; 3 day 3; 4 day 4; 5 day 5. 6-10: acid prepared whey: 6 day 1; 7 day 2; 8 day 3; 9 day 4; and 10 day 5. changes in the aggregation mechanisms operating at freezing temperature (anema and mckenna, 1996; fennema et al., 2008). results of (figure 3) sds-page for whey samples stored at 25°c are shown in figure 2. similar results were observed for other temperatures. significant differences were seen between sweet and acid whey samples. slight differences among sds-page patterns in each whey type were observed during storage. this result seems to contradict the results of nsi which indicated loss of protein solubility during prolonged storage. as explained under materials and methods, electrophoresis was performed on the supernatants of samples after centrifugation. at this stage, it is possible that some proteins precipitated due to centrifugation and did not appear in the supernatant. the fact that sdspage results show little significant difference between samples is probably related to extraction method used for preparation of samples for electrophoresis. in this procedure ~50 µg protein was loaded into each well, therefore the concentration of protein in all wells was similar. when stored at 60°c several high molecular tahmasebi et al 069 weight bands started appearing from the third day of storage of the enzymatically prepared whey samples. in contrast, in acid prepared whey samples, several of the bands were lost and diffused. low molecular weight protein bands also began to appear, the intensity of which increased as the storage time increased from 1 to 5 days. the main proteins affected by heating at 60°c are β-lg and α-la, with approximate molecular weights of 18 and 14 kda, respectively (farrel, 1988). similar results, but less drastic, were observed for samples stored at -18, 4, 25, and 40°c. other investigators have reported similar results, but more significantly occurring at higher temperatures (bryant and mcclements, 1998; de kruif and tuinier, 1999; sava et al., 2005; cavallieri et al., 2007; jovanovic et al., 2007). the combined effects of thermal treatment and ph have profound effect on whey protein solubility. while native whey proteins resist aggregation at ambient temperatures for a wide range of ph's, slight heating at 70°c at various time intervals indicate a minimum solubility at isoelectric ph (~4.5) (zhu and damodaran, 1994). drastic drop in solubility of whey protein during frozen storage as observed in this study could produce profound effects on the structural and chemical properties of proteins. several mechanisms are believed to be responsible in altering the functional properties of proteins, including acceleration and formation of ice crystals (bhargava and jele, 1998; damodaran, 2008; strasburg et al., 2008), oxidation of sulfhydryl groups to disulfide bonds (shenouda, 1980; lee et al., 1992), and increase in the rate of chemical reactions due to the concentration effects of freezing, that is, macromolecoules are forced together, making interactions more probable with resultant denaturation, insolubilization, aggregation, and precipitation of proteins. taken together, the results of the solubility studies and sdspage indicate decrease in solubility of whey proteins during storage, even on refrigeration (4°c), ambient and moderately high (40 to 60°c) temperatures. changes in solubility at these temperatures are probably the result of several types of interactions, especially electrostatic and hydrophobic interactions between whey protein molecules, the latter playing a more important role (farrel, 1980; corredi and dalgleish, 1996; sava et al., 2005). β-lg is the main protein involved in these interactions fundamentally due to its high concentration (> 50% of whey proteins) (galani and apenten, 1999; cavallieri et al., 2007), and its rather unique self association to form dimers or higher ordered polymers at reduced temperatures (farrel, 1988). at higher temperatures, the sulfhydryl-disulfide exchange reactions are responsible for decrease in solubility, aggregation and precipitation of these proteins. also, α-la might participate in these interactions to produce high mw co-aggregates (farrel, 1988; de kruif, 1999; cavallieri et al., 2007). upon heating β-lg at neutral ph, the native dimers start to dissociate into monomer, leading to the exposure of the previously buried hydrophobic amino acids and the single free thiol group. above 78°c, this is accompanied by the aggregation of the β-l molecules as a result of hydrophobic interactions, with consequent decrease in protein solubility (sava et al., 2005). at the temperatures used, this phenomena does occur leading to some degree of decrease in solubility which is expected to affect the function of whey proteins in any food system. in conclusion, the result of this study indicates that whey protein solubility is affected by storing at different temperatures, however the degree of temperature dependence of this property seems to be low. since whey proteins are routinely used in different food systems, some degree of insolubilization is expected to occur during storage, particularly when frozen or stored for a prolonged time. it has been suggested that disaccharides and polymers (such as polysaccharides and polyethylene glycol) can stabilize proteins and protect them against denaturation (srirangsan et al., 2010). use of these additives for protecting whey proteins against insolubilization can be considered in future studies on whey protein properties. acknowledgement this study was financially supported by grants (no. 89gr-vt-11) from shiraz university research council. references alting ac, hamer rj, de kruif g, paques m, visschers rw (2003). number of thiol groups rather than the size of the aggregates determines the hardness of cold set whey protein gels, food hydrocoll., 17: 469-479. anema sg, mckenna ab (1996). reaction kinetics of thermal denaturation of whey proteins in heated reconstituted whole milk. j. agric. food chem., 44: 422-428. aoac (2002). official methods of analysis. association of official analytical chemistry. airlington. bhargava a, jele p (1995). freezing of whey protein concentrate solutions and its effect on protein functionality indicators. int. dairy j., 5: 533-541. bryant 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(ed}. food chemistry, 4 t h ed, marcell dekker, new york, pp. 923-73. sun c, gunasekaran s (2010). rheology and oxidative stability of whey protein isolate-stabilized menhaden oil-in-water emulsions as a function of heat treatment j. food sci., 75: c1-c8. tsoublli mn, labuza tp (1991). accelerated kinetic study of aspartame degradation in the neutral ph range. j. food. sci. 56: 1671-1675. zhu h, damodara s (1994). heat-induced conformational changes in whey protein isolate and its relation to foaming properties. j. agric. food chem., 42: 846-855. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (7), pp. 347-351, july, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the scientific basis for the traditional use of medicinal plants among the local people in nigeria nwinyi, obinna c. 1 *, chinedu, nwodo s. 1 , ajani, olayinka o. 2 , ikpo chinwe.o 2 and ogunniran, kehinde o. 2 1 department of biological sciences, college of science and technology, covenant university. km 10 idioroko road, canaanland, pmb 1023 ota, ogun state, nigeria. 2 department of chemistry, college of science and technology, covenant university, km 10 idiroko road, pmb 1023, ota, ogun state, nigeria. accepted 11 january, 2018 the upsurge in the prevalence of side effects of many synthetic antimicrobial agents and incidence of multidrug resistant bacteria has spurred scientists on the research for plant based antimicrobial of therapeutic potentials. ocimum gratissimum and piper guineense present such potential of high medicinal value. these plants are used in nigeria traditionally as condiments and for treatment of various ailments such as pyorrhea, dysentery and bronchitis. aqueous and ethanol extracts of o. gratissimum and p. guineense leaves were screened for antibacterial activity against escherichia coli and staphylococcus aureus. both extracts were found to exhibit selective inhibition against the isolates. the diameter zones of inhibition exhibited by the extracts were between 2 + 0.01 – 10 + 0.10 the minimum inhibitory concentration (mic) determined by the agar diffusion method was between 10.00 and 2.50 mg/ml -1 . ethanol extracts showed more inhibitory effect compared to the aqueous extracts. results obtained show that the extracts of o. gratissimum and p. guineense possess some level of antibacterial activities against e. coli and s. aureus. key words: ocimum gratissimum, piper guineense, antibacterial activity, minimum inhibitory concentration (mic), escherichia coli, staphylococcus aureus. introduction there is growing interest in exploiting plants for medicinal purposes especially in africa. this stems from the fact that microorganisms are developing resistance to many drugs and as such created situation where some of the common and less expensive antimicrobial agents are loosing effectiveness (montefore et al., 1989). herbal medicine which uses medicinal plants primarily presents as an alternative to such situation (sofowora, 1993). these medicinal plants have immensely contributed to the development of human health and welfare. concomitantly, there is an increase in data and huge patronage to *corresponding author. e-mail: nwinyiobinna@gmail.com. herbal products round the world (elsenberg et al., 1990; omoseyindemi, 2003). medicinal plants such as ocimum gratissimum and piper guineense have been asserted to provide various culinary and medicinal properties. these medicinal properties exert bacteriostatic and bacteriocidal effects on some bacteria. these effects have been attributed to the peptides, alkaloids, essential oils, phenols and flavonols which are major components in these plants (okigbo and igwe, 2007). o. gratissimum belongs to the family leguminocaeae, commonly known as “alfavaca”. it is naturally used in the treatment of different diseases which includes: upper respiratory tract infections, diarrhea, headache, conjunctivitis, skin disease, pneumonia tooth and gum disorder, fever and as mosquito repellants nwinyi et. al. 348 (onajobi, 1986; ilori et al., 1996; okigbo and mneka, 2006). o. gratissimum is found in the tropical and warm temperature regions such as india and nigeria (okigbo and ogbonnaya, 2006). some of the vernacular names in nigeria include: (ncho-anwu, ahuji) igbo, (efinrin,) yoruba, (aramogbo) edo and (daidoya) hausa (effraim et al., 2000). o. gratissimum has been described to have other species in the flora of tropical west africa. these include: ocimum viride linn, ocimum suave linn, ocimum basilicum linn and ocimum canum sims. mshana et al. (2000) reported their numerous medical uses. the ocimum oil has been described to be active against several species of bacteria and fungi. these include listeria monocytogenes, shigella, salmonella and proteus, for fungi trichophyton rubrum, trichophyton mentagrophytes, cryptococcus neoformans, penicillum islandi cum, and candida albicans (begum et al., 1993; nwosu and okafor, 1995; akinyemi et al., 2004; janine de aquino lemos et al., 2005; lopez et al., 2005). from recent findings, o. gratissimum has proved to be useful in the medication for people living with human immuno deficiency virus (hiv), and acquired immuno deficiency syndrome virus aids (elujoba, 2000). in congo, o. gratissimum decoction is used for gonorrheal infection, vaginal douches for metritis and vaginitis and used in treatment of mental illness (abdulrahma, 1992). p. guineense commonly referred to as african black pepper or ashanti pepper is very similar to piper nigrum which is the true pepper of commerce from which black and white peppers are processed (isawumi, 1984). p. guineense belongs to the family piperaceae. it has more than 700 species throughout the tropical and subtropical regions of the world. it is known with different vernacular names in nigeria: igbo (uziza), and yoruba (iyere). p. guineense has culinary, medicinal, cosmetic and insecticidal uses (dalziel, 1955; okwute, 1992). p. guineense insecticidal activity against zonocerus variegatus is attributable to the piperine-amide composed by the plant. the leaves are considered aperitive, carminative and eupeptic. they are also used for the treatment of cough, bronchitis, intestinal diseases and rheumatism (sumathykutty et al., 1999). escherichia coli and staphylococcus aureus are organisms associated with the gastrointestinal tract of man and animals. they occur in other environment. s. aureus is among the invasive gram positive known as pyogenic cocci implicated in several diseases of human. it has been found to be normal flora of upper respiratory tract and vagina . s. aureus has been known to produce heat stable toxins which are implicated in illnesses and stomach upset (cdc, 1989; mosset et al., 1990). from literatures, s. aureus had shown to be very resistant to a wide variety of antibiotics (nwinyi et al., 2008). e. coli, a facultative anaerobe of wide distribution in the environment, has been implicated in the cause of urinary tract infec tions, meningitis, sepsis, wound infections, noscomial pneumonia and arthritis. a subgroup enterohemorrhagic e. coli (ehec) can cause severe potentially fatal illness known as hemorrhagic colitis with symptoms of blood diarrhea and severe abdominal pain (dolore et al., 2001). thus, this study seeks to investigate the scientific basis for the traditional use, of these medicinal plants among the local people in nigeria, for treating ailments associated with e. coli and s. aureus infections. materials and methods collection of plant material fresh samples of o. gratissimum leaves were collected from a forest at alor town in anambra state between the hours of 6-8 am at a prevailing temperature of about 28+ 2 o c while the fresh leaves of p. guineense were bought from traditional healers at a local market in awka, anambra state. all the collections were done in the month of may. the plants were identified and authenticated at the herbarium of the department of biological sciences, nnamdi azikiwe university, awka, anambra state. a voucher specimen was deposited at the herbarium for reference purposes. preparation of extracts fresh leaves of o. gratissimum and p. guineense were thoroughly washed using tap water and rinsed with distilled water. the leaves were dried for 5 min in an oven at 60 o c to stop enzyme activity (effraim et al., 2000). they were then air dried to a constant weight and milled to a fine powder with the aid of a binatone blender (model blg-401). two solvent were used for the preparation of the extracts, namely distilled deionized water and ethanol 60% conc. the aqueous extract was prepared by weighing out (250 g) of the milled powdered leaves of o. gratissimum and p. guineense and adding in 200 ml of distilled deionized water respectively in a 500 ml beaker and stirring vigorously with a glass rod. the combination was allowed to settle for 3 h using the infusion method. the extracts were then filtered using whatman no.1 filter paper. the ethanol extracts were obtained by weighing out same fraction 250 g of the different plants and wrapping it in whatman no.1 filter paper and placed in the holding chamber of the soxhlet extractor. about 500 ml of the 60% ethanol was used as solvent for the extraction of the leaves using the reflux method for a period of 48 h. this was carried out exhaustively. from literature, soxhlet extraction has been found to give a higher yield of the extracts. the extracts were then concentrated by evaporating to dryness using rotary evaporator at a temperature 40 o c. a darkgreen colored mass for p. guineense and o. gratissimum were obtained and stored in airtight bottles at 4 o c in a refrigerator until ready for use. the stored extract was reconstituted using the corresponding solvent to obtain extracts of several concentrations 10.0, 5.0, 2.5, 1.25 mg/ml -1 and stored at 4 o c prior to determination of the minimum inhibitory concentration. preparation of test organisms clinical isolate of s. aureus was obtained from glanson laboratories, awka, anambra state, nigeria while the strain of e. coli was obtained from a culture collection centre at sammlung von microorganismen (dsm) germany. the isolates were tested for viability by resuscitating the organisms in buffered peptone broth, after which it was subcultured into nutrient agar medium and incubated at 37 o c for 24 h. the isolates were subcultured in a nutrient broth at 37 o c for 8 h prior to antibacterial testing. table 1. antibacterial activity of the aqueous extracts of ocimum gratissimum and piper guineense against escherichia coli and staphylococcus aureus. plant test organism mean zones of inhibition (mm) control (water) 10.0 5.0, 2.5, 1.25 ocimum gratissimum escherichia coli 3+ 0.09 staphylococcus aureus 7.5+ 0.10 piper guineense escherichia coli 2 + 0.07 staphylococcus aureus 4+ 0.10 (-), no inhibition of growth; (10.0, 5.0, 2.5, 1.25) mg/ml -1 , different concentrations. table 2. antibacterial activity of the ethanol extracts of ocimum gratissimum and piper guineense against escherichia coli and staphylococcus aureus. plant test organism mean zones of inhibition (mm) control (ethanol) 10.0, 5.0, 2.5, 1.25 ocimum gratissimum escherichia coli 5+ 0.09 staphylococcus aureus 8 + 0.11 7.5+ 0.11 6 + 0.06 piper guineense escherichia coli 10 + 0.10 8+ 0.10 7+ 0.11 staphylococcus aureus 2+ 0.01 (-), no inhibition of growth; (10.0, 5.0, 2.5, 1.25 mg/ml -1 ), different concentration. determination of antibacterial activity of the aqueous and ethanolic extracts agar well diffusion technique as described by adeniyi et al. (1996) was used to determine the antibacterial activity of the extracts. an 18 ml of mueller hinton agar plates (mha difco, france) were seeded with 2 ml of an overnight broth culture of each bacterial isolate (equivalent to 10 7 – 10 8 cfu ml -1 ) in sterile petri-dish .the seeded plates were allowed to set after a uniform distribution of the bacte-rial isolate following slow rotation of the petri dish . a standard sterile cork borer of 8 mm diameter was used to cut uniform wells on the surface of the agar. the wells filled with 2 ml of each extracts were with the aid of a sterile pasteur pipette. the dishes were allowed to stand for 45 min at room temperature to allow proper diffusion of the extract to occur. the control experiments were setup with 2 ml of ethanol (60%) and distilled deionized water which served as controls were also put in separate wells. all the plates were incubated at 37 o c for 24 h. the assay was conducted at regular intervals of 24 h until marked decline in the potency of the extracts to inhibit the growth of the test organisms was noticed. zones of clearance round each well means inhibition and the diameter of such zones was measured in millimeter (mm). the minimum inhibitory concentration (mic) in mg/ml was determined by comparing the different concentration of the extracts that have different zones of inhibition and then selecting the lowest concentration of each extract (agaetmor, 2009). results antibacterial activity of the extracts (aqueous and ethanol) extracts. the results of the antibacterial activity of the extracts against the test organisms namely e. coli, s. aureus are shown in tables 1 and 2. the mean zones of inhibition of growth of the isolates are a function of relative antibacterial activity of the extracts. the extracts showed selective levels of activities against the isolates. the highest growth inhibitory activity was obtained from the ethanol extracts. ethanol and distilled deionized water served as control and were inactive against both test organisms. the minimum inhibitory concentration of the aqueous and ethanol extracts results of the minimum inhibitory concentration (mic) are summarized in table 3. generally, less values were obtained for the aqueous extracts. higher concentrations of the o. gratissimum extract were needed to inhibit e. coli when compared to s. aureus. the mic of the aqueous extract of o. gratissimum was 10.00 and 10.00 mg ml -1 for e. coli and s. aureus, respectively whereas that of the ethanol extract ranged between 10.00 and 2.50 mg ml -1 for e. coli and s. aureus, respectively. discussion from this investigation, the aqueous extracts of p. guineense and o. gratissimum showed minimal antibacterial activity against the isolates at 10 mg/ml -1 concentrations. this observed difference between these plants extracts may be due to insolubility of active compounds in water or the presence of inhibitors to the antimicrobial components (okigbo and ogbonnanya, 2006). amadioha and obi (1999), okigbo and ajale (2005a), okigbo et al. 349 afr. j. food sci. res. nwinyi et. al. 350 table 3. minimum inhibitory concentration (mic) of the aqueous and ethanol extracts of ocimum gratissimum and piper guineense. test organism ocimum gratissimum piper guineense aqueous extract ethanol extract (mg aqueous extract ethanol extract (mg (mg ml -1 ) ml -1 ) (mg ml -1 ) ml -1 ) escherichia coli 10.00 10.00 10.00 2.50 staphylococcus aureus 10.00 2.50 10.00 10.00 (2005b) reported that inactivity of plant extracts may be due to age of plant, extracting solvent, method of extraction and time of harvesting of plant materials. conversely, the ethanol extracts of p. guineense and o. gratissimum showed a concentration dependent gradient decrease in the level of inhibition against isolates. from the results there is variation in the degrees of antibacterial activities of the extracts on the isolates. the variation is presumed to be due to different active compounds present in these plants. ethanol extracts of o. gratissimum showed more antibacterial activity against s. aureus than e. coli. this result is in agreement to agatemor (2009) where it was reported that gram negative bacteria are more resistant than gram positive bacteria to the essential oil which are antimicrobial agents. nweze et al. (2004) reported phytochemical screening of o. gratissimum in the presence of alkaloids, tannins, glycoside, saponin, resins, cardiac glycoside, steroidal terpens and flavonoids. flavonoids are reported to exhibit antioxidant activity (ramanthan, et al., 1989) and are effective scavengers of superoxide anions (robak and gryglewski, 1988).thus this can significantly affect the cell wall of s. aureus which invariably may lead to the collapse of the cell wall and overall, affect the entire mechanism of the organism. e. coli a gram negative organism contains a high level of lipid material. these materials are thought to make a substantial contribution to the mechanism whereby injurious chemicals are prevented from reaching their sites of action within the cell. p. guineense ethanol extract showed significant inhibition to e. coli due to high content of essential oils such as monoterpenes and sesquiterpene and minimal effect was observed on s. aureus. this result agrees to obafemi et al. (2006) where sesquiterpene was tested against s. aureus. from this study, it was observed that ethanol extracts exhibited high inhibitory activity on the test organisms. this can be deduced to the ability of ethanol to extract more of the essential oils and secondary plant metabolites which are believed to exert antibacterial activity on the test organisms. this study however can justify the use of the leaves in traditional medicine practice as a therapeutic agent and can explain the long history use of these plants. references abdulrahma f (1992). studies on natural products.the moracease in african traditional medicine and management of psychiatry in borno state.m.sc.thesis, university of maiduguri, maiduguri .pp 89-95. adeniyi ba, odelola ha, oso ba (1996).antimicrobial potential of diospyros mesiliformis (ebenaceae) afr. j. med. sci. 255: 221-224. agatemor c (2009). antimcrobial activity of aqueous and ethanol extracts of nine nigerian spices against four food borne bacteria. elec. j. environ. agric. food chem. 8(3): 195-200. amadioha ac, obi vi (1999) control of anthracnose disease of cowpea by cymbopogon citrates and ocimum gratissimum .acta. phytopathol.entomol. hungerica 34 (1-2): 85-89 akinyemi ko, mendie ue, smith st, oyefolu ao, coker ao (2004). screening of some medical plants for anti-salmonella activity. j herb pharmocother. 5(1): 45-60. begum j, yusuf m, chowdhury u, wahab ma (1993). studies on essential oils for their antibacterial and antifungal properties. part 1. preliminary screening of 35 essential oils. j. sci. ind. res. 28: 25-33. dalziel im (1937). piperacease .useful plants of west tropical africa hand book. african press ibadan nigeria. pp. 16-17. dalziel im (1955). the useful plants in west tropical africa hand book 2 nd printing crown agents. dolore ge, doyle (2001). escherichia coli in diarrhea disease. ann. int. med. 247: 81-90 effraim id, salami ha, osewa ts (2000). the effect of aqueous leaf extract of ocimum gratissimum on haematological and biochemical parameters in rabbits afr. j. biomed. res. 175-179. elujoba aa (2000). studies on the antidiarrhoea activity of ocimum gratissimum. university of ile ife press. ilori m, sheteolu ao, omonibgehin ea , adeneye aa (1996). antibacterial activity of ocimum gratissimum (lamiaceae). j. diarhoeal dis. res.14: 283-285. isawumi ma (1984).the peppery fruits of nigeria. nigerian field, 49: 3744. janine de aquino l, xisto sp, orionaida de fatima lf, realino de paula j, pedro hf, hasimoto de suza lk, aline de aquino l ,maria de rosario rs (2005). antifungal activity from ocimum gratissimum l. towards cryptococus neoformans. mem. inst. oswaldo cruz 100(1): 55-58. lopez p, sanchez k, batlle r, nerin c. (2005). solid and vapour phase anti-microbial activities of six essential oils susceptibility of selected food borne bacterial and fungal strains. j. agric food chem. 53(17): 6939-6946. mshana nr, abbiw dk, addae-mensah i, adjanohoun e, ahji mra, enow-orock eg, gbile zo, odei ma, adenlami h, oteng-yeboah aa, sarppony k, sofowora a, tackie an (2000). traditional medicine and pharmacopoeia contribution to the revision of ethno botanical and floristic studies in ghana, scientific, technical and research commission of the organization of african unity montefore d, rotimi yo, adeyemi-doro, fa (1989). the problem of antibacterial resistance to antibiotics among strains from hospital patients in lagos and ibadan. niger. j. antimicro. chemother. 23: 604. mosset daa, van, nn (1990). staphylococcus aureus and related staphylococci in food ecology, proliferation, toxigenesis, control monitoring. j. appl. bacterial symp. suppl. 69(19):123-146. nweze ei, okafor ji, njoku o (2004). antimicrobial activities of methanolic extracts of trema guineensis (schumm and thorn) and morinda lucida benth used in nigerian herbal medicinal practice. j. biol. res. biotechnol. 2(1):39-46. nwinyi oc, chinedu ns, ajani oo (2008). evaluation of antibacterial activity of pisidium guajava and gongronema latifolium .j. med .plant 351 afr. j. food sci. res. res. 2(8): 189-192. nwosu mo, okafor ji (1995). preliminary studies of the antifungal activities of some medicinal plants against basidiobolus and some other pathogenic fungi. mycoses. 38: 191-195. obafemi ca, sulaimon ta, akinpelu da, olugbade ta (2006). antimicrobial activity of extracts and a germacranolide-type of sesquiterpene lactone from tithonia diversifolia leaf extract. afr. j. biotechnol. (12): 1254-1258. okigbo rn, igwe m (2007). the antimicrobial effects of piper guineense uziza and phyllantus amarus ebe-benizo on candida albicans and streptococcus faecalis. acta microbiologica .et. immunologica.hungarica. 54 (4): 353-366. okigbo rn, ogbonnanya ou (2006). antifungal effects of two tropical plants extracts ocimum gratissimum and afromaomum melegueta on post harvest yam discorea spp rot. afr. j. biotechnol. 5 (9): 727-731. okigbo rn, ajale an (2005a). inhibition of some human pathogens with the tropical plant extracts chromolineena odorata and citrus aurantifolia and some antibiotics. int. j. mol. med. adu sci 1:34-40 okigbo rn ,mbajaka c,njoku co (2005b) antimicrobial potential of (uda) xylopia aethopica and ocimum gratissimum on some pathogens of man. int j. mol. med. ad. sci. pakistan 1 (4): 392-394. okwute sk (1992). plant derived pesticidal and antimicrobial agents for use in agriculture .a review of phytochemical and biological studies on some nigerian plants. j. agric. sci. technol. 2 (1): 62-70. omoseyindemi bx (2003). plants as natural medicine .paper presented at the annual conference of botanical society of nigeria (boson) university of lagos. onajobi fd (1986). smoothe muscle contacting lipidic –soluble principles in chromatographic fractions of ocimum gratissimum .j. ethnopharmacol.18: 3-11. ramanthan r, lau kk, da np (1989). antiperoxidative action of flavonoids and related products in ground pork (abstract) proceedings of iii int. symp.on flavonoids in biology and medicine singapore: p. 56. robak k, gryglewski, rj (1988).flavonoids are scavengers of super oxide anions. biochem. pharmacol. 37: 837-841 sofowora a (1993). medicinal plants and traditional medicine. spectrum books ltd.ibadan, nigeria. pp. 224 -227. sumathykutty ma, rao jm, padmakumari kp, narayanan cs (1999) .essential oil constituents of some piper species .flavors and fragrance j. 14:279-282. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (8), pp. 224-230, august, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the effectiveness of polar and non polar solvents in african breadfruit seed oil extraction titus ugochukwu nwabueze * and kalu sunday okocha department of food science and technology, michael okpara university of agriculture, umudike, p.m.b 7267 umuahia, abia state, nigeria. accepted 22 april, 2017 the physical and chemical indices of african breadfruit (treculia africana) seed oil extracted with polar (isopropanol, hexane and butanol) and non polar (acetone) solvents were investigated. the oil (19.85%) and energy contents 452.35 (kcal) suggest that african breadfruit seeds are a high -energy food. the yields were significantly (p 0.05) different with hexane extracted (non-polar solvent) oil having 19.85 % whilst oil extracted with polar solvents ranged from 15.58 -19.30 %. melting points were 34, 27, 26, 21 0 c for oil extracted with hexane, isopropanol, butanol and acetone respectively. smoke points were within the limits of 170 – 255 0 c for the four oil samples. iodine values ranged from 14.50 (hexane extracted) t0 25.17 (acetone extracted) . saponification values ranged from 125.89-267.85 while peroxide values were 3.20 mg/kg (hexane) and 3.60-3.83 mg/kg for polar solvents. free fatty (oleic) acid of oil extracted with hexane was 1.71% and polar solvent extraction ranged from 1.651.78%. hexane had the least peroxide value (8.74 mg/kg) compared to higher values for oil extracted with the polar solvents (9.10 9.83 mg/kg). keywords: african breadfruit, treculia africana, oil extraction, saponification, fatty acids, peroxide value, thiobarbituric acid introduction african breadfruit (treculia africana) constitutes a strategic reserve of essential food nutrients that are available at certain critical periods of the year when reliable sources of these nutrients are under cultivation and are very scarce. diverse food forms could be produced from the seeds on the basis of custom, tradition, ethnic background. it is boiled and consumed as white porridge and sauce with or without fresh corn, roasted and consumed with coconut or palm kernel as snack, made into refreshing milk drink, prepared into flour as soup condiment or thickener and for bakery and confectionaries. in the past the consumption was limited to poor village dwellers for whom it supplemented their diets during times of food scarcity and substituted the more expensive rice during festivals and other ceremonies on the basis of tradition and cost (nwabueze *corresponding author email: e-mail: teeubueze@yahoo.co.uk and nwokenna, 2006). but today, african breadfruit has become a delicacy and a specialized meal not only for the rich and the urban dwellers in nigeria but has also become a foreign exchange earner. dehulled kernels are sun-dried and exported to cater for the african consumer interests overseas. african breadfruit seed is of high nutritional value. each seed contains about 14-17% crude protein, 2.5 % crude fibre, 35-60 % carbohydrate and a good supply of vitamins and minerals (akubor, 2000). the amino acid composition has also been highlighted to further buttress its nutritional potentials (nwabueze, 2007) . the oil potential of the seed has been reported (ajiwe et al., 1995). generally, plant oil serves as rich source of essential fatty acids and energy yielding approximately 9 kilocalories per gram as well as being a carrier of fat-soluble vitamins (a, d, e, and k) which help the body to absorb most of its nutrients (ajiwe et al., 1995). plant oils play other distinctive role in the natural flavour and palatability of a wide of food commodities (ihekoronye and ngoddy, 1985) nwabueze and okocha 225 while certain of its components (linoleic, linolenic and arachidonic acid) are essential in all diets for body growth and normal skin condition (ebuehi et al., 2006). it has been reported (bjorck and asp, 1983) that only 45-55 % of the oil present in raw materials could be extracted with ethyl ether after extrusion. but nierle et al. (1980) report an average oil recovery in extruded wheat to be 40 and 20 % for maize. a number of reasons were advanced for this reduction of oil extraction in extrudates. monoglycerides and free fatty acids have been reported to form complexes with amylose during extrusion processing (mercier, 1980; badrie and mellowes, 1992). such complexes are likely to cause difficulty in plant oil extraction with petroleum ether. a literature search did not reveal much information deal ing with selection of organic solvents for plants and in particular african breadfruit seed oil extraction. the objective of this research was to evaluate the effectiveness of polar and non polar solvents in african breadfruit seed oil extraction and to study their effects on the physical and chemical properties of the oil. this study is necessary to assess african breadfruit seed oil quality indices for proper application in food and industrial system. materials and methods raw materials and preparation about 10 kg of african breadfruit (treculia africana) seeds were purchased from umuahia main market, abia state, nigeria. they were cleaned by hand picking to rid them of any contaminating stones or extraneous and organic materials before being parboiled at 100 o c for 15min. parboiled and drained seeds were then threshed in a commercial attrition mill and manually dehulled to recover the kernels. the kernels were sun dried for about 17 h and then milled in a blender (moulinex 276, france, speed 1) to fine flour (2 mm particle size). the flour was preserved in a tight polyethylene bag at room temperature (28± 2 o c) from which samples were collected for different analyses. extraction and yield of african breadfruit seed oil african breadfruit seed oil was extracted from the resulting flour using four different food grade organic oil extraction solvents (boiling point of 60 80 o c). these included iso-propanol, butanol, and acetone (polar solvents) and hexane (non polar solvent). the extraction involved the use of soxhlet extraction method (aoac, 1995). about 100g of the flour were weighed into a thimble in the soxhlet extractor fitted to conical flask. african breadfruit seed oil was extracted with 250 ml of each solvent. the solvent was boiled under reflux for about 6 h. african breadfruit seed oil yield was calculated for each extraction solvent by weight difference of the sample before and after extraction and reported as mean of duplicate determinations. proximate composition of dehulled full fat african breadfruit seed flour proximate composition of dehulled-sun dried african breadfruit seed was determined in triplicate for moisture, crude protein (micro kjeldhal method), fat (soxhlet method), crude fibre, and ash according to aoac (1995) methods. total carbohydrate was determined by difference. energy was calculated using the at water factors of 4 x protein, 4 x carbohydrate and 9 x fat (nwabueze, 2006). determination of vitamins in african breadfruit seed oil vitamin a was determined by the method described by delia and meiko (2003) while the spectrophotometeric method described by pearson (1976) was used for the determination of vitamin e. physical properties of african breadfruit seed oil the physical characteristics of african breadfruit seed oil determined included yield, colour (photometric system), specific gravity, melting and smoke points were determined by the standard methods as described by aoac (1995). specific gravity was determined by use of specific gravity bottles at a temperature of 28±2 o c. photometric colour index (pci) of african breadfruit seed oil was determined on 1g sample according to the method described by pike (2003). the sample was weighed and dissolved in 20 ml water/ethanol mixture. the mixture was filtered after standing for 30 min. the absorbance of the filtrate was measured at 400, 550, 620 and 670 nm using spectrophotometer (unican he 105y, england). the solvent was used as blank. photometric colour index was calculated as pci = 1.29 (a400) + 69.70 (a500) + 41.20 (a620) –56.41 (a670) (2) where a = absorbance. chemical properties and acid concentrations the acid values, iodine value, saponification value, peroxide and thiobarbituric acid values were determined by the standard methods of aoac (1995). fatty acid composition of the oil was determined by the method described by christie (1980) using a flow-mac flame ionization gas liquid chromatography. the methylation of the fatty acids was prepared according to standard methods of aoac (1995) . the equipment was set at oven temperature of 200 o c with a 1.8 m by 0.32 cm stainless column packed with 10 % degs on material support of silicon mesh size. nitrogen was used as a carrier gas at a flow rate of 25 ml/min and the quantification of the fatty acid was obtained by automatic integration with linear response attached to the system. effect of ambient storage on african breadfruit seed oil extracted oil samples from different extraction solvents were stored in plastic bottles tightly corked to practically prevent entrance of air at (ambient) room temperature (28± 2 o c) for 25 days. during this period the thiobarbituric acid and peroxide values were determined at 0, 8, 19 and 25th days for each extract and reported as mean duplicate determinations. statistical analysis differences between means were assessed by students t – test, while the levels of significance of the data were calculated by table 1. proximate composition and energy values of dehulled full fat african breadfruit seed flour. moisture (%) protein (%) fat (%) ash (%) carbohydrate (%) energy (kcal) 9.425±0.002 8.76±0.002 19.85 ±0.025 2.30±0.001 59.67 ±0.006 452.35±0.041 table 2. oil yield and vitamin content of african breadfruit seed oil extracted with different solvents. extraction solvent yield (%) vitamin a ( g/g) vitamin e (mg/100g) isopropanol 18.10 c ± 0.01 21.32 b ± 0.01 3.11 b ± 0.01 butanol 19.30 b ± 0.01 16.09 c ± 0.03 2.05 d ± 0.01 hexane 19.85 a ± 0.02 27.39 b ± 0.01 5.67 a ± 0.04 acetone 15.58 d ± 0.06 12.78 d ± 0.03 2.56 c ± 0.03 means not followed by the same superscripts along the same column are significantly different (p 0.05). analysis of variance at 0.05 % level of significance according to steels and torrie (1981). results and discussion proximate composition of dehulled full fat african breadfruit seeds flour the proximate composition of dehulled sun dried full fat breadfruit seeds flour is presented in table 1. with a moisture content of 9.43 %, the african breadfruit seed full fat flour will keep well. protein content was 8.70 % which was lower than the range (15.76 -17.52 %) reported by nwabueze (2006) probably due to variety used. since african breadfruit seeds have not been classified or graded into varieties or breeds or cooking types, they are marketed as mixtures of varieties, a problem that also affects its cooking time (nwabueze and nwokenna, 2006). the ash content of 2.30 % suggested a good source of minerals (nwokolo, 1987; akubor, 2000) while the oil (19.85%) and energy contents suggest the seed to be a high-energy food. the carbohydrate content of 59.66 % could pass african breadfruit seed for composite flour in baked and confectionary products. african breadfruit seed oil yield and vitamins oil yield and vitamin content (a as carotenoid and e) of african breadfruit seed oil extracted with different solvents are shown in table 2. generally, all the african breadfruit seed oil extracts was yellow in colour and remained liquid at room temperature (28±2 o c) . the photometric colour index shows significant (p 0.05) differrences in their values. this could be attributed to the selective permeability of the different solvents used. the yields of oil extracted with the hexane (non-polar solvent) were significantly (p 0.05) different from that of the oil extracted with polar solvents (isopropanol, butanol and acetone), having a yield of 19.85% while the amount of oil extracted by the polar solvents ranged from 15.58% (acetone) to 19.30% (butanol). various authors (nierle et al., 1980; ajiwe, et al., 1995; ebuehi and avwobobe, 2006) have reported varying extraction values with differrent solvents or seeds. ajiwe, et al. (1995) reported an extraction yield of 20.83% oil from african breadfruit seed by soxhlet using petroleum ether as extraction solvent, nierle et al. (1980) reported an average oil reco-very of 20% in extruded maize while ebuehi and avwobobe (2006) reported a yield of 17.36% for water melon. in this study, hexane gave highest oil yield but the use of such oil has been questioned in terms of safety particularly if not purified (bera, et al., 2004). ethanol, methanol and acetone have been recommended as solvents for extraction of vegetable oils. thus, oil extraction yield from african breadfruit seed may correlate closely with polarity of the extraction solvent. non-polar solvents have both sides charged and are able to penetrate into the matrix of a feed. this is so, because they lack an o-h end which otherwise would interfere with the extraction process. the low extraction yield of african breadfruit seed oil reported in this study and in literature could be attributed partly to extraction solvents used, state of the food material and partly due to complex formation between fatty acids and carbohydrate breakdown components (mercier, 1980). such complexes will definitely cause difficulty in plant oil extraction with petroleum ether. following the minimum fat requirement of 6% in complementary formulation (obatolu, 2002) , the oil yields by all the solvents meet this requirement. vitamin a ( – carotene) varied significantly (p 0.05) with a range of 12.78 g/g in oil extracted with hexane to 37.39 g/g in oil extracted with acetone. vita-min a deficiency has been recognized as the second 226 afr. j. food sci. res. nwabueze and okocha 227 table 3. physical properties of african breadfruit seed oil extracted with different solvents extraction solvent specific gravity (g/ml) melting point ( 0 c) smoke point( 0 c) photometric colour index isopropanol 0.8627 c ± 0.001 27.15 b ± 0.002 149.67 d ± 0.03 148.08 c ± 0.03 butanol 0.8756 b ± .0001 26.00 c ± 0.004 255.00 a ± 0.06 367.11 b ± 0.05 hexane 0.8656 c ± .0001 34.00 a ± 0.001 250.00 b ± 0.02 476.55 a ± 0.02 acetone 0.9760 a ± .0003 21.01 d ± 0.002 180.27 c ± 0.08 140.77 d ± 0.03 means not followed by the same superscripts along the same column are significantly different (p 0.05). table 4. chemical properties of african breadfruit seed oil extracted with different solvents extraction solvent iodine value saponification value peroxide value (mg/kg) isopropanol 15.72 c ± 0.06 125.89 d ± 0.3 3.83a±0.1 butanol 21.51 b ± 0.01 238.43 c ± 0.01 3.60 a ± 0.01 hexane 14.50 d ± 0.01 259.46 b ± 0.01 3.20 b ± 0.01 acetone 25.17 a ± 0.1 267.85 a ± 0.03 3.78 a ± 0.03 means not followed by the same superscripts along the same column are significantly different (p 0.05). most common form of malnutrition. the difference in vitamin a content of the oil samples could be attributed to a stability factor since it is readily stable in non polar solvents. thus the amount extracted could be as a result of the solvent used as well as the amount of oil extracted (yield) and the concentration of the vitamin in the extracted oil. the vitamin e content varied from 2.05 mg/100g in oil extracted with hexane to 56.69 mg/100g in oil extracted with butanol. all the oil samples extracted with polar solvents (isopropanol, butanol and acetone) had low values (2.05 – 3.11 mg/100g) of vitamin e. these vitamin e values are higher than those reported (baurnfeind, 1980) for soybean oil (1.2 mg/100g) and corn oil (2.0 mg/100g). with the working solvents in this study, african breadfruit seed oil will make a good source of vitamin e and hence have an antioxidant stability effect on the oil. this is because vitamin e has been recognized as the most biological oxidant in humans. physical properties of african breadfruit seed oil table 3 shows physical properties of african breadfruit seed oil extracted with different solvents. specific gravity was significantly (p 0.05) different varying from 0.8627 g/ml isopropanol to 0.970 g/ml (acetone). this range of specific gravity of african breadfruit seed oil compares with the ranges reported for palm oil (0.91) and groundnut oil (0.84) (ebuehi, et al., 2006) and 0.91 for pumpkin (curcubita pepo) seed oil (ihediohanma et al., 2006). higher deviations from these values point to hydrolytic and oxidative changes. melting and smoke points of the oil samples were significantly (p 0.05) different with extraction solvent used. the smoke points showed that african breadfruit seed oil could be used in deep frying food systems as oils with high smoke points resist ignition. chemical properties of african breadfruit seed oil chemical properties of african breadfruit seed oil extracted with different solvents are presented in table 4. iodine values were generally low when compared to other oils of plant origin. it ranged from 14.50 (hexane extracted) t0 25.17 (acetone extracted) which functionally may not qualify the oil to be used as a drying oil. the degree of unsaturation of oil is determined by measuring its iodine value. iodine value is a measure of fat or oil stability and resistance to oxidation. oresanya et al. (2000) stated that fat oxidation which occurs during fat extraction or storage and transportation leads to hydrolysis of triacylglycerols to free fatty acids. lee (1983) strongly suggested that iodine value of oil be determined by the temperature at which the oil seeds are produced, thus the higher the temperature, the lower the iodine value and vice-versa. african breadfruit seed oil from the four different solvents showed significantly (p 0.05) different values with respect to their saponification values. they ranged from 125.89-267.85. oil extracted with isopropanol had the least value while that of acetone was the highest. high saponification value indicates high molecular weight oil good mainly for soap making and for hair shampoo (ajiwe, et al., 1995). according to champe and harvey (1994), saponification values measure the amount of alkali required to combine with the fatty acids liberated by the hydrolysis of fats and oils from which weights of fatty acids could be determined. 228 afr. j. food sci. res. table 5. acid concentrations of african breadfruit seed oil extracted with different solvents extraction solvent ph acid value (%) thiobarbituric acid (mg/kg) oleic acid (%) isopropanol 3.38 d ± 0.1 3.21 c ± 0.06 18.42 a ±0.3 1.65 b ± 0.06 butanol 4.45 c ± 0.0001 2.48 d ± 0.0001 16.63 b ± 0.0001 1.25 c ± 0.0001 hexane 5.70 a ± 0.0001 3.40 b ± 0.0001 15.69 c ± 0.0001 1.71 ab ± 0.0001 acetone 5.16 b ± 0.03 3.55 d ± 0.03 14.83 d ± 0.03 1.78 a ± 0.03 means not followed by the same superscripts along the same column are significantly different (p 0.05). t h io b a rb it u ri c a c id ( m g m a lo n a ld e h y d e /k g o il) 70 60 50 40 30 20 10 0 day 0 day 8 day 19 day 25 isopropanol butanol hexane acetone extraction solvents figure 1. effect of storage on thiobarbituric acid of african breadfruit seed oil extracted with different sovents. peroxide value of african breadfruit seed oil varied with solvent used. peroxide value is used as an indicator of deterioration of fats and oils. as oxidation takes place, the double bonds in the unsaturated fatty acids are attacked producing peroxides. this in turn decomposes releasing secondary products which cause rancidity (champe and harvey, 1994; ebuehi, et al., 2006). in this study, peroxide value of oil extracted with hexane (3.20 mg/kg) was significantly (p 0.05) lower than oil samples extracted with polar solvents (3.60-3.83 mg/kg). acid concentrations of african breadfruit seed oil table 5 shows acid concentrations of african breadfruit seed oil extracted with different solvents. mean ph values of the oils varied significantly (p 0.05) with different solvents. it ranged from 3.38 (isopropanol extraction) to 5.70 (hexane extraction). this variation could have affected the values of vitamins observed in this study as ph < 7 has been reported (ihekoronye and ngoddy, 1985) to affect the stability of vitamin a and its precursors. the table showed that there was no significant (p 0.05) difference between the free fatty acid (oleic acid) of oil extracted with hexane (1.71%) and that extracted with either acetone (1.78%) or with isopropanol (1.65%). oil samples extracted with butanol had the least free fatty acid as oleic acid and so may not be easily prone to oxidation. champe and harvey (1994) stated that several cardiovascular diseases have been implicated in human population consuming diets rich in polyunsaturated fatty acids. the acid value for the four different crude oils were high thus, the oil extracts would need some form of purifi nwabueze and okocha 229 12 10 /k g o i) 8 (m e g .o 2 6 v a lu e pe ro xi d e 4 2 0 day 0 day 8 day 19 day 25 isopropanol butanol hexane acetone extraction solvents figure 2. effect of storage on peroxide valves of african breadfruit seed oil extracted with different sovents. cation (refining) to enhance stability and storage. low acid values suggest stability of oil. oscar (2002) reported that acid value or free fatty acid (ffa) in crude oils, estimates the amount of oil that will be lost during refining. thus the higher the acid as ffa values the higher the amount of oil that could be lost during processing. effect of storage on thiobarbituric acid and peroxide values of african breadfruit seed oil effect of storage of african breadfruit seed oil extracted with different solvents on thiobarbituric acid and peroxide value are shown in figure 1 and 2, respectively. thiobarbituric acid and peroxide values are two important rancidity indices which were found to vary with storage in this study. at the end of the 25 day storage period at room temperature (28 o c), the oil extracted with hexane had the least peroxide value of 8.74 mg/kg compared to higher values for oil extracted with the polar solvents which ranged from 9.10 mg/kg (butanol) to 9.83 mg/kg (isopropanol). this observation indicates that rate of autocatalytic degradation of oil extracts, measures rancidity, increased with storage. the relatively higher peroxide values of oil samples extracted with polar solvents with storage were still lower than 20 mg/kg allowed for use olive oil (fao/who, 1993). lower values imply that the oil has lower degree of rancidity. the rate of increase which was also observed in thiobarbituric acid of oils related to extraction solvents and perhaps to vitamin e content of the oil as antioxidant. this trend has been attributed to the fact that products formed during the reaction tend to catalyze the rate of autoxidation reaction. lee (1983) reported that the reaction increases exponentially. conclusion most of the studies reported in this favoured the use of non polar (hexane) solvent as an extraction solvent. the extraction of oil with hexane gave highest oil yield but the use of such oil has been questioned in terms safety. it is recommended above other physical and chemical properties for use in industries other than food. however the oil extraction with acetone a polar solvent is recommended for food use on safety grounds. references ajiwe vie, okeke ca, agbo hu (1995). extraction and utilization of breadfruit seeds oil (treculia africana). bioresources technology, 53: 183-184. akubor pi (2000). studies on food potentials and storage properties of african breadfruit kernel flour. an unpublished msc thesis, department of food science and technology, university of agriculture, makurdi, nigeria. aoac (1995). official methods of analysis . 15th edition. association of official analytical chemists, arlington, va: association of analytical chemists. badrie n, mellowes wa (1992). cassava starch or amylose effects on characteristics of cassava (manihot esculenta crantz) extrudate. j.food science. 57: 103-107. baurnfeind j(1980). in: machlin, l.j. 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distributors. new delhi. pp. 195-204. pearson d (1976). the chemical analysis of foods: 572 churchill livingstone london. 7th edn. pike ao (2003).fat characterization. in: food analysis, 3rd edition. aluwer academic /planum. publishers new york. pp. 227 –235. steels rgd,torrie jh (1981). principles and procedures of statistics.a biomedical approach. 2nd edn. mc graw –hill intern auckland. pp.50102. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (1), pp. 001-005, january, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper rheological changes of sea cucumber stichipus japonicus during different heated times xin gao, zhaohui zhang*, lianfeng liu and qian liu college of food science and engineering, ocean university of china, qingdao, china. accepted 12 october, 2018 changes in tissue structure, weight, volume and rheological properties of sea cucumber meat were studied. sea cucumber stichopus japonicus was heated at 70°c for 1, 2, 6, 10 and 24 h, respectively. microscopic photograph revealed that the structure has significant changed. when heated from 1 to 24 h. the weight, volume, rupture strength, and elastic moduli of sea cucumber meat decreased greatly, but the relaxation time and viscosity showed positive correlation with heating time. these results confirmed that the change of texture and rheological properties of sea cucumber meat was mainly due to thermal denaturation and gelatinization of collagen during heating. key words: sea cucumber, tissue structure, rheological properties, collagen. introduction sea cucumber stichopus japonicus is a highly priced mollusk and its meat is considered great delicacies all over the world. the edible worth of sea cucumber has already been strongly certified. and the physiological functions of sea cucumber have also been recorded (su et al., 2003). in food science, studies of sea cucumber mainly focus on the nutrition values, biological activity, and pharmacological activity of the components, such as carotenoids, fatty acid, free sterol compositions, fucan sulfates, chondroitin sulfate, stiparin-inhibitor, tensilin and glycosides, these components have specially physiological functions (postma et al., 2009; chen et al., 2001; kariya et al., 2004; ismail et al., 2008; ji et al., 2008; duan et al., 2010; marques et al., 2011).but these studies do not have a rheological consideration on sea cucumber meat. previous papers on rheological and texture properties of yellowtail salmon, jellyfish and abalone suggest that elements contributing to meat hardness (tenderness) are muscle fiber and connective tissue (kimura et al., 1991; gao et al., 2001, 2007). in the case of heating treatment, changes in texture are mainly attributed to heat denaturation of protein fibers. sato et al. (2002) reported that sea cucumber meat contained a larger proportion of *corresponding author: e-mail: zhangzhh@ouc.edu.cn. tel: 86 532-82032182. fax: 86-532-85901692. collagen against the total amount of protein. there have few studies relating to the rheological properties (elastic modulus e, relaxation time , viscosity , and rupture strength rs) of heated sea cucumber meat. therefore, we studied the structural and rheological properties of sea cucumber meat, and compared the changes with different heating time. in this paper, structural changes were investigated by observing sea cucumber muscle and collagen fibril with van gieson staining, and by using texture meter measurements for the rheological properties (e, , , and rs). thermal measurement was conducted by differential scanning calorimetry. materials and methods raw sea cucumber s. japonicus, harvested in qingdao, shandong province, china (average weight was 41.25 ± 6.94 g) were purchased at a retail store. the raw sea cucumbers were cleared away theirs visceras, and then heated at 70°c for 1, 2, 6, 10, and 24 h, respectively. after cooling for 30 min, the upper part meat was used as the sample for the experiments. preparation for light microscope sea cucumber samples were cut into 5 mm cube, embedded into tissue-tek (oct compound) at-20°c,and formed into 10 mm cubes(niitu and hiramoto, 1982). these blocks were trimmed and sliced into sections of 10 μm thickness using a cryostat (model cm 1900; leica company limited, u.s.a.). st re ss （ g） 400 300 200 100 0 0 2 4 6 8 10 12 time (s) figure 1. the stress-relaxation curve of sea cucumber meat. the sections were then mounted on a glass slide and stained with van gieson stain (kageyama and watamabe, 1988). light micrographs were taken with an olympus bx51 optical light microscope. differential scanning calorimetry thermal measurements were carried out to determine the denaturation temperature of the samples with a differential scanning calorimeter (dsc) (200pc; netzsch gerätebau gmbh, germany) at heating rate of 2℃/min in the temperature range from 2 and 100°c (gao et al., 2001). the wet weight of sample in an alsample capsule was 25.9 mg. rheological properties stress–relaxation measurement stress–relaxation experiments were carried out using a uni-axial compression and elongation type texture meter (model qts-25i) at room temperature. the plunger was of a cylindrical type (0.3 cm i. d.) and was used to compress the samples (20 × 10 × 10 mm) at a constant strain of 20%. before measuring, we confirmed that the samples were suitable for linearity when the volume was larger than 10 mm 3 by using the same plunger (that is 0.3 cm i. d.). the direction of applied stress was the same as the direction of observation by microscopy. all of the measurements were repeated six times for each sample. stress–relaxation curves were analyzed using the following progressive approximate method (kimura et al., 1991; gao et al., 2001, 2007 sato et al., 2002; niitu and hiramoto, 1982; kageyama and watamabe, 1988; iso et al., 1981). the typical stress-relaxation curve of sea cucumber meat was shown in figure 1. the instantaneous modulus e0 was calculated from the load when compression reached the maximum. the approximate equation of the stress–relaxation can be expressed as: p(t)  e 0 n e i exp (t /  i ) (1) i 1 in which p(t) is the stress at relaxation time; e0 is the constant strain; t is the time; ei is the elastic modulus of the i th element; and  i is the stress–relaxation time of the i th element.  i is related to the viscosity of the i th element,  i, and ei, as shown in equation 2: i i / ei (2) e0 is defined as follows: e 0  e1  e2  en(3) rupture strength measurement rupture strength was measured at room temperature by the same apparatus used for the stress–relaxation measurement. the cylindrical plunger (0.3 cm in diameter) compressed the samples at a rate of 20 mm/s. the experiment’s results represented the average of six experimental values for each sample. statistical analysis statistical analysis on a completely randomized experimental design was performed using the spss statistics software. significantly (p﹤0.05) different property means were determined using tukey’s multiple range test. results changes in weight and volume the changes of weight and volume in sea cucumber meat during different heating time are shown in table 1, which give a comparison of significant values that obtained from different heating time. when the sea cucumber meat was heated for 1h, its weight and volume decreased approximately 44 and 42%, respectively, and table 1. the changes of the weight and volume of heated sea cucumber meat. variable raw 1 h 2 h 6 h 10 h 24 h weight (g) 41.25±6.94 a 22.92±7.52 b 14.02±4.89 c 9.96±0.33 d 9.17±0.43 d 9.00±0.93 d volume (cm 3 ) 39.90±5.04 a 22.50±7.78 b 13.00±5.66 c 8.50±0.71 d 8.63±0.36 d 8.38±0.48 d values in the same row followed by different superscipt letters are significantly different (p  0.05). when it was heated for more than 1 h, the weight and volume decreased gradually. by the heating time for 6 to 24 h, weight and volume decreased approximately 78 and 79%, respectively, although the values are close. muscle structure the structures of raw and heated sea cucumber meat observed by an optical microscope are shown in figure 2. to discriminate collagen from muscle fibers, collagen and muscle fibers were stained with van-gieson staining method. muscle fibers were dyed yellow and collagen fibrils red. neither raw nor heated meat is stained yellow in figure 2. raw sea cucumber muscles were dyed red completely. however, the heated samples did not show the red staining. from 1 to 24 h, the structures changed significantly. for heated sea cucumber meat, the size of aperture among myofibrils increased gradually with heating time, and tissue structure seemed to have collapsed when the meat had been heated for 6 to 24 h. differential scanning calorimetry in dsc thermogram (figure 3), raw sea cucumber meat showed one peak at 57°c. the enthalpy change δh for raw sea cucumber meat was calculated from the area between the peak and the baseline, the result being 519 mjmg -1 . the thermograms of heated samples (1, 2, 10 and 24 h of heating) were overlap, and no peak occurs. this confirms that collagen in heated sea cucumber was completely gelatinized after heating. rheological properties rheological parameters obtained from heated sea cucumber meat are shown in table 2. table 2 also gives a comparison of significance values that obtained from different heating time. elastic moduli (e0, e1) of heated sea cucumber meat were significantly different. when heated for 1 to 24 h, e0 remarkably decreased at the first 2 h and then remained almost the same. the relaxation time (1) and viscosity (1) of sea cucumber meats showed positive correlation with heating time. the rupture strength for sea cucumber meat was similar to elastic moduli, which decreased with heating time. discussion it is well known that sea cucumber meat contains a higher content of collagen than protein (gao et al., 2001, 2007; sato et al., 2002). the presence of collagen is assumed to contribute to tissue structure (figure 2). in dsc thermogram (figure 3), the collagen denaturation by dsc was studied. in previous studies (brenner et al., 2009; ando et al., 1992), the denaturation temperatures of among fish species, so the peak at 57°c was assumed to be due to the overlap of collagen. therefore, it was assumed that the process by which collagen changed into gelation had been completed within 1 h, and the nutritional ingredient and water-soluble components of sea cucumber meat has been drawn out during longer heating time. therefore, the heating time for 1h is suit for sea cucumber meat. rheological properties, such as elasticity e, relaxation time , viscosity , and rupture strength (rs), changed greatly in heating treatment. it was reported in a previous study that, structural diversity of muscle connective tissue existed specifically and structural differences in connective tissue among fish species are clarified in relation with muscle firmness (farahnaky et al., 2010; taskaya et al., 2010). the resumed collagen network assumes to contribute to elastic moduli (gao et al., 2001), and as discussed above, the weight and volume decreased all the heating time. so it could be considered that at the first 1 h the structure of sea cucumber muscle changed absolutely, consisting with the change in elastic moduli e0, e1 values; and from 6 to 24 h, the sea cucumber muscle structure kept relatively steady and e0, e1 values kept almost constant. the heated sea cucumber meat, which was immersed in boiling water and experienced an outflow of denatured protein and moisture due to heating, recovered its water content from the surrounding boiling water. this water exchange maintained the muscle structure and affect to the relaxation time and viscosity (binsi et al., 2009). meanwhile, (gao et al., 2004) have reported that viscosity was greater when the viscous force was smaller, whereas viscosity was smaller when viscous force was greater. therefore, we considered that sea cucumber meat became less viscous when it was heated for longer time. the rupture strength is chiefly related to the firmness and energy as the meat is being ruptured. as discussed above, collagen in sea cucumber meat can be changed into gelation with 1 h. after heated for 2 to 24 h, (a) (b) (c) (d) figure 2. light microscopy of heated sea cucumber stained with van gieson staining; (a) raw sea cucumber; (b) 1 h of heating; (c) 10 h of heating; (d) 24 h of heating. temperature (°c) figure 3. differential scanning calorimetry patterns of sea cucumber sample; (a) raw sea cucumber sample, (b) heated sea cucumber samples (1, 2, 10 and 24 h). table 2. the rheological properities of sea cucumber meat during different heating time. h e0 e1 τ1 η1 rs (×10 6 dyn/cm 2 ) (×10 6 dyn/cm 2 ) (s) (×10 6 dyns/cm 2 ) (n) raw 62.20±12.23 d 32.51±6.63 d 5.14±0.88 e 125.01±11.90 d 130.40±13.07 f 1 2.13±0.24 a 1.89±0.25 a 35.78±13.22 a 65.63±18.14 a 19.22±3.94 a 2 1.32±0.02 b 1.20±0.05 b 45.87±8.26 a 55.83±6.90 a 16.53±2.96 ab 6 1.08±0.08 c 1.05±0.09 bc 103.66±79.70 b 109.80±28.25 b 13.43±2.66 bc 10 0.91±0.12 c 0.86±0.09 c 114.29±22.71 c 98.96±23.25 b 7.54±1.17 d 24 0.96±0.19 c 0.90±0.17 c 161.04±20.27 d 144.68±77.95 c 4.21±0.53 e values in the same column followed by different superscript letters are significantly different (p﹤0.05), n=6. the denatured protein and moisture flow out under gravity. therefore, the rupture strength of meat heated 1h was smaller than raw meat, but higher than meat heated for other times. conclusion based on these results, we confirmed that the difference of rheological parameters (elastic modulus e, relaxation time , viscosity , and rupture strength rs) of heated sea cucumber meat resulted from a difference in muscle structures. in firmness, sea cucumber meat was suitable for a short heating time within 1 h. acknowledgements the study was partly supported by grants from the natural science foundation of china (no. 31071631), and 948 of chinese ministry of agriculture (2011-z26). references ando m, touohara h, sakaguchi m (1992). three-dimensional structure of collagen fibrillar network of pericellular connective tissue in association with firmness of fish muscle. nippon suisan gakkaishi, 58: 1361-1364. binsi pk, shamasundar ba ,dileep ao, badii f, howell nk (2009). rheological and functional properties of gelatin from the skin of bigeye snapper (priacanthus hamrur) fish: influence of gelatin on the gel-forming ability of fish mince. food hydrocol., 23: 132-145. brenner t, nicolai t, johannsson r (2009). rheology of thermoreversible fish protein isolate gels. food res. int., 42: 915-924. chen sg, xue ch, yin l, tang qj, yu gl, chai wg (2001). comparison of structures and anticoagulant activities of fucosylated chondroitin sulfates from different sea cucumbers. carbohydrate poly., 83: 688-696. duan x, zhang m, mujumdar as, wang sj (2010). microwave freeze drying of sea cucumber stichopus japonicus. j. food eng., 96: 491497. farahnaky a, askari h, majzoobi m, mesbahi gh (2010). the impact of concentration, temperature and ph on dynamic rheology of psyllium gels. j. food eng., 100: 294-301. gao x, ogawa h, tashiro y, iso n (2001). rheological properties and structural changes in raw and cooked abalone meat. fish. sci., 67: 643-647. gao x, tashiro y, ogawa h (2004). rheological properties and structural changes in steamed and boiled abalone meat. fish. sci., 68: 499-508. gao x, zhang yq, xu jc, sun y, zhao qx, chang yg (2007). structural changes and rheological properties of dry abalone meat (haliotis diversicolor) during the process of water restoration. j. ocean univ. china, 6: 403-406. ismail h, washui y, huang y (2008). antifungal activity of aqueous and methanolic extracts from the mediterranean sea cucumber, holothuria polii. j. med. mycol., 18(1): 23-26. iso n, mizuno h, saito t, ohzeki f, yang lc (1981). an analysis of the stress-relaxation curve obtained from a sample of raw meat sample. nippon suisan gakkaish, 49: 949–952. ji tt, dong yw, shuanglin dong sl (2008). growth and physiological responses in the sea cucumber, apostichopus japonicus selenka: aestivation and temperature. aquaculture. 283: 180-187. kageyama k, watamabe y (1988). mamual of histologic techniques, igaku syoin press, tokyo. pp. 45-53. 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(in japanese) postma j, ridzwan bh, abidi az (2009). biological control of pythium aphanidermatum in cucumber with a combined application of lysobacter enzymogenes strain 3.1t8 and chitosan. biol. control, 48(3): 301-309. sato m, kunisaki n, urano n (2002). collagen as the major edible component of sea cucumber (stichopus japonicus). j. food sci., 67: 1319-1322. su xr, lou yj, chang yq (2003). study on the nutritional components and the antineoplastic activity of po1ysaccharides of sea cucumber. acta. nutri. sinica. june, 25: 181-182. taskaya l, chen y, jaczynski j (2010). 17 color improvement by titanium dioxide and its effect on gelation and texture of proteins recovered from whole fish using isoelectric solubilization/precipitation. food sci. technol., 43: 401-408. http://www.sciencedirect.com/science?_ob=articleurl&_udi=b7rn8-4s1jk8d-2&_user=431619&_coverdate=03%2f31%2f2008&_alid=874163271&_rdoc=237&_fmt=high&_orig=search&_cdi=25772&_st=4&_docanchor=&view=c&_ct=732&_acct=c000020678&_version=1&_urlversion%20 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b7rn8-4s1jk8d-2&_user=431619&_coverdate=03%2f31%2f2008&_alid=874163271&_rdoc=237&_fmt=high&_orig=search&_cdi=25772&_st=4&_docanchor=&view=c&_ct=732&_acct=c000020678&_version=1&_urlversion%20 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b7rn8-4s1jk8d-2&_user=431619&_coverdate=03%2f31%2f2008&_alid=874163271&_rdoc=237&_fmt=high&_orig=search&_cdi=25772&_st=4&_docanchor=&view=c&_ct=732&_acct=c000020678&_version=1&_urlversion%20 in ternationa l scholars journa ls african journal of food science research issn: issn 2375-0723 vol. 4 (1), pp. 001-010, january, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the detection of salmonella heidelberg in some egyptian foods sedki hegazy botany and microbiology department, faculty of science, al-azhar university, egypt (now, on secondment to college of applied medical science, majmaah university, al-majmaah 11952, p.o.box 1816, ksa). e-mail: sedki_hegazy@yahoo.com. accepted 09 july, 2015 a total of two hundred samples of egyptian foods including chicken, beef, milk, kushary and sogok (sausage) were obtained from local markets and super markets. the foods were examined to detect salmonella heidelberg. as a result, s. heidelberg were detected in 6 of 40 (15%) chicken, 2 of 40 (5%) beef, 1 of 40 (2.5%) milk, 1 of 40 (2.5%) kushary, and 4 of 40 (10%) sogok. the effects of temperature, ph, and antibiotics on growth of s. heidelberg were studied. the results indicated that 35°c was the optimum temperature and 6.8 was the optimum ph for s. heidelberg growth. antibiotic susceptibility patterns indicated that 4 out of 14 (28.5%) of s. heidelberg isolates (e5, e7, e12 and e13) were resistant to cefazolin, bacitracin, ceftazidime and sulphamethoxazole/ trimethoprim 19:1 respectively. this study emphasized that the presence of s. heidelberg in some egyptian foods under investigation, such as chicken, beef, milk, kushary and sogok (sausage), is of concern due to the fact that they have the potential to cause human infections, and are resistant to some antibiotics. so, the current control measures on animal origin foods should be improved. key words: detection, isolation, characterization, salmonella heidelberg, egyptian foods. introduction salmonellosis is a significant health problem (kaldhone et al., 2008). in 1999, there were an estimated 1.4 million cases of salmonella infections in the united states, which resulted in 17,000 hospitalizations and 585 deaths (mead et al., 1999). since then, the overall rate of salmonellosis in the united states has decreased by approximately 9%; however, the rate of salmonella enterica serovar heidelberg infections increased by 25% over the same period (cdc. 2006). s. enteric serovar heidelberg ranks fourth among serotypes causing human salmonellosis (cdc, 2005, 2008; fda, 2010) but is often the most commonly detected serotype among turkey and chicken salmonella isolates submitted to the u.s. department of agriculture’s national veterinary services laboratory (nvsl) (ferris et al., 2002; foley et al., 2008). annually, human infections with s. enterica serovar heidelberg lead to approximately 84,000 cases of salmonellosis and contribute to approximately 7% of the salmonella-related deaths in the united states, the second highest percentage after s. enteric serovar typhimurium (hennessy et al., 2004; kennedy et al., 2004; vugia et al., 2004). in addition to that, salmonella enterica serovar heidelberg ranks among the top 3 serovars isolated from persons infected with salmonella in canada (edpnml, phac, cschah, 2009), and is more frequently reported in north america than in other regions of the world (who, 2009). although many salmonella heidelberg infections result in mild to moderate illness, the bacterium also causes severe illness with complications such as septicemia, myocarditis, extraintestinal infections, and death (burt et al., 1990; vugia et al., 2004). the most common source of s. enterica serovar heidelberg infections is likely the consumption of undercooked or mishandled poultry products, such as turkey, chicken, and eggs (hennessy et al., 2004). salmonella surveillance data collected in poultry processing plants as part of u.s. department of agriculture’s pathogen reduction-hazard analysis hegazy 002 critical control point program showed that 11.4% of broiler chickens and 7.1% of turkeys carried salmonella infections in 2006 (u.s. department of agriculture, 2007). these findings, coupled with the fact that there has been a nearly fourfold increase in the per capita consumption of poultry products in the united states over the past half century (buzby and farah 2006), indicate that the contamination of poultry products with s. enterica serovar heidelberg is a major public health concern. surveillance data show that antimicrobial resistance among s. enterica serovar heidelberg isolates has been increasing. in addition to that, sources of human salmonella heidelberg infection include consumption of poultry or eggs and egg-containing products (hennessy et al., 2004; bucher et al., 2007). in canada, s. heidelberg is commonly isolated from healthy chickens from farm, abattoir, and retail sources (diarrassouba et al., 2007; phac, 2006). it has also been isolated, although less frequently, from ground beef, pork, and turkey meat (escartín et al., 2000; zhao et al., 2008) and from clinical samples from various animal species (phac, 2006). moreover, a variety of foods, including poultry, eggs, meat, milk, fruits, and vegetables, have been implicated as vehicles of one or more of these pathogens in outbreaks of food-borne illness (beuchat, 1995; d’aoust, 1997; doyle et al., 1997). the incidence of human infections by s. heidelberg increased by 20% from 1996 to 2005, even though the overall number of cases of salmonellosis decreased by 9% (cdcp, 2006). however, since 2005, the incidence of s. heidelberg infections has decreased such that in 2009, the overall incidence had decreased by 33% as compared to 1996 baseline data (cdcp, 2010). s. heidelberg infections are likely caused by the consumption of contaminated meat, poultry, eggs, or egg containing products (bucher et al., 2007; chittick et al., 2006; hennessy et al., 2004). the foodnet data indicated that the principal risk factor for s. heidelberg infections is the consumption of eggs prepared outside the home (hennessy et al., 2004). s. heidelberg appears more invasive than other gastroenteritiscausing serovars; ≈9% of isolates of this serovar received through the canadian integrated program for antimicrobial resistance surveillance (cipars) during 2003 to 2005 were recovered from blood samples (phac, 2005). treatment with antimicrobial agents may be life-saving in the case of invasive infections (dutil et al., 2010). however, data from the national antimicrobial resistance monitoring system (narms) indicates that the percentage of s. enteric serovar heidelberg isolates from humans and poultry (chicken and turkey) that were resistant to cephalosporins increased overall from 1997 to 2003. for example, in 1997, none of the isolates from humans and 1.6% of poultry isolates were resistant to ceftiofur (tio); by 2003, the numbers increased to 5.2 and 7.4%, respectively (fda, 2006). this increase in cephalosporin resistance is likely associated with the spread of the ampc _-lactamase, which is encoded by blacmy (winokur et al., 2000; zhao et al., 2003; aarestrup et al., 2004). in addition to that, the spread of multidrug resistance among s. heidelberg isolates is a risk to the management of salmonellosis in both veterinary and human clinical practice. therefore, an increased understanding of pathogen distribution and mechanisms of antimicrobial resistance transmission is important for development of strategies to limit salmonellosis due to multidrug-resistant strains (kaldhone et al., 2008). the main objective of this study is to detect s. heidelberg in some egyptian food. as such, the isolates were characterized by studying the relation of ph and temperature to growth of the isolates and their susceptibility to different antimicrobial agents. materials and methods sample collection and analysis a total of two hundred samples of egyptian foods including (quantity of samples are given in brackets): chicken (40), beef (40), milk (40), kushary (40), and sausage (sogok) (40) were used in this study. all samples under investigation were collected from local markets and super markets (solid in cairo and assuit city, egypt) in sterile plastic bags and stored at -4°c until they were analyzed. enrichment and isolation pre-enrichments and isolation were performed as a standard method for isolation of salmonella from foods. food sample (25 g) were added to 225 ml buffer peptone water (bpw) (oxoid) incubated for 24 h at 37°c. a total of 214 bacterial isolates were obtained in the form of highly pure culture from food samples under investigation (200 samples); these bacterial isolates were screened and the suspected similar ones were placed together for the purpose of selection and identification processes. 93 isolates were collected and morphologically identified as gram negative, and subjected for dropping of 0.1 ml (three drops) of pre-enrichment on to modified semi-solid reppaport vassilliadis agar (msrv) (oxoid) and incubated for 24 h at 42°c. identification presumptive salmonella colonies were sub-cultured on tryptic soya agar (tsa) (difco) plates for 24 to 48 h at 37°c in order to obtain pure culture according to the study of stefanovicova et al. (1998). sub-culturing of positive samples was done on modified brilliant green agar (mbga) (oxoid) and xylose-lysine-desoxycholate 003 afr. j. food sci. agar (xlda) (oxoid), and incubated for 24 h at 37°c. 38 out of 93 g-ve isolates were pre-identified as salmonella. biochemical tests were carried out by bbl ® entrotube tm miniaturized diagnostic kit (bacton dikinson), and salmonella serological confirmation was investigated by slid agglutinations o, h antiserum (behring), according to the study of hendriksen (2003). effect of temperature on salmonella heidelberg growth all s. heidelberg (14) isolates were inoculated into conical flasks containing nutrient broth and incubated for 24 h at different temperatures namely 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50°c. effect of ph values on salmonella heidelberg growth the ph values of nutrient broth media were adjusted by a model jenway 3020 ph meter using 1n na2oh and 1n hcl at different values (1.2, 2.3, 3.1, 4.3, 5.4, 6.8, 7.0, 8.2, 9.1, and 10.2). confirmation of measurements was carried out using ph meter. all s. heidelberg (14) isolates were inoculated into conical flasks containing nutrient broth and incubated at 35°c for 24 h. antimicrobial susceptibility test antimicrobial susceptibility of the s. heidelberg isolates (14) was studied by the disk diffusion technique on isosensitest medium by using the zone size criteria recommended by the disk manufacturer and on the basis of the breakpoints established by swedish reference group for antibiotics (srga, 1990). the following antibiotics and antimicrobial agents (oxoid) were used: cefazolin, ceftazidime, polymxin b, tetracycline, cefonicid, chloramphenicol, amoxycillin clavulanic acid (augmentin) 2:1, bacitracin, ampicillin, gentamycin, kanamycin, penicillin g, sulphamethoxazole/ trimethoprim 19:1 and nitrofurantion. results and discussion detection of salmonella heidelberg s. heidelberg is among the most commonly detected bacteria from retail meats and food animals and ranks fourth among serovars associated with human infections, causing an estimated 84,000 illnesses in the united states annually (hennessy et al., 2004; cdcp, 2008, fda, 2009). while most salmonella infections are selflimiting and resolve within a few days, s. heidelberg tends to cause a disproportionately high percentage of invasive infections (vugia et al., 2004) for which antimicrobial therapy is often warranted, making antimicrobial resistance a significant concern (han et al., 2011). the examination of food samples of animal origin for their presence has become routine all over the world. however, salmonella enterica infections are a significant public health concern worldwide, with an estimated 1.028 million cases, 19,000 hospitalizations, and 400 deaths in the united states each year (scallan et al., 2011). human salmonellosis is typically associated with the consumption of contaminated foods, such as fresh and processed meat and poultry, eggs, and fresh produce (benenson and chin, 1995; mead et al., 1999; tauxe, 1991). moreover, sources of human s. heidelberg infection include consumption of poultry or eggs and eggcontaining products (hennessy et al., 2004; bucher et al., 2007). in addition to that, salmonella surveillance data collected in poultry processing plants as part of u.s. department of agriculture’s pathogen reduction-hazard analysis critical control point program showed that 11.4% of broiler chickens and 7.1% of turkeys carried salmonella infections in 2006 (u.s. department of agriculture, 2007). it is confirmed that, poultry, meat, milk, dairy products, person-to-person spread and pet-toperson exposures have caused many outbreaks (gangarosa, 1978; todd, 1985). in the current study, 200 food samples including, chicken (40), beef (40), milk (40), kushary (40), and sausage (sogok) (40) were collected from different supermarkets and local market solid in cairo and assuit city, egypt. total viable counts (tvcs) found in the individual food samples were recorded (data not shown). 238 bacterial isolates were obtained in the form of highly pure culture, these bacterial isolates were screened and the suspected similar ones were placed together. only 93 g-ve isolates were selected and subjected for identification processes. the isolates were identified according to bergey's manual of systematic bacteriology (krieg, 1984). only 38 out of 93 of these g-ve bacterial isolates were identified from different food samples. according to the preidentification (gram's stain, cell shape and cell arrangement), the bacterial isolates (38) were identified as salmonella sp. only 14 of salmonella isolates out of 93 g-ve bacterial isolates (15%) were suggestive of being belonging to s. heidelberg; these isolates were identified as s. heidelberg according to bergey's manual of systematic bacteriology (krieg, 1984) and bergey's manual of determinative bacteriology (hensyl, 1994). all isolates were grown as well on modified semi-solid reppaport vassilliadis agar (msrv) (oxoid), modified brilliant green agar (oxoid) and xylose-lysinedesoxycholate agar (xlda) (oxoid). biochemical tests carried out by bbl ® entrotube tm miniaturized diagnostic kit (bacton dikinson) and the s. heidelberg serological confirmation were investigated by slid agglutinations o, h antiserum (behring). hegazy 004 table 1. detection of s. heidelberg in some egyptian foods. bacterial isolate foods* chicken beef milk kushary sausage (sogok) salmonella heidelberg e1 1 (2.5%) salmonella heidelberg e2 1 (2.5%) salmonella heidelberg e3 1 (2.5%) salmonella heidelberg e4 1 (2.5%) salmonella heidelberg e5 1 (2.5%) salmonella heidelberg e6 1 (2.5%) salmonella heidelberg e7 1 (2.5%) salmonella heidelberg e8 1 (2.5%) salmonella heidelberg e9 1 (2.5%) salmonella heidelberg e10 1 (2.5%) salmonella heidelberg e11 1 (2.5%) salmonella heidelberg e12 1 (2.5%) salmonella heidelberg e13 1 (2.5%) salmonella heidelberg e14 1 (2.5%) total (%) 15 5 2.5 2.5 10 * 40 samples from each food. only 14 isolates (e1, e2, e3, e4, e5, e6, e7, e8, e9, e10, e11, e12, e13, and e14) were identified as s. heidelberg by both biochemical and serological testing as mentioned before (table 1). s. heidelberg were detected in 6 of 40 (15%) chicken, 2 of 40 (5%) beef, 1 of 40 (2.5%) milk, 1 of 40 (2.5%) kushary, and 4 of 40 (10%) sogok as shown in table 1. same isolates of s. heidelberg, by several investigators, were isolated from chickens (phac, 2006; bucher et al., 2007; diarrassouba et al., 2007), beef (escartín et al., 2000; sørensen et al., 2002; zhao et al., 2006; van et al., 2007; anonymous, 2008; zhao et al., 2008), turkey (nayak and kenney, 2002; fakhr et al., 2006; kaldhone et al., 2008); poultry (chambers et al., 1998; demczuk et al., 2000; mammina et al., 2003; van et al., 2007); eggs (chambers et al., 1998; demczuk et al., 2000). in addition to that, presence of salmonella spp. was detected in meat, poultry and shellfish. thus, it highlights the considerably high prevalence of salmonella spp. in raw meat and poultry, in which 32/50 (64%) of pork samples, 31/50 (62%) of beef samples, and 16/30 (53.3%) of chicken samples were contaminated with salmonella spp. however, the rate of salmonella contamination in shellfish (18.0%) was much lower than that in meat and poultry (60.8%) (van et al., 2007). moreover, the prevalence of salmonella in beef cattle was 4.6% (11/240), and the rate was significantly higher in fasted cattle (7.46%) than in non-fasted cattle (0.94%) (abouzeed et al., 2000). s. heidelberg strains were isolated from humans, retail meats, and food animals (kaldhone et al., 2008; lynne et al., 2009; nayak et al., 2004; oloya et al., 2009; zhao et al., 2008). a total of 298 salmonella serovar heidelberg isolates were recovered, representing 21.6% of all salmonella serovars from retail meats, of which 178 (59.7%) were from ground turkey, 110 (36.9%) were from chicken breast, and 10 (3.4%) were from pork chops; moreover, none was found in ground beef (zhao et al., 2008). in addition to that, bryan and doyle (1995) reported that turkey and chicken meat can be vehicles of food borne salmonellosis because the raw product is initially contaminated with salmonella cells when it is delivered to the consumer or due to subsequent undercooking, crosscontamination, or improper thawing. all bacterial isolates were selected to study the effect of temperature, ph and antimicrobial susceptibility on bacterial growth. effect of temperature and ph on growth of salmonella heidelberg isolates earlier studies of predictive models describing the effects of environmental factors such as temperature, ph, water activity, and preservative agents on the growth, survival, or inactivation of salmonella spp. are still limited (park et al., 2007). the effect of temperature on growth of s. heidelberg isolates were studied at different temperatures including: 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50°c respectively. the results indicated that the optimal temperature for growth of all s. heidelberg isolates was 35°c, as such the increase or decrease of temperature indicates weak growth (table 2). survival populations of s. heidelberg at low-temperature (4, 7 and 10°c) showed a maximum growth of 2 logs in brain heart infusion broth (bhi) at 10°c among all the treatments (morey and http://www.ncbi.nlm.nih.gov/pubmed?term=nayak%20r%5bauthor%5d&cauthor=true&cauthor_uid=12412915 http://www.ncbi.nlm.nih.gov/pubmed?term=kenney%20pb%5bauthor%5d&cauthor=true&cauthor_uid=12412915 http://www.ncbi.nlm.nih.gov/pubmed?term=zhao%20s%5bauthor%5d&cauthor=true&cauthor_uid=19785533 http://www.ncbi.nlm.nih.gov/pubmed/22217013 http://www.ncbi.nlm.nih.gov/pubmed?term=morey%20a%5bauthor%5d&cauthor=true&cauthor_uid=22217013 005 afr. j. food sci. table 2. growth of s. heidelberg isolates at different temperatures. bacterial isolate temperature (ºc) 5 10 15 20 25 30 35 40 45 50 salmonella heidelberg e1 w + + + + +++ + salmonella heidelberg e2 w + + + + ++ + salmonella heidelberg e3 vw w + + ++ ++ + salmonella heidelberg e4 w + + ++ +++ ++ vw salmonella heidelberg e5 vw w + + ++ +++ ++ salmonella heidelberg e6 w + ++ +++ ++ + salmonella heidelberg e7 vw w + + ++ ++ + salmonella heidelberg e8 vw w + ++ ++ +++ + vw salmonella heidelberg e9 w + ++ ++ +++ + salmonella heidelberg e10 vw w + + ++ ++ + salmonella heidelberg e11 w + ++ ++ +++ ++ salmonella heidelberg e12 vw w + + ++ ++ + salmonella heidelberg e13 w + ++ ++ +++ + salmonella heidelberg e14 vw w + + ++ ++ + -, no growth; vw, very weak growth; w, weak growth; +, moderate growth; ++, good growth; +++, abundant growth; ++++, excellent growth. singh, 2012). on the other hand, the effect of ph values on growth of s. heidelberg isolates were studied at different ph values including: 1.3, 2.2, 3.2, 4.4, 5.2, 6.8, 7.1, 8.3, 9.2, and 10.0 respectively. the results indicated that the optimal ph values for growth of all s. heidelberg isolates was 6.8, as such the increase or decrease of temperature indicates decreased growth (table 3). however, there is little information about the relation of temperature, ph and growth of s. heidelberg as mentioned before. antimicrobial susceptibility antibiotic resistance of fourteen s. heidelberg isolates recovered from food samples were tested for antibiotic susceptibility against 15 antibiotics. the antibiotic resistance rates for each source and for the whole set of isolates are shown in table 4. the isolates were studied by the disk diffusion technique on iso-sensitest medium by using the zone size criteria recommended by the disk manufacturer and based on the breakpoints established by swedish reference group for antibiotics (srga, 1990). fifteen antibiotics and antimicrobial agents were applied during this study against the growth of the fourteen s. heidelberg isolates (table 4). the following antibiotics and antimicrobial agents (oxoid) were used: cefazolin, polymxin b, tetracycline, amoxycillin clavulanic acid (augmentin) 2:1, chloramphenicol, bacitracin, ampicillin, gentamicin, cefonicid, ceftazidime, kanamycin, penicillin g, sulphamethoxazole/ trimethoprim 19:1 and nitrofurantion and streptomycin, respectively. results of the antimicrobial susceptibility patterns indicated that s. heidelberg isolate (e5) was resistant to cefazolin and s. heidelberg isolate (e7) was resistant to bacitracin. s. heidelberg (e12) was resistant to ceftazidime and s. heidelberg isolate (e13) was resistant to sulphamethoxozole/trimethoprim 19:1 as shown in table 4. briefly, in the current study, the results indicated that of the 4 out of 14 isolates, 28.5% were resistant to four antimicrobial agents tested and of the 10 out of 14 isolates, 71.5% were susceptible to other eleven antimicrobial agents under investigation (table 4). in the same way, 42% of the isolates were resistant to at least 1 of the 15 antimicrobial agents tested, and 4% of the isolates were resistant to 8 or more antimicrobial agents. resistances to streptomycin (32%), tetracycline (30%), and kanamycin (24%) were most commonly detected (kaldhone et al., 2008). conversely, the resistance rates were lower than those observed for narms isolates from turkey and ground turkey, where typically more than 50% of isolates in 199 of 298 isolates (67%) were resistant to at least one antimicrobial agent, with 16.4% (n _ 49) of the isolates being resistant to at least five antimicrobials. six isolates (3.0%), all recovered from ground turkey, were resistant to at least nine antimicrobials, including beta-lactams, aminoglycosides, chloramphenicol, sulfamethoxazole, and tetracycline. for chicken and turkey isolates combined, resistance to tetracycline was most common (39.9%), followed by streptomycin (37.8%), sulfamethoxazole (27.7%), gentamicin (25.7%), kanamycin (21.5%), ampicillin (19.8%), amoxicillinclavulanic acid (10.4%), cefoxitin (9.0%), and ceftiofur (9.0%). rare isolates were resistant to chloramphenicol http://www.ncbi.nlm.nih.gov/pubmed?term=singh%20m%5bauthor%5d&cauthor=true&cauthor_uid=22217013 hegazy 006 table 3. growth of s. heidelberg isolates in relation to different ph values. bacterial isolate ph values 1.3 2.2 3.2 4.4 5.2 6.8 7.1 8.3 9.2 10.0 salmonella heidelberg e1 salmonella heidelberg e2 salmonella heidelberg e3 salmonella heidelberg e4 salmonella heidelberg e5 salmonella heidelberg e6 salmonella heidelberg e7 salmonella heidelberg e8 salmonella heidelberg e9 salmonella heidelberg e10 salmonella heidelberg e11 salmonella heidelberg e12 salmonella heidelberg e13 salmonella heidelberg e14 + + +++ ++ + + + +++ ++ + + + +++ ++ + + + +++ ++ + + + +++ ++ + + + +++ ++ + + + +++ ++ + + + +++ ++ + + + +++ ++ + + + +++ +++ + + + +++ ++ + + + +++ ++ + + + +++ ++ + + + +++ +++ + -, no growth; +, moderate growth; ++, good growth; +++, abundant growth; ++++, excellent growth. table 4. susceptibility of some antibiotics against s. heidelberg isolates. antibiotics bacterial isolate salmonella heidelberg e1 salmonella heidelberg e2 salmonella heidelberg e3 salmonella heidelberg e4 salmonella heidelberg e5 salmonella heidelberg e6 salmonella heidelberg e7 salmonella heidelberg e8 salmonella heidelberg e9 salmonella heidelberg e10 salmonella heidelberg e11 salmonella heidelberg e12 salmonella heidelberg e13 salmonella heidelberg e14 s 1 : sensitive; r 2 : resistant. c e fa z o li n p o ly m x in b t e tr a c y c li n e a m ox yc ill in cl av ul an ic ac id (a ug m en tin )2 :1 c h lo ra m p h e n ic o l b a c it ra c in a m p ic il li n g e n ta m ic in c e fo n ic id c e ft a z id im e k a n a m y c in p e n ic il li n g s ul ph am et ho xo zo le /t ri m et ho pr im 1 9: 1 n it ro fu ra n ti o n s tr e p to m y c in s 1 s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s r 2 s s s s s s s s s s s s s s s s s r s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s r s s s s s s s s s s r s s s s s s s s s s s s s s s s s s s s s s s s (1%), nalidixic acid (1.0%), and trimethoprimamikacin, ceftriaxone, and ciprofloxacin (zhao et al., sulfamethoxazole (0.7%). all isolates were susceptible to 2008). 007 afr. j. food sci. fifty-eight salmonella enterica serovar heidelberg isolates isolated from food animals were tested for antimicrobial susceptibilities and further characterized for selecting antimicrobial resistance genes, plasmid carriage, class 1 integrons, and genetic relatedness using pulsed-field gel electrophoresis (pfge). seventy-two percent of isolates displayed resistance to at least one of the antimicrobial agents tested, while 24% exhibited resistance to eight or more antimicrobial agents. resistance was most commonly observed to tetracycline (71%), streptomycin (62%), and kanamycin (52%). isolates obtained from cattle and swine displayed the highest rates of resistance while isolates from chickens more often displayed susceptibility to the tested antimicrobials (lynne et al., 2009). in addition to that, patchanee et al. (2008) reported that all s. heidelberg isolates from swine were resistant to one or more of the antimicrobials tested and the majority (73.3%) showed multidrug resistance to streptomycin, tetracycline, and kanamycin (r-type: sttekm). about 80% of the s. heidelberg isolates of human origin were pan-susceptible, however, one isolate showed multidrug resistance to 10 of the 12 antimicrobials tested. on the other hand, some researchers were of the opinion that s. heidelberg was resistant to many antimicrobial agents including, cephalosporin (folster et al., 2010), quinolone (boscán-duque et al., 2007), ceftiofur and nalidixic acid (donado-godoy et al., 2012), gentamicin, sulfamethoxazole, tetracycline, kanamycin, and streptomycin (fakhr et al., 2006), gentamicin, spectinomycin, streptomycin, tetracycline (nayak and kenney, 2002), gentamicin, streptomycin and sulfisoxazole (abouzeed et al., 2000). this study emphasized that the presence of s. heidelberg in some egyptian foods under 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international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of two different solvent extracts of colpomenia sinuosa in vitro antioxidant activity using standard procedures lekameera. r, p. vijayabaskar and s. t. somasundaram* centre of advanced study in marine biology, annamalai university, parangipettai –608 502, tamilnadu, india. accepted 15 july 2016 the brown alga, colpomenia sinuosa was subjected to dpph (1,1diphenyl-2-picryl hydrazyl), nitric oxide radical, hydrogen peroxide, scavenging of abts radical and total antioxidant capacity (tac) inhibitory assays to assess its antioxidant property. the dimethyl sulphoxide (dmso) and methanol extracts showed highest antioxidant activity in dpph (96.56 and 88.57%), nitric oxide radical (81.2 ± 4.1% and 76.69 ± 3.1 %), hydrogen peroxide (70.7 ± 3.5 and 56.6 ± 2.0%) and abts (76.8 ± 3.8% and 74.0 ± 2.8%). the moderate activity was observed in total antioxidant capacity (60.3 ± 4.3 and 57.0 ± 2.0) assay. the activity was higher and comparable to that of commercial antioxidants butylated hydroxy anisole (bha) (87.38  1.32%) and butylated hydroxy tolune (bht) (56.05  0.19%) at 2 mg/ml concentration. the significant free radical scavenging activity of c. sinuosa indicated that it could be a potential source for natural antioxidant lead molecules. key words: abts, colpomenia sinuosa, dpph, hydrogen peroxide, nitric oxide radical. introduction a free radical is a molecule with one or more unpaired electrons in the outer orbital. many of these free radicals, in the form of reactive oxygen and nitrogen species, are an integral part of normal physiology. the importance of reactive oxygen species and free radicals has attracted increasing attention over the past decade. an over production of these reactive species can occur, due to oxidative stress brought about by the imbalance of the bodily antioxidant defense system and free-radical formation (wong et al., 2000). reactive oxygen species (ros) such as superoxide radical (o2 •– ), hydroxyl radical (oh • ), peroxyl radical (roo • ) and nitric oxide radical (no • ), attack biological molecules, such as lipids, proteins, enzymes, dna and rna, leading to cell or tissue injury associated with aging, atherosclerosis carcinogenesis (keli-chen et al., 2005) and may lead to the development of chronic diseases related to the cardio and cerebrovascular systems (halliwell and gutteridge, 1989). *corresponding author. e-mail: baski_bos@yahoo.co.in, baski_pan@rediffmail.com. tel: +91 4144 243223, 243070, ext. 251. fax: +91 4144 243555 free-radical scavengers are antioxidants which can pro-vide protection to living organisms from damage caused by uncontrolled production of reactive oxygen species and subsequent lipid peroxidation, protein damage and dna strand breaking (ghosal et al., 1996). the most commonly used synthetic antioxidants presently arebuty-lated hydoxyanisole (bha), butylatedhydoxytoluene (bht) propylgallate (pg) and test butylatedhydroquinone. however, these synthetic antioxidants have side effects such as liver damage and carcinogenesis (wichi, 1988; sherwin et al., 1990). therefore, there is a need for isolation and characterization of natural antioxidant having less or no side effects, for use in foods or medicinal materials to replace synthetic antioxidant. marine algae have received special attention as a source of natural antioxidants (matsukawa et al., 1997). seaweeds are known source of pharmacological and food additives with potential health effects like antioxi-dative and anticarcinogenic (lim et al., 2002; athukurake et al., 2003). based on the above facts, the brown seaweed colpomenia sinuosa (derb et sol) was studied for its potential antioxidant property. in the present investigation two different solvent extracts of c. sinuosa were investigated in vitro antioxidant activity using standard procedures. lekameera et al. 232 figure 1. scavenging activity of colpomenia sinuosa extract on dpph. materials and methods the brown alga c. sinuosa (derb et sol) (phaeophyta: phaeophyceae: dictyosiphonales: punctariaceae) was collected from tuticorin coast, tamil nadu, india (lat. 8º45’ n; long. 78º10’ e) and identified by the method described by umamaheshwara rao (1987). the fresh seaweed was rinsed with fresh water, air dried in shade at 60 o c and pulverized into a fine powder. 10 g of seaweed powder was extracted sequentially with 100 ml of diethyl ether (98%) and methanol (99%) in a soxhlet extractor for six hours and was repeated twice (lim et al., 2002). the crude extracts were then dried in vacuum and the resultant residues were stored in dark at 4 º c until further use. subsequently, the diethyl ether residue was dissolved in dimethyl sulfoxide (dmso) and the methanol residue in methanol and subjected to the following assays to assess the antioxidant potential. dpph radical scavenging assay dpph (1,1-diphenyl-2-picryl hydrazyl) radical scavenging was assayed as described by hwang et al. (2001). 10 l of the seaweed extract with 0.2 ml of dpph (100 µm) (sigma-aldrich) in methanol solution was incubated at 37ºc for 30 min and the absorbance of the supernatant was measured at 490 nm using elisa micro plate reader (bio rad laboratories inc., california, usa, model 550). activity calculation was done as follows: radical scavenging activity (%) = [(a0 – a1 / a0) x 100] ic50 value concentration of the sample required to scavenge 50% dpph free radical. nitric oxide radical inhibition assay nitric oxide radical inhibition was assayed by incubating 2 ml sodium nitroprusside (10 mm), 0.5 ml phosphate buffer saline and 0.5 ml (0.25 mg) of extract solution at 25ºc for 150 min. then 1 ml of sulfanilic acid reagent (0.33% sulfanilic acid in 20% glacial acetic acid) was added to 0.5 ml of reaction mixture for 5 min to complete diazotization, subsequently. 1 ml naphthyl ethylene diamine dihydrochloride (nedd) was added and allowed to stand for 30 min at 25ºc. the absorbances of these solutions were measured at 540 nm (govindarajan et al., 2003; badami et al., 2005). hydrogen peroxide radical scavenging assay ability of the seaweed extracts to scavenge hydrogen peroxide was determined as described by govindarajan et al. (2003) and gulcin et al. (2004). one ml (0.25 mg) of the extract was rapidly mixed with 2 ml of 10 mm phosphate buffered (0.1m, ph 7.4) hydrogen peroxide solution. the absorbance was measured at 230 nm in the uv spectrophotometer after 10 min of incubation at 37ºc (shimazdu, uv160a) against a blank (without hydrogen peroxide). the percentage of scavenging of hydrogen peroxide was calculated using the following formula. (a0 – absorbance of control; a1 – absorbance of sample) abts [2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation scavenging assay free radical scavenging activity of the extract was also determined by abts (sigma-aldrich) radical cation decolourization assay (re et al., 1999). abts radical cation was generated by mixing 20 mm abts solution with 70 mm potassium peroxodisulphate and allowing it to stand in the dark at room temperature for 24 h before use. 0.6 ml of extract (0.25 mg) was mixed with 0.45 ml of abts reagent and absorbance of these solutions was measured at 734 nm after 10 min. total antioxidant activity total antioxidant activity was measured by following the method of mitsuda et al. (1996). 7.45 ml sulphuric acid (0.6 m solution), 0.9942 g sodium sulphate (28 mm solution) and 1.2359 g ammonium molybdate (4 mm) were mixed together in 250 ml with distilled water and labeled as a total antioxidant capacity (tac). 100 l of extract was dissolved in 1 ml of tac absorbance was read at 695 nm after 15 min. results and discussion dietary natural antioxidants are reported to help in preventing aging and other diseases. there are some evidences that seaweeds contain compounds with a relatively high antioxidant and antiproliferative activity. seaweeds are low in fats but contain vitamins and bioactive compounds, like terpenoids, sulfated polysaccharide and polyphenolic compounds, the latter being a potential natural antioxidant not found in land plants (lahaye and kaffer, 1977). dpph radical scavenging assay the increase in scavenging activity of c. sinuosa extracts on dpph radicals was concentration dependent (figure 1). the dmso and methanol extract exhibited a strong scavenging activity on dpph (96.56 and 88.57%) at 0.25 mg concentration (figure 2). inhibition above 50% was 233 afr. j. food sci. res. figure 2. dpph radical scavenging activity of methanol and dmso dissolved diethyl ther extracts (0.25 mg/ml) compared with commercial antioxidants (2 mg/ml). dpph dpph radical scavenging assay; bhtbutylated hydroxy tolune; bha-butylated hydroxy anisole; d-dmso dissolved diethyl ether extract; mmethanol extract. figure 3. nitric oxide radical inhibiting activity of colpomenia sinuosa.d-dmso dissolved diethyl ether extract; m-methano. observed for both dmso dissolved and methanol extracts at a concentration of 0.05 mg. the ic50 values for dmso dissolved and methanol extracts were 0.03 and 0.0275 mg/ml, respectively (figure 7). the scavenging activity was greater than that of commercial antioxidant bha and bht at 2 mg/ml concentration (badami et al., 2005; heo et al., 2005). the ic50 values in the present study are comparable to that of organic and aqueous fractions of brown alga ecklonia cava. it was also found that the ic50 value of c. sinuosa was higher than that of bht but less than that of –tocopherol (senevirathane et al., 2006). the study of yuan et al. (2005) revealed that the ic50 values of palmaria palmate extract was lower than that of c. sinuosa extract. however, the extractions of active ingredients depend upon various polarity nature of the solvent system and the quantum of the active principles in the particular resources. it was also found that dpph scavenging activity of bha and bht (gulcin, 2005) are comparable to the activity of c. sinuosa extract. the ic50 activity of dmso dissolved and methanol extract of c. sinuosa was comparatively stronger than ethanolic extract of nori, kombu, wakame, hijiki and the known antioxidant vitamin e (ismail and hang, 2002). nitric oxide radical inhibition assay nitric oxide is generated from amino acid l-arginine by vascular endothelial cells, phagocytes and certain cells in the brain (moncada et al., 1991). the toxicity and damage caused by no• and o2• is multiplied as they react to produce reactive peroxynitrite (onoo ), which lead to serious toxic reactions in the biomolecules, like proteins, lipids and nucleic acids (moncada et al., 1991; yermilo et al., 1995). scavengers of nitric oxide compete with oxygen and lead to the production of nitric oxide (marcocci et al., 1994). the present investigation on c. sinuosa extract (dmso and methanol) shows better activity (figure 3). c. sinuosa extract at 8.3 mg/ml concentration showed an inhibition of 81.2 ± 4.1 (dmso dissolved) and 76.69 ± 3.1 (methanol). the ic50 values for both the extracts were 2.6 and 2.7 mg/ml, respectively (figure 7). these ic50 values of nitric oxide radical assay was comparable to the ic50 values of cassia fistula (linn) bark extract as reported by raju ilavarasan et al. (2002). badami et al. (2005) reported that, the ic50 value of ethyl acetate, petroleum ether and methanol extracts of aporosa lindleyana root inhibit the nitric oxide generation. the suppression of no• release may be partially attributed to direct scavenging by both extracts of c. sinuosa, which decrease the amount of nitrite generated from the decomposition of sodium nitroprusside in vitro (senevirathene et al., 2006). hydrogen peroxide radical scavenging assay many species of seaweed possess scavenging ability of hydrogen peroxide (athukurake et al., 2003; siriwardhana et al., 2003). the measurement of h 2o2 scavenging activity is one of the useful methods of determining the ability of antioxidants to decrease the level of pro-oxidants such as h2o2 (czochra and widensk, 2002). it can cross membranes and may slowly oxidize a number of compounds. hydrogen peroxide itself is not very reactive, but sometimes it can be toxic to cells because of rise in the hydroxyl radicals in the cells (gulcin, 2006). the inhibitive effect of seaweed extract was subjected to hydrogen peroxide scavenging assay and was found to be moderate (figure 4) when compared to other assays. the ic50 values were 2.7 and 4.2 mg/ml for dmso dissolved and methanol extracts, respectively (figure 7). lekameera et al. 234 figure 4. hydrogen peroxide radical scavenging activity of colpomenia sinuosa. bhtbutylated hydroxy tolune; bhabutylated hydroxy anisole; t--tocopherol; d-dmso dissolved diethyl ether extract; m-methanol extract; *bha, bht, t (-tocopherol) 75 mg/ml. figure 5. abts radical cation scavenging activity of colpomenia sinuosa. d-dmso dissolved diethyl ether extract; m-methanol extract. the inhibition of 70.7 ± 3.5% and 56.6 ± 2.0% observed for dmso dissolved and methanol extracts at 8.8 mg/ml concentration were lower than the activity of commercial antioxidants of bha and bht and – tocopherol (heo et al., 2005). these results indicate the antioxidant molecular leads dissociate in different proportions with differrent solvent system. nevertheless c. sinuosa proved to possess the vital antioxidant lead molecules. abts [2, 2’-azinobis-(3-ethylbenzothiazoline-6sulfonic acid)] radical cation scavenging assay the abts radical reactions involve electron transfer and the process take place faster rate when compared to dpph radicals. re et al. (1999) reported that the decolorization of the abts• + radical also reflects the capacity figure 6. total antioxidant activity of colpomenia sinuosa. bhtbutylated hydroxy tolune; bha-butylated hydroxy anisole; t--tocopherol; d-dmso dissolved diethyl ether extract; m-methanol extract; *bha, bht, t (-tocopherol) 75 µg/ml. of an antioxidant species to donate electrons or hydrogen atoms to inactivate this radical species. in the abts radical cation scavenging activity, the seaweed c. sinuosa extract showed concentration dependent scavenging activity (figure 5). the dmso dissolved extract showed activity of 76.8 ± 3.8% and methanol extract showed activity of 74.0 ± 2.8% for the concentration of 4 mg/ml. the ic50 values were 1.4 and 1.1 mg/ml for dmso dissolved and methanol extracts, respectively (figure 7). katalinic et al. (2006) found that, in this assay, abts radical cation was generated directly in stable form using potassium persulfate. generation of radical before the antioxidants added prevents interference of compounds, which affect radical formation. this modification makes the assay less susceptible capacity. the extracts of c. sinuosa showed higher scavenging activity compared to grape seed extract (keli-chen et al., 2005). abts was used to reconfirm the antioxidant property of the seaweed extract c. sinuosa. total antioxidant activity the total antioxidant activity of extract was calculated based on inhibition percentage of 60.3 ± 4.3 and 57.0 ± 2.0, respectively (figure 6). the antioxidant activity increases with increasing concentration. the ic50 values were 0.8 and 0.9 mg/ml for dmso dissolved and methanol extracts (figure 7). however, the c. sinuosa extracts showed lesser activity than the standards of bha, bht and -tocophorol. conclusion all these results denote that c. sinuosa could be an important source of antioxidant molecules.further studies are required to elucidate the compound showing antioxi 235 afr. j. food sci. res. figure 7. ic50 values for colpomenia sinuosa in different assays. dpph dpph radical scavenging assay; nora-nitric oxide radical scavenging assay; hpra-hydrogen peroxide radical scavenging assay; taa-total antioxidant assay; d-dmso dissolved diethyl ether extract; m-methanol extract. dant property in vivo and the compound could evolve as an anticancer drug in near future. acknowledgements the authors are thankful to prof. t. balasubramanian, director, cas in marine biology, annamalai university, parangipettai and to dr. s. badami, reader, jss college of pharmacy, ootacamund for the facilities provided in carrying out this work. references athukurake y, lee kw, song cb, ahn cb, shin ts, cha yj, shahidi f, jeon yj (2003). potential antioxidant activity of marine red alga grateloupia filicina extracts. j. food lipids. 10: 251-265. badami s, rai rs, suresh b (2005). antioxidant activity of aporosa lindleyana root. j. ethnopharmacol. 37(35): 1-5. czochra mp, widensk a (2002). spectrophotometric determination of hydrogen peroxide scavenging activity. j. anl. chemic. acta, 452: 177184. ghosal s, tripathi vk, chaeruhann s (1996). active constituents of emblica officinalis. part i. the chemistry and antioxidative effects of two new hydrolysable tannins, emblicanin a and b. indian j. chem. 35: 941-948. govindarajan r, rastogi s, vijayakumar m, rawat aks, shirwaikar a, mehrotra s, pushpangadam p. 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(1995). formation of 8-notroguanine by the reaction of guanine with peroxynitrite in vitro. carcinogenesis. 16: 2045-2050. yuan vy, bone ed, carrington fm (2005). antioxidant activity of dulse (palmaria palmate) extract evaluated in vitro. food chem. 91: 485494. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (1), pp. 063-071, january, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a survey of the iodine content of school-children diets fortified with marine fish amund maage 1 *, jogeir toppe 1,2 , mathilda steiner-asiedu 3,4 , ebenezer asibey berko 3 and einar lied 1,5 1 national institute of nutrition and seafood research, box 2029 nordnes, n-5817, bergen, norway. 2 fao, fisheries and aquaculture department, viale delle terme di caracalla, 00153 rome, italy. 3 department of nutrition and food science, university of ghana, legon, ghana. 4 brown university, bio-med institute of community health promotion, providence 02912, ri, usa 5 nutrimarine life science as, bontelabo 2, n-5003 bergen, norway. accepted 11 december, 2016 the present study was carried out in an iodine deficient area in northern ghana, and the purpose of the study was: (1) to undertake a food dietary survey on school children in an iodine deficient area, (2) measure the iodine content in the staple food of this area, (3) carry out an intervention study with a traditional food supplemented with marine fish as a natural iodine source. sixty school children, in the village of sekoti in the upper-east region of ghana, participated in the study. the dietary survey was performed as repeated 24 h recall, and staple foods were analysed for iodine by icp-ms. a traditional diet fortified with 10% codfish-powder was given to the children every day for two weeks, and iodine status was measured before and after the intervention. the foodstuffs normally eaten by the children contained little iodine. however, beans prepared at a market nearby had surprisingly high values of iodine, explained by the addition of saltpetre, which contained very high amounts of iodine. about 60% of the children in the study area had goitre and the prevalence was highest among girls. following the intervention period, the urinary iodine concentration was significantly increased and the tsh level in serum was significantly decreased, indicating an elevated iodine status. the children having goitre had the strongest tendency to elevate their iodine status. key words: iodine, fish, goitre, urinary iodine, tsh. introduction iodine deficiency disorders (idd) continue to be a major health problem in the world, particularly in third world countries. globally, the prevalence of goitre in 1999 was estimated to be 13% (who/unicef/iccidd, 2001). however, the prevalence of idd is likely to be greater than this. results based on prevalence of urinary iodine (ui) below 100 µg/l, estimates that 36.5% of school children worldwide have an inadequate iodine nutrition. this number is 42.3% in africa and as high as 59.9% in europe (who, 2004). in 1998, asibey-berko and co-workers reported that goitre rate of children (8 14years) in sekoti (northern ghana) was 59.1% and median urinary iodine for both sekoti and buila (also in northern ghana) was 16 µg/l. a national survey in 2001 found that *corresponding author. e-mail: ama@nifes.no. the median urinary iodine was 196 µg/l, while the national goitre rate was 20% with a range of 7% to 30% (nseid 2007). the most severe consequence of idd is irreversible brain damage, which can affect up to 5 15% of a population in severely affected areas (who, 2004). iodine deficiency is the single most important cause of preventable mental handicap in the world by the turn of the century (who/unicef/iccidd, 2001). the physiological function of iodine is as part of the thyroid hormones thyroxin (t4) and the more active form triiodotyronine (t3), which influences metabolism in all cells, including stimulation of growth and development of the central nerve system (delange and fisher, 1995). the recommendations of dietary iodine given by nrc (1989) were critically reviewed by delange (1993) and the recommendations were set at 90 µg/day for children 0 to 6 years and 120 µg/day for children 6 to 12 years. for all persons older than 12 years the recommendation is 150 maage et al. 064 µg/day. however 200 µg/day is recommended for pregnant and lactating women (who/unicef/iccidd, 2001). several indicators are used to assess idd in individuals and communities, including the size of the thyroid gland and urinary iodine. the size of the thyroid gland is inversely correlated with the average iodine intake, and indicates a person's iodine status (who/unicef/iccidd, 2001). urinary iodine excretion is a commonly used parameter to evaluate iodine status, showing a strong correlation with the iodine intake (vought et al., 1963). blood constituents are also used to assess the iodine status. the level of thyroid-stimulating hormone (tsh) and thyroglobulin (tg) increases when the iodine intake is low (delange, 1995). in 1992 united nations put forward a plan to eradicate iodine deficiency as a major health problem within the year of 2001 (fao/who, 1992) through increasing: 1) iodine fortification of foods and food ingredients such as salt, water and flour; 2) distribution of iodine capsules in areas where idd is severe, 3) use of iodine rich nutrients, like seafood, and decreasing the consumption of goitrogens. although the last decade has seen significant progress in combating idd, particularly in africa and asia, the total number of people affected by idd is still high. accoriding to who/unicef/iccidd (2001) the idd problem was underestimated in earlier studies and eradication policies have not been as effective as expected. iodine is normally obtained from food but most foods are poor sources of the element with the exeption of sea foods which are good sources of iodine (lee et al., 1994; coultate, 1996; eckhoff and maage, 1997; hou et al., 1997; karl et al., 2001; rose et al., 2001). the iodine content in fish is typically in the range of 300 to 1000 g/kg while most vegetable product had less than 30 g/kg. marine fish species low in fat, like cod and saithe have the highest iodine contents (dahl et al., 2004). in some africa countries iodine deficiency has been a common problem, especially in areas remote from the sea, and northern ghana has been no exception with reports on high goiter prevalence (asibey-berko, 1995). the recognition of severe idd problems in ghana prompted the campaign to use iodized salt. however, household iodized salt use has been low and estimated at about 28 % as of 1998 (nseid, 2007) . it is therefore also important to study further measures to improve the iodine status in communities. the present study was undertaken to study iodine nutrition and the possible use of marine foods in an intervention to improve this situation. the aim of this study was: 1) to perform a small dietary survey on school-children. 2) to collect staple foods in the study area, and measure the iodine content of these foodstuffs. 3) to perform an intervention study to evaluate changes in iodine status in school children, following a period of consuming of traditional foods fortified with marine fish. method study area and subjects sekoti, a village 35 km east of bolgatanga in the upper -east region, ghana, was chosen for the study since it is noted for rates of idd (asibey-berko, 1995). sixty school children ages 10 to 12 years (23 girls and 37 boys) were randomly selected and participated in the study. the study was approved by an institutional review board and all parents provided informed consent for their children after the study has been explained to them in the local language. the village chief together with his elders permitted the study to be carried out in the village. the study was carried out from february 26th to march 12th 1996. dietary assessment to obtain information on the iodine intake of the children, it was necessary to conduct dietary evaluation. a repeated 24 h recall was conducted each morning with each child to obtained information on the food eaten the previous day. two trained graduate nutrition students interviewed the children using open ended questionnaire. two teachers in the village were given a short orientation and served as interpreters for the period. the interviews went fast since food choices in the village are limited. samples of food portions were shown to the children to help them quantify the amount eaten. children were asked the type of foods eaten as a composite or single food item. the questionnaire covered all foods eaten through out the day, that is, breakfast, lunch dinner and all snacks. the dietary assessment was necessary since it was the key to knowing what the participants are eating to be able to determine the iodine content of the foods. both raw food materials and prepared meals eaten by the participants were collected in sekoti, or at the market in bolgatanga for iodine analysis. this was necessary since using literature values for iodine contents of the foods might not give true estimates. the materials sampled were stored in polyethylene bags, frozen transportation to the university of ghana, legon. the samples were later air freighted to nifes, bergen, norway, where they were freeze-dried, homogenised and stored in 50 ml nunc-cups prior to analysis intervention diet the intervention diet was fermented maize/fish blend and this provided each child 460 µg/day of iodine. table 1 profiles the iodine content in the raw food materials as well as the prepared meals. white maize meal (zea mays) was bought from indus, birmingham, uk. for each 5 kilos of maize meal, 4 l of tap water and 1 l of abc milk (tine norske meierier, bergen, norway), containing the bacteria lactobacillus acidophilus (a), bifidobacterium bifidum (b) and lactobacillus casei (c), were mixed to a dough in a food processor (varimixer bjørn 93/ar60, brøndby, denmark). the dough was then transferred to aluminium trays (20 l), covered with polyethylene foil and fermented at 30ºc for 48 h, until a stable ph of 3.7 was reached. the fermented product was frozen at -20ºc, then vacuumdried and fortified with 10% (w/w) codfish powder if saithe, gadus virens (toro a/s, rieber and søn, bergen, norway): the maize/fish blend was pulverised, bagged into polyethylene bags and packaged into boxes and air freighted to accra, ghana. the product was stored at the nutrition and food science department, university of ghana prior to the start of the intervention program. the fish maize blend was used to prepare breakfast (8 a.m.) and lunch (1 p.m.) for the children. breakfast was a popular porridge table 1. dry matter and content of nutrients analysed of raw materials and prepared meals of the intervention diet (dry matter base). foodstuff dry matter (%) protein ¹(%) fat¹ (%) energy¹ mj/kg) iodine (n)(mg/kg) codfish powder 95 20.5 (3) fermented maize meal 96 0.039² (4) fermented maize meal + 10 % codfish 4 15.5 3.4 17.6 2.13 (4) koko, prepared 18 12.3 0.7 17.3 1.34 (2) banku, prepared 34 15.5 1.0 1.82 (2) banku and gravy, prepared 32 15.5 6.4 18.0 1.73 (2) ¹analysed by skov (1997). ²abc milk (tine norske meierier, bergen, norway) added contained some iodine. called koko and each child received about 500 ml (2 cups) . koko was made of 4.5 kg of the raw material mixed with 30 litres of water. the mixture was boiled for about 30 min added 1.5 kg of sugar, and then distributed equally into 60 cups. lunch was also a popular dumpling called banku and each child received about 600 g. banku was made by mixing 12.0 kg of the maize/fish blend with 25 l of water, added salt (uniodized) to taste and cooked for about 1 h with constant stirring and kneading. banku was served with gravy made of 6 kg of tomatoes, 0.6 litres cooking oil (frytol), red pepper and salt (not iodised). the preparation and the serving of the food were under the supervision of a home economist/nutritionist. children ate all the food served at any given time. data collection baseline and follow-up data collection were done in the mornings before breakfast. demographic data collected were age and sex. anthropometric measures included mid-upper arm circumference as well as height and weight for body mass index determination. physical appraisal by palpation was done by an experienced person to determine goitre grades in the children (delange, 1995). physiological samples collected were urine and blood. the field conditions allowed for spot collection of urine. the urine samples were collected in 20 ml polyethylene tubes and stored at -20ºc. blood samples were collected as drops of whole blood on filter paper (schleicher and shuell, grade 903), air dried, put into a polyethylene bag, covered with aluminium foil and then stored at 20ºc. analyses the food, water and urine samples where analysed for iodine by inductively coupled plasma-mass spectrometry (icp-ms) type perkin elmer elan 5000 a icp-ms (ontario, canada) using tellurium as internal standard. the method is decribed in eckhoff and maage (1997) and further in julshamn et al. (2001). two replicate portions (0.2 g each) of the dried and pulverised food samples were added nitric acid and perhydrol before digestion in a microwave oven (milestone1200 mega, sorisole, italy), following addition of internal standard and then diluted to 25 ml. immediately before the icp-ms analysis the digested samples were diluted 1:2 with a 3% nh3 solution. the water samples brought from ghana were not digested but some droplets of concentrated nh3 were added before analyses. the urine sam-ples were thawed overnight at 18ºc. the internal standard was then added and the sample diluted 1:10 with 1% hno3 prior to the icp-ms analysis. the detection and quantification limit of the method was determined to be 0.2 and 0.6 g/l respectively. for analytical quality control of the iodine analyses, the standard reference materials (srm) 1566a oyster tissue from nist, usa and bcr 129 hay powder from bcr, belgium were used. those represented a wide range of iodine concentrations from 167 ± 24 g/kg in the hay to 4460 ± 420 in the oyster tissue. our analyses of the reference material were well within the uncertainty of the srm. since there at the time of analyses were no certified reference materials for iodine in urine, a recovery test was also performed with good results. the blood samples, collected on filter paper, where analysed by using the enzyme-linked immunosorbant assay (elisa) methodology. a tsh assay kit (spectrascreen dried blood tsh eia kit, eim diagnostics, santee, ca, usa), based on a standardised method (tseng et al., 1985; miyai et al., 1981) was used for the analysis. statistical procedures to find significant differences prior to and following the intervention period different tests for dependent variables were used. the level of significance is p < 0.01 if nothing else is mentioned. the distribution of the variables was visually checked for normality by using categorised normal probability plot. the results are given as medians and quartiles when the variables were not normally distributed. the result from normally distributed variables is given as means and standard deviations. the two variables iodine in urine and tsh in serum did not show normality, the nonparametric wilcoxon matched pairs test was therefore used to test if the changes were significant after the consumption of the intervention diet. for the normally distributed variables weight and upper arm circumference, student's t-test for dependent variables was used to check if the changes were significant. to test differences between the independent variables boys and girls mann-whitney u-test was used for the variables showing no normal distribution, and student's t -test for independent variables for the normally distributed variables. when looking at the iodine excretion in urine, and tsh level in blood, the differences between groups graded according to the grade of goitre, were tested by the non-parametric kruskal-wallis anova median test. result dietary habit during the 14 days intervention period, the food mostly consumed by the children other than the intervention diet was millet/rice balls (“tou zaafi”-tz) and this was 34% of the food-volume consumed. this was followed by rice which contributed 19%, and then tubani (cowpea paste) which contributed 15% of the food eaten during the 065 afr. j. food sci. res. maage et al. 066 table 2. dry matter, energy and iodine analysed in raw materials and prepared meals of the staple foods (dry matter base). data are presented as means of to parallel determinations. dry matter energy iodine place obtained (%) (kj/g) (µg/kg) yam dioscorea sp., roasted 58.7 15.9 63 bolgatanga, market gari (cassava masihot utilissima), raw 93.6 16.1 <16 bolgatanga, market cowpea beans vigna ungiculata, raw 95.6 25 bolgatanga, market bambara beans phaseolus sp., raw 95.4 19 bolgatanga, market rice oryza sativa, raw 92.1 16.0 4 <16 sekoti, market beans vigna ungiculata, prepared ¹ 27.2 18.1 234 bolgatanga, market rice/beans, prepared ¹ 35.4 100 bolgatanga, market sorghum sorghum vulgare, prepared 31.5 17.1 48 sekoti, school fufu (cassava + plantain musa paradisica), prepared 26.6 16.9 46 bolgatanga, bus-station kenkey (maize zea mayz), prepared 32.0 17.7 102 bolgatanga, market tubani (beans), prepared ² 41.2 19.0 74 sekoti, school tubani (beans), prepared ¹ 45.4 19.7 538 bolgatanga, market tz (millet pennisetum americanum), raw ³ 88.2 45 bolgatanga, market tz (millet), prepared ³ 19.9 17.0 93 bolgatanga, market tz (millet), prepared ³ 23.3 18.8 110 bolgatanga, restaurant okra hibiscus esculentus, dried 96.6 17.4 518 bolgatanga, market tombrown (maize + cowpea), roasted 95.1 18.0 400 bolgatanga, market tomatoes lycopercsicon esculentum, prepared 27.2 949 sekoti, school groundnutpaste arachis hypogaea, raw 93.2 20.9 135 bolgatanga, market salt 99.0 119 bolgatanga, market salpetre 98.5 50 400 ghana ¹ salpetre added under preparation of beans. ²ashed sorghum stalk added instead of salpetre. ³tz made of millet meal. rice meal is sometimes used instead. 4 value obtained from matvaretabellen (statens ernæringsråd, 1995). period. the remaining 7% is from snacks mostly wild fruits that the children picked in and around the village. food choices and household food availability is limited, hence children received one meal at school which is normally sorghum. however, during the intervention the 60 participants did not eat the school food (sorghum) but instead received the intervention diet (banku and koko) and this formed 25% of the bulk of food eaten. iodine content of foods and other food additives the iodine content of foods mostly eaten and available in the region is shown in table 2. the iodine content in the foods ranges from less than 16 µg/kg (raw cassava and rice) to 538 µg/kg (tubani). salpetre which is usually a food additive in ghana contains the highest amount of iodine (50,400 µg/ kg) and it boosted the iodine levels in foods where it had been added. the two main water sources in the area are well and borehole and both showed very low iodine concentrations; 0.2 and 0.8 µg/l respectively. antropometric indicators the mean values of weight, height, body mass index (bmi) and mid-upper arm circumference are presented in table 3. a fortnight is too short a time to see any significant change in height for children, however, weight did increase significantly on average by 0.9 kg, (p < 0.01). bmi which is an index including weight also increased significantly by 0.4 kg/m 2 (p < 0.01). midupper arm circumference also increased significantly by 0.4 cm (p < 0.01). nonetheless, when stratified by gender, no significant difference was found between boys and girls, for weight, bmi and midupper arm circumference. iodine status the prevalence of goitre was more than 60% among the children. however, 10% of them showed visible goitre (table 4). urinary iodine increased significantly, from 34 to 79 µg/l (p < 0.01) by the end of the intervention while reatinine decreased (table 5). baseline correlation bet table 3. weight, height, upper arm circumference and body mass index (bmi) of the school children in sekoti, ghana, prior to and following the consumption of the fish containing experimental diet. significant differences (p < 0.01) within a column are shown by different superscripts. data are presented as means (sd), n = 54. weight (kg) height (cm) upper arm circumference (cm) bmi (kg/m 2 ) before intervention 30.3 a (4.1) 137 (7) 18.3 a (1.3) 16.0 a (1.2) after intervention 31.2 b (3.9) 18.7 b (1.3) 16.4 b (1.1) table 4. distribution of goitre among 10 to 12 years old schoolchildren in sekoti, ghana. sex (n) no goitre (%) total goitre rate (%) goitre, invisible (%) goitre, visible (%) girls (23) 30 70 52 18 boys (37) 43 57 52 5 both (60) 38 62 52 10 table 5. iodine concentration, creatinine concentration and the ratio iodine/creatinine in urine samples from schoolchildren in sekoti, ghana, prior to and following the consumption of the fish containing diet. significant differences (p < 0.01) within a column are shown by different superscripts. data are presented as medians and precentiles (25th 75th), n = 54. iodine(µg/l) creatinine(g/l) iodine/creatinine(µg/g) before intervention 34 a (20 53) 1.37 a (0.76 1.66) 28 a (20 47) after intervention 79 b (37-122) 0.70 b (0.34-1.32) 115 b (84 156) between creatinine concentration and iodine/creatinine ratio was significant at start of the intervention (r = 0.39, p < 0.01). figure 1 depicts the relationship between iodine concentration in urine and the level of goitre. as the level of iodine increased the occurrence of goitre decreased. the concentration of iodine in urine increased after consumption of the fish diet but the increase was significant for the groups without and with invisible goitre (p < 0.01). no significant change was found in the group having visible goitre. there was no significant difference in the concentration of iodine in urine for boys and girls. figure 2 shows the tsh level in serum before and after the intervention period. the two groups having goitre, invisible and visible, showed a significant decrease in serum tsh level (p < 0.05) after eating the fish containing diet. the group without goitre did not show any significant change in serum tsh level. when stratified by sex, the decrease in serum tsh was only significant among the girls (p < 0.05). discussion food habits and iodine assessment of foods millet/rice balls (“tuo zaafi”-tz), sorghum, tubani (cowpea paste) and rice contributed more than 90% of the food normally consumed by the schoolchildren in sekoti. these carbohydrate rich foods contained low levels of iodine, and did only cover a minor part of the children's recommended daily intake of 120 µg (who/unicef/iccidd, 2001). the low levels of iodine in the raw materials of cereals and beans reflects the low iodine content in the soil in the area were they are grown (hetzel and maberly, 1986). the iodine content in the water samples was insignificant, also reflecting the low iodine content in the soil. tomatoes and okra had higher levels of iodine than the cereals and the beans, probably because they are grown on farms closer to coastal areas, where iodine levels in the soil are higher (hetzel and maberly, 1986). meals were usually served with a stew, often made of tomatoes, okra or groundnuts, which resulted in a slight increase in the iodine content in most of the dishes. dishes of beans and tubani bought at the market in bolgatanga contained surprisingly high amounts of iodine. these high levels of iodine can be explained by the addition of saltpetre during the food preparation. saltpetre was added to get the desired texture and taste, and had an iodine content of more than 50,000 µg/kg. at sekoti, however, where the children involved in the study lived, ashed sorghum stalk was used instead of saltpetre as a cheaper alternative. this explains the low levels of iodine found in the prepared beans and tubani bought there. the salt used for food preparation was not iodised, and contained therefore insignificant levels of iodine. 067 afr. j. food sci. res. maage et al. 068 io d in e co n ce n tr at io n i n u ri n e (µ g /l ) 160 before intervention 140 after intervention 120 100 80 60 40 20 a a a a b b 0 no goitre goitre, invisible goitre, visible figure 1. urinary iodine concentrations of the schoolchildren in sekoti, ghana, according to the grade of goitre. significant difference (p < 0.01) prior to and following the consumption of the fish containing diet within a group is shown by different letters. data are presented as medians with 25th and 75th percentiles. for the group having no goitre n = 20, n = 28 for the group having invisible goitre and n = 6 for the last group with visible goitre. the low levels of iodine in the staple foods could be the major explanation of the high incidence of idd in the sekoti area. however, other factors than low nutritional iodine may contribute to goitre and diseases related to iodine deficiency. goitrogenes and selenium deficiency are such factors. some foods contain anions like tiocyanate, nitrate and nitrite, which all are goitrogenes (browngrant, 1961; delange and fisher, 1995; ubom, 1991; fordyce et al., 2000). saltpetre, which had a high iodine concentration, contains nitrate which can inhibit the utilisation of iodine (tajtakova et al., 2006). in sekoti, millet was one of the most important staple foods, which contains a goitrogen called vitexin (gaitan et al., 1995; elnour et al., 1997). a probably high intake of vitexin may also contribute to the high incidence of goitre recorded in sekoti. selenium deficiency could be a factor contributing to the idd symptoms as selenium is a part of type i 5`deiodinase which converts t4 to the more active t3 hormone (meinhold et al., 1993; roti et al., 1993). however, selenium status of participants was not measured. changes in nutritional status according to túron and chew (1994) protein energy malnutrition (pem), at ages 11 to 13 years, was diagnosed based on a bmi below 15.0 kg/m 2 . in the present study, the schoolchildren had at the beginning an average bmi slightly higher than this cut-off point, and the bmi increased significantly by 0.4 kg/m 2 during the study. the improved nutritional status was also confirmed by the increase in upper arm circumference and the significant weight increase. the consumption of the experimental diets thereby showed a positive effect on the nutritional status, which might be a result of increased dietary protein quality, a higher food consume than normal and an increased intake of specific nutrients like iodine, which had an obvious positive effect on the children's iodine status. the prevalence of goitre in the sekoti area was estimated to be 59% in 1991, and the area was then defined as an area with severe iodine deficiency (who/unicef/iccidd, 1993). the same prevalence of goitre was found in this study when started in 1996 and showed that iodine deficiency was still a large problem in this area. the researchers were told that iodine capsules were distributed to the population two years prior to the present study. this seemingly did not correct the low iodine status of the schoolchildren two years after the distribution of the capsules. iodine status urinary iodine concentration is the major parameter for 069 afr. j. food sci. res. t s h l ev el i n s er u m ( µ u /m l) 10 before intervention after intervention 8 6 4 2 a a a b a b 0 no goitre goitre, invisible goitre, visible figure 2. tsh levels in serum of the schoolchildren in sekoti, ghana, according to the grade of goitre. significant difference (p < 0.05) prior to and following the consumption of the fish containing diet within a group is shown by different letters. data are presented as medians with 25th and 75th percentiles. for the group having no goitre n = 20, n = 28 for the group having invisible goitre and n = 6 for the last group with visible goitre. evaluating iodine deficiency. in casual urine samples the concentration of iodine can be related to urinary createnine (vought et al., 1963) . however, the urinary iodineiodinecreatinine ratio is not a good indicator in cases where this ratio is significantly correlated with the createnine concentration itself (furnée et al., 1994). in this study we also measured this ratio but the iodine-creatinine ratio was not used further as the correlation with the creatinine concentration was found to be significant. the urinary iodine concentrations were therefore preferred as iodine status indicator. according to bourdoux et al. (1985) the measurement of urinary iodine concentrations in fifty to one hundred casual samples gives a good index for iodine status in a population. median urinary iodine levels of 20 49 µg/l indicate moderate iodine deficiency; median values of less than 20 µg/l indicate severe iodine deficiency (who/unicef/iccidd, 2001). the 25th and 75th percentiles of iodine levels in the urine collected before the intervention was 20 and 53 µg/l respectively, which confirms that idd was a serious problem in the area. during the study period the average concentration of iodine in urine increased significantly by about two times. the increase was less than expected, because the contribution of iodine from the intervention diet was several times the contribution of iodine from the staple foods usually eaten by the children. it is, however, possi ble that the low urinary iodine after eating the intervention diets is an indication that iodine deficient children retained a considerable amount of the ingested iodine. the tsh concentration in serum decreased signifycantly as a result of the consumption of the iodine rich diet. for children aged 1 to 15 years the level of tsh in serum is expected to be between 0.6 and 6.3 µu/ml (delange and fisher, 1995). in our study, the tsh median value of the school children in the study came below the upper limit of this interval after the intervention period. the findings from the present study show a relationship between the degree of goitre, urinary iodine concentration and the level of tsh in serum. when grouping the schoolchildren according to the degree of goitre, it was seen that the urinary iodine excretion was not signifycantly increased in the group having visible goitre, while the tsh level in serum was significantly decreased in this group. in the group having invisible goitre the serum-tsh concentration also significantly decreased and further a significant increase in urinary iodine concentration was seen. the group having no goitre had no significant change in serum tsh, but had the highest increase in urinary iodine concentration. individuals having goitre seem to have a more efficient iodine trapping mechanism in the thyroid gland, resulting in less iodine excreted by maage et al. 070 the urine. more iodine is then available for producing t4 and t3, which in turn reduces the level of tsh in serum. our data showed higher goitre prevalence among females than among males when the experiment started, the same tendency is reported in other studies from mali (fish et al., 1993), malawi (furnée et al., 1994) and sudan (elnagar et al., 1995). the higher goitre prevalence among the females can explain why it was observed a significant decreased in the tsh level in serum in females while this was not observed in males. conclusion/implication the high prevalence of goitre in this area showed that iodine deficiency was a major problem. the results from the present study show that goitrous individuals had a significant ability to improve their iodine status following 14 days on a diet containing elevated amounts of iodine due to marine fish inclusion. even though the use of iodised salt is the major alleviating factor for iodine deficiency, for parts of the population it is not always efficient. diets containing marine fish could be a helpful supplement in alleviating the idd in some areas. acknowledgements mr. james asedem, regional nutrition officer, is warmly thanked for his support and acceptance of the project. mr. thomas ndanu and mr. seth aduafarwuah, depart-ment of nutrition and food science university of ghana, legon, ghana, are thanked for their invaluable help in the field study. the project was supported by the norwegian cooperation programme for development, research and higher education (nufu) under contract 14/92. reference asibey-berko e (1995). prevalence and severity of iodine deficiency disorders in ghana. in: asibey-berko e, orraca-tetteh, r (eds) proceedings of the national workshop on iodine deficiency disorders in ghana held in accra, ghana. university of ghana, ghana, pp. 15-23. aisbey-berko e, amoah ag, ado f, agyepong e (1998). endemic goitre and unrinary iodine levels in rural communities in the bolgatanga and builsa districts of the upper east region of ghana. east afr. med j. 75:5001-5003. bourdoux p, delange f, filetti s, thilly c, ermans am (1985). reliability of the iodine/creatinine ratio: a myth? 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effects of fish protein supplementation to traditional fermented maize diets master thesis, universitetet i bergen, norway. statens ernæringsråd, statens næringsmiddeltilsyn (1995). matvaretabellen (food composition table), universitetsforlaget, oslo. 071 afr. j. food sci. res. tajtakova m, semanova z, tomkova z, szökeova e, majoros j, radikova z, sebökova e, klimes i, langer p (2006). increased thyroid volume and frequency of thyroid disorders signs in school children from nitrate polluted area. chemosphere. 62(4):.559-564. tseng yc, burmann kd, baker jr, wartofsky l (1985). a rapid, sensitive enzymed-linked immunoassay for human thyrotropin. clin. chem. 7:1131-1134. túron b, chew f (1994). protein energy malnutrition. in: shils me, olson ja, shike m, (eds), modern nutrition in health and disease, 8th ed, lea and febiger, philadelphia, 1: 950-976. ubom ga (1991). the goitre-soil-water-diet relationship: case study in plateau state, nigeria. sci. tot. environ. 107:1-11. vought rl, london wt, lutwak l, dublin td (1963). reliability of estimates of serum inorganic iodine and daily fecal and urinary iodine excretion from single casual specimens. j. clin. endocrinol. 23:12181223. who/unicef/iccidd (1993). global prevalence of iodine inefficiency disorders. mdis working paper #1), who, geneva. who/unicef/iccidd (2001). assessment of iodine deficiency disorders and monitoring their elimination. a guide for programme manager (who/n/01.1),. 2nd ed, who, geneva. who (2004). iodine status worldwide. who global database on iodine deficiency. who, geneva. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (2), pp. 001-008, february, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper facilitating food crop production in lungwena, mangochi district in malawi: lessons from a farmerbased pass-on seed support model v. h. kabambe*, w. g. mhango, m. msiska, w. a. b msuku, g. k. c. nyirenda and c. masangano university of malawi, bunda college of agriculture, p.o. 219, lilongwe. accepted 22 november, 2018 lungwena extension planning area is a coastal community on the south end of lake malawi. due to a unimodal rainfall pattern, food shortages are common for two to five months before the next harvest. in order improve food production, a farmer-based seed multiplication program was initiated in 2005/06 season in three selected villages. focus group discussions (fgd’s) were used to identify important crops for the area and their related constraints. a scaling-out program was then designed and implemented with farmer participation. the main research objective was to identify and test a mechanism for accelerating food crop production in a community where availability, use and production knowledge of improved main food crops are low. the guiding principles were to identify an easy-to-administer crop production intervention within reach of resources and management capacity of the researchers, extension system and farmers. the hypothesis was that by working with a few eager farmers to multiply seeds of improved varieties of cassava and legumes, the seed would spread out through organised or informal sales and sharing. the fgd’s showed that maize (zea mays l.), cassava (manihot esculent a crantz), pigeon peas (cajanus cajan {l.}. millspaugh), cowpeas (vigna unguiculata {l.} walpers) and bambara groundnuts (vigna subterranea {l.} verd) were main food crops. poor access to seeds of improved crop varieties and knowledge of improved production technological packages were noted as main constraints. for maize, lack of inorganic fertilizers and poor soil fertility were other constraints. the results of the seed multiplication program showed that out of 63 farmers planned to participate in seed production, 21 took part. some 38 farmers were identified for further multiplication. those that participated were also trained, and indicated that they learned new skills and concepts quite useful to their farming. the low success rate was attributed to lack of commitment and unpreparedness to fence cassava and pigeon peas crops. scaling out of seed amongst farmers may be enhanced through promotion of sales, rather than sharing. it was anticipated that the knowledge that new seeds, particularly of cassava cuttings, are available would prevail and farmers would transact with it further. key words: food security, livelihoods, farmer participatory methods, community seed production, mangochi district. introduction malawi is a small south east african country with a total land area of 118,484 km 2 . there is a unimodal pattern of rainfall. maize (zea mays l.) is the staple food crop. bet *corresponding author. e-mail: kabambev@yahoo.com. ween 1998 and 2002 maize area has ranged between 1292,269 and 1,507,088 ha, with average production range of 1.137 to 1.650 t ha -1 . (ministry of agriculture, irrigation and food security (moafs, 2005). in terms of area committed to a crop, the importance of various crops are in the following order: maize, tobacco (nico-tiana tobacum) l., cassava (manihot esculenta crantz), common beans (phaseolus vulgaris l.), sweet potato (ipomea batatas {l.} lam.), groundnuts (arachis hypogea l.) and pigeon peas (cajanus cajan {l.}. millspaugh) (moafs, 2007). the area planted to maize and maize mixtures by smallholder farmers was about 65% in the early 1960s, and increased to 70 85% of the land under cultivation in the 1990’s (heisey and smale, 1995). between 2000 and 2005 the country has experienced some serious food shortages due poor rainfall and other factors such as poor storage or excess sale of harvest. it is therefore a policy of government to encourage crop diversification so that food can be available at household level throughout the year (moafs, 2005). the studies in this report were a component of the lungwena health, nutrition and agricultural multidisciplinary project, which aimed at addressing problems of poverty, food insecurity, and ill-health through a multidisciplinary approach. the project was implemented by relevant academic departments of the university of malawi, in collaboration with norwergian agricultural university (nufu). the project geographically focused on lungwena extension planning area (epa), in mangochi district, southern malawi (figure 1). in terms of administrative arrangement for extension delivery in malawi, the country is divided into eight agricultural development divisions (add), which are in turn divided into general district agricultural offices, then epa’s followed by sections. with regard to national food security and nutrition, some areas of the country may experience food shortages due to distribution shortfalls and some local food production factors, particularly in rural areas. in 2004, rural women in malawi were twice as likely to be thin as their urban women. rural women in mangochi were more likely to be thin than women in other districts (national statistical office {nso} and orc macro, 2005). nationally, 48% of children under 5 are stunted, 22% are under weight while 5% are too thin (nso) and macro 2005). according to benson (2002), 70 80% of people in mangochi, including lungwena area were under the poverty line of us $ 0.43 a day. this is the case even though mangochi, including lungwena epa, enjoys a fish-based economy and diet, as it lies along the lake. the aim of the crop production component was to identify constraints to food crop production and design and promote appropriate production technologies with high potential for adoption impact on crop yields. this report provides results of farmer-participatory activities to identify production constraints and design community based intervention programme. the research question in the program was ‘how can we accelerate a food production in a system where access, use and knowledge of improved varieties of cassava, pigeon peas or cowpeas is low? the hypothesis was that by working with a few eager farmers to multiply seeds of improved varieties of cassava and legumes, the seed would spread out through organised or informal sales and sharing. materials and methods about lungwena extension planning area lungwena extension planning area (epa) is found in mangochi district, under machinga agricultural development division in southern malawi. the total land area is 28,101 ha. of this, 16,324 is cultivated arable land, 2,836 is marginal cultivated land, and 2631 is under waterlogged conditions while the rest is under namizimu forest reserve. the area receives an average of 780 1000 mm rainfall per annum. in 2003 the epa had about 20,340 farm families, with an average land holding size of about one hectare per household. the main crops grown in the area include maize, cassava, rice (oryza sativa l.), cowpeas (vigna unguiculata {l.} walpers), sweet potato, pigeon peas, velvet beans (mucuna spp) (mr chanza, head of epa, personal communication, december 2004) determining existing crop production systems and constraints focus group discussions focus group discussions (fdg’s) were conducted in february 2005 by a research team comprising an entomologist, pathologist and three agronomists. the focus group discussions were conducted in four villages as follows:mdalamakumba and kwilasya (located away from the coast, upland) and chilonga and mtumbula (coastal land). the objective of the focus group discussions was to collect information on crops and cropping systems in the lungwena epa, constraints to increased production to be used as a planning tool in the interventions. during the discussions, farmers were asked what crops they are growing, why they grow these crops, the problems they face, and what they do about these problems. through a show of hands, percentages were calculated for some of the res-ponses. all meetings were facilitated by the local extension agent and village chiefs, who were accompanied by their assistants. there was very good participation by women in the discussions. in addition to the discussions, researchers made general observations in crop fields to identify agronomic, soil or pest situations. development of an intervention program the intervention program was conducted in two phases. the first phase was a desk design by researchers, and the second, as a discussion with farmers in fdg’s. the desk design outlined options for interventions for tabling to farmers in the fdgs. the guiding principle was to identify an easy-to-administer crop production intervention within reach of resources and management capacity of the researchers and of farmers. for resources, issues such as isolation distances in seed multiplication were considered. to increase maize seed, for example, isolation distances of 200 m are required, compared to only 5 m for groundnuts, cowpea, bambara groundnut and cassava (muliokera, 1997). the investigators held a meeting with the communities in the three villages where the intervention were targeted. the villages were mdalamakumba, chapola and chilonga. the objective was to inform the communities about the seed multiplication program and discuss issues related to criteria for selecting farmers, land for the demonstrations, sharing of seed; whether seed should be given to individuals or a group of farmers. the discussions were held in september 2005. in chilonga village a total of 39 farmers participated (17 men and 22 women), in chapola village 195 farmers participated (76 men and 119 men) while in m’dalamakumba village there were 105 participants (no gender breakdown was recorded). in all meetings, the village leaders were present. the outcomes of these approaches are presented in the results section. figure 1. map of lungwena project area. results and discussions focus group discussions (fgd’s) – crops grown and constraints to production the numbers of farmers taking part in fgd’s, major crops and proportion of farmers growing them are shown in table 1. the crops include maize, groundnuts, rice, pumpkins (cucurbita maxima duch), cowpeas and sweet potatoes. the crops are usually grown in intercropping to maximize land and labour use. the reasons for growing the crops were narrow. in most cases crops were grown for food and sale. in some cases crops were grown as a food security measure (crop can provide food or cash at a time when maize is depleted and cash is rare). for crops not commonly grown, farmers cited poor seed shortage as reasons for not growing these, and said they would grow these for food as well (table 2). another serious problem was that of freerange goats during the dry season. the goats, which are set lose after the rains, destroy crops such as cassava and pigeon peas, which extend into the dry season. other problems were wild animals, lack of storage technology on crops such as cassava, and poor access to storage insecticides, lack of markets and prolonged dry spells during the season. farmers mitigate food shortages by growing some short season crops or crop varieties in the dry season, using bucket irrigation. the irrigation water is drawn from ma table 1. crops grown and proportion of farmers growing them, by four villages, according to focus group discussions. village sample size (n) and % farmers growing crop crops grown mdala makumba chilonga kwilasya mtumbula village n = 51 n = 67 n = 113 n = 57 maize 100 100 100 10 cassava 68 12 yes+ 21 groundnuts 95 83 yes 24 rice 100 73 yes 57 tomatoes 82 50 yes 14 sweet potato 91 12 no 43 sorghum 64 63 yes 0 finger millet 9 22 yes 0 cotton 0 0 yes 0 vegetables 5 0 no 0 cowpea 95 56 no 0 bananas 36 60 yes 0 bambara nuts 68 0 no 0 sugarcane 41 48 no 0 velvet bean 65 0 yes 24 pumpkins 91 95 yes 85 pigeon pea 48 0 no 14 dolichos beans 36 0 no 45 + it was not possible to determine percentages due to continuous departures and arrivals of participants table 2. reasons for not growing certain crops according to focus group discussions. reason for not growing crops associated with reason constraint classification goats and wild animals cassava, dolichos bean, sugarcane, natural, social attacking crop in dry season pigeon peas, sorghum lack of seed tomatoes, sugarcane, pigeon peas, irish technical, economic potatoes, cassava, finger millet, bambara groundnuts, cowpea, vegetables shortage of family labour rice, sweet potatoes social, economic bird attack sorghum natural, social mid-season dry spell sweet potatoes, rice biophysical, technical new crop for them cotton technical heavy clay soil not suitable groundnuts natural lack of market rice, pigeon pea, velvet beans economic nually dug wells, in low river banks or areas with low water tables (dimbas) while other farmers irrigate using water from the lake. in addition to problems noted in table 2, farmers identified several pest problems, such as wireworms and stalk borers in maize, bean beetles, mice, crickets, cassava mosaic virus, and cassava mealy bug. farmers also mentioned lack of low lying wet lands (dimbas) for winter crop production. to further understand and analyse the production constraints, the problems stated by farmers were re-classified according to nature of problem, such as natural, biophysical economic and social, or in terms of possible solution such as technical, social, economic administrative. notably, lack of seed was a constraint prevailing across many cross, and was considered technical and economic problem. the prevailing cropping system was mixed cropping. maize and pumpkin intercropping was the most predominant, followed by maize and cowpeas, dolichos beans (lab lab), or velvet beans. amongst the many crops, only rice or cassava was planted in pure stands, because these are grown under water-logged conditions. for the cassava, very large mounds are used for planting in order to avert the water logging. researcher observations field observations indicated that crops were differently affected by the dry spell. early maturing hybrid maize varieties that were planted with first rains and grain legumes such as cowpea performed better than local maize or late planted crops. in maize there was general lack of knowledge on improved production practices such as importance of early planting, plant spacing within the row was too narrow, and plants per station were greater than four. the recommendation is to plant maize in ridges at 75 cm apart, 75 cm between stations with three seeds per station. local, late-maturing maize varieties were commonly planted and there was lack of knowledge on improved varieties of cassava and legumes. the observations showed that a training component on general principles of crop production, such as use of improved varieties, early planting, weeding and fertilizer use and management would be necessary. such training would have to show how these management factors link to drought escape, improved fertilizer use efficiency, pest management and higher yield. it was also clearly indicative that the introduction of any crops to be harvested beyond the month of may would suffer from goat attack. developing and intervention strategy for 2005/06 season the research team then considered the results of fgd discussions and field observations and identified areas where feasible interventions were possible, with reference to table 2. shortage and lack of access to improved seed was a pertinent problem for many crops. as a technical constraint, the team considered that it was feasible to furnish certain seeds to the community, thereby simultaneously providing the economic empowerment needed to kick-start the systems. therefore the team proposed to multiply seed for various crops in the 2005/2006 cropping season. at the design stage, seed multiplication involved cassava, cowpeas, bambara groundnuts and pigeon peas. the objective was to increase availability of seed for the various technologies to the people in lungwena. although maize is the staple for the area, the research team believed that the type of food shortage required the use of alternate crops. one common reason for food shortage is that farmers sell their crops soon after harvest, since many traders go into households to lure farmers into selling. under such circumstances, crops such as cassava, which can actually be harvested during the food insecure periods of september to february, may be advantageous as they may not be prone to such unplanned sell, and that farmers are much better minded not to sale at such times. also, the crops chosen do not require inorganic (chemical) fertilizer inputs (moaifs, 2005) and would therefore be easier to administer. further, these crops required minimum isolation distances for permissible seed production purposes (muliokela, 2005) . the legume crops were also preferred by women in that they were valuable relish for the home. in terms of nutrition, they are recommended as complimentary diet to starchy cereals or cassava. maize was also excluded because, being a staple food, it is already promoted through many other national wide programs, such as the starter pack program (blackie and mann, 2005) and the fertilizer subsidy in 2005/06 and 2006/07 (moaifs, 2007). many agricultural programs tackle food productions issues from the poverty point of view (blackie and mann, 2005). lack of seed has previously been reported as the main reason for farmers’ failure to adopt improved crop varieties (masangano and miles, 2005). the design of a seed production must be guided by the objective of the program (setimela et al., 2004). in this case, the aim was not to organize commercial seed production, but rather to introduce seeds into the area, to be circulated by a range of systems, which could include selling, gifts and facilitated sharing (pass-on system). the research team established tentative seed output targets and the estimated land requirements. it was planned that seed would be multiplied in lungwena by farmers themselves to ensure farmer participation in the project and dissemination of the technologies. however, some seed would also be multiplied at bunda college crop science research farm and at lungwena epa office field plot in order to provide a back up, in case demand would be very high. the planned total area was 2 hectares for cassava, bambara nuts and cassava and 3 hectares for pigeon peas. researchers noted the need to discuss with the chiefs on issues related to land, criteria for selecting farmers, sharing of seed and whether seed should be given to individual or a group of farmers. as a framework it was envisaged that suitable farmers would be willing to participate, have land that is accessible to the community so that people should be able to visit the plots, and be able to explain to others what technologies are being shown and the associated cultural practices. participatory program design during the program development discussions, all the 3 villages (chilonga, chapola and m’dalamakumba) accepted that a seed multiplication program, involving the proposed crops (cassava, cowpeas, bambara groundnuts and pigeonpea should start in the 2005/2006 crop season. it was also agreed that all crops would be grown in pure stand. participants indicated that the programme would assist them to acquire seed of various crops and were willing to actively participate. they expressed gratitude to the researchers for taking their problems into consideration. the discussions also included issues on table 3. the agreed plot sizes and numbers of farmers to be involved for each crop. crop plot area number of total number of plots total area in the 3 (ha) plots/village (farmers) in the 3 villages villages (ha) cassava 0.12 5 15 1.8 bambara nuts 0.12 5 15 1.8 cowpea 0.12 5 15 1.8 pigeonpea 0.12 6 18 2.2 total 21 63 7.6 table 4. the sharing arrangements for the pass-on system. s/n crop % to owner % to pass-on 1 cassava i). roots: all roots go to owner i). roots: none ii). stems: 30% ii). stems: 70% 2 bambara 70% 30% 3 cowpea 70% 30% 4 pigeonpea 70% 30% land area to be allocated to the various crops, criteria for selection of fields for seed multiplication, sharing of crop produce/seed, criteria for selection of participating farmers, organizational and model for sharing seed yield. the agreed plot sizes and numbers of farmers to be involved for each crop were as shown in table 3. regarding criteria for selection of participating farmers, all communities agreed that the participating farmers should be willing to participate, hard working and energetic, be able to follow all cultural practices as instructed, have land that is accessible to the community so that people should be able to visit the trials and should be able to share with others the technologies that are being shown and the associated cultural practices. on organisational structure, farmers agreed to have their names written including those on the waiting list. farmers also decided to form groups but would work individually on their plots. each group would have a chairperson, secretary and treasurer. farmers resolved that a fence should be built on cassava and pigeon pea plots to protect the crops from animals such as goats. the participating farmers agreed to complete land preparation by mid october, 2005. after debate, it was agreed that seed would be passed on as shown in table 4. in addition, farmers requested that seed should be delivered timely (that is, by end of october 2005 for all the 3 legumes) and onset of rains for cassava. farmers also permitted the researchers to visit lungwena in midoctober to assess progress on land preparation. implementation of the seed multiplication activities all seeds, except bambara groundnuts, were delivered on time. as there is no research programme on bambara groundnuts in malawi, it was difficult to find large quantities of seed. the objectives of this exercise was to increase production of basic or foundation seed of cassava, cowpea and pigeon peas, hence these were obtained from research responsible institutions. the ultimate goal was to extend the period in which the seeds can be used, and to provide seeds of high yield potential and improved pest resistance. for cassava the variety supplied was mbundumali. it is high yielding variety with sweet taste. it is suitable for local markets, since most farmers consume cassava in raw form or after direct boiling. cassava in the area is not processed into flour, hence bitter varieties would not be suitable. the cowpeas varieties used were pinkeye and 418, obtained from bunda college. the pigeon pea variety was icp9145, with resistance to fusarium wilt, a fungal disease which causes die-back of stems. field technicians from lungwena epa supported the farmers with advice on management of their plots. after delivery of inputs, a monitoring trip was undertaken, which revealed that only a small proportion of the planned number of farmers actually participated (table 5). many registered farmers did not pick up seed for planting at all. for crops such as pigeon pea and cassava, farmers cited the need to fence as cause for lack of interest. no clear reason was given for cowpeas. a visit was conducted in august 2006 to get feedback from the farmers involved in seed multiplication and assess their yields, and to facilitate sharing arrangements. a summary of actual seeds grown and estimated yield is shown in table 6. training of participating farmers a one-day training session was conducted for all partici table 5. the number of farmers involved in seed multiplication and the land area planted, including field at lungwena epa centre. village crop total area planted no. of farmers by gender male female chilonga cassava 0.64 ha 3 1 chapola cassava 0.53 ha 6 2 cowpeas 0.2 ha 0 11 pigeon peas ? 1 0 mdala-makumba cassava 0.79 3 3 cowpeas 0.72 1 5 pigeonpeas 0.10 0 1 total 15 15 table 6. numbers of seed growers, estimated yield, and sharing arrangements at end of season. village name crop no. of estimated sharing arrangements farmers production chilonga cassava 2 7 farmers identified to share seed with chapola cowpea 9 141 kg no farmers identified for sharing. farmers to plant in own fields cassava 3 one farmer identified for sharing mdala makumba cassava 2 seed already shared with 11 farmers. encouraged to find 3 other farmers. cowpeas 3 150 kg encouraged to find 8 farmers. pigeon peas 1 not harvested then, no farmer identified for sharing pating farmers. the aim of the training was to improve the knowledge of farmers on general principles of crop management and to explain to them the importance of these for higher yields. farmers were also taught general principles of pest and disease management. the crop plots at the epa were used for field demonstration. the link between pest management and general management was emphasised. after the training session, farmers indicated that the training session was beneficial to their knowledge of integrated crop and pest management. since the training took part within the growing season, it was not possible to monitor its impact on the way farmers manage their crops or on yields. discussion and conclusion the paper has presented a description of the complete process of problem identification and identification of possible solutions. a review of the project shows that only 20 growers took part, out of a planned 63 growers. many seed programs tend to be community based. farmers grow seed as a club, or schools are used (setimela et al., 2004). in this study, farmers opted to grow seed individually, the main reason being to avoid ‘free riders’ and ‘drop-outs’. the view of the researchers is that farmers did not have commitment and desire for improved seed, and researchers and development agents should develop mechanisms to identify such farmers and communities. one possible way to raise enthusiasm would be to conduct awareness campaigns on the potential yields and other advantages of improved varieties, market opportunities, utilization, and storage. another lesson learnt was that having effective local leadership is important for such programs. for example, the chief in m’dalamakumba village helped to coordinate our activities, and it ensured initial farmer and confidence in the program. as a contrasting example, there was the smallest number of participants in group discussions in chilonga village, where we also had the least number of farmers growing seed. some of the farmers that took part planted out larger areas than planned, indicating that the programme was most suitable for them. it was also noted that farmers had high enthusiasm with cassava because it has a double role as a food security crop and cash crop. even then, the need to fence fields of cassava and pigeon peas against goats deterred some farmers. some farmers transferred their cuttings to dimba (wet areas) as a means of seed preservation. cowpea was easy to manage in this respect, however, in the field it was very susceptible to a range of insect pests and weevil attack in storage. acknowledgement we would like to acknowledge the financial support of the norwegian centre for international coorperation. we also like to thank staff of lungwena epa and mangochi district agriculture office for their splendid support and collaboration during the project. references benson td (2002). malawi: an atlas for social statistics. national statistical office, zomba and international food policy research institute. blackie mj, mann ck (2005). the origin and concept of the starter pack. in levey s (ed) starter packs: a strategy to fight hunger in developing countries? lessons from the malawi experience. cabi publishing, reading, uk. pp. 15-27. heisey pw, smale m (1995). maize technology in malawi: a green revolution in the making? cimmyt research report no. 4. mexico city: international maize and wheat improvement centre (cimmyt). ministry of agriculture and food security (moafs) (2005). guide to agriculture and natural resource management. agricultural communications branch, lilongwe, malawi. ministry of agriculture and food security (moafs) (2007). annual agricultural statistical bulletin. planning division. government of malawi. lilongwe, malawi. masangano cm, miles ca (2005). factors affecting the adoption of kalima bean (phaseolus vulgaris l.) variety in malawi. j. sustain. agric. 24(2): 117-129. national statistical office (malawi), orc macro (2005). malawi demorgraphic health survey. cleveton, maryland: nso and orc macro. muliokera sw (1997). zambia seed production handbook. ministry of agriculture, food and fisheries, zambia. berlings, sweden. setimela ps, monyo e, banziger m (2004). successful communitybased seed production strategies. mexico, d.f.: cimmyt. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (4), pp. 289-296, april, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of the chemical composition and functional properties of bambara groundnut and cooking banana ijarotimi oluwole steve*, oyewo mary temitope and oladeji babatunde stephen department of food science and technology, federal university of technology, akure, ondo state, nigeria. accepted 06 may, 2017 the cooking banana and bambara ground nut were processed into flour, mixed in ratios of 90:10, 80:20, 70:30 and 60:40 respectively and chemical, functional and sensory properties were evaluated. the protein contents of formulated samples were significantly higher than ogi, but lower than nutrend (p < 0.05). also, the energy values of cbr4 (384.4  0.3 kcal.) was significantly lower than the nutrend (397.1 ± 1.8 kcal.) (p < 0.05) and there was no significant difference with the ogi (383.1  0.1 kcal.) (p < 0.05). the mineral composition of cbr4 had the highest amount of calcium, magnesium, iron, potassium, sodium and phosphorous when compared with other formulated food samples. the cbr4 had the highest water absorption capacity, least gelation capacity and swelling capacity, while cbr1 had the least values of the formulated food samples. the overall acceptability of the formulated food samples were significantly lower than the ogi and nutrend (p < 0.05). the amount of cbr3 and cbr4 needed to meet the rda for energy, protein and minerals of infant were comparable with that of nutrend, but lower than ogi. the study concluded that the nutrient composition of cbr4 was better than ogi, but lower than nutrend. hence, it may be used as a substitute for ogi and the expensive commercial weaning formula. key words: chemical composition, functional properties, cooking banana, bambara groundnut, infant mix. introduction childhood malnutrition is very common in developing countries (plahar and hoyle, 1991; kim, 2000; fao, 2004). this is because infants at this stage of develop-ment require higher energy and proteins in their diet so as to meet increasing demand for metabolism. the nu-triational status of children less than 5 years of age is of particular concern, since the early years of life represent the period for optimal growth and development (prechu-lek et al., 1999). their nutritional well-being reflects household, community and national investments in family health thereby contributing both directly and indirectly to overall country development and in particular, develop-ment of human resource (central bureau of statistic, 1999). malnutrition contributes directly or indirectly to more than 60% of ten million child deaths each year (who, 2002). it has been reported that over 226 million children *corresponding author. e-mail: soijarotimi@gmail.com. tel.: 2348035670760. below 5 years old are stunted, 67 million are wasted and 183 million weight less than they should for their age (unicef, 1998). the prevalence of malnutrition incre-ases dramatically from the age of 6 18 months as a re-sult of poor feeding practices and low purchasing power that characterized many homes. diets in developing cou-ntries are frequently deficient in macronutrients (protein, carbohydrates and fat, leading to protein-energy malnutrition), micronutrients (electrolytes, minerals and vitamins, leading to specific micronutrient deficiencies) or both (brabin and coulter, 2003; pinstrup-andersen et al., 1993; levin et al., 1993; millward and jackson, 2004). in order to solve this nutrition problem many researchers have worked extensively on cereal-legume combinations in nigeria. for example, fashakin and ogunsoola (1982) formulated nutogi (a mixture of corn gruel and peanut), akinrele and edwards (1989) formulated soya-ogi (corn gruel plus soya bean).the traditional weaning foods could be improved upon by combining locally available foods that complement each other in such a way that new pattern of amino acids created by this combination is similar to that recommended for infants (fashakin et al., ijarotimi et al. 290 bambara groundnut seed cleaning/sorting soaking (24hours) draining roasting (180 0 c, 15minutes) winnowing milling sieving roasted bambara groundnut flour figure 1. flow chart showing roasted bambara groundnut flour. cooking banana cleaning/sorting peeling slicing drying (60 0 c, 24hours) milling sieving (0.4mm mesh) cooking banana flour figure 2. flow chart showing cooking banana flour. 1986). in view of this, the present study therefore was undertaken to develop low cost formulations, using cooking banana fruits (musa spp., abb genome) and roasted bambara groundnut seeds (vigna subterranean l. verde) with locally available technology and an attempt was made to evaluate the chemical composition and functional properties of the food samples. materials and methods materials the materials, that is, raw green cooking banana (musa spp, abb genome), bambara groundnut (v. subterranean l. verdc), nutrend (a commercial weaning food) and ogi (a traditional weaning food) were purchased from a local market in akure town, ondo state, nigeria. methods preparation of samples the bambara groundnut seeds were sorted, soaked for 24 h in hot water, drained and roasted in hot fine sand at 180 ° c for 15 min. the roasted seeds were winnowed, dry milled, sieved through 0.4 mm wire mesh screen (figure 1). cooking banana fruits were peeled, sliced into pieces, oven-dried at 60 ° c for 24 h, milled and sieved through 0.4 mm wire mesh screen. the flours were stored in sealed cellophane bag at room temperature (figure 2). the cooking banana flour and roasted bambara groundnut flour were mixed in ratios of 90:10, 80: 20, 70: 30 and 60:40 respectively and the flour were then stored in cellophane bag at room temperature prior to analysis. chemical analyses the nutrient composition of the food samples was determined using the standard procedures of association of official analytical chemists (1990) . triplicate samples of each sample were determined for moisture content in a hot-air circulating oven (galenkamp). ash was determined by incineration (550 ° c) of known weights of the samples in a muffle furnace (gallenkamp, size 3). crude fat was determined by exhaustively extracting a known weight of sample in petroleum ether (boiling point, 40 to 60°c) in a soxhlet extractor. protein (n × 6.25) was determined by the kjeldahl method. crude fiber was determined after digesting a known weight of fat-free sample in refluxing 1.25% sulfuric acid and 1.25% sodium hydroxide. the carbohydrate content was determined by subtracting the total crude protein, crude fiber, ash and fat from the total dry weight (100 g) of the food sample differences. the gross energy was determined with a gallenkamp ballistic bomb calorimeter (gallenkamp ccb-330-010l uk). minerals content (sodium, potassium, calcium, magnesium, iron, copper, zinc, manganese and selenium) of the flour samples was determined using association of official analytical chemists (1990) method. flour was digested with a mixture of concentrated nitric acid, sulfuric acid and perchloric acid (10:0.5:2, v/v and analysed using an atomic absorption spectrophotometer (gbc 904aa; germany). the total phosphorus was determined as orthophosphate by the ascorbic acid method after acid digestion and neutralization using phenolphthalein indicator and combined reagent (apha, 1995). the absorbance was read at 880 nm (spectronic 21 d, miltonroy, new york, usa) and kh2po4 (merck, mumbai, india) served as a standard. anti-nutrient determinations phytic acid was extracted from each 3 g flour sample with 3% 291 afr. j. food sci. res. trichloro-acetic acid by shaking at room temperature followed by high speed centrifugation as described by wheeler and ferrel (1971). the phytic acid in the supernatant was precipitated as ferric phytate and iron in the sample was estimated. phytate-phosphorus (phytate-p) was calculated from the iron results assuming a 4:6 iron: phosphorus molecular ratio. the phytic acid was estimated by multiplying the amount of phytate-phosphorus by the factor 3.55 based on the empirical formula c6p6o24h18. tannin contents were determined by the modified vanillin-hcl methods (burns 1971; price et al., 1978) . a 2 g sample was extracted with 50 ml 99.9% methanol for 20 min at room temperature with constant agitation. after centrifugation for 10 min at 653 x g, 5 ml of vanillinhcl (2% vanilli and 1% hcl) reagent was added to 1 ml aliquots and the colour developed after 20 min at room temperature was read at 500 nm. correction for interference light natural pigments in the sample was achieved by subjecting the extract to the conditions of the reaction, but without vanillin reagent. a standard curve was prepared using catechin (sigma chemical, st. louis, mo) after correcting for blank and tannin concentration was expressed in g/100 g. oxalate was determined by aoac (1990) method. 1 g of the sample was weighed into 100 ml conical flask. 75 ml of 3 m h2 so4 was added and the solution was carefully stirred intermittently with a magnetic stirrer for about 1h and then filtered using whatman no.1 filter paper. the sample filtrate (extract) (25 ml) was collected and titrated against hot (80 90 ° c) 0.1 n kmno4 solution to the point when a faint pink colour appeared that persisted for at least 30 s. the concentration of oxalate in each sample was obtained from the calculation: 1 ml 0.1 permanganate = 0.006303 g oxalate. trypsin inhibition activity (tia) the trypsin inhibition activity was assayed in terms of the extent to which an extract of the defatted flour inhibited the action of bovine trypsin (ec 3.4.21.4) on the substrate benzoyl-dlarginine-p-nitrianilide (bapna) hydrochloric (kakade et al., 1974). the samples (1 g each) were extracted continuously at ambient temperature for 3 h with 50 ml, 10 mm naoh using a mechanical shaker (gallenkamp orbital shaker surrey, uk). the ph of the resulting slurry was adjusted to 9.4 9.6 with 1 m naoh. after extraction, the suspendsion was shaken and diluted with distilled water such that 1 cm 3 of the extract produced trypsin inhibition of 40 60% at 37 ° c. the respective dilutions were noted. consequently, tia was calculated in terms of mg pure trypsin (sigma type lll, lot 20h0868) tia = 2.632da mg pure trypsin inhibited g -1 sample s where d is the dilution factor, a is the change in absorbance at 410 mm due to trypsin inhibition per cm 3 diluted sample extract and s is the weight of the sample. functional property determination water absorption capacity was determined using the method of salunkhe (1985) modified by adebowale et al. (2002). 10 ml of distilled and deionized water was added to 1.0 g of the sample in a beaker. the suspension was stirred using magnetic stirrer for 5 min. the suspension obtained was centrifuge at 3500 rpm for 30 min and the supernatant was measured into a 10 ml graduated cylinder. water absorbed was calculated as the difference between the initial volume of water added to the sample and the volume of the supernatant. least gelation property was determined using the method described by coffman and garcia (1977). sample suspendsions of 2 16% were prepared in distilled water. 10 ml of each of the prepared dispersions was transferred into a test tube and heated in a boiling water bath for 1 h, cooled rapidly in a cold water bath and allowed to cool further at 4 ° c for 2 h. the least gelation concentration was determined when the sample from the inverted test tube did not slip or fall. swelling capacity was determined by weighing 20 g of the food sample into a cleaned, dried graduated cylinder. the cylinder was tapped 3 times on the table and then 80 ml of distilled water was poured into the cylinder. the cylinder was allowed to stand for 1 h after which the final volume of the food sample was noted. the ratio of the final volume to initial volume gave the swelling capacity on volume basis. the supernatant was decanted and the weight of food sample and the cylinder was obtained and the ratio of final weight to initial weight of the food sample gave the swelling capacity on weight basis. sensory evaluation the 4 formulated samples obtained from the different fractions of cooking banana and bambara groundnut mixes were made into light gruels, using about 20 g and 60 ml of water. the reconstituted formulated and control food samples (that is, nutrend, a commercial weaning food and ogi, a traditional weaning food) were coded and presented to 30 panelists that were familiar with the product. the samples were rated based on colour, aroma, taste, mouth feel and overall acceptability using 9 point hedonic scale scored from dislike extremely (1) to like extremely (9). statistical analysis the data were analysed using spss version 13.0. the mean and standard error of means (sem) of the triplicate analyses were calculated. the analysis of variance (anova) was performed to determine significant differences between the means, while the means were separated using the new duncan multiple range test. results and discussion the proximate composition of the formulated food samples are shown in tables 1. the formulated diet containing 60% cooking banana fruit flour and 40% bambara groundnut flour (cbr4) had the highest energy value (384.4  1.3 kcal.), while the sample containing 90% cooking banana and 10% bambara groundnut flour (cbr1) had the least energy value (361.5  1.0 kcal.). the energy value of cbr4 was insignificantly high when compared with other formulated diets and ogi (traditional weaning diet, corn gruel) (p > 0.05), but significantly lower when compared with the nutrend (commercial weaning diet) (p < 0.05). the protein content of cbr4 (13.8  0.01 g) was significantly higher than ogi (5.60  0.02 g) and other formulated foods, but also significantly lower than nutrend (16.00  0.2 g). in this present study it was shown that the protein content of the formulated diet increases progressively as the % of bambara groundnut increases. also, it was observed that the protein content of cooking banana diet supplemented with roasted bam bara groundnut (13.8  0.01 g) was lower when compared with the cooking banana and fermented bambara ground nut mixed (ijarotimi, 2008). quite a number of studies have reported that fermentation method improves the nutritive values of food products compared with other processing methods, such as roasting, cooking, etc (paredes-lópez and harry, 1988; adams, 1990; obizoba ijarotimi et al. 292 table 1. mean (sem) of proximate composition of cooking banana and bambara groundnut formulated diets. nutrients rcb rbg cbr1 cbr2 cbr3 cbr4 ogi nutrend energy (kcal) 361.5  1.0d 387.8  1.9b 359.7 1.5d 368.8  1.0c 382.5  3.0b 384.4  0.3b 383.1  0.1b 397.1  1.8a moisture (g) 8.9  0.2a 4.6  0.3e 5.9  0.01d 6.4  0.03c 6.7b  0.03c 6.9  0.02b 2.9  0.06f 4.8  0.02e protein (g) 3.3  0.1h 18.9  0.1a 6.7  0.18f 8.6  0.02e 11.5  0.02d 13.8  0.01c 5.6  0.02g 16.2  0.2b fat (g) 1.5  0.1f 6.6  0.2b 2.4 0.02e 3.3  0.06d 3.6  0.23d 5.9  0.05c 0.80 ± 02g 8.5  0.03a fibre (g) 1.2  0.2e 3.8  0.2b 3.9  0.01b 2.8  0.03c 2.7  0.01c 2.3  0.01d 0.5  0.01f 4.5  0.01a ash (g) 2.6  0.2bc 3.5  0.1a 3.4  0.4a 3.1a  0.03b 2.9  0.02ab 2.3  0.06cd 1.7  00.02d 2.7  0.02bc carbohydrate(g) 83.7  0.1b 63.3  0.7f 77.8  0.2c 76.1  0.2d 75.9  0.3d 68.9  0.2e 88.4  0.01a 64.0  0.2f cbr1 cooking banana + roasted bambara groundnut (90:10) cbr2 cooking banana + roasted bambara groundnut (80:20) cbr3 cooking banana + roasted bambara groundnut (70:30) cbr4 cooking banana + roasted bambara groundnut (60:40) nutrend commercial weaning (nutrend) ogi traditional weaning food rbg raw bambara groundnut rcb raw cooking banana. and atii, 1991; aletor, 1993). the fibre content of the formulated diets decreases as the % of bambara groundnut increases, but cbr1 diet con tained the highest fibre content (3.9  0.2 g) when compared with the remaining formulated diets, while cbr4 contained the least fibre content (2.3  0.01 g). the mineral composition of the diets is pre- sented in table 2. the mineral composition of the formulated diets showed that cbr4 sample contained the highest mineral content, while cbr1 contained the least in virtually all the minerals determined. in this study, it was also observed that the mineral content of the food samples increases as the % of bambara groundnut increases. the increased in vital nutrients as supplementation of bambara ground increases could be attributed to the fact that bambara groundnut is a good source of protein and minerals. quite a number of studies have reported that legumes and nuts seeds are good sources of protein and minerals and that these seeds are usually consumed in many parts of the developing countries, particularly, where animal proteins are scarce or very expensive (brough et al., 1993; lalude and fashakin, 2006). the anti-nutritional factors of the formulated diets are presented in table 3. the formulated diet containing 60% of cooking banana and 40% bambara groundnut flour (cbr4) contained the highest amount of oxalate (1.16  0.06 mg/100 g), tannic acid (0.24 mg/100 g), phytin acid (27.25  0.46 mg/100 g), phytin-phosphorus (7.5  0.3 mg/ 100 g) and trypsin inhibitor (7.40  0.5 mg/100 g), while cbr1 diet contained the least concentration of oxalate (0.3 mg/100 g), tannic acid (0.05 mg/100 g), phytic acid (7.2 mg/100 g), phytin-phosphorus (1.9 mg/100 g) and trypsin inhibitor (1.9 mg/100 g) of the formulated food samples. it is evident in the present study that the concentration of antinutrients increased as the % of bambara groundnut increased. several studies have reported that legumes, such as soybean, bambara groundnut etc., contain antinutritional or toxic components (grant et al., 1989; hwei-ming et al., 1997), such as protease inhibitors, lectins, goitrogens, antivitamins, saponins, tannins, phytoestrogens, flatulence factors (rachis, 1975), lysinoalanine, allergens, phytate (leiner, 1994), soytoxin (vasconcelos et al., 1997). legumes consumption has been related to various deleterious effects, such as growth retardation (martinez et al., 1995a), lowered digestibility and absorption of dietary nutrients (pusztai et al., 1995) and physiological, metabolic and immunological disturbances (hajobs et al., 1995; martinez et al., 1995b). however, it is evident that the antinutrient concentration in legumes can be eliminated or reduced to tolerable level through processing methods like roasting, fermentation, soaking etc. (grant et al., 1989; leiner, 1994; vasconcelos et al., 1997; agbede and aletor, 2003; khokhar and chauham, 1986). the functional properties of the formulated food samples are shown in table 4. the water absorption capacity of food materials is an index of the maximum amount of water that it can take up and retain, hence determine the 293 afr. j. food sci. res. table 2. mean (sem) mineral composition of cooking banana and bambara groundnut formulated diets. nutrients rcb rbg cbr1 cbr2 cbr3 cbr4 ogi *nutrend calcium (mg) 30.45  0.06e 60.0  0.30a 19.79  0.11g 27.11  0.26f 39.38  0.56d 45.95  0.24c 51.85  0.65b 39  0.00d magnesium (mg) 62.98  0.23e 57.19  0.21f 65.69  0.08d 102.28  0.37c 136.44  0.51b 184.80  0.90a 14.29  0.39g nd zinc (mg) 0.28  0.03c 5.70  0.20a 0.013  0.002d 0.15  0.02cd 0.016  0.002d 0.016  0.002d 0.7  0.00b iron (mg) 2.60  0.02b 5.50  0.2a 0.15  0.01d 0.24  0.02d 0.22  0.01d 0.24  0.02d 0.34  0.04d 1.0  0.00c potassium (mg) 51.47  0.09g 33.94  0.16h 54.72  0.06e 68.14  0.15c 69.69  0.10b 71.95  0.07a 52.81  0.39f 57.0  0.00d sodium (mg) 26.72  0.04d 7.6  0.01f 26.52  0.06d 27.56  0.17c 27.73  0.57c 30.89  0.08b 40.27  0.13a 22.0  0.00e phosphorus (mg) 94.06  0.12b 33.32  0.21g 41.93  0.16f 47.22  0.30e 70.19  0.84d 75.31  0.89c 97.55  0.15a 22.0  0.00h manganese (mg) 0.93  0.06 nd copper (mg) 0.12  0.01 nd aluminum (mg) nd lead (mg) nd chromium (mg) nd mercury (mg) nd *the values as specified by the manufacturer. table 3. mean (sem) antinutritional contents of cooking banana and roasted bambara groundnut mixed. sample oxalate (mg/100 g) tannic acid (mg/100 g) phytic acid(mg/100g) phytin phosphorous (mg/100 g) trypsin inhibitor (mg/100 g) cbr1 0.32  0.02d 0.06  0.01b 7.0  0.02d 2.05  0.15d 1.75  0.15d cbr2 0.54  0.04c 0.08  0.01b 14.12  0.25c 4.10  0.20c 3.30 0.20c cbr3 0.81  0.02b 0.16  0.02a 19.70  0.80b 6.10  0.20b 5.00  0.20b cbr4 1.16  0.06a 0.24  0.03a 27.25  0.46a 7.5  0.30a 7.40  0.50a energy and nutrient dense of a food. recently, several studies have reported that the high pre-valence of proteinenergy malnutrition in many parts of developing countries is as result of low-dense energy and other vital nutrient intakes (levin et al., 1993; pinstrup -andersen et al., 1993; brabin and coulter, 2003; milward and jackson, 2004). the least gelation concentration of the for-mulated food samples increased with successive substitution levels and there were no significant difference between the formulated samples be-yond 20% substitution levels and the ogi and that of nutrend samples respectively (p > 0.05). the high gelation value implies that the diet would require more energy consumption to cook and hence the gel strength of the diets would be weak and undesirable (enujiugha, 2006). the swelling capacity increased with the % increase of bambara groundnut flour. however, the swelling capacities of the formulated diets were significantly higher than ogi but lower than the nutrend (p < 0.05). table 5 shows the results of sensory attributes of prepared formulated diets, ogi and nutrend. all the sensory attributes of the formulated diets samples were significantly low compared to the nutrend (a commercial weaning food) and ogi (a traditional weaning food) (p < 0.05). the disparity between the flavour and taste of the formulated diets samples and the control food samples could be attributed to the characteristics beany aroma and taste of bambara groundnut flour. the poor rating of the formulated diets samples in term of aroma, taste, texture and colour compared with the ogi and nutrend could also be attributed to the familiarity of the panel of judges to these food ijarotimi et al. 294 table 4. functional properties of the formulated weaning formulation and the controls. sample % water absorption capacity (wac) % least gelation concentration (lgc) % swelling capacity (sw) cbr1 178.3  0.98 fg 4.0  1.15 c 11.9  0.31 f cbr2 183.7  0.82 f 5.3  0.67 bc 13.7  0.41 e cbr3 205.4  0.62 e 6.0  1.15 abc 18.9  0.08 c cbr4 207.2  1.13 e 6.0  1.20 abc 23.2  0.51 b ogi 123.5  0.29 k 6.0  1.13 abc 13.7  0.41 e nutrend 346.5  3.25 a 8.0  1.16 ab 31.6  0.48 a table 5. sensory qualities of the reconstituted formulated weaning foods and control samples. samples colour aroma taste mouth feel overall acceptability cbr1 5.00  0.82 c 6.00  0.69 bc 6.00  0.62 c 5.57  0.81 b 6.29  0.47 b cbr2 5.14  0.55 c 5.71  0.29 bc 5.43  0.43 c 5.86  0.55 b 5.29  0.52 b cbr3 6.29  0.36 bc 5.71  0.42 bc 5.71  0.18 c 5.43  0.75 b 5.71  0.29 b cbr4 6.14  0.59 bc 5.43  0.78 c 4.86  0.40 c 6.00  0.54 b 5.57  0.48 b ogi 7.43  0.29 b 7.14  0.34 b 7.14  0.34 b 7.86  0.63 a 7.71  0.52 a nutrend 9.00  0.00 a 9.00  0.00 a 8.86  0.14 a 8.43  0.20 a 8.86  0.14 a table 6. the amount of formulated, ogi and nutrend food samples to meet recommended daily allowance (rda) of infant. nutrients rda (< 1 years) cbr3(70:30) cbr4(60:40) ogi nutrend energy (kcal) 3444 214 212 212 207 protein (g) 14 116 101 249 88 carbohydrate (g) 95 140 65 107 149 fat (g) 30 439 502 3947 355 calcium (mg) 270 789 591 527 692 phosphorus (mg) 275 579 370 282 1058 sodium (mg) 370 472 1200 922 1682 potassium (mg) 700 818 972 1336 1228 zinc (mg) 3 30000 30000 429 magnesium (mg) 75 52 40 511 copper (mg) 0.5 iron (mg) 1.1 5000 5000 3667 1100 manganese (mg) 0.6 231 samples. the amount of formulated, ogi and nutrend food samples that needed to meet the recommended daily allowance (rda) of infant (< 1 year) is shown in table 6. the amounts of cbr 3 and cbr4, which are needed to meet the daily energy and protein requirement of infants, were absolutely within the same range with that of nutrend, but lesser amount of cbr3 and cbr4 were needed rda for the protein, phosphorous and potassium requirement of the infant compared with ogi and nutrend. however, the amount of the formulated diets that were needed for rda for iron and zinc were high compared with the nutrend food 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90:10; 80:20; 70:30; 60:40; 50:50) was evaluated for proximate composition and physico-chemical properties. physicochemical properties of dough as well as sensory and physical properties of the bread produced from the different flour samples were also evaluated. the proximate composition of the flour samples showed a reduction of about 14 to 38% in protein content but with a significant enhancement in the fibre content (167 to 967%) depending on the level of substitution. the fat and ash contents, as well as the ph of the flour samples increased with increase in the proportion of the tigernut. dough with good viscoelastic properties and acceptable bread with qualities similar to 100% wheat bread was produced from 10% tigernut flour addition. key words: wheat, tigernut, composite flour, bread, proximate composition. introduction bread is an important staple food in nigeria. it is how-ever, relatively expensive, being made from imported wheat that is not cultivated in the tropics for climatic reasons (olaoye et al., 2006) .the idea of substituting part of wheat with other starchy crops is not new. several institutions, including fao, have carried out research designed to find ways of partially substituting wheat flour with other sources of flour or replacing wheat altogether (bokanga, 1995). with the constant increasing con-sumption of bread and other baked products in many countries, the composite flour programme promises to save significant amount of foreign exchange, provide a traditional nutritious food to more people at lower cost and to utilize indigenous crops to a greater extent. previous efforts in nigeria on the use of composite flour for bread have concentrated on the use of flour from cassava. different levels of success have also been recorded with the use of flours from legume, cereals, roots and tubers in baked goods (kure et al., 1998; dhingra and jood, 2002; basman et al., 2003). *corresponding author. e-mail: adeomowaye@yahoo.com. tigernut (cyperus esculentus), an underutilized crop, was reported to be high in dietary fibre content, which could be effective in the treatment and prevention of many diseases including colon cancer, coronary hearth diseases, obesity, diabetics and gastro intestinal dis-orders (anderson et al., 1994). tigernut flour has been demonstrated to be a rich source of quality oil and con-tains moderate amount of protein. it is also an excellent source of some useful minerals such as iron and calcium which are essential for body growth and development (oladele and aina, 2007). its tubers are also said to be aphrodisiac, carminative, diuretic, emmanogogue, stimu-lant and tonic (chopra et al., 1986; chevallier, 1996). tigernut has also been reported to be used in the treatment of flatulence, indigestion, diarrhoea, dysentery, and excessive thirst (chevallier, 1996). in addition, tiger-nut has been demonstrated to contain higher essential amino acids than those proposed in the protein standard by the fao/who (1985) for satisfying adult needs (bosch et al., 2005). therefore, tigernut, with its inherent nutritional and therapeutic advantage, could serve as good alternative to cassava in baking industry. with the progressive increase in the consumption of bread and other baked products in many countries, the ade-omowaye et al. 207 table 1. proximate composition of the composite flour samples. sample moisture (%) protein (%) fat (%) ash (%) fibre (%) carbohydrate(by diff) a 14.7+0.1 c 15.1+0.3 f 1.3+0.3 a 0.45+0.01 a 0.3+0.1 a 68.7 b 14.4+0.1 bc 12.9+0.5 de 4.6+0.2 b 0.8+0.02 b 0.8+0.1 ab 66.5 c 14.4+0.0 bc 12.0+0.4 cd 7.7+0.3 c 1.1+0.02 c 1.4+0.4 bc 63.4 d 14.2+0.0 b 11.1+0.3 bc 11.0+0.3 d 1.2+0.01 d 2.0+0.4 cd 60.5 e 13.8+0.2 a 10.2+0.3 ab 14.0+0.2 e 1.4+0.01 e 2.6+0.3 de 58.0 f 13.8+0.1 a 9.4+0.5 a 17.3+0.4 f 1.6+0.01 f 3.2+0.1 e 54.7 means followed by different superscript within a column are significantly different (p 0.05) a-100%wf; b 90%wf,10%tf; c80%wf,20%tf; d-70%wf,30%tf; e60%wf, 40%tf; f-50%wf, 50%tf composite flour programme, especially in developing countries, has the potential to conserve foreign exchange, provide nutritious food to more people at affordable cost and widen the utilization of indigeneous crops in food formulation. information on the use of tigernut in baked goods is scanty. thus, this study evaluated the proximate composition, physico-chemical and visco-elastic properties of flour and sensory properties of bread produced from wheat-tigernut composite flour at varying levels of tigernut substitution (10 50%). materials and methods dry tigernut (brown variety) was purchased locally. wheat flour and other principal ingredients like yeast, sugar, fat and salt were also obtained from a local market in the southwestern part of nigeria. preparation of tigernut flour the method of adeyemi (1988) was used in the preparation of tigernut flour. dry tigernuts were sorted to remove unwanted materials like stones, pebbles and other foreign seeds, before washing with tap water. the cleaned nuts were dried in a cabinet dryer at 60 o c for 24 h to a moisture content of about 13%. the dried nuts were milled and sieved through 600 µm aperture size. the resultant flour was packed and sealed in polythene bags until analyzed. preparation of the various flours blends flour blends containing varying proportions of tigernut flour (10 50%) together with wheat flour were prepared by mixing required amounts of respective flours. production of bread the procedure for the production of bread in aacc (1984) was used, which employs a bulk fermentation process. two hundred gram of each of the composite flour sample was weighed along with the required amount of water to obtain dough, which was kneaded on a pastry-board to smoothen it. the dough was initially fermented for 2 h at 30 o c before being subsequently knocked back by kneading to expel carbon dioxide and tighten-up the dough to improve the texture of the final product. the secondary fermentation also lasted for 2 h at 30 o c. the dough were then sized and moulded into the baking pans for final proving at 30 o c for 2 h. dough baking was carried out in the oven at a temperature of 230 o c for 25 min. proximate analysis protein (n* 6.25), moisture, fat, ash and crude fibre contents were determined using aoac (1990) methods. carbohydrate content was determined by difference. other analyses the ph and titratable acidity (tta) of the composite flour were determined by the methods of aoac (1990). water absorption capacity (wac) was determined using the method of mbofung et al (2006) . bulk density was determined following the method describe-ed by wang and kinsella (1976). the visco-elastic property of dough from the composite flour was determined using alveograph mixer as described by gilles and medcalf (1965). sensory evaluation the baked, cooled bread samples were presented to 10 semi trained panelists using the method described by taiwo et al. (1997). the panelists were asked to indicate their observations using a 6 point hedonic scale for crust colour, crumb colour, crumb grain texture, aroma, taste, flavour, symmetry, and overall acceptability. excellent and very poor were ranked 6 and 1 respectively. statistical analysis all analyses were carried out in duplicates and the data collected were analyzed using plot it software (spe, 1993). data were subjected to analysis of variance (anova) and turkey’s test was used for comparison of means. significance was accepted at p = 0.05. results and discussion proximate composition proximate composition of the flour samples are presented in table 1. the protein content (%) of the composite flour decreased from 12.9 to 9.40 with increase in tigernut flour substitution. this may be attributed to low protein content of tigernut (addy and eteshola, 1984). although there was a reduction in the protein content of substituted table 2. physico-chemical properties of the flour samples. sample titratable acidity (%) ph water absorption capacity (%) bulk density (g/ml) a 0.057 e 5.80 a 85.17 a 0.57 a b 0.054 e 5.95 b 85.36 ab 0.59 ab c 0.045 d 6.02 bc 85.5.0 b 0.60 bc d 0.041 c 6.06 bcd 85.61 bc 0.61 bc e 0.036 b 6.10 cd 85.85 cd 0.62 cd f 0.024 a 6.16 d 86.01 d 0.64 d means followed by different superscript within a column are significantly different (p 0.05) a, b, c, d, e, and f are as defined in table 1 table 3. viscoelasticity properties of dough from wheat-tiger nut flour mixes. samples p (mmhg) l (mm) g w (j) a 130 e 79 e 19.8 e 402 f b 106 d 63 d 17.7 d 276 e c 90 c 52 c 16.1 c 190 d d 76 b 42 b 14.4 b 134 c e 62 a 38 b 13.7 b 100 b f 58 a 22 a 10.5 a 60 a presistance to deformation of the dough; ldough extensibility; gindex of swelling; w-deformation work. means followed by different superscript within a column are significantly different (p 0.05) a, b, c, d, e, and f are as defined in table 1 wheat flour samples with tigernut flour, a notable enhancement of fiber content in the range of 167 967% was achieved. tigernut addition also resulted in 71 to 256% improvement in the ash content of the composite flour. the fat content (%) of the composite flour increased from 1.3 to 17.3 proportionately as tigernut flour increaseed. this might be due to high fat content (32.88%) of the tigernut flour (addy and eteshola, 1984). hence, defatting the nut before utilization may yield better result. the carbohydrate content (%) of the composite flour decreased from 68.7 to 54.7% indicating low carbohydrate content of the tigernut flour. physico-chemical properties of the flour water absorption capacity (wac) of the composite flour ranged from 85.17 to 86.01% as indicated in table 2. the rate at which each sample absorbed water increased with increase in tigernut addition. no significant difference was found between pure wheat flour and substituted wheat flour with 10% tigernut flour. however there were significant differences among 100% wheat flour and other substituted wheat flour samples. alobo and ogbogo (2007) reported that the wac of gelatinized tigernut starch was 70%. the increase in wac of the composite flour may be attributed to higher fibre and starch contents with increasing tigernut substitution. the ability of flour to absorb water was reported to have a significant correlation with its starch content (mbofung et al., 2006). result of the bulk density of the samples is also presented in table1. the values ranged from 0.57 to 0.64 g/ml which showed an increasing trend with increase in tigernut flour addition. earlier work has reported bulk density of 0.48 0.66 g/ml for raw and malted wheat flour (magnesia and wafflemix, 2007), which is comparable to that obtained in this study. there was increase in ph values with corresponding decrease in titratable acidity values of the flour samples as the content of tigernut flour increased (table 2). the titratable acidity values ranged from 0.024 to 0.057% while the ph values were between 5.80 and 6.16. an earlier report has shown that the ph of cassava flour was 6.55 (chinma and ocheme, 2007). visco-elastic properties of dough from the flour samples and physical characteristic of bread result of the visco-elastic characteristics of the dough is presented in table 3. the index of swelling of the dough ranged from 10.5 to 19.8 with the dough from pure wheat flour having the highest value and the sample substituted with the highest level of tigernut had the least value. a decreasing trend (60 402 j) in the deformation work was observed with increase in the level of tigernut addi-tion. the extensibility of the dough ranged from 22 to 79 mm while the dough resistance to deformation ranged between 58 and 130 mmhg. pure wheat dough had the highest values in all the parameters evaluated. wheat produces white flour that has a unique property of wheat proteins, which can produce dough with sufficient strength and elasticity required to produce low density bread. this observation suggests that an increase in tigernut flour addition result in reduced dough extensibility. the high fat and fibre contents in tigernut may be responsible for the reduction in the dough elasticity of the substituted wheat flour with tigernut flour. however, the visco-elastic property of dough produced from flour substituted with 10% tigernut flour was close to that of pure wheat flour, suggesting its potential utilization in the baking sector. the loaf volume ranged from 145 to 350 cm 3 as shown 208 afr. j. food sci. res. ade-omowaye et al. 209 table 5. scores for sensory attributes of bread samples from the composite flour. quality attributes sample code crust crumb crumb grain symmetry taste aroma overall attributes colour colour texture acceptability a 5.3 a 5.3 a 5.4 a 5.8 a 4.7 a 4.3 b 5.8 a b 5.3a 5.2 a 5.3 a 5.6 a 4.8 a 4.6 a 5.6 a c 4.4 b 4.3 b 4.8 b 5.4 a 5.3 a 5. 0a 5.5 a d 4.0 b 4.0 b 4.1 c 4.7 b 4.9 a 5.0 a 4.9 b e 3.6 b 3.6 b 3.5 c 4.3 b 4.8 a 5.0 a 4.6 b f 2.8 c 3.0 c 3.0 c 3.4 c 5.1 a 5.2 a 3.4 c means followed by different superscript within a column are significantly different (p 0.05) a, b, c, d, e, and f are as defined in table 1. table 4. loaf volume (100 g dough) of bread produced from the composite flour samples. samples volume (cm 3 ) a 350 f b 315 e c 290 d d 240 c e 175 b f 145 a means followed by different superscript within a column are significantly different (p 0.05) a, b, c, d, e, and f are as defined in table 1. in table 4, with 100% wheat bread having the highest value. this result showed reduced loaf volume as proportion of tigernut increased in the flour mixes. however, volume of bread produced from 90:10 wheattigernut mixes was comparable to those produced from pure wheat flour. sensory properties the mean sensory scores for the bread produced from the composite flour are presented in table 5. the result of the sensory evaluation revealed that bread from pure wheat flour and those produced from composite flour with 10% and 20% tigernut flour were rated alike in almost all the quality attributes evaluated indicating the feasibility of adding tigernut to baked goods. this result suggests the potential application of tigernut flour either as full fat or defatted flour in baking industry. conclusion inclusion of tigernut flour in wheat flour at levels of 10 to 50% resulted in notable increase in fibre and ash contents while protein content decreased. the significant increase in the fibre content (167 to 967%) could be nutritionally advantageous in nigeria, where white bread is one of commonest staples among all classes of people. evaluation of the viscoelastic properties of dough from the composite flour, physical and sensory properties of bread revealed that a 10% wheat flour substitution with tigernut flour yielded bread product that was similarly rated with that produced from pure wheat flour. from the result of this study, it could be suggested that tigernut flour might find useful application in the nigerian baking industry. references aacc (1984). approved methods of analysis. st. paul. minnesota: the american association of cereal chemists p.1298 aoac (1990). association of official analytical chemists, 15 th edition. wincosin u.s.a. p. 1298 adeyemi ia (1988). a research note: ogi quality sorghum dry milled from fermented sorghum grains. j. cereal sci. 53: 640-642. addy eo, eteshola e (1984). nutritive value of a mixture of tigernut tubers and baobab seeds. j. science, food agric. 35: 437-440. anderson jw, smith bm, gustafson nj (1994). health benefits and practical aspects of high fibre diets. ame. j. clinical nutr. 59: 1242s1247s. alobo ap, ogbogo po (2007). selected properties of tigernut starch as affected by physical and chemical modifications. proceeding of the 31 st annual nifst conference, abuja, nigeria. 22 nd – 25 th october, 2007. basman a, koksel h (2003). utilization of transgluranase use to increase the level of barley and soy flour incorporation in wheat flour 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(www.powderandbulk.com/resources/material_bulk_density_charts.ht m) mbofung cmf, abuobakar yn, njintang a, abduo boubak, balaam f (2006). physico-chemical and functional properties of six varietes of taro (colocasia esculenta l.schott) flour. j. food technol. 4(2): 135142. oladele ak, k aina jo (2007). chemical composition and functional properties of flour from two varieties of tigernut (cyperus esculentus). afr. j. biotechnol. 6(21): 2473-2476. olaoye oa, onilude aa, idowu oa (2006). quality characteristics of bread produced from composite flours of wheat, plantain and soybeans. afr. j. biotechnol. 5 (11): 1102-1106. spe (1993). scientific program enterprises. computer software plotit graphics and statistics version 2.0 (ics gmbh, frankfurt). taiwo, k.a., akanbi, c.t., ajibola, o.o. (1997). establishing processing conditions for canning cowpea seeds in tomato sauce. inter. j.food sci. technol. 32: 313-324. wang yd, kinsella je (1976). functional properties of novel protein. j.food sci. 41(2): 286-294. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (9), pp. 208-216, september, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of solid-liquid extractive techniques and an innovative solid-liquid extraction technology using the naviglio extractor ® daniele naviglio 1* , fabiana pizzolongo 1 , raffaele romano 1 and lydia ferrara 2 1 dipartimento di scienza degli alimenti, università degli studi di napoli “federico ii” via università 100 80055 portici napoli – italy. 2 dipartimento di chimica farmaceutica e tossicologica, università degli studi di napoli “federico ii”, via domenico montesano 49 80131 napoli – italy. accepted 29 march, 2015 this document is a review on solid-liquid extractive techniques and describes an innovative solid-liquid extraction technology using the naviglio extractor ® . also explained is an application for the production of alcoholic extract from lemon peel. the alcoholic extract, mixed with a sugar and water solution in the right proportions is used to make a well known italian lemon liquor commonly named "limoncello". lemon liquor is obtained utilizing the naviglio extractor ® ; the procedure used is fast and efficient and uses about half the weight of lemon peel per volume of ethyl alcohol used for the extraction of the odorous and taste responsible compounds, compared to the commonly used extraction procedures. to test the consumer’s preference and compare the taste of the liquor obtained with that obtained by peel maceration from the same lot of lemons and obtained using the traditional recipe, a consumer test has been carried out. one hundred people, chosen from among frequent consumers of limoncello, tasted the two lemon liquors, and evaluated intensity of aroma, colour, alcohol taste and sweetness. in about 81% of the preferences, the liquor obtained using the naviglio extractor ® was preferred. the extraction process used allows the ethanol from used up lemon peel to be totally recovered so that these can be disposed of as non toxic waste or used in agriculture or as cattle feed. key words: lemon liquor, lemon peels, naviglio extractor, maceration, solid-liquid extraction, limoncello. introduction the most important and delicate phase in the preparation of alcoholic drinks deriving from blending an alcoholic extract with water and sugar is represented by the maceration in ethyl alcohol of matrixes of a vegetable nature, like flowers, leaves, berries, roots, fruits rind etc. the maceration is a very slow process of solid-liquid extraction that occurs at room temperature; it is realized by soaking the solid to extract with an opportune liquid, in a container called a macerator with the aim to extract soluble compounds responsible for odour and taste. the extractable compounds are extracted from the solid matrix of the extracting solvent through diffusion and because of osmosis due to prolonged * corresponding author. e-mail: naviglio@unina.it. contact. the solution must be desultorily remixed, during the whole period of extraction, with the purpose of allowing the diffusion of the drawn out substances and avoiding the phenomenon of super saturation in proximity of the solid surface; this event has two negative effects: the extraction process can be blocked and some oxidative processes can be permitted (nota et al., 2001; dugo et al., 1998). the lemon liquor commonly called “limoncello” is produced in italy mainly in the campania region from the alcoholic extract of the external yellow part of the lemon peel which is known as “flavedo”. in recent years the production of this alcoholic beverage has exponentially grown (douglas, 2000) and so many researchers have begun to study the chemical and physics characteristics naviglio et al. 209 of “limoncello” (bocco et al., 1998; bonomi et al., 2001; dugo et al., 2000; moio et al., 2000; nota et al., 2001; naviglio et al., 2002; versari et al., 2003; crupi et al., 2007). experimental taste-testing has shown that, maintaining a peel/alcohol ratio of 30% (w/v) at least two days of maceration are required to produce liquor that can be considered appreciable, this is because the smaller components (compounds present under 2% in weight with respect to the total weight of the extractable compounds in ethyl alcohol) of the peel of the lemon are released more slowly in comparison to the larger components (limonene, beta-pinene and gamma-terpinene); the presence of the smaller components is essential to confer the liquor the organoleptic characteristics necessary to make it appreciable to frequent consumers of lemon liquor (nota et al., 1999; nota et al., 2001). in these conditions it is inevitable that the alcoholic extract becomes full of water; all the water contained in the flavedo rapidly diffuses in to the solution during the extractive process so that the alcoholic content is lowered from 96% (v/v) to about 80% (v/v), in the case in which the peel/alcohol ratio is 30% (w/v) as quoted above; this is in accordance with the measured water content of the flavedo that is equal to 70% (w/w) (nota et al., 1999; nota et al., 2001). the process of dissolution of water in ethyl alcohol is rapid and it is completed in the first three hours of maceration; because in the maceration the mixing is irregular, during the long phase of stasis of the solution, strong gradients of concentration of water are generated in proximity of the surface of the peels, which in turn lower the alcoholic content locally, favouring hydrolysis reactions of the essential oil compounds (mazza, 1987; verzera et al., 1992 a, b). everything leads to the conclusion that during the maceration of the lemon peel in ethyl alcohol, with the purpose of obtaining an extract containing the whole component of the essential oils, a lowering in the initial alcoholic content is inevitable and this fact causes the collateral reactions of hydrolysis in the essential oils. finally, it must be noted that maceration is not an exhaustive extractive procedure with regard to the lemon peels; in fact, even extending the maceration long-er, over a month, the peels still preserve part of the essential oils that are no longer extracted because the concentration of the essential oils becomes so high that it reaches an equilibrium between the molecules that enter the solid and those that are released from it (steady state). maceration is an extractive process that is based on two fundamental effects, diffusion and the osmosis; actually, it is the most economical process among all known extractive techniques due to a simple apparatus required: a sealable container. currently, no other existing technique of solid-liquid extraction is applicable to the extraction of essential oils from lemon peel at room temperature for the production of lemon liquor; maceration is carried out industrially in the same manner as it is done at artisan artisan or family level. this is one of the main reasons for which the “limoncello” is loved by a great number of consumers of alcoholic drinks and the public; the naturalness of the product fully preserves the lemon’s beneficial properties (bocco et al., 1998; grandi et al., 1994; horowitz and gentili, 1997; middleton and kandaswami, 1994) and grants the drink strong digestive power, in fact its consumption is preferred especially consumed after meals. this paper has two aims: firstly as an innovative technology of solidliquid extraction, the naviglio extractor (naviglio, 2000; naviglio, 2003) is briefly introduced and compared to the current techniques of extraction, as far as extraction times are concerned, the extractive efficiency and quality of the extracts, particularly with maceration; the other to compare the lemon liquor produced in the traditional manner with that produced using the naviglio extractor, which allows the total recovery of ethanol from the used up lemon peels. experimental materials the lemons used for the experimentation were taken from the same production stock as the “ovale of sorrento” cultivation. ethyl alcohol at 95% (v/v) is the one commercially used and commonly used for the preparation of alcoholic drinks and it was purchased at the market. the sugar and the water with a low mineral content (fixed residue<200 mg/l) were purchased at the local market. traditional procedure for the preparation of the alcoholic extract of the lemon peels lemons (about 7 kg) were washed and dried; only the most external part of the lemon rind (flavedo) was recovered using a proper utensil; 600 g of lemon peel were transferred to a bowl (macerator) and covered with two litres of ethanol (peel/ethanol ratio: 30 g/100ml). the system was left to soak for 48 h and it was occasionally shaken. the alcoholic extract was recovered and paper filtered to eliminate any solid impurity. a sugary solution was prepared by dissolving 2 kg of sucrose in 4 l of water (sugar/water ratio: 50% (w/v)). two litres of this solution were added with one litre of alcoholic extract and the resulting mixture was gently stirred for 15 min to obtain the complete homogenisation. the exhausted lemon peels were undergone to the distillation in vapour stream for the gravimetric measure of the residue ethyl alcohol by means of hydrostatic balance. innovative procedure for the preparation of the alcoholic extract of lemon peels lemons (about 3.5 kg) were washed and dried; only the most external part of the lemon peel (flavedo) was recovered using a proper utensil; 300 g of peel were put in a porous bag and then it was introduced in the extraction chamber of the naviglio extractor (mod. 2 litres capacity, nuova estrazione s.a.s., naples, italy) and 2 l of ethyl alcohol were added (peel/ethanol ratio: 15 g/100ml). to allow the total recovery of the essential oils, 30 extractive cycles of 4 min each for a total of 2 h (2 min in static phase and 2 min in dynamic phase) were performed obtaining at the end the alcoholic extract to be used for the preparation of the liquor. as a side pro 210 afr. j. food sci. res. product used up peels were obtained and treated as follow. recovery of ethanol and essential oils from exhausted peels with water to recovery additional ethanol and essential oils from the used up peels the naviglio extractor was filled with two litres of water; two washing extractive cycles (8 min each) were performed to obtain water enriched in ethanol and essential oils. the enriched water was collected and the exhausted lemon peels were undergone to the distillation in vapour stream for the gravimetric measure of the residue ethyl alcohol by means of hydrostatic balance. preparation of the lemon liquor to prepare the lemon liquor a solution containing 1.5 kg of sugar completely dissolved in 3 l of the enriched water was prepared. the alcoholic extract (about 1.9 l) was mixed with a double volume of the aqueous sugar solution enriched in alcohol and essential oils. consumer test one hundred people, 65 males and 35 females from eighteen to forty years old, frequent consumers of lemon liquor, were chosen to carry out the organoleptic test of the comparison of two liquors. the test consisted of blind smelling and tasting of the two lemon liqueurs obtained, one produced following the traditional recipe and one made with the innovative technique. after tasting each sample, consumers were asked to rate the intensity of aroma, colour, alcohol taste and sweetness on a six-point scale. consumers were also asked to express their global preference. the subjects were advised to use water to rinse their mouths between samples. the samples were presented coded by three-digit numbers and in a randomised order. they were evaluated at room temperature following the indications as reported in literature (meilgaard et al., 1999). a t-test was used to determine significant differences between products at p< 0.05 level, using xlstat – pro 7.5.2 (microsoft, usa) for statistical analysis of data. results and discussion solidliquid extraction: an overview in many industrial processes, the initial phase of the preparation of a product requires the application of a solid-liquid extraction technique to isolate the extractable material contained in the most varied vegetable-type matrixes. the most important example is the medicinal plant sector, from which the active principles with pharmacological properties for the treatment of several human pathologies and illnesses are obtained; similar fields are herbalist, cosmetics and the perfume industry; even in these cases the principal ingredients of theirs products are obtained by submitting parts of plants such as flowers, leaves, roots etc. to solid-liquid extraction. also in other industrial sectors like the beverage industry, a solid-liquid extraction is used to get alcoholic extracts from the peel of citrus fruit, flowers, leaves etc. that are then mixed to water and sugar to obtain the finished product. the list could continue mentioning many other industrial applications. at the of solid-liquid extraction there is, without doubt, an experimental observation which anyone can verify: if, a solid matrix containing extractable material is simply dipped in a liquid, this begins to enrich with certain chemically similar substances (“similia similibus solvuntur”) in the phenomena of diffusion and osmosis. this technique of extraction, maceration, is the most simple and most economic and therefore it is widely used. unfortunately, it is not always applicable because it requires a long period of contact between the solid and the liquid; for example vegetables cannot be left to soak in water due to rapid fermentation processes that lead to putrefa-ction of the extract. the productive demands of the industry, which impose the production of large quantities of extracts in short times, have found an application in the extraction for percolation; in this case it is possible to treat large quantities of solid material with large volumes of liquid and obtain the extractable substances in a short times, but this process sacrifices the efficiency of the extraction that is kept low because of the limited contact between the solid and the extracting liquid. the current techniques of solid-liquid extraction use diffusion and osmosis as principles with which it is possible to manage the processes to reduce the time and to increase the yield of extraction. in fact, the actual trend is that of increasing the temperature of the extractive system or extending the contact of the extracting solvent with the solid to achieve better results. maceration maceration is the extractive technique largely used in limoncello production (dugo et al., 2000; moio et al., 2000; nota et al., 2001; naviglio et al., 2002; versari et al., 2003; crupi et al., 2007) that consists in covering the solid to extract with the liquid and to leave the system in this condition for a prolonged time. the extraction occurs at room temperature and accordingly there is not alteration of the thermo labile compounds; on the other hand the time of extraction is on average quite long given that extraction occurs mainly by diffusive effect, in that it is necessary to occasionally shake the system to favour the diffusion of the extracted components and to avoid super saturation in the immediate proximity of the surface of the solid to be extracted, it leads to a deceleration of the global extractive process. in this way it is possible to obtain lemon liquor and many other very similar alcoholic beverages. maceration is also a technique widely used in many official methods of analysis (see aoac methods). a last effect, ultrasounds or microwave assisted extraction, was introduced recently to enhance maceration , but the related techniques are nowadays under investigations (stanisavljevi et al., 2007). percolation percolation reduces times by treating big quantities of naviglio et al. 211 solid matrix, but it is not used in limoncello production; this technique requires the realization of a column of solid material packed in the percolators that are big steel cylinders capable of containing tons of material with volumes ranging from 0.5 to 5 cubic metres. in the percolators the extracting liquid flows from the top to the bottom; since in only one passage the yield is not high, it is necessary to recycle the liquid different times to enrich it more than possible in extractable compounds. to increase the efficiency of the process it is possible to heat the percolator and in this case the thermo labile substances are subject to thermal degradation. gene-rally, the efficiency of the global process is not high, but due to the large quantities of solid employed, the extract results proves to be sufficiently rich in extractable com-pounds. also in this case the extractive principle is based essentially on diffusion and on osmosis (hansen and hinrichsen, 1992). this technique is widely used for the production of pharmaceuticals. supercritical fluid extraction (sfe) a recent technique that requires a particular extractive system is extraction with supercritical fluids especially with carbon dioxide. the limitation of this technique, that requires contact of the solid with the carbon dioxide at the fluid state, is due to the fact that when carbon dioxide is in the supercritical state it acquires the chemical-physic properties of n-hexane. with this method it is therefore possible to extract non polar compounds from solid matrixes. the advantage of this technique is that at the end of the extraction the solvent, the carbon dioxide, is removed under the form of gas giving the possibility to recuperate the extractable concentrate. in on one side it is difficult to use this technique for laboratory uses, from the other this technique finds some important applications at the industrial level like the extraction of oil from seeds, caffeine from coffee, nicotine from tobacco etc., but it is however very expensive and not universally applicable (mehr et al., 1996; ching et al., 1998; giannuzzo et al., 2003; rajaei et al., 2005). extraction using vapour stream for particular applications such as the production of essential oils and, generally, compounds with high vapour tension, the technique of distillation in vapour stream can be used. this technique of solid-liquid extraction is peculiar because it requires the transport of volatile compounds from a vapour stream. in any case, since the extractive system is submitted to strong heating, the thermo labile substances undergo some transformations and they are not entirely recuperated. some examples include the separation of tocopherols from soya sludge and extraction of citrus essential oils (bannwart de moraes et al., 2006; parshikova, 2006). ultrasound or microwave assisted extraction (uaemae) these examples are to show that the techniques of solidliquid extraction currently used are not universally applicable because they are limited. besides, the extractive principle on which they are based is essentially linked to the phenomena of diffusion and osmosis of the substances contained in the solid; these tend to occupy the whole volume of the extracting liquid after they have been extracted. to raise the efficiency of these extractive systems a higher temperature is used, which causes diffusion to increase with the purpose of reducing extraction times and obtaining higher efficiency. this expedient is not applicable to vegetable matrixes because they contain substances that degrade themselves by effect of heating. the use of ultrasounds or microwaves (luque-garcía and luque de castro, 2004; stanisavljevi et al., 2007) for the extraction of active principles from medicinal plants leads to obtaining the same results of extraction by squeezing, or even worse results because the system is heated for the prolonged treatment. in these cases the solid matrix becomes completely shattered and gives a mixture that is impossible to separate in its constituents and therefore renders these techniques inapplicable at industrial level. soxhlet method for solid-liquid extraction another extractive technique from the laboratory is soxhlet, which is reported as the official method of extraction for numerous analytical methods in which an initial preparation of the extract of a solid sample is required (see aoac methods). the soxhlet also uses the heating of the system, since it is based on the principles of diffusion and osmosis, for which it cannot be used for the substances that are degraded by the effect of heating. it is a process very similar to maceration; the difference consists in the fact that the extractive solvent in contact with the solid matrix is fresh due to its evaporation. even if accelerated soxhlet are on the market, in literature some papers that compare this technique with others are reported with the aim of replacing it for the retrieval of different substances (crespo and yusty, 2005; hawthorne et al., 2002; bowyer and pleil, 1997). this technique is mainly performed on a laboratory scale because of the difficulty of constructing very large glass containers. accelerated solvent extraction (ase ® ) to raise the yield of extraction and reduce the extraction times it is possible to use the ase ® extractor (accelerated solvent extraction) patented by the american dionex. the material to extract is inserted in a cylindrical steel container and the extracting solvent is introduced; 212 afr. j. food sci. res. table 1. comparison of the main features related to the current techniques of solid-liquid extraction. extractive technique granulation solvent efficiency time extract quality extract stability squeezing not important indifferent complete minimum unsatisfactory poor maceration important fundamental complete long very good very good percolation important fundamental partial medium good good soxhlet important fundamental complete long unsatisfactory poor distillation in vapour stream not important indifferent partial medium unsatisfactory poor supercritical gas important fundamental partial medium very good very good ultrasounds not important indifferent complete medium unsatisfactory poor accelerated solid-liquid extraction ase important fundamental complete minimum unsatisfactory poor extractor naviglio ® not important indifferent complete minimum very good very good the temperature of the system is brought above the boiling point of the solvent, which is maintained in the state of liquid thanks to a contemporary increase in pressure. after a brief contact the solid matrix is completely exhausted. also for this technique it is not possible employ unstable matrixes to the heat but for many analytical applications, especially when many samples are to be analysed, it is very useful (zuloaga et al., 1998). as with the soxhlet extractor, ase is a laboratory technique and currently it is impossible to set up large plants because of very high pressure requested. naviglio extractor or rapid solid-liquid dynamic extractor this document introduces an innovative technology of solid-liquid extraction, which can be used as valid alternative to the existing ones: the naviglio extractor ® . it completely changes the philosophy of solid-liquid extraction thanks to the discovery of a new principle of extraction: naviglio’s principle (naviglio, 2003) . the extraction happens because of the generation of a negative gradient pressure from the inside toward the outside of the solid matrix, therefore it can be conducted at room temperature or sub room temperature. an extractive cycle is constituted by a static phase and a dynamic one; during the static phase the liquid is put under pressure at about 8 10 atm on the solid to extract and it is left for sufficient time to allow the liquid to penetrate the inner solid and to balance the pressure between the inside and the outside of it. after this time, the pressure is immediately removed and falls quickly to the atmospheric pressure; in this moment the principle of naviglio causes which appears to be as a suction effect of the liquid from the inside toward the outside of the solid; this rapid movement of the extracting solvent transports the extractable material toward the outside. the cycles can be repeated until the solid is exhausted. experimental trials carried out until today on more than two hundred vegetable species showed that, working at a pressure of 9 atm, the greatest part of the solid matrixes, independently from the degree of crumbling, can be extracted using about thirty extractive cycles which occur in about two hours. besides, the reproducibility of the extraction has been proved on the same matrix in ponder terms and comparative experiments have been conducted with the other extractive techniques, which have underlined the highest recovery in favour of the naviglio extractor ® , as well as a superior quality of the extract and in any case an alteration of the thermo labile substances has been induced (cutillo et al., 2006; micali et al., 2004; naviglio et al., 2002; naviglio et al., 2006). finally, after the validity of naviglio’s principle was demonstrated, experiments in the pharmaceutical sector of medicinal plants and experiments for the improvement of herbalist products, cosmetics and perfume industry are in progress at the faculty of pharmacy of the università degli studi di napoli “federico ii” while the research in the field of food science at department food science is performed nowadays (naviglio et al., 2006). in table 1 the comparison between the different existing solid-liquid extractive techniques is shown and, as it is possible to observe, the naviglio extractor ® offers in one solution everything that the operator would like to receive from a solid-liquid extraction. the new philosophy of rapid solid-liquid dynamic extraction the rapid solid-liquid dynamic extractor represents an innovative technology of solidliquid extraction that allows the solid matrixes containing extractable substances in an organic or inorganic solvent and in their mixtures to be exhausted in a short time, compared to the other cur-rently existing extractive techniques. the novelty of the extractor is in the fact that it changes the extractive philosophy because it reverses the tendency of the cur-rent methods, which aim to heat the extractive system to raise efficiency and to shorten the extraction times; the naviglio extractor carries out the extraction at room or sub-room temperature and it uses an increase of pressure of the extracting liquid on the solid matrix to extract. the importance of extracting at low temperatures naviglio et al. 213 figure 1. the first experiment production of “limoncello” in the two-syringe system. lies in the fact that thermal stress is avoided on the thermo labile substances in this way. therefore it is possible, for instance, to faithfully reproduce the composition of the compounds contained in the medicinal plants without inducing transformations of the active principles, which generally are the more “delicate” substances to extract because they are susceptible to the effects of the temperature. moreover, extraction is independent from chemical features of the extractive liquid and it makes possible to extract in heterogeneous phase compounds that do not dissolve themselves like essential oils in water (naviglio et al., 2003). naviglio’s principle the extractor works by alternating a static phase with a dynamic phase; during the static phase the extracting solvent is brought to a pressure of about 9 atm and it is kept in this state for sufficient time to reach an equilibrium between the pressure of the liquid to the outside of the solid matrix and the liquid within it; at the end of the static phase a dynamic phase begins, which is produced by a sudden movement of the pistons; it contributes to a rapid decrease of pressure of the system to lower values. in this moment the extraction of the solid matrix is realized. it becomes possible because a delta of negative pressure is generated from the inside toward the outside of the solid matrix, due to the sudden fall of the pressure of the liquid to the outside of the solid, that is at the base of the new extractive principle (naviglio’s principle): the extrac table substances of the solid are dragged out for an absolute suction effect, in every cycle of extraction constituted by a static phase and dynamic. the last phase allows, at the same time, a rapid and complete remixing of the solid matrix and an immediate diffusion of the drawn out substances in the whole mass of the liquid avoiding the phenomenon of local super saturation or others unpleasant effects (naviglio et al., 2005). the naviglio extractor ® represents a valid alternative in comparison to existing extractives techniques and it mainly finds application in the extraction of the active principles medicinal plants, in the extraction of essential oils of citrus fruit, in the extraction of essences and perfumes, in the extraction of fats etc. it can be dimensioned according to the demands starting from a bench apparatus size for analytical uses up to an industrial dimension for the production of large quantities of extract. from the principle to the realization of the machine: evolution of the naviglio extractor ® the functioning of the naviglio extractor, the statement of naviglio’s principle and the related considerations are discussed elsewhere (naviglio, 2003). in this paragraph we briefly want to resume the history of the production of the final naviglio extractor machines. the intuition of the principle explained above came to dr. d. naviglio in 1993, when like a vision a movement of pistons similar to that which happens in a car engine appeared in his mind. if a suction effect was achieved this movement could allow the extraction of extractable compounds from solids. to verify the efficiency of extraction, elementary trials were performed connecting two syringes through a duct and producing pressure only using the force of hands (figure 1); in this way it was possible to have the initial approach in demonstrating the validity of the principle in the production of a minimum quantity of “limoncello”. these first results gave the possibility to obtain the patent of both the new extractive process and the extractor (naviglio, 2000). in figure 2 the bench-top prototype of naviglio extractor ® is shown with which it was possible to scient-ifically verify the validity of naviglio’s principle. it is composed of two cylinders (syringes) that act as extrac-tive chambers, in which two pistons moved by compress-ed air run. this prototype works as elsewhere reported (naviglio, 2003) and it allows the production of about 50 ml of extract. in figure 3 the industrial prototype is shown; it allows the treatment of about 1 5 kg of solid (this quantity depending on the value of density of the solid) and obtains about 40 l of extract. the optimization of variables and the necessity of limiting encumbrance to a minimum have been resolved at the same time leading to the construction of the final version of the naviglio extractor model of nominal capacity ranging from 0.5 to 2 l (figure 4). currently there are models of naviglio extractor market ranging from a capacity of 0.5 to 400 l. moreover, 214 afr. j. food sci. res. figure 2. bench-top naviglio extractor ® prototype. figure 4. last model of bench-to 0.5 (in the figure), 1 and 2 litre naviglio extractor ® . figure 3. industrial 50 litre naviglio extractor ® prototype. it is possible to realize the same above mentioned principle using solely one extracting chamber and one cylinder lowering the efficiency of the extractive process in respect of a two cylinders-two pistons system with the aim of reducing the production costs. finally, the initial working pressure was of about 6 atm but new models of naviglio extractor ® working at higher pressures (9 10 atm) have already been built; higher pressures allow a reduction in extraction times contemporarily obtaining an exhaustive process of extraction. alcoholic content of the extract at the end of the maceration process, since the lemon peels contain about 70% (w/w) of water, the alcoholic content of the extract has been lowered in an appreciable manner from 96% (v/v) to about 80% (v/v) and in this case the ratio of dilution to achieve a final liquor to the thirty percent in alcohol is 2.67; in the case of the extract obtained with the naviglio extractor ® the alcohol content is lowered to about 90% (v/v) and accordingly the ratio of dilution is 3.00. this is due to the fact that the quantity of peel is smaller and, because of washing the peels with water, ethanol and essential oil are totally recovered. in this way it is possible to get about 12% (v/v) more lemon liquor which represents a source of profit considering the cycle of total production performed in one year. disposing of used lemon peels at the end of the process of maceration, used lemon peels, submitted to the analysis of alcoholic content by distillation in vapour stream, showed a ethanol content of about 15% (v/v) and then they were not disposable as a manure; while the peels deriving from the process of production of lemon liquor using the naviglio extractor were lacking in ethanol and then they can be disposed as manure or employed as feed after a drying process at room temperature or in hot air. evaluation of the results of the consumer test results of consumer test are reported in figure 5 as a histogram. the two liquors were significantly different (p<0.05) for aroma, colour, alcohol taste and sweetness. naviglio’s liquor exhibited a more intense aroma, colour and sweetness than the other one. this shows that by means of this innovative extraction technology it is possible to extract more compounds that increase aroma and colour; moreover, the brief contact of lemon peel with ethanol avoids hydrolysis phenomena of some extracted compounds and in this way the aroma is closer to the fresh lemon aroma. these features result in a preference for naviglio’s liquor; in fact the results of the preference test showed that 81% of the judges clearly preferred the lemon naviglio et al. 215 6.00 5.00 in te n s it y 4.00 3.00 2.00 1.00 0.00 a ro m a c o lo u r al co ho lta st e s w e e tn e s s naviglio liquor com m arcial figure 5. histogram of data obtained from the consumer test with regard of colour, sweetness, aroma and alcohol for the compared lemon liquors. lemon liquor produced with the naviglio extractor ® , while the remaining part found the two liquors equivalent. conclusions the naviglio extractor introduces some notable advantages in the production of lemon liquor. first of all it reduces the times of extraction of the essential oils from the lemon peel bringing the extractive time from a minimum of 48 h to about two hours; besides the peels are completely exhausted from the essential oils in this period of time, which does not happen in maceration but extends the phase to a long extractive time. using half the peels that are normally used, in the same way a pleasant drink is obtained similar to that produced according to traditional recipes that require one or two weeks of extraction time. using a smaller quantity of peels the alcoholic content is not excessively lowered; finally, ethyl alcohol, which is normally lost during the maceration phase (about 10 15% v/v), is completely recovered using two rapid washing cycles (16 min.) with potable water that subsequently is used for the preparation of the sugary solution. the total recovery of ethanol in the extractive cycle allows the production of a major quantity of lemon liquor and brings the dilution ratio of 2.67 in the maceration to a value of 3.00. the current research in finding a best extraction technique did not at the end prove to be as widely reported in recent literature; 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(2-3): 33-36. publisher: kubanskii gosudarstvennyi tekhnologicheskii universitet. punín crespo mo, lage yusty ma (2005). comparison of supercritical fluid extraction and soxhlet extraction for the determination of pcbs in seaweed samples. chemosphere 59(10): 1407-1413. rajaei a, barzegar m, yamini y (2005). supercritical fluid extraction of tea seed oil and its comparison with solvent extraction. european food res. technol. 3: 401-405. stanisavljevi it, lazi ml, velikovi vb (2007). ultrasonic extraction of oil from tobacco (nicotiana tabacum l.) seeds. ultrason. sonochem. 14(5): 646-652. versari a, natali n, russo mt, antonelli a (2003). analysis of some italian lemon liquors (limoncello). j. agric. food chem. 51: 49784983. verzera a, del duce r, stagno d’alcontrones i, trazzi a, daghetta a (1992a). trasformazioni dei componenti degli aromi solubili agrumari in ambiente acido acquoso. nota i: monoterpeni aciclici e monociclici. industrie delle bevande 21: 217-222. verzera a, stagno d’alcontrones i, donato mg, del duce r, gigliotti c (1992b). trasformazioni dei componenti degli aromi solubili agrumari in ambiente acido acquoso. nota ii: monoterpeni biciclici. industrie delle bevande 21: 379-386. zuloaga o, etxebarria n, fernández la, madariaga jm (1998). comparison of accelerated solvent extraction with microwaveassisted extraction and soxhlet for the extraction of chlorinated biphenyls in soil samples. trends in anal. chem. 17(10): 642-647. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (2), pp. 001-004, february, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper testing the sensory acceptability of biltong formulated with different spices flávia edite j. m. dzimba, josé de assis f. faria* and eduardo henrique m. walter. department of food technology, faculty of food engineering, cp 6121, state university of campinas – unicamp, 13083-862 campinas-sp, brazil. accepted 24 september, 2018 biltong is a traditional meat product widely consumed in south africa and other countries. in this work different formulations of biltong were prepared, using either traditional south african or brazilian (salt, sugar, nitrite, pepper, allspice, aniseed, garlic, onion, coriander and, or, pineapple juice) spices and sensory evaluated. the appearance acceptability of the alternative formulations was judged as being more accepted them the traditional one, possibly due their lighter colour. there were no statistical differences (p  0.05) among them with respect to flavour. some consumers cited that the tenderness of formulation with pineapple juice was softer. in fact, 77% of the consumers reported liking this formulation slightly or more. the main reason cited by consumers for liking the flavour of biltong was their pleasant seasoning. the product, in general, was described as being hard, but readily softened by the saliva when chewed. overall, the results suggested that, with a little adaptation of its original formula, it would be possible to introduce biltong as an acceptable snack or delicatessen in brazil and different south american markets. key words: biltong, sensory acceptability, spices, pineapple. introduction biltong is a well known uncooked dried meat product wid-ely consumed in south africa and many others countries (van der riet, 1982; attwell, 2003). in brazil three ethnic products derived from beef are widely consumed: carne-de-sol, charque, and jerked beef (carvalho jr, 2003). although produced using the same principles of presser-vation used in south american’s dried meat products, lowering of its water activity by salting and dehydration, there are differences in the formulation and method of preparation of biltong that make it quite peculiar and unique. unlike south american’s dried meat products, biltong does not needs rehydration or cooking treatment prior to consumption. it is a ready-to-eat meat product and seems to be a potential snack in several popular diets, where consumption of increasing amounts of pro-teins and less carbohydrates are encouraged. biltong can be prepared from beef or other similar meat *corresponding author. e-mail: assis@fea.unicmp.br. phone: 55 19 3521-4016. fax: 55 19 3289-3617. source. it is flavoured with spices such as aniseed, coriander, allspice or garlic. the condiments can be mixed with pepper and added to the salt, but other optional ing-redients are nitrite, sugar, and saltpetre, which promote a red colour to the product (thomas, 1975; osterhoff and leistner, 1984; prior, 1984) . many of these formulations are passed from generation to generation. the product is microbial stable at the barrier combination of water acti-vity 0.77 and ph 5.5 (osterhoff and leistner, 1984), althouugh many consumers prefer “moist” biltong (att-well, 2003) with a aw usually ranging from 0.85 to 0.93 (osterhoff and leistner, 1984). besides many african countries, biltong has found markets in australia, portugal, united kingdom, and united states of america (attwell, 2003). in spite of the fact that south american food habits are strongly influenced by african culture, this product is completely unknown in south america. growth of meat consumption in south america is strongly related to the emerging middle class of urban populations. for these consumers, traditional methods of meat preservation, such as salting and drying, play an important role in the meat marketing figure 1. acceptability of biltong appearance. structure, nowadays considered a delicacy food type. the success or failure of new food products depends on many factors, but one interesting aspect is the cultural elements. cultural elements such as food habits, cooking techniques, and health concepts, strongly influence the acceptability of a new product among consumers. however, of all the variables influencing food acceptability, the sensory attributes of the product are the most important. therefore the objective of this work was to evaluate the sensory acceptability of biltong formulated with the traditional south african spices and compared with typical brazilian spices. materials and methods processing of biltong the manufacture of biltong was carried out following the prior (1984) methodology. hindquarter beef muscles were cut into strips of approximately 15 20 x 2 x 1.5 cm along the grain of the muscle. three different formulations were used for the biltong manufacture. formulation i biltong was prepared with spices from south africa, safari biltong seasoning® (crown national, chempet, south africa). in this formulation, the cuts were sprinkled on both sides with sea-soning and laid on a tray. every 30 min, the cuts were turned over, and after 4h they were hung to dry at 35 ± 3 °c for 36 h in a drying cabinet. formulation ii biltong was processed using typical south american spices. the formula was adapted to local flavour expectations by using the following ingredients: salt (3%); sugar (0.72%); nitrite (150 mg per kg meat); pepper, allspice, aniseed, garlic, and onion (0.024%) and coriander (0.50%). the meat was dry salted and left overnight under refrigeration (10 ± 2°c) to allow the salt to penetrate. the meat was then dried in a drying cabinet at 35 ± 3°c for 36 h. formulation iii biltong was prepared as described in formulation ii, but with added pineapple juice as a source of the enzyme bromelain. the first step of the process consisted of immersing the meat in the pineapple juice (ananas comosus l. merr) for 15 min at 5°c. the meat was then dry salted with the mixed spices as in formulation ii, left overnight and dried. sensory evaluation a panel of 55 untrained judges of both sexes was recruited among students and staff of the faculty. each panellist was selected accord-ing to his potential as a consumer of the product. initially panellists were asked to evaluate the appearance of the samples and then decide if they would like to taste them or not. they were requested to justify their option. for panellists who decided to taste the samples, the three formulations were served in random order. panellists eval-uated the samples seated in individual booths equipped with red lights to mask colour differences. panellists used a 9-point hedonic scale (9 = like extremely; 1 = dislike extremely) to grade how much they liked or disliked each sample (stone and sidel, 1993). statistical analysis the statistic of the data was done by analysis of variance (anova) and tukey test, using sas software (sas 1985). a significance level of 5% was used for all comparisons. results figure 1 shows the acceptability by consumers based on the appearance of tested biltong. it can be seen that while 18% of the consumers liked formulation i, nearly 65% liked formulation ii and 60% liked formulation iii, with a grade of 6 (liked slightly) or higher on the hedonic scale. table 1. acceptability means of biltong appearance and flavour. acceptance formulation i formulation ii formulation iii appearance 3,31 b 5,05 a 4,98 a flavour 5,62 a 5,60 a 6,02 a * means are based on 9-point intensity scale (1=dislike extremely; 5=neither like nor disliked; 9=like extremely). in the same line formulations with common letter (s) did not differ significantly (p  0.05) among them. figure 2. acceptability of biltong flavour. means for appearance acceptability of the samples and results of the tukey test are presented in table 1. in fact, formulation i was judged as being statistically different (p  0.05) from formulations ii and iii, being the least accepted among them. there were no statistical different (p  0.05) between formulations ii and iii, and they showed mean acceptability close to the term “neither like nor dislike” on the hedonic scale. figure 2 shows the results of flavour acceptability for the three formulations. nearly 67% of the consumers liked the flavour of formulations i and ii. formulation iii was more acceptable showing 77% consumer acceptability. none judge attributed the value 1 (dislike extremely) to the flavour of any formulation and nearly 2% of them liked formulation i extremely, while 6% liked formulations ii and iii extremely. formulations ii, iii, and i were not statistically different (p  0.05) among them with respect to flavour acceptability. discussion the majority of the consumers invited to evaluate biltong liked to taste it. they referred to curiosity as the reason to taste biltong. the appearance acceptability of the alternative formulations ii and iii was judged as being more accepted them the traditional formulation i, possibly due to their lighter colour. the main reason cited by consumers for liking the flavour of biltong was their pleasant seasoning. the product, in general, was described as being hard, but readily softened by the saliva when chewed. some consumers cited that the tenderness of formulation iii was softer. it is possible that the pineapple protease, the bromelain, was successful in improving meat tenderness. it is known that pineapple enzymes is one of the most commonly used meat tenderizers for food processing in home cook ing and industrial processes (lizuka and aishima, 1999). this work has shown that, even though biltong is a product completely unknown to south american consumers, the sensory characteristics of the product are acceptable among them. an alternative biltong formulation, using both pineapple juice to soften the meat, and popular south american spices instead of typical south african condiments better fulfilled the consumers expectations towards the snack sensory attributes; obtaining the best acceptability values. this study clearly indicated that, with some further limited improvements in the biltong formulation, it would be possible to introduce this meat product as an acceptable snack or a premium delicacy in brazil and others markets. acknowledgements the authors thank the conselho nacional de desenvolvimento científico e tecnológico (cnpq) of brazil, which provided the scholarship for the first author. appreciation is extended to council for scientific and industrial research (csir) republic of south africa, for their permission to use their facilities for this research, and crown national, for the safari biltong seasoning. references attwell e (2003). biltong wakes up. food rev. 30:11-13. carvalho jr. bc (2003). the ethnical origin of the main meat processed products of brazil. in: 49th international conference of meat science and technology held at campinas, brazil, pp. 121-127. lizuka k, aishima t (1999). tenderization of beef with pineapple juice monitored by fourier transform infrared spectroscopy and chemometric analysis. j. food sci. 64:973-977. osterhoff dr, leistner l (1984). south african biltong another close look. j. s. afr. vet. assoc. 55:201-202. prior ba (1984). role of micro-organisms in biltong flavour development. j. appl. bacteriol. 56:41-45. sas (1985). sas/stat user’s guide version 5.0. cary, north carolina, usa: sas institute inc. stone hs, sidel jl (1993). sensory evaluation practices, 2nd ed., academic press, san diego, ca, usa. thomas pl (1975). dried meat products. csiro food res. quarterly. 35:73-78. van der riet wb (1982). biltong a south african dried meat product. fleischwirtschaft. 62:1000-1001. african journal of food science research vol. 2 (10), pp. 142-148, october, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper gender limitation in agricultural land access: effects for food security in ondo state, nigeria okojie martins, ekiosa osaro and yobosa duke department of agriculture, faculty of food science and technology, ambrose alli university, ekpoma, edo state, nigeria. accepted 15 september, 2014 this study assessed gender discrimination in agricultural land access: implications for food security in ondo state nigeria. specifically, it analysed men and female accessibility to forms of land holding and the factors affecting agricultural land accessibility in the study area. multistage sampling technique was used in selecting 240 respondents used for this study. data collected were summarized using descriptive statistics such as frequency counts and percentages and correlation analysis was used to test the hypothesis stated. the results revealed that the mean age of the male respndents was 48.3 while that of female was 43.7 with the standard deviation of 14.9 and 11.3, respectively. also, at p ≤ 0.05, there was significant relationship between accessibility to agricultural land and male and female socio-economic characteristics such as age (r = 0.484), marital status (r = 0.568), farm size (r = 0.504), farming experience (r = 0.479), household (r = -0.668), access to credit facility (r = 0.476), and membership of social organization (r = 0.593). this study therefore concluded that gender differentials, especially with regards to land favour the males. it is therefore recommended that redesigning and redeveloping the structure of land policies to be more gender sensitive and inclusive. key words: gender, land acess, food security, correlation, discrimination. introduction in a rapidly changing world, food and agricultural land holding systems in developing countries are facing new and increasingly complex challenges (derman et al., 2007). fighting poverty, ensuring food and nutrition security while protecting the environment still remains a major challenge facing global development practitioners today. in discussing agriculture, two key factors that cannot be ignored are land and labour. land ownership, accessibility, and the sustainability of this access are very crucial for any meaningful agricultural development. either by design or circumstance, women constitute a large proportion of farm labour and agricultural workers. these women usually control very little or no amount of land, which is very important in determining farm productivity *corresponding author. e-mail: okojie700@gmail.com and labour welfare (afonja et al., 2002). land is the most valuable form of property in agrarian societies because of its economic, political symbolic and ritual importance (bioye et al., 2006). it is the basis of political power and social status in most societies of the world. it is a productive wealth-creating and life sustaining asset which every human being craves for and provides a sense of identity and rootedness within a community (argwal, 1994). land is used for production of biomass, ensuring food, fodder, renewable energy and raw materials for existence of human and animal life. it is a base for settlement and industrial use and a store of our cultural heritage and is actually a source of raw materials like minerals, clay, energy and water (blum, 1998). land stands for continuity of ownership since it is a burial ground where all clansmen are buried and consequently a central place for the spirits of their ancestors for example in african countries. http://www.internationalscholarsjournals.org/ okojie et al. 142 in most developing countries, land is not only the primary means for generating livelihoods but often the main vehicle for investing, accumulating wealth, and transferring it between generations. thus, the ways in which access to land is regulated, property rights are defined, and ownership conflicts are resolved has broad implications for food security (deininger and binswanger, 1999, fao, 2006, 2008, 2009). access to land can be through right of ownership, through informal concessions granted by individuals to kin or friends. legal ownership is normally accompanied by legal restrictions on disposal, that is, there is no effective control here. in most african societies, women have land use priorities from husbands but have no independent rights which allow them control or produce from the land. the direct advantage of land rights are that a woman can use it to grow food crops, fodder for animals, keeping livestock, practicing sericulture, growing trees and vegetable gardening (cousins and claassens, 2006). land rights facilitate access to credit and strengthen support that the women receive from relatives. access to land means reliable food supply, better healthcare, better housing and reliable income in most cases. the percentage of men and women employed in agricultural sector decreased between 1997 and 2008 (due to the increasing industrialization of the considered countries), the percentage of women employed in agriculture is still higher in almost all developing regions as shown in apendix 1. in the last years, migration of men towards the cities led to a gradual feminization of smallscale agriculture, with an increasing percentage of womenheaded households in rural areas (fao, 2008). the relevance of women’s agricultural labour can be appreciated if we consider that, for instance, the agricultural sector in sub-saharan africa contributes about 30% of the gnp of the continent, employing from 60 to 90% of the population and producing from 25 to 90% of the income deriving from exports (fao, 2009) and fao (2002) lawanson (2010) opined that nigeria is a typical patriarchic society where male superiority and dominance originated from historically rooted culture and religion. in pre-colonial times, females generally were accorded less value and lower social status. western culture reinforced this anomaly. however, bruce and lloyd (1991) stated that the western culture has failed to address gender inequality in access to land, in spite of the role that land plays in the lives of the women who are increasingly being saddled with the responsibility of heading and maintaining households, especially in developing countries. women constitute over 50% of the nigerian population, make up about 37% of the formal sector (world bank, 2001), and dominate the informal economic sector which is principally made up of home-based enterprises (soetan, 2002); moreso, lawanson (2010) stated that their relative powerlessness both economically and politically, are unable to exercise control over resources, particularly land. some cultures in nigeria, through marriage and inheritance practices, prohibit women from owning land. however, soetan (2002) posited that marital status increases the women ability to own land. at rural level, women work mainly on their own, linking their activities to the family needs, and just a small percentage of them, everywhere lower than men’s receives a wage. in latin america, for instance, only 2.3% of women in agriculture get a wage against 20.9% of men; in southern asia salaried rural women are 11.9%, while men are 21.8%7 (alice, 2008). with the increasing roles of rural women in agriculture and contributions to food security and the consequence inequality on land access across gender, there is therefore the need to access the gender discrimination in land accessibility and implications to food security in nigeria the specific objectives for the study were to: 1. examine the socio-economic characteristics of the respondents 2. analyse male and female accessibility to forms of land holdings in the study area 3. assess the factors affecting male and female accessibility to land. 4. profile the security of tenue over land across gender. hypothesis for this study was stated in the null form as follows: there is no significant relationship between selected socio-economic characteristic of male and female respondents and their accessibility to land in the study area. the study area the study was conducted in ondo state of nigeria. the state was carved out of the old oyo state on the 3 rd february, 1976 with the capital in akure. the state covers an area of approximately 15,500 km 2 and it is bounded in the south by the bight of benin and atlantic ocean; north by ekiti and kogi states; east by edo and delta states and west by osun and ogun states (figure 1). the state lies between longitude 5°45′ and 7°52′ on the north – south pole, and longitude 4°20′ and 6°5′ on the east – west pole. according to analytical report of the national population commission (npc) (2006), ondo state has 3,441,024 million people with eighteen (18) local government areas. the tropical climate of the state is broadly of two seasons: rainy season (april to october) and dry season (november to march). a temperature throughout the year ranges between 21 to 29°c and humidity is relatively high. the annual rainfall varies from 2,000 mm in the southern areas to 1,150 mm in the northern areas. the soils derived from the basement complex rocks are mostly well drained, with a medium to fine texture. the state enjoys luxuriant vegetation with high forest zone (rain forest) in the south and sub-savannah forest in the northern fringe. the indigenes of the state belong to the yoruba ethnic group 143 afr. j. food sci. res. figure 1. map of the study area. and are composed of the akokos, the ondos, the ikales/ilajes and the apoi/ijaw arogbos. however, non – indigenes from every part of the country and outside reside in the state. yoruba and english are the languages of the people for official and business transactions. the state is basically agrarian with large scale production of cocoa, palm produce and rubber. other crops like maize, yam and cassava are produced in large quantities. sixty-five percent of the state labour force is in the agriculture sub-sector (coastalnews, 2012). the state is also blessed with very rich forest resources where some of the most exotic timber in nigeria abounds. ondo state is equally blessed with extensive deposits of crude oil, bitumen, glass sand, kaolin, granites and limestone. therefore, the state has great potentials for rapid industrial growth in view of its raw materials base. reasonable segment of the populace are also traders and artisans. other occupations of the people include weaving, mat – making, dying, soap making, wood okojie et al. 144 table 1. distribution of respondent’s demographic characteristics. variable male (n= 160) female (n=160) frequency percentage frequency percentage age >30 42 27.2 34 21.3 31-60 90 56.3 102 63.8 60 years and above 28 16.5 24 14.9 level of education no formal education 19 11.9 31 19.4 primary school 56 35.0 61 38.1 secondary education 63 39.3 50 31.2 tertiary education 22 13.8 18 11.3 farming experience < 5 10 6.3 5 3.1 6-10 32 20.0 40 25.0 11-20 68 42.5 79 49.4 >20 50 31.2 36 22.5 household size >2 15 9.4 12 7.5 2-5 35 21.9 89 55.6 6-9 84 52.5 40 25.0 >10 26 16.2 19 11.9 farm size <1 12 7.5 62 38.8 1-3 91 56.8 83 51.9 >3 51 35.7 15 9.3 source: field survey, 2010. carving, among many others. the state lies entirely within the tropics of 162 mm per annum. sampling procedure and percentage while the inferential statistics were correlation analysis and chi-square. data were analysed with the aid of statistical package for social sciences (spss) version 14.0 and costat analytical software. a multi-stage sampling technique was adopted to select respondents for this study. the first stage involved purposive sampling of six local government areas based on their degree of involvement in farming. the local government areas were irele, okitipupa, odigbo, owo and akoko, ose and akoko south-west. in the second stage, the local government areas were grouped into communities. in the third stage, two communities were randomly selected from each of the local government areas. in the final stage, twenty respondents with equal number of male and female were sampled from each of the communities primary data were used for this study. data were collected using structured questionnaire and interview schedule for both the literates and illiterates respondents respectively. a total of 240 respondents were used for this study. analytical technique data were analyzed using descriptive statistics and inferential statistics. the descriptive statistics employed were frequency counts results and discussion the result of the socio-economic characteristics as shown in table 1 revealed that 27.2% of male respondents were less than 30 years, 56.3% were found within the age bracket of 31 to 60 years while only 16.5% were 61 year sand above. in the female category, 21.3% of the respondents were less than 30 years, 63.8% were within the age group of 31 to 60 years while just 14.9% of the respondents were 60 years and above. the mean ages of male and female respondents were 48.3 and 43.7 years respectively. the findings revealed that majority (56.3 and 63.8%) of respondents were in their middle active ages, an indication that they will still be active to access land. furthermore, 35.0% of male respondents had primary education, 39.3% had secondary education and only 13.8% had tertiary education while 11.9% did not have 145 afr. j. food sci. res. table 2. distribution of respondents’ accessibility to forms of land holdings. gender male (n=160) female (n=160) variable frequency percentage frequency percentage access to community land 152 95.0 38 23.8 access to inherited land 138 86.3 52 32.5 access to land by purchase 160 100.0 72 45.0 access to land by lease 160 100.0 61 38.1 access to land by gift 115 71.9 29 18.1 source: field survey, 2010. multiple responses were given. table 3. chi-square analysis showing the difference between gender and land accessibility. variable x 2 c x 2 t df c gender 5.83* 3.84 1 0.57 p-values at 0.05 level of significance; c= contingency coefficient; x2c = chisquare calculated; x2t = chi-square formal education. in the female group, 38.1% had primary education and only few (11.3%) had tertiary education. this implied that male respondents were more educated than their female counterparts in the study area. for farming experiences in years, majority (42.5%) of male respondents had between 11 to 20 years and 31.2% had greater than 20 years. more so, 49.4% of female respondents had 11 to 20 years of farming experience while only 22.5% were found to have more than 20 years of farming experience as shown in table 1. in addition, majority (52.5%) of male respondents had between 6 to 9 household sizes while majority (55.6%) of female respondents had between 2 to 5 household sizes. this implied that male respondents had higher household sizes than the female. this could be as a result of male having more than one wives. also, 56.8% of male respondents had between 1 to 3 hectares farm size, 35.7% had more than 3 hectares farm size while 51.9% of female respondents had between 1 to 3 hectares and a few (9.3%) cultivated more than 3 hectares of land in the study area. this analysis shows that male respondents had higher farm size than their female counterparts in the study area. results in table 2 revealed that majority (95.0%) of male respondents had access to communal land while few (23.8%) of female respondents had access to community land. also, 86.3% of male had access to inherited land while only 32.5% of female respondents had access to inherited land. more so, 100.0% of male respondents were found to have access to land by purchase while only 45.0% of female could access land by purchase. in addition, majority (71.9%) of male could access land through gift. the above analyses indicated that male had more access to any form of land than their female counterparts. also, the result of chi-square analysis revealed that there was a significant difference between gender and accessibility to land at 0.05 level of significance as shown in table 3. gender was found to exert a little above avarage (57%) strenght of association on land accessibilty. this significant difference further revealed that male have more access to tabulated; df = degree of freedom; source: field survey, 2010. land than female in the study area. this finding was in conformity with lawanson (2010), bruce and lloyd (1991) and afonja et al. (2002). the low accessibility of female to agricultural land could be as result of sociocultural factors that could hinder female from owning land (lawanson, 2010). results in table 4 revealed that 70.0% of male respondent indicated that income level affects land holding, while majority (100.0%) of female indicated that marital status is the major determinant to hold land in the study area. more so, about 84.5 percent of the female respondents viewed cultural belief as a factor that affects land holding while a few (38.8%) of the male respondents indicated that cultural belief is a factor that affects land holding among men in the study area. this finding revealed that men were less affected by the various factors indicated, an indication that female were not having equal access to agricultural land despite their contributions to the food production in sub-saharan africa. this affirms the position of alice (2008) in the study ‘’effect of land tenure system on women’s knowledge-base and resource management in manjiya county, uganda’’. the study indicated that a very low percent of women in agriculture gets a wage as aginst the percentage of men. the pearson correlation analysis revealed that marital status was positively correlated with land accessibility. this conforms with soetan (2002). multiple responses were given on the security of tenure over land, results in table 5 indicated that about 83.1 percent of male owned land while only few (17.5%) of women owned land in the study area. more so, about 74.4 percent of male had been in possession of their land for more than 5 years while about 1.3% of women had held land for the same duration with men. in addition, about 88.1% of male had the rights to transfer land while none of the female respondents had the same priviledge of transferring land like their male counterparts. this analysis reveals that gender differential okojie et al. 146 table 4. factors affecting land ownership. variable male (n=160) female (n=160) frequency percentage frequency percentage income 112 70.0 129 80.6 marital status 70 43.8 160 100.0 access to credit facility 132 82.5 108 67.5 age 125 78.1 115 71.9 cultural belief 62 38.8 135 84.5 source: field survey, 2010. table 5. security of tenure over land. variable male female frequency percentage frequency percentage land ownership owned 133 83.1 28 17.5 otherwise 27 16.9 132 82.5 duration of land use <2 years 39 24.3 3-5 years 41 25.6 118 73.8 >5 years 119 74.4 3 1.9 rights to land use only 19 11.9 160 100 transfer 141 88.1 source: field survey, 2010. table 6. results of correlation analysis showing the relationship between male and female socio-economic characteristics and accessibility to agricultural land. variable correlation coefficient (r) coefficient of determination (r 2 ) age 0.484* 0.064 marital status 0.568* 0.059 level of education 0.115 0.013 farm size 0.504* 0.039 farming experience 0.479* 0.121 household size -0.668* 0.072 access to credit facility 0.476* 0.141 membership of social organization 0.593* 0.154 source: field survey, 2010. critical value of r = 0.427, level of significance= 0.05. exists in security of tenure over land in the study area. this findins conform with lawanson (2010), who stated that women relative powerlessness both economically and politically in a typical african setting limits their control over resources, particularly land and also that male superiority and dominance over resources in nigeria originated from historically rooted culture and religion. the results of the hypothesis stated and tested on table 6 revealed that there were positive and significant correlation between accessibility to agricultural land and age (r = 0.484); marital status (r = 0.568); farm size (r = 0.504); farming experience (r = 0.479); access to credit (r = 0.476) and membership of social organization (r = 0.593). the r values were compared with the critical value of r = 0.427. household size had negative but significant relationship with respondents’ socio-economic characteris tics and accessibility to agricultural land. only level of education was found to have non significant relationship. the above information established that an increase in the value of the independent variables would result in corresponding relationship between respondents’ socioeconomic characteristics and accessibility to agricultural land. the values of coefficient of determination (r 2 ) further revealed the percentage contribution of the independent variables to agricultural land accessibility. the higher the values of r 2 , the stronger the influence as reflected in the percentage contribution of the significant variables. this information therefore justified these variables as highly significant between respondents’ socio-economic characteristics and accessibility to agricultural land. conclusion and recommendations based on the findings of this study, most of the respondents were within their productive age of 31 to 60 years. there was gender differences in land accessibility in the study area as male were found to have more access to agricultural land than their female counterparts. female were more affected by the factors affecting land holding in ondo state. age, marital status, farm size, farming experience; access to credit facility and cultural beliefs were found to determine accessibility to agricultural land in the study area. there is therefore the need for an intensive effort and emphasis on mainstreaming gender in agricultural programmes. this will facilitate the entry of women as active decision makers on issues that relate to food security and income generation. women are important links to development: they maintain food security and the general well-being of their families/households. therefore, improving women's status and control over land should be considered as strategically important efforts at all levels in combating the state of food security in the country. it is therefore recommended that society and government should consider gender in agriculture by restructuring the system of land holding in order to include the vulnerable group as this will have a significant effect on food production in ondo state and in nigeria at large. also, land tenure policies should be restructured to ensure that farmers (male and female) have equal access to agricultural land especially for perennial crops and are able to obtain land on a more permanent basis would be helpful in combating the state of food insecurity in the country. 147 afr. j. food sci. res. references afonja s, mills-tettey r, amole d (2002). gender differentials in access to land and housing in a nigerian city, being monograph of the center for girder and social policy studies, obafemi awolowo university, ile-ife. alice mk (2008). the effect of land tenure system on women’s knowledge-base and resource management in manjiya county, uganda. argwal b (1994). a field of one’s own: cambridge university press. fao corporate document repository: current and emerging issues for economic analysis and research, 2010 blum cl (1998). land tenure and administration in africa: land tenure and resource access in africa, iied/fao, london. bioye ta, abdul ra, joseph bo (2006). women and land rights reforms in nigeria: promoting land administration and good governance 5th fig regional conference. bruce j, lloyd c (1991). family research and policy issues for the 1990's’, in understanding how resources are allocated within households. cousins b, claassens a (2006). ‘more than simply ‘socially embedded’: recognizing the distinctiveness of african land rights’. keynote address at the international symposium on the frontier of land issues: social embeddedness of rights and public policy’ montpellier, may 17-19. coastalnews (2012). profile of ondo state, nigeria. deininger k, binswanger h (1999). the evolution of the world bank’s land policy: principles, experience and future challenges’, the world bank research observer 14:247-276. derman br odgaard o, sjaastad e (2007). conflicts over land and water in africa. oxford: james currey. the world bank research observer 14:247-270. food and agricultural organization (2002). assessment of the world food security and nutrition situation. background document for the 38th session of the fao, rome, pp. 34-39. food and agricultural organization, (2006). the state of food insecurity in the world rome. food and agricultural organization (2008). gender, property rights and livelihoods in the era of aids. food and agricultural organization (2009). gender equity in agriculture and rural development. a quick guide to gender mainstreaming in fao’s new strategic framework. lawanson to (2010). gender differentials in nigeria: implications for sustainable development. j. extension. syst. 21(1):46-57. soetan r (2002). ‘women, small scale enterprises and social change: implications for changes in industrialization strategy”, in afonja.s and aina o (eds) gender and social policy studies. obafemi awolowo university, ile ife the world bank (2001). integrating gender into the world bank's work: a strategy for action. www.worldbank.org okojie et al. 148 appendix appendix 1. percentage of women and men employed in the primary sector. region 1998 2007 male women male women world 39.4 42.9 33.1 36.4 eastern asia, south-eastern asia and pacific 44.3 51.6 36.4 41.2 latin america and caribbean 26.4 12.6 22.1 9.7 southern asia 53.7 74.4 41.5 65.1 sub-saharan africa 65.1 71 60.3 65.1 source: global employment trends for women, ilo, 2009-data for 1998 and 2007 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (4), pp. 001-003, april, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. short communication control of ochratoxin a (ota) in kunu zaki (a nonalcoholic beverage) using daniellin tm adegoke, g. o1*, odeyemi, a. o.1, hussien, o.1 and ikheorah, j.2 1 department of food technology, university of ibadan. ibadan. nigeria. 2 nigeria stored product research institute, ibadan. nigeria. accepted 10 february, 2019 kunu-zaki, a non-alcoholic beverage, was produced using modified traditional method incorporating daniellin tm . treated samples kept for 5 days at ambient condition (26 + -2 c) while untreated samples kept for only 1 day. protein contents and calorific values of kunu-zaki treated with daniellin tm (0.5 to 5.0%, w/v) were between 5.76 to 5.93% and 1606.47 to 1626.8 kj/100g while values for untreated samples were 5.72% and 1547 kj/100g respectively. ochratoxin a (ota) in raw materials used for kunu-zaki production was reduced from 50 mg/kg to <1.5 mg/kg with the incorporation of daniellin tm at 1.5, 2.0 and 2.5%. key words: ochratoxin a, kunu-zaki beverage, daniellin tm , food safety. introduction kunu-zaki is a cereal-based, non-alcoholic fermented beverage. the nutritional and medical importance of kunu-zaki have been reported (gaffa and ayo, 2002). kunu-zaki has poor keeping qualities (osuntogun and aboaba, 2002) owing to faulty processing and storage methods and it is known to be prone to microbial contamination. microbial contamination of plant products has been reported elsewhere (beuchat and brackett, 1990). bacterial species associated with the contamina-tion of kunu-zaki have been reported (ayo et al., 2004; umoh et al., 2004). fungi notably aspergillus and penicillium species have also been isolated from kunu-zaki (osuntogun and aboaba, 2004). to date, there are no data on mycotoxin contamination of kunu-zaki (a cereal-based beverage). in cereals and oil seeds, mycotoxins of public health significance include aflatoxins, citrinin and ochratoxin a (pillet, 1998; janardhane et al., 1999). the adverse effects of mycotoxins in man include genotoxicity, carcinogenicity, mutagenicity, teratogenicity and immunotoxicity (iarc, 1993). ochratoxin a (ota) is a moderately stable molecule that can withstand most food processing techniques like malting and brewing (boudia and lebars, 1995; baxter, 2001). as ota may appear in finished cereal products like beverages (scott, 1996), this present work was carried out in order to extend the shelf life of kunu-zaki *corresponding author. e-mail: goadegoke@yahoo.com and also control the level of ota contamination of the beverage using daniellin tm (adegoke, 2005; 2006). the multifunctional profiles of aframomum danielli from where daniellin tm was obtained have been reported (adegoke and gopalakrishna, 1998; ashaye et al., 2006; adegoke et al., 2006). materials and methods preparation of kunu-zaki the method described by gaffa and ayo (2002) was modified as follows: about 300 g of millet (pennisetum typhodeum) cleaned and washed thrice with potable water was steeped together with 300 g of peeled sweet potato ( ipomoea batata) for about 3 h at 62  1 o c. the soaked grains were removed, washed again with potable water and wetmilled into a paste which was divided into two parts in a ratio 1:3 (v/v). to the larger part (3 parts), boiling hot water was added to gelatinize the milled grains to which the smaller part (1 part) was added with vigorous and constant stirring. the suspension was left to ferment at room temperature (26  2 o c) for 8 10 h after which it was filtered using a sterile muslin cloth. chemical additives or spice like ginger, pepper were not added to the filtrate (kunu-zaki). treatment of kunu-zaki with daniellin tm to 100 ml of freshly-prepared kunu-zaki was added in duplicate, 100 ml of cold, aqueous solution of daniellin tm to give final concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5% ( w /v) respectively (table 1 were used for analytical works. daniellin tm was not added to the control samples. table 1. percentage reduction of ota in kunu-zaki using daniellin tm sample ota level (µg/kg) % reduction untreated kunu-zaki * 10 kunu + 0.5% daniellin tm 5 50 kunu + 1.0% daniellin tm < 2.5 75 kunu + 1.5% daniellin tm < 1.5 100 kunu + 2.0% daniellin tm < 1.5 100 kunu + 2.5% daniellin tm < 1.5 100 *sorghum used for kunu-zaki production had 50 µg/kg of ota was added to 50 g of each of control and treated samples of kunuzaki followed by shaking for 5 min. the resulting mixture was extracted with 50 ml of chloroform over a bed of anhydrous sodium sulphate. sodium bicarbonate (6.0 g) and celite mixture (25 ml of 5% aqueous nahc02 solutions, celite and 50 g) were used for the clean-up process. thereafter, 70 ml of n-hexane and 70 ml chloroform were used for washing the column. elution was done using 700 ml of acetic acid: benzene solution (2:98). the eluate was evaporated to dryness and was later used for thin layer chromatography after dissolving each sample residue in 500 ml of benzene: acetic acid (98:1). spotting was done using 5-50ml (5, 10, 20, 30, 40, 50 ml) of ota standard (courtesy, iaea/nafdac, lagos) and 20 ml of each sample residue on tlc plates (20 x 20cm) in a developing solvent (benzene: methanol: acetic acid; 10:1:1). spotted control and test samples were viewed under uv light after which quantification of ota in each sample was calculated (aoac, 2002). figure 1. proximate composition of kunu-zaki using daniellin tm chemical analysis crude protein and moisture contents were determined using standard methods (aoac, 1984). ph measurements were done using ph meter (3305 jenway) after initial standardization with ph 4 and 7 buffers solutions and titratable acidity (ta % lactic acid) was done by titrating 10 ml of kunu-zaki against o.1n naoh to phenolphthalein end point. mineral contents of kunu-zaki were determined using atomic absorption spectrophotometer (ashing process) as described by cerwyn (1995) while caloric content of kunu-zaki was estimated using oxygen bomb calorimeter (anon, 1961). determination of ota level in kunu-zaki the method for ota qualification described by aoac (2002), was modified as follows: 5 ml of aqueous solution of sodium bicarbonate results and discussion within 1 day of storing kunu-zaki, microbial deterioration appeared while samples treated with daniellin tm (0.5 to 2.5% w/v) remained stable after 5 days of storage at 26  2 o c. our findings are thus in agreement with the report on the poor keeping qualities of kunu-zaki (osuntogun and aboaba, 2004). upgrading the processing of kunuzaki in line with haccp principle will therefore involve controlling hazards associated with raw materials and processing methods. hazards can arise from processing and handling operations of household foods (abdulsalam and kaferstein, 1993). osuntogun and aboaba, (2004) isolated some moulds from kunu-zaki. moulds in addition to visible spoilage, can spoil foods through the formation of mycotoxins (huis int’ veld, 1996). in this present study, daniellin tm stabilized kunu-zaki for 5 days and also eliminated ota with consistent lowering of ph of the beverage (figure 1). the calorific and protein values of kunu -zaki treated with daniellintm are shown in figure 1. millet used for kunuzaki production was found to be contaminated with ota (50 mg/kg). however, using daniellin tm at 1.5, 2.0, 2.5 (w/v) and a 100% reduction of ota was found in treated kunuzaki (table 1). the presence of ota in kunu-zaki, reported for the first time, is of public health significance. nephrotoxicity and carcinogenity have been associated with ota (iarc, 1993). while the control of growth and synthesis of some mycotoxins can be achieved using synthetic chemicals (thompson, 1992; nesci et al., 2003), the safety of some of these additives is of public health concern (miller, 1989; adegoke et al., 1998). the european commission proposed 5 mg/kg of ota in raw cereals and 3mg/kg in cereal-derived products (ec, 2002). the control of ota to zero level (table 1) and absence of aflatoxin b1 (unpublished data) in kunu-zaki shows that daniellin tm can be used for controlling ota in kunu-zaki. our study has also confirmed the reports of ashaye et al. (2006), aroyeun and adegoke (2007) that aframomum danielli can be used in food processing /preservation. references abdulsalam m, kaferstein l (1993). safety of street foods. world health forum 14: 191-194. adegoke go (2005). production of antioxidants adde, adm, dfadm from the spice aframomum danielli, patent no rp 16224, federal republic of nigeria. adegoke g.o (2006). daniellin food grade multipurpose powder in class 30. trade mark tp/162938/06, trade marks section, federal ministry of commerce, abuja, nigeria. adegoke go, fasoyiro sb, skura b (2000). control of microbial growth, browning and lipid oxidation by the spice aframomum danielli. eur. food res. technol. 211: 342-345. adegoke go, kumar mv, gopalakrishna ag, varadaj mc, sambaiah k, lokesh br (1998). antioxidants and lipid oxidation – a critical appraisal. j. food sci. technol. 35: 283-298. anon (1960). oxygen bomb calorimetry and combination method, technical manual 1300, pure instr. co. iii. usa. aoac (1984). official methods of analysis. association of official analytical chemists, washington dc. aoac (2002). official methods of analysis. association of official analytical chemists, washington dc. aroyeun so, adegoke go (2007). reduction of ochratoxin a (ota) in spiked cocoa powder and beverage using aqueous extracts and essential oils of aframomum danielli. afr. j. biotechnol. 6: 612-616. ashaye oa, taiwo oo, adegoke go (2006). effect of local preservative (aframomum danielli) on the chemical and sensory properties of stored warankasi. afr. j. agric. res. 1: 10-16. ayo ja, umianze h, gaffa t (2004). microbiological evaluation of “kunu-zaki” and “zoborodo” drink (beverage) locally produced and sold in polytechnic community in nigeria. nig. food j. 22: 119-126. baxter de (2001). behaviour of ochratoxin a in brewing. j. am. soc. brewing chem. 59: 98-100. beuchat lr, brackett re (1990). survival and growth of listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and temperature. j. food sci. 55: 755-780. boudia h, lebars j (1999). thermostability of ochratoxin in wheat under two moisture conditions. appl. microbiol. 6: 1156-1158. cerwyn sj (1995). analytical chemistry of foods. blackie academic, london, new york. ec (2002). european commission regulation no 472/2002 of 12 march, 2002. gaffa t, ayo ja (2002). innovation in the traditional kunu-zaki production process. pakistan j. nutr. 1: 202-205. huis int’ veld jhj (1996). microbial and biochemical spoilage of foods: and overview. int. j. food microbiol. 3: 1-18. iarc (1993). some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins, international agency for research on cancer, lyon, france. 56: 489-521 janardhane gr, raveesha, ka, shetty hs (1999). mycotoxin contamination of maize grown in karnataka (india). food chem. toxicol. 37: 863-868. miller s (1989). new technologies and the future of food and nutrition, john wiley and sons inc. new york. nesci a, rodriguez m, etcheverry m (2003). control of aspergillus growth and aflatoxin production using antioxidants at different conditions of water activity and ph . j. appl. microbiol 95: 279-287. osuntogun b, aboaba oo (2004). microbiological and physico-chemical evaluation of some non-alcoholic beverage. pakistan j. nutrit. 3: 188192. pillet a (1998). natural occurrence of mycotoxins in foods and feeds: an updated review. rev. med. vet 149: 479-492. pillet a (2001). natural occurrence of mycotoxin in and foods and feeds: a decade review. mycotoxin and phytotoxins in perspective at the turn of the mellineum. proceeding of the x th international iupac symposium on mycotoxins and phytotoxins, editors – dekoe wj, samson ra, van egmond hp, gilbert j, sabino m, wageningen, the netherlands, pp. 153-172. scott pm (1996). mycotoxin transmitted into beer from contaminated grains during brewing. jaoac intl. 79: 675-682. thompson dp (1992). inhibition of mycelial growth of mycotoxigenic fungi by phenolic antioxidants. mycologia 84: 791-793. umoh vj, oranusi su, kwaga jkp (2004). the public health significance of pathogens isolated from kunu-zaki sold in retail outlets in zaria, nigeria, nig. food j. 22: 10-17. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (9), pp. 373-377, september, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of the quality parameters of malted and unmalted maize and soybean blends adetuyi, f.o.*, badejo, o.f., ikujenlola, a.v. and omosuli, s.v. department of food science and technology, rufus giwa polytechnic, p.m.b 1019, owo, nigeria. accepted 11 april, 2017 the complementary blends of maize ( zea mays l ssp mays) and soybean (glycine max l merrill) were stored for a period of 12 weeks. the maize grains were malted by subjecting it to cleaning, washing, steeping, sprouting, drying, desprouting, milling and packaging. a portion of the maize flour was blended with soybean flour at the ratio of 70:30 (malted maize flour: soybean flour; unmalted maize flour: soybean flour). the resulting products were stored in polyethylene and plastic containers. the functional properties (bulk density, viscosity, water and oil absorption capacity, swelling capacity, peroxide value and least gelation concentration) of the products were determined. the result showed that the bulk density and the peroxide value of both malted and unmalted maize flour reduced significantly (p>0.05) when blended with soybean. the swelling capacities of the malted products were lower than those of the unmalted blends and it also decreased though not significantly (p>0.05) with the period of storage. the malting process reduced the viscosity of the products, however; inclusion of soybean flour increased the viscosity. the viscosity and the peroxide value increased significantly (p>0.05) in the two storage containers as the storage period increased. the result revealed that the packaging materials had no significant effects on the parameters assessed. key words: unmalted maize, malted maize, soybean blends, bulk density, swelling capacity, water absorption capacity. introduction complementary foods, in most developing countries, are mainly from cereal with animal protein being used as supplements. however, because of the high cost of the animal protein, attempts have been made to look into alternative sources (obatolu and cole, 2000). in order to improve the nutrition of children in region of chronic and acute malnutrition, various kind of economical protein-rich plant mixtures are used for different area in africa (mosha and svanberg, 1990) . the combination of such food ingredients often alter the food composition of the food product and may change the functional and sensory pro-perties (kinsella, 1976). it has been recommended that, for long period of conservation, flour should be stored in closed atmosphere. in this condition, flour acidity in-creases owing to accumulation of a linoleic and linolenic *corresponding author: e-mail: foluadetuyi@yahoo.co.uk. tel: +2348034257996. acids, which are slowly oxidized; solubility of gluten protein decreases (kent, 1978). the hazards to flour in storage include mould and bacterial attack, insect infestation, also oxidative rancidity. the optimum moisture content of the storage of flour must be in relation to the length of storage envisaged, and to the prevailing ambient temperature and relative humidity, remembering that due to hygroscopicity, flour will gain or lose moisture to the surrounding atmosphere, unless packed in hermetically sealed containers (kent, 1978). package is a means of providing the correct environmental conditions for food or any other product, in order to protect the product against any deterioration be it microbiological, chemical or physical in nature (komolafe, 2005). previous workers in the field of nutrition and food science and technology had worked on the effects of malting on the quality parameters of complementary food, however, scanty information is available on the effect of storage on the quality parameters of malted maize and soybean blends. the aims of this work were therefore to adetuyi et al. 374 evaluate the quality parameters of malted and unmalted maize and soybean blends and also to determine the effect of storage in polyethylene and plastic container on the qualities of the blends, because these are the common storage containers readily available to greater populace of nigeria. materials and methods maize (zea mays l ssp mays) and soybean (glycine max l merrill) grains used for this work were purchased from ‘oja oba’ market in akure, ondo state. the maize grains were malted using the method described by kulkarni et al. (1991) for cereal germination with some modifications. the maize was soaked for 12 h in a volume of water three times its weight and drained. it was spread on a wide wooden box for germination under ambient temperature (30± 2 0 c) for 72 h and watered twice daily. the germinated grains were washed and dried to a moisture content of 10%. it was then milled using hammer mill. the unmalted maize grains were washed and dried to a moisture content of 10% and were milled using hammer mill. the soybean flour was also produced according to the method of kulkarni et al. (1991). the soybean seeds were cleaned and washed. they were boiled for 30 min and later soaked for 24 h with the changing of soaking water at 4 h interval. the testa on each grain was removed and the seeds were dried. the dried soybean seeds were milled into powder using hammer mill. it was formulated thus: unmalted maize 100% (umm); 70% unmalted maize + 30% soybeans (umms); malted maize 100% (mm); 70% malted maize + 30% soybeans (mms). the formulations were divided into two each. one part was stored in polyethylene bag while the other was stored in a plastic container for 12 weeks at room temperature of 30±2 0 c. the functional and the physicochemical properties were determined every three weeks during the storage period. all determinations were done in triplicates. the bulk density was determined according to the method described by okaka and potter (1977). the bulk density was calculated as mass of flour per unit volume (g/cm 3 ). the water and oil absorption capacity (wac) and (oac) were determined using the method described by beuchat (1977). a sample (1 g) each was mixed with 10 ml of distilled water for wac and 10 ml of oil for oac and blended for 30 s. the samples were allowed to stand for 30 min and centrifuged at 3500 x g for 30 min at room temperature. the supernatant was decanted. the weight of water or oil absorbed by the flour was calculated and expressed as wac or oac. the method of ukpabi and ndimele (1990) was used for the swelling capacity. flour (20 g) was put into a washed, dried and weighed graduated measuring cylinder. 100 ml of distilled water was added and allowed to stand for one hour. the supernatant was discarded and the cylinder with its content weighed to obtain the weight of the net sample. the difference in final to initial volume of the sample gave the swelling capacity on volume basis. the method of coffman and gracia (1977) was used in the determination of least gelation concentration. appropriate sample suspensions were weighed into 5 ml distilled water each to make 2-20% (w/v) suspension. the test tubes containing these suspensions were heated for 1 h in boiling water (bath) followed by rapid cooling under running tap water. the test tubes were further cooled for an hour under the running water, the least gelation concentrations (lgc), were determined as concentration when the sample from the inverted test tube did not fall or slip. the viscosity was determined using the association of official analytical chemists aoac (1990) method. ten grams of the sample was mixed with 150 ml of deionized water (heated to 95 0 c for 5-10 min) to gelatinize, it was cooled and the viscosity was measured using the ostwald viscometer. the pearson (1976) method of analysis was used for the ph analysis, the sample (1 g) was taken in separate test tube and 10 ml of distilled water was added. it was then shaken properly and measured with the aid of iso-electric ph meter. the peroxide value was determined according to the method of aoac (1990). statistical analysis data collected were subjected to the analysis of variance (sas, 2002). mean separation were done where there were significant differrences using duncan multiple range test procedure as described in the sas software. significance was accepted at p>0.05. results and discussion the results presented in table 1 showed that the bulk density of the flour from both malted and unmalted maize decreased after it had been blended with soybean flour umm from 0.77 to 0.66 g/m 3 while mm from 0.83 to 0.81 g/m 3 . this result agrees with the finding of akubor and obiegbuna (1999) . they reported that the bulk density of flour from malted maize and soybeans blend reduced significantly. as the storage period increased, the bulk density reduced in the two storage containers. there was no significant difference (p>0.05) in the bulk densities of the samples during the 12 weeks period of storage in the two storage containers. the reduction in the bulk density of the germinated flours would be an added advantage in the preparation of supplementary foods (akubor and obiegbuna, 1999). low bulk density food is desired where packaging is a serious problem (ikujenlola, 2008). swelling capacity is the volume of expansion of molecule in response to water uptake which it possessed until a colloidal suspension is achieved or until further expansion and uptake is prevented by intermolecular forces in the swelled particle (houssou and ayernor, 2002). the swelling capacity (swc) of the malted and unmalted maize increased after it had been blended with soybean. the swc of the unmalted maize blend (1.0 cm 3 ) was higher than the malted maize blend (0.50 cm 3 ). this result differs from the observation of ikujenlola and fashakin (2005) that there was reduction in the swelling capacity of the diet prepared from germinated and ungerminated maize cowpea bean blend. the increase in the swelling capacity could be attributed to the increase in the carbohydrate content of the blend because the carbohydrate content of the soy bean might have caused the increase in the carbohydrate content of the blend. the swc decreased though not significantly (p>0.05), during storage in the two containers under observation for the 12 weeks storage period. gelation is one of the most important functional properties which determine the suitability of incorporation of a particular substance into food products (adebowale and adebowale, 2008). the least gelation concentration (lgc) of the flour increased after the maize (malted and unmalted) had been blended with soybean umm 6-8 w/v and mm 48 w/v. obatolu and cole (2000) observed reduction in the lgc of cowpea and malted maize blend. the lgc was not consistent in storage but remained the 375 afr. j. food sci. res. table1. bulk density, swelling capacity and least gelation concentration of malted and unmalted maize soybean blend. parameters week 0 week 3 week 6 week 9 week 12 plastic polyethylene plastic polyethylene plastic polyethylene plastic polyethylene plastic polyethylene umm 0.77(a)a 0.77(a)a 0.70a 0.70a 0.80a 0.78a 0.68a 0.68a 0.76a 0.74a bd umm 0.66(b)a 0.66(b)a 0.60a 0.70a 0.80a 0.72a 0.61a 0.60a 0.62a 0.64a g/cm 3 mm 0.83(a)a 0.83(a)a 0.76a 0.78a 0.80a 0.76a 0.77a 0.73a 0.72a 0.76a mms 0.81(a)a 0.81(a)a 0.76a 0.78a 0.70a 0.76a 0.67a 0.70a 0.69a 0.72a umm 0.80(b)a 0.80(b)a 0.60a 0.30a 0.80a 0.80a 0.60a 0.60a 1.00a 0.60a swcumms 1.00(a)a 1.00(a)a 0.70a 0.60a 0.80a 0.60a 0.60a 0.60a 0.80a 0.60a cm 3 mm 0.40(b)a 0.40(b)a 0.40a 0.30a 0.60a 0.40a 0.80a 0.80a 0.50a 0.40a mms 0.50(b)a 0.50(b)a 0.30a 0.80a 0.60a 0.60a 0.60a 0.80a 0.60a 0.50a umm 6(b)a 6(b)a 6a 6a 4a 6a 6a 6a 4a 6a lgcumms 8(b)a 8(b)a 8a 8a 6a 8a 8a 8a 6a 6a w/v mm 4(c)a 4(c)a 4a 4a 8a 6a 6a 6a 6a 6a ms 8(a)a 8(a)a 8a 8a 6a 8a 4a 6a 4a 6a value represents mean of triplicate. values with the same letter along the same row are not significantly different (p>0.05) while value with the same letter inside bracket along the column are not significantly different (p>0.05). umm-unmalted maize 100%, umms70% unmalted maize + 30% soybean, mmmalted maize 100%, mms70% malted maize + 30% soybean. bdbulk density, swswelling capacity, lgcleast gelation concentration. table 2. water absorption capacity and oil absorption capacity of malted and unmalted maize soybean blend. plastic polyethylene plastic polyethylene plastic polyethylene plastic polyethylene plastic polyethylene umm 240(b)a 240(b)a 220a 260a 240a 220a 240a 240a 240a 240a wacumms 280(a)a 280(a)a 240a 260a 260a 260a 240a 240a 240a 240a % mm 200(d)a 200(d)a 240a 260a 180a 220a 220a 240a 240a 260a mms 220(c)a 220(c)a 240a 220a 200a 200a 220a 240a 240a 260a umm 220(a)b 220(a)b 220a 200a 220a 220a 240a 240a 280a 280a oacumms 160(c)b 160(c)b 260a 260a 240a 240a 260a 240a 260a 280a % mm 120(d)b 120(d)b 280a 200a 240a 220a 240a 220a 280a 280a mms 180(b)b 180(b)b 200a 200a 240a 220a 220a 220a 280a 280a value represents mean of triplicate. values with the same letter along the same row are not significantly different (p>0.05) while value with the same letter inside bracket along the column are not significantly different (p>0.05). umm-unmalted maize 100%, umms70% unmalted maize + 30% soybean, mmmalted maize 100%, mms70% malted maize + 30% soybean. wacwater absorption capacity, oacoil absorption capacity. same in the first 6 weeks; though there was no statistical significant difference (p>0.05) in the values in the two storage containers. water absorption capacity is important in the development of ready to-eat-food cereal grains, since a high water absorption capacity may assure product cohesiveness (houssou and ayernor, 2002). the water absorption capacity (wac) as shown in table 2 was higher in the unmalted maize (240%) than malted (200%). these values were higher than the value reported by yusuf et al. (2007) for snake gourd seed flour (130%). the reduc-tion in the wac of malted maize agreed with the findings of tatsadjieu et al. (2004) in the study of germination of sorghum. the wac increased after the maize had been blended with soybean, umms-280% and mms-220%. this could be attributed to the added protein from the soybean since protein are mainly responsible for the bulk of the water uptake and to less extent the starch and cellulose at room temperature (houssou and ayernor, 2002). the unmalted blends had a higher wac than the malted blends. this result agreed with the report of badifu and ebegonye (1999) and ikujenola and fashakin (2005). when the flour was stored for 12 weeks in plastic container and polyethylene, it was observed that there was no statistical significant difference (p>0.05) in fresh samples of week 0 and stored samples week 12.oil absorption capacity is a critical assessment of flavour retention and increases the palatability of foods (kinsella, 1976). the oil absorption capacity (oac) of the flour of unmalted maize (220%) was higher than that of the malted maize (120%). the values reported in this work was higher than the oac value of snake gourd seed flour (54%) (yusuf et al., 2007). the oac reduced in unmalted maize blends while it increased in malted maize blend, also the oac of malted maize blend 180% was higher than the unmalted maize blend 160%. this result conforms to the findings of padmashree et al. (1987), mosha and svanberg (1990) and obatolu and cole (2000) that table 3. viscosity, ph and peroxide value of malted and unmalted maize soybean blend. parameters week 0 week 3 week 6 week 9 week 12 plastic polyethylene plastic polyethylene plastic polyethylene plastic polyethylene plastic polyethylene umm 3.69(b)b 3.69(b)b 3.86b 4.11b 4.36b 4.90a 5.96a 6.32a 6.25a 6.66a vis umms 4.16(a)b 4.16(a)b 3.96b 4.62b 4.36b 4.76b 6.10a 6.67a 6.18a 6.76a g/m2 mm 3.12(c)d 3.12(c)d 3.62b 3.72b 4.06c 4.21c 5.14a 5.70a 6.36a 6.40a mms 3.46(c)c 3.46(c)c 3.51b 3.57b 3.82b 4.18b 5.11a 5.92a 6.75a 6.82a umm 6.13(a)a 6.13(a)a 5.69a 5.99a 6.11a 6.14a 5.97a 5.99a 5.88a 5.93a ph umms 6.01(a)a 6.01(a)a 5.98a 5.93a 6.04a 6.06a 5.85a 5.93a 5.98a 5.57a mm 5.78(a)a 5.78(a)a 5.77a 5.72a 5.74a 6.76a 5.57a 5.51a 5.56a 5.53a mms 5.83(a)a 5.83(a)a 5.78a 5.80a 5.83a 5.86a 5.66a 5.69a 5.60a 5.67a umm 42.50(a)c 42.50(a)c 56.16a 52.16b 48.17b 49.38b 60.14a 50.16b 62.15a 60.16a pvumms 23.45(b)c 23.45(b)c 26.14c 50.36a 30.10b 40.31a 40.34a 40.38a 41.26a 43.26a mm 44.76(a)b 44.76(a)b 28.56d 60.14a 36.13c 38.36c 50.11a 49.37b 51.26a 50.17a mms 24.67(b)d 24.67(b)d 30.28c 33.14c 30.36c 33.18c 48.36b 50.33a 50.18a 52.56a value represente mean of triplicate. values with the same letter along the same row are not significantly different (p>0.05) while value with the same letter inside bracket along the column are not significantly different (p>0.05).umm-unmalted maize 100%, umms70%unmalted maize + 30% soybean, mmmalted maize 100%, mms70% malted maize + 30% soybean. vsviscosity, pvperoxide value. showed increase in oac of germinated base blends. during storage in two containers, the oac increased significantly (p>0.05) in the first 3 weeks of storage but there was no significant difference in the oac of the flour of week 3 to week 12. the viscosity of unmalted maize flour (3.69 g/m 2 ) was significantly higher (p>0.05) than the malted maize flour (3.12 g/m 2 ). this result agreed with the finding of ayernor and ocloo (2007) in the study of malted rice. the decrease in the viscosity could be attributed to the action on the starch by the hydrolyzing enzymes that were producing during malting (ayernor and ocloo, 2007). however, badifu and ebegonye (1999) reported increase in the viscosity of germinated melon kernel flour. table 3 showed that the viscosity of the unmalted and malted maize products increased after blending with soy bean (umm: 3.69 – 4.16 g/m 2 , mm: 3.69 – 4.16 g/m 2 ). the viscosity also increased significantly (p>0.05) as the storage period increased in the two storage containers. the result of the ph showed that there was no significant (p>0.05) difference in the ph values of the flour from both unmalted and malted maize reduced after it has been blended with soybean, umm6.13, umms-6.01, mm5.78 and mms5.83. there was no significant (p>0.05) difference in the p h values of both unmalted and malted maize blends in the two storage containers throughout the storage periods. peroxide value usually used as an indicator of deterioration of fats. as oxidation takes place, the double bonds in the unsaturated fatty acid break down to produce secondary oxidation products which indicate rancidity (ihekoronye and ngoddy, 1985). after blending of the maize (malted and unmalted) with soybean, the peroxide value decreased significantly, umm: 42.50 23.45 while mm: 44.76 24.67. during storage, the peroxide value increased significantly (p>0.05) in the two storage containers as the storage period increased. this agreed with the observation of gahlawat and sehgal (1994) that the peroxide value and fat acidity of weaning food developed from locally available food stuffs increased with increase in storage period. conclusion it was observed during this work that the two storage containers used behaved similarly, therefore the use of these storage containers has no significant effect on the parameters estimated at the end of the storage period. it is therefore concluded that malted and unmalted maize blends can be stored in plastic or polyethylene container and there will be no compromise on the functional and physicochemical properties of the blends. it is also recommended that work should be done on the microbial content of the blends in storage. references adebowale ya, adebowale ko (2008). evaluation of the gelation characteristics of mucuna flour and protein isolate. electronic j. environ. agric. food chem. 7(9):3202-3222. akubor pi, obiegbuna je (1999). certain chemical and functional properties of ungerminated and germinated millet flour. j. food. sci. technnol. 36: 241-243. aoac (1990) official methods of analysis 14th edition. association of official analytical chemist. washington dc, usa. ayernor gs, ocloo fck (2007). physicochemical changes and diastatic activity associated with germinating paddy rice (psb.rc 34). afr. j. food. sci. 1:37-41. badifu gio, ebegonye gm (1999). effect of germination on chemical, physical and functional properties of 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ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (3), pp. 001-009, march, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review pork production system and its development in mainland china heng-wei cheng1*, you-ming wang2, qing-ping meng2, jia guo2 and yi-zheng wang2 1 livestock behavior research unit, usda-ars, west lafayette, in 47906, usa. 2 institute of feed science, key laboratory of molecular animal nutrition, ministry of education, zhejiang university, hangzhou 310029, p. r. china. accepted 11 april, 2019 livestock production and marketing have been driven by the growing consumer demand for high quality and low cost animal protein. as a result, intensive livestock industries have been rapidly developing globally. international trade, especially with china, creates new opportunities and challenges for the emerging international agriculture market. china provides an enormous market for international trade by stimulating its import and export activities. pigs are the primary farm animals raised for human consumption in china, and the development of pork production systems in china will affect its trade globally. to meet the need for developing international animal agriculture and trade, this article describes the current status of the swine industry, its production in china and the factors that may affect its development. urbanization, available farmland, grain production, water resources and management practices encompass some of these factors. in addition, the effects of the culture, religion and education of the populations on the development of swine sector in china are also discussed. understanding the development of the swine production system and its associated factors is critical to assessing china’s impact and implication in the emerging international agricultural market. key words: animal agriculture, swine farm, pork production, international trade, china. introduction china is the world‟s largest country by population (more than 1.3 billion citizens, approximately 22% of the world‟s population in 2008; nbsc, 2009). its economy has been the fastest growing in the world for the past quarter of a century, especially following its market-oriented economic reforms in 1978 and open trade since joining the world trade organization (wto) in 2001 (rozelle and swinnen, 2004; wang and hu, 2007; carter et al., 2009). these changes, especially the transformation of agriculture from soviet-type centrally planned economy (government control system) to a market-oriented economy (household responsibility system) and urbanization, have significantly impacted china‟s economic structure and its patterns of international trade in most areas including the agricultural sector (lin et al., 1996; lohmar et al., 2009). *corresponding author. e-mail: hwcheng@purdue.edu. with the development of china‟s economy, its per capita income has grown rapidly. increase in household incomes have led to significant changes in the patterns of food consumption which is shifting from grains, legumes, root crops and soybean to livestock derived products. meat and egg consumption shows a consistent increase while grain consumption of rice, barley and wheat is either steady or declining (huang and bouis, 2000). the demand for livestock, especially pork, has and will be further influenced by urbanization, as a processassociated increase in urban populations and higherincome groups (lu, 2007). taking advantage of the economic scale, the swine sector has integrated production systems to become an industry. types of swine farms in china in general, there are three types of swine farms in china based on the numbers of pigs produced on the farm; table 1. the size of chinese pig farms in 2003. size (number of pigs) number of pig farms changes from percentage of farms (%) 2002 2003 2002 (%) 2002 2003 1-9 99,894,369 101,963,901 2.07 94.81 94.48 10-49 4,438,302 4,815,474 8.50 4.21 4.46 small farms 104,332,671 106,779,375 2.35 99.02 98.94 50-99 790,307 851,429 7.73 0.75 0.79 100-499 212,909 249,016 16.96 0.20 0.23 500-2,999 27,495 33,844 23.09 0.03 0.03 medium farms 1,030,711 1,134,289 10.05 0.98 1.05 3,000-9,999 3,242 3,388 4.50 0.00307 0.00314 10,000-49,999 862 911 5.68 0.00082 0.00084 over 50,000 28 30 7.14 0.00003 0.00003 modern farms 4,132 4,329 4.77 0.00392 0.00401 total 105,367,514 107,917,993 2.42 100 100 sources: ministry of agriculture of the people‟s republic of china; china animal agriculture association; china animal husbandry yearbook of 2004; china agricultural information network; food and agriculture organization of the united nations. small (including backyard farms, < 50 head), medium (503,000 head), and large (> 3,000 head) (table 1). with urbanization and production efficiency, china pork and pork production has shifted from backyard and small farms to specialized household (or local community) farms as well as modern intensive farms, especially in the urban areas close to big cities, such as beijing, shanghai, and chengdu (figures 1a and b). small size pig farm in china, the most popular swine holders are small size pig farms including the scattered pig farms (table 1). a small size pig farm has average herds with less than 50 pigs, while a scattered farm has even fewer pigs. it is estimated that over 80% of the rural household, or around 180 million farmers, have pigs with an average of 2-3 head per householder. hogs housed in small swine farms (also called „safety net‟ based on its economic function) are usually low-input pigs of indigenous breeds or hardy crossbreds. pork production is low, mostly used for home consumption (farm families) and/or to supply predominantly rural markets (local community) with limited competition. the purpose of traditional pig farming is „feeding hogs to fertilize crops and have a recipient for table scraps‟ with little emphasis on quality and efficiency of pork production. therefore, hogs in the traditional farms frequently consume large amounts of green roughage, based on the location, such as water plants, grass, vegetable leaves, tubers, carrots, pumpkins, fruits, and various crop stalks; and only fed little grain at early and late stages (fang and fuller, 1998). this diet lacks certain essential nutrients, resulting in a low feed efficiency. it results in low costs to raise a hog, but it takes great time of about 200 days for pigs to reach slaughter weight, generating a low net income. in small pig farm, the farmers and family members are the major stock-keepers. pig care and welfare is largely dependent on their animal-related experiences, economical status, religion, and education. hogs housed in the traditional farms are facing several major welfare issues, such as an unbalanced diet, uncontrollable climate conditional change, and disease. in addition, the small pig farmers are under increasing internal and external pressures arising from social, economic and environmental factors. to overcome these problems and to meet consumers‟ demand for lean and “healthful” meat, pork production is moving towards specialization by adopting more modern management technologies for improving feed efficiency and increasing pork production and food safety. with this movement, the chinese swine industry has quickly changed the denomination from backyard production of a few pigs to „large‟ size operations with more than 50 head annually. medium size swine farm the second type is the medium size pig farm that raises herds from 50 to 3,000. currently, this production system houses approximately 25% of the total pigs in china. there was an approximate 10% increase in the medium a. b. figure 1. distribution of swine farms and the factors affecting their development in mainland china. a) the geographic population distribution in mainland china. each dot represents 5,000 persons; b) the geographic distribution of swine farms in mainland china; and c) main rivers in mainland china. (sources: noll, p. and b. noll, 2009; editional board of the series national atles of china, 1996; fabiosa (2005); and the international institute for applied systems analysis, 1999). size pig farms from the year 2002 to 2003, especially in farms with pigs from 100 to 499 and 500 to 2,999 (17 and 23.1%, respectively; table 1). in total, from 2002 to 2006, the numbers of medium size swine farms were increased to approximately 204%.taking advantage of increased demand for pork and pork products in local markets, many rural households have shifted their focus from crop production to hogs and thus increased their swine herds. the aim of farming has also been shifted from “fertilizer for crop cultivation” to capital gain, with the emphasis on increasing grain feed, improving feed efficiency and reducing time to reach slaughter weight. to increase pork production, the farmers‟ focus is placed upon improving pig welfare by reducing and controlling managementassociated stressors, such as housing environments, stock densities, climate conditions and disease incidence. farmers, and specially trained stock-keepers, play major roles in maintaining the pigs‟ health and welfare base on their experiences, economical status, religion, and education. modern intensive swine farm the last farm type is the modern intensive swine farm (also called „industrialized‟ swine farm) with 3,000 and more pigs. in contrast to the previously discussed systems that focus on providing animal products for farm families and the local community, modern intensive swine farms are specialized economic enterprises. pork production relies on grain-based feed and modern technological advances as well as management skills. in the modern intensive swine farm, hogs are kept purely as a commercial venture by a firm. they are intensively reared under strictly regulated conditions and are provided with all necessary inputs, including new technologies for transportation, food preservation, disease prevention, feed efficiency and advance management technology. there is a trend in the swine sector to simplify and specialize with an ability to grow more pigs in less space. chinese government encourages the development of modern swine farms to meet the rising demand for meat consumption. in 2007, the state council announced the “regulation on implementing the law of the people‟s republic of china on corporate income tax” to reduce corporate income tax from 33 to 25%. in addition, swine holders have received great profits resulting from the currently high prices of pork products. to take advantage of emerging opportunities for growth, china‟s modern intensive swine farms have been developed rapidly, some of them reaching size and scope similar to those in the u.s. for example, the china national cereal, oils and foodstuffs corp (cofco) has invested $1.36 billion in hubei province to build a 500,000 head reproducible sow breeding base (the pigsite, 2008). additionally, several centers for pig breeding and swine disease prevention research have been established in china (usda‟s grain report, 2008c). foreign companies are also encouraged to invest in swine rearing farms in china through joint ventures. a u.s. investment bank, for example, has bought over 10 farms in jiangxi and fuijan provinces; and the tangrenshen company in hunan province recently undertook a new 10 million swine project (the pigsite, 2008). currently, modern intensive pig farms account for less than 1% of the total pig farms and less than 5% of total hog production in china (table 1). with rising demand for pork and pork products, the modern intensive production system will be continuously developed and will be the dominant producer in the pork market in china. these developments will translate into more concentrated swine rearing firms in the future, pushing backyard and small operators out of the swine business. large-scale pork production enterprises are generally located around big metropolitan area (high density of population), such as beijing, shanghai, and guangzhou, to serve the growing demand of urban consumers (for example, labor migration and urbanization) (figure 1b). similar to the swine farms in other countries, the modern swine farms in china are facing several major pig welfare and environmental issues, such as housing condition, group size, stock density, air quality (odor, ammonia production), climate conditions, disease and food safety, and environmental contamination as well as worker availability and safety. one way to overcome some of these challenges is to move the large production units out of the cities, away from the areas with dense populations. it could be similar to the relocation of animal agriculture made by the u.s. livestock sector in the mid-20 th century. during that time, the u.s. red meat processing was moved out of the cities to small towns in the midwest and great plains. effects of available resources on swine industry in china the high demand for animal derived products has important implication for the development of animal agriculture, especially modern intensive swine farms. however, whether china can feed itself, providing enough livestock productions including pork and pork productions, is affected by multiple factors such as population growth (massive growth is unavoidable, projected about 1.48 billion in 2050), dietary change (reduce feed crop demand for meat), urbanization (increase in city population and activities), available arable land (decline, degradation, and soil erosion), water supply (deficit and pollution), disasters (drought and flood), policies and economic arrangements (increase or decrease production efficiency), modern marketing institutions and infrastructure, and scientific and technological development and adaptation (increase efficiency and productivity) (fao, 1998). the question being asked is: what is china‟s capacity to increase its agricultural production and at what costs? population, arable land, and swine farm relationship in china china has the largest population in the world, approximately 1.3 billion people in 2008, and projected to be close to 1.5 billion in 2020. such a huge population imposes substantial stress on its natural resources including arable land. china has about 22% of the world population, but only 7% of the world‟s arable land (geissler, 1999). china‟s farmland is 10 times less per capita compared to that of the u.s. (knapp, 2009). the average per capita availability of farmland could be less than 0.08 ha/person by the year 2025, resulting from conversion of farmland to industrial, residential and transportation uses and soil erosion as well as desertification (smil, 2006). china‟s economy and animal agriculture are further affected by the uneven distribution of the population and farmland throughout the entire country (figures 1a and b). the limitations in topography, soil condition and temperature have restricted cultivation to only about 15% of the total land area in china. china‟s farmland has been further reduced by urbanization. urbanization attracts rural labor and increase urban population and allocation area. as at 2005, china has over 60 metropolitan areas with populations greater than 750,000, most of them are at the east (coast) and central areas (figure 1a). the unremitting urbanization, such as conversion of farmland to industrial, residential and transportation uses, has devoured massive amounts of fertile farmland, resulting in threatening grain supply and development of animal agriculture. for example, in 2008, a total of 191.6 thousand ha of cultivated land was used for construction purpose (nbsc, 2009). in addition, land degradation (resulting from intensive chemical, fertilizers and pesticides application), land erosion (stressed land by farming steep hillsides or damaged surface covers), and the increasing risk of floods are growing threats to china‟s food supply (long et al., 2006; yu, 2008). between 1950 and 1999, 9.2 billion tons of soil spilled into the yellow river, raising its bed by as much as 4 m. this is a consequence of a severe soil erosion problem. in 2008, 24800 ha of cultivated land was destroyed by disasters (nbsc, 2009). now, china has more than 3.5 million sq km of eroded land, of which 1.6 million sq km is due to water and 1.9 million due to wind. in 2000, land erosion caused economic losses of a total of 200 billion yuan ($29 billion), 2.25% of the country‟s gdp. if the erosion continues as its current rate in northeastern china, crop output could fall by as much as 40% over the next 50 years, and in the southwest, about 100 million people will be at risk of losing their land over the next 35 years. based on the limitation of arable farmland and high demand for quality food and meat, the chinese government has encourage development of animal agriculture, especially the swine industry, to secure economic and society stability in urban areas. modern swine farms are rapidly developing and are highly concentrated at the provinces with high percentage of arable land and feed mills, that is, the middle and low yangtze river region and south-east china. the modern swine farm distribution is more economically efficient when reducing costs for transportation and securing feed supply. however, urbanization and development of animal agriculture have reached an environmental carryingcapacity, that is, the modern intensive swine farms located at a narrow region with high density of human population and limited water resources. environmental debt is growing daily; china is now faced with the challenging prospect of increasing the productivity of its animal agriculture, while simultaneously reducing the level of pollution from this activity, such as air quality, manure waste, and soil and water contamination (brubaker, 2004). swine farming and grain production relationship in china in china, it is estimated that 72% of total grain products are used for feed animals (usda‟s grain report, 2007). the rapid increase in china‟s population and a great demand for meat has an enormous impact on feed-grain market (bingsheng, 2001; zhou et al., 2008). however, china is faced with severe challenges in safeguarding grain security due to rising living standards, decreasing arable land, increasing water shortages and climate changes (natural disaster). the chinese government has made a midand long-term grain security plan, aimed at achieving grain production of at least 500 billion kg by 2010 and more than 540 billion kg by 2020 to meet increasing population and developing economic needs (china daily, 2008). to meet these goals, the chinese government has issued several policies which include reduced agricultural taxes, raised subsidies, and introduced minimum state grain-purchasing prices. in 2007, grain output was 501.5 million tons and further rose to 505 million tons in 2008 (grains new, 2009). the selfsufficiency rate was 98%. currently, china‟s grain supply and demand are basically balanced, but the situation is expected to worsen in the long-term (zhou et al., 2008). however, there is limited room to increase arable land because of industrialization and urbanization that are taking farmland away from grain production (china daily, 2008). self-sufficient levels could be down to less than 90% by 2020. loss of farmland, coupled with the increase in animal production means that feed-grain supplies, will be continuously dwindling and the price of corn will climb. facing the changing corn market, familybased rural backyard and local community-based small pork productions are losing ground to more concentrated and specialized pork operations. to stabilize the grain market, the chinese government has increased sown area for grain; however, china‟s agricultural land area is very limited at only 0.42 ha of arable land per person (fabiosa, 2005). in addition, available farmland may reach or close to its limit because of heavy use of fertilizer and irrigation, growing multiplecropping, and raising two or three crops per year. china‟s government exhausts all avenues to improve grain production as well as its productivity in the use of grain.one promising area for raising pig production is to increase feeding efficiency through balanced diets from commercially mixed feed formulations and application of advanced technologies to reduce production costs such that the production cost for one pound pork carcass weight was about $0.35 0.39 in the u.s., while it went up to $0.60 in china in the year 2000 (amponsah and qin, 2000; pig international, 1998). although the current changes of the economy affect hog prices and pork production through the changes of ethanol prices and corn prices, there is still a huge gap to be improved in pork production in china. on commercialized swine farm, feed cost is about 6070% of the total swine production cost in china (amponsah and qin, 2000; usda gain report, 2008c). to increase feed efficiency and decrease feed cost, the chinese government is encouraging the development of feed mills. in china, most mills are collectively owned or state owned. in order to subsidize pork production, the chinese government operates feed grain mills at a lesser cost, thereby indirectly subsidizing domestic hog producers to meet economic developmental needs. however, limited arable land prevents china from becoming a large-scale feed-grain producer (fuller, 1997). according to food and agriculture organization (fao) data (1998), currently, china must feed 13.0 people for each hectare of arable land, whereas europe must feed 4.1 people, and the u.s. needs to feed only 1.4 people with the same arable area. land scarcity limits china's ability to continue increasing its livestock production to meet the growing domestic demand without increasing its imports of livestock feedstuffs. however, china's uncertain direction in biotechnology policy could limit feed grain imports, since the u.s. and other suppliers make wide use of genetically modified varieties of feed grains and oilseeds. currently, the use of genetically modified (gm) crops in the food supply is not a major concern issue in china. however, the government is developing regulations to control gmo products. if china imposes stringent labeling or traceability requirements that apply to feed grains and feed products, it will raise internal feed costs to china's livestock producers and slow the sector's growth (fuller et al., 2001). water resources, grain production, and swine farm relationship in china another major limitation of agriculture in china is water. water shortage has become a bottleneck restricting sustainable development of the economy in china (brown and halweil, 1998). the regional distribution of water resources does not match the agricultural demand, and massive water diversion projects have dislocated the entire country. based on water supply, agricultural zones in china can be subdivided into three natural zones: the monsoon zone in the east, the arid inland zone in the northwest, and the qinghai-tibet plateau inland zone in the southwest. these subdivisions account for 45, 30 and 25% of all land, respectively. the eastern monsoon zone is agricultural; the northwest and southwest are pastoral. in the north and west regions, large parts of the existing agricultural areas cannot be cultivated at their full potential due to insufficient rainfall, and water shortageassociated desertification. agricultural land in china is farmed intensively but in many areas, it could be more productive on a unit of labor basis if water supply were sufficient. extensive irrigation is the key to china‟s ability in maintaining agricultural production. in china, there are four major rivers, yellow river (north), yangtze river (central), pear river (south), and huai river (between yellow river and yangtze river), as well as multiple small rivers in the east and south of china (figure 1c) (the iiasa, 1999). these rivers drain most parts of the eastern and central regions but not the western regions of china. however, due to climate change, these rivers flood yearly, resulting in land erosion. water resources, like in some regions of the world, is the major factor affecting swine farm development through multiple factors. these factors include maintaining and developing farmland, planting and growing of grain, and urbanization and industrialization by population growth. theses factors cause rivers to run dry and ground water table to fall. especially in the west, northeast, and north provinces of china, arid and semi-arid regions are facing great problem of water shortage. china‟s arable land can be increased if enough water supplies existed. for example, in 2008, over 1.179 million ha of farmland was developed with effective irrigation and another additional 1,390 million ha of farmland was guaranteed by water-saving irrigation system (news from grain, 2008). however, in china, swine farms are concentrated at the same geographic regions that also allocate numerous farmlands and a great density of humans, that is, the middle and low yangtze river region and south-east china (figures 1b and c). the geographic distribution of developing swine farms has resulted in pollution of many rivers and lakes, leading to further reduction of usable water resources. even the water quality is continuously improved. in 2008, monitoring of water quality on 409 sections of the seven major water systems in china showed that 45.0% of the section was below the national quality standard grade iii for surface water; and 20.8% were worse than grade v (nbsc, 2009). it is a critical issue for china to improve the quality and availability of water for developing animal sectors, such as swine production. china will provide a huge market for internal pork exports including u.s. pork producers (amponsah et al., 2003). effect of religion on development of the swine industry in china throughout history, animals, including pigs, have played an important role in human lives. in china, since ancient times, chinese people have recognized animals as their properties, and animal husbandry has played important economic roles. animal meat, especially pork and pork products is a main source of food for chinese people. however, the relationship between humans and animals is affected by culture and religion. as at 2004, the country had five officially recognized religions, buddhism, taoism, islam, catholicism, and protestantism. among them, buddhism and islam have different restrictions regarding killing animals and eating animal meat, especially pigs. buddhism buddhism teaches that; animals are sentient beings, no different from humans in their ability to feel suffering; animals possess buddha nature and therefore an equal potential to become enlightened; rebirth, any human could be reborn as an animal, and any animal could be reborn as a human. based on the doctrine, if a human looked far enough back in one's infinite series of lives, he or she would eventually perceive a relationship with every animal in some way. the buddhist belief in rebirth (luen hiu) which teaches that ultimately humans and animals are part of a single family since we are to be reborn into one existence (human or animal) after another until it becomes the permanent; that is, a god (harvey, 1990). particularly in china, an important way of demonstrating buddhist piety is to release animals (fang sheng) that one bought from markets, particularly birds or fish, into their natural environments. the hui hui community in china evolved into ten ethnic groups and two systems: the uighurs, the kazaks, the khalkhas, the uzbeks, the tajiks and the tatars living mainly in the xinjiang uighur autonomous region, and the huis, the salas, the dongxiangs and the bao‟ans live mainly in the chinese inland. they inhabit six major regions, such as nanjing, suzhou, xiningr, guanzhong, yunnan, and beijing (vipassana foundation, 2008). in china, based on the different analyses, the percent of the population that adheres to buddhism ranges from 16 to 21% (about 200 millions) (wu, 2007), although not all of them adhere to a vegetarian diet. these widely held religious believes should be taken into account in further development of chinese animal husbandry, including the swine sector. islam the primary religion of the ten of fifty-six nationalities in china, such as hui, uigur, kazak, uzebek, kirgiz, tartar, tajik, dongxiang, baoan, salar, is islam. nearly all of the nation's muslims are members of ethnic minority nationalities; most belong to the sunni branch, but there are also large numbers of citizens from tajik that are shia decent. the total population of muslim citizens is approximately 20 million (other sources estimate the islamic population of china to be over 37 million) (pervis, 2005). the distribution of the chinese muslim population is characterized by being „scattered widely and concentrated in small groups. in the northwest, particularly shaanxi, gansu, ningxia, qinghai, and xinjiang, they live and intermarry with local people, and eventually become permanent inhabitants. muslims are prohibited from eating pork and the flesh of other animals that have not been ritually slaughtered. pork is forbidden in islam according to the tex of qur an; and chinese muslims call those meats „diet‟. such religious beliefs should also have a great impact on chinese animal husbandry, especially the swine industry development. this is currently evident in the regional relative popularity of swine farming in china. in the western regions of china, the majority of the population is muslim and subsequently, there are fewer settled pig farms in that region (figure 1b). pork consumption is low in ningxia where the islam religion plays a predominant role. conclusion since 1978, china has introduced structural and economical reforms through decentralization and privatization that have increased both domestic private investment and direct foreign investment, resulting in rapid development of its economy. china‟s agricultural economy becomes the largest in the world and china leads import and export of agricultural products globally. high income has led to changes in chinese dietary habits, which shifted from grain to meat-based. animal husbandry, mainly the swine sector, has greatly expanded to meet this rising demand. swine production is currently undergoing a drastic change from small farms and backyard to large production facilities (modern intensive swine farms). however, with the farmland and water resources stretched to the limit and the labor costs rising, china is facing great challenge to maintain the rapid growth in its economy. it is the authors‟ opinions that the best way to develop and improve animal husbandry in china should be to implement a cost-effective maintenance system to meet human needs but without abuse of animals, and based on its special situation and limited resources. the most important issues encompass improvement of nutrition, genetics and breeding, housing, health maintenance and disease prevention, handling facilities and techniques, training of stock-keepers and environmental protection, as some of those mentioned in the u.s. and other countries globally. references amponsah w, qin xd (2000). pork. in: colyer d, kennedy pl, amponsah wa, fletcher sm, jolly cm (eds.), competition in agriculture: the united states in the world market. ny: the haworth press, inc., pp. 235-250. amponsah w, qin xd, peng xh (2003). china as a potential market for u.s. pork exports. rev. agr. econ., 25: 259-269. brown l, halweil b (1998). china‟s water shortage could shake world food security. world watch, 11: 10-21. brubaker d (2004). trade and growth of animal agriculture in the world international competition: the china model. in: local and global considerations in animal agriculture: the big picture. washington, dc., pp. 5-8. carter ca, zhong f, zhu j (2009). development of chinese agriculture since wto accession. eurochoices, 2: 10-16. china daily (2008). china outline first multi-year grain security plan. http://www.chinadaily.com.cn/bizchina/200811/13/content_7202034.h tm2008(accessed 25 april 2009). pg 1 fabiosa jf (2005). the impact of ownership structure on the performance of china‟s feed mill sector. midwest agribusiness trade research and information center, iowa state university. pp 126 fang c, fuller f (1998). feed-grain consumption by traditional porkproducing households in china. the center for agricultural and rural development. iowa state university. pp 1-20 fao (food and agriculture organization of the united nations) (1998). poverty alleviation and food security in asia lessons and challenges. regional office for asia and the pacific, food and agriculture organization of the united nations, bangkok. annex 3 fuller f (1997). china's food and feed grain supply and demand. the workshop entitled challenges and opportunities for sustainable agriculture in china, beijing, china. p.10 fuller f, tuan f, wailes e (2001). rising demand for meat: who will feed china‟s hogs? china‟s food and agriculture: issues for the 21st century, pp. 17-19. geissler c (1999). china: the soybean-pork dilemma. p. nutr. soc., 58: 345-353. grain news (2009). china commodities report: 2008 domestic grain output up 0.7% -four straight years of growth. http://www.grainnet.com/articles/china_commodities_report__2008 _domestic_grain_output_up_0_7___fourth_straight_year_of_grow th-69508.html2009 (accessed 12 march 2009). p. 1-4 harvey p (1990). introduction to buddhism. cambridge university press, cambridge. p. 120. huang jk , bouis h (2000). structural changes in the demand for food in asia: empirical evidence from taiwan. agr. econ., 26: 57-69. knapp r (2009). china: a teaching workbook. columbia university press, columbia, pp. 1-12. lin jy, cai f, li z (1996). the lessons of china‟s transition to market economy. cato j., 16: 201-231. lohmar b, gale b, tuan f, hansen j (2009). china‟s ongoing agricultural modernization: challenges remain after 30 years of reform. usda econ. inf. bull., p. 51. long kl, heiig g k, wang j, li xb, luo,m, wu,xq, zhang m (2006). land use and soil erosion in the upper reaches of the yangtze river: some socio-economic considerations on china‟s grain-for green program. land degrad. dev., 17: 589-603. lu a (2007). china's rural population shrinks to 56% of total. http://news.xinhuanet.com/english/2007-10/22/content_6925292.htm 2007 (accessed 12 march, 2009). nbsc (national bureau of statistics of china) (2009). statistical communique of the people‟s republic of china on the 2008 national economic and social development. http://chinadataonline.org/freesource/zixunshow.asp?id=4872009 (accessed 12 march, 2009). news from grain (2008). the 2008 land grab for food and financial security. http://www.grain.org/nfg/?id=6102008(accessed 16 march, 2009). pervis d (2005). opportunities in the food market of china. agri-food trade service, agriculture and agri-food canada. pp. 1-73. pig international (1998). the production cost comparison. watt publishing company. mt. morris, illinois. usa, 28: 18-24. rozelle sa, swinnen j (2004). success and failure of reform: insights from the transition of agriculture. j. econ. lit., 42: 404-456. smil v (2006). japan and china: the next fifty years. japan focus, september 5, 2006. the iiasa (the international institute for applied systems analysis) (1999). main rivers in china. http://www.asu.edu/clas/asian/china%20physical%20geography%20 (for%20academic%20decathlon).ppt1999(accessed 10 march, 2009). the pigsite (2008). people‟s republic of china: agricultural situation: livestock and products 2008. http://www.thepigsite.com/articles/2425/peoples-republic-of-chinaagri cultural-situation-livestock-and-products-2008(accessed 20 march, 2009). usda‟s gain report (2007). people‟s republic of china, grain and feed. gain report number: ch7015, pp. 1-34. usda‟s gain report (2008). people‟s republic of china, agricultural situation livestock and products. gain report number: ch8078. pp. 1-21. vipassana foundation (2008). buddhists around the world. http://thedhamma.com/buddhists_in_the_world.htm2008(accessed 16 july, 2008). wang y, hu a (2007). multiple forces driving china's economic development: a new analytic framework. china world econ., 15: 103-120. wu j (2007). religious believers thrice the estimate. china daily, p. 1. yu x (2008). land erosion “threat to food supply”. http://www.chinadaily.com.cn/bizchina/200811/22/content_7230508.h tm2008(accessed 20 november, 2008). zhou zy, tian w, malcolm b (2008). supply and demand estimates for feed grains in china. agric. econ., 39: 111-122. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (7), pp. 186-189, july, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of breadfruit processing and its performance in biscuit production olaoye o. a 2* , onilude a. a 1 and oladoye c. o 2 1 department of botany and microbiology, university of ibadan, nigeria. 2 division of food sciences, school of biosciences, university of nottingham, uk. accepted 23 march, 2015 breadfruit was processed into flour and used to supplement wheat flour, in the percentages of 0, 5, 10, 15, 20 and 25, for biscuit production. the proximate composition, sensory evaluation and microbiological quality, in terms of aerobic plate count, were evaluated. the crude fibre and ash contents (%) increased with increase in the proportion of breadfruit flour (bf) level, with the 25% bf level having the values of 4.98 and 6.78 respectively. lowest values of 11.54, 24.15, and 40.80 were however observed for the biscuit with 25% bf level in terms of crude protein, ether extract and carbohydrate respectively, showing decrease with corresponding increase in the bf levels. the microbiological analysis indicated that the aerobic plate count (apc, log cfu/g) ranged between 1.00 and 1.75. the sensory evaluation showed that breadfruit supplemented biscuits (bsbs) were not significantly different (p<0.05) from whole wheat biscuits (wwb) with respect to sensory attributes of texture, appearance and general acceptability, at all levels bf supplementation. in terms of crispiness, aroma and taste, significant differences were not observed only up to 20% bf level, at the same level of probability (p<0.05). key words: breadfruit flour, wheat flour, proximate composition, composite biscuit, sensory evaluation, aerobic plate count. introduction biscuits are nutritive snacks produced from unpalatable dough that is transformed into appetizing product through the application of heat in an oven (kure et al., 1998). they are ready-to-eat, convenient and inexpensive food product, containing digestive and dietary principles of vital importance (kulkarni, 1997). the principal ingre-dients are flour, fat, sugar and water; while other ingre-dients include milk, salt, flouring agent and aerating agent (wade, 1988). biscuits are a rich source of fat and carbohydrate, hence are energy giving food and they are also a good source of protein and minerals (kure et al., 1998). breadfruit is a fruit tree that is propagated with the root cuttings and the average age of bearing first crop is between 4 to 6 years (amusa et al., 2002). it produces its fruit up to three times in a year and the number of fruits produced is very high. the fruit has been described as an important staple food of a high economic value (soetjipto and lubis, 1981). it is commonly cultivated in several *corresponding author. e-mail: olaayosegun@yahoo.com. other tropical countries like west indies, ghana, sierra leone, jamaica and nigeria. the breadfruit pulps are made into various dishes; it can be pounded, fried, boiled or mashed to make porridge; it can also be processed into flour and used in bread ands biscuit making (amusa et al., 2002). breadfruit has also been reported to be rich in fat, ash, fibre and protein (ragone, 1997). despite the importance of this fruit, its production is faced with several problems including short shelf life and poor yield due to diseases (cook, 1975). the fruits are thus utilized in nigeria within 5 days of harvesting because of their short shelf lives. the use of composite flours in bread making has been reported by many researchers. olaoye et al. (2006), investigated the use of supplementation of flours of soybean and plantain in wheat in the production of bread. the research workers were able to obtain acceptable bread samples with up to 15% supplementation of wheat with plantain flour. also mepba et al. (2007), produced composite breads and biscuits from mixed flours of wheat and plantain, with up to 30% supplementation of plantain flour. however, the quality attributes of the products tend to decrease with corresponding increase in the percent olaoye et al. 187 b r e a d fr u it s c l e a n in g p e e lin g s l ic in g b l a n c h in g d r y in g d r y m il l in g s ie v in g s c r e e n in g b r e a d f r u it fl o u r figure 1. flow chat for the production of breadfruit flour age substitution with plantain flour. notwithstanding a successful substitution of up to 15% of composite flour in the production of baked products will go a long way in reducing cost and enhance utilization. in their own findings giami et al. (2004), were able to successfully make acceptable bread from composite flour of kernels of roasted and boiled african breadfruit. if such researches are encouraged and commerciallized, it will help in reducing wastage normally associated with our local crops in west africa and africa as whole, as well as maximizing their utilization. it is therefore a serious challenge to researchers in africa to help in finding out how many of our crops could be maximally utilized to prevent associated waste due to lack of proper storage facilities. this study was aimed at processing breadfruit into flour and to examine its performance in biscuit production, in terms of proximate composition, sensory and microbiological qualities, when used as composite with wheat flour. materials and methods materials the breadfruits used for this study were collected from a local market in ileife, south-western nigeria. they were taken to the laboratory for immediate use and processing, in clean polythene bags. processing of breadfruit into flour the breadfruits seeds were processed into flour as shown in figure 1. the breadfruits were thoroughly washed to remove any dirt and unwanted materials. they were then peeled and washed with clean water. the breadfruits were sliced, blanched for about 5 min and ingredients weighing mixing cutting baking cooling packaging figure 2. flow chart for biscuit production. then dried in the oven at 105 o c for 1 3 h, after which they were milled into flour. the flour was screened through a 0.25 mm british standard sieve (model bs 410) (giami et al., 2004) blend formation six blends were prepared by mixing breadfruit flour with wheat flour in the percentage proportions of 0:100, 5:95, 10:90, 15:85, 20:80 and 25:75 respectively, using machine food processor (kenwood km 201, england). biscuit production biscuits were produced from the six blend formulations using the two stage creaming-up method (figure 2). the baking formula is as described by ihekoronye (1999) (modified) . all ingredients except flour and sodium bicarbonate were added with continued mixing. the dough was then placed on a cutting board, rolled out until uniform thickness and textures were obtained. biscuit cutter was used to cut the sheet of rolled dough into desired shapes and sizes. the shaped dough pieces were then baked at about 220 o c for 15 min, allowed to cool, packed and stored (kure et al., 1998). proximate analysis the proximate composition of the breadfruit supplemented biscuit (bsb) samples was determined using the methods of egan et al. (1981). the samples were analyzed for moisture, ash, crude fibre, crude protein, crude fat and carbohydrate (by difference). sensory evaluation the sensory attributes, including crispiness, aroma, texture, taste, appearance and general acceptability, were evaluated by a semi trained 10-member panel, using a 9-point hedonic scale with 1 representing the least score (dislike extremely) and 9 the highest score (like extremely). analysis of variance (anova) was perform 188 afr. j. food sci. res. table 1. proximate composition and aerobic plate count of whole wheat and breadfruit supplemented biscuit samples parameter/biscuit sample a b c d e f crude protein (%) 12.50 12.35 11.90 11.75 11.67 11.54 crude fibre (%) 1.03 1.36 2.27 2.73 3.45 4.98 ether extract (%) 26.50 26.35 25.27 24.81 24.23 24.15 ash (%) 3.09 4.37 5.11 5.90 6.15 6.78 moisture (%) 11.50 11.45 11.40 11.49 11.67 11.75 carbohydrate 45.38 44.12 44.05 43.32 42.83 40.80 apc (log cfu/g) 1.00 1.15 i.25 1.25 1.65 1.75 a – biscuit produced from 100% wheat flour. b biscuit produced from composite flours of 95% wheat and 5%breadfruit. c biscuit produced from composite flours of 90% wheat and 10% breadfruit. d biscuit produced from composite flours of 85% wheat and 15% breadfruit. e biscuit produced from composite flours of 80% wheat and 20% breadfruit. f biscuit produced from composite flours of 75% wheat and 25% breadfruit. apc – aerobic plate count. ed on the data gathered to determine differences, while the least significant test was used to detect significant differences among the means (ihekoronye and ngoddy, 1985). microbiological examination the aerobic plate count was carried out on the bsb samples using the method of fawole and oso, (1988). 10 g of each sample was taken aseptically and homogenized in 90 ml sterile distilled water; in a blender (philips type hr 2815i) for about 2 min. serial dilutions (using 1 ml of homogenates) were made in 9 ml sterile distilled water, dispensed in test tubes. one milliliter of each dilution was pour plated in sterile petri dishes, using the plate count agar (pca, oxoid), incubated at 37 o c for 24 36 h. counts of visible colonies were made and expressed as log cfu/g sample. results and discussion the proximate composition and aerobic plate count (apc) of the whole wheat biscuits (wwb) and breadfruit supplemented biscuits (bsb) samples are as presented in table 1. the crude protein decreased with increase in the proportion of the breadfruit flour (bf) level in the biscuit samples. the highest crude protein (%) of 12.50 was recorded for the wwb (that is, 0% bf level) and this value decreased gradually to the lowest value of 11.54 recorded for the 25% bsb. the protein content of wheat has been reported to be higher than its breadfruit counterpart and this could be responsible for the lower values of crude proteins in the biscuit samples, as the amount of breadfruit flour increases (morton and miami, 1987; asiedu, 1989; udio et al., 2003). the crude fibre (%) ranged between 1.03 and 4.98 and this showed a corresponding increase with increase in the proportion of breadfruit flour. breadfruit has relatively higher crude fibre than wheat and this could justify the result obtained for the different biscuit samples. this observation is in support of the findings of esuoso and bamiro (1995) as well as amusa and co-workers (2002) . crude fibre is known to aid the digestive system of human (ihekoronye and ngoddy, 1985), indicating that the bsbs could attract good acceptability by many people as well as health organizations. the fat content (%) of the biscuits followed the same trend with crude protein, though the incremental values were minimal. the highest value of 26.50 was recorded for the wwb while lowest value of 24.15 was obtained for the 25% bsb. fat plays a significant role in the shelf life of food products and as such relatively high fat content could be undesirable in baked food products. this is because fat can promote rancidity in foods, leading to development of unpleasant and odorous compounds (ihekoronye and ngoddy, 1985). the values obtained for the ash contents indicated that the 25%bsb had the highest value of 6.78. the ash contents were observed to decrease with the percentage decrease in the bf level. breadfruit contains comparatively higher ash content than wheat (ragone, 1997) and this could be responsible for the higher ash contents recoded for the bsbs than the wwb counterparts. it then follows that incorporation of breadfruit flour in the process of biscuit making could enhance the mineral intake of many people, as ash is indicative of the amount of minerals contained in any food sample. the moisture contents of the biscuits ranged between 11.50 and 11.75, with the wwb having the highest value. these values were minimal and may not have adverse effect on the quality attributes of the product (kure et al., 1998). the carbohydrate contents were highest for the biscuits in terms of all proximate composition parameters (that is, crude protein, crude fibre, ether extract, ash, moisture and carbohydrate) determined in this study. this was expected as the ingredients composed of mainly carbohydrate rich materials, which are wheat and breadfruit flours. the microbiological examination, in terms of aerobic plate counts (apc, cfu/g), ranged from 1.00 to 1.75. the counts were minimal and are within acceptable limits (fawole and oso, 1988). microorganisms play significant role in the determination of shelf lives of food products. they are usually responsible for spoilage of many food items. a high aerobic olaoye et al. 189 table 2. hedonic sensory mean scores of the whole wheat and breadfruit-wheat composite biscuit samples attribute/biscuit sample a b c d e f crispiness 6.7 a 6.9 a 7.2 a 6.8 a 6.2 ab 5.7 b aroma 6.7 a 7.0 a 7.2 a 6.7 a 6.2 ab 5.6 b texture 6.8 a 6.8 a 7.1 a 7.0 a 6.9 a 6.8 a taste 7.1 a 7.3 a 7.5 a 7.1 a 6.6 ab 6.1 b appearance 7.1 a 7.0 a 7.3 a 7.1 a 6.9 a 6.7 a general acceptability 6.7 a 7.1 a 7.2 a 6.9 a 6.6 ab 6.3 a mean scores in rows with same letters are not significantly different (p<0.05). a, b, c, d, e and f are as defined in table 1 plate count (apc) could indicate the presence mixed population of microorganisms, which may consist of spoilage types. limits of microbial counts have been recommendded in most foods to keep them safe for consumption (porter, 1978). the product should however be well kept after processing in suitable packaging materials capable of preventing contamination and hence subsequent proliferation of spoilage microorganisms. the sensory hedonic mean scores of the bread samples are shown in table 2. the mean scores decreased with increase in the proportion of bf in all biscuit samples in terms of all the sensory attributes tested. the analysis of variance showed that the bsbs were not significantly different (p<0.05) from the wwb up to 20% in terms of all sensory attributes, and up to 26% with respect to texture, appearance and general acceptability. in terms of crispiness, aroma and taste, the 20 and 25% bsb were not significantly different from each other at the same probability level. conclusion this study showed that the quality of biscuits could be improved with supplementation of breadfruit flour, in terms of crude fibre and ash. the whole wheat biscuits were not significantly different from the breadfruit supplemented biscuits up to 15% in all sensory attributes tested. the use of breadfruit in biscuit making, and other food products, would greatly enhance the utilization of this crop in developing countries like nigeria and west africa in whole, where the crop has not been optimally utilized. further work is necessary in terms of the microbiological examinations of the products. in dept analysis into the microbiological qualities and shelf life will help in improvement in the product. references amusa na, kehinde ia, ashaye oa (2002). bio-deterioration of breadfruit (artocarpus communis) in storage and its effects on the nutrient composition. afri. j. biotechnol. 1(2): 57-60. asiedu jj (1989). processing tropical crops. mcmillan education ltd.london. 1 st edn. p. 21. cook aa (1975). disease of tropical and sub-tropical fruits and nuts. longman co. ltd. uk. p. 34. egan h, kirk rs, sawyer r (1981). pearson’s chemical analysis of foods. 8 th ed. churchill livingstone, medical division of long group ltd. great britain. p. 591. esuoso ko, bamiro fo (1995). studies on the baking properties of nonwheat flours, breadfruit (artocarpus artilis). int. j. food sci. nutri. 46(3): 267-73. fawole mo, oso ba (1988). laboratory manual in microbiology. 3 rd ed, spectrum books ltd. ibadan. giami sy, amasisi t, ekiyor t (2004). comparison of bread making properties of composite flour from kernels of roasted and boiled african breadfruit (treculia africana decne) seeds. j. material res. 1(1): 16-25. ihekoronye a (1999). manual on small scale food processing. 1 st ed, academic publishers nsukka. p. 32. ihekoronye ai, ngoddy po (1985). integrated food science and technology for the tropics. 1st ed. mcmillan publishers. pp. 261291. kulkarni sd (1997). roasted soybean in cookies: influence on product quality. j. food sci. technol. 34: 503-505. kure oa, bahago ej, daniel ea (1998). studies on the proximate composition and effect of flour particle size on acceptability of biscuit produced from blends of soyabeans and plantain flours. namida tech-scope j. 3: 17-21. mepba hd., eboh l, nwaojigwa su (2007). chemical composition, functional and baking properties of wheat-plantain composite flours. afri j. food nutri devt.,7(1):1-22 morton jf, miami fl (1987). breadfruit. in: fruits of warm climates. pp. 50-58. olaoye oa, onilude aa, idowu oa (2006). quality characteristic of bread produced from composite flour of wheat, plantain and soybeans. afri. j. biotechnol. 5(11): 1102-1106. porter nn (1978). food science. 3 rd ed. avi. publishers. pp. 406-415. ragone d (1997). breadfruit artocarpus altilis (parkinson) fosberg. promoting the conservation and use of underutilized and neglected crops. institute of plant genetics and crop plant research, gatersleben international plant genetic resources institute, rome, italy soetjipto nn, lubis as (1981). vegetables: ibpgr secretariat, rome. p. 330. udio aj, osuntogun ba, falade mo, adewusi sa (2003). chemical analysis of breadfruit (artocarpus communis forst) from southern western niger. j. food technol. 1(2):29-35. wade p (1988). biscuits, cookies and crackers: the principles of craft. 1:1-4. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (3), pp. 078-082, march, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the physico-chemical properties of cashew tree gum e. gyedu-akoto 1 , i. oduro 2 , f. m. amoah 1 , j. h. oldham 2 , w.o. ellis 2 , k. opoku-ameyaw 1 and rasheed bin hakeem 1 1 cocoa research institute of ghana, akim-tafo, ghana. 2 department of biochemistry, kwame nkrumah university of science and technology, kumasi, ghana. accepted 11 october, 2016 the physico-chemical properties of cashew gum (cg) collected from four cashew growing districts, sampa, wenchi, bole and jirapa in ghana were studied to help promote the utilization of cashew gum in the food industry. the gum was collected from trees of two different age groups, those that were 10 years and below and those above 10 years. physicochemical properties of cg were compared to those of gum arabic. parameters studied included ph (3.8 4.2), total ash (0.5 1.2%), protein content (1.27 1.80%), total sugars (0.96 2.10 mg/g), total phenols (0.21 2.26%), moisture content (9.8 13.2%) and insoluble matter (1.9 4.8%). gum from mature trees was generally found to have higher levels of protein, moisture, sugars and phenols than that from young trees, with the exception of ph which was lower in gum from mature trees. there were also variations in some of the physico-chemical properties of the cg from the different locations. the predominant minerals in cashew tree gum were ca, k, na and fe and their nutritional benefits is discussed. this study showed that cg possesses good physico-chemial properties and high levels of minerals key words: cashew gum, physico-chemical, gum arabic, nutritional benefits. introduction ghana has a land surface area of about 23.9 million km 2 and six main ecological zones put into two categories, the closed forest area which covers about 34% of the country (8.22 million ha) and the savanna area which covers 15.62 million ha or about 66% of the land area (nsiah-gyabaah, 1995). the climate is tropical in ghana and rainfall decreases from south to north (dickson and benneh, 1988). plant exudates are gums from various plant species obtained as a result of tree bark injury. they are normally collected as air-dried droplets (fitwi, 2000). synthesis of these gums generally occurs in all organs of the plant with different qualitative composition, appearing to be genetically controlled and influenced by environmental conditions (glicksman, 1969). cashew gum, which is the exudate from anacardium occidentale l have been found to have many lucrative possibilities for industrialization. the art of its use began in china centuries ago, reaching its climax of develop-ment during the period of 1368 – 1644 ad (glicksman,. *corresponding author. e-mail: akua_akoto2004@yahoo.co.uk. 1969). the gum is a complex polysaccharide of high molecular mass comprising 61% galactose, 14% arabi-nose, 7% rhamnose, 8% glucose, 5% glucuronic acid and 2% other sugar residues (glicksman and sand, 1973). cg is similar to gum arabic and can be used as a substitute of liquid glue for paper, in the pharmaceutical and cosmetic industries as agglutinant for capsules and pills and in food industry as a stabilizer of juices (smith and montgomery, 1959). thus, cg extraction can represent one more source of revenue for the producer, in addition to the nut. therefore, there is the need to study the physico-chemical properties of cashew gum. materials and methods collection of cashew tree gum farms were selected from two districts each within the guinea savannah (bole and jirapa) and the transitional belt zones (wenchi and sampa). selection was done based on two different age groups. these are farms that were 10 years and below (young farms) and those above 10 years (mature farms). these age groups were used because cashew production was introduced into ghana by the adventist development and relief agency (adra) in the early 1990s and this made most of the farms fall within the ages of gyedu-akoto et al. 079 table 1. mean values of physico-chemical parameters of cashew gum. parameter sampa wenchi bole jirapa young mature young mature young mature young mature phenol (%) 0.21 0.50 0.29 0.51 0.35 1.35 0.34 2.26 0.002 0.008 0.001 0.04 0.001 0.03 0.01 0.02 mc (%) 11.8 12.0 11.2 13.2 9.8 11.3 11.5 12.5 0.29 0.00 0.27 0.29 0.76 0.29 0.02 0.00 im (%) 3.1 1.9 2.8 3.8 2.0 4.8 2.4 2.2 0.56 0.42 0.57 0.20 0.28 0.28 0.42 0.28 sug(mg/g) 2.10 0.89 1.20 1.37 0.96 0.85 1.64 1.36 0.30 0.29 0.37 0.27 0.30 0.30 0.10 0.16 ph 4.1 3.9 4.2 3.8 4.1 3.8 4.0 3.8 0.00 0.01 0.00 0.01 0.01 0.01 0.01 0.00 ash (%) 1.0 1.0 1.0 0.5 1.0 0.8 1.0 1.2 0.00 0.00 0.00 0.00 0.00 0.29 0.00 0.29 protein(%) 1.56 1.35 1.40 1.41 1.80 1.27 1.58 1.38 0.10 0.00 0.04 0.01 0.28 0.01 0.20 0.39 *std dev in italics 4.3 4.22 4.1 4.1 4.2 4 4.1 3.9 4 3.8 3.8 3.8 young ph 3.9 3.8 mature 3.7 3.6 3.5 sampa wenchi bole jirapa area of collection figure 1a. effect of maturity on ph. 10 20 years. under each age group a maximum of five farms were selected and six trees selected from each farm. the gum exudates collected were then sorted to remove pieces of bark and other foreign matter and dried in an oven at 30 o c for two weeks. the dried and cleaned gum samples were then milled with a ken wood blender (uk) for physico-chemical analyses. physico-chemical analysis this included the determination of ph, moisture, and ash contents by aoac (1990) methods. protein content was by the kjedahl’s method (aoac, 1990) and insoluble matter determined according to british pharmacopoeia (1993) method. the total sugar concentration was determined using phenol-sulphuric acid method (dubois et al., 1956). total phenols were extracted with 80% aqueous acetone and phenolic content was determined with folinciocalteu reagent, using catechin as a reference compound (singleton et al., 1999). ca, fe, k, zn and na contents were determined by atomic absorption spectroscopy using unicam 929 aas model (uk). data obtained were analyzed using statgraphic plus for analysis of variance (anova) and multiple comparison tests. results and discussion the average values of the physico-chemical parameters determined are presented in table 1. the ph of cg ranged from 3.8 to 4.2 with gum from mature trees being more acidic than that from young trees (figure 1a). this compared favourably with that of gum arabic which is reported to be between 4.0 and 4.8 (belitz et al., 2000). the ph of gums coming from trees of the different age groups and also from the different locations were found to be significantly different (p < 0.05) from each other with some significant interaction between the locations and the ages of the trees (p < 0.05). the ash contents of cg also ranged from 0.5 to 1.2% and these fall within the acceptable level of less than 4% for gum arabic (belitz et al., 2000) . ash content is an important property considered as a purity parameter in gums (glicksman, 1969). the very low values of ash show that cashew gum has a good quality of mineral content. gums from both young and mature trees from sampa had the same ash content but those from mature trees in jirapa had higher ash content than that from young trees. for bole and wenchi, gum from mature trees had lower ash contents (figure 1b). there were significant differences in the ash contents for gums from trees of the different age groups and also from the different locations (p < 0.05). these findings were supported by multiple comparison tests, which showed that gums from sampa and wenchi were significantly differrent from those from bole. however, there was no interaction between the locations and the ages of the trees. 080 afr. j. food sci. res. 1.2 1 1 1 1 0.8 1 1.2 1 0.8 0.5 %ash 0.6 0.4 young 0.2 mature 0 sampa wenchi bole jirapa area of collection figure 1b. effect of maturity on ash 2 1.56 1.8 1.58 1.35 1.4 1.41 1.5 1.27 1.38 %protein 1 young 0.5 mature 0 sampa wenchi bole jirapa area of collection figure 1c. effect of maturity on protein. 2.26 2.5 2 1.35 % phenol 1 .5 0.5 0.51 young 1 0.21 0.29 0.35 0.34 mature 0 .5 0 sampa wenchi bole jirapa area of collection figure 2a. effect of maturity on phenols. the protein content of cg ranged from 1.27 to 1.80% (figure 1c). it was observed that cashew gum from young trees had higher protein content than that from mature trees with the exception of that from wenchi (figure 1c). gums from the two age groups showed significant differences (p < 0.05) but the differences in the locations were not significant. the total sugar concentrations of the gums were between 0.96 and 2.10 mg/g and these results compares well with that for gum arabic, which is found to be in traces (tic gum, 2001). cashew gum from young trees 14 12 13.2 12.5 11.8 11.3 11.5 12 11.2 9.8 %moisture 10 young 8 mature 6 sampa wenchi bole jirapa area of collection figure 2b. effect of maturity on moisture content of cashew gum. traces generally had higher sugar content than that from mature trees with the exception of that from wenchi. there were no significant differences in the sugar concentrations of gums from the different locations and also from trees of different ages. the total phenolic contents of the gums were between 0.21 and 2.26%. gum from mature trees had higher phenolic content than that from young trees for all the locations (figure 2a). the phenolic contents of gums from the different locations and from trees of different ages were significantly different (p < 0.05) with the phe-nolic contents of gums from sampa and bole being significantly different from that from jirapa. the moisture content of cg was found to be between 9.8 and 13.2% and this results falls within the acceptable level for gum arabic (12 17%) (belitz et al., 2000). gum from mature trees have higher moisture content and this may be due to the fact that they produced more gum and would need longer time to dry (figure 2b). there were significant differences in the moisture content for gums from trees of the two age groups coming from the different locations (p < 0.05). however, there was no interaction between the locations and the ages of the trees indicating that they were independent of each other. multiple comparison tests again showed that cashew gums from sampa, wenchi and bole were significantly different from that from jirapa. the insoluble matter content was found to range from 1.9 to 4.8% and was mainly debris. this agrees with the report of glicksman (1969) that most exudate gums yield some amount of insoluble residue when mixed with water. the low levels of insoluble matter indicate that cg is highly soluble in water. the levels of insoluble matter in gums from young trees in sampa and jirapa were found to be higher whilst those in bole and wenchi were lower. the differences may be attributed to the abilities of the gums to trap dirt and loose bark during and after exudation. however the differences were not significant for the locations and the age groups of the trees. cashew gum was found to have very high levels of calcium, sodium, and potassium, which are the essential minerals required by the body to meet metabolic needs cashew gum was gyedu-akoto et al. 081 2000 1755 1750 1358 1470 1405 1152 1269 1500 1012 ca 1000 young (mg/kg) mature 500 0 sampa wenchi bole jirapa area of collection figure 3a. effect of maturity on calcium. 1397 1400 1200 908 k 1000 554 800 385.5 386 young (mg/kg) 600 275 139 294.5 400 mature 200 0 sampa wenchi bole jirapa area of collection figure 3b. effect of maturity on potassium. 398.25 368 313 400 294 300.5 285.25 264 350 263 300 fe 250 200 young (mg/kg) 150 mature 100 50 0 sampa wenchi bole jirapa area of collection figure 3c. effect of maturity on iron. found to have very high levels of calcium, sodium, and potassium, which are the essential minerals required by the body to meet metabolic needs (welch and graham, 2003). the levels of minerals ranged from 1012 to 1750 mg/kg for calcium, 139 to 1397 mg/kg for potassium (figure 3a and 3b) and 114 to 301 mg/kg for sodium. the calcium contents of the gums showed significant differences between the locations and the ages of the cashew trees (p < 0.05) with gums from wenchi being significantly different from those from jirapa (p < 0.05). levels of potassium also showed significant differences between the locations and the ages of the trees (p < 0.05) with gums from sampa, wenchi and bole being significantly different from that from jirapa. however, there were no significant differences between gums from trees of the different ages and from the locations in terms of the sodium content. the levels of iron and zinc in cg were also found to be 258 to 398 mg/kg and 6.3 to 35.5 mg/kg respectively (figure 3c). zinc showed no significant differences between the gums from cashew trees of different ages and also from the different locations. iron also showed some significant differences (p < 0.05) in gums from cashew trees from the different locations and different age groups. gums from mature trees were generally found to have higher levels of protein, phenols, moisture and minerals than those from young trees and this may be due to the storage of starch and the production of cellulose, hemicellulose and proteins in the mature trees (rayburn, 1993). also much of the soil nutrient is stored in the mature trees. the variations in the physico-chemical properties of cashew gums from the different locations may also be due to the mixed species of cashew trees found in the collection areas, the different soil composition of the different regions together with the varying climatic conditions. conclusion cumulatively, this study explains that cg possesses good physico-chemical properties and high levels of minerals. therefore, it can be used in meal replacers, nutritional beverages and weight-loss products. healthconscious consumers demand natural ingredients and hence, this offers cashew gum tremendous potential. location and maturity also showed significant effects on the physico-chemical properties of cashew gum. references aoac (1990). official methods of analysis, 15 th ed., association of official analytical chemists, inc., arlington, va. belitz hd, grosch w, schieberle p (2004). food chemistry, 3 rd edition, springer, berlin. pp. 309-314. british pharmacopoeia (1993). cambridge university press, london. pp. 578-579. dickson kb, benneh g (1988). a new geography of ghana. london: longman. dubois m, gilles ka, hamilton jk, rebers pa, smith m (1956). analyt. chem. 28: 350. fitwi g (2000) the status of gum arabic and resins in ethiopia, report of the meeting of the network for natural gums and resins in africa (nagra), kenya college of communications technology, nairobi, kenya. 29-31 may, 2000. fao/kepri. pp. 14-22. glicksman m (1969) gum technology in food industry, academic 082 afr. j. food sci. res. press, usa. pp. 11-16. nsiah-gyabaah k (1996) bush fires in ghana, iffn no. 15: 24-29. rayburn be (1993) plant growth and development as the basis of forage management, factsheet toc, west virginia university, extension service. singleton vl, orthofer rm, lamuela-raventos rm (1999). analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu, methods enzymol. 299: 152-178. smith f, montgomery r (1959). the chemistry of plant gums and mucilages, reinhold publishing corporation, new york. pp 1-30. tic gums inc (2001). focusing on gum arabic – prepared foods: 49. welch rm, graham rd (2003). breeding for micronutrients in staple food crops from a human nutrition perspective. j. experimental bot. 55 (396): 353-364. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (2), pp. 263-266, february, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the effect of heat treatment on furazolidone residues in liver and muscle tissues of chicken anakalo a. shitandi 1 *, oketch aila 2 , stellah ottaro 1 , leakey aliong’o 1 , grace mwangi 1 , harish kumarsharma 3 and matofari joseph 1 1 guildford institute, egerton university, p.o. box 536 egerton 20107 kenya. 2 ministry of health, p.o. box 42 oyugis kenya. 3 food technology department, sant longowal institute of technology, longowal. sangrur-148106. punjab, india. accepted 02 july, 2017 this study investigated the effect of deep frying (210 o c/15 min) on furazolidone residues in liver and muscle tissues of chicken. furazolidone was administered (2 mg/kg body weight) orally to chicken daily for five days. the hens were then sacrificed at 1, 5, 24 168 and 264 h after treatment stopped and liver and muscle tissue samples obtained. the samples were deep fried, blended with distilled water and then centrifuged at 6000 rpm for five minutes. the supernatant was analyzed for the concentration of the drug using a using the delvotest sp microbiological assay. a detection limit of 11.0 g/ml was obtained with spiked liver tissues contaminated with furazolidone. furazolidone residues were detected in fried liver and muscle tissues 264 h post treatment. it was concluded that furazolidone drug residues in chicken liver and muscle tissues were not destroyed by deep frying. key words: furazolidone, depletion rate, delvotest test, poultry, deep frying. introduction various antibiotics, sulfonamides and coccidiostats are usually administered via feed or drinking water for the prevention and treatment of infectious diseases in laying hens. they also enhance feed efficiency, promote growth and improve productivity (hermes, 2003; gaudin et al., 2004; bergwett, 2005). the uncontrolled and unlimited use of anticoccidial drugs may however lead to the accumulation of undesirable residues in the animals treated and their products. these residues may have adverse effects on both the animals and human beings (lee et al., 2001; mccracken et al., 2005). in high income countries, birds whose residues exceed tolerance levels are removed from the distribution line (lee et al., 2001). however, in many low income coun-tries such as kenya, monitoring of drug residues is not done (shitandi and sternesjo, 2001). hence, the risk consumers of poultry products are exposed to is not known. furazolidone is a nitrofuran that has been used for many years for treatment of bacterial and protozoan *corresponding author. e-mail: ashitandi@lycos.com. phone: +254 51 62454; fax: + 254 51 62527. infections in poultry. it was banned in usa and european countries, but it’s widely used in veterinary practice in low income countries like kenya (ali, 1999; cooper et al., 2005). furazolidone produces adverse reactions in both man and animals as it’s carcinogenic and mutagenic (hoogenboom et al., 2002). when administered, furazoli-done metabolizes to its metabolite 3-amino-2-oxazolidi-none (aoz). the delvotest sp system is a broad spectrum screening test for the detection of antibiotic residues and sulphonamides in milk. this is a microbial inhibition assay that is based on the international dairy federation (idf) reference method. it is the standard test method used for the detection of antibiotic residues in liquid milk at the pasteurizing plants (fallon et al., 1995, 1996). the delvotest sp system is dependent on rapid growth and acid production of bacillus stearothermophilus var. calidolactis. being a microbial assay, it can be used to test a broad spectrum of antibiotics (food safety / autho-rity of ireland / 2002). in a previous study the potential of the b. stearothermophilus var calidolactis c953 as a test organism to detect a broad spectrum of drugs residue was demonstrated in unprocessed poultry meat (shitandi et al., 2006). the fate of drug residues during heat pro-cessing is however unclear. this study thus investigated the effect of shitandi et. al. 263 table 1. positive, doubtful and negative rates of detection for furazolidone contaminated liver tissues. conc. % % positive/ % ( g/ml) positive negative negative 0.1 0 30 70 0.2 10 40 50 0.4 20 30 50 0.6 30 30 40 0.8 40 30 30 1.0 50 20 30 2.0 60 20 20 4.0 60 30 10 6.0 70 20 10 8.0 80 10 10 10.0 90 10 0 12.0 100 0 0 14.0 100 0 0 20.0 100 0 0 nb: the responses were determined from replicates of ten for each drug concentration. heat treatment on furazolidone residues in liver and muscle tissues of chicken. it further evaluated the depletion rate of furazolidone so as to establish appropriate withdrawal period for the drug using the delvotest sp system materials and methods site of experiment the experiment was carried out at the taton farm and microbiology laboratory of dairy and food science technology department, egerton university. all experimental animals were acquired, retained, and used in compliance with the national laws and regulations of the research institution(s) of the authors. experimental animals were properly housed and used in accordance with the research plan. in vitro studies preparation of standards 100 mg of furazolidone (cosmos, kenya ltd) were weighed into a 100 ml flask and dissolved in 100 ml distilled water to produce a solution of 1000 g/ml (stock solution a). stock a was further diluted with distilled water (stock b) producing a solution of 100 g/ml. stock b (1 ml) was added to 4 ml of liver homogenate prepared at a dilution of 1:2 (tissue: distilled water) producing a solution of 20 g/ml. standards of 14, 12 10, 8, 6, 4, 2, 1, 0.8, 0.6, 0.4, 0.2 and 0.1 g/ml were prepared from stock solution b using blank tissue solutions. determination of limits of detection a sample of each standard homogenate was placed in a delvotest sp tube and replicated ten times (a total of 10 tubes). following the manufacturers instructions, the delvotest sp tubes were incubated for thee hours in a water bath at a constant temperature of 64 ± 1 o c. the number of positive (purple), doubtful (partly purple, partly yellow) and negative (yellow) colour changes were noted for each standard. percentages positive, doubtful and negative were calculated for each standard tissue homogenate. percent positive responses were plotted against tissue homogenate drug concentration to determine the detection limit of the delvotest sp. the limit of detection was defined as the concentration where 95% of the test results were positive. liver and muscle tissues that were free of antimicrobial drugs were used as the negative control. liver and muscle tissue spiked at 50 g/ml were used as positive controls. in vivo studies eighteen broiler chicken (ross breed) one day old were purchased from kims poultry farm (nakuru, kenya) and reared for six weeks at the poultry section of animal science department, egerton university. the birds had access to water and antibiotic free broiler feed ad libitum (the feed was obtained from unga limited, nakuru). the birds were gavaged with a commercial oral suspension of furazolidone once daily for five days at a dosage of 2 mg/kg of body weight. after the last treatment, the birds were sacrificed by decapitation in groups of thee at intervals of 1, 5, 24, 168 and 264 h. the liver and breast muscle samples were collected and frozen at -20 o c until analyzed for furazolidone residues. thee birds served as controls and were killed before the treatment began. the liver and muscle tissues were deep fried at 210 o c for 15 min. the tissues were homogenized with distilled water at a ratio of 1:2 (tissue: distilled water). the homogenates were centrifuged for five minutes at 6000 rpm to eliminate tissue debris which appeared to inhibit diffusion of the drug into the medium. supernatant from the homogenates were analyzed for residues using the delvotest sp system as earlier described. the percentages of positive, doubtful and negative responses were calculated for the tissues at the different concentrations and plots of concentration versus percentage positive made. semi quantification of furazolidone residues was done by comparing the obtained growth inhibition of samples and that of standards. data analysis data was analyzed using chi square test at 95% confidence interval. the test was used to compare depletion rates of the drug in liver and muscle tissues. results in vitro studies the results of furazolidone contaminated liver tissues are shown in table 1. a detection limit of 11.0 g/ml (figure 1) was obtained with spiked liver tissues contaminated with furazolidone. 100% positive delvotest response was achieved for concentrations greater than or equal to 11 g/ml. in vivo studies the delvotest sp showed a 30% positive response at 1 h 264 afr. j. food sci. res. 100 p o s it iv e 80 60 p e rc e n t 40 m e a n 20 0 .2 .4 .6 .8 1.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 20.0 concentration in ug/ml cases weighted by percent positive figure 1. limit of detection of furazolidone in liver tissues by delvotest sp. table 2. in vivo results for liver tissues in delvo test withdrawal % % % conc hours positive negative doubtful ( g/ml) 1 30 30 40 0.6 5 20 50 30 0.4 24 20 40 40 0.4 168 10 20 70 0.2 264 10 10 80 0.2 nb: the responses were determined from replicates of ten for each sample. post treatment and 10% at 264 h post treatment. the 30% positive response did correspond to a furazolidone concentration of 0.6 ug/ml in deep fried liver tissues at 1 h after treatment. the concentration of furazolidone in liver tissues declined steadily between 1h and 264 h post treatment. more doubtful samples appeared 5 h post treatment at 50%.the least concentration determined in liver was 0.2 g/ml 264 h post treatment (table 2). the results indicate the presence of residues 11 days post treatment. the results were also presented graphically in figure 1 the delvotest sp detected a maximum furazolidone concentration of 0.8 g/ml in deep fried muscle tissues at 1h after treatment. the concentration of furazolidone in muscle tissues declined rapidly between 1h and 24 h post treatment. afterwards the concentration remained relatively constant between 24 and 168 h post treatment. this was followed by a decline to 0.2 g/ml at 264 h post treatment (table 3) . like for liver tissues, the muscles also had residues 11 days post treatment. the depletion rate of the drug in liver and muscle tissues were compared using chi – square test which gave a p value of 0.227 (table 4). the findings suggest that table 3. in vivo results for muscle tissues in delvo test withdrawal % % % conc hours positive negative doubtful ( g/ml) 1 40 30 30 0.8 5 30 50 20 0.6 24 10 80 10 0.3 168 10 90 0 0.3 264 10 30 60 0.2 nb: the responses were determined from replicates of ten for each sample. table 4. chi – square test. value df asymp. sig. (2-sided) pearson chi-square 7.500 a 6 0.277 likelihood ratio 7.777 6 0.255 linear-by-linear association 3.104 1 0.078 no. of valid cases 5 a. 12 cells (100%) have expected count less than 5. the minimum expected count is 20. there is no significance (p < 0.05) difference between the depletion rates of the drug in liver and muscle tissues. discussion the delvotest sp system is a broad spectrum screening test for the detection of antibiotic residues and sulphonamides in milk (idf, 1991; kroll, 1999). the present research suggests that in addition to detecting antibiotics in milk, the delvotest sp system is could also be capable of detecting the furazolidone residues in fried chicken tissues. the in vitro studies revealed positive responses of the system to furazolidone residues greater than or equal to 11 g/ml in liver homogenates. however, the response of the delvotest sp system to liver homogenates was unpredictable between 2 and 10 g/ml, but concentrations below 2 g/ml were not detected. these levels of detection seem to be higher than those for another b. stearothermophilus based test, the two tube test which showed much lower detection levels in experiments carried out in the same laboratory (shitandi et al., 2006). in the in vivo experiment, the delvotest sp system detected a maximum furazolidone concentration of 0.6 ug/ml in deep fried liver tissues at 1 h after treatment. the concentration of furazolidone in deep fried liver tis-sues declined steadily between 1 h and 264 h post treat-ment. the least concentration determined in deep fried liver was 0.2 g/ml 264 h post treatment. the system detected a maximum furazolidone concentration of 0.8 g/ml in deep fried muscle tissues at 1 h after treatment. shitandi et. al. 265 the concentration of furazolidone in deep fried muscle tissues declined rapidly between 1h and 24 h post treatment. afterwards the concentration remained relatively constant between 24 and 168 h post treatment. this was followed by a decline to 0.2 g/ml at 264 h post treatment. the concentration detected after 1h post treatment in deep fried muscle tissue was higher than that detected in deep fried liver tissue. however, the depletion rates were not significantly different at 95% confidence interval. the delvotest sp system microbial assay contains a pre-defined number of b. stearothermophilus spores in agar wells which enables the test to be carried out in 2 h 30 min (at the time the negative control has been changed to yellow). the growth of the spores at 64°c initiates an acidification process which causes the turning of a ph indicator from purple to yellow. the presence of antibacterial substances will cause delay or inhibition of the spores, depending on the concentration of the residues. in the presence of residues the spores will not multiply and the ph indicator will remain purple (food safety authority of ireland, 2002). the bioavailability and effect of cooking on aoz was studied by mccracken and kennedy (1997). there were no significant effects on total concentration of aoz due to grilling, micro waving or frying. humans may be exposed to aoz if animals treated with furazolidone have no adequate withdrawal periods before slaughter. drug residues in food animals being raised for human consumption may pose a public health concern. in low income countries such as kenya, monitoring of drug residues is not done (shitandi and sternesjo, 2001) and hence the risk consumers of animal products are exposed to is not known. consumer protection can be ensured by screening such animals for residues (anadon and martinez, 1998; kozarova and mate, 2000). most food containing drug residues is consumed after cooking or processing, yet surveillance for these residues is almost always conducted on raw tissue (rose et al., 1997). according to the manufacturer’s instructions (cosmos, kenya limited), the animals to which the drug has been administered should be consumed after ten days (240 h), which is considered the safe withdrawal period. however it is apparent from the present research that even after eleven days (264 h), the drug still persists in the deep fried tissues. the fact that the delvotest system was able to detect furazolidone residues in deep fried liver and muscle tissue for longer periods of time may reflect the ability of these tissues to retain furazolidone or its metabolites for long periods. furazolidone is rapidly metabolized in vivo (cooper et al., 2005). however, protein bound metabolites are formed and are detectable several weeks after administration mccracken et al.,1997; okeeffe et al., 2004). the side chain residues (aoz) persist longer than intact parent compounds. furazolidone residues have been shown to accumulate in eggs from birds administered therapeutic dose of furazo furazolidone in their daily feed (mccracken et al., 2001). the side chain accounts for the long lasting residues detected in deep fried chicken tissues such as muscle and liver. the general public is becoming increasingly aware of food safety issues and the potential for chemical and microbiological hazards in foods. there is a possibility of health risk due to protein bound metabolites of drugs that are formed during metabolism as they are characterized by long half lifes (hoogenboom et al., 2002). considering the withdrawal time of ten days for furazolidone in chicken as prescribed by the manufacturer, residues can be expected to persist in treated animals even after cooking, indicating the consumers are not given adequate protection. from the present research, it was apparent that furazolidone drug residues in chicken liver and muscle tissues were not destroyed by deep frying. this study further demonstrated that the depletion rates in both tissues were not significantly different at 95% confidence interval. the depletion studies suggests that the withdrawal periods for furazolidone in liver and muscle tissues may need to be adjusted above the manufacturer’s recommended periods. it may be of future interest to also investigate the possible elution of the drug residues into oil that has been used to deep fry chicken liver and muscle tissues. acknowledgments we are grateful to the food science and technology department, egerton university for financial support through the internal research funds. we appreciate the animal science department for support and advice in rearing the chicken used in this project, in particular mr. maritim, mr. kibe and mr. amwayi. references ali bh (1999). pharmacological, therapeutic and toxicological properties of furazolidone: some recent research. vet. res. commun. 23: 343-360. anadon a, martinez-larranaga mr (1998). residues of antimicrobial drugs and feed additives in animals products: regulatory aspects. livestock prod. sci. 59: 183-198. bergwett aa (2005). rapid assays for detection of residues of veterinary drugs. in: rapid methods for biological and chemical contaminants in food and feed. wangeningen 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anticoccidial residues in poultry. j. vet. sci 2006. 7(2):177-180. african journal of food science research vol. 2 (9), pp. 129-138, september, 2014. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper control measures in nigerian traditional food manufacture: some useful properties of extruded millet-soybean for the ‘fura’ harshanti o. and abubakar bukar department of food technology, kaduna polytechnic, kaduna, kaduna state, nigeria. accepted 19 august, 2014 ‘fura’ is a traditional thick dough ball snack produced principally from millet or sorghum which is common in northern nigeria. it is consumed with ‘nono’ (local yoghurt produced from cow milk) or mashed in water before consumption in the form of porridge. the effects of extrusion conditions feed composition (ratio of soybean to millet), percentage moisture wet basis and screw speed (rpm) on the water absorption index (wai); water solubility index (wsi) and viscosity from millet/soybean flour mixtures were studied using response surface methodology (rsm) for ‘fura’ processing. models were developed and appropriate statistical analysis adopted to test the adequacy of the models. the models showed r 2 = 0.73, 0.76, 0.74 for wai, wsi and viscosity, respectively, indicating that the model could be used to navigate the design space. because some of the stationary points were outside the range of the experiment, graphical optimization was adopted to determine the optimum and predicted values. the optimum levels and the corresponding predicted values were obtained. the optimal combination of feed composition (17.7% soybean), feed moisture (27.3% wet basis) and screw speed (161 rpm) resulted in optimal wai of 4.6 gwater per gsample . the optimal combination of feed composition (14.77% soybean), feed moisture (15.23%) and screw speed 220 rpm) resulted in optimal wsi of 6.15%. the optimal combinations of feed composition (46.97%), feed moisture (29.34%) and screw speed (174.52rpm) resulted in optimal viscosity of 4.58 nsm -2 . key words: millet, soybean, extrusion, optimization, ‘fura’, water absorption index, water solubility index, viscosity. introduction pearl millet (pennisetum glaucum (l.) r.br.) is grown extensively in the dry areas of western and southern india and along the west african sub region including nigeria, where it is used as food for an estimated 400 million people (hoseney et al., 1992). more than 80% of the production is used for human consumption, parti-cularly in the semiarid tropic region of africa and asia. ‘fura’ is one of the several indigenous food preparations made from pearl millet in west africa particularly in nigeria. it is a thick dough ball snack produced principally from millet or sorghum. the mode of preparation varies *corresponding author.e-mail: h_shanti@yahoo.co.uk. only slightly among different communities in the region, but the basic ingredient remains the same (that is, millet or sorghum). depending on the com-munity, it is traditionally consumed with ‘nono’ (local yog-hurt produced from cow milk) or mashed in water before consumption in the form of porridge. however, the production method lacks process specifications governing functionality, composition, ingredients, additives and shelf life. processing has remained a home-based or artisanal activity that is carried out with rudimentary equipment and techniques, which is characterized by inconsistent product quality, poor hygiene, very short shelf life and unacceptable standards. fura has a limited storage life with a range of 3-4 days at refrigeration storage (5 o c), 1-2 days at room temperature (25 o c) and 18 h at 35 o c (jideani et al., http://www.internationalscholarsjournals.org/ harshanti and abubakar 129 2002); being a single cereal based product it is limiting in the essential amino acid lysine. the inclusion of soybean as a basic ingredient in producing ‘fura’ through extrusion can improve its protein content and functionality. interest in soybean foods has increased with consumer awareness of its health benefits, especially with soybean related ingredients being utilized as one of the major sources of high-protein fortification (united soybean board, 2006; yeu et al., 2008). extrusion cooking has some unique features compared to other heat processes. it is capable of breaking covalent bonds in biopolymers and facilitating reactions otherwise limited by diffusion of reactants and products (iwe et al., 2001). extrusion alters the nature of many food constituents, including starches and proteins, by changing their physical, chemical and nutritional properties. high temperature short time (htst) extrusion cooking technology has limitless applications in processing of cereal based products. extrusion cooking is a popular means of preparing snacks and ready to eat foods. extrudates are microbiologically safe, can be stored for long periods because of low moisture without need for refrigeration and requires less labour for handling and less packaging materials and storage space (filli and nkama, 2007). extrusion technology could be applied for ‘fura’ processing for the purpose of improving its shelf life and value addition to the product by making it a convenient food item (nkama and filli, 2006; filli and nkama, 2007). the water absorption index (wai) measures the volume occupied by the extrudate starch after swelling in excess water, which maintains the integrity of starch in aqueous dispersion (qing-bo et al., 2005). the water solubility index (wsi) describes the rate and extent to which the component of powder material or particles dissolves in water. it depends mainly on the chemical composition of the powder and its physical state. the wsi often is used as an indicator of degradation of molecular components (kirby et al., 1988), measures the degree of starch conversion during extrusion which is the amount of soluble polysaccharide released from the starch component after extrusion. binoy et al. (1996) reported that water solubility index increased with severity of screw configuration. pelembe et al. (2002) reported increased nitrogen solubility index in extruded sorghum-cowpea porridge. among functional properties, water holding capacity is important because of the hydro-gen bonds formed between water and polar residues of protein molecules. an increase in shear energy inputs during extrusion can cause deploy-merization and degradation of component. high shear can also cause fragmentation of products from both starch and proteins; the degradation products are small molecules and gene-rally water soluble. water is absorbed and bound to the starch molecule with resulting change in the starch granule structure (binoy et al., 1996). the viscosity of a fluid reflects its resistance to flow and influence acceptability of liquid foods. viscosity depends on solubility and water holding capacity as well as the structure of components in food. viscosity profile can be thought of as a reflection of the granular changes in the starch granule that occur during gelatinization, (thomas and atwell, 1997) . extrusion can induce starch dextrinzation which can result in the reduction of viscosity in gruels and a concomitant increase in caloric and nutrient density (jansen et al., 1981). in africa, due to deforestation by utilisation of wood for fuel, there is a great need for pre–cooked foods. high temperature, short-time (htst) extrusion cooking could be used to produce such foods of high nutritional quality and ready to eat products, (pelembe et al., 2002). despite incre-ased use of extrusion processing, extrusion is still a complicated process that has yet to be mastered. small variations in processing contions affect processs variables as well as product quality (qing-bo et al., 2005). the objectives of this work was to study the effects of feed composition, feed moisture and screw speed on the water absorption index, water solubility index and viscosity of extruded ‘fura’ from pearl millet and soybean flour mixtures using response surface methodology. materials and methods flour preparation from millet traditional method of flour preparation was used in this study. the process consists of dry cleaning of millet, that is, winnowing using an aspirator. the kernels were thereafter dehulled after mild wetting of the grain using a rice dehuller, india at the jimeta main market, yola, nigeria. after dehulling, the grains was washed and then dried in a convection oven at 50°c for 24 h to 14% moisture content. the dried grain was milled using the roller mill equipped with a 150 m screen. flour preparation from soybean the soybean seeds were steeped in tap water at 28°c for a period of 24 h in a plastic bowl. the kernels were thereafter dehulled using traditional pestle and mortar. the dehulled mass was winnowed using an aspirator. the dehulled soybean kernels were ground in a laboratory disc mill to fine flour. the flour was sieved using a150 m screen, and the underflow was used for further research work. flour preparation from soybean the soybean seeds were steeped in tap water at 28°c for a period of 24 h in a plastic bowl. the kernels were thereafter dehulled using traditional pestle and mortar. the dehulled mass was winnowed using an aspirator. this was ground in a laboratory disc mill to fine flour. the flour was sieved to pass through a 150 m screen size. spice preparations kimba (negro pepper) and ginger were sorted and cleaned manually before being kept in a convection oven at 60°c for five 130 afr. j. food sci. res. table 1. independent variables and levels used for central composite rotatable design. variable symbols coded variable level -1.68() -1 0 1 1.68() feed composition (%) x1 3.2 10 20 30 36.8 feed moisture (%) x2 16.6 20 25 30 33.4 screw speed (rpm) x3 116 150 200 250 284 table 2. experimental design extrusion experiment in their coded form and natural units. design point independent variables in coded form experimental variables in their natural units (x1) (x2) (x3) (x1) (x2) (x3) 1 -1 -1 -1 10 20 150 2 -1 +1 -1 10 30 150 3 -1 -1 +1 10 20 250 4 -1 +1 +1 10 30 250 5 +1 -1 -1 30 20 150 6 +1 +1 -1 30 30 150 7 +1 -1 +1 30 20 250 8 +1 +1 +1 30 30 250 9 -1.68 0 0 3.2 25 200 10 +1.68 0 0 36.8 25 200 11 0 -1.68 0 20 16.6 200 12 0 +1.68 0 20 33.4 200 13 0 0 -1.68 20 25 116 14 0 0 +1.68 20 25 284 15 0 0 0 20 25 200 duplicate tests at all design point except the centre point (0, 0, 0) which was carried out five times. experiment was carried out in randomized order. (x1) =feed composition (x2) = feed moisture and (x3) = screw speed. hours. the seeds were then pounded using the traditional pestle and mortar. the mass was ground and sieved using a 150 m screen size. blend preparations and moisture adjustment millet flour (mf) and soybean flour (sf) were mixed at various weight ratios, and the total moisture contents of the blends adjusted to the desired values with a mixer as described by zasypkin and tung-ching (1998). weights of the components to be mixed were calculated using the following formula zasypkin and tung-ching lee (1998): csf = [rsf × m × (100 w)] / [100 × (100 wsf)] cmf = [rmf × m × (100 w)] / [100 × (100 wmf)] wx = m csf cmf where csf or cmf are the masses of either soybean flours (sf) or millet flour (mf), respectively, r sf or r mf are respective percentages of either soybean flours (sf) or millet flour (mf) in the blend, d.b. (rsf + rmf = 100%); m is the total mass of the blend; w, the moisture content of the final blend, percentage wet weight basis (w.w.b.); w x is the weight of water added; and wsf and wmf are the moisture contents of sf and mf, respectively. the blends were mixed in a plastic bowl and packed in polyethylene bags and kept in the refrigerator overnight to allow moisture equilibration. the samples were however, brought to room temperature before extrusion process. experimental design (central composite rotatable designs) the response surface methodology (rsm) is a widely adopted tool for the quality of optimizations processes (myers and montgomery, 1995). the rsm, origially described by box and wilson (1951) is effective for responses that affect many factors and their interactions. the central composite rotatable composite design (ccrd), (box and hunter, 1957) was adopted to predict responses based on few sets of experimental data in which all factors were varied within a chosen range. a three factors and three level experimental design was adopted for this work (table 1) . the independent variables considerd were feed composition x1 (%), feed moisture content x2 (%) and screw speed x3 (rpm) . the independent variables and their variation levels are shown in table 1. the levels of each variables were established according to literature information and preliminary trials. the outline of the experimental layout with the coded and natural values is presented in table 2. extrusion exercise extrusion cooking was performed in a single screw extruder, model harshanti and abubakar 131 (brabender duisburg dce-330), germany equipped with a variable speed d-c drive unit, and strain gauge type torque meter. the screw has a linearly tapered rod and 20 equidistantly positioned flights. the extruder was fed manually through a screw operated conical hopper at a speed of 30 rpm which ensures the flights of the screw filled and avoiding accumulation of the material in the hopper. this type of feeding provides the close to maximal flow rate for the selected process parameters (constant temperature, constant die and screw geometry but with three variable screw speeds) and three designed feed composition and feed moisture contents. a round channel die with separate infolding heater was used. the die used was a cone shaped channel with 45 degrees entrance angle, a 3 mm diameter opening and 90 mm length. the screw was a 3:1 compression ratio. the inner barrel is provided with a grooved surface to ensure zero slip at the wall. the barrel is divided into two independent electrically heated zones (that is feed end and central zone) . there is a third zone at the die barrel, electrically heated but not air cooled. the extruder barrel has a 20 mm diameter with lenght to diameter ratio (l:d) of 20:1. the extruder consists of two electrically heated zones. desired barrel temperature was maintained by a circulating tap water controlled by inbuilt thermostat and a temperature control unit. the feed material was fed into a hopper mounted vertically above the end of the extruder which is equipped with a screw rotated at variable speed. the rotating hopper screw kept feed zone completely filled to achieve a 'choke fed' condition. experimental samples were collected when steady state was achieved, that is, when the torque variation of plus or minus 0.28 joules (nm) or about (0.5%) of full scale (likmani et al.,1991). the extrusion process consisted of 15 individual runs. water absorption index (wai) and water solubility index (wsi) the wai and wsi were determined using the method described by qing-bo et al. (2005). the ground extrudate was suspended in water at temperature 30°c for 30 min; it was stirred gently during this period and centrifuged at 3000 × g for 15 min. the supernatant was decanted into an evaporating dish of known weight. the wsi was considered as the weight of dry solids in the supernatant expressed as a percentage of the original weight of sample. the wai was considered as the weight of gel obtained after removal of the supernatant through a strainer (pore size = 500 µm) per unit weight of original dry solids (gh20/1g sample). determinations were made in triplicate and the average taken. viscosity viscosity was determined with the aid of rotational viscometer model (rheotest 2 type) made in hungary, equipped with concentric cylinders. the system has provision for tempering vessel, that is, connecting a liquid circulation thermostat to the correct temperature was ensured. viscosity measurement was carried out at 30°c. triplicate determination was carried out and the result averaged. statistical analysis homogeneous variances or homoscedasticity is a necessary prerequisite for (linear) regression models. therefore, a reduction in variability within the objective response (dependent variables) was by transforming the data to standardized scores ( z  x  x s where x = dependent variable of interest; x = mean of dependent variable of interest and s = standard deviation). for each standardized scores, analysis of variance (anova) was conducted to determine significant differences among the treatment combinations. also, data were analyzed using multiple regression procedures (matlab 1984-2000,). to estimate feed composition, feed moisture and screw speed effects each objective response, the standardized scores were fitted to a quadratic polynomial regression model by employing a least square technique (gacula and singh, 1984; wanasundara and shahidi, 1996). the model proposed for each response of y was: y  b0  b1 x1  b2 x 2  b3 x 3  b11 x 2 1b22 x 2 2  b33 x 3 2  b12 x 1 x 2  b13 x1 x 3  b23 x 2 x 3 (1) where y = the response, x1 = feed composition, x2 = feed moisture, x3 = screw speed, b0 = intercepts, b1, b2, b3 are linear, b11, b22, b33, = are quadratic and b12, b13 and b23 are interaction regression coefficient terms, respectively. coefficients of determination (r 2 ) were computed. the adequacy of the models was tested by separating the residual sum of squares into pure error and lack of fit. for each response, response surface plots were produced from the equations, by holding the variable with the least effect on the response equal to a constant value, and changing the other two variables. results and discussion description of model the model did not show a significant lack of fit (p = 0.0915), with r 2 = 0.73, 0.76, and 0.74 for wai, wsi and vcscity, respectively. the probability of the f value for the model = 0.05 which can explain the reason for variation. filmore et al. (1976) reported that r 2 value less than 0.8 makes the model unsuitable for any further analysis. however, yagci and gogus (2008) reported that with significant probability values (p< 0.001) and nonsignificant lack of fit, the models could be adequately be used as predictor models, regardless of low coefficient of determinants. if a model has a significant lack of fit, it is not a good indicator of the response and should not be used for prediction (myers and montgomery, 2002). we may probably conclude that the proposed models approximates the response surfaces and can be used suitably for prediction at any values of the parameters within experimental range. water absorption index (wai) and water solubility index (wsi) the effects of treatment variables as linear, quadratic or interaction coefficients are detailed in table 3. the design point 1 (10% feed composition, 20% feed mois-ture and 150 rpm screw speed) recorded the maximum wai value of 5.62 gh2o/g sample. the lowest value of 4.26 gh2o/g was however, recorded for design point 10 (36.8% feed composition, 25% feed moisture and 200 rpm screw speed). the model did not show a significant lack of fit (p = 0.0915) with r 2 = 0.73, and the 132 afr. j. food sci. res. table 3. effect of extrusion variables on wai, wsi and viscosity of samples. coefficient wai wsi vscsty experimental runs wai wsi vcscity x1 x2 x3 obsd. prdt. obsd. prdt. obsd. prdt. linear (10 : 20 : 150) 5.62 5.73 6.42 6.44 7.44 7.12 b0 -0.3780 -0.1177 -0.4230 (10 : 30 : 150) 4.77 4.73 5.84 5.84 7.78 8.23 b1 -0.5860* -0.3781 -0.7553* (10 : 20 : 250) 5.47 5.23 6.26 6.34 5.70 5.66 b2 -0.4960* -0.5936* 0.3928 (10 : 30 : 250) 5.22 5.43 5.93 6.12 8.34 8.23 b3 -0.2210 -0.3138 -0.2150 (30 : 20 : 150) 5.34 5.21 6.45 6.34 5.99 6.34 quadratic (30 : 30 :150) 4.69 4.76 5.62 5.54 5.33 5.45 b11 -0.0353 -0.2303 0.1738 (30 : 20 : 250) 4.49 4.48 5.74 5.46 4.34 4.23 b22 0.1588 -0.1224 0.2160 (30 : 30 : 250) 4.44 4.42 5.16 5.17 5.98 6.12 b33 0.4306* 0.5253* 0.2301 (3.2: 25 : 200) 5.16 5.18 5.84 6.20 7.80 7.65 (36.8:25:200) 4.26 4.21 5.21 4.89 4.60 4.45 0.1096 -0.1269 -0.1984 (20:16.6:200) 5.34 5.65 6.16 6.26 5.52 5.55 -0.3836 -0.2793 0.0198 (20:33.4:200) 4.58 4.77 5.18 5.43 7.18 7.34 b23 0.3288 0.1269 0.4564 (20 : 25: 116) 5.48 5.48 6.83 6.45 6.85 6.77 r 2 0.7274 0.7592 0.7406 (20 : 25 : 284) 5.14 5.23 6.31 6.12 5.94 6.23 adjusted r 2 0.4821 0.5425 0.5071 (20 :25: 200) 4.75 4.80 5.84 5.66 5.58 6.34 lack of fit ns ns * model * * * interaction b12 b13 a y  b0  b1 x1  b2 x 2  b3 x 3  b11 x 2 1b22 x 2 2  b33 x 3 2  b12 x1 x 2  b13 x1 x 3  b23 x 2 x 3 , x1 = feed composition, x2 = feed moisture, x3 = screw speed b *, ** significant at p < 0.05 and p < 0.01, respectively; ns, not significant. probability of the f value for the model = 0.05 which can explain the reason for variation. the r 2 value measures the total variation about the mean response as explained by the regression. since the fitted second-order model provides a good fit, it was used to search for optimum levels of feed composition, feed moisture and screw speed. the effects of treatment variables as linear, quadratic or interaction coefficients are detailed in table 3. the correlation coefficient of r = 0.85 was significant at (p 0.05); therefore, the model could be used to navigate the design space. hence, the second-order model was judged to be adequate at 0.05%. the effect of extrusion condition on wai was influenced by linear and quadratic terms significantly (p < 0.05) table 3. the interaction term did not show significant (p > 0.05) influence. high water absorption capacity is required to produce ‘fura’ with acceptable flexibility of consistency. the maximum wsi value of 6.83% was observed for design point 13 (20% feed composition, 25% and 116 rpm screw speed), while the lowest observed value of 5.18% was observed for sample 12 representing 20% feed composition, 33.4% and 200 rpm screw speed (table 3). the wsi was equally influenced significantly by linear and quadratic terms, (p < 0.05), the model did not show a significant lack of fit and recording the coefficient of determinant of r 2 = 0.76 wsi. wsi is often used as an indicator of degradation of molecular components (yang et al., 2008), which measures the degree of starch conversion during extrusion which is the amount of soluble polysaccharide released from the starch component after extrusion process. wai has been generally attributed to the dispersion of starch in excess water, and the dispersion is increased by the degree of starch damage due to gelatinization and extrusion–induced fragmentation, that is, molecular weight reduction of amylose and amylopectin molecules (yagci and gogus, 2008). the relationship between depended and independent variables were shown in three dimensional presentation, the response indicated that, increasing feed moisture significantly decreased the wai of extrudate; increasing the level of soybean flour tends to decrease the wai figure 1. this is expected because of oil in soybean which interfered with water uptake. singh et al. (2007) observed a decrease in wai with addition of pea grits in extrusion of rice. they explained that a decrease in wai was due to the dilution of starch in rice pea blends. the wai measures the volume occupied by the extrudate starch after swelling in excess water, which maintains the integrity of starch in aqueous dispersion (qingbo et al., 2005). mercier and feillet (1975) observed that higher amylose results in a higher wai. colonna et al. (1989) indicated that wai decreases with the onset of dextrinization. pelembe et al. (2002), reported increased wai as the percentage of cowpea increased in sorghum harshanti and abubakar 133 w a i (% ) 7.0 6.0 5.0 4.0 3.0 -2 -1 0 1 -2 ) 1 n(% io t i 2 s o p m o c d e e f figure 1. effect of feed moisture and feed composition on wai. wsi (%) 8.0 7.0 6.0 5.0 -42.0 0 2 1 0 2 -1 -2 figure 2. effect of screw speed and feed moisture on wsi. extrudates. cowpea proteins have relatively higher water solubility than sorghum proteins (chavan and kadam 1989b). the 3d plot for the wsi indicates that increasing screw speed appears to increase wsi while increasing the feed moisture decreased wsi (figure 2) significantly. altan et al. (2008) reported increase in wsi with increase in screw during the extrusion of barley–tomato blends. the increase in wsi with increasing screw speed was 348 afr. j. food sci consistent with the results reported for corn meal and corn and wheat extrudates (jin et al., 1995; mezreb et al., 2003). mezreb et al. (2003) reported that the increase of screw speed induced a sharp increase of specific mechanical energy, the high mechanical shear degraded the macromolecules, and so the molecular weight of starch granules decreased and hence increased wsi. it could be expected that wsi would decrease since ) n(% io t i s o p m o c d e e f figure 3. effect of feed moisture and feed composition on viscosity. extrudates of high legume content contain more starch aggregates or microgels which will be suspended in water (gomez and aguilera, 1983) . this suggests that water solubility index (wsi) is not only due to starch contents but also due to water-soluble components, like proteins which are present in soybean. mercier and feillet (1975) reported increase in soluble starch with increasing extrusion temperature and decreasing feed moisture. wsi often is used as an indicator of degradation of molecular components (kirby et al., 1988). gelatinization, the conversion of raw starch to a cooked and digestible material by the application of water and heat, is one of the important effects that extrusion cooking has on the starch component of foods (qing-bo et al., 2005). water is absorbed and bound to the starch molecule with resulting change in the starch granule structure. it has been established (pomeranz, 1991; wolf and conan, 1971) that proteins are the most reactant components in foods and some of their reactions are essential for functionality. among functional properties, water holding capacity is important because of the hydro-gen bonds formed between water and polar residues of protein molecules. viscosity the viscosity of a paste depends on to a large extent on the degree of gelatinization of the starch granules and the rate of molecular breakdown. the maximum viscosity value of 8.34 nsm -2 was observed for sample 4 (10% feed composition, 30% feed moisture and 250 screw speed), while the least value of 4.34 nsm -2 was recorded for design point 7 representing 30% feed composition, 20% feed moisture and 250 rpm screw speed table 3. the viscosity of samples was influenced by linear terms significantly (p < 0.05). the coefficient of determinant for the viscosity was r 2 = 0.74. increase in the amount of soybean flour and increase in feed moisture decreased the apparent viscosity of extrudate (figure 3). in addition to the effect of extrusion, the reduction in viscosity may be attributed to the high level of oil from the soybean flour which consequently decreased the shear effect as a result of lubrication in the metering zone. increase in moisture on the other hand, will further lubricate the dough leading to less shearing effect. low moisture in the feed can possibly increase frictional damage, particularly when the residence time is high due to low screw speed. viscosity generally depends on solubility and water holding capacity as well as the structure of components in a food system. viscosity profile can be thought of as a reflection of the granular changes in the starch granule that occur during gelatinization (thomas and atwell, 1997). extrusion can induce starch dextrinization resulting in reduction of viscosity in gruels and a concomitant increase in caloric and nutrient density (jansen et al., 1981). arambular et al. (1998) reported decreased apparent viscosity of extruded instant corn flour when temperature was increased. davidson et al. (1984) reported that viscosity over a heating and cooling cycle have been used to characterize the changes in extruded products in numerous studies. this characteristic is affected by both physical modifications of the granule structure as well as changes to the structures of the starch polymers. they further reported that, the characteristics of the paste viscosity curves were significantly altered by extrusion processing with extrudates showing low values. pelembe et al. (2002) reported that, reduced viscosity of protein – rich sorghum – cowpea extrudate could be very beneficial for infant feeding. the high bulk (low nutrient density) of cereal weaning porridges is a major cause of infant malnutrition in africa, since it harshanti and abubakar 135 table 4. optimum levels of independent variables for wai; wsi and viscosity. independent feed feed screw *predicted nature of stationary variable/responses composition (%) moisture (%) speed (rpm) value point *wai gwater per g sample 17.67 27.27 160.8 4.55 saddle *wsi (%) 14.77 15.23 219.8 6.15 saddle *viscosity(nsm -2 ) 46.973 29.34 174.52. 4.58 saddle limits nutrient intake (da et al., 1982). hagenimana et al. (2006), reported that viscosity values of extruded rice flours were far less than those of their corresponding unprocessed rice flour dispersed in the micro visco amylo graph (mvag), indicating that their starches have been partially pregelitinized by extrusion process. they reported that peak viscosity indicated a high positive correlation with hot paste viscosity and cold paste viscosity with r > 0.70 (p < 0.01). ozcan and jackson (2005) reported that during extrusion cooking of corn starches, extruded starch had higher water absorption and water solubility indices, and they had lower rapid viscoamylograph viscosity profiles when compared with raw starch. this can be attributed to the fact that degradation of the starch occurred during extrusion. it is suggested that starch degradation in the extruded product is a likely significant factor associated low viscosity profiles. the mixtures of raw and extruded starches have potential applications in the industry for functional properties. arambular et al. (1998) reported decreased apparent viscosity of extruded instant corn flour when temperature was increased; though the temperature used in this study was not varied, it was however in the high regime. likimani et al. (1991) indicated that the degradation of molecular bonding of starch during extrusion influenced the characteristics of the extruded product and was used to characterize the target parameters (solubility and viscosity). the high bulk (low nutrient density) of cereal weaning porridges is a major cause of infant malnutrition in africa, since it limits nutrient intake (da et al., 1982). increased screw speed resulted in an increase in input energy which caused stretching and sometimes fracture of protein-protein matrix, thus, making product less viscous when reconstituted, the screw speed from this study influenced viscosity. linkimani et al. (1991) reported that extrusion induced starch dextrinization which resulted in reduction of viscosity in gruels and a concomitant increase in caloric and nutrient density. adeyemi and beckley (1986), reported that a high level of damaged starch would reduce peak viscosity of flour or ‘ogi’. starch dextrinization during extrusion cooking, however, occurred mostly under processing conditions at very high temperature and low moisture (gomez and aguilera, 1983) where shear effects were significant. general increase in water absorption of sorghum extrudates was reported by gomez et al. (1988). extrudate photographic responses the effects of extrusion variables (feed composition, feed moisture and screw speed) can be seen as shown in plates 1-15. the effect of the independent variables on the expansion ratio of extrudates is evident. the results shown in the photographs describes the changes occurred during extrusion as influenced by the extrusion variables. dependent variables and their predictive models experimental values were obtained for individual responses y for the design points. multiple regression coefficients were obtained by employing a least squares technique to predict quadratic polynomial models for the responses y. the quadratic regression model for the influenced variables are presented in the equation, where x1, x2, and x3 are the coded values for feed composition, feed moisture and screw speed levels, respectively. the coefficients with one factor represent the effect of the particular factor, while the coefficients with two factors and those with second order terms represent the interaction between the two factors and quadratic effect, respectively. the positive sign in front of the terms indicates synergistic effect, while negative sign indicates antagonistic effect. wai = -0.3780-0.5860x1-0.4960x2-0.2210x3-0.0353x1 2 + 0.1588x2 2 +0.4306x3 2 +0.1096x1x2-0.3836x1x3 + 0.3288x2x3 wsi = -0.1177 0.3781x1 0.5936x2 0.3138x3 0.2303x1 2 0.1224x2 2 +0.5253x3 2 -0.1269x1x2-0.2793x1x3 + 0.1269x2x3 vscsity = -0.4230 0.7553x1 + 0.3928x2 0.2150x3 + 0.1738x1 2 + 0.2160x2 2 + 0.2301x3 2 0.1984x1x2 + 0.0198x1x3 + 0.4564x2x3 eqws=0.8209+0.5413x1 0.0510x2 + 0.3823x3 0.0549x1 2 0.4342x2 2 -0.7142x3 2 +0.2001x1x20.0150x1x3 0.0650x2x3 optimum conditions the optimum levels of independent variables and the corresponding predicted values of responses are shown in table 4. the optimal combination of feed composition (17.7%), feed moisture (27.3%) and screw speed (161 136 afr. j. food sci. res. plate 1 15. shows the physical state of extrudate responses.( 1) 10% soybean, 20% feed moisture, 150rpm screw speed; (2) 10% soybean, 30%moisture, 150rpm screw speed; (3) 10% soybean, 20% feed moisture, 250 rpm screw speed; (4) 10% soybean,30% feed moisture, 250rpm screw speed; (5) 30% soybean, 20% feed moisture, 150 rpm screw speed; (6) 30% soybean, 30% feed moisture, 150rpm screw speed; (7)30% soybean, 20% feed moisture, 250 rpm screw speed; (8) 30% soybean, 30% feed moisture, 250 rpm screw speed; (9) 3.2% soybean, 25% feed moisture, 200 rpm screw speed;(10) 36.8% soybean, 25% feed moisture, 200rpm screw speed; (11) 20% soybean, 16.6% feed moisture, 200 rpm screw speed; (12)20% soybean, 33.4%moisture, 200rpm screw speed; (13)20% soybean, 25% feed moisture, 116 rpm screw speed; (14) 20% soybean, 25%, feed moisture, 284 rpm screw speed; (15) 20% soybean, and 25% feed moisture, 200rpm screw speed. rpm) resulted in optimal wai of 4.6 gwater per gsample. all the extrusion variables were located within the range of experimental values of the independent variables; hence, the fitted response equation was adequate for depicting responses near the stationary point. the optimal combination of feed composition (14.77% soybean), feed moisture (15.23%) and screw speed (220 rpm) resulted in optimal wsi of 6.15%. the optimal combination of feed composition (46.97%), feed moisture (29.34%) and low screw speed (174.52) resulted in optimal viscosity of 4.58 nsm -2 . conclusion extruded ‘fura’ was produced from pearl millet and soybean blends using a single screw extruder through designed experiments using rsm. the decision to include soybean flour and to adopt extrusion cooking was motivated by the need to improve its protein content and quality, physical state, functionality, safety, shelf-life of the end product. the (rsm) was effective in explaining the effects of process variables (feed composition, feed moisture and screw speed) during the optimization of harshanti and abubakar 137 water absorption index, water solubility index and the viscosity of ‘fura’ from millet/soybean flour mixtures by extrusion cooking. results indicated that the variables were significant on target parameters. the importance of process variables on target parameters could be ranked in the following order: feed composition(x1) > feed moisture (x2) > screw speed (x3). response variables predicted with model equations under optimum conditions were in general agreement with experimental data. the results from this study could be projected to explore the possibility by interested processors for effective prediction of a known process conditions for the purpose of achieving desired product quality. extrusion cooking of ‘fura’ constituted a great improvement on the traditional product that is usually at high moisture content of between 60 75% and readily deteriorates on storage at room temperature. extrudates obtained in this study had moisture content less than 7 g/100g and would not require refigeration for storage. acknowledgement 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food sci., 63(6): 10581061. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 3 (8), pp. 203-207, august, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper physico-chemical changes and diastatic activity associated with germinating paddy rice g. s. ayernor 1 and *f. c. k. ocloo 2 1 department of nutrition of food science, university of ghana, legon, ghana. 2 biotechnology and nuclear agriculture research institute, ghana atomic energy commission, p. o. box lg 80, legon, ghana. accepted 07 july, 2015 paddy rice was germinated for 0, 5, 7 and 9 days period at 32°c. physical and chemical characteristics, as well as diastatic activity were determined using standard methods. drastic decreases in thousand-grain weight, starch content, and viscosity of malt paste with significant increases in sugar content and diastatic activity were observed during germination (p<0.05). the 9-day malted rice had a diastatic activity of 94.5-degree lintner, with shorter starch conversion time. the paddy rice studied was therefore suitable for germination and use as an alternate source of enzymes for starch hydrolysis. key words: physico-chemical, diastatic activity, germination, malted rice. introduction malting of cereals other than hulled barley has been in research domain in recent years (dewar et al., 1997; suhasini and malleshi, 1995; hammond and ayernor, 2001). the reasons being economic considerations and local availabilities. in ghana, various cereals such as maize, sorghum, rice and millet have been malted for use as sources of enzymes in sugar and brewing industries. research has shown that malted rice has the potential for achieving high starch conversion rate in sugar production (hammond and ayernor, 2000) . malting is a process involving germination and drying of cereal seeds, the pri-me objective being to promote the development of hydro-lytic enzymes that are not active in raw seeds (dewar et al., 1997). during malting, the seeds undergo various changes of modification such as increase in the quanti-ties of and amylases present in the grain and partial degradation (hydrolysis, catalysed by enzymes) of reser-ve substances (cell wall, gums, protein, starch) in the starchy endosperm (dewar et al., 1997). malted rice is, however less commonly used in brewing than the ordinary raw rice. whereas barley, wheat and rye produce relatively large amounts of both and amylases during malting, rice essentially produces amylase during this process (egwim and oloyede, 2006). since the combination of both amylolytic enzymes results *corresponding author. e-mail: ocloofid@hotmail.com in a more rapid and complete degradation of starch to fermentable sugars, malts from other cereals are preferred materials for most users. the important role of amy-lase in rice seeds stays in the hydrolytic breakdown of re-serve starch in endosperm tissues during germination. since barley, wheat and rye are not usually grown in ghana, there is a need to explore other locally grown cereals especially rice for the production of malt for local sugar and brewing industries. however the quality of malt for use is greatly influenced by the physical, chemical and enzymes activities within the seeds and those that are produced during germination. the purpose of this contribution is to investigate physicochemical changes and diastatic activity associated with germinating of local paddy rice as a source of enzy-mes in starch conversion and brewing industries. materials and methods paddy rice (psb. rc. 34) was purchased from the irrigation development authority (ida) at ashaiman near accra, ghana. the seeds were stored dry at 4°c prior to analysis. germination process viable seed were used for the germination process. about 300 g of paddy rice was cleaned, washed thoroughly in water and soaked in a volume of water 3 times the weight of seeds for 24 h. the soaked seeds were placed on a jute sack in a basket and kept under ambient temperature (25 – 35°c) and watered 2 – 3 times a day to en ayernor and ocloo 204 table 1. properties of paddy rice and malt. property paddy rice (0 day malting) 5-days malting 7-days malting 9-days malting lsd0.05 moisture (%) 11.92 ± 0.14 9.06 ± 0.16 13.51 ± 0.10 12.51 ± 0.06 reducing (%) 3.26 ± 0.01 a 6.72 ± 0.06 b 12.20 ± 0.08 c 12.44 ± 0.00 d 0.025 non-reducing (%) 0.30 ± 0.05 a 1.24 ± 0.01 b 3.56 ± 0.01 c 3.92 ± 0.06 d 0.03 diastatic power (°l) 0.00 ± 0.00 a 52.20 ± 0.00 b 72.00 ± 1.27 c 94.50 ± 1.27 d 2.33 values are averages of triplicate ± standard deviation on dry matter basis. a-d = values with different letters in the row are significantly different at p<0.05 table 2. correlation between starch content and some malt characteristics of paddy rice. malt characteristics starch content thousand-grain weight + 0.99* viscosity at 75°c + 0.98* viscosity at 36°c + 0.97* diastatic power of malt 0.99* significant at p< 0.05. f.c.k. ocloo. hance germination process. samples were taken for days 0, 5, 7 and 9 for analysis. sample preparation for analysis samples taken for days 0, 5, 7 and 9 were solar dried (38°c) for about 20 – 25 h. the dried malt samples were milled using a disc attrition mill (straub model 4e grinding mill, straub co phila, usa). the milled rice malt samples were packaged and stored at cold room temperature (4°c). physical and chemical analysis samples were analyzed for moisture using aoac method (aoacn°925.10, 1990) . starch, reducing and non-reducing sugars were determined using aoac (aoacn°923.09 and 930.45, 1990) methods. starch conversion time was determined using the method described by cecil (1995). a drop of mash prepared from the samples was stirred with iodine solution in test tubes and time taken for the blue-black colour to disappear recorded. weighing the rice seeds before and after malting. the weights of 50 grains were recorded before malting, after malting and drying the plumule and the radicle (roots) were removed by hand and weight of malted seeds recorded. malting losses were expressed as percentage on dry matter basis. viscosity determination the viscosity of the pastes of rice malts was determined using brookfield viscometer (model dv + iv viscometer version 4.1, brookfield engineering labs inc., stoughton, usa). ten grams of sample was dissolved in 500 ml distilled water (2% slurry). the suspension was heated on a water bath with constant stirring for 30 min at a temperature of 90°c. the viscosity of the resulting gelatinized material was determined at 75 and 36°c at 50 rpm using spindle number 1. determination of diastatic activity the diastatic activity was determined using association of america cereal chemists method 22-15 (aacc, 2003). statistical analysis anova and regression analyses were performed on the data using statgraphics computer software (statistical graphics corp., stst inc; usa) with probability, p < 0.05. significant means were separated using least significant difference (lsd0.05). results moisture thousand-grain weight determination hundred (100) grains of paddy rice malted at 0, 5, 7 and 9 days rice were randomly selected from the bulk and weighed using balance. each weight was multiplied by 10 to obtain the 1000 kernel weight (esiape, 1994). determinations were done in triplicate. malt yields and malting loss malt yield after each germination was determined by weighing the seeds with plumule after malting. the weights of 50 grains were recorded before malting, after malting and drying the radicle (roots) were removed by hand and the yield of the malt determined by weighing. malt yields were expressed as percentage on dry matter basis. malting loss after each germination period was determined by table 1 shows chemical and physical properties of paddy rice and malt. moisture content varied from 9.06 to 13.51 %. however, these moisture contents depended upon the duration of drying and the temperature. malt yields and malting loss the yield and loss of malted paddy rice grains are presented in figure 1. there were marked differences in the malting loss and malt yield of the grains during germination. malting loss was highest in 9-day malt (about 59.75%) . the malting loss for 5-day malt was lower than in 7-day malt, recording 16.8% as compared to 25.90% respectively. malt yield was invariably higher (92.81%) when germination was for 5-days. 205 afr. j. food sci. res. 120 70 m al tin gl os s( % dr yb a si s) m a lt yi el d (% d ry b a si s) 100 60 80 50 malt yields 40 60 malting loss 30 40 20 20 10 0 0 5 7 9 malting time (days) figure 1. yield and loss of malted paddy rice grains. 40 70 35 60 30 1000 grains wt. w ei gh to f1 00 0g ra in s( g ) starch content 50 s ta rc hc on te nt of gr ai ns (% ) 25 40 20 30 15 20 10 10 5 0 0 0 5 7 9 malting time (days) figure 2. effect of malting on the weight and starch content of paddy rice grains. thousand-grain weight and starch content figure 2 shows the effect of malting on the weight and the starch content of paddy rice grains. drastic changes in the thousand grains weight and the starch content of the paddy rice grains were observed during germination. thousand grains weight decreased from 34.00 to 15.36%, about 54.8% of weight loss. the starch content also decreased from 64.03 to 23.74%. the starch content for the 9 day malt was 37% of the original starch content of the paddy rice. germination significantly (p<0.05) affected both thousand grains weight and the starch content of the paddy rice grains. regression analysis of the (m in ) 6 ti m e 5 h yd ro ly si s( c on ve rs io n ) 4 3 y = -0.525x + 7.5417 2 r 2 = 0.9728 s ta rc h 1 0 4 5 6 7 8 9 10 malting time (days) figure 3. effect of malting on the starch conversion / saccharification time of rice malt. data obtained showed that thousand grains weight significantly correlated with starch content of the grain (r = + 0.99, p<0.05) (table 2). reducing and non-reducing sugars the main sugar of rice embryo and endosperm is glucose, together with small amounts of raffinose, and fructose. reducing and non-reducing sugars also increased significantly (p<0.05) during germination (table 1). saccharification / starch conversion time starch conversion time is the time taken to convert starch into sugars. sacccharification time decreased with increasing malting time (figure 3). it decreased from the average values of 4.9 to 2.8 min. regression analysis of the conversion time data produced the following linear regression model equation: y= -0.525x + 7.5417 where y = conversion time and x = malting time. the correlation coefficient (r) of this expression was 0.986 and the coefficient of determination (r 2 ) was 97.28 %. the model indicated significant (p<0.05) effect. viscosity of pastes of rice malt figure 4 shows the effect of germination on viscosity of malt paste at temperatures of 75 and 36°c. at both tem ayernor and ocloo 206 cp s 120 100 viscosity at 75°c p a st e, viscosity at 36°c 80 m a lt 60 o f 40 v is co si t y 20 0 0 5 7 9 malting time (days) figure 4. effect of germination on viscosity (cps) of malt paste at temperatures 75 and 36 °c. peratures, viscosity decreased during malting. regression analysis of the data showed that viscosity at both 75 and 36°c correlated with starch content of the grain (r = + 0.98 and r = + 0.97 respectively, p<0.05) (table 2). diastatic activity of malted rice the amylolytic activity of the paddy rice malt increased with germination. the diastatic activity increased signifycantly (p<0.05) during germination from 0.00 to 94.50°l (table 1). regression analysis of the data showed that diastatic activity correlated with starch content of the grain during malting (r = 0.99, p<0.05) (table 2). discussion the values obtained for moisture are generally low and therefore may enhance shelf-life of the malt samples produced. moisture content gives an index of quality and consistency of the milled grains. moisture content of grains would be low if high temperature is used and also when the time of drying is extended for medium temperature drying. it is important to dry the malt to a slightly lower moisture level if one intends to store the malt for any considerable length of time. malting loss is the material lost, as per cent dry weight in converting the grain into malt. the results obtained suggest that as the malting loss increases with increasing duration of germination, the malt yield decreases. suhasini and malleshi (1995) reported that malting loss was caused by metabolic activity and separation of vegetative growth (root and shoot) and increased with the increase in duration of germination. similar trends were also reported by hammond (2001). these observations could be attributed to the chemical changes that occurred during the malting process (table 1). the partial degradation of high molecular weight materials in the starchy endosperm of the paddy rice during malting resulted in the observed increases in malting loss and decrease in malt yields. the thousand grains weight of cereals is also used to determine the suitability of grains for malting. it also reflects the density and the seed size of the commodity. starch serves as carbohydrate source, which provides energy to the seed during germination. there were general decreases in the thousand grains weight and starch content of the paddy rice grains. since there was a positive correlation, whenever there was a reduction in starch content of the paddy rice grains during germina-tion, there would be equivalent reduction in the thousand grains weight. similar changes were observed by suhasini and malleshi, (1995). the decrease in the thou-sand grains weight observed could be attributed to the physiological activities. during germination, physiological activeties in the grains increased due to the action of enzymes leading to the utilization of food reserves for energy and growth. also, the duration of malting resulted in losses in grain constituents. furthermore, leaching of minerals and other grain constituents during steeping and germination might in part have accounted for the reduction in the thousand grains weight. the decrease in the starch content of the grains during germination was also due to the action of hydrolytic enzymes such as and amylases, which hydrolyze starch into low molecular weight carbohydrates such as maltose, glucose and dextrins. such gradual decrease in starch during germination is well known. the longer the duration of malting, the smaller the residual starch and consequently higher simple sugars. high starch content tends to be associated with large grain size. the breakdown in the starch content of the paddy rice germinated for 0, 5, 7 and 9 days might have contributed to the observed increase in malting loses and decrease in malt yield (figure 1), as well as the observed sugar values. a considerable increase in the sugar contents in both embryo and endosperm was detected in early germinating sorghum (gill et al., 2003). the starch conversion time of 5day malt was longer than that of 7 and 9-day malts, possibly due to the lower amylolytic activity of the 5-day malt. since the model obtained had a significant effect, the equation arrived at could be used to predict the conversion time for paddy rice malt for a particular malting time. the r 2 observed showed that about 97% of the variability of conversion time (y) could be attributed to differences in malting time (x). viscosity determination of malt paste is important because it is one of the physicochemical attributes of malt content in hydrolytic enzymes. the results obtained were predicted because the decrease in starch content would result in less gelatinisation occurring and hence decrease in the viscosity of the paddy rice grains. 207 afr. j. food sci. res. the decrease in the viscosity of the malt paste was due to the action on the starch by hydrolysing enzymes that were produced during malting. starch breakdown proceeds by the combined actions of -amylase, debranch-ing enzyme (pullulanase like enzyme), -amylase and glucosidase in germinated cereal seeds (zeeman et al., 2007). sumathi et al. (1995) reported lower viscosities in malted legumes with corresponding increased in amylase activity. diastatic power/activity gives a measure of the saccharifying power of the malt prepared. it was originally considered to give a relative measure of starch-converting power of malt. this is however partly true as the and -amylases, which together constitute “diastase” and other enzymes such as amyloglucosidase and isoamylase may be present in malt and are differentially affected by heat. liquefying and dextrinising power are associated mainly with -amylase activity and saccharifying power mainly with amylase activity. the trend observed corresponded positively with that reported by hammond (2001). conclusion the 9day malt of high diastatic activity had the highest rate of starch conversion as compared to 0, 5 and 7 days malting. there were decreases in starch content and viscosity of malt paste, which was accompanied by increase in reducing sugar content and diastatic activity of malt (p<0.05) during malting. starch conversion time of the rice malt decreased with increasing germination time. the physical and chemical changes observed were as a result of increase in the activity of hydrolytic enzymes present in the grain. the paddy rice studied was suitable for germination. however further studies need to be conducted on other varieties of rice available in the country for their malting characteristic references american association of cereal chemists (2003). approved methods of analysis. 10 th ed. aacc, inc. st. paul 4, minnesota, u.s.a. association of official analytical chemists (1990). official methods of analysis. 15 th ed. vol. 1 and 2. washington d.c. cecil je (1995). the use of cassava starch in the artisanal production of maltose. in: agbor egbe t, brauman a, griffon d, trèche s (eds), transformation alimentaire du manioc. pp. 500-502. dewar j, taylor jrn, berjak p (1997). determination of improved steeping conditions for sorghum malting. j. cereal sci. 26: 129-136. egwim ec, oloyede ob (2006). comparison of -amylase activity in some sprouting nigerian cereals. biokemistry. 18(1): 15-20. esiape j (1994). a study of the characteristics of amylase (starch hydrolysing enzymes) of the red sorghum (sorghum bicolor) variety available in ghana and its suitability for brewing. bsc. project. university of ghana, legon. gill pk, sharma ad, singh p, bhullar ss (2003). changes in germination, growth and soluble sugar contents of sorghum bicolor (l.) moench seeds under various abiotic stresses. plant growth regulation. 40(2): 157-162. hammond tk, ayernor gs (2000). combination of malted cereals and cassava starch in the production of sugar syrup. j. ghana sci. association. 2 (1): 87-92. hammond tk (2001). factors affecting the production of fermentable sugars from cassava flour using a combination of rice malt and commercial enzymes. m.phil thesis, university of ghana, legon. hammond tk, ayernor gs (2001). characteristics of malted rice for the production of sugar syrup. j. ghana sci. association. 3 (3): 91-99. suhasini aw, malleshi ng (1995). influence of malting conditions on amylase activity, physical characteristics and nutrient composition of wheat malt. j. food sci. technol. 32(2): 98. sumathi a, malleshi ng, rao sv (1995). elaboration of amylase activity and changes in paste viscosity of some common indian legumes during germination. plant foods for human nutr. 47(4): 341-347. zeeman sc, delatte t, messerli g, umhang m, stettler m, mettler t, streb s, reinhold h, kötting o (2007). starch breakdown: recent discoveries suggest distinct pathways and novel mechanisms. functional plant biology. 34(6): 465-473. in ternationa l scholars journa ls african journal of food science research issn: issn 2375-0723 vol. 4 (2), pp. 011-017, february, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of processing on hemagglutinin activity of lectin extract from selected green vegetables (vernonia amygdalina, gnetum africanum, petrocarpus soyauxil and telifera occidentalis) *obeta n.a., ugwuona f. u and edward q.a. department of food science and technology, michael okpara university of agriculture, umudike, abia state, nigeria. received 20 february, 2016; accepted 01 april, 2016 lectins are carbohydrate binding proteins present in most plants and plant foods. they are toxic, inflammatory, and resistant to cooking and digestive enzymes. in this study, lectins were extracted from okazi leaf (gnetum africanum), bitter leaf (vernonia amygdalina), fluted pumpkin leaf (telferia occidentalis), and oha leaf (pterocarpus soyauxii). partial purification of the lectin extracts was achieved by precipitation with ammonium sulphate and the extracts were tested for hemagglutinin activity using 4% suspension of human erythrocytes in lectin buffer. the blood specifity of the different blood groups was observed in the variation of lectins activities in a0, a1 and a2. the hemagglutinin activities of the vegetable lectins decreased slightly at 60°c when exposed to processing (moist heating and oven drying) but at 100°c there was no agglutination activate in onugbu and okazi vegetables except in a1 for blood group a and b in fluted pumpkin and okazi leaves respectively. loss in agglutinations activity suggest that lectins from these vegetables are heat labile and can be deactivated by moist or dry heating. key words: agglutinations, hemagglutinin , lectins, vernonia amygdalina, gnetum africanum, petrocarpus soyauxil and telifera occidentalis. introduction lectins are proteins of non-immune origin that recognize and bind to carbohydrate without modifying them (meagher et al., 2005; sigh et al., 2014). lectins can be found in plants, animal, microorganisms and viruses. ingestion of lectins by animals may cause antinutritional effects, degeneration of cells membranes, inhibition of digestive enzymes and immune (allergic) reactions (vasconcelos and oliveira, 2004). the resulting interference with absorption of nutrients may give rise to serious physiological consequences (silva et al., 2010). they can cause “food poisoning” by passing the gut wall and deposit themselves in the _________________________________ corresponding author. e-mail: amybeta2@gmail.com. tel. 07060787491 distant organs thereby causing real life disease (puztai et al., 1989; wang et al., 1998 freed, 1999). they are able to disrupt small intestine metabolism and damage small intestinal villi because of the ability of lectins to bind with brush border surfaces. considering the vast effect of lectins, it is necessary to investigate the processing effect on haemagglutinin activity of lectin extract from selected green vegetables using human blood groups (a, ab, b and o). circumstantial argument in favour of the defense role of plant lectins is their marked stability under favourable conditions. most lectins are stable over a wide range of ph and are able to withstand heat, insect proteases and resist animal attacks, to this respect they are believed to have strong defense-related proteins such as protease inhibitors, anti fungal proteins, alpha amylase obeta et al. 012 inhibitor and thionins (erdman and foryce, 1999). many plant lectins with diverse biological activities have been purified and characterized. lectins activate cells and characterized several cytokines produced by immune cells (yamamoto and irimaru 2011). the vegetable leaves contain anti-nutritive factors such as high level of tannic acid, saponin, lectin etc. akwaowo et al. (2000) reported that young leaves often preferred for human consumption contain higher cyanide and tannin content than matured leaves. while some of the anti-nutrients in the leaves are above safety limit for human consumption, most are not harmful but rather have some health benefits to its consumers (ladeji et al., 1995; ajibade et al., 2006). processing techniques can increase the nutritional quality of the plant foods by reducing specific antinutrients (imaobong et al., 2013). the level of lectins decreased to a considerable extent during heat processing especially moist heating (cooking, extrusion, autoclaving and microwave) (habiba, 2002; wang et al., 2008, embaby, 2010). most lectins are known to have higher thermal stability (70 0 c > 30 mins) and do not completely degrade with insufficient and low temperature cooking. while some lectins degrade others pass through the gut, about 1% 5% absorbed into the blood stream of animals which is a significant amount sufficient to cause an immune response resulting in hemolytic anemia and jaundice (breneman, 1984; rabal and akbar, 2009). this lectin toxicity is due to an inhibitory effect on plasma membrane repair (miyake et al., 2007). the main objective of this study is to evaluate the processing effect (moist heating and oven drying) on haemagglutinin activities of lectin extracted from selected green vegetables using human erythrocytes (a, b, ab and o). lectin extracted from processed (moist heated and oven dried) selected green vegetables will help to unveil the processing effect on haemagglutinin activities using human blood in vitro. also this work will be useful in advising food processors, food chemist, food handlers and consumers on the effective method to adopt during food processing/preparation of these vegetables and which vegetables are to be avoided by various blood groups. preparation of samples moist heated samples: the vegetables were blended fresh into slurry and 25g were measured into a beaker. it was then heated in a water-bath at two temperature regimes (60 and 100°c) for a period of 10 mins. the heated mash samples were completely washed and immersed in 1dm 3 of distilled water contained in a conical flask. thorough mixing was done by stirring at intervals for about 1hour. the homogenate was filtered through two layers of muslin cloth and the filtrate allowed to stand for 12hours at room temperature after which the supernatant were decanted and stored in an air tight container in a refrigerator for further analysis while the settled residue were discarded oven dried samples: the green leafy vegetables were dried for 10 hours each at 60°c and 100°c in an electric oven. the samples were then pulverized using the electric rotor blender (emel, em-242 jumia). a 25g of the samples were completely immersed in 1dm 3 of distilled water contained in a conical flask. thorough mixing was done by stirring at intervals for about 1hour. the homogenate was filtered through two layers of muslin cloth and the filtrate allowed to stand for 12hours at room temperature after which the supernatant were decanted and stored in an air tight container in a refrigerator for further analysis while the settled residue was discarded preparation of lectin buffer lectin buffer was prepared according to the method described by brooks et al., (1997). a 6.057g of tris base, 8.70g of sodium chloride, 0.203g of magnesium chloride and 0.11g of calcium chloride were weighed out. the tri base and the salts were then mixed in a 1000ml volumetric flask and dissolved with 100mls of distilled water. concentrated hydrochloric acid (hcl) was added to adjust the ph to 7.6 and the volume is made up to 1000mls. partial purification of lectin the method described by brooks et al., (1997) was used in recovery of the protein (lectins) from the supernatant. the crude protein in the supernatant was precipitated by stirring ammonium sulphate ((nh4)2so4) into the liquid to give a 10%w/v solution. (1g of ammonium sulphate in 10mls). the mixture was allowed to equilibrate for 1hr at room temperature. the precipitate formed were separated at 2000rpm for 10mins using a bench top centrifuge and kept at 0 0 c in ice bath. the recovered supernatant was further saturated with ammonium sulphate to give 20% weight per volume (w/v) solution with the same procedure. the supernatant was removed and the precipitate was put into a test tube and placed inside an ice bath. this process was repeated to precipitate the remaining proteins at 30%, 40% and 50% saturation. the precipitate obtained at different ammonium sulphate concentration were combined in 250ml measuring cylinder and dissolved in 50mls of lectin buffer, and was use for analysis the same day. preparation of red blood cells (erythrocytes) the red blood cells were prepared according to the method described by diehl et al., (1998). a 5mls of fresh whole blood cells from human blood group a, b, ab, and o were collected from healthy donors at daughters of mary private hospital, umuahia in abia 013 afr. j. food sci. state. the blood cells were obtained with 5ml syringe and transferred into sterile edta bottle. the ethylenediaminetetracetic acid (edta) in the bottle serving as anti-coagulant. the bottles were vigorously shaken to properly mix the blood with edta. the whole blood was transferred into a test tube to which 10mls of lectin buffer (ph 7.6) was added. it was then centrifuged at 2000rmp for 10 mins to enable sedimentation of the red blood cells at the bottom of the centrifuge with a pipette. another 10mls of lectin buffer was reintroduced into the blood samples and the washing was repeated 3 times to obtain a clear supernatant. this was carefully removed with a pipette leaving behind the red blood cells. a 2mls of the red blood cells (washed) each from the blood types o, a, ab and b were placed into 100mls measuring cylinder and diluted with 50mls of lectin buffer to obtain 4% suspension of erythrocytes in lectin buffer solution. the supernatant (plasma and buffer) layer was carefully removed. haemagglutinin test for agglutinating activity the lectin activity of the crude extracts was monitored using the method described by brooks et al., (1997). the agglutination was carried out with serial dilutions of the lectin solution in improved wells of microtitre plates using human erythrocytes. about 0.1ml of lectin buffer was placed in each of the wells of multi well titre plates, and then 0.1ml of lectin solution (crude extract in lectin buffer) was added to first well. about 0.1ml of the first dilution was removed from the first well and added to the next well to obtain the second dilution. to each of the dilution in the well, 0.1ml of 4% suspension of the erythrocytes was carefully layered on top of the lectin solution. after the addition of the red blood cells suspension in all the cells, the mixture was agitated and allowed to rest for one hour at room temperature and was observed for agglutination. the result was monitored visually. a positive agglutination test was indicated by the formation of a uniform layer of solution over the surface of the well while a negative test was indicated by the formation of a discrete bottom at the bottom of the well. results and discussion the results show that both moist heating and oven drying affected the agglutinating activities of the vegetable extracts. from table 1 moderate agglutination was observed in a0 (concentrated lectin extract) at 60 0 c for blood groups b and ab (in pumpkin) a, b and ab (in oha) but onugbu and okazi showed slight agglutination at the same temperature. one fold dilution (a1) at 60 0 c showed a slight agglutination for all the blood groups in pumpkin and group o, groups a,b,o for oha and onugbu respectively. okazi showed no agglutination for blood group a and b. in two fold dilution at 60 0 c there were no agglutination except in ab for pumpkin. at 100 0 c for a1 and a2 no agglutination in all the vegetables except for blood group a in pumpkin. the negative agglutination in some of the blood groups might be as a result of agglutinating inhibitors. some lectins can bind to cell but would not cause agglutination. agglutinating inhibitors as described by lis and sharon (1986) are known for their hydrophobic and hydrophilic force which is able to alter the carbohydrate-lectin interaction. the carbohydrate lectin complex is stabilized by intermolecular hydrogen bonds and van der waal forces. the destabilization of such forces by inhibitors hinders agglutination. it also indicates that as the dilution progresses, there is loss of activity of the lectin as observed in loss of hemagglutinin activity at a1 and a2 dilutions. according to lis and sharon (1986), erythroagglutination by lectin is affected by the molecular properties of lectin, cell surface properties, metabolic state of cells and conditions of assay such as temperature, cell concentration and mixing. agglutinating activity was devoid at a2 for all blood groups in all the vegetables such loss in activity may be due to the presence of agglutination inhibitors. the drastic reduction of hemagglutinin activities of the selected vegetables at 100 0 c (moist heating) in almost all blood groups shows that hemagglutinin activities of the studied vegetables are heat-labile. heat labile hemagglutinin is growth depressant at minute level in foods and tends to be toxic at high concentration (liener, 1994). table 2. shows hemagglutinating activities of oven dried vegetables. fluted pumpkin oven dried at 60 0 c gave scanty agglutination. in blood group o, agglutinating activity was lost and at 100 0 c there was total destruction of lectins except for blood group ab that showed slight agglutination at a0. in oha leaf oven drying at 60 0 c had slight agglutination as observed at a0 for blood group a and ab and loss of activity at a1 and a2. also weak agglutination was shown by blood group b and o at a0 and a1 and loss of activity was equally observed at a2. at 100 0 c, there was loss of activity for blood group a and o. blood group b and ab showed weak agglutination at a0. in bitter leaf oven dried at 60 0 c, weak agglutination was observed at a0 for blood group a and o and at ab there was total loss of activity. also at a0 and a1 slight or weak agglutination was observed by blood group b. at 100 0 c there was total destruction of lectin activity in all blood groups except for blood group b that shows slight agglutination at a0. oven dried okazi leaf at 60 0 c has agglutination activity at a0 for blood group a, o and ab. in blood group b there was total loss of activity at 100 0 c, the lectins lost agglutinating activity as negative agglutination was observed. these losses of activity may be due to the presence of agglutinating inhibitors and it varies with the blood groups which agrees with the previous findings on lectin. okamoto et al., (1990) who reported that heat obeta et al. 014 stability of these proteins differ from lectin to lectin. hernandez-infante et al., (1998) reported also that microwave heat treatment significantly reduced hemagglutinin lectin activity in beans and legumes. conclusion in this work moist heating at 60 0 c and 100 0 c and oven drying at 60 0 c and 100 0 c used in determining efficiency of heat processing in reducing this anti-nutrient (lectin) reveals that at a high heat treatment the level of lectin activity was reduced or eliminated. the degree of lectin binding activity differed from one blood type to the other and also with difference in temperature. from the study vegetables should be processed by boiling at 100 0 c and also oven drying at 100 0 c to ensure adequate removal of these anti-nutrients from the leaves. recommendation it is recommended that further research should be done to evaluate the effect of other types of heat treatments on the hemagglutinin activity and their specificity in regards to different blood types in human as well as other animals. this will facilitate the knowledge on the prevailing processing method best suitable for these vegetables thereby helping the food industries, food processors and consumers to be aware of the best time and heat treatment required for effective processing of the above selected green leafy vegetables. references ajibade sr, balogun md, afolabi oo, kupolati md (2006). sex differences in the biochemical contents of teltaira occidentalis. j. food agric environ. 4:155-156. akwaowo eu, ndon ba, etuk eu (2000). minerals and anti-nutrients in fluted pumpkin (telfaria occidentalis hook f). food chem.., 70:235-240. breneman (1984). basics of food allergy. spring field, iii, thomas, p 10-29 brooks s.a., leathem a.j.c., schumacher u. (1997). lectin histochemistry – a concise practical hand book. bios, oxford, uk. 1 st edition pp.16-152. brooks s.a., leathem ajc, schumacher u. (1997). lectin histochemistry – a concise practical hand book. bios, oxford, uk 1 st ed. pp 16-152 diehl ja, chend m. roussel mf, sherr cj (1998). glycogen synthase kinase-3β regulates cyclin d1 proteolysis and sub cellular localization. genes dev 12(22): 3499-3511. embaby he (2010). effect of soaking, dehulling and cooking methods on certain anti-nutrients and in vitro protein digestibility of bitter and sweet lupin seeds. food sci. biotechnol., 19, 1055-1062. erdman jw, foryce (1999). characterization of a novel, antifungal, chitin-binding protein. 181 (24): 74217429. freed, d.j. (1999) allergist. british medical journal 318 (7190):1023-1024. habiba ra (2002). changes in anti-nutrients, protein solubility, digestibility, and hcl-extractability of ash and phosphorus in vegetable peas as affected by cooking methods. food chem., 77, 187-192. hernandez-infante m, sousa v, montalvo i, tena e (1998). impact of microwave heating in hemagglutinins, trypsin inhibitors and protein quality of selected legume seeds. plant foods hum. nutr., 52: 199 -208. imaobong, iyang udousoro, roland, u., ekop, efiok johnson udo (2013). effect of thermal processing on anti-nutrients in common edible green leafy vegetables. j. nutr. 12 (2) 162-167. ladeji o, okoye zs, ojobe t (1995). chemical evaluation of the nutritive value of leaf (fluted pumkin telferia occidentalis) food chem 53:353-355. liener ie (1994). implication of antinutritional components in soybean foods. crit. rev. food sci. nut., 34, 31-67. lis h, sharon n (1986). biological properties of lectins. in: the “lectins: properties, functions and applications in biology and medicine” ed. by i.e. liener, n. sharon and i.j. goldstein. academic press, orlando, pp 265-291. meagher jl, winter hc, ezell p, goldstein ij (2005). crystal structure of banana lectin reveals a novel second sugar binding life. glycobiology is 1033-1042. miyake, y; asano, k; kaise, h; emaru,m; nakayama, m and tanaka, m (2007). critical role of macrophages in the marginal zone in the suppression of immune responses to apoptotic cell associated antigens. jornal of clinical investigation issn 0021-9738. 1558-8238 – online http://www.jci.org/articles/view/31990 okamoto y, minamoto s, shimizu k, mogami h, taniquchi t (1990). interleukin 2 receptor beta chain expressed in an oligodendroglioma line bonds interleukin 2 and delivers growth signal. proc. natl. acad. sci. usa 87(17): 8684-6588. pusztai a, greer f, grant g (1989). specific uptake of dietary lectins into the systemic circulation of rats. biochem soc trans. 17:481–482. rabia h, akbar m (2009). dietary lectin as disease causing toxicants. pak. j. nutr. 8 (3) 293-3003. sigh ss, devi sk, ng tb (2014). banana lectina brief review. molecules 19:18817-18827 silva mc, angelita dc, santos dd (2010). flavis cristina almedia marcus and celester-maria patto de abreu. partial purification of leaf lectin from manihot esculenta and screening its fungicidal activity. j. agric. biotec and sustain. dev. pp136-141. vasconcelos im, oliveira jt (2004). antinutritional properties of plant lectins. toxicology 44: 385-403. wang n, hatcher dw, gawalko ej (2008). effect of variety and processing on nutrients and certain anti-nutrients in field peas (pisum sativum). food chem., 111: 132-138. wang q, yu l-g, campbell bj, milton j, rhodes jm (1998). identification of intact peanut lectinin peripheral venous blood. lancet. 352:1831–1832. [pubmed]. http://www.jci.org/articles/view/31990 http://www.ncbi.nlm.nih.gov/pubmed/9851393 015 afr. j. food sci. appendix result on effect of moist heating on hemagglutinin activity of crude lectin extract of the selected vegetables table 1. hemagglutinin activity of crude lectin extract from moist heated vegetables. blood group dilution for heated fluted pumpkin c o n t r o l dilution for heated oha leaf c o n t r o l dilution for heated bitter leaf (onugbu) c o n t r o l dilution for heated okazi leaf c o n t r o l 60 0 c 100 0 c 60 0 c 100 0 c 60 0 c 100 0 c 60 0 c 100 0 c a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a + + + + ++ + + + + + b ++ + + ++ + + + + + + + o + + + + + + + ab ++ + + ++ ++ + + + + + ++ = moderate agglutination, + = slightly agglutination, =no agglutination, a0=concentrated lectin extract, a1= 1 fold dilution, a2 = 2 fold dilution, control =without erythrocytes. obeta et al. 016 result on effect of oven drying on hemagglutinin activity of crude lectin extract of the vegetables. table 2. hemagglutinin activity of crude lectin extract from oven dried vegetables. blood group dilution for oven dried fluted pumpkin c o n t r o l dilution for oven dried oha leaf c o n t r o l dilution for oven dried bitter leaf c o n t r o l dilution for oven dried okazi leaf c o n t r o l 60 0 c 100 0 c 60 0 c 100 0 c 60 0 c 100 0 c 60 0 c 100 0 c a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a0 a1 a2 a + + + + b + + + + + + + + o + + + + ab ++ + + + + ++ = moderate agglutination, + = slightly agglutination, =no agglutination, a0= concentrated lectin extract, a1= 1 fold dilution, a2 = 2 fold dilution, control =without erythrocytes 017 afr. j. food sci. micro titre plates containing hemagglutinin assay in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (6), pp. 327-330, june, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the chemical composition and sensory attributes of fermented yellow maize (zea mays) processed by two methods grace, t. o. otitoju department of home science nutrition and dietetics, university of nigeria, nsukka. e-mail: graceebaiyeri@yahoo.com. accepted 05 march, 2017 the study compared the chemical composition and sensory attributes of fermented yellow maize (zea mays) processed by two methods. the grains were divided into two equal portions and fermented for 48 h. one portion was drained, sun -dried, dry-milled and subdivided into two equal parts: one was unsieved-dried-milled flour (udm) and the other was sieved-dried-milled flour (sdm). the second portion of the grains was drained, wetmilled and subdivided into two equal parts unsieved-wet-milled flour (uwm) and sieved-wet-milled flour (swm). the flours of udm, sdm, uwm and swm were analyzed for crude protein, crude fat, carbohydrate, crude fibre and ash. gruels were prepared from the flour samples and their organoleptic attributes were tested for colour, texture, flavour and general acceptability. the nutrient content of udm and sdm flour were significantly higher than the uwm and swm in terms of protein, fat, ash and fibre contents. there was no significant difference in the carbohydrate levels based on the processing techniques used. organoleptic attributes of gruels also showed no significant difference for sdm, uwm, and swm. however, the effect of dry-milling without sieving seems to offer more benefits in conserving and improving the proximate (nutrient) levels of fermented maize – ‘ogi’. key words: nutrient composition, fermented maize, organoleptic attributes. introduction cereals, such as maize, sorghum, millet, rice among oth-ers are processed to detoxify the anti-nutritional factors, increase palatability and improve bioavailability of nutri-ents. there are many household food technologies used in the processing and preparation of foods and they inclu-de soaking, fermentation, milling (dry and wet milling), and sundrying among others (eka, 1984). fermentation is one of the household food technologies reviewed extensively as means by which the nutritive val-ue of plant foods could be improved (obizoba, 1998; nnam 2000; obadina et al., 2008). food samples such as maize, sorghum, millet, rice can be fermented to incre-ase the nutrient content, carbohydrate digestibility, and energy densities of gruels, increase the bio-availability of amino acids and also improve their shelf life under con-trolled environment (who, 1998). fermentation can also reduce the high bulk of the traditional complementary foods by reducing the viscosity of the cereal gruel or por-ridge (potter and hotchkiss, 1995). sun-drying of food removes water, reduces moisture content and concentrates nutrients (baiyeri, 2004). sundrying of foods such as fermented maize is a cheap traditional method of food preservation, because solar radia-tion (free gift of nature from sunlight) does the drying and enhances the shelf life of foods products. however, a greater loss of nutrient has been found associated with wet-milling of maize-ogi. akingbala et al. (1981), found a decrease in protein, fat, ash and crude fibre in wet-milled maize-ogi as compared with maize-ogi that was processed by dry-milling: dry-milling of fermen-ted maize has shown that the nutrient content of comple-mentary foods can be improved by conserving the nutri-ent contents as well as enhancing the shelf life while wet-milling results mostly into nutrientloss, yield mainly star-ch and allows contamination from dirty water. it is there-fore advisable to dry-mill fermented maize because, it is more hygienic, retains nutrient contents and improves shelf life. (ruud and rosa, 2002; dal et al., 2007) maize (zea mays) is an important cereal crop produced exten-sively in nigeria. maize is the third most important cereal crop in the world otitoju 328 and ranked the second most important cereal crop in nigeria (enwere, 1998). in some countries, maize constitutes a substantial part of the diet of most of the population (fao, 1992). the carbohydrate levels of maize grains are very high and the protein quality of common maize is similar to that of rice and wheat with lysine as the most limiting amino acids (fao, 1992). maize is reasonably fair in sulphur containing – amino acids (methionine and cystine) and vitamin a precursor, beta-carotene (obi, 1991; obiakor, 2001). however, maize-ogi is still the usual first traditional complementary food given to children in nigeria. it is called ogi, akamu, koko, pap (enwere, 1998). adequate processing and preparation of fermented maize-ogi using dry-milling method will reduce loss of nutrient, and improved the shelf life as well as decrease the antinutrient factors hence, the objective of this study. materials and methods preparation of samples two kilograms of yellow maize that were purchased at nsukka market in enugu state, nigeria, were cleaned by hand picking to remove dirt, stones and unwanted materials. furthermore, maize grains were steeped in deionised water in a ratio of 1:3 (w/v) and allowed to ferment at 28 + 2 o c for 48 hr. at the end of fermentation, grains were divided into two equal portions; one portion was drained, sundried, dry-milled and sub-divided into two equal parts, one part was not sieved (unsieved dried-milled flour) udm while the other part was sieved (sieved dried milled flour) sdm. both flours were packaged separately in airtight polythene. the second portion of the fermented maize was also drained, wet-milled and sub-divided into two equal parts. one portion of the wet-milled flour was wet-sieved (using 70mm mesh screen) and the bran was washed away from the endosperm with adequate water. the filtrate collected (cornstarch sediment) was poured into a muslin bag, tied and pressed to dewater sample. both sieved and unsieved samples were dried to produce unsieved wet-milled flour (uwm) and sieved-wet-milled flour (swm) respectively. these were also packaged separately in airtight polythene for chemical analysis and sensory evaluation. chemical analysis the proximate composition of the flours (udm, sdm, uwm and swm) was determined by aoac (1995) procedure. protein determination was done by micro kjeldahl procedure, crude fat was by soxhlet extraction, carbohydrate was determined by difference. ash was determined by dried ashing method in a muffle furnance and crude fibre by a modified weende method.gruel (pap) was prepared from the flour samples for organoleptic evaluation. preparation of gruels for organoleptic evaluation a recipe was developed and used to prepare gruel from the four samples of fermented maize flour of udm, sdm, uwm and swm respectively. 100g of flour was mixed with 100 ml of deionised cold water to make the paste; then slurry was made by boiling with 500 ml of deionised water. the deionised water was boiled and samples were mixed with cold deionised water. the hot water was then poured into the cold paste and covered for 5 minutes. organoleptic attributes the sensory evaluation of the gruels was conducted in the maternal and child health centre (mchc) of bishop shanahan hospital, nsukka. during one day, a panel of 20 nursing mothers was used by random sampling from the mchc to determine the organoleptic attributes of the products. each of the panelists was seated comfortably with window open for proper ventilation, fluorescent lighting and free from distraction. the judges evaluated the samples using a nine point hedonic scale, where 9 was the highest score and 1 the lowest score. the degree to which a product was liked was expressed as like extremely (9 points), like very much (8 points), like moderately (7 points), like slightly (6 points), neither like nor dislike (5 points), dislike slightly (4 points), dislike moderately (3 points), dislike very much (2 points), dislike extremely (1 point). the gruels were presented to each of the panelist as coded in the hedonic scale. each panelist was given a take-away serving bowl, with spoon and a cup of water to rinse the mouth at interval of testing the gruels to avoid carry over effect. an ambient room temperature was maintained throughout the testing sessions. the 20 panelists for flavor, texture, colour and general acceptability evaluated the four products (tests and control). data analysis data obtained from the study were analyzed statistically. analysis of variance (anova) was used to test for treatment effect (obi, 1986). duncan’s new multiple range test (dnmrt) was used to test the significance of the differences among means (p < 0.05). results table 1 presents the proximate composition (% dry matter) of dried and wet-milled fermented yellow maize flours. the protein levels of the flours ranged from 0.89% in swm (control) to 11.48% in udm (unsieved dried-milled flour) of the fermented yellow maize. the dried-milled flours of the fermented yellow maize (udm and sdm) had higher crude fat levels of 3.57% and 3.55% than the wet-milled flours (uwm and swm) having low crude fat levels of 1.67% and 0.67%. the carbohydrate levels ranged from 80.4% to 99.22%. the wet-milled flours (uwm and swm) had higher levels of carbohydrate (99.22% and 98.22%). the crude fibre levels of sieved dried milled flour and sieved wet-milled flour (sdm and swm) was in trace amount (0.00%) whereas, the unsieved dried-milled flour and the unsieved wet-milled flour (udm and uwm) had appreciable quantity of crude fibre (0.66% and 0.50%). ash levels of the four samples (tests and control) ranged from 0.22% in swm flour (control) to 1.89% in udm. ash level was low in the wet-milled flours of uwm and swm (0.22%). however, the highest level of ash was 1.80% to 1.89% found in the unsieved-dried-milled flours (udm and sdm). table 2 showed the sensory scores of gruels made table 1. proximate composition of dried and wet-milled floursmade from fermented yellow maize (% dry matter) nutrient 100 g/samples udm sdm uwm swm* protein 11.48 a 10.48 a 1.89 b 0.89 b fat 3.57 a 3.55 a 1.67 b 0.67 b carbohydrate 82.4 b 80.4 b 99.22 a 98.22 a fibre 0.66 a trace 0.50 a trace ash 1.89 a 1.80 a 0.22 b 0.22 b * control for each nutrient, values with similar letter in each row are statistically similar (p > 0.05) while those with different letters are significantly different (p< 0.05). table 2. sensory scores of gruels made from dried and wet-milled flours of fermented yellow maize udm sdm uwm swm* flavor 7.6 a 8.2 a 8.5 a 7.5 a texture 7.2 b 8.4 a 8.4 a 8.0 a colour 6.6 a 8.6 a 8.7 a 8.3 a general acceptability 5.0 a 8.4 a 8.5 a 7.7 a means not followed by the sample letter in a row are significantly different. udmunsieved dried-milled flour made from fermented yellow maize; sdmsieved dried milled flour made from fermented yellow maize; uwm -unsieve wet-milled flour made from fermented yellow maize; swm*sieved wet-milled flour made from fermented yellow maize (control). from dried and wet-milled flours of fermented yellow maize. the flavour of the gruels was statistically similar for tests and control and the products were liked moderately by the judges. there was no significant difference in flavour among the samples. the texture of udm was liked moderately while sdm, uwm and swm texture were liked very much. the colours of sdm, uwm and swm gruels were significantly different from that of udm. however, judges liked sdm, uwm and swm very much. the general acceptability of gruels in terms of flavour, texture and colour attributes were comparable (p>0.05). although, udm acceptability was significantly different from others, yet the judges like the products moderately. discussion the higher crude protein levels of the dried-milled fermented yellow maize flours (udm and sdm) than the wet -milled flours (uwm and swm) was attributed to the beneficial effect of dry-milling processing method. this agreed with the observation made by (akingbala, 1981; olatunji, 1982; obizoba and atti 1991; baiyeri, 2004) that fermented maize nutrients can be improved and conserved when processed adequately. the low levels of protein in the control (swm) and (uwm) flours may be due to processing and preparation method of fermented maize – ‘ogi’. as a result, uwm and swm had a greater loss of nutrient through wet-milling. nnam (2002), showed that vital nutrients are leached in the residue sieved out, leaving the sediment (filtrate) as cornstarch. nkama, (1994) also reported the same loss of nutrients during the production of ‘0gi’ with wet milling and sieving. this leads to considerable loss of essential nutrients, especially protein and dietary fibre of the food content. the lower levels of crude fat in the wet-milled flours (uwm and swm) than the dried-milled (udm and sdm) could be attributed to effect of processing on the fermented maize – ‘ogi’ flo-ur. but, adequate preparation and processing through dry milling conserved the crude fat levels of udm and sdm. crude fibre was trace in the wet-milled samples because of sieving, leaving the residue with bulk of fibre and the sediment as corn-starch. the same loss of crude fibre was observed by (baiyeri, 2004) when maize ‘ogi’ was used as a negative control of complementary foods. ash levels of all the samples varied. this could be attributed to the low level of mineral contents of maize. although uwm and swm flours had higher quantity of carbohydrate, the difference was not significant in all the samples. the varying degrees of sensory scores observed for flavour, texture and colour of the gruels made from fermented yellow maize (dried and wet-milled) offers promise for their use at household level. however, the acceptance of all the gruels made from the four samples indica 329 afr. j. food sci. res. otitoju 330 te that without sieving and wet-milling as it is practiced traditionally, the products could gain popularity with increase use of dry-milling without sieving. this is because wet-milling and sieving reduces the nutrient to ordinary corn-starch and chaff while dry-milling without sieving conserved the nutrient as well as improving the shelf life. the flours of unsieved dried-milled and sieved driedmilled (udm and sdm) fermented maize – ‘ogi’ showed nutritional adequacy over the wet-milled flours (uwm and sdm) in terms of crude protein, crude fat, crude fibre and ash. the control (swm) had the poorest nutrient except carbohydrate level (corn-starch sediment) . the effect of drymilling without sieving was beneficial in conserving and improving the proximate (nutrient) levels of fermented maize – ‘ogi’. this may be of importance in terms of nutritive and economic values in developing nations where assess to adequate protein in the diet is not easy to come by amongst the poor population. we recommend that the flours of udm and sdm could be used as an improvement to the popular traditional maize – ‘ogi’ (swm). similarly, dry-milling without sieving could be more beneficial for use in the preparation of fermented maize – ‘ogi’, therefore household method of preparing fermented maize – ‘ogi’ (wet-milling and sieving) could be change to dry-milling without sieving especially in formulating feed for infants and growing children who need adequate protein. references akingbala jo, rooney lm, faubion jm (1981). a laboratory procedure for the preparation of “ogi” a nigerian fermented food. j. food sci. 14: 1523-1526. aoac (1995). association of official analytical chemists. official methods of analysis. washington dc. baiyeri gt (2004). evaluation of multimixes and porridges made from maize (zea mays), soya bean (glycine max) and plantain (landrace and hybrid – musa aab) flour for use as complementary food, m.sc thesis, department of home science nutrition and dietetics university of nigeria, nsukka. pp.1-46. dal bf, clarke ci, ryan lam, ulmer h, schober tj, ström k, sjögren j, van sinderen d, schnörer j, arendt ek (2007). improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain lactobacillus plantarum fst 1.7. j. cereal sci. 45 (3): 309-318. eka ou (1984). a review of studies on changing in nutrient composition during fermentation. niger. j. nutr. sci. 5: 9-21. enwere nj (1998). foods of plant origin. snapp press, nigeria ltd., enugu. pp. 24-102. fao (1992). maize in human nutrition. food and agriculture organization of the united nations (fao), rome, pp. 1-105. nnam nm (2000). chemical evaluation of multimixes formulated from some local staples for use as complementary foods in nigeria. plant foods for human nutrition. 55:255-263. nnam nm (2002). evaluation of complementary food based on maize, groundnut, paw paw and mango flour blends. niger. j. nutr. sci. 23 (182): 8-16. nkama (1994). traditional production and chemical of ndaleleyi. a nigeria fermented pearl millet food. plant food hum. nutr. 46: 109 116. obadina ao, oyewole ob, awojobi tm (2008). effect of steeping time of milled grains on the quality of kunnu-zaki (a nigerian beverage. afr j. food sci. 2: 033-036. obi iu (1986). statistical methods of detecting differences between treatment means. snapp press (nig.) ltd., enuguobi iu (1991). maize, its agronomy, diseases, pests and food values, pp. 131-140. obiakor pn(2001).nutritional and organoleptic evaluation of fermented baobab maize. m.sc thesis, department of home science nutrition and dietetics university of nigeria, nsukka. pp. 5-18. obizoba ic (1998). fermented foods. inc: osagie, a.u. & eka, o.u. (eds), nutritional quality of plant foods. benin city, nigeria. post harvest research unit. university of benin, pp. 160-198. obizoba jc, atti jv (1991). effect of soaking, sprouting fermentation and cooking on the nutrient composition and some antinutritional factor of sorghurm (guinesia) seeds. plant food hum. nutr. 42: 1323. olatunji o (1982). sorghum and millet processing and uses in nigeria cereal. fd. wld. 27: 277-280. potter nn, hotchkiss hj (1995). fermentation and other uses of microorganisms. food science. cbs publishers india. pp. 254-268. ruud v, rosa sr (2002). an overview of small-scale food fermentation technologies in developing countries with special reference to thailand: scope for their improvement. int. j. food microbiol. 75 (3): 231-239. who (1998). complementary feeding of young children in developing countries: a review of current scientific knowledge. world health organization (who) geneva, pp. 80. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 4 (5), pp. 044-048, december, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of hybrid gas-liquid electrical discharge on liquid foods (milk) g.m.el-aragi and y. m. abedel rahman plasma physics and nuclear fusion department, nuclear research center, aea, po 13759 cairo, egypt. accepted22 november, 2015 pulsed high voltage discharges have been recently developed as sterilization method to replace the traditional thermal processes for the sterilization of liquid foods such as milk. the gas-liquid hybrid discharge (hd) reactor consists of plexiglass cylinder containing the liquid of high voltage electrode above liquid’s surface and grounded electrode submerged in the liquid. the hd could produce both arc discharge in gas and liquid phases. the high energy plasma arc produces a pressure shock wave, which kills the targeted micro organisms by causing physical damage to their cellular matrix either by the sudden recoil of the cell or by the micro-eddies created on the internal cell structure. bacterio assay, has shown that the number survivor micro organisms after treatment by 13.5 msec pulse discharge n = 0.388 x 10 9 cfu/ml versus the initial number of viable microorganisms in the control sample before treatment (no) = 1.456 x 10 9 cfu/ml which translates to log reduction of about 0.57. the applied electric field strength (of about 2.5 mv/m ) has more pronounced effect on inactivation the micro oraganisms than the length of the treatment time. the decimal reduction time or time required for a 1-log cycle reduction in the microbial population is about 24 msec. bacterol analysis, has shown that the proteus mirabilis and enterobacteri cloacae (enterobacteriaceae) that were appearing in the control sample before treatment, were disappeared from treated sample. key words: pulse discharge, sterilization, hybrid discharge, thermal processes, shock wave. introduction the application of a pulsed high voltage discharge in an air gas phase over de-ionized water changes the liquid in several ways. electrical discharges in air produce ozone and small amounts of nitrogen oxides. in liquids, the ozone is converted into hydrogen peroxide while the nitrogen oxides are converted into nitric acid. the corona discharges strike the water surface and dissociate water molecules into hydroxyl radicals and hydrogen atoms. hydroxyl radicals may recombine to form hydrogen peroxide. hydrogen atoms react with dissolved oxygen to formulate hydroperoxyl radicals, which recombine to yield hydrogen peroxide and oxygen. recombination of hydrogen atoms produces hydrogen molecules. also, the metal of the anode tip may be sputtered due to the high electric field strength and formation of anode hot spots and hence elementary metal or metal oxides may be found in the liquid after discharge is completed. in general, the pulsed high voltage discharges in liquid generate plasma arcs that initiate a variety of physical *corresponding author. e-mail: elaragi@gmail.com. and chemical effects as high electric field, intense ultraviolet radiation, overpressure shock waves and, especially, formation of various reactive chemical species acting on biological cells and chemical compounds. these effects have various important roles in different application regions in the liquid and the magnitude of their contributions strongly depends upon the energy of the discharge (lukes, 2001) in pulse power discharges of several mw (sun et al., 1998; willberg et al., 1996; lang et al., 1998) a large part of the energy is consumed in the formation of a high temperature (several tens of thousands of degrees kelvin) plasma channel, which emits light in a wide range of wavelengths (sun et al., 1998). this radiation can cause photolysis effect, leading to dissociation of liquid molecules and formation of hydroxyl radicals. in addition, the expansion of the plasma channel against the surrounding liquid generates an intense shock wave with pressure on the order of hundreds of mpa (shah et al., 1999). the resulting shockwave can indirectly induce pyrolytic and free radical reactions via electrohydraulic el-aragi and rahman 045 figure 1. experimental arrangement and circuit diagram of the hybrid gas-liquid electrical discharge system. cavitation (suslick et al., 1999). as the plasma channel cools, thermal energy is transferred to the surrounding liquid, resulting in the formation of a steam bubbles, in which the temperatures and pressures are high enough for the formation of transient supercritical liquid. therefore, the oxidative degradation of organic compounds by such type of discharge is attributed mainly to the ultraviolet photolysis, electrohydraulic cavitation and supercritical liquid oxidation (willberg et al., 1996). milk provides sufficient nutrients needed to support the growth of a host of microorganisms. bacteria are capable of utilizing the proteins, fats, carbohydrates and vitamins in milk for their growth and metabolism. different bacterial species may differ in regard to the enzymes they have or need to break down milk components. new cold sterilizing techniques include sterilizing with pulsed electric field (pef), ultrasonic and microwaves, high voltage, infrared and strong impulse of light in the visible wavelength. however, ultraviolet radiation is only effective in surface, and microwave can produce very high temperature not feasible for cold sterilization techiques. pulsed electric field (pef) can easily to be managed, and is now widely applied as a methods that we can be expected to be feasible at the industrial level (ayhan et al., 2001). the aim of this work is to examine the use of hd treatment system and the capability of pulsed plasma arc discharge directly in the liquid (milk), in killing microorganisms. experimental setup the experimental apparatus is shown in figure 1. the high-voltage electrode (discharging electrode) is 8 mm diameter copper rod and is connected to the pulse forming network “pfn”and to the power supply via a rotating spark gap switch. the ground electrode is submerged in the liquid, it is made of stainless steel and has a semi-spherical shape with a diameter of 4 cm. the distance between the tip of the discharging electrode and the liquid surface was set to 1.0 cm while that between the water surface and the submerged grounded electrode was set to 0.8 cm. the reactor nf capacitor charged through a 50 k ohm resistor by a negative dc high-voltage power supply and rotating spark gap switch. the vessel has an inner diameter of 12 cm. the circuit consists of a 20 capacitor discharges when the voltage on the capacitor reaches the spark-over voltage of the spark gap switch, producing a narrow positive high-voltage pulse. the pulse generator provides a voltage pulse of 25 kv with about 8 ns rise time. the voltage is measured by a high voltage probe and the discharge current is monitored by a rogowskii coil. the voltage and current waveforms are recorded using a digital storage oscilloscope (lecroy model 9300c) the electrical circuit for production of high voltage pulses is based on discharging the capacitor into the liquid sample inside of the reaction chamber. the capacitor is negatively charged via a resistor and the negative side of the capacitor is grounded via the triggered rotating spark gap switch; hence, a positive pulse appears on the anode. the main advantage of a spark gap connected to the ground is that the trigger circuit can be kept at low voltage. a pulse-forming network (pfn) accumulates electrical energy over a comparatively long time, and then releases the stored energy in the form of pulse. in practice a pulse-forming network pfn is charged by means of a high voltage power source, and then rapidly discharged into a load via a high voltage switch such as a spark gap or thyratron. a pulse-forming network (pfn) consists of a series of a high voltage energy storage capacitors and inductors. the combination of high voltage source, pfn, high voltage switch is sometimes called a power modulator or pulsar. a common form of a pulse-forming network (pfn) used in our experiment is the guillemin type e-network, in which the capacitance and inductors are the same in each mesh and there is mutual inductance between adjacent coils. the rise time is determined by the rise time of the first mesh in the network, closest to the load. the value of a capacitor (c) and the inductance (l) of one a pulse-forming network (pfn) section are 5 nf and 4.5micro h respectively. the experiment is conducted by injecting the sample (raw milk) into the treatment chamber, which is pre-cleaned and sterilized.the high voltage pulse generator parameters are adjusted to provide a 25 kv output voltage at 25 hz discharge frequency for 300 s. after the treatment the sample is taken out of the chamber and placed in a sterile bottle. a control sample of raw milk is also placed in a sterile bottle. results and discussion a lecroy 200 ms/s 4-channel digital storage oscilloscope model (9300c) was used to recorded voltage and current waveforms, via a high voltage probe and a pulse current transformer, respectively; and to calculate the pulse energy and discharge power. the measured peak value of the discharge current was v o lt a g e (k v ) 30 25 20 15 10 5 0 -200 0 200 400 600 800 1000 1200 time (ns) c u r r e n t ( k a ) 2 1 0 1 2 2 0 0 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 t i m e ( n s ) figure 2. typical discharge current and the voltage waveforms. figure 3. fourier transformation of the discharge current pulse. approximately 750 a during the pulse. figure 2 shows the current and voltage waveforms measured as a func-tion of time at an input energy of 9 j (maximum applied voltage 30 kv) at atmospheric conditions (air pressure of 1bar). fourier transform of the current pulse, as shown in figure 3, shows the different frequency components in the pulse. it can be seen that most of the energy in the discharge current is between 100 khz and 50 mhz. the spectrum have several peaks at different frequencies with the fundamental frequency component at 100 khz and the harmonic frequency components at 6.25, 11.7 and 35.2 mhz. it is clear that the energy is spread over a wide frequency range, which is characteristic of short pulse discharges. in an electrohydraulic discharge, cavitated bubbles are generated by the pulsed arc discharge and induce shock like pressure, which rises in the cavitation zone near the discharge electrodes. these cavitated bubbles are a major source of ultraviolet emission and radical species such as ozone, oh, h2o2, etc. such chemical and physical reactions occur simultaneously in the cavitation zone and are expected to affect the treatment of the liquid (milk). thus electrohydraulic discharge processes provide multiple mechanisms to achieve microbial inactivation. the standard approach to describing changes in microbial population as a function of time uses the survivor curve equation: log [n / n0 ] = -t / d where: n = microbial population at any time, t n0 = initial microbial population d = decimal reduction time, or time required for a 1-log cycle reduction in the microbial population. when microbial populations are exposed to pulsed electric fields (pef), the applied electric field intensity should be above specific threshold determined by the critical electric field intensity for the target microorganism. a model similar to those for temperature and pressure can be used to describe the influence of electric field intensity on the rate of microbial population reduction. the proposed model would be (peleg, 1995; peleg and cole, 1998; anderson et al., 1996): log [d / dr ] = -( e – er ) / z where: dr = decimal reduction time at a reference electric field intensity (er). the electric field coefficient in this model is defined as: z(e) = the increase in electric field intensity (e) required 046 afr. j. food sci. res. el-aragi and rahman 047 figure 4. rate of inactivation in log-reduction as a function of energy release (j) to reduce the decimal reduction time (d) by 1-log cycle at a specific temperature and pressure. a similar model has been proposed and used by hulsheger and others (1981) (hülsheger, 1981) and was applied by jeyamkondan and others (1999) (jeyamkondan et al., 1999), given by log [n / n0 ]= log [t / tc ] {-( e – ec ) / k } where: t = treatment time, which is the product of number of pulses and pulse width. tc = critical treatment time or treatment time below which no inactivation of microorganisms occurs ec = critical electric field strength or electric field strength below which no inactivation of target microorganism occurs. k = specific rate constant (a coefficient with magnitude based on the slope of the survivor curve obtained at several levels of electric field intensity.) larger magnitudes of the coefficient k would suggest a higher resistance to changes in electric field intensity. the left hand side is the standard log-reduction and one log reduction refers to 90% reduction in the initial population of microorganisms. the electric field strength has more pronounced effect on inactivation of the microorganisms. the effect of electric discharges on the microorganisms (bacteria) in the raw milk is shown in figure 4. changes in the populations of bacteria (n) in the treated milk relative to the initia l populations (no ) as a function of discharge energy (j) released during the treatment. raw milk with electrical conductivity < 10 -12 ohm -1 cm -1 was used, pulse repetition frequency less than 50 hz and the initial concentration of bacteria was 1.5*10 9 colony-forming units ml -1 . pulsed high voltage discharge generates high electric field intensities ,which is considered superior to traditional heat treatment of foods because it avoids, or greatly reduces, the detrimental changes of the sensory and physical properties of foods (quass, 1997). the large field intensties are achieved through discharging a capacitor into the reaction container. the energy at the reactor is generally converted into high e-field intensities, ultraviolet radiation, shock wave, and radical species such as o3 , h 2o2 , o and oh by intense spark discharge. the liquid will also experience temperature rise due to thermal effects from thew arc, the joule heating is eminent and large fraction of energy will also be transferred to heat. the temprature of the liquid was measured using thermometer about 42°c the strong arc disharge needs strong e-field and this field is proportional to the shock wave intensity. therefore, these results mean that the shock wave is an effective parameter of the destruction of microorganisms in food. discharge characteristic depend largely on the voltage capacitance and milk conductivity. during the discharge, it is noted that the phenomenon of sonoluminescence that is, emission of light by bubbles in a liquid (milk) excited by sound waves. cavities are generated in a fluid (that is, bubbles) when the tensile phase (negative pressure) of the acoustic wave is sufficiently strong to rip the fluid apart. the tensile phase of the shock wave is large enough to generate violent cavitation events. shock waves of sufficient intensity (function of the discharge circuit, pulse duration, liquid conductivity, electrode size, gap distance and reactor configuration) will break the attractive forces in the existing molecules and create gas bubbles (hart and henglein, 1985; shah et al., 1999). these gas bubbles grow until they reach a critical size. upon reaching the critical size, the gas bubbles implod or collapse. the implosion of microscopic gas bubbles of a liquid ruptures the cell membrane and collisions with other organisms and particular matter can also cause further mortality within the target area (pétrier and francony, 1997; suslick et al., 1999). conclusion the hd treatment was effective in the destruction of different types of bacteria. enterococcus spp (gram-positive) short chains or pairs of cells. “fecal” strepto-cocci (but are not coliform); common in fecal matter, but also in the dairy farm environment. used as indicator organisms in some foods. acid producers. some strains have some heat resistance. also proteus mirabilis spp (gramnegative) destructed through generation of ultra-violet radiation, shock wave, ozone and free radicals. acknowledgements i wish to express my thanks to prof. dr h. m. soliman head of division of material and nuclear manufacturing for all her support and encouragement throughout this work and my thanks to all colleagues in the plasma department specially to mr. t. abdel razek for his efforts during this work. references anderson wa, mcclure pj, baird-parker ac, cole mb (1996). the application of a log-logistic model to describe the thermal inactivation of clostridium botulinum at 213b at temperatures below 121-1 °c. j. appl. bacteriol. 80: 283-290. hart ej, henglein a (1985). free radical and free reactions in the sonolysis of aqueous iodide and formate solutions. j. phys. chem. 89 (20): 4342-4347 hülsheger h, pottel j, niemann eg (1981). killing of bacteria with electric pulses of high field strength. radiat. environ. biophys. 20: 53-65. jeyamkondan s, jayas ds, holley ra (1999). pulsed electric field processing of foods. rev. j. food protect. 62(9) : 1088-1096. lang ps, ching wk, willberg dm, hoffmann mr (1998). oxidative degradation of 2,4,6-trinitrotoluene by ozone in an electrohydraulic discharge reactor. environ. sci. technol. 32 (20): 3142-3148. peleg m (1995). a model of microbial survival after exposure to pulse electric fields. j. sci. food agric. 67(1): 93-99 peleg m, cole mb (1998). reinterpretation of microbial survival curves. crit. rev. food sci. 38(5): 353-380 pétrier l (2001). water treatment by pulsed streamer corona discharge, ph.d. thesis, prague. pp. 9-14 pétrier ch, francony (1997). ultrasonic waste-water treatment: incidence of ultrasonic frequency on the rate of phenol and carbon tetrachloride degradation, ultrason. sonochem. 4 (4): 295-300 quass dw (1997). pulsed electric field processing in the food industry. a status report on pef. palo alto ca. electric power research institute. cr-109742. shah yt, pandit ab, moholkar vs (1999). cavitation reaction engineering. new york: kluwer academic plenum publishers. sun b, sato m, harano a, clements js (1998). non-uniform pulse dischargeinduced radical production in distilled water. j. electrostatics 43 (2): 115-126. suslick ks, didenko y, fang mm, hyeon t, kolbeck kj, mcnamara iii w, mdleleni mm. wong m (1999). acoustic cavitation and its chemical consequences. philosophical transactions of the royal society of london. 357: 335-353. willberg dm, lang ps, hochemer rh, kratel a, hoffmann mr (1996). degradation of 4-chlorophenol, 3,4-dichloroaniline and 2,4,6trinitrotoluene in an electrohydraulic discharge reactor. environ. sci. technol. 30(8): 2526-2534. zehra ayhan, hye won yeam, howard zhang q (2001) . flavor, color, and vitamin c retention of pulsed electric field processed orange juice in different packaging materials. j. agric. food chem. (49): 669 674. 048 afr. j. food sci. res. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (9), pp. 378-381, september, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of mushroom species using sawdust of different economic tree species abbot o. oghenekaro*, john a. okhuoya and emmanuel o. akpaja mushroom biology unit, department of plant biology and biotechnology, university of benin, p. m. b. 1154, benin city, nigeria. accepted 06 may, 2018 lentinus squarrosulus (mont.) singer was cultivated on the sawdust of five economic tropical tree spe-cies; chlorophora excelsa, celtis zenkeri, guera cedrata, nesogordenia papaverifera and brachystegia, collected during processing at the sawmill. the highest yield of fruiting bodies (16.17 ± 1.25 g) was observed on b. nigerica sawdust supplemented with 1% caco3, 1% sugar and 20% wheat bran. the lowest yield (4.56 ± 1.84 g), was observed on n. papaverifera sawdust without supple-mentation. the results of this study demonstrate that l. squarrosulus mushroom did not show any particular trend with increase in supplementation level. the study underscores the need for guided use of sawdust types along with the required appropriate supplements. key words: agro-industrial waste, bioconversion, cultivation, edible mushroom, sawmill waste. introduction production of edible mushrooms in nigeria is almost at the level of commercialization. however, the practical field application of many laboratory results still leaves much to be desired. many still depend largely on the mushrooms harvested from the wild. in nigeria, many mushrooms are highly prized, not only as food but also in traditional medicine (oso, 1977; alabi, 1990). lentinus squarrosulus (mont.) singer is one of the edible mushrooms very common in the southern part of nigeria (nicholson, 1989; oso, 1975; akpaja et al., 2003). the ibo people of nigeria have even devised a peculiar way of tenderizing the usually tough overripe fruit bodies by dressing it, robbing it with edible oil and thereafter wrapping it in leaves. the wrap will then be placed in a fire place. this mushroom is very popular in nigeria and has been highly recom-mended for commercialization (okhuoya, 1997). a study by wuyep et al. (2003) on this mushroom indicated its production of lignin and cellulose degrading enzymes. it is also reported to be widespread throughout equatorial africa, south-east asia, the pacific islands and australia (neda and noi, 1998). despite the widespread acceptance of this mushroom as an edible species in *corresponding author. email-abbotkaro@yahoo.com. tel.: +2348036362036; fax: +23452602370 nigeria, there is lack of information about its cultivation. more importantly cultivation on sawdust being the most readily available and cheapest organic waste in nigeria has not been investigated. this study reports on the cultivation of this mushroom species using sawdust of different economic tree species. materials and methods spawn preparation pure l. squarrosulus (strain ls001ubnig) was obtained from the mushroom biology unit of the department of plant biology and biotechnology, university of benin, benin city, nigeria. the spawn was prepared using sorghum (sorghum bicolor) grains as substrate. empty salad cream bottles (275 ml) (readily available as discards) were filled with parboiled sorghum grains (75% water) to three quarter full and covered with cotton wool. the bottles were autoclaved at a temperature of 121°c and 1.05 kgcm 2 pressure for 20 min, allowed to cool down overnight and were inoculated. the inoculated grains were incubated at room temperature (25°c). after the grains have been fully colonized, they were kept in the refrigerator (5°c) until used. collection of substrates sawdust from the following economic trees; chlorophora excelsa (welw.) benth. (moraceae), celtis zenkeri engl. (ulmaceae), guera cedrata (a.chev.) pellegr. (moraceae), nesogordenia papaverifera (a. chev.) r. capuron (sterculaceae) and brachystegia nigerica oghenekaro et. al. 379 table 1. effect of sawdust type on the time of lentinus squarrosulus mycelial colonization. substrate mixture time of full mycelial colonization (days) c1 c2 10% wb 20% wb chlorophora excelsa 13.80±2.35* 9.80±0.58 17.50±6.50 17.00±0.00 celtis zenkeri 9.60±0.51 7.80±0.49 13.00±0.58 n.a guera cedrata 10.40±0.68 9.60±0.51 13.40±2.16 16.33±2.91 nesogordenia papaverifera 10.20±0.37 10.25±0.25 12.75±1.93 15.00±0.00 brachystegia nigerica 10.00±0.32 10.00±0.78 16.67±0.33 17.00±0.00 *= mean of five replicates ± standard error. c1 = 100% sawdust c2 =98% sawdust, 1% caco3, 1% sugar. 10% wb = 88% sawdust, 10% wheat bran, 1% caco3, 1% sugar. 20% wb = 78% sawdust, 20% wheat bran, 1% caco3, 1% sugar. n.a = not available. hoyle and a. p. d. jones (fabaceae) were obtained during timber processing from uwasota sawmill, benin city, nigeria. wheat bran was bought from an animal feed dealer at textile mill road stores in benin city. calcium carbonate (caco3) was bought from a chemical dealer while sugar was bought at uselu market benin city. sawdust and wheat bran were sun dried on daily basis. it was done in such a way that the layer of material was not thicker than 4 cm allowing its proper and even drying. both materials were mixed regularly during the process. substrate preparation the experiment had two controls. in order to determine if the supplement addition, will affect the mushroom’s growth and development. the first control (c1) was just the individual sawdust types measured into the cellophane bag on 300 g oven dry weight basis. the second control (c2) was set up by supplementing each sawdust type with 1% calcium carbonate and 1% sugar. two different treatments were prepared. the first treatment (10% wheat bran) was done by supplementing each sawdust with 1% caco3, 1% sugar and 10% wheat bran. the second treatment (20% wheat bran) was supplemented with 1% caco3, 1% sugar and 20% wheat bran. moistened sawdust (300 g) oven dry weight and 75% water was loaded into a polythene bag (15 x 30 cm). five replicate bags (15 x 30 cm) were prepared for each treatment. thereafter, polyvinyl chloride ‘neck’ (5 wide x 3 cm long) was passed through the filled bag and the mouth of the bag covered with cotton wool. bags were steamed for three hours on two consecutive days. substrate spawning and incubation after cooling bags were randomly picked up and inoculated with mushroom spawn at 5% spawn level (quimio et al., 1990). spawned bags were incubated for 30 days in a clean room well shaded from direct sunlight and protected from the infiltration of rodents and other pests. fruit body induction, harvesting and post harvest handling fruit body induction was achieved by reducing the temperature of the environment by spraying water into the room and opening of the mouth of the bag, followed by spraying water on the surface of the substrates. fruiting bodies were harvested four days after primordial emergence. fresh weight of each harvest/flush was recorded. mushrooms were dried at 105°c for 48 h, cooled in desiccator and their weight determined. analysis of data this was done by determining the mean and standard error for each parameter. analysis of variance was calculated for each set of data. the experiment, comprising five types of sawdust and two supplement levels (10 and 20% wb) was replicated five times in a completely randomized design. results and discussion l. squarrosulus mycelium was able to colonize the differrent sawdust types irrespective of the supplementation level. the only exception to this observation was found in the 20% wb sawdust of c. zenkeri (table 1). this exception may have occurred due to an imbalance in the carbon/nitrogen ratio required for mycelial growth (chang, 1978) . the fastest mycelial colonization of (7.8 ± 0.49 days) was observed in (c2) c. zenkeri sawdust mixture, while it took as long as 17.50 ± 6.50 days for the mushroom to completely colonize the 10% wb sawdust of c. excelsa (table 1). this result suggests c. zenkeri sawdust with minimal upgrading (1% caco3 and 1% sugar) is more appropriate for mycelial growth of l. squarrosulus. the differences in the treatments as per total time of colonization were significant (p = 0.05). primordial emergence was first observed in the pure b. nigerica sawdust after 20.60 ± 0.06 days (table 2) whereas the longest time of primordial emergence was found in n. papaverifera 20% wb. the differences in the mean time for first primordial emergence were significant (p = 0.05). mean fresh weight of dry fruiting bodies ranged from 16.17g ± 1.25 g in b. nigerica (20% wb) to 4.56g ± 1.84 g in n. papaverifera (c2). the differences in dry fruiting bodies yield (table 3) on different sawdust and supplement combinations were significant (p = 0.05). the highest organic matter loss due to the activity of the mushroom on the different sawdust substrates with their 380 afr. j. food sci. res. table 2. effect of substrate supplementation on the first time of primordial emergence (days). substrate mixture c1 c2 10% wb 20% wb chlorophora excelsa 31.00±4.04* 27.30±2.85 34.00±0.00 35.33±2.40 celtis zenkeri 25.40±0.51 27.40±1.91 30.50±3.18 n.a guera cedrata 31.25±0.85 28.20±0.74 39.00±2.00 33.67±0.33 nesogordenia papaverifera 23.50±2.18 21.67±1.20 34.75±0.75 41.00±1.00 brachystegia nigerica 20.60±0.16 22.25±1.60 39.00±0.71 42.00±1.00 *= mean of five replicates ± standard error. c1 -100% sawdust c298% sawdust, 1% caco3, 1% sugar. 10% wb-88% sawdust, 10% wheat bran, 1% caco3, 1% sugar. 20% wb-78% sawdust, 20% wheat bran, 1% caco3, 1% sugar. n.a = not available. table 3. sporophore yield of lentinus squarrosulus cultivated at different supplementation levels. substrate mixture supplementation with wheat bran (%) c1 c2 10% wb 20% wb chlorophora excelsa 15.93±0.98* 6.72±2.21 12.70±0.00 9.69±1.78 celtis zenkeri 15.97±2.17 16.02±2.21 11.17±2.97 n.a guera cedrata 6.60±2.23 6.36±1.30 12.13±0.00 13.01±1.44 nesogordenia papaverifera 4.56±1.84 7.16±3.17 14.85±0.65 14.70±0.60 brachystegia nigerica 12.65±2.42 12.41±2.53 12.56±0.70 16.17±1.25 * = mean of five replicates in grams. c1 -100% sawdust c298% sawdust, 1% caco3, 1% sugar. 10% wb-88% sawdust, 10% wheat bran, 1% caco3, 1% sugar. 20% wb-78% sawdust, 20% wheat bran, 1% caco3, 1% sugar. n.a = not available. table 4. percent organic matter loss due to the production of lentinus squarrosulus on sawdust of selected economic trees. substrate mixture level of wheat supplementation (%) c1 c2 10% wb 20% wb chlorophora excelsa 19.93* 33.96 13.88 10.86 celtis zenkeri 29.23 21.61 25.53 13.07 guera cedrata 21.51 32.59 33.51 12.66 nesogordenia papaverifera 28.86 24.99 15.06 15.85 brachystegia nigerica 25.19 16.33 29.19 15.96 *= mean of five replicates. c1 -100% sawdust c2 98% sawdust, 1% caco3, 1% sugar. 10% wb-88% sawdust, 10% wheat bran, 1% caco3, 1% sugar. 20% wb-78% sawdust, 20% wheat bran, 1% caco3, 1% sugar. n.a = not available. their respective supplement ranged between 33.97% in c. excelsa (c2) and 12.66% in g. cedrata 20% wb mixture (table 4). l. squarrosulus (mont) singer grew successfully with sporophore production on the various sawdust types. different treatments and controls gave different results. this may be due to effective substrate utilization (can-. non, 1986; laborde, 1992) in several substrate mixtures. the results of this study indicate that even though sawdust is readily available and cheap, there is the need to be guided regarding its sources. this is necessary because sawdust type strongly influences mushroom yield. also, as shown by royse et al. (1990), amendment of synthetic substrates may enhance mushroom yield. oghenekaro et. al. 381 also, an ethnoknowledge obtained from some individuals in sawmills suggests that there is the need to be cautious in the use of berlinia sp. sawdust. it is believed that this tree imparts toxicity to edible mushrooms (personal communication). at least, two individuals claim they had seen persons affected with some ailments eating mushrooms grown on this type of wood. a study on the empirical confirmation of this ethno-knowledge is ongoing and will be reported in the nearest future. it may be necessary to conduct further research into the use of local sawdusts for cultivation of this mushroom specie so as to increase the yield. acknowledgement uwasota sawmill workers are highly acknowledged for providing sawdust. references akpaja eo, isikhuemhen os, okhuoya, ja (2003). ethnomycology and usage of edible and medicinal mushrooms among the igbo people of nigeria. in. j. medicinal mushrooms. 5: 313-319. alabi ro (1991). mycology and nigerian culture: past, present and future. in: proceeding of the first conference on african mycology. mauritius, 10(15):13-52. alexander hs (1978). morphology and classification of edible mushrooms in: chang, s.t and hayes, w.a. (eds). the biology and cultivation of edible mushrooms: academic press, new york, p. 819. cannon pf (1986).international commission on the taxonomy of fungi (itcf): name changes in fungi of microbiological, industrial and medicinal importance. part 1. microbiological science 3: 168-171. laborde j (1992). required qualities of indoor composition. mushroom world. 3: 23-30. neda h, doi y (1998). notes on agarica in kyushu district!! memoirs of the national science museum, no 31, tokyo. pp. 89-95. nicholson ra (1989). common mushrooms found in akwa ibom state. the nigerian field. 54: 19-32. okhuoya ja. (1997). mushroom cultivation: the nigerian experience. in: food processing technologies for africa. hamid dirar a (ed). emerging technologies series. united nations industrial developing organization, vienna. p. 196. oso ba (1975). mushroom and yoruba people of nigeria.mycologia, 67: 311-319. oso ba (1977).pleurotus tuber-regium from nigeria.mycologia, 69: 271279. quimio th, chang st, royse dj (1990). technical guidelines for mushroom growing in the tropics, plant production and protection paper 106. food and agriculture organization, rome, 1: 55. royse dj, bahler bd, bahler cc(1990). enhanced yield of shitake by saccharine amendment of the synthetic substrate. appl. environ. microbiol.56: 479-482. stamets p (2002a). growing gourmet and medicinal mushrooms. 3 rd edition. ten speed press berkerly. p. 574. stamets p (2002b). mycomedicinals. an informational treatise on mushrooms. 3 rd edition.mycomedia productions, olympia. p. 96. wuyep pa, khan au, nok aj (2003). production and regulation of lignin enzymes from lentinus squarrosulus (mont) singer and psathyrella atroumbonata pegler. afr. j. biotechnol. 2: 444-447. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (1), pp. 001-005, january, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper comparative effects of crude oil on juveniles clarias gariepinus and clarias anguillaris awoyinka o. a.1*, atulomah e.1 and atulomah n. o. s.2 1 department of biochemistry, babcock university, ilisan-remo, nigeria. 2 public and allied health unit, babcock university ilisan-remo, nigeria. accepted 21 october, 2019 this study was based on biochemical assessment of the effect of crude oil spillage on marine life. hence, life sustaining indices such as ph, dissolved oxygen (do), and free co2 were analyzed in aquaria. however, a 96 h static acute toxicity test was also investigated on the juveniles clarias gariepinus (african catfish) and clarias anguillaris (mudfish) on exposure to different concentrations of crude oil polluted water. over the period of the experiment there was an overall decrease in ph of the test vessel from 8.64 ± 0.03 to 5.50 ± 0.08. decrease trend was also found in dissolved oxygen from 6.28 ± 0.07 to 3.01 ± 0.06 and free co2 levels from 8.35 ± 0.55 to 11.25 ± 0.17. however as the concentration of crude oil increases the haematological parameters such as red blood cell (rbc), packed cell volume (pcv), and white blood cell (wbc) reduced significantly (p ≤ 0.05) in both fish species. after 96 h, probit analysis showed the lc50 of crude oil for c. anguillaris to be 1.22 mg/l while that of c. garieinus was 2.19 mg/l. overall, less insults was observed from the crude oil on c. gariepinus compare to c. anguillaris that tend to elicit a low tolerance to the crude oil. key words: clarias gariepinus, clarias anguillaris, crude-oil, lc50, and static-acute. introduction the activities of man have been responsible for introducing contaminants into the environment in the last few decades. the resulting pollution has led to serious consequences on aquatic life (clair et al., 2003). apart from the presence of industrial wastes that are sources of constant threat to marine life, the ecological disasters caused by the oil industry while exploring crude oil and accidental discharge of crude oil on high seas is a serious cause for concern (speight, 1999). the main threat posed to living resources by the persistent residues of spilled oils and water-in-oil emulsions (“mousse”) is one of physical smothering that may consequently lead to depletion of biodiversity in the aquatic environment if adequate remediation is not provided (cooney et al., 2001). the animals and plants most at risk are those that could come into contact with a contaminated sea surface (longhurt, 1961). these *corresponding author. e-mails: woyinka@yahoo.com, oawoyinka@babcockuni.edu.ng. include aquatic mammals, reptiles and birds that feed by diving or form flocks on the sea as well as aquatic lives on shorelines ( suchanek, 1993). juvenile clarias anguillaris and clarias gariepinus were selected as the aquatic organisms for this experiment due to the fact that their response and sensitivity to toxicants is high (sunmonu and oloyede, 2006). both are species of the family clariidae, the air breathing catfishes, found throughout africa and the middle east. they live in freshwater lakes, rivers, swamps, as well as human-made reservoirs, such as ponds or even urban sewer systems (teugels, 1986). c. gariepinus commonly refer to as african catfish is a large, eel-like usually of dark gray or black colouration on the back, fading to a white belly. it has a slender body, a flat bony head and abroad terminal mouth with four pairs of barbell (andrews and matsuda, 1975). however, c. anguillaris otherwise known as mudfish have an oval-shaped to rectangular in dorsal outline with a broadly rounded snout (teugels, 1986). previous studies have shown that crude oil can have both lethal and sub-lethal effects on a wide range of organisms. these include the observation reported by in juvenile pink salmon (oncorhyncusgorbuscha) (wang et al., 1994); while drastic changes in liver enzyme activities of catfish (c. gariepinus) were reported following exposure to crude oil ( sunmonu and oloyede, 2006). against the foregoing this work was set to carried out comparative study on the effect of crude oil exposure on some physical parameters, haematological parameters and lethality in c. gariepinus and c. anguillaris under experimental conditions. materials and methods collection of crude oil the crude oil was obtained from chevron depot site, lagos nigeria. collection of test organism juvenile c. anguillaris and c. gariepinus were collected from a fresh water fish farm in iperu remo and ilishan remo respectively. the acute toxicity test was carried out at the department of chemical and environmental sciences animal laboratory, babcock university nigeria. experimental methods modified method of american public health association (apha, 1992) was used to carry out the static bioassay procedure. this involved carefully controlled environmental conditions to define the response of the test organism to crude oil. twelve rectangular glass tanks of the dimension 50 × 30 × 45 cm and capacity of 100.5 l were used as aquarium. the test organisms were brought into the laboratory and the weight of the fishes was taken. this ranged between 1.6 and 1.87 g and the length of the fishes ranged between 5.5 to 7.3 cm. the length and weight of the biggest was not more than twice that of the smallest, to avoid errors due to size. the stock populations were held for one week in the laboratory, inside these aquaria, filled with 30 litres of unchlorinated borehole water. the animals were held at a temperature range of 26 to 28°c. the water in the tanks was changed once every three days in order to avoid the accumulation of toxic metabolites and wastes secreted by the fishes. juvenile c. anguillaris and c. gariepinus of fairly equal length and weight were taken randomly from the stock population using a hand net and carefully transferred to the glass test vessel containing the test solution. static acute toxicity study preliminary studies carried out using several concentrations provided the basis for the spread of test concentrations along pre determined toxicity range of test samples. reference chemical used as a negative control is sodium dodecyl sulphate (gulley and west, 1996). fish tanks filled with only freshwater were used as positive control. concentrations of crude oil prepared as 0.6, 1.0, 1.4 and 1.8 mg/l were added to the c. anguillaris labelled aquarium while 1.2, 2.0, 2.8 and 3.6 mg/l were added to the c. gariepinus labelled aquarium for two hours to allow aeration. thereafter, a total number of twenty of the fish species were added to each test vessel and covered with wire mesh to prevent the organisms from jumping out of the tanks. mortalities of the test organisms were recorded at 24, 48, 72 and 96 h according to ospar offshore industries committee (oic, 2002). the experimental animals were taken as dead when there were no opercula and other forms of body movements even on prodding with a glass rod. determination of physicochemical parameters samples of the test solutions were taken from each vessel for the determination of physico-chemical parameters. the following parameters were measured after the placement of each test solutions in each tank: ph, dissolved oxygen and temperature using the horiba u10 and the dissolved oxygen meter. the level of carbon dioxide in each aquarium was determined by the titrimetric method (maissonneuvve and larose, 1988). determination of hematological parameters the method of dacie and lewis (1975) as modified by alexandra and griffiths (1993) was used in the determination of the haematological parameters. after harvesting from the aquaria, the fishes were allowed to stay in a dissecting tray for about ten minutes to reduce the slime on their bodies. they were thereafter dissected and blood sample was collected with a disposable syringe and needle and immediately transferred into sterile ethylene diamine tetra-acetic acid (edta) embedded vials for haematological analysis. the automated haematologic analyzer (sysmex kx – 21, uk) was used to analyze the haematological parameters namely packed cell volume (pcv), white blood cell (wbc) and red blood cell (rbc). results and discussion one of the major problems of the inhabitants of the niger delta region of nigeria is contamination of water and aquatic lives by crude oil spillage (sunmonu and oloyede, 2008). the severity or degree of the problems in the inhabitants of the area is dependent upon the point of contact with the polluted water (omoregie, 1998). this observation is reflected in the physicochemical parameters of this study. there were overall decreases in ph of all the test solutions after a 96 h period of the experiment (tables 1 and 2). the decrease in ph which poses a lethal effect on the marine life is indicated to be from the organic pollution from the fishes (swingle, 1961; godswill, 1989). however, similar trends were also observed in the values of dissolved oxygen (do) in all the aquaria. a significant decrease (p ≤ 0.05) in dissolved oxygen over 96 h period of the study was recorded. it is known that low do causes anaerobic decomposition of organic matter in water, forming noxious and toxic substances such as hydrogen sulphide and methane which ultimately would have deleterious effect on the aquatic life (mason, 1991; dprm, 2000). there was a corresponding increase in free co2 in the aquaria containing c. anguillaris from 8.35 ± 0.23 to 11.25 ± 0.17 over a period of 96 h. and 8.69 ± 0.33 to 11.48 ± 0.15 for aquaria containing c. gariepinus. this observation can be attributed to the oil film formation that reduces the table 1. physicochemical parameters determined in test solutions with crude oil sample containing clarias anguillaris (mudfish) 24 to 96 h into the experiment. concentration duration positive negative 1.2 2.0 2.8 3.6 (mg/l) (h) control control 0.6 1.0 1.4 1.8 ph 24 7.86 ± 0.02 a 7.25 ± 0.04 b 7.96 ± 0.03 c 8.20 ± 0.02 d 8.43 ± 0.05 e 8.64 ± 0.03 f ph 96 6.23 ± 0.03 a 6.89 ± 0.02 b 5.68 ± 0.06 c 5.50 ± 0.08 d 5.63 ± 0.04 e 6.42 ± 0.07 f dissolved oxygen 24 6.17 ± 0.1 a 6.28 ± 0.0 a 6.18 ± 0.1 a 6.21 ± 0.1 a 6.28 ± 0.1 a 5.96 ± 0.1 a dissolved oxygen 96 3.27 ±0.04 a 5.46 ± 0.05 b 3.27 ± 0.07 c 4.81 ± 0.04 d 4.28 ± 0.06 e 3.01 ± 0.06 f carbon (iv) oxide 24 8.53 ± 0.1 a 8.45 ± 0.1 a 8.35 ± 0.2 a 8.46 ± 0.3 a 8.67 ± 0.5 a 8.35 ± 0.5 a carbon (iv) oxide 96 11.00 ± 0.14 a 9.25 ± 0.15 b 10.86 ± 0.22 c 10.00 ± 0.25 d 9.69 ± 0.37 e 11.25 ± 0.17 f values are means ± sem for 20 catfish. a,b,c,d,e,f column values with different superscript are significantly different (p ≤ 0.05). table 2. physicochemical parameters determined in test solutions with crude oil sample containing clarias gariepinus (african catfish) 24 to 96 h into the experiment. concentration duration positive negative 1.2 2.0 2.8 3.6 (mg/l) (h) control control 0.6 1.0 1.4 1.8 ph 24 7.34 ± 0.05 a 7.34 ± 0.07 a 9.50 ± 0.02 b 9.44 ± 0.05 c 9.68 ± 0.02 d 9.70 ± 0.08 d ph 96 5.43 ± 0.08 a 6.34 ± 0.06 b 6.28 ± 0.06 c 5.87 ± 0.08 d 6.43 ± 0.02 e 6.67 ± 0.04 f dissolved oxygen 24 6.21 ± 0.28 a 6.16 ± 0.24 a 6.20 ± 0.13 a 5.96 ± 0.13 a 6.00 ± 0.23 a 6.36 ± 0.33 a dissolved oxygen 96 3.59 ± 0.15 a 4.06 ± 0.14 b 3.29 ± 0.23 c 3.76 ± 0.10 d 3.64 ± 0.09 e 3.36 ± 0.12 c carbon(iv) oxide 24 8.36 ± 0.34 a 8.27 ± 0.23 a 8.69 ± 0.33 a 8.45 ± 0.25 a 8.56 ± 0.23 a 8.76 ± 0.25 a carbon(iv) oxide 96 11.45 ± 0.12 a 10.67 ± 0.14 b 11.50 ± 0.09 a 11.23 ± 0.10 c 11.36 ± 0.12 c 11.48 ± 0.15 a values are means±sem for 20 fishes. a,b,c,d,e,f column values with different superscript are significantly different (p≤0.05). table 3. hematological parameters determined from clarias gariepinus and clarias anguillaris exposed to vary concentrations of crude oil. concentration (mg/l) positive control 1.2 2.0 2.8 3.6 0.6 1.0 1.4 1.8 pcv(%)catfish 39.0 ± 1.45 a 36.0 ± 1.23 a 34.0 ± 1.46 a 30.0 ± 1.45 a 27.0 ± 1.26 a pcv(%)mudfish 38.0 ± 2.18 a 34 ± 1.98 b 31 ± 1.46 b 27 ± 1.50 b 17 ± 1.31 b wbc(10 9 /l)catfish 290.5 ± 2.78 a 285.0 ± 1.56 a 266.0 ± 2.25 a 250.0 ± 2.30 a 233.0 ± 2.56 a wbc(10 9 /l)mudfish 286. ± 2.50 a 282.0 ± 2.15 b 256.0 ± 1.98 b 230.0 ± 3.50 b 222.0 ± 2.75 b rbc(10 12 /l)catfish 2.11 ± 0.05 a 2.08 ± 0.02 a 2.00 ± 0.02 a 1.66 ± 0.05 a 1.00±0.03 a rbc(10 12 /l)mudfish 2.11 ± 0.05 a 2.11 ± 0.07 b 1.85 ± 0.02 b 1.06 ± 0.03 b 0.47±0.05 b a,b, column values with different superscript are significantly different (p≤0.05). dissolution of atmospheric oxygen that comes in contact with the test solutions (basau, 1959; mason, 1994), thereby leading to reduction in the dissolved oxygen in the aquarium. table 3 shows hematological parameters determined from the two fish species after 96 h exposure to different crude oil concentration. there was significant difference (p ≤ 0.05) in all the haematological parameters determined between the two fish species. as the concentration of crude oil increases the values of wbc (white blood cell) also increases. however, decline in the values of pcv (packed cell volume) and rbc (red blood cell) were observed after exposure to increasing levels of crude oil concentrations. there was a significant difference (p ≤ 0.05) in the effect of toxicant on the pcv of both species of fish after 96 hours of exposure to the toxicant. the pcv of the c. anguillaris appeared to be more reduced compared with c. gariepinus at double concentration of toxicant. the significant reduction (p ≤ 0.05) in rbc count may be attributed to cytotoxic effect and suppression of erythropoiesis caused by constituents of the crude oil (suchanek, 1993). this would imply reduction in the level of oxygen that would be carried to the tissues and the level of carbon dioxide returned to the probit transformed responses p ro b it 1.5 1.0 0.5 0.0 lc50=1.22mg/l r sq linear = 0.865 -0.25 0.00 0.25 log of concentration figure1probit1. transformprobit respotransformesoftheresponses96hracutetoxicityofof thecrude96oilonhclacuteriasanguillaristoxicity. of crude oil on c. anguillaris. lc50=2.19mg/l figure 2 probit transform responses of the 96hr acute toxicity of crude oil on clarias gariepinus. figure 2. probit transform responses of the 96hr acute toxicity of crude oil on c. gariepinus. lungs would also be reduced (sunmonu and oloyede, 2008). the major functions of wbc are to fight infection, defend the body by phagocytosis against invasion by foreign organisms and to produce or at least transport and distribute antibodies in immune response. a significant reduction (p ≤ 0.05) in wbc count with increase in crude oil concentration may suggest that the species are exposed to high risk of infections with mudfish being most susceptible based on its comparative level of white blood cell count. figures 1 and 2 show probit transformed response of the 96 h acute toxicity of crude oil on c. anguillaris and c. gariepinus respectively. from the probit analysis, the lc50 of c. gariepinus was 2.19 mg/l (p ≤ 0.05) while the lc50 c. anguillaris (mudfish) was 1.22 mg/l (p ≤ 0.05), suggesting that c. gariepinus showed better ability to resist adverse environmental condition than c. anguillaris. conclusion considering the observations on c. gariepinus in this study based on parameters selected. it is concluded that the specie is relatively more tolerant to high level of crude oil compared to c. anguillaris. references alexandra rr, griffiths, jm (1993). haematocrit basic biochemical methods, 2 nd ed. john wiley and sons inc. new york, pp. 186-189. apha (1992). standards 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3.5. fish physiology and toxicology laboratory. department of zoology and physiology. university of wyoming. longhurt ar (1961). report on the fishries of nigeria. federal department of fishries, ministry of economic development, p. 42. maissonneuvve c, larose s (1988). physico chemical parameters in a fish pond. macmillan publishing company, london, pp. 72-73. mason cf (1991). biology of freshwater pollution 4 th ed longman sci. technical co-publisher, united states, pp 56-76. mason cf (1994). biology of freshwater pollution 6 th ed. longman sci. technica co-publisher, united states pp. 54-70. omoregie e (1998). changes in the hematology of the nile’ tilapia (oreochromis niloticus) under the effects of crude oil. acta. hydrobiol., 84: 287-292. oic (2002). ospar offshore industries curial committee: guidance on the assessment of the toxicity of substances (reference number: 2002-2004). speight jg (1999). the chemistry and technology of petroleum. marcel dekker, pp. 215-216. suchanek th (1993). oil impacts on marine invertebrate populations and communities am. zoologist, 33: 510-523. sunmonu to, oloyede ob (2008). haematological response of african catfish (clarias gariepinus) and rat to crude oil exposure. the internet j. hematol., 4: 1-2. sunmonu to, oloyede ob (2006). changes in liver enzyme activities in african catfish (catfish gariepinus) exposed to crude oil. asian fish. sci., 19: 104-109. swingle h (1961). relationship of ph of pond waters to their suitability of fish culture. congress, 910: 72-75. teugels gg (1986). a systematic revision of the african species of the genus clarias. annual museum report african central science of zoology. 247: 199. wang sy, lum jl, carls mg, rice sd (1994). the relationship between growth and total nucleic acids in juvenile pink salmon (oncorhyncus gorbuscha) fed crude oil contaminated food. canadian j. fish. aqua. sci., 50: 996-1001. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (7), pp. 211-216, july, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the level of arsenic in serum, urine, bile and tissues of goats and camels infected with trypanosoma evansi f. m. youssif 1 , t. hassan 2 and o. s. a. mohammed 3 1 central veterinary research laboratories (cvrl) khartoum-sudan. p. o. box 8067, sudan 2 department of medicine, pharmacology and toxicologyfaculty of veterinary medicine. university of khartoum. p. o. box 32, sudan. 3 department of medicine, pharmacology and toxicologyfaculty of veterinary medicine. sudan university of science and technology. p. o. box 407, sudan. accepted 02 april, 2017 ninety out of hundred nubian goats were experimentally infected with trypanosoma evansi and twenty out of twenty five camels naturally infected with the same parasite were used in this experiment. single i / m doses of cymelarsan were given to four groups at rates of 0.125, 0.25, 0.625 and 1.25 mg/kg. other four groups were also given the drug via i / m route at rates of 0.125 and 0.25 mg/kg / week for two weeks and daily for 8 days. three groups of camels were given cymelarsan in single i / m doses at rate of 0.25, 0.612 and 0.125 mg/kg weekly for three successive weeks. specimens of different tissues, serum, urine and bile were collected immediately after death or slaughter. a weekly slaughter program 14 days post cessation of the drug for surviving animals was conducted for successive five weeks (two goats/ week and one camel / week). total arsenic of cymelarsan r residues detected in samples was measured. the concentration of arsenic was increased in serum, urine, bile and tissues of goats infected with t. evansi and given cymelarsan in single i / m doses (0.125 0.625 mg/kg) and gradually returned to normal by the end of the experiment. in the remaining groups arsenic concentration in collected samples did not returned to normal level. in camels, the amount of arsenic in the different tissues, serum, urine and bile returned to normal by day 42 of last cessation of the drug. we conclude that camels tend to excrete arsenic from the body more rapidly than goats. key words: nubian goats, camels, trypanosoma evansi, cymelarsan, residues (arsenic). introduction trypanosomosis is by far the most important protozoan disease of camels and is probably the most important health problem of all. death may reach 3%, declining milk production; abortion and chronic poor conditions are the classic symptoms of the disease in camels (wilson, 1984). mornet (1954) considered that goats and sheep made up 4% of all the animals infected with trypanosoma congolense. lewis, (1949) reported that goats could be infected when kept close to infected cattle. cymelarsan ® has a rapid trypanocidal activity. trypanosomes are destroyed within a few hours; it achieves a *corresponding author. e-mail: rozavet@gmail.com. peak plasma concentration by about 15 min post-injection, irrespective of the route of injection, after which plasma levels decline rapidly. the absolute bioavailability is a 100% when given by the intramuscular route (bujon, 1990). the product has a short half-life in horses and monkeys, and the toxicological profile or a residue is low. the proposed withdrawal time for meat, milk and edible tissue is provisionally recommended at 2 weeks (raynaud et al., 1989). cymelarsan r in milk and meat was measured by determination of the total arsenic in infected camels in kenya using recommended route (sones, 1991). groups of arsenical compounds (phenyl arsenoxides, arsphenamines, arsonic acids, acid-substituted phenylarsenoxides and inorganic arsenicals) in plasma were elucidated by detection of total arsenic in urine, stool, blood and tissues youssif et al. 212 (ralph et al., 1944). no significant difference between the pharmacokinetics and the routes of administration or the different formulation was reported (toutain, 1991). the aim of this study was to investigate the level of arsenic in serum, urine, bile and tissues of goats and camels infected with trypanosoma evansi and given cymelarsan at different dosages rate and frequency. sudan has an immense animal wealth, which satisfies all local needs of meat and milk and produces an export surplus constituting 20% of foreign currency earnings (aoad, 1992). materials and methods animals nubian goats hundred nubian goats of both sexes, 8 – 10 month-old and weighing 9 10 kg, were purchased from sheikh abu zaid market, omdurman, khartoum state. animals were housed in pens at the college of animal production and veterinary medicine, sudan university, hillat koko. each animal was fed daily on green forage consisting of 3 kg lucerne (medicaqo sativa), 1.5 kg sorghum (sorghum vulgare) and 2 kg millet ( pearl millet) one time weekly as a concentrate, and had free access to water. twenty goats were chosen randomly. group (1) was uninfecteduntreated (control negative group), while group 2, infected with t. evansi, was infected-untreated (control positive group). the remaining animals were infected with t. evansi and were used for drug treatment. they were divided randomly into ten groups of 10 goats each. each goat in groups 3, 4, 5 and 6 was given single intramuscular (i/m) dose of cymelarsan ® at the rate of 0.125 mg/kg (halftherapeutic dose), 0.25 mg/kg (therapeutic dose), 0.625 mg/kg (two and half times the therapeutic dose), 1.25 mg / ml (five-times the therapeutic dose), respectively. each goat in groups 7 and 8 was given cymelarsan ® intramuscularly (i / m) at rate of 0.125 mg/kg (half-therapeutic dose) twice/week for two weeks, 0.125 mg/kg (half-therapeutic dose) daily for 8 days, respectively. goats in groups 9 and 10 were each given the drug (i / m) at the rate of 0.25 mg/kg (therapeutic dose) twice / week for two weeks and 0.25 mg/kg (therapeutic dose) daily for 8 days, respectively. camels (camelus dromedarius) twenty-five onehumped camels (camelus dromedarius) 1 – 3 years-old, of both sexes and weighing (350 400 kg) were obtained from el gadarif state and were stabled in elmewelh market pens, omdurman, khartoum state. each camel was fed daily on 4 kg lucerne (m. sativa), 3.5 millet (p. millet) and 2.5 kg sorghum (s. vulgare); with free access to water. they were divided randomly into five groups of 5 camels each. group 1 was uninfected-untreated (control negative group), while group 2 was naturally infected-untreated (control positive group). the remaining animals were naturally infected with t. evansi. each camel in groups 3 and 4 was given a single intramuscular dose of cymelarsan at the rate of 0.25 mg/kg (therapeutic dose), and 0.6125 mg/kg (two and halftherapeutic dose), respectively. camels in group 5 were each given a single dose of the drug at rate of 0.125 mg/kg (half-therapeutic dose) weekly for three successive weeks. fourteen days post cessation of the drug, a weekly slaughter programs for survival animals (2 goats and camel/week) was conducted for five successive weeks. parasite and infection albino rats of two months old, and weighing 250 g, were inoculated intraperitoneally with 0.2ml camel’s blood containing 3 5 parasite/field. these camels were infected naturally with t. evansi strain gad trip (1) which was obtained from el gadarif state, eastern sudan. when parasitaemia developed in rats, each goat (except goats in group 1) was injected intravenously with 0.75 ml of rat's blood containing 5 x 10 5 organisms. the parasites were activated by adding phosphate glucose solution (pgs) buffer before inoculation. collection and preservation of samples serum each animal was bled from the jugular vein at 1, 3, 24 h and 3 days, 7, 14, 21, 28, 35, 42 days post-treatment in a test tube con-taining no anticoagulant. samples were left to clot, centrifuged at 3000 rpm and were collected and kept in a deep freezer at -20 o c for determination of residues of cymelarsan. urine, bile and tissues a weekly slaughter program for survival animals was conducted for successive five weeks (2 goats and camel / week) after cessation of the drug. samples of urine and bile were taken in plain containers (wols, u.k). specimens of heart, brain (cerebellum and cerebrum), kidney, spleen, liver, lung, ovary, uterus, testis, muscle, fat and site of injection, were kept in plastic containers. collected samples of urine, bile and tissues were kept in a deep freezer at -20 o c for determination of residues of cymelarsan. determination of total arsenic as residue of cymelarsan total arsenic residues of cymelarsan were measured by aoac, (1995) method using spectrophotometer (unicam 8625 / u.v. vis u.k.) at wavelength 450 nm. statistical analysis all data were computerized using mstat-c program (michigan state university), for the analysis of variance and for means separation. results goats the infected untreated goats group 2 died 11days post infection, while goats in groups 3 and 10, died 15 days post treatment (pt). goats in groups 6, 7, 8 and 9 died 17 days pt, goats in groups 7 and 8 died 28 31pt and goats in group 9 died 22 days pt, and animals in groups 1, 4 and 5 survived till the end of the experiment (day 56 post treatment). table (1) summarizes the concentration of arsenic in serum, tissues, urine, and bile in goats infected with t. evansi and treated with single or multiple dosages of 213 afr. j. food sci. res. table 1. the concentration of cymelarsan arsenic in different organs ( µg / g) and serum, bile and urine (µg / ml) in nubian goats infected with t. evansi and given single or multiple dosages of cymelarsan (m± se). organs/ serum liver kidneys heart lungs spleen cerebellum cerebrum fat site leg uterus ovary testis bile urine dose of muscle injection group (1) 0.07 a 0.07 group (2) 0.07 a 0.07 group (3) 0.10 0.2±0.22 0.07 0.07 0.07 0.07 0.07 0.07 0.1±0. 0.2±0.0/ 0.07 0.07 0.07 0.07 0.07 0.07 0.125mg/kg ±0.14 b / 0.07 02/ 0.07 0.07 group(4) 0.12±0.1 3± 0.0/ 5±0.2/ 0.1± 0.07 0.07 0.07 0.07 0.2 0.5±0.1/ 0.07 0.7±0.0/ 0.1±0.0/ 0.2±0.0 1.5±0.0/ 0.07 0.25mg/ kg 4 b 3.5±0.0/ 0.3±0.2/ 0.01 ±0.01/ 0.07 0.07 0.07 / 0.07 0.07 0.07 0.07 0.07 group (5) 0.15 5±0.0/ 3±0.1/1± 0.07 0.07 0.07 0.1 ±0.0 0.5±0.02/ 0.1±0. 0.5±0.2 0.07 0.5±0.0/ 0.2±0.1/ 1.5±0.0 1±0.1/ 0.07 0.625 ±0.19 b 3.5±0.0/± 0.2/0.5± / 0.07 0.07 0/ /0.2±0.0/ 0.5/±0.0 0.1±0.02/ 1/ 0.07 mg /kg 0.0/ 0.0/ 0.07 0.1±0. 0.1±0.0 /0.2±0.00 0.07 0.07 0.07 0/ / 0.07 1/ 0.07 0.07 group (6) 2.7 30 ±1.1 35±0.2/ 40± 20±1.0 20±0.2/15 10±0.4/ 15±0.1/ 3±0.1/ 5±0.01/ 25±0.2/ 10±1.0/ 5±0.1/ 15±0.2/ 10±0.01/ 5±0.01/50 1.25mg/kg ±0.09 c /25±1.0 30 ±0.4 0.1/33 1/25±1 ± 1±0.1 10± 0.3 2± 0.0 5.5 ±0.2 20 ±0.5 8± 0.7 5± 0.0 20 ±0.3 15 ±0.02 .01 ±0.9 .2 0.1 group (7) 2.5 50±1.5/ 70±0.5/ 30±1.5/ 15±0.9 40±0.3/35 10±0.4/ 5± 0.4/ 50±0.4 80±0.2/ 40±0.3/ 20±0.2/ 10±1.0/ 30±0.5/ 20±0.2/ 25±0.01/2 0.125 ±0.02 c 60.5± 1.3 85± 0.2 35 ±1.2 / ± 0.5 15 ±0.1 5± 0.7 / 95 ±0.3 30± 0.4 25± 0.4 15± 1.2 25 ±0.4 40 ±0.3 5 ±0.01 mg / kg 20±0.3 45 0.5 twice / week for 2 weeks group (8) 105±2.2/ 130 ±0.3 90±1./ 30±0.5 80± 0.2/ 75±0.2/ 45±0.8/ 105±0. 130±0.1/ 100 50±0.3/ 30±1.4/ 60±0.6/ 50±0.7/ 25±0.0/ 0.125 mg 3.5±0.01 c 140± 2.2 /110±0.2 95 ±1.4 / 85 ±0.5 70± 2.5 40 ±0.3 11/ 120 ±0.2 ±2.1 55 ±0.3 40± 1.1 75± 0.7 70± 0.5 25±0.01 / kg daily 40±0.7 40±0.6 /105± 2.3 for 8days group (9) 3.00 100±2.4/ 140±0.5/ 160±0. 85±1.2 120± 115±3.4/ 160±0.8/150 200±3. 180±0.4/ 250±2.3/ 90±0.6/ 110±1.3/ 120±0. 100±1.1/ 15±00.33/ 0.25 mg / ±0.001 c 105± 2.3 155± 0.6 1/140± / 0.1/120± 180 ±3.5 ±0.4 0/150 200± 0.5 300± 2.0 100± 0.8 120 ±1.5 3/ 110± 1.0 16±0.46 kg twice / 0.7 801.9 0.2 ±1.5 150± week for 0.4 2 weeks group 250±1.9/ 300±1.7/ 250±2. 120±1. 350±2.3/3 350±3.1/ 150±0.1/160 305±1. 450±0.1/ 300±2.5/ 200±1.3/ 150±1.1/ 120±1. 180±1.2/ 25±00.37/ (10) 0.25 5.9±0.03 d 300±2.5 305± 1.8 4/300± 1/130± 60± 2.7 250 ±3.2 ± 0.1 1/ 250 ±0.2 250 ±2.2 250± 1.4 180± 1.7 2/ 200 ±1.5 30±0 0.7 mg / kg 2.0 2.0 355± 200 daily for 1.7 ±1.5 8days the different letters in one columns show significant changes p < 0.05, week 1 (w1) = 14 day post treatment (d.p.t), w2 = 21 d.p.t, w 3 =28 d.p.t, w 4 = 35 d.p.t and w 5 = 42 d.p.t. figure 1. the mean of arsenic in serum of t. evansi infected nubian goats and treated with single dosages or cymelalarsan. table 2. the concentration of cymelarsan arsenic in different organs (µg / g), serum and urine (µg / ml) in camels infected naturally with t.evansi and given single or multiple dosages of cymelarsan within the withdrawal period (m ± se). groups/organs group (1) group (2) group (3) group (4) group (5) 0.125 mg / kg 0.25 mg / kg 0.625 mg / kg weekly / 3weeks serum 0.07 0.07 2.3±0.01 8.3±0.02 5.6±0.00 liver 0.07 0.07 0.07 0.07 0.07 lungs 0.07 0.07 0.07 0.07 0.07 kidneys 0.07 0.07 10 ±0.05 /3 ±0.01 / 0.07 10 ±0.01 /5 ±0.02 / 0.07 10 ±0.02 /4 ±0.00 / 0.07 heart 0.07 0.07 0.5 ±0.01 /0.6±0.00 / 0.07 0.5±0.01 /0.5±0.00 / 0.07 0.8±0.00 /0.3±0.00 / 0.07 spleen 0.07 0.07 0.1 ±0.02 / 0.07 0.5 ±0.00 / 0.07 0.5 ±0.01 / 0.07 cerebellum 0.07 0.07 0.07 0.07 0.07 cerebrum 0.07 0.07 0.07 0.07 0.07 fat 0.07 0.07 0.5 ±0.05 / 0.07 0.5 ±0.00 / 0.07 0.5 ±0.00/ 0.07 site of injection 0.07 0.07 5 ±0.2 / 0.07 5 ±0.02 / 0.07 5 ±0.02 / 0.07 leg muscle 0.07 0.07 0.07 0.07 0.07 uterus 0.07 0.07 2.2 ±0.01 / 0.07 2.5 ±0.02 / 0.07 2.02 ±0.01 / 0.07 ovary 0.07 0.07 0.5 ±0.02 / 0.07 0.7 ±0.00 / 0.07 0.8 ±0.00 / 0.07 testis 0.07 0.07 0.2 ±0.01 / 0.07 0.5 ±0.01 / 0.07 0.9 ±0.01 / 0.07 cymelarsan. goats in groups 6-10 showed increased amounts of arsenic in the different tissues at levels up to (450 µg/g) while, urine(30 µg/ml), bile (200 µg/ml)and in serum(5 9 µg/ml) did not return to normal levels( 0.07 µg/g) or ( 0.07 µg/ml) till the end of the experiment. the increase was found to depend upon the dose and the frequency of drug administration. goats of groups (3 5) had values ( 0.07 µg/g) in tissues and ( 0.07 µg / ml) in serum (figure 1), urine and bile by the end of experiment (day 42 pt) equal to that of control goats in groups 1and 2. camels no death was observed in experimental camels naturally infected with t. evansi and treated with single or multiple dosage of cymelarsan, till the end of the experiment but the infected untreated control group (group 2) died 22 days post infection (22 days after found the camels in the field). table (2) summarizes the concentration of arsenic in tissues, serum, and urine in camels. all groups of treated animals tend to record 0.07 µg/ml arsenic in serum (figure 2), urine and 0.07 µg / g in different tissues samples by the end of the experiment. discussion the purpose of the study was to investigate and compare the residues of the drug cymelarsan (arsenical compound) in the different tissues, serum, bile and urine in youssif et al. 214 215 afr. j. food sci. res. µ g /m l 16 14 12 10 8 6 4 2 0 1 2 1h 3h 24h 3 7 14 21 28 35 42 days hours days group (1)control negative group (1) control positive group (3) 0.25mg/kg group (4)0.625mg/kg group (5)0.125 mg/kg weekly/3weeks figure 2. the mean of arsenic in serum of t. evansi infected camels and treated with single or multiple doses of cymelarsan nubian goats and one-humped camels infected with t. evansi. the study also investigated the relationship between the accumulation pattern of the drug and its toxicity effect in the two species of animals. ralph et al. (1944) mentioned that the toxicity of arsenical compounds is primarily a function of the degree to which they are bound by tissues, and that the red blood cells form a valid model for the body tissues constituting a reliable index of systemic toxicity, as the sequence of events in the blood stream reflects what occurs in body tissues. after leaving the blood, toxic compounds were firmly bound by the organs and relatively high levels were found in the liver and kidneys 48 h after injection. this supports our findings that goats tissues such as liver, spleen, kidneys, heart and lungs, can accumulate considerable amounts of arsenic as these organs contain high concentrations of the enzymes containing thiol groups. the accumulation of arsenic at high levels in the muscles and site of injection is comparable to the findings of youssif, (2005) who studied the toxic effect of different dosages of cymelarsan in nubian goats characterized by irritating effect of arsenic on tissues. it was noticed that in camels the serum, liver, kidneys, and site of injection contained the highest values compared to other organs. sones, (1991) studied the residues of as in infected camels in kenya and treated with the recommended dose of cymelarsan and found that the residues of total arsenic were detected in fat, liver and kidneys were less than 2 mg / kg with no residues in plasma of one camel 7 days and others slaughtered on days 14 or 21. in goats which received half recommended therapeutic dose twice a week for two weeks or once weekly for three weeks in camels the picture is surprising where goats recorded higher values of as than that recorded in camels. this may be due to species difference. cymelarsan with arsenical atoms, has a high affinity to bind to rbc in goats compared to that of camels; this was supported by the increased level of arsenic in goats especially those which received the recommended dose twice a week for two weeks and also by the high concentration of as in kidneys and urine in goats compared to that of camel. this might be attributed to unknown other route of excretion of arsenic. excretion could be through saliva or faeces or as might be deposited in bones. another possible explanation is that camels might have a high capacity to excrete arsenic through the kidneys rapidly in less than 28 days post treatment or in goats drug accumulation might have taken place due to the fact that large and repeated doses are beyond the capacity of the vital organs responsible for the arsenic detoxification and excretion. the nervous system contains large amounts of fats and repeated administration of the drug revealed a tendency of the drug to accumulate in the fat and nervous tissues of the goats more than camels. the accumulative effect in goats is more evident than in camels which might explain why camels can tolerate five times the recommended therapeutic dose and also supports the findings of bujon, (1990) who mentioned that camels can tolerate 12 20 times the recommended therapeutic dose. high doses of inorganic arsenical compound given to pregnant experimental animals produced various malformations in fetus and offspring. youssif et al. 216 however, such effects have not been noted in humans with excessive occupation exposure to arsenic compounds (curtis and john, 2003).joseph, (2001) mentionned that arsenic produces pathological effect on the reproductive system, which is confirmed by our findings that testis and the uterus have the tendency to accumulate as in large quantities. we conclude that goats are more susceptible to accumulation of as in the tissues and express clinicopathological performance than camels. further studies are needed to elucidate the physiological pathways that make camels more tolerable to excess accumulation of as than goats. references aoac official methods of analysis, (1995). drugs and feed additives in animal tissue, charles j. barnes, chapter 23, edit. 2. supplement march (1993). aoad (1992). arab orgnization for agriculture development. the role of animal resources in arab food security, fifth vet. conf. 12-16.jan. 16. bujon b (1990). cymelarsan, a new trypanocide for treatment of camel trypanosomosis. rhône mérièux, lyon. pp. 1-18. curtis dk, john, bw (2003). essentials of toxicology. mcgraw-hill co. inc. new york-usa. p. 352. joseph dr (2001). the practical veterinarian, veterinary toxicology. first edit. butter worthheinemann. pp. 87-314. lewis d j(1949). sudan notes and records.30: 179-211. mornet p (1954). the pathogenic trypanosomes of french west africa. their distribution and frequency and the incidence of domestic animal infestation. isctr 5th meeting, pretoria. pp. 37-51. ralph b, hogan md, harry eagle md (1944). the pharmacologic basis for the widely varying toxicity of arsenicals. j. pharm. exp. ther. 80: 93-113. raynaud jr, tiytaubm ol, baltz, p, senses kr (1989b). plasmakinetic , toxicity and tolerance of cymelarsan in horses, cattle and camels. twentieth asc-trc meeting, mombassa, kenya, april 10 14. sones k (1991). residues of total arsenic in meat and milk following treatment with cymelarsan in the camels. experimental report. rhone merieux, lyon-france. toutain pl(1991). bioequivalence study of cymelarsan in the dromedary. experimental report. rhone merieux, lyonfrance. wilson rt (1984). the camel. first edit. longman group ltd. london and new york, pp. 122-127. youssif mf (2005). pharmacotoxicity of some trypanocidal drugs in food animals (camelus dromedarius and nubian goats). a thesis submit for ph.d. university of khartoum-sudan. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (3), pp. 267-270, march, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper production of kokoro with better nutritional content and sensory quality from maize flour p. i. uzor-peters 1 , n. u. arisa 2* , c. o. lawrence 1 , n. s. osondu 1 , and a. adelaja 1 1 department of food technology, university of ibadan, ibadan, nigeria 2 department of food technology, bells university of technology, ota, nigeria. accepted 09 july, 2017 kokoro a local maize snack was made from substitution of maize flour with either defatted soybean or groundnut cake flour (9:1, 7:3, 5:5, 3:7 and 1:9 substitution ratios) were evaluated for proximate and sensory qualities. increase in substitution ratio produced kokoro with higher nutritional content (28.46 and 35.10 percent protein for those made from 1:9 substitution ratio respectively). increase in substitution with the two substitutes progressively increased protein and fat content but reduced the ash, crude fibre and carbohydrate contents. more over the crispiness and over all acceptability of the kokoro decreased with increasing substitution. products made from 9:1 substitution were well accepted and compared favourably with whole maize product. key words: maize, soybeans, groundnuts, defatted, substitution, nutrition. introduction cereal grains are used for production of different classes of foods; these include breakfast cereals such as corn flakes, breads, and pastries brewing of both alcoholic and nonalcoholic drinks. in different cultures and societies staple foods are also produced for use as accompa-niments for soups, gravies and stews and they supply the basic energy requirement of the consumers. . they are also used for the production of different snack foods which are eaten to prevent hunger before main meals or just (as relish) for the fun of eating them, but they are deficient in some essential amino acids like lysine. these amino acids can be supplied to the food by compli-menting the maize with legumes such as groundnuts and soybeans which are better sources of the sulphur amino acids (okaka, 2005). there is usually improved balance of amino acids in the products made from such combina-tions (ameida-dominguez et al., 1990). most often snack foods do not provide nutrients in adequate quantities needed by the body (omueti and morton, 1996). this may be due to their composition or due to the production process they went through. what ever is responsible for their poor nutritional content, it is necessary to ensure that every food consumed by an *corresponding author. e-mail chizaramekpere2006@yahoo.co.uk individual contains required nutrients in adequate amounts. this is especially important due to the fact that many people now work outside their homes and are becoming more dependent on snacks for the supply of part of their daily nutritional requirements. it is therefore necessary to produce a highly acceptable snack with high nutritional quality that could be useful in nutritional programmes to combat malnutrition and nutrient deficien-cies (rosa et al, 2003). kokoro is a popular local snack made and consumed during the day, alone or with roasted groundnuts, and ‘washed down’ with soft drinks or just water by the people of south western nigeria. it is made from maize flour which contains primarily carbohydrates. as a product that is consumed on such a wide scale it would be important to enhance its nutritional value. addition of vegetable protein such as textured vegetable protein could be one way of raising the nutritional value of the product by intro-ducing more protein into it (rosa et al, 2003). the possibility of producing acceptable kokoro, with better nutritional content and sensory quality from maize flour mixed with either defatted groundnuts or soybeans flour was therefore investigated. this was hoped, will increase the nutritional content of the snack as well as increase the use to which the defatted products (soybean and groundnuts cakes) can be put to, in addition to their use as animal feed. table 1. proximate composition of kokoro treatment subst moisture protein content fat content ash content cf content cho content ratio content (%) (%) (%) (%) (%) (%) whole mazie 1 7.58+0.376 7.03+0.473 1.43+0.441 1.55+0.318 1.26+0.441 76.19+0.408 means of triplicate determinations and standard deviations note: cho, carbohydrates cf, crude fibre maize (zeamays indenta) cleaned mill (dry milling 100µm size mesh) mixing maize with either defatted groundnut or soybean cake flours boil (continuous stirring) cool (40 o c) knead (5 minutes) roll into shape fry (3 minutes) cool and pack 1:9 (figure 1). the kokoro snacks were made from the produced from 100% maize flour. kokoro production was as follows: the 250 g of composites flour, 30 g of sugar and 10 g of salt were gently stirred into 500 cm 3 boiling water in a stainless steel pot. the mixture was cooked with continuous stirring until stiff dough was formed. the dough was cooled to a temperature (40 o c) at which it could be kneaded by hand for 5 min. the kneaded dough was cut into pieces, rolled into cylindrical shapes and fried in 1000cm 3 of hot refined vegetable oil for 3 min and then cooled and packed in sealed polyethylene bags. proximate analysis moisture and protein content analysis were determined using the procedure described by aoac (1990). the ash content was determined using the procedure described by pearson (1976). the sample (5g) was dried in an oven at 100 o c for 2 h. the dried sample was placed in a furnace and ignited at 550 o c for 2.5 h, cooled and weighed. the soxhlet extraction method was used for the fat content determination. oil in 5 g sample was extracted using hexane in soxhlet extraction equipment for 2.5 h under reflux. the crude fibre content was determined using the procedure described by kirk and sawyer (1991). carbohydrate content was determined using the method described by pearson, (1976). kokoro figure 1. flow chart of kokoro production materials and methods maize (zea mays indentata) was obtained from bodija market ibadan, oyo state, nigeria. partially defatted soybeans and groundnut cakes were sourced from adom feed mills limited and sudit oil and chemical limited ibadan, nigeria, respectively. cleaned maize was dry milled using a hammer mill and sieved with a 100µm sieve and stored for use in polyethylene bags at room temperature (28 o c). the partially defatted soybeans and groundnut cakes were separately milled with a hammer mill, and classified using a 100 µm sieve and then stored in polyethylene bags at room temperature (28 o c) for 1 week within which they were used for kokoro production. kokoro production composite flours of the maize flour and legume flour (partially defatted soybean or groundnut flour) were produced using different levels of the legume flours in the following ratios: 9:1, 7:3, 5:5, 3:7 different composites flours a control kokoro sample was also and sensory evaluation a nine point hedonic scale and multiple comparison preference test described by larmond (1977) was used to test the acceptability of the kokoro made from maize and defatted soybean or groundnut cake flour against a whole maize kokoro.. a panel of 10 judges who were conversant with the sensory qualities of kokoro was chosen. the panelists were each given six samples at a time to evaluate the effect the different substitution ratios will have on the appearance, aroma, taste, crispiness, and over all acceptability of the kokoro at 5% confidence level. results and discussions the proximate composition of kokoro made with whole maize flour is shown in table 1. the production of kokoro with 1:9 ratio flours (maize to either defatted soybean or groundnut cake flour) resulted in products with 14% or 11% (tables 2 and 3) protein contents respectively. this showed an increase in the level of protein in the products .percentage protein in the products increased as the level of flour substitution increased table (35.10 and 28.46% protein respectively for 90% substitution with defatted soybean and groundnut cake flours). similar trend was shown by the fat content. increased protein and fat con uzor-peters et al. 268 269 afr. j. food sci. res. table 2. effect of partially defatted groundnut cake flours on the proximate composition of kokoro treatment subst ratio moisture protein fat content ash content cf content cho content content (%) content (%) content (%) (%) (%) (%) (%) maize /dgcf 9:1 5.79+0.334 11.82+0.425 13.00+0.163 0.98+0.358 0.90+0.358 67.51+0.425 7:3 7.52+0.237 15.32+0.245 16.58+0.367 0.95+0.261 0.63+0.089 59.00+0.628 1:1 9.64+0.318 15.32+0.245 18.74+0.278 0.92+0.141 0.52+0.089 49.05+0.249 3 :7 11.24+0.327 26.71+0.412 29.97+0.278 0.94+0.384 0.22+0.171 30.92+0.396 1 :9 8.82+0.335 28.46+0.327 34.06+0.325 0.90+0.365 0.16+0.065 27.06+0.433 means of triplicate determinations and standard deviations note: dgcf, defatted groundnut cake flour cho, carbohydrates cf, crude fibre table 3. effect of partially defatted soybeans on the proximate composition of kokoro treatment subst ratio moisture protein content fat content ash content cf content cho content content (%) content (%) (%) (%) (%) (%) (%) maize 9:1 7.80+0.335 14.00+0.140 12.70+0.466 1.80+0..310 1.12+0.279 62.68+0.335 /dgcf 7:3 7.60+0.190 22,30+0.272 18.40+0.360 1.00+0.197 0.88+0.141 49.82+0.358 1:1 5.20+0.180 26.20+0.082 21.90+0.114 0.84+0.253 0.82+0.256 45.00+0.274 3 :7 4.90+0.220 31.07+0.348 23.20+0.237 0.84+0.253 0.54+0.171 39.50+0.204 1 :9 3.40+0.290 35.10+0.224 26.8+0.343 0.72+0.188 0.49+0.139 33.49+0.269 means of triplicate determinations and standard deviations note: dscf, defatted soybean cake flour cho, carbohydrates cf, crude fibre contents were due to the incorporation of either the defatted soybean or groundnut cake flours in the in the formulation. also this inclusion may have also increased the oil absorption capacity of the dough thereby resulting in the product’s higher fat content of 26.80 % and 34.06 for 9:1 ratio flours (maize to either defatted soybean or groundnut cake flour) respectively. however, the higher level of substitution resulted in lower ash, crude fibre and carbohydrate contents of the resultant kokoro. the reduction in the carbohydrates was due to the increasing level of flour with higher protein and fat contents and the tendency of the dough mixture to absorb more oil than that made solely from maize flour (table 2 and 3). the reduced crude fibre and oil content in the products may have made the products to have reduced level of crispiness. sensory evaluation increase in the level of substitution of the maize flour with either defatted soybean or groundnut cake flour resulted in decrease in the acceptability of the aroma rating of the kokoro especially for that made with defatted soybean cake flour (table 4 and 5). there was no significant difference between the products made from 1:9, 3:7 and 5:7 ratios of maize to defatted soybean cake flour in terms of taste and aroma. the level of crispiness decreased with increase in substitution for both treatments (defatted soybean or groundnut cake flour). there was significant difference in the crispiness of products made from 1:9, 3:7 and 5:5 substitutions ratios. the taste of the products made from 1:9 ratios of maize to either defatted soybean or groundnut cake flours was preferred. in terms of overall acceptability of the products, those made from 1:9 ratios of maize to either defatted soybean or groundnut cake flours were well accepted in terms of all the sensory characteristics studied. however, products made from 3:7 ratio of maize to defatted groundnut cake flour received good aroma ratings (table 5). the acceptability of the kokoro made from 1:9 and 3:7 substitution could have been due to the fact that at this level of substitution the taste of legumes did mask the taste of the maize but just complimented it, giving it a slightly more desirable different taste compared to kokoro made with whole maize flour this is in line with the findings of onweluzo and morakinyo (1997) who said that flour prepared from seeds of afzelia africana dehulled by different treatments used to replace 10, 20, 30, 40 and 50% wheat flour in biscuits and doughnuts had sensory scores of high overall acceptability for products made from 10–30% level of substitution. in terms of the crispiness and the general appearance of the kokoro at that level (1:9 and 3:7) of substitution ratio they still resembled those of whole uzor-peters et al. 270 table 4. effect of partially defatted soybean cake flour on the sensory characteristics of kokoro treatment subs.ratio appearance aroma taste crispiness oaa maize/dscf 9:1 5.10 a 5.10 a 5.10 a 4.60 a 5.20 a 7:3 4.30 a 4.30 a 4.50 a 3.80 b 4.40 a 1:1 3.00 b 3.70 b 3.80 b 3.20 b 3.90 b 3 :7 2.40 c 2.60 b 2.40 c 1.50 d 2.10 c 1 :9 1.30 d 1.80 c 1.80 c 1.26 d 1.40 d note: dscf: defatted soybean cake flour subs. substitution oaa, over all acceptability table 5. effect of partially defatted groundnut cake flour on the sensory characteristics of kokoro treatment subs. ratio appearance aroma taste crispiness oaa maize 9:1 5.00 a 4.93 a 5.40 a 4.80 a 4.90 a /dgcf 7:3 3.37 b 4.43 a 3.62 b 3.10 b 3.50 b 1:1 2.25 c 3.31 b 2.93 b 2.00 c 2.40 c 3:7 1.50 d 2.37 b 1.93 c 1.40 d 1.50 d 1:9 1.18 d 2.06 b 1.37 d 1.10 d 1.10 d dgcf: defatted groundnut cake flour subs. substitution oaa , over all acceptability maize products. however as the substitution level increased the flovour, aroma, taste, crispiness and general appearance of the products became different from the standard whole maize kokoro. this difference could have been as a result of; the higher oil content which would have affected the taste, flavour, crispiness and appearance of the products, the increase would have also resulted in increased masking of the taste, aroma and flavour of the kokoro especially those made from substitution with defatted soybeans which would have become increasingly more beany in flavour, aroma and taste. conclusion kokoro with higher nutritional content can be made with composite flours from defatted soybean or groundnut cake flour, but the maximum level of substitution which is sensorially acceptable is a ratio of to 3 maize:7 either defatted soybeans or groundnut cake flours. references ameida-dominguez ng, valencia me, higuera-capra i (1990). formulation of corn based snack with high nutritional and biololgical and sensory evaluation. j. food sci. 55. 228. aoac (1990). association of official analytical chemist, official method of analysis 14 th edition, washington. kirk rs, sawyer r(1991). pearson’s composition and analysis of foods. 9 th edn. longman group. p 646. larmond,, e. (1977). laboratory method for sensory evaluation of foods. canadiandepartment of agricolture (1637). ottawa. pp. 7479. matz, s. a. (1976). snack food technology.avi publ. company,westpoint connecticut. pp. 337-339. okaka, jc (2005). basic processing of grain cereals and legumes in: handling, storage and processing of plant foods. ocj academic publishers, enugn, nigeria. pp. 30-60 omueti o, morton id (1996).development by extrusion of soybean snack sticks a nutritionally improved soya-maize products based on the nigeria snack (kokoro). inter. j. food sci. nutri. 47: 5-13. onweluzo jc, morakinyo ao (1997). effect of pre-dehulling treatments on the composition of seeds of the legume afzelia africana and its potential use in pastries. j. plant foods for human nutri. springer netherlands. 50 (3): 203-210. pearson d (1976). chemical analysis of foods.7 th edition, churchill livingston, edinburgh, united kingdom. rosa n, chávez-jáuregui rac, maria em, pinto es, jose aga (2003). acceptability of snacks produced by the extrusion of amaranth and blends of chickpea and bovine lung. international j. food sci. technol. 38(7): 795-798. usda (1971) textured vegetable products. notice 209. food and nutrition service, washington d.c. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (4), pp. 001-008, april, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review the role of dietary phytase in formulation of least cost and less polluting fish feed for sustainable aquaculture development in nigeria u. u. gabriel1*, o. a. akinrotimi2, p. e. anyanwu2, d. o. bekibele2 and d. n. onunkwo2 1 department of fisheries and aquatic environment, rivers state university of science and technology, p. m. b. 5080, port harcourt, nigeria. 2 african regional aquaculture centre/nigerian institute for oceanography and marine research, p. m. b. 5122, port harcourt, nigeria. accepted 16 january, 2019 the production of fish meal in nigeria from the wild sources has for some period now been dwindling and when available are very expensive, because most of the fish meal used in fish feed formulation are imported. hence viable alternatives have to be found for the sustainability of the aquaculture industry in the country. plant based protein sources, which are relatively cheap, readily available and easily accessible hold the solution to this, but with limitations in utilization, due to the presence of phytic acid, an anti-nutritional agent present in virtually all plant ingredients. phytic acid reduces bioavailability and digestibility of nutrients like proteins, phosphorus and other minerals, there by promoting accumulation of dissolved solids which ultimately leads to pollution. hence, the need to create awareness of the efficiency of phytase treated diets, for the survival of the industry in the nearest future is imperative for the overall success of aquaculture venture. the role of dietary phytase in the formulation of fish feed using plant based protein sources which is cost effective, and reduces the incidence of aquatic pollution by making the nutrients and minerals in the feed more available to fish are thoroughly discussed in this paper. key words: fish feed, phytase, cost effective, diet, sustainable aquaculture. introduction the last few years has witnessed spectacular growth in aquaculture, in nigeria, due to spurred interests of many people in fish farming, and this will continue to play an increasingly important role in meeting the demand for fish (aifp, 2004). most production in nigeria is realized from pond based, tanks and water re-circulatory culture systems using polyculture farming techniques. however, the bulk of high-value fresh and brackish water finfish in these parts of the world are produced by intensive farming systems using inputs in the form of nutritionally complete formulated diets (hassan, 2001). according to heindl (2002) about 8 million tonnes of fish are produced in china alone with the use of mixed or manufactured feed. while fagbenro et al. (2003) reported about 0.46 million *corresponding author. email: ugwemg@yahoo.com tonnes of fish was produced in nigeria by using artificial diet. as aquaculture production becomes more and more intensive in nigeria (table 1) . fish feeds will be a significant factor in increasing the productivity and profitability of aquaculture. according to jamu and ayinla (2003) feed management determines the viability of aquaculture as it accounts for at least 60 percent of the cost of fish production. the level of growth and intensification witnessed in aquaculture in recent times has raised several issues that need to be addressed for the sustainability of the industry. the high cost of fishmeal due to depletion of catch from the wild and competitive demand from other industries such as poultry and livestock, necessitating the need for the development of fish feed from high quality inexpensive sources and methods for reducing the level of anti-nutritional factors to the barest minimum (baruah et table 1. domestic fish production by section (tonnes). year artisanal industrial aquaculture 1990 291,864 36,226 15,840 1992 283,943 39,365 20,041 1994 201,176 30,486 27,112 1996 180,112 26,117 49,364 1998 340,116 27,114 86,122 2000 120,121 23,121 121,112 2002 100,126 21,121 300,114 source: adapted from eyo (2003) table 2. fish feeds production (tones) in nigeria feed type farm-made commercial total tilapia 14,258 6,554 20,812 catfish 10,552 4,206 14,758 total 24,810 10,760 35,570 source: fagbenro et al. (2003). et al., 2004). interestingly, cheng and guillaume (1984) reported a beneficial effect of purified phytic acid on growth and exoskeleton development in shrimp penaeus japonicus. similarly, mcclain and gatlin (1988) reported reduced zinc bio availability in tilapia oreochromis aureus fed 1.5% phytate; fish growth was improved compared with control fish fed with no phytate. more over, recent studies with mammals, suggested that phytic acid have a beneficial effects within the animal body by suppressing the incidence of colonic cancer by acting as a food antioxidant and forming iron-chelates that inhibit ironcatalyzed hydroxyl radical formation and lipid peroxidation (empson et al., 1991; graf and eatin, 1993; tacon, 1995). hence, this paper critically discusses dietary phytase as one of the best options in formulating a cost effective growth promoting and low polluting fish feed for sustainable aquaculture development. fish feed development the pivotal role of nutrition in aquaculture cannot be over emphasized as demonstrated in several studies (nrc, 1983; falaye, 1983; adiukwu, 1999; fasakin et al., 2003; gabriel and keremah, 2003; ibiyo and olowosegun, 2004). for any aquaculture venture to be viable and profitable it must have a regular and adequate supply of balanced artificial diets for the cultured fishes. this is so because the dissolved nutrients that promote primary and secondary production in the natural environment are seasonal and might be insufficient or may not occur in required proportion to meet the nutritional demand for culture fish es. supplementary feeding satisfies this need and ensures that the fish gets the appropriate spectrum of its basic food requirement for maximum growth (ugwumba and ugwumba, 2003). artificial feeds also known as supplementary feeding are well-compounded mixture of feedstuffs in mash form or pellets that could be fed to fish. mash feeds are fed to fry and pellets to fingerlings, juveniles and adults depending on pellet size (eyo, 2003). artificial feeding of fish has many known advantages which include, enhancement of high stocking density especially in polyculture system resulting in high yield, promotion of growth and enables the farmer to observe the behaviour of his fish during feeding in order to detect any abnormality (gabriel et al., 2000; gabriel and keremah, 2003). unlike in the past, when fish depends on natural food in the pond, the production of fish feed is becoming popular with each passing day in the country. about 35,370 tonnes of feed were produced in 2003 (table 2), using locally available feed ingredients. nutrient requirements of fish fish like other animals have a requirement for essential nutrients in order to grow properly (table 3). irrespective of the culture system in which they are grown, growth, health and reproduction of fish is primarily dependent upon an adequate supply of nutrient both in terms of quantity and quality. supply of inputs (feed) has to be ensured so that the nutrients and energy requirements of the species under cultivation are met so that the production goals of the system can be achieved (tacon, 2000). the essential nutrient requirements of fish are proteins, lipids, carbohydrates, vitamins and minerals. protein is a major constituent of fish diet. knowledge of the protein requirement of fish is essential for the formulation of a well-balanced artificial diet for economical fish feeding (lovell, 1989). protein requirement is linked with the general energy requirement of the fish at a given water temperature and the ability of the fish to gain weight at its inherent capacity (eyo, 2003) . according to gabriel and keremah (2003) protein and energy levels significantly influenced food conversion efficiency of heterobranchus bidorsalis , however the efficiency was not high enough to influence carcass composition and condition of fish. necessity for plant based protein source research in fish nutrition in recent years seems to focus on the replacement of animal protein sources by plant based proteins (table 4) with the aim of reducing the cost of supplemental feeds (de silva, 2001). the aquaculture feed industry relies heavily on the use of fish meal because of its balanced amino acid profile that closely matched the fish’s requirement pattern. the commonly used rates in fish feeds ranges from between 25 and 65% (average 35%). however, the increasingly scarce supply of fish table 3. protein requirement of commonly cultured fish species in nigeria. species fry to fingerling to adults to brood reference fingerlings juveniles stock oreochromis nilotics 35-40 30-40 20-30 ballarin and haller (1982) satherodon galilaeus 35 35 35 omoniyi and fagate (2003) clarras gariepinus 37.5 32.5 40 ayinla and akande (1988) heterobranchus sp 36-40 35 40 ayinla (1991) hybrid catfish heteroclarias 42.5 35 35 eyo and faloyi (1999) common carp 40 38 38 fagbaro et al. (2000) heterotis niloticus 30.1 25.30 25 otubusin (1987) table 4. animal and alternative plant based feedstuff in fish diets. nutrient conventional feedstuffs percent ration alternative feedstuffs maximum inclusion rate (%) protein fish meal 40 groundnut cake 25 palm kernel cake 15 cottonseed cake 20 jackbean 10 soyabean 45 cod liver oil 10 corn oil 20 soybean oil 10 palm oil 10 source: (eyo, 2003). meal with its concomitant rise in price and the increased competition from other livestock industry necessitates seeking a cost-effective replacement to supply dietary protein in aquaculture feeds. this aspect of fish feed development research is centered on the search for inexpensive, readily available and nutritious protein sources which can supply all the nutritional needs of the fish. one obvious approach involves the greater utilization of proteins from plant sources, which has been extensively investigated with promising results (eyo and olatunde, 1996; desilva, 2001; olufeagba et al., 2002; eyo, 2003; alegbeleye et al., 2004). however, the results from such studies reviewing the fact that these plant materials are not maximally utilized by the fish when compared with animal protein sources (ofojekwu and kigbu, 2002). this is commonly attributed to anti-nutritional factors especially phytic acid. limiting factors to the utilization of plant based protein sources phytic acid or the hexaphosphate of myoinositol occurs naturally throughout the plant kingdom (table 5) and is present in considerable quantities within many of the major legumes and oils seeds (tacon, 1995). according to matyka et al. (1993) 46.73% of the total phosphorus within plant based ingredients is organically bound phytin phosphorus which is largely unavailable to fish. this is due to the absence of the enzyme phytase within the digestive tract of fish (lovell, 1989). besides, it acts as a strong chelator, forming protein and mineral-phytic acid complexes; the net result being reduced protein and mineral bioavailability (davies and gatlin, 1991; hossain and jauncey, 1993; nrc, 1993). furthermore, most fish lack intestinal phytase that can digest the salts of phytic acid (phytins), hence they are released into the environment in the form of phytate phosphorus which in turn are acted upon by micro-organisms that release the phosphorus leading to pollution (alvarado, 1997). interestingly, pollution from phytic acids in plant protein sources can be reduced to the barest minimum by including phytase in plant based protein ingredients when formulating fish feed. need for phytase inclusion in formulation of fish feed phytase is an enzyme which is microbial in nature, and as most monogastric animals like fish cannot produce this enzyme; hence it must be supplied by inclusion in formulated diet of plant origin. over the years, plant products such as oil seed cakes and meal have been evaluated as fish feed ingredients. with advanced processing techniques, their nutritive values have been enhanced to such an extent that they are now considered conventional ingredients in aquafeeds (ayinla and akande, 1988; table 5. toxic constituents of selected plant feedstuffs. source toxic factor preventative treatment soya bean trypsin inhibitor toasting, steaming or boiling haemagglutinins saponins phytic acid phytase treated groundnut cake aflatoxins proper storage phytic acid phytase treated cotton seed gossypol phytic acid pre-press thoroughly to extract the oil phytase treated pigeon pea linamarin boiling for 20 minutes phytic acid phytase treated kidney bean heamagglutinin phasiolin phytic acid boiling for 20 minutes auto claving phytase treated lima bean (phaseolus lonatus) saponnis phytic acid toasting phytase treated source: jackson et al. (1996). figure 1. action of phytase. ayinla, 1991; fagbenro 1999; eyo et al., 2003). addition of microbial phytase either in powdery or liquid form has been reported to improve the utilization of plant phosphorus in fish diets considerably (forst et al., 1999; robinson et al., 2002; debnath, 2003). its inclusion improves optimal utilization in plant based protein diet for fish thereby enhancing fish growth, leading to maximum profit in aquaculture. therefore, inclusion of dietary phytase will play a major and significant role in increasing the level of production and utilization of plant protein based artificial diet used mostly in the poor and developing nations of the world. phytase pathway of action phosphorous in plants normally remains in an associated form with a molecule called phytic acid (phytate). phytic acid consists of a sugar (similar to glucose) called myoinositol, to which phosphate (po -4 ) groups are covalently linked. phytase releases these phosphates from the inositol ring as shown in (figure 1) (baruah et al., 2003). release of phosphorus depends on the ph of the intestine. the optimum microbial phytase activity occurs phytase (ftu) is defined as the quantity of enzyme that liberates 1 µmole of inorganic phosphorus per minute from table 6. average percentage apparent availability of phosphorus determined for catfish using dietary phytase. feed stuff non supplemented phosphorus availability % supplemented dietary phytase average) diet % wheat middling 28 40 corn 25 38 soyabean meal 50 75 cotton seed meal 43 56 ground nut cake 46 70 palm kernel cake 36 46 sorghum 31 42 jack been 38 47 source: yan and reigh (2002) table 7. average apparent protein digestibility coefficients of phytase supplemented and non-supplemented atlantic salmon diet. feed stuff non supplemented (digestibility) % phytase supplemented digestibility % corn 60 72 cotton seed meal 61 84 soya bean meal 75 89 wheat bran 68 82 rice bran 68 78 ground nut cake 70 82 sorghum 63 80 palm kernel cake 69 76 source: debnath (2003). 0.0015 mol/l sodium phytate at ph 5.5 and 37 c (von sheuermann et al., 1988). roles of dietary phytase in fish feed formulation the important roles of dietary phytase as veritable option in the preparation of least cost diets from locally available and cheap plant protein sources cannot be overemphasized. the use of phytase may revolutionise the future of aquaculture industry, world-wide. this is because fish meal, a major source of protein in aquafeed is produced from fish caught from the wild. with the rapid depletion of fish in the wild and stocks reaching its maximum biological limits, the aquaculture industry is already in crisis. hence the need for plant based protein sources as viable alternative. the roles of phytase in plant protein based diets are as follow: increased bio availability of phosphorus and other minerals phytic acid is reported to chelate metal ions such as calcium, magnesium, zinc, copper and iron to form and secondly at ph 2.5 (simons et al., 1990) . one unit of at two ph values; the highest activity being at ph 5.0 5.5 insoluble complexes that are not readily absorbed from the gastrointestinal tract of fish (hendricks and bailey, 1989). microbial phytase is effective in enhancing the bioavailability of phosphorus considerably, thereby reducing the faecal phosphorus output (baruah et al., 2004). it also improves the apparent absorption of magnesium, zinc, copper and iron in plasma, bone and the whole body of fish (vielma et al., 1998). yan and reigh (2002) further assert that phytase supplementation at 500 units per kilogram of diet was sufficient to improve the retention of calcium phosphorus and manganese by catfish, ictalurus punctatus fed on an all-plant protein diet (table 6). improvement of protein digestibility phytase supplementation in fish diet has been reported to improve protein digestibility and retention (storebakken et al., 1998). it was further confirmed by debnath (2003) that apparent net protein utilization and digestibility in atlantic salmon was significantly improved by the enzyme supplementation (table 7), while non-enzyme supplemented groups showed a low digestibility. many researchers (kornegay, 1995; vielma et al., 1998; baruah et al., 2004) have also observed that phytase-supplementation in plant-based practical diets has been reported to increa table 8. growth response of clarias gariepinus fed on phytase treated soybean meal based diet. parameter rate of phytase inclusion (%) 0 2 4 6 8 10 mean initial weight (g) 7.25 7.28 7.28 7.39 7.40 7.60 mean final weight (g) 10.32 12.41 14.32 17.92 18.11 19.01 mean weight gain 3.07 5.13 7.04 10.53 10.71 11.41 feed conversion ratio (fcr) 16.28 9.74 7.10 4.73 4.66 4.38 specific growth rate (sgr) 0.14 0.24 0.33 0.50 0.51 0.54 source: van weerd et al. (1999) table 9. physico-chemical conditions of farm effluents in rainbow trout tank culture fed phytase treated soyabeans meal with untreated diet. parameters phytase treated diet untreated diet ph 6.7 6.0 dissolved oxygen (d.o mg l -1 3.3 1.1 nh3-n (mg l -1 ) 0.18 2.01 no2 –n (mg l -1 ) 0.02 0.09 po4 3 (mg l -1 ) 0.6 3.01 source: hassan (2001). se protein digestibility by breakdown of phytin-protein complexes. enhancement of fish growth and performance incorporation of microbial phytase, in the diet of fish fed predominantly on plant based protein, have been reported to result in increased weight gain in i. punctatus (jackson et al., 1996). weight gain and feed consumption was increased by 23.52 and 11.59 percent, respectively compared to a control group in pangasius pangasius (debnath, 2003). also van weerd et al. (1999) observed that african catfish, clarias gariepinus performed better on phytase supplemented diets (table 8). reduction of incidence of aquaculture pollution the environmental impact assessment of the aquaculture industry is getting increasing attention and rigorous restrictions are being set on this industry by governments and environmentalists (baruah et al., 2004). given the fact that feed is the biggest source of nutrient loading in aquaculture production, a clear understanding of its impact is essential for sustainable development either in intensive or semi-intensive aquaculture (hassan, 2001). most discharge of farm effluents is majorly phosphorus, which leads to eutrophication. dietary phosphorus in plant protein source is generally unavailable to finfish, hence, making fish to excrete predominantly phosphorus in soluble and particulate forms. these soluble forms of organic phosphorus and phos phates affect water quality directly (de silva and anderson, 1995). however, addition of microbial phytase in the diet of fish overcome this problem (table 9), by making the chelated phosphorus available to fish and lessen faecal excretion thereby reducing environmental pollution (alvarado, 1997; hassan and ahmed, 2001; and baruah et al., 2004). enablement of least cost feed formulation least cost feed is a feed formulation in which there is optimum utilization of the contents of feed ingredients by careful combination resulting in maximum yield at minimal cost (de silva and anderson, 1995) . it involves production of feed at the most cost effective level in terms of resources -material, time, energy and money by minimizing input while maximizing output (sadiku, 2003). inclusion of phytase in fish diets have over the years enabled the fish nutritionists to formulate a cheaper feed based on plant source protein. protein from plants origin is cheaper and readily available, and easily accessible than the animal protein sources. despite its cheapness their inclusion in fish feed formulation is limited due to some activities of anti-nutritional factors. but with inclusion of phytase which reduces the effects of these anti-nutritional factors the plant protein sources can be maximally utilized. fish nutritionist can therefore, formulate and produce feed, majorly, from plant source ingredients which is far cheaper than the fish meals, thereby reducing the cost of feed, which will ultimately leads to profit maximization in the entire farming enterprise. conclusion heavy dependence on plant based feed in aquaculture is inevitable in the nearest future as this is essential to the sustainability of this industry. increasing demand for fishmeal by the livestock and poultry, industry, coupled with declining harvest of fish stock from the wild, demand that fish nutritionists find alternative sources of qualitative fish feed that will enhance fish performance in aquaculture practices. this now shifts the emphasis on plant source protein as a viable option. on the other hand, plant ingredients limited by the presence of phytic acid, which makes the phosphorus unavailable, thereby limiting their inclusion levels in most fish feed formulation, can be maximally used in fish formulating by adding dietary phytase. however it is evident that phytase supplementation improves the bioavailability of the phosphorus and nitrogen (protein) which leads to reductions in feed costs. therefore awareness of the efficiency of these enzymes has to be created among fish culturists, as an effective and efficient approach in the formulation of cost effective, growth promoting and low polluting fish feed for profitable aquaculture venture. references aifp (2004). farming nigerian water newsletter of the aquaculture and inland fisheries project of the national special programme for food security in nig. f.a.o office abuja, nigeria 3(4): 2-4. adiukwu ia (1999). aquaculture in nigeria: problem and prospects. j. fishery technol. 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(2002). effects of fungal phytase on utilization of dietary protein and minerals, and dephosphorylation of phytic acid in the alimentary tract of channel catfish ictalurus punctatus fed an allplant-protein diet. j. world aqua. soc. 33:pp. 10-12. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (6), pp. 321-326, june, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of functional properties, mineral content and amino acids of fermented maize flour blended with bambara groundnut t. i. mbata 1,3* , m. j. ikenebomeh 2 and s. ezeibe 3 1 department of applied microbiology and brewing, nnamdi azikiwe university, awka, nigeria. 2 department of microbiology, university of benin, benin city, nigeria. 3 department of microbiology, federal polytechnic, nekede, owerri, nigeria. accepted 21 july, 2017 the mineral content, essential amino acids and selected functional properties of composite blend of maize flour (mf) and boiled bambara groundnut flour (bgf) in the ratio 70:30 w/w were investigated using standard processing technique. results obtained showed that maize flour blended with 30% bambara groundnut in addition to germination significantly improved the mineral and amino acids profile of the composite blend. functional properties (bulk density, water absorption capacity and foam capacity) were also affected by fermentation, which significantly lowered the water absorption capacity and bulk density increased the foam capacity of bambara-maize‘ogi’. sensory evaluation indicated that the bambara-maize‘ogi’ was generally acceptable. the application of bambara groundnut blend to traditional foods suggests a viable option for promoting the nutritional qualities of africa maize-based traditional foods with acceptable cooking qualities. key words: fermented maize flour, bambara groundnut, mineral content, functional properties. introduction consumption rate of maize and maize based product is grossly increasing in nigeria. the sudden change could be attributed to eating habits as a result of poverty, which has become a chronic problem in developing countries, whereby parents are unable to afford high quality foods for their families (cole et al., 1989). many nigerians are now consuming less quality foods at the expense of what they need for a healthy life. se-veral studies have fortified local starchy foods with legumes like soybean, groundnut, bambara groundnut e.t.c, in order to improve their nutritional qualities. bam-bara groundnut is essentially grown for human consump-tion. the seed makes a complete food, as it contains suf-ficient quantities of protein, carbohydrate and fat. several researchers have examined the biochemical composition of the seed (owusu domfeh et al., 1970; oluyemi et al., 1976; oliveira, 1976; linnemann, 1987). on the average, the seeds were found to contain 63% *corresponding author. e-mail: theoiyke@yahoo.com. tel.: +2348032618922. carbohydrate, 19% protein and 6.5% oil. its protein con-tent can be used to fortify our mostly starchy foods like ‘ogi’, made from maize (mbata et al., 2007). maize (zea mays) grains are used in the production of several traditional foods, unfortunate they lack adequate micronutrients. in order to help alleviate the ever-increas-ing problems of malnutrition in developing countries, the need for fortification of popularly consumed low protein staple foods with inexpensive sources of plant proteins cannot be overemphasized. the united nation’s children fund (unicef) (1998) estimated that approximately one out of three children younger than 5 years of age are chronically malnourished and are thus trapped early in life in a pattern of ill health and poor development. malnutrition is thus associated with more than half of all deaths of children worldwide (sobo and oguntona, 2006). according to onyezili (1999), malnutrition contributed to more than half a mil-lion death of babies born in nigeria in 1999. these nutria-tional deficiencies are also known to lead to a high death rate, disabling diseases and retardation in physical growth and mental development (banigo et al., 1986). mbata et al. 322 this tends to reduce the population of the affected country. growth of infants in the first two years of life is very rapid. food dependency is typically on breast milk which is reputed to be the best for human infants. however, the rapidity of growth mentioned earlier shows the need to supplement breast milk in meeting the nutritional requirement of the older child, hence, the need for nutritional supplements (martorell et al., 1994). weaning foods in most african countries are usually in the form of cereal gruels which are watery suspensions of cooked maize, rice or sorghum (onilude et al., 1999) that are very much low in quality protein. in nigeria and other parts of west africa, cereal grains lack two essential amino acids, lysine and tryptophan (fao, 1985; hoshiai, 1991), thus making their protein quality poorer compared to that of animals (chavan and kadam, 1989). earlier studies have documented increased lysine and tryptophan in germinated corn (tsai et al., 1975), improved vitamin content in germinated sorghum and maize (asiedu et al., 1993), increased amino acid and vitamins in fermented blends of cereals and soybeans (onilude et al., 1999). many brands of low-cost proprietary weaning foods have been developed from locally available, highcalorie cereals and legumes in tropical africa (price et al., 1978). most cereals are low in essential minerals such as calcium, potassium, iron and zinc (oyenuga, 1969) and blending cereals with legumes rich in proteins and essential amino acids has also been perfected (livingstone et al., 1993). to be useful and successful in food application, fermented maize flours fortified with a legume protein, bambara groundnut, should possess desirable functional and micronutrients properties. however, there are no information on the functional properties and micronutrient status of fermented maize flour blended with bambara groundnut. this study, therefore, aimed at the determination of functional properties, mineral content and amino acids of fermented maize flour blended with bambara groundnut. materials and methods collection of sample yellow maize (zea mays l) and cream coat bambara groundnut (vigna subterranean l) used were purchased from an eke awka market in anambra state, nigeria. the samples were thoroughly cleaned by picking all broken kernels, stones, together with other foreign particles and the good ones were sorted out. the samples were then stored in sterile polyethylene bags and taken to the laboratory for mineral content and functional analyses. pre-treatment of bambara groundnut the bambara groundnut was first thoroughly cleaned by picking all the stones and other foreign particles present in them while sorting out the good ones. the cleaned bambara groundnut were soaked in water for 1 h and boiled at a temperature of 100°c for 20 min. the seeds were dehulled manually, sun-dried for 2 3 days. the dried seeds were then dry-milled into flour using a disc attrition mill (hunt no. 2a premier mill, hunt and co, uk) to an average particle size of less than 0.3 mm. the milled grain was then sieved through a fine mesh (0.5 m) to obtain the bambara groundnut flour. preparation of traditional fermented maize flour two hundred grams of the cleaned maize samples were soaked in plastic bucket containing 300 ml of distilled water and steeped for 24 h at room temperature (28 ± 2°c). the steep water was discarded by decantation and the steeped grains were germinated (48 h) by spreading on a clean grease free tray pan and thereafter it was sun dried for 2 3 days by putting it in a sterilized tray pan. the maize grains were then milled using a disc attrition mill (hunt no. 2a premier mill, hunt and co, uk) to an average particle size of less than 0.3 mm. the milled grain was then sieved through a fine mesh (0.5 m) to obtain the maize flour. supplementation of fermented sorghum with soybeans the bambara groundnut and maize flours were mixed together in the ratio 30:70 (w/w) (bressani and elias, 1974) (figure 1). chemical analysis calcium (ca), copper (cu), iron (fe), iodine (i2), zinc (zn), and magnesium (mg) were determined using the atomic absorption spectrophotometer method as described by aoac (1998). amino acid analysis lysine concentration in the sample was determined in triplicates, by digestion under vacuum with 6 m hcl in sealed ampules at 110°c for 22 h. the hydrolysates were derivatized and analyzed for amino acids on a water hplc system controlled by millenium 2010 software (water div, millipore corp, milford, ma, usa) (millipore, 1987). tryptophan was determined according to the aoac (1998) method. functional properties the bulk density was determined using the method of wang and kinsella (1976). ten grams of the sample material were placed in a 25 ml graduated cylinder and packed by gentle tapping of the cylinder on a bench top 10 times from a height of 5 8 cm. the final volume of the test material was recorded and expressed as g/ml. the method described by cegla et al. (1977) was used in the determination of water absorption capacity (wac). 6 g of materials were weighed in a 100 ml beaker. a known volume of water was pipetted into the beaker. the wet material was carefully stirred and allowed to equilibrate for 1 h at 26°c. after complete water absorption, the sample was further treated with 0.01 ml water portions with 10 min interval before visual observation. the volume that gave a complete absorption of water (no visible free water) was recorded. wac was calculated as the ratio of maximum volume in g absorbed by 100 g dry material. the foam capacity (fc) was determined as described by naranyana and narasinga-roo (1982). in this method, the volume of foam at 60 s after whipping was expressed as fc. sensory evaluation fresh samples of cooked porridge prepared with each of the pro maize bambara-groundnut cleaning cleaning soaking (24h) soaking (1h) germination (48h) boiling (20 min) dehulling sundrying (48h) sundrying (48h) milling milling sieving 70% 30% traditional mixing fermented maize flour sieving bambara-groundnut blended fermented maize flour figure 1. flow diagram for the production of bambara-groundnut-blend and traditional fermented maize flour samples. ducts by boiling 10% (w/v) slurry of the dough for 15 min were assessed for colour, texture, flavour (aroma), taste and overall acceptability. an 8 point hedonic scale, where 8 represented the highest score and 1 the lowest was employed to test the product for flavor, taste, texture, colour and general acceptability. the hedonic scaling used is thus: no. 2 2007 8 = like extremely 7 = like very much 6 = like moderately 5 = like slightly 4 = dislike slightly 3 = dislike moderately 2 = dislike very much and 1 = dislike extremely each treatment was evaluated three times by each panelist. consumer panel which was randomly selected from students and lecturers of the department of applied biochemistry and food technology, nnamdi azikiwe university awka, nigeria, participated in the testing sessions. the assessments were conducted in a well lit room designed for sensory evaluation. the food was stored safely in a cool and dry place and served immediately or soon after preparation. each judge was presented with a glass of water after each testing session to rinse their mouths so as to prevent carryover effect. statistical analyses the data were subjected to analysis of variance in a completely randomized design using the method of stell and torrie (1980). significance difference was accepted at p 0.05 levels. results the mineral composition of fermented maize flour and bambara groundnut-maize fortified flour of the diets are presented in table 1. the zinc, iron, iodine, magnesium, and calcium contents of the flours ranged from 62.84 78.20, 49.72 58.80, 3.00 18.12, 460.02 475.20 and 115.64 128.40 mg per 100 g dry flour for fermented maize and bambara groundnutmaize fortified flours respectively. the flours especially those of bambara-maize ‘ogi’ supplied adequate amounts of most minerals except copper (24.60 29.75 mg). the functional properties (bulk density, water absorption capacity and foam capacity) of the diets are presented in table 2. the bambara groundnutmaize flour has a lower bulk density (0.35 g/cm 3 ) than the fermented maize flour (0.55 g/cm 3 ). the water absorption capacity was also lower (41.5 g/g) in bambara groundnut-maize flour than in fermented maize flour (44.5 g/g). foam capacity values increased in bambara groundnutmaize flour (44.5 g/g) than in fermented maize flour (41.5 g/g). table 3 shows the amount of bioavailable lysine and tryptophan in fermented maize and bambara groundnutmaize flours. the result showed an appreciable increase in lysine and tryptophan concentration in bambara groundnut maize flour. the overall acceptability scores of the various sensory attributes are shown in table 4. 323 afr. j. food sci. res. mbata et al. 324 table 1. the mineral content of the bambara groundnut-maize blended flour (mg/100g). elements fermented maize flour (mf) bambara groundnut-maize blended flour (bgf) copper (cu) 29.75 a 24.60 b zinc (zn) 62.84 c 78.20 d magnesium (mg) 460.02 e 475.20 f calcium (ca) 115.64 g 128.40 h iron (fe) 49.72 i 58.80 j iodine (i2) 3.00 k 18.12 l means of three independent determinations. mean values within a row with different superscripts differ significantly (p < 0.05). table 2. functional properties of the bambara groundnut-maize blended flour. properties flour (bgf) fermented maize flour (mf) blended bambara groundnut maize bulk density (g/cm 3 ) 0.55 a 0.35 b water absorption capacity (g/g) 44.5 c 41.5 d foaming capacity (%) 0.16 e 0.61 f means of three independent determinations. mean values within a row with different superscripts differ significantly (p < 0.05). table 3. amino acid content (g/16gn) of bambara groundnut-maize blended flour (bgf) properties traditional bambara groundnut-maize blended fermented maize flour flour (bdf) lysine 0.50 ± 0.11 a 4.20 ± 0.21 b tryptophan 0.1 ± 0.08 c 0.86 ± 0.06 d means of three independent determinations. mean values in the same column with different superscripts differ significantly (p<0.05). table 4. organoleptic characteristics and acceptability of bambara groundnut-maize blended dough mean scores a, b supplements colour texture flavour taste overall 1 6.9 6.9 6.9 6.8 6.9 2 6.7 7.0 6.8 6.9 6.9 3 6.7 7.0 6.8 6.8 6.8 4 6.6 6.4 6.7 6.8 6.6 5 6.7 6.8 6.1 6.4 6.5 a means are scores of 20 judges and not significant (p>0.05) different b/w supplementary foods. b panelists used 8 point hedonic scale. discussion the study highlighted the need for fortification of maize fermented flour with bambara groundnut. in tropical africa, weaning foods based on cereals (maize) are deficient in essential amino acids such as lysine and tryptophan e.t.c, thus making their protein quality poorer compared to that of animals (chavan and kadam, 1989). however, most cereals are also low in essential minerals such as calcium, potassium, iron and zinc (oyenuga, 1969) and blending with legumes rich in proteins and essential amino acids has been reported (livingstone et al., 1993; mbata et al., 2007). despite the reported studies in the nutrient status of cereal based diets in sub-saharan africa, the nutrient needs of infants and sick adults are still not being met. an earlier report by mbata et al. (2007) considered the nutritional status (proximate, amino acid and rheological properties) of fermented maize meal by fortification with bambara groundnut. in the study, the effect of fortification 325 afr. j. food sci. res. of maize based traditional foods with legume protein bambara groundnut at 10% and 20% replacement levels on the rate of fermentation and organoleptic product quality were investigated. though the nutritional status was enhanced, the composite blends of maize and bambara groundnut (70:30 w/w) as in the study, gave a better nutritional and supplemental relationship in the production of bambara-maize‘ogi’. addition of 30% bambara groundnut into bambara-supplemented maize improves the mineral and essential amino acid contents of maize ‘ogi’. ‘ogi’ has a poor biological value thus, children weaned entirely on ‘ogi’ made solely from maize are known to suffer from malnutrition and generally provide insufficient amounts of certain key nutrients (particularly iron, zinc and calcium) to meet the recommended nutrient intake during the age range of 6 – 24 months and so the need for enrichment (dewey and brown, 2003). results obtained from this study showed that there were significant improvement in the mineral and essential amino acid contents of maize blended meal with bambara groundnut. the minerals especially zinc, calcium, magnesium, iron and iodine increased with 30% addition of bambara groundnut. this increase could be due to the combination effect because maize and most cereals are deficient in essential micronutrients. the essential amino acids composition at the end of 72 h fermentation differed significantly (p < 0.05) from those of fermented maize meal. however, a varied pattern was noticed in the improvement of each amino acid in the blended sample. lysine and tryptophan are essential amino acids which are vital for growth and maintenance of the body and are often limiting in some cereals (fao, 1985; asiedu et al., 1993a). bambara-groundnut is very good nutritionally, rich in lysine. the incorporation of bambara-groundnut to maize meal increased these amino-acids content. this agrees with the finding of wu and wall (1980), who showed that germination of cereals followed by other processing techniques is essential to improving lysine content. the findings from this study have showed that the blended food (bambara-maize ‘ogi’) was nutritious, since the product met the recommended dietary allowance (rda) with respect to mineral and essential amino acids as recommended by the food and agriculture organization/world health organization (1985) and the national institute of nutrition (1992) for children, pregnant and lactating mothers. the functional properties of the maize fermented meal and their blends are shown in table 2. bulk density (bd) of the formulations was low in bambara-maize‘ogi’ (0.35 g/ml) compared to that of maize fermented meal (0.55 g/ml). the water absorption capacities (wacs) of the bambara-maize‘ogi’ (which indicated the volume of water needed to form a gruel with a suitable thickness for child feeding) was also low (41.5 g/100 g) compared to that of maize fermented flour (44.5 g/100 g). the water absorp-tion capacities (wacs) of maize flour were significantly (p < 0.05) different from the water absorption capacities of bambara groundnut-maize‘ogi’. however, a weaning food should have low wac and bd in order to produce a more nutritious and suitable weaning food. this could be achieved by reducing the viscosity of the starchy components by malting (malleshi and desikachar, 1982). a low viscosity (less bulky) food contains higher nutrient contents since the volume of the food is low. the foam capacity (fc) was increased by fermentation and thus had a similar addition effect on the flour blends (onimawo et al., 2007). akubor and obiegbuna (1999) reported similar effects on millet flour by germination. fermentation may have caused surface denaturation of bambara groundnut proteins and reduced the surface tension of the molecules which gave good formability. in the organoleptic evaluation, the bambara-maize‘ogi’ were accepted by the trained panelists. mean scores were not significantly (p > 0.05) different. this shows that the product was liked by the trained panelists. conclusion the study showed that fermentation could improve both the amino acids, mineral contents and some selected functional properties of bambara groundnut. this improvement invariably improved the essential amino acids, mineral contents and functional properties of the blends made from bambara groundnut and maize flours. this supplementation of ‘ogi’ with bambara groundnut increases the biological value of ‘ogi’ which reduced the occurrence of malnutrition and micronutrient deficiency in children weaned with ‘ogi’. the bambaramaize ‘ogi’ produced from the blend was safe and generally acceptable and could be used in weaning, and improving birth weight. references akubor pi, obiegbuna je (1999). certain chemical and functional properties of ungerminated and germinated millet flours j. food sci. technol. 36(3):241-243. aoac (1998). official methods of analysis 16th edn. arlington, va: association of official analytical chemists. vol 1 and 2. asiedu m, lied e, nilsen r, sandnes k (1993). effect of processing (sprouting and /or fermentation) on sorghum and maize11: vitamins and amino acid composition. biological evaluation of maize protein. food chem. 48:201-204 banigo eoi, ihimoyan ki, osai gea (1986). development of soy beverage for nigeria. niger. food j. 4(1): 55-64. bressani r, elias lg (1974). legume foods in atscul am (ed), new protein foods. new york: academic press. 4: 231-297. cegla gf, meinke ww, mattil kf (1977). composition and characteristics of aqueous extracted textured vegetable protein flours: soy and cotton seed. j. food sci. 42: 807-811. chavan jk, kadam ss (1989). nutritional improvement of cereals by fermentation crc crit. rev. food sci. nutr. 28: 349400. 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of market women in abeokuta, nigeria. nig. j. nutr. sci. 1and 2: 62-66 stell rg, torrie jh (1980). principles and procedure of statistics, 2 nd edn. mcgraw hill books, new york. unicef (1998). united nations children's fund. the state of the world’s children. oxford university presswang jc, kinsella je (1976). functional properties of novel proteins: alfalfa leaf protein. j. food sci. 41:286-292 wu yv, wall js (1980). lysine and tryptophan increased during germination of cereal grains. cereal chem. 53: 222-226. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (6), pp. 095-200, june, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of cassava varieties and roasting methods on the chemical composition and pasting properties of tapioca grits adebowale, a.a.*, sanni, l. o. and onitilo, m.o. department of food science and technology, university of agriculture, abeokuta, nigeria accepted 10 february 2016 this study investigated the chemical composition and pasting properties of tapioca grits from different cassava varieties and roasting methods. tapioca grits (partially gelatinized irregular flakes from roasted cassava starch), were produced from three different cassava varieties (odongbo, tms 30572 and oko-iyawo) and two roasting methods (traditional and rotary dryer). the chemical composition and pasting properties of the tapioca grits were evaluated. the results showed that the effect of cassava variety and roasting methods on chemical composition of tapioca grits was significantly different (p<0.05). the principal component analysis (pca) of variation in the chemical properties of the tapioca grits indicated that moisture, sugar and starch accounted for 83% of the variation in the chemical properties of tapioca grits. this study showed that peak and hot paste viscosities are the key pasting parameters in characterizing tapioca grits from the cassava varieties and roasting methods studied and that variation in peak viscosity of the tapioca grits might be due more too varietal influence than the roasting method. key words: cassava, variety, roasting, tapioca grits, chemical, pasting. introduction cassava is one of the most drought tolerant crops and can be successfully grown on marginal soils, giving reasonable yields where many other crops do not grow well. the collaborative study on cassava in africa (cosca) revealed that between 1961 and 1999, total cassava production in africa nearly tripled from 33 million tonnes per year from 1961 to 1965, and to 87 million tones per year from 1995 to 1999, in contrast to the more moderate increases in asia and latin america (nweke et al., 2002). most of the increases in cassava production in africa were achieved in ghana and nigeria. one of the advantages cassava has over other starchy crops is the variety of uses to which the roots can be put. apart from being a staple food for humans (especially in africa), it additionally has an excellent potential as livestock feed, and in textile, plywood, paper, brewing, chemical and pharmaceutical industries. originally, the research focused on improved yields, cultivation practices and *correspondence author. e-mail: deboraz2002@yahoo.com. crop protection. cassava research has, since 1985, also focused on processing, quality control and new product development (dufour et al., 2002). a major constraint to cassava utilization is that cassava deteriorates rapidly. cassava has a shelf life of 24 – 48 h after harvest (wenham, 1995). hence, fresh cassava roots must be processed into a more shelf -stable form within 2 to 3 days from harvest. one of such cassava product is tapioca grit. tapioca grit is a partially gelatinized dried cassava starch, which appears as flakes or irregularly shaped granules. it is consumed in many parts of west africa, and widely accepted as a convenient diet (hollesman and ates, 1956). a number of reports are available in the literature on tapioca (oyewole and obieze, 1995; sanni et al., 1997; oyewole et al., 2003; adebowale et al., 2006, 2007). there is no published work on the effect of cassava variety and roasting methods on chemical and pasting of tapioca grits. the main objective of this study therefore was to evaluate the effect of cassava varieties and roasting methods on the chemical composition and pasting properties of tapioca grits. adebowale et al. 096 table 1. chemical components (%) of tapioca grits from different cassava variety and roasting method. tapioca samples moisture sugar starch carbohydrate amylose protein fat odongbo traditionally roasted 9.30 b 2.25 b 92.08 bc 94.34 b 23.12 a 0.24 a 0.25 c odongbo rotary dryer roasted 10.53 b 2.59 c 94.75 d 97.34 c 22.95 a 0.23 a 0.21 bc tms 30572 traditionally roasted 7.80 a 2.75 c 87.59 a 90.34 a 24.20 b 0.23 a 0.20 bc tms 30572 rotary dryer roasted 7.20 a 2.09 ab 93.81 cd 95.90 bc 24.30 b 0.26 b 0.20 bc oko-iyawo traditionally roasted 9.53 b 2.82 c 86.96 a 89.79 a 23.19 a 0.24 a 0.15 ab oko-iyawo rotary dryer roasted 7.40 a 1.88 a 89.96 b 91.85 a 23.27 a 0.23 a 0.12 a range 7.20-10.50 1.88-2.75 86.96-94.75 89.79-97.34 22.95-24.30 0.23-0.26 0.12-0.25 sd 1.35 0.38 3.32 3.08 0.59 0.01 0.05 cv 7.4 5.94 1.51 1.43 1.01 3.64 18.85 p of variety *** ns *** *** *** ns *** p of roasting method ns *** *** *** ns ns ns p of variety x roasting methods *** *** ns ns ns *** ns *** interactions significant at p<0.01 ns. interactions not significantly different at p>0.05 mean values having the same superscript with column are significantly different (p<0.05) materials and methods three cassava varieties (one improved variety; tms30572, and two traditional varieties; odongbo and oko-iyawo) were obtained from the teaching and research farm of the university of agriculture, abeokuta, nigeria. the varieties were grown under rain-fed conditions. no fertilizer or herbicides were used, and hand weeding was done when necessary. all the cassava varieties were harvested at 12 months after planting. the harvested roots were processed within 60 min after harvesting. preparation of tapioca grits fifty kg of freshly harvested cassava roots were peeled, washed with water and grated with a diesel engine powered mechanical grater (action zone made of 3 mm stainless steel, nigerian made). the resultant pulp was immediately sieved through a screen and suspended in 70l of water. this separates the fibrous and other coarse root material from the starch pulp. the starch pulp was allowed to settle for 4 6 h before decanting. the thick starch cake at the bottom of the bowl was pressed to remove water. this was screened through a screen (20mesh/inch size) to produce coarsegrained moist starch flour. in the traditional method, the moist starch flour was roasted over an open firewood flame in a large, shallow stainless steel pan for 20 min at temperature range 120 150°c with constant stirring using a piece of stainless steel plate. vegetable oil was used to rub the pan before roasting to prevent stickiness and burning (adebowale et al., 2006). in the rotary dryer method, the moist starch flour was roasted in a locally fabricated electrically powered rotary dryer. charcoal was used as heating source (figure 1) . roasting temperature was 100 o c and an elapsed time of 20 min. the dryer has a suction fan that removes steam generated during roasting. the fan was switched off during the first 10 min after loading the flour into the dryer to effect partial gelatinization. the fan was switched on thereafter to effect drying of the partially gelatinized starch granules (sanni et al., 2006). the rotary dyer has a drying capacity of 250 kg/man day. analytical determinations the moisture, protein and fat contents of the samples were deter mined using the aoac (1990) method. the starch and total sugars content were determined using a colorimetric method (dubois et al., 1956). absorbance was read at 490 nm using a spectrophotometer (model spectronic 601, milton roy company, usa). the amylase content was determined using the method of williams et al (1958) involving the preparation of stock iodine solution and iodine reagent. absorbance was read using a pectrophotometer at 620 nm. a blank was used to standardize the spectrophotometer. pasting properties were determined using a rapid visco analyser (rva) (model rva 3d+; network scientific, 5australia). the sample was turned into slurry by mixing 3 g with 25 ml of water inside the rva can and inserted into the tower, which was then lowered into the system. the slurry was heated from 50 to 95°c and cools back to 50°c within 12 min, rotating the can at a speed of 160 rpm with continuous stirring of the content with a plastic paddle. parameters estimated were peak viscosity, setback viscosity, final viscosity, pasting temperature and time to reach peak viscosity. statistical analysis each analytical determination was carried out in three replicates. data were subjected to analysis of variance (anova) and principal component analysis (pca) using sas version 8e software (sas institute inc., cary, nc, usa) at p<0.05. means were separated using duncan’s multiple range test. results and discussions chemical composition of tapioca grits from different cassava varieties and roasting methods the effect of cassava varieties and roasting methods on the chemical properties of tapioca grits from different cassava varieties and roasting methods are presented in table 1. rotary dryer roasted tms 30572 tapioca grits had the least moisture while traditionally roasted tapioca grits from odongbo cassava variety had the highest moisture content. the lower the initial moisture content of a product to be stored the better the storage stability of table 2. percentage variance explained by each chemical component of tapioca grits. component number component name eigenvalues % of variance cumulative % 1 moisture 2.57 36.72 36.72 2 sugar 2.11 30.11 66.83 3 starch 1.16 16.53 83.36 4 carbohydrate 0.68 9.68 93.05 5 amylose 0.33 4.68 97.73 6 protein 0.16 2.27 100.00 7 fat 1.35e-07 1.93e-06 100.00 extraction method: principal component analysis. the product. also, the lower the initial moisture content of the product the more the efficiency of the drying method because this shows that much of the water contained in the fresh sample or product had been removed during drying (pierre, 1989). the moisture content of all the tapioca grits were below the 10% stipulated standard of the revised regulation of the standard organization of nigeria (son, 1988; sanni et al., 2005). all the tapioca grits can be stored for up to 7 months because their moisture contents were below the levels reported by ukpabi and ndimele (1990) who found that gari samples with a moisture content of < 16% but > 13% could be stored for 27 months without mould infestation. the varietal effect on the moisture content of tapioca grits was significantly different (p<0.01), but the effect of roasting methods was not significantly different (p>0.05) . this indicates that irrespective of either traditional or rotary dryer roasting, tapioca grits with low moisture and hence longer shelf life can be produced from the cassava varieties processed. tapioca grits from tms 30572 traditionally roasted had the highest sugar content while rotary dryer roasted tapioca grits from oko-iyawo cassava contained the minimum level of sugar. traditionally roasted tapioca grits from oko-iyawo cassava variety had the least content of starch while the rotary dryer roasted odongbo variety tapioca grits had the highest starch content. similar trend was displayed by the carbohydrate content. tapioca grit is essentially a starchy product from moistened roasted cassava starch. the starch and sugar content for all tapioca grits were higher than values recorded by oyewole and obieze (1995). the amylose content of the tapioca grits ranged between 22.95% and 24.30%, with rotary dryer roasted tapioca grits recording the highest amylose content and rotary dryer roasted odongbo tapioca grits containing the highest amount of amylose. the protein and fat content of the tapioca grits ranged from 0.23 to 0.26% and from 0.12 to 0.25% respectively. the amylose content of the tapioca grits were higher than that obtained by oyewole and obieze (1995). raja and ramakrishna (1990) reported that heat treatment causes a reduction in the amylose content of cassava, but this study showed little variation in the amylose content of tapioca grits from the two roasting methods. also, the interactions between cassava variety and roasting methods on amylose content of the tapioca grits were not significantly different (p>0.05), but the varietal effect was significantly different (p<0.01). the parameters of principal component analysis (pca) are as shown in table 2. the results showed that moisture, starch and sugar content accounted for 83% of the variations in the chemical properties of the tapioca samples. hence, in characterizing the chemical composition of tapioca grits from different cassava variety and roasting methods, moisture, sugar and starch are the key components of variance assessment. pasting properties of tapioca grits from different cassava varieties and roasting methods the pasting profile of tapioca grits from different cassava varieties and roasting methods are shown in table 3. tapioca grits are usually cooked into paste referred to as tapioca meal before consumption, although some consumer’s prefer eating tapioca grits as snack. the pasting properties become important in predicting the behaviour of tapioca paste during and after cooking. when starch or starch-based foods are heated in water beyond a critical temperature, the granules absorb a large amount of water and swell to many times their original size. over a critical temperature, which is characteristics of a parti-cular starch, the starch undergoes an irreversible process known as gelatinization. when the temperature rises above the gelatinization temperature, the starch granules begin to swell and viscosity increases on shearing. the temperature at the onset of this rise in viscosity is referred to as the pasting temperature. the pasting temperature of the tapioca samples was lower than values reported for other cassava products (sanni and jaji, 2003; sanni et al., 2004; adebowale et al., 2005). the lower pasting temperature exhibited by tapioca grits is as a results of the partial gelatinization of starch granules during roasting. the pasting temperature is a measure of the minimum temperature required to cook a given food sample, it can have implications for the stability of other 097 afr. j. food sci. res. adebowale et al. 098 table 3. pasting properties of tapioca grits from different cassava varieties and roasting methods. tapioca peak trough breakdown final set back peak pasting samples (rvu) (rvu) (rvu) viscosity (rvu) viscosity (rvu) time (min) temperature (°c) odongbo 449.30 b 162.55 a 285.91 b 210.89 a 48.33 a 3.85 bc 63.53 b traditionally roasted 352.86 a 179.03 c 173.84 a 244.16 b 65.14 b 4.27 d 63.60 b odongbo rotary dryer roasted 477.83 bc 176.78 bc 282.44 b 233.89 b 57.11 ab 3.76 b tms 30572 63.07a traditionally roasted 476.52 bc 165.42 b 282.25 b 228.05 ab 62.64 b 3.63 a 63.17 a tms 30572 rotary dryer roasted 513.55 c 178.44 c 325.19 c 237.14 b 58.70 ab 3.82 bc 63.17 a oko-iyawo traditionally roasted 467.30 bc 167.97 ab 294.17 bc 226.81 ab 58.83 ab 3.89 c 63.17 a oko-iyawo rotary dryer roasted range 352.86162.55173.84210.89-244.16 48.33-65.14 3.63-4.27 63.07-63.60 513.55 179.03 325.19 6.48 2.63 10.28 4.59 11.56 1.13 0.21 p of variety *** ns *** ns ns *** ** p of roasting ** ns ** ns ** *** ns method p of variety x ns *** ** ** ns *** ns roasting methods *** interactions significant at p<0.01 **interactions significant at p<0.05 ns interactions not significantly different at p>0.05 components in a formula and also indicate energy costs (newport scientific, 1998). the pasting temperature of the tapioca grits is generally lower than the boiling temperature; hence the tapioca grits can form a paste in hot water below boiling point. this, at commercial level, is a remarkable cost saving. as the temperature increases during the pasting test, the granules rupture and amylose leaches into the solution, followed at a slower rate in some cases by the amylopectin fraction. the series of processes that follow gelatinization is collectively referred to as the pasting profile. peak viscosity, which is the maximum viscosity, developed during or soon after the heating portion of the pasting test (newport scientific, 1998), is also lower compared to values obtained for dried fufu by adebowale et al. (2005). this is also because tapioca is partially gelatinized. varietal differences exist in the peak viscosity of the tapioca grits at p<0.01, while the effect of different roasting methods is significant at p<0.05.the statistical significant (p>0.05). therefore, variation in peak viscosity of the tapioca grits might be due more to genetic variation interaction between variety and roasting method is not than processing variable. peak viscosity is often correlated with the final product quality. it also provides an indication of the viscous load likely to be encountered during mixing (maziyadixon et al., 2004, 2005). it is customary to measure the peak time that occurs with peak viscosity. the peak time is a measure of the cooking time (adebowale et al., 2005). the time to attain peak viscosity obtained for tapioca grits is considerably lower than that reported for other cassava product by sanni et al. (2004) and adebowale et al. (2005). this might be due to the fact that tapioca is partially gelatinized. during the hold period of a typical pasting test, the sample is subjected to a period of constant temperature (usually 95°c) and mechanical shear stress. this further disrupts the starch granule and amylose molecules generally leach out into solution and align in the direction of the shear (maziya-dixon et al., 2005). the period is sometimes called shear thinning, holding strength, hot paste viscosity or trough due to the accompanied breakdown in viscosity. it is the minimum viscosity value in the constant temperature phase of the rva profile and measures the ability of paste to withstand breakdown during 099 afr. j. food sci. res. table 4. percentage variance explained by each of the pasting parameters. component number component name eigenvalues % of variance cumulative % 1 peak 4.09 58 58 2 trough 1.81 26 84 3 breakdown 0.54 8 91 4 final viscosity 0.43 6 98 5 set back 0.12 2 99 6 peak time 8.93e-03 0.13 100 7 pasting temperature 1.91e-08 2.72e-07 100 extraction method: principal component analysis. cooling (newport scientific, 1998). the effect of cassava variety as well as the roasting method on the hot paste viscosity of tapioca grits is not significant (p>0.05). but the interaction between variety and roasting method is significant at p<0.01. large values indicate little breakdown of sample starches. the rate of starch breakdown depends on the nature of the material, the temperature, and the degree of mixing and shear applied to the mixture (newport scientific, 1998) . the ability of a mixture to withstand heating and shear stress that is usually encountered during processing is an important factor for many processes especially those requiring stable paste and low retrogradetion/seneresis. the breakdown viscosity recorded by traditionally roasted tapioca grits is higher than those of the rotary dryer method for all the three varieties. there are significant differences in the breakdown viscosity for tapioca grit from different cassava varieties and roasting methods (p<0.05). the final viscosity indicates the ability of the starchbased food to form a viscous paste or gel after cooking and cooling. the viscosity after cooling to 50°c represents the setback or viscosity of cooked paste. it is a stage where retrogradation or re-ordering of starch molecules occurs. sanni et al., (2004) stated that lower setback during the cooling of the paste indicates greater resistance to retrogradation. principal component analysis (pca) for the pasting parameters of the tapioca grits showed that peak viscosity and hot paste viscosity accounted for 84% of the variance in the pasting profile of tapioca grits from different cassava variety and roasting methods (table 4). conclusion this study has revealed that in characterizing the chemical composition of tapioca grits from different cassava variety and roasting methods, moisture, sugar and starch are the key component of variation in chemical composition of tapioca. the pasting temperature of the tapioca grits is generally lower than the boiling temperature; hence the tapioca grits can form a paste in hot water below boiling point. this, at commercial level, is a remarkable cost saving. this study found out peak and hot paste viscosities as the key pasting parameters in characterizing tapioca grits from the cassava varieties and roasting methods studied and that variation in peak viscosity of the tapioca grits might be due more to genetic variation than roasting method. acknowledgement the authors wish to acknowledge the financial support by eu/incdev/unaab cassava sme project and the integrated cassava project, international institute of tropical agriculture, ibadan, nigeria. references adebowale aa, sanni lo, awonorin so (2005). effect of texture modifiers on the physicochemical and sensory properties of dried fufu. food sci. technol. inter. 11(5): 373-382. adebowale aa, sanni lo, kuye a (2006). effect of roasting methods on sorption isotherm of tapioca grits. electronic j. environ. agric. food chem. 5 (6): 1649-1653 adebowale aa, sanni lo, awonorin so, daniel io, kuye a(2007). effect of cassava varieties on the sorption isotherm of tapioca grits. inter. j. food sci. technol. 42 (4): 448-452. aoac. 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centre for agriculture and rural cooperation wageningen, the netherlands. pp. 1-2, pp. 3076. raja kcm, ramakrishma sv (1990). compositional and pasting characteristics of plain-dried and parboiled cassava (manihot esculenta, crantz). food chem, 38: 79-88. sanni lo, adebowale aa, filani ta, oyewole ob, westby a (2006). quality of flash and rotary dried fufu flour. j. food agric. environ. 4(3and4): 74-78. sanni lo, maziya-dixo b, akanya ci, alaya y, egwuonwu cv, okechukwu ru, ezedinma c, akoroda m, lemchi j, ogbe f, okoro e, tarawali g, mkumbira j, patino m, ssemakula g, dixon a(2005). standards for cassava products and guidelines for export. international institute of tropical agriculture, ibadan, nigeria. pp. 1139. sanni lo, atere c, kuye a (1997). moisture sorption isotherms of fufu and tapioca at different temperatures. j. food eng. 34(2), 203 212 sanni lo, jaji ff (2003). effect of drying and roasting on the quality attributes of fufu powder. inter. j. food properties. 6(2): 229-238. sanni lo, kosoko sb, adebowale aa, adeoye rj (2004). the influence of palm oil and chemical modification on the pasting and sensory properties of fufu flour. inter. j. food properties 7(2): 229– 237. standard organization of nigeria (son) (1988). nigeria industrial standard for gari. 188: 189 ukpabi uj, ndimele c (1990). evaluation of the quality of gari produced in imo state, nigeria, niger. food j. 8: 105-110. wenham je (1995). postharvest deterioration of cassava, a biotechnology perspective. fao plant production and protection fao, rome. paper 130. williams vr, wu w, tsai hy, bates hg (1958). varietal differences in amylose content of rice starch. j. agric.food chem. 6: 47-48. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (11), pp. 001-004, november, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper proximate composition and amino acid profile of two non-conventional leafy vegetables (hibiscus cannabinus and haematostaphis barteri) d. kubmarawa*, i. f. h. andenyang and a. m. magomya department of chemistry, federal university of technology, p. m. b. 2076, yola, nigeria. accepted 22 june, 2019 two non-conventional leafy vegetables namely hibiscus cannabinus and haematostaphis barteri consumed largely by the rural dwellers in adamawa state of nigeria were subjected to standard chemical analysis to determine proximate nutrient content, amino acid composition and anti-nutritional factors. the results of the proximate analysis showed high crude fibre in h. barteri (33.04%), h. cannabinus had percentage crude fibre (29.61%). the protein contents were relatively high in both vegetables (13.78 and 12.40%), respectively. carbohydrate, lipid, ash and moisture contents were within the range of values expected for dry leafy vegetables. all the essential amino acids were present in good quantities in both vegetables. only cysteine and methionine ranked below 50% in comparison to the who/ fao reference protein. the anti-nutritional factors analysed in the vegetables namely, tannins, oxalates, and phytates were lower than the range of values reported for most vegetables. key words: hibiscus cannabinus, haematostaphis barteri, vegetables, nutrients, anti-nutrients. introduction green leafy vegetables constitute an indispensable constituent of human diet in africa generally and west africa in particular (oguntona, 1986). the varieties of leafy vegetables utilized are diverse, ranging from leaves of annuals and shrubs to leaves of trees. leafy vegetables are generally good sources of nutrients. they are important protective foods and highly beneficial for the maintenance of health and prevention of diseases as they contain valuable food ingredients which can be utilized to build up and repair the body. the fight against malnutrition and under -nourishment continues to be a basic goal of development and a variety of strategies are being applied. strategies based on nutrientrich foods like vegetables are considered essen-tial (susane, 1996). in nigeria, most rural dwellers rely on leaves gathered form the wild as their main source of leafy vegetables. throughout the year, these non-conventional vegetables play an important role in every-day cooking. although, commonly eaten in rural areas, they are also consumed by urban people who buy from *corresponding author. e-mail: dkubmarawa@yahoo.com. traders who also collect them from the wild. in addition, these vegetables supply calories and nutrient during the dry season when there is shortage of cultivated green vegetables and other food resources. the inclusion of these non–conventional vegetables in the diet of most rural communities therefore is critical to their survival. until recently, little attention has been given to the role of these wild and semi-wild vegetables in nigeria. by learning more about the nutritional and anti-nutritional contents of such plants, one can better assess their importance in the well-being of the communities that consume them. two such plants are hibiscus cannabinus and haematostaphis barteri. h. cannabinus, commonly known as kenaf, is an annual tough herbaceous plant belonging to the malvaceae family. its leaves have an acid flower and are used for soups. edible oil is obtained from the seed. con-centrated food for cattle in the form of seed cake comes from the residue after oil extraction. kenaf has been reported to be anodyne, aperitif, aphrodisiac, fattening and purgative (duke and wian, 1989). h. barteri is a tree belonging to the anacardiaceae family. its fruit is a red purple drupe. it has a thin acidic pulp with a resinous flavour. the tree is found wild in borno and adamawa states of nigeria (bokhari and table 1. proximate composition of h. cannabinus and h. barteri leaves. hibiscus cannabinus haematostaphis barteri moisture (%) 11.82 ± 0.45 10.25 ± 0.70 ash content (%) 5.11 ± 0.15 9.83 ± 0.18 crude fibre (%) 29.61 ± 0.22 33.04 ± 0.02 lipids (%) 2.33 ± 0.34 1.66 ± 0.86 crude protein (%) 13.78 ± 1.17 12.40 ± 0.82 carbohydrate% 37.67 ±1.03 34.94 ± 0.43 values are means ± s.d. for three (3) determinations. table 2. anti-nutritional components of h. cannabinus and h. barteri leaves. antinutrients hibiscus cannabinus haematostaphis barteri phytic acid (mg/100g) 19.78 ±1.80 17.80 ± 0.20 tannins (mg/100g) 2.74 ± 0.47 4.92 ± 0.34 oxalates (mg/100g) 158.5 ± 0.07 122.0 ± 2.14 values are means ± s.d. for three (3) determinations. ahmed, 1979). tender leaves are used to prepare soups; oily seed kernels are also edible. this paper reports the proximate nutrient contents, amino acid composition and antinutritional factors of the leaves of h. cannabinus and h. barteri consumed in adamawa state of nigeria mostly by the rural dwellers. materials and methods collection and treatment of samples h. cannabinus and h. barteri leaves were sampled randomly from farmlands in yola, adamawa state, nigeria in the month of september 2005. they were destalked, air dried, ground into fine powder and stored in airtight containers for the various analysis. proximate analysis the samples were analyzed for proximate composition (moisture, crude fat, fibre, carbohydrate and protein). moisture and lipid were determined by cocks and pede. method (1996). the method of nelson (1994) was adopted for the determination of ash and crude fibre. determination of crude protein was by the micro-kjedahl procedure. the carbohydrate content was obtained by difference that is 100-(sum of percentages of moisture, ash, protein, lipid, fibre). amino acid analysis the amino acid profile was determined using the method described by sparkman et al. (1958). each sample was dried to constant weight, defatted, hydrolyzed, evaporated and loaded into the techno sequential multi-sample amino acid analyzer (tsm) which is designed to separate and analyze free acidic, neutral and basic amino acids of the hydrolysate. determination of anti-nutrients tannin content was estimated using the vanillin-hcl method modified by price and butler (1977) . oxalates were analyzed using the method of ukpabi and ejidon (1989). the reddy and love method (1999) was adopted for the estimation of phytates. results and discussions the proximate composition of h. cannabinus and h. barteri are given in table 1. crude protein contents were 13.78 and 12.40% for h. cannabinus and h. barteri, respectively. these values though higher than in most conventional leafy vegetables are close to the lower end of the range expected for dry leafy vegetables (15-30%) (aletor and adeogun, 1995) . fibre, which is known to promote softer stools with increased frequency and regularity of elimination, was most abundant in h. barteri (33.04%). h. cannabinus had crude fibre content of 29.61%. in the diet therefore, both vegetables will be highly beneficial in easing bowel movement. lipids, carbohydrate and ash contents of both vegetables were within values expected for dry leafy vegetables. the major anti-nutritional factors commonly found in green leafy vegetables are phytic acid and oxalic acid (osagie and offiong, 1995) (table 2) . tannins may also be present in significant amounts in certain vegetables especially those of leguminous origin. high levels of phytates and oxalates have long been known to inhibit the absorption and utilization of minerals by animals including man (taylor, 1975). tannins decrease protein quality by reducing digestibility and palatability. other anti-nutritional effects attributed to tannins include damage table 3. amino acid composition of the leaves of h. cannabinus and h. barteri (g/100g protein). amino acids h. cannabinus h. barteri fao ref. protein lysine 3.96 3.02 4.20 threonine 3.25 2.26 2.80 cysteine 0.90 0.83 2.00 valine 3.85 3.25 4.20 methionine 0.91 0.86 2.20 isoleucine 2.81 3.03 4.20 leucine 7.05 5.66 4.20 tyrosine 3.06 2.86 2.80 phenylalanine 4.55 3.86 2.80 histidine 2.41 2.01 arginine 5.02 5.11 aspartic acid 7.02 6.69 serine 1.45 1.72 glutamic acid 11.11 9.52 proline 2.50 2.06 glycine 0.72 1.02 alanine 1.65 2.25 table 4. percentage comparison of the essential amino acid composition of the vegetables with the who standard (sample/standard multiply by 100)%. amino acids h. cannabinus h. barteri lysine 94 72 threonine 116 81 cysteine 45 42 valine 91 77 methionine 41 39 isoleucine 67 72 leucine 167 134 tyrosine 109 102 phenylalanine 162 137 to the intestinal tract, interference with the absorption of iron and a possible carcinogenic effect (butler, 1989). the amounts of phytates, oxalates and tannins in the vegetables (table 2) were well below the range of values that would adversely affect their nutritional values or cause any of the toxic effects associated with the antinutrients. table 3 shows the amino acid composition of the h. cannabinus and h. barteri. seventeen amino acids were found in varying proportions in the vegetables. all the essential amino acids were present. the proportions of the essential amino acids in the vegetables were compared with the who/fao protein standard (table 4). with few exceptions, the essential amino acids in both vegetables compared favourably with the who protein standard. h. cannabinus had a ranking of above 100% for threonine, leucine, tyrosine and phenylalanine in comparison to the who standard. lysine, valine and isoleucine also had high scores: 97, 91 and 67%, respectively. only methionine and cysteine in h. cannabinus ranked below 50%. in h. barteri the essential amino acids were also present in good quantities. leucine, tyrosine and phenylalanine scored above 100%. with the exception of cysteine and methionine, the remaining essential amino acids had a ranking of above 50% in comparison to the standard. these results support the claim that the protein in leafy vegetables although low, is of a very high grade (okaka et al., 2002). conclusion this study reveals that the leaves of h. cannabinus and h. barteri can serve as good sources of food nutrients for man. fibre diets promote the wave-like contractions that move food through the intestine, thus easing the passage of waste. it also lowers cholesterol level in the blood, reduces the risk of various cancers, bowel disease and improves general health and well being (eromosele and eromosele, 1993). the high fibre contents of h. barteri (33.04%) therefore, will make it highly beneficial in the diet. both vegetables contained high percentages of the essential amino acids except methionine and cysteine, which are commonly deficient in green leafy vegetables. the anti-nutritional levels of the leaves were generally lowtherefore the nutritional value of the vegetables will not be affected. these results re-enforce the growing awareness that wild and semi-wild vegetables can contribute useful amounts of essential nutrients including amino acids to human diets. although this study has revealed much about the nutritional value of the leaves of h. cannabinus and h. barteri, additional knowledge remains to be secured. there is need to determine vitamins and minerals present in the vegetables. the effect of cooking procedures on nutrient availability of the vegetables also needs to be studied. these investigations would provide a more complete picture of the nutritional significance of the vegetables. reference aletor mva, adeogun oa (1995). nutrient and anti-nutrient components of some tropical vegetables. food chem. 53: 375-379. bokhari mh, ahmed mj (1979). food plants in borno state nigeria, university press, maiduguri, nigeria pp. 20-43. butler lg (1989). effects of condensed tannin on animal nutrition, in: “chemistry and significance of condensed tannin” hemmingway rw, karehesy jj, edition plenum press new york, u.s.a. pp. 391402. cocks lv, pede c (1996). laboratory handbook for oil and fat analysis”, london, new york, academic press pp. 30-130. duke ja, wian f (1981). ecosystematic data on economic plants. quart. j. crude drug res. 17: 3-5. eromosele ic, eromosele co (1993). studies on the chemical properties and physio-chemical properties of seeds of some wild plants: j. plant food hum. nutr. (netherland). 43: 251-258. fao (1970). amino acid content of foods and biological data on proteins, food and agricultural organization of the united nations, rome, italy p. 285. nelson ss (1994). introduction to the chemical analysis of foods, jones and bartletes publishers, london pp. 93-201. oguntona tcrb (1986). proximate composition of three leafy vegetables commonly consumed in north eastern nigeria. paper presented at the 1 st national workshop on food composition at ibadan, nigeria pp. 1-3. okaka jc, akobundu ent, okaka nc (2000). “human nutrition, an integrated approach”, 2 nd edition, oc janco academic publishers, enugu, nigeria pp. 1-66. osagie au, offiong ue (1998). nutritional quality of plant foods”, ambik press, benin city, edo state, nigeria pp. 131-221. price mc, butler lc (1977). anti-nutritional content of some forage crops j. agric. food chem. 25 (6):1268-1273. reddy mb, love m (1999): the impact of food processing on the nutritional quality of vitamins and minerals, adv. exp. biol. 15: 4591006. sparkman dh, stein eh, moore s (1958). automatic recording apparatus for use in chromatography of amino acids, anal. chem. 30; 119. susane g (1996). a challenge for urban and rural development, agric. rural dev. 3: 42-44. who/fao (1973). reports: energy and protein requirements. geneva world health organisation (who) technical report series 522: 1113. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (2), pp. 255-262, february, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the nutritional composition of complementary foods micronutrients improved cassava varieties o. o. onabanjo 1, 2 *, c. r. b. oguntona 1 , bussie maziya-dixon 2 , i. o. olayiwola 1 , e. b. oguntona 3 and a. g. o. dixon 2 1 department of nutrition and dietetics, university of agriculture, abeokuta, ogun state, nigeria, 2 international institute of tropical agriculture, ibadan, oyo state, nigeria, 3 department of animal nutrition, university of agriculture, abeokuta, ogun state, nigeria. accepted 24 august, 2017 complementary foods were formulated and their nutritional composition analyzed from three yellow and one white cassava varieties. the composites were analyzed for proximate, mineral, fatty acids and amino acids. the mean values for the proximate composition of the diets are moisture (41.4 g kg -1 ) wet weight, ash (47.4 g kg -1 ), crude protein (145.8 g kg -1 ), crude fat (106.7 g kg -1 ), crude fiber (21.1 g kg -1 ), total carbohydrate (633.5 g kg -1 ), and energy (409.2 kcal) . the diets supplied adequate amounts of most minerals, except iron (about 65% of the standard). the composites contained all the essential amino acids with adequate chemical score for most amino acids. the complementary diets contained moderate to high proportions of -linolenic acids (18:3n3) and a high proportion of linoleic acid (18:2n6), which resulted in a high total n-6/total n-3 ratio and a high linoleic/ -linolenic acids ratio. micronutrient improved cassava varieties can be effectively used to formulate complementary food as acceptable micronutrients supplement. key words: micronutrient-improved, cassava, complementary foods, children. introduction childhood malnutrition is widely prevalent in many parts of the world, particularly in the developing countries like nigeria (unicef, 2006). protein, energy and micronutrient malnutrition are the commonest form. the period from birth to 2 years of age is a critical period for the promotion of optimal growth, health, and behavioral development. studies in nigeria have indicated a high prevalence of malnutrition among under-five children of which inappropriate breastfeeding and complementary feeding practices coupled with high rates of infectious diseases are the principal proximate causes of malnutrition during the first two years of life (nfcns, 2003; ndhs, 2003). breast milk is the perfect food for the infant during the first 6 months of life (unicef, 2006), because it contains all the nutrients and immunological factors an infant requires to maintain optimal health and growth. however, at about 6 months, the supply of energy and some nutrients from breast milk is no longer adequate to meet *corresponding author. e-mail: oonabanjo@cgiar.org. an infant’s needs, hence, need for complementary food (who/unicef, 1998; dewey, 2003; dewey and brown, 2003). the amount of nutrients required from complementary foods depends on the quantity provided by human milk and varies markedly by nutrient, ranging from nearly 100% for iron to 0% for vitamin c. the calculation of the nutrients required from complementary foods is based on the recommended intake for each nutrient minus the amount of the nutrient consumed daily from human milk (who/unicef, 1998; dewey and brown, 2003). the adequacy of complementary diet to meet nutrient requirements depends on the nutrient content of foodstuff used in the formulation. the food-based dietary guideline for nigeria has recommended the use of available foodstuff in various communities and home levels to prepare nutritious diets for infants and children. cassava, soybean, groundnut and carrot are readily available foods in nigeria. they have promising nutritional attributes. cassava is one of such staples with potential to meet this requirement, especially in the sub-saharan africa. the importance of cassava to the livelihoods of many millions of poor people has made the commodity a target onabanjo et. al. 256 for interventions. new partnership for africa development (nepad) has adopted the slogan “cassava: a powerful poverty fighter in africa” for its pan-african cassava initiative. the normal white tubers contain only small amounts of -carotene (bradbury and holloway, 1998), but yellow cassava contains up to about 100 times as much (mcdowell and oduro, 1993). in addition, cassava leaves are good sources of -carotene and are consumed as vegetables in many parts of the third world, especially in the democratic republic of congo, and senegal. cassava could be a vehicle for delivering micronutrientrich food to alleviate deficiencies. use of multimix might help to ensure nutrition security in infants and young children. the nutrient potentials of the food (cassava, soybean, groundnut and carrot) make it imperative that scientific studies be made on their composite for possible use as complementary food. the purpose of the study was to formulate and analyze the nutritional composition of complementary foods micronutrients improved (3 yellow and one fleshed) cassava varieties. materials and methods characteristics of raw ingredients cassava roots and leaves (manihot esculenta), soybean (glycine max.), groundnut (arachis hypogaea), and carrot (daucus carota) were used to formulate four composite diets. the cassava roots and leaves, and soybean were obtained from the experimental fields of the international institute of tropical agriculture, (iita) ibadan, nigeria. groundnut and carrot were purchased from local markets in ibadan. cassava roots were peeled manually with a stainless steel knife, washed thoroughly with potable water to remove all dirt and adhering sand particles. the roots were then grated into mash using a stainless steel grater driven by a petrol engine, packed into hessian sacks, and dewatered immediately using a hydraulic press to express liquid from the mash as well as to prevent fermentation. the mash was then sieved manually with a stainless steel sieve to pulverize the pressed cake and separate fibrous materials. the resulting flour was dried in an oven dryer at 40 0 c, and milled using a stainless steel milling machine before being packaged into polyethylene bags and stored at a cool dry room at -20 0 c. soybean and groundnut were cleaned and roasted in a preheated stainless steel pot on an electric stove for 15 min. the roasted seeds were dehulled manually by rubbing gently with hand and winnowed to remove the hulls. the dehulled seeds were then ground into fine flour with a domestic grinding machine. cassava leaf flour was obtained by washing fresh leaves and pressing them between the palms to remove excess water. the leaves were heattreated in an empty dry pot, placed over a source of heat. the leaves were tossed constantly with a wooden spoon to ensure even distribution of heat for 5 min. the heat–treated leaves were pounded in a wooden mortar for 30 min. the pounded leaves were put in twice their weight of water, gradually brought to a boil, and cooked for 1 h 30 min. the processed leaves were dried at 60 0 c for 48 h, milled, and stored in air-tight zip-lock bags until analysis (bokanga, 1994). the carrot powder was obtained by washing the carrot roots with water and then slicing and drying them at 40 0 c, and milling them into flour with a stainless steel milling machine. lastly, the processed food commodities were analyzed for their chemical proximate and mineral composition, fatty and amino acids profile and diets formulated based on the who/fao codex standard (fao/who, 1991). formulation of composite flours the composite flours were formulated by blending all the ingredients in different proportions using the owl tech wizard software tm , version 3 to develop and optimize the different formulations. the composite flours of the four complementary foods were formulated based on the optimization given by the software as follows: diet 1: cassava root (variety 01/1371), 67%: soybean, 14.7%: groundnut, 10.5%: cassava leaves, 5.3%: carrot, 2.5% (w/w). diet 2: cassava root (variety 01/1235), 67.6%: soybean, 13.5%: groundnut, 10.5%: cassava leaves, 5.7%: carrot, 2.7% (w/w). diet 3: cassava root (variety 94/0006), 67.7%: soybean, 14.1%: groundnut, 9.7%: cassava leaves, 5.7%: carrot, 1.8% (w/w). diet 4: cassava root (variety tme 1), 68.9%: soybean, 13.8%: groundnut, 9.3%: cassava leaves, 5.7%: carrot, 2.3% (w/w). chemical analysis the composite flours were analyzed for their proximate composition, minerals, amino acids, and fatty acids profile to ascertain their conformity with the standards. proximate composition standard procedures (aoac, 2005) were used to determine the moisture content, crude fat, fibre and ash content, crude protein (n * 6.25) by (hach, 1990), and total carbohydrate (dubois et al., 1956) from total starch and sugar. energy value was calculated using the atwater’s conversion factors, protein and carbohydrate (4 kcal/g) and fat (9 kcal/g). methods validation the accuracy of the concentrations determined in this study was checked by measurements of the reference materials srm no. 2383 baby food composite from the national institute of standards and technology of the department of commerce, united states of america. amino acid analysis amino acids composition was quantified after hydrolysis with hplc in reversed-phase column, after a derivatization with 9fluorentylmethyl chloroformate and a fluorescence detector at the irene analytical services, south africa (aoac, 2005). the concentration of the individual amino acid was calculated in relation to the protein content. chemical score was calculated as follows using egg as the reference protein. chemical score = mg amino acid in 1g of test protein × 100 mg amino acid in 1g of reference protein fatty acid analysis fatty acids were separated and quantified using a hewlett-packard gas chromatograph equipped with hp chemstation software (6890 series ii) after extraction with chloroform: methanol (2:1, v/v) as described by (christopherson and glass, 1969). 257 afr. j. food sci. res. table 1. proximate composition of the composite diets g kg -1 dry weight* nutrients diet 1 diet 2 diet 3 diet 4 yellow white moisture 41.7± 0.14 40.5± 0.07 42.1± 0.02 41.1± 0.13 ash 50.2± 0.07 49.4± 0.22 44.4± 0.08 45.4± 0.06 crude protein 147.7± 0.22 145.6± 0.38 145.0± 1.02 145.0± 0.86 crude fat 108.9± 0.57 107.0± 0.21 106.7± 0.08 104.1± 0.33 fiber 21.0± 0.08 21.7± 0.11 20.1± 1.01 21.7± 0.88 sugars 62.2± 0.54 58.9± 0.22 61.0± 1.09 46.5± 0.76 starch 581.3± 1.31 600.1± 0.21 558.7± 0.97 577.9± 0.54 total carbohydrate 643.5± 1.80 658.9± 2.17 619.7± 1.17 624.4± 1.11 energy (kj) 414500±12.33 418900± 11.10 401900± 9.44 401500± 14.11 *values are means ± sd for three determinations complementary foods, diet 1 (cassava variety 01/1371), 2 (variety 01/1235), 3 (variety 94/0006), 4 (variety tme 1) table 2. mineral contents (mg kg -1 )) of the diets. mineral diet 1 diet 2 diet 3 diet 4 (mg kg -1 ) yellow white iron 75.4 73.8 74.2 73.9 zinc 63.0 62.6 62.2 62.0 manganese 29.0 28.0 27.0 27.0 copper 89.0 87.0 87.0 90.0 calcium 2700.0 2480.0 2380.0 2510.0 boron 13.0 13.0 14.0 13.0 molybdenum 0.8 0.9 0.9 1.0 cobalt 0.2 0.2 0.2 0.2 nickel 1.9 1.7 1.7 2.0 magnesium 1490.0 1490.0 1440.0 1470.0 sodium 210.0 230.0 168.0 210.0 potassium 9100.0 9000.0 8600.0 8600.0 phosphorus 2400.0 2500.0 2600.0 2600.0 silicon 1480.0 1480.0 1490.0 1460.0 aluminium 10.0 1.4 9.0 1.1 titanium 0.6 0.7 0.6 0.6 chromium 0.0 0.0 0.0 0.0 cadmium 0.1 0.1 0.1 0.1 lead 1.0 1.0 1.0 1.0 selenium 4.0 4.0 4.0 4.0 analysis of minerals the minerals were determined at waite analytical services. school of agriculture and wine, university of adelaide, australia using an inductively coupled plasma atomic emission spectrometry (aoac, 2005) (icpaes) made in switzerland by arl model 3580 b. replicates of three samples were analyzed to check on the accuracy and reproducibility of the method. results the proximate compositions of the diets are presented in table 1. the mean compositions are moisture (41.4 g kg -1 ), ash (47.4 g kg -1 ), crude protein (145.8 g kg -1 ), crude fat (106.7 g kg -1 ), crude fibre (21.1 g kg -1 ), total carbohydrate (633.5 g kg -1 ), and energy (409.2 kcal). the mineral compositions of the four diets are presented in table 2. the iron, zinc and calcium contents of the flours ranged from 73.8 75.4 mg, 62.0 63.0 mg and 2380 – 2700 mg per kg dry flours respectively. the flours supplied adequate amounts of most minerals except for iron and zinc contents, which supplied about 65 and 67% of the codex standard (fao/who, 1991) respectively. again, a dry weight of 65 g of the composite flours were calculated and compared to recommended dietary allowances (rda) for the same age groups (dewey, 2001). the comparison result is presented in table 3. all the four complementary foods were found to provide more than adequate intake levels of selenium (4 mg/kg/day) and magnesium (957.1 mg/kg/day), and relatively low amounts of calcium (1636 mg/kg/day), iron (48.31 mg/kg/day), and zinc (40.59 mg/kg/day). the composites contained all the essential amino acids (table 4). the comparison of the essential amino acids to the fao reference values (who/fao, 1985), and percentage chemical score are presented in table 5. the complementary foods contained higher chemical scores of most essential amino acids. the chemical score ranged from 37.7% in formula 4 for methionine to 310% in formula 1 for leucine. six (6) of the essential amino acids, leucine, lysine, phenylalanine, threonine, tyrosine and valine, met the fao/who reference values in all the diets. the fatty acids compositions of the diets are presented in table 6. the four diets contained all the fatty acids between tetradecanoic acid tetracosanoic acid (c-14 to c-24) with the exception of hexanoic acid (c-6) through tridecanoic acid (c-13), pentadecanoic acid (c-15), and nicodecanoic acid (c-21). odd-chain fatty acids, such as heptadecanoic acid (c17:0) and 10heptadecenoic acid (c17:1), were present in very small concentrations, varying from 0.4 g kg -1 of total fatty acids to 1.1 g kg -1 . compared with the who recommendation onabanjo et. al. 258 table 3. daily consumption of the four complementary diets compared to the recommended daily allowance. mineral diet 1 diet 2 diet 3 diet 4 mean rda † (6-11 (mg kg -1 /day) yellow white months) iron 49.01 47.97 48.23 48.035 48.31 90.0 * zinc 40.95 40.69 40.43 40.30 40.59 41.0 *† manganese 18.85 18.20 17.55 17.55 18.04 copper 57.85 56.55 56.55 58.50 57.36 calcium 1755.00 1612.00 1547.00 1631.50 1636.00 4000.0 boron 8.45 8.45 9.10 8.45 8.61 molybdenum 0.52 0.59 0.59 0.65 0.59 cobalt 0.13 0.13 0.13 0.13 0.13 nickel 1.24 1.11 1.11 1.30 1.19 magnesium 968.50 968.50 936.00 955.50 957.10 530.0 sodium 136.50 149.50 109.20 136.50 132.90 potassium 5915.00 5850.00 5590.00 5590.00 5736.00 phosphorus 1560.00 1625.00 1690.00 1690.00 1641.00 silicon 962.00 962.00 968.50 949.00 960.40 aluminium 6.50 9.10 5.85 0.72 5.54 titanium 0.39 0.46 0.39 0.39 0.41 chromium 0.00 0.00 0.00 0.00 0.00 cadmium 0.07 0.07 0.07 0.07 0.07 lead 0.65 0.65 0.65 0.65 0.65 selenium 2.60 2.60 2.60 2.60 2.60 1.00 † fao/who (2002). * rda, assuming a moderate bioavailability of 10% iron absorption. *† rda, assuming a moderate bioavailability. table 4. amino acid composition of the diets (g kg -1 protein). amino acids fao/who diet 1 diet 2 diet 3 diet 4 reference.protein* yellow white isoleucine 42 40.0 38.4 39.5 41.4 leucine 42 130.2 72.9 71.9 72.7 lysine 42 65.4 61.4 64.1 62.6 methionine 22 10.6 11.5 10.2 8.3 phenylalanine 28 50.1 50.5 50.3 50.8 threonine 28 35.3 35.8 35.9 36.1 valine 42 46.5 46.1 47.3 48.5 tryptophan 14 8.8 12.8 12.0 13.6 alanine 45.9 44.8 45.5 47.3 arginine 106.0 108.1 108.4 109.9 aspatatic acid 111.9 113.2 11.08 114.1 cysteine 20 11.2 9.0 9.0 08.9 glutamic acid 177.3 181.7 179.1 178.5 glycine 47.7 48.6 46.7 48.5 histidine 37.1 27.5 33.0 26.0 proline 48.3 48.6 48.5 47.9 serine 44.8 47.3 45.5 44.9 tyrosine 28 41.8 41.6 41.9 40.2 * (fao/who, 1985) 259 afr. j. food sci. res. table 5. percentage chemical scores of the essential amino acids in the diets. amino acid diet 1 diet 2 diet 3 diet 4 yellow white isoleucine 95.2 91.4 94.0 98.6 leucine 310.0 173.6 171.2 173.1 lysine 155.7 146.2 152.6 149.0 methionine 48.2 52.3 46.4 37.7 phenylalanine 178.9 180.4 179.6 181.4 threonine 126.1 127.9 128.2 128.9 valine 110.7 109.8 112.6 115.5 tryptophan 62.9 91.4 85.7 97.1 table 6. fatty acids composition of complementary diets as g kg -1 total fatty acids. fatty acid diet 1 diet 2 diet 3 diet 4 yellow white saturated fatty acid tetradecanoic acid (14:0) 0.7 0.5 0.4 0.4 hexadecanoic acid (16:0) 97.3 97.2 97.7 98.1 heptadecanoic acid (17:0) 1.0 0.9 0.9 1.1 octadecanoic acid (18:0) 0.6 29.8 30.3 30.2 eicosanoic acid (20:0) 13.1 13.1 13.7 13.3 docosanoic acid (22:0) 0.6 20.5 20.8 21.0 tetracosanoic acid (24:0) 1.6 11.4 11.7 11.6 monosaturated fatty acids 9-tetradecenoic acid (16:1) 0.7 0.6 0.6 0.7 10-heptadecenoic acid (17:1) 0.4 0.4 0.5 0.4 9octadecenoic acid (18:1) 529.0 518.7 512.9 515.8 11-eicosenoic acid (20:1) 10.0 9.8 9.9 9.7 13-docosenoic acid (22:1) 0.6 0.6 0.6 0.6 polyunsaturated fatty acids 9,12-octadecadienoic acid (18:2) 270.6 282.2 285.9 283.2 9,12,15-octadecatrienoic acid (18:3) 12.5 13.3 13.2 13.0 9,12,15-eicodecatrienoic acid (20:3) 0.6 0.7 0.6 0.6 9,12,15,18-eicodecatetranoic acid (20:4) 0.4 0.5 0.6 0.4 diets contained moderate (12.5 13.3 g kg -1 ) to high proportions of -linolenic acids (18:3n3) and high proportion of linoleic acid (18:2n6), which resulted in a high total n-6/total n-3 ratio and a high linoleic/ -linolenic acids ratio (20.10 22.10) (table 7). discussion in assessing the overall nutritive intake of infants, the combined composition of the breast milk and complementary food needs to be taken into consideration. some nutrients, such as fatty acids, are abundant in breast milk. however, low concentrations of other nutrients, such as iron, calcium, and zinc, may lead to clinically significant deficiencies if without supplementation (fao/who, 1995). also, the recommendation of the world health organization that children should be exclusively breastfed until 6 months, with introduction of complementary foods and continued breastfeeding thereafter (fao/who/unu, 1985), was considered in analyzing the nutritional value of the complementary foods. the potential disadvantages of fiber in foods for infants and young children are an important nutritional issue (jansen, 1980). possible undesirable aspects of high fiber levels in weaning foods include increased bulk and lower caloric density, irritation of the gut mucosa, and onabanjo et. al. 260 table 7. fatty acid composition (g kg -1 wt/wt) of the complementary diets compare to who recommendations. fatty acid diet 1 diet 2 diet 3 diet 4 recommended † yellow white linoleic acid 18:2n-6 270.6 283.2 110-120 282.2 285.9 linolenic acid 18:3n-3 12.5 13.3 13.2 13.0 8-9 arachidonic acid 20:4n-6 0.4 00.5 0.6 0.4 5-6 total unsaturated 824.8 826.8 824.8 824.4 448-524 total saturated 174.9 173.4 175.5 175.7 452-535 unsaturated/saturated ratio 5:10 5:10 5:10 5:10 0.84:1-1.16:1 18:2n-6/18:3n-3 ratio 20:10 21:10 22:10 22:10 5:1-10:1 total n-6/total n-3 ratio 22:10 22:10 22:10 22:10 2.4:1-2.7:1 † recommended data from koletzko et al. (1992). medians calculated from average fa values reported of human milk in europe and africa. ratio of 18:0 to 18:3 as suggested by the fao/who joint consultation, 1995. adverse effects on the efficiency of absorption of various nutrients of significance in diets with marginal nutrient content. a maximum level of 5% of fiber for infants and young children is set by the fao/who/unu codex standards (fao/who, 1998). children should have lower dietary fiber intakes than adults, with the recommended amount proportional to body weight (hegazy et al., 1989). the fat content of the complementary foods was within the fao/who codex standard (fao/who, 1991). this could be attributed to the inclusion of soybean and groundnut in the blends, and tends to agree with the recommendations (fao/who, 1998), that vegetable oils be added in foods meant for infants and children. this will not only increase the energy density but also be a transport vehicle for fat soluble vitamins. brown et al. (1998) have concluded that infants aged 6 8 months who are consuming an average amount of breast milk/day (674 g/day) do not need the dietary supplementation of fatty acids to achieve 30% of the daily intake. if the breast milk has a low fat concentration, as may happen in developing countries, infants require an additional 10 24% of the energy from fat. by the time infants reach 9 months of age, their complementary food diet should provide 13% of the energy from fat (dewey and brown, 2003) . the four complementary formulas supply appreciable quantities of fat to the blends. the fat can also provide essential fatty acids, such as n-3 and n6 polyunsaturated fatty acids (pufa), needed to ensure proper neural development. fatty acids, especially the essential ones (efa), linolenic acid and linoleic acid, are precursors to docosahexaenoic acid and arachidonic acid that are critical for growth and development, particularly of the nervous system in the first 6 months of life (salem et al., 1996). both are necessary components of myelin, synaptic cell membranes, and photoreceptor cells. the report of who on complementary feeding concludes that diets low in fat may be adequate, as long as the minimum requirements for efa are met (brown et al., 1998). fernandez et al. (2002), and his colleagues also were of the same opinion, as the findings of their study on the fatty acid composition of nigerian weaning foods revealed that the foods were devoid of arachidonic and decosahexanoic acids, but high in linoleic and linolenic acids. the linoleic acid to linolenic acid ratio observed in this study is higher than the recommendation. the concern is that the relatively high levels of linoleic acid might inhibit the synthesis of docosahexaenoic acid from -linolenic acid (ballabriga, 1994). the energy values of the complementary foods are in accordance with the recommendations of fao/who/unu (1985), which specify 1000 kj/g (1.0 kcal/g) as safe for small children aged 2 to 5 years. the food and agriculture organization and the world health organization (fao/who, 1998) have also recommended that foods fed to infants and children should be energydense. this, according to the recommendation, is necessary because low energy intake foods tend to limit total energy intake and the utilization of other nutrients. this suggests that infants may not have to consume larger quantities of the formulas to meet their energy needs. the requirements for the maintenance of body protein equilibrium as well as the optimum pattern of individual essential amino acids change little between the ages of 6 and 24 months (reeds and garlick, 2003). according to the codex standard, the amounts of protein that should be supplied by complementary foods are between 60 and 110 g kg -1 of dry food. the protein values in this study agrees with the recommended values of the codex standard, meaning that all the formulas provide adequate levels of protein for a child who consumes an average amount of breast milk. these values are slightly below the recommendation of the fao/who/unu (1985), which recommends a minimum protein content of 261 afr. j. food sci. res. 150 g kg -1 dry weight. both soybean and groundnut have been recommended for infant feeding (badamosi et al., 1995; temple et al., 1996; gibson et al., 1998; nnam , 2002) due to their positive contribution to protein nutrition levels. the amino acid content of complementary foods is a particularly relevant issue in infant feeding, where protein-energy malnutrition (pem) has continued to pose challenges in developing countries. this, according to other researchers, is due to poor feeding practices and the low quality protein commonly associated with plantbased single diets (badamosi et al., 1995; temple et al., 1996). all the essential amino acids were present in the complementary foods, but quite a number did not meet the fao/who reference values. a lower score for any of the essential amino acids designates the limiting characteristics of the amino acid and gives an indication of the protein quality. complementary foods are important sources of micronutrients for developing infants. iron, zinc, phosphorus, magnesium, and calcium have been identified as problem nutrients from six months of age and must be supplemented by the addition of complementary food (brown et al., 1998). according to brown and co-workers (who, 1998), breast milk can adequately supply the nutrients need of infants 9 to 11 months old for vitamin c, folate and b12. in contrast, complementary foods should provide approximately 12% of the vitamin a, 25% to 50% of the cu, riboflavin, 50% to 75% of thiamin, mn, and 75% to 100% of niacin, zn and fe, assuming an average composition and intake of breast milk, meaning that the composition of these micronutrient is low in human breast milk. the calcium, zinc, copper, magnesium, and phosphorus contents of the four complementary foods are adequate to meet the needs of children aged 6 to 23 months. this supported the findings (bond et al., 2005). however, the iron standards were not met, although about 65% of the fao/who codex standards were supplied (fao/who, 1991). these local complementary foods would have to be further fortified with iron to adequately complement breast milk. the absorption and utilization of the iron and zinc in complementary foods which contain plant materials are significantly lower than in those from animal sources. the bioavailability of non-heme iron found in plant sources is 2 8%, compared to 25% bioavailability of heme iron found in animal products (brown et al., 1998). phytic acid is a potential inhibitor of both iron and zinc in plant food materials. it affects the bioavailability of minerals by forming an insoluble complex with the metals of iron, zinc, and calcium, inhibiting their absorption (gibson et al., 1998). the inhibitory effect of phytic acid on zinc absorption is notably dose-dependent (sandstrom and lonnerdal, 1989). hence, any strategies that reduce the phytic acid content of these complementary foods might markedly enhance zinc bioavailability and simultaneously increase absorption of non-heme iron and calcium to some extent. the bioavailability of non-heme iron or zinc can also be improved by enriching the complementary foods with sources of absorption enhancers such as ascorbic acid (for non-heme iron), other organic acids and cellular animal protein (for non-heme iron and zinc), and fat (for retinol and provitamin a carotenoids). these enhancers can be added in the form of fresh fruits (citrus fruits), vegetables (tomatoes, green leaves), legumes (groundnut flour) or small amounts of meat, poultry, or fish (perhaps as dried flours) and their inclusion in complementary foods should be encouraged (gibson et al., 1998). from this study, it would be concluded that utilization of micronutrients improved cassava varieties particularly to formulate complementary food in various cassava agroecologies in the region will contribute to an increase in the intake of micronutrients and hence help in combating deficiencies, primarily of iron, calcium, and zinc. acknowledgements we thank the international institute of tropical agriculture, ibadan, nigeria, for support to this research. references aoac (2005) official methods of analysis association of official analytical chemists, washington d.c, usa. badamosi ej, ibrahim lm, temple vj (1995) nutritional evaluation of a locally formulated weaning food, juth-pap. west afr. j. biol. sci. 3: 85-93. ballabriga a (1994) essential fatty acids and human tissue composition. an overview. acta paediatrica scandinavica. 402: 63-68. bokanga m (1994). processing of cassava leaves for human consumption. international workshop on cassava safety. acta horticulturae 375: 203207. bond b, fernandez diane r, vanderjagt dorothy j, williams margaret, huang yung-sheng, chuang lu-te, millson mark, andrews ronee, glew robert h (2005). fatty acid, amino acid and trace mineral analysis of three complementary foods from jos, nigeria. j.food comp. anal. 18: 675-690. bradbury jh, holloway wd, (1988). chemistry of tropical root crops; significance for nutrition and agriculture in the pacific. aciar monograph series. ramsay ware printing, melbourne. p.890 brown kh, dewey kg, allen lh (1998). complementary feeding of young children in developing countries: a review of current scientific knowledge. who/nut/98. world health organization, geneva, switzerland. christopherson sw, glass l (1969). preparation of milk fat methyl esters by alcoholysis in an essential nonalcoholic solution. j. dairy sci. 52: 1289-1290. dewey kg, brown kh (2003). update on technical issues concerning complementary feeding of young children in developing countries and implications for intervention programs. food nutr. bull. 24: 5-28. dewey kg (2003). nutrient composition of fortified complementary foods: should age-specific micronutrient content and ration sizes be recommended? j. nutr. 133: 2950s-2952s. dewey kg (2001). nutrition, growth and complementary feeding of the breastfed infant. ped. clin. n. amer. 48: 87-104. dubois m, gilles ka, hamilton jk, rebers pa, smith f (1956). colorimetric method for determination of sugars and related substances. anul. chem. 28: 350-356. onabanjo et. al. 262 fao/who (1991). codex standard for processed cereal-based foods (including guidelines on formulated supplementary foods for older infants and young children). world health organization, geneva, switzerland. fao/who (2002). human vitamin and mineral requirements. report of a joint fao/who expert consultation. rome, italy fao/who (1998). preparation and use of food-based dietary guidelines. report of a joint fao/who consultation. technical report series 880. who, geneva, switzerland. fao/who/unu (1985). energy and protein requirements. reports of a joint fao/who/unu expert consultation. world health organization technical report series 724. who. geneva, switzerland. fernandez de, vanderjagt dj, williams m, huarg ys, chuang lut-te, millson m, andrew r, pastuszyn a, rh glew (2002). fatty acids, amino acids, and trace mineral analyses of five weaning foods from jos, nigeria. plant foods for human nutrition. 57: 257-274 gibson rs, ferguson el, lehrfeld j (1998). complementary foods for infants feeding in developing countries: their nutrient adequacy and improvement. eur. j.clin. nutr. 52: 764-770. hach (1990). systems for food, feed and beverage analysis procedures. hach company, loveland, colorado. 80539-0389, usa. hegazy sm, hussien nm, bressani r (1989). nutritional quality of lowcost food supplements for infant feeding. egypt j. food sci. 17:1-13. jansen gr (1980). a consideration of allowable fiber levels in weaning foods. food nutr. bull. 2:38-47. koletzko b, thiel i, abiodun po (1992). the fatty acid composition of human milk in europe and africa. j. paed. 120: 62-70. mcdowell i, oduro ka (1993). investigation of the bcarotene content of yellow varieties of cassava (manihot esculenta crantz). j plant foods. 5: 169-171. nigeria demographic and health survey (ndhs) (2003). in nutrition of young children and mothers in nigeria, usa. nigeria food consumption and nutrition survey summary. iita, ibadan, nigeria (2003). nnam nm (2002). evaluation of complementary foods based on maize, groundnut, pawpaw and mango flour blends. nig. j. nutr. sci. 22(23): 8 -18. reeds pj, garlick pj (2003). protein and amino acid requirements and the composition of complementary foods. j. nutr. 133: 2953s-2961s. salem norman jr, wegher brent, mena patricia, uauy ricardo (1996). arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants. proc. natl. acad. sci. usa sandstrom, b and lonnerdal, b. (1989). promoters and antagonists of zinc absorption, in zinc in human biology ed. cf mills 57-58. international life sciences institute. human nutrition reviews, springer-verlag. london, uk. temple vj, badamosi ej, ladeji o, solomon m (1996). proximate chemical composition of three locally formulated complementary foods. west afr. j. biol. sci. 5: 134-143 unicef (2006). united nations children's fund. state of the world children, brodock press limited. who/fao (1995). (world health organization/food and agriculture organization). who and fao consultation: fats and oils in human nutrition. nutrition rev. 53: 202-205. who/unicef (1998). complementary feeding of young children in developing countries: a review of current scientific knowledge. who/nut/98.1. world health organization, geneva. pp. 1-228. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (5), pp. 001-004, may, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of steeping time of milled grains on the quality of kunnu-zaki (a nigerian beverage) obadina, a.o1, oyewole, o.b2, and awojobi, t.m2 1 department of food science and technology, bells university of technology, p.m.b. 1015, ota, ogun state, nigeria. 2 department of food science and technology, university of agriculture, abeokuta, p.m.b. 2240, ogun state, nigeria. accepted 17 march, 2019 millet grains were steeped in water for varying period of time during “kunun zaki” production in order to study the effect of the duration of steeping on the quality of “kunu zaki”. other processing factors were kept constant in the course of this study. kunun zaki produced from millet grains steeped for 36 h was rated best in terms of sensory characteristics. the steeping period had no significant effect on the specific gravity of the produced “kunun zaki”. as expected the titratable acidity and ph were inversely proportional, with the latter decreasing and the former increasing during the fermentation-steeping period. the protein content increased between 12 and 48 h steeping time. during the steeping period, the carbohydrates decreased rapidly in the first 12 h. however, the rate of carbohydrates decrease reduced beyond the first 12 h. this may be due to the decrease in the rate of fermentable sugars. key words: kunun-zaki, millet, steeping, quality, ph, titratable acidity, sensory. introduction the soft drink segment of the nigerian beverage industry is heavily dependent on imported raw material and nige-ria is presently passing through a developmental stage in which there is a strong emphasis on local sourcing of raw materials. this awareness has transformed into a general interest in commercial processing of indigenous foods in order to conserve the scarce foreign exchange by limiting importation of raw materials. cereals are the major local raw materials in the production of beverages in nigeria which include pito, burukutu, local gin and kunnu-zaki. cereals are eaten in large quantities and are the main sources of both major and minor nutrients (adeyemi and umar, 1994). due to their high viscosity on cooking when made into gruel, large amount of water is used during preparation to obtain the right consistency; this obviously decreases the nutrient density (mosha and svanberg, 1983). kunnu-zaki is a traditional non-alcoholic fermented beverage widely consumed in the northern part of nigeria. it is however becoming widely consumed in the southern parts among low and middle income workers who cannot afford industrially produced beverages like coca-cola, pepsi etc. its popularity is due to its characteristic sweet-sour taste, its refreshing *corresponding author. e-mail: obadinaw@gmail.com. quality as well as the creamy or milky appearance and also its flowing consistency. adeyemi and umar (1994) described the traditional process for the manufacture of kunnu-zaki which involves steeping of millet grains, wet milling with spices (ginger, cloves, and pepper), wet sieving and partial gelatinization of the slurry, followed by addition of sugar to taste and bottling. as a cereal product kunnu-zaki can supply carbohydrates, proteins, fats, minerals and vitamins if the nutrients are not destroyed by over processing (chapman and cater, 1976). according to hamad and fields (1979) there is significant increase in the relative nutritive value of several cereals after natural fermentation. fermentation in the case of kunnu-zaki is by lactic acid bacteria and yeast (adeyemi and umar, 1994). unlike other traditional cereal-based alcoholic beverages, information is scanty on the manufacturing process, quality and rheological characteristics of kunnu-zaki. in its traditional manufacture, the basic processes are not standardized and levels of ingredients such as spices and sweetener are not quantified. one of the unit operations that vary from one processor to the other is the steeping period. the effect of these variations on the quality of kunnu-zaki is yet to be inves tigated. the objective of this work was to study the effect of steeping time on the physico-chemical, nutritional quality and consumer acceptability of the kunnun-zaki materials and methods the materials used are millet ( pennisetum typhoides ), spices-ginger (zingiber officinale), black pepper (piper niger), red pepper (capsian annum) and cloves (syzygium aromaticum), and granulated sugar. these materials were obtained from local markets in abeokuta, they were cleaned and sorted. preparation of kunnu-zaki 500 g of dehulled millet grains were cleaned and sorted, weighed and steeped in 1000 ml of water at room temperature for 48 h. the same procedure was repeated four times after steeping each sample was milled with 3.25 g ginger, 0.25 g cloves, 1.25 g red pepper and 0.25 g black pepper .the resulting slurry was sieved using a sterile sieve until all the starch is extracted, the shaft were then discarded. the filtrate was allowed to settle, the supernatant decanted and the sediment was divided into 2 parts (ratio 3:2). the larger portion was cooked by the addition of 500 ml boiling water for 5 min while 500 ml cold water was added to the second part. the two slurries were thoroughly mixed and sweetened with 50 g granulated sugar (i.e. 10% sugar w/w millet used). proximate analysis moisture content the moisture content was determined according to (aoac, 1990) method. ash content determination the ash content was determined according to (aoac, 1990) method. crude fibre determination 2 g of the sample was accurately weighed into a fibre glass and 100 ml of 0.255 n h2so4 was added. the mixture was heated under reflux for 1 h with the heating mantle. the hot mixture was filtered through a fibre sieve cloth. the filterate obtained was thrown off and the residue was returned to the fibre glass to which 100 ml of 0.31 n naoh was added and heated under reflux for another 2 h. the mixture was again filtered through a fibre sieve cloth and 10 ml of acetone was added to dissolve any organic constituent. the residue was washed with about 50 ml hot water twice on the sieve cloth before it was finally transferred into the crucible. the crucible and the residue were oven dried at 105ºc overnight to drive off moisture. fat determination gravimetric rose-gottlieb method as described by pearson (1981) was used. protein determination the protein content of the sample was determined by using formal titration method as described by pearson (1981). ph determination the ph was measure using electrometric ph meter (genway, 4590). this was calibrated by the use of prepared buffer solutions of accurately known ph (ph 4 and ph 9). 30 ml of the sample was measured into a curvette and the glass electrode of the ph metre was dipped into the sample. total titratable acidity the titratable acidity of the product was measure by direct titration as described by pearson (1981).10 ml of the sample was pipetted into each of two beakers labeled c and s. to the colour control beaker, 1 ml of rosanilline solution was added and stirred. to sample beaker s, 1 ml of phenolphthalein indicator was added and titrated with 0.1 m naoh, with continuous stirring until the colour matched the pink colour of c. the acidity was calculated as the lactic acid (percent m/v). specific gravity determination the specific gravity of each sample was determined using the picnometer specific gravity bottle. the bottle was washed, rinsed and dried. the empty bottle was weighed and mass recorded as m. it was filled with sample and weighed, mass recorded as m1. the bottle was emptied, rinsed and filled with water and weighed, mass recorded as m2. the specific gravity was calculated sensory evaluation the four different kunun zaki steeped for varying period of time were subjected to sensory evaluation. a total of twenty untrained panelist drawn from the university of agriculture, abeokuta based on their familiarity with the product were used for the evaluation. the parameter evaluated includes taste, colour, flavour, sweetness and overall acceptability. the coded samples were served in clean transparent plastic bottles at room temperature (25 o c) in individual booths with adequate florescent lights. sample presented to the panelists was at random and one at a time. the panelists were given enough water to rinse their mouths between each sample. the nine-point hedonic scale (larmond, 1977) was used for the evaluation and the resulting data were analyzed using analysis of variance (anova) to establish significant differences among treatment. duncan multiple range test was used to separate means where significant differences existed (duncan, 1955). results and discussion sensory evaluation the mean sensory scores for mouth feel, colour, taste, flavour, sweetness and over all acceptability of kunun zaki samples prepared from millet grains steeped for varying period of time are given in table 1. the highest mean values of preference were obtained from 36 h steeping. the analysis of variance revealed no significant difference in all the samples in terms of mouth feel, flavour and sweetness at 5% probability level. however, sample steeped for 36 h was rated highest in mouth feel and flavour. relatively equal rating of the samples in terms of sweetness and flavour were probably as a result table 1. mean of sensory scores. duration of steeping parameters 12 hours 24 hours 36 hours 48 hours mouth feel 5.45 a 1.9 6.20 a 1.3 6.20 a 1.29 5.45 a 1.76 overall acceptability 5.65 b 2.0 6.65 ab 1.7 6.80 a 0.9 6.35 ab 1.3 colour 6.00 b 2.0 6.65 ab 2.0 7.40 a 1.1 7.25 a 1.2 taste 5.45 b 1.6 6.40 ab 1.6 6.70 a 0.9 5.95 ab 1.7 flavour 5.75 a 1.6 6.40 a 1.2 6.20 a 1.7 5.70 a 1.7 sweetness 5.90 a 1.9 6.05 a 1.9 6.85 a 1.1 5.90 a 1.4 1 values are mean standard deviation of 20 participants 2 mean along the same row with different superscript are significantly different from each other at 1% probability level. table 2. proximate composition of kunun-zaki on dry basis. duration of steeping parameters 12 hours 24 hours 36 hours 48 hours crude protein 5.16d 0.003 5.78c 0.005 5.86b 0.007 6.35a 0.002 fats content 19.02c 0.009 22.67b 0.002 24.08a 0.001 24.13a 0.005 ash content 2.20a 0.002 2.19a 0.007 2.17b 0.007 2.13c 0.007 carbohydrates 73.59a 0.007 69.38b 0.003 67.84c 0.009 67.44d 0.003 mean along the same row with different superscript are significantly different from each other at 1% probability level. figure 1. variation in protein, fats, carbohydrates and ash contents of kunun-zaki with different steeping time. of equal amount of sugar and spices added to each sample after fermentation, during the production to ensure that there is no variation in the factors of production other than the steeping time. there were significant differences in the colour of the samples at probability level of 5%, with samples steeped for 36 and 48 h rated relatively equal and better. there were no significant differences in the taste and over all acceptability; however from the mean separation, 2 homogenous sub-sets were obtained with sample steeped for 36 h rated highest (best) in taste and overall acceptability. proximate composition table 2 shows the proximate composition of kunun zaki on dry basis. the result of the analysis of variance revealed significant difference between the values of crude protein, fats, ash and carbohydrates contents obtained for each steeping time. a plot of the variation in crude protein, carbohydrates, fats and ash contents with steeping time was made (figure 1). kunun zaki steeped for 12 h contains 5.2% protein, while that of 36 h has 6.4% protein. in general, a gradual increase was observed in trend of protein with steeping time. this agrees with the report of hamad and fields (1979), au and fields (1981), kazanas and fields (1981), that there was significant increase in the relative nutritive value of proteins after natural fermentation (occurring during steeping). kazanas and fields (1981) also reported increase in average nutritive value of proteins by 10.67% lysine and iso leucine increase from 8.60 to 10.50 mg/g and nitrogen from 8.33 to 109.58 mg/g after fermentation. suberu (2001) also reported that a gradual increase in total soluble proteins of kunun zaki accompanied natural fermentation. figure 1 also shows the variation in carbohydrates with steeping time. 73.6% was reported in the product steeped from 12 h while 69.4% was recorded for 24 h. the rapid decrease was probably as a result of decrease in starch content during fermentation according to usha et al. (1996) and high consumption of sugar by microbes 7 6 5 p h 4 ph 3 2 1 0 12 18 24 30 36 42 48 54 time (h) figure 2. variation in ph of kunun-zaki with different steeping time. 0.04 0.035 a c id it y 0.03 0.025 t it ra ta b l e 0.02 tta 0.015 0.01 0.005 0 12 18 24 30 36 42 48 54 time (h) figure 3. variation in total titratable acidity kunun-zaki with different steeping time. which are mainly active at this stage. carbohydrates recorded for the subsequent steeping time decrease at a lower rate. consumption of sugar in the production of organic acids was probably slower due to the feedback inhibition of the microbes by accumulated acidity. the variation in fats content with steeping time was also demonstrated in figure 1. from 12 h of steeping time there was a gradual increase in fats content of the products. the ash content with corresponding steeping time was also recorded in figure 1. the ash content of product steeped for 12 h was 2.2%, for 24, 36 and 48 h lower value were obtained. figure 2 shows the value for ph, titratable acidity and specific gravity of the kunun zaki steeped for different period. the result of analysis of variance indicates that there was a significant difference between the values. decrease in ph was a result of increasing hydrogen ion content probably due to the microbial activity on the carbohydrates and other food nutrient to produce organic acids. the range agrees with the report of adeyemi and umar (1994). analysis of variance of the results of the titratable acidity shows that there was a significant difference at (p<0.05) between the values in general; there was increase in titratable acidity with increasing steeping time. 0.0154% was obtained at 12 h steeping, 0.0262% at 24 h and 0.0340% at 48 h (figure 3). conclusion the relationship between the physical, chemical properties and nutritional parameters of kunun zaki and the steeping time of the millet grains was described. there were significant differences in the colour of the samples at probability level of 5%, with samples steeped for 36 and 48 h rated relatively equal and better. it can be concluded that in conclusion section, the relationship between the physical, chemical properties and nutritional parameters of kunun zaki and the steeping time of the millet grains was described. it can be concluded that kunun zaki from millet grains steeped for 36 h produced the best sensory and nutritional characteristics. references adeyemi la, umar s (1994). effect of method of manufacture on quality characteristics of kunu-zaki, a millet based beverage. niger. food j. 12: 34-42. au pm, fields ml (1981). nutritive quality of fermented sorghum. j. food sci. 46: 652. chapman rs, carter pl (1976). crop production principles and practices. freeman and company, san francis and co., pp. 67-69. hamad a, fields ml (1979). evaluation of protein quality and available lysine of germinated and fermented cereals. j. food sci. 44: 456. mosha ac, svanberg ed (1983). preparation of weaning foods with high nutrient density using flour of germinated cereals foods. nutr. bull. 5: 10 -14. suberu pa, kassum a (1992). rheological characterization of nigerian liquid and semi-liquid foods, kunu-zaki and kunungyada. niger. food j. 10: 23-33. usha as, chandra ts (1996). effect of fermentation on the primary nutrients in finger millet (eleusine coracana). j. agric. food chem. 44: 2616-2618. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (5), pp. 300-307, may, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper chemical composition of tubers of eight genotypes and the effect of domestic cooking methods on their nutritional quality p. e. akin-idowu 1,2 , r. asiedu 1* , b. maziya-dixon 2 , a. odunola 1 , a. uwaifo 1 1 cancer research and molecular biology laboratories, department of biochemistry, university of ibadan, ibadan, nigeria. 2 international institute of tropical agriculture, oyo road, p. m. b. 5320, ibadan, oyo state, nigeria. accepted 05 september, 2017 raw and processed tubers from eight genotypes of yellow yam (dioscorea cayenensis) were evaluated for their contents of iron, zinc, calcium, phosphorus, total carotenoids, vitamin c, phytic acid and tannin. the mean values obtained (in mg/kg on dry weight basis) were 7.2 for iron, 9.2 for zinc, 169.3 for calcium, 1331.3 for phosphorus, 181.8 for phytate and 353.6 for tannin. similarly, 6.31 µg/g of total carotenoids and 37.3 mg/kg of ascorbic acid were obtained on fresh weight basis. the genotypes differed significantly (p < 0.05) in tuber contents of zinc, calcium, phosphorus, total carotenoids and vitamin c but they were similar with respect to iron, phytate and tannin contents. genotype tdc 95-65 had the highest levels of zinc, calcium, total carotenoids and phytate while tdc 95-294 had the highest levels of iron and phytate. tubers of tdc 95-65, tdc, 95-294, tdc 04-168 and tdc 98-136 are good sources of iron, zinc and phosphorus. the two cooking methods had little effect on the minerals but significantly reduced the levels of total carotenoid, vitamin c, phytate and tannin contents. key words: antinutrients, boiling, dioscorea cayenensis, phytic acid, pounding and tannin. introduction yam, dioscorea species is an important staple in much of west africa (omonigho and ikenebomeh, 2000). annual world production of yam is about 40 million tonnes and per capita consumption is estimated to be 256.4 g per day in the major production zones (faostat, 2005). yam is of higher nutritional value than some other root and tuber crops such as cassava (latham, 1969) . its pro-tein content is about 3 6% as compared to 1 2% in cassava (charles et al., 2004). yams are reported to contain relatively high levels of minerals (afoakwa and sefa-dedeh, 2001). iron is an important trace element in the human body. it plays crucial roles in haemopoiesis, control of infection and cell mediated immunity (beard, 2001). iron deficiency anaemia is the most prevalent nutritional deficiency and is estimated to affect more than one billion people worldwide (trowbridge and martonell, 2002). the nigeria food consumption and nutrition survey 2001 2003, showed that 27.45% of children less than *corresponding author. e-mail: r.asiedu@cgiar.org. tel: +234 2-2412626. fax: +234-2-2412221. five years, 24.3% of mothers and 35.3% of pregnant women are suffering from iron deficiency (maziya-dixon et al., 2004). zinc is an essential micronutrient necessary for human growth and immune functions (black, 2003). an estimated 20% of the world population is reported to be at risk of inadequate zinc intake (hotz and brown, 2004). the nigeria food survey showed that zinc defi-ciency affects 20% of children less than five years, 28.1% of mothers and 43.9% of pregnant women (maziyadixon et al., 2004). yellow yam (dioscorea cayenensis) is an important species of cultivated yam based on the sig-nificant role it plays in the diet of many people in coastal west africa, selected countries in east and central africa, and in the caribbean region. it is known to contain carotenoids which represent the most widespread group of naturally occurring pigments in nature (martin and ruberte, 1975). carotenoids are primarily of plant origin and -carotene which predominates (chandler and schwartz, 1988), serves as an important nutritional component in foods. it is a major precursor of vitamin a and provides pleasant yelloworange colors to foods (si-mon, 1997). dietary vitamin a deficiency causes debilitating health problems such as xerophthalmia, corneal akin-idowu et al. 301 lesions, keratomalace and in very severe cases blindness (passmore and eastwood, 1986). the world health organization (1995) reported these problems affecting young children in africa. yams are also known to contain some antinutritional components that may have adverse effects on human nutrition (dipak and mukherejee, 1986). these are mainly tannins, phenols and phytic acid. phytic acid (inositol hexaphosphate) is an organic acid found in plant materials (heldt, 1997). it combines with some essential elements such as iron, calcium, zinc and phosphorus to form insoluble salts called phytate which are not absorbed by the body thereby reducing the bioavailability of these elements. two principal traditional methods used for preparing yams for consumption in coastal west africa, especially nigeria, are boiling tuber pieces and pounding into a dough after boiling (omonigho and ikenebomeh, 2000). several traditional household food-processing methods can affect the bioavailability of nutrients in plant-based diets. these include thermal processing, mechanical processing, soaking, fermentation and germination, (hotz and gibson, 2007). in spite of the importance of yellow yam as a food source, only few studies have been done to provide information on the mineral, total carotenoid, vitamin and antinutritional contents of ready-to-eat yams, that is, either the boiled or pounded product. the purpose of this study was to evaluate the chemical composition of tubers of eight genotypes of yellow yam and the effect of domestic cooking methods on their nutritional quality. materials and methods tubers from eight genotypes of yellow yam, tdc 04-167, tdc 04168, tdc 04-169, tdc 04-170, tdc 95-293, tdc 95-294, tdc 95-65 and tdc 98-136 were planted at the experimental field of the international institute of tropical agriculture (iita), onne, nigeria. the design was a randomized complete block with three replications. the tubers were harvested nine months after planting by which time all leaves had senesced. five healthy tubers were selected per genotype per plot from each of the three replications. sample preparation and cooking methods the tubers were washed and each was split into four longitudinal sections with a stainless steel knife. sub-samples containing a section of each yam tuber were selected and divided into three portions. the first portion was used for analysis as raw. the second portion was boiled in distilled water (1:2 w/v) for 20 min, excess water was drained off as is the usual household practice and samples were left to cool. the third portion was cooked in a yam pounder (model sd-900y, national electronic co. ltd., tokyo, japan) by adding distilled water (1:2 w/v) for 15 min and then pounded for another 10 min. the weight of yam and volume of water used were enough to give pounded yam of same consistency as that of the conventionally prepared pounded yam using mortar and pestle. fresh samples were used for determination of ascorbic acid and total carotenoid contents. for other analyses, samples were dried in a convection oven (gallenkamp hotbox oven, size 2, gallenkamp, uk) at 60°c for 48 h. the dried samples were milled into flour using an analytical mill (analysenmuhle type a10, 79219 staufen, janke and kunkel, gmbh and co. kg, ika labortechnik, germany.) and stored in airtight plastic bags at -4°c until used. all determinations were carried out in duplicate, giving six values per genotype for each parameter (considering the three replications in the field). chemical analyses total carotenoid content was determined spectrophotometrically as described by rodriguezamaya (1999). ascorbic acid content was determined according to the aoac, 1990 method. for analyses of minerals, dried and milled yam samples were sent to the waite analytical service laboratory, adelaide, australia and analyzed there using inductively coupled plasma atomic emission spectrometry (icpaes), (made in switzerland by arl model 3580 b), (zarcinas et al.,1987). phytic acid (phytate) content was determined by the method of wheeler and ferrel (1971). tannin was determined using the vanillin-hcl method as modified by chang et al., (1994), using catechin as the tannin standard. the tannin content was expressed as ‘catechin’ equivalents. statistical analysis samples from each of the three replicates for each genotype was taken and analyzed in duplicate. data were subjected to analysis of variance (anova) using statistical analysis system (sas) version 8.02 (sas, 2000) and duncan’s multiple range test. results and discussion the tuber iron contents ranged from 6.7 7.9 mg/kg on dry matter (dm) basis with a mean value of 7.2 mg/kg dm (table 1). these are similar to values reported by bell (1984); bradbury and holloway (1988) and usda (2003). there were no significant differences among the genotypes (p = 0.79) for iron content. the iron content varied little between the raw and processed products, showing inconsistency in the changes (increase or decrease) among the eight genotypes studied (table 2). increase in the iron content may be due to contamination of iron from the cooking utensils. our observations are similar to reports by bell (1984) which showed that iron content increased slightly when tubers were peeled and boiled. the recommended dietary allowance (rda) for iron is 10 mg/day (rda, 2008) since iron is not significantly reduced by processing, genotypes tdc 95-294, tdc 04-168 and tdc 95-65 will contribute 39.5% of the rda requirement for iron if a 500 g yam meal is consumed. yam is also not eaten alone but often with a vegetable sauce, additional minerals obtained from the sauce can increase the iron content of the meal. the zinc content ranged from 8.4 10.9 mg/kg dm with a mean value of 9.2 mg/kg dm (table 1). results obtained are similar to reports by bell (1984) and usda (1999). tdc 95-65 had the highest level (p = 0.05) of zinc (10.9 mg/kg dm) followed by tdc 95-294 (10.2 mg/kg). the reduction in the zinc content due to primary processing was slight (table 2). bell (1984), reported similar findings in peeled and boiled tubers of d. cayenensis. the rda 302 afr. j. food sci. res. table 1. micronutrient content (mg/kg) a in raw, boiled and pounded b tubers from eight genotypes of yellow yam. genotype iron zinc raw boiled pounded raw boiled pounded tdc 04-167 6.7 ± 0.3a 7.1 ± 0.8 7.0 ± 0.4 8.6 ± 0.7b 9.1 ± 2.5 7.7 ± 0.9 tdc 04-168 7.8 ± 1.9a 9.1 ± 1.8 8.7 ± 0.2 9.6 ± 1.9ab 10.4 ± 2.2 10.3 ± 1.4 tdc 04-169 7.0 ± 0.9a 7.5 ± 1.3 8.2 ± 1.4 9.0 ± 0.3b 8.5 ± 0.5 9.0 ± 0.7 tdc 04-170 7.2 ± 1.2a 6.1 ± 0.8 6.3 ± 0.8 8.7 ± 0.9b 8.1 ± 0.9 8.2 ± 0.7 tdc 95-293 6.7 ± 1.4a 7.0 ± 0.8 7.8 ± 0.8 8.4 ± 0.3b 8.3 ± 0.3 8.8 ± 1.2 tdc 95-294 7.9 ± 0.3a 8.0 ± 0.2 8.7 ± 1.8 10.2 ± 1.5ab 8.7 ± 1.1 9.9 ± 2.6 tdc 95-65 7.3 ± 1.1a 7.2 ± 0.7 7.8 ± 1.6 10.9 ± 1.2a 9.4 ± 2.4 10.1 ± 2.5 tdc 98-136 6.9 ± 1.1a 6..9 ± 1.4 7.2 ± 1.4 8.5 ± 0.8b 7.6 ± 1.6 7.9 ± 1.4 range 6.7 7.9 6.1 – 9.1 6.3 – 8.7 8.4 – 10.9 7.6 – 10.4 7.7 – 10.3 mean 7.2 7.4 7.7 9.2 8.8 9.0 a means of two determinations in each of three replicate samples expressed on dry weight basis, ± standard deviation. values with same subscripts in the same column are not significantly different at p < 0.05 b pounded refers to a combination of the processes of boiling and kneading into a dough. for zinc is 15 mg/day (rda, 2008) . consumption of 500 g of tdc 95-65 and tdc 95-294 will contribute 36.3% of the rda. values obtained in this study shows that d. caye-nensis is a good source of zinc. the calcium content ranged from 75.2 263.2 mg/kg on dry matter basis with a mean value of 169.3 mg/kg dm (table 3) and are similar to values reported for several cultivated yam species (bradbury, 1988) . the highest value was recorded for tdc 95-65 (263.2 mg/kg) which was significantly different (p < 0.05) from values for tdc 04-169, tdc 04-170, tdc 04168 and tdc 95-294. the calcium content increased significantly for most of the genotypes (tdc 04168, tdc 04-169, tdc 04-170, tdc 95-293, tdc 95-294 and tdc 98136) when tubers were boiled and pounded (table 2). bradbury et al., (1988) reported similar findings in which an increase in the cal-cium content was observed when yam tuber was boiled. the rda for calcium is 800 mg/day (rda, 2008), con-sumption of a 500 g yellow yam meal will contribute 16.5% of the rda. the yellow yam genotypes are not good sources of calcium. phosphorus content in this study ranged from 1200 1576.7 mg/kg dm with a mean value of 1331.2 mg/kg (table 3) which was about six times the calcium content in the tuber. this is similar to earlier reports by obigbesan and agboola (1978). tubers of tdc 98-136 were significantly different from tdc 04167, tdc 04-168, tdc 04-169, tdc 04-170, tdc 95-293 and tdc 95-294 (p < 0.05). tdc 98-136 was observed to have the highest level of phosphorus, (1576.7 mg/kg) followed by tdc 95-65 (1373.3 mg/kg) and tdc 95-294 (1350.0 mg/kg). boiling resulted in slight decrease (6.98 – 19.50%) in phosphorus content of all the genotypes studied (table 2). the rda for phosphorus is 500 mg/day (rda, 2008). consumption of a 100 g yam meal will contribute 31.6% of the rda. from faostat, 2005, yam consumption per capita is estimated to be 256.4 g per day. yellow yams are good sources of phosphorus. the total carotenoid contents ranged from 3.40 10.86 µg/g on fresh matter basis with a mean value of 6.31 µg/g (figure 1). significant variation in the carotenoid content was observed among the genotypes (p < 0.05). duncan’s multiple range tests grouped the genotypes into two, based on their carotenoid content. tdc 95-293 (10.58 µg/g), tdc 95-294 (8.65 µg/g) and tdc 95-65 (10.86 µg/g) had significantly higher carotenoid content than tdc 04-167 (4.33 µg/g), tdc 04-168 (4.73 µg/g), tdc 04-169 (4.45 µg/g), tdc 04-170 (3.40 µg/g) and tdc 98-136 (3.44 µg/g) (p < 0.05). the yellow coloured tubers of yam generally do contain useful amounts of carotene or provi-tamin a as has been identified by martin and ruberte (1975). large variation in total carotenoid content observ-ed among the eight genotypes was a reflection of the wide spectrum of the colour of flesh of the yellow yam tubers. these results agree with earlier conclusion that carotenoids, especially -carotene are largely responsible for the yellow or orange-fleshed colour in dioscorea cayenensis (martin and ruberte, 1975). a similar obser-vation has been reported in sweet potato (de almeida-muradian et al., 1992). the rda for vitamin a is 800 1000 µg retinol equivalent (re)/day for adults, whereas children and infants require 500µg re/day (rda, 2008). low et al., (1997) suggested that cultivars having more than 100 µg retinol equivalent (re) per 100 g fresh roots were good sources of vitamin a. tubers of tdc, 95-293, tdc 95294 and tdc 95-65 having high carotenoid con-tent can be said to be good sources of this micronutrient. khachik et al. (1992) reported that various cooking proce-dures affected the carotenoid content of green vege-tables. this study recorded a decrease of 27.65 42.92% of total carotenoids on boiling and 31.18 – 47.05% on boiling followed by pounding (table 2), thus affecting the nutritional value of those genotypes known to be poten-tially high in carotenoid content. this loss is due to the akin-idowu et al. 303 table 2. changes (%) in the content of nutrients and antinutrients of boiled and pounded b tubers of yellow yam as compared with raw samples. genotype boiled pounded tdc tdc tdc tdc tdc tdc tdc tdc tdc tdc tdc tdc tdc tdc tdc tdc 04167 04-168 04-169 04-170 95-293 95-294 95-65 98-136 04167 04168 04-169 04-170 95-293 95-294 95-65 98-136 iron +5.97 +16.67 +7.14 -15.28 +4.48 +1.27 -1.37 0 +4.48 +11.54 +17.14 -12.50 +16.42 +10.13 +6.85 +4.35 zinc +5.81 +8.33 -5.56 -6.90 -1.19 -14.71 -13.76 -10.59 -10.47 +7.29 0 -5.75 +4.76 -2.94 -7.34 -7.06 calcium -13.97 +43.03 +98.40 +56.30 +63.55 +64.33 +27.93 +120.94 -35.31 -29.29 +52.79 +33.39 +6.65 +8.88 -8.59 +29.00 phosphorus -10.26 -13.39 -11.11 -17.39 -10.56 -19.50 -17.72 -6.98 -7.89 +3.54 +5.29 -9.97 +0.83 -2.96 -3.64 -1.27 total carotenoids -40.42 -42.92 -34.61 -27.65 -28.64 -42.31 -28.73 -35.17 -41.11 -43.55 -38.20 -31.18 -39.98 -47.05 -38.67 -37.21 vitamin c -43.41 -47.33 -47.92 -48.41 -43.30 -36.39 -38.81 -41.30 -42.44 -49.56 -52.08 -49.09 -47.13 -40.82 -43.91 -44.02 phytate -28.33 -32.49 -25.51 -13.01 -22.94 -32.07 -26.22 -33.01 -32.18 -33.21 -28.34 -18.95 -29.22 -34.27 -28.22 -35.15 tannin -38.89 -33.05 -32.02 -34.26 -43.85 -45.05 -45.73 -49.59 -36.05 -34.11 -36.94 -39.76 -53.97 -45.93 -51.29 -51.11 + = % increase; = % decrease b pounded refers to a combination of the processes of boiling and kneading into a dough. table 3. macronutrient content (mg/kg) a in raw, boiled and pounded b tubers from eight genotypes of yellow yam. genotype calcium phosphorus raw boiled pounded raw boiled pounded tdc 04-167 209.0 ±105.0ab 179.8 ± 66.7 135.2 ± 76.0 1266.7 ± 125.0b 1136.7 ± 15.3 1166.7 ± 212.2 tdc 04-168 155.0 ± 50.1bc 221.7 ± 37.5 109.6 ± 36.1 1320.0 ± 122.9b 1143.3±145.7 1366.7 ± 130.5 tdc 04-169 75.2 ± 16.1d 149.2 ± 45.4 114.9 ± 25.2 1260.0 ±160.9b 1120.0 ± 26.5 1326.7 ± 35.1 tdc 04-170 122.2 ± 54.3cd 191.0 ± 60.7 163.0 ± 41.8 1303.3 ±170.1b 1076.7±232.9 1173.3 ± 176.7 tdc 95-293 197.0 ± 80.3abc 322.2 ± 175.2 210.1 ± 17.1 1200.0 ±137.5b 1073.3 ± 90.2 1210.0 ± 43.6 tdc 95-294 144.1 ± 47.6bcd 236.8 ± 81.3 156.9 ± 125.3 1350.0 ± 72.1b 1086.7 ± 15.3 1310.0 ± 60.0 tdc 95-65 263.2±291.7a 336.7 ± 120.6 240.6 ± 130.4 1373.3± 27.2ab 1130.0±150.0 1323.3 ± 140.1 tdc 98-136 188.6 ± 46.4abc 416.7 ± 100.2 243.3 ± 45.1 1576.7 ± 89.6a 1466.7 ± 86.2 1556.7 ± 83.3 range 75.2 – 263.2 149.2 – 416.7 109.6 – 243.3 1200.0 – 1576.7 1073.3-1466.7 1166.7–1556.7 mean 169.3 275.4 171.7 1331.3 1154.2 1304.2 a means of two determinations in each of three replicate samples expressed on dry weight basis, ± standard deviation. values with same subscripts in the same column are not significantly different at p < 0.05. b pounded refers to a combination of the processes of boiling and kneading into a dough. that carotenoids are heat-labile compounds and undergo oxidation and degradation upon exposure to heat, light, acids, metals and enzymes (k’osambo et al., 1998). carotenoids are easily oxidized because of the large number of conjugated double bonds in the compounds (krinsky et al., 1990) . the loss in carotenoid content was higher for pounding as carotenoids degrade with longer processing time at higher temperatures and cutting or maceration of the food (rodriguezamaya, 1997). the ascorbic acid contents ranged from 18.4 52.2 mg/kg on fresh weight basis (fw) with a mean of 37.3 mg/kg fw (figure 2). tdc 95293 had the highest level of ascorbic acid (52.2 mg/kg). tdc 04-168 and tdc 04-170 recorded 45.0 and 44.0 mg/100g respectively. the ascorbic acid content in the fresh yam tubers as found to vary greatly between genotypes. high variability 304 afr. j. food sci. res. genotype figure 1. total carotenoid contents in raw, boiled and pounded tubers from eight genotypes of yellow yam. a s c o rb ic a c id ( m g /k g ) genotype figure 2. ascorbic acid content in raw, boiled and pounded tubers from eight genotypes of yellow yam has been reported to exist in the ascorbic acid content of fruits and vegetables (mozafar, 1994) . the values obtain-ed were similar to those reported by bradbury and holloway (1988); bradbury and singh (1986). boiling and pounding reduced the ascorbic acid content in all the genotypes (table 2). the reduction observed was bet-ween 36.39 48.41% for boiling and 40.82 52.08% for pounding. ascorbic acid is the least stable of all the vita-mins and is easily destroyed by heat, light, oxidation and alkalinity (sood and malhotra, 2001) . the recommended dietary allowance (rda) for ascorbic acid is between 40 60 mg/day for infants and adults (rda, 2008). consump-tion of a 500 g meal of tubers of tdc 95-293, tdc 04-168 and tdc 04-170 will provide about 21 26 mg of ascorbic acid of which a significant proportion (almost half the content) is lost during processing. phytate content ranged from 166.2 194.9 mg/kg on dry matter basis with a mean of 181.8 mg/kg) (figure 3). the t o ta l c a ro te n o id c o n te n t ( g /g ) akin-idowu et al. 305 genotype figure 3. phytate content in raw, boiled and pounded tubers of eight genotypes of yellow yam. t a n n in c o n te n t (m g / k g ) genotype figure 4. tannin content in raw, boiled and pounded tubers from eight genotypes of yellow yam. highest levels were obtained in tdc 95-294 (194.9 mg/kg) and tdc 95-65 (194.9 mg/kg) both having same values. these values are lower than the phytate level (360 mg/kg) reported for nigerian yam species (osagie et al.,1996), higher than the 37.0 mg/kg reported for d. cayenensis tubers by udoessien and ifon (1992), and much lower than 4520 mg/kg reported by adeyeye et al., (2000) . results of our study showed that d. cayenensis tubers have fairly low phytate contents. no significant dif-ference was observed among the genotypes (p = 0.98). p h y ta te c o n te n t (m g /k g ) 306 afr. j. food sci. res. this is important because high phytate content is of significance as it lowers the availability of many essential minerals. phytate could be substantially reduced or eliminated by soaking, germination and cooking (martin-cabrejas et al., 2004). our study showed that all the genotypes studied recorded a loss of between 13.01 and 33.01% on boiling and 18.95 and 35.15% on boiling fol-lowed by pounding (table 2). reports by beal and mehta (1985) showed cooking reduced phytate content of peas by 13%. marfo et al., 1990, reported that cooking had a greater reducing effect on phytate levels in the tubers (yam, cocoyam and cassava) than in the cereals and legumes. the tannin content ranged from 297 427.2 mg/kg on dry matter basis with a mean of 353.6 mg/kg dw (figure 4). these values are comparable to those reported for yellow yam (300 mg/kg) by udoessien and ifon (1992), but lower than those reported for water yam (1300 mg/kg) by osagie et al. (1996) and for other widely consumed food crops, for example, 2600 23700 mg tannin per kg in sorghum (ford and hewitt, 1979). no significant dif-ference was observed for tannin content among all the eight genotypes (p = 0.63). boiling and pounding of all the genotypes studied resulted in decreases of 32.02 49.59% and 34.11 53.97% respectively of tannin con-tent (table 2). the decrease in the levels of these antinu-trients during heat treatment might be due to thermal degradation and denaturation of the antinutrients as well as the formation of insoluble complexes (kataria et al., 1989). tannin content of most food is usually reduced by processing and this has been reported to enhance the bioavailability of iron. pounding resulted in greater loss of the antinutrients and this might be attributed to heat and disruption during the pounding process. conclusion the variations observed in zinc, calcium, phosphorus, vitamin c and total carotenoid contents can be of advan-tage in breeding programmes designed to improve the nutritional quality of yam. tubers of tdc, 95-65, tdc 95-294, tdc 04-168 and tdc 98-136 are good sources of iron, zinc and phosphorus since they can provide a sub-stantial amount of these mineral requirements in west africa where daily consumption may exceed 1 kg/person. additionally, boiling and pounding slightly affected only the iron, zinc and phosphorus contents of yellow yam tubers. vitamin c and total carotenoid contents, though present in substantial amounts that can contribute signi-ficantly to the recommended dietary allowance, were observed to be reduced greatly by both food preparation methods with the reduction more pronounced on pound-ing. yellow yam was found to have low levels of antinu-trients when compared to values recorded in some le-gumes and pulses. these antinutrients were further re-duced during processing and should therefore not pose a problem to human health. both methods of food pre-paration were effective in reducing the 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(2003). usda national nutrient data base for standard reference, release 16. wheeler el, ferrel re (1971). a method for phytic acid determination in wheat and wheat fractions. cereal chem. 48:312-316. who (world health organization) (1995). micronutrient deficiency information system. global prevalence of vitamin a deficiency. mdis working paper 2. who/nut/95.3, geneva, switzerland. zarcinas ba, cartwright b, spouncer lr (1987). nitric acid digestion and multi-element analysis of plant material by inductively coupled plasma spectrometry. commun. soil sci. plant anal. 18:131-146. http://faostat.fao.org/site/346/default.aspx http://www.anyvitamins.com/rda.htm in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (4), pp. 001-006, april, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper hypolipidemic and antioxidant potency of heat processed turmeric and red pepper in experimental rats hanumanthappa manjunatha and krishnapura srinivasan* department of biochemistry and nutrition, central food technological research institute, mysore – 570 020, india. accepted 16 january, 2019 the hypocholesterolemic and antioxidant potency of both raw and pressurecooked turmeric and red pepper were evaluated in experimental rats rendered hypercholesterolemic by feeding 0.5% cholesterol enriched diet and maintained for 8 weeks on 5% spice diet. dietary turmeric and red pepper, either raw or heat processed significantly countered the extent of hypercholesterolemia. serum total cholesterol was 31 and 32% lower as a result of feeding raw and heat processed turmeric. the same was lower by 16 and 23% in animal groups fed raw and heat processed red pepper. the reduction in blood cholesterol brought about by these two dietary spices was predominantly in the ldl-cholesterol fraction. dietary red pepper, both raw and heat processed fully countered the increase in serum triglyceride content of hypercholesterolemic rats. increase in hepatic cholesterol in hypercholesterolemic animals was moderately countered by dietary red pepper, either raw or heat processed. both dietary turmeric as well as red pepper significantly countered the increase in hepatic triglyceride level in hypercholesterolemic rats. total thiols in serum were slightly but significantly increased by raw turmeric and raw red pepper both in basal and in hypercholesterolemic rats, but not by heat processed spices. serum -tocopherol was significantly enhanced (81 113%) by both dietary turmeric and red pepper in hyper-cholesterolemic animals. hepatic lipid peroxides were significantly lower (9 15%) as a result of dietary turmeric and red pepper in hypercholesterolemic situation. thus, the results of this animal study suggested that although heat processing of turmeric and red pepper by pressure cooking resulted in a considerable loss of the active principles – curcumin and capsaicin, the hypolipidemic potency or the antioxidant potency of the parent spices were not significantly compromised. key words: antioxidant effect, heat processing, hypocholesterolemic effect, red pepper, turmeric. introduction spices are a group of esoteric food adjuncts, which have been in use for thousands of years. by virtue of their pleasing colour, flavour or pungency, they can transform our food into attractive and appetizing meal. in addition to these organoleptic properties, few spices are also known to possess several medicinal properties (nadkarni and nadkarni, 1976) and are effectively used in the indigenous systems of medicine. in the past three decades, it has been experimentally documented that several common spices can also exert health beneficial physiological effects (srinivasan, 2005). these physiological effects of *corresponding author. e-mail: ksri.cftri@gmail.com. tel.+91 821-2514876. fax: # +91-821-2517233. of spices in most instances have been attributed to the main spice active principles present in them. among these physiological influences spices are documented to exhibit, their hypolipidemic and antioxidant properties have far-reaching health implication. the active principles of the spices turmeric (curcuma longa) and red pepper (capsicum annuum) have been evidenced in several animal studies to exert hypolipidemic and antioxidant properties (srinivasan, 2005). it is also evidenced that heat processing of spices – turmeric, red pepper and black pepper results in a significant loss of their active principles (srinivasan et al., 1992; suresh et al., 2007). hence, it is desirable to characterize the altered compounds formed from spice active principles during heat processing of parent spices, and to ascertain the extent to which these spices retain their health beneficial potency in spite of significant chemical alteration of active principles. especially the hypolipidemic potency and the antioxidant potency exerted by turmeric and red pepper by virtue of their respective active compounds – curcumin and capsaicin need to be evaluated in heat-processed spices. in this investigation, elaborate animal studies were made to quantitate the relative potency of cooked turmeric and red pepper with regard to their hypolipidemic and antioxidant properties. materials and methods heat processing of spices spices – turmeric (curcuma longa) and red pepper (capsicum annuum) were locally purchased and powdered to pass through no.50 mesh sieve. these spice powders were suspended in distilled water (100 g / l) and subjected to pressure cooking for 10 min at 15 p.s.i. at the end of heat treatment, the samples were cooled to room temperature. appropriate controls were also included wherein the samples did not undergo any treatment. these samples were lyophilized and stored at 4c. quantitation of spice principles by tlc spice samples in the lyophilized food samples were extracted with ethyl acetate in a soxhlet apparatus for 4 h. the extracts were concentrated in a flash evaporator to a known volume (2 ml) and stored in dark at -20ºc until further analysis. the individual spice principles were quantitated after separation by appropriate tlc procedures as described below (ravindranath et al., 1981; srinivasan et al., 1981). care was taken to minimize the exposure to light during the extraction procedure and tlc separation. curcumin aliquots of the ethyl acetate extracts (quadruplicate) of the lyophilized food material and reference curcumin (6 g) were spotted on silica gel -g coated plates (20 x 20 cm). the plates were developed with the upper phase of the solvent system: benzeneethanol-water-acetic acid (100: 27.5: 7.5: 0.5 v/v/v/v) in a chamber pre-equilibrated with the above solvent system for 2 h. the yellow curcumin bands were scraped off and quantitatively transferred to centrifuge tubes. curcumin in the scrapings was extracted with 4 ml acetone, centrifuged at 2000 rpm for 5 min and 2 ml of the clear supernatant was used for the rubrocurcumin reaction. two ml of acetonic extract was transferred to another test tube into which were successively added, 1 ml 7.5 mg% boric acid (in acetone) and 1 ml 5 mg% boric acid (in acetone). the contents of the tube were evaporated to dryness over a hot water bath. the residue was redissolved in 2 ml ethanol and absorption of the purple colour was measured at 550 nm. capsaicin aliquots of ethyl acetate extracts (quadruplicate) were spotted on silica gel -g coated plates (20 x 20 cm) along with reference capsaicin (40 g). plates were developed with petroleum ether (60 – 80ºc) acetone (65: 35 v/v) in a chamber pre-equilibrated with the same solvent for 90 min. the developed plates were air-dried and then sprayed uniformly with fresh gibb`s reagent (0.1% 2,6 dichloroquinone-4-chlorimide in methanol). the plates, when dry, were exposed to ammonia vapours in a closed chamber for exactly 1 min. the blue-coloured zones of capsaicin thus visualized were scraped off and quantitatively transferred to centrifuge tubes containing 2 ml water. the tubes were vortexed for 10 min to extract the colour and centrifuged at 2000 rpm for 2 min. absorption of the clear blue supernatants was read at 610 nm. animal treatment female wistar rats (8 per group) weighing 110 120 g and housed in individual stainless steel cages were maintained on various experimental diets ad libitum for 8 weeks. the basal diet consisted of (%): casein, 21; cane sugar, 10; corn starch, 54; nrc vitamin mixture, 1; bernhardt-tommarelli modified nrc salt mixture, 4, and refined peanut oil, 10. the hypercholesterolemic diet consisted of 0.5% cholesterol and 0.125% bile salts at the expense of an equivalent amount of corn starch in the basal diet wherein the peanut oil is also replaced with hydrogenated vegetable fat. the test material was incorporated into the basal diet/high cholesterol diet at 5 g / 100 g replacing an equivalent amount of corn starch. at the end of the experimental duration, overnight fasted animals were sacrificed under light ether anesthesia. blood was collected by heart puncture and serum separated by centrifugation. liver was quickly excised, weighed and stored frozen till lipid extraction. lipid profile total lipids were extracted according to folch et al. (1957) and estimated gravimetrically. cholesterol (searcy and bergquist, 1960), triglycerides (fletcher, 1968) and phospholipids (charles and stewart, 1980) were determined in the lipid extracts of serum and liver by using standard procedures. serum cholesterol and triglyceride associated with hdl fraction were determined after precipitation of apolipoprotein-b containing lipoproteins with heaprin-manganese reagent according to the method of warnick and albers (1978). ldl-vldl precipitate was extracted with chloroform: methanol (2:1 v/v) and used for cholesterol and triglyceride determination. lipid peroxides plasma lipid peroxides were estimated by the fluorimetric measurement of thiobarbituric acid complex by the method of yagi (1984). the fluorimetric measurement was carried out at an excitation wavelength of 515 nm and emission wavelength of 553 nm and compared with the standards prepared by reacting 0.5 nmole 1,1,3,3-tetraethoxy-propane with tba reagent. lipid peroxide in liver tissue was determined by the method described by ohkawa et al. (1979) involving photometric measurement of thiobarbituric acid complex extracted into butanol. absorbance of the butanol extract was measured at 532 nm and compared with that of standard tetraethoxypropane, treated similarly. antioxidant molecules total thiols in serum/liver were measured spectrophotometrically by using ellman’s reagent according to the method described by sedlock and lindsay (1968). glutathione in liver was estimated by using ellman’s reagent according to beutler et al. (1963). ascorbic acid in serum/liver was estimated spectrophotometrically by measuring the 2,4-dinitrophenyl-hydrazone derivative of dehydroascorbic acid according to omaye et al. (1973). -tocopherol in serum was determined by hplc method described by zaspel and table 1. influence of dietary turmeric and red pepper on serum lipid profile. animal group cholesterol triglycerides phospholipids total ldl-vldl hdl basal-control 70.2 ± 1.58 47.4 ± 1.64 22.8 ± 0.30 100.3 ± 4.92 93.7 ± 3.82 basal-turmeric (raw) 71.9 ± 3.39 49.6 ± 2.30 22.3 ± 0.39 107.8 ± 7.17 95.7 ± 1.31 basal-red pepper (raw) 77.3 ± 2.93 49.4 ± 1.84 27.9 ± 1.36* 99.6 ± 4.68 101.7 ± 3.27 hcd-control 440.8 ± 14.4 429.5 ± 24.1 11.3 ± 1.14 117.1 ± 5.64 112.5 ± 6.50 hcd-turmeric (raw) 304.7 ± 28.6** 291.3 ± 18.2** 13.4 ± 0.91 116.1 ± 8.88 156.5 ± 9.92* hcd-turmeric (cooked) 298.0 ± 38.7** 285.7 ± 20.8** 12.4 ± 0.53 110.4 ± 4.23 154.3 ± 9.39* hcd-red pepper (raw) 369.4 ± 11.1** 358.2 ± 25.2** 11.2 ± 0.51 101.6 ± 5.60** 139.5 ± 6.46* hcd-red pepper (cooked) 340.7 ± 29.3** 328.2 ± 28.0** 12.5 ± 0.94 98.5 ± 4.74** 150.7 ± 8.95* values expressed as mg/dl are mean ± sem of 8 rats in each group. ldl: low density lipoprotein; hdl: high density lipoprotein; vldl: very low density lipoprotein; hcd: high cholesterol diet. * significant increase compared to corresponding control. **significant decrease compared to corresponding control. csallany (1983) using ods column (c18) and an uv-visible detector (295 nm) and a solvent system acetonitrile – methanol (1:1). statistical analysis results are expressed as mean (sem and comparisons between groups were made by means of an unpaired student’s t-test (snedecor and cochran, 1976). differences were considered significant when p < 0.05. results and discussion influence of dietary turmeric and red pepper on serum lipid profile in normal as well as high cholesterol fed animals is presented in table 1. high cholesterol feeding for 8 weeks resulted in a significant increase in serum total cholesterol concentration and this increase was observed predominantly in the ldl-associated fraction. the increase in serum total cholesterol was as much as 6.25-fold. the increase in serum ldl-cholesterol is about 9-fold. dietary turmeric and red pepper, either raw or heat processed significantly countered the extent of hypercholesterolemia. serum total cholesterol was 31 and 32% lower as a result of feeding raw and heat processed turmeric. the same was lower by 16 and 23% in animal groups fed raw red pepper and heat processed red pepper. the reduction in blood cholesterol brought about by these two dietary spices was predominantly in the ldl-cholesterol fraction. about 32% decrease in ldlcholesterol was evidenced in turmeric fed animals, whereas dietary red pepper produced 17 and 24% decrease in the same. the hdl-cholesterol fraction essentially remained unchanged as a result of treatment with turmeric or red pepper. hypercholesterolemic rats registered a 17% increase in serum triglyceride concentration (table 1). while dietary turmeric, either raw or heat processed did not affect the serum triglyceride content in these hypercholesterolemic rats, dietary red pepper, both raw and heat processed fully countered the increase in serum triglyceride content. serum phospholipid concentration was 20% higher in hypercholesterolemic rats compared to basal control. dietary turmeric, both raw and heat processed produced further increase in serum phospholipid concentration (38 39%) in hypercholesterolemic rats. similarly both raw and heat processed red pepper feeding increased serum phospholipid concentration by 24 and 34% respectively. thus, there was practically no difference between raw and heat processed turmeric with regard to beneficial influence on serum cholesterol concentration in hypercholesterolemic rats. even the influence of raw and heat processed turmeric on serum phospholipid content was also similar in hypercholesterolemic animals. the beneficial effect of red pepper on serum cholesterol concentration in hypercholesterolemic rats was even better in the case of heat processed spice when compared to the effect produced by raw red pepper. similarly, the influence of heat processed red pepper on the serum phospholipid concentration in hypercholesterolemic rats was higher than that of corresponding raw spice. dietary turmeric or red pepper generally did not have any influence on serum lipid profile in basal rats expect for a small increase in hdl-cholesterol by raw red pepper. liver lipid profile of normal and hypercholesterolemic rats as influenced by dietary turmeric and red pepper is presented in table 2. liver cholesterol was increased by 14.6-fold as a result of high cholesterol feeding for 8 weeks. this increase in hepatic cholesterol was moderately countered by dietary red pepper, either raw or heat processed by 17 and 20% respectively. dietary raw red pepper also decreased hepatic cholesterol (by 14%) in basal rats. dietary turmeric did not influence hepatic cholesterol level. hepatic triglyceride concentration was elevated by 108% as a result of feeding the cholesterol enriched diet for 8 weeks. both dietary turmeric as well as red pepper significantly countered the increase inhepatic triglyceride level in hypercholesterolemic rats. table 2. influence of dietary turmeric and red pepper on hepatic lipid profile. animal group total lipids cholesterol triglycerides phospholipids basal-control 51.1 ± 1.89 4.92 ± 0.11 25.4 ± 1.74 21.2 ± 1.66 basal-turmeric (raw) 47.2 ± 0.96 5.07 ± 0.29 27.7 ± 1.19 23.8 ± 1.87 basal-red pepper (raw) 44.1 ± 1.51** 4.24 ± 0.19** 18.5 ± 1.26** 20.6 ± 1.65 hcd-control 138.3 ± 6.24 72.3 ± 2.78 52.8 ± 2.71 25.8 ± 1.63 hcd-turmeric (raw) 147.3 ± 5.71 71.8 ± 2.57 32.1 ± 1.37** 22.9 ± 1.88 hcd-turmeric (cooked) 153.4 ± 7.41 69.6 ± 3.41 27.9 ± 1.44** 23.6 ± 1.41 hcd-red pepper (raw) 135.9 ± 5.97 60.2 ± 2.98** 32.1 ± 1.41** 23.3 ± 0.89 hcd-red pepper (cooked) 147.9 ± 4.68 57.6 ± 3.20** 29.8 ± 2.99** 25.7 ± 1.61 values expressed as mg/g liver are mean ± sem of 8 rats in each group. hcd: high cholesterol diet **significant decrease compared to corresponding control. table 3. influence of dietary turmeric and red pepper on serum antioxidant molecules and lipid peroxides. animal group total thiols ascorbic acid -tocopherol lipid (µmole/dl) (mmole/dl) peroxides (mg/dl) (µg/dl) basal-control 17.3 ± 1.16 6.23 ± 0.20 0.783 ± 0.027 1.24 ± 0.024 basal-turmeric (raw) 19.8 ± 0.73* 5.56 ± 0.27 0.852 ± 0.029 1.11 ± 0.039 basal-red pepper (raw) 19.7 ± 1.19* 5.32 ± 0.09 0.800 ± 0.032 1.20 ± 0.033 hcd-control 16.0 ± 1.30 5.20 ± 0.32 0.528 ± 0.037 1.01 ± 0.009 hcd-turmeric (raw) 20.0 ± 1.02* 5.22 ± 0.25 0.956 ± 0.050* 0.973 ± 0.008 hcd-turmeric (cooked) 14.6 ± 0.58 4.96 ± 0.22 1.127 ± 0.084* 0.978 ± 0.026 hcd-red pepper (raw) 20.8 ± 0.84* 5.89 ± 0.35 0.965 ± 0.025* 1.11 ± 0.019 hcd-red pepper (cooked) 19.7 ± 094 4.93 ± 0.08 1.001 ± 0.050* 0.928 ± 0.047 values are expressed as mean ± sem of 8 rats in each group. hcd: high cholesterol diet * significant increase compared to corresponding control. the decrease in hepatic triglyceride concentration evidenced was 39 and 47% by raw and heat processed turmeric respectively, while it was 39 and 44% in the case of raw and heat processed red pepper. hepatic phospholipid concentration was not altered as a result of high cholesterol feeding, and the same was also not influenced by either of the spices. liver total lipid was increased in high cholesterol fed rats by 171%. the same was not beneficially countered by either dietary turmeric or red pepper. raw red pepper however decreased heaptic total lipid by about 14% in basal rats. the significant decrease in hepatic total lipid and triglyceride content caused by dietary 5% red pepper in normal rats observed in this investigation is in agreement with a similar observation reported earlier (sambaiah and satyanarayana, 1982). influence of dietary turmeric and red pepper on the concentration of various antioxidant molecules and lipid peroxides in serum is presented in table 3. total thiols in serum were slightly but significantly increased by raw turmeric and raw red pepper both in basal rats as well as in hypercholesterolemic rats. this effect was not seen in the case of heat processed spices. among the antioxi dant molecules, while serum ascorbic acid concentration was not influenced by either of the dietary spices; -tocopherol was significantly enhanced by both dietary turmeric and red pepper in hypercholesterolemic animals. the extent of increase in serum -tocopherol produced by dietary raw and heat processed turmeric was 81 and 113% respectively. serum -tocopherol was increased by 83 and 90% in hypercholesterolemic rats by dietary raw and heat processed red pepper. serum lipid peroxide concentration was not influenced by dietary turmeric or red pepper either in basal rats or in hypercholesterolemic rats. influence of dietary turmeric and red pepper on the concentration of various antioxidant molecules and lipid peroxides in liver is presented in table 4. hepatic lipid peroxides were significantly lower as a result of dietary turmeric and red pepper both in normal and hypercholesterolemic situation (table 4). the decrease in hepatic lipid peroxides produced was 28 and 13% by dietary raw turmeric and red pepper respectively in normal rats. a decrease in the same by 15 and 11% raw and heat processed turmeric was seen in the case of hypercholesterolemic rats, while raw and heat processed red pep table 4. influence of dietary turmeric and red pepper on liver antioxidant molecules and lipid peroxides. animal group total thiols glutathione ascorbic acid lipid peroxides (mmole/mg protein) (µg/mg protein) (µg/mg protein) (nmole/mg protein) basal-control 49.1 ± 1.47 0.425 ± 0.020 1.775 ± 0.062 0.652 ± 0.036 basal-turmeric (raw) 58.1 ± 3.39* 0.423 ± 0.025 1.983 ± 0.101* 0.469 ± 0.020** basal-red pepper (raw) 60.0 ± 4.23* 0.434 ± 0.026 2.913 ± 0.231* 0.570 ± 0.009** hcd-control 47.4 ± 2.05 0.497 ± 0.023 2.620 ± 0.109 0.503 ± 0.023 hcd-turmeric (raw) 39.6 ± 1.30 0.487 ± 0.029 2.090 ± 0.070** 0.426 ± 0.025** hcd-turmeric (cooked) 42.3 ± 0.96 0.480 ± 0.024 2.310 ± 0.087** 0.446 ± 0.010** hcd-red pepper (raw) 41.5 ± 1.28 0.448 ± 0.040 1.870 ± 0.083** 0.459 ± 0.015** hcd-red pepper (cooked) 45.1 ± 1.14 0.510 ± 0.014 2.170 ± 0.076** 0.448 ± 0.021** values are expressed as mean ± sem of 8 rats in each group. hcd: high cholesterol diet * significant increase compared to corresponding control. **significant decrease compared to corresponding control table 5. influence of dietary turmeric and red pepper on body weight gain and liver weight in hypercholesterolemic rats. body weight (g) liver weight initial final gain (g) (g/100 g body) basal control 116.2  1.05 195.2  4.95 79.0  2.00 4.98  0.12 2.55  0.06 basal – turmeric (raw) 116.3  0.98 186.2  6.86 69.9  2.57 5.27  0.15 2.83  0.08 basal – red pepper (raw) 116.3  0.87 172.7  4.19 56.4  1.36** 4.39  0.10 2.54  0.05 hcd control 124.5  1.14 182.3  2.85 57.8  1.90 8.02  0.23 4.39  0.14 hcd – turmeric (raw) 124.6  1.08 188.1  4.45 63.5  1.35 8.60  0.25 4.61 0.13 hcd – turmeric (cooked) 124.9  0.97 182.3  2.44 57.4  0.76 8.44  0.17 4.62  0.09 hcd – red pepper (raw) 124.8  1.03 182.3  2.80 57.5  0.88 7.24  0.21 3.97  0.15 hcd – red pepper (cooked) 124.9  1.05 187.5  3.71 62.6  1.23 7.64  0.26 4.07  0.13 values are  sem of 8 animals in each group. **significant decrease compared to corresponding control. red pepper produced a decrease of 9 and 11% respecttively. ascorbic acid concentration in liver was favourably influenced by dietary turmeric and red pepper in normal rats (table 4). the extent of increase in hepatic ascorbic acid was 12 and 64% in the turmeric and red pepper diet groups. these dietary spice principles did not show any beneficial effect on liver ascorbic acid in hypercholesterolemic rats. on the other hand, there were decreases in hepatic ascorbic acid of hypercholesterolemic rats in dietary turmeric and red pepper groups. the decreases in hepatic ascorbic acid were of the order of 12 to 29% in these spice groups. hepatic glutathione content remained unaffected by dietary turmeric and red pepper in both basal as well as in hypercholesterolemic rats. hepatic total thiols were higher as a result of dietary turmeric and red pepper only in basal rats (table 4). the increase in total thiols was 18 and 22% in the respective diet groups as compared to control. this is the first observation on the beneficial influence of dietary turmeric and red pepper on the antioxidant status of experimental animals with respect to antioxidant molecules and lipid per oxides in blood and liver, although such an effect has been documented for their active principles curcumin and capsaicin. the beneficial influence of dietary turmeric and red pepper on the antioxidant status of experimental animals with respect to antioxidant molecules and lipid peroxides in blood and liver is practically the same irrespective of whether the spice is fed raw or after heat processing. thus, the results of this animal study sug-gested that although heat processing of turmeric and red pepper by pressure cooking resulted in a conside-rable loss of the active principles – curcumin and cap-saicin, the hypolipidemicpotency or the antioxidant potency of the parent spices were not significantly compromised. acknowledgement the first author (hm) is grateful to council of scientific and industrial research, new delhi for the award of research fellowship. references beutler e, duron o, kelly bm (1963). improved method for the deter mination of blood glutathione. j. lab. clin. med. 61: 882-888. charles j, stewart m (1980). colorimetric determination of phospholipids with ammonium ferrothiocyanate. anal. biochem. 104: 10-14. fletcher mj (1968). a colorimetric method for estimating serum triglycerides. clin. chim. acta. 22: 393-397. folch j, lees m, sloane-stanley gh (1957). a simple method for the isolation and purification of total lipids from animal tissues. j. biol. chem. 226: 497-509. nadkarni km, nadkarni ak (1976). in: indian materia medica, 3rd edn., popular prakashan ltd., bombay. ohkawa h, ohishi n, yagi k (1979). assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. anal. biochem. 95: 351-358. omaye st, turnbull jd, sauberlich he (1973). selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. meth. enzymol. 62: 3-11. ravindranath v, satyanarayana mn, rao mvl (1981). rubrocurcumin reaction and its use in micro-determination of certain organic acids. indian j. chem. 20: 907-909. sambaiah k, satyanarayana mn (1982). influence of red pepper and capsaicin on body composition and lipogenesis in rats. j. biosci. 4: 425-430. searcy rl, bergquist lm (1960). a new color reaction for the quantitation of serum cholesterol. clin. chim. acta 5: 192-197. sedlock j, lindsay rh. estimation of total, protein bound and nonprotein sulfhydryl groups in tissues with ellman’s reagent. anal. biochem. 1968; 25: 192-205. snedecor gw, cochran wg. in: statistical methods. 6 th edn., iowa state univ. press, ames, p.298. srinivasan k, sambaiah k, chandrasekhara n (1992). loss of active principles of common spices during domestic cooking. food chemistry. 43: 271-274. srinivasan k (2005). role of spices beyond food flavouring: nutraceuticals with multiple health effects. food rev.int. 21: 167-188. srinivasan mr, satyanarayana mn, rao mvl (1981). a thin layer chromatographic method for the estimation of capsaicin and related compounds. j. res. industry, 26: 180-183. suresh d, manjunatha h, srinivasan k (2007). effect of heat processing of spices on the concentrations of their bioactive principles: turmeric (curcuma longa), red pepper (capsicum annuum) and black pepper (piper nigrum). j. food comp.anal. 20: 346-351. warnick gr, albers jj (1978). a comprehensive evaluation of heparinmanganese procedure for estimating hdl-cholesterol. j. lipid res. 19: 65-76. yagi k (1984). assay of lipid peroxides in blood plasma or serum. meth.enzymol. 105: 328-331. zaspel bj, csallany as (1983). determination of -tocopherol in tissues and plasma by hplc. anal.biochem. 130: 146-150 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (4), pp. 083-090, april, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of the textural and sensory properties of cookies in order to improve quality sukhcharn singh 1* , c. s. riar 2 and d. c. saxena 3 department of food engineering and technology, sant longowal institute of engineering and technology, longowal, india. accepted 05 march, 2017 textural and sensory properties of cookies were studied by supplementing various proportion of sweetpotato flour (0 100%) to the wheat flour. the cookie dough was subjected to rheological analysis and texture profile analysis by using ta-xt2i (sms) in order to determine dough hardness, cohesiveness, springiness, adhesiveness etc. spread factor, puncture force and fracture strength of the cookies were also determined. water absorption increases from 29 to 169% with increasing the proportion of sweetpotato flour. the control dough had cohesiveness and adhesiveness values of 0.295g/s and 19.314g/s. the cohesiveness of dough with 20% sweetpotato flour and 80% of wheat flour increased to 0.480 g/s and then decreased with increasing percentage of sweetpotato flour. sweetpotato flour lowered the spread factor from 6.666 to 6.150 with increasing its level from 0-100%. sensory evaluation revealed that increasing levels of sweetpotato flour (60%) lowered the overall acceptability (2.7) because of taste and distinct flavor developed during baking. key words: sweetpotato flour, cookies dough, rheological characteristics, texture. introduction sweet potato (ipomoea batatas lam) is the seventh most important food crop in the world. it is grown in many tropical and subtropical regions. among the world's major food crops, sweetpotato produces the highest amount of edible energy per hectare per day (horton and fano, 1985). india has a long history of sweetpotato cultivation. in india, the area covered under this crop in 2005, was 100,000 ha and production was 900,000 mt (fao, 2006). sweet potato has a large potential to be used as a food in developing nations with limited resources because of its short maturity time and ability to grow under diverse climatic condition and on less fertile soil. options for sweetpotato products are numerous, and based on recent diagnostic assessments carried out in developing countries; dried chips, starch, and flour were identified as among the most promising (collins, 1989) . sweet potato flour can serve as a source of energy and nutrients (carbohydrates, beta-carotene (provitamin a), minerals (ca, p, fe, and k)), and can add natural sweetness, color, flavor and dietary fiber to processed food products (woolfe, 1992; ulm, 1988). traditionally cookies are *corresponding author. e-mail: sukhcharns@yahoo.com. made from wheat flour and there is little record of rice being used in cookie type products. small quantities of other cereal flours or starches can be added to give special flavors or structural properties. dough is the intermediate stage between flour and the bakery product. the rheological characteristics of the dough are very important, as they influence the machinability of the dough as well as the quality of the finished product (faridi and faubion, 1986). these characteristics depend on several factors such as ingredient quality and quantity, mixing conditions, resting time and temperature of the wheat flour dough (bloksma and bushuk, 1988). in recent years the interest in high fiber content in foods has greatly increased and brown flours or high extraction flours are being used. abd and moniem (1994) studied high dietary fiber cookies by replacing wheat flour with 6 – 24% of cereal by-products like corn bran, rice bran or barley husk but no objective evaluation was carried out. because of the distinct properties, the use of sweetpotato flour in the preparation of bread is restricted. most of the researchers in this aspect found a substitution level of 10 – 15% for wheat flour on a dry weight basis as the most acceptable (el-sahy and siliha, 1988; lopez and villagarcia, 1984). hagenimana et al. (992) reported that he addition of orange-fleshed sweetpotato in buns, cha singh et al. 084 table 1. different combinations of sweetpotato flour and wheat flour for cookie preparation. s.n sweetpotato flour (%) wheat flour (%) 1 00 100 ( control) 2 20 80 3 40 60 4 60 40 5 80 20 6 100 00 pattis, and mandazis greatly increased the content of total carotenoids in these products. most of the technical research on sweetpotato flour has been focused on the development of new products using sweetpotato flour rather than on efficient methods to produce and store the flour (lizado and guzman, 1982). addition of various proportion of sweetpotato flour in wheat flour can increase the nutritive values in terms of fibre and carotenoids. this also helps in lowering the gluten level and prevent from coeliac disease (tilman et al., 2003). our approach in the present study was to replace the wheat flour in cookies by sweetpotato flour (gluten-free flours) in order to increase the fiber and other nutrients. the objective was to develop cookies with good taste, texture and appearance, which resembles as closely as possible to the wheat flour based product. the textural property and sensory quality of cookies are taken into consideration to improve the quality of cookies. materials and methods sweet potatoes of variety psp-21 were procured locally from the field after harvesting. roots were washed, trimmed and cured to make them free from soil and other foreign materials, rotting, insect damage. trimming was carried out manually and curing was done at 35°c for 2 3 days, stored at 12 -15°c at 80%rh till further use. laboratory grade potassium metabisulphite (kms), nacl and citric acid were purchased from m/s. brightways agencies, chandigarh, india. flour preparation treated sweet potatoes were peeled and cut into thin slices manually. slices were directly immersed into 1% nacl solution and then immersed in solution containing kms (1%) and citric acid (0.5%) for 30 min. drying of sweetpotato slices was done on perforated trays in a tray dryer (m/s. balaji enterprises, saharanpur, india) at 55°c till 7 8% moisture content and then stored in air tight container till further use. the dried chips were milled into flour using the laboratory grinder (m/s. sujata, new delhi, india) and passed through 80 mesh sieve to obtain flour of uniform size. the flour was then packed in air tight container and stored under refrigeration condition till further used. wheat flour, sugar and shortening were procured from the local market. the wheat flour used had protein and ash content of 9.2 and 0.65%, respectively. the sugar used was ground coarsely in a laboratory grinder (sujata, india). analysis of sweetpotato flour sweetpotato flour was analyzed for moisture, protein, ash, lipids, crude fiber and falling number according to aacc (1995) methods. starch and sugars (total and reducing) were determined according to methods of aoac (1981). water absorption water absorption of flour was determined with little modification to the method reported by anderson et al. (1969). five gram flour of each sample was weighed into a centrifuge tube and 30 ml of distilled water was then added and mixed thoroughly. this was allowed to stand for 30 min and centrifuged at 3,000 rpm for 15 min. the supernatant was then decanted and the sample weighed again. the amount of water retained in the sample was recorded as weight gain and was taken as water absorbed. the results were expressed as weight of water absorbed in grams per 100 g dry matter of the sample. odor evaluation sweetpotato flour odor was subjectively evaluated by a panel of ten members. in this test, distilled water was added to sweetpotato flour to give a final moisture content of 40%. after homogenization, the flour was put aside in a closed transparent plastic container for two hours and then presented to each panelist in a randomized complete block design. during evaluation, each panelist placed the plastic container near their nose, removed the cover and took a deep breath to smell the solution. the perceived odor intensity of the flour was scored on an interval category scale ranging from low intensity to high intensity. this was repeated three times using different code numbers on each occasion (owori and hagenimana, 2000) color measurement color of flour and cookies samples was measured by colorimeter cr-300 (ramsey, n.j., u.s.a.) and recorded in the l* a* b* color system. the colorimeter was calibrated using a standard white plate. samples were placed in the sample holder for measurement. color values were recorded as ‘‘l*’’ (lightness), ‘‘a*’’ (redness), and ‘‘b*’’ (yellowness). from a* and b* values, the hue angle tan 1 b* a* and chroma (a*) 2(b*) 2were calculated. cookie formulation cookie dough was prepared according to the following formula: 100 g flour (contain different proportion of sweetpotato flour and wheat flour, table 1), 30 g sugar, 35 g shortening, 1 g sodium chloride, 0.5 g sodium bicarbonate, 1 g ammonium bicarbonate, 0.5 g baking powder, and various proportion of water to make required consistency of dough. dough preparation cookie dough was made in a laboratory mixer (hobart mixer, model n50, canada). fat and sugar was creamed in a mixer with a flat beater for 2 min at slow speed. dough water containing the baking chemicals and sodium chloride was added to the resulting cream, and mixed for 5 min at high speed to obtain a homogeneous mixture. finally, flour containing various proportion of sweetpotato 085 afr. j. food sci. res. figure 1. typical texture profile curve for dough. flour, which had been sieved twice with baking powder was added, and mixed for 3 min at medium speed. the dough was rested for 30 min before evaluation. rheological characteristics of dough texture profile analysis texture profile analysis of the dough was carried out by texture analyzer (model ta-xt2i, stable micro systems, haslemere, u.k.). under the following conditions: probe (p75)/75mm ; load cell, 50 kg; pre test speed,3.0mm/s; post test speed, 10.0mm/s. dough samples were obtained by sheeting with a rolling pin over a rectangular platform and frame of height 1.0 cm to give a sheet of 1.0 cm thickness. the sheeted dough was cut into a circular shape using cutters of 4.5 cm diameter. the cylindrical dough of 4.5 cm in diameter and 1.0 cm thickness was used for these studies. the real-time plots (figure 1) were analyzed for the following: (1) dough consistency (ns), the combined area of the two resistance peaks; (2) dough hardness (n), the maximum resistance to the first compression peak (height of peak 1); (3) dough cohesiveness, the ratio of the areas of the two resistance peaks (a2/a1); (4) dough adhesion (ns), the area of the first adhesion peak; (5)springiness is length 2/ length 1. (6) resilience (area 2/ area1). cookies preparation the flow sheet of the cookies making process is shown in figure 2. the dough was then sheeted to a thickness of 4.5 mm with the help of a rolling pin and an aluminum frame of standard height. the cookies were cut with a cookies die of diameter 55 mm and transferred to a lightly greased baking tray. the cookies were baked at 205°c for 12 min in a baking oven (continental, india). the baked cookies were cooled and stored in air tight container for 12 h before further analysis. evaluation of cookies physical characteristics spread factor: the cookies were allowed to cool for 30 mins. the aacc (1995) method: 10-50d (200) was used to determine cookies width, thickness, and spread factor. cookie width (w) was measured by placing 6 cookies edge-toedge to get the average width in millimeters. cookie thickness (t) was measured by stacking 6 cookies on top of each other. width divided by the thickness gave the spread factor. fracture strength (snap test) the 3-point bending rig of texture analyzer (model ta-xt2i, stable micro systems, haslemere, u.k.) was used to determine fracture strength. the distance between the two beams was 40 mm. another identical beam was brought down from above at a pre-test speed: 2.0 mm/s, test speed: 0.5 mm/s, posttest speed: 10.0 mm/s, distance: 5 mm to contact the cookie. the downward movement was continued till the cookie breaks. the peak force was reported as fracture strength. density density was determined by standard seed displacement method and result was expressed as g/cc (aacc 1983). sensory evaluation and statistical analysis the cookies were evaluated by a panel of 15 members. samples were evaluated on a desk placed in the air-conditioned laboratory, which provided a quiet and comfortable environment. the cookies were served on a white disposable plastic tray and tap water was provided for rinsing. mean of 15 evaluations was reported. the de singh et al. 086 sweet potato grading and washing peeling 8 ± 0.3 mm slicing treatment (55°c) drying grinding sieving (size 80 mesh) ingredients mixing kneading sheeting cutting baking packing and storage figure 2. flow diagrams for preparation of sweetpotato flour cookies. 180 160 140 (% ) 120 a b s o r p t i o n 100 80 w a te r 60 40 20 0 0 20 40 60 80 100 sweetpotato flour (%) figure 3. water absorption of the sweetpotato flour samples at 30°c. tails of sensory attribute, definitions are given in table 2. statistical analysis was done using minitab statistical software (minitab inc. usa) results and discussion proximate analysis of sweetpotato flour is presented in table 3. the moisture content of sweetpotato flour was 8.7%. sweetpotato flour had very low protein content (2.3%) and high fiber content (9.4%). this high fiber increases the utility of sweetpotato flour in various food products. the starch content in sweetpotato flour was 74.5 (dwb). ulm (1988) mentioned that the total carbohydrates of the sweetpotato in roots are comprised of approximately 80% starch and 20% simple sugars. woolfe (1992) divided digestible carbohydrates into starch and total sugars with levels of 87.5 and 12.5% respectively. sweetpotato flour has distinct odor. odor intensity of sweetpotato flour is summarized in table 4. it was observed that the controlled sample has lower odor intensity and the intensity of odor increased significantly from 2.5 to 4.2 with the increased proportion of sweetpotato flour. this may be due to the volatile flavor constituents in sweetpotato flour which gave characteristics flavor and enzyme reaction also involved in the change in odor as reported by purcell et al. (1980). the water absorption of sweetpotato flour was 172%. this high percentage of water absorption was because of more fiber and sugar content in the flour. water absorption results (figure 3) showed that increasing the proportion of sweetpotato flour increased the absorption of water. increase in water absorption lead to the weakened dough and decrease dough development and dough stability. a similar trend also observed by hamed et al. (1973). addition of sweet potato flour to wheat flour caused a decrease in extensibility of the dough. effect of sweetpotato flour on color values of flour and cookies is shown in tables 5 and 6. the l* value of flour decreases significantly from 94.43 to 82.38 with increasing the proportion of sweetpotato flour but this value decreased from 62.26 to 48.43 in the case of cookies. the b* value which shows yellowness of flour increased significantly from 9.23 to 21.59 with increasing the proportion of sweetpotato flour. the yellow-orange color of sweetpotato flour was caused by the presence of carotenoid pigments, which affect the red-green chromaticity (van hal, 2000). texture profile analysis the textural characteristics of the doughs made from varying proportion of sweetpotato flours, are given in table 7. varying the sweetpotato flour and water content changed the textural characteristics of dough signifycantly. the hardness increased significantly from 1105 to 4864.3g in dough prepared from control sample to 100% sweet potato flour. a similar trend was observed for the biscuit dough by (manohar and haridas, 1999). 087 afr. j. food sci. res. table 2. sensory attribute, definitions and extremes. sensory trait definition initial bite (texture) crisp to hard. (4.9 to 1) 1)crisp cookie suddenly breaks when a small amount of force is applied 2)crumbly the cookie breaks easily, forming loose fragment in the mouth. 3)tender the cookie is easily broken down upon chewing. 4)hard cookie withstands substantial force on initial bite mouth feel crunchy to teeth clogging ( 4.9 to 1 ) 1)crunchy require repeated chewing to break cookie down. crunchy sound is heard. 2)granular small particles are detectable when the cookie is chewed 3)flaky on chewing a rough paste is formed, containing large irregular pieces. 4)teeth clogging particles stick in the mouth after the paste is swallowed. taste sweet to burn or caramel. ( 4.9 to 1) 1)sweet sweet with good baked taste. 2)slightly sweetpotato taste sweet with giving some sweetpotato taste. 3)off taste giving off taste after eaten. 4)burnt or carmel taste burnt or not good after taste color light brown to very dark brown. ( 4.9 to 1 ) 1)light brown give uniform light brown color 2) medium brown give patches of light and dark brown 3) brown uniform brown color. 4)dark brown very dark brown color overall acceptability outstanding to unacceptable ( 4.9 to 1 ) 1)outstanding 4.0-4.9 2)acceptable 3.0-3.9 3)marginal 2.0-2.9 4)unacceptable 1.0-1.9 table 3. physicochemical analysis of sweetpotato flour* s. no constituents percentage (%) 1 moisture 8.7±0.1 2 starch 74.5±0.85 (dwb) 3 reducing sugar 6.3±0.1 (dwb) 4 total sugar 11.4±0.26 (dwb) 5 ash 1.56 ±0.19 (dwb) 6 protein 2.3±0.07 (dwb) 7 lipids 0.52±0.06 8 falling number 134±1.0 9 water absorption 172±1.5 10 fiber 9.4±0.08 *average of three reading dwb = dry weight basis table 4. effect of sweetpotato flour on odor intensity. s. no spf: wf (%) mean panelist score (odor intensity*) 1 00:100 1.3 a 2 20:80 2.5 b 3 40:60 2.6 b 4 60:40 3.1 b 5 80:20 4.0 c 6 100:00 4.2 c the values denoted by different letters in the same column are significantly different (p< 0.05) *average of ten panelist 1-1.9 = none, 2-2.9 = slight, 3-3.9 = moderate, 4-4.9 = strong adhesiveness, of dough’s increased significantly (p < = 0.05) up to 340.15 at 80% sweetpotato flour addition, but at 100% sweetpotato flour the values decreased to 260.30. the cohesiveness increased significantly from 0.295 to 0.425 up to 40% sweetpotato flour addition and then decreased to 0.317 with increasing sweetpotato flour to 100%. quality characteristics of cookies physical characteristics of cookies the spread value of cookies from different flours was not affected significantly (table 8). the spread was greater 6.67 for cookies made from control, and decreased significantly with increasing the proportion of sweetpotato table 5. effect of sweetpotato flour (spf) on color values. spf:wf(%) l* a* b* chroma hue angle 00:100 94.43 d 2.73 a 9.23 a 9.63 a 73.50 c 20:80 89.71 c 4.66 b 13.22 b 14.02 b 70.55 c 40:60 85.78 b 6.75 c 17.93 c 19.16 c 59.34 b 60:40 85.21 b 7.23 c 19.84 c 21.12 c 59.96 b 80:20 84.94 b 8.10 d 21.59 d 23.05 d 59.43 b 100:00 82.38 a 8.52 d 21.59 d 23.21 d 58.44 a the values denoted by different letters in the same column are significantly different (p< 0.05) *average of three reading. table 6. effect of sweetpotato flour (spf) on color values of cookies. spf: wf(%) l* a* b* chroma hue angle 00:100 62.26 e 17.30 d 27.89 d 32.88 d 58.18 d 20:80 61.45 d 17.32 d 27.58 d 32.57 d 57.86 c 40:60 58.23 c 15.98 c 24.45 c 29.21 c 56.81 c 60:40 54.14 b 14.35 bc 20.36 b 24.91 b 54.80 b 80:20 53.89 b 13.47 b 19.04 b 23.32 b 54.71 b 100:00 48.43 a 11.03 a 13.49 a 17.43 a 50.70 a the values denoted by different letters in the same column are significantly different (p< 0.05) *average of three reading. table 7. effect of different ratios of sweetpotato flour dough on texture profile analysis. spf: wf (%) hardness force (g) springiness resilience cohesiveness adhesiveness(g/s) (l2/l1) mm a5/a4 g/s (a2/a1) g/s 00:100 1105.0 a 0.364 a 0.151 a 0.295 a 19.31 a 20:80 1688.2 b 0.537 c 0.129 c 0.380 b 164.27 b 40:60 2649.1 c 0.522 c 0.132 c 0.425 c 209.69 c 60:40 2955.3 c 0.529 c 0.135 b 0.382 b 323.27 e 80:20 3684.7 d 0.443 b 0.138 b 0.365 b 340.15 e 100:00 4864.3 d 0.356 a 0.150 a 0.317 a 260.30 d the values denoted by different letters in the same column are significantly different (p< 0.05) *average of three reading. flour. there was significant decrease in thickness of cookies with marginal increases in sweetpotato flours. the density of the cookies, decrease gradually from 0.587 to 0.540g/cc and the decrease was significant. however, the fracture strength of cookies increased with addition of sweetpotato flour up to 40% and further decreased with increase in sweetpotato flour. this may be due to the higher fiber content in the flour. this indicated that the cookies became soft with increased sweetpotato flour content. sensory characteristics of cookies the cookies formed with addition of 40% sweet potato flour got overall acceptability score of 3.6 which is very much acceptable to consumer (table 9). sensory evaluation shows that the cookies made from 100% sweetpotato flour was marginal acceptable with an overall score of 2.1. the score of taste reduced significantly to 1.8. this is because of typical flavor component and caramalization of free sugar in sweetpotato flour during baking. however the texture of cookies made from 100% sweetpotato flour was tender and acceptable. the sensory score shows that sweet potato flour @ 40% is acceptable with reference to taste mouth feel, color and texture. cookies can be made from sweet potato flour by addition of some flavor and improver. singh et al. 088 089 afr. j. food sci. res. table 8. effect of incorporation of sweetpotato flour into wheat flour on the physical characteristics of cookies. spf: wf(%) width(mm) thickness(mm) spread density fracture strength force (g) factor (g/cc) 00:100 320 d 48 e 6.666 e 0.587 e 2632 a 20:80 310 c 47 d 6.595 d 0.560 d 3730 d 40:60 301 b 46 c 6.543 c 0.551 c 3800 d 60:40 293 b 45 b 6.511 c 0.549 bc 3722 d 80:20 280 a 44 a 6.363 b 0.543 ab 3535 c 100:00 275 a 44 a 6.150 a 0.540 a 3072 b *the values denoted by different letters in the same column are significantly different (p< 0.05) *average of three reading. table 9. sensory characteristics of cookies made by adding various proportion of sweetpotato flour into wheat flour*. spf: wf (%) initial bite (texture) mouth feel taste color overall 00:100 4.6 d 4.1 d 4.7 c 4.3 c 4.7 d 20:80 3.9 c 3.8 cd 3.7 b 4.1 b 3.9 c 40:60 3.7 c 3.5 c 3.5 b 4.0 b 3.6 b 60:40 3.4 a 2.3 b 2.1 a 3.7 a 2.7 b 80:20 3.2 a 1.9 b 1.9 a 3.7 a 2.3 a 100:00 3.2 a 1.3 a 1.8 a 3.6 a 2.1 a * the values denoted by different letters in the same column are significantly different (p 0.05). conclusion the out come of the present research can be used as valuable information for the development of high fiber low gluten cookies. incorporation of 40% sweetpotato flour yielded approximately similar results compared with wheat flour cookies with improved nutritional value and texture. industrial relevance this work showed the peculiar characteristics (water absorption capacity, moisture, protein, ash, lipids, crude fiber and falling number) of sweetpotato flour. the results obtained could be very valuable in decision making for industries that want to take nutritional advantage of sweetpotato flour as alternative or supplement to cereal flours. sweetpotato flour could be useful in the manufacture of highly nutritious cookies. references aacc (1983) approved methods, 8th ed. st. paul, mn: american association of cereal chemists aacc (1995) approved methods of the american association of cereal chemists. 10 th ed. method 44-15a, 44-13, 08-01, 30-10 and 32-10.. the association: st. paul, mn. abd el, moniem gm (1994) mathematical models for maximum improvement of in vitro protein digestibility of high dietary fiber cookies, nahrung. 38 (1): 32-37 anderson ra, conway hf, pfeifer vf, griffin el (1969). gelatinization of corn grits by roll and extrusion cooking. cereal science today. 14: 4-6. aoac (1981). official methods of analysis of aoac. 14 th ed. washington, association of analytical chemists. bloksma ah, bushuk w (1988). rheology and chemistry of dough. in: pomeranz y (ed) wheat chemistry and technology, 3rd edn. american association of cereal chemists, minnesota. pp. 131–217 chiappe l, lopez pe, villagarcia m (1984). yield and flour production of twelve white sweet potato cultivars for industrial use. in: proceedings of sixth symposium of the international society for tropical root crops (edited by j.e. wenham). lima, peru: faculty of agriculture, national agrarian university. pp. 587589. collins m (1989).economic analysis of wholesale demand for sweetpotatoes in lima, peru, m.sc. thesis, department of agricultural and resource economics, university of florida, gainesville, usa.pp.1-164. el-sahy km, siliha h (1988). use of sulphite treated sweet potato flour in manufacturing of wheat bread. getreide mehl und brot. 42 (7): 215-217 faridi h, faubion jm (1986). dough rheology: its benefits to cereal chemists. in: faridi h, faubion jm (eds) fundamentals of dough rheology. american association of cereal chemists, minnesota. pp. 1-9. hagenimana v, carey e, gichuki st, oyunga ma, imungi jk (1992) changes in carotenoid content after drying and processing sweetpotato products. ecological food nutrition, 37: 450-473 hamed mge, siliha h, sandy sk (1973). preparation and chemical composition of sweet potato flour. cereal and bakery products. 50 (2): 133-139. horton d, fano h (1985). potato atlas, international potato center (cip), lima, peru. p. 136. lizado mlc, guzman mp (1982). development of new products using singh et al. 090 sweetpotato flour home econ. j. 10(1): 62. manohar rs, haridas r. (1999). effects of water on the rheological characteristics of biscuit dough and quality of biscuits. eur food res technol (209):281-285 owori c, hagenimana v (2000). quality evaluation of sweetpotato flour processed in different agro-ecological sites using small scale processing technologies. african potato association conference proceedings. 5: 483-490. purcell ae, later wl, lee ml (1980). analysis of the volatile constituents of baked, “jewel” sweetpotato. j. agric. food chem (28): 939-941. ranganna s (1999). handbook of analysis and quality control for fruits and vegetable products. tata mcgraw hill publication, 2nd ed. new delhi. pp. 1-15 tilman jc, colm mob, denise mc, anja d, elke ka (2003). influence of gluten free flour mixes and fat powder on the quality of gluten free biscuits eur food res. technol 216: 369-376 ulm sg (1988). the effect of storage conditions on selected quality attributes of sweetpotato flour, thesis of the university of tennessee, knoxville. p. 7-26 van hal m (2000). quality of sweetpotato flour during processing and storage. food rev. inter. 16: 1-37. woolfe ja (1992). sweetpotato: an untapped food resource, cambridge university press, cambridge, 292-389. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (5), pp. 308-313, may, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of some physical, chemical and sensory attributes of three pineapple (ananas comosus) varieties grown in ghana wisdom wardy, firibu kwesi saalia, matilda steiner-asiedu * , agnes s. budu and samuel sefa-dedeh department of nutrition and food science, university of ghana, box lg 134, legon – accra, ghana. accepted 23 september, 2017 the role of fruits and fruit juices in nutrition and health cannot be overemphasized. and as consumers look for better quality of fruits with regard to certain characteristics, new varieties are coming up to meet demand. it is against this background that this present study was carried out to assess the quality of md2 pineapple variety with respect to the other varieties on the ghanaian market. this study examined the physical, chemical and sensory quality of the md2 pineapple variety compared to those of the sugarloaf and the smooth cayenne varieties on the ghanaian market. sugarloaf had the highest juice volume of 205.72ml/kg fruit, followed by md2 (134-191) with smooth cayenne having the smallest volume. these differences were statistically significant, p<0.05. there were no significant flavor differences (p> 0.05); although there were significant differences (p< 0.05) in the overall preference for fruit juices which indicated that md2 was most preferred pineapple fruit. md2 compares very well with pineapple varieties already on the ghanaian market. its cultivation in ghana has potential both for the local and international market and should be encouraged. key words: fruit, pineapple, md2, sensory evaluation, chemical attributes, fruit juice. introduction since its introduction to the european market in 1996, the md2 pineapple variety has increasingly gained more attention than smooth cayenne, sugarloaf and other traditional pineapple varieties of commerce. the md2 has been described as super -sweet, self ripening and having a longer storage life with a value twice as much as that of the smooth cayenne variety (achuonjei et al., 2003). the production of md2 in ghana has been estimated to be 2% of total pineapple production with the smooth cayen-ne at the forefront (trienekens, 2003). it has there-fore become necessary for ghanaian growers to take ad-vantage of the increasing popularity of md2 if they are to maintain their share of the european market. in ghana, the volume of pineapple export in 2005 was 46694 tonnes as against 71804 tonnes in 2004. this marks a percent change of -34.97%. pineapple export in 2005 had a value of $12,784,300 ($12.7 million) as against $22,068,600($22 million) in 2004 representing a *corresponding author. e-mail: tillysteiner@ug.edu.gh. tel: +233-24 5447875. fax: + 233-21 500629. percent change of -42.07%. these earnings from pineapples stand out as markedly high when compared to its closest competitor, banana. in 2005, the volume of bana-na export was 1116 tonnes and in 2004, 725 tonnes re-flecting a change of 53.93% with a value of $458,000 and $ 208,900 in 2005 and 2004, respectively, marking a change of 119.24% (isser, 2006). it has been suggested that, the md2 demands a cold chain supply in order to maintain a long shelf life. its presence on the ghanaian market with tropical weather conditions may likely affect quality attributes. the competition with other varieties like the sugarloaf and smooth cayen-ne that are more familiar to ghanaian consumers should not be underestimated. its potential on the local market will largely be based on its quality attributes. a major question arises as to whether the md2 can signifycantly alter the preferences of consumers in ghana in a trend akin to that observed in europe. further, should the characteristics of the md2 be perceived as superior to that of the already existing cultivars in ghana and it is possible that, inevitable shifts in local market trends may well demand an increased production of the md2 locally. this prospect may also create a significant economic re wardy et al. 309 percussion for the horticultural sector considering the fact that pineapple is among the top-five nontraditional export commodities in terms of foreign earnings in ghana. an assessment of the quality of md2 with respect to the other varieties on the market will aid on-going resea-rch targeted at establishing the quality of md2 pineapple in ghana. this study examines the physical, chemical and sensory quality of the md2 pineapple variety com-pared to those of the sugarloaf and the smooth cayenne varieties on the ghanaian market. the implications for industry, nutrition and health are discussed. methods freshly harvested pineapples of three varieties; md2 at two different ripening stages, m1 (one-fourth of the shell colour yellow) and m3 (three-quarters of the shell colour yellow); smooth cayenne and sugarloaf were obtained from blue skies products company limited and kings farms (ghana). samples were transported in paper cartons to the laboratory and kept refrigerated at 5-8ºc prior to analyses which run over a four-day period. four replicates each of the three varieties were sampled for physical and chemical analyses. physical analyses fruits were weighed using a top loading balance (salter-and model, japan), the weight of the samples were determined. the volumes of the samples were also determined by the displacement method after coulter and lorenz (1991) and the density determined as weight per volume (g/cm 3 ). using a pineapple slicer, the flesh of the pineapples was scooped, passed through a juice extractor and the juice collected. volume of juice (mls) was measured using a measuring cylinder. chemical analyses vitamin c was determined by the dichloroindophenol method (aoac, 1990). acidity (ph) of the juices was evaluated using an electronic toa ph meter (hm 305 model, japan) at 27ºc. titratable acidity was assessed as outlined by aoac, (1984). soluble solids were determined using an abbe refractometer (aoac 932.12; 1995). total carbohydrates were estimated by the anthrone method (plummer, 1987). sweetness index (si) was derived as the ratio of the percent soluble solids to titratable acidity and astringency index (ai) was expressed as the ratio of titratable acidity (g citric acid/100 g fruit) to soluble solids content (%) (table 3b). sensory analyses organoleptic characteristics of the three pineapple juices were carried out. thirty (30) panelists were randomly assigned the samples blindly for evaluation for sweetness, color, flavor and overall preference. the samples used for the sensory evaluation included 100% juice from each of the three varieties in addition to md2 juice blended with smooth cayenne or the sugarloaf (all at the m3 maturity stage) in the following proportions: 80:20, 60:40, 40:60, 20:80. a total of 11 juice samples were evaluated using a seven-point hedonic scale. the sensory evaluation (table 3a) was done in 2 days; day 1 was between md2 and smooth cayenne and day 2 was between md2 and sugarloaf. this was necessary to reduce respondent burden (and the risk of sensory fatigue) and also to ensure a fair comparison of md2 to either variety but not between smooth cayenne and sugarloaf. data analysis statgraphics plus, version 3.0 (statistical graphics corporation, stsc inc, u.s.a.) was used to analyze the data, using one factor anova (for pineapple) at three levels (smooth cayenne, md2 and sugarloaf). multiple range test (lsd) was used to assess differences between the varieties at 95% confidence level. results physical evaluation the densities of the md2 samples ranged between 0.92 1.10 g, that of sugarloaf was 0.99-1.06 g/cm 3 . the smooth cayenne recorded the lowest densities ranging from 0.94-0.99 g/cm 3 . these differences, however, were not statistically significant (p>0.05). the volume of fruit juice obtained from the samples were significantly different (p<0.05). sugarloaf had the highest juice volume of 205. 72 ml/kg fruit, followed by md2 at m3 stage, with values ranging from 143.12-191.43 ml, md2 at m1 stage with a range between 134-182 ml. smooth cayenne had the least volume between 91.7-108.65 ml. chemical evaluation the chemical characteristics of the pineapples are shown in table 1. the md2 at the m1 and m3 stages had an ascorbic acid content of 54.17 and 49.59mg/100ml respectively. this was followed by sugarloaf (42.11 mg/100 ml) with smooth cayenne having the least value (21.04 mg/100ml). the differences observed were statistically significant (p<0.05). titratable acidity was different among samples (p< 0.05). smooth cayenne showed the highest titratable acidity value, followed by the md2 at the m1 stage. sugarloaf had the highest ph and smooth cayenne had the lowest values. the varietals differences were significant (p<0.05). however, ph variations between the two maturity stages of the md2 (m1 and m3) were not statistically significant. total carbohydrates were highest for the md2 at m3 followed by the sugarloaf, smooth cayenne and then md2 at m1. the differences were significant (p<0.05) and further analysis revealed that the sugar content of md2 at m3 and of sugarloaf are the same but higher than those of smooth cayenne and md2 at m1. the total sugar content was significantly different for the md2 at the two maturity stages. differences in the soluble solids content between the varieties were significant (p<0.05). sugarloaf had the highest sweetness index of 15.14; followed by the md2 (m3: 12.72 and m1:12.65) and the smooth cayenne had the lowest index of 6.98. these differences between the varieties were significant (p<0.05) but not between the 310 afr. j. food sci. res. table 1. chemical attributes profile of the pineapples (means (sd). varieties ph soluble solids (%) acidity (%) vitamin c (mg/100 ml) total sugars (g/100 ml) sweetness index astringency index md2 5.40 (0.06) 14.5 (0.00) 1.15 (0.01) 51.88(3.24) 16.90 (8.61) 12.69 (0.05) 0.08 (0.00) md2 m1 5.35 (0.33) 14.50 (1.00) 1.16 (0.09) 54.17(4.49) 10.81 (1.02) 12.65 (1.81) 0.08 (0.01) m3 5.44 (0.33) 14.50 (1.00) 1.14 (0.06) 49.59(4.31) 22.98 (7.39) 12.72 (0.57) 0.08 (0.00) sl 4.96 (0.02) 16.75 (0.96) 1.11 (0.05) 42.11(2.85) 17.50 (94.88) 15.14 (1.47) 0.07 (0.00) sc 5.80 (0.02) 11.59 (2.30) 1.67 (0.08) 21.04(4.26) 12.39 (5.41) 6.98(1.67) 0.15 (0.03) table 2a. organoleptic profile of md2 and sugarloaf juice (mean rank). colour sweetness flavour overall preference 100 %md2 2.17 3.73 3.20 3.97 100 % sl * 3.97 4.27 4.03 4.33 80%md2-20%sl 2.37 2.70 3.20 2.63 60%md2-40%sl 2.73 3.67 3.43 3.33 40%md2-60%sl 3.47 3.47 3.43 3.17 20%md2-80%sl 3.83 3.13 3.70 3.17 *sl= sugarloaf. table 2b. organoleptic profile of md2 and smooth cayenne juice (mean rank). color sweetness flavor overall preference 100%md2 2.17 3.17 2.97 3.27 100%sc** 3.97 3.30 3.17 3.73 80%md2-20%sc 2.37 3.03 3.37 3.23 60%md2-40%sc 2.73 2.87 3.47 3.50 40%md2-60%sc 3.47 3.10 3.20 3.63 20%md2-80%sc 3.83 3.57 3.30 3.63 **sc= smooth cayenne. like extremely, 1; like very much, 2; like slightly, 3; neither like nor dislike, 4; dislike slightly, 5; dislike very much, 6; dislike extremely, 7. varieties were significant (p<0.05) but not between the two maturity stages of md2. the smooth cayenne had the highest astringency index of 0.15. the sugarloaf had the lowest index of 0.07, whilst md2 at m1 and m3 each had 0.08. the astringency index of the smooth cayenne was signifi cantly different (p<0.05) from that of the sugarloaf and md2 at the two maturity stages. no significant difference was found between the sugarloaf and wardy et al. 311 table 3a. anova summary table for sensory tests [f-ratios (p-value)]. source of variation color sweetness flavor overall preference variety mix 5.77 (0.0168) 3.44 (0.0644) 2.25(0.1345) 0.00 (0.9499) ratios 9.84 (0.0000) 1.95 (0.0855) 0.77(0.5683) 3.05 (0.0103) *values represent f-ratios (p-value). table 3b. anova summary table for chemical and physical analyses. source of variation ph soluble solids acidity vitamin c total sugars sweetness astringency density vol. of juice per kg (%) (%) (mg/100ml) (g/100ml) index index (g/cm 3 ) pineapple (ml/kg) pineapple variety 8.77 8.74 56.39 52.96 4.47 22.44 17.56 1.62 20.81 (0.0024) (0.0024) (0.0000) (0.0000) (0.0251) (0.0000) (0.0001) (0.2358) (0.0000) *values represent f-ratios (p-value). the md2 at m1 and m3. there was no significant differrence between the md2 at the two maturity stages sensory evaluation for organoleptic characteristics of the pineapples, the colour of 100% md2 (m3) juice was most preferred (tables 2a and 2b). for fruit juice blends, samples containing higher proportions of md2 at m3 were most preferred. the md2-smooth cayenne juice blends generally had lower rankings than the md2sugarloaf blends. 80% md2-20% sugarloaf was the most preferred of the md2sugarloaf blends whilst 60% md2-40% smooth cayenne was the most preferred of the md2smooth cayenne products. blends of md2 and sugarloaf were more preferred to products containing 100% of either the md2 or the sugarloaf. blends of the sugarloaf and the md2 were less preferred to md2-smooth cayenne as judged by their higher mean rank sums. product composi tions with higher amounts of the md2 were preferred to those with higher concentrations of sugarloaf or the smooth cayenne, but the differences were not significant. there was no significant effect on flavour (p> 0.05), however, there was significant difference (p< 0.05), in the overall preference for flavor. md2 was most preferred. addition of sugarloaf at 20% to the md2 is seen to create detectable differences in overall preference. preferences for the smooth cayenne-md2 blends were seen to decrease with increasing amounts of the smooth cayenne. discussion specific gravity may be useful as a fruit grading index for differentiating eating quality as well as maturity. a greater density corresponds to a greater fruit weight that may possibly imply a larger serving size and a more compact texture. translucency has been correlated with specific gravity although the latter is a better indicator of eating quality than the former (smith, 1988). since the desire of some consumers is for more of the juice than the flesh, the volume of juice extractable from a particular variety becomes important indicating which of the varieties may be most profitable in commercial pineapple juice manufacture. from the results, sugarloaf is seen to have more juice, less fibre and more sugar presenting the best prospects and advantages in this enterprise. however, sugarloaf with a greater juice volume may be prone to bruising and other post harvest mechanical injuries to greater extents than the md2 and the smooth cayenne. ascorbic acid content of fruits in general is an important quality index due to its health significance as a vitamin and cellular antioxidant. furthermore, in food processing, ascorbic acid functions as an antioxidant by being preferentially oxidized (bartholomew et al., 2003, ngoddy and ihekoronye, 1985). ascorbic acid value of pineapple is largely variable depending on factors such as 312 afr. j. food sci. res. the cultivar, stage of maturity, conditions of storage and the part of fruit. it ranges from 20/100 to 34.44 mg/100 ml (collins, 1968; ngoddy and ihekoronye, 1985). the vitamin c content of the md2, sugarloaf and smooth ca-yenne is consistent with the entire varieties meet the die-tary requirement of vitamin c but md2 has the best potential due to its very high levels on weight for weight basis than the other varieties. the amount of vitamin c in a fruit has been linked with internal browning associated with post harvest chilling injury (miller, 1951). internal browning is a minor problem if fruit ascorbic acid is grea-ter than 500 µm (teisson and combres, 1979). the md2 with a higher ascorbic acid concentration may have an advantage over the sugarloaf and the smooth ca-yenne in reducing post harvest quality changes linked to chilling injury (paul and rohrbach, 1985). fruit acidity increases with maturity (gortner and singleton, 1965); however, md2 at maturity stages m1 and m3 appear to have little or no effect on titratable acidity (kotey, 2006). the higher acidity of the smooth cayenne may imply a longer storage life and a possibly higher astringency index. under high temperatures, gell-ing, properties may be affected when acidity is high due to the hydrolysis of sucrose and pectin methoxyl groups. sugarloaf with a high ph may be less suitable for jam manufacture than the smooth cayenne and the md2 since its glycoside linkages would remain relatively less stable at low acidity. the ph values obtained reflects to a significant extent the microbial stability of the various varieties. the md2 and smooth cayenne with lower phs’ will be expected to keep better than the sugarloaf. acidi-fiers may be needed to enhance the keeping properties of sugarloaf products. senescence is enhanced by increasing sugar content in fruits. this has implications for sugarloaf and md2 at m3 maturity stage which puts them at an increased risk of deterioration than the smooth cayenne. soluble solids are also known to impact sweetness index than does the total sugars (bartholomew et al., 2003). the soluble solids content is also used as an indication of fruit maturity and quality (paul, 1993) and for pineapples, they range bet-ween 10.8 17.5% (dull, 1971) with very little variations between varieties. however, the slightly higher values for sugarloaf are consistent with the sweetness rankings. studies by sideris and krauss (1934) indicate that, fruits which have a sweetness index greater than 19 are regar-ded as sweet and less acid by taste. the data for sugar-loaf are within these limits. the implication is that, sugar-loaf has an intense flavour and possibly, a higher consu-mer preference than the md2. smooth cayenne would most definitely have the lowest consumer preference. astringency or tartness was similar for the md2 and the sugarloaf. this is probably due to the nullifying impact that acidity has on sweetness. these values indicate that, though the sweetness of the sugarloaf is appreciably higher, their eating quality may be similar. the smooth cayenne with a light yellow colour complemented the gol den brown colour of md2 to give a darker rich yellow colour for their blends. it is likely that, 20% of md2 pre-sent in the product was just sufficient to balance out any contrast between the smooth cayenne and the sugarloaf indicating that the colour of md2 is sufficiently deep enough to cancel out any disparities. the md2 thus signi-ficantly impacts on colour with its golden brown colour being the preferred choice colour. conclusion/implication md2 compares very well with pineapple varieties already on the ghanaian market. its cultivation in ghana has po-tential both for the local and international market and should be encouraged. due to its rich dark yellow colour, it could be added to juices with pale yellow colour to en-rich them. md2 variety has a great potential in the horti-cultural industry. references achuonjei p, boschma s, happe g, hoogendoorn b, meekma i, pilkes j, waardenburg r (2003). ghana: sustainable horticultural export chain. michigan state university, michigan. aoac (1984). official methods of analysis. 14 th ed. association of official analytical chemists, washington d.c. p. 985. aoac (1990). official methods of analysis. 15 th ed. association of official analytical chemists, washington d.c. p.1005. bartholomew dp, paull re, rohrbach kg (2003). the pineapple: botany, production and uses. cabi publishing, uk. pp. 253-274. collins jl (1968). the pineapple, botany, utilisation, cultivation. leonard hill ltd, london. pp. 200-294. coulter la, lorenz k (1991). extruded corn grits – quanoa blends: ii. physical characteristics of extruded products. j. food proc. preserv. 15: 243-259 dull gg (1971). the pineapple. in: hulme ac (ed.) the biochemistry of fruits and their products. academic press, new york. 2: 303-324. gortner wa, singleton jl (1965). chemical and physical development of the pineapple fruit ii. carbohydrate and acid constituents. j. food sci. 30(3): 19. kotey end (2006). quality of md2 pineapples from different farms. unpublished dissertation, department of nutrition and food science, university of ghana, legon. miller ev (1951). physiological studies of the pineapple, ananas comosus. l. merr. with special reference to physiological breakdown. plant physiol. 26:66-75 ngoddy po, ihekoronye ia (1985). integrated food science and technology for the tropics. macmillan publishers, london. pp. 73303. paull re (1993). pineapple and papaya. in: seymour, taylor gj, tucker g (eds.) biochemistry of fruit ripening. chapman and hall, london, pp. 291-323. paull re, rohrbach kg (1982) juice characteristics and internal atmosphere of waxed ‘smooth cayenne’ pineapple fruit. j. am. soc. horticult. sci. 107: 448-452. plummer dt (1987). introduction to practical biochemistry. 3 rd ed. mcgraw hill book company ltd, london. pp. 179-180 sideris cp, krauss bh (1938). physiological studies on the factors influencing the quality of pineapple fruits. i. physico-chemical variations in the tissue of ripe pineapple fruits (continued). pineapple q. 4:48-57. smith lg (1988). indices of physiological maturity and eating quality in smooth cayenne pineapples. 1. indices of physiological maturity. queensland j. agric. ani. sci. 45: 213-228. teisson c, combres jc (1979). le brunissement interne de wardy et al. 313 l’ananas. iii. symptomatologie. fruits 34: 315-339. trienekens jh (2003). market induced innovations through international supply chains development: klct tr-207 a working paper. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (7), pp. 331-337, july, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of fufu flour and dough from 43 cmd resistant varieties etudaiye, h.a. 1 , nwabueze, t.u. 2 and sanni, l.o. 3 1 national root crops research institute (nrcri), umudike, p. m. b 7006, umuahia, abia state, nigeria. 2 michael okpara university of agriculture, umudike, p. m. b 7267, umuahia, abia state, nigeria. 3 international institute of tropical agriculture, ibadan, p. m. b 5320, ibadan, oyo state, nigeria. accepted 19 march, 2018 cassava fufu flours were produced from 43 cassava mosaic disease resistant varieties. the cassava roots were washed, peeled and steeped in water in a 1:2 (v/v) for 48 h to ferment. the fermented pulp was sieved and mechanically milled into flour of 3.0 mm particle size. proximate composition, functional properties and pasting characteristics of the ‘fufu’ flours were assessed. moisture content ranged from 5.52 to 12.25%, protein 0.35 to 2.80% and dry matter 87.75 to 94.48%. water absorption capacity ranged from 0.70 to 2.20 g/ml. the cmd varieties 92b/00068 and m98/0068 had the highest water absorption capacity values. bulk density ranged from 0.61 in the variety 98/2226 to 0.70 g/ml in varieties 98/0505, 98/0510, 95/0379 and 95/0289 while final viscosity of the fufu flours ranged from 76.00 in 96/1569 to 295.00 rvu in the 99/6012 and pasting temperature from 77.55 to 81.60 o c. given the economic and nutritional importance of cassava, the development and subsequently processing the cmd resistant varieties into fufu flour will help to improve food security and diversity not only in nigeria but also beyond. key words: cassava mosaic disease (cmd), fufu, functional properties, pasting, characteristics. introduction cassava (manihot esculenta) is an important staple food crop for millions of people in the tropical areas of african, asia and latin america. it is estimated (iita,1990) that the crop provides about 40% of all the calories consumed in africa and ranks second only to cereal grains as chief source of energy in nigerian diet (ngoddy, 1989). cassava roots contain mainly carbohydrates, of which 80% is starch and >1% fat (goomez, 1979). by this, cassava plays important role in alleviating african food crisis though poor in protein (1.20%) and rich in cyanide (> 10 mg/100 g fresh weight) in some varieties such as tms 50395 (iita,1990). the nr8082 cassava variety with low -cyanide highyielding quality developed by the national root crops research institute, nigeria, has commonly been distributed to nigerian farmers (nwabueze and odunsi, 2007) . however, nigeria’s cassava output is now threatened by a virulent form of the cassava mosaic dis-ease (cmd) advancing rapidly from east africa (iita, 2005). this is what initiated the pre-emptive management *corresponding author: email: etudaiyehuss@yahoo.com of cmd project by the international institute of tropical agriculture (iita) primarily to develop cassava mosaic disease resistant varieties. consequently, about 45 new cmd resistant varieties have been developed (nwabueze and anoruoh, 2008). in nigeria, rapid urban growth and development place a dynamic challenge to cassava products and market development for cassava foods will continue to increase. although cassava roots are processed by several traditional methods, which vary widely from region to region into products such as gari, lafun, landang, fufu, flour, chips, starch akara, okpokpo garri, meal, ighu, syrups, dextrins, and alcohol (nwabueze and odunsi, 2007), high quality cassava flour that can replace wheat and other imported flours in tropical countries (wheatley and best, 1991) has been reported. production of fufu of acceptable standard from the cmd resistant varieties has not been adequately reported in literature. the objective of this research was to produce and evaluate fufu flour and dough from 43 cmd resistant varieties, in terms of their proximate composition, functional and pasting properties. it is expected that the result obtained from the study will contribute in providing information on these cmd resistant varieties etudaiye et. al. 332 table 1. forty three 10-12 months old cmd resistant varieties harvested from the field trial of nrcri, umudike. cmd cmd cmd s/n varieties s/n varieties s/n varieties 1. 97/4769 16. tme 419 30. 92/0057 2. 99/6012 17. 96/0603 31. 91/0166 3. 94/0561 18. 98/2226 32. 96/1089a 4. 97/0162 19. 82/005 33. 96/1314 5. 94/0026 20. 97/0211 34. 97/3200 6. 96/1642 21. 95/0289 35. 98/0040 7. 98/0510 22. 92/0326 36. tms 30572 8. 98/0505 23. 4(2)1452 37. 99/2123 9. 99/3037 24. 98/0002 38. 92/0067 10. 98/2101 25. 97/4779 39. 97/0039 11. 97/4763 26. 96/1632 40. 95/0379 12. 97/2205 27. 96/0523 41. 92b/0061 13. 98/1565 28. m98/0068 42. 98/0581 15. 92b/0068 29. m98/0028 43. 96/1569 nrcri = national root crops research institute and cmd = cassava mosaic disease resistant varieties. and also serve as a guide for future research and improvement of these new cassava varieties. materials and methods source of raw materials forty three cassava mosaic disease resistant (cmd) varieties were harvested at 10 12 months old from the field trial of national root crops research institute (nrcri), umudike, abia state, nigeria (table 1). production of fufu flours cassava fufu flours were produced from each of the cmd resistant varieties using the processing methods described by okpokiri et al. (1984). the cassava roots were washed, peeled and re-washed with clean borehole water. they were steeped in water in a 1:2 (v/v) for 48 h. at the end of the steeping period the cassava samples were re-washed and grated into pulp using the iita mk powered grater (3.5hp petrol engine, lambourn, ltd, corydon, cr93ee, united kingdom). each cassava pulp sample was resteeped in water for another 24 h to ferment. the fermented pulp was sieved with endescotts laboratory test sieve with an aperture size of 2.0 mm (endescotts laboratory test sieve london, united kingdom). recovered samples were packed in bags and de-watered using a john willy and sons hydraulic press (7.5hp, john willy and sons ltd, united kingdom) . the resulting cassava cake was pulverized by hand and sun-dried on a wide opaque water-proof spread (jiffy bags macro packaging co., united kingdom). the sun-dried sample was mechanically milled into flour of 3.0 mm particle size using a disc attrition mill (2a premier mill, hunt and co., united kingdom). further sieving was done manually with a muslin cloth to obtain fine fufu flour. the fufu flours obtained from the 43 batches of cmd resistant varieties were properly packaged and sealed in grip-seal polyethylene bags (gl-model, 2.25” x 2.25”, jiffy bags macro packaging co., united kingdom). packaged samples were stored at room temperature (28 ± 2 o c) until ready for analysis. proximate composition of fufu flours proximate composition of the 43 cmd fufu flours were determined in triplicates for moisture, crude protein (kjeldhal method), fat (soxhlet method), and ash according to aoac (1990). total carbohydrate was determined by difference. the dry matter content of the flours were calculated and reported as mean values in table 2 while the proximate values reported on wet matter basis is shown in table 3. functional properties water absorption capacity (wac) of the fufu flour samples was determined by the method described by okaka and porter (1979) while the bulk density was determined using the method described by okezie and bello (1988). pasting properties pasting properties of the fufu flours were determined with the aid of a rapid visco analyzer (rva 3d+, network scientific unit, snw 2102, australia). parameters determined were final viscosity, set back viscosity, pasting time and pasting temperature. statistical analysis data obtained from the analyses of the fufu flour samples were subjected to statistical analysis using statistical analytical system (sas, 1999) software package. analysis of variance (anova) was done and means separation using fischer lsd to determine significant differences at 5 % probability was done. results and discussion proximate composition table 2 shows the moisture, dry matter and energy content of fufu flours produced from 43 cmd resistant varieties in umudike. moisture content ranged from 5.52% in fufu flour made from the cmd 97/4769 variety to 12.25% in the 96/1569 variety. the reverse was the case for their dry matter contents being 94.48 and 87.75% respectively. the generally low moisture content of the fufu flours is an indication of a good stable shelf life if packaged and stored. this is because with this moisture range, the quality of the final product will not be adversely affected. furthermore, high moisture products require further costly drying operations to allow easy handling and storage (sefa – dedeh and saalia, 1997). values of moisture contents were within the recommended standard of 13 % (m/m) for edible cassava flour (sanni et al., 2005). the high dry matter is an indication of desirable quality attributes in the cmd resistant varieties. such attributes like good yields, diseases and pest tolerance, high root 333 afr. j. food sci. res. table 2. moisture and dry matter content of fufu flours produced from 43 cmd resistant varieties in umudike cassava cultivar moisture content (%) dry mattercontent (%) energy (kcal/g) 97/4769 5.52 ± 0.02 94.48 ± 0.02 375.08± 0.03 99/6012 6.17 ± 0.01 93.83 ± 0.01 375.40± 0.04 94/0561 6.51 ± 0.01 93.49 ± 0.01 374.95± 0.03 97/0162 6.97 ± 0.02 93.03 ± 0.02 373.74± 0.02 94/0026 6.99 ± 0.04 93.01 ± 0.04 373.48± 0.04 96/1642 7.02 ± 0.02 92.98 ± 0.02 376.20± 0.12 98/0510 7.26 ± 0.04 92.74 ± 0.04 371.83± 0.04 98/0505 7.29 ± 0.02 92.71 ± 0.02 371.40± 0.02 99/3037 7.45 ± 0.02 92.55 ± 0.02 362.82± 0.03 98/2101 7.46 ± 0.02 92.54 ± 0.02 361.94± 0.06 97/4763 7.48 ± 0.01 92.52 ± 0.01 369.55± 0.04 97/2205 8.19 ± 0.03 91.81 ± 0.03 367.68± 0.12 98/1565 8.19 ± 0.01 91.81 ± 0.01 365.06± 0.02 92/0325 8.22 ± 0.01 91.78 ± 0.01 367.28± 0.06 92b/0068 8.32 ± 0.02 91.68 ± 0.02 365.54± 0.04 tme 419 8.32 ± 0.56 91.68 ± 0.56 366.79± 0.03 96/0603 8.37 ± 0.02 91.63 ± 0.02 377.27± 0.06 98/2226 8.62 ± 0.02 91.38 ± 0.02 365.30± 0.03 82/0058 8.70 ± 0.02 91.3 ± 0.02 370.50± 0.12 97/0211 8.70 ± 0.20 91.3 ± 0.20 361.00± 0.04 95/0289 9.09 ± 0.03 90.91 ± 0.03 364.78± 0.02 92/0326 9.13 ± 0.02 90.87 ± 0.02 364.60± 0.04 4(2)1452 9.14 ± 0.03 90.86 ± 0.03 361.44± 0.03 98/0002 9.32 ± 0.01 90.68 ± 0.01 362.85± 0.12 97/4779 9.35 ± 0.02 90.65 ± 0.02 360.66± 0.03 96/1632 9.35 ± 0.12 90.65 ± 0.12 363.44± 0.02 96/0523 9.43 ± 0.03 90.57 ± 0.03 365.21± 0.03 m98/0068 9.53 ± 0.01 90.47 ± 0.01 361.77± 0.12 m98/0028 9.55 ± 0.01 90.45 ± 0.01 362.40± 0.04 96/1089a 9.57 ± 0.02 90.43 ± 0.02 359.91± 0.03 91/0166 9.63 ± 0.02 90.37 ± 0.02 357.60± 0.02 92/0057 9.66 ± 0.06 90.34 ± 0.06 361.19± 0.03 96/1314 9.78 ± 0.01 90.22 ± 0.01 358.33± 0.03 97/3200 9.78 ± 0.05 90.22 ± 0.05 361.72± 0.02 98/0040 9.96 ± 0.03 90.0 ± 0.03 359.94± 0.04 tms 30572 10.13± 0.05 89.87 ± 0.05 359.25± 0.03 99/2123 10.26 ± 0.03 89.74 ± 0.03 359.61± 0.06 92/0067 10.38 ± 0.02 89.62 ± 0.02 358.36± 0.04 97/0039 10.39 ± 0.01 89.60 ± 0.01 357.22± 0.03 95/0379 10.45 ± 0.02 89.55 ± 0.02 358.31± 0.12 92b/0061 10.65 ± 0.01 89.35 ± 0.01 316.80± 0.04 98/0581 11.26 ± 0.04 88.74 ± 0.04 312.78± 0.03 96/1569 12.25 ± 0.02 87.75 ± 0.02 350.25± 0.12 yields (fresh and dry) meet end-users characteristics (iita, 2005). dry matter is a practical approach to improving the shelf life and marketability of fufu flour (akingbala et al., 1991). the energy content ranged from 357.22 in the 97/0039 cmd variety to 377.27 cal/kg in the 96/0603 variety. these varieties have 10.39 and 8.37% moisture and 89.60 and 91.63% dry matter contents respectively. protein, ash and fat contents were generally low while carbohydrate contents were high (table 3). the composi etudaiye et. al. 334 table 3. proximate composition of fufu flours processed from cmd resistant varieties in umudike. cassava variety protein (%) ash (%) fat (%) fibre (%) carbohydrate (%) 97/4769 2.45 ±0.04 0.64 ± 0.01 0.41± 0.06 0.07 ± 0.03 90.91 ± 0.04 99/6012 1.93 ±0.03 0.64 ± 0.03 0.24± 0.03 0.06 ± 0.01 91.38 ± 0.02 94/0561 1.4 ±0.02 0.22 ± 0.01 0.51± 0.02 0.07 ± 0.02 91.19 ± 0.01 97/0162 2.07 ±0.06 0.32 ± 0.03 0.58± 0.03 0.05 ± 0.02 90.06 ± 0.04 94/0026 1.40 ± 0.02 0.24 ± 0.02 0.56± 0.01 0.04 ± 0.01 90.71 ± 0.01 96/1642 2.55 ± 0.05 0.39 ± 0.01 0.40± 0.02 0.04 ± 0.02 90.60 ± 0.02 98/0510 1.82 ± 0.03 0.07 ± 0.02 0.27± 0.03 0.05 ± 0.01 90.53 ± 0.02 98/0505 2.45 ± 0.02 0.05 ± 0.03 0.20± 0.02 0.06 ± 0.01 89.95 ± 0.03 99/3037 2.15 ± 0.01 0.59 ± 0.01 0.42± 0.01 0.01 ± 0.01 87.61 ± 0.03 98/2101 1.93 ± 0.05 2.52 ± 0.02 0.42± 0.01 0.06 ± 0.05 87.37 ± 0.03 97/4763 2.41 ± 0.13 0.37 ± 0.04 0.23± 0.03 0.05 ± 0.02 89.46 ± 0.02 97/2205 1.40 ± 0.02 0.34 ± 0.01 0.40± 0.01 0.05 ± 0.01 89.62 ± 0.02 98/1565 0.70 ± 0.02 0.51 ± 0.04 0.34± 0.01 0.07 ± 0.01 89.80 ± 0.03 92/0325 1.75 ± 0.02 0.10 ± 0.01 0.12± 0.02 0.01 ± 0.01 89.80 ± 0.30 92b/0068 1.23 ± 0.01 0.20 ± 0.02 0.66± 0.01 0.03 ± 0.03 88.67 ± 0.02 tme 419 0.70 ± 0.04 0.41 ± 0.01 0.39± 0.02 0.06 ± 0.01 90.12 ± 0.03 96/0603 0.98 ± 0.02 0.22 ± 0.02 0.39± 0.01 0.08 ± 0.01 89.96 ± 0.01 98/2226 2.80 ± 0.02 0.45 ± 0.03 0.34± 0.04 0.03 ± 0.02 87.76 ± 0.03 82/0058 1.78 ± 0.01 1.58 ± 0.08 0.34± 0.01 0.02 ± 0.01 87.58 ± 0.03 97/0211 2.80 ± 0.20 1.53 ± 0.31 0.56± 0.01 0.03 ± 0.03 86.19 ± 0.01 95/0289 1.78 ± 0.01 0.22 ± 0.03 0.42± 0.01 0.02 ± 0.01 88.47 ± 0.02 92/0326 2.80 ± 0.02 0.34 ± 0.04 0.56± 0.01 0.08 ± 0.06 87.09 ± 0.02 4 (2) 1452 1.40 ± 0.02 1.28 ± 0.01 0.64± 0.01 0.02 ± 0.01 87.52 ± 0.02 98/0002 1.78 ± 0.01 0.43 ± 0.01 0.41± 0.02 0.04 ± 0.03 88.01 ± 0.01 97/4779 2.10 ± 0.01 0.97 ± 0.02 0.42± 0.01 0.04 ± 0.02 87.12 ± 0.01 96/1632 2.80 ± 1.58 0.48 ± 0.07 0.56± 0.01 0.01 ± 0.01 86.80 ± 0.02 96/0523 1.21 ± 0.06 0.44 ± 0.02 0.17± 0.01 0.04 ± 0.02 89.71 ± 0.01 m98/0068 1.05 ± 0.04 0.20 ± 0.02 0.17± 0.01 0.04 ± 0.02 89.01 ± 0.01 m98/0028 0.35 ± 0.03 0.37 ± 0.02 0.44± 0.03 0.03 ± 0.01 89.26 ± 0.02 96/1089a 2.80 ± 0.02 0.94 ± 0.02 0.43± 0.01 0.06 ± 0.01 86.2l ± 0.02 91/0166 2.55 ± 0.05 2.31 ± 3.45 0.30± 0.02 0.03 ± 0.02 86.18 ± 0.04 92/0057 1.05 ± 0.01 0.57 ± 0.02 0.43± 0.01 0.01 ± 0.01 88.28 ± 0.03 96/1314 2.00 ± 0.01 3.34 ± 4.30 0.29± 0.02 0.08 ± 0.01 86.93 ± 0.03 97/3200 1.40 ± 0.04 0.17 ± 0.06 0.36± 0.02 0.07 ± 0.06 88.22 ± 0.02 98/0040 1.05 ± 0.04 0.53 ± 0.01 0.42± 0.06 0.05 ± 0.03 87.99 ± 0.04 tms 30572 1.75 ± 0.02 0.77 ± 0.06 0.33± 0.02 0.03 ± 0.02 87.32 ± 0.56 99/2123 2.80 ± 0.10 0.42 ± 0.02 0.53± 0.02 0.08 ± 0.01 85.91 ± 0.01 92/0067 1.08 ± 0.02 0.77 ± 0.05 0.60± 0.02 0.04 ± 0.01 87.16 ± 0.01 97/0039 2.10 ± 0.01 0.52 ± 0.03 0.50± 0.03 0.06 ± 0.04 86.08 ± 0.03 95/0379 1.23 ± 0.03 0.47 ± 0.02 0.43± 0.02 0.04 ± 0.03 87.38 ± 0.03 92b/0061 1.23 ± 0.03 0.08 ± 0.01 0.48± 0.06 0.08 ± 0.03 76.89 ± 0.04 98/0581 1.26 ± 0.02 0.53 ± 0.03 0.02± 0.01 0.04 ± 0.01 76.89 ± 0.03 96/1569 2.10 ± 0.01 0.52 ± 0.01 0.29± 0.01 0.03 ± 0.02 84.81 ± 0.01 means and standard deviation of triplicate determinations of moisture and dry matter content. tion showed varied significant differences with the mean values of ash and crude fibre being less than the maximum codex standard for edible cassava flour (3.0 and 2.0% respectively) (fao, 1995). the protein content ran ged from 0.35% (m98/0028) to 2.88% in some varieties. the low protein of fufu flours is not a serious issue as fufu is usually consumed accompanied with different protein sources both of animal and vegetable origin. 335 afr. j. food sci. res. table 4. functional properties of fufu flours processed from 43 cmd resistant varieties in umudike cassava variety wac (g/ml) bd (g/ml) 96/1569 1.20 ± 0.03 0.72 ± 0.01 tms 30572 1.10 ± 0.03 0.67 ± 0.02 m98/0068 2.20 ± 0.10 0.72 ± 0.01 82/0058 1.10 ± 0.10 0.72 ± 0.04 98/0002 0.70 ± 0.20 0.63 ± 0.02 96/1632 0.70 ± 0.10 0.66 ± 0.01 97/0211 0.94 ± 0.15 0.66 ± 0.02 82/0058 1.00 ± 0.10 0.67 ± 0.04 4(2)1452 0.90 ± 0.02 0.71 ± 0.05 97/0162 1.10 ± 0.10 0.70 ± 0.02 99/3037 0.90 ± 0.30 0.67 ± 0.01 92b/00061 1.40 ± 0.30 0.72 ± 0.02 98/2226 1.00 ± 0.20 0.61 ± 0.01 92b/00068 2.20 ± 0.02 0.72 ± 0.02 91/0166 1.00 ± 0.10 0.67 ± 0.02 96/1642 1.07 ± 0.15 0.65 ± 0.01 98/0581 1.30 ± 0.10 0.67 ± 0.01 98/0505 1.20 ± 0.10 0.77 ± 0.01 97/2205 1.50 ± 0.02 0.63 ± 0.03 97/0039 1.30 ± 0.10 0.66 ± 0.04 96/0603 1.30 ± 0.20 0.66 ± 0.01 97/3200 1.20 ± 0.10 0.67 ± 0.01 96/1314 1.10 ± 0.10 0.63 ± 0.02 tme 419 1.20 ± 0.10 0.72 ± 0.01 99/3037 0.90 ± 0.30 0.67 ± 0.01 98/0510 0.90 ± 0.40 0.77 ± 0.03 92/0067 1.13 ± 0.06 0.63 ± 0.02 98/1565 1.07 ± 0.25 0.68 ± 0.02 97/4763 1.30 ± 0.01 0.66 ± 0.01 92b/00061 1.40 ± 0.30 0.72 ± 0.02 92/0057 0.90 ± 0.20 0.72 ± 0.01 95/0379 1.20 ± 0.10 0.77 ± 0.02 97/4769 1.10 ± 0.10 0.67 ± 0.02 95/0289 1.30 ± 0.03 0.77 ± 0.02 m98/0028 1.30 ± 0.20 0.62 ± 0.02 94/0561 1.30 ± 0.10 0.67 ± 0.02 92/0325 1.10 ± 0.10 0.67 ± 0.03 98/2101 1.20 ± 0.01 0.67 ± 0.01 98/0040 1.40 ± 0.10 0.67 ± 0.01 94/0026 1.30 ± 0.02 0.67 ± 0.02 92/0326 1.30 ± 0.20 0.72 ± 0.02 99/6012 1.50 ± 0.10 0.67 ± 0.02 means and standard deviation of triplicate analysis. noting the minimum fat requirement of 6% in complementary formulation (obatolu, 2002), none of the varieties in their flour form could meet this requirement. however, they met the codex standard of fao (1995) for cassava products. generally variations in proximate com position could be attributed to processing and varietal differences. from the proximate values it is easy to single out variety 96/1632 as having the best nutritional value. this confirms the carbohydrate as the main nutritional component of cassava roots with about 80% as starch (purseglove, 1991). african countries are faced not only with problems of food security but also with nutritional insecurity which are contributing towards dietary micronutrient deficiencies. fortunately cassava has been recognized as a suitable crop for micronutrient intervention in africa (oyewole and asagbara, 2003). functional properties the functional properties of the 43 cmd resistant varieties are reported in table 4. water absorption capacities (wac) ranged from 0.70 to 2.20 g/ml. the cmd varieties 92b/00068 and m98/0068 had the highest wac values. water absorption capacity is a useful indication of whether protein can be incorporated with aqueous food formulations, especially those involving dough handling. dough handling is an important processing operation in processed cheese, sausages and confectioneries. the interaction of proteins with water is important to properties such as hydration, swelling, solubility and gelation. it is a function of ionic strength, ph, temperature, size and shape of the protein molecules. gelatinization of carbohydrates and swelling of crude fiber may also occur during heating, leading to increased water absorption. other processing factors that increase water absorption of flours include fermentation and germination. during fermentation, proteolytic activity takes place which causes increase in the number of polar groups. this development would increase hydrophilicity of the seed or flour proteins. bulk density (bd) ranged from 0.61 in the variety 98 / 2226 to 0.70 g/ml in varieties 98/0505, 98/0510, 95/0379 and 95/0289. bulk density is the ratio of the mass per unit volume of a substance. it is an indication of the porosity of a product which influences package design. the bulk densities of the fufu flours will help us in determining suitable packaging requirements of the flours as it relates to the load the sample could carry if allowed to rest directly on one another. bulk density also relates to mouth feel and flavor of the food to which the flour is incorporated. bulk density is affected by moisture and reflects particle size distribution of the flour. unlike the wac, fermentation and germination are possible processing factors that cause decrease in bulk density. these factors which were employed as process methods in this work might have contributed greatly to the low bd values obtained. pasting properties table 5 shows the final viscosity, set back, peak time and etudaiye et. al. 336 table 5. pasting properties of fufu flours processed from cmd resistant varieties in umudike. cassava variety final-viscosity (rvu) set-back(rvu) peak-time (min) pasting-temp ( o c) 96/1569 76.00 28.17 4.07 79.10 tms 30572 104.75 29.75 4.07 79.15 m98/0068 102.83 34.75 4.07 77.55 82/0068 103.25 28.67 4.13 80.05 98/0002 133.25 37.00 4.20 79.15 96/1632 143.75 40.42 4.13 79.25 97/0211 154.25 41.5f 4.33 79.20 82/0058 154.83 37.83 4.27 78.40 4(2)1452 17125 48.25 4.33 79.15 97/0162 169.58 41.08 4.27 79.20 99/3037 183.92 54.33 4.27 79.20 92b/00061 187.08 55.92 4.13 79.15 98/2226 189.67 59.17 4.07 77.55 92b/00068 190.50 47.58 4.47 79.25 91/0166 191.50 43.83 4.40 79.15 96/1642 193.33 42.67 4.47 78.55 98/0581 193.33 47.75 4.47 80.85 98/0505 202.58 47.67 4.47 79.20 97/2205 202.67 54.25 4.40 78.50 97/0039 202.75 57.00 4.33 78.30 96/0603 204.58 50.50 4.47 79.15 97/3200 208.08 58.08 4.33 80.05 96/1314 210.25 58.33 4.27 78.45 tme 419 212.42 53.00 4.40 79.15 99/3037 220.25 51.33 4.47 79.20 98/0510 212.42 53.00 4.40 79.15 92/0067 221.00 56.67 4.67 80.10 98/1565 223.58 61.08 4.33 77.60 97/4763 226.67 55.92 4.33 77.75 92b/00061 228.00 61.42 4.40 79.95 92/0057 232.33 58.50 4.67 81.60 95/0379 233.83 59.00 4.87 80.05 97/4769 235.50 64.17 4.47 80.05 95/0289 239.42 61.92 4.67 78.45 m98/0028 245.17 61.00 4.73 79.9b 94/0561 245.50 53.00 4.53 78.35 97/0325 246.42 63.58 4.73 80.85 98/2101 249.50 61.25 4.80 79.15 98/0040 283.92 70.42 5.07 79.25 94/0026 286.58 64.42 4.93 78.45 92/0326 293.58 64.25 5.00 80.90 99/6012 295.00 62.08 5.33 80.05 means of triplicate analysis. pasting temperatures of the flours of 43 cmd varieties. it is known that fufu flour is cooked into paste before consumption; hence, the pasting properties of fufu flour are an important quality index in predicting the behavior of fufu paste during and after cooking. final viscosity is the most commonly used parameter to determine a particular starch-based sample quality. it gives an idea of the ability of a material to gel after cooking. final viscosity of the fufu flours processed from 43 cmd resistant varieties ranged from 76.00 in 96/1569 to 295.00 rvu in the 99 / 6012 variety. fufu flours with high viscosities showed that the associative forces between the starch molecules are 337 afr. j. food sci. res. relatively weak. the molecules are able to penetrate their starch granules much easier, and the granular swell enormously leading to weakening of associated forces which in turn makes them susceptible to breakdown. breakdown is responsible for long cohesive nature of the cassava paste. set back viscosities showed variations in the fufu pastes ranging from 28.17 in the paste made from 96/1569 variety to 70.42 rvu in the 98/0040 variety. setback value is the difference between final viscosity and hot paste viscosity or trough. it is a measure of the stability of the paste after cooking. it is the cooling phase of the mixture during pasting in which a re-association between the starch molecules occurs to a greater or lesser degree. it therefore affects retrogression or re-ordering of the starch molecules. set back pasting property has been reported to correlate with texture of fufu flours. it is also associated with synergism and weeping (sanni et al., 2006). low set back of fufu paste indicates high stability. hence, fufu paste obtained from cmd resistant varieties 96/1569 with set back value of 28.17 rvu will be most stable after cooking. peak time is the time at which the viscosity peaks. it measures the time it takes for the fufu pastes to gel during cooking. peak time of the fufu paste obtained from cmdresistant varieties ranged from 4.00 to 5.33 min, which was obtained at a temperature range of 77.55 to 81.60 o c. conclusion the proximate composition of fufu flours processed from cmd resistant varieties in umudike showed low moisture, high carbohydrate and high dry matter contents. these are indications of stable shelf life, cheap and available source of calories to the consumers most especially in the rural areas. it showed ease of reconstitution during preparation into fufu dough. water absorption capacity of the fufu flours enables us to know the extent to which water is added during dough preparation and to improve handling characteristics and maintain freshness in the dough. bulk densities of the fufu flours from the cmd resistant cultivars will guide the processors to determine the packaging requirement of the flours as it relates to the load the sample could carry if allowed to rest directly on one another. pasting properties will help the consumers to know the cassava varieties with ease of reconstitution and consistency of dough. therefore, this work will help the farmers, consumers and industrialists to have idea on the cmd resistant varieties with desirable qualities for fufu production in umudike location. therefore, processing of cassava in various food forms like fufu flour has the potential to help nigeria improve its food security, diversify its manufacturing base, generate more income, raise employment and achieve trade balance. references akingbala jo, oguntimehin b, abas ab (1991). effects of processing methods on the quality and acceptability of fufu from low cyanide cassava. j. sci food agric. 5a (1): 151-154. aoac. (1990). official methods of analysis 15 th ed. association of official analytical chemists , washington d .c us a . fao (1995). codex standards for edible cassava flour. codex 176 – 1989 (rev. 1 – 1995), codex legal alimentarius 133 36, canadian legal info. institute. vol. 7. iita (1990). cassava in tropical agric: a reference manual. iita, ibadan, nigeria, pp. 87-120 (1990). iita (2005). cassava illustration guide book, growing cassava commercially in nigeria. international institute of tropical agriculture, ibadan, nigeria. pp. 21-22 ngoddy po (1989). processing and storage of cassava. proc. of a course held at university of nigeria nsukka, 10–23 april. 1988. dotam publications ltd. pp.51-57. nwabueze tu, odunsi fo(2007).optimization of process conditions for cassava (manihot esculenta) lafun production. afr. j. biotechnol. 6 (5) 603-611. nwabueze tu, anoruoh ga (2008). evaluation of flour and extruded noodles from eight cassava mosaic disease (cmd) resistant varieties. http://fabt.edmgr.com/ obatolu va (2002). nutrient and sensory qualities of extruded malted or unmalted millet / soybean mixture. food chem. 76:129-133 okaka jc, potter nn (1979). tropical plant perishables: handling, storage and processing. silicon valley publishers, enugu. okezie bo, bello ab (1988). physico – chemical and functional properties of winged bean flour and isolate, compared with soy isolate. j. food sci. 53: 450-454. okpokiri ao, ijeoma bc, alozie so, ejiofor m.an(1984). production of improved cassava fufu. niger. food j. 2:145-148. oyewole ob, asagbara y (2003). improving traditional cassava processing for nutritional enhancement. food nutr. agric 32: 17-21. purseglove jw (1991). tropical crops: dicotyledons: longman scientific and technical. co-published in the united states with john wiley and sons. newyork. sanni lo, maziyadixon b, akanya ci, alaya y, egwuonwu cv, okechukwu ru, ezedinma c, akoroda m, lemchi j, ogbe f, okoro e, tarawali g, mkumbira j, patino m, semakula g, dixon a (2005). standard for cassava products and guidelines for export. iita, ibadan, nigeria, pp. 11-39. sanni lo, adebowale aa, filani ta, oyewole ob, westby a (2006). quality of flash and rotary dried fufu flour. j. food agric. environ. 4 (3& 4), july – october, 2006. sas (1999). statistical analytical system. copyright version. sas institute inc., cary nc, usa. sefa-dedeh s, saalia fk (1997). extrusion of maize – cowpea blends in a modified oil expeller. j. sci. food agric 73: 160 -168. wheatley cc, best r (1991). how can traditional forms of nutrition be maintained in urban centres: the case of cassava. entwicklung landlicher raum. 25(1):13-16. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (8), pp. 352-355, august, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the mineral concentration of some nigerian packed fruit juices, concentrate and local beverages dosumu o. o., oluwaniyi o. o.*, awolola g. v. and okunola m. o. department of chemistry, university of ilorin, p.m.b. 1515, ilorin, nigeria. accepted 17 march, 2018 time stability and mineral content of some nigerian packet fruit juices, concentrate and local beverages (sobo drink, kunnu drink, pito and burukutu ) were determined. the ph of the beverages was fairly stable throughout the 15 days the experiment lasted. the acid contents were within suitable range for human consumption and ranged from 0.041 m – 0.205 m. the concentrate has the highest titratable acidity ranging from 0.098 m on day 1 to 0.205 m on day 15. elemental analysis of the minerals showed that the beverages were rich in macronutrients. potassium was found to be the most abundant with content ranging from 4.35 – 5.85 mg/ml while manganese and copper were not detectable. sodium content was between 1.300 – 1.335 mg/ml; calcium 0.560 – 0.925 mg/ml; magnesium 0.005 – 0.06 mg/ml; iron 0.005 – 0.025 mg/ml and zinc 0.005 – 0.010 mg/ml. the results showed that the drinks contain safe levels of mineral elements for human consumption and are sufficiently stable and safe. key words: stability, fruit juices, beverages, concentrate, mineral content. introduction if the importance of a nutrient is judged by how long we can do without it, water ranks as the most important. a person can survive only eight to ten days without water, whereas it takes weeks or even months to die from a lack of food. while water has no caloric value and therefore is not an energy source, without it in our diets we could not digest or absorb the foods we eat or eliminate the body’s waste. water is replenished by drinking liquids like fruit juices and beverages, preferably those without caffeine or alcohol, both of which increase the output of urine and thus dehydrate the body (worthington-roberts, 2007). various types of drinks/beverages are often consumed as sources of fluids. fruit juices are directly extracted from fruits and consumed without further processing. some of these are packed in bottles or laminated papers and sold. fruit drink concentrates are a concentrated form of the juice extracted from natural fruits. since it is expensive to package and store single strength juice, it is often desirable to remove a part or all of the water from the juice (kim et al., *corresponding author. e-mail: laraoluwaniyi@yahoo.com. 1988). such concentrates are diluted with water prior to consumption. concentration reduces the storage volu-mes, thereby reducing the transport costs, and facilitates preservation which is achieved by improved shelf life with increased relative solid concentration. however during the process of concentration, a large part of the characte-ristics determining the quality of the fresh products under-goes remarkable modification which could reduce the nutritional value of the drink (hur and choi, 1993; lee and sohn, 2003; choi et al., 1995). fruits have been a part of human diet and food supplement over the years. they are considered as healthy food supplements because they contain high quantity of water, carbohydrates, proteins, vitamins a, b1, b2, c, d and e; and minerals such as ca, mg, k, zn and fe (wenkam, 1990; okwu and emenike, 2006). besides their dietary importance, they are also useful as nutrient supplements and recommended internationally as supe-rior to processed foods (wenkam, 1990). fruit consump-tion has been reported to be beneficial to health and to contribute to the prevention of degenerative processes, particularly lowering the incidence and mortality rate of cancer and cardioand cerebrovascular diseases (rapisararda et al., 1999). dosumu et al. 353 table 1. ph variation of beverages over 15 days. beverage ph values / days samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 foj 3.2 3.1 3.3 3.2 3.2 3.1 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.1 3.1 coj 3.9 3.7 3.6 3.7 3.7 3.6 3.7 3.6 3.5 3.4 3.4 3.4 3.4 3.3 3.3 boc 2.6 2.6 2.7 2.7 2.7 2.6 2.7 2.6 2.4 2.4 2.4 2.4 2.4 2.1 2.1 sd 2.8 2.8 2.7 2.7 2.7 2.7 2.6 2.6 2.6 2.6 2.5 2.6 2.6 2.6 2.3 ku 3.3 3.0 2.9 2.8 2.7 2.7 2.7 2.7 2.7 2.5 2.5 2.5 2.4 2.4 2.3 pt 3.7 3.7 3.6 3.6 3.5 3.4 3.0 3.0 2.9 3.0 2.9 3.0 2.9 2.8 2.8 bk 3.4 3.3 3.3 3.5 3.7 3.4 3.1 3.0 2.9 3.1 3.0 3.0 2.9 3.0 3.0 apart from fruit juices and concentrates, other plant materials are often used in the preparation of alcoholic and non-alcoholic beverages. the simplicity of production, availability of raw plant materials and abject poverty in many rural communities, as well as the new economyrevamping policies of the government have resulted in increased production and consumption of many local beverages at cottage levels in nigeria. sobo drink and kunnu drink are ready local alternatives to imported refreshing drinks. they are also reported to have various medicinal values (onoja, 1996). sobo drink is an aqueous extract of the dried reddish-brown petals (calyxes) of hibiscus sabdariffa which is usually sweetened with sugar and sometimes flavoured with spices like ginger, hot pepper etc along with natural flavours like pineapple, orange or lime juice (odebunmi and dosumu, 2007). other artificial flavourings like strawberry, vanilla, banana flavours etc, may also be added depending on individual taste (bola and aboaba, 2004). kunnu is a drink made from ground millet or/and guinea corn and it is basically a refreshing, non-alcoholic drink. pito and burukutu are locally brewed alcoholic drinks from ground and fermented millet or/and guinea corn. pito is the liquid part that floats while burukutu is the part that settles in the bottom with some sediment in it. in spite of the high consumption rate of these beverages, their stability and nutritional quality are not well documented. this work therefore aims to examine and provide information on the keeping qualities as well as the mineral composition of some of these drinks. experimental two popular brands of orange juices packed in laminated paper were used in this study and labelled foj and coj. a fruit drink concentrate was also used (boc) while the local beverages used are sobo drink (sd), kunnu (ku), pito (pt) and burukutu (bk). foj and coj represent the ready-made fruit juices and were purchased from a store within ilorin metropolis, boc is a concentrate that is diluted before consumption and was also purchased from a store, sd and ku were locally-prepared non-alcoholic beverages while pt and bk are alcoholic local beverages and they were obtained from local brewers. all samples were kept in sample bottles with tightfitting lids and kept at room temperature. stability studies the stability of the samples was monitored by measuring the ph at 25°c for 15 days at 24 h intervals. the acidity was also determined by titrating 10 ml of samples with 0.1 m naoh solution using 1% phenolphthalein as indicator. the titration was carried out on a daily basis for a period of 15 days. mineral content determination each drink sample was digested by the wet ashing method prior to mineral content determination as described by abulude et al. (2007). 1 ml of sample was measured into a beaker and 10 ml of 1% hcl was added and the mixture heated on a hot plate until the content was reduced to about 1 ml. the solution was then made up to 50 ml with 1% hcl and stored in plastic containers until analyzed for k, na, ca, mg, mn, fe, cu and zn using atomic absorption spectrophotometer. results and discussion the ph values obtained for all the drinks ranged between 2.1 and 3.7 (table 1) and there was an observed gradual reduction in the ph values as the days progressed (figure 1) except for bk which increased slightly until day 5 after which there was a steady decrease in the ph. this could imply that the samples were becoming more acidic and therefore less susceptible to bacterial action but more susceptible to the action of yeasts and moulds (adeyeye and agesin, 1999). the samples were also observed to develop characteristic ‘fermented’ smell as the days progressed. this may be as a result of the fermentation of the drinks to alcohol and then to vinegar due to the action of yeasts. foj had relatively constant ph values (3.1 – 3.3) compared with other samples and boc had the lowest ph values (2.1 – 2.7). the results of the titratable acidity of the beverages presented in table 2 shows that the different beverages behaved differently as the days progressed. generally, the highest titratable acid concentrations were obtained in the packed juices (foj and coj) and burukutu. the acid content of foj was observed to gradually increase as the days progressed until after 5 days when the concentration started reducing gradually to a minimum concen 354 afr. j. food sci. res. table 2. titratable acidity (m) of the beverages. days titratable acidity (m) foj coj boc sd ku pt bk 1 0.111 0.098 0.068 0.041 0.071 0.060 0.100 2 0.111 0.101 0.066 0.042 0.071 0.062 0.091 3 0.109 0.103 0.065 0.043 0.080 0.065 0.090 4 0.109 0.111 0.067 0.044 0.085 0.075 0.087 5 0.128 0.128 0.068 0.044 0.090 0.077 0.086 6 0.125 0.132 0.066 0.043 0.102 0.090 0.097 7 0.121 0.132 0.066 0.047 0.104 0.092 0.101 8 0.120 0.134 0.064 0.048 0.108 0.095 0.105 9 0.119 0.138 0.063 0.048 0.105 0.115 0.111 10 0.114 0.137 0.064 0.049 0.111 0.117 0.118 11 0.113 0.137 0.064 0.049 0.112 0.126 0.132 12 0.111 0.144 0.064 0.050 0.114 0.136 0.143 13 0.106 0.153 0.065 0.053 0.117 0.147 0.148 14 0.105 0.204 0.063 0.054 0.120 0.156 0.154 15 0.101 0.205 0.063 0.056 0.123 0.159 0.158 figure 1. ph values of beverages over a period of 15 days. figure 2. variation in titratable acidity of beverages over 15 days. tration of 0.101 m. the other packed juice exhibited different characteristics with the concentration increasing gradually from the 0.098 m on day 1 to 0.205 m on day 15. boc which is a concentrate showed a fairly constant titratable acidity (0.063 – 0.068 m). this is expected since the concentrate contains some preservatives which are expected to inactivate the enzymes in the drink and therefore prevent the deterio-ration or any chemical reaction in the drink (fellers et al., 1986). sobo drink has the least titratable acid concentrations (0.041 – 0.056 m) and these increased gradually as the number of days increased. ku and pt also showed increasing acidity as the experimental days increase while bk recorded a slight decrease in the acidity during the first 5 days and then a continual increase during the rest of the days which was attributed to the breakdown of the natural antioxidants and antimicrobial agents. the mineral compositions of the beverages are shown in table 3. potassium is the most abundant element in all the beverage samples followed by sodium and then calcium. copper and manganese were not detectable in the beverages while magnesium, iron and zinc are present in trace amounts. this result is in agreement with the result reported by onibon et al. (2007) who worked on some nigerian fruits and also reported that potassium was the most abundant mineral in the fruits while magnesium, iron and zinc were present in minute quantities the potassium and sodium are macroelements required dosumu et al. 355 table 3. mineral composition (mg ml -1 ) of the beverage samples. beverage mineral composition (mg ml -1 ) samples k na ca mg fe zn mn cu foj 5.100 1.305 0.730 0.010 0.010 0.010 nd nd coj 5.850 1.320 0.925 0.005 0.025 0.005 nd nd boc 5.215 1.300 0.805 0.005 0.010 0.010 nd nd sd 4.750 1.305 0.875 0.010 0.015 0.005 nd nd ku 4.850 1.320 0.595 0.010 0.010 0.005 nd nd pt 4.350 1.300 0.640 0.005 0.010 0.010 nd nd bk 5.550 1.335 0.560 0.060 0.005 0.005 nd nd nd = not detectable. for the maintenance of cellular water balance, acid-base balance and nerve transmission and are required in large amounts in the body (wardlaw, 1999; worthington-roberts, 2007). deficiencies of these macronutrients lead to muscle cramps, mental confusion, loss of appetite and irregular cardiac rhythm (worthington-roberts, 2007). however, there is a direct relationship between sodium intake and hypertension in humans (dahl, 1972) therefore it is desirable that the concentration of sodium in foods is not as high as that of potassium. these results therefore indicate a desirability of the consumption of these beverages because they contain a high concentration of sodium and potassium but the concentration of sodium is lower than that of potassium. calcium is essential in bone and teeth formation (wardlaw, 1999) and a deficiency of it causes rickets and osteoporosis (hunt et al., 1980) . consumption of these beverages is therefore desirable as they serve as good sources of this macronutrient. conclusion this work has shown that except for coj (a packed fruit juice) and bk (a local alcoholic beverage), the beverages have fairly stable titratable acidity throughout the days of the experiment and the acid contents are suitable for consumption even without refrigeration. the shortcoming is the ‘alcoholic smell’ emanating from the drinks due to fermentation as the days of the experiment progressed. this was because the drinks were kept at room temperature. the mineral ion contents of these drinks are not too high as to call for caution in the consumption of the drinks but the concentrations make these drinks good natural sources of these metal ions. references adeyeye ei, agesin (1999). nutritional composition of chrysophyllum albidum, malus pumila and psidium guajava fruits. bangladesh j. sci. ind. res. 34: 452-458. bola o, aboaba oo (2004). microbiological and physico-chemical evaluation of some non – alcoholic beverages. pak. j. nutr. 3: 188 192. choi hd, kim kt, hong hd, lee by, kim ss (1995). rheological properties of pear juice concentrates. korean j. food sci. technol. 27: 845851. dahl (1972). salt and hypertension. am. j. clin. nutr. pp.25-238. hunt sm, groff jl, holbrook jm (1980). nutrition: principles and clinical practice. john wiley and sons. new york, pp. 451452. hur ss, choi yh (1993). studies on the efficient concentration process of apple juice with reverse osmosis process. korean j. food sci. technol.25: 321-326. kim kh, park hj, kim dm (1988). per-concentration of apple juice with different reverse osmosis membranes. korean j. food sci. technol. 20: 419425. lee jh, sohn ks (2003). effect of concentration methods on the quality of single and blend juice concentrates. j. food sci. nutr. 8: 225-229. odebunmi eo, dosumu oo (2007). fermentation studies and nutritional analysis of drinks made from water extract of hibiscus sabdariffa calyx (sobo), juices of citrus sinensis (orange) and ananas comosus (pineapple). j. food technol. 5(3): 198204. okwu de, emenike in (2006). evaluation of the phytonutrients and vitamin content of citrus fruits. int. j. mol. med. adv. sci. 2: 16. onibon vo, abulude fo, lawal lo (2007). nutritional and antinutritional composition of some nigerian fruits. j. food technol. 5(2): 120 -122. onoja r (1996). chemical constituents of yakwua hibiscus sabdariffa. university press, jos. pp. 4 -5. rapisararda p, anthonio t, rossella lc, prancesco b, anna op, antonella s (1999). antioxidant effectiveness as influenced by phenolic content of fresh orange juice. j. agric. food chem. 47: 47184723. wardlaw gm (1999). perspectives in nutrition. (4 th edn.). mc graw-hill companies, usa., p. 532. wenkam a (1990). utilization and processing of fruits. macmillan press, london, 5: 388 -506. worthington-roberts b (2007). human nutrition. microsoft student 2008 [dvd]. microsoft corporation. abulude fo, elemide ao, ogunkoya mo, adesanya wo (2007). design and performance evaluation of a juice extractor constructed in nigeria. res. j. appl. sci. 2: 1-34. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (1), pp. 250-254, january, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the gelatin from the skin of the flying gurnard and how it interacts with cassava starch l. d. abbey 1,2 *, p-n. t. johnson 1 and n. k. howell 2 1 food research institute (csir),p. o. box m 20, accra, ghana. 2 school of biomedical and molecular sciences,university of surrey,guildford, surrey gu2 7xh, uk. accepted 27 july, 2017 gelatin extracted from the skin of the flying gurnard (dactylopterus volitans l,) was characterised and its interaction with cassava starch mixtures of different ratio concentrations was investigated by rheology and differential scanning calorimetry (dsc) . protein and moisture were the main proximate composition, whilst glycine was the main amino acid. the gelatin had high bloom (275 g) strength with high imino acids, proline and hydroxyproline, content of 217/1000 residues. gelation temperatures increased as more starch is used in the mixtures, ranging from 19.0°c for gelatin to starch ratio 4:1 to 70.1°c for mixture 1:4. inclusion of cassava starch also increased the elastic modulus of the gelatin phase. therefore, gelatin from flying gurnard skin has a potential to serve as an alternative source of non-bovine or porcine gelatin for food applications. key words: gelatin, amino acids, bloom strength, storage modulus, starch. introduction gelatin is widely used in the food industry at an estimated quantity of 200,000 metric tonnes (choi and regenstein, 2000). it is derived by hydrolytic degradation of collagen. the functional properties of gelatin are related to their chemical characteristics; molecular weight, number of each kind of amino acid residues, number of polypeptide chains and on the position of the breaks. the gel strength, viscosity, setting behaviour and melting point of gelatin depends on their molecular weight distribution and the amino acid composition. the imino acids, proline and hydroxyproline are important in the denaturation of gelatin subunits during gelling (johnston-banks, 1990). gelatin with high levels of imino acids tends to have higher gel strength and melting point. the molecular weight distribution is also important in determining the gelling behaviour of gelatin. in general the quality of the gelatin depends on the composition of the raw material and factors including the species, breed, age, manner of feeding the animal, storage condition of raw materials and to some extent the manufacturing processes *corresponding author. e-mail: abbeyld@yahoo.com. (hinterwaldner, 1977). commercially, gelatin is obtained from mammals like bovine and porcine through acid and alkaline hydrolysis. however, periodic outbreaks of bovine spongiform encephalopathy (bse) coupled with religious prohibitions have created a demand for alternative sources of gelatin (choi and regenstein, 2000). gelatin can also be obtained from fish skins and bones, which constitute about 30% (gòmez-guillèn et al., 2002) of the total weight of side streams obtained after filleting of fish. the side streams constitute about 75% of the total weight of fish during normal fish processing (shahidi, 1994). studies on properties of fish skin gelatins showed that fish gelatins have lower melting and gelling temperatures than those from mammals (choi and regenstein, 2000; fernández-diaz et al., 2001; leuenberger, 1991). in addition, gelatin from low temperature fish species contains a lower amount of proline and hydroxyproline; lower level of hydrogen bonds and lower melting points than the species from a tropical environment (gilsenan and ross-murphy, 2000; arnesen and gildberg, 2002). however these properties may be modified by the use of gel enhancers like salts, glycerol and variations of ph (sarebia et al., 2002). abbey et al. 251 production of gelatin from skins and bones of fish could therefore be a very profitable value addition during general fish processing. in ghana, the flying gurnard (dactylopterus volitans) (linnaeus, 1758) is abundantly harvested during the main fishing season. unfortunately, it has very little market value as fish for home consumption. it is therefore important to find alternative uses for it. one possible good use will be the production of gelatin from the side streams. there is a wide variety of foods that contain gelatin and starch and for that matter the properties of gelatin and starch in aqueous systems have been studied extensively, and remain the focus of substantial current research (ross-murphy, 1992, 1994; keetels and van vliet, 1994). however, it has been known that phase separation occurs in aqueous mixtures of gelatin and starch for various types of starch at concentrations above -1 wt% (ostwald and hertel, 1929) and this could possibly affect the functional properties of gelatin in starchy foods. cassava starch has high amylose content, usually in the range of 13.6 to 19.1% (defloor et al., 1998) which helps it to retrograde easily at room temperature (thomas and atwel, 1999) and therefore helps in stabilization/ solidification of other food components added to it. it is expected this potential of cassava starch will tremendously assist in the transforming of the gelatin from the flying gurnard into a semi-solid form which helps in food preparation and packaging unfortunately, there is paucity of information of the interactions of the starch and gelatin obtained from this fish skin. this study therefore concerns the characterisation of the gelatin from the skin of the flying gurnard and how it interacts with cassava starch. materials and methods materials freshly landed flying gurnard fish (d. volitans. l) was purchased from fishmongers at the canoe landing beach of tema in ghana. the fish were held in ice at –30°c. fish skin was removed from the thawed flying gurnard. methods gelatin preparation gelatin was prepared from the skins of the flying gurnard according to the method of gudmundsson and hafsteinsson (1997). proximate composition and amino acid analysis the moisture, crude fat, ash, colour and ph were determined according to the methods outlined in the gelatin manufacturers of europe monograph (gme) version 1, july 2000. protein digestion was by methods of to ensure complete hydrolysis of collagen (eastoe and eastoe, 1952). the gelatin was hydrolysed with 6 n hcl (20 ml) at 110°c for 24 h to yield amino acids. the hydrolysed samples and amino acid standards (20 l) were derivatized with phenylisothiocyanate (pitc) according to the waters pico-tag method (1986). the amino acids were then were measured in triplicate by hplc (bildlingmeyer et al., 1987). bloom strength a gelatin concentration of 6.67% (w/v) was prepared and brought up to 22°c for 3 h. then after it was brought to 60°c (but not exceeding) on a magnetic heater stirrer for approximately 15 min, for the gelatin to dissolve completely and immediately introduced into standard bloom jars and covered after 2 min. the bloom jars were left to condition for 16 h in a water bath at 10°c. the bloom jars were centrally placed under a 0.5 mm radius cylinder probe attached to a texture analyser (ta-xt2i) stable micro systems. the bloom strength of the gelatin was determined according to the method described by stable micro systems, in accordance with gme 2000. interaction studies with cassava starch samples of the flying gurnard gelatin and cassava starch were mixed in the ratio 0:10; 1:4; 1:1; 4:1 and 10:0 (w/v in distilled water) and subjected to rheological and differential scanning calorimetry (dsc) studies to evaluate the components interactions for potential development of products. these ratios were selected since phase separation occurs in aqueous mixtures of gelatin and starch at starch concentrations above -1 wt% (ostwald and hertel, 1929). rheological measurements the blended samples of gelatin and cassava starch at the various proportions were subjected to small-deformation oscillatory measurements with a rheumatics controlled stress rheometer and a 40 mm parallel plate geometry with a gap of 0.3 mm and frequency of 1 radian per second, to obtain sufficient data without compromising the measurements of entanglements. a dynamic temperature sweep was performed between 20 and 2c at a rate of 1c /min and then heated to 20c. the applied stress was 1 pa to keep the oscillatory strain at about 1%, sufficiently low to ensure the measurements were within the linear viscoelastic region (hamann, 1991). the sample was surrounded by silicone oil to prevent evaporation of solvent during the temperature sweep. the viscoelastic properties of the gels measured included the storage modulus (g’) and loss modulus (g”) as a function of time within the linear viscoelastic range of the gels (mitchell, 1980). the cross-over point g’/g” at which gel formation occurred and the gelation temperature were noted. differential scanning calorimetry (dsc) measurements eight hundred milligrams of gelatin samples as prepared for the rheological studies were placed in a pre-weighed dsc cell (setaram micro dsc vii). an equal weight of distilled water was also introduced into the reference cell to obtain a flat base line. a temperature scan of 2 to 90°c and heating rate of 0.5 °c/min were maintained through out the study. heat absorbed or released by the transformation of the samples resulted in an endothermic or 252 afr. j. food sci. res. table 1. proximate and amino-acid compositions of the flying gurnard gelatine. composition constituents % amount proximate moisture 9.7±1.4 crude fat 0.2±0.05 ash 0.9±0.2 protein 89.6±1.9 amino-acid aspartic 3.57±0.43 glutamic 6.30±0.72 hydroxyproline 7.04±0.49 serine 2.93±0.2 glycine 28.43±2.21 histidine 0.76±0.02 arginne 4.28±0.37 threonine 2.58±0.28 alanine 22.57±2.13 proline 14.70±0.85 tyrosine 0.31±0.00 valine 1.98±0.20 methionine 1.45±0.06 cystine 0.25±0.08 isoleucine 0.87±0.09 leucine 1.97±0.15 phenylalanine 1.95±0.20 trptophan 4.09±0.37 lysine 3.64±0.21 values are means of three determinations ± s.d exothermic peak as a function of temperature. the transition temperature (tm) was measured at the tip of the peak. peak areas showing transition temperatures and enthalpies ( ) were calculated automatically by integrating the area under the peak. each sample was scanned in triplicate. results and discussions table 1 show that the proximate composition of the gelatin is mainly made up of protein and water. the low ash content makes its suitable for use in food applications (jones, 1977). table 1 also indicates that the key amino acids in the gelatin of the skin of the flying gurnard are alanine, glycine and proline. in particular, the 28.4% of glycine in gelatin of the skin of the flying gunrand compares favourably with 31.6 and 31.13% found in the horse mackerel and the commercial gelatin, respectively (muyonga et al., 2004). proline and hydroxyproline residues, referred to as the imino acid content of the gels, were expressed as the number of the residues in 1000. this was found to be 217/1000 residues. these are comparable to the residues of commercial gelatin but significantly different m o d u lu s 60 50 lo ss g' (storage modulus) pa 40 an d (p a) g" (loss modulus) pa (p a) 30 g ' g " m od ul us 20 s to ra g e 10 0 0 20 40 60 80 100 temp°c figure 1. gelation profile of flying gurnard skin gelatin with varying proportion of cassava starch (ratio) during a dynamic temperature ramp. from those of the horse mackerel residues. the imino acid content is important in the renaturation of gelatin subunits during gelling as the turns of the helix and the stability of the triple helix structure of the gelatin depend largely on these acids (johnston-banks, 1990). as a result, gelatin with high levels of imino acids tends to have higher gel strength and melting point. the bloom gel strength of the gelatin of the flying gurnard fish skin was estimated as 275 g. in comparison, the bloom strength of gelatin of catfish is 150 g whilst that of commercial pig gelatin is 290 g (muyonga et al., 2004). interaction studies with cassava starch rheological studies figure 1 shows a typical profile obtained for the gelation of the gelatin with the cassava flour as a function of temperature. the profiles for the various mixtures followed similar trends. all the gel mixtures showed marked increase in g’ and g” values respectively after 79ëc but these values dropped before reaching 90ëc during indicating a weakening of the gel structure (tsai et al., 1997). the storage modulus and loss modulus of the mixtures (table 2) show that at 90ëc there was a drop of viscosity and loss of rigidity of gelatin : starch (10:0) but upon cooling to 20ëc high values of g’ (2530 pa) and g” (261 pa) were obtained. similar observations were made for the other mixtures with the exception of the 1:1 gelatin: starch mixture which had a significantly (p<0.05) decrease in value of g’ (22 pa) and g” (5.4 pa) at 90ëc to g’ (16 pa) and g” (1.7 pa) on cooling to 20ëc respectively. these observations are in line with the fact that the viscosity of gelatin decreases with temperature. abbey et al. 253 table 2. storage modulus (g’ pa) and loss modulus (g” pa) of mixtures of the flying gurnard gelatin and cassava starch during a dynamic temperature sweep at 90ëc and 20ëc* composition gelatin: cassava starch 90 ºc 20 ºc g’ g” g’ g” 10:0 0.01±0.00 0.01±0.00 2530±191 261±29 4:1 0.05±0.02 0.17±0.04 74.9±8.25 21.7±13.97 1:1 22.2±0.94 5.4±0.18 16.1±3.71 1.7±0.21 1:4 101.8±13 30.4±8.1 156±14 28.5±8.17 0:10 246±22 84.3±6.50 729±5 99.2±15.00 * values are means of three determinations ± s.d. table 3. transition temperatures and enthalpy change mixtures of the flying gurnard gelatin and cassava starch. composition gelatin: flour transitions onset temp (to) ºc peak temperature (tm) ºc enthalpy/ j/g ( h) 10:0 1 8.98 18.89 2.95 4:1 1 12.70 19.02 0.17 2 43.31 50.66 0.03 3 65.01 69.69 0.97 1:1 1 27.41 29.74 0.005 2 51.35 44.44 0.0007 3 65.35 70.07 0.313 1:4 1 48.17 53.83 0.0005 2 65.61 69.71 0.52 0:10 1 63.82 69.47 0.67 e n d o th er m ic h ea t f lo w ( j/ g ) 2e-14 0 -2e-14 first scan -4e-14 second scan -6e-14 -8e-14 -1e-13 0 20 40 60 80 100 temperature ºc during the cooling period may be attributed to the various constituents present and in particular the swollen granules and fragments which associate or retrograde as a result of molecular interactions (hoover, 2001). the changes in the gelation temperatures as shown in table 2 suggest some interactions between the gelatin and the starch granule and are attributed to charges on the proteins and starch (takeuchi, 1969). with increasing starch concentration, the crosslink density in the gel may have increased, yielding higher values of g’. a number of studies have elucidated this mechanism of interaction and the main effect of the starch component is to raise the modulus of the gelatin phase. this property could be employed in food applications (white et al., 1993). differential scanning calorimetry (dsc) figure 2. dsc thermograms of the gelatin of the flying gurnard skin scanned from 0 to 90ëc cooled and rescanned. this explains why gels are easily formed at lower temperatures (ferry, 1948). the increase in viscosity the thermograms for the flying gurnard gelatin gel are shown in figure 2. the transition temperature and enthalpy change for gelatin from flying gurnard were found to be 18.89ëc and 2.95 j/g, respectively. the enthalpy reflected the amount of energy required to denature the samples and is related to the number of junction zones 254 afr. j. food sci. res. 0.2 10% gelatin 0.15 (j /g ) 0.1 8% gelatin + 2% 0.05 cassava fl o w cassava 5% gelatin + 5% h ea t 0 2% gelatin + 8% -0.05 en do th er m ic -0.2 cassava -0.1 10% cassava -0.15 -0.25 -0.3 0 20 40 60 80 100 temperature ºc figure 3. dsc thermograms for mixtures of gelatin of the flying gurnard gelatin and cassava starch scanned from 0 to 90ëc. involved in the gelation of each sample (arnesen and gildberg, 2002) . figure 3 also shows that all the thermograms of the gelatin and cassava flour mixtures were endothermic. the dsc thermograms show that starch on its own (0: 10) indicated a single transition (tm values 69.5 ëc). however when mixed with gelatin, three thermal transitions (tm values of 19.0 ëc; 50.7 ëc; 69.7 ëc) and (tm values 29.7ëc; 44.4ëc; 70.1ëc) were respectively obtained for gelatin and starch mixtures 4:1 and 1:1. however the 1:4 (gelatin: starch mixture), indicated two transitions (tm values of 53.8 o c; 69.7ëc). these results tend to offer support to the type of interactions observed in the rheological studies in this report. the mixtures which show interaction with the starch may enhance the potential of using a mixture of gelatin and cassava starch in food applications where such properties exhibited by the mixtures are required. conclusions gelation properties of the flying gurnard gelatin especially the high bloom strength and high melting point indicate that fish gelatin could be used to replace mammalian gelatins in some food applications without significantly altering the texture. the changes in gelation temperatures when fish gelatin was mixed with cassava starch due to weak electrostatic interactions confer textural changes and melting point close to that of mammalian gelatins which would be an added advantage in food applications. developing gelatin from the flying gurnard skin would reduce the waste skin which would otherwise be discarded, to produce a value added product that may be of economic benefit to the fish industry in ghana. references arnesen ja, gildberg a (2002) preparation and characterisation of gelatin from the skin of harp seal (phoca groendlandica) bioresource technol 82: 191-194 bildlingmeyer, b.a, cohen, s.a, tarvin, l. & frost, b.a, (1987).new rapid high sensitive analysis of amino acids in food type samples. j. association official analytical chemist. 59: 241-247 choi ss, regenstein jm (2000) physicochemical and sensory characteristics of fish gelatin. j. food sci. 65:194-199. defloor i, dehring i, delcour ja (1998) physiocochemical properties of cassava starch. starch/starke, 50: 58-64 eastoe je, eastoe b (1952), a method for the determination of total nitrogen in proteins. the british gelatin and glue research association res. report, series b 5:1-17. ferry j (1948) protein gels, advances protein chemistry 4: 1-78 . gilsenan pm, ross-murphy sb (2000) rheological characterisation of gelatins from mammalian and marine sources. food hydrocol 14: 191-196. gudmundsson m, hafsteinsson h(1997). gelatin from cod skins as affected by chemical treatments. j. food sci. 62: 37–39. hamann dd (1991). rheology. a tool for understanding thermally induced protein gelation, in interactions of food proteins; ed by pariss n and barford r, american chemical society: washington, dc, pp. 212-227. hinterwaldner r (1977). in science and technology of gelatin, ed by ward ag and courts a, academic press london, 9: 295. hoover r (2001) composition, molecular structure, and physicochemical properties of tuber and root starches: a rev. carbohydrate polymers, 45: 253-267. johnston-banks fa (1990). gelatin. food gels, new york: elservier applied food science series, pp.233-289. keetels cjam, van vliet t (1994). in gums and stabilisers for the food industry 7, eds g.o. phillips,.williams pa, wedlock dj. irl press, oxford, pp. 271-280. leuenberger bh (1991). investigation of viscosity and gelation properties of different mammalian and fish gelatins. food hydrocol 5:(4) 453-461. mitchell jr (1980). the rheology of gels j. texture studies 11: 257-270. muyonga jh, cole cgb, duodo kg (2004), extraction and physicochemical characterisation of nile perch (lates niloticus) skin and bone gelatin. food hydrocol 18: 581-592. ross-murphy sb (1994). in gums and stabilisers for the food industry 7, eds phillips go, williams pa, .wedlock dj. irl press, oxford, pp. 243-255. sarebia ai, gómez-guillén mc, montero p (2002) the effect of added salts on the viscoelastic properties of fish gelatin. food chem.70: 71-76. takeuchi i (1969). interaction between protein and starch, cereal chem. 46: 570 –579. thomas dj, atwell wa (1999). starches. aacc inc. st. paul. minn. tsai ml, li cf, lii cy (1997) effects of granular structure on the pasting behavior of starches. cereal chem.74: 750–757. white jp, davidson vj, otten l (1993). a capillary viscometer for characterization of fluid foods. canadian institute of food science and technology. 26:109-114 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (9), pp. 368-372, september, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of fermentation on the proximate composition of tigernut flour and its potentials in food formulation adejuyitan, j. a.*, otunola, e. t., akande, e. a., bolarinwa, i. f. and oladokun, f. m. department of food science and engineeringladoke akintola university of technology, ogbomoso, oyo state, nigeria. accepted 09 august, 2017 yellow variety of tiger nut used was obtained from sabo market, in ogbomoso, nigeria the nuts were cleaned, sorted, washed, and were soaked in water and left to ferment for 24, 48 and 72 h respectively. the nuts were drained, dried in an oven and ground into flour. the flour samples were passed through a 45 m mesh size sieve. the flour was analyzed for proximate composition and some functional properties. there were changes in some constituents of the flour with fermentation time. there was an increase in protein content (7.73 9.23%) and reduction in fat content, likewise with the ash, and starch content. there was also an increase in the sugar content over the fermentation time (7.31 9.69%). for the functional properties, 0.56 0.62 g/ml were recorded for loose bulk density, within the fermentation time of 24 – 72 h. for packed bulk density 0.83 0.91 g/ml were recorded, 123 – 141 g/100 g for water absorption, 67.6 71.3 g/ml for oil absorption capacity within the fermentation time of 24 72 h. changes in the pasting characteristics of the flour samples were also noticed. key words: tigernut flour, fermentation, proximate composition, functional properties. introduction tigernut ( cyperus esculentus) is an underutilized crop of the family cyperaceae which produces rhizomes from the base and tubers that are somewhat spherical. it is commonly known as “earth almond”, “chufa”, “chew-fa” and “zulu nuts”. it is known in nigeria as “ayaya” in hausa, “ofio” in yoruba and “akiausa” in igbo where three varieties (black, brown and yellow) are cultivated. among these, only two varieties, yellow and brown, are readily available in the market. the yellow variety is preferred over others because of its inherent properties like its large size, attractive colour and fleshier nature. the yellow variety also yields more milk, contains lower fat and higher protein and less anti nutritional factors espe-cially polyphenols (okafor et al., 2003). tigernut can be consumed raw, roasted, dried, baked or made into a refreshing beverage (cantalejo, 1997). in addition, tiger-nut is used for making oil, soap, starch and flour. although many researchers have worked on tiger nut (eteshola and oraedu, 1996), there is little information on the effect of fermentation on the flour properties. fermentation a value addition to the food product.this project therefore aimed at determining the effect of fermentation on the proximate composition of tigernut flour in order *corresponding author. e-mail: jadejuyitan@yahoo.com. to be able to explore its potentials in food formulation. materials and methods sample preparation yellow variety of tiger nut used was obtained from a local market in ogbomoso, oyo state, nigeria. the nuts were cleaned, sorted, washed, and were soaked in water and left to ferment for 24, 48 and 72 h respectively. the nuts were drained, dried in an oven and ground into flour. the flour samples were passed through a 45 m mesh size sieve. proximate analysis kirk and sawyer (1991) methods were used to determine moisture, protein, fat, crude fibre and ash contents while carbohydrate was calculated by difference. water and oil absorption capacities water and oil absorption capacities of the flour samples were determined by beuchat (1977) methods. one gram of the flour was mixed with 10 ml of water or oil in a centrifuge tube and allowed to stand at room temperature (30 ± 2°c) for 1 h. it was then centrifuged at 200 x g for 30 min. the volume of water or oil in the sediment water was measured. water and oil absorption capacities were calculated as ml of water or oil absorbed per gram of flour. adejuyitan et al. 369 table 1. effect of fermentation period on the proximate composition of tiger nut flour. period (hours) properties 0 h 24 h 48 h 72h crude protein 6.67±0.16d 7.73±0.08c 8.40±0.09b 9.23±0.09a fat 14.41±0.04a 13.66±0.02b 11.86±0.04 c 11.24±0.04c dry mater 90.10±0.04a 89.80±0.02ab 89.67±0.04 ab 89.57±0.02 ab starch 21.25±0.02a 19.07±0.04b 17.93±0.03c 15.88±0.04d crude fiber 2.04±0.03a 1.90±0.04ab 1.73±0.02c 1.64±0.02cd sugar 6.58± 0.04d 7.31±0.02c 8.77±0.03b 9.69±0.02a ash 2.72±0.04 a 2.60±0.02a 2.49 ±0.04a 2.37±0.03 a moisture content 9.93±0.04ab 10.22±0.02a 10.31±0.04a 10.42±0.02a means followed by different letters within a row are significantly different (p < 0.05). foam capacity and foam stability the method described by narayana and narasinga rao (1982) was used for the determination of foam capacity (fc) and foam stability (fs). two grams of flour sample was added to 50 ml distilled water at 30 ± 2°c in a 100 ml measuring cylinder. the suspension was mixed and properly shaken to foam and the volume of the foam after 30 s was recorded. the fc was expressed as a percentage increase in volume. the foam volume was recorded 1 h after whipping to determine the fs as a percentage of the initial foam volume. bulk density a 50 g flour sample was put into a 100 ml measuring cylinder and tapped to a constant volume. the bulk density (g cm -3 ) was calculated as weight of flour (g) divided by flour volume (cm 3 ) (okaka and potter, 1979). swelling power this was determined with the method described by leach et al. (1959) with modification for small samples. one gram of the flour sample was mixed with 10 ml distilled water in a centrifuge tube and heated at 80°c for 30 min. this was continually shaken during the heating period. after heating, the suspension was centrifuged at 1000 × g for 15 min. the supernatant was decanted and the weight of the paste taken. the swelling power was calculated as: swelling power = weight of the paste / weight of dry flour. pasting properties three grams of the flour sample was mixed with 25 ml distilled water in the canister of a rapid viscoanalyzer (rva, model 3d; newport scientific, sydney, australia) monitored with rva control software and operated. the following parameters were obtained from the plotted graphs: peak viscosity, pasting temperature, and setback viscosity, breakdown viscosity, final viscosity and time to reach the peak viscosity. statistical analysis all analyses were carried out in duplicates and the data collected were analyzed using plot it software (spe, 1993). data were subjected to analysis of variance (anova) and turkey’s test was used for comparison of means. significance was accepted at p = 0.05. results and discussion chemical composition the results of the proximate composition are shown in table 1. there are changes in some constituents of the flour with fermentation time. there was an increase in protein content and reduction in fat and ash content. there was an increase in the sugar content along the fermentation days. this could be attributed to metabolic activities of the microorganisms during fermentation. for instance the decrease in the starch and increase in sugar contents may be due to breaking down of starch to sugar by some microorganisms containing enzyme amylase. this could further enhance the exploration of its potential in food formulation. the flour samples from fermented tigernuts contained higher amounts of protein than the unfermented flour. as shown in table 1, the protein content increased with fermentation period. at 0 h fermentation the protein content of the flour was 6.67% while for 24, 48, and 72 h fermentation the flour contained protein of 7.73, 8.40 and 9.23% respectively. this could be compared favourably with the value of 9.8% reported for wheat flour (akubor and badifu, 2004), 6.34 – 8.57% reported for jackfruit seed flour (mukprasirt and sajjaanantakul, 2004) but lower than the values 26.3, 22.5 and 11.4% reported for conophor nut flour (odoemelan, 2003), benniseed flour and pearl millet flour (oshodi et al., 1999), respectively. the fat content decreased with fermentation period from 14.41% to 11.21%. this is contrary to the report of oladele and aina (2005) who obtained fat content of 32.13 to 35.43% for flour from different varieties of tigernut. however this is relatively high when compared to pearl millet (7.6%) and quinoa (6.3%) (oshodi et al., 1999) missing in reference pigeon pea flour (1.80%; okpala and mammah, 2001) and wheat flour (3.10%; akubor and badifu, 2004) but low compared to some 370 afr. j. food sci. res. table 2. some functional properties of tigernut flour. properties a b c d loose bulk density (g/ml) 0.56±0.03a 0.62±0.02a 0.59±0.04a 0.59±0.03a packed bulk density (g/ml) 0.83±0.02a 0.91±0.04a 0.83±0.03a 0.91±0.02a water absorption capacityg/100 g 141±0.02c 156±0.03b 164±0.03a 123±0.03d oil absorption capacityg/100 g 67.6±0.03c 69.8±0.03b 71.3±0.02a 61.2±0.04d gelation capacity (%) 21.5±0.03c 23.2±0.02b 24.5±0.03a 19.2±0.01d dispersibility (%) 69.2±0.02b 67.3±0.04c 65.4±0.04d 72.5±0.03a viscousity(ml/s) 0.132±0.03a 0.126±0.04a 0.121±0.02a 0.143±0.04a means followed by different letters within a row are significantly different (p = 0.05) a flour from 24 h fermentation of seed. b flour from 48 h fermentation of seed c flour from 72 h fermentation of seed d control (no fermentation) commonly consumed oil seeds in nigeria; pentaclethra macrophylla (46.0%; achinewhu, 1982), telfairia occidentalis (49.2%; fagbemi and oshodi, 1991). the ash content decreased with fermentation time from 2.72% (0 h) to 2.31% (72 h). the ash content is an indication of mineral elements present in the flour. however oladele and aina (2007) has reported varieties of tigernuts especially the yellow variety to have high amounts of calcium, sodium and copper potassium, magnesium, manganese and iron. according to oladele and aina (2007), the values of calcium found in the flours, 140 and 155 mg/100 g are adequate for bone and teeth development in infants. the presence of other minerals such as iron is highly important because of its requirement for blood formation. the reduction in the ash content may arise as a result of usage of these minerals by inherent microorganisms for metabolic activities which may also be the reason for the reduction in the starch content (21.25 15.88%) which has been broken down to sugar thereby causing an increase in the sugar content (6.58 9.69%). functional properties water absorption capacity describes flour – water association ability under limited water supply .the water absorption capacity was 141,156 and 164 g/100 g for the flour from 24, 48 and 72 h fermentation respectively as shown in table 2. the water absorption for flour from 0 h fermentation was 123 g/100 g. the water absorption capacity increased with increasing fermentation time. however water absorption capacity of 3.4 ml/g has been reported for raw conophor flour (odoemelam, 2003 check reference for spelling) and 1.7 ml/g reported for african yam bean (eke and akobundu, 1993). this result suggests that tigernut flour may find application in the production of some baked products. the flours were found to have oil absorption capacity of 67.6, 69.8, and 71.3 g/100 g for the 24, 48 and 72 h fermentation respectively. the flour from the unfermented tigernut was found to have oil absorption capacity of 61.2 g/100 g. result shows that tigernut may be a lower flavour retainer than raw winged bean (1.4 ml/g) and (1.2 ml/g) flours (narayana and narasinga, 1982) and african yam bean (1.42 ml/g) flour (eke and akobundu, 1993). the lower oil absorption capacity of tigernut flour might be due to low hydrophobic proteins which show superior binding of lipids (kinsella, 1976). bulk density is a measure of heaviness of a flour sample the 72 h fermentation has the highest bulk density of 0.83 g/ml. this is also for packed bulk density as shown in table 2. the 0.55 g/cm 3 obtained for compares favourably with 0.54 g/cm 3 reported for african breadfruit kernel flour but lower than 0.71 g/cm 3 reported for wheat flour (akubor and badifu, 2004). pasting properties as shown in figures 1 to 4 the pasting temperature, peak viscosity, breakdown viscosity, final viscosity and setback viscosity of the flours from 24 and 48 h fermentation are lower than 0 h fermentation. flour from 72 h fermentation has the highest in these parameters. the peak viscosity of 18.75 and 18.17 rvu were recorded for the flours from 24 and 48 h fermentation respectively, while the peak time was 5.13 and 5.07 min respectively. the peak viscousity of flour from 0 h fermentation was 38.17 rvu at peak time of 5.6 min. the setback viscousity and breakdown viscousity were 33.08 and 6.50 rvu. however oladele and aina (2007) has recorded the peak viscosity of 9.75 rvu for flour from yellow tigernut and 16.33 rvu for flour from black variety of tigernut and the time to reach peak viscosity at, 6.53 min and 5.33 min respectively and both have low setback and breakdown viscosities. this probably was attributed to the high fat content of the samples. conclusion tigernut flour is a rich source of oil and contains mode adejuyitan et al. 371 800 r v u 600 v is co s ity 400 200 peak = 18.75 final = 27.67 0 newport scientific pty ltd hold = 14.08 0 3 6 9 12 15 time mins a figure 1. pasting characteristics of flour from 24 h fermentation of tigernut. 800 r v u 600 v is co si ty 400 200 peak = 18.17 final = 25.42 0 newport scientific pty ltd hold = 13.58 0 3 6 9 12 15 time mins b figure 2. pasting characteristics of flour from 48 h fermentation of tigernut. rate amount of protein. fermentation can also beused to improve the nutritional content of this seed. further work needs to be carried out on the microorganisms associated with the fermentation and to develop starter culture to ferment this seed and probably see changes on the resultant flour for uses in food formulation. 800 r v u 600 v is co si ty 400 200 peak = 46.75 final = 66.08 0 hold = 38.33 newport scientific pty ltd 0 3 6 9 12 15 time mins c figure 3. pasting characteristics of flour from 72 h fermentation of tigernut. 800 r v u 600 v is co si t y 400 200 final = 64.75 peak = 38.17 0 hold = 31newport.67 scientific pty ltd 0 3 6 9 12 15 time mins control figure 4. pasting characteristics of flour from 0 h fermentation of tigernut. references achinewhu s.c. (1982). composition and food potential of african oil bean pentaclethra macrophylla) and velvet bean (mucuna uriens). j.food sci. 47: 1736–1737. akubor pi, badifu gio (2004). chemical composition, functional properties and baking potential of african breadfruit kernel and wheat flour blends. int. j. food sci. technol. 39: 223–229. 2476. afr. j. 372 afr. j. food sci. res. biotechnol. beuchat lr (1977). functional and electrophoretic characteristics of succinylated peanut flour protein, j. agric. food chem. 25: 258-261. cantalejo mj (1997). analysis of volatile components derived from raw and roasted earth almond (cyperus esculentus l.). j. agric. food chem. 45: 1853-1860. de vries, femke t (1991). chufa (cyperus esculentus, cyperaceae): a weedy cultivar or cultivated weed? econ. bot. 45: 27–37. eke os, akobundu ent (1993). functional properties of african yam bean (sphenostylis stenocarpa) seed flour as affected by processing. food chem. 48: 337-340. eteshola e, oraedu aci (1996). fatty acid composition of tigernut tubers (cyperus esculentus l.), baobab seeds (adasonia digitata l.) and their mixture. j. am. oil chem. soc. 73(2): 255–257. fagbemi tn, and oshodi aa (1991). chemical composition and functional properties of full fat fluted pumpkin seed flour. niger. food j. 9: 26-32 kinsella je (1976).functional properties of protein foods. crit. rev. sc. nutr. 1:219229 kirk sr, sawyer r (1991). pearson’s composition and analysis of foods, 9th edn., longman scientific and technical, uk leach hw, mccowen ld, schoch tj (1959). structure of the starch granules. in: swelling and solubility patterns of various starches. cereal chem. 36: 534-544. mukprasirt a, sajjaanantakul k (2004). physico-chemical properties of flour and starch from jackfruit seeds (artocarpus heterophyllus lam.) compared with modified starches. int. j. food sci. tech. 39: 271-276. narayana k, narasinga rao ms (1982). functional properties of raw and heat processed winged bean flour. j. food sci. 47: 1534-1538. odoemelan sa (2003). chemical composition and functional properties of conophor nut flour (tetracarpidium conophorum) flour. int. j. food sci. technol. 38: 729-734. okafor jnc, mordi ji, ozumba au, solomon hm, olatunji o (2003). preliminary studies on the characterization of contaminants in tigernut (yellow variety). in proceedings of 27th annual nigerian institute of food science and technology (nifst) conference.13-17 october, 2003. pp. 210-211. okaka jc, potter nn (1979). physicochemical and functional properties of cowpea powders processed to reduce beany flavour. j. food sci. 44: 1235-1240. okpala lc, mammah en (2001). functional properties of raw and processed pigeon pea (cajanus cajan) flour. int. j. food sci. nutr. 52: 343-346. oladele ak, aina jo (2007). chemical composition and functional properties offlour produced from two varieties of tigernut (cyperusesculentus) afr. j. biotechnol. 6 (21): 2473-2476, spe (1993). scientific program enterprises. computer software plot it graphics and statistics version 2.0 (ics gmbh, frankfurt). adejuyitan et al. 055 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (7), pp. 338-346, july, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of the fungal populations and their toxins associated with coffee bean seeds fardos bokhari* and magda mohammad aly faculty of science, biology department, king abd el-aziz university, p. o. box 12161, jeddah 21473, saudi arabia. accepted 03 april, 2018 thirty samples of coffee beans were collected from different places of jeddah, saudi arabia to determine and identify fungal population. twenty six species belonging to 7 genera were isolated using potato dextrose agar (pda) and malt extract agar (mea) media at 28°c. the most prevalent genera were aspergillus and penicillium. aspergillus was present in 73 and 100% of the samples but penicillium was present in 86.6 and 100% on the two mentioned media, respectively. also, fusarium, mucor, rhizopus and alterneria were recovered in moderate incidences on the two media. out of the thirty samples of coffee beans collected, thirteen were contaminated with mycotoxin (43.3%). mycotoxin profiles were also determined in these samples. it was found that aflatoxin g1 (afl g1) showed the highest incidence rates of occurrence. it occurred in about 23.3% of all samples analyzed and in 54% of the mycotoxin contaminated samples. the other toxins detected were aflatoxins b1 (16.6%), b2 (10%), g2 (6.6%), ochratoxin (10%), patulin (16.6%) and sterigmatocystin (6.6%). the factors affecting the afl g1 production by aspergillus flavus were studied. the results clarified that addition of caffeine up to 1 g/l reduced the toxin synthesis (afl g1) . the effect of some medicinal plants and spices added singly or in combination to the malt extract, on fungal growth and aflg1 production by a. flavus was also studied. cinnamon and cloves affected fungal growth and mycotoxin production. no growth or toxins were detected in the presence of the two plants. saffron and ginger did not affect fungal growth or prevented toxin production by a. flavus. in conclusion, coffee beans in saudi arabia are highly contaminated with toxigenic fungi, specially, a. flavus, which was found in this study to be the main producer of afl g1. medium, temperature, vitamin c, caffeine and some medicinal plants or spices which are used as a traditional additive in saudi arabia may affect fungal growth or/and toxin production. key words: aflatoxins, mycotoxin, aspergillus, coffee beans, secondary metabolites, medicinal plant, spices, additives. introduction aspergillus is a filamentous fungus that produces mycotoxins in many food and feed crops. aflatoxins are polyketide-derived, highly toxic, mutagenic, tetratogenic and carcinogenic secondary metabolites for higher vertebra-tes and other animals (bokhari and shaker, 2008). they are produced primarily by aspergillus flavus, aspergillus parasiticus, aspergillus nomius, aspergillus toxicarius and aspergillus parvisclerotigens (holmes et al., 2008) on crops such as corn, peanuts, cotton seeds, and coffee beans. furthermore, the natural occurrence of the toxige-nic aspergillus species and aflatoxin production in coffee beans was suggested by many authors (levi, 1980; daivasikamani and kannan, 1986; abdelhafez and el *corresponding author. e-mail: fmbokh@kau.edu.sa. maghraby, 1992; nakajima et al., 1997; batista et al., 2003). regulatory guidelines issued by the u.s. food and drug administration (fda) prevent sale of commodities if contamination by these fungi or mycotoxins exceeds certain levels. aflatoxins b1 and b2 are produced by a. flavus and a. parasiticus, but the latter species also have been confirmed to form g1 and g2 toxins. on the other hand, ochratoxin was produced by aspergillus ochreceus, aspergillus niger and aspergillus carbonarius (bhatnagar et al., 2004). the fungal isolates were analyzed for toxins production in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. bokhari (2007a) reported that coffee seeds were highly contaminated with toxigenic fungal isolates and toxins especially ochratoxin a. the occurrence of fungal contamination and mycotoxin production started at the beginning of har bokhari and aly 339 vest due to high moisture content of the seeds and increased during transport, storage, or marketing. within the last decade, significant advances have been made in mycotoxin detection methods and control strategies as well as in studying the effect of environ-mental factors on toxin synthesis (patterson, 1984; nakajima et al., 1997) . the most significant environmental factors that influence toxin synthesis are media used especially car-bon and nitrogen sources, ph, temperature, water acti-vity, and plant metabolites (bhatnagar,et al., 2003; calvo, et al., 2002). natural plant metabolites affect toxin prod-uction and fungal development. earlier research efforts in this, as well as other labs, have shown that plant com-ponents or volatile extracts can alter either aspergillus growth or aflatoxin production, i.e. volatile aldehydes, vitamin c (clevstrom et al., 2004), caffeine (buchanan et al., 1983b) and other compounds from neem leaf (zerin-gue and bhatnagar, 1999), cotton leaf (zeringue and mccormick, 1990) and corn-leaf (wilson et al., 1981). anthocyanins and related flavonoids also affect aflatoxin biosynthesis (norton, 1999). in some cases, growth was not significantly aff-ected by various metabolites, while aflatoxin biosynthesis and fungal development were sig-nificantly decreased (juglal et al., 2002; basilico and basilico, 1999). the plant linoleic acid, its derivatives and their precursor affect sexual and asexual sporulation in aspergillus nidulans, sclerotial development, and toxin synthesis (champe and zayat, 1989). the conversion of oleic acid (18:1) to linoleic acid (18:2) is a critical biosyn-thetic step in the generation of sporogenic psi factors. similarly, hitokoto et al. (1980) reported that addition of cloves or cinnamon inhibited aspergillus growth but aillium cepa or green tea leaves inhibited toxin production. the objectives of the present study were to identify the fungal populations and their toxins associated with coffee bean seeds, collected from jeddah, saudi arabia and to evaluate the potential of the toxigenic isolate (a. flavus) to produce aflatoxins g1 which was more dominant mycotoxin compared with the other toxins detected. no studies were found dealing with the occurrence of afl. g1 in coffee bean seeds or the factors affecting its production. different factors witch affect aflatoxin g1 production and the effect of different traditional additive (saffron, ginger, cinnamon, cloves and cardamom) of coffee used in saudi arabia were also studied. materials and methods collection of samples thirty coffee bean samples with different varieties of coffee arabica l. (about 1-1.5 kg each) were collected from different markets of jeddah governorate, where coffee beans were sold in open bags. each sample consisted of three replicates. the samples were collected in a sterile polyethylene bags to minimize the loss of water content and provide sufficient aeration, sealed, transferred immediately to the laboratory, kept at 4ºc until mycological and mycotoxins analysis. moisture content of the green coffee bean samples the moisture content of the samples was directly determined by dry weight method (aziz, 1987). about 10 g of each sample was transferred to an oven at ° under vacuum for 1224 h and until a constant weight. percentage of water content was calculated. fungal isolation and identification isolation was carried out by the method described by batista et al. (2003) . to isolate fungi associated with the coffee bean seeds, the seeds were placed directly on filter paper, moistened with sterile dist. water. then, thirty beans were collected randomly from each coffee bean sample. a total of 15 seeds were plated directly on agar plates (3 seeds/plate) and the other were disinfected with 1% sodium hypochloride for 2 min in order to permit isolation of fungi present in the interior of the beans. the media used are either potato dextrose agar or malt extract agar (merck, germany) for isolation of different types of fungi. the compositions of malt agar were as the following: malt extract, 20 (g/l); glucose, 20 (g/l); peptone, 15 (g/l); agar, 20 (g/l). ten plates for each sample (five plates for each medium) were used. the plates were incubated at 28°c for 7-10 days and the developing fungi were counted and identified according to macro and microscopic characteristics as described in raper and fennell (1965), moubasher (1993) and samson et al. (1995). the total counts (t.c.) of each species of fungi were calculated and were divided by the weight of the coffee bean seeds used in milligram (tc/mg). extraction of mycotoxins from coffee bean seed samples a one hundred grams of each sample was extracted with 100 ml chloroform twice times and following the procedures described by sorenson et al. (1967). the chloroform extract was dried over anhydrous sodium sulfate, filtered, then concentrated under vacuum and the dry material was transferred to a dark vial with a small amount of chloroform, which was evaporated to near dryness. detection of mycotoxins thin layer chromatographic technique of the clean extract was done on percolated silica gel plates (merck, silica gel 60, 25 mm, 20 x 20). detection of the different mycotoxins was carried out according to standard procedures described by roberts and patterson (1984) and el-shanawany et al. (2005). a mixture of toluene-ethyl acetate90% formic acid (50:40:10) was used as a mobile phase. mycotoxins were visualized under ultraviolet light at 366 nm in a chromatovisor. aflatoxin, ochratoxin a, patulin and sterigmatocystin, isolated from fungi contaminated the coffee beans, had a retention time and fluorescent spot similar to the standard mycotoxins being tested (sigma). chemical confirmation of mycotoxins chemical confirmation of mycotoxins was performed directly on the developed tlc plate. two spraying reagents were used to visualization and increase the fluorescence intensity of the mycotoxins (grabarkiewicz-saczesna et al., 1985) . the plates were then sprayed with either 20% alcl3 solution or 20% sulfuric acid, heated to 110ºc and examined under uv light (365 nm) as described by bokhari (1993). 340 afr. j. food sci. res. cultivation of a. flavus isolated from coffee bean seeds in liquid media a. flavus was cultured in liquid media to study different factors affecting growth and toxin production. each treatment had three replicates. for inoculum preparation, a. flavus was grown on czapex dox agar containing rose bengal and chloramphenicol (25 mg/ml) in petri dishes at 28°c until extensive formation of conidia was observed. the use of a combination of rose bengal at a concentration of about 130,000 and chloramphenicol in fungus plating media to prevent growth of bacteria and restrict size of colonies was found to be far superior to the older standard procedure. thus, a disc of 10 days old culture was used as a source of conidia for inoculating of the different flasks. extraction of aspergillus toxins after incubation, the content of each flask (medium + mycelium) was homogenized for 5 min in a high-speed blender with 100 ml chloroform. the extracts procedure was repeated three times. the chloroform extracts were combined, washed, dried, filtered, and concentrated near to dryness, cleaned and mycotoxins detected as previously described (dos santos et al., 2003). effect of different temperature on growth and aflatoxin g1 production erlenmeyer flasks (250 ml) containing 50 ml of sterile malt extract medium were inoculated with about 3 ml of heavy spore suspension containing 2 x 10 4 spore/ml (optical density, 0.65). the flasks were incubated at different temperatures (4, 10, 16, 22, 28, 30, 34°c). after 7 days of incubation, the contents of each flask were mixed with 120 ml of chloroform: water (100:10, v/v) and were shaken vigorously by a rotary shaker (200 rpm) overnight. the extract was sequentially filtered through anhydrous sodium sulfate. the chloroform extract was collected, dried and afl. g1 was detected as a fluorescent green color under the uv light. effect of different media on growth and aflatoxin g1 production by a. flavus a. flavus was cultured in different media. the media used were coffee broth, coffee dextrose, potato dextrose or malt extract. after incubation at 28°c for 7 days, the fungal growth and the quantity of afl. g1 were determined as described above. effect of different concentrations of caffeine or vitamin c on growth and aflatoxin g1 production by a. flavus different concentrations of either vitamin c or caffeine were added to the growth media of a. flavus in 250 ml erlenmeyer flasks containing 50 ml of sterile malt extract medium. the vitamin c concentrations were 0, 1.1, 2.2, 3.3 and 4.4 g/l as well as the caffeine concentrations ranged from 0 to 1 g/l. the growth and the quantity of afl g1 were determined and compared with the results of control (containing no caffeine or vitamin c). the effect of different medicinal plants on a. flavus growth and aflatoxin g1 production the ability of a. flavus to grow and produce afl g1 in a medium containing different types of medicinal plants or spices was investigated. the medium used was malt extract; and ph of the medium was adjusted to 5.5. the plants used were cardamom, cinnamon, cloves, saffron, and ginger. they were collected, washed, cut into pieces, dried at 60ºc, milled and sieved with 1 mm mesh and added to the fungal medium (1 g/l). after 10 days of incubation at the appropriate temperature, the growth and afl g1 were quantified.. statistical analysis each experiment has three replicates and three determinations were conducted. the numerical data were presented as mean + standard deviation. the student t-test was used to compare between numerical data of control and treated. p-value < 0.05 was considered statistically significant. results and discussion the results in table 1 clearly show that coffee beans samples, collected from jeddah, were highly contaminated with fungi which were represented by seven genera and twenty six species. more fungal species were counted by abdel-hafez and maghraby (1992) in egypt and bucheli et al. (1998) in thailand. abdel-hafez and maghraby (1992) could isolate 26 fungal species belonging to 16 genera from coffee beans samples. the total viable counts of mold in all coffee beans samples were 419 and 346 colonies/mg dry coffee seeds on each malt extract agar and potato dextrose agar, respectively. the levels or occurrences of the different fungal population encountered in the current study ranged from high, moderate to rare and were almost in agreement to those observed in coffee beans studied by batista et al. (2003). examination of coffee bean samples for the presence of toxigenic and non toxigenic fungi was carried out. it has been found that aspergillus and penicillium followed by fusarium and mucor were the most prevalent genera on the two used isolation media. these results were similar to a great extent to results obtained by panneerselvam et al. (2001). they studied microflora of coffee beans and reported that the genera aspergillus, penicillium, cladosporium, trichoderma and mucor were the most dominant genera. the same findings were obtained by many other (nunnes et al., 2001; bokhari, 2007a). aspergillus was the commonest genus in all samples examined. it was represented by 9 species and appeared in 73 and 100% of the samples tested on the two used media. it was represented by 63-65 % of the total fungi examined. a. flavus was the commonest species. it was represented by 24 and 14% of the total aspergillus species recovered on malt extract and potato dextrose agar, respectively. a. candidus, a. fumigatus, a. niger, a. ochraeceous, a. sydowii, a. terreus, and a. versicolor were less dominant compared to a. flavus. abdel-hafez (1984) reported that coffee seeds were highly contaminated by the genus aspergillus, followed by penicillium and rhizopus. the second highest incidence rate was represented by genus penicillium. it was recovered from 86 to 100% of coffee beans seeds samples. most of penicillium species were prevalent on malt extract agar medium and with mode bokhari and aly 341 table 1. total counts (tc per mg), number of cases of isolation out of 30 samples and occurrence remarks of fungal genera and species recovered from coffee bean samples at 28ºc for 10 days at two types of media (malt dextrose agar and potato dextrose agar). isolated fungi potato dextrose agar malt extract agar occurrence no. of t.c. /mg occurance no. of t.c. remarks appearance dry remarks appearance /mg /30 sample seed /30 sample dry seed aspergillus h 30 234 h 22 275 a. candidus m 9 21 m 6 11 a. flavus h 22 33 h 19 67 a. fumigatus h 19 22 h 14 33 a. melles r 3 11 r 1 5 a. niger h 25 7 r 2 22 a. ochraeceous h 18 33 h 17 45 a. sydowii m 10 36 r 3 31 a. terreus h 19 40 h 19 32 a. versicolor m 11 31 m 9 29 penicillium h 30 50 h 26 70 p. variable h 15 13 h 19 34 p. canescens r 0 0 r 5 7 p. chrysogenum h 30 7 r 3 7 p. citrinum r 4 11 m 6 11 p. glabrum r 0 0 r 1 4 p. rubrum m 12 16 r 1 2 p. oxalicum r 0 0 r 2 5 p. italicum r 5 3 r 0 0 fusarium h 18 8 h 19 23 f. oxisporium m 7 5 r 5 3 f. moniliforme m 11 3 h 14 20 mucor m 21 14 m 15 21 m. racemosus m 9 10 m 7 3 m. circinelloides 0 0 0 h 7 11 m. hiemalis m 12 4 r 1 7 rhizopus m 8 5 r 4 9 r. stolonifer m 8 5 r 4 9 alterneria m 9 25 m 11 11 a. chlamydospora m 6 9 m 11 11 a. .solani m 8 5 r 4 9 a. alternata m 9 25 m 9 9 h, high occurrence (more than 15); m, moderate (less than 15 and more than 5); r, rare (less than 5). rate incidence or completely absent on pda medium. from the genus penicillium, 8 species were identified of which penicillium chrysogenum; penicillium duclauxii and penicillium janczweskii were the most prevalent. fusarium and mucor were also common genera, recovered at the average of 63 and 50% of all samples examined, constituting, 5.5 and 5% of total fungi on malt extract agar medium and at the average of 37 and 50% of all samples, constituting 3.4 and 6% of the total fungi examined on pda. the remaining genera and species were remarked in low or rare frequencies of occurrence on one or the two isolation media (table 1). as shown in table 2, thirty samples of coffee beans were collected from different places and % of water content was determined. the contamination of seeds with different mycotoxins was determined qualitatively. it was found that mycotoxin production capability by fungi can be limited or impeded by water activity. increasing water content of the sample increased the number of toxins detected. four samples (no. 1, 5, 10, and 26) had moisture contents ranged from 13.6–14% and contaminated by at least two mycotoxins. the % of moisture content of samples 18 342 afr. j. food sci. res. table 2. moisture content, number of toxigenic isolates and natural occurrence of mycotoxins recorded in the different coffee bean samples, collected from various sources. sam % no. of no. of % of quality of the toxin detected no. moisture fungal toxigenic toxigenic b1 b2 g1 g2 och. a patulin streg. content isolates isolates isolates 1 13.6 22 3 13.6 + + 2 12.9 9 1 11.1 + + 3 13.4 6 0 0.0 4 12.8 12 2 15 + 5 13.8 21 2 9.5 + 6 12.4 12 0 0.0 7 14.1 19 1 5.2 + 8 13.0 20 0 0.0 9 13.9 24 4 16.0 10 13.6 14 4 29 + + 11 12.8 10 0 0.0 12 12.9 7 0 0.0 13 12.4 11 0 0.0 14 13.8 3 1 33.5 15 12.8 5 0 0.0 16 12.8 14 0 0.0 17 12.8 9 0 0.0 18 12.0 27 3 11.1 + + + 19 11.8 13 0 0.0 20 12.9 19 0 0.0 21 11.7 9 0 0.0 22 12.8 11 0 0.0 23 12.3 12 0 0.0 24 12.0 12 2 11.1 + 25 12.1 5 1 20.6 26 14.0 4 2 50 + + 27 12.8 8 0 0.0 28 11.2 4 0 0.0 29 12.0 14 0 0.0 30 12.6 6 2 33.5 + sam: sample number, b1, b2, g1 and g2: aflatoxin b1, b2, g1 and g 2, och.a :ochratoxin a, strg. : stergmatocystin, +: detected. from 12-12.6% and found to be contaminated with four different toxins. all the 30 coffee bean samples were tested qualitatively for naturally occurring mycotoxin contaminations and showed that 57% of the samples were mycotoxin-free (table 2). it was found that five samples were contaminated by aflatoxins b1 and/or b2 while, afl. g1 was recovered in the extract of seven coffee bean samples, thus, it was selected for more studies. only samples (no. 4, 18) as in table 2 were contaminated with afl. g2. also, the results revealed that 17% of the tested samples (5 out of 30) were contaminated by aflatoxins b1 and/or b2; 30% contains aflatoxins g1 or g2; 10%, ochrotoxin; 17%, patulin; but only 7%, sterigmatocystin. bucheli et al. (1998) recorded that a. flavus, a. niger, a. fumigatus were isolated from the coffee seeds contaminated by fungi. on contrast, fusarium solani was the common est fungus in coffee seeds (muthappa, 1984) but a. flavus, a. niger, a. funiculosum and cladosporium were the commonest in coffee bean samples collected from egypt. nunes et al. (2001) reported that coffee seeds were contaminated by the genus aspergillus, especially, a. ochraceus (10.9%) and a. niger (22.9%). out of 79 fungal isolated belonging to the genus aspergillus and penicillium, only 8.9% were toxigenic isolates, 7,6% belonging to genus aspergillus, especially, to a. flavus which was represented by 38% of the total toxigenic isolates of the genus aspergillus. similar results were obtained by sinha and sinha (1991), who found that out of 48 strains isolated in india, 33 belonged to toxigenic isolates of the genus a. flavus. the total toxigenic isolates of the genus aspergillus. similar results were obtained by sinha and sinha (1991), who found that out of 48 strains isolated in bokhari and aly 343 table 3. the effect of different media on growth and aflatoxin production by aspergillus flavus, grown for 10 days at 28ºc. media used fungal growth (mg/ml) aflatoxin g1 quantity of aflatoxin production produced ng/g coffee dextrose 7.3 + 1.3 ++ 9.6 + 1.3 potato dextrose 8.0 + 2.4 11 +0 .9 malt extract 13.0+ 1.0* +++ 16+ 1.3* sabouraud 3.4+ 0.6 + 6.4+ 0.3 czapek'sglucose 5.0+ 1.3 + 6.0+ 0.9 *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production table 4. the effect of different temperature on growth and aflatoxin production by aspergillus flavus grown on malt extract for 10 days. temperature fungal growth aflatoxin g1 quantity of aflatoxin (mg/ml) production produced ng/g 4 °c 1.4 + 1.3* nd nd 10 °c 6.8 + 2.4* nd nd 16°c 8.2+ 1.0* + 11.5* 22 °c 11.4+ 0.6* ++ 12.0 25 °c 12.0+ 1.3 13.0 28 °c 13.3 + 1.3 +++ 16.0 30°c 8.0 + 2.4* ++ 12.0* 35 °c 7.0+ 1.0* + 4.8* 38 °c 5.4+ 0.6* nd nd *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production. nd: not detected india, 33 belonged to toxigenic isolates of the genus a. flavus. a. flavus, which was isolated from coffee beans was the best producer for aflatoxins especially g1. the factors affecting the growth and production of afl. g1 were studied. the effect of five different media on fungal growth of a. flavus and afl. g1 production was detected (table 3). it was found that the semi synthetic malt extract broth was the best for both fungal growth and afl. g1 production, followed by potato dextrose and coffee dextrose. on the other hand, inoculation of a. flavus in sabouraud broth medium decreased the growth and afl. g1 by 30 and 40% respectively compared to inoculation in malt dextrose broth. natural media, especially pda increased mycotoxin production compared with the synthetic media (abramson et al., 1990; cortes-esposa et al., 2006; alarjani, 2008). as can be seen in table 4, temperature affects the growth and toxin production by a. flavus . increasing the tem-perature enhanced the growth up to 28ºc where the gro-wth was maximum and got to 13.3 g/l. increasing tem-perature more than 28ºc decreased both fungal growth and afl. g1 production. no toxin was recorded at 4, 10 and 38ºc. davis and diener (1978) recorded that the aflatoxins b1 and g1 were produced by a. flavus on a semi-synthetic medium and their quantities were affected by the media used and temperature of incubation. the ideal temperature for aflatoxin production is 29–30ºc (schroeder, and hein, 1967; payne, 1998). aflatoxin production is significantly decreased at temperatures below 25ºc, but is completely inhibited at 37ºc or above. as shown in table 5, addition of caffeine to the growth medium up to 1 g/l decreased the fungal growth and aflatoxin g1 production to about 67 and 23%, respectively. similar results were obtained by soliman (2002), who reported that the highest concentrations of the aflatoxins produced by a. flavus were detected in the decaffeinated green coffee bean compared with the normal green coffee bean samples. he added that addition of 1-2% caffeine reduced the growth of a. flavus in liquid medium by 50% and the level of aflatoxin in the medium was undetectable. complete inhibition of ochratoxin a, produced by a. ochraceus, was carried out by addition of 1 g/l caffeine to yes growth medium (nehad et al., 2005), but 2 g/l caffeine inhibited aflatoxins production by a. parasiticus in the same medium (buchanan and lewis, 1984). similar reductions in afl. b1, ochratoxins and zearalenone were observed by a number of authors (tsubouchi et al., 1987; buchanan et al., 1982, 1983a; nunes et al., 2001). table 6 shows that addition of vitamin c to the growth 344 afr. j. food sci. res. table 5.the effect of different concentration of caffeine on growth and aflatoxin production by aspergillus flavus grown on malt extract for 10 days at 28ºc. caffeine concentration fungal growth aflatoxin g1 quantity of aflatoxin g/l (mg/ml production produced ng/g 0.0 (control) 13.2+1.3 +++ 16.5 0.2 11.3 + 1.3* ++ 11.0* 0.4 10.6+ 0.3* 7.0* 0.6 8.3 + 0.9* + 4.8* 0.8 8.3 + 1.1* + 4.0* 1.0 8.2+ 2.3* nd 2.8* *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production. nd: not detected table 6. the effect of different concentrations of vitamin c on growth and aflatoxin production by aspergillus flavus grown on malt extract for 10 days at 28ºc. vitamine c concentration fungal growth aflatoxin g1 quantity of aflatoxin g/l (mg/ml) production produced ng/g 0.0 (control) 13.2+ 2.3 +++ 16.0 1.1 13.3 + 1.3 ++ 13.7 2.2 14.6+ 0.3 14.0 3.3 16.3 + 0.9* +++ 15.8 4.4 17.3 + 1.1* +++ 17.0 *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production. nd: not detected table 7. the effect of different addition of medicinal plants or spices on growth and aflatoxin production by aspergillus flavus grown on malt extract for 10 days at 28ºc. type of spices fungal growth aflatoxin g1 quantity of flatoxin (mg/ml) production produced ng/g control 13.2+ 2.3 +++ 16.0 saffron 13.3 + 1.5 ++ 13.7* ginger 13.6+ 0.3 14.0* cinnamon 6.3 + 0.6* nd nd cloves 7.3 + 1.1* nd nd cardamom 11.2+ 2.3 + 10.0 cloves+ cinnamon 6.3 + 1.3* nd nd *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production. nd: not detected medium up to 4.4 g/l enhanced the fungal growth and mycotoxin production by a. flavus. similar results were obtained by clevstrom et al. (2004). they found that aflatoxins produced by a. flavus which was either isolated from cereal, barley, or coffee were enhanced up to 800 times with the presence of vitamin c in the culture medium. the increase in growth and patulin production got to 30% by the addition of vit. c to the czapex dox broth medium (podgorska, 1992). similarly, the effect of additions of five different plant materials to the growth medium of a. flavus was studied as can be seen in table 7. it was found that cinnamon, cloves or both inhibited both growth (50%) and afl. g1 production (100%) of a. flavus . on contrast, the presence of either saffron or ginger did not significantly affect the aspergillus's growth or toxin production, but cardamom decreased fungal growth with no significant effect on the quantity of afl.g1 production. similar results were obtained by hitokoto et al. (1980) who reported that addition of some spices or medical plants or some plant extracts to the fungal growth media may inhibit or enhance fungal growth and toxin production. cinnamon, cloves inhibited growth of a. flavus, a. ochraceus and a. versicolor. in contrast, allium cepa, green tea and other additive completely prevented toxin production by toxigenic fungi. likewise, inhibitions in mycotoxins biosynthesis bokhari and aly 345 were noticed by juglal et al. 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sollenberger2, l.e.; karue3, c. n.; and musimba3, n. k. r. 1 kenya agricultural research institute, p. o. box 450, kitale, kenya, 2 agronomy department, university of florida, 2183 mccarty hall, p.o. box 110300, gainesville, fl, 32611-0300, 3 department of range management, university of nairobi p.o. box 30197, nairobi, kenya. accepted 12 april, 2020 two experiments were conducted to evaluate the feed value of acacia (acacia brevispica) and leucaena (leucaena leucocephala seedpods (whole fruit) during the dry season. in experiment 1, treatment diets were rhodes grass (chloris gayana) hay (control) and the hay supplemented with either acacia or leucaena seedpod meal. in experiment 2, maasai lovegrass (eragrostis superba) hay (control) was offered alone or with a supplement of leucaena seedpod meal. in experiment 1, calves supplemented with leucaena meal had higher gain (486 g d -1 ) than the control (239 g d -1 ) or those receiving acacia pod meal (250 g d -1 ). in experiment 2, calves on leucaena meal had higher gains (559 g d -1 ) than the control (276 g d -1 ) confirming the results obtained in experiment 1. seedpods of a. brevispica contained only 65% of their seeds, resulting in a lower digestible energy concentration than leucaena seedpods. these data confirm that cattle productivity can be increased if pods of l. leucocephala are collected during periods of abundance, stored and used as supplements for growing calves. key words: acacia, leucaena, seedpods, grass hay, calf weight gain introduction in many parts of the tropics, arid and semi-arid lands constitute a large proportion of the area. inadequacy of livestock feed in the dry season, particularly lack of protein, is a major constraint to livestock production in these areas (minson, 1990). this is because conventional supplements such as oilseed cakes and meals made from animal byproducts are expensive and not readily available. under these circumstances, the most practical supplement may be locally available legume trees (topps, 1992). acacia, a genus of indigenous woody legumes, occupies vast areas in semi-arid and arid areas of tropical and subtropical countries (nrc, 1979). studies have indicated that seedpods of some acacia species, such as a. tortilis and a. albida, as well as leaves of a. brevispica, when offered as supplements to poor quality roughages, give live-weight gains in livestock comparable with those fed oilseed cake or *corresponding author’s e-mail: elkananyambati@yahoo.co.uk. lucerne (medicago sativa) supplements (ilca, 1988; 1989; tanner et al., 1990). sawe et al. (1998) showed that goats (capra species) supplemented with leaves or pods of a. brevispica, a. tortilis and a. nilotica grew faster than unsupplemented controls. in a recent study to evaluate the nutritive potential of some acacia tree leaves, abdulrazak et al. (2000) found that leaves from a. nubica, a. tortilis, a. mellifera, a. brevispica, a. seyal and a. nilotica had moderate to high crude protein (cp) concentrations. based on their observation of goats browsing, the authors concluded that leaves have potential as livestock fodder. however, tannins in acacias (woodward and reed, 1989; reed, 1995) may bind protein, making it unavailable to rumen microbes and thus affecting dry matter (dm) intake and digestibility (kumar and d’mello, 1995). these effects on animal performance have been shown to be both positive (ben saleem et al., 1999) and negative (degen et al., 1998; maasdorp et al., 1999). leucaena leucocephala is a versatile, drought tolerant, tree legume recommended for use in tropical and subtropical countries (gutteridge and shelton, 1994). several studies have demonstrated that leucaena forage, when fed as a supplement to poor quality roughages, resulted in body weight gains comparable to those obtained using conventional supplements such as oilseed cakes, animal byproduct meals and cereal by-product (saucedo et al., 1980; and manidool, 1983). chemical analysis of l. leucocephala seedpods from nigeria (adeneye, 1979) and india (damothiran and chandrasekaran, 1982) revealed that they are of high nutritive value indicating the possibility of them as a source of cattle feed. naseeven et al. (1989) showed that cracked l. leucocephala seeds are as good a protein source for fattening cattle as cottonseed cake. both a. brevispica and l. leucocephala are prolific producers of seedpods which can be harvested and fed to cattle. there is limited information on the nutritive value of seedpods, particularly those from a. brevispica and l. leucocephala, and their value as dietary supplements for calves is unknown. objectives of this study were to assess the effects of maturity on nutritive value of seedpods of these legumes and to determine the effect of feeding seedpods as a dry-season supplement on intake and weight gain of calves in smallholder crop-livestock mixed farming and agro-pastoral farming systems. materials and methods nutritive value of seedpods seedpods of a. brevispica and l. leucocephala were sampled at the immature (5 weeks after pod formation), dough (9 weeks), mature (13 weeks), and dry (15 weeks) stages. bulk samples were taken from a group of trees, oven dried at 60 0 c for 2-3 days, and ground before being analyzed for crude protein (cp), ash, neutral detergent fiber (ndf) and acid detergent fiber (adf). dry fruits only were separated into seed and pod fractions, were ground in a christy norris hammer mill to pass 1-mm screen and analyzed for cp, ash, ndf, adf, ether extract (ee), and in vitro dry matter digestibility (ivdmd). analytical procedures are described below. data were not analyzed statistically because chemical analyses were conducted on bulked samples not samples from replicated plots. experimental animals and treatments two feeding experiments were conducted at the university of nairobi field station, kabete, kenya, using boran (bos indicus) cross-bred calves balanced for sex. in experiment 1, 18 calves aged between five and nine months with an average weight of 132 kg (range 91 to 150 kg) were used. the calves were assigned to three treatment groups balanced for age, initial live weight and sex. the three experimental diets were: grass hay harvested from the university field station supplemented with wheat (triticum aestivum) bran (control-1); grass hay supplemented with either acacia or leucaena seedpods mixed with wheat bran (asm and lsm-1, respectively). in experiment 2, 16 calves aged between 5.5 and 9 months, with an average weight of 131 kg (range 102 to 162 kg) were used. the calves were assigned to two treatment groups balanced for age, initial live weight and sex. the treatments were grass hay supplemented with wheat bran (control-2) or with leucaena seedpod meal plus wheat bran (lsm-2). experimental feeds rhodes grass (chloris gayana) and maasai lovegrass (eragrostis superba) hays were used as basal diets in experiments 1 and 2, respectively. both hays were harvested at the university of nairobi dryland field station, kibwezi, kenya, in a semi-arid area classified as being in ecological zone five (pratt et al., 1966). maasai lovegrass is a native species in arid and semi-arid areas and is an important source of feed. a predominantly maasai lovegrass hay was harvested from a native stand. rhodes grass is a high yielding planted grass used in reseeding denuded grassland areas. the rhodes grass was harvested from a reseeded pasture that had not been cut for two growing seasons. both hays were mature and consisted mainly of stem. they were chaffed into approximately 510 cm pieces to facilitate handling and feeding. dry seedpods of a. brevispica and l. leucocephala, collected at the national range research center, kiboko, kenya, in ecological zones four and five (pratt et al., 1966), were sun-dried for 2-3 d before grinding to make a meal. pods were collected using hooked sticks or by pruning and lopping trees, in which cases some branches were left for continued growth. the a. brevispica seedpods collected contained approximately 65% of their seeds. most of the l. leucocephala seedpods collected contained all their seeds. experimental procedures all calves were dewormed using valbazen and sprayed against ectoparasites using stelladone. calves were confined in individual pens throughout the experimental period. the basal hays were offered ad libitum between 0600 and 1430 hours each day. the seedpod meal supplements were mixed with 0.6 kg of wheat bran and a mineral mixture. supplements were fed at 0500 hours at 1.5 kg d -1 (asm) or 1.2 kg d -1 (lsm-1 and lsm-2) to supply equal amounts of n. at the same time the control calves received 0.6 kg d -1 of wheat bran, equivalent to the amount used in formulating the supplements. water was freely available at all times. daily records were kept of hay and supplement offered and orts. calves were weighed weekly using a weighbridge following a 13-hour overnight food and water fast. there was a 14-day adjustment period to the diets at the start of both experiments. the experimental period lasted for five and four weeks in experiments 1 and 2, respectively. chemical and statistical analyses chemical analyses were carried out on samples of seedpods at four phenological stages to monitor changes in nutrient profiles with maturity and on separated seedpod components to determine the contribution made by seed and the empty pods to the nutritive value of the whole seedpod. total nitrogen concentration was determined by the kjeldahl technique and cp calculated as n x 6.25. ash and ee were determined by the conventional methods of aoac (1990) and adf and ndf by the methods of van soest et al. (1991). the ivdmd was carried out using the method of tilley and terry (1963). tannins were determined using the vanillin-hydrochloric acid method of burns (1963; 1971). weekly live weights of individual animals were adjusted using table 1. concentrations of cp, ash, ndf and adf in l. leucocephala and a. brevispica seedpods at four phenological stages. cp ash ndf ‡ adf ‡ --------------g kg -1 ---------- a. brevispica immature (5) † 186 44 423 361 dough (9) 184 50 490 366 mature (13) 178 45 509 342 dry (15) 143 41 550 335 l. leucocephala immature (5) 272 54 292 200 dough (9) 208 55 468 344 mature (13) 203 59 533 422 dry (15) 186 57 561 421 † number in parenthesis denotes age in weeks after pod formation. ‡ ndf = neutral detergent fiber, adf = acid detergent fiber. covariance analysis based on initial live weights (sas, 1996). computed average daily weight gain and hay intake data were subjected to one-way analysis of variance using the general linear model of sas and the treatment means separated using duncan's multiple range test (sas, 1996). results chemical analysis concentrations of cp, ash, ndf and adf in a. brevispica and l. leucocephala seedpods were determined at four phenological stages (table 1). for both species the concentration of cp declined while that of ndf increased with maturity. in leucaena seedpods adf followed the same trend as ndf, but with the acacia adf was greatest at the dough stage and least at the dry stage. there was no consistent trend in total ash in both seedpods, and the levels did not fluctuate much with age. nutrient composition in separated dry seedpod components of both a. brevispica and l. leucocephala are shown in table 2. seeds of both trees contained more cp and ee and had greater ivdmd and estimated digestible energy (de) concentration than empty pods, but seeds were lower in fibre and total ash than empty pods. seedpods of l. leucocephala had a higher concentration of tannins than a. brevispica, where most of the tannin was found in the empty pods. nutrient composition of feed ingredients, supplements and hay used in experiments 1 and 2 are reported in table 3.in experiment 1, leucaena seedpod meal and formulated lsm1 supplement (including wheat bran) had higher total ash, lower fiber, higher ivdmd and greater estimated de concentration than acacia seedpod meal and the formulated asm supplement, respectively. the lsm-2 supplement used in experiment 2 had slightly lower cp, higher ndf and lower ivdmd than lsm-1 supplement. both hays used contained similar concentrations of cp, ash, and fiber, but ivdmd was higher in maasai lovegrass in experiment 2 than rhodes grass in experiment 1. intake and live-weight gain calves on the lsm-1 diet had significantly (p <0.01) higher average daily gains (adg; 486 g d -1 ) than those on the asm (250 g d -1 ) and control-1 (239 g d -1 ) diets (table 4). average daily gain of calves on the asm diet was not statistically different p >0.05) than the control. in experiment 2 calves receiving lsm-2 had greater adg (559 g d -1 ) than control-2 calves (276 g d -1 ) (p < 0.01) (table 5). supplementation of hay diets with legume seedpod meal maintained or tended to increase grass hay intake in experiment 1 (table 4). total dm intake was 2490, 3400, and 3360 g d -1 for control, asm, and lsm-1 treatments, respectively. this resulted in average cp and de intakes being 65 and 34% above that of control-1. in experiment 2, adg responses to lsm-2 (table 5) were similar to those for lsm-1 in experiment 1 (table 4). grass hay intake was not different for control-2 and lsm2 suggesting that the effect of supplementary feeding was primarily additive (table 5). the relative increases in cp and de intake of the lsm-2 vs. control-2 treatments were 40 and 9%, respectively, in experiment 2. discussion in agreement with the literature (fao, 1981; getachew et table 2. chemical composition of l. leucocephala and a. brevispica separated dry seedpod components. constituent (dry matter basis) † cp ash ee ndf adf ivdmd de ‡ tannin * ------------------------g kg -1 ------ kcal kg -1 g kg -1 whole pod l. leucocephala 186 57 44 561 421 530 2.41 17.6 a. brevispica 143 41 38 550 335 547 2.50 0.90 empty pod l. leucocephala 57 78 23 737 620 212 0.63 20.3 a. brevispica 85 51 22 785 581 215 0.65 1.80 seeds l. leucocephala 282 42 91 413 193 726 3.51 15.5 a. brevispica 186 33 42 307 160 809 3.97 0.50 † cp = crude protein, ee = ether extract, ndf = neutral detergent fibre, adf = acid detergent fibre, ivdmd = in vitro dry matter digestibility, de = digestible energy ‡ estimated using regression equation, de (kcal kg -1 dm) = -0.559 + 0.056x; r 2 = 0.966 and se = 0.083 (heaney and pigden, 1963) where, x = digestible organic matter in g 100 g -1 dm * expressed as catechin equivalent table 3. nutrient composition of feed ingredients, experimental diets and hay used in experiments 1 and 2. constituent (dry matter basis) † dm cp ash ndf adf ivdmd de ---------------------g kg -1 ------------ kcal kg -1 experiment 1 acacia seedpod meal 891 121 49 637 490 400 1.68 leucaena seedpod meal 899 177 55 527 386 548 2.51 wheat bran 889 160 69 535 164 725 3.50 rhodes grass 942 41 87 792 493 418 1.78 asm supplement (asm) ‡ 918 140 65 590 326 560 2.58 lsm supplement (lsm-1) ‡ 921 166 73 528 251 669 3.19 experiment 2 leucaena seedpod meal 906 153 59 597 477 486 2.16 wheat bran 892 190 68 439 138 753 3.66 maasai love grass 914 36 83 780 530 534 2.43 lsm supplement (lsm-2) ‡ 887 172 72 507 294 646 3.06 † dm = dry matter, cp = crude protein, ndf = neutral detergent fibre, adf = acid detergent fibre, ivdmd = in vitro dry matter digestibility, de = digestible energy (kcal kg -1 dm) = -0.559 + 0.056x; r 2 = 0.966 and se = 0.083 (heaney and pigden, 1963) where, x = digestible organic matter in g 100 g -1 dm. ‡ supplements were seedpod meals plus wheat bran. al., 1994) cp of legume seedpods decreased and fibre generally increased with advancing maturity. the decline in cp was greatest between 13 and 15 weeks after pod formation for a. brevispica, and between 5 and 9 weeks for l. leucocephala. these results suggest that a. brevispica could be utilised best not later than 13 weeks after pod formation, whereas l. leucocephala pods can be utilised even at the dry stage. this is because dry acacia seeds are damaged by the bruchid beetle (southgate, 1983) and the pods of a. brevispica dehisce their seeds easily when table 4. calf body weight gain and intake of rhodes grass hay, supplement, crude protein and estimated digestible energy (dei) in experiment 1. treatment response control-1 asm lsm-1 se cv body weight gain (g d -1 ) 239 a† 250 a 486 b 42 32 intake hay (g dm d -1 ) 1940 a 2060 a 2250 a 135 36 hay (g dm kg -1 bw 0.75 d -1 ) 50.8 a 50.3 a 53.3 a 6 11 supplement (g dm d -1 ) 554 1340 1110 nd ‡ nd total cp (g d -1 ) 165 271 275 nd nd estimated dei (kcal d -1 ) 5.48 7.12 7.53 nd nd † treatment means within a row followed by the same letter superscript do not differ significantly (p <0.01). ‡ nd = not determined. table 5. calf body weight gain and intake of masaai lovegrass hay, supplement, total cp and estimated digestible energy (dei) in experiment 2. treatment response control-2 lsm-2 se cv body weight gain (g d -1 ) 276 a† 559 b 51 34 intake hay (g dm d -1 ) 3210 a 3130 a 104 35 hay (g dm kg -1 bw 0.75 d -1 ) 79.8 a 77.4 a 6 7 supplement (g dm d -1 ) 533 1060 nd ‡ nd total cp (g d -1 ) 212 296 nd nd estimated dei (kcal d -1 ) 9.93 10.9 nd nd † treatment means followed by the same letter superscript in the same row do not differ significantly (p <0.01). ‡ nd = not determined. dry (lamprey, 1967). acacia pods contained less cp and higher ndf and adf than that reported for leaves (abdulrazak et al., 2000), but data were generally within the range found in other studies (tanner et al., 1990; sawe et al., 1998; abdulrazak et al., 2000). the lower cp concentration of intact dry pods of a. brevispica compared to the value reported by sawe et al. (1998) could be due to location and seasonal differences (larbi et al., 1998) and stage of maturity at harvest. dry pods of l. leucocephala also contained lower cp and higher fibre than reported for leaves (wiegand et al., 1996), but the cp was similar to leucaena forage reported by larbi et al. (1998). the nutritive value of feeds depends mainly on the digestible energy concentration. whole a. brevispica pods had a lower cp concentration and lower ivdmd than l. leucocephala. acacia seedpod meal used in experiment 1 contained approximately 65% of its seeds a likely cause of the lower de concentration compared to leucaena seedpod meal, which contained most of its seeds. the daily intakes of cp by calves on asm and lsm-1 diets were similar, yet differences (p <0.01) were observed in calf gain, most likely attributable in part to the higher intake of de of calves on lsm-1 (7.53 vs. 7.12). crude protein concentrations of the grass hays in these experiments were well below requirements of growing calves. supplementation of low quality basal roughage diets with legume forage increases essential nutrients, especially nitrogen, available to the rumen microbes, thereby increasing the amount and rate of rumen breakdown and fermentation, resulting in increased rates of passage of both particulate and liquid matter phases and increases in intake and, consequently, in animal performance (norton and poppi, 1995). the voluntary dm intake of grass hay by calves supplemented with lsm-1 in experiment 1 was 16% greater than those fed the control-1 diet. additive effects of supplementation on hay intake suggest that lsm-1 increased gain by overcoming the protein deficiency resulting in greater intake of cp, dm, and de compared to control-1. the higher intake of hay and hence total cp and de in experiment 2 was expected because the maasai lovegrass hay had higher ivdmd than the rhodes grass hay used in experiment 1. however because the hay used in experiment 2 had higher ivdmd and the hay was offered ad libitum, supplementation with lsm-2 did not increase intake of the basal hay, but resulted in higher cp intake, and significantly (p <0.01) increased adg. this positive response could be attributed to the supplement overcoming the depressing effect of the low cp concentration of hay on intake (minson and milford, 1967). the greater gain by calves supplemented with lsm-2 than the control-2 calves in experiment 2 confirmed the results obtained in experiment 1, and is in agreement with the results of naseeven et al. (1989), who found that cracked seeds of leucaena were comparable to cottonseed cake as a supplement for cattle. similar live-weight gain responses have been obtained when l. leucocephala forage was offered as a supplement to low quality roughages (masama et al., 1997; kaitho et al., 1998). conclusion on communal african rangelands, which are often chronically overstocked, under nutrition leads to reduced growth rates and poor reproductive performance of cattle during the dry season. the results of this study indicate that seedpods of l. leucocephala can be used by smallholder crop-livestock farmers and agropastoralists in arid and semi-arid areas to provide a locally available feed that is cheap and high in protein. these seedpods can be used as supplements to low quality forages, resulting in better utilisation of the forage and improved live-weight gains in cattle. the lower cp and ivdmd of a. brevispica pods, due in part to lower seed retention, than in l. leucocephala, limit its potential for use as a supplement, especially as dry pods. less mature pods are higher innutritive value and may have greater impact, although they were lower in nutritive value than l. leucocephala pods across the entire range of maturities evaluated. further work is needed to determine the effect of mature seedpods on cattle gains when most of the seeds are retained before they dehisce their seeds and also to determine the effect of feeding increasing levels of seedpods of both legumes on animal performance. acknowledgements the senior author thanks the university of nairobi and the kenya agricultural research institute (kari) for supporting his studies at the university of nairobi. references abdulrazak sa, fujihara t, ondiek jk, orskov er (2000). nutritive evaluation of some acacia tree leaves from kenya. anim. feed sci. tech. 85:89-98. adeneye ja (1979). a note on the nutrient and mineral composition of leucaena leucocephala in western nigeria. anim. feed sci. tech. 4 (3):221-225. aoac (association of official analytical chemists) (1990). official methods of analysis. 15 th edition. washington, dc. ben saleem h, nefzaoui a, ben saleem l, tisserand jl (1999). intake, digestibility, urinary excretion of purine derivatives and growth by sheep given fresh, air-dried or polyethylene glycol-treated foliage of acacia cyanophylla lindl. anim. feed sci. technol. 78: 297-311. burns re (1963). methods of tannin analysis for forage crop evaluation. univ. of georgia agr. exp. sta. tech. bull. n.s. 32. p. 14. burns re (1971). a method for estimation of tannin in grain sorghum. agron. j. 63:511-512. damothiran l, chandrasekaran nr (1982). nutrition studies with leucaena forage. leucaena res. rep. 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ethiopian highland sheep. anim. prod. 51:127-133. tilley jma ,terry ra (1963). a two stage technique for the in vitro digestion of forage crops. j. brit. grassl. soc. 18: 104-111. topps jh (1992). potential, composition and use of legume shrubs and trees as fodder for livestock in the tropics (a review). j. agric. sci. (camb.). 118: 1-8. van soest pj, robertson jb, lewis ba (1991). methods for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition. j. dairy sci. 74: 3583-3597. wiegand ro, reed jd, combs dk (1996). leaves from tropical trees as protein supplements in diets for sheep. trop. agric. 73(1): 62-68. woodward a , reed jd (1989) the influence of polyphenols on the nutritive value of browse: a summary of research conducted at ilca. ilca bull. 35: 2-11. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (3), pp. 001-007, march, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effects of feed restriction on production and reproductive performance of rhode island red pullets etalem tesfaye1*, berhan tamir2, aynalem haile3 and tadelle dessie3 1 debre zeit agricultural research center, debre zeit, ethiopia. 2 faculty of veterinary medicine, addis ababa university, ethiopia. 3 international livestock research institute (ilri), addis ababa, ethiopia. accepted 23 january, 20019 the study examined the effect of skip a-day feed restriction on rhode island red chicken and evaluated the effect in relation to age, body weight, egg quality, weight of reproductive tract and liver at the onset of egg laying. two hundred 40 day-old chicks with mean body weight of 48.92 ± 1.53 g were randomly distributed to 12 pens each with 20 chicks, representing 4 feeding regimen of t1 (unrestricted, control), t2 (restricted at 7, 14, 21 and 28 days of age), t3 (restricted at 35, 42, 49 and 56 days of age) and t4 (restricted at 63, 70, 77 and 84 days of age). feed restriction was based on skipping a-day once in a week and the next day’s feed offer was based on the previous day’s feed intake. the average day to lay first egg was significantly longer (p < 0.05) for t3 treatment groups compared with the others. the values were 140.8 ± 0.8, 140.17 ± 2.5, 147 ± 2.2 and 143.83 ± 2.3 days for t1, t2, t3 and t4 groups, respectively. there was also highly significant (p < 0.01) difference in body weight (1305 ± 13.2, 1374.3 ± 23.2, 1352 ± 23.5 and 1429.23 ± 44.5 g for t1, t2, t3 and t4 groups, respectively) and the values were higher for birds restricted at later age of growth. egg weight at the onset of egg lay for the respective treatments were 42.68 ± 0.72, 42.43 ± 1.91, 47.9 ± 0.87 and 41.88 ± 0.77 g. pullets of t4 group had significantly (p < 0.01) heavier uterus weight and longer length in absolute term than the control. except for yolk diameter and shell thickness (p < 0.05), the egg quality parameters considered were not significantly (p > 0.05) different between the treatment groups. the result revealed the conclusion that feed restriction by skip a-day feeding system at t3 resulted in higher egg weight and can be considered as the optimum age for pullet rearing. additionally, feed restriction at t4 could also be recommended due to heavier uterus weight and longer length in absolute term as these attributes indicate good consistency for the subsequent egg production. keywords: body weight, egg quality, feed restriction, onset of egg laying, skip a-day. introduction in commercial poultry production system, profit can be attained by minimizing feed cost which accounts for more than half of the total cost of production. according to wilson and beyer (2000) feed cost accounts for 60 70% of the cost for poultry production. any attempt to improve commercial poultry production and increase its efficiency therefore, needs to focus on better utilization of available feed resources (dzarc, 1997). one of such means is restricting the amount of daily feed offer for sometime and stimulating compensatory growth (dunnington et al., 1992). hurwitz and plavink (1989) applied feed restriction in grow *corresponding author. e-mail: etalemtes@yahoo.com. ing egg type pullets and reported the delay in the onset of egg production leading to some increase in egg weight and improved overall feed efficiency. at the onset of egg production, egg weight is not large enough for commercial purpose. a delay in the onset of egg production caused by restricting the duration of light and/or feed increased egg size (bornstein and lev, 1984). this increase is in concomitant with increased body weight (plavink and hurwitz, 1983). hurwitz and plavink (1989) concluded that egg weight is a function of both age and body weight at the onset of egg produc-tion. feed restriction has been used to regulate rate of growth and to control age of sexual maturity in replacement pullets. it has led less consistently to other benefits such as lower laying hen mortality and increased as lower laying hen mortality and increased egg production (mbugua, 1985). feed restriction usually involves regulating the consumption of a well balanced diet or limiting the intake of specific nutritional factors as energy or amino acids. this paper reports the effect of feed restriction at different growth stages on the performance of rhode island red chicken in relation to age, body weight and egg weight at the onset of egg lay. materials and methods animals, experimental design and treatments the experiment was conducted at poultry farm of haramaya university, located at an altitude of 1980 m.a.s.l, 9 0 26 ' n latitude and 42 0 3' e longitude (aua, 1998). a total of 240 chicks with mean body weight of 48.92 ± 1.53 g were randomly distributed into 12 pens each with 20 chicks. the chicks were randomly assigned to the four feeding treatments [t1 (unrestricted, control), t2 (restricted at 7, 14, 21 and 28 days of age), t3 (restricted at 35, 42, 49 and 56 days of age) and t4 (restricted at 63, 70, 77 and 84 days of age] using a completely randomized design (crd). the birds maintained in deep litter system 15 cm depth and pen was designed 1.5 m x 1.5 m in width and length each with 2 round feeder and drinker throughout the experimental period. the chicks reared together until 84 days of age the period when the skipping days ended and then the male birds were culled from the pens as there is no reason keeping them till point of egg lay. after 12 weeks of age till the point of egg lay a total of 108 female birds were fed ad lib by measuring the daily intake and refusal per pen. feeds and feeding birds were fed on a commercial starter layer diet until the age of 6 weeks and on commercial grower layer diet up to the end of the experiment (at age of 22 weeks) . feed was offered to the birds twice a day except at days of feed restriction for t2, t3 and t4. the laboratory chemical analysis results of feeds used in the experiment revealed the crude protein and energy contents of the commercial feeds of 20.22% and 3144 me kcal/kg dm for the starter diet and 18.52% and 2883 me kcal/kg dm for grower’s layer feed offered, respectively. the cp and me contents of the diets were within the range of the recommended cp and me levels of starters and growers of 18% and 2950 me kcal/kg and 17% and 2850 me kcal/kg, respectively (leeson and summers, 2001). feed restriction was based on skipping aday (restrict daily ration) once in a week, totally 4 days at different age of development for each treatment for the whole experimental period and the next day’s feed offer was based on the previous day’s feed intake. the feed restriction was done after the completion of one treatment then the other was followed (t3 started when t2 was finished and then t4 was followed). on the feed restriction day only water was given. the daily feed intake was measured as the difference between feed offered and refused. measurements and observations data on feed intake, age at first egg laying, body weight, egg quality and weights of reproductive tract and liver were recorded. age at onset of egg laying (aoel) was fixed as the age at which the first egg was laid by 5% of the flock and the average date was taken for each treatment. the weight of the first laid egg in the trial in each group was determined by weighing the egg using the sensitive ba lance of 0.01 g precision and the date the egg was laid, pen number and egg weight were marked on it for later identification. egg quality was assessed in terms of egg weight, shell thickness, egg yolk color, albumen height, yolk diameter and haugh unit score (hus). for the measurements, 12 eggs per treatment and 4 per replication were taken and the average was computed for each quality parameters. the shell thickness was the average of the blunt, middle and sharp points of the egg and was measured using a micrometer gauge. albumen height was measured by tripod micrometer unit and hus was read from haugh unit. the yolk color was described by the so-called roche color fan as a range scale consists of a series of fifteen colored plastic strips, with one rated as very pale yellow and 15 a deep intense reddish orange. a description of the fan and the colors comprising it is given by vuilleumier (1969). at the end of the feeding trial (22 weeks), 6 randomly selected pullets from each treatment group were starved for 12 h and weighed immediately before slaughter. weight of ovary including yellow and white follicles as a whole, excluding white follicles (less than 6 mm in diameter) and excluding yellow follicles (generally greater than 6 mm in size or 0.3 g in weight) were taken. the diameter of a large yellow follicle was also measured by centimeter tape taking from each 2 slaughtered pullets and the averages were computed for the treatments. the number of yellow follicles was counted. absolute and relative (relative to body weight) weights and lengths of infundibulum, magnum, isthmus, uterus and vagina were also measured separately. the oviduct was measured as a whole using the sensitive balance. statistical analysis data were analyzed using the general linear model (glm) procedures of sas (sas, 2002). the fixed effect fitted in the model included the effect of treatment (control, t2, t3 and t4). tukey kramer test was used to separate means which were significant in the least squares analysis of variance (sas, 2002). the following model was used for the analysis. yij = µ + ti + eij where, yij = an observation (experimental unit) µ = overall mean ti = feed restriction effect of ith restriction level and eij = error term results and discussion dry matter intake and body weight the feed restriction of skip a-day program didn’t result in a significant (p > 0.05) difference in average daily dm intake between treatments and the control group (table 1). the non significant difference in daily feed intakes between the control and treatment groups were not in agreement with the findings of nir and nitsan (1979), pinchasove et al. (1985) and dunnington et al. (1992) who reported that intermittently fed group consumed considerably more on the following day than did ad libitum fed chickens. the similarity in daily dm intakes might be due to the fact that the amount of feed offered to birds on the next day was based on the previous day’s feed intake of the control group. the total dm intake where table 1. least squares means (± se) for dry matter intake and body weight change as affected by feed restriction. treatments parameters t1 t2 t3 t4 se p-value number of observation = 240 daily dry matter intake (g) 70.54 ± 3.19 66.68 ± 3.13 68.33 ± 1.32 64.69 ± 3.77 0.98 ns total dm intake (g) 11.17 a ±0.51 10.56 ab ± 0.50 10.82 ab ± 0.21 9.97 b ± 0.34 0.17 * initial body weight (g) 48.51 ± 0.34 48.48 ± 0.77 49.03 ± 0.67 49.65 ± 1.00 0.23 ns daily body weight gain (g) 8.933 ± 0.23 8.967 ± 0.60 9.133 ± 1.22 9.067 ± 0.61 0.19 ns final body weight (g) 1433.00±82.23 1438.00 ± 95.02 1464.00 ± 89.30 1451.00 ± 92.31 28.94 ns a, b, c = means within a row with different superscripts are significantly different; * t1 = unrestricted, control, t2 = restricted at 7, 14, 21 and 28 days of age, t3 restricted at 63, 70, 77 and 84 days of age. = p < 0.05; ns = non-significant; se = standard error; = restricted at 35, 42, 49 and 56 days of age and t4 table 2. least square means (± se) for effect of feed restriction on age at onset of egg laying, body weight and egg qualities. treatments parameters t1 t2 t3 t4 se p-value number of observation = 108 140.80 ± 0.80 bc 140.17 ± 2.50 c 147.00 ± 2.20 a 143.83 ± 2.30 b age at onset of egg laying (days) 0.96 * body weight (g) at first egg 1305.00 ± 13.20 b 1374.30 ± 23.20 ab 1352.00 ± 23.50 ab 1429.23 ± 44.50 a 15.18 ** egg weight (g) 42.68 ± 0.72 b 42.43 ± 1.91 b 47.9.00 ± 0.87 a 41.88 ± 0.77 b 0.79 ** shell thickness (mm) 0.26 ± 0.01 ab 0.25 ± 0.00 b 0.26 ± 0.01 ab 0.29 ± 0.01 a 0.01 ** height of albumen (mm) 8.38 ± 0.38 7.84 ± 1.26 8.65 ± 0.41 7.60 ± 0.09 0.21 ns diameter albumen (cm) 6.69 ± 0.63 6.72 ± 0.28 7.12 ± 0.28 7.03 ± 0.16 0.11 ns diameter of yolk (cm) 3.47 ± 0.06 ab 3.44 ± 0.06 ab 3.49 ± 0.01 a 3.38 ± 0.06 b 0.02 * yolk color (roche fan) 4.33 ± 0.58 4.33 ± 0.29 4.50 ± 0.50 4.67 ± 0.29 0.11 ns hus 95.43 ± 2.03 93.07 ± 4.75 95.75 ± 2.25 86.52 ± 9.38 1.75 ns a, b, c = means within a row with different superscripts are significantly different; ** = p < 0.01; * = p < 0.05; ns= non-significant; se= standard error; t1 = unrestricted, control, t2 = restricted at 7, 14, 21 and 28 days of age, t3 = restricted at 35, 42, 49 and 56 days of age and t4 restricted at 63, 70, 77 and 84 days of age. as were significantly (p < 0.05) different between the groups. birds under t4 consumed less feed during the period compared to the control group (table 1). the reason might be due to the fact that the feed restriction applied at later age and at the time the daily requirement was high and it was reduced the total consumption than the other groups relatively due to skipping feed. the results obtained in the present experiment were similar to the findings of zubair and leeson (1994) and mcmurtry et al. (1988) who reported that significantly less feed was needed per unit of weight gain as did controls that after undergoing nutritional stress by consuming a diluted diet. there were no significant (p > 0.05) differences in initial body weight, final body weight as well as average daily weight gain of the birds under feed restricted and the unrestricted groups (table 1). the results indicated that the feed restriction at different age groups did not affect the performance of the birds in terms of body weight change. these findings were in agreement with the reports of zubair and leeson (1994), who suggested that increased growth was due, in some way, to better nutrient utilization. in addition, the results obtained in the present study were also in agreement with the findings of plavink and hurwitz (1985), calvert et al. (1989), fontana et al. (1992), jones and ferrell (1992) and scheilder and baugnma (1993) who reported the lower food intake of food restricted birds than that of birds given ad libitum access, but when access to feed was again unrestricted the birds exhibited an accelerated rate of body weight gain typical of compensatory growth. age at onset of egg laying and egg quality there was a difference (p < 0.05) in age at the onset of egg laying among the treatment groups (table 2) . the feed restricted birds in t3 delayed significantly (p < 0.05) to lay their first egg among the treatment groups. similar to the present findings, different researchers (hocking and whitehead, 1990; hocking, 1990, 1992) indicated the linear relationship between the number of yellow follicles and body weight in broiler breeders, turkeys and ducks which had been fed throughout rearing to achieve different proportions of the body weight. the reason might be due to the distribution of available energy to different organs during the feed restriction to make priority for tissue table 3. least square means (± se) for effect of feed restriction on relative weight of the different parts of oviduct. parts of oviduct treatments t1 t2 t3 t4 se p-value number of observation = 108 ± 0.01 b ± 0.01 b ± 0.02 a ± 0.01 b infundibulum (%) 0.08 0.07 0.12 0.08 0.01 ** magnum (%) 1.45 ± 0.01 a 1.13 ± 0.10 b 1.58 ± 0.15 a 1.33± 0.08 ab 0.06 ** isthmus (%) 0.35 ± 0.01 a 0.23 ± 0.01 c 0.31± 0.04 b 0.31 ± 0.01 b 0.01 ** uterus (%) 1.20 ± 0.04 1.10 ± 0.07 1.23 ± 0.16 1.32 ± 0.10 0.04 ns vagina (%) 0.25 ± 0.01 0.20 ± 0.01 0.23 ± 0.03 0.23 ± 0.01 0.01 ns oviduct (%) 3.55 ± 0.12 a 2.83 ± 0.08 b 3.60 ± 0.42 a 3.44 ± 0.20 ab 0.11 * a, b, c = means in the same row with different superscript are significantly different from each other, ** = p < 0.01, * = p < 0.05, ns = non-significant, se = standard error; t1 = unrestricted, control, t2 = restricted at 7, 14, 21 and 28 days of age, t3 = restricted at 35, 42, 49 and 56 days of age and t4 restricted at 63, 70, 77 and 84 days of age. growth. sahota and bhatti (2001) showed a reduction in feed intake and body weight of pullets and delayed age of sexual maturity due to feed restriction. body weight at the onset of egg laying was also significantly (p < 0.01) different between t4 and the control groups. similarly, egg weight was higher (p < 0.01) for t3 compared to t2, t4 and the control group (table 2). these results agreed with the findings of bornstein and lev (1984), who reported delayed onset of egg laying and increased egg size as the result of feed restriction. in addition, it was in agreement with the reports of hurwitz and plavink (1989) who found that feed restriction resulted in delay in the onset of egg production, leading to some increase in egg weight. however, baloch et al. (2001) found no significant differences in egg weight as a result of starvation or feeding regime. restricting caloric and/or protein intake before sexual maturity delays the onset of egg production and increases the number of ova that are packaged in to complete eggs throughout the laying cycle (etches, 1996). the present study revealed that birds that attained higher body weight at earlier age laid smaller eggs. the results concur with findings of summers et al. (1990) who indicated that the effect of early maturity is associated with the depression in egg weight produced subsequently. however, the results of plavink and hurwitz (1983) indicated that body weight was positively correlated with age at onset of egg production and egg weight. birds can start egg laying if higher body weight is attained at earlier ages (bornstein and lev, 1984) which is in agreement with the present results. the initial similarity of the sexual maturity plots for birds feed restricted at t2 and t4 with the control might be due to a similar number of birds in both feeding regimens having already reached the necessary body weight or body composition threshold for reproductive development (brody et al., 1980, 1984). shell thickness significantly (p < 0.01) varied between the feed restricted and unrestricted groups (table 2). the pullets reared under feed restriction at early (t2) had thin egg shell than t4. the results of the present study were not in agreement with the findings of kar et al. (1977), who reported that shell thickness of eggs were not influenced by feed restriction. the higher shell thickness in the groups might be due to the fact that the birds might have accumulated adequate amount of ca and developed bones well for later ca mobilization when feed restriction had occurred than the chicks feed restricted at early age of development (t2). height and diameter of albumen, yolk color and haugh unit score (hus) did not significantly (p > 0.05) differ between feed restricted and the unrestricted birds (table 2). yolk diameter differed (p < 0.05) between treatments and birds in t4 had performed poorly than t3. the result might imply improvement in the subsequent performance of the laying house in terms of egg weight for the group (t3). it could be noted that larger yolk diameter associated more with the large egg size. development of the reproductive tract weight and length of the different parts of oviduct: the least squares means of the effect of feed restriction on the relative weight and length of the different parts of oviduct are given in tables 3 and 4. the overall percent (p < 0.05) weight of oviduct was significantly lower in birds kept under t2 group compared with the control and t3. this indicated that the thickness of the wall of the oviduct might be associated with secretary activity and was much more in birds under feed restriction group t3 and the control. this higher proportional weight of the oviduct possibly indicates that adequate and preferential development of reproductive organs instead of other organs occurs at t3, t4 and control groups compared to early age (t2) restriction. the weight of infundibulum was higher (p < 0.01) in birds restricted at t3 (table 3). the relative to body weights of table 4. least squares means (±se) for effect of feed restriction on relative length of the different parts of oviduct. parts of oviduct treatments t1 t2 t3 t4 se p-value number of observation = 108 infundibulum (%) 0.49 ± 0.03 0.50 ± 0.02 0.54 ± 0.07 0.55 ± 0.05 0.01 ns magnum (%) 2.02 ± 0.06 2.08 ± 0.12 2.15 ± 0.20 2.08 ± 0.18 0.04 ns isthmus (%) 0.77± 0.04 ab 0.67± 0.03 bc 0.61 ± 0.06 c 0.83 ± 0.06 a 0.03 ** uterus (%) 0.60 ± 0.04 b 0.53 ± 0.04 b 0.49 ± 0.07 b 0.77 ± 0.06 a 0.04 ** vagina (%) 0.44 ± 0.02 0.38 ± 0.08 0.41 ± 0.05 0.43 ± 0.01 0.01 ns total (%) 4.32 ± 0.13 4.16 ± 0.18 4.19 ± 0.43 4.65 ± 0.35 0.09 ns a, b, c, d = means in the same row with different superscript are significantly different from each other, ** = p < 0.01, ns = non-significant, se = standard error, t1 = unrestricted, control, t2 = restricted at 7, 14, 21 and 28 days of age, t3= restricted at 35, 42, 49 and 56 days of age and t4 restricted at 63, 70, 77 and 84 days of age. table 5. least square means (± se) for effect of feed restriction on ovarian follicles. treatments parameters t1 t2 t3 t4 se p-value number of observation = 108 percentage weight of ovary 55.00 ± 2.00 48.90 ± 11.40 51.03 ± 3.20 50.77 ± 7.80 2.09 ns weight of white follicles (g) 5.80 ± 0.30 a 5.70 ± 0.30 ab 5.20 ± 0.10 b 6.10± 0.30 a 0.12 * weight of yellow follicles (g) 48.80 ± 2.00 43.10 ± 1.80 45.70 ± 3.30 44.70 ± 2.50 0.89 ns number of yellow follicles (no) 9.50 ± 1.00 8.30 ± 0.80 8.20 ± 0.60 9.00 ± 1.30 0.29 ns diameter of yellow follicle (cm) 2.60 ± 0.10 2.40 ± 0.26 2.90 ± 0.13 2.70 ± 0.20 0.07 ns percentage weight of liver 2.34 ± 0.20 2.48 ± 0.22 2.75 ± 0.32 2.52 ± 0.33 0.08 ns a, b = means in the same row with different superscripts are significantly different; * = p<0.05; ns= non-significant, se= standard error; t1=unrestricted, control, t2= restricted at 7, 14, 21 and 28 days of age, t3= restricted at 35, 42, 49 and 56 days of age and t4 restricted at 63, 70, 77 and 84 days of age. magnum, which is normally responsible for thick egg white synthesis was significantly (p < 0.01) higher for birds under t3 and control compared to t2. the recorded higher egg weight for t3 reflected the higher magnum weight that weight that could synthesis larger egg components. lakhotia (2002) reported that magnum contributes all the thick albumen of the egg weight and in turn results in higher egg weight. the lengths of magnum was not significantly (p > 0.05) different between treatment groups and the control. absolute as well as relative weight and length of isthmus, which is responsible for egg shell membrane formation, was significantly (p < 0.01) different between treatments. the t4 birds had greater weights of isthmus relative to body weight. pullets of t4 group had significantly (p < 0.01) longer length than t2, t3 and the control group. this might indicate that this group of birds may have good consistency for the subsequent egg production since they had showed good feed efficiency. information in the literature on the effect of feed restriction on weight of oviduct, infundibulum, magnum, isthmus, uterus and vagina is generally scanty and therefore, comparison with our findings could not be made sufficiently. in general, the overall absolute length of oviduct was significantly (p < 0.01) different between treatments. feed restriction at t3 tended to improve the egg size by promoting the development of cumulative oviduct part than other feed restriction stages. this finding probably indicates that a threshold of oviduct weight must be achieved before sexual maturity is attained, whatever the feeding regimen might be. weight of ovary and number of ovarian follicles: the mean weight of ovary including yellow and white follicles from the 4 feeding regime is shown in table 5. there was no difference (p > 0.05) in mean weight of ovary between treatments and the control. the white follicles were smaller (p < 0.05) in t3 as compared to the control and t4 group of the treatments. this might be due to the fact that birds under t4 may produce enough follicle stimulating hormones which are responsible for follicle development as a result of improved feed utilization. the weight, diameter and number of yellow follicles were not significantly (p > 0.05) affected by feed restriction. the entry of number of follicles into the growing phases was similar in both feed restricted and the control group. similarly, hocking (1993a) confirmed that the numbers of yellow follicles at the onset of lay were unaffected by the degree and age of restriction from 14 weeks of age. liver weight pullets reared under t3 had significantly (p < 0.05) heavier liver and there was no significant (p > 0.05) difference on liver percentage among the pullets reared on feed restriction and the unrestricted feeding regimen (table 5). the increase in liver weight was due to the reason for effective synthesis of available energy to lipid for egg yolk formation during the re-feeding time. the deposition of yellow yolk in the egg requires participation of the liver and the adrenal gland may participate in the control of ovarian function. interactions between these tissues might require to coordinate the assembly of the yolk and to prepare the largest follicle for ovulation (etches, 1996). the reason might be due to the fact that the increase in liver size may increase the effectiveness of yolk synthesis for large egg weight (etches, 1996). the same reason might be given for the fact that birds in t3 laid comparably larger egg than the other feed restricted groups and the control. pinchasove et al. (1985) found that intermittent feeding was accompanied by a consistent increase in the relative weight of the liver. however, katanbaf et al. (1989), susbilla et al. (1994) and jones (1995) reported a non significant difference in relative weights of liver by the feeding regime at the slaughter. the inconsistency of the result might be due to the effect of age at which feed restriction applied and type of feeding management (jones and ferrell, 1992). conclusions the findings of this study revealed that feed restrictions by skip a-day feeding system at middle age of development (t3) resulted in higher egg weight and this can be considered as the optimum age for pullet rearing. additionally, feed restriction at latter ages (t4) could also be recommended due to heavier uterus weight and longer length in absolute term as these attributes indicate that this group of birds has good consistency for the subsequent egg production. future work may consider the incorporation of additional days on skip a-day feeding system by increasing the total days either twice per week or more to obtain better performance on pullet rearing without sacrificing the bird’s production efficiency. in addition, the feed restriction method may also be tested at different laying phase of the birds and on total egg production. acknowledgements we are greatly indebted to the ethiopian institute of agricultural research (eiar) and haramaya university for covering the research cost. references aua (1998). alemaya university of agriculture. proceedings of the 15th annual research and extension review meeting, 2 april 1998, alemaya, ethiopia. pp. 29-30. baloch wa, suzuki h, onoue y (2001). effect of different fasting and feeding regimes on body weight and productive performance of layers after first induced molt. pakistan j. zool. 32 (3):275-277. bornstein s, lev s (1984). body weight and/or fatness as potential determinants of the onset of egg production in broiler breeder hens. brit poultry sci. 25:323-341. brody t, eitan y, soller m, nir i, nitsan z (1980). compensatory growth and sexual maturity in broiler females reared under severe food restriction from day of hatching. british poultry sci. 21:437-446. brody tb, siegel pb, cherry ja (1984). age, body weight and composition requirements for the onset of sexual maturity of dwarf and normal chickens. british poultry sci. 25: 245-252. dunnington ea, gross wb, siegel pb (1992). restricted feeding and broiler performance: age initiation and length of restriction. poultry sci. 71:440-447. debre zeit agricultural research center, dzarc (1997). annual research report 1996/1997. debre zeit, ethiopia. p 86. etches rj (1996). reproduction in poultry. cab international, ontario, canada n1g 2w1.p 318. hocking pm (1990). influence of the male on the decline of fertility with age in broiler breeder flocks. pp 213-219. in: j. p. brillard (ed). control of fertility in domestic birds. inra, paris, france. hocking pm, whitehead cc (1990). relationship between body fatness, ovarian structure and reproduction in mature females from lines of genetically lean or fat broilers given different food allowances. british poultry sci. 31:319-330. hocking pm (1992). effects of photostimulation at 18, 24, and 30 weeks of age on the productivity of female turkeys fed ad lib, or restricted until point of lay. british poultry sci.. 33:253. hocking pm (1993a). effects of body weight at sexual maturity and the degree and age of restriction during rearing on the ovarian follicular hierarchy of broiler breeder females. british poultry sci. 34:793-801. hurwitz s, plavink i (1989). severe feed restriction in pullets during the early growing period: performance and relationship among age, body weight and egg weight at the onset of production. poultry sci. 68: 914-924. jones gpd, ferrell dj (1992). early life food restriction of broiler chickens. ii. effect of food restriction on the development of fat tissues. british poultry sci. 33:589-601. jones gpd (1995). manipulation of organ growth by early life food restriction: it’s influence on the development of ascites in broiler chicken. british poultry sci. 36:135-142. kar rr, quisenberry jh, bradley jw (1977). egg quality and performance as influenced by restricted feeding of commercial caged layers. poultry sci. 56:1914-1919. katanbaf mn, dunnington ea, siegel pb (1989). restricted feeding in early and late feathering chickens on organ size and carcass composition. poultry sci. 68:359-368. lakhotia rl (2002). poultry eggs. al winton and kw winton (original) ed. pp 1-4. pub agrobios (india). (updated version). mbugua pn, austic re, cunningham dl (1985). effect of feed restriction on growth and metabolism of replacement pullets. poultry sci. 64:1950-1958. pinchasove y, nir i, nitsan z (1985). metabolic and anatomical adaptation of heavy bodied chickens to intermittent feeding. food intake, growth rate, organ weight, and body composition. poultry sci. 64:2098-2109. plavink i, hurwitz s (1983). organ weights and body composition in chickens as related to the energy and amino acid requirements: effect on strain, sex and age. poultry sci. 62:152-163. sahota aw, bhatti bm (2001). effect of feed restriction during growing period on laying performance of white leghorn hens. pakistan vet j 21(3):145-147.sas (2002). statistical analysis systems for mixed models. sas institute inc., cary, nc, usa. summers jd, spart d, atkinson jl (1990). restricted feeding and compensatory growth for broilers. poultry sci. 69:1855-1861. susbilla p, frankel tl, parkinson g, gow cb (1994). weight of internal organs and carcass yield of early food restricted broilers. british poultry sci. 35:677-685. vuilleumier jp (1969). the “roche yolk color fan” an instrument for measuring yolk color, pp. 457-481. in: p., hunton (ed). world animal science, poultry prod elsevier, tokyo. wilson kj, beyer rs (2000). poultry nutrition information for the small flock. kansas state uni agric exp stat coop ext serv. http://www.oznet.ksu.edu. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (1), pp. 001-010, january, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper is the ph drop profile curvilinear and either monophasic or polyphasic? consequences on the ultimate bovine meat texture abdelghani boudjellal2*, samira becila1, gerald coulis1, carlos hernan herrera-mendez1, laurent aubry1, jacques lepetit1, khaled harhoura 4, miguel angel sentandreu3, hamid aïtamar**and ahmed ouali1 1 inra, ur370 quapa, 63122 saint genes champanelle, france. 2 inataa, université de constantine, route de aïn el bey, 25000, constantine, algérie. 3 department of food science, instituto de agroquimica y tecnologia de alimentos (c.s.i.c.), apt. 73, 46100 burjassot, valencia, spain ** usthb, laboratoire de génie des procédés – environnement, b.p. 32, 16111 el-alia, bab-ezzouar, algérie. 4 ecole nationale vétérinaire d'el harrach, 12 av. hacène badi, 16010 el harrach, algérie. accepted 09 november, 2018 in meat science, ph has always been considered as an important determinant of meat quality including juiciness and tenderness. muscle acidification is generally believed to be a linear continuous and monophasic process. from examples provided in the literature, we showed that this was not the case and concluded that the ph profile is either exponential or sigmoidal but never linear. in addition these examples demonstrate that the profile is polyphasic. on this basis the ph drop profile was analysed in bovine longissimus muscle from 100 animals of different age, gender and breed, a choice aiming at increasing the variability of the ultimate tenderness. the results clearly indicate that most if not all animals present one or two transient decreases in the rate of ph fall leading to the appearance of plateau -like discontinuities. these transient stability of the ph are always observed soon after death (<8-9h) and within a ph range of 6.2 to 6.8. in beef, ultimate toughness of longissimus muscle from animals showing one ph stability step is significantly lower than that observed for animals showing two steps. animals with one step further showed a lower initial rate of ph drop and a lower extent of ph fall. animal age and sex affect tenderness which is higher for females and decreases with animal age. this relationship between the number of ph stability steps and the ultimate toughness of meat was confirmed using these other sources of variation in the ultimate quality of meat. regarding the different groups of age, a significant and linear relationship was found between the ultimate meat toughness and the percentage of animals showing either one step or two steps (r = 0.92). similarly, a significantly greater number of animals present one ph stability step in females than in males. taken together, these results demonstrated that muscle from animals showing only one stability step will provide more tender meat irrespective of their breed, sex and age. the potential origins of the ph stability steps and their relationship with meat toughness were then discussed. key words: ph, muscle, ph stability steps, meat toughness, cell death. introduction the rate and the extent of post mortem ph decline have been always considered to be of prime importance to *corresponding author. e-mail : boudjellal@caramail.com. tel : (213) 31 66 18 84.fax : (213) 31 66 18 84. ultimate meat qualities. according to bendall (1973) and warriss, bevis and ekins (1989), the characteristics of the ph decline are determined by the physiological condition of the muscles at the time of stunning, and can be related to lactate production, or to be more specific of the capacity of the muscle to produce energy in the form of table 1. bovine animals used in the present experiment: classification according to their breed, gender and age. breeds gender age (months) total 15 19 24 54 78 102 aubrac f 4 2 4 10 m 3 2 7 12 charolais f 2 5 6 13 m 4 4 7 15 limousin f 4 4 6 14 m 3 4 6 13 salers f 2 1 6 9 m 2 5 7 14 total 12 15 27 12 12 22 100 of atp. it has been shown that genotype as well as muscle type and amount of stress imposed on the animals in connection with pre-slaughter handling are major causes of the variations observed in ph decline. consequently, the capacity of the muscle to produce atp at the time of stunning is a result of an interaction between genotype, muscle type and environmental factors. (monin et al., 1986; rahelic and puac, 1980-81; fuji et al., 1991; le roy et al., 1990; monin et al., 1986). in vivo, atp is the immediate source of energy for muscle contraction. although a muscle fibre contains only enough atp to power a few twitches, its atp "pool" is replenished as needed. there are three sources of highenergy phosphate to keep the atp pool filled, i.e. creatine phosphate, glycogen and cellular respiration in the mitochondria. the pool of creatine phosphate in the fibre is about 10 times larger than that of atp and thus serves as a modest reservoir of atp. skeletal muscle fibers contain about 1% glycogen. the muscle fibre can degrade this glycogen by glycogenolysis producing glucose-1phosphate. this enters the glycolytic pathway to yield two molecules of atp for each pair of lactic acid molecules produced. not much, but enough to keep the muscle functioning if it fails to receive sufficient oxygen to meet its atp needs by respiration. however, this source is limited and eventually the muscle must depend on cellular respiration. after bleeding, very limited amounts of blood is present in muscle and oxygen supply from blood is very likely negligible. in such hypoxic environment, glycolysis and to a much lower extent, creatine phosphate, will constitute the last resort for energy production but cells cannot maintain anaerobic respiration for an extended length of time. glycolysis generates lactic acid which is the major cause of the concomitant postmortem acidification of muscle tissue. according to a large set of reports and in contrast to the conclusions of bendall (1973), ph drop is mostly consi dered as a pseudolinear process and the rate of ph fall was determined by linear regression analysis using the first experimental values (henckel et al., 2000) . some others estimate the rate of ph fall by measuring the ph value at predifined times and most commonly at 45 min, 3 or 6 h postmortem (klont et al., 2000). the two questions arising are (a) is the ph drop profile linear or curvilinear? and ((b) is it monophasic or polyphasic? in the present report, we provide evidence supporting that the ph drop profile is curvilinear and polyphasic using data taken from published works and personal data. the consequence of the number of phase on ultimate beef toughness was further analysed and discussed. materials and methods animals and sampling amongst 164 bovine animals of different breeds, sex and age slaughtered at the abattoir of the inra research centre for another experiment, 100 were used for the present study. the number of animals within each group (sex and age groups) is depicted in table 1. after captive-bolt stunning, exsanguination and dressing, m. longissimus was excised within 45-50 min after stunning. one longissimus muscle was used for toughness measurement whereas biochemical and physicochemical parameters were assessed on the other muscle. five 5 cm thick transversal slices (300 to 400 g each) were cut off from the last muscle. two of them were used for ph, osmolarity and water retention measurements during the 10 first hours following animal bleeding (day 0), one at times 1, 2 and 3 h post-mortem and the other at time 4, 6 and 8 h post-mortem. four of the five cuts were immediately vaccum packed and immersed in a water bath at 12°c. from the last sample, we took 20 to 30 g piece of muscle and vacuum packed the remaining part. three pieces of 1 g of muscle (3 replicates) were cut off from this sample, immersed in 9 ml of cold 5 mm iodoacetate and homogenised with a polytron homogeniser for subsequent ph measurement. this procedure was repeated at each sampling times. at 24 h all samples were transferred to the cold room at 4°c until subsequent use. no difference in temperature was found between meat samples from one experiment to another and 12°c was reached on average figure 1. curve fitting efficiency using an exponential decay of the ph for a muscle showing only one ph stability step (insert figure 1a). (a) fitting of the whole curve to determine the constant rate vtotal. open circles represent the experimental values and the thick black line is the fitted curve. the insert located the first ph stability step (closed circles) between 1 and 3 h post mortem. (b) determination of v1 by fitting the curve at the level of the ph stability step. (c) determination of vfinal by fitting the rest of the ph drop curve after translocation of the points to time 0, a modification enabling the verification of the maximum value estimated by the exponential model. at 80 ± 3 min after animal exsanguinations. this parameter was therefore no more considered. ph measurement 1 g of muscle cut into small pieces with a scalpel was immersed in 9 ml of 5 mm cold iodoacetate and homogenised with a polytron homogeniser. muscle ph was then measured with a combined glass electrode using a wtw 537 ph meter (amilabo, lyon, france). rheological assessment of meat toughness muscle toughness was assessed on raw meat according to lepetit and buffiere (1995). this measurement is independent on the collagen content enabling comparison of muscles with various amounts of connective tissue (lepetit, 1991). fitting of the ph profile for rate constant determination this was performed using the following exponential equation: phtime t = [(phmax-phmin)* exp(-kph * t)]+phmin where phmax and phmin were the maximum and minimum ph values; kph, the rate constant of ph fall, and t, the length of post-mortem storage (hours). rate constant were determined for all parts of the polyphasic ph drop curve (figure 1) including the first (v1) and the second step (v2) when present, the part of the profile between the last step and 72 h post-mortem (vfinal) and the whole profile (vtotal). fitting was carried out with the microsoft excel xp solver using the least square method. assuming that glycolysis is the only one process taking place in post-mortem muscle, we can effectively expect a linear ph decline. however, this assumption does not agree with the modern view of cell biology which is far different from the scientific basis of the proposal of bendall (1973) . our actual understanding of cell biology strongly supports the idea that glycolysis will be faced to antagonist processes which will preserve cells from acidification and fight for cell survival. hence, considering that ph is a discontinuous and polyphasic process, we are allowed to determine the rate of ph fall within each phase identified even if the identity of the underlying active antagonist process remain unknown. data collection from the literature published profiles of ph decline were scanned and curves digitized using the unscanit program from ritme informatique (paris). statistical analysis comparison of mean values was carried out using the student's t test and differences were significant for p<0.05. anova was performed using xlstat from addinsoft (paris). results is the ph drop profile curvilinear and either monophasic or polyphasic? to analyse these aspects, we first refer to published data found in the literature. bond and warner (2007) recently reported the ph drop profile observed in longissimus muscle from lambs subjected or not (control) (n=6) to exercise (n=6) before stunning. each point of the plot presented in figure 2a is the mean value for 6 lambs. both profiles are clearly not linear. the ph decrease is indeed either sigmoidal (control group) or exponential (exercised group). refined examination of the profiles showed that in control animals, the ph value is quite similar between times 30 and 60 min (grey closed circles). the only one linear region of the profile was indicated by a square between 3 and 5 h post-mortem. in exercised animals, a transient change in the rate of ph fall can be detected between 120 and 150 min as compared to previous points (grey closed triangles). the slope of the curve increased between these sampling times. in most published works, the starting ph value, i.e. the physiological value corresponding to time 0, which is assumed to be about 7.0, was almost always not included in the profiles. inclusion of the starting physiological value as the first data point will make it possible to estimate the initial speed of ph fall assuming an exponential decay (inserts figure 2a) . the transient changes in the rate of ph fall observed at times 30 min and 2 h for control and exercised animals respectively, are all the more realistic that each point is the mean value for 6 animals, a phenomenon known to tighten the differences. a second example was found in the paper from henckel et al. (2000) who analysed the effect of exercise and adrenaline on the ph drop in pig longissimus muscle. for our purpose, we considered only the exercise effect and compared the profile of exercised and control animals. figure 2b shows the plots of ph decline versus time for exercised and resting animals without (figure 2b) or with (insert figure 2b) the initial value, i.e. ph 7.0, respectively (adapted from henckel et al., 2000). as depicted in figure 2b, it can be stressed that the ph profile is overall not linear but rather exponential. in addition, the ph decrease seems to be regular and without any transitional change in the rate. magnification of the first points region by expanding the time scale and by introducing the initial physiological value revealed that a transient increase in the rate of ph fall can be detected at 15 and 45 min for exercised and control animals, respectively (large insert of figure 2b). furthermore, the initial rate of ph fall appears much higher in the exercised animal as compared to the control animal (small insert of figure 2b). although our analysis concerned only one animal from each group, the present finding is different from the conclusion of these authors who reported a similar rate of ph fall for both groups of animals. the reason is very likely that, in the cited work, these rates were determined by using the first experimental values corresponding to 1, 15, 30 and 45 min post-mortem assuming a linear ph decline (henckel et al., 2000), suggesting that all ph profiles must include the initial physiological value and the profile analysis must be carried out more accurately. hence, we might suspect that differences between these two groups of animals are very likely larger than those reported by henckel et al. (2000). the next example was from klont et al. (2000) who analysed the veal quality characteristics of animals classified into three groups (fast, intermediate and slow) according to their rate of ph decline (n=10 for each group). in that experiment, ph measurements were performed on rectus abdominis veal muscle at 0.75, 3, 24 and 48 h post-mortem. although a detailed analysis of the profile is impossible because of the low number of points, this example has been chosen to emphasize that in addition to a transient stagnation of the ph, a slight increase can be observed between 0.75 and 3 h postmortem within the "slow" group (figure 2c). expansion of the time scale confirmed the pseudo-stability of the ph in the "slow" group between 0.75 and 3 h post-mortem (insert figure 2c). finally, the last example concerned data published in 1996 by zamora et al. dealing with a kinetic analysis of ph drop in three bovine muscles including m. longissimus (l), triceps brachii caput longum (tb) and supraspinatus (ss). measurement of ph was carried out at 2, 4, 7, 10, 15, 24, 48 and 72 h post-mortem. as shown in figure 2d, the ph drop profile is sigmoidal for l and tb muscles and exponential for ss muscle. furthermore, in l and tb muscles, ph does not move to much between 2 and 4 h post-mortem, a feature emphasized by expanding the time scale and including the initial physiological value (grey closed symbol in the insert of figure 2d) . by contrast, a continuous exponential ph drop profile was obtained for ss muscle which is mainly composed of type i fibres. taken together, the examples presented clearly demonstrate that, in most cases, ph decline is not linear. it is either exponential or sigmoidal. in addition, in most muscle examined, a transient stability of the ph and even a slight increase can be observed supporting that ph drop is a discontinuous process. on this basis, we attempted to analyse the ph drop profile of bovine longissimus muscle from 100 animals of different age, sex and breed. similar investigation was also carried out on five lamb muscles including m. semimenbranosus (sm), briceps femoris (bf), rectus femoris (rf), longissimus lombarum (ll) and longissimus thoracis (lt). we further examine the consequence of the number of transient ph stability on the ultimate toughness of bovine longissimus muscle assessed by a mechanical method (lepetit and buffiere, 1995). polyphasic profile of ph drop in beef longissimus muscle kinetic analysis of post-mortem ph drop in beef longissimus muscle revealed a polyphasic process with one or two transient ph stability steps in the first hours post-slaughter (figure 3a). this feature was observed in all animals analysed in that experiment irrespective of their breed, sex and age. figure 3a showed the ph profile of 3 animals. these were a 54 months old charolais cull cow (f-ch-54), a 102 months aubrac cull cow (f-au 102) and a 24 months old salers young bull (m-sa-24). one transient ph stability step was observed between 2 and 3 h post-mortem for the 54 months old charolais cull cows whereas the 102 months old aubrac cull cows showed two steps between 1 and 3 h post-mortem for the first one and between 4 and 8 h post-mortem for the second. the salers 24 months old young bull exhibited only one stability step between 1 and 3 h post-mortem. the initial rates (first step) of ph drop were estimated to be 2.7 (f-ch-54), 1.1 (m-sa-24) and 0.7 (f-au-102) phunit/h. the configurations (1 or 2 steps) described here were observed for all breeds irrespective of animal age and sex. analysis of the post mortem ph drop profile needs therefore a relatively high frequency of time course measurements. one or two ph stability steps: what consequences on the ultimate toughness? in order to increase the variability in ultimate quality traits, especially tenderness, the present experiment was carr figure 2. profile of ph drop taken from the literature. (a) ph drop profile of lamb longissimus muscle from resting and exercised animals. inserts correspond to different expanded time scales for the first sampling times. each point is the mean value for 6 animals (adapted from bond and buttler, 2000) . (b) ph drop profile of pig longissimus muscle from resting and exercised animals. inserts correspond to different expanded time scales for the first sampling times (adapted from henckel et al., 2001). (c) ph drop profile of bovine rectus abdominis muscle from slow, intermediate and fast glycolysing animals. resting insert corresponds to an expanded time scales for the first sampling times. each point is the mean value for 10 animals. (adapted from klont et al., 2000). (d) ph drop profile of bovine longissimus muscle. insert corresponds to an expanded time scales for the first sampling times (adapted from zamora et al., 1996). figure 3. ph drop profiles in bovine muscle: ph profile of longissimus muscle from three different animals including a charolais 54 months old cull cow (f_ch_54m), an aubrac 102 months old cull cow (f_au_102m) and a 24 months old salers young bull (m_sa_24m). figure 4. toughness of bovine longissimus muscle (mean ± sd) from two sets of animals showing one ph stability step (1 step; n= 53) or two ph stability steps (2 steps; n=47). the difference between the two groups is highly significant. (p <0.001). ied out on 100 animals of different breeds, age and sex. ultimate toughness of longissimus muscle was estimated mechanically after 14 days of storage at 4°c and the mean value of the set of animals showing one ph stability step (n=53) was compared to the mean value of the other set with two ph stability steps (n=47). as shown in figure 4, muscles from animals with one ph stability step are significantly less tough after 14 days of ageing than those with two ph stability steps (p<0.001). the question arising is to know what could be the relationship with the rate and extent of ph fall? to answer this question, different rate constants characterising the ph profile were determined by fitting the experimental curve to an exponential equation. the first one take into account the whole set of ph values from 1 to 72 h postmortem (vtotal). the second (v1) and the third (v2) rate constants correspond to the rate of ph fall during the first and second stability steps, respectively. the last value (vfinal) represents the rate of ph fall after the last stability step up to 72 h. on the other hand we determined the extent of ph fall using the 24 h ph value as the end point of the rigor process since, for longer periods, the ph increased more or less. as shown in table 2, the set of animals with one ph stability step exhibited a significantly lower magnitude of ph drop (1.47 versus 1.50 for one and two steps respectively; p<0.05) and initial rate of ph fall (v1) (1.25 versus 1.61 for one and two steps respectively; p<0.05). by contrast no significant differences between the two groups were found for vtotal and vfinal. the data of table 2 fit quite well with the above statements since animals with 1 step are more tender and exhibited higher ultimate ph and lower rate of ph fall in the first hours post-mortem than those with 2 steps. number of ph stability steps in males and females: relationship with ultimate toughness although still very controversial, most studies suggested that females provide more tender meat than males. hence, we tested here this assumption and found that longisimus muscle from females was significantly more tender (p=0.031) than the same muscle from males (figure 5a). as the above results suggested that animals with one ph stability step would give more tender meat, figure 5. toughness of longissiumus muscle from female and male as affected by the presence of one or two ph stability steps. (a) comparison of female (n=46) and male (n=54) ultimate toughness of longissimus muscle; male are significantly tougher than female (p<0.05). (b) percentage of animals showing one or two ph stability steps within each group. steps. a major proportion of females exhibited only one step and conversely a greater proportion of males showed two ph stability steps. (c) toughness of bovine longissimus muscle (mean ± sd) from two sets of animals showing one or two ph stability step within each group (females and males). longissimus muscle from males with two steps are significantly tougher than the same muscle from animals with one step (p<0.001). no significant difference was observed in the female group. figure 6. toughness of longissiumus muscle from female and male of different ages (a) and relationship with the percentage of animals showing two ph stability steps (b). .(a) as affected by the presence of one or two ph stability steps. we tested whether the presence of only one ph stability step is more frequently observed in females than in males. as depictured in figure 5b, 67% of the females showed only 1 ph stability steps versus 40% in males supporting that the presence of only one ph stability step is often associated with more tender meat. accordingly, males with one step were significantly (p<0.01) more tender than those with two steps (figure 5c). although not significant, females with one ph stability step exhibited lower ultimate toughness (figure 5c). variability in the number of ph stability steps with animal age as for sex, in the present experiment we tested the hypothesis suggesting that ultimate tenderness of meat decreased with animal age. results of figure 6a comforted this statement since, for males and females, ultimate toughness of meat increases with animal age. the fact that this change occurs more sharply in males can be probably explained by the reduced range of age considered for male (15 to 24 months) as compared to females (54 to 102 months). plotting of the percentage of animal with two ph stability steps (figure 6b) versus ultimate toughness showed a positive linear relationship with a high correlation coefficient (r = 0.92). in agreement with the results reported above, this would suggest a great impact of the number of ph stability steps in the first hours postmortem on the ultimate meat toughness. the presence of one step would be synonymous of a better ultimate tenderness. table 2. significance of the difference between animals showing 1 or 2 ph stability steps in the extent of ph drop (amplitude), the rate of ph drop over the whole rigor process (vtotal), the rate of ph drop over the first ph stability step (v1) and over the second stability step (v2) and finally over the last part of the curve between the last ph stability step and 72h post-mortem (vfinal). amplitude vtotal v1 v2 vfinal 1 step 1.47 ± 0.07 0.12 ± 0.07 1.25 ± 0.79 0.17 ± 0.11 2 steps 1.50 ± 0.06 0.13 ± 0.08 1.61 ± 0.62 1.22 ± 0.82 0.21 ± 0.15 p 0.022* 0.51 ns 0.0129* 0.125 ns table 3. variance analysis of the effect of gender, breed and age on the magnitude of ph drop at 1h post-mortem. anova performed with xlstat (addinsoft, paris). source ddl sum of squares mean of squares f pr > f gender 1 0.258 0.258 6.439 0.012 breed 3 0.374 0.125 3.116 0.028 age 4 0.202 0.051 1.265 0.286 discussion the conversion of muscle into meat starts with the onset of the rigor mortis, a phase during which energy stores (phosphocreatine, atp and glucose derivatives) are exhausted. rigor is associated with a drop of the ph and a loss of the muscle elasticity which reached its maximum toughness (bendall, 1973). muscle acidification has always been considered as a major determinant of meat quality traits including juiciness and tenderness. however, this characteristic assessed through ph measurement, is the consequence of complex and not well understood processes. as soon as blood flow ceases, muscle cells will be subjected to hypoxia and quickly after to hyperosmotic conditions. the cell responses to these stresses are multiples. first of all, the cytosolic ph drops from ph 7.47.5 to 6.7-6.8 within 15 min after exposure to either hypoxia or hyperosmotic conditions or both (satre et al., 1989; pintsch et al., 2001). when a tissue experiences hypoxia, small amounts of oxygen can exert a vitally important influence on cellular energetics (wilson et al., 1988; gnaiger et al., 1995). gnaiger et al. (2001) thus reported that oxidative phosphorylation is more efficient under oxygen limitation suggesting that mitochondria will be more efficient to produce atp. another feature reported under low oxygen content is the phosphoryation of myosin light chain after activation of the myosin light chain kinase (kuwayama et al., 1996, 1998; oyama, 1996), a change noticed in post-mortem muscle and associated with tender meat (bouley et al., 2004). in cancer chemotherapy, hypoxia-induced acidification may even cause resistance to the drugs used for this purpose (greijer et al., 2005). all these events probably occur in post-mortem muscle. with the development of hypoxic conditions after bleed ing, the first modification will be very likely a rapid decline of the ph. this feature is probably the major cause of the amplitude of the ph drop always observed upon the first sampling time (in general 30 to 60 min post-mortem). the ph value at the first sampling time is highly variable and depends on numerous factors including animal species, individuals, muscle type and others. assuming an initial physiological ph of 7.0, a ph drop ranging from 0.1 to 0.44 ph unit can be calculated at 1 min post-mortem from the data reported by henckel et al. (2000). the variability depends on the muscle considered and the treatment the animals were subjected to before stunning (exercised or not). for the three muscles studied (m. longissimus, semimembranosus and biceps femoris ) the magnitude of the ph drop were, on average, 0.16 ± 0.07 and 0.39 ± 0.06 ph unit for the control and the exercised groups of pigs, respectively. in the present experiment, mean values for each group of animals in the ph at 1 h post-mortem (different sex, age and breed groups) ranged between 0.32 to 0.50 ph unit. variance analysis indicates a significant effect of sex (p = 0.012) and breed (p = 0.028) on the ph at 1 h post-mortem whereas no effect was observed for age (p=0.286) (table 3). in that case, the very fast drop of ph immediately after bleeding can probably not be ascribed to glycolysis and the subsequent accumulation of lactic acid. the high level of protons originated very likely from hydrolysis of atp to adp, one proton released being associated to the hydrolysis of one atp, suggesting an intense metabolic activity of the cell just after death. this was supported by the increase in the level of enzymes involved in the oxidative and the glycolytic pathways within 20 min after slaughter, two pathways providing the energy needed to preserve cells from death and/or to setup the program cell death machinery (jia et al., 2006a,b). the findings of jia et al. (2006a, b) further demonstrate that protein syn thesis takes place after animal bleeding and prove the intense metabolic activity of post-mortem muscle cells suggested before. although not exhaustive, the few examples from the literature included in the present paper clearly indicate that ph decline is not linear but rather exponential or sigmoidal with a linear part limited to few experimental points. it is therefore incorrect to estimate the rate of ph decline assuming a linear decline. this discontinuity in the time course profile is strongly supported by one of these examples (klont et al., 2000) which showed a slight increase in the ph of beef muscle between 45 min and 3 h post-mortem. such transient limited increases in the ph were sometimes noticed in our experiment. evidences provided therefore mostly agree with transient changes in the rate of ph fall and transient stability of this variable called plateau. it can be further stressed that the causes of muscle acidification are multiple and proceeds through a series of antagonist processes acting towards or against ph drop. hence, the mechanisms established in the 70's must be reviewed in the light of the recent improvements in our understanding of cell biology. on the basis of these different observations supporting a non linear and discontinuous drop of ph, we analysed here the ph profile of longissimus muscle from 100 bovine animals of different age, sex and breed and try to clarify the consequences of the plateaux on the ultimate tenderness of meat assessed mechanically after 14 days of storage. the selection of animals of various age, sex and breed in the present experiment aimed at increasing the variability of ultimate meat tenderness. in bovine longissimus muscle, we observed one or two ph stability steps occurring essentially in the first ten hours post-mortem and in a ph range of about 6.2 to 6.8. animals with one step showed a significantly lower magnitude of ph drop (p<0.05) and a lower initial rate of ph fall (p < 0.05). results of previous studies about the relationship between early-postmortem muscle ph and extent of ph fall with beef tenderness have been inconsistent (reviewed in monin and ouali, 1990; eilers et al., 1996). in the present study, higher initial rates of ph fall determined by curve fitting were found to provide tougher meat, a result in agreement with those of zamora et al. (1996 and 2005). this contrasted with the controversial and inconsistent relationship between ph 3h post-mortem and ultimate meat toughness (eilers et al., 1996) . this discrepancy might originate from the fact that ph 3 h underestimates the initial rate of ph drop since this point is, in most cases, included in the first ph stability step. similarly to the findings of zamora et al. (1996; 2005), a greater extent of ph fall leading to more acidic meat cuts provides also tougher meat. in addition, animals with one stability steps provide significantly more tender meat irrespective of sex, age and breed ((p<0.001). to comfort this finding, two other sources of variability in the ultimate meat toughness were then analysed in cluding sex and animal age. although very controversial, meat from female cattle was found to be more tender than males while tenderness seemed to decrease with animal age. the number of reports supporting these assumptions is quite similar to the number providing converse findings (see prost et al., 1975). hence, faced to these uncertainties, we tested the effect of age and sex on ultimate meat tenderness. ultimate toughness was thus found to increase with age and to be lower for female than for male. on this basis, we then analysed the effect of age and sex on the number of ph stability steps in longissimus muscle. regarding the animal gender effect, the percentage of animals showing one ph stability step is much greater for females than for males comforting our previous findings suggesting a greater tenderness in animals with only one stability step. for males, the proportion of animals with two ph stability steps is even much higher than those with one step. although the biological causes of this difference between males and females are not clearly known (reviewed in monin and ouali, 1990), a better understanding of the origins of these stability steps would be very likely helpful. the second source of ultimate toughness variability investigated was animal age. it is well recognized that animal age is an important determinant of meat quality including tenderness (reviewed in monin and ouali, 1990). our results clearly showed that, for both males and females, ultimate toughness increased with animal age. the plot of ultimate toughness versus the percentage of animals with two ph stability steps, a characteristic previously related to tougher meat, showed a high positive correlation between these variables (r=0,92). the present findings therefore provide a new predictor of meat tenderness based on the transient ph stability steps, one step corresponding to more tender meat and conversely, two steps are indicative of tougher meat. how the presence of ph stability steps in the first hours post-mortem can be explained? on the basis of the current understanding of the conversion of muscle into meat, it is difficult to find out answers to the related questions. indeed, the current concept is based on the following view. when phosphocreatine stores are exhausted, the required energy is mainly produced through the anaerobic degradation of glycogen by glycolysis. the rate of the process first depends on the efficiency of the glycolytic pathway, on the level of glycogen stores and on the buffering capacities of muscle cells. as long as glycolytic enzymes are not inhibited by acidic ph, acidification will proceed regularly and proportionally to the glycolytic pathway efficiency. the discontinuity in the ph fall observed in the present experiment cannot be explained by a transient reduction in the activity of phosphocreatine kinase and other enzymes of the glycolytic pathway but rather by a modification of either the buffering capacity of muscle cells or a transient more effi cient control of muscle cell ph. a more efficient control of the ph is supported by the fact that a transient slight increase in the ph was sometimes observed instead of a flat plateau. in recent review papers, we proposed that the first step of the conversion of muscle into meat is not the rigor mortis but the onset of the cell death programme or apoptosis (ouali et al., 2006; herrera-mendez et al., 2006). the onset of the apoptotic process is energy dependent and includes the synthesis of a series of proteins involved in different cellular regulation pathways and necessary for the completion of the cell death process. these include caspases, a large set of kinases, hsps, etc (jia et al., 2006ba). this was supported by the clear shift in energy metabolism in the post mortem muscle resulting from an increase in enzymes involved in the glycolytic pathway as well as in the tri-carboxylic acid cycle indicative of greater aerobic and anaerobic energy metabolism in the first hours post-slaughter (jia et al., 2006b). amongst these over-expressed proteins, some contributes to cell defense and survival whereas others act for the progression of the apoptotic process, the balance between these antagonist pathways shifting rapidly to the onset of apoptosis. in addition, the intense metabolic activities of muscle cells just after exsanguination need a precise regulation of the intracellular ph which could counteract the acidification process. we further suggested, that the membrane polarity inversion might led to the replacement of acidic charges (phosphatidyl-serine) by basic charges (phosphatidylethanolamine, phosphatidyl-choline) counteracting the acidification process. in addition this membrane polarity inversion will cause very likely important modifications in the membrane fluidity which will become more permeable to salts and small metabolites molecules. membrane fluidity is indeed a strictly controlled process (simkiss, 1998; vigh et al., 1998). acknowledgements thanks to the commissariat au développement economique et à l’aménagement du massif central who funded this project in collaboration with f.n.a.d.t. references bouley j, meunier b, culioli j, picard b (2004). analyse protéomique du muscle de bovin appliquée à la recherche de marqueurs de la tendreté de la viande. rencontres recherches ruminants 2004. eilers jd, tatum jd, morgan jb, smith gc (1996).modification of earlypostmortem muscle ph and use of postmortem aging to improve beef tenderness. j. anim. sci. 74: 790-798. gnaiger e, steinlechner-maran r, mendez g, eberl t, margreiter r (1995). control of mitochondrial and cellular respiration by oxygen. j. bioenerget. biomembr. 27: 583-596. greijer ae, de jong mc, scheffer gl, shvarts a, van diest pj, van der wall e (2005). hypoxia-induced acidification causes mitoxantrone resistance not mediated by drug transporters in human breast cancer cells. cell. oncol. 27: 43-49. herrera-mendez carlos hernan, samira becila, abdelghani boudjellal and ahmed ouali. 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zamora f, aubry l, sayd t, lepetit j, lebert a, sentandreu ma, ouali a (2005). serine peptidase inhibitors, the best predictor of beef ageing amongst a large set of quantitative variables. meat sci. 71: 730-742. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (7), pp. 001-005, july, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper scoparia dulcis protects against trypanosoma brucei-induced immunosuppression in experimentally infected rabbits n.e.j. orhue1*, e. a. c. nwanze1 and a. okafor2 1 department of biochemistry, faculty of life sciences, university of benin, p.m.b 1154, benin city nigeria. 2 department of molecular, cell and developmental biology, university of california, los angeles, 405 hilgard avenue, p. o. box 951361, los angeles, ca. 900095-1361. accepted 10 march, 2019 the present paper summarizes our findings on the effect of scoparia dulcis on the population of immune cells during a 28 day experimental trypanosoma brucei infection in rabbits. the result obtained showed that infection resulted in an initial rise in both total white blood cells (wbc) and the absolute number of circulating lymphocytes followed by a progressive decrease in total wbc and all wbc subtypes namely; lymphocytes, monocytes and granulocytes, although the % lymphocytes (lymphocytes expressed as % of total wbc) remained consistently higher than normal throughout the study period. these changes are consistent with the development of trypanosome-induced immunosuppression in their mammalian host. treatment with s. dulcis at a daily oral dose of 25 mg/kg body weight significantly reduced the severity of the observed lesions (p < 0.05) when compared with untreated infected animals. thus the herb demonstrates significant potency in protecting against the parasite induced decrease in the population of immunologically active cells. key words: trypanosoma brucei, immunosuppresion, scoparia dulcis, rabbit. introduction african trypanosomiasis is a disease complex caused by several species of the parasitic protozoan trypanosoma. the disease can affect both wild and domesticated mammals (ashcroft et al., 1959). infection of humans with either trypanosoma brucei gambiense or trypanosoma brucei rhodesiense results in sleeping sickness a debilitating and often fatal disease (truc et al., 1992). although there is massive immune response mounted by the host secondary to infection, this is often overpo-wered by the ability of the parasite to express variable surface glycoproteins and thus evades the host’s immune response (spriggs, 1985). the result is a profound immunosuppression that lowers the host’s resistance, paving way for other infections which further complicates both the clinical and pathological features of the disease. the immunosuppression that develops is the result of both a progressive loss in the population of immunolo-gically active cells as well as the ineffectiveness of the *corresponding author. e-mail: jerryorhue@yahoo.com. humoral immune response owing to the phenomenon of antigenic variation. the more complex phenomenon of antigenic variation may be viewed as a survival strategy and consist of spontaneous, periodic changes in the molecular architecture of the variant surface glycoprotein that serves as a protective blanket on the parasite cell membrane (cross, 1975, 1976; borst and greaves, 1987; pays et al., 1989; borst et al., 1996, 1998; barry, 1997; cross et al., 1998; deitsch et al., 1997; pays and nolan, 1998). the severity of the clinical signs as well as the degree of mortality depends on several factors including the dose and virulence of the parasite as well as the specie and breed of affected animals (moulton and sollod, 1976; suliman and feldman, 1989). current research efforts in our laboratory have been focused partly on studying the biochemical lesions associated with experimental african trypanosomiasis, including the possibility of managing the disorder using locally available medicinal plants. the present paper focuses on the amelioration of trypanosome induced loss in the population of immune cells using scoparia dulcis (vassourinha, bitterbroom or sweetbroom weed), a widely table 1. effect of scoparia dulcis on trypanososmeinduced decrease in total white blood cells. groups total wbc (x 10 -3 /u/l) day 0 day 7 day 14 day 21 day 28 group i 8.541 a 8.397 a 8.613 a 8.428 a 8.602 a 0.216 0.304 0.195 0.206 0.324 group ii 8.422 a 9.860 b 9.276 a 6.851 b 5.030 b 0.304 0.231 0.165 0.202 0.154 group iii 8.643 a 9.610 b 10.463 b 5.820 c 3.012 c 0.278 0.164 0.241 0.136 0.102 values are mean ± s.e.m. values on the same column, but with different superscripts differ significantly (p < 0.05). table 2. effect of scoparia dulcis on trypanosome-induced decrease in total circulating lymphocytes groups total lymphocytes (x 10 -3 /u/l) day 0 day 7 day 14 day 21 day 28 group i 5.114 a 5.050 a 5.340 a 5.033 a 5.264 a 0.118 0.130 0.124 0.116 0.120 group ii 5.091 a 6.920 b 6.740 b 4.503 b 3.420 b 0.130 0.125 0.126 0.100 0.108 group iii 5.066 a 7.188 b 8.383 c 4.653 b 2.258 c 0.128 0.132 0.138 0.090 0.112 values are mean ± s.e.m. values on the same column, but with different superscripts differ significantly (p < 0.05). distributed herb reputed for numerous ethnomedicinal values (branch and dasilva, 1983; denis, 1988). many aspects of the several speculated pharmacological properties of s. dulcis have been validated by scientific research and includes the hypoglycaemic, anti-tumour promoting and anti-viral activities (jain, 1985; nashino, 1993; hayashi, 1990). phytochemical screening of the herb revealed that it is rich in flavonoids and terpenes and the pharmacological actions of s. dulcis are believed to be due to the presence of these phytochemicals (hayashi, 1987, 1990, 1991; kawasaki, 1987; ahmed, 1990). materials and methods animal treatment a total of 15 healthy rabbits of the new zealand white breed (initial average weight = 1.50 kg) obtained from a private farm in benin city were used for the experiment. these were randomly divided into 3 groups of n = 5 and with all groups of animals allowed a 2 week orienttation prior to the commencement of experiment. animals in groups 1 served as controls while those in groups 2 and 3 where inoculated with t. brucei. inoculation was by intraperitoneal injecttion of 0.5 ml of a 1:1 (infected whole blood: normal saline) preparation and with each inoculum containing about 2 x 10 6 of the parasite. parasite estimation was by the rapid “matching” method (herbert and lumbsden, 1976) the original stock of t. brucei was obtained from the nigerian institute for trypanosomiasis research table 3. effect of scoparia dulcis on trypanosome-induced decrea-se in total monocytes. groups total monocytes (x 10 -3 /u/l) day 0 day 7 day 14 day 21 day 28 group i 1.707 a 1.770 a 1.760 a 1.622 a 1.726 a 0.082 0.078 0.102 0.124 0.095 group 1.773 a 1.540 a 1.394 a 1.198 b 0.889 b ii 0.120 0.116 0.110 0.104 0.076 group iii 1.913 a 0.984 b 0.852 b 0.638 c 0.465 c 0.115 0.087 0.096 0.010 0.102 values are mean ± s.e.m. values on the same column, but with different superscripts differ significantly (p < 0. vom, plateau state, nigeria, group 1 animals (control) were each given intraperitoneal injection of 0.5 ml of normal saline instead of parasite. all animals were allowed unlimited access to food (growers pellets, product of bendel feeds and flours mill, ewu, edo state, nigeria) and clean drinking water. in addition, animals in group 2 were given scoparia dulcis at a daily oral dose of 25 mg/kg body weight throughout the 28 day study period. the required weight of the air-dried and pulverized herb was administered as an aqueous suspension by gavage. blood samples were collected prior to infection on day 0 and analyzed for baseline values. subsequent data obtained on day 7, day 14, day 21 and day 28 were compared with these pre infection values within group in order to ascertain the pattern of observed changes. in addition, comparisons were also made across groups to evaluate the extent of trypanosome induced changes as well as the degree of s. dulcis-mediated amelioration. for the avoidance of doubt, blood samples were collected in edta bottles and the subsequent analysis carried out within a few hours of sample collection. haematological analysis all haematological parameters were auto analyzed by means of an automated beckam coulter autoanalyzer. statistical analysis statistical analyses were done using the analysis of variance (anova) and post test multiple comparisons were by the turkeykrammer method. in all instances p values < 0.05 were considered statistically significant. results the results obtained from this study are presented in tables 1 5. the data are supportive of previous findings that t. brucei is immunosuppressive. whereas the number of circulating monocytes and granulocytes decreased on day 7 post-infection, and continue to do so steadily for the rest of the study period, total white blood cell count (wbc) and lymphocyte patterns showed an initial progressive increase on day 7 and day 14 and thereafter followed a drastic downward slide throughout the rest of the study period. by day 21and beyond, all parameters investigated had reached dangerously low levels, falling to table 4. effect of scoparia dulcis on trypanosome-induced decrea-se in total granulocytes. groups total granulocytes (x 10 -3 /u/l) day 0 day 7 day 14 day 21 day 28 group i 1.635 a 1.577a 1.510 a 1.773 a 1.512 a 0.058 0.032 0.092 0.063 0.052 group ii 1.556 a 1.410 a 1.146 b 1.149 b 0.712 b 0.069 0.054 0.072 0.045 0.021 group iii 1.661 a 1.438 a 1.228 b 0.629 c 0.257 c 0.072 0.047 0.081 0.026 0.034 values are mean ± s.e.m. values on the same column, but with differrent superscripts differ significantly (p < 0.05). table 5. effect of scoparia dulcis on trypanosomeinduced decrease in % lymphocytes. groups lymphocytes (% of total white blood cells) day 0 day 7 day 14 day 21 day 28 group i 59.874 a 60.138 a 62.041 a 59.720 a 61.200a 1.906 2.104 2.241 2.018 2.120 group ii 60.460 a 70.136 b 72.632 b 65.731 a 68.172 a 2.015 2.302 2.612 1.782 2.008 group iii 58.631 a 74.800b 80.124 b 78.603 b 75.272 b 2.112 3.062 3.182 2.560 2.141 values are mean ± s.e.m. values on the same column, but with different superscripts differ significantly (p < 0.05). as low as 34.84% for total wbc, 44.57% (lymphocytes), 64.59% (monocytes) and 31.73% for granulocytes on day 28 when compared with their respective preinfection values. the only exception to this trend was % lymphocytes (lymphocytes expressed as % of total wbc) which remained persistently above normal and may be explained on the basis of sustained or thriving infection. discussion as stated earlier, there was an initial increase in lymphocyte count and % lymphocyte (lymphocyte as % of total wbc). these changes became significant as early as day 7 (p < 0.05). this is certainly indicative of the animal’s swift immunological response to infection. although the % lymphocyte remained persistently higher than the pre-infection levels throughout the 28 day study period, the absolute number of lymphocytes began to decrease on day 21 and continued progressively so throughout the experiment. other wbc parameters investigated such as % monocyte and % granulocyte also progressively decreased to varying degrees. the pattern obtained is assertive of progressive immunosuppressoin characteristic of t. brucei infection in mammals. several other studies have shown that t. brucei is capable of evading the host immune response by the process of antigenic variation, by which process the parasite switches the identity and specificity of antigenic determinants expressed on its cell surface. this phenomenon, made possible by means of trypanosomal glycolsylated phosphatidyl inositol (gpi)-anchored variable surface glycoproteins thus puts the parasite way ahead of the host immune response. this subject has been widely researched and reviewed (agur et al., 1989; askonas, 1985; barry and turner, 1991; blum et al., 1993; bulow and overath, 1985, 1986; bulow et al., 1989b). the results obtained from this study show that within 7 days of infection with the parasite, the population of immunologically active cells began to decrease and indeed continued to do so for as long as infection persisted. the complexity here is that, not only is the host’ immune system rendered ineffective occasioned by rapid switching of antigenic surface glycoproteins, but even the means of doing so progressively decreases as the number of immunologically active cells (notably lymphocytes and macrophages) decreases. the persistently higher than normal levels (p < 0.05) of % lymphocytes observed in this study should not be misconstrued as a sparring effect on this class of wbc, but rather a possible preferential production of lymphocytes relative to other classes of wbc. this is readily understood when it is recalled that the absolute number of lymphocyte continued to decrease throughout the study. it is probable that the aetiology of trypanosome-induced leucopenia in the rabbit may be similar to the case with trypanosome-induced anaemia. there are striking indications that the onset of anaemia in african trypanosomiasis may be strongly related to disruption of erythrocyte membrane caused directly by parasite attack on red cells (banks, 1979, 1980; anosa and kaneko, 1983). it has also been suggested that products secreted by the parasite may play a significant role in the disruption of red cell membrane (huan et al., 1975; tizard et al., 1978; pereira, 1983; knowles et al., 1989). reduction in red cell membrane sialoglycoprotein secondary to elevated activity of plasma sialidases promotes the rapid destruction of erythrocytes (esievo et al., 1982; aminoff, 1988; olaniyi et al., 2001). a role for parasite and macrophagederived free radicals and proteases in the pathogenesis of trypanosome-induced anaemia has also been postulated (knowles et al., 1989; igbokwe et al., 1994). it is note worthy that there was no significant difference (p > 0.05) observed in all parameters investigated in control animals. it is also interesting to note that these deleterious changes were much less severe in the s. dulcis treated infected animals relative to those that were not treated. the possible mechanisms by which the herb carry out this role remain a subject of great speculations. several possible mechanism working separately or in concert may account for the observed effect. the possibility that s. dulcis or certain components of the herb may help stabilize the membrane of blood cells cannot be out rightly dismissed. specifically, it is not out of place to suggest that the anti-oxidant or free radical scavenging properties of s. dulcis previously reported by other workers (babincova and sourivong, 2001; sourivong et al., 2007; babincova et al., 2008), may play vital roles in this regard especially against the backdrop of the role of free radicals in the pathogenesis of t. brucei infection it is also probable that increased production of blood cells helps in replenishing of these cells. in the absence of any evidence of possible trypanocidal activity for the herb, it does not seem an attractive option to speculate that the higher level of immunological cells in treated animals could be due to the destruction of the parasite by agents native to the plant. be that as it may, the need to screen the plant for possible trypanocidal activity cannot be over emphasized. whatever the possible mechanism may be, the present study under scores the fact that s. dulcis provides a measure of immunological boost during experimental t. brucei infection in rabbits. acknowledgements this project was funded by the university of benin (uniben), benin city, nigeria and 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(1985) antigenic variation in trypanosomiasis: genomes in flux. j. inf. dis. 152: 855-856. suliman hb, feldman bf (1989). pathogenesis and aetiology of anaemia in trypanosomiasis with special reference to t. brucei and t. evansi. vet. bull. 59: 99-107. sourivong p, schronerova k, babincova m (2007). scoparone inhibits ultraviolet radiation-induced lipid peroxidation. z. naturforsch c. 62(1-2): 61-64. tizard ir, nielsen kh, seed jr, hall je (1978). biologically active products from african trypanosomes. microbiol. rev. 42: 661-681. truc p, aerts d, mcnamara jj, claes y, alligham r, leray d, godfrey, dg (1992). direct isolation in vitro of trypanoma brucei from man and other animals and its potential values for the diagnosis of gambian trypanosomiasis. trans. r. soc. trop. med. hyg. 86: 627629. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (2), pp. 001-007, february, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper nutritional status of undergraduates in a nigerian university in south-west nigeria o. b. adu1*, a. m. falade 1, e. j. nwalutu1, b. o. elemo1 and o. a. magbagbeola2 1 department of biochemistry, lagos state university, p. m. b 1087, apapa, lagos, nigeria. 2 department of biochemistry, college of medicine, university of lagos, idi-araba, lagos, nigeria. accepted 23 november, 2018 the study evaluated the nutritional status and eating habits of undergraduate students in a nigerian university. one hundred undergraduates (ages 15 to 40 years) of the lagos state university, ojo participated in the study. general information, anthropometric data, as well as a 7-day dietary recall were obtained by means of questionnaire. venous blood samples were collected from the respondents and analyzed for vitamins a and c, creatinine, zinc, iron, total and differential blood counts. mean body mass index (bmi), mid arm circumference (mac), vitamins a and c, iron (fe), zinc (zn) and creatinine concentrations were 24.56 ± 3.3 kg/m 2 , 26.7 ± 3.0 cm, 20.5 ± 14.3 µg/dl, 1.5 ± 71.27 µg/dl, 19.62 ± 5.65 µmol/l, 6.6 ± 1.9 mg/kg and 1.24 ± 1.53 µg/dl, respectively. mean pcv, wbc and hb were 39.2 ± 4.9%, 5.34 ±1.73 × 10 6 /l and 12.22 ± 1.93 g/dl, respectively. 53% of the respondents were over-weight; 6%, obese and 15% under weight. pcv, hb and lymphocytes were significantly higher in males than in females. there was positive correlation between serum vitamin c and zn concentrations (r = 0.203), fe and zn (r = 0.539), zn and neutrophil (r = 0.210) and vitamin a and basophil (r = 0.559). there was however a negative correlation between zn and eosinophil count. number of meals and milk intake had no effect on the status of the subjects. however, fruit intake positively affected neutrophil count (r = 0.202); vegetable intake positively affected serum fe concentration (0.256); and intake of nutritional supplements positively affected serum zn concentration. also, serum vitamin a concentration in both male and female students was low. key words: adolescent nutrition, anthropometry, body mass index, hematology, nutritional status, serum zinc, vitamin a status, undergraduates. introduction recent trends in the results of major examination in the country have indicated that there is a decline in academic performance of students at all levels (ighodalo, 2004). this decline has been attributed to some major factors like poor academic background, attitude of students towards examinations and attitudes of teachers to work. another remote cause of poor academic performance of students could be linked to the worsening socio-economic condition of the country, which has affected the *corresponding author. e-mail: tosin.adu@lasunigeria.org. feeding habit of students (ighodalo, 2004). malnutrition is a major problem in both developed and developing countries and deficiencies in some nutrients have been reported to cause diseases which could lead to impaired cognitive development (simeon and mcgregor, 1989). other studies have related lifestyle of students, particularly breakfast consumption, to their cognitive abilities as reflected in their academic performance (pollit, 1982, 1987; lisa, 1998). however, most of these studies have excluded young adults in the tertiary institution. in developing countries, many children with mild to moderate malnutrition survive to reach adolescence, when malnutrition tends to remain mild but chronic, being de table 1. mean ± sd of some nutritional parameters of undergraduate students. parameters male (n= 41) female (n= 59) total (n= 100) weight (kg) 65.38 ± 10.43 a 63.54 ± 7.65 a 64.00 ± 9.09 height (m) 1.66 ± 0.26 a 1.61 ± 0.18 a 1.63 ± 0.23 bmi (kg/m 2 ) 24.19 ± 3.46 a 24.83 ± 2.92 a 24.53 ± 3.34 mac (cm) 27.15 ± 3.22 a 26.58 ± 2.71 a 26.70 ± 3.00 vitamin a ( /dl) 21.94 ± 12.7 a 19.41 ± 15.41 a 20.49 ± 14.34 vitamin c (mg/dl) 1.73 ± 1.34 a 1.47 ± 1.24 a 1.57 ± 1.27 iron ( mol/l) 19.64 ± 5.62 a 19.79 ± 5.70 a 19.62 ± 5.65 zinc (mg/kg) 6.79 ± 1.90 a 6.44 ± 2.09 a 6.57 ± 1.97 creatinine (mg/dl) 1.05 ± 0.84 a 1.39 ± 1.88a 1.24 ± 1.53 pcv (%) 41.45 ± 3.80 a 37.56 ± 5.03b 39.22 ± 4.88 wbc (x10 6 /l) 5.68 ± 1.65 a 5.02 ± 1.77a 5.35 ± 1.74 hb (g/dl) 12.95 ± 1.18 a 11.66 ± 2.22 b 12.22 ± 1.93 n (%) 66.15 ± 8.04 a 64.02 ± 9.08 a 64.95 ± 8.61 l (%) 26.31 ± 6.46 a 29.64 ± 8.24 b 28.21 ± 7.63 m (%) 3.15 ± 2.58 a 2.41 ± 2.00 a 2.69 ± 2.27 e (%) 3.49 ± 1.80 a 3.36 ± 2.25 a 3.46 ± 2.05 b (%) 0.05 ± 0.22 a 0.13 ± 0.38 a 0.09 ± 0.33 abv alues with different superscripts within row are statistically significant at p < 0.05). tectable only by anthropometric measurements. on the other hand, relatively well-nourished children may develop malnutrition in adolescence as a result of acquired dietary habits, influenced by obsession with thinness (who, 1986; matsuhashi, 2000; ryan et al, 1998). several studies, mainly from developed countries, have demonstrated that, despite the increasing trends in the prevalence of overweight and obesity, fatness phobia is common during adolescence, especially in females (thompson and chad, 2003; jones et al., 2001; weinshenker, 2002). nutritional status is the combination of an individual’s health as influenced by intake and utilization of nutrients and determined from information obtained by physical, biochemical and dietary studies (durning and fidanza, 1985). information on the nutritional status and dietary habits of the adolescent population in nigeria is however scanty. this study was therefore intended to evaluate the nutritional status and eating habits of adolescents and young adults in a nigerian university. materials and methods subject the study was conducted using undergraduate students of the lagos state university, ojo campus. subjects were randomly selected from across the 6 faculties on ojo campus. subjects who had taken ill in the last 4 weeks and those currently on medication were exempted from the study. in the end, 100 students (ages 15 – 40 years), which included 41 males and 59 females participated in the study. data collection data on general information, socioeconomic status, and eating habits were obtained by the administration of structured questionnaires. anthropometric assessment was carried out according to the method of scrimshaw and gleason (1992). dietary assessment was done using a 7 day dietary recall. ten millilitre of venous blood was drawn from the forearm of the subjects and evenly divided into three bottles. one bottle contained edta and the two were plain bottles. the blood sample in the edta bottle was used for the haematological analysis and the other two bottles were centrifuged in a haereus labofuge for 5 min at 2000 rpm. the serum obtained was used for the mineral and biochemical analyses. analyses packed cell volume (pcv), haemoglobin (hb), total white blood cell (wbc) and differential white blood counts were determined according to the method described by baker and silverton (1985) . serum creatinine was determined according to the modified jaffe method using the creatinine kit (quimica, sa). serum vitamins a and c con-centrations were determined as described by baker and silverton (1985) . serum iron (fe) and zinc (zn) concentrations were deter-mined by atomic absorption spectrophotometry using a phillip pux 1000 atomic absorption spectrophotometer. statistical analyses sample mean and standard deviation values were calculated and means were compared using student’s t test. pearson’ correlation was used to test for association. all statistical analyses were done using the spss version 11.0 software. results table 1 shows the values of some anthropometric, biochemical and haematological parameters of subjects. there was no statistical difference between male and female undergraduate students except pcv, hb and lymphocyte values which were significantly higher (p < 0.05) in male students. age, bmi and mac distribution of the undergraduates in this study are shown in figures 1 to 3. 83% of the students’ populations were aged between 2125 years, 12% were 26-30 years, 3% were 1520 years and 2% were 31-40 years. 53% of the student populations were overweight, 36% had normal weight, 15% were under-weight and 6%, obese. only 3% of the student population involved in the study had mac lower than normal (< 22 cm). figure 4 shows the frequency of milk, fruit and vegetable and nutritional supplement intake by respondents. 41% of the students consumed milk daily; 24%, weekly, and 28%, occasionally. about 7% of the students did not take milk at all. well over half (55%) of the respondents consumed fruit occasionally, 31% weekly and 11% daily. about 3% of them did not consume fruits at all. consumption of vegetables was also low among the students. 50% of them consume vegetables occasionally; 39% of them, weekly and only 11% of them daily. intake of nutritional supplements among the students was also very low. up to 30% of them did not take any form of nutritional supplements, 44% took supplements occasionally and only 7% took their supplements daily. table 2 shows the pearson correlation values for some of the parameters measured. there was no corre-lation between milk and frequency of meals intake, and nutritional status of the students. however, there was a positive relationship between fruit intake and neutrophils count (r = 0.202); vegetable intake and serum fe concentration (r = 0.205); and supplements intake and serum zn concentration. there was also a positive correlation between serum vitamin c and zn concentrations (r = 0.203); zn concentration and neutrophils count (r = 0.210); and vitamin a and basophil count (r= 0.559). over 80% of the student population had low vitamin a concentration (<24 g/dl). discussion the present study is a preliminary report of ongoing efforts by the authors to elucidate from the nutritional point of view, the underlying cause of the increasing poor academic performance among undergraduates in tertiary institutions. results from studies like this are important for effective policy formulation and implementation, especially where nutritional interventions are required. previous reports (ryan et al., 1992) have indicated essential roles of diet and nutrition in determining health status. a good number of the study population ate at least 3 meals daily, but the dietary recall showed that they ate mostly carbohydrates, with very little protein (data not shown) . this was in agreement with the report of umoh (1977) in his study of nutritional and health problems in south-eastern nigeria. bmi and mac were used to assess leanness and these two parameters are indicators of the long term dietary history of the respondents. in this study, bmi value greater than 31.5 kg/m 2 was considered as an indication of obesity and below 18.0 kg/m 2 as leanness (who, 1995). leanness as a result of under nutrition causes reduced metabolism, reduced energy production and non availability of free glucose which is both required for studying and stress management. about half of the students who participated in the study (53%) were over weight, 6% obese and 15% underweight. obesity has been associated with an increased propensity for the development of kidney, heart and circulatory diseases; diabetes and complications during pregnancy and child birth in females (guthrie, 1979; wardlaw and kessel, 2002; who, 2003). a recent report (cannon and leitzmann, 2005) indicated a staggering rise of obesity in young people in middle income countries, and this has serious implications in later life. with the exception of serum vitamin a concentration, all other serum metabolites and haematological parameters were within normal range. mean serum vitamin concentration for both male and female subjects was low. consumption of fruits, vegetables and nutritional supplements among the undergraduates in the study was low. consumption of fruits and vegetables among adolescents between the ages of 12 and 19 years has been reported to decrease with age (aihw, 2007). this trend could be as a result of waning parental influence as adolescents get into tertiary institutions or the problem of easy accessibility to these foods on the university campus. this factor might also have contributed to the low vitamin a status of the subjects. the positive correlation between zinc and iron was contrary to previous reports (solomon and jacob, 1981) where ingestion of zinc and iron in various ratios led to reduced zinc absorption. davidson et al. (1995) however reported that iron ingestion had no effect on zinc absorption. there was a positive relationship between zn, vitamins a and c, and some cells of the immune system. vitamin c, apart from having antioxidant activity, also helps in immune function. white blood cells which are the major immune cells contain the highest vitamin c concentration of all body constituents (wardlaw and kessel, 2002). several reports have also shown the importance of zn in both specific and non specific immune responses (cunningham et al., 1990; shankar and prasad, 1998). over 80% of the subjects had low serum vitamin a con f re q u e n c y 60 50 40 fr eq ue nc y 30 male 20 female 10 0 15-20 21-25 26-30 31-40 age (yrs) figure 1. age distribution of respondents by gender. 35 30 25 20 male 15 female 10 5 0 underweight normal overweight obese bmi range figure 2. bmi distribution of respondents by gender. 50 45 40 35 f re q u e n c y 30 25 20 15 10 5 0 below normal normal mac range figure 3. mac distribution of undergraduate students. 60 50 40 s u b j e c t s 30 o f n o 20 10 0 daily weekly occasionally none frequency of intake male female above normal milk intake fruit intake vegetable intake nutritional supplement figure 4. frequency of milk, vegetables and supplement intake by respondents. table 2. pearson correlation between nutrient intake, nutritional and immunological status of undergraduates. parameter correlation vitamin a vitamin c creatinine zinc iron n l m e b pearson correlation -0.154 0.102 -0.034 -0.021 -0.055 0.071 -0.087 0.078 -0.065 0.002 no of meals sig. (2-tailed) 0.133 0.313 0.735 0.838 0.593 0.484 0.388 0.443 0.518 0.983 n 96 100 99 97 97 100 100 100 100 100 pearson correlation 0.011 0.035 0.022 -0.121 -0.051 0.057 -0.05 -0.026 -0.098 -0.003 sig. (2-tailed) 0.917 0.73 0.831 0.238 0.55 0.575 0.624 0.798 0.333 0.974 milk intake n 96 100 99 97 97 100 100 100 100 100 pearson correlation 0.054 -0.038 -0.092 0.004 0.12 .202* -0.127 -0.104 -0.153 0.094 fruit intake sig. (2-tailed) 0.603 0.707 0.367 0.968 0.24 0.044 0.209 0.301 0.128 0.353 n 96 100 99 97 97 100 100 100 100 100 vegetable pearson correlation -0.023 0.056 -0.005 0.166 .256* -0.071 -0.006 0.057 0.004 -0.035 intake sig. (2-tailed) 0.825 0.582 0.958 0.105 0.011 0.486 0.956 0.574 0.965 0.732 n 96 100 99 97 97 100 100 100 100 100 supplement pearson correlation 0.136 0.138 0.041 0.041 .221* 0.039 -0.031 0.093 -0.065 0.055 intake sig. (2-tailed) 0.187 0.169 0.686 0.686 0.03 0.703 0.756 0.358 0.523 0.584 n 96 100 99 99 97 100 100 100 100 100 pearson correlation 1 -0.133 -0.115 -0.057 -0.107 0.004 -0.018 -0.035 0.128 .559** vitamin a sig. (2-tailed) 0.275 0.268 0.588 0.305 0.971 0.861 0.732 0.213 0 n 96 96 95 97 93 96 96 96 96 96 pearson correlation -0.133 1 -0.094 .203* 0.097 0.082 -0.033 0.117 -0.167 -0.173 vitamin c sig. (2-tailed) 0.275 0.354 0.046 0.346 0.418 0.742 0.247 0.097 0.084 n 96 100 99 97 97 100 100 100 100 100 pearson correlation -0.115 -0.094 1 -0.163 -0.148 0.162 0.162 0.002 0.045 0.051 creatinine sig. (2-tailed) 0.268 0.354 0.113 0.15 0.11 0.11 0.986 0.656 0.616 n 95 99 99 96 96 99 99 99 99 99 pearson correlation -0.057 .203* -0.163 1 .539** .210* -0.119 0.067 -0.249* -0.107 zinc sig. (2-tailed) 0.588 0.046 0.113 0 0.039 0.245 0.517 0.014 0.298 n 97 97 96 98 97 97 97 97 97 97 pearson correlation -0.107 0.097 -0.148 .539** 1 0.163 -0.052 0.003 -0.151 -0.164 iron sig. (2-tailed) 0.305 0.346 0.15 0 0.112 0.613 0.974 0.14 0.108 n 93 97 96 97 97 97 97 97 97 97 pearson correlation 0.021 0.06 -0.008 0.027 -0.136 0.081 -0.091 0.173 -0.01 -102 pcv sig. (2-tailed) 0.844 0.567 0.941 0.798 0.194 0.438 0.385 0.095 0.927 0.327 n 90 94 93 92 92 94 94 94 94 94 pearson correlation 0.19 0.042 -0.063 -0.071 -0.189 .250* -0.264* 0.004 -0.082 0.146 wbc sig. (2-tailed) 0.075 0.686 0.554 0.502 0.072 0.016 0.011 0.966 0.433 0.162 n 89 93 92 91 91 93 93 93 93 93 pearson correlation 0.067 0.034 -0.009 0.004 -0.137 0.084 -0.099 0.121 0.026 0.018 hb sig. (2-tailed) 0.529 0.745 0.933 ..971 0.194 0.42 0.341 0.243 0.801 0.86 n 90 94 93 92 92 94 94 94 94 94 centration, despite the claims by most respondents that they took fruit, milk and vegetables regularly. although these foods are good sources of the vitamin, the method of storage and processing and the adequacy of intake could be responsible for the low serum concentration. vitamin a deficiency has been associated with nonaccidental blindness, impairment of growth and immunity and follicular hyperkeratosis (wardlaw and kessel, 2002). nutritional education and intervention therefore is expedient in order to prevent cases of visual impairment among the students. conclusion there was a high incidence of over weight and vitamin a deficiency among the student population. in curbing this trend, there is therefore an urgent need for sustained nutritional education among young adolescents. references aihw (2007). profile of nutritional status of children and adolescents. cat. no. phe 89. australian institute of health and welfare, canberra. baker ft, silverton re (1985). introduction to medical laboratory technology, 6 th ed, butterworth, london pp. 320-335. cannon g, leitzmann c (2005). the new nutrition science project. public health nutr. 8: 673-694. cunningham rs, bockman rs, lin a, giardina pv, hilgartner mw, caldwell-brown d, carter dm (1990). physiological and pharmacological effects of zinc on immune response. ann. new york acad. sci. 587: 113-122. davidson l, almgren a, sandstrom b, hurrell rf (1995). zinc absorption in adult humans: the effect of iron fortification. br. j. nutr. 74: 417 – 425. durnin jvga, fidanza f (1985). evaluation of nutritional status. biblthca nutr. dieta 35: 20-30. guthrie ha (1979). introductory nutrition. 4 th edn. the cv mosby company, st. louis p. 503. ighodalo d (2004). reasons for yearly weakness observed in waec candidates. punch newspaper p. 48. jones jm, bennett s, olmsted mp, lawson ml, rodin g (2001). disordered eating attitudes and behaviours in teenaged girls: a school-based study. can. med. assoc. j. 165: 547-552. lisa ms (1998). the correlation between eating breakfast and school performance. am .j. clin nutr. 65: 7795-9845. matsuhashi y. (2000).thinness: drives and results. j. adolesc. health 27: 149-150. pollit e, lewis ns, garza c, shulman r (1983). fasting and cognitive function j. psychiatr. res. 17: 168-174. pollit e, watkins we, husaini ma (1997). three month nutritional supplementation in indonesia infants and toddlers, benefit child memory function 8years later. am. j. clin. nutr. 66: 1357-1363. ryan sa, craig ld, finn sc (1992). nutrient intakes and dietary patterns of adult americans. a national survey. gerontol. soc. amer. m145-m150. ryan ym, gibney mj, flynn ma (1998). the pursuit of thinness: a study of dublin schoolgirls aged 15 y. int. j. obes. relat. metab. disord. 22:485-487. schrimshaw ns, gleason or (1992). rapid assessment procedures. qualitative methodologies for planning and evaluation of health related programmes int. nutr. foundat. dev. countries. boston. shankar ah, prasad as (1998). zinc and immune function: the biological basis of altered resistance to infection am. j. clin. nutr. 68:447s-463s. simeon dt, grantham–mcgregor sm (1990). nutritional deficiencies and children’s behaviour and mental development. nutr. res. rev. 3: 1-24. solomon nw, jacob ra (1981). studies on the bioavailability of zinc in humans: effects of heme and non heme iron on the absorption of zinc. am. j. clin. nutr. 34: 475-482. thompson am, chad ke (2003). the relationship of social physique anxiety to risk for developing an eating disorder in young females. j. adolesc. health 31: 183-189. umoh z (1977). nutritional and health problem in southern eastern nigeria. nig. med. j. 2: 56-57. wardlaw gm, kessel m (2002). perspective in nutrition 5 th edn. mcgraw – hill. london p. 322. weinshenker n (2002). adolescence and body image. school nurse news. 19:12-16. world health organization (1986). young people’s health. a challenge for society; report of a who study group on young people and health for all by the year 2000.. geneva: world health organization. technical report series no. 731, 1-117. world health organisation (1995). field guide on rapid nutritional assessment in emergencies, who, regional office for eastern mediterranean, alexandria. world health organisation (2003) . diet, nutrition and the prevention of chronic diseases. report of a joint who/fao expert consultation. who technical report series no 916. geneva. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (2), pp. 001-003, february, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. short communication the microbial quality of pre-packed mixed vegetable salad in some retail outlets in lagos, nigeria r. e. uzeh1*, f. a. alade1 and m. bankole2 1 department of botany and microbiology, university of lagos, lagos, nigeria. 2 nigerian institute of medical research, lagos, nigeria. accepted 15 august, 2019 pre-packed mixed vegetable salad and salad ingredientscarrots, cucumber, cabbage, and lettuce were analyzed for their microbial quality. the salads were obtained from fast food outlets (well packaged at 4 o c) and open markets (exposed at 35 o c) within lagos metropolis. the analysis was both qualitative and quantitative. microorganisms isolated from salad samples from fast food outlets include aspergillus fumigatus, trichoderma spp, staphylococcus aureus and proteus mirabilis, while those isolated from open market samples include mucor spp, a. fumigatus, aspergillus niger, trichoderma spp, neurospora crassa, proteus vulgaris, s. aureus, citrobacter freundii, proteus mirabilis, and corynebacterium spp. those from salad ingredients include mucor spp, a. fumigatus, trichoderma spp, n. crassa, rhizopus spp, a. niger, p. vulgaris, p. mirabilis, s. aureus, pseudomonas aeruginosa and c. fruendii. the total viable count was highest in salad samples from open markets (5.9×10 6 cfu/g) and lowest in salad samples from fast food outlets (2.6 × 10 4 cfu/g). the total viable counts obtained from the salad ingredients were generally lower than those obtained from salads. among the salad ingredients the highest count was however obtained from carrot (3.0 × 10 2 cfu/g) and lowest count from cucumber (1.3 × 10 2 cfu/g). gentamicin, chloramphenicol, cotrimoxazoleoflaxacin were most effective against the bacterial isolates yielding greater zones of inhibition. the storage temperature and the dirty nature of the open markets must have been responsible for the occurrence of more microorganisms in salad samples from open markets than those from fast food outlets. the need for safe salad can not be overemphasized. key words: vegetable salad, salad ingredients, microorganisms, fast food outlets, open markets. introduction salad is a term broadly applied to many food preparations that have a mixture of chopped or sliced ingredients which may be mainly fruits or vegetables. the inner tissues of healthy plants and animals are free of microorganisms. however, the surfaces of raw vegetables and meats are contaminated with a variety of microorganisms and this depends on the microbial population of the environment from which the food was taken, the condition of the raw product, the method of handling, the time and conditions of storage (pelczar et al., 2006). bacteria involved in spoilage of vegetables are usually pectinolytic species of the gram negative genera of erwinia, pseudomonas, clostridium, and xanthomonas *corresponding author. e-mail: roseline_uzeh@yahoo.com. and the non-sporing gram positive organisms like corynebacterium (adams and moss, 1999) . salads containing raw vegetables have been identified as vehicles of traveller’s diarrhea, an illness sometimes experienced by visitors to developing countries (beuchat and larry, 1996). the microbial flora of partially processed vegetables as found in pre-packed mixed vegetable salads should be of great concern since both food spoilage and safety are involved. the presence or absence as well as the fate of both human pathogens and plant tissue spoilage organisms are important. the objective of this study therefore is to isolate and identify the microorganisms present in pre-packed mixed vegetable salads from different retail outlets in lagos, nigeria, as this will help in establishing the effect of environment where the food was taken, type of handlingand type of storage condition on the microbial quality of the product. table 1. total viable count of salad samples and salad ingredients. salad samples locations total viable count (cfu/g) salad ingredients total viable count (× 10 2 cfu/g) yaba market 5.9 × 10 6 cucumber 1.3 bariga market 5.7 × 10 6 carrot 3.0 fast food outlet 1(f1) 3.3 × 10 3 lettuce 2.0 fast food outlet 2(f2 ) 2.6 × 10 4 cabbage 2.1 table 2. occurrence of microorganisms in salad and salad ingredients. fast food open markets salad ingredients isolates outlets f1 f2 yaba bariga cucumber carrot lettuce cabbage mucor spp + + + a. fumigatus + + + + + + trichoderma spp + + + + n. crassa + + + + rhizopus spp + + + a. niger + + + + p. vulgaris + + + + s. aureus + + + + + + c. freundii + + + p. mirabilis + + + + p. aeruginosa + + corynebacterium spp + + materials and methods collection of samples pre-packed mixed vegetable salads were obtained from fast food outlets stored at 4 o c in refrigerators located in clean and enclosed buildings (f1 and f2 ) and dirty open markets (yaba and bariga) where wares and food items are displayed in the open at temperature of 35 o c. salad ingredients: carrots, lettuce, cabbage, and cucumber were also obtained from the market. the fast food outlets are in better hygienic condition than the open markets which are very dirty. isolation of microorganisms from each salad sample, 25 g was aseptically weighed and macerated and 225 mls of sterile distilled water was added. serial dilution was carried out using sterile distilled water as diluents. from each dilution 1 ml was plated using the pour plate method. the culture media used were nutrient agar, macconkey agar and potato dextrose agar. plating was done in duplicates. nutrient agar plates and macconkey agar plates were incubated at 37 o c for 2448 h while the potato dextrose agar plates were incubated at 25 o c for 48-72 h. for the salad ingredients the procedure explained above was done for each of them. after incubation, colonies that developed on the plates were counted to obtain total viable count. pure cultures of the isolates were obtained by subsequent sub-culturing on fresh agar plates. identification of microbial isolates this was done based on cultural, morphological and biochemical characteristics of the isolates using standard methods (buchanan and gibbons, 1974; talbot, 1971). antibacterial susceptibility testing broth culture of each bacterial isolate was swabbed on mueller hinton agar plates with sterile swab sticks. commercial antibiotic discs were placed on the inoculated plates, which were subsequently incubated at 37 o c for 24 h. after incubation, the plates were examined for zones of inhibition. the degree of sensitivity was expressed as a measure of the diameter of zones of inhibition of growth in millimeters. results the total bacterial count was higher in salad samples from the open markets than those from fast food outlets. the salad ingredients analyzed had the least bacterial count (table 1). the microorganisms isolated from salads and salad ingredients include citrobacter fruendii, proteus vulgaris, proteus mirabilis, staphylococcus aureus, mucor spp, aspergillus fumigatus, trichoderma, neurospora crassa and aspergillus niger. corynebacterium spp was isolated from only salad samples collected from open markets. pseudomonas aeruginosa and rhizopus spp were isolated from only salad ingredients. more microorganisms occurred in salads obtained from the open markets than in salads obtained from fast food outlets (table 2). most bacterial isolates were more sensitive to gentami table 3. antibiotic sensitivity of isolated gram positive bacteria. antibiotics zones of inhibition (mm) staphylococcus aureus corynebacterium sp amoxycillin 25 18 erythromycin 25 19 tetracycline 27 26 cloxacillin 16 14 chloramphenicol 30 30 cotrimoxazole 30 17 augmentin 27 17 gentamycin 29 24 table 4. antibiotic sensitivity of isolated gram negative bacteria. antibiotics zones of inhibition (mm) c. freundii p. vulgaris p. mirabilis p. aeruginosa gentamicin 26 27 24 21 nalidixicin acid 19 28 oflaxacin 29 30 29 29 augmentin 15 14 tetracycline 28 16 16 amoxycillin 17 cotrimoxazole 17 15 nitrofurantoin 16 18 -: no sensitivity. cin, chloramphenicol, contrimoxazole and oflaxacin (tables 3 and 4). discussion the higher bacterial counts obtained from salad samples as compared to salad ingredients with lower bacterial count may be due to handling and non hygienic practices during salad preparation which may have led to introduction of additional microorganisms into the salad. exposure of salad to contaminants during storage and retail may have also contributed. on the other hand, growth of the initial contaminants during storage of salad may have contributed to the higher bacterial counts, especially with salad in the open markets which were stored at ambient temperatures. salad samples from the fast food outlets yielded lower numbers and types of microbial isolates than the market samples. this could be as a result of the relatively clean environment in the fast food outlets when compared to the dirty open markets. the storage temperature of 4 o c in the fast food outlets compared to 35 o c in open markets may also be responsible. from results obtained from this work, different types of bacteria and molds were isolated from vegetable salads. vegetables, fruits, pulses, oil seeds and their products carry microorganisms such as fungi, actinomycetes and bacteria (majumder, 1994). pseudomonas spp is a prominent inhabitant of soil and water. the organism is responsible for diseases of vegetables like angular leaf spot of cucumber. s. aureus is of health significance as it can cause food poisoning. however, the need for good hygienic practices, proper handling, storage and retail of salads in clean environment and at refrigeration temperature can not be over emphasized to ensure good quality and safe salads. references adams mr, moss mo (2000). food microbiology. r. soc. chem. sci park, cambridge p. 447. beuchat lr, larry r (1996). pathogenic microorganisms associated with fresh produce. j. food protect. 13: 204-216. buchanan re, gibbons ne (1974). bergeys manual of determinative microbiology, 8 th edition. the williams and wilkins co, baltimore. majumder sk (1994). effect of pests of foods. central food technol. res. institute mysore. 570013 india. pelczar mj, chan ecs, krieg nr (2006). microbiology 5 th edition. tata mcgraw-hill publishing company limited, new delhi.. talbot phr (1971). principles of fungal taxonomy. macmillan press, london p. 274. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (3), pp. 001-014, march, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper identification, characterisation and composition of scavengeable feed resources for rural poultry production in central tanzania e. h. goromela1*, r. p. kwakkel2, m. w. a. verstegen2 and a. m. katule3 1 natonal livestock research institute, p.o. box 202 mpwapwa, tanzania. 2 animal nutrition group, wageningen university, p.o box 338, 6700 ah wageningen, the netherlands. 3 sokoine university of agriculture, p.o. box 3004 morogoro, tanzania. accepted 05 january, 2019 a participatory study was carried out in four villages of central tanzania to appraise existing and potential scavengeable feed resources available for rural poultry. in addition, proximate analysis of selected scavengeable feed resources including chicken crop and gizzards contents was carried out to quantify their feeding value. results indicate that the most important scavengeable feed resources in the dry season were cereal grains and their byproducts, oil seeds and oil seed cakes and in the wet season were forage leaves, flowers, seeds, garden vegetables, insects and worms. changes in seasonal conditions, farming activities, land size available for scavenging and the flock size had a major influence on the feed availability. the mean dry matter (dm) of the feed resources was 888 ± 1.8 g per kg. gross energy ranged from 17.1 to 29.3 mj kgdm -1 and crude protein (cp) from 64.5 to 418 g kgdm -1 . crude fibre (cf) ranged from 33.3 to 230 g kgdm -1 and ether extract (ee) ranged from 16.0 to 488 g kgdm -1 . the mineral composition ranged from 1.5 to 18.4 g kgdm -1 for calcium (ca); 3.6 to 17.3 g kgdm -1 for phosphorus (p); 9.5 to 34.5 g kgdm -1 for potassium (k) and 0.2 to 8.5 g kgdm -1 for magnesium (mg). physical analysis of crop and gizzard contents indicated that the diets consumed by scavenging chickens consisted of cereals and cereal by-products (29.0%), vegetables and forage materials (1.8%), seeds and seed by-products (3.4%), insects and worms (0.2%), egg shells, feathers and bones (0.3%), unidentified feeds (41.5%), inert materials (0.8%) and sand/grit (23.0%). the diet consumed as determined from the crop/gizzard contents had dm of 479 ± 9.6 g per kg and metabolizable energy (me) of 10.1 ± 0.5 mj kgdm -1 . nutrient composition in kgdm -1 of the crop and gizzard contents was: 80.4g cp; 70.7g ee; 45.7g cf; 234g ash; 6.6g ca; 6.5g p; 12.1g k and 2.6g mg. the study showed that the nutrient concentrations of scavengeable feed resources consumed by rural poultry were below the recommended levels for optimum growth and egg production. key words: central tanzania, rural poultry, scavengeable feed resources, nutrient composition, crop and gizzard. introduction the livestock industry in tanzania contributes about 18% of the national gdp and about 30 % of the agricultural gdp, and provides food which is consumed in the form of meat, milk and eggs (urt, 2005). the livestock industry *corresponding author. e-mail: egoromela@yahoo.com is divided into a traditional and commercial sector. cattle and poultry are the major livestock species. the poultry sub-sector consists of traditional small-scale poultry production in the rural areas and commercial small to largescale poultry production in peri-urban and urban areas. the traditional smallscale poultry production is the largest sub-sector and comprises various poultry species such as chickens, ducks, turkeys, geese, pigeons and guinea fowls. most of these species are local ecotypes and account for about 90% of the total poultry population in tanzania (moac, 1995) . due to their high degree of adaptation to a wide range of environments, rural poultry or village poultry are widely distributed throughout the country. chickens are the most common poultry species in the country, and account for about 95% of total poultry in the traditional sector (moac, 1995). these rural poultry are owned by individual smallholder farmers that practise a wide range of management strategies, mainly characterized by hardly any or no inputs for housing, feeding and health care. free-ranging is the dominant poultry production system practiced by most of the households in rural areas. in this system, poultry usually are allowed to scavenge their own food around homesteads and from the surrounding environments during the daytime. this low-input/low-output system has been a major component of the smallholder farming in the country for centuries. in spite of the social and economical importance of the free-range system, few developments have been undertaken in tanzania to improve the overall productivity of rural poultry. for example, in the past, breeding programmes were introduced to the villages to improve the productivity of local birds by crossing with exotic cockerels (goromela, 2000). such programmes have also been reported in many other african countries (tadele and ogle, 1996; kitalyi, 1998). however, these programmes placed more emphasis on rapid genetic improvement with the justification that improvement in feeding would be inefficient when animals of low genetic potential are raised (sonaiya et al., 1999). as a result, the majority of these programmes failed due to lack of management skills that were needed for the crossbreds and the management changes that should be adopted by the rural people. in recent years, there has been a growing awareness of the need to balance the rate of genetic improvement with improvement in feeding and in management (kitalyi, 1998; sonaiya et al., 1999; minga et al., 2000). moreover, there has been a renewed interest in rural poultry production and in the potential of rural poultry as efficient converters of locally available scavengeable feed resource base (sfrb) into eggs and meat (kitalyi, 1998; sonaiya et al., 1999; minga et al., 2000). in the free-range system, rural poultry obtain their diets mainly from sfrb. however, the amounts and availability of sfrb are not constant throughout the year (cummings, 1992). such feed resources tend to vary with factors such as seasonal conditions, farming activities, life cycle of insects and other invertebrates (roberts, 1995; tadele, 1996; sonaiya, 2004). if the supply of sfrb and the seasonal variations is known, efficient strategies for production of scavenging poultry can be developed (gunaratne, 1999). thus the objectives of this study were to appraise existing and potential scavengeable feed resources available for rural poultry and quantify their feeding value. the second aim was to determine the constraints with regard to the availability of these feed resources for rural poultry in free-range systems in central tanzania. materials and methods the study area this study was carried out in four villages of bumila, chitemo and kisokwe in mpwapwa district and chamkoroma in kongwa district in dodoma region. administratively, central tanzania comprises dodoma and singida regions with a total human population of 2.7 million. about 80% of this population depend on crop and livestock production as the major basis of their livelihoods. central tanzania is categorised as a semi -arid zone with a savannah climate characterized by a short rainy period of four months between december and may. the average annual rainfall is 450 mm. the average maximum temperature is 28 o c and the minimum temperature is 17 o c with an average relative humidity of 70%. the villages were selected essentially based on the farming systems being practiced. bumila and chamkoroma villages are located in the maize-beanssunflower based farming system while kisokwe and chitemo villages are located in the sorghum-pearl millet-groundnut based farming system. identification of available scavengeable feed resources a reconnaissance survey was carried out in november, 2005 to obtain background information of the above mentioned four villages. a group of 20 30 key informants in each village was interviewed. using this background information, the questionnaire for a formal survey was developed and pre-tested for consistency of questions. then a formal survey was carried out in december, 2005 in the above mentioned villages and the questionnaire addressed feed resources and nutrients eaten by rural poultry in the villages at different times of the year; factors affecting quantity and quality of sfrb and interventions/strategies that should be used to improve feed resource availability. a total of 318 households from the four villages were interviewed with a sample size of 70 80 households per village. most of the respondents were adult women who had responsibilities for the daily management of local birds as their main domain. in addition, a balanced sub-sample of 40 farmers involved in the formal survey from each village was used in group interviews to obtain qualitative information on seasonal availability of sfrb and constraints with regard to the availability of these feed resources. pra tools such as pair-wise ranking; matrix scoring and direct observations were used. samples of existing and potential feed resources were collected from interviewed farmers during the household survey. these samples were sorted out in the laboratory and pooled together based on types and varieties of the feed resources. the final feed samples were sub-sampled for the determination of chemical composition. characterization of scavengeable feed resource base characterization of scavengeable feed resources for rural poultry was carried out to assess the relative availability of each feedstuff separately during the wet and dry season. a relative qualitative scale was developed by farmers and ranged from abundantly available to scarcely available. in addition, a pairwise ranking method was used to rank the most important feedstuffs which had significant impact on the rural poultry production. feed resources which had the highest scores and ranked from 1 to 6 were considered as the most important feedstuffs in each feed category. moreover, the factors affecting quantity and quality of sfrb with respect to the variation of energy and protein sources were investigated using matrix scoring method. using a total of 120 groundnut seeds, farmers assigned these seeds to each month of the year drawn on a flip-chart where 10 seeds per a particular month indicated maximum availability of the feed resources. the number of seeds assigned on each month was counted as scoresor percentage of the feed resources that were more likely to be available and the underlying factors were discussed. quantitative assessment of sfrb from chickens’ crop and gizzard contents a quantitative assessment of sfrb in the chickens’ crop, proventriculus and gizzard contents was carried out between december 2005 and january 2006. a total of 141 scavenging chickens of both sexes with an average liveweight of 1.2 kg ± 0.3 were randomly purchased from farmers in the four villages for the analysis of crop and gizzard contents. the crop and gizzard contents were used to determine the amount and composition of the diets consumed by scavenging chickens. the chickens were collected directly from the farmers between 11.00 and at 16.00 h. chickens were weighed before slaughtering using an electronic balance. the chickens were slaughtered according to normal practice on farms and the feathers were removed after dipping the body into hot water. the carcasses were eviscerated and the whole gastro-intestinal tracts were removed and packed in the cool boxes containing ice-packs. samples of the gastro-intestinal tracts were taken to the laboratory where they were frozen at -30 o c. later the frozen gastro-intestinal tracts were opened up after thawing in the air for a few hours and the crop, proventriculus and gizzard were eviscerated. the ingested feed materials from crop, proventriculus and gizzard were identified visually using magnifying lenses. chyme of crop, proventriculus and gizzard was physically separated into different individual feed components. these individual feed components were weighed using electronic balance. in order to determine their proportions on dry weight basis the samples were partially dried at 60 o c in the laboratory using an oven. chemical analyses of scavengeable feed resources and chickens’ crop/gizzard contents samples of scavengeable feed resources and samples of chickens’ crop and gizzard contents were sub-sampled according to the villages from which they had been collected. all the samples were ground using laboratory mills to pass through a 2 mm screen in order to produce homogenous samples for the analysis of chemical composition. the homogenous samples of feed resources and crop/gizzard contents were analysed for dry matter (dm), crude protein (cp), ether extract (ee), crude fibre (cf), ash, calcium (ca), phosphorus (p), potassium (k), and magnesium (mg) according to the procedures of aoac (1985). nfe was estimated from organic matter (om) minus the sum of amounts of cp, ee and cf (g/kg dm). gross energy (ge) was determined using a regression equation of the chemical composition of the feed resources as described by wiseman (1987): ge (kcal/kgdm) = 57.2 cp% + 95.0 ee% + 47.9 cf% + 41.7 nfe%. the metabolizable energy (me) of the crop/gizzard contents was determined by calculating the true metabolizable energy (tme) with the assumption that tme was 8% higher than the me (wiseman (1987). data analysis all the data collected were coded and analysed for descriptive statistics for each variable investigated using spss programme version 11.0 for windows 2003. results scavengeable feed resources available for rural poultry in the villages the scavengeable feed resources available for rural poultry in the 4 villages of mpwapwa and kongwa districts are presented in tables 1 4. the results show that feed resources consisted of energy, protein and mineral/vitaminrich feedstuffs. about 52.9% of the respondents in kisokwe, 64.8% in bumila, 61.6% in chamkoroma and 46.7% in chitemo reported that energy-rich feedstuffs were slightly available during the wet season while 80.79% of the respondents in kisokwe, 82.28% in bumila, 68.66% in chamkoroma and 72.6% in chitemo reported that these feedstuffs were abundantly available in the dry season. regarding the availability of protein and mineral/vitamin-rich feedstuffs, 64.8% of the respondents in kisokwe, 70.0% in bumila, 68.1% in chamkoroma and 55.7% in chitemo reported that such feedstuffs were highly available in the wet season while 56.8% of the respondents in kisokwe, 60.0% in bumila, 50.7% in chamkoroma, and 53.9% in chitemo reported that these feedstuffs were slightly available in the dry season. the study revealed that both conventional and unconventional feedstuffs in all the four-villages were generally similar and abundantly available in certain periods despite the differences in farming systems and climatic conditions. characterization of scavengeable feed resources available for rural poultry the relative availability of scavengeable feed resources during different seasons is shown in tables 1 4. the results indicate that the availability of various sfrb between dry and wet seasons varied widely. much of this variation is in amounts and in flora and fauna and origin of species available as food for the rural poultry. most of the energy-rich feedstuffs and some protein-rich feedstuffs particularly sunflower seed cakes and groundnut seeds were abundantly available during the dry season. however, at the end of the dry season and during the wet season (january april) the availability of these feed resources were relatively low. the protein-rich feedstuffs (insects and earthworms); and mineral/vitamin-rich feedstuffs were abundantly available during the wet season while in the dry season these feedstuffs were relatively low. among the energy-rich feedstuffs: maize bran, maize brew wastes, maize thick porridge (ugali), maize grains and sorghum grains ranked highest in bumila village and chamkoroma village while in kisokwe village and chitemo village sorghum ugali, maize ugali, pearl-millet ugali, pearl millet brew wastes, sorghum brew wastes and maize brew wastes ranked highest. in bumila village and chamkoroma village the protein/mineral feedstuffs: forage leaves, forage flowers, garden vegetables, tree table 1. relative availability of scavengeable feed resources for rural poultry during wet and dry season as assessed by farmers at bumila village in mpwapwa district relative feed availability during wet relative feed availability during dry pair-wise ranking by season 1 season 1 farmers categories of scavengeable feeds total dec jan feb mar apr may jun jul aug sept oct nov scores rank 1. cereal grains maize xx xx x x xx xxx xxx xxx xxx xxx xxx xx 5 4 sorghum xx xx x x xx xxx xxx xxx xxx xxx xxx xx 4 5 2. cereal by-products maize bran xx xx x x xx xxx xxx xxx xxx xxx xxx xx 8 1 maize brew-waste xx x x x xx xxx xxx xxx xxx xxx xxx xx 7 2 sorghum bran xx xx x x xx xxx xxx xxx xxx xxx xxx xx 2 7 sorghum brew-waste xx x x x xx xxx xxx xxx xxx xxx xxx xx 0 9 3. other cereal and tuber products maize ugali xx xx xx xx xx xxx xxx xxx xxx xxx xxx xx 6 3 sorghum ugali xx xx xx xx xx xxx xxx xxx xxx xxx xxx xx 1 8 cassava 3 6 1. crop and animal protein feeds sunflower seed cake x x x xx xxx xxx xxx xx 9 5 groundnuts seeds x x x xx xxx xxx xxx xxx xxx xx 8 6 sesame seeds x x 0 14 fish meal x xx x 3 11 meat meal x x 1 13 2. insects and worms earth worms xxx xx xx xxx xxx xx x 4 10 maggot larvae xx xxx xx xxx xxx xx x 2 12 termites xx xx xxx xxx xxx xx xx xx xx x x xx 7 7 grasshopper xx xxx xxx xxx xxx xx xx xx xx xx xx xx 6 8 cut worms xxx xxx xxx xxx xxx xx x 5 9 3. minerals and vitamins garden vegetables xx xxx xxx xxx xxx xxx xx xx 11 3 mtps/forage leaves xx xxx xxx xxx xxx xxx xx xx x 13 1 forage flowers x xx xxx xxx xxx xxx xx 12 2 tree seeds x xx xx xxx xxx xxx xx x 10 4 1 relative scale: xxx= abundant, xx = average, x= low, and = very low/none table 2. relative availability of scavengeable feed resources for the rural poultry during wet and dry season as assessed by farmers at chamkoroma village in kongwa district relative feed availability during wet relative feed availability during dry season 1 pair-wise ranking by season 1 farmers categories of scavengeable feeds total dec jan feb mar apr may jun jul aug sept oct nov scores rank 1. cereal grains maize xxx xx x x xx xxx xxx xxx xxx xxx xxx 7 3 sorghum xx xx xxx x 6 4 rice xx x x xx xxx xxx xxx xxx xx xx 3 7 2. cereal by-products maize bran xx x x xx xxx xxx xxx xxx xxx xx 8 2 maize brew-waste xx x xx xx xxx xxx xxx xxx xxx xx 5 5 sorghum bran xx xx xxx xx 2 8 sorghum brew-waste xx xx xxx x 1 9 rice bran/polishings xx x x xx xxx xxx xxx xxx xx xx 0 10 3. other cereal and tuber products maize ugali xxx xx x x xx xx xxx xxx xxx xxx xxx xx 9 1 sorghum ugali xx xx xx xx xx xxx xxx xxx xxx xxx xxx xx 4 6 1. crop and animal protein feeds sunflower seed cake xx x x xx xxx xxx xxx xxx xx 8 4 groundnuts seeds x x xx xxx xxx xxx xxx xxx xxx xx xxx xx 6 6 fish meal x x xx xx xx xx xx xx xx 2 10 2. insects and worms earth worms xx xxx xx xxx xxx xx x 4 8 maggot larvae xx xxx xxx xxx xxx xx x 1 11 termites xx xxx xx xxx xxx xxx xxx xx x x x x 7 5 grasshopper xx xxx xxx xxx xx xx x x x x x x 3 9 cut worms xx xxx xxx xxx xxx xx xx x 5 7 3. minerals and vitamins garden vegetables xx xxx xxx xxx xxx xxx xx xx x 11 1 mtps/forage leaves xx xxx xxx xxx xxx xxx xx xx x x 10 2 forage flowers x xx xxx xxx xxx xx x x 9 3 tree seeds x x x x xx xxx xxx xxx xxx xx 0 12 1 relative scale: xxx= abundant, xx = average, x= low, and = very low/none table 3. relative availability of scavengeable feed resources for the rural poultry during wet and dry season as assessed by farmers at kisokwe village in mpwapwa district. categories of scavengeable feeds relative feed availability during wet relative feed availability during dry season 1 pair-wise ranking by farmers season 1 dec jan feb mar apr may jun jul aug sept oct nov total scores rank 1. cereal grains maize xx x x xx xxx xxx xxx xxx xx xx 0 12 sorghum xx x x xx xxx xxx xxx xxx xx xxx 3 9 pearl-millet xx x x xx xxx xxx xxx xxx xxx xx 1 11 2. cereal by-products maize bran xx x x xx xxx xxx xxx xxx xxx xx 4 8 maize brew-waste x x x xx xxx xxx xxx xxx xxx xx 6 6 sorghum bran xx x x xx xxx xxx xxx xxx xxx xx 5 7 sorghum brew-waste x x x xx xxx xxx xxx xxx xxx xx 7 5 pearl millet bran x x x xx xxx xxx xxx xxx xxx xx 2 10 pearl-millet brew waste x x x xx xxx xxx xxx xxx xxx xx 8 4 3. other cereal and tuber products maize ugali xx x x xx xxx xxx xxx xxx xxx xx 10 2 sorghum ugali xx x xx xxx xxx xxx xxx xxx xx 11 1 pearl-millet ugali xx x x xx xxx xxx xxx xxx xxx xx 9 3 1. crop and animal protein feeds sunflower seed cake x x xx xxx xxx xx xx 0 12 groundnuts seeds x x xx xxx xxx xxx xxx xx xx 5 7 fish meal xx x x x x xx xx xx xx 1 11 2. insects and worms earth worms xx xxx xx xxx xx x 4 8 maggot larvae xxx xxx xxx xxx xx xx x 2 10 termites xxx xxx xxx xxx xxx xx x x x x x x 7 5 grasshopper xx xx xxx xxx xxx xx xx xx x x x x 6 6 cut worms xx xxx xxx xxx xxx xx x x 3 9 3. minerals and vitamins garden vegetables xx xxx xxx xxx xxx xx xx x 8 4 mtps/forage leaves xx xxx xxx xxx xxx xx xx x 9 3 forage flowers xx xx xxx xxx xxx xx xx x 11 1 tree seeds xx x xx xx xxx xxx xxx 10 2 1 relative scale: xxx= abundant, xx = average, x= low, and = very low/none table 4. relative availability of scavengeable feed resources for the rural poultry during wet and dry season as assessed by farmers at chitemo village in mpwapwa district relative feed availability during wet relative feed availability during dry season 1 pair-wise ranking by season 1 farmers categories of scavengeable feeds total dec jan feb mar apr may jun jul aug sept oct nov scores rank 1. cereal grains maize xx x x xx xxx xxx xxx xxx xxx xx 1 12 sorghum xx x x xx xxx xxx xxx xxx xxx xx 2 11 rice 0 13 pearl-millet xx x x xx xxx xxx xxx xxx xxx xx 4 9 2. cereal by-products maize bran x x x xx xxx xxx xxx xxx xx 5 8 maize brew-waste x x x xx xxx xxx xxx xxx xxx xx 7 6 sorghum bran xx x x xx xxx xxx xxx xxx xx 6 7 sorghum brew-waste x x x xx xxx xxx xxx xxx xxx xx 8 5 pearl millet bran x x x xx xxx xxx xxx xxx xxx xx 3 10 pearl-millet brew waste x x x xx xxx xxx xxx xxx xxx xx 9 4 3. other cereal and tuber products maize ugali xx x x xx xxx xxx xxx xxx xxx xx 11 2 sorghum ugali xx x x xx xxx xxx xxx xxx xxx xx 12 1 pearl-millet ugali xx x x xx xxx xxx xxx xxx xxx xx 10 3 1. crop and animal protein feeds groundnuts seeds x x x xx xxx xxx xxx xxx xxx xx 2 9 fish-meal x x x x x x x x x x x x 4 7 meatmeal 0 11 2. insects and worms earth worms xx xxx xxx xxx xxx x 7 4 maggot larvae xxx xxx xxx xxx xxx xx x 5 6 termites xxx xxx xxx xxx xxx xx x 10 1 grasshopper x xx xxx xxx xxx xxx xxx xx x x x x 9 2 cut worms xx xxx xxx xxx xxx xxx xx x 8 3 3. minerals and vitamins garden vegetables xxx xxx xxx xxx xxx xx x x 6 5 mtps/forage leaves xxx xxx xxx xxx xxx xx xx x 1 10 forage flowers xx xxx xxx xxx xxx xx xx x 3 8 1 relative scale: xxx= abundant, xx = average, x= low, and = very low/none table 5. factors affecting availability of scavengeable feed resource base for rural poultry in the four villages of mpwapwa and kongwa districts. % frequency of the factors affecting availability of sfrb factors kisokwe bumila chamkoroma chitemo pearson chi square ( 2 ) season 24.5 26.1 25.2 24.2 2 (4) =12.87, p<0.05 land preparation 25.8 24.2 25.2 24.8 2 (4) = 6.66, p>0.05 harvesting 25.6 25.6 25.2 23.6 2 (4) =14.59, p<0.05 flock size 24.6 26.5 26.1 22.8 2 (4) = 9.75, p<0.05 feed management 20.6 28.1 28.1 23.1 2 (4) =7.49, p>0.05 land size for 2 (4) =34.76, p<0.001 scavenging 22.4 27.0 28.2 22.4 seeds, sunflower seed cakes and groundnuts ranked highest while in kisokwe village and chitemo village garden vegetables, termites, grasshopper, cutworms, earthworms and maggot larvae ranked highest. factors affecting availability of scavengeable feed resource base for rural poultry factors affecting availability of sfrb are presented in table 5. results indicate that season, harvesting of crops, poultry flock size and land size for scavenging had significant association (p<0.05) with the availability of sfrb while land preparation and management of feed resources at household level had no significant association (p>0.05). further analysis of the data revealed that there was highly significant interaction effects (p<0.001) among the factors affecting availability of scavengeable feed resources. the effect of season and farming activities on the availability sfrb could evidently perceived by farmers as shown in figures 1 4. chemical composition of scavengeable feed resources available for rural poultry results on the chemical composition of scavengeable feed resources are presented in table 6. the results show that the chemical composition of scavengeable feed resources varied considerably. the dm contents of cereals, oil seeds and their by-products and fish-meal were higher than those of dried cassava chips. gross energy (ge) ranged from 17.1 to 29.3 mj kgdm -1 . cereal grains and their by-products had low energy content compared to oil seeds and their by-products. maize bran and pearl millet brewers’ wastes had high crude fibre contents. also sesame seeds, sunflower seeds and sunflower seed cakes had high crude fibre content owing to the presence of husks. crude protein contents of cereal grains and cereal bran were similar. they ranged between 113.3 and 145 g cp kgdm -1 but cereal bran had higher values than grains. rice grains and rice bran/polishings had the lowest protein values of 108 and fe ed st uf fs 12 energy 10 protein fo r 8 6 s c o re s 4 q u al it a tiv e 2 0 jan feb mar apr may jun jul aug sep oct nov dec months figure 1. effect of season and farming activities on the availability of scavengeable feed resource base at kisokwe village. fe ed st uf f s 12 10 fo r 8 sc or e 6 q ua lit at iv e 4 energy 2 protein 0 jan feb mar apr may jun jul aug sep oct nov dec months figure 2. effect of season and farming activities on the availability of scavengeable feed resource base at chitemo village. 89 g cp kgdm -1 respectively. the oil seeds, their byproducts and fish meal had higher protein contents than cereal grains and their by-products. cereal grains and fe ed st u ff s 12 10 fo r 8 sc or e 6 q ua lit at iv e 4 energy 2 protein 0 jan feb mar apr may jun jul aug sep oct nov dec months figure 3. effect of season and farming activities on the availability of scavengeable feed resource base at bumila village fe ed st uf f s 12 10 fo r 8 sc or es 6 q u al it at iv e 4 protein 2 energy 0 jan feb mar apr may jun jul aug sep oct nov dec months figure 4. effect of season and farming activities on the availability of scavengeable feed resource base at chamkoroma village their by-products had low crude fat contents (ee) compared to the oils seeds and their by-products. the ash contents of scavengeable feed resources were fairly high except for grains of maize, red sorghum (improved variety) and rice polishings. the nitrogen-free extract (nfe) content was high in all energy-rich feedstuffs than protein feedstuffs. the mineral contents of potential feed resources were generally low. physical composition of scavengeable feed resources found in chicken crop and gizzard results on physical composition of the individual feed components found in the crop, proventriculus and gizzard (table 7) show that there was diversity in the composition of the diets consumed by scavenging chickens. the aggregate analysis of individual feed materials in the crop and gizzard consisted of: cereals and cereal by-products (29.0%), vegetables and forage materials (1.8%), seeds and seed by-products (3.4%), insects and worms (0.2%), egg shells and bones (0.3%), unidentified feeds (41.5%), inert materials (0.8%) and sand/grit (23.0%). generally, the proportion of energy feedstuffs was higher than protein and mineral/vitamin-rich feedstuffs. average dry weight of both crop and gizzard contents (g) did not vary very much between villages where in bumila village was 22.1 ± 7.9, chamkoroma village was 20.6 ± 11.0, chitemo village was 24.8 ± 16.0 and kisokwe village was 20.0 ± 13.4. chemical composition of scavengeable feed resources found in chickens’ crop and gizzard the results on chemical composition of scavengeable feed resources found in crop, proventriculus and gizzard of scavenging chickens are given in table 8. the dm concentration of the crop and gizzard contents ranged from 472.3 to 493.7 g dm per kg and the mean dm was 478.7 ± 9.6 g per kg. the crude protein content of the crop and gizzard contents was generally low with an overall mean of 80.4 ± 2.3 cp g kgdm -1 . the crude fibre content varied among the villages and the overall mean was 70.7 ± 15.7 cf g kgdm -1 . ether extract content was relatively low with an overall mean of 45.7 ± 21.3 ee g kg dm -1 . kisokwe had the lowest value of 19.9 g kgdm -1 . the ash content ranged from 210.2 to 258.0 g kgdm -1 2 and the overall mean was 234.2 ± 20.5 g kgdm -1 . the nfe content had high values and the overall mean was 555.0 ± 32.5 nfe g kgdm -1 . the mineral content was generally low but had constant values. discussion scavengeable feed resources for rural poultry and the factors affecting their availability energy-rich feedstuffs such as grains of maize, sorghum, pearl-millet and their by-products appeared the most important scavengeable feed resources in all villages. the cereals are the most important crops grown for human consumption in the semi-arid areas of central tanzania. their availability is high during the dry season when they are harvested. these energy-rich feedstuffs are used mostly as basal feeds or as supplements to the diets scavenged by rural poultry in most of rural households in the villages. supplementation with available feedstuffs is sometimes done by giving cereal grains and their by-products and household wastes generally in the morning or evening depending on their availability in the households. however, supplementation of these feedstuffs decreases and sometimes disappears during the wet season in most of the households due to their scarcity. protein-rich feedstuffs such as sunflower seeds, groundnut seeds, sesame seeds, sunflower seed cakes are also the most important poultry feeds in the villages. table 6. chemical composition of feed resources for rural poultry obtained from interviewed farmers in the four villages of mpwapwa and kongwa districts dm chemical composition (g kgdm -1 ) ge g/kg cp cf ee ash nfe ca p k mg (mj/kg 1. cereal grains maize 877 113 52.1 41.8 14.5 779 1.5 5.0 11.0 1.0 19.0 sorghum 1 874 136 157 77.7 25.0 604 3.9 5.2 10.1 2.1 20.0 sorghum 2 875 121 136 48.1 33.3 662 3.2 5.4 11.7 1.3 19.1 sorghum 3 865 111 33.3 15.6 17.2 823 3.6 3.5 9.5 0.8 18.3 pearl-millet 4 875 123 37.0 51.3 69.0 720 3.4 5.8 11.4 1.4 18.3 pear-millet 5 869 130 63.8 140 76.6 590 3.8 6.5 15.4 2.2 20.2 finger-millet 865 122 106 16.0 73.4 683 6.0 3.6 11.4 0.8 17.5 rice grains 875 108 62.6 16.2 90.1 723 6.2 4.8 12.0 1.0 17.1 2. cereal bran maize 6 897 145 229 92.8 53.4 480 2.6 7.7 17.1 3.6 20.1 maize 7 874 142 186 105 62.7 504 2.1 8.1 16.2 3.7 20.1 sorghum 8 871 137 62.3 51.5 53.6 696 3.9 6.7 16.2 2.4 18.8 pearl-millet 875 122 72.8 52.2 84.1 669 2.6 5.8 11.4 1.6 17.7 rice polishings 879 89.2 48.9 35.6 15.3 811 6.1 6.4 20.1 2.8 18.7 3. cereal brewers waste maize & p/millet 890 275 97.7 27.6 36.4 563 12.7 7.9 22.1 3.5 19.5 sorghum 9 875 287 70.7 140 53.6 449 12.6 9.4 21.8 4.5 19.8 pearl millet 906 294 184 62.9 84.1 375 11.4 9.2 20.1 4.2 19.8 4. other energy feeds cassava chips 10 856 64.5 74.7 68.1 64.0 729 3.5 2.6 9.9 0.2 19.5 5. oil seeds & by-products sunflower cakes 915 303 202 145 38.1 312 15.4 13.0 25.9 6.1 22.5 sunflower seeds 912 284 230 399 35.4 51.6 8.8 17.1 26.0 8.3 28.1 sesame seeds 900 328 146 279 67.2 180 10.3 17.3 30.0 8.5 24.9 groundnut seeds 922 322 17.8 488 64.7 108 8.5 15.0 25.6 7.7 29.3 6. animal protein feeds fish meal 910 418 109 105 37.8 330 18.4 16.5 34.5 7.5 22.1 note: 1 red sorghum (local variety); 2 white sorghum (pato variety); 3 red sorghum (improved variety); 4 pearl-millet local variety; 5 pearl-millet improved variety; 6 maize (hand processed); 7 maize (machine processed); 8 sorghum bran, 9 white sorghum local variety (lugugu) and 10 dried cassava chips, ge = gross energy (mj/kg dm). again, these are important oil seed crops found in the farming systems of semi-arid areas of central tanzania. like the energy feedstuffs, these feedstuffs are given to the birds as supplements. in some households, ground sunflower meals from screw pressed cakes are given to the poultry in combination with cereal byproducts. other proteinrich feedstuffs such as fish-meal and meat-meal are not commonly available in the villages. however in recent years, fish-meal has been gradually taking an important place in poultry feeding in chamkoroma, bumila, and kisokwe and chitemo villages. unconventional feedstuffs such as earthworms, maggot larvae, termites, and grasshopper and cut worms are another potential scavengeable feed resource in the study area. availability of these feedstuffs is seasonal dependent. higher quantities are available during the wet season and they are always obtained from the surround ding environments. similar findings have been reported by tadele (1996) in ethiopia and mwalusanya et al. (2002) in tanzania. the study revealed that heaps of cattle dung in the kraal or bomas is used as media for earthworms, maggot larvae and cutworm production and they provide an excellent source of protein rich-diets for the rural poultry. in africa: insects, maggot larvae and earthworms are the most important protein sources for scavenging chickens (smith, 1990; sonaiya, 1995) . in ghana, maggot larvae are produced by farmers from fermented mixture of blood, rumen contents and cattle dung (smith, 1990). in colombia a 100 kg of cattle manure could yield between 0.9 and 2.6 kg of earthworms (beteta et al., 1996). the study also showed that mineral and vitamin-rich feedstuffs particularly garden vegetables, forage leaves, flowers and forage seeds are also important feed table 7. physical composition of sfrb in the dry season expressed as percentage of total dry weight of the chickens’ crop and gizzard contents in the four villages of mpwapwa and kongwa districts physical components of crop and gizzard contents (% dry basis) bumila chamkoroma chitemo kisokwe overall (n = 35) (n = 30) (n = 41) (n = 35) (n= 141) 1. energy feedstuffs maize grains 8.0 7.0 4.1 6.3 6.3 maize bran 4.0 6.0 0.8 2.3 3.3 sorghum grains 0.3 0.0 3.5 0.8 1.2 sorghum bran 0.2 4.4 13.7 11.4 7.4 pearl-millet grains 0.0 0.0 1.4 0.0 0.4 pearl-millet bran 0.0 0.0 0.1 0.0 0.0 local brew wastes 3.0 0.0 1.2 2.8 1.8 cassava chips 0.0 0.1 0.0 0.9 0.2 kitchen wastes 11.0 9.3 7.8 5.5 8.4 2. protein feedstuffs insects 0.1 0.3 0.4 0.1 0.2 earthworms 0.0 0.0 0.0 0.1 0.0 feathers 0.1 0.1 0.1 0.0 0.1 sunflower cake 0.5 4.3 0.1 5.5 2.5 watermelon seeds 0.0 0.0 0.0 0.2 0.1 tree seeds 0.0 0.2 0.1 0.0 0.1 ground nuts 0.1 2.2 0.3 0.7 0.7 3. mineral/vitamin feedstuffs grass leaves/stems 0.0 0.3 0.0 0.0 0.1 fruits 0.0 0.7 0.0 0.4 0.4 forage leaves 0.2 0.8 0.1 0.4 0.4 onions 0.0 0.1 0.0 0.0 0.3 egg shells 0.0 0.0 0.2 0.0 0.1 tree flowers 0.0 0.6 0.0 0.2 0.2 fibrous materials 0.7 0.9 0.2 0.1 0.5 bones 0.1 0.2 0.2 0.0 0.1 4. inert materials metal materials 0.2 0.3 0.4 0.9 0.4 trouser/shirt button 0.1 0.0 0.0 0.2 0.1 shoes materials 0.1 0.0 0.0 0.0 0.0 plastic materials 0.5 0.4 0.2 0.2 0.3 5. other feed materials sand/grits 24.1 28.7 17.3 22.2 23.0 unidentified feeds 46.7 32.2 47.8 39.4 41.5 table 8. average chemical composition of crop and gizzard contents of scavenging chickens in the four villages of mpwapwa and kongwa districts village dm chemical composition (g kgdm -1 ) me g/kg cp cf ee ash nfe ca p k mg (mj/kg bumila 472 80.3 83.1 52.2 258 517 7.1 7.3 13.6 2.9 9.4 chitemo 474 82.1 51.5 39.9 242 563 7.2 7.2 14.3 2.9 10.5 kisokwe 494 77.3 64.2 19.9 227 594 7.2 6.8 13.2 2.4 9.9 chamkoroma 479 82.1 83.8 70.7 210 545 7.8 8.0 13.3 3.3 10.5 overall mean 479 80.4 70.7 45.7 234 555 7.3 7.3 13.6 2.9 10.1 ±9.6 ±2.3 ±15.7 ±21.3 ±20.5 ±32.5 ±0.3 ±0.5 ±0.5 ±0.4 ±0.5 resources for the rural poultry. leaves, flowers and seeds from trees species such as leucaena leucocepahala, caliandra colothyrus, acacia tortilis and citrus vulgaris are collected by some farmers for their poultry. however their availability is limited by the seasonal nature as most of them are available during the wet season. this indicates that during the dry season, rural poultry can develop mineral and vitamin deficiencies because of the scarcity of the succulent forage materials on the range. apart from season, the study also revealed that availability of scavengeable feed resources for rural poultry was significantly associated wit the farming activities. in the study area, land preparation involves clearing and burning of cropresidues and natural weeds in the fields. these are the ecological niches for termites, grasshopper, reptiles and other invertebrates. such invertebrates are usually destroyed by fire during land preparation and in some cases they migrate to bushy areas where they cannot be scavenged by poultry. in addition, during ploughing and planting, farmers use hand-hoes and animal drawn implements such as ox-drawn ploughs. as a result, some surface vegetation and crop-residues are incorporated into the soil and the soil borne insects are exposed to the surface where they become available to the rural poultry. moreover, during planting some grains and oil seeds sown in the fields become available for scavenging poultry. as previously stated, crop harvesting and post harvest activities had also significant effect on the availability of sfrb. the study noted that there are substantial amounts of cereal grains, oil seeds and their by-products spilled on the ground during harvesting and threshing of these crops which are picked up by the rural poultry. moreover, availability of sfrb is associated with the land size available for scavenging and the size of the flock biomass in the village. the study revealed that land size available for scavenging is directly related to the density of village houses. in all the four villages, housing density was relatively high and most of the family flocks had to mix with flocks from their neighbours when scavenging. such situation in most cases could result into high competition for sfrb among the poultry and the chicks are more likely to suffer from this competition. management of the feed resources at farm level is another factor reported to contribute to the availability of sfrb. it was noted that most of the household wastes discarded out by the family members could be available for other livestock species such as pigs, goats, pets and wild birds. nutritional composition of scavengeable feed resources for rural poultry in the villages the high gross energy values in the cereal grains and their byproducts indicate that these feedstuffs are good sources of digestible energy and metabolizable energy for poultry (mcdonald et al., 2002). despite the high energy content, most of the grains and their by-products had relatively low protein contents ranging from 8 12 cp g kgdm -1 . the low protein values in cereal grains and their by-products could have been associated with the high fibre content in these feedstuffs. high fibre content has been reported to have diluent effect on the grains as whole and reduces the energy and protein value proportionally (mcdonald et al., 2002) . as a result of this, most of the cereal grains and theirby-products have moderately to low content of lysine, tryptophan and threonine for monogastric animals and methionine for poultry (mcdonald et al., 2002). regarding the mineral contents, the results showed that scavengeable feed resources in the villages had very low mineral contents particularly calcium (ca) and magnesium (mg). phosphorus (p) and potassium (k) contents seem to be comparatively high, although much of it was present in oil seeds and their byproducts and fish meal than in cereal grains and their byproducts. the low mineral contents in cereal grains could have been associated with the marginal soil fertility in the study area coupled with very low fertilization inputs and erratic rainfall pattern. the high content of ether extract and crude protein in the oil seeds and their by-products shows that these feedstuffs can have a significant contribution to the energy and protein diets for rural poultry. however, in general oil seed proteins are deficient in essential amino acids in particular cystine, methionine and lysine and the feed materials have high fibre content (mcdonald et al., 2002). thus the high fibre content and the relative low levels of the essential amino acids in these feed materials is a constraint to their inclusion in poultry feeds because they cannot provide adequate supplementation of the cereal bran. therefore they should be used in combination with other protein sources such as soya bean and fish meal. physical and chemical composition of chickens’ crop and gizzard contents visual observations of the crop and gizzard contents revealed that the scavenged feed had a diversity of feed ingredients. the diversity in the composition of the diets consumed by scavenging birds showed that they can select from various feed ingredients available and compose their own diets. thus a free-range system is of particular importance to smallscale poultry producers in developing countries such as tanzania because it reduces substantially the costs of commercial feeds which most of the farmers can not afford to buy. also the system offers an effective way to make use of locally available grains such as maize, sorghum and pearl millet, oilseeds and their by-products. however, as indicated in table 8, most of the sfrb available for scavenging are not concentrated enough particularly in terms of protein. the crop and gizzard contents in the present study contain on average 80.4 g of cp per kgdm. the low crude protein in the scavenged feeds could be explained by the lower percentage of protein feedstuffs in the diets consumed by birds in the dry season. similar results were obtained by tadele (1996) and rashid et al. (2005) who reported low cp content of 76 and 102 g per kgdm respectively, in the crop contents of scavenging hens in the dry season. however, pousga et al. (2005) in burkina faso reported that the cp in the crop contents was higher in the dry season (115 g kgdm -1 ) compared to the wet season (105g kgdm -1 ) for both local and crossbred birds, resulting from higher consumption of green grass/leaves, larvae, insects and worms due to occasional showers occurred at the end of dry season. the metabolizable energy (me) concentration of 10.1 mj kgdm -1 in the crop and gizzard contents was approximately similar to the energy content of around 11.4 mj kgdm -1 reported by pousga et al. (2005) in burkina faso, rashid et al. (2005) in bangladesh and tadelle (1996) in ethiopia in the dry season. however these energy values were lower than the energy content of around 12.4 mj kgdm -1 reported by the same authors during the rainy season. these results confirm the earlier findings that birds kept in the free-range system can certainly not find all the nutrients they need for optimum growth and egg production all the year round (goromela et al., 2006). supplementation with the energy and protein sources can complement what birds need for optimum growth and egg production in central tanzania. conclusion from the present study, it can be concluded that in central tanzania cereal grains and their by-products, the oil seeds and their by-products are the most important scavengeable feed resources available for rural poultry. unconventional scavengeable feed resources such as forage leaves and flowers; forage seeds, garden vegetables, insects and worms are also the most important protein and mineral-rich feedstuffs for rural poultry. however, their availability is seasonal in nature. considerable amounts of cereal grains and their byproducts and some of their by-products were available during the dry season while large amounts of protein and mineral/vitamin feedstuffs were available during the wet season. thus any attempt to improve the diet of scavenging birds should take into consideration what the birds are essentially consuming depending on season and farming activities. the chemical composition of sfrb indicates that cereal grains and their by-products had high energy contents and low contents of crude protein and minerals particularly calcium and magnesium. the low protein and mineral/vitamins contents in these energy-rich feedstuffs require supplementation with locally available protein and minerals/vitamins sources. the high crude fibre levels of above 20% and the low content of amino acids in oil seeds and their by-products can potentially limit the inclusion of this feed material in poultry diets where the recommended maximum fibre level of inclusion rate is 10% of the diet for adult poultry. thus they should be combined with other protein sources such as soya-bean and fish-meals when given to poultry. the low concentrations of energy, protein and minerals in the crop and gizzard contents indicates that diets consumed by birds could not meet optimum requirements of scavenging birds for growth and egg production. supplementation of these nutrients would be necessary to attain reasonable growth and egg performance for rural poultry. however, the major part of the present study has evaluated the available sfrb and the nutrient contents which may not represent the actual diets and nutrients scavenged by rural poultry. thus further studies should be undertaken to determine the physical and chemical composition of the crop and gizzard contents and thus feed consumed by scavenging birds in different seasons and farming systems of central tanzania and investigate their interaction effects with poultry manage-ment practices on the productivity of rural poultry. acknowlegements the authors wish to thank the netherlands organization for international cooperation in higher education (nuffic), the netherlands for the great financial support which enabled us to undertake this field study. the authors would like to express their heartfelt thanks for the dedication and generous support from the local leaders and farmers in bumila, chamkoroma, kisokwe and chitemo villages of mpwapwa and kongwa districts during the study period. all the enumerators who participated in household survey and entering data into the spss data editor are very much thanked for their commitment in this study. lastly the authors are very grateful to the research staff at the national livestock research institute mpwapwa and the nutrition laboratory staff at the central veterinary laboratories in dar es salaam, tanzania for the support of research materials; analysis of feed samples and transport facilities rendered during the study period. references aoac (1985). association of official analytical chemists. official methods of analysis 12 th ed. washing, d.c beteta th (1996). experience of recycling manure in colombia. m.sc. thesis. swedish university of agricultural sciences, department of animal nutrition and management, uppsala sweden. cummings rb (1992). the advantages of free choice feeding for village chickens. proceedings of the xix world’s poultry congress. 3 (20-24 september 1992): 627-630 amsterdam, the netherlands. goromela eh (2000). a century report of monogastric research and development activities in tanzania 1900-2000. a century report submitted to the tanzania commission for science and technology, dar es salaam, tanzania. 6 th september. goromela eh, kwakkel rp, verstegen mwa katule am (2006). strategies to optimize the use of scavengeable feed resource base by smallholders in traditional poultry production systems in africa: a review. afr. j. agric. res. 1 (3): 091-100. gunaratne sp (1999). feeding and nutritional of scavenging chickens. in: the scope and effects of family poultry research and development. international network for family poultry developmentinfpd.http://www.fao.org/ag/againfo/subjects/en/infpd/documents/ec o nf_scope/add_paper2.html. kitalyi aj (1998). village chicken production systems in rural africa household food security and gender issues. fao animal production and health paper 142:http://www.fao.org/docrep/003/w8989e/w8989e00.htm mcdonald p, edwards ra, greenhalgh ffd, morgan ca (2002) animal nutrition. sixth edition pp. 583-604 minga um, yongolo mgs, mtambo mma, mutayoba s, lawrence p, mwalusanya na, katule am mlozi mrs (2000). the potential for rural poultry production and health in africa. proc. of the workshop on the possibilities for smallholder poultry projects in eastern and southern africa, morogoro, tanzania ministry of agriculture and co-operatives (1995). national sample census of agriculture 1994/95. report volume ii. mwalusanya na, katule am, mutayoba sk, minga um, mtambo mm, olsen je (2002). nutrient status of crop contents of rural scavenging local chickens in tanzania. br. poult. sci. 43: 64-69. pousga s, boly h, lindberg je, ogle b (2005). scavenging pullets in burkina faso: effect of season, location and breed on feed and nutrient intake. tropical animal health and production, 37: 623-634. rashid mm, islam mn, roy bc, jakobsen k, lauridsen c (2005). nutrient concentrations of crop and gizzard contents of indigenous scavenging chickens under rural conditions of bangladesh. livestock research for rural development 17(2) .http://www.cipav.org.co/lrrd/lrrd17/2/rash17016.htm. roberts ja (1995). assessing the scavenging feed resource base for smallholder poultry development. in: sustainable rural poultry production in africa. proceedings of an international workshop held on june 13-16, 1995 at the international livestock research institute, addis ababa, ethiopia. pp. 40-52. sonaiya eb (1995). feed resources for smallholder poultry in nigeria. world anim. rev. 82 (1): 25-33. sonaiya eb, branckaert rds guèye ef (1999). the scope and effect of family poultry research and development. research and development options for family poultry. first-infpd/fao electronic conference on family poultry. sonaiya eb (2004). direct assessment of nutrient resources in free range and scavenging systems. regional report: in: worlds poult. sci. j. 60: 523-535 tadele d (1996). studies on village poultry production systems in the central highlands of ethiopia m.sc. thesis. swedish university of agricultural sciences. uppsala, sweden. united republic of tanzania/urt ( 2005). www.tanzania.got.tz/livestockf.html. wiseman j (1987). feeding of non-ruminant livestock. english translation © butterworth & co. (publishers) ltd, 1987. pp. 9-10. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (2), pp. 001-012, february, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper physical properties of tylosemia esculentum and the effect of roasting on the functional properties of its flour v. a. jideani*, j. van wyk and m. h. cruywagen department of food technology, cape peninsula university of technology, p.o. box 652, cape town 8000, south africa. accepted 26 november, 2018 the physical properties of two varieties of morama beans and the effect of roasting on the functional properties of their flours were studied. majority (65, 92.5, 79%) of the seeds has medium dimensions (17.0 19.99, 14.0 19.99, 12.0 14.99 mm) of length, width and thickness respectively. the size of the morama bean (mb) follows a normal distribution, being much heavier than most other oil seeds and legumes with 1000-seed weight of 2.2 2.3 kg. significantly higher angle of repose was observed for the cement surface (9.76 o ). mb sphericity (85.7-86.8%) and aspect ratio (91.9 92.5%) indicates that the bean will roll rather than slide. morama flour (mf) is rich in protein (31.7 33.1%), fat (36.4 39.0%) and ash (2.9%). roasting significantly increased the protein and ash. roasting increased the water absorption capacity of mf significantly (p < 0.05) (211% increase). roasting significantly increased the oil absorption capacity (112% increases) . emulsifying activity ranged from 37.90 38.20% respectively for lightand dark-brown morama flour. emulsion stability ranged from 70.62% (light brown) to 72.18% (dark brown). the colour of unroasted mf is light yellow (l = 82.71, hue angle = 88.05, chroma = 26.21), turning darker and less saturated on roasting. key words: morama bean, physical properties, functional properties, lightness, hue, chroma. introduction tylosemia esculentum is a woody prostrate trailing plant which produces an oil seed legume called morama beans, is endemic to the southern parts of africa where it grows in the grasslands and bushveld (coetzer and ross, 1977), with large populations in botswana (around the central kgalagadi) and namibia, smaller populations in the provinces of limpopo, north-west and gauteng of south africa (national research council, 2006). the plant thrives in poor quality soil and harsh climatic con-ditions and has creeping stems which are up to 6 meters long, protecting it from the destructive windstorms of the kalahari desert (hartley, 1997). morama beans grow in a large, flat woody pod in groups of approximately six beans per pod. the beans are chestnut to dark brown in *corresponding author. e-mail: jideaniv@cput.ac.za. abbreviation: mb; morama bean, mf; morama flour, wac; water absorption capacity, oac; oil absorption capacity. colour, normally spherical and weigh 23 g each. each bean has a hard inedible outer shell with an edible two lobed seed inside. the inner flesh is cream coloured (national research council, 2006) . t. esculentum has a long history as a food resource. it is believed that mora-ma beans, and other edible parts of this plant, may have been in our diet as long as food was in existence, and even today it is an important component for some living in the southern parts of africa which include the herero, tswana, khoi san and other bantu speaking people (national research council, 2006). the composition of the morama bean is similar to that of peanuts and soybeans. it has a protein content of 29.6 41.8% and an oil content of 32.1 45.3% (monagham and hallovan, 1995). in addition to its high protein and oil content, morama beans also contain minerals and vitamins which include potassium, phosphorus, thiamin, riboflavin and nicotinic acid. morama bean is a source of clear golden yellow oil, which has a nutty odour with an agreeable taste similar to almond oil. the taste of the morama bean is soapy with a slimy texture due to its high figure 1. (a) morama seed varieties, leftdark brown; rightlight brown. (b) characteristic dimensions of morama bean: length (l), width (w) and thickness (t). water activity (hartley, 1997). the bean is not eaten raw but rather roasted and eaten as a snack or boiled and consumed as a vegetable (mmonatau, 2005). when roasted morama beans resemble the taste of roasted cashew nuts or chestnuts (mmonatau, 2005). it has been reported that morama beans contain trypsin inhibitors; these are however destroyed by heat which would usually be applied during the cooking or roasting processes (mmonatau, 2005). malnutrition is an increasing global issue. the morama bean is widely believed to potentially play a significant role in solving the world’s malnutrition problems due to its high content of unsaturated fatty acids, proteins and other nutrients. despite the high nutritional value of the morama bean, the only food application is roasting the dry beans and consuming it as a snack. the acceptability of legume flours in foods depends on their nutritional value and functional properties (ahenkora et al., 1999). some of these properties useful in food product development are water and oil absorption, emulsification, foam capacity and gelation (wu et al., 2009). besides, physical properties of foodstuffs are important in the design, handling, storage and drying systems, as well as daily operation of the foodstuff. unfortunately, information about the physical and functional properties of morama bean is lacking. to tap into the high nutritional content of the morama bean our objective was to assess the physi cal properties of morama bean, functional properties of the flour and effect of roasting with a view to obtain an indication of what possible products the flour can be applied to. materials and methods source of morama beans the morama beans were purchased from ghanzi, botswana. the beans were kept at a temperature of 5°c and removed 1 h before the physical properties of the beans were measured. the beans were sorted into two varieties based on colour: lightand darkbrown seeds (figure 1a) . all tests were conducted separately on the seeds and flours from these two groups. physical properties of morama beans linear dimensions the method stated of mpotokwane et al. (2008) was adopted to measure the linear dimensions of the beans. hundred (100) seeds were selected from each variety by obtaining a hand full of beans from a random location in a bowl containing the beans. the length (l), width (w) and thickness (t) as illustrated in figure 1b of each selected bean were measured to an accuracy of 0.001 mm using a vernier caliper. from the results obtained in these measurements, the geometric diameter, sphericity, volume, surface area and aspect ratio were determined. geometric diameter, sphericity, volume, surface area and aspect ratio the equations of mohsenin (1986) were used to determine the geometric diameter (dg) and sphericity ( ) of the bean. (1) (2) where; l = bean length, w = bean width and t = bean thickness in mm as shown in figure 1b. the bean volume (v) and surface area (s) were determined using equations adopted from jain and bal (1997). v  b 2 l 2 (3) 6(2l  b) s  b 2 l 2 (4) 2l  b where; ; l = length of the seeds; w = width of the seeds; t = thickness of the seeds in mm as shown in figure 1b. one thousand bean weight the method reported by mpotokwane et al. (2008) was adopted to determine one thousand bean weights. the mass of one thousand seeds of each variety were obtained using an electronic balance weighing to an accuracy of 0.0001 g. this was done in triplicate. bulk – and true density the method of asoegwu et al. (2006) reported by mpotokwane et al. (2008) was used to determine the bulk density of the beans. a 500 ml cylinder was filled to the 500 ml mark with beans and weighed on an electronic balance. the bulk density was calculated as the ratio of bulk weight and volume of the cylinder (g/ml). the true density was calculated using the liquid displacement method of asoegwu et al. (2006). all weights were measured using a top loading balance. a 500 ml beaker was filled to the 350 ml mark with water. approximately 100 g of beans were immersed in the water. the mass of the water displaced is the balance reading with the seed submerged minus the mass of the beaker with water. the seed volume was estimated by dividing the mass of displaced water (g) by the density of water (g/cm 3 ). seed density was determined by dividing the seed mass by the measured seed volume. this was repeated thrice for each group of beans. seed porosity: seed porosity is a property of grain which depends on its bulkand kernel densities. seed porosity (p) was determined using the equation stated by mpotokwane et al. (2008):  p b 100 p  1 p t where pb = bulk density (kg/m 3 ) and pt = seed density (kg/m 3 ). angle of repose the method stated by mpotokwane et al. (2008) was adopted for determination of the repose angle. a topless and bottomless cylinder, with a height of 17 cm and a diameter of 10.5 cm was used. the cylinder was filled to the top with beans. the cylinder was then slowly raised until the beans formed a natural heap. the angle of repose was calculated from the diameter and height of the heap formed on cement -, glassand woodensurface. the angle of repose determined in replicates was determined using the formula tan 1 ( 2h l) where h = height of heap and l = length of the heap. preparation of morama bean flours roasting of morama beans the method reported by mmonatau (2005) was used for roasting morama beans (light and brown). the beans were roasted for 15 min at 160°c using defy 731 multifunction thermofan oven. cracking of morama beans the unroasted and roasted morama beans from the light and brown varieties were separately cracked using a hammer. the edible part of the beans was separated from the shell by hand picking. preparation of unroasted and roasted morama bean flour unroasted morama bean flour was prepared following the method reported by chau and cheung (1998) with modifications. the dehulled unroasted beans were blanched at 100°c for 30 min followed by soaking in distilled water at 25  3°c for 3 h. the soaked beans were washed, drained and dried at 50°c for 72 h. the dried beans was milled into flour using a hallde blender and sieved to a mesh size of 500 micron. the unroasted flour was packaged in polyethylene bags, sealed and stored at 5°c until required. roasted beans were milled into flour using a hallde (sweden) blender at high speed and sieved to a mesh size of 500 micron. the roasted flour was packaged in polyethylene bags, sealed and stored at 5°c until required. proximate composition of morama bean flours the aoac (1990) method was used to determine the moisture, fat, protein and ash content of the unroasted and roasted morama flours. the energy content of the flours was estimated using the atwater factor as reported by marero et al. (1988). energy content = [(4 × protein) + (4 × carbohydrate) + (9 × fat)] × 4.2 where protein, carbohydrate and fat are contents of the sample expressed in g/100 g sample, 4, 4 and 9 are kilocalories from protein, carbohydrate and fat respectively, 4.2 is a factor for converting from calories to joules. the determination was carried out in triplicate. functional properties of morama bean flour water absorption capacity the method described by mc walters et al. (2002) was adopted to measure the true-and apparent water absorption capacities of unroasted and roasted morama bean flours. each flour, (2 g) [m0] was suspended in 20 ml of distilled water and agitated in a mechanical shaker for 1 hour. the slurry was then centrifuged at 31304 × g for 1 h and the sediments dried on whatman filter paper (m1 + h2o) for 1 h. the proportion of water absorbed was calculated as apparent water absorption capacity using the formula reported by mc walters et al. (2002). waa  m1  h2o m0 100 (5) m0 protein solubility protein solubility of the morama bean flours were determined using mc walters et al. (2002) method. the filtrated obtained from the sample while measuring water absorption capacity was used to determine protein solubility. the invritrogen quibit fluorometer was used to measure the amount of soluble proteins present in the filtrate. protein solubility was reported as percentage soluble proteins. the measurement was done in triplicate. fat absorption capacity a method adopted by sogi and chandi (2007) was used to determine the fat absorption capacity of morama bean flours. flour (0.5 g) was mixed with 10 ml canola oil and centrifuged at 750 x g for 15 min. the supernatant was then drained off for 30 min using cheese cloth and the weight gain reported as oil absorption capacity in triplicates. ph morama bean flour (0.6 g) was dissolved in 20 ml distilled water to obtain a 3% suspension. the ph of the suspension was measured using a ph meter. least gelation concentration the least gelation concentration was determined using the method adopted by sathe and salunke (1981). a test tube containing suspensions of 2, 4, 6, 8; 10, 12, 14, 16, 18 and 20% weight per volume material in 5ml distilled water was heated in boiling water for 1 h. this was followed by cooling in ice and further cooling for 2 h at 4°c. the least gelation concentration stated was the tube with the lowest concentration suspension of which the content did not fall down or slip when the test tube was inverted at ph 7. this was done in triplicates. emulsion capacity and stability the method reported by naczk et al. (1985) with modifications was used to determine the emulsion capacity and stability of the morama flours. flour (3.5 g) was mixed using a mechanical mixer for 1 min. canola oil (25 ml) was added and the mixture was mixed again for 1 min. another 25 ml of canola oil was then added and the mixture was mixed for 3 min. the emulsion was divided evenly into two centrifuge tubes and centrifuged at 1100 x g for 5 min. the emulsifying activity was calculated by dividing the volume of the emulsified layer by the volume of the emulsion before. the emulsion stability was determined using samples prepared for measurement of emulsifying activity. these samples were heated for 15 min at 85°c, cooled, divided evenly into two 50ml centrifuge tubes and centrifuged at 1100 × g for 5 min. the emul sion stability was expressed as the percentage of emulsifying activity remaining after heating at ph 7. colour measurement the colour of the different varieties of unroasted and roasted morama bean flour was measured using the hunter lab instrument in daylight colour mode (d65) and 10° standard observer. the flour samples (30 g) were place in the sample holder and the surface color was measured at three different positions (senthil et al., 2002). the colour readings were displayed as l* a* b* values where l* represents lightness/ darkness dimension, positive and negative a* value indicates redness and greenness respectively and b* indicates yellowness for positive and blueness for negative values. the colour measurement was repeated four times. the colour values were converted to hue tan 1 b * a * and a * 2 b * 2 chroma . data analysis analysis of variance (anova) and one sample t-test was used to determine mean differences in treatment. duncan’s multiple range tests was used to separate means where differences existed (spss, 2007). results physical properties of morama beans linear dimensions of morama beans the length, width and thickness of morama bean (mb) are detailed in table 1. the morama varieties did not differ significantly (p > 0.05) in their linear dimensions. morama seeds have a mean length of 18.6 mm and a range of 12.0 23.2 mm. the width of morama seeds ranged from 12.0 27 mm with a mean of 17.0 mm and the thickness ranged from 9.80 19.20 mm with a mean of 13.1 mm. table 1 details the l/w, l/t and l/d g ratio for morama bean (mb) . the varieties did not differ significantly in the ratios. l/t exhibited the highest value in both varieties. the l/dg and l/w ratios were very similar. figure 1 shows the frequency distribution for the length, width and thickness of morama bean. majority (65%) of the seeds has medium length of 17.0 19.99 mm. in terms of width, 92.5% of the seeds have a medium width of 14.0 19.99. with respect to thickness 79% of the seeds have a medium size of 12.0 14.99 mm. geometric properties of morama beans the geometric properties of morama beans are presented in table 2. the morama bean varieties did not differ significantly (p > 0.05) in geometric properties. the geo table 1. effect of variety on linear dimensions of morama beans*. variety dimension mean (mm) range (mm) l/w l/t l/da light length 18.6  1.5 12.0 22.8 1.11  0.16 1.42  0.16 1.19  0.09 width 17.1  2.0 12.0 27.0 thickness 13.2  1.1 10.0 15.7 dark length 18.6  1.8 15.5 – 23.2 1.10  0.12 1.45  0.18 1.20  0.08 width 17.0  1.5 13.6 20.8 thickness 12.9  1.4 9.8 19.2 total length 18.6  1.6 12.0 – 23.2 1.10  0.14 1.44  0.17 1.20  0.08 width 17.0  1.8 12.0 – 27.0 thickness 13.1  1.3 9.8 – 19.2 *values are mean  standard deviation. f re q u en cy ( % ) 100 90 small medium 80 large 70 60 50 40 30 20 10 0 length width thickness seed size (mm) figure 2. morama bean seed dimension distribution of at morama bean. length (small <17 mm; medium 17.00-19.99 mm, large >20 mm); width (small <14 mm, medium 14.00-19.99 mm, large >20 mm); thickness (small <12, medium 12.00-14.99 mm, large >15). table 2. geometric properties of morama beans*. variety geometric diameter seed volume surface area sphericity aspect ratio (mm) (mm 3 ) (mm 2 ) (%) (%) dark 15.5  0.93 1143  210 10752  2293 83.7  5.7 91.9  9.8 light 15.7  0.96 1183  222 11219  2360 84.4  6.8 92.5  14.5 total 15.6  0.95 1163  217 10986  2333 84.1  6.2 92.2  12.3 *values are mean  standard deviation. table 3. effect of variety on gravimetric properties of morama beans*. 3 true density repose angle variety 1000-seed weight (kg) bulk density (kg/cm ) (kg/cm 3 ) (°) porosity (%) dark 2.3  0.1 652  21 996  6 9.2  0.5 34.6  2.0 light 2.2  0.2 668  21 1000  1 9.4  0.5 32.6  2.0 *values are mean  standard deviation. metric diameter of the beans was 15.84 and 15.65 mm respectively for the dark and light variety. the mean volume of the beans was 1143 and 1182 mm 3 respectively for the dark and light variety. the mean volume and surface area did not differ significantly among the varieties. the mean sphericity of the dark brown variety was 85.7% and that of the light brown variety was 86.8%. the sphericity of the light variety of morama beans was higher than the dark brown beans. the mean aspect ratio of the dark brown bean was 91.9% and that of the light brown beans was 92.5%. there were no significant differences between the aspect ratio of the different varieties of morama beans. table 4. effect of variety and surface on repose angle of morama bean*. material repose angle (°) total dark brown light brown glass 8.74  0.43 9.15  0.12 8.95  0.37 a cement 9.55  0.21 9.97  0.29 9.76  0.33 b wood 9.27  0.24 9.05  0.12 9.16  0.21 a total 9.19  0.45 a 9.39  0.47 b *values are mean  standard deviation. different superscripts in each row or column indicate significant difference (p < 0.05). gravimetric properties of morama beans the effect of variety on gravimetic properties of the morama beans are reported in table 3. there were no significant differences in gravimetric properties between the varieties. the dark brown beans had a mean 1000 seed weight of 2.3 kg and the light brown beans a mean of 2.2 kg. the mean bulk densities of the beans were 668 and 652 kg/cm 3 respectively for the light and dark brown morama beans. the mean true densities of the beans were 999 and 996 kg/cm 3 respectively for the light brown and dark brown beans. the porosity for the dark and light brown beans was 34.6 and 32.6% respectively. the porosity of the dark brown beans was higher than that of the light brown bean, though not significant. frictional properties the effects of variety and surface on angle of repose are detailed in table 4. the mean angle of repose for the dark brown beans on a glass-, cementand woodsurfaces were 8.74, 9.55 and 9.27° respectively and that of light brown beans on glass-, cementand woodsurfaces were 9.15, 9.97 and 9.05° respectively. the effect of variety and surfaces on angle of repose differed significantly (p < 0.05). the light brown morama was significantly higher in angle of repose than the dark brown morama. significantly higher angle of repose was observed for the cement surface (9.76°). the glass and wood surfaces did not differ significantly in their effect on angle of repose. comparison of morama bean physical properties with other seeds table 5 details the comparison of mean properties of the morama bean with that of bambara groundnut, chickpea, jack bean, egyptian peanut and african bread fruit. one sample t-test with 95% confidence interval on the linear dimensions indicates that morama bean is significantly (p < 0.05) longer than bambara, chickpea, and african bread fruit; but shorter than jack bean and the egyptian peanut. in terms of width morama bean is significantly wider than all the other seeds. mb is thicker than the other seeds except for egyptian peanut. mb has a higher aspect ratio than bambara groundnut, chickpea, jackbean and breadfruit. sphericity of mb is higher than chickpea, jackbean and african breadfruit, but the equal to bambara groundnut. porosity of morama is the same as that of jackbean but higher than bambara. effect of variety and roasting on the nutritional composition of morama bean flour. the nutritional composition of unroasted and roasted morama flour is detailed in table 6. the moisture content ranged from 6.4% for dark brown variety to 6.9% for the light brown variety. the fat content ranged from 36.4% (light brown) to 39.0% in dark brown. protein content ranged from 31.7% in light brown variety to 33.2% in dark brown variety. the ash content ranged from 2.9% in dark brown morama to 2.9% in light brown morama. the varieties differed significantly (p < 0.05) in moisture and protein content. the multivariate tests indicated that variety did not have significant (p = 0.133 with partial eta squared = 0.643) contribution to the multivariate model. however, the high partial eta squared indicated that variety had sig table 5. comparison of morama bean physical properties with other seeds. morama bambara chickpea b jackbean c egyptian african property bean groundnut a peanut d breadfruit l (mm) 18.6 11.48 10.2 18.66 35.86 11.91 w (mm) 17.0 9.66 7.74 13.14 14.96 5.69 t (mm) 13.1 9.17 7.66 10.22 16.34 4.64 true density (kg/m 3 ) 998 1003 1190 20.54 979 bulk density (kg/m 3 ) 660 775 778 614 porosity 33.56 22.7 32.6 aspect ratio (%) 92.5 84.6 75.6 70.6 47.8 sphericity (%) 84.1 85.8 82.9 72.7 57.1 geometric mean diameter 15.9 9.81 8.46 13.56 6.80 (mm) a mpotokwane et al. (2008); b tabatabaeefar et al. (2003); c eke et al. (2007); d alfaro et al. (2004); e omobuwajo et al. (1999). table 6. effect of variety and roasting on the nutritional properties of the morama flour 1 . p ro x im a te ( g /1 0 0 g d ry b a s is ) composition morama variety type of flour 2 dark brown light brown unroasted roasted moisture 6.4  0.5 a 6.9  0.1 b 6.5  0.5 6.8  0.3 f-ratio 7.61 1.86 p-value 0.0222 0.2062 partial eta squared 0.4580 0.1710 fat 39.0  4.1 36.4  6.0 35.2  6.2 40.3  1.6 f-ratio 1.01 3.88 p-value 0.3407 0.0804 partial eta squared 0.1011 0.3012 protein 33.2  1.3 a 31.7  1.1 b 31.6  0.8 a 33.3  1.3 b f-ratio 11.25 16.49 p-value 0.0085 0.0028 partial eta squared 0.5555 0.6469 ash 2.9  0.5 2.9  0.3 2.6  0.2 a 3.2  0.2 b f-ratio 0.01 30.19 p-value 0.9193 0.0004 partial eta squared 0.0012 0.7704 carbohydrate 18.5  4.3 22.0  7.1 24.2  6.2 a 16.4  1.3 b f-ratio 2.06 10.13 p-value 0.1846 0.0111 partial eta squared 0.1866 0.5296 energy (kj/100 g) 2343  89 2279  124 2265  139 2358  34 f-ratio 1.258 2.609 p-value 0.2911 0.1407 partial eta squared 0.1226 0.2248 1 values are mean  standard deviation. in each column different superscripts in each row indicate significant difference (p < 0.05). 2 combined effect of unroasted (dark and light) and roasted (dark and light). nificant effect on some of the nutritional composition of morama flour. on the contrary, the effect of roasting on the nutritional composition of morama flour was significant (p = 0.001, partial eta squared = 0.936). the between-subjects effects showed that roasting significantly increased the protein (p = 0.003) and ash (p = 0.000), while the effect on carbohydrate (p = 0.011) was a decrease. effect of variety and roasting on the functional properties of bean flour the multivariate tests showed that both varieties (p = 0.041 with partial eta squared = 0.912) and roasting (p = 0.000, partial eta squared = 0.999) contributed significantly to the multivariate model (table not shown). water and oil absorption capacity the effect of variety and roasting on the water absorption capacity (wac) and oil absorption capacity (oac) of morama flour is stated in table 7. wac of unroasted morama flour ranged from 10.8% (light brown) to 11.8% (dark brown). varieties differed significantly (p < 0.05) in water absorption capacity (wac). dark brown morama had significantly higher wac when compared with light variety. roasting increased the wac of morama flour significantly (p < 0.05) in both varieties, a 5.5% for unroasted flour to 17.1% for roasted flour (211% increases). oil absorption capacity (oac) of unroasted morama ranged from 27.3% (dark brown) to 27.6% (light brown). there was no significant difference in oac between the dark and the light brown morama flour. however, significant difference (p < 0.05) existed between the unroasted and roasted flours. for each variety roasting significantly increased the oac resulting in 112% increase. emulsifying activity and stability the effects of variety and roasting on the emulsion activity and stability of morama flour are detailed in table 7. emulsifying activity ranged from 37.9 for light brown to 38.2% for dark brown morama. emulsion stability ranged from 70.6% (light brown) to 72.2% (dark brown). the two varieties did not differ significantly in emulsifying activity and stability. the unroasted and roasted flours differed significantly (p < 0.05) in emulsifying activity, but no difference was observed for emulsion stability. ph, protein solubility and least gelation concentration the effect of variety and roasting on the ph and protein solubility of morama flour is detailed in table 7. the ph of the unroasted flour from the two varieties ranged from 6.54 (light brown) to 6.68 (dark brown). although the p value for ph indicate significant difference (p < 0.05), between the varieties and the flours, the very low partial eta squared value for variety (0.4127) and flour type (0.3878) suggests that the differences in ph was by chance. the ph of roasted morama flour negatively correlated with wac (r = -0.855, p = 0.030), oac (r = 0.836, p = 0.038), emulsifying activity (r = -0.814, p = 0.049) and emulsion stability (r = -0.945, p = 0.004). the protein solubility ranged from 83.3% (light brown) to 86.8% (dark brown). the varieties differed significantly (p < 0.05) in protein solubility. roasting significantly increased the protein solubility in both varieties. protein solubility of unroasted morama flour positively correlated with wac (r = 0.951, p = 0.003), oac (r = 0.948, p = 0.004) and ea (r = 0.838, p = 0.037). protein solubility of roasted morama flour negatively correlated with protein content (r = -0.843, p = 0.035). the least gelation concentration for both varieties of morama flour was 4%. effect of variety and roasting on the colour of morama flour the multivariate tests showed that both variety (p = 0.038 with partial eta squared = 0.520) and roasting (p = 0.000, partial eta squared = 0.995) contributed significantly to the multivariate model (table not shown). the effect of variety and roasting on the lightness, hue angle and chroma of morama flour is shown in table 8. lightness (l) of morama flour ranged from 82.4 for the dark brown to 83 for the light brown variety. the hue angle for the dark variety was 88.1 o and 88 o for the light variety. the chroma values were 25.6 for the dark variety and 26.80 for the light variety. although the multivariate tests (table 8) indicated that the effect of variety on the lightness and hue of morama colour is significant, the partial eta squared values (< 0.5) suggests that the difference may be by chance. however, significant difference exists between the varieties in chroma. the lightness of 82.7, hue angle of 88.1 and chroma of 26.2 suggest that the colour of unroasted morama flour is light yellow. discussion linear dimensions and geometric properties of morama beans the similarity of l/dg and l/w ratios (table 1) indicates that thickness of the seeds is closely related to its length. baryeh (2002) reported similar results for millet and co kuner and karababa (2007) for coriander seeds and ogunjimi et al. (2002) for locust bean seed. the following general expression can be used to describe the relationship among the dimensions of morama seed l = 1.10w = 1.44t (6) the size of the morama bean follows a normal distribution table 7. effect of variety and roasting on the functional properties of morama flour 1 . functional property (%) morama variety nature of flour 2 dark brown light brown unroasted roasted wac 11.8  6.3 a 10.8  6.4 b 5.5  0.7 a 17.1  0.6 b f-ratio 42.40 5136.47 p-value 0.0001 0.0000 partial eta squared 0.8249 0.9983 oac 27.3  9.1 27.6  10.5 18.5  0.7 a 36.4  1.1 b f-ratio 0.35 1030.66 p-value 0.5700 0.0000 partial eta squared 0.0372 0.9913 emulsifying activity 38.2  1.8 37.9  2.2 36.2  0.6 a 39.8  0.3 b f-ratio 1.23 180.32 p-value 0.2963 0.0000 partial eta squared 0.1202 0.9525 emulsion stability 72.2  0.8 70.6  2.2 70.6  2.2 72.2  0.8 f-ratio 3.16 3.00 p-value 0.1090 0.1171 partial eta squared 0.2601 0.2502 protein solubility 86.8  2.6 a 83.3  2.0 b 83.5  2.1 a 86.6  2.8 b f-ratio 14.44 11.42 p-value 0.0042 0.0081 partial eta squared 0.6160 0.5593 ph 6.7  0.1 a 6.5  0.1 b 6.7  0.1 a 6.6  0.2 b f-ratio 6.32 5.7 p-value 0.0330 0.0407 partial eta squared 0.4127 0.3878 1 values are mean  standard deviation. in each column different superscripts in each row indicate significant difference (p < 0.05). 2 combined effect of unroasted (dark and light) and roasted (dark and light). (figure 1). the geometric mean diameter values were lower than the length and width, but higher than the thickness of morama bean. on the contrary co kuner and karababa (2007) reported that the geometric mean diameter of coriander seeds were lower than the length, and higher than its width and thickness. a sphericity higher than 70% gives an indication of how close the shape of the morama beans are to that of a sphere (eke et al., 2007). such a higher sphericity enables the beans to roll rather than slide which is a property required in the design of hoppers, chutes and other storage facilities (mpotokwane et al., 2008) . a similar trend had been reported for the bambara groundnut (mpotokwane et al., 2008) and jackbean (eke et al., 2007). the high aspect ratio indicates that morama bean is more oblong in shape (mpotokwane et al., 2008). the values of aspect ratio which are higher than 70% indicated that the beans are likely to roll rather than slide. the sphericity and aspect ratio of the morama beans are similar to that reported for the bambara groundnut (mpotokwane et al., 2008), jackbean (eke et al., 2007) and the soybean (polat et al., 2006). gravimetric and frictional properties of morama beans morama beans are much heavier than most other oil seeds and legumes (table 3). knowledge of the specific mass of the beans is important as it is an essential parameter for storage, handling and processing of the product and it is required for calculation of the critical velocity, table 8. effect of variety and roasting on the colour of morama flour 1 . colour parameter morama variety nature of flour 2 dark brown light brown unroasted roasted lightness (l) 82.4  1.2 a 83.0  0.6 b 83.5  0.1 a 81.9  0.6 b f-ratio 9.99 85.32 p-value 0.0075 0.0000 partial eta squared 0.4344 0.8678 hue angle (tan -1 (b/a)) 88.1  1.0 a 88.0  0.8 b 87.3  0.1 a 88.8  0.2 b f-ratio 8.71 737.36 p-value 0.0113 0.0000 partial eta squared 0.4011 0.9827 chroma [(a 2 + b 2 ) 1/2 ] 25.6  1.1 a 26.8  0.1 b 26.7  0.1 a 25.7  1.2 b f-ratio 13.61 9.37 p-value 0.0027 0.0091 partial eta squared 0.5114 0.4190 1 values are mean  standard deviation. in each column different superscripts in each row indicate significant difference (p < 0.05). 2 combined effect of unroasted (dark and light) and roasted (dark and light). should pneumatic method be used in processing (alcali et al., 2006). the considerable difference between the bulk and true densities of both the dark brown and light brown beans is a good indication of compressibility (alcali et al., 2006). the porosity is a property of the beans which depends on its bulk and kernel densities (mpotokwane et al., 2008). the implication of the higher repose angle from the cement surface is that the storage of morama on a cement surface will lead to the maximum utilization of space. effect of variety and roasting on the nutritional composition of morama bean flour the high fat content of morama bean was within the range reported in the literature by earlier researchers (keegan and van staden, 1981; monagham, 1995; amarteifo and moholo, 1998). the high protein content was within the range reported in the literature (keegan and van staden, 1981; bower et al., 1988; monagham, 1995). morama flour is therefore rich in protein, fat and ash, hence it can contribute significantly to the nutritional needs of consumers of products made from it. the relatively high ash and fat content indicates that it could be a good source of minerals and oil. roasting did not affect the moisture and fat content significantly. this perhaps was because the bean was roasted with the shell intact thereby reducing moisture and oil loss. effect of variety and roasting on the functional properties of morama bean flour water and oil absorption capacity: the ability of a substance to associate with water under a limited water condition is wac (singh, 2001). the wac of morama bean obtained in this study is less than that reported in literature for other legume (ghavidel and prakash, 2006; ahenkora et al., 1990). differences in wac of different legumes could be due to the differences in the content of polar amino acid residues of proteins or charged side chains which have an affinity for water molecules (kinsella, 1976; jitngarmkusol et al., 2008). it is also known that polysaccharides, which are hydrophilic greatly, affect water absorption (ghavidel and prakash, 2006). the high lipid content in morama could also exert high negative effect on its wac. increased wac as a result of heating have been reported for winged bean (narayana and narasinga rao, 1985), cowpea (giami, 1993), velvet bean (ahenkora et al., 1999), kernel flour (akuboret al., 2000), green gram, cowpea, lentil and bengal gram (ghavidel and prakash, 2006), benniseed and bambara (yusuf et al., 2008) . cheftel et al. (1985) reported that wac of protein can be improved by partial denaturation. thus the increase in wac of roasted morama flour may be due to partial denaturation of proteins. furthermore, major proteins dissociate into subunits during roasting (akubor et al., 2000), which might have more binding sites than the native or oligomeric proteins. in addition, gelatinization of carbohydrates and swelling of crude fibre may occur during roasting, leading to increased wac (akubor et al., 2000). this increase in oac may be attributed to the solubilisation and dissociation of the proteins into subunits and subsequently increasing the number of binding sites (ghavidel and prakash, 2006). the higher the moisture content of unroasted morama flour the higher the oac (r = 0.835, p = 0.039). emulsifying activity and stability it is known that the emulsification properties of legume flours may result from both soluble and insoluble protein, as well as other components, such as polysaccharides (mcwatters and cherry, 1977; jitngarmkusol et al., 2008). protein can emulsify and stabilize the emulsion by decreasing surface tension of the oil droplet and providing electrostatic repulsion on the surface of the oil droplet (sikorski, 2002; wong, 1989), while some types of polysaccharides can help stabilize the emulsion by increasing the viscosity of the system (dickinson, 1994). the emulsifying activity of unroasted morama flour positively correlated with moisture content (r = 0.0.915, p = 0.011). however, emulsifying activity of roasted morama flour negatively correlated with protein content (r = -0.848, p = 0.033). the negative correlation of emulsifying activity with protein content implies that the emulsification activity of roasted morama flour does not only depend on the quantity of protein, but may also rely on protein characteristics, such as its hydrophilichydrophobic portion. the enhanced emulsion stability of morama flour could be due to the unfolding of the protein during heating exposing the hydrophobic residues causing the protein molecules to be better adsorbed on the oil-water interface. in addition, viscosity increase due to enhanced hydration and swelling of starch and nonstarch polysaccharides in the heated sample could occur, thereby retarding coalescence (jitngarmkusol et al., 2008). ph and protein solubility the acidity of morama bean flour was in agreement with the ph values of many legumes flours. the negative correlation of ph of raosted morama flour with wac suggests that using roasted morama flour in a more acid medium may improve its wac, oac, emulsifying activity and stability properties. the positive correlation of unroasted morama flour protein solubility with wac, oac and emulsifying activity suggests that the wac, oac and emulsifying activity of unroasted morama flour may be due to solubilized proteins. a similar relationship has been reported for soybean, groundnut and guar proteins (yusuf et al., 2008). least gelation concentration the least gelation concentration (4%) of morama bean flour is very low compared to the 10% reported for soybean flour (padilla et al, 1996). roasting had no significant effect on the least gelation concentration. a minimum protein concentration is required for gelation to take place, as the concentration is increased above this minimum, the time required for gelation is reduced (alfaro, alvarez, el kho and padilla, 2004). gelation is however not only related to the quantity of protein, but also the type of protein and non-protein components of the flour. effect of variety and roasting on the colour of morama flour roasting had a significant effect on the lightness, hue and chroma values of morama flour. however, the effect on chroma with partial eta squared of less than 0.5 appears to be by chance. roasting resulted to a decrease in the lightness and chroma value of the flour and an increase in the hue angle. the lower lightness (81.9) and chroma (25.7) values of the roasted flour indicates that the colour of the roasted flour is darker and less saturated than that of the unroasted flour. conclusion morama bean is longer than bambara, chickpea, and african bread fruit; 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277-282. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 5 (7), pp. 217-223, july, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of fungal profiles of cocoa produced in côte d’ivoire in order to discover whether toxigenic and pathogenic moulds are present tagro s. guehi 1* , n’dri d. yao 1 , niamien p. manizan 2 , koffi r. nevry 3 , louis b. koffi 4 and yao m. konan 3 1 laboratoire de biochimie et de technologie alimentaire. u.f.r-s.t.a, université d’abobo-adjamé, 02 bp 801 abidjan 02 côte d’ivoire. 2 laboratoire d’environnement et de biologie aquatique (leba). ufr-sge université d’aboboadjamé, 02 bp 801 abidjan 02 côte d’ivoire. 3 laboratoire de microbiologie et de biologie moléculaire. u.f.r-s.t.a, université d’abobo-adjamé, 02 bp 801 abidjan 02 côte d’ivoire. 4 laboratoire de biotechnologie et de microbiologie. programme cacao. cnra. 08 bp 33 abidjan 08 côte d'ivoire. accepted 09 may, 2017 the degree of fermentation and incidence of contaminating fungi were determined in 90 raw cocoa bean samples. excessive moisture content and low percentages of brown beans are recorded in cocoa samples from soubré and alépé, corresponding to maximum value of percentages of purple, mouldy and insect infested beans. differences in mould counts were detected in cocoa beans according to the producing regions; cocoa from alépé was most contaminated by moulds. six fungi were commonly isolated that is, absidia corymbifera, rhizopus oryzae, aspergillus tubingensis, aspergillus tamarii, aspergillus flavus and penicillium chrysogenum. nearly all of the fungi recovered can be considered as storage fungi. studies of cocoa samples revealed that raw cocoa beans collected from duékoué appeared to be of better quality than that from alépé and soubré. key words: cocoa, fermentation, fungi, quality. introduction nowadays, one of the most widespread concerns in advanced technological countries is food quality and safety. the economy of most developing countries, based primarily on their agricultural resources, is strongly dependent on the often rigorous and rigid quality standards set by developed countries. cocoa is a very important ingredient in several kinds of foods, such as cakes, biscuits, child-foods, icecreams and sweets. cocoa beans are an important source of cocoa powder and food worldwide and constitute an inexpensive fat source and are the principal raw material of chocolate from africa and both central and south america (tafuri et al., 2004). export of raw cocoa beans is of great economic importance in several tropical countries. west africa produces two-thirds of the world's cocoa, with côte d'ivoire and ghana as the major producers (anon, *corresponding author. email: g_tagro@hotmail.com 2004). cocoa beans are seeds from fruit pods of the tree theobroma cacao l. each pod contains 30 40 beans, embedded in a mucilaginous pulp. following opening of the pods, cocoa beans become contaminated naturally with a variety of micro-organisms, originating from workers' hands, containers for transport, knives, pod sur-faces, etc. fresh cocoa beans have an astringent, unpleasant taste. such product has to be fermented, dried and roasted in order to obtain raw cocoa beans with desired characteristic flavour and taste (thompson et al., 2001) . beans are piled in either heaps, boxes, trays or small baskets, covered with plantain leaves and left to ferment for 5 7 days (fowler, 1999). during cocoa bean fermentation, sugars in the mucilage are converted into alcohol by yeasts which proliferate after the sterile mass from the pods is exposed to the surrounding air (ardhana and fleet, 2003) . the mass then becomes runny and drops away from the beans. an enzymatic reaction takes place killing the embryo and contributing to the formation of flavour and the colour changes in cotyledons from purple to brown. guehi et al. 218 fermentation reactions have been reviewed by fowler (1999) and beckett (2000) . at the end of cocoa fermentation, changes in colour are widely used to predict flavour potential of cocoa beans. during the quality control, a cut test to observe changes in cotyledon colour during fermentation has been considered as a good indicator (shamsuddin and dimmick, 1986) when determining the degree of fermentation of cocoa beans (pettipher, 1986; misnawi et al., 2003). thereafter, drying to reduce moisture content of beans from 60 to 8% limits mould growth during transportation and storage. as is common with agricultural food commodities, raw cocoa bean quality after harvest is influenced by a wide variety of abiotic and biotic factors. mould contamination of food and feed is difficult to predict because it depends on a complex interaction of factors, such as temperature, moisture, endogenous fungal species, storage history and storage time (chelack et al., 1991). important factors include processing technologies and storage conditions for preventing the development of mould (magan et al., 2003). exposure of cocoa beans to high humidity is most likely to occur at stages between harvest and final consumption. then, poor post-harvest management can lead to rapid deterioration in the quality of cocoa, severely decreasing of commercial and nutritional values. fungal activity can also cause undesirable effects in food commodities including discolouration and deterioration in quality, and can result in contamination with mycotoxins (magan et al., 2003). it is important to note that ivorian raw cocoa bean quality did not escape being degraded since the liberalization of the cocoa-producing chain in 1999 and the reasons were unspecified (dgtcp, 2004). therefore it is important to identify the factors that reduce commercial value by studying the degree of fermentation and incidence of contaminating fungi in some samples of ivorian cocoa beans because moulds can produce mycotoxins. the objective of this study did not focus on post-harvest handling and technology processing of cocoa beans at the farmer’s level in côte d’ivoire. first, we purchased raw cocoa beans from three main ivorian producing regions in order to compare their degree of fermentation. secondly, the study explored the fungal profiles of cocoa produced in côte d’ivoire in order to discover whether toxigenic and pathogenic moulds are present. materials and methods cocoa-producing regions three cocoa producing regions in côte d’ivoire, characterized by differing climatic conditions during harvesting and raw cocoa bean yield, were selected for study. the regions were as follows: (i) alépé, a southern area near abidjan, a moderate hot rainy region with an average of 28 29°c during the harvest season, a low altitude, below 500 m, 70 80 mm/month rainfall and moderate cocoa yield; (ii) duékoué a western area, a relatively cold, high rainy region with an average of 23 24°c and 70 mm/month rainfall, with relatively high altitude of 800 1000 m and relatively high cocoa produce, (iii) soubré a western center region, with temperate, relatively high rainy region with an average of 25 28°c and 65-70 mm/month rainfall, with moderate altitude below 600 m and very high cocoa yield. sampling during the first 3 months of the 2007 harvest period, 90 raw cocoa bean samples belonging to three types (brown, black and clustered) of bean was collected from three farms in each of three selected regions at the end of the drying stage or during storage. currently when cocoa beans processing technologies were carefully conducted both cocoa beans surface and inside colour is brown. but in some case such as when a) cocoa beans were extracted from rotten or decayed pods, b) beans were dried in iron surface or c) they are get wet by the rains, their only surface’s colour changed from brown in to black. clustered beans came from fermentation of beans with the rachis together. thereafter drying of cocoa, the mass of beans were obtained and constituted clustered beans type. they are characterized by current high moisture content up to 8% and by several beans up to two in the same cluster. ten samples (each approximately 1 kg) of each descriptive type of bean were taken from each farm at a single sampling time and kept inside in polythene bags during transport from the farm to the laboratory. the period from sampling to mycological analysis did not exceed a week. the farmers were questioned on processing history and storage time of their products. cocoa beans quality analyses moisture content moisture in the beans was determined using the international organisation for standards (iso) method (hamid and lopez, 2000). approximately 10 g of ground bean sample was placed into a pre-weighed dish (w1) with a lid and re-weighed to the nearest mg (w2). dishes and contents were put in an oven at 103 ± 2°c for 16 h. after cooling in desiccators they were weighed (w3) and the percentage moisture content was calculated using the following formula: moisture content (%)  (w 2 w 3 ) 100 (w2 w1 ) cut test score (%) the cut test for sanitary and fermentation quality of beans was performed according to the international method described by hamid and lopez (2000). random samples of 300 beans for each cocoa sample were cut lengthwise using a sharp knife to expose a maximum cotyledon surface. both halves of the cotyledon were examined visually in daylight. observations were made for insect damage, mould infestation, germination as well as of the colour of the beans (slate, fully purple and fully brown). slate bean characteristics include rubbery cotyledon, blackish colour, and resistance to cutting. purple beans occur when the fermentation has been terminated prematurely. fully brown beans are well-fermented beans. results were expressed as a percentage and all analyses were done triplicate. mycological analysis of the cocoa beans sub-samples of cocoa beans were stirred for 5 min at room tempe 219 afr. j. food sci. res. table 1. cut test scores (%) of each type of 10 cocoa beans samples collected from alépé. values are means of 10 samples of each type of beans. within columns, values followed by the same letters are not significantly (p = 0.05) different according to the fisher protected l.s.d. test. types of cocoa quality/characters beans samples average slaty fully purple purple brown brown germinated mouldy insect infested moisture content brown beans 6.72±0.60 a 1.52±0.69 bd 1.71±0.58 ac 0.77±0.12 a 87.19±3.88 bd 2.74±0.87 a 2.31±0.92 a 1.83±0.54 a black beans 7.94±0.69 ac 0.81±0.44 a 1.64±0.71 a 0.79±0.19 ac 62.45±4.15 a 4.57±1.38 cd 20.62±2.66 ce 4.00±0.67 bd clustered beans 9.58±0.30 bd 2.43±1.24 ce 4.29±1.38 bd 2.12±0.61 bd 72.76±6.15 ac 3.57±0.49 bd 8.40±1.72 bd 2.33±0.85 ad temperature in 0.1% peptone water (1:10 w/v) and triplicate 0.1 ml of each dilution was spread on three 90 cm petri plates of dichloran rose bengal chloramphenicol agar (drbc) (samson, 1991). the results (n) were expressed in cfu.g -1 using the following formula (afnor, 2002): n  c v  (n  0.1 n )  d 1 2 where: c is the sum of colonies count on all retained successive dilution petri dishes; v is the volume of inoculums spread-plated onto drbc; n1 is the number of petri dishes retained to the first dilution; n2 is the number of petri dishes retained to the second dilution; d is the dilution rate of the first retained dilution. calculations are carried out with a probability of 95%. the confidence interval was calculated by the following formula:   c 1,96 c  1 bbd  where b  v (n1  0,1n2 ) moulds detected were isolated and sub cultured on czapeck-dox agar for identification purposes. all isolated moulds were identified firstly by the macroscopic characters and then according to the identification keys for common food-born fungi (pitt, 1985; pitt and hocking, 1997). fungal isolates were sent to bccm tm /mulc culture collection (leuven catholic university, b-1348 louvain-laneuve, belgium) for to complete or to confirm the identity of moulds. statistical analysis data were subjected to oneway analysis of variance (anova) using jmp software version 5 (sas institute, 2002) and significant differences between means were performed by fisher protected l.s.d test at p = 0.05. results measurement of the degree of fermentation of cocoa beans results of cut test analysis of cocoa beans collected from alépé are summarized in table 1. they showed that clustered cocoa beans samples had high percentage of fully purple beans with 4.29 ± 1.38%. black beans samples presented very high percentage above 20% of mouldy beans. percentage of insect infested beans was low except for black beans type. table 1 also showed that only clustered beans presented moisture content above 8%. cocoa collected from duékoué showed the same percentage of fully purple beans for all studied types of beans (table 2). but black and clustered beans samples had higher percentages of mouldy beans of 12.32 ± 9.64% and of 9.43 ± 4.47% respectively than brown beans (0.84 ± 0.69%). low percentage of insect infested beans was found in all cocoa samples from duékoué whatever the quality (ranged from 0.33 to 2.14%). clustered beans showed relatively high moisture content of 8.41 ± 1.28% and lower percentage of purple beans (3.88 ± 1.09%) than black beans. table 3 presents the results of measurement of moisture content and of the degree of fermen-tation of cocoa collected from soubré. all tested samples showed moisture contents above 8% whatever their physical characteristics: 11.11, 11.44 and 12.69% in respectively brown, black and clustered beans. in others respects, clustered beans showed higher percentage of fully purple beans (7.10%) than all others beans. highest percentage of mouldy beans were obtained only in black and clustered beans of 8.86 ± 2.96% and 5.17 ± 1.84% respectively. the percentage of insect infested beans is low whatever the physical quality of cocoa beans analysed (ranged from 0.27 to 2.60%) . results of cut test scores of cocoa collected from each cocoa producing region are shown in table 4. the highest moisture content (11.74%) is measured in cocoa from soubré. cocoa collected from duékoué and soubré guehi et al. 220 table 2. cut test scores (%) of each type of cocoa beans samples collected from duékoué. values are means of 10 samples of each type of beans. within columns, values followed by the same letters are not significantly (p = 0.05) different according to the fisher protected l.s.d. test. types of cocoa beans samples quality/characters average moisture content slaty fully purple purple brown brown germinated mouldy insect infested brown beans 7.47±1.14 ab 2.07±1.01 ab 4.07±1.38 ab 1.88±0.53 a 82.60±3.21 c 0.74±0.68 a 0.84±0.69 a 0.33±0.24 a black beans 7.23±0.89 a 2.82±2.12 b 4.36±3.21 b 3.05±1.26 b 64.68±5.61 a 3.40±1.93 c 12.32±9.64 c 2.14±0.76 c clustered beans 8.41±1.28 b 2.02±1.09 a 3.88±1.09 a 4.53±2.31 b 70.18±4.19 ab 0.79±0.70 ab 9.43±4.47 bc 0.76±0.58 ab table 3. cut test scores (%) of each type of cocoa beans samples collected from soubré. values are means of results of 10 samples of each type of beans. within columns, values followed by the same letters are not significantly (p = 0.05) different according to the fisher protected l.s.d. test. types of cocoa beans samples quality/characters average moisture content slaty fully purple purple brown brown germinated mouldy insect infested brown beans 11.11±0.92 a 1.98±0.36 a 8.26±3.42 b 4.61±0.47 b 78.96±3.21 b 0.52±0.46 a 0.29±0.22 a 0.27±0.22 a black beans 11.44±0.98 a 2.05±0.69 a 3.64±0.69 a 1.77±0.68 a 77.49±1.68 b 0.93±0.69 b 8.86±2.96 c 2.60±1.19 b clustered beans 12.69±0.79 b 3.33±0.90 b 7.10±2.10 b 8.68±1.27 c 71.65±2.13 a 0.43±0.36 a 5.17±1.84 b 0.56±0.53 a showed highest percentage of fully purple beans. the highest percentage of mouldy beans was recorded in cocoa purchased from alépé with 10.43 ± 8.00%. but the same percentage of slaty beans (1.56 to 2.41%) and the same level of brown beans (70%) were recorded in cocoa whatever the origin. in average, all cocoa samples studied presented 9.78% moisture content, 4.17% of fully purple beans, 7.18% of mouldy beans. moulds counts and fungal profile in each type of beans. overall, levels of fungal contamination of tested cocoa samples purchased from each cocoa producing region are summarized in tables 5. mould counts in cocoa analysed reached maximum values of 541.98 ± 0.14×10 5 and 186.16 ± 0.70×10 4 cfu.g -1 in black beans and clustered beans respectively at alépé. there was a quailtative uniform distribution of filamentous fungi in samples tested. at duékoué, higher levels of mould counts (434.59 ± 0.55×10 4 and 167.1 ± 0.60×10 4 cfu.g -1 ) were recovered in black and clustered beans respectively than brown beans (2.60 ± 1.35.10 3 cfu.g -1 ). six strains of filament-tous fungi such as absidia corymbifera (cohn) saccardo and trotter, rhizopus oryzae went and prinsen geerlings, aspergillus tubingensis (scho-ber) mosseray, aspergillus tamarii kita, a. flavus link : fries, penicillium chrysogenum thom, were found in black beans while a few fungi ( asper-gillus tubingensis, absidia corymbifera) were recorded in brown beans from duékoué. there was a qualitative variable distribution of filament-tous fungi in cocoa samples tested in accordance with their quality. cocoa collected from soubré showed most infected beans. indeed black beans and clustered beans cont-ained 341.01 ± 0.35 × 10 4 and 96.20 ± 0.554 × 10 4 cfu.g -1 respectively. at this region, several strains of filamentous fungi were found in black beans samples while two strains were recorded in clustered beans and only one strain was isolated from brown beans sam ples. discussion in order to evaluate the commercial value of cocoa according to the type of beans 90 samples were collected from three ivorian mains cocoa producing regions. the degree of fermentation and moisture content were measured. moisture content of clustered beans is higher than tolerable limits (8%) whatever the producing area. that high moisture content is due to the agglomeration of several beans. beans clustering could has been caused either by harvesting of unripe pods, or by poor separation of beans and placenta during cocoa pods opening. so beans would be remained clustered within the placenta during and after fermentation. therefore, drying induces clustered beans which were not dried properly as previously demonstrated by barel (1998). indeed, the hot air in contact with external cocoa beans 221 afr. j. food sci. res. table 4. cut test scores (%) of all cocoa beans samples collected from each producing region. values are means of results of all 30 samples from each selected region. within columns, values followed by the same letters are not significantly (p = 0.05) different according to the fisher protected l.s.d. test. cocoa quality/characters producing average moisture slaty fully purple brown germinated mouldy insect regions content purple brown infested alépé 8.08±1.31 a 1.56±1.05 a 2.53±1.53 a 0.97±0.61 a 70,21±5.21 a 3.62±1.28 c 10.43±8.00 b 2.60±1.19 c duékoué 7.71±1.19 a 2.10±0.80 b 4.03±1.19 b 2.29±1.03 b 76,02±7.35 b 1.01±0.99 a 6.31±5.20 a 0.56±0.53 a soubré 11.74±1.11 b 2.41±0.90 c 5.96±2.87 c 3.09±1.11 c 71,23±6.21 a 0.56±0.54 a 4.79±3.97 a 0.22±0.27 a total samples 9.78±0.25 a 2.02±0.25 b 4.17±0.47 b 2.16±0.12 b 71,84±3.56 a 1.73±0.29 b 7.18±0.81 a 1.12±0.18 b table 5. total mould count (cfu.g -1 ) on drbc agar and main fungal isolates in each type of cocoa beans collected from alépé, duékoué and soubré. values are means of results of 10 samples of each type of beans. within columns of each cocoa producing region, values followed by the same letters are not significantly (p=0.05) different according to the fisher protected l.s.d. test. cocoa producing regions types of cocoa beans mould count (cfu.g -1 ) main isolates strains isolated (ranked in order of relative importance) brown beans 6.27±2.05×10 3a aspergillus tubingensis, a. flavus, absidia corymbifera, penicillium chrysogenum alépé black beans 54198.37±13.82×10 3c absidia corymbifera, aspergillus tubingensis, a. tamarii, a. flavus, penicillium chrysogenum clustered beans 1861.61±7.03×10 3b absidia corymbifera, aspergillus tamarii, 2.60±1.35×10 3a a.tubingensis, penicillium chrysogenum brown beans aspergillus tubingensis, absidia corymbifera 4346.92±5.55×10 3b absidia corymbifera, rhizopus oryzae, duékoué black beans aspergillus tubingensis, a. tamarii, a. flavus, penicillium chrysogenum clustered beans 1671.86±5.97×10 3c absidia corymbifera, aspergillus tamarii, a. 1.53±0.82×10 3a tubingensis, penicillium chrysogenum brown beans absidia corymbifera soubré black beans 3410.14±3.53×10 3c absidia corymbifera, aspergillus tubingensis, a. 961.95±5.44 ×10 3b tamarii, penicillium chrysogenum clustered beans absidia corymbifera, rhizopus oryzae would dry residual pulp and causes the formation of internal and external crusts. these crusts slow down the departure of most of water from internal beans. at the end of drying stage, the internal beans contain moisture contents largely higher than those of external beans. consequently, whole clustered beans show moisture content higher than the critical moisture content (8%) for mould growth (hansen, 1975). highest moisture contents were recorded in cocoa beans collected from soubré. these moisture contents could be due to insufficient or inadequate drying system of cocoa linked to two raisons. firstly, according to the investigations that we have carried out, the growers do not take any more time to dry their product until the water content reached 8% because of hard competition between the exporters of cocoa since the liberalization of the cocoa chain. at farmer’s level, some exporters did not inspect cocoa beans correctly for moisture content determination and often preferred to buy wet cocoa beans (moisture content above 8%) for fear it would be sold with the competitors. in the others respects, at soubré farmers did not pay too much attention to the moisture content of cocoa beans sold, because they want to get money quickly. secondly, the high moisture content observed in cocoa collected from soubré could be due to the high rainfall (65 70 mm/month) which can increase the relative humidity in this region during the harvest. indeed, according to hamid and lopez (2000) the beans exchange moisture in accordance with the site. therefore, properly dried cocoa beans could absorb moisture during the storage. in others regions such as alépé and duékoué, cocoa’s growers seemed to observe carefully official recommendations about the conduction of cocoa drying in order to obtain final moisture content around of 8%. although the percentage of brown beans was above 60% in all samples collected, the percentage of fully purple beans was highest in clustered beans (4.29%) collected from alépé; brown and clustered beans from soubré in a discordance with beans appearance. according to misnawi et al. (2003) high percentage of fully purple cocoa beans is not considered as a severe default of cocoa quality because some countries such as malaysia guehi et al. 222 are currently making use of the unfermented and partly fermented cocoa beans especially for cocoa liquor, powder and cocoa butter production. but because of the excessive astringency and bitterness of such cocoa beans, fully purple beans are undesirable in west africa where fermentation process is strongly recommended to be made during 5 7 days. this treatment aims to reduce considerably the content of polyphenols, to lead to the change of beans internal colour from purple to brown (pettipher, 1986) and to confer to cocoa a good chocolate’s flavour potential (misnawi et al., 2003). indeed, during cocoa fermentation polyphenols are subjected to biochemical modification through polymerization and complexation with protein, hence decreasing astringency (bonvehi and coll, 1997). at the same time anthocyanins are hydrolysed to anthocyanidins and usually disappear rapidly during the fermentation process, e.g. wollgast and anklam (2000) have reported a lost of 93% after 4 days fermentation. so at duékoué, fermentation pro-cessing seems not properly conducted. according to the growers of this region, the fermentation process was stopped sooner (below 4 days) than necessary because of the thieves since the increasing of insecurity due to the war situation in côte d’ivoire since 2002. some cocoa farmers of region of soubré recognize that they fermented their cocoa during short duration likely the growers at duékoué because of the high yield of cocoa per week. high percentage of fully purple beans in clustered beans collected from alépé could be due to the formation of clusters probably due to the absence of the manually daily turning of the fermentation mass. however, according to crespo (1986) many forastero beans often have a purplish tone even after proper fermentation. as any information about the genotypic origin of collected cocoa beans is unavailable, no conclusion can be made on the causes of formation of purple beans in samples studied. but on the basis of current results, the cocoa collected from soubré is poorly fermented compared to the cocoa collected from duékoué and alépé. differences in mould counts of cocoa beans have been detected between cocoa producing regions. higher percentages of mouldy beans were recorded in black and clustered beans samples whatever their origins than brown beans. but cocoa collected from alépé is more contaminated by moulds than cocoa produced at soubré and duékoué. differences in moulds contamination level seem to be linked to meteorological differences between cocoa growing. surprising observation of an inverse relationship in soubré between moisture content and mouldiness was made. this relationship could be linked to the fact cocoa beans were freshly prepared and then shortly stored at framers level when they we re collected. in the others respects, at this region cocoa beans were not properly dried before selling because of the abundance of the cocoa’s production and the increasing competition between the multinational cocoa companies’ traders. highest percentage of mouldy beans in black beans could be attributed to their probable origin from rotten pods or pods which were stored during long time before opening. these results agreed with those of renaud (1954) who reported that such beans are easily contaminated by the moulds during fermentation and drying stages. they confirmed also that poor post-harvest management can lead to rapid invasion of stored agricultural commodities by moulds (lacey and magan, 1991). moulds could penetrate the beans through the germ sections and spoil cotyledons, the flavour and the whole bean (crespo, 1986). mechanical damages are also conducive to entry of spoilage fungi in insufficiently dried grains. according to bopaiah (1992) the presence of germinated and broken beans could also favour the formation of mouldy beans. percentage of mouldy beans in clustered beans could be linked to their high moisture contents as stated by magan and lacey (1988) . analysis of percentage of mouldy beans of each type of cocoa carried out that percentage of mouldy beans seems to be positively correlated with the percentage of insect infested beans. those observations would confirm that some storage insects disseminated storage fungi as previously suggested by sinha (1971). as cut test is usually adopted at the international market allowing the maximum mouldy beans of 3% for grade 1 and 4% for grade 2 respectively, only brown cocoa beans can consider as good quality cocoa. highest mould count up to 4% was observed in black and clustered beans whereas brown beans showed good quality characteristics whatever the cocoa producing region. these observations are not surprising considering the fact that black beans could be obtained from rotten pods as reportedly by renaud (1954) and clustered beans presented high moisture content (hansen, 1975). black and clustered beans could be again considered as poor quality beans because highest mould counts (that is, 10 6 ufc/g) with the risks of the production of mycotoxins (gourama and bullerman 1995). as brown beans are smooth, hard skins, they were impermeable to most fungi. although mouldy beans were determined based on two different methods, namely cut test and plating methods on drbc, results of mouldy beans seem to be positively correlated with mouldy beans. cocoa collected from alépé is most contaminated by moulds. this strong contamination could be due to the fact that culture of cocoa would have passed in the second plan to the profit of others crops such as hevea. consequently ferment and dried cocoa beans could be remained probably in storage for a long time at the farmer’s level under inappropriate conditions. six species of moulds were isolated from cocoa beans collected that is, a.corymbifera, r. oryzae, a. tubingensis, a. tamarii, a. flavus, p. chrysogenum. all fungi generaly identified had already been recorded in cocoa from different cocoa producing areas (hansen, 1975; guénot et al., 1976; niles, 1981, bopaiah, 1992; dharmaputra et al., 1999). nearly all species of fungi re 223 afr. j. food sci. res. covered in the present work can be considered as common saprophytic soil organisms or storage fungi. post harvest moulds development in cocoa can be expected to relate to unfavourable climates for drying and storage: poor drying practice or quality control, inadequate or poor storage conditions. no ochratoxin aproducing fungi were isolated from any samples tested but unfortunately one potential aflatoxin producing fungus such as a. flavus was recorded in some samples from alépé and duékoué. as mycotoxin-producing abilities of fungi isolated from raw cocoa beans were not elucidated in this study, very high mould counts in black and clustered beans showed good evidence to support their 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(eds) food microbiology fundamentals and frontiers, asm press, washington, pp. 721-733. wollgast j, anklam e, (2000). review on polyphenols in theobroma cacao: changes in composition during the manufacture of chocolate and methodology for identification and quantification. food res. inter. 33:423-447. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (3), pp. 001-011, march, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of flour production methods on the yield, physicochemical properties of maize flour and rheological characteristics of a maize -based non-fermented food dumpling mathew k. bolade department of food science and technology, federal university of technology, p. m. b. 704, akure, ondo state, nigeria. e-mail: matbolade@yahoo.co.uk. accepted 12 september, 2019 the study was aimed at investigating the influence of flour production methods (grit soaking method [gsm], grit non-soaking method [gnm] and pestle and mortar dehulling method [pmdm]) on the yield, physicochemical properties of maize flour and rheological characteristics of a maize-based non-fermented food dumpling. the flour yield from the production methods ranged between 56.6 and 62.3% with gsm and gnm giving the lowest and highest values respectively. there were variations in the mean particle size (203.3 220.5 µm) and damaged starch content (12.2 14.5%) of flours obtained from the three production methods. the flours from the production methods also exhibited variations in the pasting temperature (73.2-74.6 o c), peak viscosity (84.2 110.6 rvu) and final viscosity (111.4 147.4 rvu). the lightness indices (l*-values) of the flours (88.8 90.0) and maize ‘tuwo’ (66.9 67.7) were affected by the flour production methods while the chroma (c-values) of the flours (14.6 14.9) and maize ‘tuwo’ (8.9 9.1) were also affected though not significantly different (p<0.05). the cohesiveness index (15.1 15.9%) and softness index (17.2-17.8 mm) of maize ‘tuwo’ prepared from the flours also exhibited variations. key words: dumpling, flour, maize, tuwo, rheological characteristics. introduction maize (zea mays l.) is an important cereal grain in the world and it has a diverse form of utilization including human food uses, animal feed formulation and as a basic raw material for industrial purposes (courtois et al., 1991). in africa, a greater proportion of maize grains be-ing produced is used as human food whereas industrial processing of the bulk of maize grains are carried out in the developed countries (anon, 1997). however, there has been a tremendous increase in maize grain utilization for animal feed formulations in the developing countries in recent times due to a rapid increase in poultry consumption (okoruwa, 1997). maize is consumed in diverse forms in different parts of africa, from boiled or roasted maize cobs to dry-milling of grains to obtain flour or wet-milling to obtain starch (rooney and serna-saldivar, 1987). many traditional african food products that can be obtained from maize include ogi, eko or agidi, maize tuwo or tuwon masara, bantu, aliha, abari, kenkey, injera, guguru, kokoro, elekute, egbo, chenga, etc. (okoruwa, 1997). dry-milling is an age-long processing procedure for cereal grains and its primary objective is to separate the kernel into its anatomical parts (that is endosperm, bran, and germ) which is essentially a physical change in the cereal grains (johnson, 1991). different classes of end-products of dry-milling of cereal grains include flaking, coarse, medium or fine grits; coarse or fine meal; flour and germ (kent and evers, 1994). the production of flour from maize grains via drymilling, particularly in africa, is usually accomplished through diverse production methods. one method involves grinding the whole maize grains using motordriven grinder to obtain whole maize flour (nago et al., 1997) or the maize grains may first be dehulled using pestle and mortar after which the dehulled grains are ground or milled into flour (mestres et al., 2003). the maize flour from this form of production method is usually rich in lipids, which are present in the germ, thereby leading to a rapid development of rancidity in the flour (andah, 1976; nago et al., 1997). another method of maize flour production involves initial dehulling and degerming of the maize grains using a decorticating equipment to obtain the maize grits. it is these grits that are finally milled into flour (mestres et al., 2003). maize flour, one of the end-products of drymilling of maize grains, and the food products derivable from the flour have been observed of able to exhibit variations in their physical, chemical and/or rheological properties due to such factors as maize varieties (mestres et al., 1991; pan et al., 1996; yuan and flores, 1996; nago et al., 1997; sandhu et al., 2007), milling methods (nghi and narasimha, 1994; martinez-flores et al., 1998), particle size differential of flour (bolade et al., 2009) and differences in the drying temperatures of maize grains (hardacre and clark, 2006). however, there are limited research works on the effect of flour production methods, with variation in the unit operations, on the flour properties and textural characteristics of food products prepared from such flour. housson and ayernor (2002) studied the physicochemical properties of whole maize flour and dehulled-degermed maize flour so as to predict the functional properties of products derivable from such flours. mestres et al. (2009) also investigated the influence of maize flour, produced through the use of disc and hammer mills, on the quality attributes of thick pastes commonly consumed in benin, a west african country. the objective of this study therefore was to investigate the influence of flour production methods on the yield, physicochemical properties of maize flour and rheological characteristics of a non -fermented maize dumpling (maize tuwo) commonly consumed within hausaspeaking communities of west africa sub-region. materials and methods materials the maize variety used for the study was dmr-lsr-w, which is white maize and was obtained from the institute of agricultural research and training (iar & t), moor plantation, ibadan, nigeria. the maize grains were two months old after harvest. production of maize flour three different methods were used to produce maize flour, namely: grit non-soaking method (gnm), grit soaking method (gsm) and pestle and mortar dehulling method (pmdm). fifteen kilograms of maize grains were used for each method to produce the flour. grit non-soaking method (gnm): this involves initial cleaning of the maize grains manually by removing the stones, damaged kernels and other extraneous materials. the cleaned grains were then tempered by sprinkling 5% water (v/w) on the grains coupled with thorough mixing. this was followed by decortication of the grains on a grantex decorticating machine, which removed the brans and the germ to obtain the grits. the grits were finally milled using a disc attrition mill (agrico model 2a, new delhi, india) to obtain the flour followed by sieving using a sieve with 300 µm aperture and then kept in airtight polythene bags until needed. grit soaking method (gsm): the maize grains were subjected to similar unit operations as that of grit non-soaking method (gnm). however, the grits obtained were soaked in cold water (submerged) for 3 h after which they were drained and ovendried at 55°c for 16 h. the dried grits were finally milled and sieved in a similar manner as that of grit non-soaking method (gnm). pestle and mortar dehulling method (pmdm): the manually cleaned maize grains were tempered (5% water addition, v/w) followed by manual dehulling using wooden pestle and mortar through pounding. the pounded grains were manually winnowed to remove the adhering brans and other light-weight materials. the grits eventually obtained were finally milled and sieved in a similar manner as that of grit non-soaking method (gnm). evaluation of mass balance in maize flour production the mass balance in maize flour production was based on the basic principles of conservation of mass (earle, 1983). mathematically, this can be expressed as follows: wmg = wsf + whg + wss + wot + wlm where; wmg = weight of maize grains used wsf = weight of sieved maize flour whg = weight of hulls and germ removed wss = weight of soluble solids in the soaking water wot = weight of over-tails from flour sieving operation wlm = weight of other lost materials incurred during flour production. weight of hulls and germ removed was estimated as the difference between weight of maize grains used and weight of maize grits obtained after decortication. weight of soluble solids in the soaking water was evaluated by weighing 10 ml of the soaking water in a glass petri dish followed by drying in an oven at 105°c for 3 h. the weight of remaining solids in the petri dish was used to estimate the total soluble solids in the soaking water. weight of over-tails from flour sieving operation was estimated as the difference between weight of unsieved flour and weight of sieved flour. weight of other lost materials incurred during flour production represents losses that could not be accounted for during processing. determination of mean particle size of maize flour the particle size distribution of maize flour (250 g) from each production method was carried out using a sieve analysis technique with the aid of endecotts test sieve shaker (model 1 mk11-11381, london, uk). sieves of different apertures (that is 425, 300, 150 and 75µm) and pan were used by placing them in the shaker for 10 min. the flour retained on each sieve was weighed and the mean particle size (mps) of the flour determined using the method of arambula et al. (1998) as follows: mean particle size (mps) = (w1d1+w2d2+..... +w5d5)/r where; w1-5 = weight of flour through each sieve d1-5 = diameter of mesh for each sieve r = total recovery. determination of damaged starch of maize flour the damaged starch of maize flour from each production method was determined according to the method of farrand (1964). the test was based on starch susceptibility to –amylase digestibility. the extract obtained from the digestion was subjected to series of chemical treatments followed by ultimate titration against sodium thiosulphate solution from which an equivalent maltose figure was estimated. damaged starch (%) was calculated as follows: [(maltose figure – 3.5) × 6]. determination of pasting properties of maize flour the pasting properties of maize flour from each production method were determined using a rapid visco-analyzer, rva-series 4 (anon, 1996). a sample of 4.0 g maize flour (14% moisture-basis) was transferred into a canister and approximately 25± 0.1 ml distilled water was added (correction factor was used to compensate for 14% moisturebasis). the slurry was heated to 50 o c and stirred at 160 rpm for 10 s for thorough dispersion. the slurry was held at 50°c for up to 1 min followed by heating to 95°c over about 7.3 min and held at 95°c for 5 min, and finally cooled to 50°c over about 7.7 min. the parameters calculated from the pasting curve include the pasting temperature, peak viscosity, time to peak, breakdown, holding strength or trough, setback, and final viscosity. production of maize ‘tuwo’ maize ‘tuwo’ was prepared from flour of each production method using a method as described by bolade et al. (2002). the overall ratio of flour to water used in maize tuwo preparation was 1:3.5 (w/v). cold slurry of the flour was first prepared by mixing 20% of the desired quantity of flour (1.5 kg) with 25% of the desired quantity of water (5.25 l). this was followed by bringing 60% of the water into boiling and the cold slurry initially prepared was added to this boiling water coupled with vigorous stirring, using a wooden flat spoon, to form a pap-like consistency. the remaining quantity of the flour (80% of the desired total) was then added gradually to the boiling pap-like paste with continuous stirring so as to facilitate nonformation of lumps and to ensure a homogenous gel formation. the remaining quantity of water (15% of the desired total) was finally added to the formed gel, covered properly without stirring, and allowed to cook for about 7 min after which it was stirred vigorously to ensure smoothness of the gel. the final product so obtained is called maize ‘tuwo’. determination of colour characteristics of maize flour and tuwo the colour of flour and tuwo obtained through each production method was measured using a colour measuring instrument (colortecpcm, model sn 3000421, usa) and the values expressed on the l*, a*, b* tristimulus scale. the instrument was initially standardized (l*=90.29, a*=1.37, b*=0.06) using a white reference standard (white duplicating paper sheet, 80 g/m 2 ); where l* represents the colour lightness while a* and b* represent the degree of redness/greenness and yellowness/blueness respectively. the results from three replicates per sample were averaged. the colour intensity, expressed as chroma (c), was calculated from (a 2 +b 2 ) 1/2 while one of the following equations was used to calculate the hue angle: if a>0 and b>0, then h o = tan -1 (b/a); if a<0 and b>0, then h o = [tan -1 (b/a)] + 180 o ; if a >0 and b<0, then h o = [tan -1 (b/a)] + 360 o (mcguire, 1992). determination of cohesiveness index of maize tuwo the cohesiveness index of maize tuwo prepared from flour obtained through each production method was determined using the universal testing machine (model m500-50kn, testometric, england). maize tuwo was initially prepared inside a cylindrical plastic container (with a diameter of 50 and 96 mm in height), the internal surface of which was first oiled with edible vegetable oil to facilitate easy removal after solidification. the hot maize tuwo inside the cylindrical container was allowed to cool under ambient condition (30 ± 2°c) and after about 4 h it was extruded from the container and the cylindrical tuwo mould subjected to compression test. the cylindrical tuwo mould was placed between two parallel flat stainless steel circular plates each having a diameter of 100 mm. the machine was set at a speed of 20 mm/min and allowed to compress the cylindrical tuwo mould until the food sample began to rupture. the cohesiveness index of the food dumpling (maize tuwo) was calculated as the strain at peak (%) which is the extent to which the cylindrical tuwo mould could be deformed before it ruptured. determination of softness index of maize tuwo the softness index of maize tuwo prepared from flour obtained through each production method was determined using precision cone penetrometer (benchtop model, pioden controls ltd., uk). freshly prepared hot maize tuwo was scooped inside a clean cylindrical tin container having only one end opened and a dimension of 6 cm (diameter) by 6 cm (height) . after filling, the opened end was covered with an aluminium foil to prevent scale formation of tuwo and the container was thereafter allowed to cool under ambient condition (30 ± 2°c). after cooling, tuwo inside the container was subjected to penetrometer evaluation by positioning its centre perpendicularly to the falling probe of the penetrometer. the probe was finally released to fall freely from a standard distance to penetrate into the product in the cylindrical tin container. the total depth of penetration of the probe was then read on the penetrometer scale and the reading, expressed in millimetre (mm), was taken as an index of the product softness. statistical analyses all determinations reported in this study were carried out in triplicates. in each case, a mean value and standard deviation were calculated. analysis of variance (anova) was also performed and separation of the mean values was by duncan’s multiple range test at p<0.05 using statistical package for social scientists (spss) software, version 10.0; on a personal computer. results and discussion mass balance in maize flour production from different methods the mass balance in the production of maize flour from three different methods is presented in table 1. the proportion of materials added, recovered or removed during tempering and decortication steps were the same for both grit soaking method (gsm) and grit non-soaking method (gnm). the quantity of water used for tempering table 1. mass balance in the production of maize flour from three different methods. proportion (%) 1 operation component in the mass balance grit soaking grit non-soaking pestle and mortar method (gsm) method (gnm) dehulling method (pmdm) starting material maize grains (9.8 ± 0.5%, moisture content) 100 100 100 tempering average quantity of water used for 5.0 a (v/w) 5.0 a (v/w) 5.0 a (v/w) tempering the grains prior to decortication. decortication 2 i) average quantity of maize grits recovered after 69.6 ±1.4 a 69.6 ±1.4 a na 4 decortication. ii) average quantity of hulls, germs and grain brokens 30.4 ±1.2 a 30.4 ±1.2 a removed. na pestle and mortar i) average quantity of maize grits recovered after manual dehulling. na na 71.2 ± 1.8 dehulling ii) average quantity of hulls, germs and grain brokens na na 28.8 ± 1.1 removed. soaking average quantity of soluble solids in grit-soaking water 1.6 ± 0.1 na na oven-drying i) average quantity of maize grits (10.1±0.3%, moisture content) recovered after oven-drying. 64.1 ±1.3 na na ii) average loss during oven-drying operation. 3.9 ±0.4 na na milling 3 i) average quantity of unsieved flour after milling. 60.5 ± 0.9 c 65.8 ±1.1 b 67.3 ± 0.7 a ii) average loss during milling. 3.6 ± 0.6 a 3.8 ±0.4 a 3.9 ± 0.6 a sieving (< 300 µm) i) average quantity of sieved flour recovered. 56.6 ±1.4 b 62.3 ± 1.3 a 59.7 ± 1.1 a ii) average quantity of over-tails. 3.9 ± 0.5 b 3.5 ± 0.4 b 7.6 ± 0.3 a 1 average values within the same row having the same letter are not significantly different at p < 0.05; 2 temperature of maize grits during decortication= 45 – 50°c; 3 temperature of flour during milling operation: 1 st run (max.) = 58 o c, 2 nd run (max.) = 64 o c; 4 na = not applicable. was 5% (v/w) for all the methods while the average quantity of maize grits recovered after decortication was 69.6% for gsm and gnm respectively. the hulls, germs and grain brokens removed during decortication step accounted for 30.4% for gsm and gnm respectively. however, for pestle and mortar dehulling method (pmdm), the quantity of maize grits recovered after manual dehulling was 71.2%; higher than that of gsm and gnm respectively. the reason for this observation may be attributed to a possible ineffective manual dehulling operation which might have left substantial quantity of bran and germ unremoved. the average quantity of soluble solid that leached out during grit soaking, quantity of maize figure 1. effect of flour production method on the mean particle size of maize flour. grits recovered after oven-drying and the loss incurred during oven-drying were 1.6, 64.1 and 3.9% respectively specifically for the gsm. the quantity of unsieved flour obtained after milling from the production methods ranged between 60.5 and 67.3% with gsm and pmdm giving the lowest and highest values respectively. the significant difference (p < 0.05) in the flour output is attributable to variation in the grit input prior to milling. the loss incurred during milling ranged between 3.6 and 3.9% for all the production methods with no significant difference (p<0.05). the ultimate quantity of sieved flour (< 300 µm) obtained from the production methods ranged between 56.6 and 62.3% with gsm and gnm giving the lowest and highest values respectively with a significant difference (p < 0.05). the proportion of over-tails recovered from all the production methods also ranged between 5.5 and 7.6% with gnm and pmdm giving the lowest and highest values respectively with a significant difference (p < 0.05). the observed highest over-tails from pmdm may be attributed to a seeming large quantity of fibre (from bran) present in the flour due to a possible ineffective manual dehulling operation through pestle and mortar. effect of flour production methods on the mean particle size of maize flour the mean particle size of maize flour obtained from different production methods is presented in figure 1. it ranged between 203.3 and 220.5 µm with gsm and gnm giving the lowest and highest values respectively with a significant difference (p < 0.05). the lowest mean particle size of flour from gsm may be attributed to the weakened associative forces binding the endosperm together thereby giving rise to smallersized particle during milling. akingbala et al. (1987) and nche et al. (1996) observed that grain/grit soaking has the ability to stimulate enzyme activity thereby causing partial dextrinization of amylose/amylopectin molecules which in turn can lead to the weakening of associative forces within the starch structure (moorthy et al., 1996). mean particle size of flour has been observed to influence the physicochemical properties of the flour and textural characteristics of food products derived from such flour (hebrard et al., 2003). effect of production methods on the damaged starch content of maize flour the damaged starch content of maize flour from different production methods ranged between 12.2 and 14.5% with gnm and pmdm giving the lowest and highest values respectively with a significant difference at p < 0.05 (figure 2). the highest damaged starch content observed in flour from pmdm may be due to the manual pounding which might have inflicted greater damage to the starch molecules. the relative high damaged starch content in flour from gsm may also be due to the soaking figure 2. effect of flour production method on the damaged starch content of maize flour. and re-drying operations peculiar to the method which might have toughened the grits thereby making them to exhibit more resistance to milling, hence the relative high damaged starch value. damaged starch is an important parameter particularly in flour-related food processing operations because it could influence so many processing variables including water absorption by the flour, dough or batter stickiness, fermentation rate, loaf volume, colour and stability (craig and stark, 1984; hoseney, 1994). many factors responsible for variations in damaged starch content is cereal flours include type of cereal grain being handled, grain processing procedures, type and set-up of the milling machine and adjustment made during the milling stage (kent and evers, 1994). pasting properties of maize flour as influenced by different production methods the effect of flour production methods on the pasting properties of maize flour is presented in table 2. the pasting temperature of maize flour from gsm was the lowest (73.2 o c) while that from gnm was the highest (74.6 o c). the implication of this observation is that flour from gsm would gelatinize faster than that from others. this is most probably because the seeming stimulated enzymes in the soaking maize grits are capable of breaking down the matrix embedding starch granules, thus allowing the granules to swell freely and gelatinize faster (nche et al., 1996). the peak viscosity of maize flour from different production methods ranged between 84.2 and 110.6 rvu with gsm and pmdm giving the lowest and highest values respectively with a significant difference (p < 0.05). the differences observed in the peak viscosities of the maize flours may be attributed to different rates of water absorption and swelling of starch granules of these flours during heating (ragaee and abdel-aal, 2006). ayernor (1985) also observed that peak viscosity usually occurs at the equilibrium between granule swelling, which increases viscosity and granule rupture, and alignment due to mechanical shear, which causes its decrease. the breakdown viscosity of the flour samples ranged between 19.6 and 43.3 rvu with gsm and pmdm giving the lowest and highest values respectively with significant difference (p < 0.05) . the breakdown viscosity is regarded as a measure of the degree of disintegration of starch granules or paste stability during heating (dengate, 1984). the implication of this observation is that the flour from gsm with the lowest breakdown value was more resistant to heat and shear force during heating and that there was less starch granule rupture table 2. effect of flour production methods on the pasting properties of maize flour 1 . pasting factor 1 flour source pasting peak trough breakfinal setback, version i time to setback, version ii temperature viscosit (rvu) down (rvu) viscosity (difference between reach peak (difference between (°c) y (rvu) 2 (rvu) final viscosity and viscosity final and peak trough) (rvu) (min) viscosity) (rvu) grit soaking method (gsm) 73.2 b 84.2 b 64.6 b 19.6 c 111.4 b 46.8 b 9 a 27.2 c grit non-soaking method (gnm) 74.6 a 108.3 a 71.4 a 36.9 b 147.4 a 76.0 a 9 a 39.1 a pestle and mortar dehulling method (pmdm) 74.2 a 110.6 a 67.3 ab 43.3 a 144.5 a 77.2 a 9 a 33.9 b 1 results are mean values of triplicate determinations. mean values followed by different superscripts in each column are significantly different at p < 0.05. 2 rvu=rapid visco unit. which could therefore guarantee a more stable cooked paste (farhat et al., 1999). the final viscosity of the flour samples ranged between 111.4 and 147.4 rvu with gsm and gnm giving the lowest and highest values respectively. higher value of final viscosity has been attributed to the aggregation of the amylose molecules in the paste (miles et al., 1985). therefore, the lowest final viscosity value of gsm may be due to a seeming enzymatic activity that might have occurred during the grit soaking stage (nche et al., 1996), thus reducing the aggregation of its amylose molecules. the setback viscosity (version i) of the flour samples ranged between 46.8 and 77.2 rvu with gsm and pmdm giving the lowest and highest values respectively with a significant difference (p < 0.05). the lowest setback (version i) observed in gsm may be attributed to partial dextrinization of the starch molecules. akingbala et al. (1987) observed that the stimulated enzymes in the soaking grains are capable of causing partial hydrolysis of the starch molecules thereby reducing the amount of starch to be gelatinized, thus lowering the setback viscosity. the setback viscosity (version i) can be used to predict the storage life of a food product prepared from the flour (zaidul et al., 2007). therefore, the implication of this observation is that the food dumpling prepared from gsm is most likely to exhibit lower retrogradation tendency than that of others due to lower setback viscosity (version i). the setback viscosity (version ii) of the flour samples ranged between 27.2 and 39.1 rvu with gsm and gnm giving the lowest and highest values respectively. the reason for the lowest setback viscosity (version ii) in gsm is the same as that postulated for setback (version i) and this parameter can be used to predict the textural characteristics of food products prepared from the flour (otegbayo et al., 2006). therefore, the food dumpling prepared from gsm would most probably exhibit a higher cohesiveness than that from others due to the lowest setback viscosity, version ii (bolade et al., 2009). effect of flour production methods on the colour characteristics of maize flour and ‘tuwo’ the lightness index (l*-value) of flours from different production methods ranged between 88.8 and 90.0 with gsm and gnm having the lowest and highest values respectively (table 3). the additional unit operations (soaking and re-drying) involved in gsm might have contributed to the marginal decrease in the lightness index of the flour. the lightness index of maize ‘tuwo’ also ranged between 66.9 and 67.2 with gsm and gnm giving the lowest and highest values respectively. the partial non-enzymatic browning which might have occurred during oven-drying of maize grits from gsm may also be responsible for the lowering of the lightness index of both flour and ‘tuwo’. mcdonough et al. (2004) observed that high temperature heat treatments could increase the brown colour of cereal grains and meals prepared from them due to browning (maillard) reactions. the chroma, c-value, of the flour samples ranged between 14.6 and 14.9 with no significant difference (p < 0.05). the c-values of ‘tuwo’ samples also ranged between 8.9 and 9.1 with no significant difference (p < 0.05). the chroma is a measure of colour intensity of the samples (deman, 1990). the hue angle (h°) of both maize flour and ‘tuwo’ were also affected by the production methods. the hue angle (h o ) of the flour samples ranged between 90.2 and 90.3° while that of ‘tuwo’ samples ranged between 99.7 and 102.8°. however, it has been observed that the hue angle (h°) seems not to be a useful indicator for describing table 3. effect of flour production methods on the colour indices of maize flour and tuwo 1 . sample source maize flour maize tuwo l* a* b* chroma, c hue angle, h o l* a* b* chroma, c hue angle, h o grit soaking method (gsm) 88.8 b -0.09 a 14.6 a 14.6 a 90.3 a 66.9 b -2.0 a 8.7 a 8.9 a 102.8 a grit non-soaking method (gnm) 90.0 a -0.08 a 14.7 a 14.7 a 90.3 a 67.7 a -1.9 a 8.9 a 9.1 a 102.1 a pestle and mortar dehulling method (pmdm) 89.6 ab -0.06 a 14.9 a 14.9 a 90.2 a 67.2 ab -1.5 b 8.8 a 8.9 a 99.7 b 1 results are mean values of triplicate determination. mean values within the same column having the same letter are not significantly different at p < 0.05. figure 3. cohesiveness index of maize tuwo from three distinct flour production methods. figure 4. softness index of maize tuwo from three distinct flour production methods. the colour change in white maize grain processing as redyellow-green colour indices are seldom applicable (bolade et al., 2009). effect of flour production methods on the cohesiveness index of maize ‘tuwo’ the cohesiveness index of maize ‘tuwo’ samples ranged between 15.1 and 15.9% with pmdm and gsm giving the lowest and highest values respectively with no significant difference at p < 0.05 (figure 3). the marginal increase in the cohesiveness index of maize ‘tuwo’ from gsm may be attributed to changes that had occurred to the physical and chemical properties of the flour from gsm. these include the resultant relative low mean particle size of the flour and the weakening of associative forces within the starch molecules (gibbon et al., 2003) by virtue of seeming partial hydrolysis through the stimulated enzymes during grit soaking (akingbala et al., 1987; nche et al., 1996). gomez et al. (1987) observed that relatively small particle size of flour was responsible for acceptable level of cohesiveness in tortilla, among others. cohesiveness of food dumplings like maize ‘tuwo’ is an important quality attribute that usually influences consumer acceptability of the product (osuji, 1983; aboubacar et al., 1999) . moulding of food bolus with fingers and palm is one of the preliminary actions that usually precede maize ‘tuwo’ consumption and good hand-mouldability is therefore a factor that influences the overall satisfaction. thus, an enhanced cohesiveness index of maize ‘tuwo’ will predispose it towards good hand-mouldability and increased psychological satisfaction during consumption. softness index of maize ‘tuwo’ as influenced by flour production methods the softness index of maize ‘tuwo’ samples ranged between 17.2 and 17.8mm with pmdm and gsm giving the lowest and highest values respectively with a significant difference at p > 0.05 (figure 4). additional unit operations of soaking and re-drying involved in the production of flour from gsm might have weakened the associative forces within the starch structure (gibbon et al., 2003) as postulated earlier thereby leading to a greater softness index of maize ‘tuwo’ prepared from such flour. similarly, the lowest softness index of maize ‘tuwo’ from pmdm might have been caused by a possible presence of additional fibre and fat in the flour due to a seeming ineffective manual dehulling operation. hardacre and clark (2006) observed that a food gel or dumpling (e.g. maize ‘tuwo’) usually exhibits variation in the rheological characteristics as a result of different degrees of interactions of flour components (that is, protein, fat, fibre and non-starch polysaccharides) with solubilised amylose and amylopectin components of the starch granules during heating. the softness index of maize ‘tuwo’ can be used to simulate the force required to compress the food product between the tongue and palate which is normally a preliminary action usually carried out in the mouth during consumption and can lead to whether the food product will eventually be chewed or swallowed. maize tuwo, like many other traditional food gels or dumplings, is consumed by swallowing rather than being masticated or chewed and it is the prevailing textural characteristics of the product, at the point of consumption, that usually determine whether such food is swallowable or chewable prinz and lucas, 1995; szczesniak, 2002). therefore, lower softness index can predispose the food product towards being masticated or chewed while relative high value encourages swallowability. conclusion it may be concluded that different production methods of maize flour play an important role in the flour yield, physicochemical properties of flour and rheological characteristics of food dumpling (e.g. maize ‘tuwo’) prepared from the flour. flours from different production methods showed variation in the particle size, damaged starch 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with different phosphorus contents. food chem. 102: 1105-1111. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (8), pp. 361-367, august, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of the effect of precipitated whey protein (pwp) and fermentation time on the quality of iranian lavash bread hossein jooyandeh department of food science and technology, ramin agricultural and natural resources university, ahvaz, iran. e-mail: hosjooy@yahoo.com. accepted 04 february, 2018 flat bread is the main staple food for most iranians and lavash is commonly consumed flat bread. in this investigation, the effects of addition of precipitated whey protein (pwp) and fermentation time on the physical properties and sensory characteristics of lavash breads were studied. dough was prepared with four substitution levels of pwp (25, 50, 75 and 100%, instead of water used for dough making) and fermented at three different fermentation times (30, 60 and 90 min). increasing level of supplementation with pwp generally caused an increase in all sensory scores of lavash samples. this improvement was more obvious when fermentation time was increased. penetrometer values of lavash samples showed that increasing fermentation time and supplementation up to 75% created significantly softer breads compare to control. in general, increase in the level of supplementation and fermentation time significantly increased bread yellowness (b* value) and redness (a*) and decreased lightness (l* value). key words: flat bread, lavash, precipitated whey protein, fermentation. introduction cereals in generals, and wheat, in particular, are principal foods around the world that provide more nutrients than any other single food source. human beings mastered the use of wheat and the art of bread making thousands of years ago. nowadays, different breads are produced which can be divided into three categories with respect to their specific volume (volume/weight): those with high specific volume such as pan breads, those with medium specific volume such as french and rye breads, and those with low specific volume such as flat breads (faridi, 1988). flat bread can be divided into two major groups accor-ding to their cross section: single-layered and double-layered. the most important processing difference bet-ween these two kinds of bread is the second proofing period which might exceed 30 min in double-layered types. flat breads differ in the type of flour, manner of sheeting or in the appearance of the end product. these products have many characteristics that even appeal to the u.s population (qarooni et al., 1992). flat breads are a major form of wheat consumption in many middle eastern and north african countries (paulley, 1998). faridy (1981) demonstrated several rea sons for the increased popularity of flat breads. first, these products are good source of dietary fiber because flours of high extraction rate, usually more than 78%, generally are used. second, the formulas are simple, and there are few ingredients. third, they are excellent food carriers either by incorporating foods such as meat or vegetable in the dough or as a final product in which food can be placed in the pocket of pita or rolled by the single-layered flat breads. and fourth, flat bread production of-fers some real advantages for the baking industry. the ingredients are few, so there is a significant reduction in cost and storage space. the equipment requirements are relatively few compared to those of pan bread production. because flat breads are produced directly on the conve-yor belts, substantial savings are recognized by elimina-ting the need for baking trays and pans. flat breads require less cooling time and space because of their large surface areas. and finally, a large number of pro ducts can be made with only slight modifications to an existing production line. today in iran, many types of flat breads, such as lavash, sangak and barbari are produced and consumed. lavash bread is single-layered bread leavened with sour dough jooyandeh 362 table 1. composition (%) of wheat flour and pwp used for lavash production. moisture protein fat carbohydrate ash flour 11.94 10.42 1.45 75.71 0.48 pwp 89.06 4.22 1.73 2.41 0.58 /yeast or chemically with sodium bicarbonate. in recent years, many researchers have tried to improve baking properties, organoleptic characteristics, nutritional value and extension of the shelf-life of loaf bread. whey and whey products are considered as appro-priate ingredients for promoting nutritive value and organoleptic characteristics of many food products in-cluding bread and other bakery products (goecmen, 1993; rierarodriguez, 2002; drokan et al., 2003; jooyandeh, 2006; rantamaki, 2006). once considered a waste product in the cheese manufacturing process, whey and whey protein products today are used for a wide range of functional and nutritional properties (hoch, 1997). whey proteins are an important functional compo-nent in bread formulations. they enhance crust browning, crumb structure and flavor, improve toasting qualities and retard staling. whey-based ingredients can be customiz-ed to meet specific protein, minerals and lactose compositions. this is important because composition and degree of denaturation affect whey ingredient functionality (burrington, 2004). according to literature, there are scarcely any reports concerning the utilization of pwp in flat bread making. in pervious work (jooyandeh, 2006) different ingredients including whey powder, soy flour, and potato powder at different level of supplementation were used in lavash production to improve its quality. in this study the effect of using pwp and fermentation time on the quality of iranian lavash bread is evaluated. matrials and methods preparation of pwp pwp was prepared from sweet feta cheese whey by heat treatment and fermentation to coagulate and precipitate whey proteins (jooyandeh et al., 2009). after collection of whey during preparation of feta cheese, the whey was heated at 85ºc for 15 min. this heat treatment caused denaturation and coagulation which was required for proper precipitation of whey proteins during fermentation. then, the whey was cooled to 44ºc and inoculated with 1 2% of yoghurt starter cultures (yo-mix 532, danisco, germany) containing streptococcus thermophilus and lactobacillus delbruckii subsp. bulgaricus. after about 4 h fermentation at 42ºc, the ph of fermented whey reduced to 4.4 and fermented whey protein was precipitate as a separate phase. the upper phase was pouring out and remaining part was used as pwp. preparation of lavash bread wheat flour (triticum aestivum) with 84.1% extraction rate was obtained from a local supplier. lavash bread samples were produced by a technique involving double rising. in this study, pwp were incorporated in lavash breads at four substitution levels 25, 50, 75, and 100% instead of water during dough making. lavash bread samples without these ingredients were prepared and considered as control. the formula used in the preparation of experimental breads was wheat flour, 0.5% dried yeast, 1% salt, and water (optimum as assessed by dough feel at sheeting) or mixture of water and pwp. for all the recipes, the yeast was prefermented by suspending it in a sugar solution for 30 min for revitalization. for standard processing, ingredients were mixed with simultaneous addition of water or/and whey ingredients at a low disk speed (44 rpm for 1 min) and then at higher disk speed (130 rpm for 7 min) in a hunt 30 dough mixer (lancashire, uk) until a cohesive dough mass was obtained. the amounts of water needed for optimum dough consistency are shown in table 1. the resultant bulk dough was fermented at different fermentation times 30, 60 and 90 min in an incubator and maintained at 30°c at 85% relative humidity. the fermented dough was divided into balls of 200 g each. the dough pieces were dusted with flour of the same formulation (~10 g) and rounded into ball shape. the balls were covered with a wet cloth and fermented for 15 min at 30°c and then flattened into elliptical sheets 1.7 mm thickness and baked in commercial automated oven at 470°c to optimum crust color (~50 70 s), as reported by maleki and daghir (1967). after baking, lavash breads were cooled for 15 min, placed in polyethylene bags to prevent moisture loss, and stored at room temperature (30°c). all baking parameter and sensory evaluation were measured 2 h after baking. chemical analysis protein contents of flour (n × 6.25) and pwp (n × 6.38) were determined by a nitrogen analyzer (leco corporation, st. joseph, mi). moisture content of flour was determined by oven drying for 1 h at 130°c using aacc (2000) by approved method of 44-15a, ash content by dry combustion for 16 h at 580°c according to approved method 08-01, and fat content by petroleum ether extraction followed by evaporation to constant weight under vacuum using approved method 30-25. total carbohydrate content (%) was calculated by difference. for pwp, moisture content was determined at 102°c for 4 5 h according to aoac (2000) by official method 925.23, ash using official method 945.46 (at 550°c), and fat by gerber’s method. texture analysis sur pnr-6 penetrometer with 218 g of total test weight was used for determine hardness of breads as indicated by basman and koksel (1999) color measurement crust color was measured with a minolta chroma meter (cr-300, minolta, osaka, japan). this defines colour numerically in terms of lightness or l* value, (0 = black, 100 = white), a* value (greenness 0 to –100, redness 0 to +100) and b* value (blueness 0 to -100, yellowness 0 to +100) . penetrometer and crust color values of each lavash sample were determined at five different points. all mea-surements obtained with the three lavash breads from one batch were averaged into one replicate value. sensory evaluation twelve panelists (five female, seven male; aged 20-30) who had completed a graduate course in food quality and were familiar with 363 afr. j. food sci. res. crust color (5) creamish yellow color with light brown patches (4) cream color with light brown patches (3) light cream color with light brown patches (2) lighter gray color with pale or dark brown patches (1) gray color with pale or dark brown patches crumb color (5) whitish cream color (4) light yellowish cream color (3) yellowish cream color (2) light grayish cream color (1) gray color external appearance and shape (5) crust is smooth with few blisters and few cracks on the edges (4) 75% of crust is smooth with small amount of blisters and cracks on the edges (3) 50% of crust is smooth with moderate amount of blisters and cracks on the edges (2) 25% of crust is smooth with high levels of blisters and high levels of cracks on the edges (1) crust is not smooth, very high levels of blisters and cracks taste and aroma (5) characteristic lavash aroma and taste (4) light smell and taste from additive (3) perceptible smell and taste from additive (2) definite undesirable taste and smell from additive (1) very definite unacceptable smell and taste mouthfeel and texture (5) very pleasant and easy to chew (4) pleasant and easy to chew (3) slightly sticky when chewing (2) sticky when chewing (1) very sticky and doughy when chewing (waxy texture) which lavash samples do you consider acceptable? figure 1. ballot sheet for lavash samples (qarooni et al., 1987; williams, 1988; saxena and rao, 1996) with some modifications. lavash bread were chosen. all panelists were nonsmokers. instructions were given in full to panelists beforehand. examination took place in tasting booths under normal white illumination. lavash samples supplemented with pwp were evaluated on a scale of 1 5 for five quality parameters: crust color, crumb color, external appearance and shape, taste and aroma, and mouth feel and texture. a ballot sheet (figure 1) was prepared to evaluate sensory attributes of lavash breads after modifying parameters and scores of various flat breads to lavash (qarooni et al., 1987; williams, 1988; saxena and rao, 1996). consistency of the panel was checked by subjecting data for the indicated attributes from three replicate rating of nine bread samples to principal component analysis (kwan and kowalski, 1980; powers, 1984). the results (data not shown) revealed a cluster of ten assessors indicating agreement in evaluation, and two outliers. consequently, sensory measurements were conducted with the consistent panel and final judgment was obtained by averaging the scores given only by these panelists. samples, selected at random from the different treatments, were removed from polyethylene bags before evaluation. the breads were rated in comparison to regular wheat bread (without pwp). statistical analysis all determinations were made in triplicate, and mean values are presented. the data collected from studies were analyzed using spss version 11.5 (spss inc., chicago, il). one way analysis of variance was performed for determination of individual differences between 15 treatments and two way analysis was carried out to determine significant differences in each factor. pwp concentration and fermentation time, the wholeplot factors, were applied in a 5 × 3 factorial treatment structure. a probability of <0.05 was used to establish statistical significance for fixed effects and interactions using duncan’s multiple-comparison test. results and discussion chemical composition the chemical composition of wheat flour and pwp are presented in table 1. pwp had a higher fat and ash, and lower protein than wheat flour. the ph and acidity of pwp were 4.5 ± 0.2 and 63 ± 6.2 °d (0.63 g percent lactic acid), respectively. this fermented product which rich in protein can be used in different product. kadharmestan et al. (1998) reported with heat denaturation, whey protein becomes insoluble and loses its functionality like emulsifying and foam ability but it can be used as a non table 2. the mean sensory scores of lavash samples supplemented with pwp and different dough fermentation time. level of fermentation crust crumb external appearance taste and mouth feel overall pwp time (min) color color and shape aroma and texture acceptability 30 3.87 h 4.73 ab 4.27 de 4.00 h 4.07 e 4.18 h 0% (control) 60 4.20 fg 4.53 bcd 4.60 ab 4.20 g 4.30 d 4.37 f 90 4.37 ef 4.63abcd 4.20 e 4.40 ef 4.50 c 4.42 ef 30 4.13 g 4.73 ab 4.40 cd 4.13 gh 4.03 e 4.28 g 25% pwp 60 4.33 ef 4.73 ab 4.70 a 4.30 fg 4.50 c 4.51 d 90 4.40 e 4.63abcd 4.50 b 4.60 cd 4.60 bc 4.57 d 30 4.30 efg 4.63 abcd 4.60 ab 4.20 g 4.47 c 4.44 e 60 4.57 cd 4.83 a 4.50 bc 4.50 de 4.70 b 4.62 c 50% pwp 90 4.87 ab 4.73 ab 4.40 cd 4.70 bc 4.90 a 4.72 a 30 5.00 a 4.53 bcd 4.60 ab 4.50 de 4.60 bc 4.67 bc 75% pwp 60 4.83 b 4.63 abcd 4.70 a 4.60 cd 4.90 a 4.73 a 90 4.60 c 4.43 ed 4.23 de 4.90 a 4.97 a 4.67 c 30 4.77 b 4.63 abcd 4.60 ab 4.80 ab 4.50 c 4.69 ab 100% pwp 60 4.43 de 4.43 ed 4.20 e 4.97 a 4.70 b 4.57 d 90 4.27 efg 4.23 e 3.90 f 4.70 bc 4.67 b 4.40 ef a,b,c,d means within the same column having different letters are significantly different (p< 0.05). table 3. probability values from anova of the effects of different pwp supplementations and fermentation time on sensory properties of lavash breads. source fermentation crust crumb external appearance taste and mouth feel overall time (min) color color and shape aroma and texture acceptability pwp f value 67.538 10.275 16.378 53.191 63.000 154.301 significance 1 0.000 0.000 0.000 0.000 0.000 0.000 f value 3.325 4.650 40.902 40.085 69.939 48.619 fermentation time significance 0.050 0.017 0.000 0.000 0.000 0.000 pwp × fermentation f value 21.919 2.962 11.421 5.830 5.432 42.919 time significance 0.014 0.000 0.000 0.000 0.000 0.000 1. significance at the 0.05 level. functional ingredient for fortification of baked goods. many other researchers (vetter, 1984; erdogdu-arnoczky and pomeranz, 1996; kenny et al., 2001) also emphasized that whey protein denaturation is compulsory for production of a high quality bread. sensory properties the mean sensory scores of lavash samples supplemented with pwp and different dough fermentation time are presented in table 2. results showed that both supplementation and fermentation time have significant effects on all organoleptic characteristics of lavash breads. there were also significant interactions between incorporation levels and fermentation time in all sensory attri butes (table 3). supplemented breads had higher acceptability and increase in the supplementation level usually caused an increase in sensory scores. lavash samples made with 100% incorporation had better taste, but lower appearance and crumb color than other samples, including control. with respect to crust colour and taste, all levels, including control, were classified in different groups. control lavash (without pwp) significantly had lower scores than supplemented samples in terms of crust color, taste and aroma, texture and mouth feel, and overall acceptability. sensory scores of lavash samples made with fermented dough at different fermentation times also revealed that increase in fermentation time cause an increase in all sensory parameters. no differences was observed among jooyandeh 364 figure 2. influence of pwp supplementation and fermentation time on penetrometer values of lavash breads. 60 and 90 min fermentation times on the sensory parameters of lavash samples except for taste and mouth feel which were higher in samples fermented at 90 min. the one-way analysis of sensory scores (p<0.05) indicated that the best overall acceptability can be obtained by 50, 75 and 100% supplementations at different fermentation times 90, 60 and 30 min, respectively (table 2). this proved that with increase the level of supplementation, fermentation time can be considerably reduced. bread texture results indicated that supplementation and fermentation time have significant effects on the softness of lavash samples in different manner (figure 2). in supplemented lavash samples, the softness increased up to 50% and thereafter declined. however, all supplemented samples were softer than control and there was no difference between samples fortified with 50 and 75% pwp. the penetration rates for control and supplemented samples were 18.9, 21.2, 23.3, 23.4, and 19.3 respectively. the increase and then decrease in softness might be attri-buted to the mutual effect of addition of pwp. supple-mentation at higher level of pwp decreased softness and increased fermentation rate. the decrease in softness due to higher level of pwp may be related to higher amount of ash and calcium available in pwp. the amount of calcium plays a significant role in the rate of firming of bread (burrington, 2004). the effect of fermentation time on the softness of lavash samples was more obvious than that of the level of pwp supplementations. in-crease in the fermentation time caused significant in-crease in lavash softness. burrington (2004) expressed that decreasing fermentation time is detrimental to bread quality when whey protein concentrates are used. typi-cally, the shorter the fermentation time, the more sensi-tive the bread is to whey proteins. this effect was obviously observed in this study. during sourdough preparation, lactic acid bacteria produce a number of metabolites which have been shown to have a positive effect 100 l a b v a lu e 80 60 c o lo r 40 20 0 0% 25% 50% 75% 100% level of pwp figure 3. influence of pwp on crust color (l, a, and b values) of lavash breads. on the texture and staling of bread, e.g. organic acids, exopolysaccharides and/or enzymes (arendt, 2007). organic acids affect the protein and starch fractions of flour. additionally, the drop in ph associated with acid production causes an increase in the proteases and amylases activity of the flour, thus leading to a softer texture. samples of lavash supplemented with 50 and 75% pwp and fermented at 90 min were significantly softer than the rest of the supplementation levels and the control. the penetration rates for these samples were 27.6 and 28.7 dmm, respectively. the penetration rates for lavash control samples fermented at 30, 60 and 90 min were 16.4, 18.6 and 21.6 dmm, respectively. bread color a distinctive characteristic of the supplemented samples was their crust color. all samples which contained pwp, particularly at higher amount of incorporation, exhibited a white-yellowish color and differences among the samples were already visible without instrument aid. supplementation with higher amount of pwp and increase in the fermentation time significantly increased bread yellowness (b* value) and redness (a*) in the lavash samples; whereas lightness (l*) adversely was affected by these factors (figure 3 and 4). results showed that the crust color changes were significantly higher in samples contain 75 and 100% substitutions than others (p=0.003 and p=0.000; respectively). the reason can be explained by maillard reaction. the maillard reaction is quite universal in the food industry, and this reaction occurs when most foods are heated and results in reactions that promote browning of cookies, bread, and other baked goods. whey proteins contain a high amount of the amino acid lysine (trierum, 2004) which is typically the most reactive amino acid in regard to the maillard reaction because it possesses the -amino group (deman, 1999). on the other hand, reducing sugars which are another important factor in reaction are present in pwp. therefore, increase in pwp incorporation in lavashs led to increase in maillard reac 365 afr. j. food sci. res. jooyandeh 366 100 l a b v a lu e 80 60 c o lo r 40 20 0 30 min 60 min 90 min fermentation time figure 4. influence of fermentation time on crust color (l, a, and b values) of lavash breads. tion. the effect of level of pwp supplementation on the crust color of lavash samples was more obvious than the effect of different fermentation times. however, both these factors created significant differences on crust color. in lavash samples, as the percent of pwp incorporation or fermentation time increased, l values shifted from white to gray, a values shifted from gray to red, and b values shifted from gray to yellow, significantly. the l value for control (80.7) was higher than samples prepared with 50, 75, and 100% pwp (79.5, 77.7, and 76.3, respectively) and lower than sample with 25% pwp (81.1). however, there was not significant difference between control and samples contain 25 and 50% pwp. the a and b value of lavash samples varied from 0.9 to 3.6 and 15.8 to 18.4 respectively; the highest and lowest were related to control and samples supplemented with 100% pwp, respectively. conclusion supplementation with pwp generally led to quality improvement of lavash breads and softer bread. increasing the level of pwp was significantly increased penetration rates and it ranged from 18.9 (control) to 23.4 (75% pwp). however, at the highest level of supplementation, that is, breads with 100% pwp, the penetration rate was noticeably reduced and reached to 19.3 dmm. on the other hand, with increasing the fermentation time, the softer bread with higher sensory scores was produced. the penetration rates for breads fermented at 30, 60 and 90 min were 17.3, 21.1 and 25.2 dmm; respectively. in summarize, samples contained 50 or 75 % pwp and fermented for 90 min had better quality. it was also obviously observed that by increasing the rate of supplementation and decrease in the fermentation time, lavash bread with a good quality can be produced. the results indicated that the process time and preconditioning cost can be considerably lowered by adopting the approach suggested in this paper, while the quality of lavash breads is 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w, srivastava j (1988). evaluation of wheat quality for the baking of syrian-type two-layered flat breads. j. cereal sci. 7: 195-207. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (9), pp. 001-005, september, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of refining on the quality and composition of groundnut oil e. o. aluyor1*, p. aluyor2 and c. e. ozigagu1 1 department of chemical engineering, university of benin, nigeria. 2 department of vocational and technical education, ambrose alli university, ekpoma, nigeria. accepted 09 july, 2019 crude groundnut oil was obtained by solvent extraction from groundnut seeds and refined. the crude and refined oils were then analyzed. the results obtained show that the acid value decreased from 2.890 to 0.420 mg/koh. free fatty acid decrease after refining from 2.82 to 2.02%; phospholipids decreased from 23.11 to 5.53%; copper content decreased from 0.37 to 0.0361% and iron decreased from 3.98 to 0.31%. these can be viewed as an improvement in the quality of the oil after refining. refining did not have much effect on the fatty acids composition except for slight inconsistent decrease in saturated and unsaturated fatty acids. key words: free fatty acids, phospholipids, crude, refined, gas chromatography. introduction arachis oil (peanut oil; groundnut oil) is derived from groundnuts (seed of arachis hypogaea linn). groundnut oil is a vegetable oil which contains only a small propor-tion of non-glyceride constituents. its fatty acid compo-sition is complex including saturated fatty acids covering a wide range of molecular weights. groundnut oil is excellent food oil, with good flavor and high quality with its low free fatty acid value. vegetable oils generally primarily consist of triglycerides but several other com-pounds are also present. some of these additional compounds such as diglyceride, tocopherols, sterols and sterols ester need not necessarily be removed during processing. compounds such as phosphatide, free fatty acids, odiferous volatiles, colorant, waxes and metal compounds negatively affect taste, smell, appearance and storage stability of the refined oil and hence must be removed. carefully separated, however, some of these additional compounds, particularly the phosphatides are valuable raw materials (belcher, 2008). although there are many methods that are described in the literature that may be used to analyze fatty acids of vegetable oils, high precision in gas chromatographic *corresponding author. email: eoaluyor@yahoo.com. tel.: +2348023350667. analysis of fatty acids is possible with careful attention to details during sample preparation, injection, and chromatograph and data collection. small theoretical correction factors only should be necessary in most circumstance (christie, 1993). also the qualitative and quantitative determination of the constitution is done by gas chromatography of the compound or their derivatives (cert et al., 2000). a lot of works have been done in the past and many are still on by lipid analysts to explore the potentials of vegetable oils. the importance of analyzing vegetable oils cannot be overemphasized because in analyzing vegetable oil, the major feature influencing the physical and chemical properties, their application and uses are got. the analysis of phospholipids is important because they contribute to the stability and quality of edible oils, fats and fatty foods through their anti-oxidative activities. they are also responsible for oil discoloration during deodorization and steam distillation so that their determination is necessary to evaluate the efficiency of degumming (mounts and nash, 1990; monte et al., 1992; nzai and proctor, 1998). metals catalyze oxidation and they are responsible for both an increase rate of oxidation and colour fixation. with the analysis of phospholipids, cu and fe, the adverse effect of product stability and colour can be estimated. with the analysis of free fatty acids (ffa), suitability of vegetable oils for edible purposes can be determined (esuoso and odetokun, 1995). this research was to ascertain how the refining of groundnut oil affects its quality characteristics and its constituent experimentation seed oil exraction groundnut oil seed was purchased from lagos state, south–west, nigeria. the groundnut seeds were separated from the shaft by hand picking method. the seeds that were freed of the dirt were collected into a separate precleaned beaker. the samples were weighed and kept for pulverization. the grinding machine of make janke and kunkel (ika-labortechnic) was used for grinding until the samples were finely grinded. the funnel and the hollow parts of the machine were cleaned before another sample was grinded to avoid cross contamination. the pulverized samples were then extracted using the soxhlet extractor arrangement equipped with thimble. hexane was used as the solvent. the duration of the extraction was 6 h. the process was repeated twice to ensure that most of the oil in the seed was removed using fresh solvent. the crude oil with solvent extracted was collected into the pre-cleaned beaker for distillation. the extract was poured into the round bottom flask of the rotary evaporator arrangement. it separated by driving the hexane off the extract. then the extracted oil was dried of water by using an anhydrous sodium sulphate. steps used in refining the groundnut oil the crude groundnut oil has to be refined before it can be used for different applications either in the edible or industrial application. the crude oils contain small amount of naturally occurring materials such as proteinaceous free fatty acids and phospholipids. the method used for the removal of the ffa was by reacting it with sodium hydroxide solution (alkali) in a read stoichiometric ratio to ensure complete reaction and to make sure that no alkali was left over in the oil. the intermediate then reacted with phospholipids. these reacted products and the proteinaceous materials were then removed by centrifuge. following the alkali refining, the oil was washed with water to remove residual soap caused by saponification of small amount of the triglycerides oil. colour producing substances within oil, for example carotenoids and chlorophyll, were removed by bleaching process. acid activated alumina was employed for bleaching by allowing some contact time with the oil. the refined oil was then filtered and kept for usage. esterification procedure the oil sample was air dried by blowing the air gently on it. 1.0 g of oil was weighed into the beaker. the oil sample was heated in a borosilicate beaker container at 140 o f with pump running to allow homogeneity of the sample. some of drops of the acid were added to the oil in the container in a fairly fast manner of proper distribution. the samples are homogenized while the acid was being added with the aid of the mixer. about 3 ml of the methanol was put in a pre-cleaned beaker. the heater and the pump were off to allow the methanol to be added to the mixture. the methanol was added in a fast way. the mixture was mixed properly and fan was allowed to blow the fumes away. the mixture was covered properly and temperature was allowed to drop to ambient tempe rature (65 o f). the pump and fan were on occasionally for about 4 h. after cooling, the sweet fragrance ester was decanted in to a clean borosilicate container before injecting into the gas chromatography. fatty acids methyl ester gas chromatogaphy analysis condition gc: hp 6890 powered with hp chemstation rev. a 09.01 [1206] software. initial temperature: 60 o c for 3.0 min. first rate: 8 o c/min to 140 o c for 10.0 min, constant at 140 o c for 5.0 min. second rate: 10 o c/min to 250 o c for 11 min, constant at 250 o c for 10.0 min. detection temp: 275 o c. injection temp: 230 o c. detector: fid. carrier gas: nitrogen. column: hp-innowax (cross-linked peg). column length: 30.0 m. column i.d: 0.32 mm. film thickness: 0.50 µm. nitrogen pressure: 30.0 psi. hydrogen pressure: 22 psi. comp. air pressure: 28 psi. condition of analysis for phospholipids column used: zb-5 column. column length:30 m. column id: 0.25 mm. column film: 0.25 µm. detector temp: 300 o c. detector: pfpd (pulse flame photometric detector). initial temp: 30 o c for 3 min. first rate: 4 o c/min for 20 min and maintained for 1 min second rate: 15 o c/min for 10 min and maintained for 1 min mobile phase or carrier:hydrogen. hydrogen column pressure:25 psi. hydrogen pressure: 22 psi. compressed oil pressure: 28 psi. atomic absorption analysis of the sample sample was sub sampled into the conical flask. the content of the flask was treated with 5% nitric acid. the samples were prepared for different metal analysis. the solution was allowed to cool at room temperature. standard iron (fe) and copper (cu solutions of 0.20, 0.40, 0.60, 0.80 and 1.00 mg/l) were made from each of the standard heavy metals. the sets of standard solutions and filtrate of the sample were analysed by aas. the detection limits of the heavy metals in the sample were 0.0001 mg/l by means of unicam 929 london, atomic absorption spectrophotometer powered by solaar software. iron and copper cathode lamps were used for the analysis of fe and cu ions, respectively. airacetylene gas mixture was used in the generating flame. determination of physico-chemical properties saponification value of oil sample was determined by dissolving 1 g of the oil in 12.5 ml of 0.5% ethanolic koh and the mixture of refluxed for 30 min. 1 ml of the phenolphthalein indicator was added table 1. physico-chemical properties of crude and refined groundnut oil. parameters groundnut oil crude refined specific gravity at 20°c 0.916 0.915 refractive index at 40°c 1.462 1.460 saponification value (mgkoh/g) 192.00 188.00 acid value (mgkoh/g) 2.890 0.420 iodine value (mg/100g) 91.00 86.00 odour agreeable agreeable and the hot soap solution titrated with 0.5 n hcl. a blank determination was also carried out under the same condition and saponification value determined using the equation: to ascertain the iodine value, 0.1 g of the oil was weighed into the 200 ml bottle capacity. 5 ml of carbon tetrachloride was added and the 2 ml of wij’s solution stood for an hour in the dark before the analysis was carried out. the blank was prepared as the sample. 5 ml of the 10% potassium iodide solution and 50 ml of water to each bottle and titrated against the 0.1 n sodium thiosulphate using starch as the indicator until colour change to permanent pale yellow. to find the acid value 0.1 g of the dissolved in 2.5 ml of 1:1 v/v ethanol: diethyl ether solvent and titrating with 0.1n sodium hydroxide while swirling using phenolphthalein as indicator. the specific gravity was measured by means of a cleaned pycnometer which was weighed. the pycnometer was filled with cooled distilled water and was stoppered. it was kept in the water bath for 30 min. it was removed and the drops of water were wiped properly and the weight was measured. the water was thrown away, oven dried and the pycnometer was filled with oil that has been previously dried over sodium sulphate. it was stoppered and kept in the water bath for 30 min. it was then removed, water wiped properly and the weighted was measured. to find the refractive index, the surface of the prisms was cleaned up with ether. 2 drops of the oil was applied at the lower prism and the prisms were closed up. water was passed through the jacket at 45 0 c. the jacket was adjusted for reading to be taken. results and discussion the physicochemical properties of crude and refined groundnut oil obtained are shown in table 1. the specific gravity of groundnut oil decreases from 0.916 to 0.915 after refining. the decrease was not significant hence refining does not have significant effect on the specific gravity. however, the decrease may be due to the removal of gumming materials and some coloring matters which affected the weight of the oil after refining. according to paul and palmer (1972), the specific gravity of different refining oils varies with their molecular weights which are affected by refining process involved. the refractive index of groundnut oil decreased from 1.462 to 1.460 after refining. the decrease was not significant hence refining does not affect the refractive index significantly. in addition, the oil did not differ significantly in refractive index both in crude and at the refined forms. the decrease could be as a result of continuous removal of impurities during the refining process. according to pearson (1991), the amount of impurities that are contained in the oil affects the degree of reflection caused by a ray of light during refractive index determination of the oil. the odour in the sample of oils is agreeable. this means that they all have no offensive smell. this shows that refining does not affect the odour of the oil. this could be as a result of the low free fatty acids present (groundnut oil: crude, 2.821644% and refined, 2.017714% in the oil (kirk and sawyer, 1991). the saponification value of the groundnut oil decreased from 192 to 188 mg/koh after refining. this decrease generally could be due to the neutralization of fatty acids which may have resulted from the hydrolysis of the oil sample. according to kirk and sawyer (1991), the number of milligram of potassium hydroxide (koh) used to neutralize the fatty acid determine the degree of hydrolysis of the oil sample. the iodine value for groundnut oil decreases from 910 mg/100 g to 860 mg/100 g after refining. the decrease was not quite significant. hence, refining did not affect iodine value significantly. the decrease in iodine value denotes decrease in the degree of unsaturation of the oil caused by the extent of oxidation and degree of heat treatment given to the oil during refining process (kirk and sawyer, 1991). the acid value for groundnut oil decreased after refining from 2.890 to 0.420 mgkoh/g. the decrease was significant for the groundnut oil. acid value had the highest decrease after refining (85.47%). according to demian (1990), the acid value is used to measure the extent to which glyceride in oil has been decomposed by lipase and other actions such as light and heat and that it determination is often used as general indication of the condition and edibility of oils. this decrease is an improvement in the quality of the oil. it is an expected result, since a reduction in acid value is targeted in the refining process. the fatty acid composition in crude and refined groundnut oil is as shown in table 2. the oil shows a moderate decrease in fatty acid composition from its crude form to refined form. according to achinewhu and akpapunam (1985), refining does not have much effect on fatty acid composition except for some slight inconsistent decrease in saturated and unsaturated fatty acid. this inconsistency was observed in this work with palmitic and palmitoleic acid. however, caprylic acid shows the highest decrease after refining (75.96%). oleic acid was the major unsaturated fatty acid and it recorded 58.7% in crude and 57.7% in refined followed by palmitic acid, 8.23% in crude and 11.74% in refined and linoleic acid 21.8% in crude and 21.5% in refined. the percenttage of saturated fatty acid was recorded to be16.82% in table 2. fatty acid composition of crude and refined groundnut oil. fatty acid crude composition (%) refined composition (%) caprylic acid (c8:0) 0.013346 0.003208 capric acid (c10:0) 0.008544 0.005542 lauric acid (c12:0) 0.275816 0.116076 myristic acid (c14:0) 0.115270 0.1061 palmitic acid (c16:0) 8.2280 11.7378 palmitoleic acid (c16:0) 0.1073 0.1296 stearic acid (c18:0) 2.4581 2.0606 oleic acid (c18:1) 58.6871 57.6784 linoleic acid (c18:2) 21.7656 21.5413 linolenic acid (c18:3) 0.3446 0.2810 arachidic acid (c20:0) 1.8313 1.4804 behenic acid (c22:0) 3.8852 2.36610 table 3. composition of crude and refined free fatty acids and phospholipids of groundnut oil after refining. composition crude refined free fatty acid composition in groundnut oil (%) 2.82 2.01 phospholipids composition in groundnut oil (%) 5.53 23.12 crude and 20.37% in refined with palmitic acid recording the highest both in crude (8.23%) and refined (11.74%) respectively followed by stearic acid. capric acid recorded the least reduction in saturated fatty acid in its crude form with 0.0085%. the free fatty acid for groundnut oil is given in table 3. the free fatty acid for crude groundnut oil (2.82%) is slightly greater than that in the refined oil (2.01%) . from table 3, it is vividly seen that groundnut oil shows higher percentage decrease after refining (28.49%). crude oils with high free fatty acid content result in higher refined losses (international conference on palms and palm products, 1989) . according to esuoso and odetokun (1995) free fatty acid of oil suitable for edibility purpose should not exceed 5%. in view of this, groundnut oil both crude and refined is edible. the amount of free fatty acid present is a measure of the quality of the unrefined as well as the refined oil (international conference on palms and palm products, 1899). therefore, in view of the low free fatty acid in groundnut oil, both crude and refined, it makes higher quality oil. the decrease in free fatty acid could be as a result of removal of some fatty materials and free fatty acids during refining process. according to kirk and sawyer (1991), the presence of free fatty acid and other fatty materials in oil brings about the offensive odour and taste in the oil on long storage. the phospholipids composition of the groundnut oil is also shown in table 3. the amount of phospholipids on crude groundnut oil (23.12%) is higher than that recorded for refined groundnut oil. phospholipids contribute to the stability of edible oil fats and fatty food through their antioxidation activity (singleton, 1993; carelli et al., 1997). this shows that the natural antioxidative activity of refined groundnut oil may be lower than crude groundnut oil. phospholipids are responsible for oil discoloration during deodorization so that their determination is necessary to evaluate the efficiency of degumming (international conference on palms and palm products, 1989; mounts and mash, 1990, mounts et al., 1992; nzai and proctor, 1998). on heating crude groundnut oil to deodorization temperature, the brown coloration that will be observed will be more than observed for refined groundnut oil. the copper concentration of groundnut oil decreased from 0.37 to 0.036 mg/kg after refining and the iron concentration of same oil decreases from 3.98 to0.31 mg/kg after refining as shown in table 4. the percentage decrease of iron in groundnut oil after refining is the most significant (92.19%). metals such as iron and copper catalyze oxidation and therefore are responsible for both an increase rate of oxidation (international conference on palm and palm materials products, 1989) . therefore, in view of the higher concentration of metals in crude groundnut oil will be higher than that in refined groundnut oil. table 4. copper and iron concentration in the groundnut oil samples. concentration of metals in the oils crude refined % decrease in metals after refining concentration of copper in groundnut oil (mg/kg) 0.3738 0.0361 91.07 concentration of iron in groundnut oil (mg/kg) 3.9832 0.3109 92.19 conclusion refining slightly affects the physical and chemical characteristics of groundnut oil. there was decrease from the crude form to the refined form and there was a significant decrease in acid value. this significant decrease confirms the presence of oleic acid which is significant in groundnut oil. as for the major and minor constituents, refining did not have much effect on the fatty acid except for some slight inconsistent decrease in saturated and unsaturated fatty acids. references achinewhu sc, akpapunam ma (2005). physical and chemical characteristics of refined vegetable oils from rubber seed and bread fruit. j. plant food 35: 103-107. belcher wm (2008). method for refining vegetable oils and byproduct. am. oil chem. soc. pp 110-115. carelli aa, brevedan miv, crapiste gh (1997). “quantitative determination of phospholipids in sunflower oil”. j. am. oil chem. soc. 74: 511-514. cert a, moreda w, perez c (2000). chromatographic analysis of minor constituents in vegetable oils. j. chromatogr. a 881: 131 -148. chen bh, christie ww (1987). high-performance liquid chromatography and lipids, a practical guide, pergamon press, oxford. demian mj (1990). principles of food chemistry. 2 nd ed. van nostrond reinhold international company limited, london england pp. 37-38. esuoso ko, odetokun sm (1995). riv. ital. sostanze grasse 72: 311313. international conference on palms and palm products. (1989).the impart of research and development on the production, processing and utilisation of palms and palm products 2: 707-708. kirk rs, sawyer r (1991). pearson’s composition and analysis of foods.9 th ed. longmand sci. tech. engl. pp. 607-617. mounts tl, nash am (1990). hplc analysis of phospholipids in crude oil for evaluation of soybean deterioration. j. am. oil chem. soc. 67: 757-760. mounts tl, abidi sl, rennick ka (1992). hplc analysis of phospholipids by evaporative laser light-scatter detection. j. am. oil chem. soc. 69: 438-442. nzai jm, proctor a (1998). phospholipids determination in vegetable oil by thin-layer chromatography and imaging densitometry. paul pc, palmer hh, (1972). food theory and applications. john wiley palmer, hh, sons incorporated, new york pp. 217-219. pearson d (1991). the chemical analysis of foods.7th ed. church livingstone longman group limited pp. 493-490. singleton ja (1993). enrichment of phospholipids from neutral lipids in peanut oil by high performance lipid chromatography. j. am. oil chem. soc. 70: 637-638. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 6 (3), pp. 271-280, march, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper properties of the starches from cassava cultivars and cocoyam grown in malawi in industrial utilization davies e. mweta 1* , maryke t. labuschagne 1 , elizma koen 1 , ibrahim r. m. benesi 2 and john d. k. saka 3 1 university of the free state, department of plant sciences, p. o. box 339, bloemfontein, 9300, south africa. 2 chitedze agricultural research station, p. o. box 158, lilongwe, malawi. 3 university of malawi, chancellor college, p. o. box 280, zomba, malawi. accepted 01 september, 2017 the physicochemical and functional properties of starch from cocoyam and ten varieties of cassava grown in malawi were studied to unravel their potential in industrial application. the properties of starch varied significantly with crop and among varieties. cocoyam starch granules exhibited polygonal truncated shapes and small sizes (average of 7 µm) while the cassava starch granules were rounded, irregular with oval and truncated ellipsoidal-granules with average size of 10.14 µm. the cocoyam starch gave lower values of amylose values content and paste clarity but higher phosphorus content, maximum wavelength of iodine complex absorption and blue value than cassava starches. cassava starches gels were more stable to freeze thawing releasing 28.40 – 46.92% of the water than cocoyam which released 54.06% of its water. swelling and solubility of both cocoyam and cassava starches increased with temperature (60 90c) . cocoyam starch exhibited lowered swelling power and solubility than cassava starches. the studies of gelatinization and retrogradation characteristics indicated similar enthalpy values of gelatinization of cocoyam and cassava starch; cocoyam starch displayed higher retrogradation tendencies than cassava starches. key words: cocoyam, cassava, amylose, blue value, swelling, solubility, paste, freeze-thaw, gelatinization, retrogradation. introduction starch is a major component of plant foods and an important raw material for industry. in malawi starch is widely used to manufacture various products such as food, textiles, pharmaceuticals, dry cells and adhesives. utilization of native starch in industries is limited by its functional and physicochemical properties. botanical source, environmental conditions and varietal differences are known to influence the functionality of starches (peroni et al., 2006; srichuwong et al., 2005; riley et al., 2006; tester et al., 2004). starches used in malawian industries largely constitute those of maize, potato and wheat, which are imported from zimbabwe, south africa, the netherlands, united *corresponding author. e-mail: davmweta@yahoo.com. tel: +27 727390713. fax: +27 (051)4446318. kingdom and tanzania. importation of starch leads to loss of large amounts of foreign currency, and increased unemployment (nso, 1999; masumbu, 2002). therefore, there is need for an indigenous crop locally grown by subsistence farmers that will help bring direct economic benefits to those who need it most. starch has two major components: amylose and amylopectin. these polymers are very different structurally. amylose is a relatively long, linear polymer -glucan containing around 99% (14)and 1% (16) linkages while amylopectin is a much larger molecule and a heavily branched structure built from about 95% (1 4) and 5% (1 6)linkages. the structures of these polymers play a critical role in the functionality of native and modified starches. viscosity, shear resistance, gelatinization, solubility, gel stability and retrogradation are some of the functional properties that depend on the amylose/amylopectin ratio of the mweta et al. 272 the starches (tziotisa et al., 2005; chakraborty et al., 2004). cassava (manihot esculenta crantz.) and cocoyam (colocasia esculenta) are potentially important sources of starch in malawi. unlike cassava, cocoyam starch has not been exploited in local industries. lack of information on the properties of starches from cassava and cocoyam produced in malawi has contributed to limited utilization of these starches in industry. knowledge on properties of the starches from these crops therefore would unravel the opportunities offered by these root crops and help their utilization. this paper, therefore, concerns some characteristic properties of the starches from cassava cultivars and cocoyam grown in malawi that will help in industrial utilization of the starches. materials and methods materials the study used starch isolated from ten cassava varieties. cassava varieties included were the local recommended varieties: mbundumali, maunjili, silira and mkondezi, locally screened varieties: sauti and ch92/082, and introduced varieties from iita: 83350, tms4(2)1425, 81/00015 and lcn 8010 (benesi, 2005). cocoyams were purchased from masasa, mzuzu, malawi and starch was extracted as follows: fresh tuberous roots were washed, peeled, washed again, chopped to about 1 cm 3 cubes and transferred into a heavy duty blender (waring commercial, model cbcsa 33bl34). one litre of water was added to 500 g of the chopped tubers, and the chopped tubers were pulverized at a high speed for 5 min. the suspension was then filtered using a double cheese (muslin) cloth. the filtrate was allowed to stand for four hours to facilitate starch sedimentation and the top liquid was decanted and discarded. the sediment was resuspended in 1 l of water and the whole process was repeated three times. the sediment was then washed and dried in the sun (open air) for two days. methods moisture content moisture content was determined in duplicate by drying starch samples of 2 3 g in an air oven at 105c overnight. the samples were cooled in a desiccator for 1 h and weighed to 1 mg to determine weight loss. moisture content (mc) was calculated in percent (%) of sample. ph starch samples (5 g) were weighed in duplicate in a beaker, mixed with 20 ml of distilled water, the resulting suspension stirred for 5 min and left to settle for 10 min. the ph of the water phase was measured using a calibrated ph meter (benesi, 2005). ash content duplicate sets of crucibles containing dried starch samples (2 3 g) were placed in a muffle furnace at 525c for 5 h, then cooled in a desiccator and weighed. ash content was determined by weight difference and expressed as a percentage. phosphorus content phosphorus was determined by a modified method of riley et al. (2006). starch samples (2 g) were weighed into 150 ml ceramic beaker, ashed at 550c for 5 h, cooled in a desiccator, mixed with 100 ml of 1 m hcl followed by addition of 1 ml of 1m hno3. the resulting mixture was heated to boiling, cooled, transferred to a 100 ml volumetric flask and made up to volume with water. aliquots (1 ml) in triplicates were transferred to test tubes mixed with 9 ml of murphyriley solution (murphy and riley, 1958), left to stand for 15 min and absorbance at 880 nm read against a reagent blank. granule morphology granule morphology of the native starches was studied using scanning electron microscopy (sem). starch samples were mounted on circular aluminium stubs using adhesive and then coated with thin layer of gold using bio-rad sputter coating system. the samples were examined and photographed in a jeol scanning microscope (jsm-6400, tokyo, japan) at an accelerating voltage of 5 kv and a magnification of x1000, x2000 and x3000. twenty granules were selected randomly and their size measured using a ruler. water holding capacity water holding capacity (whc) was determined by centrifugal procedure. starch samples (1 g) were suspended in 15 ml of distilled water in pre-weighed centrifuge tubes, slurry shaken on a rocking shaker for 1 hour and centrifuged at 12,100 x g for 10 min. the free water removed carefully from the wet starch and the weight of wet starch was determined. whc was calculated as the ratio of mass of water absorbed to mass of dry starch. swelling power and solubility patterns swelling power was determined by dispersing 0.5 g of starch samples in 20 ml of distilled water in a pre-weighed centrifuge tubes. the slurries were heated in a thermostatically controlled water bath at 60, 70, 80 and 90oc for 30 min with shaking every 5 min to keep the starch granules suspended. the heated slurries were then cooled to room temperature and centrifuged at 12,100 x g for 10 min to separate gel and supernatant. the supernatant was decanted carefully and poured into dish for subsequent analysis of solubility pattern. weight of swollen starch was determined and swelling power was determined as the ratio of the weight of the swollen starch to the weight of the starch sample. to determine solubility of the starches, supernatant was transferred into a pre-weighed evaporating dish and dried in air oven at 105 c for 4 h. weight of the residue was determined and the water solubility index was calculated from the amount of dried solids recovered by evaporating the supernatant and expressed as gram dried solids per 100 gram of sample on dry weight basis (chiang et al., 2007). paste clarity and stability the paste clarity was determined according to the method of craig et al., (1989). a 1% aqueous suspension was made by suspending 0.2 g (db) starch in 20 ml of distilled water in a stoppered centrifuge tube and vortex mixed. the suspension was heated in a boiling water bath for 30 min thoroughly shaking every 5 min. after cooling, clarity of starches was determined by measuring percent transmittance at 650 nm against a water blank on a spectrophotometer (spectronic unicam, he ios, cambridge, united kingdom). samples used to determine paste clarity were stored at 4c (in a refrigerator) for six days and the percentage transmittance was measured after every 24 h for the period of storage. 273 afr. j. food sci. res. freeze-thaw stability (syneresis) net syneresis of gels from starch pastes was determined according to the method of (schmitz et al., 2006). starch paste was prepared by heating a 5% starch suspension (1.0 g starch in 20 ml of distilled water) at 95c for 30 min with constant shaking (every 5 min). starch paste was weighed into polypropylene centrifuge tubes and submitted to successive cycles of freezing and thawing. the gels were stored at -18c for 18 h (two tubes per sample) and thawed to 30c for 90 min in a water bath. the thawed gels were vortexed for 15 s then centrifuged at 24,200 x g for 10 min. the amount of water eliminated from the gel was weighed. four freezing and thawing cycles were performed and syneresis was calculated as a percentage of the total weight of gelatinized samples. amylose contents amylose content was determined using an amylose/amylopectin assay kit (megazyme international ireland ltd., bray, ireland). in this method starch samples are completely dispersed by heating in dimethyl sulphoxide, and lipids are removed by precipitating the starch in ethanol. precipitated starch is recovered by centrifugation and dissolved in acetate buffer solution. amylopectin is specifically precipitated by addition of concavanalin (con a) and removed by centrifugation. amylose in the aliquot is determined using glucose/peroxidise reagent (gopod) after enzymatic hydrolysis to glucose. total starch in separate aliquot is determined using gopod reagent after hydrolysis to glucose and concentration of amylose is estimated as the ratio of gopod absorbance at 510 nm of the supernatant of con a precipitated sample, to that of total starch sample (gibson et al., 1997). iodine absorption spectra and blue value iodine spectra of the native starches were determined by dmsourea method of van hung and morita (2005). starch (40 mg) was dispersed in 10 ml of 10%urea containing dimethyl sulphoxide (90% dmso, 10% 6m urea) by vigorous vortexing, heated in a boiling water bath for 20 min and cooled to room temperature. aliquots (0.5 ml) of the dispersion were transferred in triplicates to a 25 ml volumetric flask, mixed with about 20 ml of distilled water and 1 ml of iodine-potassium iodide (200 mg i2 and 2 g ki in 100 ml distilled water) solution,and filled to the mark. the mixture was stood for 20 min at room temperture and an absorption curve was measured from 500 to 800 nm with a spectrophotometer (spectronic unicam, he ios, cambridge, united kingdom). blue value (bv) of the starches was determined as absorbance measured at 680 nm. differential scanning calorimetry thermal analyses of starch samples were obtained using differential scanning calorimetry (dsc 822e, mettler, toledo, switzerland) using starch: water ratio of 1:1. starch samples of 3.0 mg were weighed into dsc aluminium pans. distilled water, 3 µl, was added to starch sample using a transfer pipette and the pans were then sealed, and the samples were left to stand for 2 h at room temperature for moisture equilibration. the sealed pans were then heated from 25 to 120c at a heating rate of 10c/min to gelatinize the starch samples. from the dsc thermograms onset temperature (to), peak temperature (tp), conclusion temperature (tc) and enthalpy of gelatinization ( hg) were determined using instruments software (star sw 9.00). temperature range and peak height index (phi) were also calculated as tc to and as the ratio hg/(tp-to) respectively. retrogradation of the starch was also determined using differential scanning calorimetry. the gelatinized samples were stored at 4c (refrigerator) for periods of 1 and 7 days. after each period the samples were equilibrated at room temperature for 2 h, and then rescanned in the calorimeter from 20 to 120c at 10c/min to measure the retrogradation transition temperatures and enthalpy. the degree of retrogradation was determined as the ratio of enthalpy change of retrograded starch to enthalpy change of gelatinized starch (gunaratne and hoover, 2002). statistical analysis data obtained was subjected to analysis of variance (anova) at the significant level of 5% ( 0.05) using statistix 8 for windows software (analytical software, tallahassee, usa). when statistical differences were found, the least significant difference (lsd) was used to compare means at the 5% significance level. correlations between physicochemical properties were also evaluated. results and discussion composition of the starches results of analysis of moisture, ash, amylose and phosphorus contents the starches are presented in table 1. moisture content of the starches ranged from 10.42 to 11.13%. ash contents of the starches ranged from 0.10 to 0.20%. cocoyam starch had ash content (0.14%) similar to the cassava starches. ph of cocoyam starch (6.3) was higher than that of cassava starches (5.1 6.0). amylose content of the starches ranged from 16.27 to 28.82% and varied significantly (p < 0.001) with its source. cocoyam starch had lower amylose content than cassava starch. amylose content ranged from 16.96 to 28.83% for cassava varieties, with silira having the lowest and 81/0015 the highest. phosphorus in starch exists mainly in two forms: phosphate-monoesters and phospholipids. root and tuber starches contain phosphorus in the form of mono phosphate esters covalently bonded to starch while phospholipids are predominant in cereal starches. phosphorus contents of starches vary with botanical source, maturity as well as growing environment (jane et al., 1996). phosphorus content of the starches ranged from 65.35 to 128.72 mg/kg (0.0063 to 0.013%) and varied significantly (p < 0.001) with source. cocoyam starch had higher phos-phorus content (119.15 mg p/kg, 0.012%) than most of the cassava varieties. mkondezi, silira and ch92/082 cassava varieties had the lowest phosphorus contents while tms4(2)1425 had highest. value ranges of 13.6 to 23.8% and 3 43% for amylose content, and, 0.007 0.012% and 0.006 – 0. 013% for phosphorus contents have been reported for cassava and cocoyam respect-tively (moorthy, 2002). our results are therefore similar to those reported except for 83350, ch92/082, 81/0015 cassava varieties which had amylose content and tms4(2)1425 had higher phosphorus content. mweta et al. 274 table 1. granule size distribution, moisture content (mc), ash content (ac), ph, amylose content (am), phosphorus content (p), water binding capacity (wbc), paste clarity (pc), maximum wavelength ( max, ) and blue value (bv) cocoyam and cassava variety starches. starch granule size distribution mc %) ash ph am p wbc pc max bv source (%) (%) (mg/kg) (g /g) (%t) (nm) size range (µm) mode (µm) average size (µm) cocoyam 2.78 – 13.88 3.33 7.00 10.42 0.14 6.3 16.27 g 119.15 a 0.90 22.83 e 614.5 a 0.329 a 81/00015 4.44 – 17.78 10.00 10.66 10.85 0.14 5.6 28.25 a 95.32 b 0.93 44.16 ab 583.5 c 0.234 d 83350 4.44 – 15.56 11.67 10.52 10.57 0.13 5.1 24.27 c 73.34 cd 0.93 48.64 a 602.7 b 0.272 bc ch92/082 4.44 – 15.00 6.11 9.38 10.75 0.15 5.5 26.56 b 65.34 d 0.98 41.24 bc 596.5 b 0.248 cd lcn8010 5.56 – 17.22 10.00 10.56 10.74 0.16 5.7 21.20 e 72.88 cd 1.08 49.51 a 598.0 b 0.276 b maunjili 4.17 – 13.89 7.78 9.61 10.73 0.10 5.1 19.20 f 76.59 bcd 0.99 27.74 e 597.5 b 0.270 bc mbundumali 5.00 – 16.67 12.22 12.07 10.76 0.19 5.7 19.48 f 79.56 bcd 1.00 35.76 cd 596.7 b 0.279 b mkondezi 2.78 – 22.22 13.33 11.01 10.63 0.14 5.7 19.73 f 70.18 cd 0.94 43.96 ab 596.8 b 0.289 b sauti 2.78 – 15.56 5.56 8.38 10.87 0.10 5.1 22.68 d 87.58 bc 0.97 40.66 bc 598.8 b 0.247 cd silira 3.32 – 12.50 10.56 8.15 11.13 0.16 5.5 16.96 g 68.18 cd 1.01 33.81 d 597.0 b 0.264 bc tms4(2)1425 5.56 – 14.44 14.44 9.82 10.88 0.12 5.3 18.61 g 128.72 a 0.86 34.67 d 598.3 b 0.277 b nb: means followed by the same letter within the same column are not significantly different from each other (p = 0.05). microscopy of starch granule shapes and size the size and distribution of starch granules is important for specific applications (wang, 1983) and this depends on botanical source and may vary among varieties of crop (moorthy, 2002; moorthy et al., 1993) . results of morphological appearance and size of cocoyam and cassava starches as determined by sem are presented in figure 1 and table 1. cocoyam starch showed small and medium polyhedral (polygonal) and truncated granules (figure 1a) and exhibited smooth surfaces with some portions of the surface being irregular. granule size ranged from 2.78 to 13.88 µm with a mode of 3.33 µm and average of 7.00 µm. shapes mostly spherical, 5to 6-sided polygonal, small rounded, medium ellipsoidal-truncated and large polyhedral with size ranges of 0.05 0.08 µm, 2.96 – 5.19 µm and 0.5 to 5.0 µm have been reported (maeda et al., 2004; sefa-dedeh and kofi-agyir sackey, 2002; moorthy et al.,1993; pérez et al., 2005). the range of size of the granule in this study was comparatively large. cassava starches showed small to large rounded, irregular with oval and truncated ellipsoidal-granules within the same sample (figure 1 b-k). just like cocoyam the starch granules had smooth surfaces with some portions being irregular. granule size distribution varied substantially among the cassava varieties. granule size ranged from 2.22 to 22.22 µm with an overall average of 10.14 µm. mbundumali, and mkondezi had the largest granule size while silira and sauti had the smallest. using light microscopy, benesi (2005) reported mostly round or oval shapes, with a flat surface on one side containing a conical pit which extended to a well for cassava starches. granule sizes ranged from 4.3 to 24.3 µm. other researchers have reported round, oval, truncated, cylindri cal and spherical shapes for cassava starch granules with size ranges from 4 to 25.0 µm (gunaratne and hoover, 2002; sriroth et al.,1999; mishra and rai, 2006). our results are in agreement with these findings. wbc, maximum wavelength ( max) and bv whc of the starches ranged from 0.86 to 1.08 g h2o/ g starch (table 1) and no significant variations with starch source were observed. starch source had a highly significant (p < 0.001) influence on both the max and blue value of the starches. cocoyam starch had higher values of max and bv than cassava starch (table 1). the value of max for the starch-iodine complex is related to the chain length of the starch mole cules. as the chain length increases, so does the value of max (tetchi et al., 2007). the results 275 afr. j. food sci. res. figure 1. scanning electron micrographs of the starch samples therefore indicate the presence of longer chain starch molecules in cocoyam starch compared to cassava starches. freeze-thaw stability freeze-thaw stability, measured by degree of syneresis, describes release of water by gels that have been kept for longer periods refrigerated or frozen. this is an important factor to be considered when formulating refrigerated and frozen foods (thomas and atwell, 1999). results of four cycles of freeze-thawing are shown in figure 2. the amount of water released by the frozen starches differed significantly (p < 0.001). in the first cycle cocoyam starch, presented the highest tendency for water lost compared to the cassava starches draining 54.0% of the water, however in the second, third and fourth cycles, the amount of water released was within the range of cassava varieties. among the cassava starches, lcn8010, mbundumali, 83350 and silira showed greater tendency to lose water draining 46.3, 45.3, 45.0 and 43.8% of the water while 81/00015 presented the least (28.4%). the percentage of water separated in the second, third and fourth cycles were low for all the starches indicating high stability at those cycles. lcn 8010 and 81/00015 cassava starches consistently showed high tendency to release water in the second, third and fourth cycle. considering total amount of water released in all four cycles, gels from cassava starches exhibited lowest tendency to syneresis compared to cocoyam except lcn8010 starches. these results indicate that cassava starch is more stable to freeze-thawing than cocoyam starch and hence could be better suited for use in freeze products than coco yam starches. among cassava starches, ch92/082, sauti and maunjili would be more suitable as they presented the highest freeze-thawing stability. paste clarity and stability starch gel clarity is a much desirable functionality of starches for its utilization in food industries since it directly influences brightness and opacity in foods that contain it as thickeners. paste clarity and significantly (p < 0.001) varied with source (table 1). cocoyam starch paste exhibited lower clarity than the cassava starch pastes. within the cassava cultivars, paste from lcn 8010, 83350, 81/0015 and mkondezi starches had the highest clarity while maunjili starch had the lowest. amylose content is known to influence the clarity of starch pastes as lower amylose starches are easily dispersed, increasing transmittance and clarity. however our results are contrary to this observation. cocoyam starch which showed the lowest clarity had comparatively lower amylose content (table1). on the other hand, 83350 and 81/00015 starches which showed the highest paste clarity had higher amylose content except for maunjili which had both lower paste clarity and amylose content. correlation analysis showed a significant positive corre-lation of paste clarity with amylose content and granule (table 4). these results suggest that paste clarity was influenced by so many factors, not only amylose to amylopectin ratio (craig et al., 1989). usually processed foods are subjected to storage by refrigeration and during storage, retrogradation process occurs. thus unaltered transparency during storage is a much desirable characteristic of starches used in food products (schmitz et al., 2006). this functionality depends on the retrogradation characteristics of the starch. retrogradation is a process where molecules comprising gelatinized starch reassociate into an ordered structure to retrieve a crystalline order, and during this process, the starch paste becomes increasingly more opaque (thomas and atwell, 1999). for the ten days of refrigerated storage applied, opacity of the cocoyam starch gel significantly increased compared with cassava starches indicating higher retrogradation tendency during storage (figure 3). s y n e re s is ( % ) 83350 81/0015 ch92/082 cocoyam lcn8010 maunjili mbundumali mkondezi sauti silira tms4(2)1425 60 50 40 30 20 10 0 1 2 3 4 cycle figure 2. syneresis (%) during freeze-thawing cycles of the starches. 83350 81/0015 ch92/082 cocoyam lcn8010 maunjili mbundumali mkondezi sauti silira tms4(2)1425 t ra n s m it ta n c e ( % ) 55 45 35 25 15 5 1 2 3 4 5 6 7 8 10 day figure 3. effect of storage on gel light absorbance of some of the starches at 650nm. swelling power and solubility patterns solubility patterns of all starches at different temperatures are shown in figure 4. solubility increased as the tempe rature increased from 60 to 90c. generally cassava starches showed higher solubility at all temperatures compared to cocoyam starch. silira and sauti varieties showed significantly higher solubility than the other varie ties at 70, 80 and 90c however no cassava varieties showed consistently lower solubility at all temperatures. swelling power of all starches showed similar pattern as the solubility (figure 5) and source had significant effect mweta et al. 276 277 afr. j. food sci. res. 60°c 70°c 80°c 90°c s o lu b il ty ( g 1 0 0 g -1 d ry s a m p le ) 35 30 25 20 15 10 5 0 8 3 3 5 0 8 1 /0 0 1 5 c h 9 2 /0 8 2 c o co ya m l c n 8 0 1 0 m a u n jil i m b u n d u m a li m ko n d e zi s a u ti s ili ra t m s 4 (2 )1 4 2 5 starch figure 4. solubility patterns of the starches 60°c 70°c 80°c 90°c s w e ll in g p o w e r (g g -1 d ry s a m p le ) 50 45 40 35 30 25 20 15 10 5 0 8 3 3 5 0 8 1 /0 0 1 5 c h 9 2 /0 8 2 c o c o y a m l c n 8 0 1 0 m a u n jil i m b u n d u m a li m k o n d e z i s a u ti s ili ra t m s 4 (2 )1 4 2 5 starch figure 5. swelling profiles of the starches. on swelling power. cocoyam starch exhibited lowest swelling power at all temperatures. maunjili, 81/00015, sauti, lcn8010 and sauti starches showed consistently higher swelling power than other cassava varieties while tms4(2)1425 starch exhibited lowest swelling power. thus botanical source variety had significant effect on swelling power and solubility of the starches under study. the differences in solubility and swelling behavior indicate differences in the structure of starches. differences in solubility could be attributed to different chain length distributions in the starches (bello-pérez et al., 2000). mweta et al. 278 table 2.thermal properties of the native starches to (c) tp (c) tc (c) hg (j/g) r (c) phi cocoyam 59.88 cde 70.03 a 83.48 ab 11.43 c 20.31 a 1.12 e 81/0015 59.22 de 63.78 d 72.30 d 12.24 bc 9.12 f 2.68 b 83350 58.56 eg 66.74 bc 82.00 abc 12.74 abc 16.36 b 1.56 de ch92/082 61.96 ab 66.96 bc 79.94 abc 11.41 c 9.99 ef 2.28 bc lcn8010 57.63 f 63.77 d 76.32 cd 14.10 ab 12.28 de 2.30 bc maunjili 58.68 ef 62.84 d 85.08 a 13.73 abc 8.31 f 3.30 a mbundumali 62.48 a 67.41 b 79.70 abc 13.54 abc 9.86 f 2.76 ab mkondezi 60.80 bc 65.79 c 76.48 cd 13.63 abc 9.98 ef 2.74 ab sauti 59.46 cde 65.62 c 77.16 bcd 12.63 bc 12.32 de 2.05 cd silira 59.35 de 66.73 bc 80.08 abc 15.04 a 14.76 bc 2.04 cd tms4(2)1425 60.14 cd 66.68 bc 76.32 cd 11.77 bc 13.08 cd 1.80 cd nb: means followed by the same letter within the same row are not significantly different from each other (p = 0.05). table 3.thermal properties of the retrograded starches to (c) tp (c) tc (c) hg j/g) r (c) phi % retrograd cocoyam 50.81 a 61.01 a 72.10 7.04 a 20.40 0.70 a 61.82 a 81/0015 48.71 bcd 57.44 b 65.10 4.44 b 17.48 0.51 bc 36.25 bcd 83350 48.67 bcd 58.24 ab 66.25 5.00 b 19.14 0.52 bc 39.25 bcd ch92/082 50.01 ab 57.88 ab 64.40 4.63 b 15.75 0.60 ab 40.88 bc lcn8010 48.74 bcd 58.26 ab 66.17 5.17 b 19.04 0.54 bc 36.67 bcd maunjili 48.01 cd 57.66 ab 67.24 4.86 b 19.29 0.50 bc 35.66 bcd mbundumali 47.54 d 56.66 b 63.85 4.24 b 18.22 0.46 bcd 31.28 cd mkondezi 47.71 d 57.10 b 64.73 4.08 b 18.78 0.44 cd 29.89 d sauti 49.60 abc 58.80 ab 64.05 4.10 b 18.28 0.45 bcd 32.46 bcd silira 47.86 cd 56.40 b 62.64 2.83 c 17.09 0.33 d 18.90 e tms4(2)1425 49.72 abc 58.60 ab 65.76 4.97 b 17.76 0.56 abc 42.40 b nb: means followed by the same letter within the same column are not significantly different from each other (p = 0.05) swelling power of starch depends on the capacity of starch molecules to hold water through hydrogen bonding. after gelatinization these hydrogen bonds between starch molecules are broken and are replaced by hydrogen bonds with water. amylose content and proportion of outside-chains of amylopectin are thought to be major players in the water retention capacity of gel (tang et al., 2005). positive and negative significant correlations have been reported between swelling power with amylopectin unit-chain ratio and amylose content respectively (srichuwong et al., 2005; sasaki and masuki, 1998). in this study swelling power had a positive significant correlation with amylose content (table 4). gelatinization and retrogradation thickening and swelling are desired functionalities governing the use of starch in food industries as thickening agent in food products such as sauce. these two functionalities depend on gelatinization characteristics of starches (karim et al., 2007) . starch gelatinization refers to the collapse of molecular order within the starch granule manifested in irreversible changes in properties as granular swelling, native crystalline melting, loss of birefringence and starch solubilization (thomas and atwell, 1999). transition tem-peratures (to, tp, and tc) and hg for the gelatinized and retrograded starches are presented in tables 2 and 3. thermal properties varied significantly (p < 0.001) among the starches. cocoyam starch exhibited signify-cantly higher tp, and tc values than the cassava starches however to was within the same range of cassava starches. mbundumali exhibited high t o, tp, and tc values among the cassava varieties while lcn8010 and 81/00015 consistently showed lower to, tp and tc values. hg was significantly lower for cocoyam starch compared to cassava starches but similar to that of ch92/082 va-riety. silira and ch92/082 exhibited the highest and low est hg respectively. phi is the ratio of enthalpy of gelatinization to the temperature range and measures unifor 279 afr. j. food sci. res. table 4. correlations of the starch properties amy gs paste to tp tc hg range phi hr dr syn sp s gs 0.2247 paste 0.552 * 0.509 * to -0.029 0.312 -0.219 tp -0.307 -0.298 -0.406 * 0.570 * tc 0.401 * -0.178 -0.522 * 0.284 0.415 * hg -0.467 * 0.324 0.142 -0.022 -0.246 0.198 range -0.327 -0.647 ** -0.258 0.308 0.606 * 0.205 -0.254 phi 0.026 0.616 ** 0.117 0.255 -0.563 * 0.030 0.560 * -0.899 ** hr 0.120 -0.203 -0.325 -0.049 0.333 0.258 -0.411 * 0.431 * -0.375 dr 0.056 -0.363 -0.364 -0.032 0.400 * 0.142 -0.738 ** 0.494 * -0.568 * 0.908 ** syn 0.759 ** -0.143 -0.328 0.034 0.470 * 0.568 * 0.360 0.512 * -0.215 0.420 * 0.201 sp 0.444 * 0.229 0.455 * -0.452 * -0.818 ** -0.483 * 0.263 -0.499 * 0.500 * -0.361 -0.430 * -0.507 * s -0.213 -0.087 0.109 -0.076 -0.160 -0.162 0.507 * -0.112 0.165 -0.735 ** -0.733 ** -0.081 0.104 p -0.029 -0.320 -0.438 * 0.048 0.363 -0.065 -0.634 ** 0.374 * -0.510 * 0.448 * 0.618 ** -0.121 -0.490 * 0.018 *= p<0.05 ;**= p<0.001 amy = amylose content; gs = granule size; to = onset temperature; tp = peak temperature; tc = conclusion temperature; hg = enthalpy of gelatinization; range = temperature range; phi = peak height index; hr = enthalpy of retrogradation; dr = degree of retrogradation; syn = syneresis; sp = swelling power (80c); s = solubility at 80c; p = phosphorus mity in gelatinization (sandhu et al., 2005). average phi values ranged from 1.12 to 3.30. r values ranged from 8.31 to 20.31c. cocoyam had the highest r values compared to cassava starches. among cassava cultivars maunjili and 81/0015 cassava varieties had the lowest r values while 83350 had the highest. the differences in gelatinization properties of starches are attributed to factors such as starch composition (amylose to amylopectin ratio, amount of lipid complexed, amylose chains, phosphorus content), molecular structure of amylopectin (unit chain and extent of branching), and granular architecture (crystalline to amorphous ratio) (gunaratne and hoover, 2002). gelatinization enthalpy was significantly negatively correlated with amylose and phosphorus contents of the starches (table 4). gelatinization temperature is considered reflection of the degree of orderly arrangement of molecules in the starch granule. to is influenced by short amylopectin branch-chains and low gelatinization temperatures are characteristic of starches with larger proportions of short amylopectin branch chains (jane et al., 1999). tp is an indication granular architecture (crystalline quality) and high peak temperature might be due to higher proportion of longer chains in the amylopectins as these require higher temperatures to dissociate completely than required for shorter double helices (karim et al., 2007). higher peak temperature for cocoyam starch compared to cassava starches suggests the presence of higher proportions of long amylopectin chains in cocoyam starch. thus among cassava starches, mbundumali could have higher proportion of long-chain amylopectins than the other varieties. retrograded starches showed lower gelatinization temperatures (to, tp, and t c) and smaller enthalpy than raw starches (tables 2 and 3) indicating weaker starch crystallinity (sasaki, 2005). to, tp, and phi values for retrograded cocoyam starch were significantly (p<0.05) higher than for cassava starches while tc and r values were similar for all retrograded starches. enthalpy and degree of retrogradation for cocoyam starch were significantly (p < 0.001) higher for cocoyam starch compared to the cassava starches suggesting rapid retrogradation for cocoyam starches. retrogradation properties of starch paste are often related to structures of amylose and amylopectin. higher retrogradation tendencies are attributed to crystallization involving small amylose molecules and long chain amylopectin (peroni et al., 2006). our results therefore suggest that cocoyam starch contain amylose and amylopectin molecules with such characteristics. among the cassava varieties, mbundumali, mkondezi and silira exhibited lower thermal properties of the retrograded starches compared to other cassava varieties. conclusion results of this study have revealed differences in chemical compositions and functional properties of the starches with crop and within varieties of the crop. cocoyam showed small to medium polyhedral starch granules while cassava starches showed small to large rounded, irregular with oval and truncated ellipsoidal-granules. cocoyam starch had lower amylose content but higher phosphorus content, maximum wavelength of starchiodine complex and blue value than the cassava starches. cocoyam starch showed lower swelling power and solubility. the gelatinization ranges for cocoyam and cassava starch were similar however cocoyam starch showed higher degree of retrogradation than cassava mweta et al. 280 starches. starch from cocoyam starch was more opaque than cassava starch gels and paste clarity of cocoyam starch decreased significantly with storage. starches from the 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press. p. 4 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (5), pp. 001-008, may, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper extraction of pure lycopene from industrial tomato waste in water using the extractor naviglio ® daniele naviglio1*, fabiana pizzolongo1, lydia ferrara2, biagio naviglio3, alejandro aragòn4, and antonello santini1 1 dipartimento di scienza degli alimenti, università di napoli federico ii, via università 100, 80055 portici (napoli), italy. 2 dipartimento di chimica farmaceutica e tossicologia, università di napoli federico ii, via montesano 49, 80131 napoli, italy. 3 stazione sperimentale per l’industria delle pelli e delle materie concianti, via poggioreale 39, 80143 napoli, italy. 4 facultad de ciencias agrarias y forestales, universidad de la plata, calle 60 y 119, 1900 la plata (buenos aires), argentina. accepted 15 march, 2019 in this paper an innovative process for the extraction of pure lycopene from tomato-waste in water that uses the extractor naviglio ® and water as solvent is presented. the use of water as extracting solvent considerably reduces the cost of the entire process if compared with the commonly used solvent-based procedure or with the newer supercritical extraction process of lycopene from tomato-waste. exhausted tomato-waste treated with water can be then easily dried at room temperature and further used, e.g. in agriculture or as food ingredient in animal nutrition. lycopene, not soluble in water, was recovered in a quasi-crystalline solid form and purified by spe (solid phase extraction) using a small amount of organic solvent. the all trans lycopene was obtained at a very high grade of purity, not less than 98% (w/w), with an average recovery from tomato waste of 14% (w/w). the availability of high purity all trans lycopene allowed us also to measure the molar absorption coefficient, unique for each molecule. an alternative procedure for the hplc analysis, that uses a phenyl-hexyl silicone stationary phase as inverse phase and a linear gradient in water and acetonitrile, is also described. key words: lycopene, extractor naviglio ® , tomato-waste, hplc-diode array, solid-liquid extraction, chromatography, solid phase extraction. introduction lycopene, whose structural formula is reported in figure 1, is a fat soluble carotenoid with 11 conjugated double bonds in the molecule, and it is a precursor of the -carotene with a well known antioxidant activity, reported as at least twice that of the -carotene (sies and stahl, 1998; di mascio et al., 2002). lycopene can be easily degraded by atmospheric oxygen and by light and converted from the all trans to the cis forms, that show a decreased biologic activity (lee and chen, 2002 wang and chen, 2006.). for these reasons it must be stored at a very low temperature ( -70°c) away from the light and from the atmospheric oxygen. in the last years the impor *corresponding author. e-mail: naviglio@unina.it. tel./fax: +39 81 2539348. tance of lycopene rapidly increased due to its pharmacological and anti carcinogenic properties (franceschi et al., 1994; giovannucci, 1999; livny et al., 2002) and antioxidant activity (di mascio et al., 2002; heber and lu, 2002; sies and stahl, 1998). epidemiological studies indicate a protective effects of lycopene against some types of cancer, e.g., stomach and prostate cancer (stacewicz – sapuntzakis and bowen, 2005; stahl and sies, 1996), so the demand for this molecule and for the carotenoids in general, is rapidly growing (ulrich, 2000). in the literature, different synthetic pathways for this molecule have been reported, but in all cases the synthesis of lycopene seems to be a very expensive and economically not convenient procedure (zhuo-cai et al., 2006). on the other hand, good amounts of lycopene are contained in many natural products, like tomato (lycopersicon esculentum mill.), water-melon, red pepper, papaya etc. products from tomato industry, the relevant interest for this molecule and its biological activity, and starting from the consideration that better methods for the characterization and determination of lycopene are needed (roldán-gutiérrez and luque de castro, 2007), the main aim of this work is to propose a new method for the extraction and characterization of lycopene from tomato wastes. the extraction procedure was realized using the naviglio extractor (naviglio, 2003) that uses a new solidliquid extraction technology (naviglio, 2000), and tap water as extracting solvent followed by a simple and rapid purification step, that requires a minimum quantity of organic solvent. figure 1. structural formula of lycopene. (britton, 1991) this molecule is also responsible for the red intense colour of these vege-tables, and a lycopene content in the range between 5.40-1500 mg/kg in tomato paste (wet weight) (roldán-gutiérrez and luque de castro, 2007) and between 20-30 mg/kg in tomato peels as raw material (rozzi et al., 2002) were determined, respectively. it is worth noting that conventional methods for the extraction of lycopene from natural vegetal origin sources use great quantities of organic solvents and the extraction procedure is usually followed by a complex purification step (olives barba et al., 2005 sadler et al., 1990.). in the case of lycopene recovery from the tomatowaste, the extraction can also be conducted using carbon dioxide in supercritical state (rozzi et al., 2002), and this method allows to recover a greater quantity of carotenoids even if partly mixed in an oleoresin containing around 6% (w/w) in lycopene (vági et al., 2007). this method, unfortunately, is actually quite expensive, making the entire procedure economically not suitable for a large scale process. biotechnological production of lycopene using microorganisms (sandmann, 1994), has been proposed too, but always at the bench scale and, in most cases, with a weak specific yield, even if recently a new approach that can be scaled-up to an industrial application has been proposed (lopez-nieto et al., 2004). since tomato by-products quantities deriving from industrial processes are growing annually, e.g. in europe it is evaluated that about ten million tons of tomatoes are processed by the food industry, the deriving wastes can be quantified in approximately 0.1 million tons, and represent an interesting low cost source of lycopene. italy is among the main producers of tomatoes in europe and the by-products and the residues of the industrial processing of tomatoes that can be reincorporated in low quality tomato products or used as an ingredient in animal food, could be more efficiently used as a source of pure lycopene. considering both the availability of the by experimental reagents acetone, methanol, acetonitrile, chloroform, dichloromethane, nhexane (all solvents were of analytical grade from fluka, bucks, switzerland); de-ionized water of hplc grade was produced by means of milli-q (millipore); lycopene standard was purchased from sigma aldrich; extractor naviglio ® mod. 500 cc (nuova estrazione s.a.s., naples, italy); spectrophotometer uv 1601 (shimadzu corp., tokyo, japan); hplc-diode array, mod. 1100 (agilent technologies, santa clara, ca, usa); cartriges octadecyl solid phase extraction (spe-c18) 10 g (restek corp., bellefonte, pa, usa); filters 0.20 micron (millipore, bedford, ma, usa); phenylhexyl silicone hplc column 250x4.6 mm, 5 micron particle size (phenomenex, torrance, ca, usa). brief description of the naviglio extractor ® in this work the naviglio extractor ® shown in figure 2 and described elsewhere (naviglio, 2003) was used. this device is a rapid and dynamic solid-liquid extractor that applies the principle that in a suitable solvent, generating a negative pressure gradient and letting it to go to equilibrium between outside and inside of a solid matrix, that contains compounds that can be extracted in the solvent followed by a rapid equilibrium condition restoring, a forced extrac-tion of the not chemically bound compounds contained in the solid matrix is produced (naviglio’s principle). the naviglio extractor ® can operate at room temperature or at sub-ambient temperature, and it works applying a pressure increase on the surface of the liquid phase containing the solid material (matrix) to be extracted. the use of low or room temperature greatly reduces the thermal stress for any heat susceptible substances present in the matrix, e.g. lycopene that can be degraded when using high temperature for the extraction (lee and chen, 2002). the device used consists of one extracting chamber equipped with a cylinder and a piston where, at the bottom, one porous set let the liquid phase and liquid soluble substances pass through, while the solid particles are blocked. the solid raw material is put in the chamber that is then filled with the solvent (organic, inorganic or a mixture of solvents). pressure gradient is applied allowing the system to reach equilibrium (static phase) at a pressure of about 8 atm. when the piston is moved from its equilibrium position, the dynamic phase starts; this step is performed for five times and for a brief period of time with aim of remixing the solutions and to allow the diffusion of the extracted compounds. the movement of the piston and hence the static and dynamic steps alternate till the figure 2. naviglio extractor ® , mod. 500 cc. (depurex88, padua, italy). extraction process was efficiently completed. one extraction cycle is formed by one static and one dynamic step; repeating more times these operations, complete exhausting of the solid matrix can be obtained. when the maximum value set for the pressure is reached, the device stops for 2 min, allowing the outside and inside part of the solid matrix to equilibrate (static phase) with the solvent pressure. immediately after this step, the piston moves, the air quickly leaves the pneumatic chambers and the system causing a lowering of the pressure inside the extraction chamber. this induces the starting of the dynamic phase and at the beginning of this step the substances that are soluble in the used solvent and substances not chemically bonded to the matrix are extracted from the solid matrix and transferred to the solvent. extractor naviglio was recently employed in the innovative production of lemon liquor (naviglio et al., 2007). chemical extraction of by-products of tomato to compare the recovery of carotenoids in the starting material obtained using the naviglio extractor with those obtained with a conventional chemical solvent based extraction procedure, chemical extraction of lycopene from tomato by product was performed as reported by others (rozzi et al., 2002). a 2 g sample of tomato byproducts was placed in an extraction tube; 20 ml of chloroform were added and the tube was treated with ultra sounds for 30 min. the sample was then centrifuged for 15 min at 2000 rpm, and an aliquot was analysed by high-performance liquid chromatography (hplc) to determine all trans lycopene. the extraction procedure was repeated to recover residual lycopene in the sample. exhaustive extraction of tomato seeds and skins with additional volumes of chloroform did not result in additional recovery of lycopene. extraction by means of extractor naviglio ® ten samples each constituted of 100 grams of tomato peels and by-products from different farms in the naples area, were placed in a bag made of 50 µm filtering membrane and transferred into the chamber of the extractor naviglio mod. 500 cc. where 500 ml of tap water was added. extraction parameters used are the following: the total extraction time was set to four hours for a total of 60 cycles. the dynamic phase used 5 cycles (piston was idle for 12 s in the up and down positions) and for the static phase a 2 min time was used. after extraction the bag was removed and strongly pressed in order to completely recover the water (about 450 ml). the aqueous extract was loaded on octadecyl solid phase extraction (spe-c18) 10 g column vacuum packed; column was washed with methanol, that does not dissolve lycopene, in order to remove pigments and then the minimum quantity of acetone was used to recover lycopene. the acetone extract was analysed by spectrophotometric and hplc analysis to confirm the presence of lycopene. to verify if the use of tap water could affect the extraction, the same extraction procedure was repeated using deionised water (see table 1). spectrophotometric analysis extracts were filtered through 0.20 micron membrane (millipore, bedford, ma, usa) and anhydrified on sodium sulphate anhydrous in order to eliminate water traces and rough impurities and analysed by spectrophotometer in the 350-550 nm wavelength range. the spectrophotometric analysis allowed to identify the all trans lycopene molecule, by comparing the spectra of absorbance and the absolute and relatives maxima peak of absorbance with spectra of absorbance and maxima of absorbance exhibited by lycopene standard and the relative values reported in literature. hplc analysis in order to better verify the lycopene purity, filtered extracts were analysed by high-performance liquid chromatography (hplc) system equipped with diode array detector mod. 1100 (agilent technologies, waldbronn, germany) . a phenyl-hexyl silicon column (250 x 4.6 mm i.d. 5 micron particle size) (phenomenex, torrance, ca, usa) was used for the separation. the flow rate was 1 ml/min and the gradient water-acetonitrile was: time 0-3 min, acetonitrile 0%; time, 20 min, acetonitrile 100%; hold for 5 min. lycopene standard was purchased from sigma-aldrich (milan, italy). calibration curve an amount of lycopene extract was dried under a nitrogen current. a stock solution of the extracted lycopene was prepared by dissolving 10.0 mg of lycopene in 100 ml of dichloromethane. from this standard solution by diluting 5 solutions containing from 1 to 5 ppm were prepared. the same procedure was used to prepare the stock solution and the diluted solutions from the purchased standard of lycopene. in this case 1.0 mg of lycopene was used. correlation coefficient of calibration line was 0.998. in the same conditions a calibration line was prepared for the lycopene standard and in this case the correlation coefficient was 0.996 and angular coefficient was 5.8% lower than the previous one because its lower purity. the observed linear response for lycopene was comprised in table 1. lycopene recovery from tomato by products using tap and demineralized water at different values of the extraction time. lycopene content (mg/kg) demineralized water tap water tap water tap water tap water sample (4 h) (4 h) (2 h) (6 h) (8 h) 1 2.3 3.2 1.5 2.6 2.5 2 3.4 3.0 1.9 2.8 3.2 3 2.7 3.5 1.1 2.5 3.3 4 1.9 1.5 0.9 1.4 1.6 5 2.8 3.9 2.1 2.9 3.5 6 1.7 1.7 1.2 1.8 1.8 7 2.9 2.5 1.8 2.4 2.6 8 3.9 3.6 1.8 2.9 3.5 9 2.2 2.0 1.4 1.7 1.9 10 2.6 2.9 1.5 2.0 2.8 table 2. recovered lycopene (a) and lycopene standard (b) maxima wavelenght of absorbance in four different solvents and specific coefficient of absorption and molar extinction values. solvent 1 (nm)  2 (nm) 3 (nm) e 1% (l/g . cm)  (l/mol*cm) n-hexane 444.50.5 470.50.5 502.00.5 310530 1667741610 acetone 448.50.5 473.50.5 505.00.5 285530 1532401610 dichloromethane 456.00.5 482.00.5 515.00.5 254030 1363081610 chloroform 457.50.5 484.00.5 517.00.5 285030 1529891610 (a) solvent 1 (nm)  2 (nm) 3 (nm) e 1% (l/g . cm)  (l/mol*cm) n-hexane 445.50.5 471.00.5 502.00.5 314050 1685712684 acetone 449.50.5 474.00.5 505.50.5 280350 1504792684 dichloromethane 455.00.5 482.00.5 516.00.5 248050 1331392684 chloroform 456.50.5 485.00.5 518.00.5 281050 1508552684 (b) the range 0-100 ppm. determination of molar absorption and specific extinction coefficient of lycopene in different solvents four solutions of lycopene extracted with the extractor naviglio were prepared in n-hexane, chloroform, dichloromethane and acetone, at a concentration of 1.00 mg/l starting from the previously prepared stock solution (100 ppm) . table 2 reports the maximum value for absorbance measured at a wavelength between 350-550 nm for each solution, the relative molar absorption coefficient and specific extinction coefficient. results and discussion use of the extractor naviglio ® for the recovery of lycopene from tomato waste the naviglio extractor was used to recover a solid lycopene fraction in a para-crystalline form from tomatowaste in tap water, using the pressure and depressure effect that operate in the solid-liquid extractor device. in this case the extraction is allowed because of the effect of depression generated in the extractor and lycopene is figure 3. visible spectra of licopene in dichloromethane (1), acetone (2), n-hexane (3) and chloroform (4). found in heterogeneous phase. for this reason during the following purification on spe-c18, all-trans lycopene present in the solid form, was separated on the filter column, and a little fraction of the other pigments present in the starting material were retained on the column head of the octadecylsilicone stationary phase. pigments were removed by washing with methanol, solvent in which lycopene is totally not soluble, while washing with acetone allowed the lycopene, very soluble in this solvent, to be completely separated. purity of extracted lycopene was checked by hplc showing the presence of one major peak corresponding to the all-trans lycopene, while its quantification was possible through the calibration curve. table 1 reports the recovery obtained for the lycopene using deionized and tap water, respectively. as it can be observed, there is no significant difference between the two liquids, suggesting that it is preferable to use tap water to keep low the cost of the entire industrial process. optimum extraction time was 4 h and a recovery between 1.5 and 3.9 mg/kg of lycopene was obtained; it can also be observed that continuing the extraction beyond this time period, recoveries did not increase and degradation events could occur, suggesting that 4 h is the optimum extraction time. qualitative and quantitative analysis of lycopene the elution of lycopene from the c18-spe can be made directly using acetone or washing the column with methanol first ad then eluting with acetone. in the first case, due to the contemporary presence of other carotenoids like carotene and lutein, lycopene at not elevated purity grade was obtained while washing the column with methanol first and allowed to obtain lycopene at a higher purity. at this point, it is worth noting that the washing of lycopene is made after the extractive procedure and the quantity of solvent employed is minimal; however the great quantity of by products are only touched with water and do not come in contact with solvent. lycopene purity was investigated byspectrophotometric uv-vis and hplc analysis, allowing to observe the presence of one peak corresponding to the all trans pure lycopene. figure 3 reports figure 4. hplc chromatograms of extracted lycopene at three different wavelengths, 413.4, 474.4, 506.4 (lower part), and dad acquisition between 200 and 600 nm of the peak measured at a retention time of 16.363 min. (upper part). visible spectra of lycopene in dichloromethane, acetone, n–hexane and chloroform respectively. the comparison of the maxima with those observed for the lycopene standard shows an excellent agreement (tables 2a and 2b) and is consistent with data reported in the literature (davis, 1949; roldàn-gutiérrez and luque de castro, 2007). figure 4 reports hplc results at three maxima wavelengths 413.4, 474.4 and 506.4 nm. one minor component, an impurity, was detected before the elution of lycopene peak and it was identified as beta-carotene. the impurity did not exceed the 2% of total signal intensity, allowing us to conclude that lycopene is at least of 98% purity (at  = 474.4 nm). the dad acquisition confirmed that no other contaminant was present in the range of 200-600 nm. chromatographic conditions reported in this paper are different from the ones used in literature; in this case a phenyl-hexyl silicone as stationary phase and a simple water-acetonitrile gradient were used and for this reason the method here reported could be proposed as a valid alternative to the ones reported in literature (brumann and grimme, 1981; olives barba et al., 2005). this procedure allows us to reduce times of analysis in the case of a high purity lycopene containing solutions for which it is not necessary the use of complex gradients of solvent for the elution. chemical extraction on the ten samples of raw materials gave an average value of 20.3 mg/kg. table 1 reports recovery from tomato by products using tap water and demineralized water at different values of the extraction time. at the optimum extraction time (4 h) the recovery in tap water was comprised between 1.5 and 3.9 mg/kg, corresponding to a percentage recovery of 7.5 and 19.5% (w/w), respectively, compared with chemical extraction. in demineralised water, the recovery ranged in the same conditions, from 1.7 and 3.9 mg/kg, corresponding to a recovery percentage of 8.5 and 19.5% (w/w), respectively. the possibility of using tap water as extracting liquid notably reduces the costs of industrial processes. the efficiency of the proposed process is lower than the other procedures proposed in the literature, because of minor quantity of lycopene present in para-crystalline form in tomato waste. but when considering the cost of the tomato waste and the easy dumping of the exhausted material, it turns to be cost reducing since no organic solvent is used in the extractive phase, moreover a higher grade pure lycopene (>98% (w/w)) is achieved in the extraction using the extractor naviglio to be compared with the chemical extraction procedure that allowed a 20-30 mg/kg recovery at a 10-20 % (w/w) purity. finally, the exhausted material can be dried at room temperature and the weight reduces about 95% (w/w). in this way it can be employed as cattle feed or as a manure in agriculture. determination of molar absorption coefficient and relative specific coefficient of absorption the possibility of isolating pure lycopene from tomato wastes gave us the possibility to measure the molar absorption coefficient () and the relative specific coefficient of absorption (e 1% ) in some of the most commonly used solvents, namely dichloromethane, acetone, nhexane and chloroform. table 2 reports the obtained values for  and e 1% . in the literature different values of  and e 1% for lycopene (davis, 1949; davis et al., 2003; roldàn-gutiérrez and luque de castro, 2007) are reported, and the difficulty of obtaining accurate values principally derives from the lack of a technique to obtaining all-trans licopene at high purity. moreover spectrum of absorbance and molar absorption coefficient, unique for each molecule, allows the unique identification of the lycopene and can be used to confirm the presence of alltrans lycopene. the high value of the measured molar absorption coefficient can be correlated to the high number of double bonds and the high conjugation of double bonds present in the lycopene molecule, that is one of the compounds with the highest molar absorption coefficient value. conclusions in this work an extractive process is described that could be easily scaled-up to become an industrial application for the production of very high grade of purity (98%) alltrans lycopene, whose demand is constantly increasing. the average recovery of lycopene from tomato byproducts obtained using extractor naviglio ® was of 14% (w/w) in respect of chemical extraction at a purity of 98% at the optimum extraction time of 4 h. this result could be further improved using a different value for the pressure applied in the extraction process. the described procedure uses tap water to extract lycopene from tomato byproducts and a new pressured extraction technology. considering that the costs are lower if compared with the cost of a conventional solvent or supercritical fluid phase extraction procedure, and since no special by-product to be wasted are produced, the overall process appears to be economically convenient. the material obtained after the extraction of the lycopene e.g. the exhausted tomatowaste residues can be easily dried and used in agriculture or as feeding for cattle or animals after the recovery of a high economical value molecule like lycopene. compared to the use of water as extracting liquid, the existing procedures to extract lycopene from vegetal sources that use organic solvents present some disadvantages: (i) organic solvents are generally toxic, so they have to be completely removed from exhausted material making necessary to use a complex purification step before dumping it; (ii) exhausted matrixes must be dumped as a special residue after extraction and they cannot be reused; (iii) organic solvents are not specific for lycopene, but at the same time other pigments or hydrophobic compounds (e.g. carotenes, xanthophylls, fat, etc.) present in the starting material are simultaneously extracted. moreover, the purification of lycopene is obtained using hplc on inverse phase, the most used technique, is particularly difficult and time consuming, since long time gradient and ternary or quaternary solvent gradients must be used (brumann and grimme, 1981) due to the co-presence of unwanted compounds in the organic extract. finally, the high purity of the lycopene obtained with use of the extractor naviglio makes the industrial process very appealing. the pure all-trans lycopene extracted makes it possible to use it, at a known dosage, as a drug and not only as a food integrator. references britton g (1991). carotenoids: in methods in plant biochemistry. london: academy press. 7: 473-518. brumann t, grimme lh. (1981). reversed phase high performance liquid chromatography of chlorophylls and carotenoids. biochem. biophys. acta. 637: 8-17. davis ar, fish ww, perkins-veazie p (2003). a rapid spectrophotometric method for analyzing lycopene content in tomato and tomato products. postharvest biol. technol., 28: 425-430. davis wb (1949). preparation of lycopene from tomato paste for use as a spectrophotometric standard. anal. chem. 21(10): 1226-1228. di mascio p, kaiser sp, sies h (2002). lycopene as the most efficient biological carotenoid singlet oxigen quencher. arch. biochem. biophys. 274: 179-185. ferrara l, montesano d, naviglio d (2003). estrazione rapida del licopene dai pomodori. ingredienti alimentari. 2(1): 6-10. franceschi s, bidoli e, la vecchia c, talamini r, d’avanzo b, negri e (1994). tomatoes and risk of digestive-tract cancers. int. j. cancer 59: 181-184. giovannucci e (1999). tomatoes, tomato-based products, lycopene and cancer: review of the epidemiological literature. j. natl. cancer inst. 91: 317-331. heber d, lu qy (2002). overview of mechanism of action of lycopene. exp. biol. med. 227: 920-923. lee mt, chen bh (2002). stability of lycopene during heating and illumination in a model system. food chem. 78: 425-432. livny o, kaplan i, reifen r, polak-charcon s, madar z, schwartz b (2002). lycopene inhibits proliferation and enhances gap-junction communication of kb-1 human oral tumor cells. j. nutr. 132: 37543759. lópez-nieto mj, costa j, peiro e, méndez e, rodríguez-sáiz m, de la fuente jl, cabri w, barredo jl (2004). biotechnological lycopene production by mated fermentation of blakeslea trispora. appl. microbiol. biotechnol. 66: 153-159. naviglio d (2000). rapid and dynamic solid-liquid extractor working at high pressures and low temperatures for obtaining in short times solutions containing substances that initially were in solid matrixes insoluble in extracting liquid. italian patent 1,303,417. naviglio d (2003). naviglio’s principle and presentation of an innovative solid-liquid extraction technology: extractor naviglio . analytical letters. 36(8): 1647-1659. naviglio d, pizzolongo f, romano r, ferrara l, naviglio b, santini a (2007). an innovative solid-liquid extraction technology: use of the naviglio extractor for the production of lemon liquor. afr. j. food sci. 1: 42-50. obermullet-jevic uc (2003). lycopene inhibits the growth of normal human prostate epithelial cells in vitro. j. nutr. 133: 3356-3360. olives barba ai, cámara hurtado m, sanchez mata mc, fernandez ruiz v, saenz de tejada m, lopez sd (2005). application of a uvvis detection-hplc method for a rapid determination of lycopene and carotene in vegetables. food chem. 95(2): 328-336. roldàn-gutiérrez jm, luque de castro md (2007). lycopene: the need for better methods for characterization and determination. trends anal. chem. 26(2): 163-170. rozzi nl, singh rk, vierling ra, watkins ba (2002). supercritical fluid extraction of lycopene from tomato processing byproducts. j. agric. food chem. 50: 2638-3643. sadler g, davis j, deyman d (1990). rapid extraction of lycopene and beta-carotene from reconstituted tomato paste and pink grapefruit homogenates. j. food sci. 55: 1460-1465. sandmann g (1994). carotenoid biosysnthesis in microorganism and plants. eur. j. biochem. 223: 7-24. sies h, stahl w (1998). lycopene: antioxidant and biological effects and its bioavailability in the human. proc. exp. biol. med. 218: 121124. stacewicz-sapuntzakis m, bowen pe (2005). role of lycopene and tomato products in prostate health. biochim. biophys. acta. 1740: 202-205. sthal w, sies h (1996). lycopene: a biologically important carotenoid for humans? arch. biochem. biophys. 336: 1-9. ulrich m (2000). business report: the global market for carotenoids. business communication. norwalk: england. vági e, simándi b, vásárhelyine kp, daood h, kéry á, doleschall f, nagy b (2007). supercritical carbon dioxide extraction of carotenoids, tocopherols and sitosterols from industrial tomato by-products. j. supercritical fluids. 40(2): 218-226. wang cy, chen bh (2006). tomato pulp as source for the production of lycopene powder containing high proportion of cis-isomers. eur. food res. technol. 222: 347-353. zhuo-cai l, bo l, hong y, zhi-rong c (2006). advances in research for chemical synthesis of lycopene. hecheng huaxue. 14(2): 118-121. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 9 (4), pp. 001-012, april, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a study of intraspecific hybrid lines derived from the reciprocal crosses between wild accessions and cultivated cowpeas (vigna unguiculata (l.) walp.) b. lelou1,2* m. diatewa2 and p. van damme1 1 laboratory of subtropical and tropical agriculture and ethnobotany, faculty of bioscience and engineering, ghent university, coupure links 653, be 9000 gent, belgium. 2 faculté des sciences de la santé; institut de développement rural, université marien ngouabi, b.p. 69, congo-brazzaville. accepted 08 july, 2020 cowpea (vigna unguiculata (l.) walp.) is an essential food legume in the (sub) tropical areas. reciprocal crosses were performed using wild accessions for transferring genes of interest to cultivars (524-b/it84s-2049). of these, a low number of seeds were obtained because of crossing barriers. a seed sample of 17 f1 hybrids germinated after germination was 69.09%. a subdivision into seed sterility, partial seed fertility and seed fertility was observed. seed sterility was possibly due to chromosomal disturbances that occur in endosperms and embryos during early seed development. in partial seed fertility, vigorous plants flowered about 128 dae but no pods were formed because of floral abscission at anthesis. in plants of (524-b (♀) × tenuis (♀)) combination, partial sterility was caused by incomplete male sterility. in 13 f1 hybrids, viability among populations was reduced (67.25%). eleven f1 hybrids were grown in the greenhouse to produce f2 seeds by natural self-pollination. plants were characterised through using cowpea descriptors. variability, in terms of morphological characters in adult f1 plants, was established. differences in vegetative, inflorescence and fruit characters were described after full plant development. the results show that the adult f1 plants appear to be dominant for wild vegetative and inflorescence characters expressed by one or two of the parents in bi-parental and reciprocal crosses, for exception plants derived from crosses in which alba wild forms served as male parent showing the same morphological characteristics as the cultivated female parents, as evidenced by the traits inherited from cultivars. the heterotic status exhibited by emerged plants, supposes that the latter types inbred at the first generation confirm that the wild parents involved in these hybrids represent a wide diversity of germplasm. seed characteristic studied in f2 has not shown segregation because of recessive type absence, suggesting that morphological traits should be monitored. key words: reproductive barriers, seed fertility, introgressive hybridisation, wild character inheritance. introduction cowpea (vigna unguiculata (l.) walp.) (2n = 22) is an essential grain legume crops in tropical and subtropical *corresponding author. e-mail: blelou@hotmail.com. tel: +242/04-420-17-77. regions (rachie and roberts, 1974). it provides inexpensive proteins and can be prepared easily in a number of edible forms, such as tender green shoots and leaves, immature pods, and green and dry seeds (fery, 1980; ehlers and hall, 1997). in small-scale agriculture, the major problem is the extremely severe damage (up to 90%) caused by 2 post-flowering insect pests in cowpea, including the legume pod borer maruca testulalis geyer and the complex of pod-sucking bugs, including clavigralla tomentosicollis sthal, anoplecnemis curvipes f. and riptortus dentipesf. (ehlers and hall, 1997; olufajo and singh, 2003). wild taxa of v. unguiculata identified within the primary gene pool are known as a potential source of resistance to major insect pests (padulosi and ng, 1990). reciprocal crosses were performed between wild accessions and cultivated cowpeas for transferring postflowering insect pest resistance to cultivars (524-b/it84s2049). according to the total number of 487 flowers that were pollinated during the hybridisation programme in 1999, compatible cross number (that is, mature pod number/total number of reciprocal crosses) produced 78 (16.01%) mature pods in 20 to 25 days after pollination (lelou and van damme, 2006). the number of incompatible crosses was about 391 (80.7%), revealing the presence of reproductive barriers after fertilisation/seed development, as reported by barone and ng (1990); lelou and van damme (2006). viability is assayed using the tests of germination (scott et al., 1998). these present attempts were conduc-ted to estimate seed fertility yielded in f1 hybrid lines derived from intraspecific reciprocal crosses between wild accessions x cultivars in order to identify some hybrid lines with excellent response after the experimental tests in 2000/2001 crop seasons. the incorporation of genes of one species into the gene pool of another is termed introgressive hybridisation (yoshinari, 2007). to illustrate multi-step processes by which genetic materials from wild accessions infiltrate into cowpea cultivars and vice-versa, the characterisation in each of f1 cowpea adult-hybrids was conducted in order to examine the extension of variability range after different morphological character inheritance. the former characters are primitive and the latter are derived types. during hybridisation programme, the derived types are expressed in these hybrids and in order to make favourable genetic material. thus three morphological types (vegetative, inflorescence and fruit characters) were characterised amongst f1 cowpea hybrid lines. from a number of the f1 cowpea hybrid lines that emerged, the expression of characters was variable, indicating that environmental conditions as well as the segregation of minor genes influence penetrance. it is known that characters are not phenotypically identical. it is possible e.g. that leaf texture type can be possessed less advantage in cultivation under greenhouse conditions than it apparently possesses in nature. the objective of this experimental study was, with regard to post-zygotic barriers that build up after successful fertilisation, to determine whether the expression of different genes derived from cultivars and wild accessions grown in the greenhouse could provide an excellent opportunity to study evolutionary processes in all f1 hybrid populations. on the basis of these results, it can be expected to select new cultivars of cowpea by selection for plant resistant to insect-pest flowering and adaptations to variable environments over several generations. materials and methods hybrid seed materials the seed samples of the original f1 cowpea hybrid lines obtained during a hybridisation programme were executed in a tropical greenhouse between wild accessions and cultivated cowpeas during the winter season of 1999 at the laboratory of tropical and subtropical agriculture and ethnobotany, faculty of bioscience and engineering (ghent university, belgium). in table 1, we characterised wild and cultivated parental accessions of cowpea manipulated in this breeding programme. seed germination experiments the seed samples of 17 f1 hybrid lines of cowpea were subjected to laboratory germination tests, as described below during the spring season of 2000 to 2001. in table 2, the different 17 reciprocal f1 hybrid combinations of cowpea, pollination number/hybrid combination, aborted ovule number and their pod and seed production, are reported respectively. these seed samples collected in all f1 hybrid lines of cowpea exhibited seed coat dormancy, as wild parental accessions of cowpea. the methods of seed preparation and sowing were an adaptation of the techniques described by lelou (1997). physical dormancy is caused by water impermeable layers of palisade cells in the seed or fruit coat which control water movement. prior to the conduction of the germination tests, these seeds were treated to break physical dormancy. seeds were surface-sterilised in 70% ethanol for one minute and disinfected by (clo3)2ca (5%) for five minutes, rinsed three times in distilled water. to induce a better permeability, seed coats were mechanically scarified mildly with a scalpel in order to overcome dormancy during germination. to prevent contamination by micro-organisms, these above mentioned seeds treated were immersed for a minute in the fungicide thiram (c6h12n2s4). after treatment, all seed samples were sown and randomly dispersed across moistened filter paper into petri dishes. water was introduced in sufficient and enough quantities into the closed petri dishes and kept in a ‘philips’ incubator: 24°c day and 20°c night temperatures at 12 h photoperiod/day. the results of germination test were recorded daily for 7 days during laboratory experiments. seedling emergence the durations from sowing to emergence of the radicle through the seed coat of seeds treated, were observed among seed samples constituting the cowpea f1 hybrid lines. petri dishes placed into an incubator were checked daily in order to detect: a) radicle elongation, that is, observation of radicle visible through seed testa, when it exceeded 4 or 5 mm out extended cotyledons; and b) seedlings destroyed, that is, seeds ungerminated in which seedlings were regarded to have died when it feel over a most of the seedling turned yellowish or brownish. once radicle definitively emerges, seedling is subsequently potted in small plastic pots (diameter 5 cm) and placed into the growth chamber with same climatic characteristics that the incubator. at the two leave-stages, the young plantlets belonging to adult f1 hybrids of cowpea were individually grown in plastic pots of 3 table 1. origin, biological status and some morphological and pollen male fertility characteristics of parental genotypes of cowpea (vigna unguiculata (l.) walp.) used in our breeding programme conducted under greenhouse at lab. of tropical agriculture in faculty of bioscience (ghent university, belgium) in 1999/2000/2001. parental genotypes and biological biological country plant terminal leaflet flower pod testa plant pollen fertility growth habit leaf texture leaf marking number/ accession number status cycle of origin hairiness shape colour colour texture (%) genotype subsp. burundiensis ni wild allogamous burundi indeterminate glaberscent cariaceous sub-globose absence violet pale tan rough 8 79.0 456 and prostrate subsp. baoulensis wild allogamous cameroon indeterminate glaberscent cariaceous sub-globose absence violet pale rough 8 83.5 ni 749 and prostrate tan subsp. pawekiae wild allogamous malawi indeterminate glaberscent cariaceous sub-globose absence violet dark smooth 8 86.0 mt 53 and prostrate brown to rough subsp. letouzeyi wild allogamous cameroon indeterminate glaberscent cariaceous sub-globose absence violet pale rough 8 84.5 ni 1420 and prostrate tan subsp. alba wild allo congo indeterminate glaberscent cariaceous sub-globose presence violet pale tan rough 8 88.0 sp 145 autogamous and prostrate subsp. tenuis wild allosouth indeterminate glaberscent cariaceous hastate presence violet pale rough 8 92.5 sp 167 autogamous africa and prostrate tan subsp. pubescens wild allo kenya indeterminate pubescent cariaceous sub-hastate presence violet pale tan rough to 8 92.5 ni 979 autogamous and prostrate hairs wrinkled var. spontanea weed autogamous niger indeterminate glaberscent cariaceous sub-globose presence violet pale smooth 8 78.0 ni 945 and prostrate tan to rough var. unguiculata 524-b cultivar autogamous usa indeterminate glaberscent membranous globose absence white dark smooth 4 87.0 and spreading tan var. unguiculata it84s cultivar autogamous usa indeterminate glaberscent membranous globose absence white dark smooth 3 87.0 2049 and spreading tan diameter 20 cm containing the same standard potting soil in the greenhouse, in order to produce the f2 generation by the natural self-fertilisation. data measurement the tests of germination were immediately carried out after incubation. the samples composed of 110 seeds considered as well-developed in moistened derived from 17 hybrid combinations variant from 4 to 10 seeds/hybrid linewere manipulated, according to ista procedures (1976). variability of seed number tested was due to the difference yielded in seed samples. the percentage of germination was calculated by use of the following formula: [σi n ni/σi n ni] × 100 (1) where: ni is the number of germinated seeds; and ni is the total number of seed placed for germination. pollen fertility was assessed by staining fresh pollen grains with aceto-carmine, according to belling (1921), and calculating the percentage of deeply stained pollen grains for hybrid line. the sample for determining the pollen fertility was approximately 200 pollen grains from 2 flowers/adult hybrid plant. the pollen viability image was captured and counted with a ‘zeiss axioskop’ optical microscope (10 × objective). pollen measurement was made on the f1 plants. pollen size measurement was subjected through using the ocular scale on the microscope, according to microscopic observation techniques described 4 table 2. distribution of f1 materials obtained, according to the different 17 hybrid combinations derived from reciprocal crosses between wild accessions and cultivated cowpea (vigna unguiculata (l.) walp.). hybrid combinations pollinated flower aborted ovule harvested mature pod yield of harvested seed hybrid ♀ parents × ♂ parents number/hybrid combination number/hybrid combination number/hybrid combination number/hybrid combination 524-b × pawekaie 18 16 1 15 pawekiae × 524-b 29 26 2 6 524-b × alba 35 30 5 26 alba × 524-b 19 14 3 15 524-b ×tenuis 20 18 2 4 524-b × pubescens 7 2 5 27 524-b × spontanea 3 1 1 5 it84s-2049 × baoulensis 21 19 2 22 it84s-2049 × pawekiae 12 7 5 63 pawekiae × it84s-2049 30 20 10 53 it84s-2049 × alba 47 36 10 38 alba × it84s-2049 33 23 7 75 it84s-2049 ×tenuis 21 16 4 14 it84s-2049 × pubescens 33 22 11 73 pubescens × it84s-2049 18 13 11 73 it84s-2049 × spontanea 23 22 1 8 spontanea × it84s-2049 12 7 5 42 by rapilly (1968). phenotypic expression of morphological traits to have an idea whether wild gene incorporation into cultivated forms of v. unguiculata was yielded after some artificial hybridisations in the f1 generation, the stable adult plants derived from reciprocal crosses between wild accessions and cultivated cowpeas were examined. an experimental study was conducted in the greenhouse. nine morphological characters in which five vegetative, two inflorescence and two fruit characters were characterised, according to the descriptors for cowpeas. the cowpea f1 hybrid line constituting the sample-adult plant was composed of 3 plants, with exception for 3 f1 hybrid lines derived from (524-b (♀) × tenuis (♂) and 524-b (♀) × spontanea (♂) combinations in which 2 plants were formed and in it84s-2049 (♀) × baoulensis (♂) combination 1 plant was obtained. fourteen hybrid combinations constituting the biodiversity at the first generation susceptible to inherit the latter types were examinedduring vegetative and reproductive stages. biodiversity range description distribution of the f1 adult hybrids, five qualitative and morphological, two inflorescence and two fruit characters as following as: growth habit, plant hairiness, leaf texture, terminal leaflet shape, leaf marking, flower colour, gametophytic male, pod colour and texture of testa seed characters were studied. amongst these selected characters, the frequencies of distribution were used to determine for each continuously varying traits with exception for pod colour and texture of testa seed characters in which 36 f1 adult plants were controlled growth habit this character was visually evaluated, 6 weeks after planting, taking into consideration in the indeterminate spreading traits of hybrid plants. the scale used was: (i) indeterminate spreading and not climbing, that is, lower branches touch the ground. (ii) indeterminate spreading and prostrate i.e. plants flat on ground; branches spread several metres. (iii) indeterminate spreading and climbing. (iv) indeterminate spreading and climbing it was observed that the adult vigour plants in hybrid accessions lines tended to have an indeterminate 5 conformation. distribution of the hybrid accession lines over the three-growth habit groups was respectively 78.58 and 21.42% for groups 2 and 3. plant hairiness according to descriptors for cowpeas, stems, leaves and pods were either glabrescent, that is, lacking hairs or tendency to become hairless vs. pubescent short hairs. these both characteristics were assigned the indexes 3 and 5, respectively. from 14 hybrid accession lines observed, only 1 hybrid line (3 sample-adult plants/f1 hybrid line of cowpea) was assigned the index 5, that is, pubescent and short hairs. in other words, almost 92.59% of hybrid accessions had glabrescent stems, leaves and pods. leaf texture leaf texture was visually observed 6 weeks after planting, according to the following scale: (i) cariaceous (ii) intermediate (iii) membranous the distribution of the hybrid accession lines over the three groups of leaf texture was respectively 78.58 and 21.42% for groups 1 and 3. terminal leaflet shape a visual evaluating of the terminal leaflet shape took place 6th week after planting, according to the following scale: (i) globose (having shape as a globe) (ii) sub-globose (iii) sub-hastate (iv) hastate (triangular, shaped like a spear point) the distribution of the hybrid accessions over the four groups of terminal leaflet shape was 71.42, 21.42 and 7.14%, for groups 2, 1 and 3, respectively. leaf marking observations were done for this character for 6 weeks after planting and the presence vs. absence of v mark was observed on leaflets: 71.42% of samples are free of leaf marking and 28.57% are leaf marked. flower colour flower pigmentation was visually evaluated using the following scale: (i) white (ii) violet (iii) mauve-pink (iv) other about 38 sample-adult hybrid plants were inspected from 45 to 150 days with the first flowers appearing 1 week earlier. the distribution of the hybrid accessions over the four groups of flower colour was 14.28 and 85.72% for groups 1 and 2, respectively. gametophytic male the results of pollen fertility from 14 cowpea f1 hybrid lines were presented in range of 60, 74 and 86.5% for groups 1, 2 and 3 respectively. pod colour a visual rating of pod colour took place after harvesting and drying, according the following scale: (i) pale tan or straw (ii) dark tan (iii) dark brown (iv) black or dark purple (v) other the number of hybrid accessions was reduced to 36 hybrid plants. because of 2 adult plants form (524-b (♀) × tenuis (♂)) combinations flowered and collapsed after emergence. distribution frequency of hybrid accessions over the five groups of pod colour was respectively 46.15, 38.46 and 15.38% for groups 1, 2 and 3. testa seed texture nine textures of testa seed were identified in the collection. these textures presenting in the cowpea descriptors are: (i) 1 – smooth (ii) 3 – smooth to rough (iii) 5 – rough (fine reticulation) (iv) 7 – rough to wrinkled (v) 9 – wrinkled (coarse folds on the testa) distribution frequency for texture of testa concerned notably 13 hybrid accessions, shown for f2 seeds in 16.66, 33.33, 41.66 and 8.33% for groups 1, 3, 5 and 7, respectively. experimental results and discussion seed germination evaluation according to the texture of seed testa derived from genetic materials of the reciprocal crosses between wild accessions × cowpea cultivars, it was provided evidence that all hybrid seeds inherited wild female parent characteristics at the first generation. the texture of seed testa was phenotypically identical, according to the origin of wild parental strains. genetically, the effects expressed in these f1 seeds allowed to show that the success of germination test depends in part on wild parent material source. from these data reported, the germination of dominant seed coat dormancy identified amongst 17 cowpea intraspecific f1 hybrids obtained is summarised in table 3. seeds germinated there was a wide variation in fertility level in the seeds, in accordance with the cross-results. the rate of germinated 6 table 3. results of germination tests, of f1 seeds derived from reciprocal crosses between wild accessions and cultivated cowpeas (vigna unguiculata (l.) walp.), according to ista procedure (1976), germination was conducted in petri dishes containing a moistened filter paper and placed in a philips incubator with specific characteristics (day and night temperatures (24/20°c), fluorescent illumination system (black and light, 365 nm, 4 watt) and photoperiod/day (12 h) hybrid combinations total number of f1 seeds sown / number of germinated seeds at the beginning and total number of seeds germinated seeds f1 ♀ parents x ♂ parents hybrid combination final periods of the tests germinated after 7 days percent/hybrid combination at the beginning period at the final period of the of the test test 524-b × pawekiae 6 2 4 6 100 pawekiae × 524-b 6 3 3 6 100 524-b × alba 6 2 4 6 100 alba × 524-b 6 1 5 6 100 524-b × tenuis 4 0 2 2 50 524-b × pubescens 7 0 0 0 00 524-b × spontanea 5 0 2 2 40 it84s-2049 × baoulensis 8 0 1 1 12.5 it84s-2049 × pawekiae 6 2 4 6 100 pawekiae × it84s-2049 8 2 6 8 100 it84s-2049 × alba 7 3 4 7 100 alba × it84s-2049 7 2 5 7 100 it84s-2049 × tenuis 6 0 0 0 00 it84s-2049 × pubescens 9 0 0 0 00 pubescens × it84s-2049 5 2 3 5 100 it84s-2049 × spontanea 4 2 2 4 100 spontanea × it84s-2049 10 2 8 10 100 110 seeds 23 seeds 53 seeds 76 seeds (mean) x = 69.09% seeds after the test was 69.09 germinated percentage. the highest percent of germination (100%) was distinguished in 11 hybrid lines derived from crosses. therefore, the seeds from (524-b (♀) × tenuis (♂)) combinations were second (50%) and these from (524-b (♀) × spontanea (♂)) combinations were third (40%). finally, the percentage of germinated seeds of (it84s-2049 (♀) × baoulensis (♂)) combinations was 12.5%. in this later case, the final counts made after 7 days showed that germination speed was absolute minimum. the results revealed that one seed germinated. however, the seedling was considered as normal in petri dish. seeds ungerminated the number of seeds ungerminated (30.91%) was determined after seedling counts. final counts made after 7 days showed that any radicle elongation was observed or very short and turned yellowish in the following hybrid lines derived from these combinations: 524-b (♀) x pubescens (♂), it84s-2049 (♀) × tenuis (♂) and it84s-2049 (♀) × pubescens (♂). since the results of the standard of germination test showed defects as several factors such as abnormal embryo development, physiological immature at the harvested period, seed quality and yield are directly influenced by the genetic composition of seeds (scott et al., 1998). manz et al. (2005) reported that the relationships between germination test and radicle emergence initiation 24 h after imbibition and finished germination before 96 h are excellent, when artificial 7 conditions provide favourable effect for viable seeds to germinate and to perform seedling growth. the f1 generation observation hybrid viability luo et al. (2005) reported that an individual is viable if it can survive the adult stage. sahai (2000) indicated that the late stage embryo mortality is the main case of seed sterility. in these cases of sterility evidenced, the embryological disturbances should be caused by genetic factors (rieseberg, 2001). in other circumstances, the adverse climatic effects can probably disturb the normal embryo development which varies considerably in different localities, localities, trees and species (robertson et al., 1996; hall, 2003). adverse climatic effects can cause injury in any (early or late) stage of embryo development. embryo survival depends on the severity of the injury (sahai, 2000). according to walsh et al. (2003), viability testing through germination is essential for the maintenance of a seed gene bank collection and can be a rapid way of identifying problems with the seed storage condition. after sterility extraction among f1 seeds derived from reciprocal crosses between wild accessions and cultivated cowpeas, the development of the next generation was required. fertility indicates chromosome pairing between wild accessions × cowpea cultivars. however, the taxonomic studies in cowpeas revealed the close phylogenetic relationships between cowpea parent materials (maréchal et al., 1978; pasquet, 1993; coulibaly et al., 2001). the particular (524-b (♀) × tenuis (♂)) combination was studied; hence it was discovered in progeny line that vigorous plants flowered about 128 dae (that is, days after emergence) (data no shown). this mutant was also characterised by failure of a majority of the flowers to develop beyond the bud stage with the result that it is nearly completely sterile. it is evidence that hybrid viability at the f1 generation was found to be reduced (67.25%). f1 cowpea hybrid line characterisation according to ibpgr (1982), the characterisation consists of recording those characteristics highly heritable that can be easily observed by eyes and expressed in all environments. these are usually represented by the morphological characters of plants (table 4). hybrids of indeterminate spreading and prostrate × indeterminate spreading and climbing grown: a total of 38 hybrid accessions derived from between indeterminate spreading and prostrate × indeterminate spreading and climbing parents was studied. the f1 was indeterminate spreading and prostate in grown habit. with regard to comparative parental characteristics; data analysis revealed that two lines derived (it84s-2049 (♀) × alba (♂) and 524-b (♀) × alba (♂)) had produced the same morphological and characteristics as their respective cultivated female parents. we also observed white flower colour after the propagation of reproductive organ. these confirmed the presence of two infertile or apomictic lines among cowpea f1 hybrid combinations. hybrids of pubescent short hairs × glabrescent: from 32 f1 hybrid accessions examined, all hybrid lines were almost glabrescent stems, leaves and pods. in certain hybrids and particularly under greenhouse conditions the expression of the pubescence short hairs was observed in 3 hybrid lines derived from (pubescens (♀)×it84s2049 (♂)) combination. hybrids of cariaceous × membranous of leaf texture: two different classes were studied at the first generation. one of the membranous parents was observed in these observed apomictic lines. other f1 hybrids indeterminate and prostrate inherited the cariaceous characteristic as the wild material parents. the distinction between types was clear and no difficulties were encountered during identification stage. hybrids of sub-globose and sub-hastate × globose of terminal leaflet shape: the particular sub-hastate character was discovered in allo-autogamous and perennial wild parental strain, that is, subsp. pubescens. the f1 hybrids derived from (pubescens (♀) ≤ it84s2049 (♂)) combinations were completely inherited subhastate characteristics. a typical leaf trait was shown in figure 1. in the f1 hybrids formed with allogamous and other allo-autogamous perennial wild parents, the type of sub-globose terminal leaflet shape was generated. on this other hand, the type of globose terminal leaflet shape characteristic was observed among apomictic lines. hybrids of v mark on leaflets: all combinations derived from allo-autogamous perennial wild parents × cultivated parents, the f1 hybrids shown v mark on leaflets. in combinations with allogamous perennial and annual wild parents, the leaf surfaces were leaf marking free. hybrids of yellow × purple flower colour: flower colour of the f1 plants was completely purple in f1 hybrid lines. in fact, several researches published that in f1 plant flower colour both genes control this trait. according to fery (1980) white and purple flower are codominant at the first generation. hybrids of high × low percent pollen fertility: pollen fertility in f1 plants was determined by staining. the data from male fertility parental genotypes was high over one 8 table 4. data on morphological character characterisation carried out among 14 hybrid combinations derived from reciprocal crosses between wild accessions and cultivated cowpeas (vigna unguiculata (l.) walp.), according to descriptors for cowpeas. characters studied growth habit plant hairiness leaf texture terminal leaflet shape leaf marking flower colour pod colour testa texture pollen fertility percent index index index index index index index index > 70% < 70% parental genotypes wild accessions pawekiae 2 5 1 2 2 3 3 86.0 baoulensis 2 5 1 2 2 1 5 83.5 alba 2 5 1 2 + 2 1 5 88.0 tenuis 2 5 1 4 + 2 1 5 92.5 pubescens 2 3 1 3 + 2 1 7 86.0 spontanea 2 5 1 2 + 2 1 5 78.0 cultivars 524-b 3 5 3 1 1 2 1 87.0 it84s-2049 3 5 3 1 1 2 1 78.0 f1 hybrid combinations 524-b × pawekiae 2 5 1 2 2 3 3 71.5 pawekiae × 524-b 2 5 1 2 2 3 3 72.5 524-b × alba 3 5 3 1 1 1 1 81.6 alba × 524-b 2 5 1 2 + 2 1 5 59.4 524-b × tenuis 3 5 3 1 2 70.0 524-b × spontanea 2 5 1 2 + 2 1 5 76.4 it84s-2049 × baoulensis 2 5 1 2 2 1 5 72.7 it84s-2049 × pawekiae 2 5 1 2 2 3 3 80.8 pawekiae × it84s-2049 2 5 1 2 2 3 3 73.8 it84s-2049 × alba 3 5 3 1 + 1 3 1 80.8 alba x it84s-2049 2 5 1 2 + 2 2 5 72.7 pubescens × it84s-2049 2 3 1 3 + 2 1 7 67.3 it84s-2049 × spontanea 2 5 1 2 + 2 1 5 72.3 spontanea × it84s-2049 2 5 1 2 + 2 1 5 84.3 growth habit, index 2: indeterminate and prostrate; index 3: indeterminate spreading and climbing. plant hairiness, index 3: glabrescent; index 5: pubescent and short appressed hairs. leaf texture, index 1: cariaceous; index 3: membranous. terminal leaflet shape, index 1: globose; index 2: sub-globose; index 3: sub-hastate; index 4: hastate. leaf marking, index (-): absence; index (+): presence. flower colour, index 1: pale tan; index 2: dark tan; index 3: dark brown.testa texture, index 1: smooth; index 3: smooth to rough; index 5: rough; index 7: rough to wrinkle. pollen fertility: percentage > 70 and < 70% over one; however, low pollen fertility percentage was observed in plants from infertile line as (alba (♀) ×524-b (♂)) combination and in f1 hybrid line of (pubescens (♀) × it84s-2049 (♂)) combination. high pollen fertility was identified in plants derived from two infertile lines of (524-b (♀)× alba (♂)) and (it84s-2049 (♀) × alba (♂)) combinations, in f1 hybrid lines from (it84s-2049 (♀)σ pawekiae (♂)) and (spontanea (♀) ×it84s-2049 (♂)) 9 figure 1.the f1 (pubescens (♀) × it84s-2049 (♂)) hybrid line presenting the sub-hastate terminal leaflet shape and v marks on leaflet characteristics. combinations. hybrids of dark tan and brown × straw pod colour: pod colour derived from f1 hybrids at the first generation presented the same phenotypic configuration as female parents in crosses there was maternal dominance inheritance occurred in all of hybrids (birky, 1995). hybrids of rough × smooth seed testa texture segregated: in all of hybrids the f1 textures of seed testa inherited seed characteristic of wild parental strains. after f1 adult-plant development, all morphological characteristics belong to the first generation but seeds provided by these hybrids plants constitute the f2 genetic material. in the first generation character recombination amongst all hybrid individuals revealed that wild characters expressed by one or two of the parent in bi-parental and reciprocal crosses were dominant, except in pod colour in which maternal dominant effect encountered, has shown maternal dominance inheritance, according to aliboth’s et al. (1997) works, wild character dominance in the fertile hybrid lines derived from reciprocal crosses between wild accessions and cultivated cowpeas showed a heterotic status for morphological, inflorescence and fruit traits. in the f2 generation, 16.66 smooth, 33.33 smooth to rough, 41.66 rough and 8.33 rough to wrinkled types occurred in a ratio of 2 smooth: 2 rough individuals. the deficiency in the recessive class in f2 may indicate lower viability of the mutant type. in seed testa texture character in which individuals were distinguished in 2 classes, the f2 ratio observed may indicate segregation 10 figure 2. case of vegetative characteristic occurred in the sterile f1 (524-b (♀) × tenuis (♂)) hybrid line regarded to be near to cultivated parent. absence because of progeny tests with them were not possible. in studying of evolutionary processes of traits, in mutants of (524-b (♀) × tenuis (♂)) during the distinction between vegetative and reproductive types, these obtained individuals were morphologically near to cultivated parent, that is, the 524-b cultivar (figure 2). the wild parent (tenuis subspecies) was implicated through purple flower colour, indicating probably that partial hybridisation was executed into this sterile hybrid line. berville (2002) illustrated this kind of difference in helianthus sp. studies in which hybrid plants derived from interspecific crosses between cultivated helianthus (♀) × helianthus mollis (♂) (that is, wild diploid and perennial species) through using molecular markers, the obtained f1 hybrids developed fragments from wild parental strains in flower characteristics, revealing partial hybridisation occurred in these interspecific crosses. in partial. 11 table 5. germination of f2 self-fertilised seeds produced by f1 adult-plants of cowpea derived from reciprocal crosses between wild accessions and cultivated cowpeas (vigna unguiculata (l.) walp.), according to ista procedures (1976). hybrid combinations harvested f2 seed number of f2 seeds ♀ parents x ♂ parents number/hybrid combination sown/hybrid combination number of f2 seeds germinated at the beginning and final periods of tests at the beginning of at the final period of period of the test the test percent of f2 seeds observations germinated (%) 524-b x pawekiae 395 143 75 68 100 high germination speed pawekiae x 524-b 756 168 98 70 100 high germination speed alba x 524-b 1071 193 78 115 100 high germination speed 524-b x spontanea 1113 188 100 88 100 high germination speed it84s-2049 x baoulensis 76 68 5 15 29.41 low germination speed* it84s-2049 x pawekiae 1032 168 60 99 94.64 high germination speed pawekiae x it84s-2049 1780 188 80 108 100 high germination speed alba x it84s-2049 652 118 55 63 100 high germination speed pubescens x it84s-2049 1603 193 75 118 100 high germination speed it84s-2049 x spontanea 1928 168 62 106 100 high germination speed spontanea x it84s-2049 1239 155 77 78 100 high germination speed 11645 1750 765 928 (mean) x = 93.09% *low germination speed in this (it84s-2049 × baoulensis) hybrid line was observed at the beginning and final periods of the tests. hybridisation, plants were nearly or completely sterile the f2 generation observation we examined 11 f2 hybrid populations derived from 11 parental different combinations. from seed number produced by natural self-fertilisation in the f1 adult plants, the total number of f2 plants studied in the green house was 33 individuals (table 5). after germination of self-fertilised seeds produced by 33 f1 adult-plants, we detected that post-zygotic barrier mechanisms which take effect after successful fertilisation, include seed abortion and the weakness or sterility of f1 hybrids. we have seen that f2 plants derived from (it84s2049 (♀)×baoulensis (♂)) combination exhibited abnormalities during growth and development stages. this hybrid failed to yield viable seeds by self-pollination. sterility identified in f2 generation may be suggested as being due to genetic causes acting along with the effects of cryptic structural differences (rieseberg, 2001). seed number obtained in f2 generation was more important than in the f1 generation. the difference in seed production between populations of f2 shown that plants f2 were productive and provided of viable seeds per pod and able to produce the next generation by natural selfpollination. conclusion in producing intraspecific hybrids derived from reciprocal crosses between wild accessions and cultivated cowpea through using conventional breeding systems, artificial hybridisations yielded several crossability barriers. despite dysfunctions identified in embryo development during gene transfer, in intraspecific f1 production after the germination tests, some f1 plants obtained were fertile. pollen fertility of these plants was normal (70%). even with semi-fertile seeds from (524-b (♀) × tenuis (♂)) combinations, partial sterile plants were generated. the occurrence was induced by incomplete expression of male sterility. the distribution of fertile plants was reduced. during characterisation, two lines derived from (it84s-2049 (♀) × alba (♂) and 524-b (♀) × alba (♂)) restored the identical characters of the cultivars. these lines were discarded because of apomictic lines. in fertile hybrid lines, the dominance of wild genes was evident. heterosis fixed 12 in plants at the first generation declines in the f2 generation because inbreeding influence. based on intraspecific f1 hybrids, we conclude that lines obtained in this study possessed different characters. in the second generation of segregation, it would be important to follow these works in open field under agro-ecological conditions appropriated to v. unguiculata species in order to elucidate inheritance of morphological characters in segregating hybrids. with regard to the persistence of incomplete male sterility expressed in this above indicated cross, it would be advantageous to develop suitable restorer lines with 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and this was not declared in over 75% of the products. tomato products made in nigeria were of comparable quality with those made in us and italy (p>0.05), but those imported from china were significantly higher in total sugar and starch, but significantly lower in lycopene (p< 0.05). variations in content between tin and sachet of the same brand and between different batches of the same product were observed. the need to patronize and support local made products while tightening regulatory processes of products imported into the country becomes imperative. keywords: tomato, quality assurance, biochemical analysis, free radicals, oxidative stress, antioxidants. introduction tomato is an annual vegetable commonly consumed in nigeria. it is widely distributed crop which can be consumed fresh, cooked or processed into a paste or syrup otherwise called a tomato puree. tomato crop can grow in several climates and this confers on the crop, its ability to survive at various temperatures. more than 30% of global tomato is sourced from the mediterranean [ahmet and vedat, 2009], but a greater percentage also comes from turkey which is located in the east of the mediterranean [ahmet and vedat, 2009]. according to powell et al [2003], tomato is grown with a view to *corresponding author e-mail: aaonyeaghala@gmail.com consuming it fresh or processing it into other forms such as pastes or puree. a good number of tomato paste products currently sold in nigeria are mostly supplied from china and other asian countries. studies have shown that lycopene from processed tomatoes are better absorbed than lycopene from fresh tomato [anonymous, 1997]. this suggests that processed tomato products such as tomato paste, tomato sauce and ketchup are better sources of this antioxidant. it has also been reported that lycopene from tomato paste was 2.5 times more bioavailable in humans than lycopene from fresh tomatoes especially when boiled with oil, a common medium in which tomato is prepared in this region of the world.[powell et al, 2003; ahmet and vedat, 2009]. recent reports have shown that lycopene www.internationalscholarsjournals.org 2 onyeaghala et al. 237 helps prevent the development of cancer, especially prostate cancer [anonymous, 1997]. this implies that commercially sold processed tomato paste should contain a good amount of this phytochemical which when consumed, should be able to benefit humans. several parameters have been used to assess the quality of tomato paste. these include: the consistency, total solids content, titratable acidity, ph and levels of sugar [ahmet and vedat, 2009; luzhang et al, [2014]. wellstructured studies which examine the quality of different tomato brands sold in nigeria are limited. however, available but invalidated reports have repeatedly shown that many tomato brands imported into the country were adulterated with starch and colorants. [shuehu, 2014, umeofia, 2016] umeofia [2016], a leading chief executive in one of the tomato manufacturing companies in nigeria, had lamented that the country loses huge sums of money to importation of fake tomato products. he reported that a good number of tomato products imported into the country from asia were often adulterated with starch which could have adverse effects on the health of nigerians. shehu [2014] reported that nigeria had turned into a dumping ground for fake and low quality tomato products imported from asia. the report further said that the spate of importation of adulterated tomato sauce in the name of tomato paste was of grave concern to stakeholders in the tomato industry. the infiltration of poor quality tomato products into the country has been attributed to the activities of unpatriotic marketers conniving with unethical foreign companies to haul in tomato paste loaded with starch and treated with food colour additives to achieve the deep red colour and the premium placed on national agency for food and drug administration and control (nafdac) on the scrutiny of drugs imported into the country to the detriment of food and cosmetics [shehu, 2014]. the importation of fake tomato products is not only a problem in nigeria. nkruma, [2015] has reported that most tomato brands sold in ghana contained starch and sugar which have raised safety concerns among consumers of such products. the reports showed that the products had labels which indicated that they contained modified starch, tomato paste, sugar and some acidic component, but there were no indications of the relative quantities of those ingredients, a situation which has raised concerns over the quality of the products and the possible health implications related to their consumption [nkruma, 2015]. the consequences of consuming fake and substandard products are better imagined than experienced. the effects and several deaths resulting from my pikin tee thing powder episode in nigeria can never be forgotten. the high prevalence of several diseases like cancer, hypertension, diabetes, cardiovascular disorders, kidney and liver disorders are evident within the country. several manufactures have been forced to close their businesses due to preference of nigerians to imported goods. even when such products are unhealthy and substandard compared to local made products, many nigerians still prefer imported goods to local made products. considering the lack of documented information on quality assessment of imported and different tomato products manufactured within the country, this study was conducted to bridge this gap. the objectives of the study were to determine the biochemical parameters contained in selected local and imported tomato brands, ascertain if imported tomato brand was better than those manufactured and packaged locally, determine if there were differences in content between tin and sachet of the same tomato brand and ascertain if there were variations in content between different batches of the same tomato brand. the information generated would help consumers of tomato products to make an informed decision when choosing available products. justification for the study nigeria is the biggest economy in africa. premised on her economic size and huge population, manufacturers all over the world have hinged on these to bring in several products into the country with a view to increasing sales and making more profit. the desire for imported goods has driven many nigerians to relegate to the background several products manufactured locally; even when it has been hypothesized that local made goods were better than imported products. on this account, several manufactures across the globe, have brought in fake and substandard products into the country believing that since most nigerians prefer cheap products to costlier ones, reducing the quality of such products could attract more purchase and patronage. this has predisposed the nation to become a dumping ground for several substandard, fake and unhealthy products. with poor regulatory systems, poor adherence to quality managements system fundamentals, porous borders, increase in the importation of sub-standard products into the country and corrupt practices across various agencies of government, there is no doubt that it is the responsibility of researchers to assess the quality of several products sold to her citizens with a view to generating evidence based data and information which could be helpful in strengthening regulatory agencies, formulate better polices that will benefit the nation, improve public health and quality of life of citizens. materials and methods study area and site different brands of tomato paste sold in two major markets in ibadan, agbeni and bodija were identified. in all, 28 different tomato brands were identified. of these brands, 16 of them were selected at random. five tins each among the 16 selected brands were purchased from randomly selected retailers. tomato brands, having 3 238 afr. j. food sci. res. corresponding sachet were also selected and purchased. this gave a total of 80 tins and 50 sachets of different tomato brands including hunt, a tomato paste manufactured and approved by the united states food and drug administration (usfda). both the tins and sachets of selected tomato brands were all registered with the national agency for food and drug administration and control (nafdac). products endorsed by the nigeria industrial standards (nis) were also noted and documented. the manufactured, expiry dates and batch numbers of all products were noted and recorded. justification for market selection markets located in agbeni and bodija are the melting points of major commercial activities not only for people living in ibadan, but also many other states in the south west geo political zone. these markets are major commercial nerves for consumers and major distributors in oyo state and other nearby south western states. majority of household products are deposited at these markets by different producers from where many retailers and users come to repurchase both for resale and for household consumption. sample size calculation: studies examining the quality of different brands of tomato paste sold in nigeria are limited. one of the outcomes for determining the quality of tomato is the quantity of lycopene contained in the paste. study by maria lorena et al [2014] in mexico on quality parameters and bioactive compounds of red tomatoes at different post-harvest conditions, reported a standard deviation of 1.5 lycopene in tomatoes. using this information, the number of tomato samples used for the study was calculated using the following formular: n = 2(zα + zβ) 2 δ 2 (µ1 µ2) 2 where: z α = standard normal deviate corresponding to 2 sided level of significance of 5%=1.96 zβ = standard normal deviate corresponding to 80% power = 0.84 n = number of samples δ = standard deviation (µ1 µ2) = assumed difference in means between the two groups considered significant. it is assumed to be 50% of the standard deviation. therefore, the minimum number of tomato samples used was calculated thus: n = 2 × (1.96+0.84) 2 × (1.5) 2 (0.75) 2 n = 2 × 7.84 × 2.25 0.56 n = 62.72 therefore the minimum number of tomato samples was 63. this was increased to 130 tomato samples for easy generalizability of information. selection criteria the inclusion and exclusion criteria for all tomato brands used for the study were: inclusion criteria presence of nafdac registration inscription of batch number presence of manufactured and expiry dates indication of country of production or packaging must either be in tin and or sachet exclusion criteria: no nafdac number no manufactured and expiry dates no inscription of country of origin no batch number laboratory analysis all biochemical parameters associated with this study were analyzed at the nigeria institute of science laboratory technology (nislt) samonda, ibadan. the following analyses were performed in all the samples: moisture content, total solids, lycopene, total sugar, starch content, vitamin c and total acidity. all parameters were measured using the approved and validated method of association of official analytical chemists [aoac] laboratory methods analysis of starch and sugar, [aoac, 2000] protocol summary starch was first hydrolysed to sugar. the sugar was determined using the lane and eynon method. result was expressed in gms/100gms (w/w) of tomato paste. the level of starch was determined by multiplying the sugar value by a factor of 0.9. the value obtained was expressed in g/100g of tomato paste. the presence of starch was qualitatively confirmed using the iodine method. vitamin c (rhamanet al [2006]) protocol summary ascorbic acid was oxidized to dehydroascorbic acid by bromine water in the presence of acetic acid. after 4 onyeaghala et al. 239 table 1. comparative analysis of various biochemical contents among different tin tomato products. paramet er tomatoes (n=80) for all products ** products packaged in nigeria fvalue pvalue rg br dock han gin ros tai st..rit der de-g l79 tt po m cia lun hun moisture (g/100g) 70±.09 72±2.07 71±2.1 9 70±3.2 1 73±3.1 6 70±3.3 6 71±2.8 2 69±2.6 8 73±3.1 1 69±2.5 1 70±3. 03 71± 3.51 73± 3.4 0 71± 2.68 73±3, 78 76±3.0 3 2.03 0.027* t solid g/100g) 30.49±6 .6 28.85±6 .02 22.62± 3.35 25.25± 14.01 23.55± 7.70 28.74± 5.01 24.23± 9.51 26.00± 7.76 30.67± 10.44 28.49± 5.77 26.58 ± 5.21 39.0 3±10 .66 28. 05± 3.8 4 21.69 ±6.75 24.56 ±2.15 20.53± 2.31 1.49 0.13 lycopen e (mg/100g ) 9.88±1. 38 6.50±0. 73 7.83±2 .09 6.71± 2.70 8.79±1 .26 8.57±1 .53 9.25±1 .08 7.74±1 .00 8.49±1 .87 7.54±0 .80 7.07± 2.11 9.11 ±1.2 5 8.6 3±1 .25 8.56± 0.72 9.85± 0.98 9.49± 1.47 2.23 0.007* t.sugar (g/100g) 0 21.9± 2.55 27.32± 2.55 29.58± 1.48 11.88± 1.02 30.9± 1.29 21.32± 1.82 20.92± 0.96 0 31.1± 1.80 21.16 ± 1.27 31.2 3± 1.80 0.4 6±0 .35 21.88 ± 2.12 12.62 ± 1.31 12.00± 0.00 287.1 0.000* starch (g/100g) 0 19.76±0 .73 24.62± 2.32 26.04± 0.23 10.74± 0.91 27.88± 1.16 19.24± 1.62 18.88± 0.84 0 28.02± 1.61 19.08 ±1.13 28.1 3±2. 73 0.1 4± 0.3 1 19.72 ±1.92 11.58 ±1.18 0 354.2 7 0.000* vit c (mg/100g ) 30.00±2 .60 23.44± 4.05 16.64± 3.4 20.74± 1.14 21.84± 1.85 25.26± 2.18 20.86± 3.47 17.88± 4.22 17.84± 2.85 27.30± 3.24 13.3± 2.77 27.2 3±3. 35 22. 82± 7.2 9 16.28 ±5.51 17.84 ±2.22 26.26± 2.26 8.44 0.000* t.acidity (g/l) 0.24±0. 16 0.18±0. 01 0.16±0 .03 0.17±0 .02 0.28±0 .02 0.18±0 .00 0.17±0 .03 0.19±0 ,030 0.26±0 .06 0.29±0 .09 0.31± 0.11 0.22 ±0.0 6 0.3 0±0 .03 0.25± 0.05 0.33± 0.01 0.22±0 .08 3.39 0.000* *p<0.05 (significant) rg=ric giko, br= brisk, dock= docker, han= hanno, gin= gino, ros= rosa, tai=taima, st.rit=st rita, der,=derica, deg=de gold; l79= l79; tt=tasty tom; pom= pomo; cia, ciao, lun=luna; hun=hunt. coupling with 2, 4, dinitrophenylhydrazine, dnph, the solution was treated with 85% h2so4 to produce a red complex. the colour formed was measured spectrophotometrically at 521nm. the intensity of colour formed was directly proportional to the concentration of vitamin c present in the sample. the result obtained was expressed in mg/100g of tomato paste. total solids [aoac, 2000] protocol summary the total solids content is a measure of the amount of paste remaining after all the water has been evaporated in an oven. the mass of the residue is expressed as a percentage(%) of the original mass of paste. the gravimetry, weight difference recorded after removal of moisture from the tomato paste by ovendrying is a function of the total solids. data obtained was expressed in g/100gms (w/w) of paste. moisture [aoac, 1995] summary of protocol the moisture content was determined by measuring 5 240 afr. j. food sci. res. fig 1. chart showing percentage tomato products that contained and those that did not contain sugar the mass of tomato paste before and after the water was removed by evaporation in an oven. the mass of the residue is expressed as a percentage(%) of the original mass of paste. gravimetry, weight difference after removal of moisture by oven-drying was determined. data was expressed in percentage (w/w) determination of total acidity [aoac, 2000] protocol summary the volume of 0.1m solution of naoh required to neutralize a standardized and measured amount of sample is the function of its acidic content. since citric acid is the commonest acid present in fruits, volume of naoh consumed is directly proportional to the citric acid present in the sample. the data obtained was expressed in g/l of citric acid spectrophotometric determination of lycopene [anonymous, 2016] protocol summary lycopene was extracted using hexane, ethanol and acetone mixture. the extract was measured spectrophotometrically at 503nm . the data obtained was expressed in mg/100g data analysis and management data collected from biochemical analysis were analyzed using spss version 20 statistical software. data obtained were expressed as mean and ± standard deviation (±sd) for quantitative variables. paired t-test was used to test the significance difference between mean values of laboratory results. one way and twoway analysis of variance (anova) were used for the comparison of means between groups.appropriate graphical application was used to express data and non-parametric analysis was used to determine the degree of significance between mean groups. ethical issues ethical approval for this study was obtained from the oyo state ministry of health ethical committee (approval reference ad13/479/1016 of february, 2016). all samples were blinded during the sampling and analytical phases of the study , but were unblinded during the reporting stage. this was performed to eliminate bias. confidentiality of data was maintained in all stages of the study. sugar free 2(12.5%) sugar 14(87.5%) sugar free sugar 6 onyeaghala et al. 241 fig 2. chart showing tomato products that contained and those that did not contain starch results the mean concentration and comparative analysis of various biochemical contents among different tin tomato products is shown in table 1. it was evident that there were significant differences in moisture, lycopene, total sugar, starch, vitamin c and acidity among all products (p<0.05). figure 1 shows percentage tomato products that contained and those that did not contain sugar. of all the tin tomato products analyzed, 14 (87.5%) contained sugar in varying degrees against 2(12.5%) which were sugar free. of all the tin tomato products analyzed, 13 (81.25%) contained starch in varying concentration against 3 (18.75%) which were starch free (figure 2). among the tomato brands which contained starch, the starch contained in such products was not declared. (figure 3). figures 4, 5, 6, 7 and 8 show the mean concentrations of lycopene, starch, sugar, total acidity and total solids contained in sachets of different tomato brands. a comparative analysis in biochemical parameters between tomato products made in nigeria and those imported from other countries such as china, italy and usa using anova showed thatthere were significant differences in moisture, (p= 0.008); lycopene (p=0.001); total sugar (p= 0.000) and starch (p=0.000) , table 2. post hoc analysis of tomato products made in nigeria and those from other countries showed that tomato brands made in the country were of comparable biochemical quality in lycopene , total sugar and starch (if any) when compared with products from usa and italy (p>0.05). tomato products imported from china were consistently and significantly higher in total sugar, starch, but significantly lower in lycopene content (p< 0.05) respectively, table 3. comparative analysis of tins and sachets of the same tomato brand raveled that significant differences (p<0.05) were observed among some tomato brands. in gino tomato paste, significant differences were observed in moisture, total sugar and starch content of the tomato brand (p<0.05; table 4) . in hanno tomato paste, differences were observed in the starch and total acidity contents of the tomato product (p<0.05; table 5). in tasty tom, significant differences were observed in total solid and total acidity contents of the tomato products (p<0.05; table 6). in derica, there was significant difference in the lycopene content between tin and sachet of the same product (p<0.05; table 7). there was no significant difference in starch free 3(18.75%) starch 13(81.25%) starch free starch 7 242 afr. j. food sci. res. fig 3. declaration of starch contained in different tomato brands. key: starch not declared. starch declared. both the tin and sachet of ric giko and promo tomato brands (p>0.05; tables 8 and 9) respectively. comparative biochemical analysis between different batches of the same tomato brand showed that in gino and derica tomato brands, there were significant differences in total solid content of the tomato brands (p= 0.002, tables 10 and 11) respectively. in ric –giko tomato brand, there was no observed significant batch variations in biochemical parameters (p>0.05) , table 12. in dangote tomato brand, significant differences were observed in total solid (p= 0.025), vitamin c (p= 0.011) and total acidity (p= 0.001), table 13. discussion tomato pastes are often consumed everyday by many homes including those in south west nigeria. they are good sources of lycopene and vitamin c, phytochemicals which have been implicated in the fight against cancer and other disorders initiated by oxidative stress. premised on their high demand, it becomes imperative that tomato products sold in various markets should be of the best international and acceptable standards. several biochemical parameters have been used to assess the quality of tomato. this includes the total acidity, sugar, moisture and total solids. however, it is expedient that all tomato pastes solely produced from pure tomato seed should contain a high percentage of lycopene and vitamin c. this study has shown that the various tomato brands investigated contained various levels of these biochemical substances. this could imply that each of the tomato brands was made from different species of tomato seed which depending on region and level of ripening, could contain varying levels of these biochemicals. all the tin tomato brands investigatedcontained sugar except rick giko and derica tomato products. it is important to note that the sugar and starch content of pomo tomato brand was negligible and this could be as a result of contamination during the production process. since most of the tomato products contained starch, it is possible that the presence of sugar could either be as a result of hydrolysis of starch or deliberately added during the manufacturing process with a view to improving series1, brisk, 19.76 series1, dockers, 24.62 series1, hanno, 26.04 series1, gino, 10.74 series1, rosa, 27.88 series1, taima, 19.24 series1, st rita, 18.88 series1, de gold, 28.02 series1, l79, 19.08 series1, tasty tom, 28.1333 series1, promo, 0.14 series1, ciao, 19.72 series1, luna, 11.58 m ea n s ta r c h c o n c en tr a ti o n s tin tomato brands 8 onyeaghala et al. 243 biochemical parameters in different brands of sachet tomato figure 4. mean concentration of lycopene (mg/100g) in sachets of different tomato brands. figure 5. mean concentration of starch (g/100g)in sachets of different tomato brands. quantity and taste. this is however subject to further investigation for proper elucidation. the sugar contained in hunt and brisk tomato products were declared, while that contained in other products were not declared. hunt is a tomato paste made and approved by the usfda. it is a regulatory requirement in us that all products made and sold in the united state should declare all chemicals known or unknown before such products are either approved by fda or sold in the usa. furthermore, brisk tomato paste could be made for export to us market which necessitated the declaration of the sugar contained in the paste. the findings from this study showed that most of the products investigated contained starch except ric giko, derica ,dangoteand hunt tomato products. it is worthy of note that majority of such tomato brands were those imported from china for nigeria market. previous reports have shown that different tomato brands especially those series1, gino, 9.815 series1, dangote, 10.427 series1, hanno, 7.96 series1, tasty tom, 8.812 series1, derica, 10.182 series1, promo, 8.748 series1, ric-giko, 9.812 m ea n l y c o p en e c o n c en tr a ti o n s sachet tomato brands series1, gino, 12.44 series1, dangote, 0.07 series1, hanno, 28.42 series1, tasty tom, 26.34 series1, derica, 0.09 series1, promo, 0.24 m ea n s ta r c h c o n c en tr a ti o n s sachet tomato brands 9 244 afr. j. food sci. res. figure 6. mean concentration of sugar (g/100g) in sachets of different tomato brands. figure 7. mean concentration of total acidity (g/l) in sachets of different tomato brands. figure 8. mean concentration of total solid(g/g) in sachets of different tomato brands. manufactured and imported from china contained starch [nkruma, 2015; umeofia, 2016; shehu, 2016]. this study supports their claims. derica and hunt tomato brands were products made in italy and us respectively. series1, gino, 13.79 series1, dangote, 0.08 series1, hanno, 31.52 series1, tasty tom, 29.2 series1, derica, 0.1 series1, promo, 0.28 m ea n s u g a r c o n c en tr a ti o n s sachet tomato brands series1, gino, 0.28175 series1, dangote, 0.315 series1, hanno, 0.1428 series1, tasty tom, 0.3383 series1, derica, 0.2594 series1, promo, 0.3185 series1, ric-giko, 0.1589 m ea n a c id it y c o n c en tr a ti o n s sachet tomato brands series1, gino, 26.983 series1, dangote, 32.503 series1, hanno, 29.012 series1, tasty tom, 27.048 series1, derica, 33.408 series1, promo, 24.348 series1, ric-giko, 26.294 m ea n t o ta l so li d s c o n c en tr a ti o n s sachet tomato brands 10 onyeaghala et al. 245 table 2. comparative analysis in biochemical parameters between tomato products made in nigeria and products from other countries (china, italy and usa) using anova. parameters tomatoes (n=80) for all products f-value p-value nig =15 china =53 italy=5 usa=5 moisture 71.13± 3.45 70.66 ± 3.14 73.20±3.11 75.23± 3.05 4.260 0.008* t solid 26.43± 6.97 26.72± 7.67 30.76± 10.44 20.53± 7.61 1.571 0.204 lycopene 9.66± 1.12 7.87± 1.62 8.49±1.87 9.49± 1.47 6.189 0.001* t. sugar 11.31± 9.13 22.22± 9.31 0.000 12.00± 0.00 14.838 0.000* starch 10.27± 8.25 19.97± 8.33 0.000 0.000 20.926 0.000* vit c 22.90± 5.95 20.92± 5.64 17.84± 2.85 26.26± 2.26 2.534 0.063 t.acidity 0.25± 0.11 0.23± 0.07 0.26± 0.06 0.22± 0.10 0.418 0.741 *p<0.05 (significant) table 3. post hoc analysis of tomato products made in nigeria and those from other countries variable country of origin country mean diff p-value moisture nigeria (mean sd) 71.13± 3.45 china (70.66 ± 3.14) 0.473 1.00 italy (73.20±3.11) -2.067 1.00 usa (75.23± 3.05) -4.467 0.053* lycopene nigeria 9.66± 1.12 china (7.87± 1.62) 1.791 0.001* italy (8.49±1.87) 1.170 0.887 usa (9.49± 1.47) 0.172 1.000 total sugar nigeria 11.31± 9.13 china (22.22± 9.31) -10.907 0.000* italy (0.000) 11.31 0.088 usa (12.00± 0.00) -0.6866 1.000 starch nigeria 10.27± 8.25 china (19.97± 8.33 ) -9.704 0.000* italy (0.000) 10.273 0.081 usa (0.000) 10.273 0.081 *p< 0.05 (significant) products from these countries are highly regulated and often conform to required regulatory standards prior to marketing. this finding also implies that rick giko tomato brand is consistent in content and qualitywith hunt, derica and dangote tomato products. starch should not be a component of quality tomato paste. nevertheless, when it forms part of the biochemical components, regulatory processes should mandate that such be declared to enable consumers make an informed decision. from this study, among all the products that contained starch, the starch contained in such products was not declared. this presents the products as though they only contained tomato paste, thus creating a wrong impression to the buyers of such products. in products were starch contained in tomato paste were declared, since the manufacturers of such products have already intimated buyers about the content of the product , it is the choice of the buyer to make informed decision before purchasing such products. approving products which were majorly starch with lower contents in vitamin c and lycopene is highly inimical to consumers’ health. several diseases have been linked to obesity and excessive generation of free radicals. it is possible that consumption of tomato paste loaded with starch and lower in lycopene and vitamin c contents could be contributing to this high incidence of obesity and associated medical problem prevalent in the country. this is subject to further investigation using a well-structured clinical study. all the products which contained starch had nafdac registration and endorsement, but without nis endorsement. however, it is worrisome that the nation’s regulatory agency should approve and register products which could be inimical to people’s health without adequately evaluation such products for quality and safety using established and acceptable guidelines. from this study, the need for reviewing, strengthening and tightening regulatory processes for registration of tomato products imported into the country becomes very evident. in every manufacturing process especially for food industry, it is expected that manufacturers adhere strictly to the principles of good manufacturing practice while putting in place good quality assurance measures. from 11 246 afr. j. food sci. res. biochemical analysis between tin and sachet of the same tomato brand table 4. comparative analysis between gino tin vs gino sachet. variable tin (n=5) sachet (n=10) t test p value moisture 73.0±3.16 76.5±2.17 -2.536 0.025* total solid 23.5±7.7 26.98±5.28 -1.0213 0.326 lycopene 8.79±1.26 9.47±2.23 -0.8196 0.427 total sugar 11.88±1.02 13.79±1.20 -3. 0337 0.010* starch 10.74±0.98 12.44±1.09 -2.9681 0.011* vitamin c 21.84±1.85 26.18±5.12 -1.8076 0.094 total acidity 0.28±0.02 0.28±0.03 0.1790 0.861 *p< 0.05 (significant) table 5. comparative analysis between hano tin vshano sachet. variable tin (n=5) sachet (n=5) t test p value moisture 70.4 ± 3.20 70.2± 3.11 1.000 0.3739 total solid 25.25± 14.0 29.01±5.28 -0.9030 0.417 lycopene 6.71± 2.70 7.96± 2.26 -0.7950 0.471 total sugar 29.58±1.48 31.52± 1.89 -2.4334 0.0717 starch 26.04± 0.23 28.42± 1.69 -2.8623 0.0458* vitamin c 20.74± 1.14 20.67± 1.56 0.128 0.9039 total acidity 0.17±0.21 0.14±0.11 7.3232 0.001* *p< 0.05 (significant) table 6. comparative analysis between tasty tom tin vs tasty tom sachet. variable tin (n=5) sachet (n=5) t test p value moisture 70.67± 3.51 75.7±2.51 -2.343 0.058 total solid 39.03± 10.66 27.04±2.23 2.555 0.043* lycopene 9.11± 1.25 8.81±2.63 0.1819 0.862 total sugar 31.23± 3.02 29.2± 1.16 1.398 0.211 starch 28.13± 2.73 26.34± 1.03 1.3735 0.219 vitamin c 27.23±3.35 16.44± 1.68 -0.5053 0.6314 total acidity 0.22±0.06 0.33±0.05 -2.4732 0.048* *p< 0.05 (significant) table 7. comparative analysis between derica tin vs derica sachet. variable tin (n=5) sachet (n=10) t test p value moisture 73.2± 3.11 76.0± 2.40 1.934 0.075 total solid 30.67± 10.44 33.4± 10.68 0.471 0.646 lycopene 8.49± 1.87 10.18±0.60 2.663 0.020 * total sugar 0.00±0.00 0.1±0.2 1.016 0.328 starch 0.00±0.00 0.09±0.19 1.033 0.320 vitamin c 17.84±2.85 18.25± 2.23 0.306 0.764 total acidity 0.26±0.06 0.26±0.07 -0.253 0.804 *p< 0.05 (significant) the findings from this study, it was evident that there were variations in tin and sachet of the same tomato brand except those of ric-giko. consistency in tin and sachet of the same tomato product implies that such products were processed under good manufacturing practice and are in consistent with good quality assurance measures. in products that showed variation in tin and sachet of the same tomato brand, this could be attributed to either differences in manufacturing process, limited to lack or poor implementation of established quality assurance measures. it could also be attributed to different locations where the tin and sachet of the same tomato brand were packaged . it is possible that many sachet tomato brands sold in nigeria are packaged locally while the tin of the same brand is packaged overseas before they are imported into the country for sales. this could account for the difference observed in tomato brands which exhibited such variations. lack of variations 12 onyeaghala et al. 247 table 8. comparative analysis between promo tin vs promo sachet. variable tin (n=5) sachet (n=5) t test p value moisture 73.2± 3.96 75.2±2.95 -1.291 0.266 total solid 28.05± 3.84 24.34± 4.78 1.204 0.295 lycopene 8.63±1.25 8.74±1.63 -0.113 0.916 total sugar 0.16±0.35 0.28±0.38 -0.429 0.690 starch 0.14±0.31 0.24±0.33 -0.412 0.702 vitamin c 22.82±7.29 14.78±2.27 2.244 0.08 total acidity 0.30±0.03 0.31±0.27 -1.0330 0.360 *p< 0.05 (significant) table 9. comparative analysis between rick giko tin vsric giko sachet. variable tin (n=5) sachet (n=5) t test p value moisture 69.8± 4.08 71.0±0.70 0.753 0.493 total solid 30.49±6.62 26.2± 4.92 -2.0687 0.107 lycopene 9.88± 1.38 9.81± 0.22 -0.129 0.903 total sugar 0.00±0.00 0.00±0.00 starch 0.00±0.00 0.00±0.00 vitamin c 30.00±2.6 29.2±3.57 -0.748 0.496 total acidity 0.24±0.16 0.20±0.11 -1.086 0.338 p< 0.05 (significant) biochemical parameters between different batches of the same tomato brand table 10: comparative analysis between gino tomato products of different batches variable batch 1 (n=5) batch 2 (n=5) t test p value moisture 76.4±3.21 76.6±0.55 -0.126 0.906 total solid 22.40±1.39 31.56±2.92 -7.214 0.002* lycopene 8.27±2.99 11.36±0.33 -2.289 0.083 total sugar 13.36±0.99 14.22±1.34 -0. 985 0.380 starch 12.06±0.93 12.82±1.22 -0.944 0.398 vitamin c 29.34±5.56 23.02±1.78 2.409 0.074 total acidity 0.27±0.03 0.29±0.04 -2.217 0.091 *p< 0.05 (significant) table 11. comparative analysis between derica tomato products of different batches. variable batch 1 (n=5) batch 2 (n=5) t test p value moisture 75.2±3.11 76.8±1.30 -1.000 0.374 total solid 23.93±4.73 42.88±3.13 -7.681 0.002* lycopene 10.28±0.86 10.08±0.25 0.551 0.611 total sugar 0.20 ±0.28 0.00±0.00 1.581 0.189 starch 0.18±0.25 0.00 ±0.00 1.616 0.181 vitamin c 16.74 ±2.04 19.76±1.18 -2.689 0.055 total acidity 0.22±0.09 0.30±0.03 -1.780 0.149 *p< 0.05 (significant) observed among different batches of the same tomato product could also be attributed to good consistency in quality in various batches of the same tomato brand. conclusion based on the objectives of this study and following the biochemical information obtained, it was evident that the quality of tomato brand made in nigeria compared well with tomato brands made in united states and italy.imported tomato brands especially those from china were of poorer quality than those manufactured and packaged within the country. however, in most of the tomato products investigated, there were variations in content between tin and sachet of the same brand and variations between different batches of the same product. this study provides scientific evidence that local made tomato products are better in quality and consistency in biochemical parameters than products imported from china. it was also very evident that ric-giko tomato products showed good consistency between batches as 13 248 afr. j. food sci. res. table 12. comparative analysis between ric –giko tomato products of different batches. variable batch 1 (n=5) batch 2 (n=5) t test p value moisture 71.0±0.71 69.8±4.09 0.735 0.493 total solid 26.29±4.93 30.49±6.63 -2.069 0.107 lycopene 9.81±0.23 9.89±1. 39 -0.129 0.903 total sugar 0.00 ±0.00 0.000± starch 0.00±0.00 0.00 ±0.00 vitamin c 29.2±3.57 30.0±2.60 -0.747 0.496 total acidity 0.16±0.02 0.24±0.16 -1.086 0.339 *p< 0.05 (significant) table 13. comparative analysis between dangote tomato products of different batches. variable batch 1 (n=5) batch 2 (n=5) t test p value moisture 74.6±5.86 77.0±0.71 -0.862 0.438 total solid 26.24±5.59 38.77±2.79 -3.485 0.025* lycopene 10.81±2.65 10.04±0.12 0.664 0.543 total sugar 0.00 ±0.00 0.16±0.36 -1.000 0.374 starch 0.00±0.00 0.14 ±0.31 -1.000 0.374 vitamin c 16.54±1.48 11.64±1.48 4.406 0.011* total acidity 0.34±0.01 0.29±0.02 7.913 0.001* *p< 0.05 (significant) well as between tin and sachet of the same product. unlike ric giko tomato products that had both nafdac and nigerian industrial standards (nis) endorsement, many of the products which were of poor quality had only nafdac registration, but lacked nis endorsement. the need to patronize, encourage and support local manufactures while tightening regulatory processes in the registration of products imported into the country becomes very imperative and should not be compromised if the health, consumer right and well being of the people are to be preserved. acknowledgement the authors express their gratitude to the management of erisco nigeria limited, ikeja, lagos for providing some financial assistance towards this study. the entire efforts and commitment of all scientists at the nilst laboratory, samonda, ibadan, nigeria who painstakingly performed all the laboratory analyses is also highly appreciated. disclaimer this study is free from all forms of conflict of interest. it was basically a scientific research activity performed with a view to assessing the quality of different tomato products sold and consumed by many nigerians. the mentioning of products’ name in this article was solely for providing scientific information. it is not intended to disapprove any product, but to provide information which could strengthen regulatory processes and enable consumers have adequate information to make informed decision. contribution of authors: aao designed the study, documented the proposal, managed literature search, drafted the manuscript and managed reviewers comment. ifi contributed to the drafting of the proposal, provided the facility for laboratory analysis. cok provided additional information during the drafting of proposal, managed and supervised laboratory analysis, provided laboratory technical support, ensured quality assurance of data and contributed to the drafting of the final manuscript. all authors read and approved the manuscript. references ahmetturhan and vedat eniz. 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journals author(s) retain the copyright of this article. full length research paper attitudes towards risk among maize farmers in the dry savanna zone of nigeria: some prospective policies for improving food production l. o. olarinde1*, v. m. manyong2 and j. o. akintola3 1 ladoke akintola university of technology, p. m. b. 4000 ogbomoso 210001, oyo state, nigeria. 2 (iita) – tanzania, c/o mikocheni agricultural research institute, mwenge road, mikocheni b, p. o. box 6226, dar-es salam, tanzania. 3 department. of agricultural economics, university of ibadan, nigeria. accepted 08 january, 2019 this paper applies econometric analyses to quantitatively determine the individual risk attitudes of sampled maize farmers in the dry savanna zone of nigeria. the extent of the risk attitudes are then made the basis for categorizing the farmers into three groups of low, intermediate and high risk averse maize farmers. this categorization now forms a necessary condition for improving the typology of the farmers, which is hypothesized to be influenced by socioeconomic, demographic and other extrinsic “risk factor”. the typology is essentially made possible by discriminant analyses, which re-categorized the farmers into their appropriate risk groups. a four-stage sampling technique leading to the selection of a final sample of about 350 maize farmers was adopted. results show that, about 8, 42% and 50% of the farmers are respectively lowly, intermediately and highly averse to maize risk. about 72% of the hypothesized variables were found to be responsible for the risk aversion among the sampled farmers. these variables are the basis of policy recommendation to address issues generated by four types of risks identified in maize production namely natural, social, economic and technical risks. these are important for harnessing crop technology and to alleviate hunger and poverty in africa. key words: risk attitudes and factors, dry savanna, nigeria, econometric and discriminant analyses and crop technology. introduction the importance of maize in nigeria cannot be overemphasized, with the country producing 43% of maize grown in west africa. it is especially important in the northern guinea savanna (ngs) where it is one of the two major crops in about 40% of the area under agricul-tural production (smith et al., 1997). maize production has increased in nigeria. for example, a seven-fold increase in production occurred between 1984 and 1994 (table 1). this rapid growth, in maize production occurred mainly through expansion of areas under cultivation (ajala et al., 1999) . the introduction of better performing improved varieties, availability of fertili *corresponding author: email:solarinde8@hotmail.com, lolarinde@yahoo.com. tel: +234-0803-3786774 zer at highly subsidized prices, improved extension services, and better infrastructures such as provision of good roads, further triggered a successful maize expan-sion especially in the ngs of nigeria (smith et al., 1997). the high yielding varieties that catalyzed this increased production were largely derived from nigeria composites a and served as the vehicle that moved maize research effort that began in the 70s and at the international institute of tropical agriculture (iita) in 1979. with a financial support from the federal government, iita released its first set of hybrids for testing in 1984. the first set of hybrids gave between 1.5 and 2 tones per hectare, more grain yield than the commonly grown cultivars tzsr-w-1 and tzb of that time (kim, 1995). so far, the above assertion about maize appears to be commendable. as a matter of fact, empirical evidences table 1. area („000ha) and production („000tons) figures for maize in nigeria during the 1984-2005 periods. area production year („000ha) „000 tons) 1984 653 1025 1985 758 1190 1986 810 1336 1987 3108 4612 1988 3212 3590 1989 3590 5008 1990 5101 5768 1991 5112 5812 1992 5223 5810 1993 5309 6290 1994 5426 6902 1995 5472 6931 1996 4273 5667 1997 4200 5254 1998 3884 5127 1999 3965 5476 2000 3999 4107 2001 4041 4620 2002 4490 4934 2003 4700 5150 2004 4466 4779 2005 3589 5957 source: faostat, 2007. table 2. maize yield and percentage area sown to hybrid in west, central eastern and southern africa. sub-regions and yield area sown to countries (ton/ha) hybrid maize (% of cultivable area) 1 west/africa (e.g. nigeria) 1.2 2 2 central africa (e.g. cameroon) 0.99 3 eastern africa (e.g. kenya) 1.4 62 4 southern africa (e.g. zimbabwe) 1.5 96 from the last three decades indicate a fair increase in maize yield. manyong et al. (2003), for instance, found that maize yields increased by 41% from 1970 to 2000 in west and central africa. they, however, discovered that maize yields which are 1.2 tons ha -1 and 0.99 tons ha -1 in 2000 for west and central africa respectively remain low as compared to countries in eastern africa, such as kenya (1.4 tons ha -1 ), and southern africa e.g. zimbabwe (1.5 tons ha -1 ). they opined that reasons for low yield in west and central africa could be in the little proportion of area sown to hybrid, particularly in nigeria where only 2% of cultivable area is sown to hybrid maize compared to kenya (62%) and zimbabwe (96%), (table 2). another major reason for the low yields also given by akpoko and arokoyo (1999) is in the low adoption rates of recommended practices. the above technical problems are gradually being put under control. for example non – governmental organizations such as sg 2000, through state ministries of agriculture have been providing adequate technical support to ensure that improved varieties of maize, especially hybrids are grown where the environmental conditions have been found to be appropriate for their growth. however, despite the various strategies adopted to increase maize production, the perceived susceptibility of maize varieties to risk associated with this crop has made most maize farmers to continue its cultivation with some level of skeptism. this is further evidenced by the declining trend in the area sown to maize in nigeria and the production figures from 1995 to 2005 (table 1) this paper is therefore conceived to explore the factors influencing the farmers‟ attitudes towards the risks associated with maize production in the study area. these “risk” factors are clearly exposed and employed as tools for categorizing and re-aligning these farmers in their proper and normative attitudinal domains. variables that significantly influence the risk aversion are also isolated. the remaining part of this paper is organized as follows: section 2 describes the conceptual and analytical framework, section 3 discusses methodology, and section 4 presents results from this research. section 5 makes a concise discussion of the research results and findings while section 6 gives the conclusion and policy implications. conceptual and analytical framework risk attitudes are implied by the shape of utility function (bard and barry, 2001) for example, if the utility function has a positive slope over the range of pay-offs, the implication is that more payoff is always preferred to less. preferences of this kind are normal for money, but may not apply for other things. for example many smallholder farmers may enjoy farming for pleasure (a way of life for most rural farmers in sub-saharan africa), but the utility does necessarily always increase with the size of the holding – a large size may be too exhaustive. in the language of mathematics (hardaker et al., 1997), the characteristic that more money is preferred to less may be written as; u (1) (w )  0; where u (1) (w) is the i-th derivative of the utility (u) function for wealth (w) . (income can be substituted for wealth here). so, if the first derivative of the utility func tion for wealth is positive (for all w), then it represents the situation of more is preferred to less‟. similarly, risk a version is indicated by a utility function that shows de creasing marginal utility as the level of the pay off is increased, while indifference (neutrality) to risk is represented by a linear utility function. more formally, in terms of the second derivative: 1. u (2) (w )  0 implies risk aversion (ce  emv ) 2. u (2) (w )  0 implies risk indifference (neutrality) (ce  emv ) and 3. u (2) (w )  0 implies risk preference (ce  emv ) *ce and emv are certainly equivalence and expected monetary value respectively. it is however, not feasible to go from the shape of the utility function to some quantitative measure of the degree of risk aversion (or preference) without some bit of complexity, because of the ordinal scale used for utility. this difficulty is resolved by a measure that is constant for any positive linear transformation of u, known as the coefficient of absolute risk aversion (cara). in this study however, a modification which essentially results into some bit of deviation from the above is attempted. this permits an appropriate and convincing analytical framework which captures the concept establishing the present study: cobb-douglas (log-linear) versus ridge regression risk is introduced in a model of economic decision making as a “safety-first‟ rule. according to this rule, an important motivating force of the decision maker in managing the productive resources that he controls and, in particular, in choosing among technological options is the security of generating returns large enough to cover subsistence needs. it has been indicated that, safety-first criterion tends to be followed whenever the satisfaction of basic needs may be at risk (scandizzo and dillon, 1976). assuming that the safety-first model holds, the degree of risk aversion manifested by an individual farmer can be derived from an observed behaviour. given a production technology, the risk associated with production and market condition, the observed level of factor use reveals the underlying degree of risk aversion. this method involves three stages: first is the estimation of production functions in which the relationship between the direct input vectors (x) and maize yields (y) is established. the data collected were subjected to ordinary least square method to obtain the regression coefficients, coefficients of multiple determination (r 2 ), etc. implicitly, the function is expressed as follows: y  f ( x1 , x 2 , x 3 , x 4 , x 5 , x 6 , x 7 , x 8 , u ) (1) where: y is the maize output in tons/ha; x1= the quantity of seed planted in kg/ha; x2= fertilizer (npk) in kg/ha; x3= fertilizer (urea) kg/ha; x4= labour utilization in labour day/ha; x5= insecticide in litre/ha; x 6= herbicide in litre/ha; x7= tractor in hour/ha; x8= animal (traction) in hour/ha; and u= the error term which is assumed to be normally distributed with mean zero and constant variance. this also takes care of all variables assumed constant. the second stage follows from the need to adjust the regression parameter estimates because of multicollinearity that existed between variables x2 and x3 (npk and urea). the lead equation was then subjected to a ridge regression analysis to reduce the influence of the multicollinear variables on the predictive strength of the equation. none of these variables (npk and urea) could be removed from the production function because they are individually, maize yield increasing inputs. in economic theory, multicollinearity commonly occurs because of the nature of aggregation of economic data. most of the explanatory variables tend to move up and down together over time and a number of highly correlated explanatory variables may be large relative to the number of observations. a number of methods of ameliorating multicollinearity have been identified in the literature. one of the prominent among these is the ridge regression analysis. as characterized by hoerl and kenard (1970a), ridge regression technique is a way of incorporating prior information into the estimation procedure. it adjusts the least squares estimator by reducing the influence of multicollinearity on the parameter estimates, it focuses on the achievement of small means square error as the relevant criterion. since a major reduction in variance can be obtained as a result of allowing a little bias, the ridge estimator can be defined as a function of ridge parameter, whose value can be selected by the data analyst. proper choice of the ridge parameter guarantees that the ridge estimator of the regression coefficient is more stable and accurate than the least squares estimators and less susceptible to data set variation. in this study, the use of ridge regression is therefore justified for the following reasons:  the existence of multicollinearity has been established between two important variables.  removing the multicolinear variables can render some important findings (expectation) invalid.  ridge regression normally proceeds from the original ols estimations and does not require any complex or tedious analytical procedure. results of ridge regression analysis are therefore expected to provide to a large extent, unbiased, precise and best estimates of regressors employed in this study. after the determination of the most suitable ridge parameter, the third stage was the selection of the most significant variable. the most significant determinant (in terms of direct variables) of the maize yield was then sin gled out to determine the levels of farmers‟ risk aversion based on moscardi and dejanvry (1977) econometric approach. assume that the randomness of net income that a farmer derives from yield uncertainty and assume also that from the relationship between input vector (x) and yield (y) as represented by (1) for the production function, the coefficient of variation (cv) of yield is:  y (2) y where is the coefficient of variation of yield;  y is the standard deviation of yield; y is the mean yield; and given factor prices (pi) and a given product price (py), the preference order can be maximized with respect to input levels. the resulting first order conditions are: p y f i y  p i (3) x i 1   k ( s ) where pi is the input price; xi, the input vector (most significant input);py ,the output price; fi, the elasticity of production of the i-th input; k(s) is the risk aversion parameter, while  and  are as defined in equation (2) above. the value of the risk aversion parameter was deduced from observed levels of products and inputs by solving equation (3) as follows:  1  p x i (4) k ( s ) 1 i  py f i  y equation (4) provides a measure of risk aversion k(s) that was derived for each farmer from knowledge of production function, the coefficient of variation of yields, product and factor prices and observed levels of factor use. following moscardi and dejanvry (1977), the risk aversion parameter k(s) was used to classify farmers into three (3) distinct groups: risk preferring – low risk – (0 < k(s) < 0.4), risk neutral – intermediate risk – (0.4 ks 1.2) risk aversion – high risk – (1.2 < k (s) < 2.0) discriminant analysis after grouping the farmers into various risk aversioncategories, a discriminant analysis was carried out. through discriminant analysis technique, one may classify individual farmers into two or more mutually exclusive and exhaustive groups on the basis of a set of independent variables. discriminat analysis requires interval independent variables and a nominal dependent variable. the usefulness of discriminant analysis in this study is found in its ability to validate the typology of farmers; that is their classification, given their characteristics, because of the existence of welldefined groups. it was also used to identify the minimum set of variables that were important for discrimination, and to give the probability of each individual that belonged to each group. the model as applied in the study ensures separation among the three groups of farmers that would have earlier been identified. the score on the linear combination of pxs for the i-th member (i = 1,2,….,n) of group g (g = 1,2…..g) and variables x (x = 1, 2,….p) may be written as z ig u i x ilg u 2 x i2g ....u p x ipg (5) the mean of the random variable z, that is the mean of the above linear combination, for the g th group, may be denoted by zg . the separation between the groups is expressed in terms of the variability among group means on the variable z (lindeman, merenda and golo, 1980). this variability is expressed, as in the univariate analysis of variance, by the sum of square among group means. this can be represented as follows; ss a ng  g  2 z z (6) where: ssa is the sum of squares among groups; zg , the mean of the g th group; z ,the grand mean based on n = n1+n2+…+ng. here, n1….ng are individuals in all groups combined. because variability is due in part to variability among individuals, the discriminant criterion is defined as the ratio of variability among group means, ssa to that of within groups, ssw. thus  ss a (7) ssw where ssa is the sum of square among groups; ssw, the sum of square within group. the values of the u‟s in equation (5) are chosen to maximize  . variable definition in this study, farmers‟ socio-economic, farming features and institutional characteristics were employed to define their categorical and behavioural attitudes. an explanation for the differential degree of risk aversion among the farmers can be sought from these characteristics. discriminant analyses were then conducted and their functions estimated to explore quantitatively the relation between risk aversion and the farmers‟ socio-economic, institutional and farm characteristics that are listed below. this was done to find out which of these factors (characteristics) are actually important in determining farmers‟ risk attitudes in maize production. the independent socio economic, institutional and farm (characteristics) variables and their definitions are as follows: w1 is the farmer‟s age in years; w2, the major occupation (only in farming = 1; others = 0); w3, the years of schooling; w4, the household size (no); w5, the membership of association (1 = yes ; 0 = no); w6, the leadership position (1 = yes ; 0 = no); w7, the proportion of maize farm to total farm area (% ha); w 8, the pro-portion of maize income to total farm income (% naira); w9, the proportion of maize income to non-farm income (%naira); w10, the total number of farms; w11, the maize farming experience (years); w12, the adequacy of maize market (1 = yes ; 0 = no); w13, the number of motivating traits/attributes of maize; w14, the number of visits by extension agents to farmers per cropping season; w15, the per capita household food expenditure (naira); w16, the first level probability of sales (%); w17, the second level probability of sales (%) (see olarinde, 2004 for details of the determination of the probabilities of sales); w18, the number of risk types faced by farmers. the grouping variables are the farmers‟ risk groups. they were classified as follows: group 1 – risk preferers; group 2 – risk neutral; group 3 – risk averters. the listed socio-economic and farm characteristics (variables) are essentially labeled “risk factors”, i.e they either positively or negatively influence farmers‟ riskaverse behaviours (table 6). it is expected that the discriminant function estimates would assign the maize farmers to the same group as would have done the classification variables (parameter k). methodology the study was conducted in kaduna state of nigeria. kaduna state is located in dry savanna ecological zone of nigeria. it has a rainfall range of 950-1500 mm with a growing period of 6-8 months. a multistage sampling technique with probabilities proportional to sizes was adopted to choose a final sample of about 350 farmers on which questionnaire schedule was administered [the sampling stages involved the selection of:  all agricultural development programme-adpzones in the state.  local government areas-lgasfrom the selected adps.  villages from the selected lgas and finally, selection of farm families from the selected villages]. data were collected on direct or tractable inputs that are assume- ed to affect maize yields, and on socio-economic and institutional factors, and “risk variables”. these data were analyzed with descriptive statistics, ordinary least square and ridge regressions and a two-level of step-wise discriminant analyses. results description of typical risk affecting maize production in the study area farmers listed several risk types affecting maize production, which belong to four groups: natural, social economic and technical. natural risks these were drought, flood, wind and storm, disease and pests. a large majority of respondents (73%) had their crops affected by drought, 40% by flood, 49% by wind and storms and 63% by diseases and pests. the implication of this is that crop yield could be low due to the negative effects of these natural occurrences. typical negative effects would obviously be caused by drought, disease and pests. social risks these refer to theft of produce, bush fire, invasion of farms by cows. about 58% of the respondents were affected by theft of produce, 53.7% by bush fire and 65.8% had their farms invaded by cows. that most farms were invaded by crows in the study area is expected, besides arable crop farming, the next most prominent occupation is cattle rearing and that explains why almost 2/3 of the sampled farmers had their farms invaded by crows. the invasion of farms is not without losses of crops in most cases as they (cows) tend to graze on raw crops consumed by humans. this could be a major problem facing the farmers to the extent of bringing down yields drastically, especially in the drier parts of the study area. newspapers repeatedly reported clashes among livestock owners and crop farmers in the savannas of west africa. economic risks two major types of economic risks were identified during the survey period. the most important was the maize producer price fluctuation. many of the farmers (73%) do not either get good prices for their maize output or that their particular breed of maize was not adequately patronized during the season to command moderate market prices. the other risk type in this category is that farmers (38%) had insufficient supply of maize seeds which resulted mostly in reduced output. most incidence of risks result in low income because economic risks are viable and important components in any enterprise. technical risks these are necessarily risk types that affect production process. the most important is the insufficient and untimely supply of inorganic fertilizers (84% of farmers). table 3. frequency distribution of maize farmers by risk groups (based on the risk parameter, k). risk averse frequency percentage cumulative group percentage low 21 6.03 6.03 medium 138 39.66 45.69 high 189 54.31 100.00 total 348 100.00 though fertilizer is purportedly supplied to the farmers by the kaduna state agricultural development project (kadp), the methods and channels of distribution are not efficient to respond to the farmers‟ needs. other important technical risks are poor or declining soil fertility (58%), insufficient credit (68%), inadequate processing facilities (41%), insufficient chemicals (25%) and lack of spraying equipment (24%). measurement of risk and categorization of risk averse farmers two functional forms were tested: the exponential and power functions. these were linearized by the logarithm transformations to make them amenable to ordinary least square (ols) estimation procedure. the double logari thm (log-linear) function had the best fit with r 2 = 0.8117 and se = 0.8177. a ridge regression analysis was performed to re-estimate the parameters of the double logarithmic production function in order to address the issue of multicolinearity between two variables (npk and urea) and to identify the most significant input for increasing maize yields in the study area. the results of the ridge regression are: in y= 5.3094 + 0.26778inx1 +0.35467x2 0.0027683inx3+0.17451inx4 (39.6520) (14.0937)** (10.7476)** (1.4000) (10.2653)** -0.50327inx5 + 0.07019inx6 – 0.03994inx7 + 0.00123inx8 (8) (3.5929) (6.3818)** (3.6272)* (0.4000) r 2 = 0.805; ridge parameter = 0.1. where: * = variable significant at p < 0.05 ** = variable significant at p < 0.001 figures in parenthesis are t-values. seed (x 1) was found to be the most important input affecting maize yield. based on the procedure of equation (4), a risk-aversion parameter, k for each maize farmer was estimated and used to classify maize farmers into there risk-averse groups. the results obtained show that 6.03, 39.66 and 54.31% of farmers are low, medium and high risk-averse respectively (table 3). validation of the categorization of the risk averse farmer‟s group the above classification of farmers in table 3 based on the risk parameter was validated through discriminant analysis using a set of 18 variables that were hypothesized to discriminate between the three groups of risk averse farmers (see section 2) . results show that this typology was effective because more than 60% of farmers were correctly classified into their respective riskaverse groups (table 4) . these results also indicate that the classification can be improved upon because the size of each group has changed using the predicted group membership of farmers. the number of low risk averse group of maize farmers was increased from 21 to 31, that of medium group from 138 to 148, and the high risk averse group, reduced from 189 to 169. as a result of the step-wise nature of the discriminant analysis, these grouping were considered as an improvement over the initial classification by the risk parameter, k. by implication, farmers are now better placed in their actual groups with higher probabilities of belonging to them. improvement in the typology of risk-groups of maize farmers in a discriminant analysis, each maize farmer was classified into a risk-averse group where its posteriori probability is the highest. the higher the probability, the best a farmers is typical of the group to which he belongs. using the posteriori probability of at least 80% of each farmer in one of the three groups from the level w discriminant analysis (table 4), a level 2-discriminant analysis resulted in an improvement in the classification of maize farmers into risk-averse groups (table 5). the level 2 discriminant analysis indicated an overall rate of 90.2% of farmers correctly classified in their respective groups, which is an improvement to the rate of 60% from the level 1 analysis. further analysis did not yield any better results, which could not be of significant improvement over the second step-wise discriminate analysis. therefore the result obtained in the level 2 discriminant analysis was taken as the authentic and valid risk averse farmer‟s groups. the following are the outcome: low risk = 27, medium risk = 14 and high risk = 178 (table 5). a priori behavioural expectations of discriminating variables and their benchmark characteristics a set of 18 variables, hypothesized a priori to discriminate between the three groups of farmers, were included in the discriminant analyses for both validation and improvement of typologies. these variables include socio-economic characteristics of respondents, farming features and institutional factors (these variables are as table 4. validation of groups of maize farmers (based on discriminant analysis level one). actual group membership predicted group membership group no. of cases low risk medium high risk low 21 7 (33.3%) 9 (42.9%) 5 (23.8%) medium 138 14 (10.1%) 81 (58.7%) 43 (31.2%) high 189 10 (5.3%) 58 (30.7%) 121 (64.0%) total 348 31 (8.9%) 148 (42.2%) 169 (41.7%) percentage of actual (original) farmers correctly classified = 60.1. note: read percentages along the row table 5. improvement in the typology of groups of maize farmers (base on discriminant analysis level two). actual group membership predicted group membership group no. of cases low risk medium high risk low 31 25 (80.6%) 3 (9.7%) 3 (9.7%) medium 148 0 (0.0%) 132 (89.0%) 16 (11.0%) high 169 2 (1.2%) 10 (5.8%) 157 (93.0%) total 348 27 (7.8%) 145 (41.7%) 176 (50.5%) percentage of actual 9original) farmer correctly classified = 90.2. note: read percentages along the row described in section 2). the individual expectation of these variables is based on some intrinsic and extrinsic relationship between them and risk aversion. these expectations are indicated alongside their benchmark values for the survey period (table 6). discriminating variables in order to identify the minimum set of variables that are important for discrimination, and in effect the extent of their significant in discriminating, a test was conducted on the coefficient of variables for their statistical significance. the, wilks‟ lambda and the significance level of each variable are presented in table 7. the results indicate that thirteen (about 72%) out of the eighteen variables are statistically significant at a minimum of 5% level of probability. they are: total number of farms owned by a farmer, first level probabilities of sales, number of visits by extension agents to farmers per cropping season, maize farming experience, adequacy of maize market, number of risk types faced by the farmer, age, second level probabilities of sales, household size, leadership position, number of motivating traits/attributes of maize breed, proportion of maize to total farm area, proportion of maize income to total income. discussion from our study, the critical variables that are important in distinguishing the sample farmers into low, medium and high risk averse maize farmers are equally vital in the design of any plan that can gear the farmers to accept innovations tailored towards increased maize production. among the discriminating variables that were found to be significant included first and second levels of probabilities of sales, maize farming experience, age, household size, leadership position and proportion of maize to total farm income. these variables essentially constitute socioeconomic factors of the risk averse producers. the prominence of the above variables among those that are significant cannot be overemphasized. for example the first and second levels of probabilities of sales are the extent of farmers‟ willingness (lottery levels one and two of maize market speculation) to sell their maize output in the face of estimated risk of loosing specific amount of money. these factors call for steady and consistent market conditions. fluctuations in farm incomes, most especially those that result in terrible loss may present difficult welfare problems for farmers, especially with peasants. the above “risk factors” can also impact on the social, cultural and economic status of farmers to the extent of reducing farm incomes that have negative multiplier effect on the total income and employment for many household that are directly linked to the peasants. a number of farming features were also found to be important in discriminating between the risk averse maize farmers. these were the total number of farms that a farmer possesses, number or risk types faced by farmers, number of motivating traits or attributes of maize and proportion of maize to total farm area. these features are table 6. benchmark characteristics of risk averse groups. risk averse group expected variables sign low medium high age (years) + 50.69 43.36 42.88 major occupation (farming = 1 ; others 0) + 0.74 0.74 0.74 years of schooling 6.62 5.93 6.09 household size (no) +(-) 17.50 13.05 11.70 membership of association (1 = yes ; 0 = (no) 0.46 0.59 0.50 leadership position (1 = yes ; 0 = no) 0.31 0.46 0.26 proportion of maize to total farm area (% ha) 52.31 49.59 42.46 proportion of maize income to non-farm income (% naira) 51.57 36.36 43.20 proportion of maize income to non-farm income (% naira) 361.61 251.69 299.08 total number of farms 11.42 4.82 3.76 maize farming experience (years) 23.62 13.92 15.96 adequacy of maize market (1 = yes; 0 = no) 0.12 0.47 0.25 number of motivating traits/attributes of maize 1.50 2.15 2.20 table 7. wilk‟s lambda statistics and levels of significance of discriminating variables (with 2 and 214 degree of freedom) for level two discriminant analysis. variables significance wilks lambda total number of farms* 0.600 0.000 first level probability of sales (%)* 0.704 0.000 number of visits by extension agents to farmers per cropping season* 0.808 0.000 maize farming experience (years)* 0.912 0.000 adequacy of maize market (1 = yes ; 0 = no)* 0.924 0.000 number of risk types faced by farmers* 0.925 0.000 age (years)* 0.929 0.000 second level probability of sales (%)* 0.936 0.001 household size (no.)* 0.938 0.001 leadership position (1 = yes ; 0 = no)* 0.960 0.013 number of motivating traits/attributes of maize* 0.961 0.014 proportion of maize to total farm area (% ha)* 0.969 0.035 proportion of maize income to total farm income (% naira)* 0.969 0.035 membership of association (1 = yes ; 0 = no) 0.991 0.377 years of schooling 0.994 0.523 proportion of maize to non-farm income (% naira) 0.994 0.530 per capita household food expenditure (naira) 0.996 0.622 major occupation (farming = 1 ; others = 0) 0.999 0.942 *statistically significant variables important in determining the basic attitudes of the farmers towards maize risk. for instance the larger the proportion of maize farm total farm area cultivated by farmers, the less likely the farmer would avoid taking risk. other farm ing features that are important are closely linked with the total number of farms. in this regard, spatial diversifycation of crop production is an important strategy in mitigating risks in maize production. the geographic spread of fields allows farmers to exploit better the heterogeneity in bio-physical resources (water, soil fertility, pests control, etc) for an increased production of maize. generally, mixed cropping allows more productive and sustainable crop production through complementary roles of components in the same piece of land; reduced labour, control of weeds, etc. lastly, institutional factors such as number of visits by extension agents to farmer per cropping season and adequacy of maize market are also significant. the reliance of farmers on innovational information comes into play here. this suggests a reinforcement of the extension systems, both private and public. also ready and good market for farm produce is usually an issue of worry for farmers in africa. year-in-year -out, peasants loose big sum of money because they cannot either sell their produce at an optimum price or preserve these produce for an increase in market worth. in summary the issues highlighted earlier on, pertaining to the natural, economic and technical risks are most often embedded in the agricultural sector of most developing countries. in the area of input supply, there is need to recognize that purchase of new product may be a risky decision for farmers. thus it may be possible to present information and advice in ways that better portray the risks involved and that permit a farmer to decide more easily which choices best suit his particular circumstances and risk-bearing capacity. agricultural extension or research workers may need to give more thought to farmers‟ aspects, especially those that impinge on the development of improved farming methods. agricultural policy makers and planners also need to account for risk and farmer‟s response to it. conclusion and policy suggestions farmers are facing different types of risk in maize production: natural, social, economic and those related to crop production (technical) . specifically, the following are suggested: the problem of natural risk for example, could be addressed through technological changes, such as breeding for drought tolerance, resistance to pests and diseases, and dwarf varieties. for social risks, institutional and policy interventions that regulate the movement of animals and authorized periods for bush fire could contribute to address issues generated by these types of social risks. however a sustainable solution would be in the “sedentarisation” of livestock through the intensification of crop-livestock integration. for economic risks, farmers need to get better organized in order to impact on markets. the study area represents the maize basket of nigeria. yet, maize farmers have no strong association to protect their interest. this institutional innovation, once implemented would greatly contribute to addressing the above economic risks. finally, for the risks that are related to production processes, addressing production issues from technical risks would require a combination of technological changes and policy interventions. the step-wise discriminant analyses in addition to the earlier regression criterion, used in estimating risk aversion levels and the consequent categorization of the sampled farmers, make us to conclude that farmers‟ risk attitudes are directly responsible for their levels of maize cultivation. the risk aversion levels resulting from the interaction of perceived socio economic, institutional and farm characteristics that characterize farming households. the risk aversion levels also substantiate the fact that sustainability of maize can be achieved by tailoring the design of the programme to the needs of small-holder farmers based on implications of these factors on the farmers‟ behaviours. in this study, we have identified variables that ultimately define the behaviours of the three categories of riskaverse maize farmers. since risk cannot be totally eradicated because of its intrinsic component, the above can serve as a basis to define policies that can help in reducing risk to an acceptable minimum. policies that incorporate the risk aversion indices and those that encompass the elements of natural, social, economic and technical risks, and their controlling measures as identified in this study should be put in place. such policies should also be tailored to the risk averse attitude of the three categories of maize producers on the basis of the socio-economic, institutional and farm characteristics that influence their attitudes. for example, policies that appeal to the high risk farmers, rendering them to be less averse will automatically render the low risk farmers more risk loving. in this way, it is possible to set up development plans that are important for harnessing maize technology to alleviate hunger. references ajala so, menkir a, king jg (1999). fertility requirements of openpollinated and hybrid maize genotypes. paper presented at the sasakawa global 2000 national maize workshop abu, zaria nigeira. akpoko jg, arokoyo jt (1999). maize technology adoption among farmers in kaduna adp villages. in badu-apraku, b, fakorede mab, ouedraogo m, quin fm (eds) strategy for sustainable maize production in west and central africa, proceedings of a regional maize workshop. iita-cotonou, benin republic, 21-25 april 1997. wecaman/iita. pp. 426-433. bard sk, barry pi (2001). assessing farmers‟ attitudes towards risk using the “closing-in” method. j.agric. res. econ. (food and agriculture organization), fao (2006), faostat, website 2007. 26(1): 248-260. hardaker jb, huire bm, anderson jr (1997). coping with risk in agriculture. cab international, wallingford, oxon, ox108de, uk. hoeri ae, kennard rw (1970a). ridge regression: biased estimation for non-orthogonal problems. technometrics, 18: 425-437. kim sk (1995). achievements, challenges and future direction of hybrid maize researcher and production in west and central africa. in b. badu-apraku,b, fakorede mab, ouedraogo m, quin fm (eds) contributing to food self sufficiency: maize research and development in west and central africa. proceedings of a regional maize workshop iita cotonu, benin republic. 29 may-2 june 1995. wecamn/iita pp. 42-28 lindeman rh, merenda pf, golo rz (1980). introduction to bivariate and multivariate analysis. scott, foresman and company, u.s.a. manyong vm, makinde ko, coulibaly o (2003). economic gains from maize research in west and ventral africa: an overview. in b.baduapraku, m.a.b. fakorede m ouedraogo, rj carsky, a menkir. (eds), maize revolution in west and central africa collaborative maize research network. iita, pp. 66-80. moscardi e, janvry a (1977). attitudes towards risk among peasants; an econometric approach. am. j. agric. econs. 59(4): 710-716. olarinde lo (2004). resource optimization strategies under differential risk attitudes among maize farmers in kaduna state, nigeria. unpublished ph.d. thesis, university of ibadan, nigeria. scandizzo pl, dillon jl (1976). peasant agriculture and risk preference in north east brazil: a statistical sampling approach. paper presented at cimmyt risk conference el batah, mexico, 9 th -15 th march. smith jg weber, mv manyong, mab fakorede (1997). fostering sustainable increase in maize productivity in nigeria,. in byerle d, eicher ck (eds). africa‟s emerging maize revolution. lunne rienner publishers, london. pp. 101-125. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (5), pp. 001-005, may, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper atherogenicity of cucumeropsis mannii and cucumis sativus oils from cameroon mercy bih achu1*, elie fokou1, clergé tchiégang2, martin fotso3 and felicité mbiapo tchouanguep4 1 department of biochemistry, faculty of science, p. o. box 812, university of yaoundé i, cameroon. 2 department of food science and nutrition, ensai/iut, ngaoundéré, cameroon. 3 centre for research in food and nutrition, impm, yaoundé, cameroon. 4 department of biochemistry, faculty of science, university of dschang, cameroon. accepted 15 march, 2019 the atherogenicity of cucumeropsis mannii and cucumis sativus oils was compared to that of corn oil and palm oil. female wistar albino rats of 6 weeks old weighing 58 – 65 g were randomly assigned to one of four diet groups: c. mannii and c. sativus oils (test diets) and corn and palm oil (control diets). there was weight gain in all the groups amounting to 128.65% (palm oil), 132.75% (c. mannii), 140.8% (c. sativus) and 153.45 (corn oil) with no significant difference. the weights of the livers ranged from 4.22 (palm oil) to 5.17 g (corn oil), ratio of weights of liver to that of rat, from 0.029 0.034 and percentage weight gain from 128.65 (palm oil) to 153.45% (corn oil). there was no significant difference in the values. for the atherogenic parameters measured, the triglyceride level ranged from 73 (palm oil) to 79.4 (c. sativus oil) with no significant difference. total cholesterol levels ranged from 49.6 (corn oil) to 64.2 mg/dl ( c. sativus oil) with significantly lower values in the corn oil group but similar values in the rest of the groups. hdl ranged from 18.94 (c. mannii oil) to 32.8 (palm oil) which was significantly high, ldl from 6.2 (palm oil) to 25.06 mg/dl ( c. mannii oil) and atherogenic ratio (ar) from 0.2 (palm oil) to 1.61 (c. mannii) which was significantly high. the levels of these atherogenic parameters are far below the borderline level for oils to cause atherosclerosis, indicating that they could be potential good edible oils for reducing cardiovascular illnesses. keywords: atherogenicity, oils, cucumeropsis mannii, cucumis sativus. introduction in africa, obesity has become a major problem in line with hiv. though obesity is traditionally seen as a sign of wealth, it is becoming a very serious health issue due to its complications (bbc news, 2004). in cameroon, according to information from an obesity clinic in the yaounde central hospital, 35% of the adult population is either overweight or obese and this often leads to diabetes and cardiovascular diseases. in addition to exercises and drugs (which usually have undesirable side effects), one of the ways of overcoming this health problem can be through the use of diets and foodstuffs, especially those that are locally available. cucurbitaceae (egusi) seeds are one of such foodstuffs. these seeds have been shown to be rich in proteins and oils and the *corresponding author. e-mail: lohmercy@yahoo.fr. oils contain mostly linoleic (an essential fatty acid which can only be got from the diet) followed by oleic acid (silou et al., 1999; murkovic et al., 1996; younis et al., 2000; achu et al., 2005; achu, 2006). essential fatty acids are important for normal foetal and infant growth and development, brain development and visual acuity (fao, 1994). the other essential fatty acids; linolenic acid, can be got from linoleic acid, and arachidonic acid from linolenic acid. arachidonic acid is a component of the liver, plasma phospholipids and cholesterol esters. it is an important precursor of prostaglandins, thrombosanes and prostacyclins. prostaglandins stimulate contraction of smooth muscles. prostacyclins have an antagonistic effect to that of thrombosanes by inhibiting platelet aggregation, relaxing coronary arteries and lowering blood pressure (ottaway and apps, 1984). linoleic acid (polyunsaturated) moderately reduces serum cholesterol and ldl levels. oleic acid (monounsaturated) appears to be neutral in regard to ldl but modestly raises hdl (fao, 1994). there is little data on the atherogenicity of oils from cucurbit seeds cultivated in cameroon. the main objective of the study was to investigate the atherogenic property of oils extracted from cucumeropsis mannii and cucumis sativus seeds from cameroon which can be exploited at the alimentary levels. this was in order to determine the levels of serum triglycerides, total cholesterol (tc), high density (hdl) and low density (ldl) lipoprotein cholesterol, atherogenic ratio (ar) and proteins of rats fed with oils extracted from the seeds of c. mannii (egusi melon) and c. sativus (“ibo” egusi) seeds compared to rats fed with palm and corn oils diets. materials and methods sample collection and treatment c. mannii seeds were collected from ebolowa (in the south province) and c. sativus seeds from bafia (in the centre province), which are amongst the regions of great cultivation of these seeds in cameroon. the seeds were bought already sun-dried by the farmers, transported in polyethylene bags to the laboratory, wiped with filter paper and dried in an oven at 70°c to constant weight. they were ground in an electric grinder, put in airtight bottles and stored in the desiccator for analyses. extraction of the oils oils were extracted from the ground seeds by continuous extraction in a soxhlet apparatus for 8 h using hexane as solvent (aoac, 1980). the hexane was evaporated on a rotary evaporator and the oil obtained was dried in an oven at 60°c for 24 h to remove all traces of solvent. the experimental design the experimental design was 4 types of oils tested x 4 groups of rats x 5 rats per group. that is: 4 oils x 4 groups = 4 x 4 groups. formulation of the diets given to rats the diet was formulated by weighing the various components according to the american institute of nutrition-76 (ain-76) composition, modified for casein level as follows: casein 10%, starch 36%, -cellulose 5%, vitamin mixture 1%, choline chloride 0.1%, dlmethionine 0,3%, sucrose 27.6%, salt mixture 5%, oil 5% (oil = corn oil, palm oil, oil extracted from c. mannii and c. sativus seeds). this diet was mixed with 10% of water to moisten the food for easy consumption by the rats. the food was given to the rats in the form of a paste of 20 g of food per rat. treatment of the rats 20 wistar albino rats of 6 weeks old weighing 58 – 65 g were used. one rat was put in a metabolic cage for an adaptation period of 5 days. during this time, they received the same food (that is, the food that was consumed by rats of the corn oil group which is the corn oil diet) and water ad libitum. they were then weighed and randomly distributed into 4 groups of 5 rats each, receiving 4 types of food differing only by the type of oil (corn oil and palm oil for the control groups and c. mannii and c. sativus oils for the experimental or test groups). the feeding took 3 months. this was to allow enough time to follow up the effect of the oils tested on the metabolism of the rats. the rats were weighed every 3 days. 12 h before sacrifice, these rats were left to fast. they only took water. on the morning of the sacrifice, they were weighed and sacrificed by decapitation. the blood of each rat was collected into dry 10 ml test tubes and centrifuged at 3000 rpm/min for 5 min. the separated serum was collected into 2 ml ependoff tubes. the serum was divided into 2 parts. the first part was preserved in aliquots at 4°c for hdl cholesterol analysis and the second part at -28°c for the rest of the analysis. the liver of each rat was equally collected and weighed in order to see the effect of the oils tested on the size of the liver. analysis of serum samples triglycerides, total and hdl cholesterol were assayed enzymatically according to the method on the randox kits used, while ldl levels were calculated using the formula of friedewald et al. (1972). the ar ratio was also calculated and the serum protein levels were assayed according to the method of gornall et al. (1949). statistical analysis the kruskal-wallis test (for the distribution of the sample as shown by the kolmogorov test was non normal) was used to find differences between the parameters measured in the rats that received diets prepared with the different oils. the studentnewmankeuls (s-n-k) test was used to locate these differences. the tests were done at 5% level of significance, using spss 10.1. results table 1 shows the amounts of food ingested, the evolution of the body weights of the rats and the ratio, weight of liver/weight of rat. the amounts of food ingested per rat per day ranges from 9.68 (corn oil) to 11.06g ( c. mannii oil). at the start of the experiment, the weights of the rats ranged from 58.28 (c. sativus oil) to 64.91 g (c. mannii). at the end of the experiment, they ranged from 139 (c. sativus) to 151.07g (c. mannii). there is an increase in weight in all the groups with a gain of 80 (palm oil) to 90.52 g (corn oil) amounting to 128.65% (palm oil), 132.75% (c. mannii), 140.8% (c. sativus) and 153. 45% (corn oil), showing no significant difference. the weights of the livers range from 4.22 (palm oil) to 5.17g (corn oil), ratio of weights of liver to that of rat, from 0.029 (palm and c. mannii oils) to 0.034 (corn oil). there is no significant difference between the food ingested and the weights of these rats that received diets prepared with the different types of oils. table 2 shows the serum levels of triglycerides (tg), total cholesterol (tc), hdl and ldl cholesterol, atherogenic ratio (ar) and serum proteins. no significant differrences were observed in the total tg levels among the four groups. however, total serum cholesterol was significantly higher in the rats on c. sativus compared to the rats on corn oil diet. there was no significant differrence in serum hdl cholesterol among palm oil and c. table 1. evolution of body weights of rats and the ratio, weight of liver/weight of rat. food initial weight final weight weight gain weight of liver/rat groups ingested % weight gain of rats (g) of rats (g) (g) liver (g) weight (g/rat/day) corn oil 9.68 ± 2.58 58.89± 3.82 149.40± 19.03 90.52± 16.67 153.45± 24.68 5.17± 1.26 0.034± 0.005 palm oil 9.77 ± 1.93 63.06 ± 3.36 143.73 ± 6.11 80.66 ± 8.36 128.65 ± 19.09 4.22 ± 0.37 0.029± 0.002 c. sativus oil 10± 1.67 58.28 ± 5.23 139.85 ± 6.37 81.57± 2.83 140.80± 12.58 4.42 ± 0.26 0.032 ± 0.001 c. mannii oil 11.06 ± 1.72 64.91 ± 3.17 151.07± 12.86 86.16 ± 11.49 132.75 ± 16.76 4.32 ± 0.28 0.029± 0.002 no significant differences were observed between groups in the amount of food ingested and in the total weight gain. table 2. serum levels of triglycerides, total cholesterol, hdl and ldl cholesterol, atherogenic ratio and serum proteins. 0 triglycerides total hdl ldl atherogenic 0 total groups (mg/dl) cholesterol cholesterol cholesterol ratio, proteins (g/l) (mg/dl) (mg/dl) (mg/dl) (ldl/hdl) corn oil 76.2± 12.66 49.6± 4.93 b 25.92± 2.52 ab 8.44± 1.14 b 0.33± 0.05 b 79.04 ± 13.4 palm oil 73 ± 10.2 53.6± 7.99 ab 32.8± 6.53 a 6.2 ± 4.57 b 0.20± 0.13 b 81.57± 8.01 c. sativus oil 79.4 ± 10.06 64.2± 8.64 a 27.72± 4.18 ab 20.6± 8.65 a 0.77± 0.38 a 78.17± 11.83 c. mannii oil 75 ± 6.96 59 ± 6.82 ab 18.94± 7.14 b 25.06± 7.25 a 1.61 ± 1.05 a 84.71±15.37 kruskal-wallis test: 0 = there is no significant difference (p>0.05) between values in the same column. student-newman-keuls test: values in the same column with different letter superscripts are significantly different (p<0.05). sativus oil. however, hdl cholesterol was significantly higher in the rats on palm oil compared to those on c. mannii oil (p<0.05). ldl cholesterol was significantly higher in the rats on c. mannii and c. sativus oils compared to the control diets, corn oil and palm oil. similarly the atherogenic ratio (ar) was significantly higher in the rats on c. mannii and c. sativus oils compared to corn and palm oil diets. total serum protein was not signifycantly different among groups. discussions the triglyceride (tg) levels were 75 in c. mannii and 79.4 in c. sativus oil rats compared to 73 in palm oil and 76.2mg/dl in corn oil rats, but with no significant differrence. however, from the results of analysis of the quality of c. mannii and c. sativus oils, c. sativus oil shows lower levels of free fatty acids (acid index: c. sativus oil = 1.76 and c. mannii oil = 4.56), higher degree of unsaturation (iodine index: c. sativus oil = 114.4 and c. mannii oil = 106.78; unsaturated fatty acids: c. sativus oil = 78.4% and c. mannii oil = 54.7%; saturated fatty acids or sfa: c. sativus oil = 21.7% and c. mannii oil = 42%), lower level of peroxides (peroxide index: c. sativus oil = 3.31 and c. mannii oil = 13.68), lower r1 value (r1 is the ratio of the sum of saturated to that of unsaturated fatty acids. the r1 of c. sativus oil = 0.28 and c. mannii oil = 0.77) and lower linolenic acid level (linolenic acid: c. sativus oil = 0.1% and c. mannii oil = 0.3%) (achu, 2006). these suggest that the quality of c. sativus oil is better than that of c. mannii oil and that c. sativus oil contains more unsaturated fatty acids than c. mannii oil. c. sativus and c. mannii oils have high levels of polyunsaturated fatty acids, pufa (39.3% in c. mannii oil and 62.2% in c. sativus oil) as in corn oil (58.5%) while palm oil has a low level (8.5%) (table 3). these results are similar to those of noubissi in djamen (1998), who after 2 months of experiment, found a non significant decrease in the tg level of rats fed with palm oil diet (rich in sfa) compared to those fed with cotton oil diet (poor in sfa). however, these results show that these egusi oils can be good edible oils (do not cause hypertriglyceridaemia) because their tg levels which are 75 in c. mannii oil and 79.4 mg/dl in c. sativus oil rats, are far below 150 mg/dl, the borderline level of tg, above which the person has hypertriglyceridaemia (randox assayed multisera, 2004b). the total cholesterol (tc) level of c. sativus oil (64.2) is similar to those of c. mannii oil (59) and palm oil (53.6) but significantly higher than that of corn oil rats (49.6mg/dl). c. sativus and corn oils all have high levels of pufa which are 62.2 and 58.5% respectively, due to their high linoleic acid levels. but c. sativus oil has higher levels of sfa (21.7%) than corn oil (16.5%) (table 3). hence, the high tc level in c. sativus oil compared to corn oil rats may be due to its higher sfa levels. however, the tc levels of all these four groups of rats were low when compared to levels that cause hypercholesterolaemia. secondly, other studies have shown that the hypercholesterolaemic effect of sfa is mostly due to c14:0 and c12:0 fatty acids rather than c16:0 (ng, 1994). c. sativus and c. mannii oils contain little or no table 3. fatty acid composition of oils used. corn oil palm oil c. sativus oil c. mannii oil (achu, fatty acid symbol (ngogang et al., (fao, 1981) (achu, 2006) 2006) 1996) caprylic acid c8:0 capric acid c10:0 lauric acid c12:0 1.5 myristic acid c14:0 1 3.5 4.7 palmitic acid c16:0 12 40.6 10.7 24.4 palmitoleic acid c16:1 0.5 0.8 stearic acid c18:0 2 4 10.6 11.2 oleic acid c18:1 24 43 16.2 14.6 linoleic acid c18:2 56.5 8.5 61.8 38.7 linolenic acid c18:3 2 0.1 0.3 arachidonic acid c20:0 1 0.4 0.2 gadoleic acid c20:1 0.5 arachidonic acid c20:2 0.3 0.3 behenic acid c22:0 lignoceric acid c24:0 0.5 total saturated fatty acids 16.5 48.1 21.7 42 total mufa 25 43.4 16.2 15.4 total pufa 58.5 8.5 62.2 39.3 total unsaturated fatty acids 83.5 51.9 78.4 54.7 pufa/sfa (r2) 3.55 0.17 2.87 0.93 = not found, mufa = monounsaturated fatty acids, pufa = poly unsaturated fatty acids. c14:0 and c12:0 fatty acids. their sfa are mostly c16:0 as in corn and palm oils (table 3) which do not lead to much rise in cholesterol levels. also, the non-hypercholesterolamic action of these oils is possibly due to their presence of tocotrienols, the unsaturated analogue of tocopherols. tocotrienols inhibit cholesterol synthesis in vivo, exerting a hypocholesterolamic action in humans and animals. this has been shown in palm oil by rukmini (1994). however, these results show that these oils are good edible oils (do not cause hypercholesterolaemia) because their tc levels (59 in c. mannii oil and 64.2 mg/dl in c. sativus ) are far below 200 mg/dl, the desirable blood cholesterol level, above which the person has hypercholesterolaemia (randox, 2004a). the hdl level of c. sativus (27.72) is similar to those of c. mannii (18.94), corn oil (25.92) and palm oil (32.8 mg/dl) rats. the hdl level of c. mannii oil is similar to those of c. sativus and corn oil rats but significantly lower (p<0.05) than that of palm oil rats (table 2) . this lower hdl value in c. mannii compared to palm oil rats may be due to the lower level of mufa in c. mannii (15.4%) than palm oil (43.4%) and higher pufa in c. mannii (39.3%) than palm oil (8.5%) (table 3) . this is in line with the results of a study carried out by o’callaghan et al. in during et al. (2000) who found that patients fed pufa dairy products showed lower plasma hdl levels than patients fed mufa dairy products. this might be the case with these oils where c. mannii had higher pufa, lower mufa and showed lower hdl levels than palm oil with lower pufa, higher mufa and high hdl levels. the ldl level of c. mannii oil rats (25.06) is similar to that of c. sativus (20.6) and these are significantly higher than those of corn (8.44) and palm (6.2) oil rats, with similar ldl levels. apart from increasing hdl levels, high mufa also reduce ldl levels. this high ldl level and the eventual higher atherogenic ratio in c. mannii and c. sativus oils may be due to their lower mufa and higher pufa levels as opposed to corn and palm oil rats. this is seen in the study carried out by during et al. (2000) where rats fed with experimental cheeses containing mostly mufa resulted in a significant increase of hdlcholesterol (11%) and a significant reduction of ldlcholesterol (31%). other findings have shown that, in addition to unsaturated tg, dietary phytosterols in vegetable oils can also reduce plasma ldl levels (jones et al. in during et al., 2000). hence, high hdl and low ldl levels are due to high mufa (low pufa) and dietary phytosterols (as in palm oil) and low hdl and high ldl levels are due to low mufa, high pufa and possibly low phytosterols (as in c. mannii and c. sativus oils). however, the hdl levels of c. mannii (18.94) and c. sativus oil rats (27.72mg/dl) are below the desirable hdl level (>40 mg/dl) and their ldl levels 25.06 and 20.6 mg/dl respectively are far below the borderline (<130 mg/dl) (american academy of family physicians, 2005). this means that these oils could be very good in reducing ldl and to a lesser extent, raise hdl levels. however, these oils need to be tested on humans in order to better confirm these findings that were carried out on wistar albino rats. the atherogenic ratio, ar (ldl/hdl) in c. mannii oil rats (1.61) is similar to that of c. sativus (0.77) and these are significantly higher (p<0.05) than those of corn (0.33) and palm (0.2) oil rats with similar ar levels. these high ar levels in c. mannii and c. sativus oil rats are due to their high ldl levels, caused by their low mufa levels. although the atherogenic ratio is higher in the test groups (than the palm oil and corn oil groups), these ratios are good, for they are below 3.55 in men and 3.22 in women, the borderline levels for lipids to cause atherosclerosis. above these values, the risk of appearance of cardiovascular illness is high (laboratoires fournier, 1981). the serum protein levels are 84.71 g/l in c. mannii and 78.17 in c. sativus oil rats compared to 79.04 in corn and 81.57 g/l in palm oil rats but with no significant difference in all these values. conclusion this study which was aimed at investigating the atherogenic property of oils extracted from c. mannii and c. sativus seeds from cameroon showed that these oils are good in reducing ldl cholesterol levels thereby reducing the atherogenic ratio, for the ar levels were far below the threshold values expected to cause athero-sclerosis. c. mannii and c. sativus oils could be potential good edible oils for use in the reduction of cardiovascular illnesses. there is the need to carry out this study on humans to better confirm these findings that were carried out on wistar albino rats. acknowledgement this research was supported by the international foundation for science (ifs), stockholm, sweden and the organization for the prohibition of chemical weapons (opcw), the hague, netherlands, through a grant to mrs achu mercy bih. references achu mb, fokou e, tchiégang c, fotso m, tchouanguep fm (2005). nutritive value of some cucurbitaceae oilseeds from different regions in cameroon. afr. j. biotechnol. 4(10): 1329-1334. achu mb (2006). a study of some physicochemical characteristics and nutritional properties of five cucurbitaceae oilseeds from cameroon. thesis presented and defended for the award of a doctorate phd in biochemistry. faculty of science, university of yaounde i. p. 180 american academy of family physicians (2005). cholesterol: what your level means. heart disease and stroke. http://familydoctor.org/029.xml.htm. access date 05/12/2005. aoac (1980). official methods of analysis of the association of official analytical chemists. 13 th ed. william horwitz ed. washington d.c. 7: 56-132 bbc news (2004). obesity epidemic out of control. bbc news world africa. sunday, 31 october, 2004, 15:22 gmt. http://news.bbc.co.uk/2/low/africa/3969693.stm access date 15/03/2007. during a, combe n, mazette s, entressangles b (2000). effects on cholesterol balance and ldl cholesterol in the rat of a soft-ripened cheese containing vegetable oils. j. ame. college of nutr. 19 (4): 458-466 fao (1981). le rôle des graisses et huiles alimentaires en nutrition humaine. alimentation et nutrition; 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seed and variability in fatty acid composition of seed oil. phytochem. 54 (1): 71-75 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (3), pp. 001-005, march, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper characteristics of snail farmers and constraints to increased production in west and central africa a. a. ngenwi1*, j. m. mafeni1, k. a. etchu1 and f. t. oben2,3 1 institute of agricultural research for development (irad) ekona, p. m. b. 25 buea, south west region, republic of cameroon. 2 department of crop science, university of ghana, legon, ghana. 3 faculty of agronomy and agricultural sciences, university of dschang – cameroon. accepted 12 october, 2018 snail gathering/rearing is an important source of livelihood for rural dwellers in the humid forest and derived savanna zones of west and central africa. recently, declining populations of archachatina archachatina and archachatina marginata species is being observed, with climatic conditions and changes in land use implicated as causal factors. however, these claims are still to be ascertained. a study was carried out to characterize snail farmers, identify constraints to increased snail production and suggest strategies required to conserve edible snail species and sustain livelihoods. structured questionnaires and focus group discussions were used for data collection in selected locations in cameroon and ghana. descriptive statistics (means and percentages) was used to summarize the data obtained. the results indicate that snail farmers are predominantly women with basic school level of education. between 42 and 62% of rural income is derived from sale of snails. causes of declining snail populations were attributed to habitat loss through deforestation, overexploitation, indiscriminate harvesting, climate change indicated by extremely high temperatures and low rainfall, high dependence on agricultural chemicals (herbicides and pesticides) and lack of training on improved snail husbandry. a holistic approach to biodiversity conservation and capacity building of current and would-be snail farmers is suggested to increase snail supply in markets. key words: archachatina spp, non-conventional livestock, climate change, africa. introduction snail is an important source of animal protein in many parts of west and central africa (blay et al., 2004), having good quality protein (69% dry weight) and rich in potassium, phosphorus, essential amino acids and vitamins c and b complex (baba and adeleke, 2006, okpeze et al., 2007).traditionally, rural folk scout freely in the forest and farmlands to collect snails during the rainy season for sale and domestic consumption. in view of the high quality of protein obtained from snails they have secured high demand in many cuisines both locally and internationally. other advantages in snail farming over most other livestock include low capital requirement for its establishment *corresponding author. e-mail: anna12672@yahoo.com. tel: +23775300487/ 94492072. and operation, less demand for professional knowledge, very high fecundity and low mortality, less labor requirement, the animal’s noiseless-ness, and availability of ready domestic and international markets, among others (cobbinah, 1993; baba and adeleke, 2006) . recently, the production of snail has not kept pace with demand (etchu et al., 2008) with different environmental and technical factors implicated. snail supply from the wild would be further affected by issues of climate variability and change. climate change is already having a negative impact on africa through extremely high outdoor temperatures (ipcc, 2007), which are not favorable to snail growth and development. domestication and intensive management of the edible land snail is inevitable. research on climate change has focused more in the tropics on crops, less on livestock and little on biodiversity conservation. change in table 1. characteristics of snail farmers and marketers. characteristics percentage (%) sex male 40 female 60 level of education primary and/or secondary 82 wild 30 source of snails backyard 4 wild and backyard 66 contribution to income gatherers 42 marketers 62 received training on snail husbandry 28 species abundance can provide useful insights on climate change and drivers of this change. however, there is death of information on this in most parts of the tropics. this study was therefore undertaken to identify immediate and remote causes of low production in cameroon and ghana. materials and methods the study was carried out in volta and greater accra regions in ghana and the southwest region in cameroon, between august and october, 2009. greater accra and volta regions fall within the humid forest and derived savanna zones and the southwest region of cameroon is exclusively a humid forest ecosystem. questionnaires were administered in the above-mentioned regions to 120 respondents (60 in each country). respondents were randomly selected from lists provided by agriculture officers. questions were designed to provide background information on socio-economic status of the farmers, causes of declining snail population and other production constraints. farmers were also asked if they had received any advice on snail rearing from agricultural extension officers and non governmental organizations (ngos) and how this information (if adopted) has changed their production practices. some claims of causes (e.g. low rainfall and high temperatures) in declining snail population were verified using available secondary data. climatic data for the past 40 years (19712009) for hohoe district was provided by ghana meteorological service. data collected was analyzed using spss software. descriptive statistics (means and percentages) provided insights into farmers’ perceptions of climate change. the study was exploratory and did not seek to compare findings from the two countries. results characteristics of snail farmers table 1 gives a summary of characteristics of snail farmers/marketers. these farmers were predominantly females (60%) and have basic primary and/or secondary education (82%). farmers, mostly women and children cover a distance of between 2 and 5 km from their homes at night to gather snails. thirty percent of them source snails from the wild, 4% exclusively from backyard rearing and 66% from wild and backyard. snail farming accounts for 42% of total income of rural farmers and for 62% of income of marketers. an average monthly income of 54,650 francs (us $ 121.00) is generated by small-scale snail farmers in cameroon. such information was not provided by ghanaian farmers. eight environmental factors (table 2) were identified as constraints to increasing snail population in the wild, with deforestation, high dependence of farming operations on agro-chemicals (principally pesticides), and high incidence of pests and indiscriminate harvesting of snails (small and large) as major factors. rainfall anomalies (annual rainfall amount minus 40 years average) between 1971 and 2009 are shown in figure 1. the solid line represents 5 years moving average. the data revealed an alarming decreasing trend in rainfall amount between 1976 and 1986. more frequent extreme rainfall events have been observed since 1977. between 1980 and 1986, rainfall deficit was recorded in 5 out of 7 years, with severe deficits (drought) in 1977, 1982, 1986 and 1994. the moving average clearly reveals a tendency of experiencing low rainfall amounts in the coming years. inter-annual variability in rainfall amount varied between 3 and 40%, with high variability characterizing rainfall between late 1980s and 1990s. farmers’ perception of high rainfall variability, decreasing rainfall amounts in ghana is supported by rainfall data available for one of the test sites. high temperatures were also confirmed by meteorological records (figure 2). mean annual temperatures vary between 26°c and 28°c, with a tendency of approaching 30°c in the near table 2. environmental factors implicated for reducing snail population identified by snail farmers. factor percentage of respondents (%) demographic pressure (deforestation) 38 high dependence on agrochemicals 16 high incidence of pests 14 indiscriminate harvesting 12 low rainfall 6 wild fire 6 high temperature 4 continuous cropping 2 . figure 1. annual rainfall residuals and moving average (solid line) for hohoe district (ghana). figure 2. annual temperature residuals and moving average (solid line) for hohoe district (ghana). future. with respect to intensive snail farming, 71% of respondents identified lack of training of rural farmers on improved management techniques as the major impediment to increased snail supply. all farmers interviewed in ghana reported feeding of snails to be less of a problem, as the giant snail which they rear is essentially vegetarian and browses on a wide range of leaves. in cameroon, farmers complained of slow growth rates using local diets. of all the farmers interviewed, only 28% had received advice from ngos and agricultural extension officers, and all of them reported low mortalities and increased production. discussion the low dependence of farmers/gatherers on snails from the wild is indicative of the impact of environmental and anthropogenic factors on edible snail populations. in addition, the extremely low percentage of farmers who practice exclusive snail rearing in their backyards (4%) implies that there is potential for improving supply through intensive backyard farming. significant advances have been made in snail research to establish low-cost housing (etchu et al., 2008), appropriate stocking densities (omole et al., 2006) and suitable readily available high quality feeding materials (agbogidi et al., 2008), all in a bid to improve backyard rearing. compounded feeds are available in the market in ghana for enhancing the fattening process, where large scale production is anticipated (blay et al., 2004). in cameroon there is a need to formulate compounded rations as farmers complain of very slow growth rates using local diets. given the significant contribution of snail farming to the income of rural farmers, much thought need to go into efforts to reduce deforestation, and improve farmers’ capacity of intensifying backyard production. appiah et al. (2009) suggested agro-forestry practices (with snails, bee keeping etc. as components) in view of tropical deforestation in ghana which endangers community’s dependence on forest resources. agricultural expansion and over-exploitation of forest resources are the fundamental causes of deforestation (appiah et al., 2009). because of climate change, new pests have been observed in many farmlands in addition to increasing population of existing ones, which in many cases have developed resistance to certain concentrations of pesticides forcing farmers to use higher doses and/or new formulations. in west and central africa, the agricultural sector faces several challenges of which pests and crop diseases are major bottlenecks. indiscriminate harvesting of snails would reduce the potential number of eggs to be laid, with negative effects on future populations. a. archachatina is the most priced species for consumption in ghana and is becoming rare (cobbinah et al., 2008). climatic variables are among the determinants of growth and survival of living organisms. land snails prefer humid environments for their optimum performance in the presence of their choice food (ejidike et al., 2004). decreasing rainfall and increasing temperatures would alter the conditions required for optimal growth and reproduction. average body weight gain in archachatina marginata is inversely proportional to temperature and directly proportional to rainfall (edijike et al., 2004) . a sharp decrease has been reported in body weight of a. marginata exposed to temperatures > 30°c (ejidike at al., 2004). temperatures in west africa have evolved faster than the global trend, with increases in the range 0.2 to 0.8°c since the end of the 1970s (ecowasswac/oecd, 2008). snail gatherers and marketers adopt measures (regular wetting of their enclosures) to reduce the temperatures their snails are exposed to in order to keep them alive for the market. conclusion snail farming constitutes a major part of the income (42 62%) of rural farmers, who are predominantly women and have limited alternative sources of livelihood. most of them have attained primary or secondary school education. environmental factors such as deforestation, increasing temperatures, low rainfall, bush fires, indiscriminate harvesting, high use of agrochemicals and lack of training on intensive snail rearing were identified as impediments to increased snail supply from the wild and in captivity. efforts should be made to reduce the effects of climate change through the adoption of agro forestry systems, and practices that reduce dependence on agrochemicals, as well as intensify training on improved management practices and the formulation of high quality diets for rapid growth and high nutritional quality. references agbogidi om, okonta bc, ezeana el (2008). effects of two edible fruits on the growth performance of african giant land snail (archachatina marginata swainson). j. agric. biol. sci. 3: 26-29. appiah m, blay d, damnyag l, dwomoh fk, pappinen a, luukkanen o (2009). dependence on tropical resources and tropical deforestation in ghana. environ. dev. sustainability.11: 471-487. baba km, adeleke mt (2006). profitability of snail production in osun state, nigeria. j. agric. food sci. 4: 147-155. blay et, ofori bd, heloo j, ofori jb, nartey e (2004). agrodiversity within and without conserved forests for enhancing rural livelihoods. in: gyasi ea, kranjac-berisavljevic g, baly et, oduro w (eds.) managing agrodiversity the traditional way: lessons from west africa in sustainable use of biodiversity and related natural resources. tokyo: united nations university press, pp. 203-214. cobbinah, jr (1993). snail farming in west africa. a practical guide. cta publication. cobbinah, jr, vink a, onwuka b (2008). snail farming: production, processing and marketing. agromisia foundation, wageningen. first edition pp. 78-82. ecowas-swac/oced (2008). climate and climate change. the atlas on regional integration in west africa. environment series. january 2008. www.atlas-westafrica.org. ejidike bn, afolayan ta, alokan ja (2004). observations on some climatic variables and dietary influence on the performance of cultivated african giant land snail (archachatina marginata): notes and records. parkistan j. nutr. 3: 362-364. etchu k, mafeni, m, ngenwi a (2008). comparative performance of three edible snail species using intensive cage housing system in cameroon. bull. anim. health prod. afr. 56: 345-352. ipcc (2007). climate change 2007: impacts, adaptation and vulnerability. contribution of working group ii to the fourth assessment report of the intergovernmental panel on climate change, m.l. parry, o.f. canziani, j.p. palutikof, p.j. van der linden and c.e. hanson, eds., cambridge university press, cambridge, uk, p. 976. okpeze, cn, omole, aj, ajayi ft, adebowale ea (2007). effects of feeding adult snails stylosanthes guianensisor lablab purpureus as substitute for pawpaw leaf. afr. j. biotechnol. 6: 1959-1962. omole aj, oluokun ja, fapounda jb, osayomi j (2006). the effects of different stocking rates on growth and reproductive performance of breeding snail (archachatina marginata) under intensive system of production in the humid tropics. w j. zool. 1: 33-35. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (3), pp. 001-004, march, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper qualities of commercially and experimentally sun dried fin fish, scomberoides tol jamila patterson1* and govindan ranjitha2 1 suganthi devadason marine research institute, tuticorin, india 2 marudupandiyar college, thanjavur, india. accepted 12 september, 2019 seafood is a popular food item and has been preserved by several ways. curing is one of the simplest methods to preserve fish to meet out the need of the people. in india, fishes are washed, gutted and sun dried on the ground, surface of rocks, wooden platforms, palm leaves and also on sandy beaches. the export of indian cured fishery products are reduced because of their poor quality. the nutritive quality of one of the commercially sun dried fish, somberoides tol was assessed. the same species was sun dried hygienically by keeping on fish drying rack under direct sun light and moving air and their qualities were assessed. the present study reveals that the fish was dried experimentally on fish drying rack that had good nutritional qualities and hygienic compared to the commercially sun dried fish from the same species. key words: fin fish, scomberoides tol, commercially and experimentally sun dried, nutritive quality, microbial quality. introduction salting and drying is an ancient and simplest method to preserve fish. in india about 17% of the total catch is being used for salting and drying (anon, 2001). the fish must be dried quickly and cleaned through sunlight and moving air. this protects the fish from microbes, insects and dirt. salting and sun drying of fish is a traditional method of seafood preservation employed in many countries. on the global basis, 14% of the marine landings are processed by curing (sanjeev and surendran, 1996). curing is the simplest method to preserve fish. in india, utilization of dried fish comes next to fresh fish about 8 million tons of fish (25-30%) of the world catch are being used for human consumption as dried, salted, smoked or treated by some combination of these processes (kamruzzaman, 1992) . curing is a simple and cheapest method of processing requiring least technical expertise, but it has great significance and relevance in the socioeconomic system of small-scale fisher folk. in india, most of the marine landings are being processed into cured products. this is still applied to a larger extent to preserve fish and squid (sikorski et al., 1995). fishes are washed, gutted and sun dried on ground, rocks, wooden *corresponding author. e-mail: jamilapat@gmail.com. platforms, palm leaves and also on the sandy beaches. the possibility of contamination is obvious in sun dried fishes using these traditional methods and sun drying on these substrates. the fishes dried slowly and unhygienically in direct sunlight in the absence of moving air. the cured fishery products have good potential for internal market and exported to various south and south east asian countries such as sri lanka, hong kong and singapore. during the past few years there has been a decline in the exports of indian cured fishery products (sugumar et al., 1995). the nutritional qualities of the dried fishes are the major attribute for these decline. only few studies on the biochemical and nutritional changes are available on biochemical and nutritional changes occurred during dehydration (raghunath et al., 2000). during the past few years, there has been a decline in the export of indian cured fishery products (sugumar et al., 1995) mainly because of their poor quality. this causes considerable loss to the fish curing industry in india. sugumar et al., 1995 suggest that the quality of dry fish available in the country requires much improvement. fungal contaminations are a common problem and it adversely affects the quality of cured fishes. the presence of these fungi in salted and sun dried fishes are acquiring importance in view of the safety and quality of the seafood. valsan et al. (1985) reported on the quality table1. percentage of nutritive values of raw and sun dried fin fish. species protein (%) carbohydrates (%) lipids (%) free fatty acids (%) sample a (fresh fish) 26 32.096 1.6 0.008 sample b (experimentally sun dried fish) 17 15.98 1.2 0.011 sample c (commercially sun dried fish) 8 13.872 1.4 0.014 of cured fishes in different parts of the country. chakrabarti and varma (1999) reported the quality of cured fishes of vishapatinam coast of india. in the fish curing yards of tuticorin coast, different varieties of fishes are washed in seawater and are immersed in 20% of brine for 1624 h and sun dried for 2-3 days (sugumar et al., 1995) and the same method is being followed till date with little modification. even though preservation of fishes by salt curing has long been practiced in tuticorin as a traditional technique, the ratios of salt-to-fish are too low to ensure adequate preservation (shanthini and patterson, 2002). microbiological and biochemical quality assessments are necessary to ensure the food safety of any processed products. therefore, in the present study, the biochemical and microbiological analysis were carried out to assess the qualities of commercially and experimentally sun dried fin fish, somberoides tol materials and methods the commercially valued fin fish s. tol was selected for the present study. fresh raw fish piece was bought from local market and brought to laboratory in a plastic bag. a small piece was cut from the raw fish and dried in a mechanical drier for biochemical and microbial analysis of the raw fish. fish drying rack was designed based on king and johnson, 1987. fish drying rack was made using wooden poles and nets. 4 wooden poles were taken and their one end is buried in sand and a square shape was formed. then polythene net was thoroughly washed, sun dried and tied on the top and the square area was covered. now the fish drying rack is ready for sun drying the fish. then a large piece of what was salted and sun dried experimentally (sample b) by keeping on fish drying rack. the commercially sun dried fish (sample c) piece was brought from local market. all the sun dried fish samples were powdered separately and packed in polythene bags and stored at 4°c for microbiological and biochemical analyses. estimation of biochemical composition biochemical parameters like proteins, lipids, carbohydrates and free fatty acids were estimated by following standard methods. the amount of proteins in the sample was estimated by biuret method (raymont et al., 1964), carbohydrate content was estimated by the phenol sulphuric acid method (dubois et al., 1956), lipid was estimated according to the method of bligh and dyer (1959) and the free fatty acid, which is an indication of the quality of fat, was estimated by the method of ke et al. (1976). microbial analyses the total plate count and fungal count were enumerated using the standard methods (apha, 1992) . escherichia coli was enumerated using the standard method of most probable number (mpn) technique (apha, 1992) and salmonella sp. was enumerated by the pour plate method recommended by the us food drug administration (1998). results the biochemical parameters like proteins, lipids, carbohydrates and free fatty acids content of raw fin fish s. tol, commercially and experimentally sun dried samples were estimated and the results are presented in table 1. the nutritive value of raw fish, s. tol (sample a) was found to be good. it had 26% of protein, 32.096 % of carbohydrate, 1.6% of lipid and 0.008% of ffa. the protein, carbohydrate and lipid quantity is reduced in sun dried fishes. the nutritive value of experimentally sun dried fish (sample b) was found to be 17% protein, 15.98% carbohydrate, 1.2% lipid and 0.011% ffa, while low nutritive value was observed in commercially sun dried fish (sample c) such as 8% protein, 13.872% carbohydrate, 1.4% lipid and 0.014% ffa. among the commercially and experimentally sun dried fish samples, the protein, lipid and carbohydrate contents were high in experimentally sun dried fish sample whereas the free fatty acid content was found to be high in commercially sun dried fish followed by experimentally sun dried fish. the low nutritive value in commercially sun dried fish sample is mainly due to improper handling, inadequate preservation and unhygienic mode of drying the fish samples. the microbiological analyses also showed variations among the samples. the changes in enumeration of total viable bacteria (total plate count, tpc), total fungal count (tfc), e. coli and pathogenic bacteria, salmonella sp. were assessed and the results are given in table 2. the pathogenic bacterial colonies were tested using the methods of apha (1992) and kiss (1984). total plate count was observed in all the 3 samples. it was higher in commercially sun dried fish sample table 2. microbiological analysis of sun dried samples. species tpc (cfu/ml) tfc (cfu/ml) e. coli (mpn) value salmonella sample a (fresh fish) 4 × 10 -4 1 × 10 -2 2.9 absent sample b (experimentally sun dried fish) 28 × 10 -4 0.27 absent sample c (commercially sun dried fish 60 × 10 -4 1 × 10 -2 0.26 absent (sample c) than the fresh fish (sample a) and experimentally sun dried fish sample (sample b) . total fungal count was observed in samples a and c and it was count was observed in samples a and c and it was absent in the experimentally sun dried fish samples. e. coli count was found to be high in commercially sun dried fish sample (sample c) than the other samples. (e. coli was found to be more in experimentally sun dried than commercially sun dried). salmonella sp. was not detected in all the three fish samples. the highest count of tpc and e. coli in commercially sun dried fish sample revealed the unhygienic condition prevailed during the drying process. discussion fish acts as protein supplement for the people living in 63% of countries (bangladesh dof, 2001). the protein content was found to be high in experimentally sun dried fish (sample b) than the commercially sun dried fish (sample) . the increase in drying time and loss of moisture content leads to protein denaturation. the protein content was higher in raw fish than the sun dried fish and it is mainly due to protein denaturation of reducing moisture level ref. the free fatty acid levels (1% oleic acid) of all the sun dried fish samples were higher than the raw fish. in the present study, high amount of free fatty acid content was observed in commercially sun-dried fish (sample c) than the experimentally sun dried fish sample (sample b). the increase amount of ffa is directly related to the degree of spoilage (ref). in the present study, commercially sun dried sample (sample c) shows high ffa content due to long storage period. the lower ffa in raw fish (sample a) was due to the less degree of oxidation. the protein, lipid and carbohydrate contents were high in fresh fish samples (sample a) than the experimentally and commercially sun dried samples (sample b and c) due to the prevalence of unhygienic conditions, improper handling and inadequate preservation of the fishes (the statement is vague and should be supported by proper references) the total plate count and e. coli counts were found to be higher in commercially sun dried fish (sample c) which is available in tuticorin local market than the experimentally sun dried fish. high counts of tpc and e. coli in commercial sample was due to high content of moisture and humidity in the environment and unhygienic method of preparation. salmonella was not detected in the raw and sun dried fish samples. the total fungal count was also high in commercially sun dried fish sample (sample c) due to high moisture content and humidity. the cured fish in hot humid tropical climates are liable to deteriorate during storage due to mould growth. visible colonies appeared on the fish samples due to high moisture content and high relative humidity in the atmosphere (ref.) the biochemical and microbial analysis showed that the quality of experimentally sun dried fish sample was good than the commercially sun dried fish sample. experimentally sun dried fish was properly handled and well exposed to sun light and moving air and it dried quickly and the end product was clean and hygienic. conclusion the present study reveals that the fish dried on fish drying rack had good nutritional qualities and hygienic. the results were remarkable and therefore this simple but effective method of sun drying could be popularized among local coastal population to increase the effective utilization of marine resources in a better and hygienic way in order to enhance the value in local as well as export market. acknowledgement the authors are thankful to the director of suganthi devadason marine research institute (sdmri), india for providing us the permission to work together and facilities to carry out the work in sdmri laboratory. references anon (2001). hand book of fisheries stastistics. 2000. govt. of india, ministry of agriculture, new delhi. apha (1992). compendium of methods for the microbiological examination of foods, (vanderzant c, splittstoesser, d, eds.), apha washington dc. p. 3 bligh eg, dyer ws (1959). a rapid method of total lipid extraction and purification. can. j. biochem. physiol. 37: 911-917. bligh eg, shaw sj, woyewoda ad (1988). effects of drying and smoking on lipids of fish. in: fish smoking and drying – the effect of smoking and drying on the nutritional properties of fish.(ed) burt jr. elsevier science publishers, ltd, essex, england. pp. 41-52. chakrabarti r, varma prg (1999). halotolerant fungi in salted and dried fish at lower vishapatnam coast. fishery technol. 36 (1): 28-31. dof (2001). fisheries resource information of bangladesh (19992000). in: saronika of matshya saptha. ministry of fisheries and live stock. government of the peoples republic of bangladesh. dubois m, gillers ka, hamilton jk, redbers pa, smith f (1956). colorimetric method for determination of sugars and related substances. anal. chem. 38: 350-356. shanthini f, jamila c (2002). fungi in salted and dried fishes of tuticorin, southeast coast of india, seafood safety, society of fisheries technologists(indian) cochinindia. pp. 412-417. ke pj, reyier cw, ackman rg (1976). news series circular. fisheries and oceans. p. 61. kiss i (1984). in testing methods in food microbiology, (kiss i ed.). elsevier amsterdam, oxford, new york. p. 437. king j (1987). how to make fish drying racks. natural resources institute technical report, p. 7. kamruzzaman akm (1992). qualitative evaluation of some commercially dried fish products of bangladesh. m.sc thesis, department of fisheries technology, bangladesh agricultural university, mymensingh, bangladesh. p. 37. raghunath mr, sankar mr radhakrishnan tv, suseela mathew ag, ammu k, ravishankar cn, jayan k, leema j (2000). biochemical investigations on antartic krill, ephausia superba. fishery technol. 37(1):19-24. . raymont jec, austin a, linzford e (1964). biochemicl studies on marine zooplankton. i. biochemical composition of neomysis integer. j. cons. perm. explor. 28: 354-363. sanjeev s, surendran gh (1996). fate of enterotoxigenic staphylococci in fish subjected to curing. fishery technol., 33(1): 6668. sikorski ze, gildberg a, ruiter a (1995). fish products: fish and fishery products. in: ruiter a (ed.). cab international wallingford, uk. pp.315-346. sikorski ze, gildberg a, ruiter a (1995). in fish and fishery productscomposition, nutritive properities and stability ( ruiter a(ed.). cab international wallingford, uk. p. 315. sugumar g, jawahar abraham t, jeyachandran p (1995). sanitation in fish curing yards of tuticorin, tamilnadu. fishery technol., 32 (2): 136-138. shanthini f, patterson j (2002). fungi in salted and sun dried fishes in tuticorin, southeast coast of india. presented in “symposium on seafood safety: status and strategies “ organized by society of fisheries technologists (india) and cift, cochin during may 28 – 30. us food drug adminstration (usfda) (1998). bacteriological analytical manuals. 8 th edition, revision a, aoac international, valsan ap, gaithersburg md, nambiar,vn, damle sd, garg dk, iyer ss (1985). in: harvest and postharvest technology of fish. quality of dry nonpenarid of bombay markets, pp. 661-664 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (12), pp. 001-006, december, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper rheological characteristics of food gum (cissus populnea) joseph shi-an alakali1, simon verlumun irtwange2* and moses mkavga2 1 department of food science and technology, university of agriculture, makurdi, nigeria. 2 department of agricultural and environmental engineering, university of agriculture makurdi, nigeria. accepted 11 august, 2019 the rheological characteristics (consistency and flow behavior indices) of food gum (cissus populnea) exudates obtained from the fresh leaves and stem as well as dried leaves and stem were determined at 20, 30, 40, 50 and 60°c using a rotational viscometer at shear rates of 0.1, 0.2, 0.5, 1.0 and 1.5 rpm for effective design and simulation of its momentum transfer process and system. the experimental design used for the study of consistency and flow behavior indices of c. populnea at 20 60°c was randomized complete block design (rcbd). the study revealed that consistency index (k) of c. populnea exudates generally increased with increase in temperature. the k values for the fresh were generally higher than the dried materials. the k values of the fresh stem exudates, which is more viscous, were significantly (p 0.05) different from the less viscous fresh leaves exudates, but did not show significant (p 0.05) difference with the dried leaves which became concentrated due to drying. the flow behavior index did not show any defined trend with changes in temperature, and was not significantly (p 0.05) different. the exudates generally exhibited pseudoplastic behavior at all the temperatures studied. a study of the apparent viscosity of the exudates between temperatures of 20 150°c shows that apparent viscosity increased with increase in temperature below the boiling point of 70°c. however, above the boiling point, the apparent viscosity decreased with increase in temperature. the viscosity-temperature data were fitted in arrhenius-type equation and yielded activation energies between 0.997 to 2.431 kj/mol°c, corresponding to temperature range between 20 70°c and 1.632 to 2.580 kj/mol with k corresponding to temperature range between 70 150°c. key words: rheological characteristics, consistency index, flow behavior index, cissus populnea. introduction the plant, c issus populnea is also called food gum (chukwu and okpalalzima, 1989) and okoho (idoma) or ager (tiv) which are some local dialects spoken in nigeria. it contains 79% carbohydrate, 11% moisture, 3% crude protein, 3% crude fibre, 2% ether extract and 2% ash (steffe et al. 1983). there are wide ranges of possible application of c. populnea. the gum is used for soup and as soup thickener. it is also widely used as medicine for the treatment of venereal diseases and indigestion and as drug binder (iwe and atta, 1993). an important quality index of c. populnea is sliminess or cohesiveness (degree of gelling or firmness). the sliminess of c. populnea is associated with the freshness or wholesome product by users. in quantitative terms, *corresponding author. e-mail: svirtwange@yahoo.com. sliminess can be measured in terms of consistency index or viscosity. knowledge of the rheological characteristics of c. populnea at different temperatures is crucial for effective design and simulation of its momentum transfer process and system. in particular, rheological characteristics are important in the determination of such design parameters as pipe diameter, pump selection and power requirement for its transport (mohsenin, 1986). the rheological characteristics of many foods such as kunu zaki (sapode and kassum, 1992), tilapia fish homogenate (ariahu et al., 2001), tomato (alakali and ijabo, 2003), canarium oil (alakali et al., 2003) have been reported in literature. however, very little is known about the rheological characteristics of c. populnea. the objecttives of this study are: (a) to investigate the rheological characteristics of c. populnea exudates from fresh leaves and stem, as well as dried leaves and stem; (b) to characterize the flow behavior of the c. populnea exudates and (c) to study the effect of temperature on the apparent viscosity of c. populnea exudates. theory the rate of flow (g ) is related to applied shear stress () by the power law (equation 1), often referred to as newton’s law of internal friction for newtonian fluids (lewis, 1990): t = mg (1) is consistency index at absolute zero temperature; ea is the activation energy (kj/mol), t is absolute temperature, r is the universal gas constant (8.314 x 10 -3 kj/ mol). where;  is viscosity or consistency index (ns/m 2 ). the power law model for non-newtonian fluids approximates shear rate-shear stress relationship using equation (2): t = kg n (2) where; k and n are consistency index and flow behaviour index, respectively. when n<1, the consistency index decreases with increasing shear stress. materials exhibiting these characteristics are said to be pseudoplastic. conversely, when n>1, the consistency index increases with increase in shear rate and the materials are said to be dilatants. the two-parameter power law model suffers theoretical credibility because the dimensions of consistency index (k) depend on the flow behaviour index (n). nevertheless, the model enjoys wide practical application with a large number of materials, especially in the food industry, obeying it. the power law model has the appeal for mathematical simplicity (satimehin et al., 2003). sapode and kassum (1992) used an empirical equation derived from the power law model for characterizing the flow behaviour of fluid foods. this equation expresses apparent viscosity (µa) as a power function of the consistency index in the manner shown in equation 3): ma = kg n1 (3) where; µa = apparent viscosity (ns/m 2 ), k = empirical consistency index (ns/m 2 ), g is rotational shear rate (s -1 ). a log – log plot of µa verses g gives a straight line of slope (n-1) and intercept (log k). many researchers have investigated the temperature dependency of viscosity of food materials such as palm oil (satimehin et al., 2003), sausage from broiler chicken (awonorin, 1993) and fluid foods (rao and anantheswaran, 1982). these authors observed that viscosity generally decreases with increase in temperature. an arrhenius type relationship generally represents the viscositytemperature relationship as shown in equation 4: æ e a ö k = k o exp ç ÷ (4) è rt ø plate 1. cissus populnea (a) fresh leaves; (b) dry leaves; (c) fresh stem strips; and (d) dry stem strips. materials and methods materials c. populnea fresh stem and leaves, dry stem and leaves (plate 1), sieve, brookfield rotational viscometer (model lv8 viscometer uk ltd), water bath (grant instrument ltd model suz cambridge, uk), calorimeter cup and distilled water. methods sample preparation the leaves and stems of c. populnea used in this work were obtained from the forest around the university of agriculture makurdi, nigeria, and thoroughly washed to remove adhering dirts and foreign materials. the leaves were divided into two lots. lot 1, which is fresh leaves, was squeezed between the palms in distilled water of one litre for 20 min to obtain exudates. the sample was filtered through a sieve to produce fresh leave exudate (fle) of 54% total solids (ts). lot 2 was left to dry under natural conditions in the sun for three days. the dried leaves were pounded into powder using mortal and pistil. the powder was dissolved in warm distilled water of one litre at 55 o c and filtered using a sieve to produce dry leaves exudates (dle) of 54% ts. the fresh stem was cleaned by scrapping with a kitchen knife. the ‘fleshy’ bark was removed and cut into fine strips and divided into two lots. lot 1 was soaked in one litre of distilled water at 55 o c for 20 min. afterwards the strips were squeezed under the water to remove the exudates. the sample was filtered through a sieve to produce fresh stem exudate (fse) of 54% ts. lot 2 was allowed to dry for three days under natural conditions and the strips pounded into powder. the powder was dissolved in one litre distilled water and filtered to produce dry stem exudate (dse) of 54% ts. viscosity measurement a brookfield rotational viscometer (model lv8 viscometer uk ltd) where ko table 1. consistency (k) and flow behavior (n) indices of c. populnea at 20-60°c. sample temperature o c consistency index (k) flow behavior index (n) r2 20 3.530 a 0.923 e 0.949 30 3.700 a 0.922 e 0.928 fse 40 3.800 a 0.911 e 0.972 50 4.040 ab 0.927 e 0.930 60 4.220 b 0.926 e 0.983 20 2.530 c 0.914 e 0.984 30 2.740 c 0.917 e 0.994 dse 40 2.910 c 0.906 e 0.989 50 3.130 d 0.950 e 0.953 60 3.420 d 0.939 e 0.951 20 2.210 c 0.935 e 0.942 30 2.430 c 0.916 e 0.964 fle 40 2.630 c 0.950 e 0.990 50 2.870 cd 0.944 e 0.928 60 3.110 cd 0.934 e 0.957 20 1.810 c 0.900 e 0.943 30 2.280 c 0.955 e 0.945 dle 40 2.510 c 0.933 e 0.977 50 2.690 c 0.931 e 0.902 60 3.000 d 0.936 e 0.980 mean values within a row followed by different letters are significantly (p 0.05) different. values are means of three replicated measurements. fse; fresh stem exudate, dse; dried stem exudate, fle; fresh leaves exudate, dle; dried leaves exudate, r 2 ; regression coefficient. table 2. summary of anova for consistency index (k) and flow behaviour index (n) for c. populnea at five levels of temperature. source of variation degrees of freedom consistency index (k) flow behavior index (n) 5% 1% c. populnea sample 3 279.0728** 0.825057 ns 3.49 5.95 temperature 4 72.47835** 1.035919 ns 3.26 5.41 error 12 **highly significant (p=0.01); ns non significant. was used for measuring the viscosities of the four samples (fle, dle, fse and dse) at 20, 30, 40, 50 and 60°c and five shear rates (0.1, 0.2, 0.5, 1.0 and 1.5 rev/min), using spindle two of the viscometer. viscosity readings were in centipoises (10 -3 ns/m 2 ). about 20 ml of the sample under consideration was measured in a calorimeter cup. it was then heated to 55°c and immediately transferred into the viscometer cup which was already maintained at a constant temperature in a water bath (grant instrument ltd model suz cambridge, uk). for every temperature and shear rate, viscosity readings were replicated three times. the experimental design used for the study of consistency and flow behavior indices of c. populnea at 20 60°c was randomized complete block design (rcbd). was attained. statistical analysis all statistical analyses were performed using spss for microsoft windows release 6.0 and genstat. means were analyzed for variance and separated using duncan multiple range tests. the non-linear regression procedure of spss was used to determine regression parameters k, ko, n and ea in equations 3 and 4, respectively. effect of temperature three hundred and fifty (350) ml each of the samples fle, dle, fse and dse was heated from 20 to 150°c in a calorimeter cup using a hot plate. the viscosity reading of each sample was taken at a regular interval of three minutes until the temperature of 150°c results the values of consistency (k) and flow behaviour (n) indices of the four samples of c. populnea (table 1) were obtained from log – log plot of µa versusg . the analysis of variance (anova) is presented in table 2 while linear table 3. regression equations for consistency index (k) and flow behaviour index (n) for c. populnea in the temperature range of 20-60°c. c. populnea consistency index (k) flow behavior index (n) sample a b r 2 r a b r 2 r fse 3.17 0.0172 0.986 0.993 0.9174 0.00011 0.074 0.273 dse 2.078 0.0217 0.991 0.995 0.892 0.00083 0.504 0.710 fle 1.754 0.0224 0.999 0.999 0.9254 0.000665 0.102 0.319 dle 1.342 0.0279 0.971 0.985 0.9118 0.00048 0.147 0.384 a = intercept; b = slope; r 2 = coefficient of determination; r = correlation coefficient; = standard deviation. table 4. regression parameters of arrhenius relationship between consistency and flow behavior indices of c. populnea at 20 – 70°c. regression c. populnea sample parameters fse dse fle dle n 6 6 6 6 gradient (b) 0.122 0.173 0.228 0.299 ea (kj/ml) 0.977 1.408 1.852 2.431 intercept (in ko) 1.402 1.112 0.904 0.975 ko 4.063 3.040 2.469 2.171 r2 0.966 0.976 0.900 0.970 fse, fresh stem exudate; dse, dried stem exudate; fle, fresh leaves exudate; dle, dried leaves exudate; n, number of observations; ea, activation energy; ko, temperature factor at absolute zero (intercept); r 2 , regression coefficient. table 5. regression parameters of arrhenuis relationship between consistency and flow behavior indices of c. populnea at 70 – 150°c. regression c. populnea sample parameters fse dse fle dle n 8 8 8 8 gradient 0.201 209 0.228 0.266 ea (kj/mol) 1.691 1.701 1.920 2.580 intercept (in ko) 1.620 1.414 1.298 1.119 ko 5.053 4.112 3.661 3.062 r 2 0.850 0.87 0.812 0.700 fse, fresh stem exudate; dse, dried stem exudate; fle, fresh leaves exudate; dle, dried leaves exudate; n, number of observations; ea, activation energy; ko, temperature factor at absolute zero (intercept); r 2 , regression coefficient. discussion the values of consistency (k) and flow behaviour (n) indices for the four samples (fle, dle, fse and dse) obtained from log – log plot of µa versus g show that n was consistently less than one (n<1) indicating pseudoplasticity. this is consistent with other fluid food such as tomato paste (alakali and ijabo, 2003), palm oil figure 1. apparent viscosity versus shear rate at 60 o c. regression analysis is presented in table 3. figure 1 shows the plot of apparent viscosity versus shear rate for fle, fse, dle and dse. figure 2 shows the shear stress shear rate relationship. the apparent viscosity versus temperature relationship is shown in figure 3. figures 4a and 4b show the linearized semi-log plots of consistency index versus the inverse of absolute temperature between 20-70 and 70-150 o c, respectively. the results of activation energies between 20-70 and 70 150 o c are presented in tables 4 and 5, respectively. figure 2. shear stress versus shear rate at 60 o c. temperature ºc figure 3. apparent viscosity versus temperature. (a) fle (n s /m 2 ) 2.000 dle fse dse 1.500 v is c o s it y 1.000 a p p a re n t 0.500 0.000 0.050 0.033 0.025 0.020 0.017 0.014 1/t (b) (n s /m 2 ) fle 2.000 dle fse dse v is c o s it y 1.500 1.000 a p p a re n t 0.500 0.000 3 1 0 9 8 7 6 6 1 1 1 .00 .00 .00 0 .00 .0 .0 .0 .0 0 0 0 0 0 0 0 0 1/t figure 4. plot of apparent viscosity (ln k) versus inverse of absolute temperature (1/t) (a) up to the boiling point (20-70 o c) and (b) above the boiling point (70-150 o c). (satimehin et al., 2003), kunu zaki and kunu gyada (sapode and kassum, 1993). the analysis of variance (table 2) indicates highly significant (p< 0.5) c. populnea sample and temperature effect on consistency index (k) while flow behaviour index (n) is non-significant. values of k increased with increase in temperature with very high positively correlated r values whereas values of n did not show any defined trend with increase in tempe-rature (table 3). the apparent viscosity of c. populnea exudates as indicated by consistency index (k) increased with increase in temperature between 20-60 o c (table 1 and figure 3). however, at temperatures beyond the boiling point of c. populnea (70-150 o c), the apparent viscosity decreased with increasing temperature (figure 3). usually, viscosity decreases with increase in temperature (awonorin, 1993; ariahu et al., 2001; alakali et al., 2003). however, c. populnea exudate showed a reverse trend at tempera tures below the boiling point (t  70 o c). this behavior is said to be due to its system of binding. these authors explained that before the boiling point of c. populnea, the elements are closely bound together such that the bonds are only totally broken after the boiling point (iwe and atta, 1992). bourne (1982) observed that certain foods exhibit this characteristic. the apparent viscosity of c. populnea decreased as shear rate increased (figure 1). similar trends were observed by alakali and ijabo (2003) for tomato pastes; alakali et al. (2003) for canarium oil; satimehin et al. (2003) for palm oil; ariahu et al. (2001) for tilapia fish homogenate; awonorin (1993) for smoked sausage made from broiler chicken and sopode and kassum (1992) for kunu zaki and kunu gyada. this behavior indicates shear thinning and is to be expected because an increase in rotational speed increases molecular alignment in the direction of flow, reducing resistance to flow and hence viscosity. this is typical of pseudoplastic fluids. this phenomenon has been widely reported to be common with hydrocolloid solutions and food pastes with sensitive structures (awonorin, 1993). the shear stress-shear rate relationship (figure 2) shows increase in shear rate as the shear stress increased. comparing the shear stressshear rate relationship with the classification chart by moshenin (1986), it was further established that c. populnea is non-newtonian pseudoplastic. exudates from fresh sample materials were observed to have higher values of consistency index than the dried samples. for example, the mean value of k for fresh stem exudate (fse) was 3.860 ns/m 2 compared to 2.950 ns/m 2 for dried stem exudate (dse). similarly k for fresh leaves exudate (fle) was 2.650 ns/m 2 compared to 2.460 ns/m 2 for dried leaves exudate (dle). this result was expected as drying may have tampered with the binding properties of the gum and reduced its cohesiveness. the k values for fse were significantly (p < 0.05) different from fle. however, k values for dse did not show significant (p  0.05) difference with dle. predictive models the predictive equations based on the power law model (equation 2) for the four samples are as follows: 0.92 (r 2 = 0.950) (5) fse:  = 3.860 g 0.93 (r 2 = 0.970) (6) dse:  = 2.950 g 0.94 (r 2 = 0.960) (7) fle:  = 2.960 g 0.93 (r 2 = 0.950) (8) dle:  = 2.450 g the high r 2 values indicate that the power law model is appropriate for describing the rheological characteristics of c. populnea. the suitability of equations 5 to 8 for predictive purposes was verified using the percentage root mean square of error (%rms), which were found to be generally low (< 10%). according to wang and brennan (1991), low percentage of rms values indicate good fit and suitability of a model for practical purpose. according to these authors, the smaller the percentage rms, the better a model fits experimental data. the relationship between temperature and viscosity of c. populnea was investigated using arrhenius equation (equation 3) for temperature range of 20 to 150°c. linearized semi-log plots of consistency index against the inverse of absolute temperature (figures 4a and b) gave straight lines which characteristically broke into two segments at the boiling point. the slope of each line segment gave the corresponding activation energy. the activation energies were determined between 20 70 and 70-150°c. it was observed that the activation energies of the exudates were low in the lower than the higher temperature ranges. at lower temperatures (below the boiling point), the gums were more viscous, hence more heat was needed to break intermolecular bond. the relative higher activation energies corresponding to the higher temperature range indicate that the lower viscosities above 70°c were more sensitive to heat. conclusions apparent viscosity decreased with increasing shear rate, indicating shear-thinning behavior. the flow behavior index did not show major change with temperature and its value was generally below one, hence c. populnea can be said to exhibit pseudoplastic behavior. both apparent viscosity and consistency index of c. populnea were found to increase with increasing temperature below its boiling point. above the boiling point, apparent viscosity decreased with increase in temperature. activa-tion energies were found to be higher at temperatures above the boiling point (t > 70 o c). references alakali js, ijabo oj (2003). effect of processing method on the viscosity, ph and colour of tomato (lycoporsicum escusentum) pastes. j. sustainable trop. agric. res. 5: 55-60. alakali js, ijabo oj, satimehin aa (2003). rheological characteristics of canarium (canarium schweinfurhii) oil. j. pure appl. sci. 6 (2): 298-303. ariahu cc, risikat b, tiamiyu oi (2001). influence of soyflour and temperature on tilapia fish homogenate. afr. j. environ. stud. 2:138141. awonorin so (1993). rheological and mechanical properties of bvitamin retention and sensory characteristics of sausage made from broiler chicken and guinea fowl. lebensmittal-wess-u-techol. 20: 291-300. bourne me (1982). food texture ad viscosity concept and measurements. new york academic press pp. 199-219. chukwu a, okpalalzima c (1989). primary evaluation of c. populnea gum as a binder in sodium sachy late tablet formulation. drug dev. ind. pharm. 15(2): 325-330. iwe mo, atta c (1993). functional properties of the active ingredients of c. populnea. crill. perr. pp. 29-53. lewis mj (1990). properties of foods and food processing systems. 2 nd ed. chi charter eillies horwood ltd. pp. 108-1136. mohsenin n (1986). physical properties of plants and animal materials vol. 1 structure, physical characterization and mechanical properties. science publishers, west port ny. pp. 899-900. rao ma, anantheswaran rc (1982). rheology of fluids in food processing. food technol. 37:116-126. sapode pa, kassum ai (1992). rheological characterization of nigeria liquids and semi liquid foods. kunu zaki, and kunu gyada. niger. food j. 10: 23-33. satimehin aa, akinemi om, ijabo oj (2003). rheological behavior of palm oil. j. pure applied sci. 6 (2): 291 –297. steffe jf, mohammed io, food ew (1983). rheological properties of fluid foods. asae. 83: 781-783. wang a, brennan jc (1991). moisture sorption characteristic of potato at four temperatures. j. food eng. 14: 269-274. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (1), pp. 001-005, january, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper response surface optimization of d(-)-lactic acid production from lactobacillus smi8 using corn steep liquor and yeast autolysate as nitrogen sources c. j. b. de lima*, l. f. coelho, k. c. blanco and j. contiero department of biochemistry and microbiology, institute of biological sciences, são paulo state university, unesp/rio claro, av. 24-a, 1515 bela vista, cep 13506-900, rio claro, sp, brazil. accepted 07 august, 2019 the production of d(-) lactic acid from lactobacillus lmi8 sp. was optimizated by central composite design, using two low-cost nitrogen sources: corn steep liquor (csl) and yeast autolysate (ya). the fermentation were performed in 250 ml erlenmeyer flasks containing 100 ml of production medium, maintained at 200 rpm and 37±1°c for 48 h. after data analysis by surface response method, it was revealed that lactic acid production was significantly affected by the isolated csl as well as the interaction between csl and ya and the maximal production of d(-) lactic acid was 41.42 g/l – a value located at the central point, which corresponded to 15 g/l of csl and 5 g/l of ya. key words: d(-)-lactic acid, lactobacillus, corn steep liquor, yeast autolysate, response surface methodology. introduction lactic acid is the common name given to 2-hydroxypropanoic acid (datta et al., 1995), which is a versatile prod-uct applied in diverse fields, including as an acidulant in the food and pharmaceutical industries (schepers et al., 2002). with the development and commercialization of biopolymers, poly(lactic acid) use has an increasing use in these new applications (datta and henry, 2006). the high chemical resistance of poly(lactic acid) is advantageous for the manufacture of fibers, nonwoven fab-rics, and films while the high heat resistance is suitable for producing the cutlery and deli trays/cups. thus, micro-bial production of optically pure lactic acid has extensi-vely been studied because chemically synthesized lactic acid is racemic. recently, the development of stere-ocomplex of poly(l-lactic acid) and poly(d-lactic acid) is one of the topics in the application of the poly(lactic acid) because its melting point (225ºc) is higher than that of poly(l-lactic acid) (177ºc) (ikada et al., 1987; tsuji and fukui, 2003). lactic acid bacteria make up a group of diverse microorganisms that are widely distributed in nature and associated with plants (kale, corn, barley), meat, milk products, porridge and silage. these lactobacilli are mic *corresponding author. e-mail: cristian@rc.unesp.br. ro-organisms in great need of fermentable carbohydrates, amino acids, b complex vitamins, nucleic acids and minerals for growth as well as other nutrients that are specific to each strain (gomes and malcata, 1999). a number of byproducts and raw materials from the food and/or agriculture industries have been employed to feed lactobacilli due to their considerable availability and low cost, including cheese whey, corn steep liquor, corn syrup, distillery yeast and molasses (moraes et al., 1991). in brazil, lactic acid production from sugarcane and corn residuals has considerable economic appeal due to the abundance and low cost of these byproducts. molasses, a byproduct of the sugar manufacturing pro-cess, is used as an animal feed as well as for ethanol and yeast production (focus, 1975). it can be also used for lactic acid production (el-sherbiny et al., 1986; tiwari et al., 1979). the most abundant sugar is sucrose, which raises the viscosity of the liquid at high concentration (focus, 1975). moreover, molasses and corn steep liquor are prominent culture media in fermentative processes due to the high content of sugars and nitrogen, respect-ively (beaulieu et al., 1995). the aim of the present study was to optimize the production medium of lactic acid by lactobacillus sp. lmi8 by employing a central composite design (ccd) and response surface method, using sugarcane molasses of low cost enriched with corn steep liquor and yeast autolysate as the culture medium. table 1. experimental range and levels of the independent variables (xi, i = 1 and 2) used in central composite design. independent variables range and levels -1 0 +1 + production medium optimization (g/l) corn steep liquor (csl) x1 0 4.3 15 25.7 30.1 yeast autolysate x2 1.47 2.5 5 7.5 8.52 materials and methods microorganisms the lactobacillus sp. lmi8 was isolated from a decantation tank of residuals from the flour industry (plaza starch mill santa mariasp, brazil). the culture was stored in man, rogosa and sharpe (mrs) medium with 20% (v/v) glycerol at -20ºc. isolation of lmi8 culture the successive dilution method was employed and plating was performed using the “pour plate” method. bacterial strain selection was based on the greatest production of the d(-) lactic acid isomer from the culture medium used in the fermentation process. sugarcane molasses sugarcane molasses was obtained from the santa lucia sugar processing plant located in the region of ararassp, brazil. the molasses was diluted and hydrolyzed at a ratio of 1 ml of h2so4 (20%) in 100 ml of molasses solution and heated for 20 min. after hydrolysis, the ph of the molasses was adjusted to 6.5 with 4.0 m of koh. medium and growth conditions the inoculum was prepared through the transference of 2 ml of stock culture to erlenmeyer flasks containing 100 ml of growth medium (mrs) . the mrs growth medium was made up of peptone (10 g/l), yeast extract (5 g/l), meat extract (10 g/l), glucose (20 g/l), sodium acetate (5 g/l), ammonium citrate (2 g/l), k2hpo4 (5 g/l), na2hpo4.2h2o (2 g/l), mgso4.7h2o (0.1g/l) and mnso4.4h2o (0.05g/l). the inoculated medium was incubated at 37 ± 1ºc for 18 h in a refrigerated incubator (new brunswick, usa) at 200 rpm. initial ph of the medium was adjusted to 6.7. a total of 10% (v/v) of the inoculum was transferred to 250-ml erlenmeyer flasks containing 100 ml of production medium (modified mrs medium) agitated at 200 rpm in a refrigerated incubator at 37 ± 1ºc for 48 h. calcium carbonate (100 g/l) was added to the medium in order to maintain the ph constant. the modified mrs medium consisted of the same salts as in the growth medium, with the addition of 70 g/l of molasses, corn steep liquor ranging from 0 to 30.1 g/l and yeast autolysate ranging from 1.475 to 8.525 g/l. central composite design (ccd) in order to study the influence of the corn steep liquor (csl) and yeast autolysate (ya) in the synthesis of lactic acid by the lactobacillus sp. lmi8 isolate, a central composite design (ccd) was used. for the two factors, this design was made up of a full 2 2 factorial design with its four cube points, augmented with three replications of the center points and the four star points, that is, points having an axial distance to the center of (± 1.41) for one factor, whereas the other factor is at level 0, resulting in a total of 11 experiments. thus, the influence of all experimental variables, factors and interaction effects on the response was investigated. the experimental design was determined using the statistic 7.0 software program. the independent variables, experimental range and levels investigated for the ccd is given in table 1. in performing the regression equation, the test variables were coded according to the following equation 1: x i  x i  x cp  (1)  x i where xi is the coded value of an independent variable, xi is the real value of an independent variable, xcp is the real value of an independent variable at the center point, and xi is the step change value. analytical methods substrate consumption and lactic acid concentrations (sample of 20 l) were determined using a high performance liquid chromatography system (waters co., milford, ma) equipped with a tunable uv detector set at 210 nm. an aminex hpx-87h ionexchange column (300×7.8 mm, bio-rad, hercules, ca) was eluted with 0.005 n of h2so4 as a mobile phase at a flow rate of 0.6 ml/min. column temperature was maintained at 60ºc and a refraction index (ri) detector was used. d( -)lactic acid concentrations were determined by an enzyme test kit (r-biopharm ag roche, darmstadt, germany), as reported elsewhere (yun et al, 2003). cell growth was measured by a uv160a spectrophotometer (shimadzu co., tokyo, japan) set at 650 nm. results and discussion optimization of fermentative production of lactic acid the influence of corn steep liquor and yeast autolysate on the fermentation of sugarcane molasses was studied using ccd with three replicates at the center, totaling 11 experiments. the results are displayed in table 2. analyzing table 2, the best results of all the responses evaluated were obtained in the experiments located at the central point (experiments 9, 10 and 11). comparing experiment 5 with experiments 6, 9, 10 and 11, performed with the lowest and highest concentrations of csl (0, 15 and 30 g/l) and equal concentrations of ya (5 table 2. results of lactic acid production obtained in experiments employing the lmi8 isolate. experiments csl ya lactic acid (g/l) (g/l) (g/l) 1 4.3 2.5 15.32 2 4.3 7.5 20.43 3 25.7 2.5 35.23 4 25.7 7.5 22.7 5 0 5 12.11 6 30.1 5 25.39 7 15 1.47 24.16 8 15 8.52 31.27 9 15 5 41.42 10 15 5 41.23 11 15 5 39.98 concentration of corn steep liquor (csl); concentration of yeast autolysate (ya). table 3. stationary point for lactic acid production and coded x1 and x2 values at the optimization point. p0 lactic acid coordinates lactic acid 1 -12.233 x1 0.139 2 -5.833 x2 -0.029 g/l), it is evident that lactic acid production was directly proportional to the increase in csl, reaching a maximal value of 25.39 (experiments 6) and 41.42 g/l (experiments 9), both in 32 h of fermentation. moreover, the increase in the nitrogen concentration in the fermentative medium led to a reduction in fermentation time, reaching maximal lactic acid production. wee et al. (2006) studied the effect of csl on lactic acid production using different concentrations (15 to 60 g/l) and obtained the same final concentration of lactic acid in all experiments, but the time needed to achieve this concentration decreased significantly when using greater concentrations of csl. yu et al. (2007) used sugarcane molasses ranging from 12 to 60 g/l and csl ranging from 24 to 56 g/l and report that the increase in molasses and csl provided greater lactic acid production in a shorter fermentation time. selmer-olsen and sorhaug (1998) and altaf et al. (2006) report that peptone and yeast extract are the main nitrogen sources used for the production of lactic acid because yeast extract is an excellent source of b complex vitamins and is often used to provide these factors to the bacteriological culture media, which are often considered indispensable to obtaining faster growth and production rates of lactic acid by lactic bacteria. the high cost of yeast extract, however, has a negative impact when used in industrial processes (hurok et al., 2005). the precision of the results of the experiments can be seen in the assessment of the results obtained at the central point of the design, where the difference between responses was close to 1%. the identification of significant parameters was performed through testing the hypotheses using the student’s t-test. the maximal likelihood of error on the test was established as 5%. thus, parameters with a level of significance greater than 5% were discounted. the adjusted empirical equation that represents the synthesis of lactic acid is expressed in equation 2: lactic acid = 40.876 + 5.12x1 – 4.41x1x2 – 11.01x12 – 6.53x22 (2) in experiment 5 (table 2), there was lactic acid production in the absence of csl. despite being less significant within the fermentation process (eq. 1), ya provided a good interaction with csl, generating greater lactic acid production. the fitness of the model was checked by the determination coefficient (r 2 ) and multiple correlation coefficients (r). in this case, the value of the r 2 (0.96) for equation 2 indicates that the sample variation of 96.0% for lactic acid was attributed to the independent variables and only 4% of the total variation cannot be explained by the model. the value of the adjusted determination coefficient (adjusted r 2 = 0.935) is also high, which stresses the significance of the model. the high value of r (0.98) demonstrates a high degree of agreement between the experimental observations and predicted values. this correlation is also proven by the plot of predicted versus experimental values of lactic acid in figure 1, as all the points cluster around the diagonal line, which means that no significant violations of the model were found. the effects of the independent variables and their interactions on the formation of the product are illustrated in the analysis of the response surfaces (figure 2) constructed from equation 2. analyzing figure 2, maximal lactic acid production occurred when nitrogen concentrations were near 15 g/l (csl) and 5 g/l (ya). for greater and lower concentrations of csl and ya, there was a reduction in response, likely due to either a lack or excess of nitrogen in the fermented medium. according to wood and holzapfel (1995) the genus lactobacillus has complex nutritional requirements. as lactic acid synthesis through fermentation is associated cell growth, there is no formation of product if the medium does not have an adequate concentration of nitrogen in order to promote this growth (pritchard and coolbear, 1993). on the other hand, high concentrations of nitrogen may lead to cell death and inhibition of the product. an algorithm carried out on the maple 9.5 program (waterloo maple, inc., canada) was used to calculate the stationary point (p0) for synthesis of lactic acid. these values are displayed in table 3. the values indicate that these responses have a maximal point, as they have equal and negative signs. lactic acid production was 41.52 g/l at the optimization point from the coded x1 and figure 1. predicted vs. experimental values plot for lactic acid. figure 2. response surface and contour curve representing the effects of corn steep liquor and yeast autolysate concentrations on lactic acid production. x2 values (table 3). comparing this value with lactic acid production values in experiments 9, 10 and 11 (table 2), the results were practically the same, as the conditions of the maximal point were near to those of the central point. thus, the corresponding values for the optimal concentrations of csl and ya were 16.48 and 4.93 g/l, respectively. the model predicted a maximal response of 41.38 g/l of lactic acid at this point. wee et al. (2006) produced 48.6 g/l of lactic acid using wood hydrolysate (equivalent to 50 g/l of glucose) supplemented with 60 g/l of csl in 36 h of fermentation. bustos et al. (2004) obtained maximal lactic acid production (58.9 g/l) in 96 h of culturing using 5 g/l of csl together with 3.6 g/l of yeast extract and 10 g/l of peptone. using a mathematical model furnished the response surface method, hauly et al. (2003) determined maximal concentrations values tested for sugarcane molasses (100 g/l), yeast extract (20 g/l) and peptone (4 g/l) as the best medium composition for the production of lactic acid. in practice, these conditions provided a production of 30.5 g/l of lactic acid. conclusion the central composite design was proved to be a useful and applicable tool for determining the behavior of the variables studied in the production of lactic acid. the lactobacillus sp. lmi8 strain using organic nitrogen source (corn steep liquor and yeast autolysate) achieved significant results regarding lactic acid production. under optimal conditions (16.48 g/l of csl and 4.93 g/l of ya) a lactic acid concentration of 41.52 g/l was obtained. acknowledgements we thank the fundação de amparo à pesquisa de estado de são paulo (fapesp) for fellowships and financial support. references altaf md, naveena bj, venkateshwar m, vijay kumar e, reddy g (2206). single step fermentation of starch to l(+) lactic acid by lactobacillus amylophilus gv6 in ssf using inexpensive nitrogen sources to replace peptone and yeast extract – optimization by rsm. process biochem. 41: 465-472. beaulieu m, beaulieu y, mélinard j, pandian s, goulet j (1995). influence of ammonium salts and cane molasses on growth of alcaligenes eutrophux and production of polyhydroxybutyrate. appl. environ. microbiol. 61: 165-169. bustos g, moldes ab, alonso jl, vázquez m (2004). optimization of dlactic acid production by lactobacillus coryniformis using response surface methodology. food microbiol. 21: 143-148. datta r, henry m (2006). lactic acid: recent advances in products, processes and technologies – a rev. j. chem. technol. biotechnol. 81: 1119-1129. datta r, tsai sp, bonsignore p, moon sh, frank jr (1995). technological and economic potential of poly-lactic acid and lactic acid derivatives. fems microbiol. rev. 16: 221-231. el-sherbiny ga, rizk ss, yousef gs (1986). utilization of beet molasses in the production of lactic acid. egypt j. food sci. 14: 91100. focus l (1975). focus uppslagsböcker ab, stockholm, sweden. gomes amp, malcata fx (1999). bifidobacterium spp. and lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. trends in food sci. technol. 10: 139-157. hauly mco, oliveira ar, oliveira as (2003). lactic acid production by lactobacillus curvatus in sugarcane molasses semina: ciências agrárias, londrina. 24: 133-142. hurok oh, wee wj, yun js, han sh, jung s, ryu hw (2005). lactic acid production from agricultural resources as cheap raw materials. bioresour. technol. 96: 1492-1498. ikada y, jamshidi k, tsuji h (1987). stereocomplex formation between enantiomeric poly (lactides). macromol. 20: 904-906. moraes io, capalbo dmf, moraes ro (1991). multiplicação de agentes de controle biológico. in: bettiol, w. controle biológico de doenças de plantas. brasília: embrapa, pp. 253-272. pritchard gg, coolbear t (1993). the physiology and biochemistry of the proteolitic system in lactic acid bacteria. ufems microbiol. 12: 179-206. schepers aw, thibault j, lacroix c (2002). lactobacillus helveticus growth and lactic acid production during ph-controlled batch cultures in whey permeate/yeast extract medium. part i. multiple factor kinetic analysis. enz. microb. technol. 30: 176-186. selmer-olsen e, sorhaug t (1998). comparative studies of the growth in whey supplemented with autolysate from brewery yeast biomass or commercial yeast of lactobacillus plantarum extract. milchwissenschaft. 53: 367-370. wood hydrolyzate and corn steep liquor. j. indus. microbiol. biotechnol. 33: 431-435. wood bjb, holzapfel wh (1995). the genera of lactic acid bacteria. glasgow: blackie academic, professional, usa. yu mc, wang rc, wang cy, duan kj, sheu dc (2007). enhanced production of l(+)-lactic acid by floc-form culture of rhizopus oryzae. j. chinese inst. chem. eng. 38: 223-228. tiwari kp, pandey a, mishra n (1979). lactic acid production from molasses by mixed population of lactobacilli. zentl bakteriol. 134: 544-546. tsuji h, fukui i (2003). enhanced thermal stability of poly(lactide)s in the melt by enantiomeric polymer blending. polymer. 44: 2891-2896. wee yj, yun js, kim d, ryu hw (2006). batch and repeated batch production of l(+)-lactic acid by enterococcus faecalis rky1 using 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 12 (1), pp. 001-007, january, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper yeast-based biocontrol: a novel approach to manage apple blue mold with candida membranifaciens and rhodotorula mucilaginosa jalal gholamnejad*, hasan reza etebarian and navazallah sahebani department of plant protection, aboureihan campus, university of tehran, iran. accepted 16 november, 2023 in this study two genus of biocontrol yeasts were isolated from healthy apple fruits surface. three strains of candida membranfaciens (a2, a4 and a5) and two strains of rhodotorula mucilaginosa (a1 and a7) were evaluated for the control of the blue mold of apple caused by penicillium expansum. one isolate of p. expansum was used in these experiments. dual culture, cell free metabolite and volatile test were used for in vitro assays. yeast strains of two genus inhibited growth of p. expansum, inhibition varied from 20.6 61.4%, in dual culture, in volatile metabolite from 57.2 89.1% and from 54.6 86% in cell free metabolite test. apple fruit wounds were inoculated with 40 µl of yeast cell suspension (10 7 cell ml -1 ), and after 24 h, inoculated by p. expansum (10 5 conidia ml -1 ). inoculated apples were incubated at room (20°c) and cold (5°c) temperature. all strains of two genus reduced lesion area at two temperatures. the a2 (c. membranfaciens) was the most effective strain at both temperatures, significantly. effect of cacl2 mixed with yeast (a1), was evaluated at 20°c. the different concentration of cacl2 reduced the lesion area from 185.07 1738.037 mm 2 reduction compared to 2452.84 mm 2 in control after incubation for 15 days at 20°c. at the same time, the other antagonistic yeast conditions such as yeast concentration and inoculation time were studied, and the best conditions for antagonistic yeast were determined. key words: apple, biocontrol, blue mold, candida membranfaciens, rhodotorula mucilaginosa, penicillium expansum. introduction fruits and vegetables suffer significant losses from fungal diseases after harvest (eckert et al., 1994). post harvest losses of fruits and vegetables are high, ranging from 10 and 40% depending on the commodity and technologies used in the packinghouses. postharvest fungal diseases of apple are mainly caused by penicillium expansum (gholamnejad and etebarian, 2009). traditionally, this disease is controlled by the application of synthetic fungicides. however, the potential impact on environment as well as human health largely limits their application (wisniewski et al., 1991). it is reported that some pathogens become to fungicide resistant (spotts and cervantes, 1986), and thus a fungicide’s effect on controlling fungal growth may be greatly reduced. considering the human health and pollution risks, the use of some fungicides is prohibited in *corresponding author. e-mail: jalalgholamnejad2006@gmail. com. many developed countries (e.g. usa and eu countries). recently, biological control has been developed as an alternative to synthetic fungicides (wilson et al., 1993), and has been achieved considerable success by using antagonistic microorganisms for controlling post harvest diseases. roberts (1990) discovered that cryptococcus laurentii has antagonistic activity against many postharvest pathogens. he added that the competition for nutrients may play a role in the antagonism of cryptococcus laurentii. decay caused by rhizopus spp. is reduced up to 70% when strawberries are treated with aureobasiduim pullulans before storage (lima et al., 1997). a few studies were reported in iran concerning the advantages of utilizing antagonistic micro-organisms include reducing environmental pollution, effectively controlling postharvest diseases, and producing high quality and safe food. in this study, the efficacy of some yeast species in controlling postharvest diseases of apple caused by penicillium expansum was tested, and one strain with the 2 best biocontrol effect was identified. the antagonistic conditions of that yeast and the methods of antagonism were also discussed. materials and methods fruit the apple (malus domestica var. golden delicious) at uniform size and maturity without wounds or rot, were used in this study. apples were harvested at commercial maturity and kept at 4°c until use. the apples were obtained from organically grown position in orchard in damavand, tehran. pathogen the isolate p11 of p. expansum obtained from rotted malus domestica ‘golden delicious’ from damavand, iran, was used in this study. the culture was derived from single spore isolate and maintained on potato dextrose agar (pda) at 4 °c in darkness until needed. biocontrol agents biocontrol agents were isolated from the surface of apple healthy fruits collected from organic production orchards, following methodology described by wilson et al., (1993). isolates consisted of c. membranfaciens (a2, a4 and a5) and two strains of r. mucilaginosa (a1 and a7). identification of selected strains was carried out by "identification service cbs" by molecular and morphological methods. optimum growth temperature for each strain in pda was determined for each strain. temperatures assayed were 5, 25, 30, and 37°c. in vitro biological control studies dual culture was carried out according to dennis and webster (1971). yeast strains were grown at 20°c in nutrient broth yeast dextrose for 48 h on a rotary shaker at 250 rpm. aliquots of 200 µl yeast suspension (1.0 × 10 7 cfu l −1 ) were streaked on half plates. after incubation at 20°c in the dark for 48 h, a plug (10 mm diameter) cut from the leading edge of a 14 day old culture of p. expansum on pda medium was placed on the other half of the plate. potato dextrose agar inoculated with the pathogen alone served as the control. plates were incubated at 20°c for 18 d then the colony diameters were measured. cellfree culture was carried out according to weller (1988) and volatile metabolite tests were conducted according to lillbro (m.sc. thesis, swedish university of agricultural sciences, 2005). these tests were used to observe the effect of yeast strains on mycelial growth of p. expansum. all antagonist–pathogen combinations were examined on pda in 90 mm petri plates with four replications. the plates were incubated 18 days at 25°c for both dual culture and cell free culture and 25 days for volatile metabolite. all tests were carried out in four replicates. the percent of the growth inhibition was calculated using the formula n = (a b)/a × 100, where n is the percent growth inhibition, “a” is the colony area of p. expansum growth inhibition and “b” is the colony area of treated , as described previously by etebarian et al. (2005). in vivo test the pathogen (p11) was grown on pda plates for 14 days. conidia were harvested by pouring a few ml of sterile distilled water (sdw) containing 0.05% tween 20 on the plates. the conidia suspension was adjusted to 1 × 10 5 conidia ml -1 . the apple fruits were washed in 70% ethanol for 30 s followed by dipping in 0.1% sodium hypochlorite solution and rinsed with sterile distilled water. the apples were wounded with 2.5 mm diameter nail to a depth of 3 mm in triplicate (gholamnejad et al., 2009). evaluation antagonistic effect of yeasts at different storage temperatures (5 and 20°c): the yeast inoculum was prepared in nutrient yeast dextrose broth nydb on a rotary shaker at 150 rpm for 48 h at room temperature. the produced yeast cell were collected by centrifugation at 6500 × g for 5 min and resuspended in water to obtain the desired concentration. concentration was adjusted to 1 × 10 7 conidia ml -1 with hemacytometer (gholamnejad et al., 2009). the inoculum of p. expansum (p11) was prepared as described above. the apple fruits were wounded as described above, and inoculated with 40 l of yeast cell suspension. the treated apples were placed on cardboard trays that were then enclosed in plastic bags. the inside of the bags were sprayed with sdw to maintain high relative humidity (up to 90%) in the bags. after 24 h, the wounds were inoculated with 20 µl of conidial suspension of the pathogen (10 5 conidia ml −1 ).treated apples were incubated at two temperatures, room (20°c) cold (5°c) in humid condition for 15 and 32 days, respectively. the lesion diameter was calculated after 15 days, using calipers. each apple constituted a single and each treatment was replicated 4 times. the antagonistic effect of different inoculation times: wounds were inoculated with 40 l of c. membranfaciens (a5) that was prepared as described above, the cell suspension at a concen-tration of 1 × 10 7 conidia ml -1 . after 2, 14, 32 h and 48 a 20 l p.expansum (p11) suspension in a concentration of 1 × 10 5 conidia ml -1 was added to each wound, and the apples were stored at 20°c. the antagonistic effect of the different yeast concentrations: wounds were inoculated with 20 l of c. membranfaciens (a4) that was prepared as described above, the cell suspension at the concentrations of 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 conidia ml 1 . after 24 h, a 20 µl p.expansum (p11) suspension at a concentration of 1 × 10 5 conidia ml -1 was added to each wound. the apples were then stored at 20°c. evaluation antagonistic effect of cacl 2 concentration on biocontrol activity of yeast: aliquots of 50 ml distilled water with different concentration of cacl2: 0 mm, 173 mm (2%), 347 mm (4%), and 521 mm (6%) cacl2, in 250 ml conical flasks were autoclaved (121°c, 15 min) prior to adding suspension of antagonistic yeast to some flask. the cell suspensions of antagonists were adjusted to concentrations of 1 × 10 7 cfu ml -1 in this solution. the same percentages of cacl2 without antagonistic yeast were prepared as described above. wounds were inoculated with a 40 l of the yeasts with and without cacl2 were prepared as described above. after 24 h, a 20 l of p. expansum (p11) suspension in a concentration of 1 × 10 5 spores ml -1 was added to each wound, and the apples was stored for 15 days at 20°c. the lesion diameter was calculated after 15 days, using calipers. each apple constituted a single and each treatment was replicated 4 times (tian et al., 2002). population dynamics of yeast on apple surface: growth curves were monitored in fruit wounds at 20 and 5°c. the wounds were inoculated with 40 µl of yeast (a1 and a2 strains) suspension of known concentration (10 7 cfu ml -1 ), and after 24 h wounds were inoculated with 20 µl of conidial suspension of the pathogen (p11) , then apples were incubated for 15 days at 20°c and for 32 days at 3 table 1. p. expansum (p11) growth inhibition percentage by c. membranfaciens (a2, a4 and a5) and r. mucilaginosa (a1 and a7) strains in vitro a . yeast isolates percentage of inhibition b dual culture volatile test cell free metabolite c. membranfaciens (a5) 20.59 b 73.5 c 84.3 a c. membranfaciens (a2) 28.48 b 57.18c 84.38 a c. membranfaciens (a4) 30.93 b 78.94 b 72.49 b r. mucilaginosa (a1) 61.4 a 71.86 c 83.36 b r. mucilaginosa (a7) 34.51b 89.14 a 86.03 a a values are the mean inhibition growth percentage from four replication. b values in the same column followed by the same letter are not statistically different by duncan’s multiple range test (p < 0.05). 5°c. controls were inoculated with sdw. apples were removed a block of tissue (1gr). the block was removed and shaken in 9 ml sterile water on a rotatory shaker for 20 min at 150 rpm. serial dilutions of the washings were made and one ml was plated on nyda containing 0.5 g/l streptomycin sulphate as a bacteriostat. after incubation at 25°c for 48 h the isolated viable colonies per gram of fresh weight of fruit (cfu/g) were counted for each sample. this study was carried out with four replicates per treatment and sample unit was four apples. (teixido et al., 1999) statistical analysis the in vitro and in vivo assays were analyzed by an analysis of variance (anova) with sas software (sas institute, version 9.0, cary, nc). all assays were carried in a completely randomized design with four replicates. statistical significance was judged at the level p < 0.05. when the analysis was statistically significant, duncan’s multiple-range test (ssr test) was used to test mean separations among mean values of each treatment. results effects of the antagonists in vitro the two antagonist tested yeasts could inhibit significantly the p. expansum (p11) mycelia growth of in dual culture, compared among. however there were significant differences among yeast strains. growth inhibition of p. expansum (p11) by strain a1 (r. mucilaginosa) was significantly greater than calculated by of other strains, whereas strain a4 (c. membranfaciens) had less effect on the growth of the pathogen (table 1). results shown in table 1 indicated that antifungal activity of volatile metabolites of the c. membrane-faciens strain a2 was lower compared with other strains. cell free metabolite of c. membranfaciens reduced growth of p. expansum (p11) by 72.5 84.4%, whereas r. mucilaginosa reduced the colony area of pathogen from 83.36 86.03% (table 1). effect of different storage temperatures on decay area all of strains of two genus yeasts significantly controlled blue mold on apples at 20 and 5°c (p 0.05). strains of c. membranfaciens reduced decay area from 201.76 to 973.23 mm 2 , whereas r. mucilaginosa reduced from 1584.14 1647.19 mm 2 compared to 3257.44 mm 2 in, after incubation for 15 days at 20 °c (table 2). at 5°c, c. membranfaciens strains reduced decay area from 298.4 to 881.8 mm 2 , whereas r. mucilaginosa reduced 1631.9 1908 mm 2 compared with 3151.1 mm 2 in control, after 32 days incubation at 5°c (table 2). optimum temperature growth for both two antagonistic yeasts was 25°c (data not shown). effects of different inoculation times on decay area p. expansum (p11) was added 2, 14, 32 and 48 h after the inoculation of the antagonistic yeast. then the apples were stored at 20°c, and the decay area was observed every 24 h after inoculation (figure 1). the figure 1 shown, it can be seen that the later the path-ogen were introduced after the inoculation of the yeast, the better the antagonism was. the reason may be that with enough time the yeast had reproduced and used the nutrient (competition for space and nutrition), or the yeast had secreted enough antagonistic substances. so yeast should been applied the antagonist early after the harvest to prevent the pathogens from infecting. effects of different concentration of yeast on decay area high, middle and low concentrations of yeast (lc = 1 × 10 5 and 1 × 10 6 conidia ml -1 ; mc = 1 × 10 7 conidia ml -1 ; hc = 1 × 10 8 conidia ml -1 ) were inoculated into wounds of apple. the apple fruits were stored at 20°c. decay area were recorded every 24 h after the treatment (figure 2). it was obvious from figure 2 that the antagonism was related to the concentration of antagonist. when concentration of the yeast reached 1 × 10 7 conidia ml -1 and spore suspension of the pathogens was 1 × 10 5 conidia ml -1 , distinct antagonism was observed. the postharvest biocontrol capability of the apple fruits was 4 table 2. lesion area on golden delicious apples with p. expansum (p11) challenged with c. membranfaciens (strains a2, a4 and a5) and r. mucilaginosa (strains a1 and a7) and incubated for at 20 and 5°c in vivo. lesion area b (cm 2 ) treatment a5 a2 a4 a7 a1 control 20°c 973.23c 309.23d 201.76d 1584.14b 1647.19b 3257.44a 5°c 298.4f 881.84d 490.57e 1631.94c 1907.96b 3151.16a a values are the mean lesion area from four apples. b values in the same row followed by the same letter are not statistically different by duncan’s multiple range test (p < 0.05). figure 1. effect of inoculation time of p. expansum on apple decay area. table 3. effect of different concentrations of cacl2 and candida membranfaciens (a5) on lesion area on apples a . treatment (%) main inhibition (%) b (cacl2) a5 c +cacl2 cacl2 (without yeast) 0 61. 9c 0a 2 70.5d 29b 4 86f 54.8c 6 92.4f 61.1c a values are the mean lesion area percentage from four apples. b values in the table followed by the same letter ( in the row and column) are not statistically different by duncan’s multiple range test. (p < 0.05). c one c. membranfaciens strain enhanced with the increase of the antagonist concentration. effectiveness of cacl2 on biocontrol activity of two strains of yeasts there was a significant reduction in the diseases after cacl2 was added to the yeasts treatments, as controlled to cacl2 alone (p 0.05) (table 3 and 4). the results showed that all concentrations of ca 2+ had significant effect on p. expansum lesion diameter. the control of pathogen was positively correlated to ca 2+ concentrations; cacl2 at 521 mm provided better control than 347 mmol l -1 but wasn't significant. finally, 347 mm was 5 figure 2. effects of c. membranfaciens (a5) concentration on apple decay area. table 3. effect of different concentrations of cacl2 and c. membranfaciens (a5) on lesion area on apples a . treatment (%) main inhibition (%) b (cacl2) a5 c +cacl2 cacl2 (without yeast) 0 61. 9c 0a 2 70.5d 29b 4 86f 54.8c 6 92.4f 61.1c a values are the mean lesion area percentage from four apples. b values in the table followed by the same letter ( in the row and column) are not statistically different by duncan’s multiple range test (p < 0.05). c one c. membranfaciens strain. table 4. effect of different concentrations of ca 2+ and rhodotorula mucilaginosa (a1) on lesion area in apples a . treatment (%) main inhibition (%) b (cacl2) a1 c + cacl2 cacl2 (without yeast) 0 56.5c 0a 2 61c 29.1b 4 77.9d 54.8c 6 88.2e 60c a values are the mean lesion area percentage from four apples. b values in the table followed by the same letter are not statistically different by duncan’s multiple range test (p < 0.05). c one r. mucilaginosa strain. population dynamics the population dynamics of two yeasts were evaluated after 5, 10 and 15 days at 20°c and 10, 20 and 30 at 5°c. the maximum population was recovered 10 and 20 days after the yeasts application at 20°c and 5°c, respectively. initially, 4 × 10 5 cfu g -1 inoculated on the wounds, 10 days after applications of r. mucilaginosa (strain a1) at 20°c, population were maximum, about 3.7 × 10 8 cfu g -1 , after that decrease population, until end of the experiment 6 a1+p11 population sizes of r. mucilaginosa strain a1 in apple wounds stored at 20°c b a1+p11 figure 3. population dynamics of r. mucilaginosa strain a1 ( ); and strains a1 to added pathogen ( ), in wounds of apple fruit stored at 20°c (a) , and 5°c (b). (figure 1.) . at 5°c, in the twentieth day population was maximum, about 3.1×10 8 cfu g -1 , after that decrease population, until end of the experiment (figure 3). discussion the use of biocontrol agents to manage postharvest decay of fruits has been explored as an alternative to the use of synthetic fungicides (wilson and wisniewski, 1989) and several commercial products are now available (droby et al., 1998). further identification of new antagonists is desirable because antagonists identified in specific geographic areas may be more effective against the pathogen isolates present in that locale. in the present research, we have identified two yeast antagonists that exhibit biocontrol efficacy against blue mold of apples caused by p. expansum. this is the first report of antagonists candida membranfaciens and rhodotorula mucilaginosa as antagonists of blue mold of apples. the biocontrol activity of yeasts antagonist such cryptococcus laurentii and candida ciferrii (vero et al., 2002) haven’t been attributed to the production antifungal metabolite. regardless, our data showed better inhibition, in dual culture. all antagonistic yeast strains reduced mycelia growth of pathogen by means of dual culture, cell free metabolite and volatile metabolite test. in cell free culture test all strains of yeast produced high inhibition zone on pda indicating that they synthesize compounds that are highly active against pathogen. growth curves of the antagonists demonstrated that all of strains of yeast could colonize and grow in apple wounds. even after a period of 32 days at 5°c, and 14 days at 20 ë c the number of viable microorganisms was greater than that originally introduced into the wound. etebarian et al. (2005) demonstrated that pseudomonas fluorescens population on wounded apple inoculated with p. expansum increased with 10-100 fold at wound site over 20 days at 20°c. in our case, the wound carrying capacity was approx. 3.7 × 10 8 cfu ml -1 per wound after only 10 days. this may indicate that r. mucilaginosa strain a1 has a high capacity for colonizing apple wounds and could be potentially a better biocontrol agent. these data indicate that only one application of the antagonists may be enough to prevent blue mold rot for at least a period of 32 days at 5°c, and 14 days at 20°c. in this study result for apples stored at 5°c for 32 days 7 and at 20°c for 14 days showed that these 5 strains reduced growth of p. expansum and was effective for control of pathogen. these strains have also ability for suppressing other pathogen such as green mold of oranges (penicillium digitatum), grey mould on tomato (botrytis cinerea) (dal bello et al. 2008). the adaptation of these five strains to a wide range of temperature provides great market potential for this product for control of postharvest diseases on apples in storage and transportation, as well as under arbitrary temperature in the market places and consumers’ home. the activity of both yeast strains and cacl2 against pathogen provided an opportunity to combine these agents at lower concentration while mainting a high level of effectiveness and consistency of performance are two major factors limiting the use of biocontrol agent in plant disease control (chalutz and wilson, 1990). yeasts and cacl2 had a significant synergistic effect on reduction of blue mold on golden delicious apple. cacl2 no suppressive effect on yeasts until 6% (w/v) in vitro. a combination of a biocontrol agent with a salt both significantly reduced at concentration from what would be needed if used alone may greatly reduce their residue on fruit (gholamnejad and etebarian, 2009). in conclusion, five strains of yeasts tested here reduced disease severity of blue mold of apple at 5 and 20°c. the combination of these strains and cacl2 were most effective on controlling of blue mold of apple. these strains warrant further investigation for their ability to control blue mold in commercial situation. further identification of new antagonists is desirable because antagonists identified in specific geographic areas may be more effective against the pathogen strains present in that locale. acknowledgements this research was carried out with grants from aboryhan campus of tehran university, iran. particular thanks to dr. javad gholamnejad, allah yar gholamnejad and shahram najafi, who cooperated in part of the experiments. references chalutz e, wilson cl (1990). postharvest biocontrol of green and blue mold and sour rot of citrus fruits by debaryomyces hansenii. plant dis. 74: 134-137. dal bello g, monaco c, rollan mc (2008). biocontrol of postharvest grey mould on tomato by yeasts. phytopathol., 156: 257-263. dennis c, webster j (1971). antagonistic properties of species groups of trichoderma. iii. hyphal interaction. trans. br. mycol. soc., 57: 363-369. droby s, cohen l, daus a, wisniewski me, wiess b (1998). commercial testing of aspire: a yeast preparation for the biological control of postharvest decay of citrus. biol. control, 12: 97-101. eckert jw, sievert jr, ratnayake m (1994). reduction of imazalil effectiveness against citrus green mold in california packinghouse by resistant biotypes of penicillium digitatum. plant dis., 78: 791-794. etebarian hr, sholberg pl, eastwell kc, sayler rj (2005). biological control of apple blue mold with pseudomonas fluorescens. can. j. microbiol., 51: 591-598. gholamnejad j, etebarian hr (2009). effect of calcium chloride on the biocontrol efficacy of two antagonistic yeasts against penicillium expansum on apple fruit. phytopara., 37: 255-267. gholamnejad j, etebarian hr, roustaee a, sahebani na (2009). biological control of apples blue mold by isolates of saccharomyces cerevisiae. j. p. prot. rea., 49: 270-275. gholamnejad j, etebarian hr, roustaee a, sahebani na (2009). characterization of biocontrol activity of two yeast strains from iran against blue mould of apple in order to reduce the environmental pollution. j. int. environ. appl. sci., 4(1): 28-36. lillbro m (2005). biocontrol of penicillium roqueforti on grainacomparison of mode of action of several yeast species. master theisis for the agriculture programme, animal science, performed at the department of microbiology. swedish university of agricultural sciences pp. 21. lima g, ippolito a, nigro f, salerno m (1997). effectiveness of aureobasidium pullulans and candida oleophila against postharvest strawberry rot. postharvest biol. technol., 10: 169-178. roberts rg (1990). postharvest biological control of gray mold of apple by cryptococcus laurentii. phytopathol., 80: 526-529. spotts ra, cervantes lk (1986). populations, pathogenicity, and benomyl resistance of botrytis spp., penicillium spp. and mucor piriformis in packinghouses. plant dis., 70: 106-108. teixido n, usall j, vinas i (1999). efficacy of preharvest and postharvest candida sake biocontrol treatments to prevent blue mould on apples during cold storage. international j. food microbiol., 50: 203-210. tian sp, fan q, xu y, jiang al (2002). effects of calcium on biocontrol activity of yeast antagonists against the postharvest fungal pathogen rhizopus stolonifer. plant dis., 51: 352-358. vero s, mondino p, burgaeno j, soubes m, wisniewski m (2002). characterization of biological activity of two yeast strains from uruguay against blue mold of apple. postharvest biol. technol. 26: 91-98. weller dm (1988). biological control of soilborne plant pathogens in the rhizosphere with bacteria. annu. rev. phytopathol., 26: 379-407. wilson cl, wisniewski me (1989). biological control of postharvest diseases of fruits and vegetables. an emerging technology. annu. rev. phytopathol., 27: 425-441. wilson cl, wisniewski me, droby s, chalutz e (1993). a selection strategy for microbial antagonists to control postharvest diseases of fruits and vegetables. sci. horti., 53: 183-189. wisniewski me, biles c, droby s, mclaughlin r, wilson c, chalutz e (1991). mode of the postharvest biological yeast, pichia guilliermondii . i. characterization of attachment to botrytis cinerea. physiol. mol. plant pathol., 39: 245-258. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 11 (1), pp. 001-008, january, 2023. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper fatty acid composition of meat from lambs fed diets containing moist-heat treated legume grains jamal seifdavati1 and akbar taghizadeh2 .1 department of animal science, faculty of agriculture, university of tabriz, iran. p.o. box 51664. tel: +98 4113392029 fax: +98-4113356004. 2 department of animal science, faculty of agriculture, university of tabriz, iran. accepted 14 may 2022 in recent years, an important objective of world farmers has been to enhance the use of alternative protein sources, preferably from local feedstuffs. in order to an experiment was carried out to study on nutritional manipulation of the saturated fatty acid (sfa) composition of lamb meat to reduce the concentration of sfa in meat from lambs fed legume grains and to raise the concentration of the hypocholesterolemic fa. fortytwo female hybrid lambs were divided into seven groups after weaning at about 60 days of age. the diets of the seven groups differed in the protein source used in the concentrate, soybean meal group (sbm), bitter vetch group (bv), common vetch group (cv) and chickling vetch group (clv). lambs were fed ad libitum and slaughtered at about 145 days of age. there were no differences in growth or carcass characteristics among the dietaries. the cv and clv diets had higher contents of the two essential fatty acids, linoleic and linolenic compared to the bv and sbm diets. however, the total percentage of sum of these two fatty acids was ranged for the seven diets from 56.6 g to 57.05g /100 g of fa methylesters. the fa most abundant in the meat from all legume groups was oleic acid, the amounts of which were significantly (p<0.01) different between treatments. the linolenic acid was higher (p<0.01) in meat from cv lambs than in meat from sbm, bv and clv animals. the provision of modest amounts of grain is more conducive to the oleic acid synthesis rather than high levels of grain. in conclusion the use of legume seeds such as bv, cv and clv in lamb diets positively affected intramuscular fatty acid composition. keywords: autoclaving; intramuscular fatty acid composition; legume seeds; meat lamb introduction recently, much focus has been done to increase meat with physiological functions to improve health status and prevent the hazard of diseases. the effect of the use of corresponding author e-mail: ataghius2000@yahoo.com local legume seeds in lamb nutrition has been considered. several studies showed that their use did not negatively affect growth, slaughter performances or meat quality (hadjipanayiotou, 2002; lanza et al., 2003b; lanza, 1999; purroy et al., 1992; surra et al., 1992). of the protein feeds commonly used in worldwide, peas, field beans, types of vetch and rapeseed are mainly locally produced. types of vetch, shch as, bitter vetch 2 table1. ingredient, chemical composition (g/kg dry matter) and fatty acid composition (g/100 g fatty acid methylesters) of the experimental diets. ingredient diets a sbm raw or raw or raw or heated clv heated cv heated bv alfalfa hay 417 417 416 416 barley grain 443 421 423 424 maize grain 58 53 52 51 soybeans meal(sbm) 69 – – – chickling vetch (clv) – 97 – – common vetch (cv) – – 96 – bitter vetch (bv) – – – 96 urea – 7 5 9 di calcium phosphate 5 5 5 6 salt 8 8 8 8 vitamin, mineral premix b 5 5 5 5 chemical composition dry matter/kg fresh matter 858 857 856 855 crude protein 163 160 162 163 rumen degradable protein (rdp) 107 107 108 107 rumen undegradable protein rup 57 53 54 55 metabolisable protein 104 104 104 104 ether extract 28 28 29 29 neutral detergent fibre 366 390 397 400 acid detergent fibre 228` 230 234 238 gross energy, (mj/kg dm) 18.5 18.1 18.1 18.0 metabolisable energy, (mj/kg dm) 13.1 13.1 13.1 13.1 fatty acid composition c14:0 0.65 0.5 0.64 0.52 c16:0 17.83 15.54 17.18 17.45 c18:0 3.76 3.38 3.62 3.68 c18:1 n-9 21.15 20.55 21.51 21.32 c18:2 n-6 43.58 45.49 44.15 43.96 c18:3 n-3 13.03 14.54 12.90 13.07 a clv, cv and bv diets with 30% of the total protein supplied by raw or autoclaved clv, cv and bv grains. b vitamin, mineral pre-mix (g/kg): 220 bicalcic phosphate, 220 magnesium oxide, 20 zinc sulphate, 20 ferric sulphate, 8 manganese sulphate,7.5 copper sulphate, 385 sodium bicarbonate, 0.45 of a mixture of vitamins a and and d3, 0.8 vitamin e 50%, 0.45 copper and, (mg/kg): 0.45 sodium selenite, 1.5 potassium iodide, 1.5 cobalt sulphate. (vicia ervilia.l), common vetch (vicia sativa.l) and chickling vetch (lathyrus sativus.l) seeds are the legume seeds available in the west north area of iran and are comparatively cheap despite its relatively high nutritional value. their crude protein content ranges from about 25 to 28 % of dry matter (abreu and bruno-soares,1998; gonzalez and andres, 2003; sadeghi et al., 2004, 2009; haddad et al.,2006; ramos morales et al., 2008; rezayazdi et al., 2008; abdullah et al., 2010 and razmazar et al., 2009).the use of diets based largely on types of vetch for lamb fattening gave similar growth performance and meat characteristics as traditional diets based on soybean meal as the main protein source (caballero et al., 1992; lanza et al., 1999). new feeding strategies in animal nutrition in both ruminants and non-ruminants have as principal objectives to increase polyunsaturated fatty acids(pufa), especially the ω-3 series, and reduce sfa in animal products (scollan et al., 2006; wood et al., 2008). there is little data available on the effects of feeding types of vetch on lamb intramuscular fatty acid composition. the objective of the present study was to evaluate the effect of totally replacing dietary soybean meal by bv, vc and clv seeds in the concentrate fed to lambs on the intramuscular fatty acid composition of their meat. materials and methods experimental design, animals and diets the feeding trial was conducted at an experimental farm of the university of tabriz (iran). the experiment was 3 table2. fatty acid composition (g/100 g fatty acid methylesters) of meat (longissimus dorsi muscle). fatty acid experimental diets a sem p-value s raw legume grains autoclaved legume bm grains clv cv bv clv cv bv c14:0 2.46 3.17 3.40 3.54 3.65 4.2 3.7 0.85 0.09 c16:0 23.43 a 13.22 c 13.37 c 21.28 a 13.49 c 17.73 ab 21.18 a 1.75 <.001 c18:0 15.27 b 13.66 bc 10.06 c 11.88 bc 18.14 a 11.32 bc 12.99 bc 1.94 0.001 c18:1 n-9 34.78 a 31.3 b 32.5 b 34.09 ab 35.5 a 33.34 ab 34.54 a 0.86 <.001 c18:2 n-6 11.81 c 15.23 a 13.61 b 14.59 ab 15.72 a 14.17 b 14.69 b 0.58 <.001 c18:3 n-3 0.09 b 0.09 b 0.11 a 0.06 b 0.08 b 0.10 a 0.03 b 0.03 0.01 a clv, cv and bv diets with 30% of the total protein supplied by raw or autoclaved clv, cv and bv grains. sem, standard error of the mean; different lowercase letters within a same row indicate significant differences among diets indicate significant differences (p< 0.05). conducted on forty-two female gizel×merino and moghani×merino lambs, born on the same farm. after weaning, at 60 days of age, the lambs were divided into 7 groups balanced according to their weights, with 6 animals per treatment and were stratified according to weight (kg 18-25, live weight), housed in individual pens and randomly assigned to one of seven dietary treatments [treated and untreated cv, 6 animals; treated and untreated bv, 6 animals; treated and untreated clv, 6 animals, sbm as control, 6 animals]. the lambs, after 10 days of adaptation to the experimental diet, were fed for a further 75 days. all the diets were included maize, barley, lucerne hay, salt, di-calcium phosphate and mineral-vitamin premix. seven isoenergetic and isonitrogenous experimental diets (legume grains) were formulated each one with 30% of the total protein from one of the experimental legume grains (table1). legume grains diets were included in total replacement of soybean meal and in partial replacement of maize and barley. sbm diet was considered as the control diet and included soybean meal as the main protein source. all the ingredients were ground and compounded into lucerne hay as tmr feed. the diets were formulated to meet requirements for the female lambs (afrc, 1993). the animals were slaughtered at about 145days of age. twenty-four hours after slaughter carcasses were split in left and right sides and from the right side samples of longissimus dorsi muscle were taken between the 6th thoracic rib and 4th lumbar rib to measure moisture, crude fat, protein and ash according to aoac procedures 4 table3. effect of the diets on lamb growth rate, carcass characteristics and meat chemical composition carcass characteristics experimental diets a sem p sbm raw legume grains autoclaved legume grains value clv cv bv clv cv bv body weight at 70 d (kg) 20.34 19.99 19.99 22.05 19.95 19.46 23.44 3.89 0.76 body weight at slaughter (kg) 29.87 28.53 27.90 29.19 29.31 30.24 29.55 1.76 0.83 average daily gain(70-145d) (kg) 0.121 0.104 0.098 0.113 0.111 0.126 0.119 0.03 0.83 dry matter intake (g/d) 687 723 711 756 719 798 764 115 0.08 feed/gain (kg) 6.58 6.74 7.85 7.31 6.82 6.63 7.04 1.12 0.45 dressing percentage 47.35 46.86 47.7 46.29 49.96 47.72 46.53 2.26 0.52 cold carcass weight (kg) 13.85 13.04 13.04 13.33 14.16 14.13 13.57 0.82 0.96 0.04 0.13 eye muscle area, cm 2 13.2 11.73 11.11 10.57 10.18 11.97 11.79 gr fat depth (mm) 0.99 0.85 0.53 0.65 0.99 0.88 0.89 0.29 0.62 longissimus dorsi characteristics 1.05 0.93 moisture (%) 75.66 74.88 75.34 75.45 75.74 75.78 75.57 ash (%) 1.23 1.37 1.32 1.31 1.27 1.32 1.27 0.05 0.10 1.44 0.01 crude fat (%) 2.48 b 2.61 b 4.97 a 4.82 a 3.06 b 3.96 a 2.48 b crude protein (%) 20.59 a 21.14 a 18.38 b 18.53 b 19.94 a 18.96 b 18.68 b 1.65 0.03 a clv, cv and bv diets with 30% of the total protein supplied by raw or autoclaved clv, cv and bv grains. sem, standard error of the mean; different lowercase letters within a same row indicate significant differences among diets indicate significant differences (p< 0.05). (aoac, 2000). subsamples of each concentrate mixture were collected weekly and mixed to give a final sample for ndf and adf analysis, without the use of sodium sulphite as described by van soest et al. (1991) using the ankom ndf/adf fiber system (2008), crude protein (method 984.13), ether extract (method 920.39) and ash (method 942.05) according to aoac (2000). fatty acid determination samples of longissimus dorsi muscle were taken at the level of the 13th thoracic rib, minced and vacuum-packed (50 g for each animal) and stored at −25 °c until needed. fa composition of meat was determined after chloroformmethanol extraction of total lipids (folch et al., 1957). briefly, a 5 g homogenised longissimus dorsi sample was blended with chloroform/methanol (2:1, v/v) twice, filtered, placed in a separator funnel and mixed with saline solution (0.88% kcl). after separation into two phases, the aqueous methanol fraction was discarded and the chloroform lipid fraction was washed with distilled water/methanol (1:1, v/v). after a further filtration and evaporation by means of a rotary evaporator, lipid extracts were transferred to test tubes for subsequent gas chromatographic analysis. duplicates of 100 mg of lipid were methylated using 1 ml of hexane and 0.05 ml of 2 n methanolic koh (i.u.p.a.c, 1987). separation of fame were also performed on a gas chromatograph (agilent, palo alto, usa) model hp 6890 coupled with a 5973 mass spectrometer detector (agilent) and hp 7683 series injector (agilent,2001, palo alto, ca, usa). 1 μl of the final extract was injected using pulsed splitless mode (140 kpa, 0.4 min, 250 °c).hp-5ms column (30 m × 0.25 mm × 0.25 μm) was operated at a temperature program 2.1 min at 50 °c and a subsequent increase to 200 °c at 10 °c/min and the final temperature of 200 °c to 270°c at 15 °c/min, was held for 12min with helium as a carrier gas (99.9998 %; flow rate: 0.9 ml/min; a split ratio of 50:1; inlet temperature: 250 °c; siad, bergamo, italy). fame was identified by comparing their mass-spectral data to the mass-spectral database in the library wiley 7.0 (hp mass spectral libraries, agilent, 2001, palo alto, usa). fatty acids were expressed as percent of total methylated fatty acids. statistical analysis data were analysed by a factorial variance analysis to study the effect of autoclaving on the intramuscular fatty acid composition of lamb meat each the group treatment. the model was: y ijk =µ+ feedstuff i + treatment j+ (feedstuff× treatment) ij+e ijk where principal effects, kind of feedstuff in diets (2 d.f.), autoclaving treatment (1 d.f.) and the interaction between them (2 d.f.) were compared with the residual error (5 d.f.). when there were significant differences for studied effects, differences between mean values were determined using the least significant difference test. all statistical analyses were performed by the general linear 5 model (glm) procedure using the statistical package sas (2001) after adjusting on covariate effect (initial body weight of lambs) with covariance analysis. results chemical composition of the experimental diets the ingredients, fatty acid composition and proximate analyses of the diets are presented in table 1. the seven diets had similar crude protein contents. with regard to dietary composition (table 1), bv and sbm diets had higher c16:0 and c18:0 contents compared to the cv and clv diets. the cv and clv diets had higher contents of the two essential fatty acids, linoleic (c18:2) and linolenic (c18:3) compared to the bv and sbm diets. however, the total percentage of c18:2 + c18:3 fatty acids was ranged for the seven diets from 56.6 g to 57.05g /100 g of fatty acid methyl esters. lamb growth performances and meat proximate analyses the growth performances data are shown in table 3. no significant differences were found between treatments. average daily gain was over 98 g/day in all groups resulting in a final weight of around 28–30 kg with small differences among groups. the animal growth performances and chemical composition of the meat samples are reported in table 3. no statistical differences between groups were found for any parameter measured of growth performances and carcass characteristics; thus their growth rate, the dm intakes, feed/gain and final body weight were not significantly affected by the protein source in the concentrate among the groups (table 3). although feed intake (p = 0.08) tended to be lower for the sbm group compared to the other treatments. there was no difference in the dry matter and crude ash contents between the experimental groups. however, chemical composition of the meat samples of lambs fed different diets showed a significant difference in crude fat and crude protein contents (p<0.01). unlike the crude fat, the crude protein level of the meat in the sbm and clv diets was slightly higher than in the other treatments. intramuscular fatty acid composition table 2 provides the fatty acid profile of longissimus dorsi muscle. among the saturated fatty acids, there was a significant difference in c16:0 contents, it was higher (p<0.001) in meat from sbm and bv lambs than in meat from clv and cv lambs. also c18:0 was higher (p<0.01) in meat from clv lambs compared to meat from the rest of lamb groups. among the bv and sbm groups the level of palmitic acid (c16:0) was higher (p<0.01) in meat from sbm lambs. the fatty acid most abundant in the meat from all legume groups, as the protein source in the concentrate, was cis-9 c18:1 (oleic acid), the amounts of which were significantly (p<0.01) different between treatments. similar results for this fatty acid were observed by lanza et al. (2003a), wood et al. (2008) and scerra et al. (2011) for other legume grains as the protein source in the dietary concentrate. overall, the linolenic acid (c18:3 ω-3), was higher (p<0.01) in meat from cv lambs than in meat from sbm, bv and clv animals. the lambs of the clv group showed higher levels (p<0.01) of the linolenic acid (c18:3 ω-3), compared to the lambs of the sbm and bv groups. discussion the fatty acid composition of longissimus dorsi muscle partially reflected the dietary fatty acid composition. ruminants do not deposit tissue fa in proportion to dietary lipid composition, as do non-ruminant animals, because rumen microorganisms hydrolyse the glycerides and then hydrogenate the dietary unsaturated fa (harfoot and hazlewood, 1988 and mcniven et al., 2004). thus, ruminant milk or meat has higher sfa/ pufa than non-ruminant animals. the levels of reduced palmitic (c16:0) and stearic (c18:0) acids in meat from lambs of the clv and cv groups, respectively, compared to the other groups represent (p<0.01) potential both in decreasing the harmful effects on health because they may be directly responsible for increasing total and low density lipoproteins (ldl) cholesterol in plasma and enhancing risks for human health (mcniven et al., 2004 and scollan et al., 2006). in lamb and mutton, the proportions of these two fatty acids are more similar. there is little variation between cuts in the proportion of fatty acids. an alternative strategy to improve the human health attributes of sheep meat is to decrease tissue levels of 18:0 by increasing the activity of stearoyl-coa desaturase (scd), although the response is often relatively small (sinclair,2007). sheep meat is characterized as being high in sfa and low in pufa, attributes that are regarded as being disadvantageous within the human diet (sinclair, 2007).despite early hypothesis (keys et al., 1965) on the effects of dietary fats on human health, there was complex problems and ambiguities on the implications of saturated fa in elevated blood cholesterol leading to coronary heart disease. a meta-analysis from hunter et al. (2010) of the evidence since 2000 resulted in one systematic review that covered all selected primary studies. this review was paying attention on the effect of stearic acid on cardiovascular disease (cvd) risks when replaced with for sfa, trans fatty acids (tfa), monounsaturated fat 6 (mufa), pufa or carbohydrates and provided the evidence to deal with this question (hunter et al., 2010). one goal of animal nutrition research is feasibility study nutritional manipulation of the fatty acid composition of sheep meat (mcniven et al.,2004; sinclair, 2007) to reduce the concentration of these fa in meat from lambs fed legume grains and to raise the concentration of the hypocholesterolemic fa (c18:1, c18:2, c18:3). moreover palmitic acid was higher (p<0.01) in meat from sbm and bv lambs than in meat from other lambs. the higher levels of these fatty acids in meat from bv and sbm lambs compared to clv and cv lambs partially reflected the levels of these two fatty acids in the diets. the level of c18:1 was lower (p<0.01) in meat from the raw clv group than in meat from the other groups. the amounts of intramuscular c18:1 for all diets was higher than the proportions in the dietary fatty acids. fortunately, animal cells can synthesize oleic acid and its derivatives from stearic acid. oleic acid is created by the dehydrogenation (desaturation) of stearic acid. on the contrary human, animals can be easily desaturated stearic acid (c18:0) to oleic acid by means of a δ9-desaturase enzyme (pereira et al., 2003). however, c18:2 was much lower in the muscle fat than in the diets, indicating that the dietary c18:2 was partially hydrogenated in the rumen (rizzi et al., 2002). the higher amount of linoleic acid (c18:2 n-6) (p<0.001) in meat from clv lambs than in meat from bv,cv and sbm lambs was probably related to the higher level of this fatty acid in the clv diet compared to the others. moreover linoleic acid was higher (p<0.01) in meat from the bv and cv groups than in meat from the sbm group. the endogenous biosynthesis of this fatty acid in muscle from linolenic acid is well-known (zhou and nilsson, 2001). linolenic acid (c18:3 ω-3), the precursor of long chain n-3 fatty acids that have a wide range of biological effects and which are believed to be beneficial for human health (kromhout, 1989; bonanome and grundy,1998; mcafee et al., 2010). the higher level of this fatty acid in the diets and also the lower level of this fatty acid in the muscle fat than in the diet, indicating that the dietary c18:3 was partially hydrogenated in the rumen (rizzi et al., 2002). despite the apparent negative impact of ruminal metabolism on muscle fa content, the process of biohydrogenation is often incomplete and accompanied several of the intermediaries that can have positive effects on human health. also, several studies (marmer et al., 1984; chilliard, 1993; murphy and mcniven, 1994) have shown that dietary fa composition differences can result in differences in tissue fa composition. the observed differences between the results this experiment with found other investigators can be explained with various influencing factors such as pasture compared with feedlot-finished, nature of diet in feedlot, whether the diet contained oil or oilseed, the fatty acid composition of the oil, proportion of grain and silage compared with hay, seasonal variations, animal genetics, grain type and production practices (mir et al., 2004; tilak et al., 2005). also in current study, no significantly the method of grain processing in the (c18:1, c18:2) content of meat from lambs fed diets containing moist-heat treated legume grains can be resulted from factors inherent in grain type. these results have been inconsistent with the findings of others (mcniven et al., 2004). they indicated in their report that meat from the extruded soybean-fed group had higher levels of oleic and linoleic acid compared with the other treatments. it is possible to modify the fatty acid composition of beef meat by including roasted soybean in the diet and processing exposes the oil to rumen biohydrogenation, but possibly not to the same extent as the oil from the raw soybean. so that, in contrast this found and consistent with our finding, a study of mohammed et al. (2010) clearly demonstrated that the milk content and profile of t-18:1 were more strongly influenced by the source of grain than by the method of grain processing indicating that factors inherent in grain type were responsible for the observed differences and these factors could not be modified by the routine processing methods used in farms such as anti-nutrient factors. conclusions to increase the ufa yield in lamb meat it is essential to provide lamb an appropriate substrate for formation of essential fa. dietary forage such as grass or legume hay appears to facilitate the establishment of the microflora that enhance the formation and deposition of ufa in the tissues also the provision of modest amounts of grain is more conducive to ufa synthesis rather than high levels of grain. acknowledgements the authors wish to thank the deanship and the personnel of khalat phoshan educational and agricultural research station and chemistry faculty at tabriz university of iran for the financial support and their technical assistance of this project. references abdullah y, muwalla mm, qudsieh ri, titi hh (2010).effect of bitter vetch (vicia ervilia) seeds as a replacement protein source of soybean meal on performance and carcass characteristics of finishing awassi lambs. trop. anim. health prod.42:293-300. abreu jmf, soares amb (1998).chemical composition, organic matter digestibility and gas production of nine legume grains. anim feed sci technol. 70:49-57. afrc (1993).energy and protein requirements of ruminants.cab international, wallingford. 7 agilent technologies (2001).agilent chemstation for lc 3d systems understanding your spectra module user’s guide. palo alto, ca, usa: agilent technol inc., 84 p. ankom (2008).procedures for fibre and in vitro analysis — accessed august 2008 from the website: http://www.ankom.com. aoac (2000).in: horwotz, w. 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animal nutrition.j dairy sci. 74: 3583-3597. wood jd, enser m, fisher av, nute gr, sheard pr, richardson ri (2008).fat deposition, fatty acid composition and meat quality: a review.meat sci. 78: 343−358. zhou l, nilsson a (2001).sources of eicosanoid precursor fatty acid pools in tissues. j. lipid res. 42: 1521−1542. 8 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 9 (9), pp. 001-003, september, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. short communication phytochemical screening on four edible legumes (vigna subterranean, glycine max, arachis hypogea, and vigna uniguiculata) found in eastern nigeria mbagwu f. n.*, okafor v. u. and ekeanyanwu j. department of plant science and biotechnology, imo state university, owerri, nigeria. accepted 07 may, 2021 the results of the phytochemical screening carried out on the seeds of vigna subterranea (bambara groundnut), glycine max (soya beans), arachis hypogea (groundnut) and vigna uniguiculata (black eyed peas) showed the presence of useful phytonutrients. the results showed that v. subterranea contained the highest percentage of alkaloids (0.41%) while a. hypogea showed the lowest yield of alkaloid (0.25%). v. uniguiculata had the highest saponin content with (0.44%) while a. hypogea with the lowest (0.25%). the highest yield of flavonoids was found in v. uniguiculata with 0.33% while the lowest yield was obtained in a. hypogea with 0.18%. the highest phenols yield were obtained from v. subterranea with 0.36% while g. max showed the lowest with 0.26%. the percentage of tannins was highest in v. uniguiculata but lowest in a. hypogea. the results of the phytochemical screening on the four species of legumes were discussed in relations to their usefulness to mankind. key words: bambara groundnut, soya beans, groundnut, black eyed peas, edible legumes, phytochemical screening, phytonutrients. introduction legumes also known as dried beans and pulses are the edible seeds that grow in pods of annuals, biennials and perennials which are modified in many ways to facilitate their dispersal by animals, wind, and water. they belong to the family fabaceae, which is one of the largest and economically important families of the flowering plants. legumes are simple, dry dehiscent fruit bearing pods containing one or more seeds, which split open by the two longitudinal lines into two halves at maturity. legumes are cosmopolitan in nature (kunkel, 1984). the family fabaceae is the third largest family of the flowering plants after the asteraceae and orchidaceae with 1800 species and about 650 genera (polhill and raven, 1981). legumes can be trees, woody, vines, or herbaceous plants (allen and allen, 1981). legume is a term derived from french word which has a wider meaning and refers to any kind of vegetables. though some are cultivated while others are wild (charina, 2002). according to barrett (1987), legumes are classified as forage legumes which are grown in *corresponding author. e-mail: mbagwu101@yahoo.co.uk. pastures and grazed by livestock. such legumes include the alfalfa, clovers and the grain legumes. they are cultivated from their seeds, they are also called pulses and are widely eaten (roberts and summerfield, 1986). legumes are characterized by a particular morphology. they have their inflorescences arranged in many spikelets. their flowers are usually bisexual. the flowers have five petals in which the superior ovary ripens to form a pod, which is the fruit. the roots of legumes are more developed. the stems of legumes do not have nodes, internodes and collar regions (kunkel, 1984). the flowers are slightly too strongly perigynous and zygomorphic. the perianth commonly consists of a calyx and corolla of five segments each. the androecium most commonly consists of ten (10) stamens in two groups; that is, they are diadelphous with nine (9) stamens in one brindle and the 10 th stamen is more or less distinct. legumes have the floral formula as: k (5) c (5) a (5+5) or (9) +1 g1 (barrett, 1987). the leaves and pods can be introduced into the soil as green manure or nitrogen source. they can be rotated with other crops for soil improvement. that is they are used to replenish soil that has been depleted of nitrogen (becker, 1983). some legumes are for forestry such as table 1. quantitative analysis of the phytochemicals in the four legumes investigated. legumes alkaloids (%) saponin (%) flavonoids (%) vigna subterranea 0.40 0.43 0.29 arachis hypogea 0.25 0.25 0.18 glycine max 0.33 0.39 0.25 vigna uniguiculata 0.37 0.44 0.33 leucaena leucocephala and it can also be used as a nurse tree providing nutrients and shade for some crops. legumes serve as dehydrated feed for livestock such as cattle, horse pet and laboratory animals (leventon and mcmahon, 2003). the pigeon pea (cajanus cajan) functions as a living fence. legumes are the primary protein sources of diet. they are rich in vitamin b namely thiamine, riboflavin and niacin which help to release energy from nutrients. they contain fiber and glycemic index. they also contain non-nutritive components that are anti-nutrients such as trypsin inhibitor, phytate, and saponin which are glycoside composed of a lipid soluble glycone that consists of either a sterol or a treperoids which are phytochemicals (gepts et al., 2005). vigna uniguiculata, arachis hypogea, glycine max and vigna subterranean serve as food to man and animals. their leaves, stems and pods serve as food to man. v. uniguiculata can be used for erosion control and has high quality of protein, which are the primary source of our diets especially for vegetarians. they have low glycemic indices which helps to reduce the risk of fatal and nonfatal heart attack which are caused by consuming starchy food with high glycemic index. soluble fibers from black eyed peas help to lower the cholesterol level which blocks the blood pressure. the protein gotten from legumes like soya beans helps to regulate blood sugar, body fluid, kidney, adrenaline, liver function and other aspects of our metabolism (gepts et al., 2005). black eyed peas and bambara groundnut serve as food, though they can be cooked, boiled or ground to prepare bean cake and moi-moi. they are also useful in pharmacology, in the production of medicine and are also important biotechnological tools because they grow easily in cultures. this work therefore is designed to phytochemically screen these four edible legumes obtained in eastern nigeria with the objective of observing and analyzing their respective chemical constituents. materials and methods the legumes used in this study were collected from idemili in anambara state, ubakala in abia state, orlu in imo state and nkanu in enugu state. only healthy and fresh legumes were collected. the samples were ground into uniform powder using thomas willey milling machine. the samples were kept in the chemistry laboratory of micheal okpara university of agriculture, umudike, nigeria for analysis. phytochemical screening chemical tests were carried out with the standard specimens using standard procedures to identify the constituents as described by the harbone (1988) and sofowara (1993). quantitative determination of alkaloids the alkaloid was extracted using a modified method of maxwell et al. (1995). dried sample using 100 ml of 10% action acid which was left to stand for 4 h. the extracts was filtered to remove cellular debris and then concentrated to about one quarter of the original volume. nh4oh was added drop wise until no precipitate occurred. the alkaloid was dried to constant weight in an oven and the percentage of alkaloid calculated as: weight of residue x 100 weight of sample quantitative determination of saponin the method used for the determination of saponin was that of peny et al. (1995). the dried samples were ground and 10 g of each put into a conical flask and 100 ml of 20% aqueous ethanol added. the samples were stirred at a constant temperature of 55°c for 12 h. after the sample was filtered with whatman number 1 filtered paper and the residue re-extracted with another 200 ml of 2% ethanol. the extracts were combined and reduced to 40 ml by heating in a water bath at 55°c. the purification process was repeated two more times. 4 g of nacl were added to adjust the ph of the solution to 4.5 (confirmed with a ph meter). the solution was shaken with 60 and 30 ml portions of n-butanol extracts and washed twice with 10 ml of aqueous nacl. the bath was dried to constant weight, and the saponin extracted was taken and expressed as a percentage; %saponin = weight of residue. results table 1 showed the percentage of the chemical constituents of each legume investigated. v. subterranea contained the highest percentage of alkaloids (0.41%) while a. hypogea showed the lowest yield of alkaloid (0.25%). the highest yield of saponin was found in v. uniguiculata with 0.44% while the lowest yield of saponin was contained in a. hypogea. the highest yield of flavonoids was obtained in v. uniguiculata while the lowest was obtained in a. hypogea with 0.18%. the highest phenols yield was obtained from v. subterranea with 0.36% while the lowest yield was obtained in g. max with 0.26%. the percentage of tannins was highest in v. uniguiculata but lowest in a. hypogea. legumes have the ability to block specific enzymes that causes inflammation. they also modify the prostagladin pathways thereby protecting platelets from clumping. discussion the seeds of the four legumes (v. subterranea, g. max, v. uniguiculata, and a. hypogea) analyzed are rich in phytochemicals. the results of the phytochemical screening and quantitative estimation of the four edible legumes studied clearly showed that the legumes are nutritious and contained some phytochemicals such as alkaloids, flavonoids, saponin, tannins, and phenols. all these phytochemicals present in these legumes compared favorably with those reported from some medicinal plants found in nigeria (sofowara, 1993). the quantitative estimation of the percentage of the phytochemicals in these legumes showed the percentage content of alkaloids in v. subterranea, v. uniguiculata, a. hypogea, and g. max as 0.41, 0.37, 0.25 and 0.33% yields, respectively. though the yields of the alkaloids are low, but are useful in the prolonging of the action of several hormones and acting as stimulants especially v. subterranea which has the highest value of alkaloid. flavonoids enable food to be tasty which is in line with the work of dakora (1995) that flavonoids promote peculiar taste in prepared foods. flavonoids are capable of treating certain physiological disorder and diseases. they are potent water soluble, super anti-oxidant and free radical scavenger which prevents oxidative cell damage and have strong anti-cancer activity which adds protection against all stages of carcinogenesis (okwu, 2004). saponins are present in all the legumes studied and are contained in appreciable quantities, with v. uniguiculata having the highest value of 0.44% which means it has cholesterol binding properties, and help in hemolytic activities (okwu, 2004). the highest value of tannins was in v. uniguiculata, which serves as astringent properties for healing of wounds and inflaming mucous membrane (okwu and okwu, 2004). phenols are found in different percentages and v. subterranea have the highest value of phenols with 0.36%. the presence of phenol indicated that these conclusion in conclusion, the outcome of this phytochemical screening thus suggest that these legumes are highly recommended in everyday diet of man so that each of them can be used for normal metabolic activities of living organisms. references allen on, allen ek (1981). the leguminosae, a source book of characteristics, uses and nodulation. madison university wisconsin press. pp. 32-39. barrett rp (1987). integrating leaf and seed production strategies for cowpea. ms thesis, michigan state university, east lansing. pp 3339. becker b (1983). the contribution of wild plants to human nutrition in the ferlo (north senegal). j. agro-forestry syst.. 2: 256-267. charina ja (2002). nutritional value of peanut. metro manila books, philippines. pp. 2-4. dakora fd (1995). plant flavonoids: biological molecules for useful exploitation. j. plant physiol., 22: 87-99. gepts p, beavis wd, brummer ec, shoemaker rc, stalker kt, weedan nf, young nd (2005). legumes as a model plant family. genomics for food and feed reports of the cross legumes advances through genomic conference. j. plant physiol., 137: 1228-1235. kunkel g (1984). plants for human consumption. an annotated checklist of the edible phanerogram and ferns. koelkz scientific books, pp. 10-21. leventon e, mcmahon k (2003). plants and society. mcgraw hill book company inc. new york. pp. 99-112. okwu de (2004). phytochemical and vitamin content of indigenous species of south eastern nigeria. j. sustain agric. environ., 6: 3034. polhill rm, raven ph (1981). advances in legumes systematic. royal botanic garden, kew. pp. 42-53. robert e, summerfield rj (1986). grain legume crops granada technical books, london. pp. 10-15. sofowara a (1993). medicinal plants and traditional medicine in africa spectrum books ltd, ibadan, nigeria. pp. 286-289. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 11 (1), pp. 001-006, january, 2023. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper mycological and nutrient profile of soymilk ezekiel c.n and fapohunda s.o*. department of biosciences and biotechnology, babcock university, ilisan remo, ogun state, nigeria. accepted 07 may 2022 the mycological profile during soymilk processing, nutrient profile and keeping quality of the milk were studied. two moulds–rhizopus nigricans and neurospora sitophila were implicated as processing contaminants. the freshly prepared soymilk was rich in iron, calcium and potassium. conversely, the milk contained very low amounts of total fermentable sugar (0.022%). gas chromatography analysis showed consequent low alcohol content (4%). an evaluation of the effect of inorganic chemical preservatives on fungal growth and sensory parameters showed that soymilk fortified with calcium chloride significantly (p<0.05) enhanced sensory characteristics and exhibited a high inhibitory activity against the moulds–r. nigricans and n. sitophila, especially at 3.5g/l concentration than other salts. therefore, calcium chloride may serve as a better inorganic chemical preservative over sodium salts in soymilk processing for large scale commercial purposes since it is capable of suppressing mould contamination at the same time enhancing organoleptic properties. keywords: soymilk; fungi; food quality; preservation; sensory characteristics. introduction soybean (glycine max) is a rich oilseed crop with about 18% fat and 40–48% crude protein it is particularly noted for its richness in lysine, arginine, cysteine, leucine, and methionine (rastogi and singh, 1989: riaz, 1999: (sharma., et al 1999), minerals and some vitamins like calcium, iron, vitamin a, riboflavin and other trace elements (cronan and atwood, 1972). it can be conveniently consumed as powder, milk or tofu (lu., et al 1980). however, some anti nutritional factors like proteinase inhibitors, alkaloids and tannins may sometime weigh down the benefits of its whole corresponding author email: oystak@yahoo.co.uk unprocessed consumption, as these substances are not well digested by monogastrics like man. various processing techniques generally are aimed at detoxification and the removal of flatus factors so that enhanced digestibility is achieved. soybean, an attraction for vegetarians and health enthusiasts, is considered low in saturated fats and cholesterol which may reduce the risk of heart disease and diabetes. it also alkalizes the blood. during processing into soymilk, which is a traditional non dairy protein food (6), certain microbes are resident and find nutritional support in the matrix (scott and aldrich, 1970; dobson, 1998)). to our knowledge, not much has been done on the mycological profile of soymilk processing. the keeping quality and nutrient profile of the milk, and the cultural conditions of the resident moulds were studied. 2 materials and methods sample collection and processing soybean seeds (5kg) were collected from the university’s food services department, and processed into the milk by standard conventional method (berk, 1992: no., et al 2004) with only slight modifications. mycological analysis the indigenous mycoflora at each processing stage was determined by plating out 50 surface-sterilized raw seeds, 50 steeped-peeled seeds, the paste and milk on malt extract agar (mea) and potato dextrose agar (pda) (bdh) containing 0.4g/l of streptomycin sulphate. surface sterilization of the raw seeds was performed in 3% naocl for 2 minutes prior to plating. the direct plating method and pour – plate technique were used for plating the seeds (raw and peeled), and paste and milk respectively. the inoculated plates were incubated at 35 o c for 24 to 96hrs. the fungal isolates were purified on pda plates and identified via their macroand microcharacters and by comparison with illustrations in mycological literature. assay of nutrients the mineral elements contained in the prepared milk were assayed according to (10). calcium and iron contents were analyzed using the atomic absorption spectrophotometer (aas) (cecil) while the potassium content was determined by the flame analyzer (gallenkamp). the q94 gas chromatography (1996 jones chromatograph) was used to determine the total alcohol content; a measure of possible fermentation by indigenous fungi. the crude protein and total sugar contents were determined using the kjedahl distiller and uv spectrophotometer at 490nm (cecil ce3041) respectively. the ph of the milk was also determined. basal medium the basal medium for mycological studies contained – nah2po4, 1.2g; feso4, 0.8g; cacl 2, 1.8g; streptomycin sulphate, 0.4g; distilled water, 1 liter (ph 6.5). inoculation of test fungus was by means of two 5mm agar discs cut from young actively growing region of fungal mycelia on pda. role of nutrient sources on fungal growth the effect of carbon and nitrogen sources on fungal growth was determined after 6 days of incubation at 35 o c as static cultures. sucrose, glucose and mannitol as carbon sources and isoleucine, alanine and glycine as nitrogen sources were each appropriately added as 1.2g/l to the basal medium. two control flasks were used: control flask 1 (c1) with no sugar or amino acid treatment and control flask 2 (c2) with appropriate sugar or amino acid but not inoculated with test fungus. the mycelial dry weight of each fungus was taken by drying the mycelia mats on whatman #1 filter paper to constant weight using oven and weighing with ohaus portable advance balance. results were recorded as means of triplicates. effect of metal salts on fungal growth the following inorganic salts and their concentrations were used—sodium carbonate, sodium bicarbonate, and calcium chloride as 1.5, 2.5, 3.5, 4.5, 5.5 and 6.5g/l. it is known that these salts are sometimes used as additives designed to regulate food taste, ph and at times spoilage (coral., et al 1988). the test moulds were freshly maintained on pda from where 3 mm disc each was used as inoculum on a modified water agar. the water agar was constituted by dissolving 1.5% agar (bdh) in distilled water and fortified with 7.5g sucrose. the plate was prepared by adding 2ml of each salt concentration into the 1.5% water agar as pour plate (alvindia., et al 2004). incubation was carried out in a transparent illuminated incubator at 35 o c for 5 days. growth was recorded by visual observation and recorded in factors---1, 2, 3, 4; 1 being very slow growth, indicating very high antifungal ability and 4 indicating luxuriant growth meaning low antifungal ability. results were reported as means of 3 replicates. sensory evaluation of soymilk samples sensory assessment was performed by adopting the hedonic scoring scale having a 5 – scoring range; 1 as the lowest/worst and 5 as highest/best. a 6-member panel was used to evaluate odour, taste and appearance. the soymilk samples used for the sensory evaluation included the freshly prepared soymilk without any preservatives and those fortified with 3.5g/l (the best salt concentration for antifungal activity) of the individual chemical preservatives: sodium carbonate, sodium bicarbonate and calcium chloride. statistical analysis the one-way anova and dmrt were used for the separation of means and test for significance in this study. the sensory assessment, influence of nutrient 3 table 1. some mineral elements in freshly prepared soymilk samples mean (sd) concentrations of some nutrients in soymilk calcium iron (ppm/ml) potassium (ppm/g) alcohol crude protein (%) total sugar (ppm/ml) (%) (%) soymilk 79.00 (0.03) 1677.5 (3.15) 15.00 (0.00) 4.00 (0.02) 7.99 (0.02) 0.022 (0.01) sd: standard deviation values are means of triplicate samples figure 1. gas chromatogram of soymilk sample for alcohol content sources on fungal isolate growth and inhibition rate of salts against the moulds were all tested at p = 0.05 using the spss ® 15.0 version. results and discussion two moulds—rhizopus nigricans and neurospora sitophila were isolated in this study. they were identified using already known parameters (domsch., et al 1980: koneman., et al 1992 : alexopoulos., et al 1996). r. nigricans was found to colonize more of the raw seeds than the past and milk in contrast to n. sitophila which inhabited the paste and milk samples only. the isolation of rhizopus sp. was disturbing since many cases of zygomycoses are now living with man in diverse forms e.g. through rhizopus oryzae (artis., et al 1982), r. microsporus (verdonck., et al 1993)) and mucor sp. (boelaert., et al 1993). in addition, this mould has been reported to liberate a toxic secondary metabolite – rhizonin a (wilson., et al 1984). every attempt should therefore be made to eliminate any fungal spore during processing of this milk, because once they are ingested in any form, their growth is enhanced by body fluid like serum (verdonck., et al 1993 : (gifford., et al 2002)). an evaluation of the mineral element content in the freshly prepared soymilk sample showed that it is rich in iron, calcium and potassium (table 1). conversely, the milk contained very low amounts of total fermentable sugar (0.022%) and consequently low alcohol content (table 1 and figure.1), a major reason for its consumption by diabetics. this may however have accounted for the very low incidence of fungal contaminants in the milk. although the cultural conditions for its growth were studied (figure.2 and 3), the presence of neurospora sp. is not a cause for concern. this is because certain innate attributes make it unlikely that this fungus will incite adverse effect on man. for example, it is an obligate aerobe, which cannot grow in the gut, bladder, or other tissues. it is also not a toxin producer and neither is it capable of forming ‘fungus ball’ in the lung as aspergillus sp. do. despite many opportunities of human exposure, e.g in bakeries (yassin and wheals, 4 figure 2. mycelia growth pattern on basal medium fortified with three different sugars. table 2. sensory characteristics of soymilk samples fortified with various chemical preservatives mean sensory characteristics from 6-member panelist odour taste appearance ph control soymilk 4.33±0.52 a 4.67±0.52 a 5.00±0.00 a 6.75 cacl2 fortified soymilk 4.17±0.41 a 4.50±0.55 a 5.00±0.00 a 6.81 na2co3 fortified soymilk 2.67±0.52 b 2.17±0.41 b 4.00±0.00 b 7.40 nahco3 fortified soymilk 2.50±0.55 b 2.17±0.41 b 4.00±0.00 b 7.97 means with different alphabet in a column indicate significant difference in p < 0.05 1992), sugar cane fields (pandit and maheshwari, 1996) and burnt fields (perkins and turner, 1998), no disease or infection has been linked to it. indeed it is on record that it is been put to use as a food or in processing foods and beverages like soybean-based cake, in java and oriental consumables like koji (shurtleff and aoyegi, 2000) and roquefort cheese in france (perkins davis, 2000). the uninvited presence of neurospora sitophila in this work is therefore capable of enhancing the nutritive value of the milk. in this study, three commonly used chemical preservatives in food industries were evaluated for their contribution to the enhancement of sensory parameters of the milk as well as their efficacy in inhibiting the moulds from soymilk. the soymilk sample fortified with calcium chloride had comparable odour, taste and appearance with the control soymilk (freshly prepared) while both samples showed higher significant (p < 0.05) values over those samples that were fortified with sodium carbonate and sodium bicarbonate (table 2). in addition to the enhancement of sensory characteristics, calcium chloride exhibited a high inhibitory activity against both tested moulds isolated in this study. its antifungal activity was significantly (p < 0.05) higher than the other two sodium salts especially at 3.5g/l concentration against both moulds (figure. 3 and 4). this is similar to the reports of (samapundo., et al 2010), which showed that calcium chloride exhibited the largest antifungal activity in a sodium chloride replacement test for chemical food preservatives including magnesium and potassium salts. conclusively, our findings therefore suggest that calcium chloride may serve as a better chemical agent of preservation over sodium salts in soymilk processing for large scale commercial purposes since it is capable of suppressing mould contamination at the same time enhancing organoleptic properties. references alexopoulos cj, mims cw, blackwell m (1996). introductory mycology. wiley, ny: 841pp. alvindia dg, kobayashi t, natsuaki k, tanada s (2004): inhibitory influence of inorganic salts on banana post harvest pathogens and preliminary application to control crown rot. journal of genetics and plant pathology, 70: 61–65. artis wm, fountain ja, delcher hk, jones he (1982). a mechanism of susceptibility to mucormycosis in diabetic ketoacidosis: transferin and iron availability. diabetes, 31: 1109–1114. 5 figure 3. mycelia growth pattern on basal medium fortified with three different nitrogen sources. figure 4. inhibition pattern of different concentrations of calcium and sodium salts against neurospora sitophila berk z (1992): technology of production of edible flours and protein products from soybeans. isreal institute of technology, rome: 1121pp. boelaert jr, locht m, cutsen jv, kerrels v, cantinieavy b, verdonck a, schneider j (1993). mucormycosis during deferoxamine therapy is a siderophore-mediated infection. in vitro and in vivo animal studies. journal of clinical investigation, 91: 1979–1986. coral lg, post ls, montville tj (1988). antimicrobial activity of sodium bicarbonate. journal of food science, 53: 981–982. cronan ml, atwood jc (1972). foods in home baking. chas a bunnet co, illinois: 558pp. dobson a (1998): chambers sciences and technology dictionary. chambers harrap, edinburgh: 1109pp. domsch kh, gams w, anderson t, torant-had a (1980). compendium of soil fungi. academic press, london. gifford aa, klippenstein jr, margo mm (2002). serum stimulates growth and proteinase secretion by aspergillus fumigatus. infection and immunity, 70: 19–26. koneman ew, allen sd, janda wm, schneskenberger pc, winn n (1992): atlas and textbook of diagnostic microbiology. jblippincott coy: 1154pp. lu jy, carter e, chang r.a (1980). use of calcium salts for soybean curd preparation. journal of food science, 45: 32–34. nielsen ss (2002): introduction to the chemical analysis of foods. cbs, new delhi: pp. no h.k, meyers sp (2004). preparation of tofu using chitosan as a coagulant for improved shelf life international journal of food science and technology, 39: 133–141. pandit a, maheshwari r (1996). life history of neurospora intermedia in a sugar cane field. journal of bioscience, 20: 57–79. perkins dd, davis rh (2000). evidence for safety of neurospora species for academic and commercial use. applied and environmental microbiology, 66: 5107–5109. perkins dd, turner bc (1998). neurospora from natural populations toward the population of a haploid eukaryote. experimental mycology, 12: 91–131. rastogi a, singh g (1989). effect of addition of full fat soy flour of different varieties on quality characteristics and bread making quality 6 of white flour. bulletin of grain technology, 27: 26–34. riaz mn (1999). healthy baking with soy ingredients. cereal foods world, 44: 136–139. samapundo s, deschuyffeleer n, van laere d, de leyn i, devlieghere f (2010). effect of nacl reduction and replacement on the growth of fungi important to the spoilage of bread. food microbiology, 27: 749– 756. scott wo, aldrich sr (1970). modern soybean production. schering aktiengesellschaft, hamburg: 140pp. sharma s, sekhen ks, naqvi hps (1999). suitability of durum wheat for flat bread production. journal of food science and technology, 36: 61–62. shurtleff w, aoyegi a (2000). tofu and soymilk production: the book of tofu vol. ii. soyfoods centre, new delhi. verdonck ak, boeleart jr, gordta bz, landugt hw (1993). effect of ferridoxamine in the growth of rhizopus. mycoses, 36: 9–12. wilson t, rabie cj, fincham je, steyn ps, schipper ma (1984). toxicity of rhizonin a, isolated from rhizopus microspores, in laboratory animals. food and chemical toxicology, 22: 275–281. yassin s, wheals a (1992). neurospora species in bakeries. journal of applied bacteriology, 72: 337–80. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (5), pp. 001-009, may, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper inclusion of marine fish in traditional meals improved iodine status of children in an iodine deficient area amund maage1*, jogeir toppe1,2, mathilda steiner-asiedu3,4, ebenezer asibey berko3 and einar lied1,5 1 national institute of nutrition and seafood research, box 2029 nordnes, n-5817, bergen, norway. 2 fao, fisheries and aquaculture department, viale delle terme di caracalla, 00153 rome, italy. 3 department of nutrition and food science, university of ghana, legon, ghana. 4 brown university, bio-med institute of community health promotion, providence 02912, ri, usa 5 nutrimarine life science as, bontelabo 2, n-5003 bergen, norway. accepted 27 march, 2019 the present study was carried out in an iodine deficient area in northern ghana, and the purpose of the study was: (1) to undertake a food dietary survey on school children in an iodine deficient area, (2) measure the iodine content in the staple food of this area, (3) carry out an intervention study with a traditional food supplemented with marine fish as a natural iodine source. sixty school children, in the village of sekoti in the upper-east region of ghana, participated in the study. the dietary survey was performed as repeated 24 h recall, and staple foods were analysed for iodine by icp-ms. a traditional diet fortified with 10% codfish-powder was given to the children every day for two weeks, and iodine status was measured before and after the intervention. the foodstuffs normally eaten by the children contained little iodine. however, beans prepared at a market nearby had surprisingly high values of iodine, explained by the addition of saltpetre, which contained very high amounts of iodine. about 60% of the children in the study area had goitre and the prevalence was highest among girls. following the intervention period, the urinary iodine concentration was significantly increased and the tsh level in serum was significantly decreased, indicating an elevated iodine status. the children having goitre had the strongest tendency to elevate their iodine status. key words: iodine, fish, goitre, urinary iodine, tsh. introduction iodine deficiency disorders (idd) continue to be a major health problem in the world, particularly in third world countries. globally, the prevalence of goitre in 1999 was estimated to be 13% (who/unicef/iccidd, 2001). however, the prevalence of idd is likely to be greater than this. results based on prevalence of urinary iodine (ui) below 100 µg/l, estimates that 36.5% of school children worldwide have an inadequate iodine nutrition. this number is 42.3% in africa and as high as 59.9% in europe (who, 2004). in 1998, asibey-berko and coworkers reported that goitre rate of children (8 14years) in sekoti (northern ghana) was 59.1% and median urinary iodine for both sekoti and buila (also in northern ghana) was 16 µg/l. a national survey in 2001 found that *corresponding author. e-mail: ama@nifes.no. the median urinary iodine was 196 µg/l, while the national goitre rate was 20% with a range of 7% to 30% (nseid 2007). the most severe consequence of idd is irreversible brain damage, which can affect up to 5 15% of a population in severely affected areas (who, 2004). iodine deficiency is the single most important cause of preventable mental handicap in the world by the turn of the century (who/unicef/iccidd, 2001). the physiological function of iodine is as part of the thyroid hormones thyroxin (t4) and the more active form triiodotyronine (t3), which influences metabolism in all cells, including stimulation of growth and development of the central nerve system (delange and fisher, 1995). the recommendations of dietary iodine given by nrc (1989) were critically reviewed by delange (1993) and the recommendations were set at 90 µg/day for children 0 to 6 years and 120 µg/day for children 6 to 12 years. for all persons older than 12 years the recommendation is 150 µg/day. however 200 µg/day is recommended for pregnant and lactating women (who/unicef/iccidd, 2001). several indicators are used to assess idd in individuals and communities, including the size of the thyroid gland and urinary iodine. the size of the thyroid gland is inversely correlated with the average iodine intake, and indicates a person's iodine status (who/unicef/iccidd, 2001). urinary iodine excretion is a commonly used parameter to evaluate iodine status, showing a strong correlation with the iodine intake (vought et al., 1963). blood constituents are also used to assess the iodine status. the level of thyroid-stimulating hormone (tsh) and thyroglobulin (tg) increases when the iodine intake is low (delange, 1995). in 1992 united nations put forward a plan to eradicate iodine deficiency as a major health problem within the year of 2001 (fao/who, 1992) through increasing: 1) iodine fortification of foods and food ingredients such as salt, water and flour; 2) distribution of iodine capsules in areas where idd is severe, 3) use of iodine rich nutrients, like seafood, and decreasing the consumption of goitrogens. although the last decade has seen significant progress in combating idd, particularly in africa and asia, the total number of people affected by idd is still high. accoriding to who/unicef/iccidd (2001) the idd problem was underestimated in earlier studies and eradication policies have not been as effective as expected. iodine is normally obtained from food but most foods are poor sources of the element with the exeption of sea foods which are good sources of iodine (lee et al., 1994; coultate, 1996; eckhoff and maage, 1997; hou et al., 1997; karl et al., 2001; rose et al., 2001). the iodine content in fish is typically in the range of 300 to 1000 g/kg while most vegetable product had less than 30 g/kg. marine fish species low in fat, like cod and saithe have the highest iodine contents (dahl et al., 2004). in some africa countries iodine deficiency has been a common problem, especially in areas remote from the sea, and northern ghana has been no exception with reports on high goiter prevalence (asibey-berko, 1995). the recognition of severe idd problems in ghana prompted the campaign to use iodized salt. however, household iodized salt use has been low and estimated at about 28 % as of 1998 (nseid, 2007) . it is therefore also important to study further measures to improve the iodine status in communities. the present study was undertaken to study iodine nutrition and the possible use of marine foods in an intervention to improve this situation. the aim of this study was: 1) to perform a small dietary survey on school-children. 2) to collect staple foods in the study area, and measure the iodine content of these foodstuffs. 3) to perform an intervention study to evaluate changes in iodine status in school children, following a period of consuming of traditional foods fortified with marine fish. method study area and subjects sekoti, a village 35 km east of bolgatanga in the upper -east region, ghana, was chosen for the study since it is noted for rates of idd (asibey-berko, 1995). sixty school children ages 10 to 12 years (23 girls and 37 boys) were randomly selected and participated in the study. the study was approved by an institutional review board and all parents provided informed consent for their children after the study has been explained to them in the local language. the village chief together with his elders permitted the study to be carried out in the village. the study was carried out from february 26th to march 12th 1996. dietary assessment to obtain information on the iodine intake of the children, it was necessary to conduct dietary evaluation. a repeated 24 h recall was conducted each morning with each child to obtained information on the food eaten the previous day. two trained graduate nutrition students interviewed the children using open ended questionnaire. two teachers in the village were given a short orientation and served as interpreters for the period. the interviews went fast since food choices in the village are limited. samples of food portions were shown to the children to help them quantify the amount eaten. children were asked the type of foods eaten as a composite or single food item. the questionnaire covered all foods eaten through out the day, that is, breakfast, lunch dinner and all snacks. the dietary assessment was necessary since it was the key to knowing what the participants are eating to be able to determine the iodine content of the foods. both raw food materials and prepared meals eaten by the participants were collected in sekoti, or at the market in bolgatanga for iodine analysis. this was necessary since using literature values for iodine contents of the foods might not give true estimates. the materials sampled were stored in polyethylene bags, frozen transportation to the university of ghana, legon. the samples were later air freighted to nifes, bergen, norway, where they were freeze-dried, homogenised and stored in 50 ml nunc-cups prior to analysis intervention diet the intervention diet was fermented maize/fish blend and this provided each child 460 µg/day of iodine. table 1 profiles the iodine content in the raw food materials as well as the prepared meals. white maize meal (zea mays) was bought from indus, birmingham, uk. for each 5 kilos of maize meal, 4 l of tap water and 1 l of abc milk (tine norske meierier, bergen, norway), containing the bacteria lactobacillus acidophilus (a), bifidobacterium bifidum (b) and lactobacillus casei (c), were mixed to a dough in a food processor (varimixer bjørn 93/ar60, brøndby, denmark). the dough was then transferred to aluminium trays (20 l), covered with polyethylene foil and fermented at 30ºc for 48 h, until a stable ph of 3.7 was reached. the fermented product was frozen at -20ºc, then vacuumdried and fortified with 10% (w/w) codfish powder if saithe, gadus virens (toro a/s, rieber and søn, bergen, norway): the maize/fish blend was pulverised, bagged into polyethylene bags and packaged into boxes and air freighted to accra, ghana. the product was stored at the nutrition and food science department, university of ghana prior to the start of the intervention program. the fish maize blend was used to prepare breakfast (8 a.m.) and lunch (1 p.m.) for the children. breakfast was a popular porridge table 1. dry matter and content of nutrients analysed of raw materials and prepared meals of the intervention diet (dry matter base). foodstuff dry matter (%) protein ¹(%) fat¹ (%) energy¹ mj/kg) iodine (n)(mg/kg) codfish powder 95 20.5 (3) fermented maize meal 96 0.039² (4) fermented maize meal + 10 % codfish 4 15.5 3.4 17.6 2.13 (4) koko, prepared 18 12.3 0.7 17.3 1.34 (2) banku, prepared 34 15.5 1.0 1.82 (2) banku and gravy, prepared 32 15.5 6.4 18.0 1.73 (2) ¹analysed by skov (1997). ²abc milk (tine norske meierier, bergen, norway) added contained some iodine. called koko and each child received about 500 ml (2 cups) . koko was made of 4.5 kg of the raw material mixed with 30 litres of water. the mixture was boiled for about 30 min added 1.5 kg of sugar, and then distributed equally into 60 cups. lunch was also a popular dumpling called banku and each child received about 600 g. banku was made by mixing 12.0 kg of the maize/fish blend with 25 l of water, added salt (uniodized) to taste and cooked for about 1 h with constant stirring and kneading. banku was served with gravy made of 6 kg of tomatoes, 0.6 litres cooking oil (frytol), red pepper and salt (not iodised). the preparation and the serving of the food were under the supervision of a home economist/nutritionist. children ate all the food served at any given time. data collection baseline and follow-up data collection were done in the mornings before breakfast. demographic data collected were age and sex. anthropometric measures included mid-upper arm circumference as well as height and weight for body mass index determination. physical appraisal by palpation was done by an experienced person to determine goitre grades in the children (delange, 1995). physiological samples collected were urine and blood. the field conditions allowed for spot collection of urine. the urine samples were collected in 20 ml polyethylene tubes and stored at -20ºc. blood samples were collected as drops of whole blood on filter paper (schleicher and shuell, grade 903), air dried, put into a polyethylene bag, covered with aluminium foil and then stored at 20ºc. analyses the food, water and urine samples where analysed for iodine by inductively coupled plasma-mass spectrometry (icp-ms) type perkin elmer elan 5000 a icp-ms (ontario, canada) using tellurium as internal standard. the method is decribed in eckhoff and maage (1997) and further in julshamn et al. (2001). two replicate portions (0.2 g each) of the dried and pulverised food samples were added nitric acid and perhydrol before digestion in a microwave oven (milestone1200 mega, sorisole, italy), following addition of internal standard and then diluted to 25 ml. immediately before the icp-ms analysis the digested samples were diluted 1:2 with a 3% nh3 solution. the water samples brought from ghana were not digested but some droplets of concentrated nh3 were added before analyses. the urine sam-ples were thawed overnight at 18ºc. the internal standard was then added and the sample diluted 1:10 with 1% hno3 prior to the icp-ms analysis. the detection and quantification limit of the method was determined to be 0.2 and 0.6 g/l respectively. for analytical quality control of the iodine analyses, the standard reference materials (srm) 1566a oyster tissue from nist, usa and bcr 129 hay powder from bcr, belgium were used. those represented a wide range of iodine concentrations from 167 ± 24 g/kg in the hay to 4460 ± 420 in the oyster tissue. our analyses of the reference material were well within the uncertainty of the srm. since there at the time of analyses were no certified reference materials for iodine in urine, a recovery test was also performed with good results. the blood samples, collected on filter paper, where analysed by using the enzyme-linked immunosorbant assay (elisa) methodology. a tsh assay kit (spectrascreen dried blood tsh eia kit, eim diagnostics, santee, ca, usa), based on a standardised method (tseng et al., 1985; miyai et al., 1981) was used for the analysis. statistical procedures to find significant differences prior to and following the intervention period different tests for dependent variables were used. the level of significance is p < 0.01 if nothing else is mentioned. the distribution of the variables was visually checked for normality by using categorised normal probability plot. the results are given as medians and quartiles when the variables were not normally distributed. the result from normally distributed variables is given as means and standard deviations. the two variables iodine in urine and tsh in serum did not show normality, the nonparametric wilcoxon matched pairs test was therefore used to test if the changes were significant after the consumption of the intervention diet. for the normally distributed variables weight and upper arm circumference, student's t-test for dependent variables was used to check if the changes were significant. to test differences between the independent variables boys and girls mann-whitney u-test was used for the variables showing no normal distribution, and student's t -test for independent variables for the normally distributed variables. when looking at the iodine excretion in urine, and tsh level in blood, the differences between groups graded according to the grade of goitre, were tested by the non-parametric kruskal-wallis anova median test. result dietary habit during the 14 days intervention period, the food mostly consumed by the children other than the intervention diet was millet/rice balls (“tou zaafi”-tz) and this was 34% of the food-volume consumed. this was followed by rice which contributed 19%, and then tubani (cowpea paste) which contributed 15% of the food eaten during the table 2. dry matter, energy and iodine analysed in raw materials and prepared meals of the staple foods (dry matter base). data are presented as means of to parallel determinations. dry matter energy iodine place obtained (%) (kj/g) (µg/kg) yam dioscorea sp., roasted 58.7 15.9 63 bolgatanga, market gari (cassava masihot utilissima), raw 93.6 16.1 <16 bolgatanga, market cowpea beans vigna ungiculata, raw 95.6 25 bolgatanga, market bambara beans phaseolus sp., raw 95.4 19 bolgatanga, market rice oryza sativa, raw 92.1 16.0 4 <16 sekoti, market beans vigna ungiculata, prepared ¹ 27.2 18.1 234 bolgatanga, market rice/beans, prepared ¹ 35.4 100 bolgatanga, market sorghum sorghum vulgare, prepared 31.5 17.1 48 sekoti, school fufu (cassava + plantain musa paradisica), prepared 26.6 16.9 46 bolgatanga, bus-station kenkey (maize zea mayz), prepared 32.0 17.7 102 bolgatanga, market tubani (beans), prepared ² 41.2 19.0 74 sekoti, school tubani (beans), prepared ¹ 45.4 19.7 538 bolgatanga, market tz (millet pennisetum americanum), raw ³ 88.2 45 bolgatanga, market tz (millet), prepared ³ 19.9 17.0 93 bolgatanga, market tz (millet), prepared ³ 23.3 18.8 110 bolgatanga, restaurant okra hibiscus esculentus, dried 96.6 17.4 518 bolgatanga, market tombrown (maize + cowpea), roasted 95.1 18.0 400 bolgatanga, market tomatoes lycopercsicon esculentum, prepared 27.2 949 sekoti, school groundnutpaste arachis hypogaea, raw 93.2 20.9 135 bolgatanga, market salt 99.0 119 bolgatanga, market salpetre 98.5 50 400 ghana ¹ salpetre added under preparation of beans. ²ashed sorghum stalk added instead of salpetre. ³tz made of millet meal. rice meal is sometimes used instead. 4 value obtained from matvaretabellen (statens ernæringsråd, 1995). period. the remaining 7% is from snacks mostly wild fruits that the children picked in and around the village. food choices and household food availability is limited, hence children received one meal at school which is normally sorghum. however, during the intervention the 60 participants did not eat the school food (sorghum) but instead received the intervention diet (banku and koko) and this formed 25% of the bulk of food eaten. iodine content of foods and other food additives the iodine content of foods mostly eaten and available in the region is shown in table 2. the iodine content in the foods ranges from less than 16 µg/kg (raw cassava and rice) to 538 µg/kg (tubani). salpetre which is usually a food additive in ghana contains the highest amount of iodine (50,400 µg/ kg) and it boosted the iodine levels in foods where it had been added. the two main water sources in the area are well and borehole and both showed very low iodine concentrations; 0.2 and 0.8 µg/l respectively. antropometric indicators the mean values of weight, height, body mass index (bmi) and mid-upper arm circumference are presented in table 3. a fortnight is too short a time to see any significant change in height for children, however, weight did increase significantly on average by 0.9 kg, (p < 0.01). bmi which is an index including weight also increased significantly by 0.4 kg/m 2 (p < 0.01). midupper arm circumference also increased significantly by 0.4 cm (p < 0.01). nonetheless, when stratified by gender, no significant difference was found between boys and girls, for weight, bmi and midupper arm circumference. iodine status the prevalence of goitre was more than 60% among the children. however, 10% of them showed visible goitre (table 4). urinary iodine increased significantly, from 34 to 79 µg/l (p < 0.01) by the end of the intervention while reatinine decreased (table 5). baseline correlation bet table 3. weight, height, upper arm circumference and body mass index (bmi) of the school children in sekoti, ghana, prior to and following the consumption of the fish containing experimental diet. significant differences (p < 0.01) within a column are shown by different superscripts. data are presented as means (sd), n = 54. weight (kg) height (cm) upper arm circumference (cm) bmi (kg/m 2 ) before intervention 30.3 a (4.1) 137 (7) 18.3 a (1.3) 16.0 a (1.2) after intervention 31.2 b (3.9) 18.7 b (1.3) 16.4 b (1.1) table 4. distribution of goitre among 10 to 12 years old schoolchildren in sekoti, ghana. sex (n) no goitre (%) total goitre rate (%) goitre, invisible (%) goitre, visible (%) girls (23) 30 70 52 18 boys (37) 43 57 52 5 both (60) 38 62 52 10 table 5. iodine concentration, creatinine concentration and the ratio iodine/creatinine in urine samples from schoolchildren in sekoti, ghana, prior to and following the consumption of the fish containing diet. significant differences (p < 0.01) within a column are shown by different superscripts. data are presented as medians and precentiles (25th 75th), n = 54. iodine(µg/l) creatinine(g/l) iodine/creatinine(µg/g) before intervention 34 a (20 53) 1.37 a (0.76 1.66) 28 a (20 47) after intervention 79 b (37-122) 0.70 b (0.34-1.32) 115 b (84 156) between creatinine concentration and iodine/creatinine ratio was significant at start of the intervention (r = 0.39, p < 0.01). figure 1 depicts the relationship between iodine concentration in urine and the level of goitre. as the level of iodine increased the occurrence of goitre decreased. the concentration of iodine in urine increased after consumption of the fish diet but the increase was significant for the groups without and with invisible goitre (p < 0.01). no significant change was found in the group having visible goitre. there was no significant difference in the concentration of iodine in urine for boys and girls. figure 2 shows the tsh level in serum before and after the intervention period. the two groups having goitre, invisible and visible, showed a significant decrease in serum tsh level (p < 0.05) after eating the fish containing diet. the group without goitre did not show any significant change in serum tsh level. when stratified by sex, the decrease in serum tsh was only significant among the girls (p < 0.05). discussion food habits and iodine assessment of foods millet/rice balls (“tuo zaafi”-tz), sorghum, tubani (cowpea paste) and rice contributed more than 90% of the food normally consumed by the schoolchildren in sekoti. these carbohydrate rich foods contained low levels of iodine, and did only cover a minor part of the children's recommended daily intake of 120 µg (who/unicef/iccidd, 2001). the low levels of iodine in the raw materials of cereals and beans reflects the low iodine content in the soil in the area were they are grown (hetzel and maberly, 1986). the iodine content in the water samples was insignificant, also reflecting the low iodine content in the soil. tomatoes and okra had higher levels of iodine than the cereals and the beans, probably because they are grown on farms closer to coastal areas, where iodine levels in the soil are higher (hetzel and maberly, 1986). meals were usually served with a stew, often made of tomatoes, okra or groundnuts, which resulted in a slight increase in the iodine content in most of the dishes. dishes of beans and tubani bought at the market in bolgatanga contained surprisingly high amounts of iodine. these high levels of iodine can be explained by the addition of saltpetre during the food preparation. saltpetre was added to get the desired texture and taste, and had an iodine content of more than 50,000 µg/kg. at sekoti, however, where the children involved in the study lived, ashed sorghum stalk was used instead of saltpetre as a cheaper alternative. this explains the low levels of iodine found in the prepared beans and tubani bought there. the salt used for food preparation was not iodised, and contained therefore insignificant levels of iodine. io d in e co n ce n tr at io n i n u ri n e (µ g /l ) 160 before intervention 140 after intervention 120 100 80 60 40 20 a a a a b b 0 no goitre goitre, invisible goitre, visible figure 1. urinary iodine concentrations of the schoolchildren in sekoti, ghana, according to the grade of goitre. significant difference (p < 0.01) prior to and following the consumption of the fish containing diet within a group is shown by different letters. data are presented as medians with 25th and 75th percentiles. for the group having no goitre n = 20, n = 28 for the group having invisible goitre and n = 6 for the last group with visible goitre. the low levels of iodine in the staple foods could be the major explanation of the high incidence of idd in the sekoti area. however, other factors than low nutritional iodine may contribute to goitre and diseases related to iodine deficiency. goitrogenes and selenium deficiency are such factors. some foods contain anions like tiocyanate, nitrate and nitrite, which all are goitrogenes (browngrant, 1961; delange and fisher, 1995; ubom, 1991; fordyce et al., 2000). saltpetre, which had a high iodine concentration, contains nitrate which can inhibit the utilisation of iodine (tajtakova et al., 2006). in sekoti, millet was one of the most important staple foods, which contains a goitrogen called vitexin (gaitan et al., 1995; elnour et al., 1997). a probably high intake of vitexin may also contribute to the high incidence of goitre recorded in sekoti. selenium deficiency could be a factor contributing to the idd symptoms as selenium is a part of type i 5`deiodinase which converts t4 to the more active t3 hormone (meinhold et al., 1993; roti et al., 1993). however, selenium status of participants was not measured. changes in nutritional status according to túron and chew (1994) protein energy malnutrition (pem), at ages 11 to 13 years, was diagnosed based on a bmi below 15.0 kg/m 2 . in the present study, the schoolchildren had at the beginning an average bmi slightly higher than this cut-off point, and the bmi increased significantly by 0.4 kg/m 2 during the study. the improved nutritional status was also confirmed by the increase in upper arm circumference and the significant weight increase. the consumption of the experimental diets thereby showed a positive effect on the nutritional status, which might be a result of increased dietary protein quality, a higher food consume than normal and an increased intake of specific nutrients like iodine, which had an obvious positive effect on the children's iodine status. the prevalence of goitre in the sekoti area was estimated to be 59% in 1991, and the area was then defined as an area with severe iodine deficiency (who/unicef/iccidd, 1993). the same prevalence of goitre was found in this study when started in 1996 and showed that iodine deficiency was still a large problem in this area. the researchers were told that iodine capsules were distributed to the population two years prior to the present study. this seemingly did not correct the low iodine status of the schoolchildren two years after the distribution of the capsules. iodine status urinary iodine concentration is the major parameter for t s h l ev el i n s er u m ( µ u /m l) 10 before intervention after intervention 8 6 4 2 a a a b a b 0 no goitre goitre, invisible goitre, visible figure 2. tsh levels in serum of the schoolchildren in sekoti, ghana, according to the grade of goitre. significant difference (p < 0.05) prior to and following the consumption of the fish containing diet within a group is shown by different letters. data are presented as medians with 25th and 75th percentiles. for the group having no goitre n = 20, n = 28 for the group having invisible goitre and n = 6 for the last group with visible goitre. evaluating iodine deficiency. in casual urine samples the concentration of iodine can be related to urinary createnine (vought et al., 1963) . however, the urinary iodineiodinecreatinine ratio is not a good indicator in cases where this ratio is significantly correlated with the createnine concentration itself (furnée et al., 1994). in this study we also measured this ratio but the iodine-creatinine ratio was not used further as the correlation with the creatinine concentration was found to be significant. the urinary iodine concentrations were therefore preferred as iodine status indicator. according to bourdoux et al. (1985) the measurement of urinary iodine concentrations in fifty to one hundred casual samples gives a good index for iodine status in a population. median urinary iodine levels of 20 49 µg/l indicate moderate iodine deficiency; median values of less than 20 µg/l indicate severe iodine deficiency (who/unicef/iccidd, 2001). the 25th and 75th percentiles of iodine levels in the urine collected before the intervention was 20 and 53 µg/l respectively, which confirms that idd was a serious problem in the area. during the study period the average concentration of iodine in urine increased significantly by about two times. the increase was less than expected, because the contribution of iodine from the intervention diet was several times the contribution of iodine from the staple foods usually eaten by the children. it is, however, possi ble that the low urinary iodine after eating the intervention diets is an indication that iodine deficient children retained a considerable amount of the ingested iodine. the tsh concentration in serum decreased signifycantly as a result of the consumption of the iodine rich diet. for children aged 1 to 15 years the level of tsh in serum is expected to be between 0.6 and 6.3 µu/ml (delange and fisher, 1995). in our study, the tsh median value of the school children in the study came below the upper limit of this interval after the intervention period. the findings from the present study show a relationship between the degree of goitre, urinary iodine concentration and the level of tsh in serum. when grouping the schoolchildren according to the degree of goitre, it was seen that the urinary iodine excretion was not signifycantly increased in the group having visible goitre, while the tsh level in serum was significantly decreased in this group. in the group having invisible goitre the serum-tsh concentration also significantly decreased and further a significant increase in urinary iodine concentration was seen. the group having no goitre had no significant change in serum tsh, but had the highest increase in urinary iodine concentration. individuals having goitre seem to have a more efficient iodine trapping mechanism in the thyroid gland, resulting in less iodine excreted by the urine. more iodine is then available for producing t4 and t3, which in turn reduces the level of tsh in serum. our data showed higher goitre prevalence among females than among males when the experiment started, the same tendency is reported in other studies from mali (fish et al., 1993), malawi (furnée et al., 1994) and sudan (elnagar et al., 1995). the higher goitre prevalence among the females can explain why it was observed a significant decreased in the tsh level in serum in females while this was not observed in males. conclusion/implication the high prevalence of goitre in this area showed that iodine deficiency was a major problem. the results from the present study show that goitrous individuals had a significant ability to improve their iodine status following 14 days on a diet containing elevated amounts of iodine due to marine fish inclusion. even though the use of iodised salt is the major alleviating factor for iodine deficiency, for parts of the population it is not always efficient. diets containing marine fish could be a helpful supplement in alleviating the idd in some areas. acknowledgements mr. james asedem, regional nutrition officer, is warmly thanked for his support and acceptance of the project. mr. thomas ndanu and mr. seth aduafarwuah, depart-ment of nutrition and food science university of ghana, legon, ghana, are thanked for their invaluable help in the field study. the project was supported by the norwegian cooperation programme for development, research and higher education (nufu) under contract 14/92. reference asibey-berko e (1995). prevalence and severity of iodine deficiency disorders in ghana. in: asibey-berko e, orraca-tetteh, r (eds) proceedings of the national workshop on iodine deficiency disorders in ghana held in accra, ghana. university of ghana, ghana, pp. 15-23. aisbey-berko e, amoah ag, ado f, agyepong e (1998). endemic goitre and unrinary iodine levels in rural communities in the bolgatanga and builsa districts of the upper east region of ghana. east afr. med j. 75:5001-5003. bourdoux p, delange f, filetti s, thilly c, ermans am (1985). reliability of the iodine/creatinine ratio: a myth? 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iodine inefficiency disorders. mdis working paper #1), who, geneva. who/unicef/iccidd (2001). assessment of iodine deficiency disorders and monitoring their elimination. a guide for programme manager (who/n/01.1),. 2nd ed, who, geneva. who (2004). iodine status worldwide. who global database on iodine deficiency. who, geneva. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (1), pp. 001-007, january, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review the importance of wheat in teething celebration in turkish culture suheyla saritas balikesir university, necatibey egitim fakultesi, balikesir/turkey. e-mail: ssaritas@balikesir.edu.tr, saritassuheyla@gmail.com. accepted 16 november, 2018 food is very essential to human existence and has an important role to play in social and cultural events as well. the study of food and eating has a long history in social sciences, specifically in folklore and anthropology, beginning in the nineteenth century. this article concentrates on a particular food, wheat and examines its significance in the turkish culture. since wheat has both cultural and social aspects in the turkish culture, the study mainly focuses on the ritual called di buğdayı or di hediği, thus, “teething ceremony”, an important biological development of a baby in the turkish culture. it deals with how the wheat meal is prepared or presented; how the ritual is performed and celebrated; and what functions of the event in the turkish culture are. key words: wheat, tooth, teething, ritual, turkish tradition. introduction food culture is an important research area within the discipline of folklife. the earliest studies about food culture concentrated mostly on tribal societies and were conducted by cultural anthropologists at the beginning of the nineteenth century. those studies primarily focused on the significance of food in primitive rituals. to give an example, in his 1964 book the raw and the cooked, the anthropologist french lévi-strauss explored natural and cultural relationships on the culinary level. he furthermore maintained that culinary rites are not inborn but rather acquired phenomena (lévistrauss, 1975, 586-595). 1 moreover, in the early twentieth century, freud investigated psychosexual aspects of consumption. the vital role of food in folklore was first been recognized by european folklorists at the end of 19 th century with the resulting folklife concentration. today, however, studies on food are still limited and neglected by folklorists. folklorists have touched food matters at a superficial level and mainly as “collectors” of miscellaneous recipes in collaboration with other researchers in different fields such as historians, home economists. 2 1 lévi-strauss, claude. the raw and the cooked. new york: harper & row publishers, 1975. 2 the similar statement is also made by don yoder for american folklorists. yoder, don. 1972. “folk cookery”, in folklore and folklife: an introduction, 326. food culture or as honigman’s (1961) term “foodways”, which is the trend in food lore today, carry extra meanings compared to the term “food”. 3 don yoder uses the term “folk cookery”, he defines it as “traditional domestic cookery marked by regional variation”. it stands “for the total cookery complex, including attitudes, taboos, and meal systemthe whole range of cookery and food habits in a society” (yoder, 1972, 325). foodways are fine examples for one to understand cultural and historical values extricably intertwined in a society. turkish folklore scholarship has not focused on food from a folkloric perspective since no books on traditional turkish food culture have been produced. the book yemek kitabı deals with how the old turkish sources give us concrete information about the turkish food tradition, such as divan-ü lügati’t –türk and kutadgu bilig. it also focuses on food and its relations with folklore, history and literature (koz, 2002). however, there are abundant incomprehensive studies in that field that studies on food culture have focused on a particular cuisine, ingredients as well as aspects of the production, preparation and consumption of specific foods. 3 john, j.honigman, foodways in a muskeg community: an anthropological report on the attawapiskat indians (ottowa, 1961), it is a model north american ethnography of food habits, based on fieldwork in a canadian indian community. moreover, every society uses food as a ritual even though it may not often be so obvious to understand its function in a ritual context. food is used in daily practices, occasions, festivals and celebrations in every society as a ritual. the role of food, particularly in ritual and celebrations is still largely overlooked in the turkish culture. the current paper deals with a turkish celebration which evolves around specific food, wheat, and its ritual context. the goal of the paper is to present and examine the turkish “teething ceremony” called di buğdayı, di bulguru or di hediği which is an important traditional turkish celebration for a baby’s development. furthermore, this paper deals with the studies about wheat and its significance in ceremonial contexts in the turkish culture. even though turkish folklorists have provided us with some information about teething as a tradition, there is not a single comprehensive study done on this subject. information about it one draws from studies by turkish folklorists that relate to birth traditions and beliefs. 4 even though such studies do provide an important arena for covering the subject and inspire food studies, they do not give enough information about the tradition and its ritual aspects, in particular. in this study, we have done personal interviews with women migrated to the city balıkesir, located in the northwest part of turkey. the interviews took place in a local bazaar called hanımeli where women sell their handmade materials and regional foods. after interviewing a total of 70 women, we have realized that the women from different regions of turkey reflect their cultural values, specifically on teething tradition. the women who are married with children and have experienced teething ceremony have shared their knowledge and experiences about this tradition in their lives. by taking folkloric approach, this study aims to explore the importance of wheat in teething celebration and the functions of the event in the turkish culture. the historical and cultural aspect of wheat as the most consumed grain in the world, wheat has been known all over the world since antiquity. it may challenge only with rice but it is definitely one of the few dominant and important food grains in the turkish culture. wheat is proudly accepted at the homes and in the lives of the turkish people because wheat is the 4 there are many folklorists who collected material about this tradition from different regions in turkey. the tradition is usually included in birth traditions and celebrations. for example, kadriye ilgaz in her article named “đstanbul’da doğum ve çocukla ilgili âdetler ve đnanmalar” talks about traditions and practices related birth in đstanbul. she includes her work teeth cutting ceremony that does not give much information. another study focusing on birth traditions are done by adil özder titled “doğum ve çocuk üstüne gelenekler, đnançlar” the study focuses on birth, customs and beliefs about children. mustafa aça in his book “balıkesir yöresi doğum sonrası đnanı ve uygulamalar” talk about traditions and rituals related with birth in balıkesir. ingredient for flour that is essential for making bread, a valuable, basic, daily dietary meal for the whole turkish nation. according to turks, it symbolizes human existence and “human battle with life” as expressed in the turkish proverb ekmek kavgası. home in turkish yuva representing the emotional meaning of the house gains meaning with existence of bread because as “foods, more than any other material item, evoke memories, feelings and identities that are both vast and individual” (thursby, 2008, 164). the man of the home goes for bread to make a living for his family. it is always hard to gain bread because the man of the home has to “take his bread from stones” “ekmeğini ta tan çıkarmak” the common turkish proverb. sometimes it is really hard to “earn the bread” “ekmegini kazanmak”, another turkish proverb because “the bread is in the mouth of lion” “ekmek aslanın ağzında” as it is described in the other common turkish proverb. bread, in fact, is equal to salt that both have been important elements in the turkish culture and turkish folk literature. the proverb “tuz ekmek hakkı” which is translated as “the right of salt and bread” in turkish symbolizes “the debt of fidelity.” the proverb is a “rich cliché that includes friendship, fidelity, sincerity, hospitality, chivalry, loyalty, honesty in the history of turkish literature and folklore (elçin, 1981, 74). both bread and salt unite the turkish people. the great anatolian philosopher and the father of the mevlevi sect mevlana celalledin rumi expresses “did he not share bread and salt with us? every hostility must have its cause; otherwise, our shared humanity would call for faithfulness in friendship”. moreover, rumi describes a journey that begins with death and ends in love: “buried in the earth, a kernel of wheat is transformed into tall stalks of grain. crushed in the mill, its value increases and it becomes bread, invigorating to the soul. ground in the teeth, it becomes spirit, mind, and the understanding of reason; lost in love, that spirit delights the sowers after the sowing”. like salt and bread, wheat holds a significant value in the history of turkish culture. the prominent turkish history and culture scholar, bahaeddin ögel, mentions that wheat had always been grown among turkish tribes who lived in harsh climate conditions in middle east (ögel, 1978, 158). eberhard (1996), a folklore scholar, reports the importance of wheat and other grain crops in the lives of turkish people in the past in a work entitled çin’in ßimal kom uları. this book inspired younger generation of turkish folklore scholars. the study published in 2008, documented that, during the 1989 excavations, samples of wheat dated back to c.1759 b.c. were found at karahöyük, a hittite settlement in the city of konya today. the study proceeds further and examines the cuneiform texts and the context in which wheat is used, so as to gather information about it and its general significance for that age (erkut, 2008: 1-4). furthermore, in the afore-mentioned work, it is stated that that wheat developed from a type of wild grass native to the arid lands of asia minor. it is believed that “cultivation of wheat is thought to have originated in the euphrates valley as early as 10,000 b.c., making it one of the world's oldest cereal crops. in the mediterranean region, centuries before recorded history, wheat was an important food. since wheat played a dominant role in the roman empire, the nation was often referred to as a “wheat empire” at that time. 5 as within greek antiquity, bread was an essential food, a wide variety of breads was available. the article which deals with the importance of bread and bread making in greece states that the tradition of bread making by paying respect to bread as a major part of every meal continued into hellenistic and byzantine times. it sustained even during the period of the ottoman occupation which lasted for approximately 400 hundred years (1453-1821). 6 even though wheat has always been an essential food in the history of turkish culture, it is hard to trace back the origins of its ritual context. studies on birth traditions and related celebrations have included it but only on a very superficial level. 7 thus, there has not been any exclusive study on the “teething” tradition and particularly its ritual context. a paper written by an undergraduate student deals with some ritual aspects of that but is mostly an account of possible sources (gurbuz, 1995). 8 tooth wheat in the turkish culture when a child gets its first tooth, is an occasion for joy and excitement, especially for mothers. the food made of boiled, hulled wheat plays an important role toward the accomplishment of this event. both the food and the ceremony are commonly named similarly: di buğdayı or di hediği (tooth wheat). 9 di means tooth and wheat means buğday in turkish. hedik refers to a traditionally cooked wheat dish eaten on this specific occasion. boiled and pounded wheat is called bulgur in turkish, another common name for this tradition. bulgur plays an important role in the turkish cuisine. it has a higher nutritional value because bulgur is 5 this information is retrieved from (http://www.ngfa.org/trygrains_wheat.asp). 6 dr. maria hnaraki in her article “baked realities: big fat greek breads” talks about the significance of bread and bread making throughout the history of the greek. 7 for example the study titled “silvan’da di hediği” by mustafa tatçı focuses on teeth cutting ceremony in a small district named silvan in the city diyarbakır, located eastern turkey. 8 gürbüz, elif. 1995. “di hediği geleneğinin ritüel karakteri ve fonksiyonları. milli folklor, cilt 4, sayı 28, kı . 9 sedat veyis örnek in his study geleneksel kültürümüzde çocuk,(child in traditional culture) lists the name of the tradition used in different regions in turkey: buğday dirlimesi (denizli/acıpayam), dirgit dökme (isparta), di a ı (burdur/tefenni), di dilgiti (antalya/serik), di dirgiti ( antalya/serik) di tohumu (çanakkale), duzlama (afyon), di göllesi (ankara, afyon, isparta), hedik di i (van/gürpınar), di emi (konya/karaman), di edi (ankara), di günü (tekirdağ/sandıklı). considered an ideal grain in a vegetarian diet too. 10 the favorite soup, the traditional anatolian winter soup, called tarhana, which has variations from one region to another, is usually made with bulgur or wheat seed (dövme) in most regions in turkey. 11 a detailed description of the preparations of tarhana in greece is well described by valamoti and anastasaki (2007, 75-100). bulgur is the first food produced after wheat is processed. wheat is boiled so as to taste a bit softer than bulgur. the ceremony “tooth wheat” celebrates the appearance of the baby’s first tooth. it is believed that if the ceremony is not held, the baby will have tough teething. the anonymous lines following symbolize the frustration of teething for the baby and the importance of wheat: “if my mother sells my bed, she would make the wheat; i would then get my teeth easily. if my mother does not make the wheat for me, she should keep my coffin ready.” preparation and presentation the preparation of the meal is a tiring task but definitely a joyful one. the mother of the baby is usually the one who prepares the meal with the help of her friends or relatives. the women who have a strong role and are the only authority in the turkish kitchen, prepare the meal for guests. since the occasion is an important event to be shared with others, the meal requires to be cooked in a big stewpot or even a cauldron. the main ingredient of the meal is the wheat which has to be boiled and remain a bit hard but not entirely cooed. the rest of the ingredients may be differentiated according to the traditions followed by the people living in different regions in turkey as well as their personal tastes. “tooth wheat”, di hediği is generally of two types, salty, and sweet, if granulated sugar is added to it (figures 1 and 2). apart from the wheat, other common ingredients are chickpeas and the dry beans. they are also boiled with wheat in the same stewpot. after they are cooked, they are drained and ready to be decorated. the decoration of the wheat requires a sini, thus, a big, flat circular tray with no edges. the meal is garnished with nuts, grains, raisings, grapes, walnuts, pomegranate seeds and candies. everything blends with the boiled wheat. the popular turkish delight in various flavors may also be used for decorating the meal. the visual presentation of the meal is important as turkish women work on composing images and symbols 10 a detailed account of making bulgur is given bayram m. (2000, 81-82). 11 the history of tarhana is not well-know; however i know a story about where tarhana comes from. many centuries ago, when the sultan was guest at a poor peasant house, there was only one thing that the house of wife could offer to the sultan. the wife boiled up the soup quickly and she was embarrassed and said this is a “dar hane” which means “poor house” and eventually the soup became “tarhana” soup. figure 1. an example of di buğdayı. (the photo is from meltem ekin’s blog. http://idilsuozdemir.blogcu.com). figure 2. an example of di buğdayı with candies and sweet nuts. (the photo is from a “tooth wheat” ceremony in december, 2008). that will impress the eye. the next step is to place various objects which symbolize different occupations at the edges of a tray. such items may be scissors, mirrors, pencils, books, moneys, combs, watches, gold, rulers or even the holy book of the quran (kuran-ı kerim). bread is considered as sacred in the turkish culture that is also oftentimes placed in the tray during the celebration. in the turkish culture, there are objects and herbs which bring fortune and happiness. people believe that they have powers to be protected from evil. those objects include garlic, evil eye and the herb peganum harmala known üzerlik otu (nazar otu) or “herbal evil” in turkish. the “tooth wheat” plate, surrounded by all the selected objects, is placed in the middle of the tray. the mother of the baby throws a piece of cloth and makes sit the baby sit on it. the baby may also put in a similar type of tray. in some regions it is common to have the baby sit on a prayer rug. ritual and celebration the information that the bay has gotten the first teeth is so important that it has to be celebrated with relatives and friends by inviting them home. the sources talking about this celebration do not give information about gender of the guests. however, all of the women interviewed stated that the celebration of teething has been taking place among only females today. in fact, the celebration is similar to “gün” day or kabul günü, a very common party all over turkey among the turkish women today. in this specific day, turkish women do not only gossip but also eat various foods prepared, such as cakes, croissants and pies. they a great deal of time, effort and money to prepare the party. one of the most common foods prepared on that day is called kısır which is a kind of bulgur (boiled and pounded wheat). as a kind of salad, kısır tastes like tabuleh. thus, eating food with either wheat or bulgur is necessary for socializing and celebration for the turkish women. in the ceremony “tooth wheat”, there are number of traditional practices which take place in order to ensure the celebratory atmosphere. usually, the oldest or the most popular guest initiates the ceremony. the baby, on its best clothes, may be covered by a piece of cloth, a scarf or a veil. a verse that reads like the “baby shall have teeth like wheat” is recited by the honorable guest who is taking piece of wheat on her palm, and who then throws it on top of the baby. i believe that throwing wheat is related with the old turkish religion shamanism. every tribe had its own sacred objects to sacrifice god in shamanism. for example, milk, butter and “kımız” (horse milk) were considered sacred among the nomadic turks, whereas wheat, grain and corn were considered sacred among the farmer turkish tribes (gomec, 1998). 12 furthermore, throwing wheat on top of the baby recalls the wedding customs in turkey. in shamanism the ritual is called saçı which is to sacrifice wheat, grain and corn to god, is the same word used for throwing grains and nuts from the head of the bride for fertility. it also means gifts and money given to the bride by relatives and neighbors (örnek, 1995, 1979). grains such as wheat and rice are thrown over the newlyweds in the hopes that the couple would be prosperous. at the “tooth wheat” celebration, occasionally, a small 12 saadettin gömeç states that there are two types of sacrificial ceremonies held in shamanism. the first is to serve or donate living or bloody animals to god. people usually served horse. the second type of ceremony is related with non-living objects. one of the ceremonies with objects is called saçı meaning the most sacred and valuable earned by tribes (gömeç, saadettin. 1998. “ßamanism ve eski türk dini”, pamukkale üniversitesi eğitim fakültesi dergisi, sayı 4, 44. portion of wheat may be placed in the baby’s mouth. the turkish women believe that putting wheat on the gums of the baby will make it feel better. the baby, who goes through lots of pain while teething, physical and emotional, as it was trying to chew the wheat. the women think that the wheat helps to soothe the baby’s pain. wheat thus functions as a type herb. i think the healing issue of wheat needs to be examined further and determined its place and importance in folk medicine. piece of wheat, usually seven in number, are placed to the left of the baby’s head, looking like a necklace. that “necklace” is kept till it dries out and then given to birds. people “make birds happy”, özer quotes (1935, 15), as a turkish belief wants the baby to love nature in the future. the ceremony goes on. the mother props the baby up on the table or on the floor. the objects placed on the edges of the tray are now placed in front of the baby (figure 3). whatever the objects the baby picks up from the tray it is believed they predict what occupation the baby will practice. for example, if the baby picks up scissors, it means that it will become a tailor; if it takes the ruler, it will become an engineer; if baby takes mirror, it will have happiness and richness; if the bread is picked, the baby will take care of the family; if the holy book is picked, the baby will have belief in god and religion in general. thus, the objects not only symbolize what occupation the baby will have but also what kind of a person it will be in the future. most of the time baby is baffled and confused that it may end up picking up nothing. after the ceremony, time comes for the meal to be consumed. the teething baby gets excited to taste the meal with his “new tooth” (figure 4). all the guests taste the meal with some other food prepared especially for the occasion such as cakes, salads, croissants, dolma(s) made of grape leaves stuffed with cooked rice, lamb, and onion. the dolma is marinated with olive oil and lemon. the famous dessert baklava, which is made with phyllo dough, nuts, butter, and sugar, is also prepared for this important occasion. one of the guests who must have healthy teeth takes the first piece of the wheat so as to taste the meal: this way the baby will similarly acquire strong teeth. the leftovers are placed in small plates, prepared in the same way in big trays, and in order to be distributed to neighbors and friends who are not present at the ceremony: the more “tooth wheat” is distributed, the healthier teeth the baby will have. giving the remaining food to people is important in the turkish culture, especially the ones who are not wealthy. it is a way for the turkish society to balance inequality and make everyone feel secure as a member of the society. it brings to people solidarity. the mystic number “seven” defines the minimum number of neighbors to distribute the meal. the rule is not to wash the empty plates otherwise the baby will acquire carious teeth. usually neighbors do not return the plates empty but place a small baby gift on them. the one day figure 3. the objects such as scissors, pencil, money, knife comb are put on a tray for the baby to pick of one of those. they are used for divinatory purpose for baby’s future occupations and identities (the photo is from meltem ekin’s blog. http://idilsuozdemir.blogcu.com). figure 4. teething baby is ready to taste his di buğdayı (the photo is from a “tooth wheat” ceremony in december, 2008). ceremony and the celebration end by congratulating mother and the baby. the guests deliver good wishes and present gifts to the baby before they depart. function and future the appearance of the first tooth is obvious sign for the biological development of a baby. the “tooth wheat” ritual and celebration wish to reassurance this development. its purpose is to confirm a baby of good breeding, underline the importance of fertility to the community, and wish the baby good health and progress. the ritual blesses the baby and assigns it good health and fortune. it emphasizes the concern for the future well-being of a child. like the wheat, which boils in the pan and needs only a short period of time to get ready, similarly the baby’s teeth will appear and grow fast: the meal will confirm that the baby’s teeth will painlessly and healthy grow. the “wheat and teeth” turkish rite embodies cultural values that have been at the core of the turkish food culture. the objects used at the ceremony are important means to understand these values. the objects used for divinatory purpose reflect not only the viewpoints of turkish people about their own culture but also their worldviews. they give insights to what is accepted and is considered important in human life. apart from the ceremony and festivity, the relationship between wheat and tooth underline the importance of tradition in turkish daily life. furthermore, wheat considered sacred in turkish culture has symbolic meaning related with first tooth. it symbolizes continuity of life. wheat which is being ground, pounded and milled is considered a metaphorical food. it is the first food for humans, thus, it is important in human life because it is first food to be processed. wheat will be processed into other foods and human life will flourish. all in all, wheat symbolizes human life. wheat also stands for fertility in the turkish culture. when cooked and then consumed, it will help the gums open and allow the teeth to come out and thus prepare the food process, an essential matter for survival. thus, the appearance of the first food functions like a piece of wheat and symbolizes human life. as wheat multiplies and improves human life, similarly the first food will also assist the human being flourish. without teeth, human beings can not process (food) and thus cannot evolve. all in all, the first tooth growth is a symbolization of human life ready to start processing food and, therefore, life. the ritual is not only a way to bless the food but also life, because the baby is accepted as an individual as well as a valid member of a family. the celebration and the ceremony initiate the baby’s socialization process. the traditions followed show how significant it is to have a baby and thus underline the whole notion of family in the turkish culture. they are also about blessing the baby and its family as well as understanding the importance of sharing such occasion with friends and relatives. since teething celebration is a way of honoring the baby as an individual, it plays a very significant role in the turkish culture today. even though every region has its own variations in teething ceremony, people still ascribe great appreciation and keep the same meanings about the tradition in the society. however, change is an important term to be discussed on this tradition that there are some concepts has been altering in the society today. it is stated that the celebration has become a party center on gift-giving and has been hosted by a close friend or a neighbor rather than the woman who has a first teething baby. hosting by a friend or a neighbor and becoming gift giving party, the teething celebration conjures up the tradition of baby shower, a party given women friends of somebody who is pregnant and popular all around the world, such as in the united states, china and india. whether hosted by a friend or the woman who has a first teething baby, the celebration has become a costly event nowadays because addition to the main food, the women prepare various foods for the event, just like the very common party, “gün” day or kabul günü” among the turkish women nowadays. in addition, similar to a baby shower, there is another transition of opening the gifts during the celebration rather than after all the guests leave the house since opening the gifts in front of the guests was considered as rude in the turkish culture in the past. in short, turkish people attribute great value to wheat and its usage is tied to rites and passages of human life such as wedding as well as social activities and cultural activities. “tooth wheat”, as a type of food celebration and symbolizing human progress in life, represents sociocultural and religious identity of the turkish people. it stands for love and charity in the turkish culture and carries many significant values, such as family, solidarity, friendship, fidelity, sincerity, hospitality, chivalry, honesty. thus, it seems that as a tradition it continues to be cherished. references eberhard w (1996). northern neighbors of china. trans. nimet uluğtuğ. vii. seri. no. 9a, ankara. türk tarih kurumu. elçin ß (1981). introduction to folk literature. kültür bakanlığı yayınları: 365, türk halk kültürü eserleri dizisi: 10, emel matbaacılık sanayi, ankara, p. 74. erkut s (2008). konya wheat sample from b.s. ii in konya karahoyuk. belleten, 72: 263: 1-4. gomec s (1998). shamanism and old turkish religion. pamukkale üniversitesi eğitim fakültesi dergisi, 4: 44. gurbuz e (1995). rituel characteristics and functions of teething ceremeony. milli folklor, 4: 28, 81-83. honigman jh (1961). foodways in a muskeg community: an anthropological report on the attawapiskat indians. ottawa. koz s (2002). food culture. kitabevi yayınları, edebiyat dizisi, đstanbul. lévi-strauss c (1975). the raw and the cooked. new york: harper and row publishers. ögel b (1978). introduction to turkish culture and history: agriculture tradition, 2, kültür bakanlığı. örnek sv (1979). child in traditional culture. türkiye đ bankası yayınları. ornek sv (1995). turkish folklore. kültür bakanlığı, ankara, p. 199. özer k (1935). customs on birth and child. türk dili. 17 đkinci kanun, p. 2. tatçı m (1991). teething ceremony in silvan. milli folklor, pp. 12. thursby j (2008). foodways and folklore: a handbook. westport-ct: greenwood press. valamoti sm, anastasaki s (2007). a daily bread-prepared but once a year. petits propos culinaires, 84: 75-100. yoder d (1972). folk cookery in folklore and folklife: an introduction, university of chicago press, pp. 325-350. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 9 (1), pp. 001-009, january, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper impacts of local-level utilization pressure on the structure of mopane woodlands in omusati region, northern namibia isaac mapaure1* and albertina ndeinoma2 1 department of biological sciences, university of namibia, p. bag 13301, windhoek, namibia. 2 department of integrated environmental sciences, university of namibia (ogongo campus), p. bag 5520, oshakati, namibia. accepted 18 october, 2020 a comparative analysis of mopane woodland structure under different utilization pressure was done in omusati region, northern namibia. heights, basal area, biomass and densities of trees, shrubs and stumps were compared between a game park, densely-populated (central) and sparsely-populated (western) areas. heavy utilization significantly reduced tree basal area, biomass and plant densities in central omusati, leading to an unsustainable situation. woodland recovery from previous disturbance in the game park has been slow. contrary to expectation, there were fewer dead stumps in central area because of continual harvesting due to firewood scarcity. height structure differed significantly with more than expected numbers of plants in the ≤ 1 m, 1.1 to 2 m and less than expected plants in the 3.1 to 4 m and 4.1 to 5 m height classes in central omusati. more than expected numbers of trees were recorded in 4.1 to 5 m height class in western area and in 2.1 to 3 m and 3.1 to 4 m height classes in game park. this indicates over-harvesting of medium-sized trees in central omusati. mopane regenerates profusely through coppicing. however, no relationship existed between stump size and number of shoots due to continual harvesting of shoots, especially in central area. proper coppice management and pollarding strategies should be implemented to improve the situation. key words: colophospermum mopane, coppicing, omusati, woodland structure, woodland utilization. introduction forests and woodlands play important roles in the livelihoods of many societies, especially in communal areas where direct dependence on natural resources is high. in addition to performing many ecological functions,woodlands and forests provide many goods and services that are essential for improving and maintaining human livelihoods (association of women foresters of burkina (amifob) et al., 2004; campbell and marsh, 2000; mandondo, 2001; mazambani, 1992). they also play important roles in local cultures and economies of many communities (nkem et al., 2008; fotso, 1998), especially in the developing world, where poverty levels *corresponding author. e-mail: imapaure@unam.na. tel: +264 61 2063133. are high. however, there have been growing concerns on woodland and forest decline and degradation caused by various factors including unsustainable harvesting practices by resource users. as a result, the need for sustainable use of natural resources has been placed high on the priorities of most governments as the world faces an ever-increasing human population which puts pressure on limited natural resources. this need was underlined by the world commission on environment and development (1987) and subsequently, united nations conferences on environment and development and numerous other platforms. the problem of resource degradation is more acute in africa where a significant proportion (40%) of the people live in abject poverty and rely heavily on natural resources for survival. sustainable utilization of woodland and forest resources, as well as their conservation have 2 been inherent in the practices of many local communities for centuries (niamir, 1990) but increasing population pressure and poor management practices have led to degradation of woodlands and forests in many parts of the world, especially those managed as open access resources. such resources need to be properly managed to ensure sustained provision for local household needs. the situation has been aggravated and made more complex by relatively new challenges such as hiv/aids (shackleton et al., 2006) and climate change (scholes et al., 2008; nkem et al., 2008). southern africa is endowed with woodland resources which have sustained millions of people for centuries, particularly rural peasants. these include brachystegiadominated (miombo), acacia-dominated (thorn), baikiaea plurijuga-dominated (zambezi teak) and colophospermum mopane-dominated (mopane) woodlands. most woodlands occurring in communal areas are under increasing pressure (chidumayo, 2002; musvoto et al., 2007) due to human population expansion. mopane woodlands are especially important in arid and semi-arid areas of southern africa where they cover large expanses of land (mapaure, 1994; timberlake, 1996). due to increasing awareness of the importance of mopane woodlands in these areas, many initiatives are underway to provide some degree of support to local communities whose livelihoods depend on these woodlands for both wood-based and non-wood products. such initiatives have mostly involved management of the mopane caterpillar (imbrasia belina) which is a nutritious delicacy and less so on other woodland resources. in namibia, mopane woodlands occur mostly in the northern parts of the country, which are home to at least 60% of the population. these woodlands cover about 77000 km 2 in the country (mapaure, 1994). they are more extensive in omusati region, whose name ‘omusati’ is the local name for mopane. concern has been expressed on the dwindling woodland resources due to human pressure. in omusati region in particular, the directorate of forestry expressed concern that woodland use seemed unsustainable (selänniemi et al., 2000) but no empirical data exist to substantiate this. it was in view of this concern that this study was carried out in omusati region to assess the state of mopane woodlands and get insights about the sustainability of utilization practices in the region by comparing areas under different levels of human pressure. materials and methods study area the study was done in omusati region, northern namibia (figure 1). omusati is one of the thirteen political regions of namibia and covers 26573 km 2 . kunene region bound omusati region in the west and south, oshana and ohangwena regions in the east and angola in the north. omusati has a human population of about 229000 and has a high overall population density of close to 9 persons/km 2 (national planning commission, 2007), ranging between 1.8 and 54.6 persons/km 2 . two communal sites were selected for study based on differences in the population densities and living styles of the respective ethnic groups: a sparsely populated western omusati site (occupied by predominantly ovahimba ethnic group) and a densely-populated central omusati (occupied by owambo ethnic group). the ogongo game park, a protected area without human habitation, was the third site. these sites experience different utilization levels, with central omusati been heavily utilized compared with the western area and the game park (mapaure and ndeinoma, 2008). topography is characterised by flat plains, which form part of the etosha depression. in the western part, the land rises gently to the foothills of the kaokoland. annual rainfall ranges between 450 and 500 mm in the northeast and 250 and 300 mm in the southwest (npc, 2007). mean maximum and mean minimum temperatures range between 32 and 34°c, and 6 to 8°c, respectively. the vegetation is classified into four broad types (selänniemi et al., 2000) as palm savanna, bush mopane savanna, seasonally flooded grasslands and patches of mopane and acacia and open shrub savanna of mopane and acacia. only mopane woodlands were selected for study. vegetation assessments these assessments were done only in woodlands dominated by colophospermum mopane. fifty-eight plots were demarcated, 21 in central omusati, 18 in western omusati and 19 in the ogongo game park. trees and stumps were assessed in plots measuring 20 x 20 m, while 5 x 5 m subplots nested within the larger plots were used for shrub assessments. trees and shrubs were defined according to anderson and walker (1974). a total of 752 trees, 283 shrubs, 178 dead stumps and 699 live stumps were assessed in all the three sites combined (central: 85 trees, 123 shrubs, 71 dead stumps, 547 live stumps; western: 335 trees, 58 shrubs, 81 dead stumps, 100 live stumps; game park: 332 trees, 102 shrubs, 26 dead stumps, 52 live stumps). all plants were identified; basal circumferences and heights of trees were measured using a tape measure and a ranging pole, respectively. for multi-stemmed trees, the basal circumference of each stem was measured separately and the total basal area for the tree was calculated. of all woody plants assessed, 87.2% were c. mopane. stumps were also identified and assessed whether they were dead or alive. basal circumferences of all live stumps were measured and the number of coppice shoots on each stump was recorded. shrub heights were measured and all stems on each shrub were counted. data analyses the biomass of each tree was calculated using the formula derived for mopane woodlands in northern botswana by tietema (1989): mass (in kg) = 0.0644*(basal area, in cm 2 ) 1.3341 the basal area was then expressed in t/ha for each plot. differences in biomass, basal areas and densities among the sites were tested using one-way anova after testing for normality. post hoc analyses were done using tukey’s test. differences between individual plants and stems at each site were tested using a paired t-test. linear regression analysis was used to test whether the number of coppice shoots on each stump depended upon stump size. other curve-fit models (exponential, cubic, quadratic and logarithmic) were also tested to determine the best model to describe the relationship. differences in frequency distribution patterns of woody plant heights among sites were tested by a χ 2 3 figure 1. location of omusati region (shaded area) in northern namibia. 4 d e n s it y ( p e r h a ) m e a n d e n s it y ( p e r h a ) 1100 1000 900 800 700 600 500 400 300 200 100 0 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 a individual trees stems central western game park area individual shrubs b stems central western game park area figure 2. differences in the densities of individuals and stems of trees (a) and shrubs (b) in mopane woodlands at three sites in omusati region, northern namibia. test. results differences in plant densities and height structure there were significant differences in tree densities among the three sites (f = 43.68, p < 0.001): the western area and game park had significantly higher tree densities than the central area (p < 0.001). there was no significant difference between the western area and the game park (figure 2a). stem densities were significantly higher than individual tree densities in the central area (t = 3.116, p < 0.01), western area (t = 6.240, p < 0.001) and in the game park (t = 5.775, p < 0.001) (figure 2a). stem densities also differed significantly among the three sites (f = 30.10, p < 0.001): the western area and the game park had significantly higher density than the central area (p < 0.001). there were no significant differences in stem densities between the western area and the game park. shrub stem densities were significantly higher than individual shrubs in the central (t = 3.171, p < 0.01), western (t = 2.987, p < 0.01) and game park (t = 4.305, p < 0.001) (figure 2b). there were no significant differences in each of these two variables among sites. most stumps encountered (98.6%) were c. mopane. other stumps recorded were combretum apiculatum, combretum collinum, kirkia acuminata, terminalia prunioides and acacia nilotica. densities of dead stumps differed significantly among sites (f = 4.48, p < 0.05) (figure 3), with higher densities in the western area than in the game park (p < 0.05). not all other comparisons 5 d e n s it y ( h a -1 ) 140 120 100 dead 80 alive 60 40 20 0 central western game park site figure 3. differences in the densities of dead and live stumps in mopane woodlands at three sites in omusati region, northern namibia. figure 4. differences in the height frequency distribution patterns of woody plants in mopane woodlands at three sites in omusati region, northern namibia. were significant. densities of live stumps also differed significantly among sites (f = 13.70, p < 0.001) (figure 3), with significantly higher density in the central area than at the other two sites (p < 0.001). there was no significant difference in live stump densities between the western area and the game park. densities of live stumps were significantly higher than dead stumps in the central area (t = 2.02, df = 40, p < 0.001). no significant differences were recorded at the other two sites. the highest proportion of woody plants recorded in the western area and the game park were 3.1 to 4 m tall, while the central area had the highest proportion in the ≤ 1 m height class. very few woody plants were >5 m tall at all sites (figure 4). there were significant differences in the height frequency distribution patterns of woody plant heights among sites (χ 2 = 250.12, df = 10, p < 0.001). much less than expected number of plants was recorded in the height classes 3.1 to 4 m and 4.1 to 5 m in the 6 table 1. differences in the mean tree basal area and mean biomass in mopane woodlands at three sites in omusati region, northern namibia. site mean tree basal area (±se) (m 2 /ha) mean tree biomass (±se) (t/ha) central 2.31 (±0.58) a 18.26 (±5.62) a western 9.07 (±1.26) b 96.72 (±17.58) b game park 6.89 (±0.92) b 66.95 (±11.64) b significance p < 0.001 p < 0.001 different superscripts for each variable indicate significant differences between the sites. central area and in the ≤1 m category in the western area and the game park. significantly, more than expected number of plants was recorded in the ≤ 1 m and 1.1 to 2 m height classes in the central area, 4.1 to 5 m height class in the western area and 2.1 to 3 and 3.1 to 4 m height classes in the game park. variations in tree basal areas and biomass there were significant differences in tree basal areas amongst sites (f = 14.13, p < 0.001) (table 1), with significantly lower basal area in the central than the western area (p < 0.001) and than in the game park (p < 0.01). the same pattern was true for tree biomass comparisons (f = 10.997, df = 2, p < 0.001; table 1). mopane regeneration from coppice all the 547 live c. mopane stumps recorded in the central area were coppicing. there was an average of 5.4 ± 0.2 coppice shoots per stump. all the 100 live stumps of mopane recorded in the western area were also coppicing, with an average of 5.0 ± 0.2 shoots per stump. in the game park, 52 mopane stumps were recorded and all of them were coppicing, with an average of 5.5 ± 0.4 shoots per stump. in general, large-sized stumps supported more coppice shoots than smaller-sized stumps but within a limited size range of less than 80 cm circumference (figure 5). linear regression analysis showed a weak relationship (r 2 = 0.295, f = 294.65 p < 0.001) between number of coppice shoots and stump size. all other curve-fit models tested were weaker than the linear model. discussion impact of woodland utilization on vegetation structure this study has clearly demonstrated that mopane woodlands in central omusati are structurally different from those of both the protected game park and western omusati. this trend is not unexpected in communal areas where population density is high (13.9 persons/km 2 in central omusati) and where resource management regimes are open access in nature. nevertheless, the extent to which the woodlands have changed in central omusati compared with the neighbouring two sites is a cause for concern. the fact that live stump densities in the central area were 342 and 910% higher than western area and game park, respectively, is clear testimony of heavy utilization pressure. mapaure and ndeinoma (2008) reported that, utilization pressure has negatively impacted on the species composition and diversity of woodlands in central omusati compared with the other two sites. densities of dead stumps in the central area are under-estimates because most of the stumps are also harvested for firewood, a situation that normally manifests itself under resource scarcity conditions. subsistence harvesting of woody plants by local communities in various sites in southern africa has been shown to result in significant changes in the structure of woodlands (luoga et al., 2004) which in turn negatively impacts on local livelihoods. in the current study, reductions in tree biomass in central omusati ranged between 72 and 82% which are much higher than a decrease of 32.5% mopane woody biomass in dukwe, botswana reported by tietema (1993a, b). considering that western omusati and the game park are also experiencing or have also experienced wood harvesting, changes recorded in central omusati are under-estimates of the actual situation because there is no true experimental control in this investigation. it is not reasonable to directly compare values of woodland parameters across the whole mopane woodland range because of the high variability and heterogeneity of woodland use at different sites. for instance, the biomass of unharvested mopane woodland in dukwe, botswana, recorded by tietema (1993a, b) was 117 t/ha, while values recorded from zimbabwe (under various management scenarios) ranged between 15 and 68 t/ha (kelly and walker, 1976; guy, 1981; grundy et al., 1993). cunningham (1993) emphasized that, patterns of woodland use and depletion vary according to settlement patterns, abundance and patchiness of favoured plant species and their sizes. it is conceded though, that the palisade structure of owambo huts consumes more 7 figure 5. the relationship between mopane stump circumference and the number of coppice shoots supported by the stump. wood than any other structure in southern africa (erkillä and siiskonen, 1992), a situation that explains overharvesting of pole-sized plants in central omusati. the lack of expected numbers of trees in height class 3.1 to 5 m and the dominance of younger trees (figure 5) is clear evidence of preferential harvesting of the pole-sized trees for household use. it is a well-known pattern that communities prefer certain sizes of trees for construction of timber and firewood (grundy et al., 1993). the demand for wood resources depends not only on human population densities but also on the replacement times of construction timber. the dominant growth form of mopane is usually singlestemmed (frost et al., 1987) but multi-stemmed shrub formations also occur (gelens, 1996; hempson et al., 2007). the inventoried sites had no naturally shrub-like forms (gelens, 1996) which normally occur on soils of high sodicity and high impermeability (thompson, 1960 cited in gelens, 1996). these results indicate that, the majority of trees were multi-stemmed, a situation normally resulting from resprouting in response to disturbance. resprouting allows plants to recover quickly without having to set seed. the importance of resprouting as a survival strategy in plants has been recorded from a variety of habitats worldwide (basnet, 1993; lamont and markey, 1995). however, one cost of producing multiple stems is that tree height is compromised (nzunda et al., 2007) as observed in this study. multi-stemming was lower in central omusati than at the other two sites, an observation which could indicate that in central omusati, most of the stems produced by an individual plant were harvested (due to higher demand), whereas they persisted longer in the other two areas. in western omusati, it may be a deliberate management practice to conserve woodlands by local communities, being a predominantly cattle-rearing (rather than crop-farming) ethnic group whose hut structures are more basic than those of owambo. the existence of multi-stemming and occurrence of stumps in the game park shows that, these woodlands have not fully recovered from previous disturbance since the mid-1990s when an army base moved out of the area. occassional illegal harvesting of wood from the game park by adjacent farmers still takes place. 8 tree basal area and tree biomass are positively related (timberlake, 1995) thus, reductions in biomass manifest themselves in a similar pattern as in basal area (table 1). differences in basal area should be interpreted in conjunction with the frequency distribution pattern in order to get a cleareroverall pattern. in central omusati, the pattern is indicative of a woodland dominated by smaller trees, disproportionately fewer individuals in the middle-size classes and much higher than expected numbers of large trees. this is indicative of overharvesting of certain targeted sizes of plants. as preferred plant sizes become scarcer, non-preferred sizes are also harvested, leading to woodland degradation. results indicate that c. mopane coppices profusely. other researchers (gelens, 1996; mushove, 1993; mlambo and mapaure, 2006; mushove and muchichwa, 1996; timberlake, 1995) have reported this characteristic from several sites. luoga et al. (2004) and grundy (1996) reported that, coppicing effectiveness was dependent upon plant size, stump height, age of rootstock and percentage of woodland stand removed. the study results indicate that, mopane is a very resilient species, which with proper management, can be sustainably maintained to provide required goods for local communities without significant degradation. however, the growth rate of this species is extremely slow (grundy, 1996). coppice shoots grow a little faster than growth from seedlings (tietema et al., 1988; erkkila and siiskonen, 1992), a characteristic that can be taken advantage of in managing the woodlands. sustainability of woodland use from the preceding account, it is evident that woodlands in central omusati have been over-utilized and degraded due to human pressure. species composition has changed and species diversity has declined (mapaure and ndeinoma, 2008), tree density, tree biomass and tree basal area have all declined primarily due to unsustainable human use. scarcity of wood resources is evident in central omusati and there are records of people travelling to western omusati to harvest wood and other resources. such activities were already recorded in the early 1990s (erkilla and siiskonen, 1992) but the incidences have intensified in the last few years (musvoto et al., 2007). all these are indications of resource scarcity and a non-sustainable situation. however, from this study, it is not easy to calculate sustainable harvesting regimes that would encourage mopane woodlands in central omusati to recover and provide enough for local communities. to infer sustainability, there is need to carry out repetitive measurements of these structural attributes over few years. nevertheless, the assertion by selanniemi et al. (2000) that woodland use in omusati appeared to be non-sustainable was confirmed. unless local communities start to manage these woodlands through proper coppice management, pollarding, thinning and other practices (gondo et al., 2007; musvoto et al., 2006), the situation is bound to worsen. such practices have been recommended as better technical options in similar situations in zimbabwe (gondo et al., 2007). however, the problem of woodland degradation should not be tackled in isolation; instead, it must be considered in a holistic and integrated manner with other challenges of poverty, the hiv/aids pandemic and climate change. all these are important drivers of patterns of woodland utilization by local communities. acknowledgements the authors are grateful to the african forestry research network (afornet) for providing funds to carry out this research. this research was part of a larger project involving studies of mopane woodlands in zimbabwe and namibia; we thus, thank our research counterparts in zimbabwe, connie musvoto, tendayi gondo and the late takaendesa mujawo. local communities in omusati were very cooperative during our data collection around their villages, for which we are very grateful. assistance in field data collection rendered by mr leonard hango and ms josephine ashipala is greatly appreciated. views expressed in this paper are not necessarily those of afornet. references anderson gd, walker bh (1974). vegetation composition and elephant damage in the sengwa wildlife research area, rhodesia. j. s. afr. wildlife. manage. assoc., 4: 1-14. association of women foresters of burkina (amifob), campbell b, frost p, kokwe g, le breton g, shackleton s, tiveau d (2004). making dry forests work for the poor in africa – building on success. forest livelihood briefs no. 3: centre for international forestry research (cifor), bogor, indonesia, pp. 1-4. basnet k (1993). recovery of a tropical forest after hurricane damage. vegetation, 109: 1-4. campbell 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for a woodland management and plantation scheme. norad/nir, gaborone, botswana, p. 86. timberlake j (1995). colophospermum mopane: an annotated bibliography and review. zimbabwe bullettin of forestry research no. 11, forestry commission, harare, pp. 1-49. timberlake j (1996). a review of the ecology and management of colophospermum mopane. in : flower c, wardell-johnson g, jamieson a (eds) management of mopane in southern africa. proceedings of a conference held at ogongo agricultural college, namibia, 26-29 november, 1996. oda/directorate of forestry, windhoek, pp. 10-16. world commission on environment and development (1987). our common future. oxford university press, oxford, pp. 27-66. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (10), pp. 001-006, october, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper simultaneous effect of divalent cation in hydrolyzed cassava starch medium used by immobilized yeast for ethanol production okon, anne anthony1* and u. nwabueze, titus2 1 department of food science and technology, university of uyo, akwa ibom state, nigeria. 2 department of food science and technology, michael okpara university of agriculture, umudike, p. m. b. 7267 umuahia, abia state, nigeria. accepted 09 july, 2019 response surface methodology was adopted in a central composite design to optimize ethanol production from cassava starch hydrolysate medium. starch hydrolyzate was prepared from tms 98/0581, a genetically developed cassava mosaic disease-resistant variety. the yeast whole cell, saccharomyces pastorianus, a lager brewing strain (726 x 10 6 cells/ml, 98.78% viability) and fungamyl and termamyl (-amylase enzymes), used for the 120 h fermentation, were immobilized by entrapment in calcium alginate gel. effects of three divalent cationic concentrations mg 2+ , zn 2+ and ca 2+ on ethanol yield were investigated at five variable combinations in 20 experimental runs in accordance with the experimental design. maximum ethanol concentration of 12.53 %v/v was produced in the 96 h of fermentation when the divalent cationic combination was 64, 0.48 and 30 mg/l (mg 2+ , zn 2+ and ca 2+ ), respectively. the study showed that effect of zn 2+ on ethanol yield was significantly (p 0.05) quadratic. keywords: ethanol, immobilization, saccharomyces pastorianus, divalent cations, optimization, response surface methodology, cassava mosaic disease. introduction yeast fermentative growth in simple media and carbon skeleton requires adequate nitrogen (for protein synthesis), mineral salts (metal ions), one or more growth factors and molecular oxygen (hough et al., 1982). metal ions, especially divalent cations are necessary for the activation of several glycolytic enzymes and, in practical terms, if industrial media is deficient in them, the conversion of sugar to ethanol may be suppressed leading to slow or incomplete fermentation process (walker et al., 2006). the uptake of these divalent cations into cells depends on the concentration of particular ions in the growth environment and on their bioavailability (chandrasena et al., 1997). to develop a process for the maximum production of ethanol, standardization of media and fermentation conditions is crucial (ratnam et al., 2005). optimization of *corresponding author. e-mail: annytony2002@yahoo.com the divalent cationic nutrients (mg 2+ , zn 2+ , ca 2+ ) required by yeast for fermentation is therefore very important for maximizing the yield and productivity and minimizing the production costs. response surface methodology (rsm) has been successfully applied to optimize alcoholic fermentation and other fermentation media (chen, 1996; chandrasena et al., 1997; ambati and ayyanna, 2001; ratnam et al., 2003; ratnam et al., 2005). ethanol production by immobilized yeast cells has been extensively investigated during the last few decades (rakin et al., 2009). immobilization of cells is very similar to the enzyme counterpart (wang, 2008). for fermentation, immobilization of cells has been developed to eliminate inhibition caused by high concentration of substrate and product, thereby enhancing productivity and ethanol yield (kourkoutas et al., 2004; vullo and wachsman, 2005; baptista et al., 2006). according to groboillot et al. (1994) the main advantages of the immobilization of yeast are the increase of ethanol yield and cellular stability, and a decrease of process expenses due to the ease of cell recovery and reutilization. the aim of this study was to investigate the effect of divalent cation combinations in hydrolyzed cassava starch medium for ethanol production using immobilized yeast (saccharomyces pastorianus). bioavailability of these cations in an optimum combination is vital for successful ethanol production by directly influencing sugar metabolism by yeast in the 120 h fermentation period in this study. materials and methods raw materials cassava starch: the raw material for ethanol production was starch from cassava mosaic disease (cmd) resistant variety (tms/0581) developed for food, feed and industrial use (dixon et al., 2005). the cmd variety was obtained from national root crops research institute (nrcri), umudike, nigeria and processed into starch according to the method described by the international institute of tropical agriculture (iita, 2005). the starch constituted 100% particles that passed through a 425 µm sieve. enzymes fungamyl: this is purified fungal -amylase produced from aspergillus oryzae. this enzyme hydrolyzes 1, 4--glucosidic linkages in amylase and amylopectin, the two components of starch. termamyl, an -amylase isolated from bacillus licheniformis, a soil bacterium. this enzyme hydrolyses 1, 4--glucosidic linkages in starch and possesses a high degree of heat stability. it is used for the continuous liquefaction of starch at temperatures of up to 105-110 o c, breaking them rapidly to dextrins and oligosaccharides. amyloglucosidase (amg), an exo-1, 4-dglucosidase (glucoamylase) was obtained from a selected strain of the fungus, aspergillus niger. this enzyme hydrolyses 1, 4and 1, 6--glucosidic linkages in liquefied starch in stepwise manner from the non-reducing end of the substrate molecules (alais and linden, 1999). yeasts: s. pastorianus, a lager brewing strain was used for the fermentation of cassava starch hydrolyzates. s. pastorianus is a hybrid organism of two yeast speciessaccharomyces cerevisiae and saccharomyces bayanus (rainieri et al., 2006; blieck et al., 2007; dunn and sherlock, 2008). it is thought that the combination of both parent species resulted in an organism able to out -compete other yeasts during the cold lager fermentations (dunn and sherlock, 2008). the enzymes and yeasts were gift from champion breweries plc., uyo, nigeria. enzymatic hydrolysis of starch: a 250 g of cassava starch was mixed with distilled water at a weight ratio of 1:4 (iita, 2005), stirred with a glass rod to obtain a uniform mixture (mash). the temperature of the mash was raised to 60 o c at which the starch particles hydrate, swell and gelatinize, making them susceptible to enzymatic hydrolysis (kumar et al., 1998). the mixture was then treated with enzymes in two steps: liquefaction and saccharification. the liquefaction was carried out at 90-95 o c by adding 2 ml each of fungamyl and termamyl enzymes for 1 h. the liquefied mash was cooled to 60 o c, 2 ml amg added and heated to 75 o c for saccharification. the hydrolysis was performed in flasks in a thermostatic water bath with a stirrer (tr24-a22bx, england). immobilization of s. pastorianus by entrapment in calcium alginate gel: a polymeric matrix was prepared using sodium alginate (nr 301054n, hopkin and williams ltd., england) . to obtain this, 300 ml of distilled water was pre-warmed at 60 o c and 4.5 g of sodium alginate was added with continuous stirring until a homogenous solution was obtained (no clot) (vullo and wachsman, 2005). s. pastorianus (7.26x10 8 cells/ml, 98.78% viability) was weighed 11.25 g and mechanically suspended in the already prepared 1.5 % sodium alginate solution. an erlenmeyer flask, 500 ml, was filled with the yeast suspension and then emptied by gravity, drop by drop, into 600 ml of 0.1 m cacl2 solution. the calcium cross-linking solution was agitated on a magnetic stirrer (stuart scientific magnetic stirrer sm 1, uk). gel formation was achieved at room temperature as soon as the yeast-alginate drops came into direct contact with the calcium solution. micro beads (0.81.0 mm diameter) were achieved with a 10 ml syringe/needle, to minimize the mass transfer resistance (wang, 2008) . the beads were fully harden in 15 min and were washed 4 times with distilled water to eliminate excess ca 2+ (vullo and wachsman, 2005). the drop volume was calibrated by passing distilled water through a diameter tube (10 ml syringe/needle) and weighing a fixed number of water droplets. the drop volume was 0.04 ml and all the suspension turned into immobilized yeast spheres with 10 7 cells per bead. ethanol fermentation of starch hydrolyzates: starch hydrolyzates obtained by the 2-step hydrolysis of cassava starch were cooled, filtered to remove the trub and sterilized in an oswald tm autoclave steam sterilizer (jric 39, india). ethanol fermentation by immobilized yeasts under anaerobic conditions was performed in 1l flasks with 500 ml of medium in laboratory temperature (20 o c). repeated batch fermentations were carried out with the same inoculum concentration and laboratory conditions, but different concentrations of divalent cation combinations for 5 days. the summary of ethanol production from cassava (cmd-resistant variety) tms/0581is is given in figure 1. analytical method ethanol concentrations of the fermenting hydrolyzates were determined using an anton paar gmbh alcolyzer plus (com 1, austria, europe). the samples were drawn into a flask sealed, shaken and released to degas. the degassed samples were filtered through folded whatman filter paper (1 qualitative, 10 cm, england) and the funnels covered immediately with a watch glass. the samples were swirled very well (to bring back any condensation of ethanol into the solution) and 50 ml filled into the sample vial and placed into the magazine of the sample changer (sp-1m). the sample changer is a part of the sophisticated beer analyzing system of the alcolyzer plus. ethanol concentration is displayed at 20 o c. distillation at the end of fermentation, the hydrolyzates were filtered for distillation (recovery of ethanol). a 100 ml distillation flask (clearfit 34/36, england) was filled with the fermented sample, placed in an electric heater, and connected to a clearfit distillation apparatus (ksh 4/33, england) with thermometer. ethanol was distilled off at the temperature of 78.5 o c (okwu and eneboachi, 2002). experimental design a central composite rotatable response surface design for a three cassava starch water, 60 o c, gelatinization liquefaction 90-95 o c, ph 4-4.5, 400rpm termamyl and fungamyl saccharification 55-75 o c, ph 4-4.5, amylo gucosidase (amg) enzyme cooling/filtration 30-33 o c sterilization 121 o c, 15 min fermentation immobilized micro-beads added, co2 out distillation 78-78.5 o c ethanol figure 1. flow chart for produc tion of ethanol from cassava starch hydrolyzate using immobi lized yeast cells. variable, five level combinations coded -1, -1.682, 0, 1, 1.682 (table 1) as modeled and used in literature (nwabueze and iwe, 2006; nwabueze, 2007) was used for the optimization of the divalent cations for ethanol production from the cassava starch hydrolyzates. magnesium (x1, mg/l), zinc (x2, mg/l), and calcium(x3, mg/l) were chosen as the independent variables at five levels of concentrations as shown in table 1. a total of 20 experiments were employed for the optimization of the cations in fermentation. data analysis statistical analyses were carried out on the data obtained from the fermentations. the data were statistically regressed using statgraphic computer software (statistica) to test the significance of main and interactive effects of the cations (nwabueze and iwe, 2006; nwabueze, 2007). statistical significance was accepted at 5% probability levels (p 0.05). three-dimensional response surface plots were made with matlab 7.1.0246 (r14) gibsoft software. the statistical design (multivariate regression analysis) with the model fitted to each set of data was as follows: y = 0 + 1x1 + 2x2 + 3x3 + 11x1 2 + 22x2 2 + 33x3 2 + 12x1x2 + 13x1x3 + 23x2x3 + (1) where y = dependent response variable, ethanol 0 + 1 … 23 = estimated regression coefficients. x1, x2, x3 = independent variables in the model (mg 2+ , zn 2+ and ca 2+ ). = random error. results and discussion response surface methodology is a sequential procedure with an objective of leading the experimenter rapidly and efficiently to the general vicinity of the optimum. using central composite design, a total of 20 experiments with different of the divalent cations were performed. responses were taken at 24 h interval until ethanol concentration dropped. ethanol concentration at 0 to 120 h from the cassava starch hydrolyzates were as shown in table 2. there was a general increase in the concentration from 0-120 h period of fermentation. increase in ethanol concentration of fermented media have been reported by several authors including balagopalan (1988)cassava wort; walker et al. (1996)malt wort; chandrasena et al. (1997)molasses; birch et al. (2003)wine must; vullo and wachsman (2005) -synthetic media; rakin et al. (2009)corn meal hydrolyzates. effect of divalent cation on ethanol yield optimum alcohol production of 12.53%v/v was obtained from samples with divalent cation combinations of 64, 0.48 and 30 mg/l (mg 2+ , zn 2+ and ca 2+ ), respectively at96 h period of fermentation. high concentration of mg 2+ and zn 2+ and low ca 2+ seems to favour ethanol production (table 2). yeast exhibits a high affinity for mg 2+ and increase in mg 2+ availability stimulates alcohol produc-tion. thus, mg 2+ is essential for yeast growth, metabolism and fermentation. this is in line with the report of smith and walker (2000). mg 2+ is also essential in nucleic acid synthesis and a cofactor of many enzymes in glycolysis while zn 2+ is an essential micronutrient and has stimulating effect in yeast metabolism. alcohol production table 1. independent variables in the central composite design. independent process variables coded variable levels corner points central point star points (mg/l) -1.682 -1 0 +1 +1.682 mg+2 x1 35 64 107 150 179 x2 zn+2 0.24 0.30 0.39 0.48 0.54 x3 ca+2 14.31 30 53 76 91.69 table 2. effect of the independent variables (x1, x2 and x3) on the ethanol of the fermenting hydrolyzates during fermentation. variables responses (alcohol %v/v) s/n period of fermentation (h) x1 x2 x3 mg zn ca 0 24 48 72 96 120 1 64 0.30 30 0.17 5.81 9.66 11.11 11.08 2 64 0.30 76 0.13 5.30 10.58 12.26 12.23 3 64 0.48 30 0.17 6.04 11.03 12.50 12.53 12.33 4 64 0.48 76 0.17 6.20 8.14 8.72 9.21 9.19 5 150 0.30 30 0.18 5.70 10.74 12.45 12.44 6 150 0.30 76 0.19 6.77 11.91 12.38 12.33 7 150 0.48 30 0.19 5.77 11.33 12.37 12.33 8 150 0.48 76 0.19 5.22 8.65 11.43 11.46 11.42 9 179 0.39 53 0.19 6.02 10.87 12.26 12.22 10 35 0.39 53 0.13 6.23 11.00 11.10 11.14 11.03 11 107 0.54 53 0.18 6.09 11.01 12.34 12.36 12.20 12 107 0.24 53 0.12 5.99 10.09 10.23 10.35 10.11 13 107 0.39 91.69 0.15 6.78 10.57 11.02 11.08 10.98 14 107 0.39 14.31 0.13 5.72 9.75 9.98 9.92 9.82 15 107 0.39 53 0.19 6.45 10.33 10.97 11.01 10.96 16 107 0.39 53 0.15 6.28 10.27 11.00 10.94 17 107 0.39 53 0.18 6.02 10.29 11.01 11.02 10.94 18 107 0.39 53 0.18 6.04 10.31 11.01 10.98 19 107 0.39 53 0.18 6.12 10.30 11.01 10.97 20 107 0.39 53 0.19 6.20 10.30 11.02 10.98 control 0.04 4.82 7.88 9.74 10.07 10.06 control: cassava starch hydrolyzate medium containing immobilized saccharomyces pastorianus without divalent cations. increased with high concentrations of zn 2+ (0.30-0.48 mg/l). similar results have been reported by densky et al. (1966) in brewing wort using ale yeast showed stimulating effect of zn 2+ at levels of 0.1-1 mg/l. desmartez (1993) showed that 0.45 mg/l concentration of zn 2+ promoted fermentation and consequently alcohol production. ca 2+ requirement for yeast growth, metabo-lism and alcohol production are low (30-76 mg/l). the same trends have been reported by walker (1994) and youatt (1993). minimum alcohol production was 9.21%v/v from 64, 0.48, 76 mg/l (mg 2+ , zn 2+ and ca 2+ ), respectively at 96 h period of fermentation. it was observed that where ca 2+ was higher than mg 2+ , ca 2+ exhibited its inhibitory/antagonistic effect on mg 2+ , consequently, the mg-dependent processes and yeast growth (walker et al., 1996). walker et al. (1996) showed that by altering the mg 2+ and ca 2+ ratio in favour of mg 2+ , alcohol production by yeast increased. however, it is interesting to note that the main effect of ca 2+ was not significant for a high level of mg 2+ in the fermentation medium, which indicates that yeast has a higher affinity for mg 2+ than for ca 2+ . this finding supports the views of walker et al. (1996) and chandrasena et al. (1997). the estimated regression coefficients for ethanol at 0 h table 3. estimated regression coefficient for ethanol at 0 h of fermentation using immobilized yeast cells and the variables (x1 = mg 2+ , x2 = zn 2+ ,, x3 = ca 2+ ). source coefficient standard error df p-value regression on constant 21.41241 1.55217 x1 0.00095 0.00998 1 0.9262 x2 -11.82806 5.68796 1 0.0643 x3 0.01962 0.01834 1 0.3098 x1x1 0.00001 0.00003 1 0.6924 x1x2 -0.00323 0.01784 1 0.8599 x1x3 -0.00002 0.00007 1 0.7780 x2x2 19.49185 6.44050 1 0.0128 x2x3 -0.03382 0.03335 1 0.3346 x3x3 -0.00005 0.00010 1 0.6391 r 2 0.6181 response surface plot for ethanol at 0 h using zn and ca as process on ethanol. e = 21.41241 + 19.49185 x2 2 where e = ethanol; x2 2 = quadratic order conclusion in this study, the response surface methodology was adopted in a central composite design to optimize ethanol production from cassava starch hydrolysate medium. s. pastorianus used for the 120 h fermentation was immobilized by entrapment in calcium alginate gel. effects of three divalent cation (mg 2+ , zn 2+ and ca 2+ ) combinations on ethanol production were investigated at five variable (2) levels in 20 experimental runs in accordance with the effect of zn 2+ experimental design. maximum ethanol yield of 12.53% v/v was produced in the 96 h of fermentation when the variables. fermentation are shown in table 3. there was a significant (p 0.05) quadratic effect of zn 2+ (x2) on the alcohol production. zn 2+ is essential for yeast growth and fermentative metabolism. the same has been reported by chandrasena et al. (1997) and walker et al. (2006). the response surface plot (figure 2) of the interaction between zn 2+ and ca 2+ confirms the quadratic effect of zn 2+ on ethanol yield. the multiple regression model developed from the data explained a variation of 61.81% at this period, and the resultant polynomial after removing the insignificant (p>0.05) terms becomes: figure 2. divalent cationic combination was 64, 0.48 and 30 mg/l (mg 2+ , zn 2+ and ca 2+ ), respectively. effect of zn 2+ on the ethanol production was quadratically significant (p 0.05). references alais c, linden g (1999). food biochemistry, aspen publishers inc. coaistherburg maryland p. 4. ambati p, ayyanna c (2001). optimizing medium constituents and fermentation conditions citric acid production from palmyra jaggery using response surface method. world j. microbiol. biotechnol. 17: 331-335. balagopalan c, padmaja g, naada sk, moorthy sn (1988). cassava in food feed and industry, crc press inc. boca roten florida pp. 97175. baptista cmsg, coias jma, 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experiment no. 11. http://www.glue.umd.edu/~nsw/ ench 485/lab11.htm. accessed 10/3/2008. youatt j (1993). calcium and microorganisms. critical rev. microbiol. 19: 83-97. 1 full length research paper utilizing the infrared technology to characterize soil qualities in the democratic republic of the congo's south kivu province barasa1*, owuor1 1 university of nairobi, kenya accepted 23 december, 2024 a fundamental prerequisite for effective land management is an understanding of soil properties. conventional laboratory analysis has long been used to evaluate soil qualities, but it is expensive and time-consuming. as a result, alternative, quicker, and less expensive methods for soil analysis must be developed. particular focus has been placed on chemometrics and infrared reflectance spectroscopy in recent years. mid-infrared (mir) and near-infrared reflectance (nir) spectroscopy methods are quick, easy, and non-destructive ways to measure a variety of soil characteristics. the purpose of this study is to use infrared spectroscopy to characterize soil. for soil samples taken in the democratic republic of the congo's sud-kivu province, this approach was used to forecast the soil's ph, soil organic c, total n, exchangeable al, ca, mg, and k, cec, and texture. using a spatially-stratified random sampling design, 530 composite soil samples were collected from two sites (burhale and luhihi) at two depths (0–20 and 20–40 cm) over a 100 km2 area. it takes roughly two minutes to analyze a soil's mir spectrum after minimal sample preparation. disparities in soil depth and land use (cultivated versus non-agricultural) were assessed between the two sites. standard wet chemistry techniques were used to evaluate a random subset of the samples (10%), and calibration models were created utilizing mir data to estimate the soil parameters for the entire collection of soil samples. the partial least squares regression (pls) approach produced solid predictions for all parameters with acceptable coefficients of determination ranging from 0.71 to 0.93. ir demands a large initial expenditure, despite being inexpensive for assessing soil parameters. therefore, in order to make this technology usable in underdeveloped nations, technical and material help is required. key words: spectroscopy, prediction, regression, soil nutrient, infrared. introduction author(s) retain the copyright of this article. african journal of food science research issn 2375-0723 vol. 12 (1), pp. 001-009, december, 2024. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 education soil one of the main causes of global food insecurity is the loss in soil fertility. because farmers are unable to employ appropriate practices for yield development, the lack of precise information on soil management and sustainability at the farm level lowers soil productivity. the majority of research centers lack the necessary equipment to conduct research, and the majority of information pertaining to the nutrient content of the soil is unavailable. the majority of farmers cannot afford the extremely high laboratory fees associated with soil analysis. due to the several steps required, the majority of research centers' existing wet chemistry procedures for soil analysis take a lengthy time to produce findings. particularly in ssa, where soil fertility depletion is more severe, there is a pressing need for quick and sustainable methods of soil analysis to enhance fertilizer use and boost agricultural production. the efficiency of mir in conducting quantitative analyses of soils, particularly soil carbon, has been shown in numerous investigations (reeves et al., 1999; shepherd and walsh, 2002; christy, 2009). numerous qualities indicated by mir spectra typically change only slowly in soils, meaning that once evaluated, re-analysis may not be necessary for that property or may only be necessary after a considerable period of time. faster and less costly data collection methods are needed to quantify the many soil characteristics in order to conduct a thorough analysis of the roles played by a variety of soil physical, chemical, and biological characteristics in terrestrial ecosystems. this is particularly true for applications requiring exact spatial resolution of soil parameters, such as precision agriculture. recent research has demonstrated that multivariate chemometrics and infrared reflectance spectroscopy can be used to quickly and affordably examine certain chemical and physical properties of the soil both qualitatively and quantitatively. as a result, infrared spectral investigations are perfect for inventorying soil resources (minasny et al, 2008). because of its quick, non-destructive, low-cost measurements and ability to identify multiple soil parameters at once, infrared reflection (ir) spectroscopy has drawn interest from soil scientists as a potential method for better soil analysis. the time has come to invest in soil health in order to boost productivity. even extension services and research institutions that can help farmers in ssa understand the health of their soil do not provide them with knowledge on managing soil fertility. infrared is a new technique for soil analysis that is quick and affordable, but it needs to be verified before being widely used. at the moment, there is little data to support its accuracy, which limits its application when it comes to land management decision-making. therefore, the goal of this study is to evaluate the precision of this novel approach and utilize the findings to examine variations in soil samples taken from various locations. materials and methods the study was carried out in the south kivu province's eastern drc. this area is described as by highland characteristics that receive a lot of rainfall and have a very high population density (farrow et al., 2006). figure 1 illustrates the two sites chosen for this investigation: burhale and luhihi. due to nutrient mining and erosion, overpopulation degrades south kivu's soils. it also reduces the amount of land available for raising livestock, which lowers the number of animals that can provide manure for farmers (dsrp, 2005). agriculture is the primary industry in the area. pigs, sheep, goats, chickens, and cattle are examples of traditional livestock. the uvira plain is home to south kivu's largest animal production area. however, because of overcrowding, there is less grazing space in burhale (walungu) and luhihi (kabare), which has resulted in fewer cattle. there are also large plantations of quinquina, tea, and coffee in the region (mateso et al., 1998). south kivu has a wide variety of soil types, most of which are influenced by their parent geology (mateso, 1998). based on the parent material, the soils of south-kivu can be subdivided into four major groups: 1. soils that have recently formed on volcanic substrate; 2. soil developed on old volcanic substrate, mainly basaltic ; 3. soils formed on old sedimentary and metamorphic rocks (cover extensive areas) and, 4. alluvial soils and lacustrin and fluviatile deposits of the plains of rusizi. the 530 samples that were gathered were all scanned infraredly utilizing spectra of infrared reflectance between 400 and 7000 cm–1. just a few grams of dirt (about 20 to 30 mg) were collected for the samples. using an agate pestle and mortar, air-dried materials were ground to a fine powder (around n<100 μm). aluminum micro titer plates contained 96 wells; an empty well was used to measure the background signal (figure 2) (icraf, 2009,). the samples were loaded into the plates using a micro spatula to fill the 6-mm-diameter wells and level the soil, being careful not to spill into nearby wells. to adjust for variations in temperature and air humidity, background measurements of the first empty well were made prior to each individual measurement (mevik and wehrens, 2007). since aluminum doesn't absorb infrared light, it can be used as a reference material (terhoeven et al., 2010). to reduce specular reflectance, the bottoms of the al wells were roughened. in order to account for within-sample variability as well as variations in particle size and packing density, soil samples were fed into four replicate wells, each of which was scanned 32 times (figure 2). the four spectra were then averaged. according to the afsis technique, only 60 samples (about 10% of the 530 samples collected as advised by afsis) were subjected to wet chemical analysis (vagen et al 2010). this indicates that the 60 samples underwent two analyses: wet chemistry and scanning. the pls (partial least squares regression) model, which combines the two data types for the prediction, was used to assess the prediction quality (naes et al, 2002). the quality of the prediction was assessed using the root mean square deviation (rmsd) and coefficient of regression (r2) for the measured and estimated values (tillmann, 2000; brown et al., 2005). r software version 2.7.1 was used for the computations and statistical analysis (r development core team, 2008). the strong r2 and low rmsd indicate that the calibration and prediction were successful; the estimated values are more in line with the actual values determined by direct wet chemistry. good forecasts 3 are regarded as having an r2 ≥ 0.75 for such a diversified data set (shepherd and walsh, 2002; chang et al., 2001). predictions with an r2 between 0.65 and 0.75 are deemed satisfactory, while those with a lower value are deemed bad (shepherd and walsh, 2002; chang et al., 2001; terhoeven et al, 2010). results and discussion the soil parameters prediction soil organic carbon soil organic c was well predicted for the validation set (r2= 0.72 and rmse = 1.07; (figure 4.1. ). this shows that the prediction for this parameter was good. this finding agrees with other soil studies (barthe et al. 2008; ludwig et al. 2008). the results also agree with those of rossel et al. (2006) who reported similar accuracy (r2 = 0.73) for a much less diverse validation set of 118 samples from 18 ha agricultural field in australia. similarly, from a global study, terhoeven et al. (2010) reported an r2= of 0.8 soil organic carbon (soc) is a key attribute of soil quality, which influences a variety of biological, chemical and physical properties of soils (carter, 2002). consequently, methods to accurately determine soc are necessary to achieve a better understanding of the nature and dynamics of soc (denm8ef et al., 2009). soil organic carbon is an indication of soil organic matter content, which acts as both a source and sinks for nutrients. soil organic carbon is linked to soil chemical, physical and biological health, and is strongly correlated with soil nitrogen supply in term of soil fertility management. the satisfactory prediction of soc (r2<0.75) helps to monitor the soil status from the study area for a sustainable soil management. a study in canada reported good prediction (r2=0.92) (madari et al., 2006) than the current study. however, according to shepherd and walsh (2002) and chang et al. (2001) predictions of r2 ranging from 0.65 to 0.75) are satisfactory. extractable aluminum extractable extractable aluminum was also well predicted t (r2= 0.71 and rmse = 1.07 (figure 4.2). 4 figure 4.1: correlation between measured and predicted values of ca:mg ratio figure 4.2: correlation between measured and predicted values of ca:mg ratio based on the given prediction criteria, this indicates a good forecast of this parameter (terhoeven et al., 2010). an r2 of 0.71 was obtained by several investigations (brusetal, 2002; borggaard et al., 2004). but according to madari et al. (2006), who used canadian soils, their results showed a superior prediction (r2=88) than the current study that may otherwise be lost from the soil rooting depth. better phosphorus control will be encouraged by less expensive methods of measuring iron and aluminum oxide concentration. lessen the pollution caused by nutrients. the mir's prediction of this parameter opens up a new area for soil fertility monitoring in the drc's eastern highlands. calcium: magnesium ratio the coefficient of correlation between actual measurements and predictions by pls model for ca: mg 5 figure4.3: correlation between measured and predicted values of total n ratio using the independent validation set of 60 samples was r2 of 0.81 (figure 4.3). compared to the results obtained in australia (minasny et al., 2009), which showed that mir cannot accurately predict the ca:mg from that region, the current study provided a better prediction (r2=0.81). however, dunn et al. (2002) found that the 178 samples they collected from canada showed the currency of ca/mg ratio prediction (r2=0.81), which is consistent with the current findings. exactable calcium the r2 values and rmse indicated that the extractable calcium prediction was good (figure 4.4). the model for the soil ca analysis was validated since the coefficient of regression was high (r2 = 0.93), indicating a solid prediction of the parameter. this indicates that based on the successful model prediction, ca, which was predicted in the current study, may be employed appropriately for evaluating soil aptitude. the study's validation predictions for ca are superior to those of other authors' findings. for instance, mccarty and reeves (2006) reported an r2 of 0.77 for extractable calcium in a study conducted in the usa, which is still less than the current study. similarly, r2 = 0.84 was obtained for prediction values by viscarra et al. (2006) and mccarty and reeves (2006). additionally, the model created in this study produced superior results than others published worldwide, such as terhoeven et al. (2010), which reported r2 = 0.61 with an rmse of 1.64. the current study's accurate ca prediction helps with soil nutrient evaluation and enables the recommendation of using an ir scanning approach for this element. total nitrogen (tn) in terms of the r2 and the rmse, total nitrogen provided a decent prediction (figure 4.5). there is a good correlation between the direct and anticipated values, as indicated by the regression coefficient of 0.76. this accurate tn prediction opens up a new way to quickly evaluate this soil element. the most crucial soil nutrient for agricultural development in ssa can now be analyzed, which opens up new avenues for soil nutrient assessment. these findings are consistent with those of other writers. for instance, minasny et al. (2009) found that the same ratio (r2=0.76) was found in a study that was done on australian soils. the similar value of r2=0.76 was reported by reeves et al. (2006), cozzolino and moro (2006), barthe et al. (2008), and vasques et al. (2009) in research conducted in areas with significant agroecological variability. however, given the prediction is good (r2>0.75) by the criteria, the accuracy is still good. this indicates that the prediction's accuracy is also influenced by the region's soil variability. consequently, the fact that the soil samples utilized in the canadian investigation came from the same region and belonged to the same soil type may have contributed to the high calibration performance that was noted (reeves, 2006). 6 figure 4.4: correlation between measured and predicted values of ph figure 4.7: correlation between measured and predicted values of extractable k soil ph with r2 = 0.80 and rmse = 2.21, the ph of the soil was accurately predicted (figure 4.6). these results are consistent with those of mccarty and reeves (2006), who used mir analysis to analyze 544 soil samples from 272 locations in a single field in the united states (r2 = 0.8). similarly, terhoeven et al. (2010) found that their research of the australian landscape yielded an rmse of 0.75 and an r2 of 0.8 worldwide. the biggest issue with land degradation in the drc is soil acidity, which is currently reducing agricultural output. the management of soil acidity will be substantially aided by methods that support the measurement of soil ph, the calculation of the rate of lime needed to reach an acceptable ph, and the quality of lime products. janik and skjemstad (1995) reported an r2 of value 0.72 (acceptable prediction) for 291 australian soils, which is marginally better than the results of the current study. according to reeves (2009), additional elements including organic acids and carbonates affect infrared's capacity to forecast ph. these results of satisfactory prediction suggest that mir spectroscopy may be able to forecast this parameter. extractable potassium extractable potassium was well predicted with the regression model having r2=0.87 (figure 4.7). 7 figure 4.8: correlation between measured and predicted values of extractable p figure 4.9: correlation between measured and predicted values of cec 8 these findings demonstrate how well potassium may be predicted spatially within the research area. the model is helpful for this parameter because of its accurate prediction of k (shepherd and walsh, 2002; chang et al., 2001). this is in line with the findings of hartemink (2006) and lark (2009), who discovered that 178 samples from the australian landscape had good k predictions. the possibility of evaluating the soil condition in the study area using the estimated values for k is made possible by the prediction's accuracy. extractable phosphorus in this investigation, extractable p was accurately predicted with r2=0.89 (figure 4.8). this suggests that the model's p determination is validated. these findings are in good agreement with those of a study done on australian soils by minasny et al. (2009). they also concur with those of sellitto et al. (2009) from india, who reported an r2 of 0.90, and hartemink (2006) and madari et al. (2006), who reported values of r2 = 0.85 and 0.93, respectively, from the canadian landscape. although hartemink (2006) showed a satisfactory prediction of this parameter with an r2 of 0.57, several canadian studies have reported poor predictions, such as those made by madari et al. (2006) from the canadian landscape and sellitto et al. (2009) from india. this excellent modeling of p in the eastern democratic republic of the congo creates a new avenue for evaluating this crucial crop production nutrient. cation exchangeable capacity this method's validation for cec yielded an r2 value of 0.84, suggesting that cec was accurately predicted (figure 4.8). several research (dunn et al., 2002; sellitto et al., 2009; richter et al., 2009) have shown that mir offers accurate forecasts for cec in various locations, such as india, canada, and the us, respectively. similar prediction accuracy for this measure was demonstrated by pirie et al. (2005) for 415 samples from southeast australia. in the current study area, the r2 value for cec is 0.84 (figure.4.9), indicating that the direct and estimated values for this parameter are closer to the expected values. similar to pirie et al. (2005) from soils in south australia, sellitto et al. (2009) from india likewise obtained strong predictions for this parameter. the soil science research can bypass many of the limitations of traditional laboratory analysis because to this excellent modeling of cec. soil physical particles prediction particle size effects on light transmission and reflection, and strong absorption features exhibited by water, explain the accurate predictions for texture (cécillon et al, 2009; chang et al., 2001). predictions for particle size were satisfactory for clay (r2 = 0.74 and rmes = 1.05); sand (r2;= 0.81 and rmes = 1.08) and silt (r2 = 0.84 and rmes = 1.06). these results are broadly similar to those of previous researchers (mccarty and reeves, 2006; pirie et al., 2005). terhoeven et al, 2010 got similar results globally (clay (r2 = 0.73 and rmes = 1.85). all these tree physical parameters respond to the calibration requirement (chang et al., 2001; shepherd and walsh, 2002; terhoeven et al, 2010) and validation of the results is correct. conclusion this study sought to determine whether a new method of soil characterization based on the infrared scanning (ir) technology might provide quick and accurate soil property quantification. overall, the results demonstrated a good and satisfactory prediction model with above 75% for both the physical features of the soil and all of the chemical parameters under investigation (ph, al, p, n, ca, k, psi, exchangeable acidity, and cec). phosphorus had the best prediction of any of the factors studied (r2=0.93), while all other parameters had r2 values greater than 0.75, with the exception of som and tn, which had r2 values of 0.73 and 0.71, respectively. it is suggested that the ir scanning method be used to determine the qualities of soil because it predicts all chemical parameters well. the approach is also quick and cost-effective. it took two days to scan the 530 samples, when using traditional soil analysis techniques could have lasted over two months. with r2 values of 0.81, 0.84, and 0.74 for sand, silt, and clay, respectively, the prediction of soil particles was likewise good for all three parameters. this suggests that the infrared scanning method is a trustworthy way to determine the texture of soil. this method is still relatively new, though, and few scientists have been able to apply it. even at a low level, this approach cannot be employed without the combination of wet chemistry results. to monitor soil and land degradation in underdeveloped nations, further work is required to enhance the application of infrared scanning. ir demands a large initial expenditure, despite being inexpensive for assessing soil parameters. therefore, in order to make this technology usable in underdeveloped nations, technical and material help is required. references barthe bg, brunet d, hien e, enjalric f, conche s, freschet gt, d’annunzio r, toucet-louri j (2008). determining the distributions of soil carbon and nitrogen in particle size fractions using nearinfrared reflectance spectrum of bulk soil samples. soil biol. biochem, 40: 15331537. brown dj, bricklemyer rs, miller pr(2005). validation requirements for diffuse reflectance soil characterization models with a case study of vnir soil c prediction in montana. geoderma, 129: 251–267. 9 brus dj, de gruijter jj, breeuwsma a(2002). strategies for updating soil surveyinformation, a case study to estimate phosphate sorption characteristics. journal of soil science 43, 567–581. carter mr(2002). soil quality for sustainable land management: organic matter and aggregation interactions that maintain soil functions. agron. j., 94: 3 8-47. cécillon l, cassagne n, czarnes s, gros r, vennetier m, brun jj(2009). predicting soil quality indices with near infrared analysis in a wildfire chronosequence. science of the total environment, 407: 1200-1205. chang cw, laird da, mausbach mj, hurburgh cr(2001). near infrared reflectance spectroscopy-principal components regression analyses of soil properties. soil science society of america journal, 65: 480–490. christy cd(2009). real-time measurement of soil attributes using on the-go near infrared reflectance spectroscopy. computers and electronics in agriculture, 61:10-19. cozzolino d, moro´n a(2006). potential of near-infrared reflectance spectroscopy and chemometrics to predict soil organic carbon fractions. soil tillage res, 85: 78_85. denef k, plante af, six j(2009). characterization of soil organic matter. pages 91_126 in w. kutsch, m. bahn and a. heinemeyer, eds. soil carbon dynamics: an integrated methodology. cambridge university press, london, uk. dsrp. (2005). document de strategie de reduction de la pauvrete. province du sud-kivu. republique démocratique du congo, ministère du plan, unité de pilotage de processus du dsrp, kinshasa. p96. dunn bw, beecher hg, batten gd, ciavarella s(2002).thepotentialofnear-infraredreflectance spectroscopy for soil analysis a case study from south-eastern australia. australian journal of experimental agriculture, 42, 607-614. farrow a, businye l, bugenze p(2006). characterisation of mandate areas for consortium for improving agriculture based livelihoods in central africa: pp 138. hartemink ae(2006). assessing soil fertility decline in the tropics using soil chemical data. advances in agronomy, 89: 179–225. icraf. 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journal of food science research issn 2375-0723 vol. 6 (10), pp. 382-393, october, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of the effect of some environmental parameters on the level of bacteriocin activity rowaida khalil*, fatima djadouni, yasser elbahloul and sanaa omar department of botany, faculty of science, alexandria university, alexandria, egypt. accepted 22 december, 2018 bacillus megaterium 22, a soil isolate, produced a bacteriocin that exhibited a broad range of inhibitory activity against food-spoilage microorganisms including salmonella typhimurium and staphylococcus aureus. the antimicrobial activity peaked at the early stationary phase. de man rogosa shrarpe (mrs) was the best medium for bacteriocin production, where growth of b. megaterium 22 for 12 18 h at 30 o c, ph 6 6.5 resulted in maximum inhibitory effect on the pathogenic indicator strains. supplemen-tation and/or replacement of medium nutrients demonstrated higher values of bacteriocin activity in the presence of 5 10% sucrose, 1% beef extract, and under limited aeration. bacteriocin activity was significantly stimulated at concentrations of up to 3% nacl, or 1% kcl. low levels of spices (curry, red and black pepper) synergistically stimulated the bacteriocin activity, except for garlic and rosemary where higher concentrations (1%) considerably influenced the activity. the bacteriocin was heat stable for 15 min of exposure to a wide range of temperatures, and over a ph range of 2 8 after 1 h of expo-sure. the bacteriocin was stable for up to 30 min of exposure to uv light, and when stored at 4 o c for 90 days. the activity was inhibited by proteolytic enzymes and tested organic solvents. sds-page revealed that the apparent molecular weight of the partially purified bacteriocin ranged from 3.496 to 6.512 kda. results presented here support the idea that the bacteriocin may propose some industrial advantages that render it as a good natural food bio preservative candidate. key words: bacillus megaterium, antimicrobial activity, bacteriocin, biopreservation, growth media, spices, storage, indicator strains. introduction bacteriocins are antimicrobial ribosomally synthesized peptides produced by bacteria that inhibit or kill microorganisms that are usually, but not always, closely rela-ted to the producer strain (sánchez-hidalgo et al., 2008). also include the original (first report) reference here and not just the latest one. different species of bacillus pro-duce bacteriocins, and within a species either several different kinds of bacteriocins (motta and brandelli, 2008) or bacteriocin-like substances (bls) (motta et al., 2008) *corresponding author. email: rowaida_georgia1@hotmail.com. tel: 002-3921595. fax: 002 3911794. may be produced with varying modes of action. bacteriocins produced by “food grade” lactic acid bacteria (lab) have long been the focus of exten-sive studies with the perspective of their potential and effective use and application as nontoxic natural food biopreservatives for the food industry and therapeutic agents for gastrointestinal infections (haugen et al., 2008). bacillus is an interesting genus to investigate for antimicrobial activity since bacillus species produce a diverse array of anti-microbial peptides representing several different basic chemical structures (bizani and brandelli, 2002), with a distinct diversity in their inhibitory activities against a va-riety of micro-organisms (korenblum et al., 2005). bacteriocins have been studied in different species including: bacillus subtilis, bacillus cereus, khalil et al. 382 bacillus stearothermophilus, ba-cillus licheniformis, bacillus thuringiensis , and other bacillus species (pattnaik et al., 2001). the best-charac-terized bacteriocin was thuricin produced by strains of b. thuringiensis (ahren et al., 2003). other bacteriocins have been reported such as lichenin which was produced by b. licheniformis 26-103ra strain (pattnaik et al., 2001), megacin produced by strains of b. megaterium (lisboa et al., 2006), antilisterial coagulin produced by b. coagulans (le marrec et al., 2000), and cerein produced by strains of b. cereus, (naclerio et al., 1993). moreover, some of these bacteriocins have been indicated as po-tential biopreservatives in food systems and beverages, as agents for biological control of phyto-pathogens (bais et al., 2004), and as antibiotic precursors (zuber et al., 1993). however, limited data exists on the applications of bacteriocins from bacillus spp and in particular from members of b. megaterium. in the recent time, it was reported that some strains of b. megaterium produced several bacteriocins effective against other strains of b. megaterium (holland and roberts, 1964; von tersch and carlton, 1983). but as far as we could determine, the antimicrobial properties of b. megaterium has not been fully explored for use as biopreservative, largely due to limited production techniques (reddy et al., 1984). hence, the search for different bacilli strains pro-ducing new antimicrobial agents with wider spectrum of activity and compatibility with different food systems is still desirable for many processing systems. the biopreservation capacity of a bacteriocin could be achieved either by using a bacteriocin-producing starter culture or by applying the bacteriocin itself as a food additive. therefore, a thorough study of the essential parameters affecting the inhibitory activity (zala´n et al., 2005), followed by optimization of production that is usually dependent on multiple strainspecific factors (lealsa´nchez et al., 2002) is necessarily required for introducing a bac-teriocin into foods as a potent biopreservative. this raised the need and was the driving force for our present investigation, in which we report on a bacteriocin pro-duced by b. megaterium isolated from soils of local terri-tory in alexandria, egypt. in this approach, the effect of some environmental parameters on the level of bacte-riocin activity is evaluated. we ascribe the influence of cultural conditions such as nutrients, nutrient concentra-tion, nutrient combinations/interactions, aeration, and other physical factors including heat, uv, ph, storage conditions for the purpose of obtaining better and stable bacteriocin activity. materials and methods bacterial strains, inoculum preparation, and cultural conditions: the strain of b. megaterium isolated from soils of the local territory (native lands) in alexandria, egypt was identified according to on the basis of its cultural, the morphological and biochemical properties, physiological, biochemical characteristics, and carbohydrate fermentation tests (schillinger and lücke, 1987). this was followed by partial 16s rrna analysis (maidak et al., 1999) conducted at the german culture collection of microorganisms and cell cultures dsmz (deutch sammlung von mikroorganismen und zelkulturen gmbh). the strain was described as b. megaterium 22. the test microorganisms used in our study include: escherichia coli, pseudomonas aeruginosa, proteus vulgaris, klebsiella pneumoniae, salmonella typhimurium, salmonella para-typhimurium a, salmonella para-typhimurium b and enterococcus faecalis, streptococcus pyogenes, and staphylococcus aureus were supplied from the microbiology laboratory at the faculty of medicine, alexandria university, and were used to determine the antimicrobial spectrum of bacteriocin. s. typhimurium and s. aureus were chosen as the indicator strains in all the antimicrobial assays. bacterial strains were propagated in mrs broth (de man rogosa sharpe) (biolife italiana s.v.l), kept frozen in 20% (v/v) glycerol at 20 o c until needed and subcultured twice before use. unless otherwise stated, test strain inoculum (1% v/v) consisted of cellular suspensions from 12 18 h mrs cultures, incubated at 30 o c, adjusted to an absorbance (600 nm) of 1 1.2, while indicator strains inoculum were prepared similarly but monitored spectrophotometrically till an o.d.600 corresponding to 10 5 cfu/ml was reached (naclerio et al., 1993). bacteriocin preparation the cells of b. megaterium 22 were grown as 2% in mrs broth at 30 o c, collected after 12 h at the early stationary phase by centrifugation at 10,000 x g for 20 min at 4 o c. the cell free supernatant (cfs) was passed through membrane filters (renner gmbh d67125/ germany) with a pore diameter of 0.2 m, and stored in the refrigerator for a maximum period of two weeks and periodically tested for the bacteriocin titer before being renewed. antimicrobial spectrum the agar well diffusion (awd) assay was used to determine the antimicrobial spectrum of the test strain producing bacteriocin above mentioned (lasta et al., 2008). diameters of inhibition zones were scored (korenblum et al., 2005). detection of bacteriocin activity during growth (growth kinetics) mrs broth was inoculated with 2% (v/v) of an overnight pre-culture of the test strain, incubated at 30 o c, where changes in o.d.600 were recorded every 3 h. the growth kinetics experiment (vinderola et al., 2002) was performed with a minor modification. the indicator strains (10 5 cfu/ml) were grown at 30 o c in mrs broth in the presence of the test strain cfs. optical density measurements (odm) were recorded every 3 h for 12 18 h. the bacteriocin activity was expressed by the percentage of growth reduction to the indicator strains and determined from the ratio between the optical densities of the treated cultures and untreated ones (the indicator strains without the cfs). this odm method was used in all the antimicrobial assays the influence of growth conditions on bacteriocin activity the effect of growth media, incubation temperature, initial ph, and aeration on bacteriocin activity was carried out as follows: b. mega terium 22 was grown aerobically at 30 and 37 o c in five different me table 1. influence of inorganic salts and spices on the activity of b.megaterium 22 bacteriocin. results are % of mean values of activity ± standard deviations (n=3). inorganic salt indicator strains concentration (%) 0.0 0.5 1.0 3.0 nacl s. typhimurium 87.02.1 ± 0.3 86.8 ± 0.3 95.7 ± 1.3 96.7 ± 1.5 s. aureus 88.1 ± 1.2 88 ± 0.3 96.7 ± 1.2 93.6 ± 1.3 kcl s. typhimurium 87.02.1 ± 0.3 83.9 ± 0.6 92.7 ± 0.6 91 ± 1.5 s. aureus 88.1 ± 1.2 83.5 ± 1.5 92.2 ± 0.6 91.5 ± 1.2 mncl2 s. typhimurium 87.02.1 ± 0.3 79 ± 1.0 15 ± 0.8 6 ± 0.4 s. aureus 88.1 ± 1.2 76 ± 0.9 16 ± 0.7 3 ± 0.2 curry s. typhimurium 87.02.1 ± 0.3 93 ± 0.2 53 ± 0.1 46 ± 0.1 s. aureus 88.1 ± 1.2 94 ± 0.2 54 ± 0.1 46 ± 0.1 red pepper s. typhimurium 87.02.1 ± 0.3 84 ± 0.4 82 ± 0.4 84 ± 0.4 s. aureus 88.1 ± 1.2 83 ± 0.4 82 ± 0.4 84 ± 0.4 black pepper s. typhimurium 87.02.1 ± 0.3 79 ± 0.1 69 ± 0.2 64 ± 0.2 s. aureus 88.1 ± 1.2 79 ± 0.2 69 ± 0.2 63 ± 0.1 bastermy s. typhimurium 87.02.1 ± 0.3 44 ± 0.3 -103 ± 8.4 -114 ± 4.8 s. aureus 88.1 ± 1.2 43 ± 0.2 -92 ± 5.3 -163 ± 6.4 garlic s. typhimurium 87.02.1 ± 0.3 94 ± 0.3 98 ± 8.4 77 ± 4.8 s. aureus 88.1 ± 1.2 89 ± 0.2 96 ± 0.8 75 ± 6.4 rosemary s. typhimurium 87.02.1 ± 0.3 92 ± 2.0 50 ± 0.4 34 ± 0.3 s. aureus 88.1 ± 1.2 96 ± 1.2 42 ± 0.3 23 ± 0.2 paprika s. typhimurium 87.02.1 ± 0.3 28 ± 0.2 -33 ± 2.4 -74 ± 1.3 s. aureus 88.1 ± 1.2 16 ± 0.7 -42 ± 0.3 -53 ± 1.2 dia, mrs (de man rogosa sharpe) broth, brain heart infusion (bhi; merck, darmstadt, germany) broth, m17 (difco laboratories, detroit, mi) broth, whey and molasses (2% v/v). aliquots of mrs broth were adjusted with 1 n hci or 1 n naoh to ph values of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 (ougnbanwo et al., 2003a), autoclaved, and inoculated with the test strain. the effect of aeration conditions on bacteriocin activity was studied by varying the volume of the growth medium where aliquots of 10, 20, 40 and 50 ml were inoculated with the test strain. all preparations with different conditions were propagated under the same conditions, and assayed for bacteriocin activity as described above. influence of medium components on bacteriocin activity the effect of medium ingredients on bacteriocin production was evaluated using modified mrs broth. the supplements studied were: 2 and 4% of monosaccharide (galactose) and disaccharide (fructose, lactose, maltose, and sucrose) sugars respectively, after replacing the medium sugar glucose (ogunbanwo et al., 2003b). subsequently the effect of higher sucrose concentrations, up to 10% (w/v) was studied. the effect of nitrogen sources on bacteriocin activity was also evaluated using (/lit): 4 g ammonium acetate, 4 g ammonium chloride, 4 g ammonium nitrate, 4 g ammonium sulfate, 4 g arginine, 10 g beef extract, 4 g sodium nitrate, 10 g tryptone, and 5 g yeast extract. the tested combinations of nitrogen (1.5 and 0.5%) included: beef extract plus yeast extract, beef extract plus tryptone, beef extract plus ammonium chloride, beef extract plus arginine. the activity of the cfs from each culture condition was assayed. the influence of some inorganic salts and spices on bacteriocin activity the inhibitory effect of inorganic salts (kcl, mncl2, and nacl) on bacteriocin activity was tested (karao lu et al., 2003) by mixing concentrations of 0.5, 1, and 3% of each salt to the cfs preparations for 2 h. spices (table 1) used as local food additives were also studied for their possibility of influencing the effectiveness of the bacteriocin activity (verluyten et al., 2004). each spice was dissolved in 10 ml of sterile warm distilled water, vortexed for 5 min, followed by centrifugation, filter-sterilization, and mixed for 2 h with the cfss to get a final concentration of 0.5, 1, and 3% (v/v). salts and spices-treated preparations were assayed for antimicrobial activity as previously described. the influence of heat, uv, ph, storage on bacteriocin activity for heat treatment, the cfs preparations were heated for 15 min at 30, 40, 50, 60, 70, 80, 90, 100 and 121°c (mota et al., 2004). similarly, sterile petri dishes containing 10 ml aliquots of crude bacteriocin preparations were exposed from 15 to 90 min to uv irradiation (philips bulb, wave length 340 nm, 220 240 v, 50 hz,) situated 30 cm distance from the petri dishes (wanda and bonita, 1991; ogunbanwo et al., 2003a). the effect of ph on activity was tested by adjusting the cfss to ph values from 2 to 12 (at one unit increments) with sterile 1 n naoh or hcl (albano et al., 2007). samples were incubated at ambient temperature (~25 o c) for 1 h. the crude bacteriocin was stored at –20 and 4ëc for different intervals of time (30, 45, and 60 days). the activity in all prepara 383 afr. j. food sci. res. 90 2.0 1.8 70 1.6 (% ) 1.4 60 0n m o f g r o w t h 50 1.2 r ed uc ti on 1.0 o d 0.6 30 0.8 10 0.4 0.2 -10 0.0 0 3 6 9 12 15 18 21 24 time (h) s. typhim urium s.aureus b. megaterium 22 figure 1. bacterial growth ( ) and bacteriocin activity of b. megaterium 22. the strain was grown aerobically in mrs broth at optimal conditions of temperature (30°c), for 24 h. activity of bacte riocin preparations against s. typhimurium ( ) and s. aureus ( ) was expressed as % reduction of growth. each point represents the mean ± s.e.m. of three independent experiments. tions and aliquots was determined as previously mentioned. the influence of proteolytic, non-proteolytic enzymes and solvents on bacteriocin activity proteolytic enzymes (oxford laboratory reagents) including papain, pepsin, trypsin, and non-proteolytic enzymes (lipase and amylase) were dissolved in 0.05 m sodium phosphate (ph 7.0), 0.002 m hcl (ph 7), 40 mm tris-hcl (ph 8.2), 0.1 m potassium phosphate (ph 6.0), and 0.1 m potassium phosphate (ph 7.0) respectively to a final concentration of 1 and 2 mg/ml. enzyme solutions were filter sterilized, mixed with aliquots of filter sterilized cfss of the test strain, incubated at 30 o c for 2 h, subsequently heated in boiling water for 5 min to inactivate the enzymes (bizani and brandelli, 2002), and assayed for antimicrobial activity. the sensitivity of freeze dried bacteriocin preparations to organic solvents such as acetone, chloroform, ethyl alcohol, hexane, and methanol was investigated (todorov et al., 2006) by being dissolved in each organic solvent to a final concentration of 10 mg/ml. samples were incubated at 30°c for 1 h, solvents were removed by evaporation, and dried residues from the organic phase were re-suspended in sterile mrs broth (ten brink et al., 1994) and assayed for antimicrobial activity partial purification and molecular weight determination the test strain was grown in mrs broth for 10 h at 30 o c. cells were harvested by centrifugation at 10,000 x g for 20 min at 4 o c, after which the bacteriocin was precipitated from the cfs with 45% saturated ammonium sulfate (akyptis et al., 1998). after 4 h of stirring at room temperature, proteins were removed by centrifugation, and dissolved in 20 mm tris-hcl buffer (ph 7). the mixture was then dialysed using a spectra/por membrane tubings (spectrum laboratories inc., ca, usa) of 12 kda cut off against 2 l of distilled water for 24 h at 4°c with at least 3 changes. the dialyzed preparation was lyophilized resulting in the formation of a dry precipitated residue referred to as the partially purified bacteriocin. its molecular weight was estimated as described by sambrook and russell (2001) using discontinuous sds-page (11%) performed using a double slab electrophoresis cell (cleaver scientific ltd). the molecular mass was calculated by comparison with the mobility of standard markers (bio-rad, germany) ranging from 90 1.434 kda. statistical analysis data were expressed as mean ± standard deviation. statistical significance was determined using one-way analysis of variance on the replicates, where a p-value of 0.05 was considered significant. results antimicrobial spectrum the cfs of b. megaterium 22 contained an antimicrobial compound with a wide spectrum active against representtatives of three gram -positive and gramnegative pathogenic strains (e. coli, k. pneumoniae , s. aureus, and s. typhimurium). the average diameter of inhibition zones as determined by the awd method ranged from 0.5 5 mm in size (data not shown). it is worth mentioning that the producing strain was not inhibited by its own bacteriocin. growth kinetics figure 1 depicts high bacteriocin activity against s. aureus at zero time, and during the first three hours of incubation against both indicator strains (at least 60% growth reduction). maximal antibacterial activity was achieved at the early stationary phase after 12 15 h, after which the growth started to decline gradually as determined by culture turbidity and bacteriocin activity, where no activity was recorded after 24 h of experimentation. the influence of growth conditions on bacteriocin activity in general, the best results corresponding to the highest inhibitory effect on the indicator strains were obtained using mrs broth after 12 18 h of incubation at 30 o c, followed by m17, finally by using whey (figure 2). good antimicrobial activity was recorded in the presence of 2% (w/v) molasses after 12 18 h of incubation. bhi medium was not suitable for bacteriocin production. b. megaterium bacteriocin exhibited lower activity against the indicator strains upon increasing the incubation temperature when using mrs broth as the growth medium. however, growth particularly in whey at 37 o c resulted in an excep khalil et al. 384 385 afr. j. food sci. res. a 150 100 % ) 50 o w t h ( 0 f g r 3 6 9 1215182124 n o -50 d u c t io -100 r e -150 -200 time (h) m17 bhi mrs molasses whey b 150 100 ) 50 (% 0 r o w t h -50 3 6 9 1215182124 o f g -100 ti o n -150 r e d u c -200 -250 -300 time (h) m17 bhi mrs molasses whey figure 2. influence of growth media on the bacteriocin production of b. megaterium 22 against the indicator strains s. typhimurium (a) and s. aureus (b) when grown in mrs broth at 30°c, ph: 6.2 – 6.5. results are expressed as % of mean values of activity (n=3) ± stan dard deviations. tion to this trend (data not shown). ph 6 6.5 fostered the maximum bacteriocin activity (almost 95% growth reduction) against the indicator strains, followed by ph 5, after growth for 12 18 h in mrs broth (figure 3). the acidic and the alkaline ph values 4.5 and 7.5 had significant adverse effects (p>0.05) on decreasing the bacteriocin activity. the strain proved to produce bacte 100 90 a 80 70 60 50 40 3 6 9 12 15 18 21 time (h) ph (4.5) ph (5) ph (5.5) ph (6) ph (6.5) ph (7) ph (7.5) 100 b 90 80 70 60 50 40 9 3 6 12 15 18 21 time (h) ph (4.5) ph (5) ph (5.5) ph (6) ph (6.5) ph (7) ph (7.5) figure 3. effect of the initial ph on bacteriocin production of b. megaterium 22 against s. typhimurium (a) and s. aureus (b) when grown in mrs broth at 30ëc. results are expressed as % of mean values of activity (n=3) ± standard deviations. riocin under limited or reduced aeration in the medium, where the highest bacteriocin activity (91 92% growth reduction) was attained in presence of 40 ml of the growth medium compared to the activity in presence of lesser volumes (data not shown). the influence of medium components on bacteriocin activity the best alternative sugar to glucose that yielded high bacteriocin activity was sucrose followed by maltose, fructose, then finally lactose and galactose after 15 18 h of incubation at 30 o c (figure 4). based on displaying the most significant effect on bacteriocin activity, different khalil et al. 386 a 100 80 r e d u c t i o n o f g r o w t h ( % ) 60 40 20 0 glucose lactose fructose maltose sucrose galactose 15 h 18 h 24 h b 100 80 r e d u c ti o n o f g r o w t h ( % ) 60 40 20 0 glucose lactose fructose maltose sucrose galactose 15 h 18 h 24 h figure 4. influence of different carbon sources (g/l) on the bacteriocin production of b. megaterium 22 against the indicator strains s. typhimurium (a) and s. aureus (b) when grown in mrs broth at 30°c, ph: 6.5. results are expressed as % of mean values of activity (n=3) ± standard deviations. concentrations of sucrose were tested. increasing the concentration to 5, 7, and 10% corresponded to an outstanding level of bacteriocin activity (approximately 100% growth reduction) after 12 15 h of incubation (figure 5). alteration in the nitrogen source of the growth medium had a significant visible effect (p<0.001) on promoting the bacteriocin antimicrobial activity. the highest inhibitory effect on the indicator strains were obtained after 15 24 h of growth in mrs broth supplemented with beef extract (1.0%) as the sole nitrogen source. no appreciable acti 387 afr. j. food sci. res. 150 100 a 50 t h ( % ) 0 r o w 0 3 6 9 12 15 18 21 24 f g -50 o ction-100 r e d u -150 -200 -250 time (h) 10 20 30 50 70 100 150 100 b t h ( % ) 50 0 r o w 0 3 6 9 12 15 18 21 24 f g -50 c t i o n o -100 r e d u -150 -200 -250 time (h) 10 20 30 50 70 100 figure 5. effect of different sucrose concentrations (g/l) on the bacteriocin production of b. megaterium 22 against s. typhimurium (a) and s. aureus (b) when grown in mrs broth at 30°c, ph: 6.5. vity was detected in presence of yeast extract (0.5%) or the rest of the nitrogen sources tested (data not shown). however, yeast extract and beef extract were the favourred combination of organic nitrogen compounds correlated with almost 98% growth reduction to the indicator strains (data not shown). the influence of inorganic salts and spices on bacteriocin activity statistically, high level bacteriocin activity was determined in nacl concentrations up to 3% (table 1). using a concentration of 1% kcl yielded high bacteriocin activity (92.7% growth reduction), whereas concentration of 3% resulted in an opposite behavior. on the other hand, the bacteriocin activity declined significantly ( p>0.05) upon treating the cfss with concentrations of mncl2 above 0.5%. spices used in this experiment (curry, red pepper, black pepper, and bastermy sheath) in concentrations ranging from 0.5 3% affected the bacteriocin activity in different patterns. the addition of 0.5% curry to the bacteriocin preparations significantly improved the activity against the indicator strains (up to 94% reduction of growth) compared to higher concentrations used (1 or 3%), where a sharp decline in activity was noticed. concentration of 3% of red pepper had a less pronounced effect on decreasing the bacteriocin activity compared to similar concentration of black pepper. low values of growth reduction percentages (43 44%) were recorded upon treatment with 0.5% of bastermy sheath, and hardly any measurable antagonistic activity was detected when higher concentration was used. conversely, the addition of either 1% garlic or rosemary to the bacteriocin preparations resulted in a significant activity stimulation, where 93 96% growth reduction to the indicator strains was observed. paprika had the most profound negative effect on the bacteriocin activity, which was more than halfed compared to that of the untreated preparations. the influence of heat, uv, ph, and storage table 2 summarizes the results obtained for different physical treatments of b. megaterium 22 cfss. the antimicrobial substance was stable for 15 min of exposure to use all temperatures, but lost its activity after being autoclaved at 121 o c. the bacteriocin maintained its stability up to 30 min of exposure to uv light, however exposure to longer periods resulted in significant decrease in growth reduction values to almost half. uv-treated cfss showed an overall high antagonistic activity against s. typhimurium compared to s. aureus. the bacteriocin was active over a ph range of 2 8 after 1 h of exposure, but was totally inhibited in the alkaline range. the bacteriocin was resistant to cooling storage for 90 days, where maximum growth reduction of the indicator strains was recorded (88%). freezing storage (-20 o c) negatively influenced the bacteriocin activity after 30 days of exposure, where weak to moderate growth reduction was detected against s. typhimurium and s. aureus (35 and 65% respectively). the bacteriocin was markedly distorted upon prolonged freezing storage, where no detectable activity was recorded. sensitivity to proteolytic and other enzymes, and solvents the antimicrobial activity against the indicator strains was khalil et al. 388 table 2. effect of thermal, uv light, ph, and storage temperature treatments on bacteriocin activity against (a) s. typhimurium and (b) s. aureus. results are expressed as % of mean values of growth reduction (n=3). treatment growth reduction (%) temperature a b 0ëc/15 min 70 83 30ëc/15 min 70 87 40ëc/15 min 68 85 50ëc/15 min 75 78 60ëc/15 min 78 79 70ëc/15 min 71 81 80ëc/15 min 73 80 90ëc/15 min 69 81 100ëc/15 min 71 81 121ëc/15 min -168 -284 exposure to uv light 15 min 79.5 11 30 min 72 22.5 60 min 31.5 21 90 min 26.5 7 ph 2 56.2 38.1 3 36.8 44.79 4 56.2 54.7 5 56.9 54.0 6 63.9 23.02 7 26 54.7 8 33 48.0 9 -34 -95.4 10 -39 -65.6 11 -400 -41.1 12 -400 -66.2 storage at 4 o c 30 79 88 45 76 74 90 77 76 storage at -20 o c 30 35 65 45 5 2 90 0 -1 sensitive to the tested concentrations of proteolytic enzymes (table 3) except for the non-proteolytic enzymes amylase and lipase when used at low concentrations (1 mg/ml). treatment with organic solvents (acetone, chloroform, ethanol, hexane, and methanol) led to total inactivation of the antimicrobial substance produced by b. megaterium 22 strain (data not shown). figure 6. sds-page electrophoresis of the partially purified bacteriocin of b. megaterium 22. lane a: coomassie brilliant blue-stained gel with small and large molecular weights of standard markers, lane b: single band of partially-purified bacteriocin. markers from top to bottom included: bovine serum albumin (e. coli), ovalbumin (chicken egg), carbonic anhydrase (bovine erythrocytes), -lactoglobilin (bovine milk), lyzozyme (chicken egg white), -lactalbumin, aprotinin, insulin drain oxidized, and bacitracin. sizes on the left are indicated in kda. the determination of bacteriocin molecular weight sds-page separation indicated that the bacteriocin peptide size ranged from 3.496 to 6.512 kda (figure 6). discussion a bacteriocin-producing bacterium was isolated from soil samples of native lands of alexandria, egypt. the strain was identified according to biochemical characteristics and by partial 16s rrna analysis and it was described as b. megaterium 22. the maximum production and antibacterial activity of the bacteriocin against the indicator strains s. typhimurium and s. aureus was found to be at the early stationary phase, after 12 h of growth in mrs broth indicating that the antimicrobial peptide is a seconddary metabolite (lisboa et al., 2006), a character also confirmed by its low molecular weight (abada, 2008). naclerio et al. (1993) similarly reported that the production and activity of cerein produced by b. cereus was recorded at the stationary growth. conversely, cherif et al., (2001) reported that thuricin 7 was produced by species of b. thuringiensis, and was expressed in the expo 389 afr. j. food sci. res. table 3. effect of enzyme treatment and concentration on bacteriocin activity against (a) s. typhimurium and (b) s. aureus. results are expressed as % of mean values of growth reduction. enzyme concentration 1 mg/ml 2 mg/ml growth reduction (%) enzyme a b a b papain -335 ± 47 -212 ± 17 -101.3 ± 19.9 -115 ± 90 pepsin -28 ± 11 -85 ± 13 -107.0 ± 13.1 -123 ± 50 trypsin -259 ± 94 -154 ± 17 -109.5 ± 7.1 -120 ± 40 -amylase 34 ± 11 11 ± 20 -118.1 ± 0.7 -119 ± 0.3 lipase 27 ± 80 13 ± 20 -122.8 ± 7.6 -125 ± 80 (n=3) ± standard deviations. nential growth phase. b. megaterium 22 bacteriocin exhibited a wide antimicrobial spectrum, and was capable of inhibiting the growth of some tested microorganisms both gram-positive and gram-negative. korenblum et al. (2005) described a fairly similar result in 90% of their isolated bacilli strains. according to biswas et al. (1991), modification of cultivation media nutrients should be considered for maximal production of bacteriocin that may have a potential use as food biopreservatives. hence, diverse set of experiments were designed to deduce the best available cultural condition(s) which could stimulate the activity of b. megaterium 22 bacteriocin. although bacteriocin production is often performed in complex media, which promote abundant growth and relatively high bacteriocin levels, it seemed more economical to test the use of some other media to determine the influence of their components on bacteriocin activity. our results show that the best medium for bacteriocin activity was mrs broth, whereas low production levels were recorded in bhi broth, molasses, and whey, which suggests that specific nutrients are required for bacteriocin production. this result was consistent with that found by de kwaadsteniet et al. (2005) where the highest activity of bacteriocin st15 produced by e. mundtii st15 was recorded after 14 h of growth in mrs broth at 30ëc. bizani and brandelli (2002) deduced that the relationship between growth and specific production rates, as a function of the temperature, showed different kinetics of production, where bacteriocin production from b. cereus 8a, was higher at 30°c than at 25°c and at 37°c. this observation was inagreement with our results, where the bacteriocin activity against the indicator strains was in general more apparent at 30 than at 37ëc. the maximum value of bacteriocin activity (95% growth reduction) was recorded when b. megaterium 22 was grown for 12 h in mrs broth adjusted to initial ph value of 6.5, while lower levels of activity (45 65% growth reduction) were recorded at initial ph of 4.5. this was in accordance with observations of todorov et al. (2006) and todorov and dicks (2007), where ph 5.0 and 4.5 repressed the activity of bachv219 and bacst712bz respectively. variation in supplementation of medium/concentration of constituents had a positive influence on bacteriocin activity (ogunbanwo et al., 2003a), where mrs broth supplemented with 2% sucrose released the best result and the highest inhibitory activity against the indicator strains. the significance of this finding lies in the low cost of sucrose compared to glucose, which could be employed as an economic ingredient in bacteriocin production media. todorov et al. (2006) reported that yeast extract is the most effective organic nitrogen compound for bachv219 production. surprisingly, this finding was not in coherent with our data, where beef extract was the favoured nitrogen source and resulted in accelerating the activity of b. megaterium 19 bacteriocin. the bacteriocin was produced under reduced oxygen level in the me-dium, a finding supported by the results of jurgen et al. (2003) which indicated that oxidative stress caused a higher level of bacteriocin production from l. curvatus lth 1174. the interaction between some food ingre-dients such as inorganic salts and spices with the determined bacteriocin was studied. concentrations of nacl and kcl up to 3% stimulated the bacteriocin activity, whereas concentrations of mgcl2 above 0.5% exerted the opposite effect. this could be reasoned to the ability of monovalent cations to mediate the adsorption of bacte-riocin to the indicator strain; while the divalent ions may mask such effect. moreover, in some cases osmotic stresses appear to favour the bacteriocin release. the response of bacteriocin activity towards the inorganic salts may suggest their synergistic effect when added with specific concentrations in foods. leroy and de vuyst (1999) reported that nacl and nan02 can inhibit the bacteriocinogenic effect of bacteriocin-producing cultures in a meat system. spices and herbs are used for imparting desirable sensory properties to fermented food products (verluyten et al., 2004), and hence it was important to consider their synergistic effects they may impart khalil et al. 391 on bacteriocin activity in case they were incorporated with bacteriocinogenic cultures in foods. in general, low concentrations of spices used (curry, red pepper, black pepper) were proportional to the bacteriocin activity. hugas et al. (2002) indicated that sakacin k production by l. sakei ctc 494 was not affected by the addition of 0.4% of black pepper, indicating that small amounts of additives should be preferably used in some foods to promote the activity of the bacteriocins applied. verluyten et al. (2004) pointed out that combination of spices together with bacteriocins may enable efficient syner-gism, rendering pathogens susceptible to the combined action of bacteriocin and the spices. to an extent, this fact was not reinforced by our result where the bacter-iocin activity was almost halved when mixed with 0.5% of bastermy sheath although it contains more than one spice with different amounts according to different pro-ducers (such as, garlic, salt, chili, feunogreek and pepper etc.). however, this result might be explained on the basis of presence of an unidentified compound that may have led to the inhibition of bacteriocin activity. in con-trast, 1% garlic powder had a significant bactericidal action towards the indicator strains, resulting in 98% growth reduction, while 3% of garlic did not severely decrease the bacteriocin activity. this latter concentration was higher than the inhibitory concentration to lactobacilli as recorded by gonza´lez-fandos et al. (1996), which indicates a possible synergism between nisin and garlic extract (singh et al., 2001). on the other hand, 0.5% rosemary seemed to positively influence the bacteriocin activity, which was in contrast to observations by verluyten et al. (2004) . the largest negative effect on growth of the indicator strains was ascribed to 0.5% of paprika probably due to interference with production as reported by verluyten et al. (2004). these results could be of industrial significance, as the bacteriocinogenic strain may be qualified for use in various food product applications based on its ability to stabilize the sensory quality and extend the shelf life of foods synergistically with spices included in the manufacture. heat treatment of b. megaterium 22 bacteriocin expressed an interesting feature of heat stability at wide range of temperatures. a close finding to our result was that of bacteriocin produced by lactobacillus ca44 and l. lactis subsp. cremoris ctc 204 as reported by vinod et al. (2006) and bromberg et al. (2005) respectively. the heat stability data may impart another industrial advantage to our producing strain in view of the potential use of its bacteriocin as a food additive in procedures of food preparation involving a heating step, and hence its potential effectiveness against psychrophile, thermophile and mesophile foodborne pathogens. the activity of b. megaterium 22 bacteriocin was ph dependent and was stable between ph 2 and 8 at 30 o c. a similar behaviour has been reported for pediocin f, which was found to be stable over a wide ph range between 3 and 9 (özlem et al., 1997). our ph data propose an additional benefit and a possible advantage to the bacteriocin in food industry, and suggest its effective applicability against molds, yeast, acetobacter, and other deteriorating food microorganisms in neutral and acidic foods. the preservation capacity of the bacteriocin in terms of the period and temperature of storage was quite interesting, as it maintained full stability and was equally active against the two indicator strains after 90 days of storage at 4ëc, besides its partial stability up to only 30 days at -20 o c, indicating that cold temperature may be the most appropriate medium of preservation. the protein status of the tested bacteriocin was confirmed by results of uv light, where it demonstrated high resistance to uv exposure for up to 60 min with no change of protein nature or function (ogunbanwo et al., 2003a). b. megaterium 22 bacteriocin was typical to lab in respect of its sensitivity to trypsin (jack et al., 1995), as well as to papain and pepsin. this resistance can be explained on the basis of the presence of unusual amino acids in the bacteriocin structure, or cyclic n-and / or cterminally blocked peptides, which may make cleavage sites inaccessible due to steric hindrance (eckart, 1994) . moreover, the slight bacteriocin resistance to amylase and lipase might be an indication that no lipids or carbohydrate components are involved in the antibacterial activity (torkar and matijaši , 2003). the complete destruction of activity by the tested organic solvents, suggests that the bacteriocin molecule may not share similar hydrophobic properties with the other lab bacteriocins (klaenhammer, 1993). the unique properties of the produced bacteriocin stimulated further investigation of its molecular weight which was found to range from 3.496 to 6.512 kda. this result was close with that obtained from the sds-page assay of some bacteriocins like megacin a-19213; being composed of two subunits, one of which is about 7.5 kda in mass (von tersch et al., 1983), and to that reported for enterocin crl35 (wachsman et al., 2003). conclusion results obtained in this research may aid in understanding the industrial and technological significances of bacteriocins in reducing the risk of food contamination as the growth and proliferation of food borne pathogenic bacteria. practical studies involving the type of medium/medium components that 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technol biotechnol. 43: 219-225. zuber p, nakano mm, marahiel ma (1993). peptide antibiotics. in: sonenshein al, hoch ja, losick r (eds) bacillus subtilis and other gram-positive bacteria. american society for microbiology, washington, pp. 897-916. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 9 (2), pp. 001-006, february, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper relationship between total anthocyanin level and colour of natural cherry laurel (prunus laurocerasus l.) fruits rezzan kasım*, melekber sülüẟoğlu and m. ufuk kasım kocaeli university, vocational school of arslanbey, 41285 kartepe/kocaeli/turkey. accepted 16 november, 2020 the total anthocyanins level and fruit colour were investigated in twelve naturel cherry laurel (prunus laurocerasus l.) genotypes. anthocyanins are well-noted as healty compounds due to their antioxidant properties. fruit samples of cherry laurel were examined for their total anthocyanin content by using a spectrophotometric differential ph method. cherry laurel genotypes showed differences in their total anthocyanins levels which ranged from 67.52 to 260.81 mg kg -1 . total anthocyanin content was the highest in kg 11 (260.81 mg kg -1 ) and followed by kg 8 (235.99 mg kg -1 ) and kg 6 (228.60 mg kg-1) and colour a* value (redness) of these genotypes were lower compared to other eight genotypes (5.63, 7.41 and 5.34, respectively). the total anthocyanins were strongly correlated with chromatic parameters (a* and b* value, chroma, hue angle) except for l* value. these results showed that antioxidant level of fully coloured fruits were lower so increasing total anthocyanins level alone was not effective on coloring of fruit of cherry laurel. key words: cherry laurel (prunus laurocerasus l.), anthocyanin, colour. introduction cherry laurel (prunus laurocerasus l.) is a member of rosaceae family, native to the west asia (çolak et al., 2005) and is a popular fruit (dark purple or black when mature) mainly distributed in the coast of the black sea region of turkey and is locally called “taflan” or “karayemiẟ” (alasalvar et al., 2005). it is mostly consumed as fresh fruit in local markets but may also be dried, pickled and put into pekmez, jam, marmalade and fruit juice products (liyana-pathirana et al., 2006). besides its use for food, both fruit and seeds of cherry laurel are well known as traditional medicine in turkey and have been used for many years for the treatment of stomach ulcer, digestive system ilness, bronchitis (seeds), eczemas and haemorrhoids and as a diuretic (fruits) in folk medicine in turkey (baytop, 1999). high fruit and vegetable consumption is associated with a reduced risk of several chronic diseases such as cancer, cardio vascular disease, coronary heart disease and atherosclerosis. the compounds believed to be *corresponding author. e-mail: rkasim@kocaeli.edu.tr. responsible for the protective effects of a fruit-rich and vegetable-rich diet include carotenoids and antioxidants vitamin. in this regard, attention has been focused on the significance of phenolics such as phenolic acids, flavonoids and in particular anthocyanins (shadidi and naczk, 2004). anthocyanins, one of the major group of pigments, are responsible for orange, red and blue colours in fruits, vegetables, flowers and other storage tissues in plants (chandra et al., 1992). colour is the most important indicator of maturity and quality in many fruit species (drake et al., 1982) and is mainly influenced by the concentration and distribution of various anthocyanins in the skin (gao and mazza, 1995), as well as by other factors such as light, temperature, ethylene and cultural practices (lancester et al., 1997). anthocyanins are scavenger activity of radical species (wang et al., 1997) and contributors to antioxidant activity (rapisarda et al., 1999), thus constituting useful markers to enable recognition and evaluation of quality of fresh fruits. the influence of ripeness on accumulation of anthocyanins and on the evolution of colour has been analysed in sweet cherries (gonçalves et al., 2007), grapes 2 table 1. total anthocyanins content and colour parameters (l*, a*, b*), chroma (c*) and hue angle (h). genotypes total anthocyanin (mg/kg fw) l* a* b* c* h 1 129.88 cde 21.15 d 11.69 cd 1.22 bcd 10.75 def 6.00 cdef 2 167.41 cd 21.80 cd 10.46 cde 1.25 bcd 10.54 def 6.35 cde 3 181.49 bc 21.14 d 8.14 def 0.68 cd 8.17 efg 4.60 defg 4 118.64 def 22.90 b 15.89 ab 3.31 a 16.24 abc 11.37 ab 5 155.85 cd 21.76 cd 12.51 bc 1.85 bc 12.67 bcd 7.51 cd 6 228.60 ab 21.33 cd 5.34 f 0.22 d 5.35 g 2.09 g 7 91.35 ef 23.07 b 17.72 a 3.87 a 18.15 a 12.08 a 8 235.99 ab 21.29 cd 7.41 ef 0.49 d 7.43 fg 3.69 efg 9 157.62 cd 21.62 cd 12.15 bcd 2.04 b 12.34 cde 9.00 bc 10 126.66 cdef 22.40 bc 16.80 a 3.66 a 17.22 a 11.33 ab 11 260.81 a 22.37 bc 5.63 f 0.42 d 5.65 g 3.13 fg 12 67.52 f 24.21 a 16.27 ab 3.97 a 16.77 ab 13.41 a values are means of three replicates (n = 3). different letters indicate significantly different values at p<0.05. (ryan and revilla, 2003) and plum (usenik et al., 2009). the chromatic parameters l*, a*, b*, chroma and hue angle correlated negatively with total anthocyanin levels in sweet cherry (gonçalves et al., 2007) and weakly correlated in plums (usenik et al., 2009). cherry laurel has become more popular in recent years in turkey. there has been pomological traits, physiological (nardini, 2002), physico-mechanic properties (çaliẟır and aydin, 2004), chemical composition of fruits and leaves (kolayli et al., 2003; çolak et al., 2005), medicinal effects (pieroni, 2000). according to the searched literature there have been studies on the chemical composition of cherry laurel (alasalvar et al., 2005; liyana-pathirana et al., 2006; halilova and erciẟli, 2010). but no research has been conducted on relationship anthocyanin level and colour of cherry laurel fruits. the aim of this study is to investigate the correlation between total anthocyanin level and colour parameters of naturel cherry laurel fruits. materials and methods plant material twelve cherry laurel genotypes were sampled from kocaeli in marmara reğion in turkey. about 1 kg of fruit per genotype was harvested when fully ripe state and named kg1, kg2, kg3, kg4, kg5, kg5, kg7, kg8, kg9, kg10, kg11 and kg 12. for each genotype nine samples of fruit were randomly selected at nearly same colour for the total anthocyanin level and colour measurements. after colour determination, total anthocyanins were analysed from the whole edible part of fruits. for each genotype, three replicates were measured (n = 3); each with three fruits. treated with 10 ml of buffer solution, ph:1.0 (125 ml of 0.2 m kcl and 375 ml of 0.2 m hcl) and 10 ml of buffer solution, ph: 4.5 (400 ml of 1 m sodium acetate, 240 ml of 1 m hcl and 360 ml of water), respectively. the mixture was homogenised and centrifuged twice at 4°c at 5000 g for 15 min. the supernatant was collected and its absorbance was read at 510 nm. total anthocyanin amount was determined by the following equation: c(mg kg -1 fw) = (absph: 1.0 – absph 4.5) x 484.82 x 1000/24825 x df where the term in parentheses is the difference of absorbance at 510 nm between ph 1.0 and 4.5 solution; 484.82 is the molecular mass of cyanidin-3-glucoside chloride; 24825 is its molar absorptivity (ε) at 510 nm in the ph: 1.0 solution and df is the dilution factor. colour analysis the skin colour variables were measured on three fruit samples immediately after picking. the cielab colour system (comission internationale de l’eclairage, 1986) is extensively used to evaluate food colour. colour of cherry laurel fruits was measured on the cheek area of nine fruits with konica minolta cr-400 (osaka, japan). value l*, a*, b*, chroma (c*) and hue angle (h) were measured to describe the colour of cherry laurel fruits. statistical analysis statistical analysis was conducted with the programme minitab 11. one-way anova was used for analysis of the anthocyanin content and colour parameters. differences between genotypes were estimated with duncan test (p<0.05). linear regression was performed between total anthocyanin concentration and colour parameters. the relationships were tested at p≤0.05. total anthocyanin analysis total anthocyanin content was measured using a spectrophotometric differential ph method, according to rapisarda et al. (2000) and lo scalzo et al. (2007). two samples of 1 g were results and discussions total anthocyanins content total anthocyanins content of twelve cherry laurel fruits are shown in table 1. the total anthocyanins content was 3 l* v al u e y = -0.0101x + 23.697 24.5 r2 = 0.3982 24 23.5 23 22.5 22 21.5 21 20.5 0 50 100 150 200 250 300 total anthocyanins figure 1. variation of l* value with total anthocyanins content of twelve different cherry laurel genotype. highest in kg 11 (260.81 mg kg -1 ); it was followed by kg 8 (235.99 mg kg -1 ) and kg 6 (228.60 mg/kg -1 ), and also differences between these three genotypes and the other genotypes were important statistically (p<0.05). also great variability existed among the examined cherry laurel fruit samples, regarding their anthocyanin content. similar results were obtained by halilova and erciẟli (2010). the genotypes with the significantly lowest concentration of total anthocyanins were kg 12 and kg 7 (67.52 and 91.35 mg kg -1 , respectively). the highest content of total anthocyanins was measured in genotype 11 at 260.81 mg kg -1 , extracted from the whole edible part of fruits. alasalvar et al. (2005) reported that the content of total anthocyanins measured two cherry laurel cultivar ranged from 123.8 to 174.3 mg 100 g -1 fw, ant total anthocyanin content of eight cherry laurel genotype was changed from 164.4 to 206.4 mg 100 g -1 fw found by halilova and erciẟli (2010). in the present study total anthocyanins content of twelve cherry laurel genotypes ranged from 67.52 to 260.81 mg/kg. these data are in agreement with those reported by alasalvar et al. (2005) and halilova and erciẟli (2010). the total anthocyanin content of dark-coloured sweet cherries was 82 to 298 mg 100 g -1 fw. in red grape cultivars it was estimated to 6.9 to 15.1 mg of cyanidin 3rutinose equivalents of 100 g -1 fw and in 30 genotypes of blueberries as 34 to 515 mg of cyanidin 3-glucoside equivalents 100 g -1 fw (moyer et al., 2002). our results were within these ranges and it can be concluded that cherry laurel genotypes were found to be good sources of anthocyanins among fruit species. correlation of total anthocyanins amount and colour parameters anthocyanins are a group of widespread naturel phenolic compounds in plants. there are mainly distributed among flowers, fruits (particularly in berries) and vegetables and responsible for their bright colours such as orange, red, violet and blue (hou, 2002). there were significant differences in colour parameters l*, a*, b*, c* and h between twelve cherry laurel genotypes. the chromatic characteristic of fruits are shown in table 1. in genotypes that have higher anthocyanin content; chroma and hue angle were always lower than those in genotypes that have lower anthocyanin level. the different correlation coefficients between total anthocyanins and colour parameters was obtained for genotypes of cherry laurel. the variation in the anthocyanin level in genotypes induces variation in the degree of correlation between total anthocyanins and colour parameter (gonçalves et al., 2007). our results confirm the findings of mcguire (1992) and gonçalves et al. (2007) that better correlation could be obtained when anthocyanin levels were compared directly with the values of l*, a* and b* (figures 1, 2 and 3, respectively). in all cherry laurel genotypes, total anthocyanins level was negatively correlated with chromatic parameters (a*, b*, hue angle (h) and chroma (c*)) and total anthocyanins and this correlation was statistically significant (p≤0.05). in kg 11, concentration of total anthocyanins were also highest, resulting in the lowest value in the parameter a* (figure 2) that indicates lower red colour of fruits (r 2 = 0.86). so, 4 a* v al u e y = -0.0692x + 22.749 20 r 2 = 0.8644 18 16 14 12 10 8 6 4 2 0 0 50 100 150 200 250 300 total anthocyanins figure 2. variation of a* value with total anthocyanins content of twelve different cherry laurel genotype. b * va lu e 4.5 y = -0.0216x + 5.3771 4 r 2 = 0.789 3.5 3 2,5 2 1.5 1 0.5 0 -0.5 0 50 100 150 200 250 300 total anthocyanins figure 3. variation of b* value with total anthocyanins content of twelve different cherry laurel genotype. this means that the highly coloured fruits have lower anthocyanin content. also, total anthocyanins content was strongly negatively correlated within all chromatic parameters. we can not say, however, anthocyanins are responsible for the typical colour of cherry laurel genotypes because in genotypes highly coloured the total anthocyanins content was lower. similarly a* values; b* value (figure 3), hue angle (figure 4), chroma (figure 5) and total anthocyanins content of fruits strongly negatively correlated (r 2 = 0.78, r 2 = 0.83 and r 2 = 0.84, respectively). in sweet cherry gonçalves et al. (2007) determined the strongest negatively correlation between chromatic parameters; l*, a*, b*, chroma and hue angle and total anthocyanins 5 h u e a n gl e y = -0.0597x + 17.108 16 r 2 = 0.8364 14 12 10 8 6 4 2 0 0 50 100 150 200 250 300 total anthocyanins figure 4. variation of hue angle (h) with total anthocyanins content of twelve different cherry laurel genotype. c* v al u e y = -0,0711x + 23,162 20 r 2 = 0,8409 18 16 14 12 10 8 6 4 2 0 0 50 100 150 200 250 300 total anthocyanins figure 5. variation of chroma (c*) with total anthocyanins content of twelve different cherry laurel genotype. level in sweet cherry. in the present work, the same results were obtained except for l* value (figure 1). l* value and total anthocyanin level of cherry laurel fruits were not strongly correlated in our study (r 2 = 0.39). usenik et al. (2007) found weakly correlation between chromatic parameters and total anthocyanin level in four plum cultivars. conclusion there is a wide range of variability between cherry laurel genotypes in their concentrations of total anthocyanins and chromatic parameters. the red colour of cherry laurel fruits which have high anthocyanin content was low. furthermore, with cherry laurel genotypes, the level of 6 anthocyanins were strongly negatively correlated (mainly statically significant) with colour parameters. only l* value of fruits weakly correlated with total anthocyanins level. the present study revealed that cherry laurel fruits are a significant source of anthocyanins. and also suggest that anthocyanins, alone, can not be responsible for the coloration of fruits of cherry laurel. references alasalvar cam, shadidi f (2005). compositional characteristics and antioxidant components of cherry laurel varieties and pekmez. j. food sci., 70(1): 47-52. baytop t (1999). curing with turkish plants in the past and today (turkish). i̇stanbul:çapa nobel medical books, p. 353. çalıẟır s, aydın l (2004). some physic-mechanic properties of cherry laurel (p. laurocerasus l.) fruits. j. food eng., 65: 145-150. chandra a, nair mg, lezzoni a (1992) . evaluation and characterization of the anthocyanin pigments in tart cherries (prunus cerasus l.). j. agric. food chem., 40: 967-969. çolak a, özen a, dinçer b, güner s, ayaz fa (2005). diphenolases from two cultivars of cherry laurel (laurocerasus officinalis roem.) fruits at an early stage of maturation. food chem., 90: 801-807. drake sr, proebsting el jr., spayd se (1982). maturity index for the color grade of canned dark sweet cherries prunus avium. j. am. soc. hort. sci. 107: 180. gao l, mazza g (1995). characterization quantition and distribution of anthocyanins and colourless phenolics in sweet cherries. j. agric. food chem., 43: 343-346. gonçalves b, silva ap, mauntinho-pereira j, bacelor e, rosa e, meyers s (2007). effect of ripeness and postharvest storage on the colour and anthocyanins in cherries (prunus avium l.). food chem. 103(3): 976-984. halilova h, erciẟli s (2010). several physico-chemical characteristics of cherry laurel (laurocerasus officinalis roem) fruits. biotechnol. biotechnol. eq., 24: 1970-1973. hou dx (2002). potential mechanisms of cancer chemoprevention by anthocyanins. curr. mol. med., 3 (2): 149-159. kolaylı s, küçük m, duran c, candan f, dinçer b (2003). chemical and antioxidant properties of laurocerasus officinalis roem. 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nardini a (2002). relations between efficiency of water transport and duration of leaf growth in some deciduous and evergreen trees. trees (berlin), 16: 417-422. pieroni a (2000). medicinal plants and food medicines in the folk traditions of the upper lucca province, italy. ethnopharmacol., 70: 235-273. rapisarda p, fonella f, maccarone e (2000). reliability of analytical methods for determining anthocyanins in blood orange juices. j. agric. food chem., 48: 2249-2252. rapisarda p, tomaion a, lo cascio r, bonina f, de pasquale a, saija a (1999). antioxidant effectiveness as influenced by phenolic content of fresh orange juices. j. agric. food chem., 47: 4718-4723. ryan jm, revilla e (2003). from plums to prunes. influence of drying parameters on polyphenols and antioxidant activity. j. agric. food chem., 51: 3675-3681. shadidi f, naczk m (2004). phenolics in foods and nutraceuticals. crc press, boca raton, fl, pp. 489-490. usenik v, fabčič j, štampar f (2007). sugars, organic acids, phenolic composition and antioxidant activity of sweet cherry (prunus avium l.) food chem., 107(1): 185-192. usenik v, štampar f, verebič r (2009). anthocyanins and fruit colour in plums (prunus domestica l.) during ripening. food chem., 114: 529534. wang h, cao g, prior l (1997). oxygen radical absorbing capacity of anthocyanins. j. agric. food chem., 45: 304-309. 1 full length research paper plantain (musa paradisiaca) waste flour's proximate and mineral composition: a possible source of nutrients for animal feed formulation rodríguez* and ramírez university of carabobo, carabobo, venezuela accepted 1 december, 2024 in the tropics, plantain fruit is a staple food and a significant source of energy. while the bracts, fruit stalk, and leaf are left on the farm as waste, the fruit's peel is thrown away as waste once the edible portion is consumed, endangering the environment, particularly in areas where it is often consumed. in several nations, these waste materials have been contemplated for use as organic fertilizer. the purpose of this study was to ascertain the mineral and proximate composition of plantain (musa paradisiaca) waste flour as potential sources of nutrients for animal feed formulation. the atomic absorption spectrophotometric method and standard aoac analysis techniques were used to ascertain the proximate and mineral composition of plantain wastes (bract, ripe peel, fruit stem, and leaf). 9.39 to 9.53g of moisture, 1.87 to 19.37g of crude protein, 0.73 to 1.83g of crude fat, 8.10 to 15.50g of crude fiber, and 54.00 to 68.00g of carbohydrates per 100g sample were all present in the trash. iron (10.50–14.00 mg), calcium (120.00–150.00 mg), and phosphorus (110.00–180.00 mg) are all abundant in plantain bracts per 100g sample. because plantain wastes are rich in protein, fiber, and vital minerals, they can be used as a source of nutrients when making animal feed. since the waste would have been an annoyance to the environment, this will have the twin benefits of being used as animal feed and a proper plantain waste management (waste reduction) approach. key words: proximate and mineral composition, animal feeds, plantain waste management. introduction one of the most significant tropical plant crops is the plantain. it is a member of the genus musa and the family musaceae. musa paradisiaca is a tropical plant native to india that is also called "plantain" in english, "ogede agbagba" in yoruba, "ayaba" in hausa, and "ogadejioke" in igbo. with a lifespan of roughly 15 years (philips, 1982), the plant has long, overlapping leaf stalks and a 1.22–6.10 m high stem (oladiji et al., 2010). each individual plantain in the cluster, which grows in clusters, is slightly longer than a banana fruit and has a diameter of around 1 inch. eight to twelve months after planting, or roughly two and a half to four months after shooting, are needed for plantain fruit to be ready for harvest (swennen, 1990a). because of its high fiber content, plantains can reduce cholesterol, ease constipation, and prevent colon cancer. in addition, its high potassium content has been shown to help reduce muscle cramps and elevated blood pressure (ng and fong, 2000). the plant's leaves, root, fruit stem, bract, and fruit have all been utilized for household and therapeutic reasons. the leaves serve as an arbortifacient, the fruit is eaten, and the leaf juice is applied to cuts, wounds, and insect bites. its sap is used to treat epilepsy, hysteria, dysentery, and diarrhea. anaemia and venereal disorders are treated with a cold infusion of the root. furthermore, the fruit has purportedly been used as a diuretic, aphrodisiac, and antiscorbutic (gill, 1992). 100g of plantain that was edible included 67.30g of moisture, 0.4g of crude fat, 31.15g of carbohydrates, 0.95 mg of potassium, 35.1 mg of sodium, 71.5 mg of calcium, 28 mg of phosphorus, 2.4 mg of iron, and produced 116 kcal of energy, according to adeniji et al. (2006). according to osma et al. (2007), plantain peels are byproducts of the plantain processing industry and are typically disposed of in rivers, landfills, or unregulated areas. after the fruit's fleshy inside is consumed, the peel is thrown away as waste, endangering the ecosystem, particularly in author(s) retain the copyright of this article. african journal of food science research issn 2375-0723 vol. 12 (1), pp. 001-005, december, 2024. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 areas where eating it is widespread. according to omole et al. (2008), snails may be able to replace corn starch in their diet with the peel, but bracts, fruit stalks, and leaves are occasionally discarded on farms. after being chopped, fermented, and dried, leaves, pseudostems, fruit stalks, and peels produce a meal that is marginally more nutrient-dense than alfalfa press-cake. in somalia, these waste products have been contemplated for use as organic fertilizer. pigs fed pseudostems in malaya are less likely than those fed other diets to develop liver and kidney parasites. this study was carried out to ascertain the proximate and mineral composition of musa paradisiaca wastes (bracts, fruit stalk, leaf, and peel) flour for potential use as livestock feeds due to the need for waste management, the rise in the prices of livestock feeds, and their growing demand. materials and methods plantain wastes sample preparation plantain bracts, fruit stalks, and leaves were gathered at a plantation in ilesa, osun state, nigeria, while ripe plantain fruit was bought in oje market in ibadan, oyo state. after removing the peel, bract, fruit stem, and leaves, they were cleaned with distilled water, allowed to sun-dry for 168 hours, and then oven-dried at 1050c until their weight remained constant. after being ground and run through a 0.1 mm mesh screen, the samples were kept in a polythene container until they were required for analysis. chemicals and reagents all the chemicals and reagents used in this study were of analytical grade and were products of british drug house laboratory, england. proximate composition the association of official analytical chemists' established methods of analysis were used to evaluate the samples' proximate nutritional makeup (aoac, 1995). the samples' moisture content was ascertained using the gallenkamp air oven method at 1050c. the microkjeldahl technique was used to evaluate the samples' crude protein content. petroleum ether was used as the extracting solvent in the soxhlet extraction procedure to assess the crude lipid content. a muffle furnace heated at 5500c for four hours to achieve a consistent weight of ash was used to measure the amount of ash present. the saura-calixto et al. (1983) method was used to calculate crude fiber. the difference was used to determine the carbohydrate content. mineral analysis by digesting the sample ash with nitric acid and perchloric acid, potassium and sodium were measured using a jenway digital flame photometer/spectronic 20 (bonire et al., 1990). the vanado-molybdate colorimetric method was used to determine phosphorus (ologhobo and fetuga, 1983). using a buck 200 atomic absorption spectrophotometer (buck scientific, norwalk), calcium, magnesium, and iron were measured spectrophotometrically (essien et al., 1992). the absorption of these minerals was compared to the absorption of standards. statistical analysis all determinations were carried out in triplicates. descriptive statistics, analysis of variance (anova) and duncan multiple range test were used to interprete the results obtained, and the level of significance was set at p≤0.05. results table 1 displays the findings of the plantain wastes' proximate nutritional composition. plantain wastes had a very low residual moisture content (less than 10g/100g sample). the moisture content of the fruit stem, ripe peel, and bracts was substantially lower than that of the leaf (p<0.05), although it was not significantly different from one another (p>0.05). the waste products' protein contents varied greatly from one another, with the highest protein level found in leaves and the lowest in bracts and fruit stalks. while there was no significant difference in the crude lipid content of bracts and leaves, there was a significant difference (p<0.05) between ripe peel and fruit stem. every sample had a notably varying ash value, with the leaf having the lowest value and the mature peel and fruit stem having the highest. the waste items' crude fiber content varied significantly from one another, with the highest value found in fruit stalks, followed by ripe peel, bracts, and leaves (p<0.05). the sugar the peel had the highest concentration, while the leaf had the lowest (p<0.05). table 2 displays the mineral makeup of plantain wastes. all of the mineral values for each sample varied significantly from one another. the leaf sample had the highest levels of calcium and phosphorus (p<0.05), ripe peel had the highest levels of sodium, and bract had the highest levels of potassium, magnesium, and iron. 3 table 1. proximate composition (dry matter, dm) of plantain wastes flour (g/100g) sample bract ripe peel fruit stalk leaf mean moisture 9.5b±0.1 9.5b±0.2 9.5b±0.1 9.4a±0.3 9.4±0.3 protein 11.5c±0.2 2.3b±0.6 1.9a±06 19.4d±0.3 8.7±0.5 crude lipid 1.8c ±0.1 0.9b±0.1 0.7a± 0.6 1.8c±0.6 1.3±0.5 ash 7.8b±0.6 8.9c±0.6 9.1d±0.6 7.2a±0.1 8.2±0.8 crude fibre 8.5b±0.1 10.4c±0.6 15.5d±0.1 8.1a±0.2 10.6±3.1 carbohydrates 60.9b ±0.5 68.0d±0.3 63.3c±0.2 54.6a±0.3 61.7±5.1 all data were mean ± standard deviation of triplicate determinations means within the same column with the same subscripts were not significantly different (duncan’s test) table 2. mineral composition (dry matter, dm) of plantain wastes flour (mg/100g) sample na k ca mg p fe bract 280.0b±0.0 40.0±0.0 135.0±0.0 18.0d±0.0 151.7c±2.9 14.0c±0.0 ripe peel 315.0c±12.0 35.0±0.0 120.0±0.0 14.5b±0.0 145.0b±0.0 12.3b±0.3 fruit stalk 253.3a±5.8 30.0±0.0 125.0±0.0 15.3c±0.3 110.0a±0.0 12.1b±0.1 leaf 280.0b±23.2 35.0±3.7 150.0±0.0 12.3a±0.3 180.0d±0.0 10.5a±0.0 all data were mean ± standard deviation of triplicate determinations means within the same column with the same subscripts were not significantly different (duncan’s test) discussion the samples' moisture content (table 1) was much higher than the value documented in the literature (5.43%, usda, 2009). the freshness and shelf life of foods and processed goods are indicated by their moisture content; high moisture content makes food items more susceptible to microbial spoiling and short shelf life, which can cause degradation (adepoju and onasanya, 2008). in comparison to other plant protein sources, the crude protein content of the leaf and bract samples was comparatively high. they surpass those of cocoyam leaves (adepoju et al., 2006), amaranthus, and shea butter fruit pulp (adepoju and ketiku, 2003). when compared to other commonly consumed staple roots, tubers, and fruits, the protein content of plantain ripe peel and fruit stalk powder was low (aurand, 1987; usda, 2009), significantly lower than that of leaf and bract samples (p<0.05), but higher than that of fluted pumpkin pod and pulp (essien et al., 1992). protein's primary role in nutrition is to provide an appropriate supply of necessary amino acids, making it a necessary part of both animal and human diets for survival. the samples of plantain wastes had extremely low crude fat content, and they might not be a suitable source of fat-soluble vitamins or be able to make a substantial contribution to the calorie content of the feeds made from the wastes. the fat content of the plantain fruit was highest in the bract and lowest in the stalk. their low fat content will prolong the flour's shelf life by decreasing the likelihood of rancidity. comparatively speaking, the samples' ash level was larger than that of other studies on plantain peels (adeyi and oladayo, 2010) and agricultural hulls (adebowale and bayer, 2002). the high ash readings were a sign that the wastes contained a lot of minerals, particularly macrominerals. the fruit stalk and plantain leaf had the greatest and lowest ash values, respectively. the crude fiber content of the plantain wastes was significantly higher than that of plant products such fruit pulp (4.3g/100g, adepoju and adeniji, 2012) and ube (dacryodes edulis) (2.1g/100g, adepoju and adeniji, 2008). the increase could be explained by the plantain waste's high total dietary fiber content, particularly in the peel (haslinda et al., 2009). crude fiber content was highest in the plantain fruit stalk, followed by mature peel, and lowest in the leaf. a high fiber diet has been shown to promote the removal of possible mutagens, steroids, and xenobotics by binding or absorbing to the fiber components and facilitating digestion. as a result, these wastes will have positive health effects for fish and cattle farms. the samples had a high carbohydrate content and might provide the animals with a strong source of energy. carbohydrate content was highest in the peel and lowest in the leaf. the samples had low levels of iron and magnesium but high levels of calcium, phosphorus, potassium, and sodium (table 2). the leaf had the most calcium and phosphorus content, the peel had the highest sodium level, and the bract had the highest potassium, magnesium, and iron content. iron, phosphorus, and calcium can all be found in good amounts in the feces. strong bones and teeth, growth, blood coagulation, heart function, and cell metabolism all depend heavily on calcium and phosphorus (roth and townsend, 2003; rolfe et al., 2009). potassium is a crucial basic ingredient for making soap and neutralizing soil (adeolu and enesi, 2013). the wastes had a greater iron content than fresh 4 and roasted dacryodes edulis fruit (7.0 mg and 3.0 mg/100 mg, respectively, adepoju and adeniji, 2008) and dried guinea corn leaf extracts (1.2–2.1 mg/100g, adepoju, 2007). because it is abundant in macrominerals, it can also be used to make baby food and instant flours for convalescence, as these human groups need high mineral levels for growth and healing. conclusion the plantain wastes can be used as raw materials or as ingredients in animal feed because they were high in ash, crude fiber, and carbs. if accessible, the leaf and bract samples' high crude protein content could be used as a source of protein for animal feed. the wastes can be a good supply of calcium, phosphorus, and iron because of their high concentrations. plantain wastes have the potential to be excellent sources of nutrients for animal feed production, and their use for this reason should be promoted as it will also lessen the 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lab. invest. 58(6): 2326. http://www.everynutrient.com/files/health_benefits_of_plantains.mht http://www.nal.usda.gov/fnic/foodcomp/plantain/ materials and methods plantain wastes sample preparation chemicals and reagents proximate composition mineral analysis statistical analysis results discussion conclusion in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (1), pp. 001-007, january, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper health implications of toxigenic fungi found in two nigerian staples: guinea corn and rice makun hussaini anthony1*, gbodi timothy ayinla2, akanya olufunmilayo helmina2, salako a. ezekiel3 and ogbadu godwin haruna4 1 department of biochemistry, federal university of technology, minna . . 2 department of biochemistry, federal university of technology, p.m.b 65, minna, niger state, nigeria. 3 department of crop production, federal university of technology, p.m.b 65, minna, niger state, nigeria. 4 sheda science and technology complex, federal ministry of science and technology, abuja, nigeria. accepted 17 june, 2019 a total of one hundred and forty eight fungi isolated from both guinea corn (67) and rice (81) in a previous fungal and mycotoxin survey in niger state, nigeria, were tested for toxicity potential in white albino mice. of all these, 64.2% were found to produce toxic metabolites that were lethal to mice and were mainly species of aspergillus spp, fusarium spp, penicillium spp and trichoderma spp. others include syncephalastrum spp, alternaria spp, phoma spp, curvularia lunata, colletotrichum spp, geotrichum candidum and helminthosporium spp, cladosporium werneckil, and mucor spp and the bacteria cryptococcus neoformis. the novel, most toxigenic fungi found contaminating these two staples were fusarium verticillioides (sacc.) nirenberg, previously known as f.moniliforme sheldon (cabi biosciences is imi 392668).the extract of the fungus caused lethality to mice at 40 mg /kg b. wt. the health implications of these toxic microbes in our diets were discussed. key words: guinea corn, rice, nigeria, toxigenic fungi, mycotoxins. introduction a survey for fungi, aflatoxin b1 (afb1), ochratoxin a (ota) and zearalenone (zea) contaminating mouldy field, marketed and stored guinea corn (sorghum) [makun, 2007] and rice (oryza sativa) [makun et al., 2007] during the dry harmattan (november – february), hot dry (march – may) and rainy (june – october) seasons in the four microclimatic zones of niger state, nigeria, was previously conducted. of the three studied mycotoxins, afb1 was the commonest contaminant of the grains followed by zen and ota in decreasing order. eight hundred and eighty four (844) fungal isolates were cultured and identified from a total of a hundred and sixty eight mouldy guinea corn samples while one thousand and sixty two fungi (1062) were isolated and identified from one hundred and ninety six mouldy rice samples analysed. the fungi found in the studied staples were species of twenty three genera namely; aspergillus, *corresponding author. email: hussainimakun@yahoo.com, 2348035882233. fax: 23466224482. penicillium, fusarium, mucor, rhizopus, alternaria, phoma, trichoderma, arthrium, helminthosporium, curvularia, collectritotichum, chaetomium, chrysosporium, cladosporium, and geotrichum. others include syncephalastrum, rhodoturula, scopulariopus, torula, bipolaris, gilocladium and nocardia. the screening studies determined only aflatoxin b1, ochratoxin a and zearalenone in the mouldy samples. it is possible that many more mycotoxins may contaminate grains in the state given that quite a number of moulds isolated from the grains are known to produce mycotoxins. apart from those which toxigenic strains are commonly associated with production of afb1 (e.g a. flavus; a. parasiticus), ota (e.g a.ochraceus) and zen (f. oxysporum), there were several which toxigenic strains are known to produce other mycotoxins e.g phoma sorghina, p. citrium, as well as those which are not completely associated with mycotoxin production. in order to have a better insight into the potential health implications of sorghum and rice infection by various fungi, it is necessary to determine the mycotoxigenic potentials of the fungal isolates which would be indicative of the likely mycotoxicoses arising from consumption of these staples in niger state, a leading cereal producing state in nigeria. this study was therefore undertaken to screen some fungi found in guinea corn and rice in the state for their toxigenicity using mice with a view to selecting a novel toxigenic fungi and determining the possible mycotoxin contaminants of the staples. multiples of isolates of each species found in the studied grains were selected for the toxicity screening. materials and methods culturing of fungi from guinea corn and rice the culturing and toxicity screening of fungi isolated and acute toxicity testing of the thirteen most toxic extracts were carried out as described by gbodi [1986]. to 20 g of akkad rice imported from thailand, 8 ml of distilled water was added and mixed thoroughly in a 50 ml conical flask and left overnight for moisture equilibration. the rice substrate was then autoclaved for 20 min at 120 o c and 15 psi pressure. after cooling it was aseptically inoculated with conidia or mycelium of five day old pure cultures grown on potatoes dextrose agar (pda) slant tubes and incubated for 14 days at 28 o c. extraction of toxins the 20 g rice cultures were homogenized for 2 min in 100 ml dichloromethane using blender. the homogenate was then filtered through fast fluted filter paper. two millimetres of corn oil was added to the filtrate in a round bottomed flask and concentrated in a rotatory evaporator at water bath temperature of 55 o c. when the dichloromethane was distilled off, clean empty distillate flask collector was used to replace the dichloromethane collector and rotatory evaporator turned on for another ten minutes to ensure complete removal of dichloromethane vapour. the corn oil-toxin extract was transferred into a vial and kept in deep freezer at -25 o c until used for toxicity testing in mice. screening the sorghum and rice fungal culture extracts for toxicity in mice five to six week old male white albino mice weighing 20-30 g were used for the toxigenicity screening of the isolates. the mice used for this experiment were raised from the parent animals purchased from national veterinary research institute, vom, plateau state, nigeria. the animals were housed in conventional plastic wire cages with sawdust bedding. they were given chick mash pellets and tap water ad-libitum. no extra lighting was provided in the night. three mice were kept in a cage. for the toxicity screening test and pilot acute toxicity assay, there were three mice per group. the cages were washed and the bedding changed every other day. the mice were kept for seven days to acclimatize prior to the toxicity assay. three mice each received by intraperitoneal (ip) injection 0.2 ml of the corn oil extract of culture of fungal extract from the grains. the mice were observed for 14 days for any signs of toxicity. five mice which received corn oil were used as controls. the toxicity of the extracts was arbitrarily categorized into four classes viz: very toxic (if all three of the extract treated mice died) moderately toxic (if two of three of the mice died) mildly toxic (if one of the three mice died) and non toxic (if none of the mice died. mass culturing of the selected very toxic fungi from the above toxicity screening, the thirteen fungi (aspergillus. flavus, a.niger, a.parasiticus, curvularia. lunata, fusarium spp, fusarium nwale, fusarium verticillioides, helminthosporium spp, penicillium spp, penicillium rubrum, p.verrucosum and trichoderma spp) that were found to produce the most toxic fungal metabolites were selected and further investigated with a view to finding a new, very toxic fungus contaminating the two staples. fresh pda tubes were inoculated with each selected fungus from stock cultures maintained on pda tube and incubated for five days at 28 o c for mass culturing of the thirteen most toxigenic fungi, 5 ml of triton x-100 (searle hopkin and williams, chalwell health, essex, england-) treated sterile distilled water (one drop triton x-100 to 200ml sterile distilled water) was added to each culture tube and the surface of the culture scratched with sterile inoculating wire to suspend the spores. the suspensions were then used to inoculate a 2.5 l fernbach flask containing 250 g of akkad rice, the moisture content of which had been equilibrated as earlier described. the flasks were autoclaved for 25 min and pressure of 15 psi. the cooled flasks were inoculated with the suspended spore and maintained in an incubator at 28 o c as a static culture for 21 days. extraction of toxins from culture material of the selected very toxic fungi for extraction of toxins from the thirteen selected isolates, 750 ml of dichloromethane were added to the fernbach flask with the mouldy rice chopped into small fragments and blended for three minutes. the homogenate was filtered through a bed of hyflosuper-cell in buchner funnel fitted with fast flow filter paper. the filtrate was again filtered through anhydrous sodium sulphate to remove the moisture and clarify the extract. the clarified clear solvent was removed at 50 o c by rotatory vacuum evaporator. the oily viscous residue was added to chilled swirling petroleum ether (1:15 residue/ petroleum ether v/v) and the mixture kept overnight in a deep freezer at -15 o c to complete the precipitation of the toxins. the crude toxin was recovered by filtration and dried in a fan oven at 50 o c for three hours before being used for pilot toxicity studies. acute toxicity testing with thirteen very toxic extracts a pilot test was carried out using the extracts of thirteen most toxigenic fungi selected from the screening test. to determine the dose of different extracts that would cause 0 to 100% death in mice, five groups of three mice each were used for each extract. the extract was dissolved in dimethyl sulphoxide (dmso) . a log interval of 0.60 was used to select a range of i.p doses, 40, 160, 640 and 2560 mg/kg body weight. a group of three mice which served as control received the highest dose of the dmso administered to extract treated mice. the animals were observed for 14 days for signs of toxicity. from this pilot acute toxicity test, fusarium vertillioides was shown to be the very toxic and relatively novel fungus of the fungal contaminants of the two staples. confirmation of identification of selected fungus the selected fungus was sent to cabi biosciences laboratory, surrey, u.k (former commonwealth mycological institute, kew, surrey, u.k) for confirmation of identity. it was confirmed to be f. verticillioides (sacc.) nirenberg, previously known as f. moniliforme sheldon. the identification number of the isolate in cabi biosci table 1. toxigenicity potential of fungi isolated from sorghum in niger state using mouse as test animal. very toxic (12) moderately toxic (17) mildly toxic (14) non-toxic (24) helmintthosporium spp (1/12) aspergillus flavus (3/17) a.fumigatus (1/14) a.parasiticus (2/24) fusarium spp (2/12) a.niger (1/17) a.nidulans (1/14) a.versicolor (1/24) a. niger (3/12) a.glaucus (1/17) a.niger (1/14) arthrium spp (1/24) penicillium rubrum (1/14) a.fumigatus (1/17) a.glaucus (1/14) a. alternate(1/24) a.flavus (2/12) a.versicolor (1/17) a.parasiticus (1/14) f. equiseti (3/24) a. parasiticus (1/12) colletrotrichum (2/17) a.ochraceus (1/14) f.oxysporum (1/24) p. verrucosum (1/12) p.citrinum (1/17) alternaria spp (1/14) f.semitectum (1/24) f.nwale (1/12) syncephalastrum spp (1/17) phoma spp (1/14) cladosporium spp (1/24) trichoderma spp (2/17) phoma sorghina (1/14) c. werneckil (1/24) mucor spp (1/17) trichoderma spp (1/14) chaetomium spp (2/24) curvularia lunata (2/17) f.solani (1/14) mucor spp (2/24) p.notatum (1/14) penicillium spp (2/24) syncephalastrum spp (1/14) rhizopus spp (2/24) penicillium spp (1/14) rhodotorula rubra (1/24) scopulariopsis (1/24) torula spp (1/24) trichoderma spp (2/24) values in parenthesis indicate the number of the isolates of the species in the group. summary/key: very toxic, all three mice died (3/3)= 12; moderately toxic, two of three mice died (2/3) = 17 ; mildly toxic, one of the three mice died (1/3)= 14; non-toxic = no mice died (0/3) = 24. biosciences is imi 392668 (appendix 2). results toxigenicity of fungi isolated from sorghum and rice one hundred and forty eight fungal isolates from both guinea corn (67) and rice (81) were tested for toxicity (tables 1 and 2). of all these, 95 were found to produce toxic metabolites and were aspergillus spp (41), fusarium spp (14), penicillium spp (10), trichoderma spp (8), syncephalastrum spp (4), three each of alternaria spp, phoma spp and curvularia lunata. others include two each of colletotrichum spp, geotrichum candidum and helminthosporium spp, and one each of cladosporium werneckil, cryptococcus neoformis and mucor spp. a few of the fungi which have not been known to produce mycotoxins were found to be toxigenic. for example syncephalastrum spp isolates from both guinea corn and rice were found to be moderately and mildly toxic. similarly, g. candidum and c. neoformans isolated from rice were demonstrated to be mildly toxic. different strains of the same fungal species infect sorghum in niger state. for example, among the fungi from sorghum that were tested for toxicity there were at least two strains of a. flavus (table 1). one was very toxic while the other is moderately toxic respectively. similarly, three strains of a. niger isolated from mouldy sorghum were very toxic, moderately toxic and mildly toxic, respectively. other fungi from sorghum that had multiple strains isolated include a. parasiticus (3), a. glaucus (2), a. fumigatus (2), a.versicolor (2), syncephalastrum spp (2), trichoderma spp (3), mucor spp (2) and penicillium spp (2). thirteen fungal species isolated from rice had more than one strain contaminating the grain in niger state (table 2).the fungi and the numbers of strains found were a. flavus (3), a. niger (2), a. glaucus (2), a.parasiticus (3), a.terreus (2), a.versicolor (2), fusarium spp (3), f.solani (2), f.verticillioides (2), penicillium spp (2), syncephalastrum spp (2) and trichoderma spp (2). many fungal species which are known to produce mycotoxins were found to be non-toxigenic in this work (tables 1 and 2). these include species known to produce aflatoxin (a.parasiticus), cyclopiazonic acid and sterigmatocystin (a.versicolor and bipolaris), patulin (a.clavatus, p.expansum), 3-nitropropionic acid (arthrinium spp), cytochalasins (alternaria alternate), trichothecenes and zearalenone (f.equseti, f.oxysporum, f semitectum), fumonisins and monili-formin (f.verticillioides), gliotoxin (gliocladium spp), emodin (cladosporium), chaetomin (chaetomium spp), ochratoxins, penicillic acid and rubratoxin (penicillium spp), satratoxins, gliotoxin and t-2 toxin (trichoderma spp). the mycotoxins associated with the demonstrated toxigenic fungi isolated from sorghum and rice they include aflatoxins, sterigmatocystin, fumitremorgens a and b, ochratoxin a, citrinin, patulin, cytochalasins, tenuazonic acid, gliotoxin, emodin, curvularin, trichothecenes (deoxynivalenol, niva-lenol, t-2 toxin, diacetoxiyscripenol (das) and related table 2. toxigenicity potential of fungi isolated form mouldy rice in niger state, using the mouse as test animal. very toxic (14) moderately toxic (19) mildly toxic (19) non-toxic (29) curvularia lunata (1/14) a. flavus (1/19) a. flavus (2/19) a. clavatus (1/29) helmintthosporium spp (1/14) a.niger (2/19) a.fumigatus (2/19) a.glaucus (1/29) trichoderma spp (1/14) a.glaucus (1/19) a.nidulans (1/19) a.parasiticus (3/29) fusarium spp (2/14) a.terreus (1/19) a.terreus (1/19) a.versicolor (1/29) a. niger (3/14) a.versicolor (2/19) alternaria spp (1/19) arthrium spp (1/29) penicillium rubrum (2/14) a.ochraceus (2/19) c. werneckil (1/19) bipolaris spp (1/29) a.flavus (2/14) f.solani (1/19) c. neoformans (1/19) fusarium spp (2/29) a. parasiticus (1/14) penicillium spp (1/19) g. candidum (2/19) f.oxysporum (1/29) f.verticillioides (1/14) p.citrinum (1/19) phoma sorghina (1/19) gilocladium spp (1/29) syncephalastrum spp (2/19) a. alternate (1/19) cladosporium spp (1/29) trichoderma spp (2/19) trichoderma spp (2/19) a.parasiticus (1/29) fusarium spp (3/19) f.solani (1/19) mucor spp (3/29) f.semitectum (2/19) nocardia brasiliensis (1/29) p.cyclopium (1/19) p.expansum (1/29) p.viridicatum (1/29) penicillium spp (1/29) rhizopus spp (2/29) syncephalastrum spp (1/29) rhodotorula rubra (1/29) f.verticillioides (4/29) values in parenthesis indicate the number of the isolates of the species in the group. summary/key: very toxic: all three mice died (3/3)= 14; moderately toxic = two of three mice died (2/3) = 19; mildly toxic = one of the three mice died (1/3) = 19; non-toxic = no mice died (0/3) = 29. table 3. results of the preliminary acute toxicity assay of the crude toxin extracts of the thirteen most toxigenic fungal isolates. mortality rate (number dead per 3 mice) source of crude toxin extract dose 1 dose 2 dose 3 dose 4 aspergillus niger 1/3 2/3 2/3 3/3 a.niger 0/3 0/3 1/3 3/3 a.parasiticus 0/3 1/3 1/3 2/3 fusarium oxysporum 0/3 0/3 1/3 3/3 curvularia lunata 0/3 0/3 2/3 3/3 helminthosporium spp 0/3 0/3 3/3 3/3 trichoderma spp 1/3 0/3 0/3 3/3 fusarium spp 0/3 1/3 2/3 3/3 penicillium rubrum 0/3 0/3 1/3 3/3 fusarium verticillioides 1/3 2/3 2/3 3/3 penicillium verrucosum 1/3 1/3 2/3 3/3 penicillium spp 0/3 0/3 2/3 3/3 helminthosporium spp 0/3 0/3 0/3 1/3 dose 1, 40 mg toxin extract/kg b.wt; dose 2, 160 mg/kg/b.wt; dose 3, 640 mg/kg b.wt; dose 4, 2560 mg/kg/b.wt. each dose is given i.p in dmso. ted compounds), fusarenon-x, zearalenone, fumonisins, moniliformin, fusarin c, rhizonin a, cyclopiazonic acid,penicillic acid, rubratoxin, penitrem a and b, satratoxin h, trichodermol and related trichodermal trichothecenes. preliminary acute toxicity testing twenty six fungal isolates from both guinea corn and rice (tables 1 and 2) were found to be very toxic to mice but there were different strains of same fungi species and so the isolate of a particular species that killed the mice fastest was selected for preliminary acute toxicity test as the representative fungal isolate for that species. thirteen fungal isolates emerged and were used for the pilot studies. the results of the preliminary acute toxicity assay of the crude extracts of the 13 most toxigenic fungi are summarized on table 3. at 40 mg/kg body weight four isolates (a.niger, trichoderma spp, fusarium. verticillioides and penicillium verrucosum) killed a mouse each out of the three used for the test. four fungal isolates (a.niger, a.parasiticus, f.verticillioides and penicillium verrucosum) caused death at 160 mg/kg body weight. all the thirteen fungal isolates except helminthosporium and trichoderma spp were lethal to mice at 640 mg/kg body weight. with the exception of a. parasiticus and helminthosporium spp, all other extracts caused 100% mortality at 2560 mg/kg body weight. from this result, a. niger and f. verticillioides caused the highest lethality in mice even at low concentration and therefore were the two most toxic fungi found in guinea corn and rice. f. verticillioides was selected as the novel most toxic fungi contaminating guinea corn and rice in niger state because less information about its toxicity is available in literature as compared to a. niger. discussion the results of the toxicity screening tests showed that many of the fungal isolates contaminating guinea corn and rice in niger state produced toxic metabolites that were lethal to mice and this is toxicologically significant. from the data, the profiles of more than thirty possible additional mycotoxins that may contaminate the studied grains in the state have been deduced, and their toxic effects in animals and man are well documented [gbodi and nwude, 1988; prelusky and rotter, 1994; beardall and miller, 1994; peraica et al., 1999; bankole and adebanjo, 2004; mycotoxin, 2005] of the toxic fungal isolates, aspergillus spp, penicillium, fusarium and trichoderma were the most prevalent and so the mycotoxins they are likely to produce would be of major health concern. aflatoxin b1 was the commonest toxin found during the mycotoxin screening studies [makun, 2007] and this correlates with the toxicity screening which shows that aspergillus spp, afb1 producers, are the most predominant toxigenic fungi found in the state. a host of mycotoxins [uraguchi and yamazaki, 1978; scott, 1994; mold-help, 2004], some of which are of public health significance are elaborated by species of aspergillus [gbodi and nwude, 1988; prelusky and rotter, 1994; beardall and miller, 1994; peraica et al., 1999; bankole and adebanjo, 2004; mycotoxin, 2005] but of major concern are the aflatoxins and sterigmatocystin which are naturally occurring hepatocarcinogens and have been linked to high incidence of liver cancer in some parts of the world where foods are frequently contaminated with aflatoxins [bankole and adebanjo, 2004; promed, 2004]. they are known to exacerbate hiv/aids [lane, 2005], impair growth of children [pier ac and mcloughlin 1985; gong et al., 2002; gong et al., 2004] and serve as antinutritional factors [iarc, 1976; hendrickse, 1991; carlos et al., 2004]. several of the mycotoxins ascribed to aspergillus species are also penicillium mycotoxins. however, the major penicillium toxins are ochratoxin a, citrinin, patulin, penicillic acid, roquefortine, cyclopianonic acid, verrucosidin, rubratoxin, cyclochlorotine and luteoskyrin [scott, 1994].the toxicological significances of these mycotoxins to human health, livestock production and trade have been reviewed by many scientists [gbodi and nwude, 1988; prelusky and rotter, 1994; beardall and miller, 1994; peraica et al., 1999; bankole and adebanjo, 2004; mycotoxin, 2005]. apart from aflatoxins, the three main aspergillus and penicillium mycotoxins that pose the greatest public health are ochratoxin a, patulin and citrinin. ochratoxin a causes kidney and liver impairment in animals and man especially pigs [wafa et al., 1998]. this mycotoxin has been proposed as the causative agent of endemic nephropathy that occurs among rural populations in croatia, bosnia and herzegovina, yugoslavia, bulgaria, and romania, where it has been estimated that about 20,000 people are either suffering from or are suspected to have the disease [peraica et al., 1999]. the toxin is also associated with urothelial tumours of pelvis and ureter in egypt, croatia, bulgaria and yugoslavia and chronic interstitial nephropathy in tunisia [peraica et al., 1999; jecfa, 200025]. patulin and citrinin are neurotoxic and nephrotoxic respectively [peraica et al., 1999]. a good number of known zearalenone producing fusarium species were shown in this work to be toxic to mice. zearalenone, an oestrogenic toxin causes infertility in animals and is associated with outbreaks of precocious pubertal changes in children in puerto rico and has been suggested to have a possible involvement in human cervical cancer [miller and trenholm, 1994]. the other mycotoxins elaborated by fusarium spp are; trichothecenes, culmorins, enniatins, fusarins, fumonisins moniliformin, butenolide and chlamydosporol [marasas, 2001]. trichothecenes are protein inhibitors with consequent immunosuppressive effects causing severe damage to digestive tract and death due to intestinal haemorrhage [beardall and miller, 1994]. the commonest trichothecenes are deoxynivalenol (don) and t-2 toxin. don was the causatic agent of a large-scale incident of human toxicosis in the kashmir valley, india in 1988, and acute toxicosis of don has been reported in china, japan, and korea among other countries [beardall and miller, 1994]. fumonisins especially fb1 cause liver and kidney cancer, and neural tube defects in rodents, leukoencephalomalacia in equine and pulmonary oedema in pigs [wilson et al., 1984]. the association of fb1 with elevated incidence of human oesphageal cancer in parts of south africa, north eastern iran and china, upper gastrointestinal tract cancer in northern italy and neural tube defects in human babes [wilson et al., 1984] is a major public health concern. the international agency for research on cancer classifies fumonisins as possible human carcinogens (category iib). trichoderma spps have been found as fungal contaminants of sorghum, maize, acha and rice in nigeria and japan [gbodi, 1986; uraguchi and yamazaki, 1978]. the mycotoxins produced by trichoderma spp are numerous and they include alamethicins, chrysophanol, emodin, ergokonin, gliotoxin, gliovirin, g-protein, harzianum a, heptelidic acid, isocyanocyclopentenes, koninginins a,b,c,g, aracelsin, saturnisporin, suzukacillin, trichodermin, trichorzianines a and b, trichothecenes, trichotoxin, trichoviridin and viridin [mycotoxin, 2005; uraguchi and yamazaki, 1978]. however, satratoxin h, trichodermol, trichodermin and t2 are the most elaborated and toxic. satratoxin h is an immunosuppressant that causes abortogenicity in animals while the other three are inhibitors of protein synthesis and cause damage to the gastrointestinal tract and haemoglobin of animals and man [prelusky and rotter, 1994; miller and trenholm, 1994]. the high incidence of mucor and alternaria spp in both mouldy guinea corn and rice, and their proven toxicity in mice suggest the likely presence of metabolites of these fungi in the grains. rhizonin a secreted by mucor spp has deleterious effects on the kidney and liver of mice and rats [visconti and sibilia, 1994]. moulds of the genus, alternaria elaborate many toxins but mainly cytochalasins and tenuazonic acid [mycotoxins, 1997] which have been implicated in human haemorrhage disease, ‘onyalia’ in south africa [beardall and miller, 1994]. literature search reveals that no mycotoxin has been associated with colletritrochum spp, geotrichum candidum, c. neoformins and syncephalastum spp, however, syncephalastrum spp are known to cause allergy to man [mycotoxin, 2005] while g. candidum can cause geotrichosis, a secondary infection in association to tuberculosis which is a rare disease that causes lesions of the skin, bronchi, mouth, lung and intestine [beardall and miller, 1994]. fifty three fungal isolates mainly species of aspergillus, fusarium, penicillium, alternaria and trichoderma which are known mycotoxin producing fungi were found to be non toxic in this study. these could be the non toxigenic strains of the species that do not have the genetic capacity to produce mycotoxins [who, 1999]. since sub-culturing can cause mutation which could lead to loss of ability to produce toxin [gbodi, 1986], it is possible that some of the potential toxic fungi lost their potency as a result of subculturing. they might also be the temperate strains of the species, of which the tropical climate of niger state could have been unsuitable for optimal mycotoxin synthesis. data from the preliminary acute toxicity studies shows that f. verticllioides (sacc.) nirenberg produced one of the most toxic metabolites and that of the thirteen most toxic fungi identified in guinea corn and rice in the state, available information on its toxicity in literature is the least. the fungus is a known producer of mainly fumonisins however there are reports that it also elaborates trichothecenes [mold-help, 2004]. the toxic effects of these mycotoxins have been discussed in previous paragraphs. fumonisins and any of the trichothecenes acting singly do not cause lethality in mice at low concentration of 40 mg per kilogram body weight [visconti and sibilia, 1994; who, 2000; seleyefubara and jebbin, 2007] as demonstrated in this work. it implies that the culture material of this strain of f. verticillioides is more toxic than pure fumonisin b1, don or t2 toxin and so might contain a new toxin or multiple of the above mentioned toxins in synergistic effect. further work on its toxic effects on experimental animals and the nature and number of toxins elaborated by this very toxic strain of fungus is therefore necessary. acknowledgements this project was partly funded by a grant from the university board of research of federal university of technology, minna, niger state, nigeria. we wish to express our profound gratitude to mallam kudu of the microbiology departmental laboratory, f.u.t, minna. the technical assistance offered by staff of the government veterinary clinic, bosso with the intraperitoneal administration of the fungal extracts to the experimental animals is highly appreciated. references bankole sa, adebanjo a (2004). review: mycotoxins in food in west africa: current situation and possibilities of controlling it. beardall jm, miller jd (1994). disease in humans with mycotoxins as possible causes. in miller jd, trenholm hl (eds) mycotoxins in grains: compounds other than aflatoxin. eagan press. usa. pp. 487-539. carlos am, todd s, marek b, tomasz, ts, amanda, sp (2004). mycotoxins: mechanisms of toxicity and methods of detection for identifying exposed individuals. j. land use. 19(2):537-549. fao (1997). mycotoxins. report of the joint fao/who/unep conference on mycotoxins held in 19 th -27 th september, 1997. printed by food agricult. organiz. food nutr. p.10 gbodi ta (1986). studies of mycoflora and mycotoxins in acha, maize and cotton seed in plateau state, nigeria. a ph. d thesis, submitted to department of physiology and pharmacology, faculty of veterinary medicine, 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(4):275-281. 1 full length research paper moringa oleifera, gliricidia sepium and leucena leucocephala ensiled with molasses as additive: effect on feed intake and digestibility in yankasa rams chukwuka* and safiyanu federal university of lafia, lafia, nigeria accepted 19 april, 2025 the nutritional potential of molasses ensiled moringa oleifera, gliricidia sepium and leucaena leucocephala as additives were compared by feeding them to twenty-four yearling yankasa rams for twelve weeks. the rams were assigned randomly to four treatments. the feed intake, feed conversion ratio, body weight changes and nutrient digestibility as influenced by the four dietary treatments were evaluated. the mean weight gain of rams fed giliricidia sepium ensiled with cassava peels (2.4kg), leucaena leucocephaala ensiled with cassava peels (2.2kg) and moringa oleifera ensiled with cassava peels (2.1kg) were significantly (p<0.05) higher than those fed control diet of ensiled cassava peels only (1.8kg). rams fed diet of cassava peels ensiled only had the least feed conversion ratio of 13.35 while rams fed supplemental gliricidia sepium, leucaena leucocephala and moringa oleifera ensiled with cassava peels had feed conversion ratio of 10.35, 11.29 and 12.0 respectively. the highest feed conversion ratio was observed in rams fed supplemental gliricidia sepium ensiled with cassava peels (10.35). the nitrogen balance values for rams on treatments cp+ gs (4.29g day-1 ), cp +ll (4.02g day-1) and cp+mo (3.64g day-1) were significantly (p<0.05) higher than those rams on control diet of ensiled cassava peels only. rams on treatment cp+gs (54.1g day-1),cp+ll (50.4g day-1) and cp+mo(47.9g day1)had significant (p<0.05) higher values of n-retention than those rams on control treatment( 36.1g day1). the nutrient digestibility of dry matter (61.83%), crude protein (60.73%), ether extract (60.73%), nitrogen free extract (68.0%) and crude fibre (63.70%) of rams on control diet were consistently lower (p<0.05) than those rams fed supplemental gliricidia sepium, leucaena leucocephala and moringa oleifera ensiled materials. rams fed giliricidia sepium, leucaena leucocephala and moringa oleifera ensiled cassava peels had better feed intake, rumen digestion and growth performance compared to rams fed control diet of ensiled cassava peels only. key words: digestibility, conversion ratio, growth rate, nitrogen retention, additives introduction education sheep play a significant role in livelihoods of the rural dwellers in most less developed countries. apart from serving as a vital protein source, it provides income for meeting urgent household needs (peacock et al., 2005). yankasa sheep is a native breed of sheep which occurs in the sahel, sudan and guinea savannah zones of nigeria. they are commonly fed with grasses, and the poor feeding regime has affected the growth rate and performance of yankasa sheep. with the perpetual increase in population of livestock in nigeria, the use of non-conventional feeds is gaining ground. previously, livestock were fed on grains as the main source of energy and soybean meal, fish meal, etc. as sources of protein. this practice has put the livestock industry into direct competition with man for these feed materials (muriu et al., 2002) thereby hindering the productivity of small ruminants; hence, shortage of animal proteins. livestock production is plagued with so many problems which include; low productivity of animals, poor management systems, poor genetic make-up of indigenous animals, non-availability of feed ingredients, shortage of good quality feeds and many more. furthermore the limited supply of raw materials for livestock feed industry has resulted in a continuous increase in the cost of production. author(s) retain the copyright of this article. african journal of food science research issn 2375-0723 vol. 13 (1), pp. 001-006, april, 2025. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 however, during the dry season, scarcity of forages is common problem limiting sheep production in the tropical areas (nwaigwe, 2011).there is shortage of good quality feed needed to sustain this sheep growth and this causes a major constraints to sheep production which can be solved by finding alternative and adequate sources of protein and energy in the concentrate mixture given to these animals. browse plants can be considerably made use of and these browse plants can be moringa oleifera, gliricidia sepium and leucaena leucoceephala. experience in other countries has shown that moringa oleifera can be used as supplement for improving the productivity of sheep which are dependent on low quality roughages as their main diet (makker and becker, 1997; aregheore, 2002). moringa oleifera also known as drumstick treet is a wellknown tree in west africa especially in semi-arid areas where it is often cultivated as a living fence around people’s garden and consumed in various forms as food. the leaves are high in protein, vitamin a and c. its fruits, flowers and leaves all contain 5-10% of protein on the average. however, according to akinbamijo et al. (2004) the value of the tree and its benefits as a highly supplement to low quality roughages in ruminant feeding system have not been fully known nor widely exploited. gliricidia sepium is an extremely versatile plant which can fulfill a number of roles in human nutrition and small holder agricultural production system. gliricidia is used as a green fodder/protein supplement to low-quality forages and by-products for cattle, sheep and goats. leucaena leucoceephala is known for its nutrient density, when fed to livestock. it meets their nutrient requirements for growth and other productive purposes (ekpenyong, 1986). the leaves are palatable and preferentially eaten by animals (malini et al., 1989). molasses which is extremely palatable and an excellent source of energy with appetizer when restricted to a level of 15% (hassoun and ba, 1991). in addition to its use as source of energy, it is also used as a binder to reduce dustiness of ration, to stimulate rumen microbial activity, to serve as a carrier for non-protein nitrogen (npn) and vitamins in liquid supplement. it also serves as an additive that aids feed intake. molasses when used in the process of ensiling reduces the ensiling period, improves the quality of ensiled material, enhance the lactic acid production during fermentation and increase the nitrogen content of the ensiled material. the objective of this study is to evaluate the effect of moringa oleifera, gliricidia sepium and leucaena leucocephala ensiled with molasses as additive on performance characteristics of yankasa rams materials and methods experimental site the experiment was carried out at the small ruminants unit of the teaching and research farm of the ekiti state university, ado-ekiti. the animals were quarantined for 4 weeks and data were collected for 12 weeks. the pens used were cleaned and disinfested with germicide, cleaning of feeders and water troughs were also carried out before the arrival of the animals. procurement of yankasa rams twenty-four yearling rams were purchased from the open markets in lokoja, kogi state of nigeria. they were quarantined for 4 weeks during which routine treatment developed at napri (1984) and modified by aye (1998) was applied. experimental layout and animal management the pen was partitioned into twenty-four equi-dimentional units with plants. the rams were weighed into their experimental units; efforts were made to ensure that all the treatments were balanced in the body weight. the design of the experiment was a completely randomized design. the animals were randomly assigned to 4 treatments and each treatment has 3 replicates of 2 animals each. the rams were fed cassava peels and panicum maximum as the basal diets and experimental diets of ensiled moringa oleifera, gliricidia sepium and leucaena leucocephala with molasses as additive. the treatments were as follows: treatment 1: control diet of ensiled cassava peels only treatment 2: 50% cassava peels ensiled with 50% gliricidia sepium. treatment 3: 50% cassava peels ensiled with 50% leucaena leucocephala. treatment 4: 50% cassava peels ensiled with 50% moringa oleifera. feeds fresh cassava peels were obtained from aba ibira in iworoko ekiti. the fresh cassava peels were sun dried for about 5-7 days depending on the weather condition. panicum maximum was harvested 1metre from the base of the plant with sickle. the stem and leaves were still succulent and not lignified. it was chopped into small pieces with a cutlass so as to prevent wastage by the animals; the grass was wilted for about 2 days to prevent scouring in animals. the leaves of moringa oleifera, gliricidia sepium and leucaena leucocephala were harvested from their plantations at the teaching and research farm, ekiti state university. the leaves were ensiled with cassava peels 3 using molasses as an additive. feeding trial the feeding trials were divided into two periods viz: preliminary period and feeding trial proper. the preliminary period lasted 7 days during which the animals were allowed to adjust to the experimental diets. feeding trial period of 12 weeks when animals were fed basal diets containing cassava peels and panicum maximum supplemented with ensiled moringa oleifera, gliricidia sepium and leucaena leucocephala with molasses as an additive. measured quantities of basal diet and the experimental diets were offered to the animals each day and the quantity left were weighed the following morning to determine daily feed intake. the volume of water was taken using a measuring cylinder before and after drinking and the water intake was recorded as daily water intake. growth rate the weights of the animals were determined using salter scale. weights of the animals were taken at the beginning of the feeding trial and subsequently every week for an assessment of growth rate. nutrient utilization this was carried out by transferring rams into wooden metabolic cage fitted with facilities for separate collection of urine and faeces. each ram was allocated to individual metabolic cage for 21 days. the feed offered, faeces and urine were collected for analysis. the animals were acclimatized for a period of 14 days. collection period for balance studies lasted for 7 days. the animals were fed on basal diets of panicum maximum supplemented with moringa oleifera, gliricidia sepium and leucaena leucocephala ensiled with cassava peels. weight of the basal and experimental diets were taken before and after feeding for 7 days and the feed intake recorded. clean and fresh water volume taken before and after drinking and the water intake determined for 7 days. 20cm3 of 10% concentrated h2so4 was added to each bowl which was used to collect the urine of animals to prevent nitrogen loss and bacteria growth infestation. the volume of urine was measured and recorded daily each morning. 10% of the urine collected from each animal was poured into a well-labeled urine collection bottle and kept in freezing cabinet prior to laboratory analysis. the faeces were collected from individual animal. 10% of the faeces collected immediately the faeces were voided daily over the 7-day period were bulked and weighed and used for moisture determination. the remaining faeces were oven dried at 700c for 36 hours,milled and stored in air tight bottles. analytical procedure dried milled sample of feeds and faeces were analysed for their proximate composition according to aoac (2005). nitrogen contents of feed, faeces and urine were determined by the micro-kjeldahl technique using the marham’s distillation apparatus. results obtained were used for the calculation of digestibility of nutrients and nitrogen balance for the experimental animals. statistical analysis data collected were subjected to anova the glm (general linear model) option minitab software version 13.2 (steel and torrie, 1980). means were separated using duncan’s multiple range test (duncan, 1955). results and discussion experimental diet composition table 1. shows the proximate composition of the ensiling materials used in the study. cassava peels used in this study contained 85.7% dry matter (dm), 6.5% crude protein (cp), 22.6% fibre (cf), 1.1% ether extract (ee), 58.3% nitrogen free extract (nfe) and 3.80% ash. the gliricidia sepium used contained 78.56% dry matter (dm), 27.40% crude protein (cp), 15.80% crude fibre (cf), 5.80% either extract (ee), 39.50% nitrogen free extract (nfe) and 5.8% ash. the leucaena leucocephala used contained 84.50% (dm), 26.58% (cp), 14.96% (cf), 6.20% (ce), 37.83% (nfe) and 4.60% ash. the moringa oleifera used contained 84.32% dm, 23.58% cp, 12.83% cf, 6.30% ee, 32.64 nfe and 5.10% ash. table 2. shows the proximate composition of silage made from cassava peels, moringa oleifera, gliricidia sepium and leucaena leucocephala. the ensiled cassava peel only, used in the study contained 26.59% dry matter (dm), 5.2% crude protein (cp), 18.56% crude fibre (cf), 0.86% ether extract (ee), 60.30% nitrogen free extract (nfe), 2.85% ash, 0.32% ca(mgkg-1), 0.11% p(mg kg-1) and ph of 5.1. the gliricidia sepium ensiled cassava peels used contained 4 32.26% dry matter (dm), 22.23% crude protein (cp), 12.56% crude fibre (cf), 3.20% ether extract (ee), 36.50% nitrogen free extract (nfe), 4.2% ash, 0.30% ca(mgkg-1), 0.11% p(mg kg-1) and ph of 5.1. the leucaena leucocephala ensiled cassava peels used contained 34.56% dry matter (dm), 25.17% crude protein (cp), 12.03% crude fibre (cf), 3.56% ether extract (ee), 33.80% nitrogen free extract (nfe), 3.80 ash, 0.28% ca(mgkg-1), 0.10% p(mg kg-1) and ph of 4.7. the moringa oleifera ensiled cassava peels used contained 32.65% dry matter (dm), 20.32% crude protein (cp), 40.02% crude fibre (cf), 4.02% ether extract (ee), 34.56% nitrogen free extract (nfe), 4.26% ash, 0.19% ca(mgkg-1), 0.10% p(mg kg-1) and ph of 4.7. body weight changes table 3. shows the effect of the various dietary treatments on the live weight gain, feed conversion ratio and dry matter intake (dmi) of yankasa rams. the dry matter intake (dmi) of rams fed ensiled cassava peels (control) only, gliricidia sepium ensiled with cassava peels, leucaena leucocephala ensiled with cassava peels and moringa oleifera ensiled with cassava peels were 286g day-1, 307g day-1, 296g day-1 300g day-1 respectively with the animals fed supplemental gliricidia sepium ensiled with cassava peels having the highest dry matter intake of 307g day-1. the weight gain of the rams fed control diet of cassava peels ensiled only was significantly lower (p<0.05) (1.8kg) compared to those rams fed ensiled cp + gs, cp + ll and cp + mo (2.4kg, 2.2kg and 2.1kg respectively). the metabolic weight gain of rams were 0.6kg, 0.9kg, 0.8kg and 0.8kg for rams fed ensiled cassava peels only, ensiled cp+ gs, cp +ll and cp + mo respectively. the feed conversion ratio of rams fed experimental diets of ensiled cassava peels only, ensiled cp + gs, cp + li and cp + mo were 13.35, 10.53, 11.29 and 12.0 respectively. the supplemental ensiled materials were significantly (p<0.05) different the control treatment. feed intake table 4 shows the mean feed intake of rams fed experimental diets of ensiled cassava peels only (control), gliricidia sepium ensiled with cassava peels, leucaena leucocephala ensiled with cassava peels and moringa oleifera ensiled with cassava peels. the mean feed intake of rams fed ensiled cp + gs, cp + li and cp + mo were 2.2 + 0.49, 1.31 + 0.45 and 1.65 + 0.31 respectively which were lower than the mean feed intake of rams fed control diet of ensiled cassava peels only ( 2.55 + 0.52). this reveals that the experimental diets of ensiled cp + gs, cp + li and cp + mo enhance better performance than the control diet of ensiled cassava peels only. rumen digestibility the dry matter (dm) and nitrogen free extract (nfe) of yankasa ram fed leucaena leucocephala ensiled with cassava peels of 69.40% (dm) and 76.5% (nfe) were higher than those rams fed ensiled cassava peels only gliricidia sepium ensiled with cassava peels and moringa oleifera ensiled with cassava peels had 61.83% (dm) and 68% (nfe), 68.26% (dm and 75.32% (nfe) and 66.0% (dm) and 68.56% (nfe) respectively. the crude protein (cp) of rams fed ensiled cp + gs was significantly (p<0.05) higher (70.39%) than those rams fed ensiled cassava peels only, cp + li and cp + mo (60.37%, 65.85% and 65.0% respectively). the crude fibre of rams fed ensiled cassava peels only and ensiled cp + li at 64.48% were significantly (p<0.05) higher than those rams fed ensiled cp + gs and cp + mo (59.32% and 59.20% respectively). the digestibility coefficient of ether extract (ee) values of rams fed the experimental diet of ensiled cp + gs was significantly (p<0.05) higher 67.3% than those rams fed ensiled cassava peels only, cp + li and cp + mo that had 60.73%, 61.62% and 63.90% respectively. nitrogen utilization the nitrogen utilization of ensiled cassava peels only, ensiled cp + gs, ensiled cp + li and ensiled cp + mo were presented in table 6. the means values of faecal nitrogen output (g/day) for rams fed control diet of ensiled cassava peels was significantly (p<0.05) lower (1.05g/day) compared to those fed ensiled cp + gs, cp + li and cp + mo that were 2.36, 2.38 and 2.16 g/day respectively. the digested nitrogen (g/day) values for rams fed ensiled cp + gs, cp + li and cp + mo were not significantly different (p<0.05) but were significantly (p<0.05) higher than the mean values for rams fed control diet of ensiled cassava peels only at (4.04 g/day). the nitrogen retention values for sheep fed ensiled cp + gs, cp + li were not significantly different (p<0.05) but were significantly higher (p<0.05) than the value for sheep fed ensiled cp + mo which in turn was significantly higher than the control diet of ensiled cassava peels only. discussion the proximate values of cassava peels ensiled only, obtained in this study are at variance with the result obtained by ukanwoko and ukandu (2011). the cassava peels ensiled only used in this study were 5 26.59 g 100g-1 dry matter (dm), 5.20g 100-1 crude protein (cp), 18.56 100g-1 crude fibre (cf), 0.86 g 100-1 ether extract (ee), 60.30 g 100-1 nitrogen free extract (nfe), 2.85g 100-1 ash, 0.32 (mg kg-1) calcium, 0.11 (mg kg-1) phosphorus and ph of 5.1. the dry matter (dm) value of cassava peels ensiled only 26.59g 100-1 obtained in this study is lower compared to 65.11g 100-1 reported by ukanwoko and ukandu (2011). the crude fibre (cf) value of 18.56g 100 1 was higher in comparison to the value of 17.21g 100-1 obtained by ukanwoko and ukandu (2011). the crude protein (cp) value of 5.20g 100-1 obtained in this study was higher than 4.5 g 100-1 reported by ukanwoko and ukandu (2011). the ether extract (ee) value obtained in this study 0.86 g 100-1 was in line with the ether extract value of 0.85 g 100-1 obtained in this study was in line with the value of 2.86 g 100-1 obtained by ukanwwoko and ukandu (2011). the values of calcium and phosphorus 0.32 ca(mg kg-1), 0.11 p(mg kg-1) obtained in this study was lower compared to the values 0.85 ca(mg kg-1), 0.14 p(mg kg-1) obtained by ukanwoko and ukandu (2011). the ph value (5.1) of the cassava peels ensiled only obtained in this study was higher than (4.32) reported by ukanwoko and ukandu (2011). the difference in the values obtained from this study with those of other workers or researchers might be due to the stage of harvest of crops, soil types and processing method adopted. rumen digestibility the higher nutrient digestibility in rams fed the supplemental diets of gliricidia sepium ensiled with cassava peels, leucaena leucocephala ensiled with cassava peels and moringa oleifera ensiled with cassava peels indicates that the rumen functions were better than that of rams fed control diet of ensiled cassava peels. the significant higher nutrient in ensiled cp + gs, cp + ll and cp + mo compared to the control diet of ensiled cassava peel only as observed in this study was due to the difference in the composition of treatments which appear to favour proper functioning of rumen micro organism in sheep under these treatments. this result shows that the yankasa sheep fed ensiled cp + gs, cp +ll and cp + mo respectively had a better rumen digestibility than those fed control diet of ensiled cassava peels. feed intake the higher mean feed intake (2.55 + 0.52) observed in rams fed control diet of ensiled cassava peels only compared to rams fed gliricidia sepium ensiled with cassava peels, leucaena leucocephala ensiled with cassava peels moringa oleifera ensiled with cassava peels shows the detrimental effect of not ensiling cassava peels with either gliricidia sepium, leucaena leucocephala or moringa oleiifera. body weight changes the sheep fed control diet of ensiled cassava peels only in the study has lower relative weight gain hence, less body weights and metabolic live weight gains compared to sheep fed experimental diets of gliricidia sepium, leucaena leucocephala and moringa oleiifera with cassava peels. this shows the effect of the treatment on weight gain and general production performance. the animals fed gliricidia sepium, leucaena leucocephala and moringa oleiifera ensiled with cassava peels had higher weight gains (2.4kg) in cp + gs, (2.2kg) in cp + ll and (2.1kg) in cp + mo compared with the weight gain in control (1.8kg). conclusion this study demonstrates that sheep fed moringa oleiifera, gliricidia sepium, and leucaena leucocephala ensiled with cassava peels had better performance than those fed control diet of ensiled cassava peels only. this shows that ensiling moringa oleiifera, gliricidia sepium, and leucaena leucocephala with cassava peels have enhance the performance of the sheep by efficiently improving the feed intake and digestibility of the animal. this study clearly demonstrates that moringa oleiifera, gliricidia sepium, and leucaena leucocephala ensiled with cassava peels in feeding ruminants is a strategy in overcoming dry season weight losses and poor performance in sheep fed cut and carry fodder. references akinbamijo oo, adediran sa, novala s, saecker j(2004), moringa fodder in ruminant nutrition in the gambia. international trypernotolerance centre, banjul, the gambia. aoac (2005) official methods of analysis. 16th edu. (association of official analytical chemists arlington, va) aregheore em(2002), intake and digestibility of moringa olefiferabatiki grass mixtures by growing goats. small ruminants research46:23-28. aye pa(1998). the effect of two management system in some physiological parameters and growth rate of the west african dwarf goats (wad) m. tech thesis federal university of technology, akure, nigeria. pp.92. duncan db(1955) multiple range and multiple f tests. biometrics, 11:1-42 ekpenyoung te(1986)nutrients and amino acid composition 6 of leucanaeleucophala. anim. feed sci. and tech. 15:183-189. hassoun p, ba aa(1991). mise all point d’nue technique de fabrication de blocks multinutrionnels sams me lasse.livestock res.ruraldev., 2(2). makkar hps, becker(1996) nutritional value and antinutritional components of whole and extracted moringa oleifera leaves. anim. feed sci. technol. 63,211218. malini bp, doss rg, growda mkm, golvindaiah mg(1989) preliminary studies on the effects of feeding leucaena leucocephala to rabbits. leucaena res. reports vol. 10:21-31. muriu ji, njoka en, tuitoek jk, nanna jn(2002). evaluation of sorghum (sorghum bicolor) as replacement for maize in the diet of growing rabbits (oryctolagus cuniculus). asian-aust. j. anim. sci., 15 (4):546-569. napri (1984) highlights of research achievements on animal production. science and technology briefing lagos december 1984 pp. 3-17. nwaigwe co, nwankpa ca, nwaigwe cu(2011) the effects of sawdust/ poultry litter and brachiaria ruziziensis silages on the performance of west african dwarf goats. asian j. biotechnol. 3:581-587 peacock c, devendra c, ahuya c, roets m, hossain m, osafo e (2005) goats. in: owen e, kitalyt a, jayasuriya n, smith t (eds.) livestock and wealth creation: improving the husbandry of animals kept by resourcepoor people in developing countries. nottingham university press, united kingdom. pp 361-385 steel rgb, torrie dh(1980) principle and procedures of statistics. mcgraw hill book co. ny. ukanwoko ai, ukandu c (2011) continental j. animal production and veterinary res.3 (2) : 36-40. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 9 (6), pp. 001-007, june, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper seasonal variation of fixed and volatile oil percentage of four eucalyptus spp. related to lamina anatomy kh. s. emara1 and a. emad shalaby2* 1 department of agricultural botany, faculty of agriculture, cairo university, giza, egypt, 12613. 2 department of agricultural biochemistry, faculty of agriculture, cairo university, giza, egypt, 12613. accepted 21 january, 2021 this experiment was conducted during the four seasons: spring, summer, autumn and winter of two successive annual cycles; 2008/2009 and 2009/2010 (starting from may 2008). four eucalyptus species were under investigation; eucalyptus camaldulensis dehnh., eucalyptus cinerea f. muell. ex bentham, eucalyptus citriodora hook. and eucalyptus globulus labill. seasonal variations in the amount of fixed and volatile oils in eucalyptus spp. matured leaves were investigated. it was determined that the amount of total lipids and essential oils significantly varied by the seasons (p < 0.01). the amount of total lipids in eucalyptus spp. reached its peak mostly in spring. but the amounts of essential oils in different species were determined to be higher in summer, autumn and spring seasons, than in winter. furthermore, the amount of total lipids and essential oils was higher in e. camaldulensis and e. cinerea than in other species. the anatomical investigation in the four studied eucalyptus species, in relation to lipids percentage indicated that, the best lipids percentage amounts in this study were exhibited in e. cinerea and e. camaldulensis, for spring and winter; and were in agreement with these species highest lamina thickness. in general, fluctuation in lipids percentage is more correlated to the internal structure of lamina (duct average diameter, ducts total numbers, and open ducts numbers) in the same season; whereas, among seasons, it is thought that metabolism contributed more greatly. cuticle thickness is true correspondence to seasonal environmental fluctuation, since it increases in all species, by shifting up from spring to summer then decrease to winter. essential oils secretion which coincided with lipids percentage may be due to environmental stress influence over metabolism rather than structural adaptation. key words: eucalyptus, seasons, fixed and volatile oils, lamina, anatomy, glands. introduction eucalypti species are mostly evergreen trees of immense size; around 800 species belong to the magnoliopsidous family, myrtaceae and native mainly to australia and some neighboring islands (bailey, 1958; cronquist, 1981; heywood, 1993; brooker, 2000). they are grown for their ornamental values, as windbreaks, for timber and fuel, and for oil, distilled from leaves, which is secondary compounds with pleasant aroma used as fragrance components in soap, detergents *corresponding author. e-mail: dremad2009@yahoo.com. tel: +202-0101203313. fax: +202-37742600. and toiletries, and also have pharmacological properties (santos et al., 2008). eucalyptus oil is being extracted in many countries like china, india, south africa, portugal, brazil and tasmania on commercial scale. in 1992, the world eucalyptus oil production was estimated to be 4000 tons of which 60 to 70% was consumed in medicinal market (bhatti et al., 2007). eucalyptus leaves oil amounting to less than 20% is enough in the commercial point of view, and only 10% of these accounts for the entire world production of essential oil (peter, 2000). essential oils are complex mixtures which comprised primarily of the lower classes of terpenes (julia, 1992; langenheim, 1994). the secondary compounds are probably co-evolution result from interaction of plants 2 table 1. the effect of different seasons on the total lipids content (%) of the four studied eucalyptus spp. during the two successive annual cycles, 2008/2009 to 2009/2010. season species e. camaldulensis e. cinerea e. citriodora e. globulus spring 18.0 b 18.75 a 16.0 a 13.0 a summer 13.2 c 13.5 d 11.0 c 11.75 c autumn 13.0 c 13.75 c 10.75 d 11.25 d winter 19.0 a 15.75 b 14.75 b 12.75 b lsd at 0.01 0.431 0.183 0.236 0.174 values with different superscript letters within the same column are significantly different (p < 0.01). versus pathogens, herbivores, pollinators (croteau et al., 2000). although monoterpenes predominate in most essential oils, many also contain sesquiterpenes. many sesquiterpenes play defensive roles in plant-insect and plant-fungal interactions (samuelsson, 1999). oil concentration is limited by gland capacity and density, which in turn is linked to leaf area reduction and increment in its thickness; thicker leaves were more likely to be smaller in area than thinner leaves, suggesting the expansion of leaves in one plane to a degree mutually exclusive of expansion in the other. aside glands, probable sink of hydrocarbon monoterpenes in the leaves may be the leaf cuticle through which significant portion of monoterpenes is volatilized (king et al., 2006). thicker epidermis and presence of two or more layers of palisade cells in leaves are the xeromorphic features adapting to water limited environments like drought (iftikhar et al., 2009). mediterranean climate is characterized by the incidence of two stress periods; summer drought and winter cold (nahal, 1981). seasonal variations in the composition and concentration of sequestered oils have been observed (silvestre et al., 1997). there are several reports that the concentration of terpenoids in aromatic plants varies during the day and seasons (hendriks et al., 1997). the aim of this study is to investigate the effect of seasonal fluctuations over the year on oil yield, fixed and volatile, of perennial evergreen tree (eucalyptus), related to varying anatomical features in order to suggest best species (among studied ones) and season for harvesting oil from leaves. materials and methods this experiment was conducted during four seasons; spring, summer, autumn and winter of two successive annual cycles; 2008/2009 and 2009/2010, at the laboratories of agricultural botany and agricultural biochemistry departments, faculty of agriculture, cairo university. collection of samples the used plant materials were four eucalyptus species (quattrocchi, 2000); red gum (eucalyptus camaldulensis dehnh.), argyle apple ( eucalyptus cinerea f. muell. ex bentham), lemonscented gum (eucalyptus citriodora hook.) and blue gum (eucalyptus globulus labill.). the leaf materials were gotten from the gardens of the faculty of agriculture, cairo university. fully expanded mature leaves were cut at the mid-date of each season (starting from may 2008) and gathered early in the morning. the leaves were pre-cooled (4°c) and wrapped in kraft paper in bunches, and then transported under dry conditions to the laboratories within two hours. seasonal effects on essential oils and lipids content (percentage) of different eucalyptus species were investigated, and then correlated with lamina anatomical correspondence to affected by climatic fluctuation over the seasons. extraction and determination of total lipids lipids were extracted by a modified method described by xu et al. (1998). the cells (ca. 5 g) were extracted twice with a mixture of distilled water (h2o), chloroform and methanol (8:10:20, v/v/v) and sonicated for 10 min using a microtop of microson ultrasonic cell disrupter. then, the sonicated cells were filtered through gf/c whatman glass microfiber (47 mm). chloroform (10 ml) and distilled water (10 ml) were added sequentially to the filtrate and sonicated again for 10 min. the resultant solution was filtered under vacuum through whatman glass filter microfiber. the filtrate was washed by 30 ml of 5% nacl solution, and then, the chloroform layer was separated and dried over anhydrous sodium sulfate. the solvent was removed through evaporation at 40°c under reduced pressure. then, the total lipids were weighed and stored at -20°c until analysis. essential oil extraction plant samples (100 g) were hydro-distilled in clevenger-type apparatus (council of europe, 1997). the essential oil samples were stored in the dark at 4°c. the amount of oil (table 1) obtained from plant material was calculated as: oil (% v/w) = observed volume of oil (ml)/weight of sample (g) × 100 anatomical studies leaves were randomly chosen as specimens for anatomical study over the four successive seasons of the year. the micro technique procedures given by nassar and el-sahhar (1998) were applied. specimens were killed and fixed for at least 48 h in formalin aceto alcohol (faa) solution. then, materials were washed in 50% ethyl 3 table 2. the effect of different seasons on essential oil content (%) of the four studied eucalyptus spp. during the two successive annual cycles, 2008/2009 to 2009/2010. season species e. camaldulensis e. cinerea e. citriodora e. globulus spring 1.78c 1.35c 0.60c 0.10c summer 2.5a 1.95a 1.8a 0.66a autumn 1.92b 1.50b 1.05b 0.23b winter 0.98d 1.0d 0.25d 0.05d lsd at 0.01 0.033 0.086 0.033 0.028 values with different superscript letters within the same column are significantly different (p < 0.01). alcohol and dehydrated in normal butyl alcohol series before being embedded in paraffin wax (mp 52 to 54°с). transverse sections, which were cut on a rotary microtome to a thickness of 20 , were stained with safranin/light green before mounting in canada balsam. slides were examined; measurements of different tissues were recorded using light microscope with micrometer eye piece and micrometer stage, and then photomicrographed using microscope olympus ax70 made in japan. the following anatomical characters were measured: cuticle thickness (µ), lamina thickness (µ), opens ducts number (in 40 x field), ducts total number [in 10x (eye lens) х 4x (object lens) field] and duct diameter (µ). statistical analysis data were subjected to an analysis of variance. combined analysis was used for the two annual seasonal cycles. means were compared by calculating the least significant difference (lsd) values at alpha 1% according to snedecor and cochran (1982). results and discussion chemical studies the amount of total lipids obtained from various eucalyptus spp. during different seasons was investigated, and it was observed that values were between 10.75 to 19.0%. it scored the minimum level in autumn season and increased significantly, till it reached the maximum at spring season in all studied species, except for e. camaldulensis which had maximum value in winter (table 1). species e. camaldulensis and e. cinerea reflect best lipids amount (19 and 18.75%, respectively). the variations of total lipid contents were affected by the species, age, water temperature, nutritional condition and seasonal variation, as confirmed by folch et al. (1957), gill and weatherley (1984), lathi (1987) and christiansen et al. (1989). recent studies have shown that lipids and fatty acids composition was influenced by seasonal variations (yılmaz et al., 1995). the same trend shows that, the variations of the levels of lipids are the results of irregular seasonal variations and water temperature was determined by dutta et al. (1985), agren et al. (1987) and yılmaz et al. (1995). from table 2, it can be seen that the content of essential oils in different seasons varied from 0.05% during winter to 2.5% during summer season. best essential oils percentages were estimated in e. camaldulensis and e. cinerea (2.5 and 1.95% respectively) in the summer season. data from table 2 indicated that, the level of these components reached its maximum in all studied plant species during the summer season and had the lowest value during winter, and this may be due to the physical and chemical stress on plant especially during summer drought. this stress led to plant secretion to different defense components called secondary metabolites, as protecting agents, especially terpenoid compounds (essential oils). these results are in agreement with the results obtained by samuelsson (1999). as to the significance estimates, more consistent trend in percentages variation of eucalyptus spp. during different seasons are present for essential oils than for lipids, and this further confirm the formers are due mainly to the effect of environmental condition on plant synthesis. wildy et al. (2000) investigated four promising eucalyptus species reported 0.01 to 13.0% for oil production, from western australia at six locations. also, zafar et al. (2003) reported similar results; that is, 0.58 to 1.47%, regarding oil potential for the different eucalyptus species. these variations might be attributed to different agro-climatic regions and soil composition in the districts of punjab. anatomical studies aiming to explain the seasonal differences affecting lipids content (%) of the four studied species, microscopical counts and measurements of certain characters were recorded to investigate the internal differences in lamina of these species. the most important criteria in this study were to compare fluctuations in glands (number and size) over seasons, in random specimen’s descent by half meter from the top, in addition to measure the cuticle and lamina thickness as correspondence to heat/cold stress. microphotographs of transverse sections illustrating four studied eucalyptus spp. (plate 1) showed that 4 plate 1. transverse sections in leaves of the four studied eucalyptus spp. (x = 9). mesophyll of adult vertically positioned lamina is often isobilateral, with cells containing tannin abundant in all unlignified tissues and bicollateral vascular bundles. epidermis coated by wax lamina characterized by the presence of secretory cavities, which are found below the epidermis on either sides of the lamina. cavities are schizolysigenous in origin; lined with distinct epithelium when young which soon is obliterated by compression. cavities are responsible for secreting oily substances. data presented in table 3 and plate 1 for the leaf specimens subjected to histological investigation cleared that as typical correspondence to seasonal environmental fluctuation, cuticle thickness increase in all species, by shifting from spring to summer, then decrease to winter, 5 table 3. different tissue counts and measurements (µ) in lamina of the four studied eucalyptus spp. during the two successive annual seasonal cycles, 2008/2009 to 2009/2010 (average of 5 readings). season measurement (µ) species e. camaldulensis e. cinerea e. citriodora e. globulus average cuticle thickness 18.33 10.83 9.17 9.17 average lamina thickness 466.67 323.33 423.33 213.33 spring open ducts number 7 6 1 2 ducts total number 9 6 3 3 duct average diameter 135 152.5 125 85 average cuticle thickness 25 13.33 15 10.83 average lamina thickness 456.67 290 400 290 summer open ducts number 5 3 4 3 ducts total number 7 5 4 3 duct average diameter 157.5 172.5 115 150 average cuticle thickness 16.67 9.17 15 10 average lamina thickness 430 290 400 333.33 autumn open ducts number 5 5 4 3 ducts total number 6 6 4 3 duct average diameter 147.5 172.5 105 117.5 average cuticle thickness 13.33 8.33 15 6.67 average lamina thickness 463.33 350 486.67 290 winter open ducts number 3 3 3 4 ducts total number 5 5 5 5 duct average diameter 187.5 140 125 110 except for e. citriodora which also increase in summer (from 9.17 µ) but score the same thickness throughout the rest two subsequent seasons (15 µ). lamina thickness varied over the seasons (by shifting, in samples, from spring to winter) for various studied species by what could be named 3 types; decreasingsustaining-increasing type as in e. cinerea and e. citriodora; increasing-increasing-decreasing type as in e. globulus and finally, decreasing-decreasing-increasing type as in e. camaldulensis. in harmony with estimated lipid amount percentage, e. cinerea and e. camaldulensis exhibited their highest lamina thickness in spring and winter. in a trial to investigate the association between lipids percentage and the most direct contributed anatomical features to it, average diameter and number of glands were studied. in the same season, referring to table 3, plate 1 and figure 1, it could be realized that, average diameter, total number of ducts, and also, open ducts number have positive relation with lipids percentage, for all species understudied. over seasons, for each individual species, it could be determined that, open ducts number (also total number) showed limited range. relation over seasons could not be distinguished as clear as in the same season, due to inconsistent expression of these features. it could be interpreted that, physiological balance between anabolism and catabolism activities, due to seasonal growth rate, has the principal role in determining the amount of stored lipids (within cavities) over seasons. also, it could be noticed that, mostly, duct average diameter increases by removing from spring to summer samples, then decreased over autumn to winter. as coincide to this relation, e. citriodora decrease in summer and increase in winter, whereas, duct average diameter of e. camaldulensis increase in both summer and winter samples. the anatomical investigation in the four studied eucalyptus species, in relation to lipid percentage indicated that, fluctuation in lipids percentage is more correlated to internal structure of lamina (ducts average diameter, ducts total numbers and open ducts numbers) in same season, whereas between seasons it was thought that, metabolism contributed more greatly. best lipid percentage amounts in e. cinerea and e. camaldulensis in spring and winter, and were in agreement with these species highest lamina thickness. cuticle thickness is true correspondence to seasonal environmental fluctuation, since it increases in all species, by shifting from spring to summer, and then decrease to winter. 6 figure 1. magnified portion of transverse sections in lamina of the most higher studied eucalyptus spp. in lipids amount for optimal season (x = 28). as regards essential oils, it was clear that they coincides lipids percentage since their highest amount was estimated in summer, and then autumn, whereas lowest amounts were in winter, and spring, for all studied species. this may confirm that, essential oils secretion is due to environmental stress influence over the metabolism rather than structural adaptation. the previously mentioned structure of eucalyptus lamina is in agreement with those given by metcalfe and chalk (1950) as well as by bailey (1958), king et al. (2006), santos et al. (2008) and iftikhar et al. (2009). conclusion these studies confirmed that, eucalyptus spp. is a potential source of oil (volatile and fixed) around the year, with regard to significant fluctuation in oils yield due to subsequent seasonal climatic conditions. putting economical factor into consideration, e. camaldulensis and e. cinerea showed best amount of total lipids and essential oils than other studied species. for purposes mostly industrial, yielding these two species in spring and winter was best (biggest lipid percentages (%)), whereas, for other usages, mostly medical, extracting rich essential oil yield is more preferable in summer and autumn. the anatomical investigation indicated that, fluctuation in lipids percentage was more correlated to internal structure of lamina (ducts average diameter, ducts total numbers, and open ducts numbers) in same season, whereas between seasons it was thought that, metabolism contributed more greatly. cuticle thickness is a true correspondence to seasonal environmental stress; same applies to essential oils secretion, which increased from spring to summer, and then decrease in winter. references agren j, mute p, hannınen o, harranen j, pentila i (1987). seasonal variations of lipid fatty acids of boreal freshwater fish species. comp. biochem. physiol., 88b: 905-909. bailey lh (1958). the standard cyclopedia of horticulture. the macmillan company, new york, usa, p. 3640. bhatti hn, iqbal z, chatha sa, bukhari ih (2007). variations in oil potential and chemical composition of eucalyptus crebra among different districts of punjab–pakistan. int. j. agric. biol., 9(1): 136138. brooker mih (2000). a new classification of the genus eucalyptus l’hér. 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cholesterol of liver and muscle in some fish species, especially vendace (coregonus albula l.) in finland. arch. hydrobiol., 110: 133-142. metcalfe cr, chalk l (1950). anatomy of the dicotyledons; leaves, stem and wood in relation to taxonomy with notes on economic uses. the clarendon press, oxford, uk, 1: 790. nahal i (1981). the mediterranean climate from a biological viewpoint. elsevier, amsterdam, pp. 63-86. nassar ma, el-sahhar kf (1998). plant microtechnique. academic bookshop, egypt, p. 224 (in arabic). peter sa (2000). the eucalyptus oil industry. felton grimmwade bickford pvt. ltd. 61 – 69. clarinda road oakleigh south vic. 3169 australia. health, 29: 368-371 quattrocchi u (2000). crc world dictionary of plant names. crc press, washington d.c., usa, p. 2298. samuelsson g (1999). drugs of natural origin, a textbook of pharmacognosy, swedish pharmaceutical press, p. 256. santos ld, thadeo m, iarema l, meira rm, ferreira fa (2008). foliar anatomy and histochemistry in seven species of eucalyptus. r. árvore, viçosa-mg, 32(4): 769-779. silvestre ajd, cavaleiro jas, delmond b, filliatre c, bourgeois g (1997). analysis of the variation of the essential oil composition of eucalyptus globulus labill from portugal using multivariate statistical analysis. ind. crops prod., 6: 27-33. snedecor gw, cochran wg (1982). statistical methods. the iowa state univ. press., ames., iowa, usa, p. 507. wildy d, t. john sp, bartle jr (2000). variation in composition and yield of leaf oil from alley framed oil mallees (eucalyptus spp.) at a range of contrasting sites in western australia. for. ecol. manage., 134: 205-217 xu x, beardall j, hallam dn (1998). modification of fatty acid composition in halophilic antractic microalgae. phytochem., 49: 12491252. yilmaz ö, konar v, celik s (1995). elazig hazar lake capoeta some tissues of female and male members who umbla'nℵn capoeta total lipid and fatty acid compositions. biochem. j., 20: 31-42. zafar i, hussain i, hussain a, ashraf my (2003). genetic variability to essential oil contents and composition in five species of eucalyptus (niab), faisalabad, pakistan. pakistan j. bot., 35: 843-852. full length research paper the approximate composition of cowpea (vignaunguiculata (l.) walp) grains preserved with solutions of moringa (moringaoleifera) seed oils and neem (azadirachtaindica a.juss) adisa* and chibuike university of nigeria enugu, nigeria. accepted 6 december, 2024 the availability, cost, safety, efficacy, and quality of bioinsecticides are the main reasons small-scale farmers utilize them to manage storage insect pests. the proximate composition of the treated grains is unknown, despite the fact that neem-moringa seed oil mixes have satisfactorily preserved cowpea grains for up to 270 days. the purpose of this study was to ascertain the approximate composition of cowpea grains that had been stored for 270 days after being treated with neem-moringa oil mixes. cowpea was treated with theoil mixes in ratios of 1: 3 and 1: 2 neem:moringa at concentrations of 2.5 µl/g and 5.0 µl/g of cowpea grains. in the conclusion of each 90-day period, proximate composition data was gathered and statistically analyzed. the findings demonstrated that, in contrast to previous treatments, treating cowpea grains with a ratio of 1:3/2.5 and 5.0 µl/g mixture of neem-moringa seed oils had no discernible (p<0.05) impact on the proximate composition of cowpea even after 270 days of storage. according to the study's findings, a neem-moringa seed oil mixture at a ratio of 1:3/5.0µl/gg performed best in terms of its impacts on the proximate composition of the kept cowpea grains. as a result, it is advised to preserve cowpea grains for the greatest possible nutritional benefits. key words: proximate composition, moringa, neem, seed oils, cowpea. introduction author(s) retain the copyright of this article. african journal of food science research issn 2375-0723 vol. 12 (1), pp. 001-008, december, 2024. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ education particularly in the semi-arid west africa savannah, the cowpea (vignaunguiculata (l.)walp), a significant tropical legume crop of african provenance, has become an essential component of the tropical cropping system (asante et al., 2000). it is a drought-tolerant, heat-loving crop that requires little soil fertility (karunasena, 2001). about 70% of the world's estimated 10 million hectares are in west and central africa, with africa alone making up 7.5 million hectares of that total (singh et al., 1996). the crop is regarded as the second most significant food grain legume and provides humans with an inexpensive source of protein. cowpea grain is cheap and nutrient-dense. it has roughly 64% carbohydrates and 25% protein (kormawa, 2000). it is also used as feed for animals. high-value, nutrient-dense forage is what the haulm is (abiotech, 2002). in nigeria, cowpeas rank second in importance among protein sources, after meat (olapade et al., 2003). it is prepared simply, combined with additional items and processed into prepared meals like "akara," a deep-fat fried paste product, or "moimoi," a steamed paste. the necessity and significance of an appropriate storage system are increased by its frequent consumption. the cowpea post-production system (storage and processing) in developing nations is linked to numerous limitations, in contrast to many other crops cultivated in semi-arid tropical regions. a granary full of cowpeas can be destroyed in two or three months by postharvest pests, including weevils (abiotech, 2002). the field of postharvest and storage technology is thought to be seriously challenged by this. cowpea's nutritional value and food processing may also be impacted by insect infestation (abiotech, 2002). many populations worldwide rely on mature dried cowpeas as part of their diet (singh et al., 1995). this legume is widely accessible, pricey, and a staple of the traditional diet. along with cowpeas other legumes are acknowledged as significant protein sources (agazounon et al., 2004). grain and leaves provide β-carotene, proteins, lipids, carbs, and vitamins b and c, all of which are essential for optimum health (enwere et al., 1998). after the grain is removed, the remaining vegetative portion of cowpea plants is used to make hay, silage, and feed for cattle (mamiro et al., 2011). fresh product and processed food petty trading offers rural and urban residents, especially women, the chance to make money (singh et al., 1997). more than half of the plant protein in the human diet comes from cowpeas (vignaunguiculata l. walp). it offers a substantial amount of dietary lysine, which is limited in cereals, and is a rich source of calories, vitamins, and minerals. it gives both people and domestic animals food or a relatively high nutritional value in areas where there is a persistent protein shortage.within a 270-day storage period, the study's goal was to evaluate the nutritional makeup of cowpea grains treated with ratios of 1:2 and 1:3 neem-moringa seed oils at concentrations of 2.5 µl/g and 5.0 µl/g of cowpea grains. materials and methods the modibbo adama university of technology yola grounds provided the hand-picked neem seeds (azadirachterindica a. juss) used in this investigation. the modibbo adama university of technology yola campus in adamawa state, nigeria, and kaltungo local government area in gombe state, nigeria, were also sources of some of the moringa (moringa oleifera) seeds. the plastic storage bowls were bought at jimeta market yola in adamawa state, nigeria, and the cowpea samples (ife brown) utilized came from a farm at modibbo adama university of technology yola. cowpea grains were treated for storage using a blend of neem and moringa seed oils in 1:2 and 1:3 ratios, with a concentration of 2.5µl/g and 5.0µl/g of cowpea grains, respectively. for 270 days, the treated cowpea grains were stored at room temperature (37 oc).data on the proximate composition of the treated and untreated (control) cowpea grain samples were gathered at the conclusion of each 90-day storage period. the aoac (2006) technique was used to determine the crude protein, crude fat, moisture, total ash, and crude fiber of the treated and untreated cowpea respectively. carbohydrates were determined using the difference method. (egan and others, 1981). table 1. proximate composition of cowpea grains treated with neem and moringaseed oils mixture treatment moisture ash fat fibre protein carbohydrate control 14.60 3.36 3.13 2.11 19.71 57.17 1:2/2.5µl/g 10.69 4.89 3.88 2.39 27.10? 51.60 1:2/5.0µl/g 11.71 4.12 3.44 1.50 22.00 57.23 1:3/2.5µl/g 10.22 3.51 3.33 1.61 19.37 61.73 1:3/5.0µl/g 11.33 3.43 3.50 1.67 19.70 60.53 mean 11.71 3.86 3.46 1.86 21.58 57.65 lsd at 0.05 0.58 0.16 1.11 0.77 0.78 1.60 prob. of f <0.001 <0.001 0.732 0.116 <0.001 <0.001 results neem-moringa seed oil mixes' impact on the proximate composition of cowpea grains that are kept the moisture content of cowpea grains treated with two levels of ratios 1:2 and 1:3 mixture of neem and moringa seed oils ranged from 10.22 to 14.60%, according to the results of proximate analysis (moisture, total ash, crude fat, crude fiber, crude protein, and carbohydrate) shown in table 1. after 270 days of storage, cowpea grains treated with a ratio of 1:3/2.5µl/g had the lowest moisture level (10.22%), whereas the control sample had the greatest moisture content (14.60%). the moisture content of the samples treated with the same level of the oils, i.e. ratios 1:2/2.5 µl/g and 1:3/2.5 µl/g, did not differ significantly (p<0.05), nor did the samples treated with ratios 1:2/5.0 µl/g and 1:3/5.0 µl/g. however, the moisture content of the treated samples and the control (untreated cowpea grains) differed significantly (p<0.05). table 1 displays the results of the total ash content.the total ash concentration at the end of the storage periods varied between 3.36 and 4.89%. the control sample had the lowest amount of ash (3.36%), while cowpea grains treated with a 1:2/2.5µl/g ratio of neem and moringa seed oils had the greatest amount (4.89%). the control samples and the samples treated with a 1:3 neem-moringa seed oil ratio did not differ significantly (p<0.05). however, at the conclusion of the storage period, there were significant differences (p<0.05) between the ash content of the cowpea grains treated with 1:2 neem – moringa seed oils and the ash content of the untreated cowpea grains (control). the crude fat content of the treatment and control samples did not differ significantly (p>0.05) (table 1). similarly, there were not the fiber contents of the treated cowpea grain samples and the control samples differed significantly (p>0.05). both treated and untreated cowpea grains had protein levels ranging from 19.37% to 27.10% (table 1). the protein content of the cowpea grain samples treated with ratio 1:2 neem-moringa seed oils was considerably (p<0.05) higher than that of the untreated cowpea grains and those treated with ratio 1:3 neem-moringa seed oils. however, the protein content of cowpea grains treated with a 1:3 neemmoringa seed oil ratio did not differ significantly (p<0.05) from the control. the highest protein content was recorded by cowpea grains treated with 1:2/2.5µl/g, while the lowest protein content was recorded by cowpea grains treated with ratio 1:3/5.0µl/g (19.37%). table 1 shows that the range of the carbohydrate content was 51.60 to 61.73%. cowpeas treated with a ratio of 1:3/2.5µ/g had the greatest percentage (61.73%), followed by those treated with a ratio of 1:3/5.0µl/g of neem-moringa seed oil, which had a mean value of 60.73%. those treated with a ratio of 1:2 neem-moringa seed oils and those treated with a ratio of 1:3 neem-moringa seed oils showed significant differences (p<0.05). additionally, when compared to the control and other treated samples, the carbohydrate content of cowpea samples treated with the ratio 1:2/2.5µl/g dramatically decreased. table 2. effect of storage period on the proximate composition of cowpea grains treated with mixtures of neem and moringa seed oils period (days) moisture ash fat fibre protein carbohydrate 90 13.08 3.18 3.24 1.60 19.32 59.60 180 9.57 4.04 3.37 1.17 20.96 61.34 270 12.48 4.36 3.77 2.80 24.45 52.03 mean 11.71 3.86 3.46 1.86 21.58 57.65 lsd at 0.05 0.45 0.12 0.86 0.59 0.61 1.24 prob. of f <.001 <.001 0.438 <.001 <.001 <.001 storage duration's impact on the proximate composition of cowpeas treated with neem and moringa seed oil mixes the results of the impacts of storage duration on the proximate composition of cowpea treated with neem and moringa seed oil combinations are shown in table 2. the moisture and ash contents varied significantly (p<0.05) over the 90, 180, and 270-day storage periods, but the fat and fiber contents of the samples stored for 90 and 180 days did not differ significantly (p<0.05); however, the percentage of fiber content increased significantly at the end of the 270-day storage period. additionally, the protein composition of the samples varied significantly (p<0.05) throughout all examined periods. lastly, the carbohydrate content of the samples that were kept for 90 and 180 days did not significantly differ, however there were decrease in the samples' carbohydrate content after 270 days. effects of storage and treatment on the proximate composition of the cowpea grains stored in different concentrations of neem and moringa seed oil mixes table 3 shows how the proximate composition of the stored cowpea grains is affected by storage durations and treatment with different amounts of a blend of neem and moringa seed oils. at 180 days of storage, the cowpea grains treated with ratio 1:3/2.5 µl/g had the lowest moisture content (6.47%), followed by the one treated with ratio 1:2/2.5 µl/g, which had the highest moisture content (14.20%), while at 90 days of storage, there were no discernible differences in the moisture content of the cowpea grains treated with the various ratios and concentrations. however, after being stored for 270 days, the cowpea grains treated with ratio 1:2/2.5 µl/g had the lowest moisture content (10.27%), followed by those treated with ratios 1:3/2.5 µl/g and 1:2/2.5 µl/g. finally, the cowpea grains treated with ratio 1:2/5.0 µl/g had the lowest moisture content (13.19%). once more, the control had the highest moisture content. with the exception of the control, where the moisture content increased steadily over the course of the storage period, the moisture content often decreased at 180 days and subsequently increased at 270 days. cowpea grains treated with ratios of 1:2/2.5 µl/g and 1:3/5.0 µl/g recorded the highest ash content (3.21%) at 90 days of storage, followed by the cowpea grains treated with ratio 1:3/2.5 µl/g, which recorded 3.20% ash content. the effects of storage duration, neem and moringa seed oil concentration, and cowpea grains treated with different ratios on ash content were also examined. the cowpea grains treated with the ratio 1:2/2.5 µl/g and the control treatment had the lowest ash concentration (3.15%). the greatest percentage ash concentration at 180 days of storage was 5.40 for cowpea grains treated with ratio 1:2/2.5 µl/g neem and moringa seed oils, followed by cowpea grains treated with ratio 1:2/5.0 µl/g, which had 3.93. cowpea grains treated with a ratio of 1:3/5.0 µl/g neem-moringa seed oils had the lowest % ash level (3.73) throughout this storage time. the cowpea grains treated with a ratio of 1:2/2.5 µl/g neem-moringa seed oil had the greatest ash level (6.07%) at 270 days of storage, followed by those treated with a ratio of 1:2/5.0 µl/g. however, the cowpea grains treated with ratio 1:3/5.0 µl/g neem–moringa seed oils had the lowest ash concentration (3.33%). table 3 shows how the protein content of stored cowpea grains is affected by the storage time and the ratio of 1:2 and 1:3 neem–moringa oils at different concentrations. the highest percentage protein value (20.23%) was found in cowpea grain treated with ratio 1:2/2.5 µl/g after 90 days of storage. this was followed by one treated with ratio 1:3/5.0 µl/g, which had a percentage protein content of 20.01%.cowpea grains treated with a 1:2/5.0 µl/g ratio of neem–moringa oils had the lowest protein content. the maximum protein level (29.56%) was found in cowpea grains treated with a ratio of 1:2/2.5 µl/g neem–moringa seed oils after 180 days of storage. these grains treated with a ratio of 1:3/5.0 µl/g had a protein content of 19.08%. the cowpea grains treated with a ratio of 1:3/2.5 µl/g had the lowest protein concentration. after 270 days of storage, the similar pattern was noted. the cowpea grains treated with ratio 1:2/5.0 µl/g neem-moringa oils had the highest amount of carbohydrates (61.36%) at 90 days of storage, followed by the cowpea grains treated with ratio 1:3/2.5 µl/g, which had 60.42%, and the cowpea grains treated with ratio 1:2/2.5 µl/g neemmoringa seed oils, which had the lowest amount (57.83%). table 3.interaction between storage period andvarying concentration of mixture of neem and moringa seed oils on the proximate compos ition of cowpea grains period control neem : moringa seed oil ratios mean lsd at prob. of (days) 1:2/2.5µl/g 1:2/5.0µl/g 1:3/2.5µl/g 1:3/5.0µl/g 0.05 f moisture 90 13.20 13.27 13.00 13.13 12.80 180 14.20 8.53 8.93 6.47 9.73 270 16.40 10.27 13.19 11.07 11.47 11.71 1.01 <.001 ash 90 3.13 3.21 3.15 3.20 3.21 180 3.33 5.40 3.93 3.80 3.73 270 3.60 protein 6.07 5.27 3.53 3.33 3.86 0.28 <.001 90 19.58 20.23 18.32 18.43 20.01 180 18.51 29.56 19.13 18.54 19.08 270 21.03 31.52 28.55 21.15 20.00 21.58 1.35 <.001 carbohydrate 90 59.25 57.83 61.36 60.42 59.14 180 59.29 52.84 63.67 67.03 63.86 270 52.97 44.15 46.66 57.75 58.60 57.65 2.78 <.001 the maximum carbohydrate content (67.03%) was found in cowpea grains treated with a ratio of 1:3/5.0 µl/g neem-moringa oils after 180 days of storage and storage combined with neem-moringa seed oil treatment (1:3/2.5 µl/g). this showed 63.83% carbohydrate content. conversely, the cowpea grains treated with a 1:2/2.5µl/g ratio of neem-moringa oils had the lowest carbohydrate content (52.84%). the maximum carbohydrate content (58.60%) was recorded by cowpea grains held for 270 days and treated with ratio 1:3/5.0µl/g neem-moringa oils. these were followed by those treated with ratio 1:3/2.5µl/g, and the lowest carbohydrate content (44.15%) was recorded by cowpea grains treated with ratio 1:2/2.5µl/g. discussion the removal of water with oil absorption into the seeds may be the reason for the significantly (p<0.05) lowest moisture content loss from the cowpea sample treated with ratio 1:3/2.5 µl/g neem-moringa seed oil. this is demonstrated by the fact that the control samples had the greatest moisture content (14.60%) of any treated samples. this outcome is in line with the findings of gayan et al. (2006), who examined the impact of certain essential oils on the physicochemical alterations of cowpeas that were stored and treated to prevent callosobruchus maculatus cowpea bruchids. given that the control samples were more vulnerable to bruchid infestation than the other samples, the outcome could possibly be the result of increased insect population and metabolism absorbing moisture from the atmosphere. according to the distinctive smell found in these samples, the absorption of the treated mixture of neem and moringa seed oils into the cowpea grains in trace amounts may be the cause of the increase in the total fat content over the storage period of the cowpea grains treated with these oils. this outcome is also equivalent to gyan et al. (2006)'s findings. there was no discernible difference in the protein content between the control and treated samples with ratios of 1:3/2.5 µl/g and 1:3/5.0 µl/g. this could be because, although there were a lot of insects and their eggs in the control samples, the insects were successfully managed in the treated samples. this outcome is consistent with the findings of gayan et al. (2006). given that that amount of oil could not preserve the cowpea grain samples for more than 180 days, the significantly higher protein contents (27 and 22%) of samples treated with ratios 1:2/2.5 and 1:2/5.0 µl/g may be the result of numerous c. maculatus eggs and larvae on the cowpea grains as a result of infestation (ilesanmi and gungula, 2010). similar findings were reported by danjumma et al. (2009), who discovered that as the degree of wheat infestation rose, there was a progressive rise in total nitrogen, total protein, non-protein, and uric acid. both untreated, non-infested grains and samples treated with ratios of 1:3/2.5 µl/g and 1:3/5.0 µl/g neem-moringa seed oils showed no change in total protein content (as determined by its nitrogen content) after storage. the fact that c. maculates hollowed out the majority of endosperm, leaving just the seed coat, which is primarily composed of fiber, may be the cause of the increase in fiber content seen in the infested cowpea grains (1:2/2.5 µl/g treated). this is consistent with the findings of bamaiyi et al. (2006) and danjumma et al. (2009). the ash content of samples treated with ratios of 1:2/2.2 and 1:2/5.0 µl/g neem-moringa increased significantly, suggesting that the more infested the cowpea grain sample, the lower the volume and concentration of moringa oil in the oil treatment mixture. additionally, as highlighted by gayan et al. (2006), the amount of seed coat that remains increases with the amount of endosperm consumed. this component is rich in ash. given that the majority of carbohydrates are found in the endosperm section of cowpea grains and that these oil levels protected the grains from infestation, it is possible that the sample treated with ratios 1:3/2.5 and 5.0 µl/g had the highest carbohydrate content (60.53–61.73%). this outcome is in line with ojimelukwe et al.'s (1999) findings on how infestation affects cowpea's nutritional content and physicochemical characteristics. there was no significant difference (p<0.05) in the moisture content of the treated cowpea and the control sample at the end of 90 days of storage, but it did decrease at the end of 180 days and then increase at the end of 270 days. this variation may have been caused by changes in the relative humidity (rh) of the environment during the storage period, according to the mean effect of storage period on the percentage proximate composition of cowpea grains treated with a mixture of neem and moringa seed oils. the first ninety days had an average relative humidity (arh) of 94.34 from august to november, followed by 72.88 from november to february and 80.70 from march to may. this outcome is in line with earlier research by ilesanmi and gungula (2010), who preserved cowpea using a combination of neem and moringa seed oils at different doses. according to anderson and alcock (2013), the treated samples provided the safest moisture level for grain storage, which was between 10 and 12 percent. this is below the essential moisture value for grains, which is 14%. this can be the result of water being removed and oils being absorbed by the grains. even though the relative humidity was high, the oil may have formed a moisture barrier for the cowpea grains while they were being stored. the ash content range of 3.18 to 4.36% is consistent with the findings of owolabi et al. (2012), who examined the mineral composition, proximate, and antinutrition of five types of improved and local cowpea. additionally, it aligns with the findings of pandey and westphal (1989), who documented the chemical makeup of cowpea grain. this variation could be the result of a weevil infestation, in which the weevils consumed the grain, leaving behind primarily the seed coat, which contains the chaff, which is equivalent to ash. the number of mineral components in the sample can be determined by looking at the ash content. the fat content results for both treated and untreated cowpea samples, which range from 3.24 to 3.77, are similarly consistent with owolabi et al. (2012)'s findings. the percentage fiber content, which ranges from 1.17 to 2.80%, is consistent with the findings of aremuet al. (2006) and owolabi et al. (2012), who conducted a comparative analysis of the chemical and amino acid composition of a few underutilized legume flours in nigeria.after 270 days of storage, the sample treated with a ratio of 1:2/2.5 µl/g contained 2.39% fiber. this could be the result of a weevil infestation, which left behind seed coat, which increased the amount of fiber because the cowpea endosperm had been eaten out. this could indicate that the oil's concentration (1:2/2.5 µl/g) was insufficient to stop infection for up to 270 days. this result is in line with a study by ilesanmi and gungula (2010) that found that after 180 days of storage, 44% of cowpea treated with a 1:2 ratio of neem to moringa seed oils were infested. this could explain why, after 180–270 days, the % protein content of samples treated with a 1:2 mixture of neem and moringa seed oils increased. given how high in protein they are, callosobruchus maculatus eggs and larvae may have contributed to the higher protein content. the study's control and all other treated samples had protein values that were consistent with the findings of pandey and westphal (1989), henshaw (2008), oyeyinka et al. (2013), owolabi et al. (2012), and carvalho et al. (2012). the decrease in percentage carbohydrate content in cowpea grain samples treated with a 1:2 mixture of neem and moringa seed oils at the end of the 270-day study may not have been caused by the oil; rather, it may have resulted from weevils eating up the grain endosperm and the protein content increasing. the percentage proximate composition of store cowpea was found to be affected by the combination of neem and moringa seed oils and storage period. at 90 days of storage, there was no discernible difference between the moisture content of the treated and control (untreated) cowpea grain samples; however, at 180 to 270 days of storage, the moisture content of the control samples was significantly (p<0.05) higher (14.20 and 16.20%) than that of the treated samples, which ranged between 6.5 and 11.5%. this is consistent with the findings of gyang et al. (2006), who suggested that the removal of water from the oil-treated samples may have resulted from the oil's absorption into the grains. they also noted that the higher moisture content of the control samples might have resulted from the increased population and metabolism of insects absorbing moisture from the atmosphere. the ash content of the control and treated cowpea grain samples did not differ significantly (p<0.05) at 90 days of storage; however, after 180–270 days, a notable increase in the ash content of samples treated with a 1:2 mixture of neem and moringa seed oils was noted. the seed coat of cowpea grains contains a lot of this substance. thus, a significant infestation of cowpea bruchids, which ate the endosperm portion of the grains and left behind the seed coat, which is high in ash component, may be the cause of the increase in this feature. at the end of 180 and 270 days of storage, the protein content of cowpea treated with a 1:2 mixture of neem and moringa seed oils showed a similar trend, with a significant increase. this could be due to bruchid eggs and larvae, which are highly protein-rich insects. with regard to carbohydrate content, the trend was marginally different. the carbohydrate content of the treated cowpea and the control did not differ significantly (p<0.05) after 90 days of storage; however, after 270 days, all cowpea grains showed a significant decrease in carbohydrate content, with the exception of the sample treated with a 1:3 mixture of neem and moringa seed oils. since carbohydrates are concentrated in the endosperm region of the grains, the loss may be caused by both bruchid consumption of the cowpea endosperm and an increase in the protein content of the samples. as storage time increased, the amount of carbohydrates dropped. this outcome is consistent with the findings of danjumma et al. (2009) about the nutritional makeup of maize grains that were prepared and stored using plant powders. according to ilesanmi (2009), the degree of bruchid infestation in cowpea grain samples treated with a 1:2 mixture of neem and moringa seed oils at the end of 270 days as compared to cowpea samples treated with a 1:3 mixture of neem and moringa oil at the end of 180 days of storage may be the reason for the significant increase in percentage protein, ash, and fiber contents and the significant decrease in percentage carbohydrate content. the low concentration of moringa oil, which translates to reduced behenic acid (the active element in moringa oil), may be the cause of the high level of infection for samples treated with a 1:2 neem-moringa oil ratio. most likely, this concentration is insufficient to stop an infection that lasts more than 180 days. when compared to the control, the analysis demonstrates that there is no discernible difference (p<0.05) in the proximate composition of any of the samples treated with ratio 1:3 at either level. this result is consistent with that of ilesanmi and gungula (2010), who used a combination of neem and moringa seed oils to preserve cowpea grains. conclusion and recommendation the study found no negative effects on the proximate of the cowpea grains when cowpea samples were stored with a 1:3 mixture of neem and moringa seed oils.to maximize the nutritional value of cowpea grains and their products, a 1:3 blend of neem and moringa seed oils, namely 1:3/5.0µl/g, should be utilized for cowpea grain preservation. references anderson ja, alcock aw (2013).storage of cereal grains and their products.fourth edition. pp.515 aoac (association of official analytical chemists) (1990).official methods of analysis.15 th edition.association of official analytical chemists, washington, dc. aremu mo, olaofe o, akintayo te (2006).a comparative study on the chemical and amino acid composition of some nigerian underutilized legume flours.pakistan journal of nutrition.5(1):34-38. asante ik, adu-dapaa h, acheampong ao (2007).determination of some mineral components of cowpea (vignaunguiculata (l.)walp) using instrumental neutron activation analysis.west african j. applied ecology, vol. 2, no. 1. carvalho afu, sousa nm, farias df, rocha-bezerra lcb, silva rmp, viana mp, gouveia st, sampaio ss, sousa mb, lima gpg, morais sm, barros cc, filho frf (2012). nutritional ranking of 30 brazilian genotypes of cowpeas including determination of antioxidant capacity and vitamins.journal of food composition and analysis. 26pp.81-88. available at www.elsevier.com/locate/jfca duke ja (1983).handbook of energy crops.retrieved from http://www.hort.purdue.edu/newcrop/duke_energy/moringa _oleifera. htmi (sept 2006) egan h, kirk rs and sawyer r (1981).pearson’s chemical analysis of foods. longman group united kindom limited. pp.137-169. henshaw fo (2008). varietal differences in physical characteristics and proximate composition of cowpea (vignaungunculata).world journal of agricultural sciences 4(3):302-306. ilesanmi jo, gungula dt (2010). preservation of cowpea [vignaunguiculata (l.)] walp grains against cowpea bruchids(callosobruchusmaculatus) using neem and moringa seed oils. international journal of agronomy.vol. 2010. ilesanmi joy (2009). preservation of cowpea (vignaunguiculata (l.)walp) against cowpea bruchids(callosobruchus maculates) using neem and moringa oils – m. tech. thesis. federal university of technology, yola.pp.8088. karunasena pgcs (2001). investigation of toxic and repellent activity of essential oils of alpiniacalcarataross and cymbopogannardus against cowpea weevil. b.sc. thesis. university of kelaniya.sl. ojimelukwe pc, onweluzo jc, okechukwu e (1999).effects of infestation on the nutrient content and physiocochemical properties of two cowpea (vignaunguiculata) varieties. plant foods for human nutrition, volume 53, issue 4, pp 321-332. owolabi ao, ndidi us, james bd, amune fa (2012). proximate, antinutrient and mineral composition of five varieties (improved and local) of cowpea, vignaunguiculata, commonly consumes in samaru community, zaria – nigeria. asia j. food sci. and technology 4(2): 70-72. oyeyinka sa oyeyinka at, karim or, kayode rmo, balogun ma, balogun oa (2013). quality attributes of weevils (callosobruchusmaculatus) infested cowpea (vignaunguiculata) products. nigerian j. agric. food and environ. 9(3):16-22. pandey rk, westphale (1989).vignaunguiculata(l.)walp. [internet] record from proseabase.van dermaesen ljg, somaatmadja s (editors). prosea (plantresources of south east asia) foundation, bogor, indonesia.http://www.proseanet.org. accessed from internet: 21 aug 2011 http://www.elsevier.com/locate/jfca http://www.hort.purdue.edu/newcrop/duke_energy/moringa_oleifera http://www.hort.purdue.edu/newcrop/duke_energy/moringa_oleifera http://www.proseanet.org/ materials and methods results neem-moringa seed oil mixes' impact on the proximate composition of cowpea grains that are kept storage duration's impact on the proximate composition of cowpeas treated with neem and moringa seed oil mixes effects of storage and treatment on the proximate composition of the cowpea grains stored in different concentrations of neem and moringa seed oil mixes discussion conclusion and recommendation 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 12 (1), pp. 001-006, january, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper impact of contaminated water on african catfish (clarias gariepinus) and its effects on albino rat liver o. adeyemi1*, c. c. osubor1 and o. adeyemi2 1 department of biochemistry, university of benin, benin-city, nigeria. 2 department of environmental sciences, federal university of petroleum resources effurun, delta state, nigeria. accepted 16 november, 2023 effect of consuming clarias gariepinus cultivated in water contaminated with (10 g/ml) respectively of phthalate, benzene and cyclohexane on the liver of rats was done. serum concentrations of bilirubin, globulin and albumin were determined. standard enzyme assays were conducted for selected liver enzymes followed by histological examination of liver section. serum albumin and globulin concentrations were found to be significantly lower in rats fed with contaminated c. gariepinus than control (p < 0.05). generally, activity of enzymes in the liver of experimental rats was found to be significantly lower than that of control (p < 0.05). particularly, activities of alkaline phosphatase, acid phosphatase, alanine and aspartate transaminases in the liver of rats fed with c. gariepinus cultivated in phthalate contaminated water were found to be 9.1 ± 1.7, 113.8 ± 6, 13.4 ± 0.9 and 20.4 ± 0.9 nmol/min/mg protein respectively while those of control were 16.8 ± 2.2, 177.9 ± 5, 16.4 ± 1.3 and 23.7 ± 1.2 nmol/min/mg protein. serum concentrations of direct and total bilirubin of rats fed with c. gariepinus cultivated in benzene contaminated water were found to be 3.52 ± 0.05 and 9.24 ± 0.50 mg/dl respectively while those of control were found to be 1.06 ± 0.02 and 4.93 ± 0.20 mg/dl respectively. histological examination of section revealed distorted cellular arrangement in the liver section of rats fed with contaminated fish relative to control. experimental evidences from this study suggest hepatotoxicity which may predispose to tissue failure. increasing cases of liver problem in nigeria may not be unconnected, inta alia, with consumption of fish from contaminated water. key words: enzymes, bilirubin, phthalate, hepatotoxicity. introduction education and enlightenment programme on omega-3 fatty acids found in clarias gariepinus (penny et al., 2002) has prompted many farmers in nigeria to cultivate the fish. as demand for c. gariepinus increased more people joined the league of farmers cultivating c. gariepinus and more ponds were constructed without consideration to water quality and intrusion by external water source (e.g. runoff and leachate from wastes) (sunmonu and oloyede, 2007). there are several important common denominators to consider when building a pond, irrespective of whether *corresponding author. e-mail: yemicoash@yahoo.com. the pond is built by hand for small scale aquaculture or by machines for large scale commercial farming. these are; type of wall, basic engineering principles, design and construction criteria for the water outlet and the drainage system and the exclusion of predators (alegbeleye et al., 1991). the ponds found around do not conform to these guidelines; most ponds serve as recipient to runoff and leachate from wastes. growing evidence revealed that animals placed on water contaminated with leachate from waste gain considerable body weight (bakare et al., 1995; oloyede et al., 2003a, 2003b; oladiji et al., 2004; adeyemi et al., 2007a). mortality and weight are salient factors considered by most fish farmers in nigeria. once the fish do not die and are gaining weight, the farmers 2 spend less money on feed and make more gain at the expense of public health. public health is endangered because components of waste leachate are hazardous and when allowed to contaminate water body the water become unfit for drinking (adeyemi et al., 2007b). components of waste leachate include heavy metals, organics such as phthalate, phenol, benzene, cyclohexane etc. (brenaam, 2000; johnson et al., 2000) . consumers need to be aware of both the benefits and risks of fish consumption for their particular stage of life. children and pregnant and lactating women may be at increased risk for phthalate intoxication from fish consumption but also are at low risk for coronary heart diseases (chd). thus, avoidance of potentially contaminated fish is a higher priority for this group. acute, chronic and long term effects of chemical compounds on living systems could be studied by evaluating the biochemical and morphological changes in various organs especially the liver. the basic structural component of the liver is the liver cells or heaptocytes. the liver is the principal organ of metabolism and has a role to play in many body processes most especially detoxification of chemical compounds. research studies have shown a variety of adverse effects on the hepatocytes of rats and catfish following exposure to environmentally toxic compounds (oloyede et al., 2003b; sunmonu and oloyede, 2006). however, little or no attention has been given to effect of components of waste leachate on public health. in the present study, an attempt is made to assess the effect cat fish cultivated in water contaminated with some components of waste leachate on the hepatocytes and performance of rat. materials and methods reagents chemicals and solvents used are of analytical grade and most are products of sigma-aldrich inc, st. louis, u.s.a, while others are products of british drug house, poole, england. experimental water the experimental water for the study was collected from the supply of adekunle ajasin university, akungbaakoko, ondo state, nigeria. the water was analysed using the method described by apha (1992). the experimental water samples were contaminated with 10 g/ml of phthalate (diethylphthalate) (cas# 84-66-2), benzene (cas# 71-43-2) and cyclohexane (cas# 110-82-7) respectively and designated as follows; a: water as collected from the university supply (control). b: water contaminated with phthalate (10 g/ml). c: water contaminated with benzene (10 g/ml). d: water contaminated with cyclohexane (10 g/ml). the concentration of each pollutant is about ten times the recommended permissible limit (acgih, 1994) and a representation of the concentration found in a typical runoff from open dumps and table 1. rat feed composition using c. gariepinus cultivated in water contaminated with phthalate, benzene and cyclohexane over a period of 56 days as protein source. nutrient weight (g) protein source (c. gariepinus) 200 fat source (soy oil) 80 sucrose 60 cellulose 60 *vitamin and mineral premix 50 corn starch 550 *the vitamin/ mineral premix contains: vit. a, 3,200,000.00 iu, vit. d5 600,000i u, vit. e, 2,800 mg, niacin 6,000 mg, vit. b1 800 mg, folic acid 70,000 mg, cobalt 80 mg, iodine 499 mg, selenium 80 mg, copper 1200 mg, vit. b12 4 mg, vit. b6 800 mg, copper 1,200 mg, folic acid 70,000 mg, vit. k3 600 mg, panthothenic acid 2,200 mg, vit. b6 800 mg, vit. b2 1000mg, iron 8,400 mg, manganese 16,000 mg. leachate from landfills (adeyemi et al., 2007b). experimental animals and management institutional and/ or national guides for the care and use of laboratory animals were followed. clarias gariepinus eighty (80) african catfish (c. gariepinus) of average weight 68.56 ± 6.92 g were obtained from the department of environmental biology and fishery, adekunle ajasin university, akungba-akoko, ondo state, nigeria. they were housed in transparent plastic containers and kept in a well ventilated laboratory. these fish were fed (3%w/w) with commercial feeds obtained from livinco feeds, jubilee road, ikare akoko, ondo state, nigeria. the experimental animals were kept inside a transparent plastic container assigned into eight (8) groups of ten (10) animals each. the first two groups of fish were cultivated in uncontaminated water from the university supply and water contaminated with 10 g/ml phthalate. the third and fourth groups were placed on water samples contaminated with 10 g/ml benzene and 10 g/ml cyclohexane respectively. a replica of the four groups was set-up as the last four groups. the feeding exercise lasted over a period of 56 days preceded by 14 days acclimatization period. two fish samples were taken from each group, dried in the oven at 40°c for 6 h daily over a period of 7 days after which they were used to compound feed for rat as shown in table 1. rattus novergicus twenty albino rats (rattus novergicus) of average weight 57.5 ± 3.25 g were used for the study. they were obtained from the animal holding of the department of biochemistry university of ilorin, ilorin, nigeria. they were kept in wooden cages and fed, ad libitum, with the formulated diet over a period of 28 days. they were classified into four groups designated thus; group a rats fed with c. gariepinus propagated in water from university supply based diet (control). group b rats fed with c. gariepinus propagated in phthalate 3 contaminated water based diet. group c rats fed with c. gariepinus propagated in benzene contaminated water based diet. group d rats fed with c. gariepinus propagated in cyclohexane contaminated water based diet. the feeding exercise was over a period of 28 days preceded by 7 days acclimatization period. the rats were anaesthetized by placing them in a jar containing cotton wool soaked with chloroform before being sacrificed by jugular puncture. the blood was obtained through their jugular veins into non-heparinized bottles. the blood samples in non-heparinized bottles were spinned at 3500 b rpm for 10 mins and serum stored at 8°c until required for use. preparation of homogenate the isolated liver was weighed and a portion of it was cut out, chopped into very small pieces and then homogenized using precooled pestle and mortar in a bowl of ice cubes. the liver homogenate was diluted using 0.25m sucrose solution to the tune of 1 in 30 dilutions. the diluted homogenate was stored at temperature of 8°c until required for use. another portion of the liver was cut out and fixed in 10% buffered neutral formaline (bnf) for 72 h at room temperature for histology study. protein determination and enzyme assay the method described by tiez (1990) was adopted for albumin and bilirubin concentrations in the experimental rats. in this method, albumin reacts with the dye-bromocresol green in an acidic medium to produce a greenish coloured complex the intensity of which is proportional to the amount of albumin present. the bilirubin in the serum is coupled with diazotized sulphanilic acid to form azobilirubin. the intensity of the purple colour that is formed is measured spectrophometrically at 520 nm. globulin was calculated from the difference between total protein and albumin (doumas, et al., 1971). protein concentration was determined by the biuret reaction described by gornall et al. (1949) . in this method, copper ion (blue) is made to react with the peptide bond of protein to give a purple coloured complex, the intensity of which was measured spectrophotometrically at 540 nm. activities of aspartate transaminase (ast) and alanine transaminase (alt) were determined using the method described by reitman and frankel (1957). the method measures spectrophotometrically the intensity of the red coloured hydrazone formed from the reaction of pyruvate with 2, 4 dinitrophenylhydrazine at 546 nm. the method of bessey et al. (1946) as modified by wright et al. (1972) was employed in the determination of alkaline and acid phosphatases. in this method, the amount of phosphate ester that is split within a given period of time is taken as a measure of the phosphatase enzymes. histology histological study on tissues obtained from experimental rats was carried out following the method described by drury and wallington (1973). statistical analysis analysis of variance anova; duncan’s multiple range test (dmrt) was the statistical analysis used (duncan, 1955). p < 0.05 was regarded as significant. results and discussion the present study is aimed at unveiling some of the health hazards associated with consumption of aquatic foods from contaminated aquatic habitats. the increasing liver disease in nigeria is a cause for concern; this disease has its root partly from nutrition and partly from inheritance. the nutritional root is not unconnected with consumption of contaminated fresh water fish. fresh water fish is on high demand because of its high omega-3 fatty acid content (penny et al., 2002); however, proper care is not devoted to the quality of water where fresh water fish (e.g. c. gariepinus) is being propagated (alegbeleye et al., 1991). this is, among other reasons, why investigation into effect of inclusion of c. gariepinus propagated in contaminated water in the diet demands a biochemical approach. the liver-body weight ratio of experimental rats is presented in figure 1. a significant decrease was observed in the liver-body weight ratio of experimental rats relative to the control (p < 0.05). particularly, that of rats fed with c. gariepinus cultivated in water contaminated with phthalate was half that of control. the rats in the different groups ate their respective formulated feed freely. the reduced liver -body weight ratio of experimenttal rats observed in this study (figure 1) is an indication of possible damage to the liver. this is in agreement with earlier report (oloyede et al., 2003a; adeyemi et al., 2008). however, further liver function tests will buttress this submission. concentrations of some serum proteins of rats fed with diet formulated with c. gariepinus propagated in contaminated water are shown in table 2. generally, the concentrations of the serum albumin and globulin of rats fed with diet formulated with c. gariepinus propagated in contaminated water were found to be significantly lower than that of control (p < 0.05). conversely, serum bilirubin concentration of rats fed with contaminated fish was found to be significantly higher than that of control (p < 0.05). particularly, the concentrations of the serum albumin of rats fed with diet formulated with c. gariepinus propagated in water contaminated with phthalate and benzene were observed to be about ¼ that of control. serum bilirubin, albumin and globulin concentrations are some biochemical indices for monitoring liver function in the blood (table 2). abnormal levels of these proteins have been reported to be associated with haemolysis or increased breakdown of rbc and/ or liver damage (islam et al., 2004). bilirubin is a breakdown product of heme (a part of haemoglobin in red blood cells). the liver is responsible for clearing the blood of bilirubin. it does this by the following mechanism: bilirubin is taken up into hepatocytes, conjugated (modified to make it watersoluble) and secreted into the bile, which is excreted into the intestine. increased total bilirubin causes jaundice and can signal a number of problems. studies had shown that if direct (that is, conjugated) bilirubin is normal, then the problem is an excess of unconjugated bilirubin and the location of the problem is upstream of bilirubin excretion. anemia, viral hepatitis, or cirrhosis can be suspected. if direct bilirubin is elevated, then the liver is 4 a c b c figure 1. liver-body weight ratio of rats fed with c. gariepinus cultivated in water contaminated with phthalate, benzene and cyclohexane. results are means of five determinations ± sem. bars carrying different notations are significantly different (p < 0.05). table 2. some serum indices of liver function of rats fed with c. gariepinus cultivated in water contaminated with phthalate, benzene and cyclohexane. group d-bil (mg/dl) t-bil (mg/dl) albumin (g/dl) globulin (g/dl) control 1.06 ± 0.02 a 4.93 ± 0.20 a 13.20 ± 2.50 a 8.80 ± 0.21 a phthalate 2.78 ± 0.01 b 7.31 ± 0.40 b 3.49 ± 0.70 b 2.14 ± 0.12 b benzene 3.52 ± 0.05 c 9.24 ± 0.50 c 4.75 ± 0.60 c 3.20 ± 0.34 c cyclohexane 1.05 ± 0.04 a 4.88 ± 0.20 a 8.68 ± 1.70 d 5.71 ± 0.32 d results are means of five determinations ± sem. values in the same column with different superscripts (a, b, c....) are significantly different (p < 0.05). conjugating bilirubin normally, but is not able to excrete it. bile duct obstruction by gallstones or cancer should be suspected (sunmonu and oloyede, 2007). in this study, rats fed with fish contaminated with phthalate and benzene presented elevated serum concentrations of both total and direct bilirubin suggesting that the liver is not able to excrete bilirubin which is an evidence of liver dysfunction. decreased serum concentrations of albumin and globulin as observed in this study lend credence to the submission that the liver function may be impaired. both globulin and albumin are produced by the liver. if the liver is damaged, it can no longer produce these proteins. the results presented on serum proteins are consistent and all pointing to the fact that the liver may have been damaged by consumption of the contaminated fish. table 3 presents specific activity of selected liver enzymes of rats fed with contaminated fish. a general significant decrease of enzyme activity was observed in the liver of rats fed with contaminated fish relative to the control (p < 0.05) . additionally, enzyme activity was found to be least in the liver of rats fed with diet formulated with c. gariepinus cultivated in water contaminated with phthalate followed by rats fed with diet formulated with c. gariepinus cultivated in water contaminated with benzene. activity of enzymes assayed for in the liver of the experimental animals is consistent with the observation on serum protein concentrations. alp catalyses the hydrolysis of organic phosphates at alkaline ph. alp 5 table 3. specific activity (nmol/min/mg protein) of selected enzymes of the liver of rats fed with c. gariepinus cultivated in water contaminated with phthalate, benzene and cyclohexane. group alp acp alt ast control 16.8 ± 2.2 a 177.9 ± 5 a 16.4 ± 1.3 a 23.7 ± 1.2 a phthalate 9.1 ± 1.7 b 113.8 ± 6 b 13.4 ± 0.9 b 20.4 ± 0.9 b benzene 12.4 ± 1.2 c 131.7 ± 4 c 12.4 ± 0.9 b 19.8 ± 1.0 b cyclohexane 12.6 ± 1.6 c 141.2 ± 4 d 14.6 ± 1.1 ab 22.6 ± 0.9 c results are means of five determinations ± sem. values in the same column with different superscripts (a,b,c...) are significantly different (p < 0.05). control: no visible lesion. phthalate: gross tissue disintegration and breakage benzene: gross tissue disintegration cyclohexane: mild tissue disintegration figure 2. light micrograph (×400) of liver of rats fed with c. gariepinus cultivated in water contaminated with phthalate, benzene and cyclohexane. mild tissue disintegration to tissue breakage (see selected areas). activity gives an indication of possibility of liver diseases (nelson and cox, 2000). probable damage to the plasma membrane of the liver may be the reason for the reduced alp activity in the liver of rats fed with c. gariepinus propagated in contaminated water (table 3). acp catalyses the removal of phosphoryl group from a phosphate ester in an acidic medium. it is found throughout the body (nelson and cox, 2000). however, damage to tissues including liver, kidney, heart, red blood cells etc. causes a decrease in tissue level of acp (moul et al., 1998). the decrease in the acp activity in the liver as observed in the present study also lend credence to liver dysfunction. increased alt/ast ratio may be indicative of the extent of cellular damage (adeyemi et al., 2008). adeyemi et al. (2009) reported that loss of ast and alt activity in tissues may be interpreted as a compromise of the tissues integrity. although alt and ast are “marker” enzymes for the liver, it is believed that any alteration at the subcellular level may affect the activity of these enzymes in other tissues (adeyemi et al., 2008). the decrease in the alt and ast activity in the liver as observed in this study (table 3) suggests that consumption of c. gariepinus propagated in contaminated water possibly led to inhibition of alt and ast activity. the experimental data revealed that inclusion of c. gariepinus propagated in contaminated water in diet may alter protein metabolism, amongst others, at the subcellular 6 level and this may predispose to impairment of the function of the liver. histology examination of liver of experimental rats is presented in figure 2. no visible lesion was observed in the control rats, however, varying changes in cellular architecture was observed in the histology photograph of rats fed with contaminated fish. these changes range from mild tissue disintegration as observed in rats fed with cyclohexane contaminated fish to tissue breakage as observed for rats fed with phthalate contaminated fish. overall, all the data obtained as portrayed by changes in the biochemical indices for liver function and enzyme activities seem to be supported by the histological study (figure 2) particularly for the rats fed with c. gariepinus cultivated in water contaminated with phthalate showed gross cellular disintegration and tissue breakage. the large open spaces in the histology photograph (marked by white circles) are indicative of tissue disintegration. these open spaces had been described as areas of tissue disintegration and breakage (loekle et al., 1983; sunmonu and oloyede, 2007). conclusion this study is significant as it has been able to show that of c. gariepinus cultivated in water conta-minated with component of waste leachate (such as phthalate, benzene and cyclohexane) is hazardous to health. experimental evidence from this study reveals that consumption of such contaminated fish could damage the liver as evidenced by: a. reduced liver-body weight ratio. b. elevated serum bilirubin concentration. c. reduced serum albumin and globulin concentrations. d. decreased alp, acp, ast and alt activity in the liver. e. gross tissue disintegration observed in the histology photograph. references acgih (1994). american conference of governmental industrial hygienists. 1993-1994 threshold limit values for chemical substances and physical agents and biological exposure indices. acgih, cincinnati,oh. adeyemi o, oloyede ob, oladiji, at (2007a). physicochemical and microbial characteristics of leachate-contaminated groundwater. asian. j. biochem. 2(5): 343-348. adeyemi o, oloyede ob, oladiji at (2007b). effect of leachatecontaminated groundwater on the growth and blood of albino rats. int. j. hematol. 3(2): 1-6. adeyemi o, oloyede ob, oladiji at (2008). biochemical evaluation of oxidative damage induced by leachate contaminated groundwater on selected tissues of rats. int. j. toxicol. 4(2): 1-7. adeyemi o, ajayi jo, olajuyin am, oloyede ob, oladiji at, oluba om, adeyemi o, ololade ia, adebayo ea (2009). toxicological evaluation of the effect of water contaminated with lead, phenol and benzene on liver, kidney and colon of albino rats. j. food. chem. toxicol., 47: 885-887. alegbeleye wo, adetayo ja, ogunmoroti t (1991). simple method for year-round breeding of clarias gariepinus. aquabyte, 4: 8-8. american public health association (apha) (1992). standard methods for examinations of water and waste water, 16 th edition, washington, d.c. p. 268. bakare aa, mosuro aa, osibanjo o (1999). effect of simulated leachate on chromosomes and mitosis on roots of allium cepa. j. environ. biol., 21(3): 263-271. bessey oh, lowry md, brock a (1946). a method for the rapid determination of alkaline phosphatase with 5mm 3 of serum. j. bio. chem. 164: 321-329. brenaam mb (2000). water works: research accelerates on advanced water treatment technologies as their use in purification grows. groundwater. 2: 14-21. doumas bt, watson wa, biggs hg (1971). albumin standards and measurement of serum albumin with bromocresol green. clin. chim. acta. 31: 87-92. drury rab, wallington ea (1973). tissue histology. in: carleton’s histological technique 4 th ed. oxford university press, new york. pp 58. duncan db (1955). multiple range and multiple f test. biomet. 11: 1-10. gornall ag, bardawill jc, david mm (1949). determination of serum proteins by means of biuret reaction j. biol. chem. 177: 751-760 islam ms, lucky ns, islam mr, ahad a, das br, raham mm, siddiui msi (2004). haematological parameters of fayoumi, assil and local chickens reared in sylhet region in bangladesh. int. j. poult. sci. 3(2): 144-147. johnson r, pankow j, bender d, prince c, zogorski j (2000). mtbe: to what extent will post releases contaminate community water supply wells? j. environ. sci. technol. 34:210a-217a. loekle md, schecter aj, christian jj (1983). effects of chloroform, tetrachloroethylene and trichloroethylene on survival, growth and liver of poecilia sphenops. bull. environ. contam. toxicol. 30: 199-205. moul, jw (1998). the contemporary value of pretreatment prostatic acid phosphatase to predict pathological stage and recurrence in radical prostatectomy cases. j. urol. 935-940. nelson dl, cox mm (2000). aminotransferases. in: principles of biochemistry 3 rd ed. worth publisher. ny. pp. 628-632. oladiji at, adeyemi o, abiola, oo (2004). toxicological evaluation of the surface water of amilegbe river using rats. biokemistri, 16(2):94-101. oloyede ob, adeyemi o, sunmonu to, bakare aa (2003a). the effect of polluted oba water on selected rat liver enzymes. niseb j. (3) 91-97. oloyede ob, sunmonu to, adeyemi o, bakare aa (2003b): a biochemical assessment of the effects of polluted water from asa river in rat kidney. niger. j. biochem. mol. biol. 18 (1) 25-32. penny mk, william sh, lawrence j (2002). fish consumption, fish oil, omega-3 fatty acids and cardiovascular disease. circulation. 106: 2747-2757. reitman s, frankel s (1957). a colorimetric method for the determination of serum glutamate oxaloacetate and pyruvate transaminases. ame. j. clin. pathol. 28:56 63. sunmonu to, oloyede ob (2006). changes in liver enzyme activities in african catfish (clarias gariepinus) exposed to crude oil. asian fisheries sci. 19: 104-109. sunmonu to, oloyede ob (2007). biochemical assessment of the effects of crude oil contaminated catfish (clarias gariepinus) on the hepatocytes and performance of rat. afr. j. biochem. res. 1(5): 083089. tietz nw (1990). serum albumin determination. in: clinical guide to laboratory tests, second edn. w.b. saunders co. philadelphia, usa. pp. 554-556. wright pj, leatherwood pd, plummer dt (1972). enzymes in rat urine: alkaline phosphatase. enzymologia, 42: 317-327. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 12 (4), pp. 001-007, april, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper enhancing rotifer mass culture through algal diversity paul o. ajah institute of oceanography, university of calabar, calabar, cross rivers state, nigeria. e-mail: ajapaulo@yahoo.com. tel: +2348033707901. accepted 4 january, 2024 outdoor cultures of brachionus quadridentatus raised in 10 m 3 concrete tanks fed on co-cultured three algal species (chlorella vulgaris, eudorina elegans and scenedesmus quadricauda) feed showed statistically significant differences in population biomass growth. growth of b. quadridentatus feeding on s. quadricauda was significantly greater than when fed on c. vulgaris. no significant differences in growth were observed between brachionus cultures fed on s. quadricauda and e. elegans and between those fed on c. vulgaris and e. elegans. daily b. quadridentatus population biomass increase was highest with s. quadricauda, followed by e. elegans and lastly c. vulgaris. b. quadridentatus needed approximately 20, 48 and 63 h, respectively, with c. vulgaris, s. quadricauda and e. elegans, to double its population. key words: zooplankton, algae, culture, nutrient. introduction mass production of catfish under controlled conditions depends on the provision of live plankton food for early fry and larval stages. the importance of live food in fry and larval rearing has been reported (ovie et al., 1993; ajah and holzlöhner, 1996; ajah, 1997, 1998; hagiwara et al., 1997, 2007). the successful production of finfish or shellfish such as shrimps and crabs not only requires a starter diet such as lepadella ovalis (l-type (93.6 × 70.3 µm 2 ) or s-type (70.3 × 35.1 µm 2 ) (ajah and ajah, in press), but also a second diet such as b. quadridentatus (432.9 × 128.7 µm 2 ) and a third diet, for example, asplanchna (685 × 356 µm 2 ) (ajah, 2008). a number of authors have emphasized the importance of adequate supply of rotifer cultures with the appropriate nutritional quality, for the survival of larvae cultured in marine hatcheries (hirata, 1980; kafuku and ikonoune, 1983; watanabe et al., 1983; lubzens, 1987). previous authors like ovie et al. (1993); adeyemo et al. (1994) and ajah (1997, 1998) have established the advantages of freshwater plankton over artemia nauplii, a saltwater anostraca. it is best to culture freshwater fish with freshwater live foods (ovie et al., 1993; ajah, 1997). b. quadricdentatus, a rotiferan, could be a suitable alternative to a. nauplii, being of equivalent size to imported a. nauplii. another reason for preferring b. quadridentatus is that, it occurred quite frequently and in large numbers in ponds at the university of calabar fish farm. earlier, lubzens et al. (1990) stated that rotifers (brachionus plicatilis) constitute a major, and in most cases the only food source for larval stages of several organisms raised in marine aquaculture including fish and various invertebrates. the culture of at least 10 species of marine rotifers including b. plicatilis, brachionus rotundiformis, brachionus pterodinoides, brachionus satanicus and hexarthra jenkinae is well documented (gatesoupe and luquet 1981; lubzens, 1987; lubzens et al., 1989, 1990; hampton and starkweather, 1998). gatesoupe and luquet (1981) noted that there could be nutritional differences in the value of rotifers as food for larvae based on the feeding conditions of the rotifers themselves. watanabe et al. (1979), found that chlorella-fed and yeast-fed rotifers differed in their proximate compositions. ajah (1998) found increased growth and survivorship of heterobranchus longifilis larvae when fed on enriched zooplankton. earlier, fujita (1979) indicated the importance of long chain -3 polyunsaturated fatty acids in rotifers as food for red sea bream larvae, and found a further improvement in the dietary value of yeast cultured rotifers by secondary culture with marine chlorella for 6 h. senecio quadridentatus has been found to thrive better in freshwater cultures than b. plicatilis (ajah, 1995). one advantage of b. quadridentatus over b. plicatilis is its low failure rate during culture. b. quadridentatus 2 culture was maintained continuously for two years irrespective of seasons with little fluctuation in population size (ajah, 1995) . the main difference between the dry and wet season blooms is in the initiation of blooms which brought about a doubling of the initial nutrients for the first two days during the wet season (ajah, 1995). brachionus calyciflorus, a freshwater rotifer was successfully fed on two types of algae, scenedesmus obliquus and chlorella sp. (kennari et al., 2008). in this study, freshwater food items (chlorella vulgaris, eudorina elegans and scenedesmus quadricauda) that might be more suitable, acceptable, cheap, readily available and capable of yielding higher population densities of b. quadridentatus a freshwater rotifera were investigated. materials and methods three hard bottomed 10 m 3 circular concrete tanks with central drainage pipes and five airlift pipes, arranged in an anticlockwise pattern to keep the plankton culture afloat were used for the outdoor culture of the rotifer. axenic monocultures of micro algae (c. vulgaris beij. var. vulgaris fott, (2 12 µm), s. quadricauda (turp.) bréb (12 15 µm) and e. elegans ehr. (95 105 µm) as well as pure monocultures of b. quadridentatus (hermann, 1783), were cultured in the laboratory of the hatchery complex of the institute of oceanography, university of calabar, calabar, nigeria, using both the “gelose” and “dilution” isolation methods (harder, 1917; mcvey and moore, 1983). the algae samples were collected from the fish ponds of the institute using a plastic container and inoculated into the algal medium using pasteur pipette. axenic algal cultures were produced under highly hygienic laboratory conditions using autoclaving and inhibitory bacterial growth precursors such as 0.005 0.01% hypochlorite solution, uv radiation and antibiotics (ajah, 1995). species were identified using ward and whipple (1959), bold and wynne (1978) and jeje and fernando (1986). zooplankton culture two 36-watt daylight fluorescent tubes were suspended 60 cm above a series of 5 60 l aquaria to illuminate a wooden cupboard of 120 × 60 × 80 cm 3 (0.6 m 3 ). room temperature was kept at 28 ± 1°c and aeration using two 1.5 hp pumps was continuous. the pure zooplankton cultures received 50 mgl -1 of baker’s yeast (saccharomyces cerevisiae) or 1.75 mgl -1 of inorganic fertilizer (n: p: k) (20:10:10) every other day to release the macronutrients needed for zooplankton growth, whereas 1.0 ml of nutrient salt solution a and 0.1 ml of solution c (laing and ayala, 1990) were administered every other day to the pure algal cultures (ajah, 1995). outdoor cultures fertilization of outdoor cultures was made possible using pig manure supplied regularly by 20 weaned pigs that were fed daily on 10% body weight with pig mash (akpan and okafor, 1997). the chemical composition of samples of pig manure was determined and found to contain the essential nutrient elements for algal growth. one (1.0) kg of pig manure was introduced into each outdoor tank during the dry season and 2 kg during the wet season for the first two consecutive days. thereafter, 1 kg per tank was administered every three days and every other day, for the dry and the wet seasons, respectively. 50 ml of each axenic algal monoculture, namely, c. vulgaris, s. quadricauda and e. elegans, from the laboratory, were inoculated into the respective tanks at cell densities of 2.6 × 10 6 , 1.68 × 10 6 and 0.12 × 10 6 cells/ml. five litres each of 4 ind ml -1 from pure b. quadridentatus cultures (ajah, 1995) were introduced into each 10 m 3 tank. the feeding duration of b. quadridentatus with each of the three algae lasted for an average of 18 days (range 15 21 days). each culture was repeated three times during both the dry season (november to march/ april) and wet months (may/ june to october). the batch culture trials continued for a period of three years amounting to a total of eighteen trials, nine per season (ajah, 1995, 1997, 1998). cell counts a 1 l plastic funnel was used to scoop samples from the thoroughly mixed plankton cultures. the samples filtered through a 56 µmplankton sieve to collect and retain both phytoand zooplankton. four cell counts of 1 ml each using a haemocytometer and four zooplankton counts using a one ml counting chamber (model: ajah001) (ajah, 1995) were carried out every day or every two days, and average values were recorded. physicochemical parameters the physical and chemical factors were monitored throughout the experimental period. dissolved oxygen in the pond was determined using lectron 5509 do meter and temperature was read using standard thermometer. chlorine was assessed by the chlorosity method of rump and krist (1988). nitrite (no2 -n) by the diazotization (spectrophotometric) method; nitrate (no3 n) by the cadmium reduction/ diazotization method, ammonium (nh4 + -n) level by the nesslerization (spectrophotometric) method, phosphate by the molybdenum blue method (spectrophotometric) (parsons et al., 1984), conductivity was read using a hach 3000 spectrophotometer and turbidity by secchi disc. single classification analysis of variance (anova) was used following sokal and rohlf (1981) to compare means of population density of zooplankton counts from the replicates. correlation coefficients between b. quadridentatus and each primary producer as well as the coefficients of determination were also calculated using spss. descriptive statistics were used to compare the means. the intrinsic rate of natural increase (r) and the population doubling time in days (td) were calculated as follows: r = (in nt in no) /t = d’/t or 2.3026(log nt no)/t (james and dias, 1984). where: d’ = the dilution time in days. nt = no.e rt (james and dias, 1984). nt = final number of individuals. no. = initial number of individuals, and t = time in days . e (exponent) = 2.7183. r = the intrinsic rate of natural increase. td = 0.6931/r (james and dias, 1984). t d = doubling time of the population in days. results c. vulgaris, e. elegans and s. quadricauda showed statistically significant ( 2 ,115f3.27, p < 0.05) differences in 3 table 1. daily mean values and summary of the three replicates showing some growth characteristics of brachionus quadridentatus while feeding on the three algae. chlorella vulgaris scenedesmus quadricauda eudorina elegans date r td (time in days) nt (ind/l) r td (time in days) nt (ind/l) r td (time in days) nt (ind/l) 1 4 0.207 3.508 3,000 0.414 1.801 4,000 0.496 1.435 3,000 5 0.433 1.611 15,000 0.504 1.423 18,000 0.638 1.094 8,000 8 0.326 2.129 31,000 0.381 1.835 41,000 0.371 1.369 11,000 9 0.312 2.368 50,000 0.424 1.637 65,000 0.433 1.071 32,000 10 0.284 3.484 62,000 0.291 2.414 70,000 0.286 1.615 46,000 11 0.375 1.848 123,000 0.360 1.936 153,000 0.272 2.422 93,000 12 0.373 1.860 143,000 0.354 1.948 160,000 0.096 5.722 104,000 15 0.310 2.234 124,000 0.158 4.888 155,000 0.119 5.802 118,000 16 0.279 2.488 88,000 0.207 3.366 125,000 0.319 2.169 127,000 17 0.257 2.707 63,000 0.140 4.978 94,000 0.189 3.660 98,000 19 0.198 3.500 55,000 0.155 4.472 77,000 0.170 4.111 55,000 22 0.216 0.482 25,000 0.108 9.02 46,000 0.123 5.724 36,000 biomass indiv. × 10 6 /10 m 3 /day 366.92 489.52 289.05 peak densities(ind./l) 102,000 104,000 176,000 av. pop. (nt) (ind/day) 65,167 ± 45622 72,000 ± 51464 60,916 ± 44971 av. rate of natural increase (r) l/day) 0.149 ± 0.01 0.276 ± 0.01 0.286 ± 0.01 av. doubling time of pop. (td) in days. 0.84 ± 0.89 2.04 ± 0.77 2.61 ± 0.38 r 2 b/w brachionus and alga density 0.166, p < 0.05 0.044, p > 0.05 0.162, p > 0.05 population biomass growth and development. b. quadridentatus’ growth using s. quadricauda was significantly ( 1 ,76f6.804, p 0.01) greater than when fed on c. vulgaris. there were no significant ( 1 ,77f0.330, p 0.05; 1 ,76f3.499, p 0.05, respectively) growth differen-ces between b. quadridentatus-fed, s. quadricauda and e. elegans, and, between b. quadridentatus fed on c. vulgaris and e. elegans. correlation coefficients between b. quadridentatus densities fed on c. vulgaris and e. elegans, e. elegans and s. quadricauda and s. quadricauda and e. elegans were 0.454, 0.413 and 0.618, respectively. the population biomass of b. quadridentatus increased quite rapidly with s. quadricauda, followed by e. elegans and lastly c. vulgaris (table 1). high average population densities of b. quadridentatus achieved with c. vulgaris, e. elegans and s. quadricauda are shown in table 1. b. quadridentatus showed extremely high rates of natural increase, with correspondingly very short population doubling times (td) with s. quadricauda (table 1). the intrinsic rate of natural increase (r) and doubling time in days (td) for b. quadridentatus using e. elegans, and c. vulgaris cultures are equally represented in table 1. the overall daily mean population growth (nt) was 65,167 ± 45622, 60,916 ± 44971 and 72,000 ± 51464 for c. vulgaris, e. elegans and s. quadricauda, respectively. however, based on the three replicates, b. quadridentatus needed less time, on the average 0.84 days, to double its population when fed on c. vulgaris, followed by 2.04 and 2.61 days, respectively, was required by s. quadricauda and e. elegans to double their populations (table 1). more stable and higher growth rates were obtained using s. quadricauda and e. elegans compared to c. vulgaris. the chemical composition of the swine manure was: nh4 + -n = 178.3 mgl -1 , so4 = 85.44 mgl -1 , sio2 = 85.2 mgl -1 , no2 -n (below detectable level), no3 4 900 800 700 600 500 400 300 200 100 0 815 wet season 522 dry season 230 188 3.855.22 2.8 2.6 0.88 1 0.880.96 0.2 0.3 4.6 4 26.528.5 cond turb nh4 no3 no2 po4 cldo temp oc figure 1. showing seasonal mean physicochemical factors and nutrient contents obtained during the culture of brachionus quadridentatus in earth pond. n = 25.3 mgl-1, po43--p = 0.64 mgl-1, n = 28.7 ppm/22.7%, p = 0.20 ppm/0.66%, k = 12.5 ppm/31.96% and moisture content 62.6%. the percentages represent total available nitrogen, phosphorus, potassium and moisture content in the sample. the mean values and ranges of the physicochemical parameters and nutrient loads of the culture environments are shown in figure 1. temperature ranged from 26.5°c to 29.4°c with average of 27.74 ± 0.108°c. dissolved oxygen levels ranged from 2.7 5.4 mgl -1 with a mean of 4.238 ± 0.126 mgl -1 . mean conductivity values in the culture tank were 642.567 ± 142 s/cm ranging from 153.7 921 s/cm. the turbidity of the culture system ranged from 109 279 ftu with a mean of 218.607 ± 32 ftu. the no3 -n level range from 1.081 to 3.050 mgl -1 with a mean of 2.048 ± 0.549 mgl -1 while the phosphate levels in the tanks were 0.150 to 1.016 mgl -1 with a mean of 0.647 ± 0.149 mgl -1 . discussion the physicochemical factors and nutrient load were within the acceptable limits required for the proper management of earth ponds (wedemeyer et al., 1976; post, 1987; swift, 1988; agarwal, 1994; biswas, 1996, ajah, 2008). physicochemical values in this report that fell within the optima required for plankton growth included amongst others temperature (ifas circular, 1951), do (banerjea, 1967), the electrolyte conductivity, ph and the do (ajah, 1995, 2008), turbidity (ajah, 1995, 2008), nitrate (sachidanandamurthy and yajurvedi, 2004) and phosphate (sawyer, 1947; screenivasan, 1965). b. quadridentatus due to its extremely high fecundity doubled its population in only 0.149 days and had a td of 0.84, giving it a comparative advantage over b. plicatilis that had r = 0.84, td = 1.212 obtained with nannochloropsis sp. (ahmad et al., 1991). this puts b. quadridentatus as a more promising alternative food source to artemia. the greater preference for s. quadricauda and e. elegans to c. vulgaris by b. quadridentatus could be explained in terms of the voracious nature and size of brachionus as well as its choice of food item. kennari et al. (2008) rather observed that b. calyciflorus had better growth and fatty acid content when fed on chlorella sp. compared with s. obliquus. investigations revealed that phytoplankton abundance was controlled by zooplankton abundance and not fertilization as in a hard water pond (sierp, 2001), whereas in soft bottom ponds such as ours both zooplankton abundance and fertilization controlled phytoplankton abundance. ajah (2008) earlier reported a greater preference of asplanchna priodonta for e. elegans which confirms the need for larger prey by larger predators. the initial population growth of keratella cochlearis was significantly affected by the feeding schedule as well as the presence of competitors, while that of daphnia was affected by neither factor. population densities of both species tended to increase as the frequency of food addition increased (macisaac and gilbert, 1991). xi et al. (2002) found out variations in population growth, body size and egg size of b. calyciflorus while feeding on different algae. fabiola et al. (2005) equally observed differences in population growth of rotifers and cladocerans when raised on algal diets supplemented with yeast. mean population growth rate (r) of 0.61 and 0.44 were obtained for rotifer fed with chlorella sp. and s. obliquus , respectively (kennari et al., 2008) which are lower than values obtained in this report using three algae, c. vulgaris (0.149), s. quadricauda (0.276) and e. elegans (0.286). conover and mayzaud (1984) made the following 5 generalizations regarding selective grazing: (1) at low concentrations of food, feeding tends to be spread over a wide range of particle sizes, (2) although “tracking” or selection of biomass peaks as a strategy to facilitate capture of rapidly reproducing, and presumably more nutritious phytoplankton (poulet and chanut, 1975), and (3) if the particle has a “suitable” shape and is not chemically distasteful, only then do zooplankton tend to select bigger objects. points 2 and 3 above agree with the findings made here. my investigation showed that there was an almost equal attraction for the various food items administered, proving that none of the food items had unpleasant odour or taste to b. quadridentatus, nor did any possess an awkward shape. in conclusion, prey size, shape, and motion are implicated as probable causes for the slight differences in feeding behaviour. b. quadridentatus generally preferred the bigger prey like e. elegans, or medium size prey, but with some flagella-like structure as in s. quadricauda as well as prey in constant motion such as e. elegans. the cilia of b. quadridentatus were capable of generating waves of current for trapping its prey. distance was not a barrier for capturing prey, due to its fast motion. b. quadridentatus could easily be grown on three algal live diets such as c. vulgaris, e. elegans and s. quadricauda. acknowledgement this study was funded through a fellowship provided by the european economic community. references adeyemo aa, oladosu ga, ayinla 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between the dietary values of brachionus plicatilis and their content of -3 highly unsaturated fatty acids. bull. jpn soc. sci. fish 45: 883889. watanabe t, kitajima c, fugita s (1983). nutritional values of live organisms used in japan for mass propagation of fish. rev. aquacult. 34: 115-143. wedemeyer ga, meyer fp, smith l (1976). diseases of fishes: environmental stress and fish diseases pp. 89-106. xi yli, liu gyi, jin hj (2002). population growth, body size, and egg size of two different strains of brachionus calyciflorus pallas (rotifera) fed algae. j. freshwater ecol. 17(2): 185-190. 7 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 11 (1), pp. 001-008, january, 2023. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review bird scaring technologies in rice production: the need for policies prohibiting participation of women and children augustine odinakachukwu ejiogu and victor b.n. okoli department of agricultural economics, extension and rural development, imo state university, owerri. accepted 06 june 2022 a disruptive technology improves a product or service in ways that the market does not expect typically by being lower priced or designed for a different set of consumers. there are arrested technologies which reduce human drudgery but have not taken root and are therefore marginalized on account of the neglected sector which they serve. in nigeria, bird scaring in the agricultural sector, is to date effected manually; in the aviation sector, it is never done manually. nigeria’s total consumption stands at 4.4 million tons of milled rice, but produces only about 2.8 million tons. the deficit is augmented through rice importation. of all the problems of rice production, that of birds scaring tends to be the least discussed in literature and by extension a neglected area. this neglect has in effect arrested bird scaring technologies that upgrade the traditional tedious and laborious use of women and children in bird scaring. for 3-5 weeks during the milk stage of rice in the field, women and children spend the hours of 7am-6pm on end daily scaring birds. for the women, the activity is an additional burden to their domestic chores. for the children, their attendance to school is adversely affected. this paper presents some alternative bird scaring operations in rice farming. it also proposes some policy measures aimed at releasing these technologies for mass adoption and thereby effectively relieving women and children from manually scaring birds in rice fields. keywords: bird scaring technologies, rice production, policies prohibition. inrtroduction a disruptive technology or disruptive innovation is an innovation that upgrades a product or service in ways that the market does not expect by being typically lower priced or produced for a different category of consumers (www.disruptive technology.htm). in this sense, at least two sets of innovations are under consideration when corresponding author email: odiiejiogu@yahoo.com discussing the concept of disruptive technology: one predating the other and the more recent improving a product or service in ways unexpected by the market. in a related concept, there are technologies which reduce human drudgery but have not taken root and are therefore marginalized on account of the neglected sector which they serve. in the agriculture sector, bird scaring in rice production is to date effected manually. in the aviation sector, bird scaring is never done manually. rice (oryza sativa) is the most important food crop in the tropics. it is a major staple food for the people of 2 west africa and the fastest growing commodity in nigeria’s food basket (akande, 2003). the importance is such that it is both a ceremonial and a staple food in many homes. most countries in sub sahara africa consume more rice then they produce. for instance, nigeria’s total consumption stands at 4.4 million tons of milled rice but produces only 2.8 million tons. the deficit is made up through massive rice importation. for 3-5 weeks during the milk stage of rice production, women and especially children spend the hours of 7am – 6pm on end daily scaring birds in rice farms. for the women the activity is an additional burden to their domestic chores; for the children their attendance to school is adversely affected. in spite of the importance of rice in the economy of sub-sahara africa, manual labour still predominates the bird scaring operations. the problem is not so much lack of improved bird scaring technologies as the absence of strategic commitment to enhancing the environment enabling elimination of use of vulnerable women and children as bird scares. of all the problems of rice production, that of birds scaring tends to be the least discussed in literature. this neglect has in effect arrested bird scaring technologies that upgrade the traditional tedious and laborious use of women and children in bird scaring. it is against this background that this paper reviews some bird scaring technologies in rice production with a view to influencing and informing policy measures aimed at releasing these technologies for mass adoption and thereby effectively relieving women and children from manually scaring birds in rice fields. the rest of the paper is divided into the following sections: background on rice research in nigeria, production, consumption, and importation of rice in nigeria, traditional use of women and children in scaring birds from rice fields, the concept of arrested technologies and conclusion and recommendations. rice research in nigeria rice (oryza glaberimma stend) is indigenous to nigeria and has been in cultivation for the past 3500 years (hardcastle, 1959). the earliest cultivation of improved rice varieties (oryza sativa l) started in about 1890 with the introduction of upland varieties to the high forest zone in western nigeria. by 1960, o. sativa had taken the pride of place over o. glaberima which is now limited to some deep flood plains of sokoto rima river basin and other isolated pockets of deep swamps all over the country. active and systematic rice research started in nigeria in 1953 with the establishment of the then federal rice station at badeggi, niger state, now the headquarters of the national cereals research institute. however, improved technologies to meet rice production needs of nigeria weigh heavily on the side of varietals improvement, germplasm collection and conservation, to the exclusion of technologies that will release women and children from scaring birds in the farms. this has found expression in the continued use of women and children in rice fields as bird scarers. production, consumption and importation of rice in nigeria rice production the three important activities of an enterprise are namely: finance, production and marketing (pandey, 2000). the finance functions involve raising funds, generating returns from the capital raised and also paying returns to the suppliers of the funds. the process of transforming inputs into outputs is called production (adeyeye and dittoh, 1985). the finance production, and marketing functions in an enterprise are in fact not sequentially but are performed simultaneously and continuously. rice production is carried out over an area of about 143 million hectares in the tropical and subtropical parts of the world (kochhar, 1981). over 65% of the world’s rice production comes from china, india bangladesh, japan pakistan and the adjoining islands in the pacific. only a little under two percent of the world’s rice production comes from the united states. however, united states is a rice exporting country. ecosystems for rice production are determined by the interplay of such variables as geology, climate, soil types and hydrology vegetation and a range of socio-economic factors (barker and herdt 1979; ward, 1994). water conditions or balances and soil fertility are two major physical factors which determine productivity of ricebased cropping systems (iita, 1988; anyaegbu and iloka (1982). rice is the only crop adapted to flooded conditions; water is the most critical limiting factor of all physical requirements. the general approaches to increasing rice production are boosting the yield per hectare and expansion in area under cultivation. but increasing agricultural production comes with economic, environmental and social costs that may ultimately limit growth (bender and smith, 1997). brown (1994) argued that many of the techniques used in increasing yield over the past decades such as increased fertilizer use crop breeding and irrigation have been known for a century or more and may not bring much additional growth. this argument is fundamentally flawed to the extent that it does not recognize the strategic roles of research and development in improving and upgrading these techniques. in nigeria, about 40% of the present area under rice cultivation is rain-fed upland and 60% is under swamp (chaudhary and nanda, 1985). the average yield per 3 hectare of rain-fed upland is 0.6 metric tons. however, with the adoption of improved varieties and package of cultivation practices the per hectare yield can be increased. similarly, the yield per hectare of swamp rice of about 1t/ha can be increased. paddy rice production rose from 0.134 million metric tons to 0.344 million metric tons in 1970 cultivated on 0.156 million and 0.255 million hectares respectively (ncri undated). rice is cultivated in virtually all the agro-ecological zones in nigeria. significant improvement in rice production in nigeria occurred in 1980 when output increased to 1 million tons while area cultivated and yield rose to 550,000 ha and 1.98 tons per hectare respectively (akande, undated). accordinh to (warda, 1994),nigeria has become the highest rice producing country in west africa and third in africa after egypt and madagascar. according to bender and smith (1997) crop yields are influenced by the interaction of soils and plants the climate and socioeconomic circumstances such as prices, the quality and cost of labor and the availability of credit. strategies to increase yields in a given area of the world depend first on identifying which factors are most limiting and then searching for ways to push the limit outward. according to iita (1988), constraints to rice production include shortage of farm labour, absence of efficient farm tools and farm machinery, lack of fertilizer and credit facilities. the constraint of farm labour tends to be more of its relative cost than being in short supply. in terms of labour, nwagbo and onwuchekwa (1987) stated that 25% of the farmers used only hired labour while 55% used both family and hired labour. men, women and children all contribute to farm labour in rice production. however, some farm operations are age and gender specific. this view is consistent with nwagbo and okorji (1987) who stated that whereas men participated in all farm operations, no woman or child was involved in tillage operations. children helped in weeding and harvesting activities but were mostly employed for bird scaring. reporting on labour use, njoku (1988) stated that the total amount of labour employed per hectare was 103 man-days of swamp rice and 123 man-days for upland rice. forty-one percent of the labour came from household sources while 59% was hired in swamp rice production. for upland rice production, 33% of labour came from family sources while 67% was hired. njoku (1988) arranged the following farm operations in their ascending order of labour use: harvesting, land preparation and weeding. in the views of nwagbo and onwuchekwa (1988) out of a total of 1434 man-hours equivalent used to produce one hectare of paddy rice, bird scaring took the highest amount of labour (612 manhours). according to nwagbo and onwuchekwa (1988), wage rate varied with the nature of farm operations. tillage operations attracted the highest and bird scaring attracted the least. labour accounted for 82% of the total cost. in the views of njoku (1988) labour tended to be one of the greatest constraints to increased rice production. rice consumption rice consumption in nigeria has been on the increase (naerls, 2005). for instance, in 1989 the demand for rice was 2.31 million metric tons while the supply was just 1.38 million metric tons. according to usaid markets (2008), total consumption of rice stands at 4.4 million tons of milled rice while annual consumption per capita stands at 29kg. in 1970 the per capita consumption was 3.5kg and increased to 14kg in the 1990s. induced by income growth, the consumption per capita has continued to rise at 11% per annum. rice imports according to ncri (undated), in the 1960s nigeria was almost 99% self-sufficient in rice production and consumption. twenty years after, this self-sufficiency declined to 38% as demand over-stripped supply. this 62% deficit necessitated massive importation of rice by the federal government of nigeria. because rice importation is paid for in foreign currency, it is therefore a source of concern as this relates to the balance of payment position of the country. according to akande (undated) nigeria spent $0.1 million on rice importation in 1970. in 1999 the value was $259 million. akande stated that between 1961 and 1999, nigeria spent $4 billion on rice importation alone. according to biyi (2005), volume of rice imported in 2003 was 2.5 billion metric tons at the price of n29.85billion. in 2004, the volume imported was 0.84 million metric tons at the price of n30.31 billion. concept technology in rice production most rice in west africa is produced small-scale farmers who use little or no external resources and have varying balance between subsistence and commercial objectives (warda, 1994). these farmers largely operate under traditional rain-fed rice production technologies and move from extensive to more intensive cultivation without adapting their traditional practices to the new objectives. according to swanson (1996) technology is the application of knowledge for practical purpose aimed at improving the condition of human and natural environment and also carry out some other socioeconomic activities. it is a means of increasing and exploiting our understanding of nature for our own benefit (ikeme and uvere, 1995). establishing a symbolic relationship between science and technology, williams 4 (1985) referred to science as a systematic search for truth which provides the basis for technology. it should be pointed out that art, conceived as practical and human skill and their application, additionally forms the bedrock for technology. with respect to technology, williams (1985) referred to is as the application of techniques which leads to increase in production. in terms of production, agricultural technology adds greatly to farm productivity (salvatore and dowling, 1987).the modern miracle of technology that has increased agricultural potential throughout the world is referred to as the green revolution. according to ruttan and binswanger (1978), green revolution ordinarily refers to the development and diffusion of high-yielding cereal varieties, particularly wheat and rice, in the developing countries of the tropics and semi-tropics, beginning in the mid-1960s. according to mafimisebi (2010), two categories of technology exist: material technology and knowledge based technology. also known as “hardware” components an example of material technology is improved rice varieties. an example of knowledge based technology also known as “software” component is planting date. the development of technology be it material or knowledge–based is driven not only by the imperatives of engineering but also by the cultural and ideological values of the society concerned (ikeme and uvere, 1995). according to muller (1976) modern large-scale technology is basically a result of the cultural demand or values of western society. this concept of culturallydriven technologies has given impetus to the exploitation and adaptation of imported technologies to suit local environmental, social and economic needs. going by the argument of cultural and ideological values imprinting on the development of technology, the need arises to investigate why bird scaring technologies in rice production is still dominated by the manual use of women and children in the rice farms. since the 1920’s when rice research began in ibadan (naerls, 2005), the popularity of rice as a food has been growing in nigeria. however the same cannot be said of releasing women and children from bird scaring operations in rice farms in nigeria. the use of women and children in scaring birds the employment of women and children in scaring birds in rice farms basically involves stationing them in the farm to effect the driving away of the pests from the rice using such means as shouting, singing, dancing, throwing missiles etc to generate both visual and acoustic stimuli that scare the birds away. children also use catapult to scare away the birds. in spite of the importance of rice in the economy of nigeria, manual labour is still employed in driving away birds. the issue is not so much lack of improved bird scaring technologies as the absence of strategic commitment to the prohibition of the use of women and children as bird scares. women predominate in agriculture. any disregard to the development of labour saving technologies especially as they relate to bird scaring in rice farms has negative consequences on the working women. going by the continued increase in the per capita consumption of rice in nigeria, fixated reliance on women and children and in fact on human beings to prevent the birds from doing harm to their rice in the field will to that extent perpetually prevent the people from realizing their potentials for increasing rice production. this eventually translates to sinking further into the poverty quagmire. employing the child-bearing women as bird scarers in rice farms is an extra burden on the traditionally demanding role of parenthood. for the elderly women it is work beyond retirement. for the school-age children, they miss classes, have less time available for homework and self-study. in extreme cases the children drop out of school. they enter the labour force prematurely. according to robson (2004) children who are engaged in farming have lower school attendance. the bird scaring process one of the cultural practices in rice production is bird scaring. it involves an array of activities aimed at driving away birds from rice during specific period the crop is growing in the field. during the milk stage of rice production birds are driven away from the rice farms. during this stage, grain-eating birds invade the rice fields and suck the milky grains leaving traces of empty or poorly filled grains, broken stems and cut panicles. according to usaiad markets (2008) birds are a problem during grain filling or milk stage. birds suck the sugary semi-liquid substance leaving behind an empty husk. at later stages when the grains are ripening, birds also cause grains to shatter. birds’ invasion of rice farms causes whitehead and very low yields. in terms of bird control, naerls (2005) stated that scaring devices are used and added that human bird scares are employed from 6am to 7pm two weeks after planting and from heading to harvesting. usaid markets (2008) stated that control of birds is done manually by the use of scares and spoilt video tapes which make noise whenever the wind blows. ncri (undated) categorized control measures into (i) direct protection, (ii) chemical repellants, (iii) agronomic repellency and (iv) indirect protection. according to ncri direct protection involves the use of human bird scarers, scare crows and flash tapes, use of different forms of traditional noise making devices, noise produced by more sophisticated techniques such as 5 carbide cannons at a rate of two per hectare with positions and directions of fire changed every other day; and periodic use of shotgun to augment explosive scarers. application of chemical repellants such as thiram, is effected to repel ducks and bush fowls which damage seeds at sowing time; at the maturity the following chemicals are applied for repelling grain eating birds: mesurel (methicarb) 3kg/100l/ha, thiram 3kg/100/ha, aluminium ammonium sulphate 10kg/100/ha. agronomic repellency includes avoidance of extended sowing dates, avoidance of establishment of rice farms close to forests where birds perch. indirect protection entails destruction of the resting colonies or roost through the use of ground or aerial spraying and by use of explosives. farm radio international (2009), stated that different methods be combined to keep birds away from rice fields. it further stated that often women and children are sent to the fields to guard against birds. this practice has serious consequences because it adds to the work-load of women and keeps the children out of school. to all intents and purposes, bird scaring in nigerian rice fields is manually effected through the deployment of women and children to the rice fields. naerls (2005) called this method human bird scarers, usaid markets (2008) referred to it as manual control of birds and ncri direct protection method involves use of human bird scarers. according to nakamura, japanese farmers use many methods to protect their rice from birds, the most effective being covering the field with netting. however, nets are costly and cannot be used in expansive fields. according to nakamura, the most common method of protecting rice is to scare the birds away using stimuli which arouse aversion in the birds and eventually drive them away from the field. nakamura classified the stimuli into two main groups namely visual and acoustic. visual stimuli include plastic bags set out in fields to flutter in the wind. in addition to driving away birds already in the field, the plastic bags deter birds on the wing from coming and landing on the rice farms. other visual stimuli include human effigies, scare crows and balloons with eye spots painted on them. acoustic stimuli scare birds with sudden loud noise. according to (nakamura 2011), one of such stimuli is the wooden clappers that are banged together by pulling a string or by the wind. nakamura added that japanese farmers most commonly use banger which produce a loud noise from an explosion of gas. furthermore, recorded distress calls and alarm calls of birds are often used to scare away birds. artificially synthesized versions are also used as they are amplified and broadcast through a loudspeaker. bird scaring technologies in japan, for instance place emphasis on reducing the tedium and drudgery of manual labour. on the other hand, those available in nigeria are not just heavily dependent on manual labour but on the manual labour of women and children. problems posed by birds in rice farms controlling pest or nuisance birds is more difficult than any other pest, insect or animal known to man (www.kingtrog.com.all). according gallagher et al. (2002) birds can be very damaging to rice especially when they occur in large flocks. during the ripening period in northeast asia, some fields are protected by being covered with bird nets which are widely available. in both asia and africa various forms of bird scaring are employed to keep birds out of rice fields. while reflective ribbons or used video or cassette tape are widely utilized for birds scaring in asia, people shouting or hurling dried mud and other missiles at the birds is common in africa. in nigeria in particular the people are women and children. sound cannons and owl or hawk look-alikes are also used in developed countries. the relative ease of scaring birds depends on which of the following three situations they are found in: i. roosting birds are easy to scare. birds roost in a particular location out of habit. when this habit is broken the problem is solved. ii. feeding birds are difficult to scare. this is so because any attempt at scaring is an attempt at denying them their daily bread. iii. nesting birds are impossible to scare. reason is that their mothering instinct is so strong that they defiantly return to the nesting site. however, birds can be stopped from selecting a site in the first place. bird populations especially pest birds are increasing (www.kingtrog.comm.all). bird damage is getting worse. little research has been carried out into bird control. arrested bird scaring technologies according to ncri (undated) the following factors are constraints to increased rice production: research (breeding and selection of low level management of rice), pest and disease management, simple and cheap farm implements, institutional and infrastructural support, credit facilities and input support delivery, marketing channels, irrigation facilities and extension services. under research constraints canvassed concentrated on problems of area cultivated and technologies utilized. ncri stated that in severe cases pest and disease management account for 100% losses. however, no specific mention was made of bird pest and the less satisfactory use of women and children to drive them away from rice farms. under farm implements as a constraint, ncri stated that traditional farm operations are carried out with hand tools which make farming tedious and cumbersome. ncri concluded that research efforts should aim at developing suitable implements for 6 weeding, land preparation and planting. no reference is made to the development of farm implements for bird scaring, despite the fact that bird scaring takes the highest amount of labour and ironically the least paid operation in rice cultivation. women together with children (boys and girls) predominate in agriculture. in most social/cultural milieus, they tend to be categorized as the vulnerable group. hence issues that have direct bearing on them tend to be easily overlooked except in strict and formal discussions on the root causes of poverty and inequality. this argument is supported by donahue (2009) who stated that whether girls (women) are able to benefit from new technologies often has to do with preexisting issues of social exclusion and discrimination. in the words of eboh (2011), despite the credential of the agricultural sector as the main financier of colonial and post colonial regional and national development in nigeria, the sector has continued to suffer neglect which began with the discovery of crude oil and the oil boom. the traditional use of women and children for bird scaring operations in rice farms is as old as rice cultivation in nigeria. new and improved bird scaring technologies are available (though not necessarily affordable). however, the use of women and children in bird scaring operation in rice farms persists till date. the price component of these technologies tend not to be a strong point when it is realized that nigeria can instead afford huge foreign exchange earnings to import rice as opposed to using a fraction of it to adapt and transfer these arrested technologies to the rice farmers. the technologies can be referred to as arrested in the sense that either by omission or commission by policy makers and relevant stakeholders the technologies have not taken root. the bird scaring technologies are arrested on account of their innocuous offence of attempting to reduce the drudgery of a vulnerable group who are engaged in a near neglected sector in most sub-saharan african countries including nigeria. the technologies tend to be generally out of commission because they improve the manual bird scaring method in a way inconsequential and immaterial to policy makers. some of the arrested technologies are highlighted as follows: i. use of reflective ribbons or used video or cassette tape tied round the rice farm in a crisscross manner which the birds will think is a net. ii. use of non-coloured net placed vertically like a curtain tied to poles fixed on either side across the rice field. depending on the size of the field, several curtains of nets are fixed at different intervals across the field. iii. setting up scare crows and hanging cassette tapes in between sticks. iv. combination of these methods to check the birds learning and adapting to a particular method. because of this problem of habituation, it is advisable to also change tactics and never rely on any one for too long. v. farmers in the same area should plant at the same time as larger area of land with rice that ripens at the same time incurs less damage. vi. use of castanets is also advocated. castanets are produced using small metallic boxes like old cans. pebbles are put into them and the cans closed. the castanets are hanged on trees at different points in the rice field. the castanets are linked to one another using a strong rope. whenever the birds appear, a single person sitting under the shade of a tree can pull the rope effectively producing a loud noise that scare the bird pests. vii. a more advanced technology is the bird scaring helikites (www.gaskites.com). the scientifically designed and patented vigilante helikite is a bird control system that works well over a long period of time because birds find it extremely difficult to overcome the innate terror of predatory hawks that helikites create. the vigilante helikite can fly at great heights of up to 60 metres thus enabling them to be seen over hills or hedges and to have excellent birds-caring ability over vast areas this is far more than is usual with any other method. to buy a vigilante helikite is cheap. so also the helium gas cylinder with enough gas to fly a helikite for nearly a year. in the u.s, this goes for about $50 or less.(www/allsopp%20vigilante%20helikite%20 %20reliable%20bird%20control.htm) these technologies should be effectively released to the rice farmers and thereby relieve the boredom, drudgery and the perpetuation of poverty attendant to the use of women and children in scaring birds in rice farms. solutions and recommendations based on the discussions in this paper, the following solutions and recommendations are suggested. i. alternatives for reducing women involvement in bird scaring should be explored. ii. emphasis should be placed on research into appropriate technologies that will release women and children from the drudgery and tedium of scaring birds in rice fields. iii. similarly, for self-sufficiency in rice production in nigeria, effective and purposeful support system should exist. for example, there should be functional credit facilities, improved and timely input delivery and aggressive and effective extension services. credit facilities should be targeted at women rice farmers. this not only enables the women acquire improved technologies for their livelihood but also gives then choice over what livelihood to engage in. this argument is supported by the fact that women comprise 85% of the 95 million poorest borrowers in the microcredit sector; without collateral, they tend to face difficulty if banks 7 iv. tighten their micro-lending requirement (world bank, 2009, buvinic, 2009). v. there should be a ban on the use of children in all forms of labour including bird scaring. future research directions from agriculture to aviation, education and information and communication technologies through finance to mortgage and across sectors, industries, and disciplines, the world is being reshaped. the attendant implications are legion—less energy and less tedium for processing natural resources, less waste in processed raw materials—and these translate to desirable effects on the environment. men and women are continuously creating comfort and utilities and unyieldingly upgrading them. however, these developments have not been felt in bird scaring activities in rice farms in nigeria to the extent that women and children are used as human scares. there is the need to investigate, for instance, why in the aviation sector bird scaring is never done manually while in the rice subsector of the agriculture sector, that is the rule rather than the exception. conclusion a disruptive technology is a technology that improves a product or service in ways that the market does not expect typically by being lower priced or designed for a different set of consumers. at least two sets of technologies are under consideration when discussing the concept of disruptive technology: one predating the other and the more recent improving a product or service in ways unexpected by the market. there are technologies which reduce human drudgery but have not taken root and are therefore marginalized on account of the neglected sector which they serve. in nigeria, bird scaring in rice production is to date effected manually. in the aviation sector, bird scaring is never done manually. most countries in sub-saharan africa consume more rice than they produce. nigeria’s total consumption, for instance, stands at 4.4 million tons of milled rice, but produces only about 2.8 million tons. the deficit is made up through rice importation in spite of the importance of rice in the economy of sub saharan africa in particular, and nigeria in general, manual labour still predominates the bird scaring operation. the problem is not so much lack of improved bird scaring technologies as the absence of strategic commitment to the environment enabling elimination of use of vulnerable women and children as bird scares. this paper reviews some bird scaring operations in rice farming and points out the poverty perpetuating propensities of continual use of women and children in scaring birds in rice farms. it also proposes some policy measures aimed at releasing these technologies for mass adoption and thereby effectively relieving women and children from manually scaring birds in rice fields. in this day and age, it is more development oriented to send children to school than to prematurely send them to the world of work. definition of terms bird scaring: it involves an array of activities aimed at driving away birds from crops during specific period the crop is growing in the field. arrested technologies: these are technologies are that have defied mass adoption on account of their innocuous offence of attempting to reduce the drudgery of a vulnerable group who are engaged in a near neglected sector. references adegeye aj, dottoh js (1985). essentials of agricultural economics. card university of ibadan. akande t (2003). “the rice sector in nigeria” united nation crop project (uncp) country agricultural project on trade liberalization in agricultural sector and the environment, geneva. pp.10. akande t (undated). “an overview of the nigerian rice economy” in www.anoverviewofthenigerianriceeconomy.htm. accessed 14 february, 2011 anyaegbu sn, iloka au (1982). upland rice production agricultural extension research liaison and training national roots crops institute, umudike extension bulletin. no. 2 pp 214 barker cb, herdt wr (1979). rain-fed lowland 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(eds), improving agricultural extension: a reference manual. rome, fao. usaid markets (2008). improved packages of practices for rice production united states agency for international development funded maximizing agricultural revenue and key enterprises in targeted sites. p.16. west african rice development association warda (1994). west african rice development association research highlights warda, liberia pp 30-34. williams skt (1985). rural development in nigeria. university of ife press nigeria. world bank (2009). “protecting progress: the challenges facing low income countries in global recession”. background paper prepared by world bank group staff for the g-20 leaders meeting, pittsburgh usa 24-25. www.kingtrog.com.all accessed 16 february, 2011. www.gaskites.com accessed 14 th february, 2011. www/allsopp%20vigilante%20helikite%20%20reliable%20bird%20co ntrol.htm accessed 16 february, 2011 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (11), pp. 001-010, november, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review the need for adequate processing to reduce the anti-nutritional factors in plants used as human foods and animal feeds: a review k. o. soetan1* and o. e. oyewole2 1 department of veterinary physiology, biochemistry and pharmacology, university of ibadan, nigeria. 2 department of health promotion and education, faculty of public health, university of ibadan, nigeria. accepted 20 august, 2019 anti-nutritional factors (anf) are compounds which reduce the nutrient utilization and/or food intake of plants or plant products used as human foods or animal feeds and they play a vital role in determining the use of plants for humans and animals. apart from cyanogenic glycosides, food poisoning arising from antinutritional factors, otherwise known as plants’ secondary metabolites has not been properly addressed in most parts of the developing world. people have died out of ignorance, poverty and inadequate nutrition information and education, especially within the african societies. there are reports from time to time of deaths after consumption of some type of beans despite cooking. also, cases of renal and liver diseases are increasing and this calls for a need to properly address the issue of thorough and adequate processing of foods/feeds before consumption. the aim of this review is to emphasize on the adequate processing of foods/feeds and to educate the people on the dangers of consuming improperly processed foods especially legumes which are reported to contain very high concentrations of anti-nutritional factors. key words: processing methods, anti-nutritional factors, plants, human foods, animal feeds. introduction there is a wide distribution of biologically-active constituents through out the plant kingdom, particularly in plants used as animal feeding stuff and in human nutrition (igile, 1996). the knowledge that these compounds elicit both toxic and advantageous biological responses has given rise to several investigations in recent times as to their possible physiological implications in various biological systems (igile, 1996). it is well known that plants generally contain antinutrients acquired from fertilizer and pesticides and several naturally-occurring chemicals (igile, 1996). some of these chemicals are known as ‘‘secondary metabolites’’ and they have been shown to be highly biologically active (zenk, 1991). they include saponins, tannins, flavonoids, alkaloids, trypsin (protease) inhibitors, oxalates, phytates, haemagluttinins (lectins), cyanogenic glycosides, cardiac glycosides, coumarins *corresponding author. e-mail: soetangboye@yahoo.com. and gossypol. the list is inexhaustible. some of these plant chemicals have been shown to be deleterious to health or evidently advantageous to human and animal health if consumed at appropriate amounts (kersten et al., 1991; sugano et al; 1993) most of these secondary metabolites elicit very harmful biological responses, while some are widely applied in nutrition and as pharmacologically-active agents (oakenfull and sidhu, 1989; soetan, 2008) . the pharmacological and other beneficial effects of these antinutritional factors in plants have been reviewed by soetan (2008). food poisoning arising from plant secondary metabolites, otherwise known as antinutritional factors, other than cyanogenic glycosides, has not been properly addressed in nigeria, and indeed in most parts of the developing world/countries, that depend/feed mostly on vegetablebased diets. people have therefore died out of ignorance, poverty and inadequate nutrition education, especially within the african societies (igile, 1996). the anti-nutritional factors (anfs) may be defined as those substances generated in natural food stuffs by the normal metabolism of species and by different mechanisms (e.g. inactivation of some nutrients, diminution of the digestive process, or metabolic utilization of feed) which exert effects contrary to optimum nutrition (kumar, 1992). being an anf is not an intrinsic characteristic of a compound but depends upon the digestive process of the ingesting animal (kumar, 1992). for example, trypsin inhibitors, which are anfs for monogastric animals, do not exert adverse effects in ruminants because they are degraded in the rumen (cheeke and shull, 1985). the utility of the leaves, pods and edible twigs of shrubs and trees as animal feed is limited by the presence of anfs (kumar, 1992). much of the available data and information on the nutrient and anti-nutrient composition of the more commonly used local foods and feeds do not cover all the foods and feeds and where available, needs updating. this is because of the possible effects of variety/genetic origin, climate, soil, processing methods, pesticides and fertilizers on the chemical composition of the food plants (fao, 1966). several considerations justify the continued surveillance, knowledge and future research on antinutritional factors/toxic substances naturally present in plants used as foods and feedstuffs and ways of reducing them to safe level of consumption. firstly, the possibility that newly developed or exotic foods may contain natural toxicants must be taken into account in evaluating their usefulness (osagie, 1998). also, introduction of new plant varieties into our diets may expose humans and animals to new toxic factors with unsuspected biological effects. secondly, improper processing of plant food like beans and pulses may expose humans and animals to high concentrations of these toxic factors. an example is the production of soymilk and its use as an alternative to cow’s milk in infant formula. it is reported that soymilk, if not properly processed and supplemented with iodine, causes goitre in infants (hydowitz, 1960). raw soybeans also cause goitre in rats and chicks. another example is the report by abeke et al. (2003), that most farmers would prefer to use raw lablab purpureus (lablab beans) to feed chicken in order to eliminate the cost of transporting the raw beans to the processor and back to the farm as well as trying to avoid the processing and other handling costs involved. thirdly, these anti-nutritional factors are increasingly recognized as significant items of the diet of humans and animals (osagie, 1998) . thus, they affect the overall nutritional value of foods and feeds. fourthly, the plant breeder, in an attempt to develop higher -yielding or diseaseresistant crop varieties must, at the same time, be alert to the possible production of undesirable components (osagie, 1998). fifthly, amounts of certain food substances that are relatively safe when consumed individually can sometimes, when taken together, have serious and even fatal events (osagie, 1998). an example is the presence of tannins in a protein-marginal diet. sixthly, public health authorities and other food regulatory bodies need to be informed about the possible dangers related to the widespread, longstanding practices previously regarded as safe (osagie, 1998). for example, cassava and some legumes have been consumed in the tropics for a long time without apparent consideration for their high cyanide contents. also, information on the anti-nutritional components of foods/feedstuffs will help dieticians, veterinarians, human and animal nutritionists to avoid recommending foods/feeds that their patients cannot tolerate, possibly because of the inability to metabolize or detoxify certain substances therein. in the last few decades or more, large numbers of scientific data have emerged, linking diet and food selection patterns to the maintenance of health and the prevention of some chronic diseases (oyewole and atinmo, 2008). there is a consensus among physicians that nutrition constitutes an essential aspect of health care. the aim of this review therefore are as follows: 1) to revisit the issue of processing foods/feeds so as to reduce or eliminate these health threatening plants’ secondary metabolites whose occurrence and concentrations/levels in food plants vary according to genetic origin, soil, fertilizer, pesticides and other environmental conditions; 2) to emphasize the need to properly process human foods and animal feeds to the level which are safe for consumption. the anti-nutritional factors (plants’ secondary metabolites) the abundance of antinutritional factors and toxic influences in plants used as human foods and animal feeds certainly calls for concern. therefore, ways and means of eliminating or reducing their levels to the barest minimum should be discovered. it could be wrongly argued that since the african cultural method of preparing food involves cooking, there is no cause for alarm. this is not entirely correct because although the toxic effects of most anti-nutritional factors present in plant food and feedstuffs can generally be eliminated by proper heat treatment, it should be appreciated that conditions may prevail whereby complete destruction may not always be achieved (aykroyd et al., 1982). for example, it has been reported that phytic acids (phytates) are not destroyed by being cooked even in boiling water (ranhotra et al., 1974). korte (1973) also observed that in mixtures of ground beans and ground cereals prepared under the field conditions prevailing in africa, the haemagluttinin was not always completely destroyed, and that cooked products produced diarrhoea and other toxicity signs. a reduction in the boiling point of water in mountainous regions could also result in incomplete destruction of toxicity or incomplete elimination of the lectins. also, people do not patiently prepare or process beans to the recommended level to destroy the antinutrients because of the high and prohibitive cost of energy sources like kerosene and gas and the scarcity of firewood, especially during the rainy season. as a result of this, people are forced to eat improperly cooked nutritionally toxic beans (pusztai et al., 1993). there has also been some press reports of food poisoning leading to mortality in humans after consumption of bean meal. the adverse effect of excessive cooking on protein denaturation is also an important factor to be considered during food / feed processing. a review of the occurrence of nitrate in unprocessed foods showed that high concentrations are frequently found in vegetables (walker, 1975). nitrates are present in all plants and are an essential source of nitrogen for normal growth. the increasing incidence of various forms of cancer in the world at large and in nigeria in particular may be attributed to the levels of certain chemicals in our foods and drinks (okafor and nwogbo, 2005). the consequences/implications of toxic factors in plants used as food/feedstuffs are not their direct toxicity to man and animals alone, but also the inconvenience and the economic loss associated with poisoning of domestic animals and the cost of preventing or reducing such happenings. it is worthy of note that plant poisons can either be accumulated in the animal or in certain organs or they are metabolized and excreted in milk (liener, 1969). ruminants may convert cyanide to the less toxic thiocyanate, which is goitrogenic (jones et al., 1997). by this food chain, toxins or their metabolites thereof may become harmful to man (habermal, 1987). nutrition education should emphasize adequate and thorough preparations of human foods and animal feeds, especially in humans where there are increases in reported cases of renal diseases (salako, 2005). oxalate, which is an anti-nutritional component of plant, if consumed in large quantities is associated with blockage of renal tubules by calcium oxalate crystals and development of urinary calculi (blood and radostits, 1989) . all these can lead to renal disease and hence death in susceptible individuals. there is also the need to re-visit studies on the levels of anti-nutritional factors in our common foods due to the influence of soil, fertilizer, pesticides, and other chemicals and environmental conditions on the levels of these anti-nutritional factors in plants. methods for quantification of the antinutritional factors in plants several methods are used for the quantitative determination of anti-nutritional factors in foods based on reports by different authors. these are: trypsin inhibitor activities are determined according to liener (1979); haemagglutinatin, jaffe (1979); cyanogenic glucosides (hcn), bradbury et al (1999); oxalates, fasset, (1996); phytates, maga (1983); tannin, dawra et al. (1988); saponin, brunner (1984); and alkaloids, henry (1973). there are other new methods for quantification of antinutritional factors due to recent advances in the nutritional sciences. classification of the anti-nutritional factors the anti-nutritional factors in plants may be classified on the basis of their chemical structure, the specific actions they bring about or their biosynthetic origin (aletor, 1999). although this classification does not encompass all the known groups of anti-nutritional factors, it does present the list of those frequently found in human foods and animal feedstuffs. the anti-nutritional factors may be divided into two major categories. they are: (1). proteins (such as lectins and protease inhibitors) which are sensitive to normal processing temperatures. (2). other substances which are stable or resistant to these temperatures and which include, among many others, polyphenolic compounds (mainly condensed tannins), non-protein amino acids and galactomannan gums (osagie,1998). more often than not, a single plant may contain two or more toxic compounds, generally drawn from the two categories, which add to the difficulties of detoxification. according to aletor (1993), there are several antinutritional factors that are very significant in plants used for human foods and animal feeds. they are: (i) enzyme inhibitors (trypsin and chymotrypsin inhibitors, plasmin inhibitors, elastase inhibitors), (ii) haemaglutinnins (concanavalin a, ricin), (iii) plant enzymes (urease, lipoxygenase), (iv) cyanogenic glycosides (phaseolunatin, dhurrin, linamarin, lutaustralin), (v) goitrogens (pro-goitrins and glucosinolates), (vi) oestrogens (flavones and genistein), (vii) saponins (soya sapogenin), (viii) gossypol from gossypium species e.g. cotton, (ix) tannins (condensed and hydrolysable tannins), (x) amino acid analogues (boaa, dap, mimosine, n-methyl-1-alanine), (xi) alkaloids (solanine and chaconine), (xii) anti-metals (phytates and oxalates), (xiii) anti-vitamins (anti-vitamins a, d, e and b12) and (xiv) favism factors. biochemical effects of the anti-nutritional factors the biochemical and toxicological/adverse effects of plant’s secondary metabolites (anti-nutritional factors) have been reviewed by several authors (mclean, 1970; cheeke and shull, 1985; aletor, 1993; osagie, 1998; fu et al., 2002). however, their adverse effects will be briefly highlighted. anti-nutritional factors diminish animal productivity but may also cause toxicity during periods of scarcity or confinement when the feed rich in these substances is consumed by animals in large quantities (kumar, 1992). cyanogenic glucoside on hydrolysis yields toxic hydrocyanic acid (hcn). the cyanide ions inhibit several enzyme systems, depress growth through interference with certain essential amino acids and utilization of associated nutrients. they also cause acute toxicity, neuropathy and death (osuntokun, 1972; fernando, 1987). alkaloids cause gastrointestinal and neurological disorders (aletor, 1993) . the glycoalkaloids, solanine and chaconine present in potato and solanum spp. (saito et al., 1990; aletor, 1991) are haemolytically active and toxic to fungi and humans. some of the toxicological manifestations of potato glycoalkaloids involve gastro-intestinal upsets and neurological disorders, especially in doses in excess of 20 mg/100 g sample. coumarins, which are constituents of forage, have been associated with the so-called bleeding disease in cattle consuming spoiled or putrid sweet clover (aletor, 1993) . it is believed that cinnamic acid or its derivatives are the precursors of coumarin and that when plant tissues containing deri-vatives are disrupted by mastication, freezing, drying or microbial spoilage, coumarin is transformed to the haem-orrhagic factor, dicoumarol (aletor, 1993) . dicoumarol depresses blood prothrombin concentration and by impli-cation reduces clotting time. more detailed review of the implications of dietary coumarin and dicoumarol in lives-tock production has been published by gustine (1972). tannins cause decreased feed consumption in animals, bind dietary protein and digestive enzymes to form complexes that are not readily digestible (aletor, 1993a). they also cause decreased palatability and reduced growth rate (roeder, 1995). saponins cause hypocholesterolaemia by binding cholesterol, making it unavailable for absorption. they also cause haemolysis of red blood cells and are toxic to rats (johnson et al., 1986). saponins from bulbostermma paniculatum and pentapamax leschenaultii have also been demonstrated to have anti-spermal effects on human spermatozoa (su and guo, 1986; pant et al., 1989). they significantly inhibited acrosine activity of human sperms and the spermicidal effect was attributed to strong damage of the spermal plasma membrane (su and guo, 1986). trypsin (protese inhibitor) causes pancreatic enlargement and growth depression (aletor and fetuga, 1987). haemagluttinins are proteins known for agglutinating red blood cells. they depress animal growth by interfering with the digestion and absorption of nutrients in the gastrointestinal tract (aletor and fetuga, 1987). phytates bind minerals like calcium, iron, magnesium and zinc and make them unavailable (nelson et al., 1968). oxalates, like phytates, bind minerals like calcium and magnesium and interfere with their metabolism. they also cause muscular weakness and paralysis. oxalates also cause gastrointestinal tract irritation, blockage of the renal tubules by calcium oxalate crystals, development of urinary calculi and hypocalcaemia (oke, 1969; blood and radostits, 1989). jones et al. (1997) reported that oxalates cause nephrotic lesions in the kidney. oxalate, phytate and tannins are anti-nutrients, which could be toxic when consumed in an unprocessed food (ojiako and igwe, 2008). the bioavailability of the essential nutrients in plant foods could be reduced by the presence in these plants of some anti-nutritional factors such as oxalates and cyanogenic glycosides (akindahunsi and salawu, 2005). too much of soluble oxalate in the body prevents the absorption of soluble calcium ions as the oxalate binds the calcium ions to form insoluble calciumoxalate complexes. as a result of this, people with the tendency to form kidney stones are advised to avoid oxalate-rich foods (adeniyi et al., 2009). gossypols are reported to cause animal and human toxicity and high incidence of irreversible testicular damage. dietary gossypol can also bring about increased requirement, not only for lysine, but also for iron which it can chelate (aletor, 1993). at the physiological level, gossypol reduces oxygen availability in the blood, while skutches (1974) showed that proteins were reduced by approximately 20% in pigs fed 0.06% free gossypol in the diet. other physiological abnormalities include hypertrophy and dilution of heart muscles and changes in electrocardiogram. dietary free gossypol of up to 0.020.03% has been reported to cause death in growing pigs while poultry can tolerate fairly high dietary levels (aletor and onibi, 1990). gossypol is rapidly deposited in the eggs and has been implicated in egg yolk discolouration (aletor, 1993). plant oestrogens also cause toxicity in animals. for example, it has long been recognized that sheep grazing subterranean clover ( trifolium subterraneum) are prone to poor reproductive performance (aletor, 1993). the causal chemical agent has since been identified as the isoflavone and genistein. chronic exposure to these oestrogenic principles in plants may lead to biochemically and physiologically active levels. there is ample evidence that uncontrolled use of plant oestrogens could produce various types of tumours in animals. more detailed information on the oestrogenicity of certain forage species and the accompanying physiopathological implications have been reviewed by price and fenwick (1985). there are some anti-nutritional factors in some plants, especially leguminous plants, whose mode of action is poorly understood. these are anti-vitamin factors. raw kidney beans are believed to contain an antagonist to vitamin e as evidenced by liver necrosis in rats and muscular dystrophy and low concentration of plasma tocopherol in chicks (liener, 1980). anti-vitamin e has also been noted in isolated soya protein, which is suspected to be -tocopherol oxidase. unheated soyabean flour has been found to be deficient not only in vitamin b12, but it also contains a heat-labile factor that increases the requirement for vitamin b12 (liener, 1980).alkaloids are also reported to cause alteration of normal foetal developments resulting in foetal malformation in ewes. these are caused by teratogenic alkaloids (mulvihill, 1972). glycoalkaloids are reported to cause haemolysis and toxicity to humans (saito et al., 1990; aletor, 1991). some plant alkaloids are reported to cause infertility (olayemi, 2007). saponins are characterised by a bitter taste and foaming properties. erythrocytes lyses in saponin solution and so these compounds are toxic when injected intravenously. the anti-nutritional effects of saponins have been mainly studied using alfalfa saponins. in non-ruminants (chicks and pigs), retardation of growth rate, due primarily to reduction in feed intake, is probably the major concern (cheeke and shull, 1985). such effects have also been noted when sesbania sesban leaf meal (saponin 0.71%) was incorporated in a chick diet (shqueir et al., 1989). furthermore, because saponins may also undergo bacterial degradation in the rumen, they may not retard the growth of ruminants. nevertheless, recent studies indicate that they inhibit microbial fermentation and synthesis in the rumen (lu and jorgensen, 1987). ricin occurs in castor beans (ricinus communis) which have been reported to cause poisoning in all classes of livestock. due to ricin, deoiled castor seed cake (cp 35%) is seldom used as a livestock feed. however, the mature leaves of r. communis have been found suitable for feeding to sheep (behl et al., 1986); hence precautions against bean contamination are necessary. castor bean meal can be detoxified by autoclaving at 20 psi for 60 min for incorporation in sheep diets (rao et al., 1988). processing techniques on the antinutritional factors information on the effect of different processing techniques on the antinutritional factors in human foods and animal feeds will attract interests by human and animal nutritionists, dieticians, veterinarians, public health and food regulatory bodies/authorities on the exploitation of these techniques so that the nutritive values of foods and feeds could be efficiently maximized. although data on the necessary optimal processing techniques, if any, is scanty, this should be established to ensure optimal utilization of human foods and animal feeds. effects of different processing methods on the anti-nutritional factors gharaghani et al. (2008) reported the effect of gamma irradiation on anti nutritional factors and nutritional value of canola meal for broiler chickens. glucosinolate content was reduced to 40, 70 and 89% at irradiation dose levels of 10, 20 and 30 kgy, respectively (p<0.01). their observation indicated that the irradiation treatment has a significant effect on the glucosinolate content of canola meal. gamma irradiation treatment with its radiolytic effects can destroy glucosinolate molecules. other investigators reported that anti nutritional factors, such as protease inhibitors (farag, 1989; sattar et al., 1990; el morsi et al., 1992; farag, 1998), -amylase inhibitors (abu-tarboush, 1998; al-kahtani, 1995), phytohamagglutinins (farag, 1989; mahrous, 1992; farag, 1998), oligosaccharids (rao and vakil, 1983; ghazy, 1990) and tannin (abu-tarboush, 1998) were significantly inactivated by gamma irradiation. varietals differences and effects of methods of domestic processing and cooking khokhar and chauhan (1997) reported the anti-nutritional factors in moth bean (vigna aconitifolia).the dry seeds were given different treatments including soaking, sprouting and cooking and the changes in the level of the anti-nutritional factors were estimated. soaking the seeds in plain water and mineral salt solution for 12 h decreased phytic acid to the maximum (46–50%) whereas sprouting for 60 h had the most pronounced saponin lowering effect (46%). the other methods of processing were less effective in reducing the levels of these anti-nutritional factors. the processing methods involving heat treatment almost eliminated trypsin inhibitor activity while soaking and germination partly removed the activity. el-adawy (2002) reported the nutritional composition and antinutritional factors of chickpeas (cicer arietinum l.) undergoing different cooking methods and germination. the studies involved the effects of cooking treatments (boiling, autoclaving and microwave cooking) and germination on the nutritional composition and antinutritional factors of chickpeas. cooking treatments and/or germination caused significant (p < 0.05) decreases in fat, total ash, carbohydrate fractions, antinutritional factors, minerals and b-vitamins. germination was less effective than cooking treatments in reducing trypsin inhibitor, hemagglutinin activity, tannins and saponins; it was more effective in reducing phytic acid, stachyose and raffinose. cooking treatments and germination decreased the concentrations of lysine, tryptophan, total aromatic and sulfur-containing amino acids. however, cooked and germinated chickpeas were still higher in lysine, isoleucine and total aromatic amino acid contents than the fao/who reference. the losses in b-vitamins and minerals in chickpeas cooked by microwaving were smaller than in those cooked by boiling and autoclaving. germination resulted in greater retention of all minerals and b-vitamins compared to cooking treatments. in vitro protein digestibility, protein efficiency ratio and essential amino acid index were improved by all treatments. the chemical score and limiting amino acid of chickpeas subjected to the various treatments varied considerably, depending on the type of treatment. based on these results, microwave cooking appears to be the best alternative for legume preparation in households and restaurants. mosha et al. (1995) reported the effect of blanching on the content of anti-nutritional factors in selected vegetables. levels of both tannic acid and phytic acid were significantly (p<0.05) reduced by conventional and microwave blanching methods while oxalic acid levels were not significantly (p> 0.05) reduced in most of the treatments by either of the blanching methods. in general, they recommended blanching as an effective method for reducing the anti-nutritional factors in green vegetables; however, further investigation on the heating times for both conventional and microwave blanching methods has been suggested. alonso et al. (2000) reported the effects of extrusion and traditional processing methods on antinutrients and in vitro digestibility of protein and starch in faba and kidney beans. de-hulling significantly increased protein content and greatly reduced condensed tannin and polyphenol levels in both legumes. extrusion was the best method to abolish trypsin, chymotrypsin, -amylase inhibitors and haemagglutinating activity without modifying protein content. furthermore, this thermal treatment was most effective in improving protein and starch digestibilities when compared with dehulling, soaking and germination. van bruggen et al. (1993) reported the methods and devices for reducing the amount of anti-nutritional factors in a mixture of raw material for animal feed. the invention relates to a method for reducing the amount of antinutritional factors in a raw material mixture for producing an ingredient for animal feed such as cattle fodder or domestic animal feed, which raw material mixture contains at least rape seed in a quantity between 1 and 100%, by subjecting the raw material mixture to a steam treatment for a predetermined time at a predetermined temperature. due to the steam treatment, the antinutritional factors are at least partially broken down and determined constituents such as fats also become better accessible, whereby the nutritional value of the final animal feed increases. plant phytochemicals exhibit diverse pharmacological and biochemical actions when ingested by animals and man (amadi et al., 2006; soetan, 2008). most of the toxic and antinutrient effects of these compounds in plants could be removed by several processing methods such as soaking, germination, boiling, autoclaving, fermentation, genetic manipulation and other processing methods (soetan, 2008). food processing methods such as boiling reduces the amount of these phytochemicals in plant products (piorrock et al., 1984). the toxic effects of oxalate, phytate and tannins could be avoided, provided the plant food is cooked before consumption (enechi and odonwodu, 2003). osman (2007) reported the effect of different processing methods on nutrient composition, anti-nutritional factors and in vitro protein digestibility of dolichos lablab bean [lablab purpureus (l) sweet]. the trypsin inhibitor values were significantly reduced (p<0.05) by the different treatment methods, with cooking being the most effective. soaking of the beans overnight reduced the trypsin inhibitor activities (tia) by 6.3% and cooking of the soaked beans caused further reduction in the tia content by 66.7%. the report of osman (2007) agrees with that of marquez and alonson 1999) who reported a reduction in trypsin activity level during soaking and boiling of chickpea. also, kadam and smithard (1987) observed a significant decrease in tia in winged bean after cooking of the presoaked bean. devaraj and manjunath (1995) found that the d. lablab proteinase inhibitors activity was completely lost by 60 min cooking. roasting and autoclaving significantly reduced the amount of tia by 23.05 and 12.09%, respectively. their data agree with that found by kapoor and gupta (1978), carlini and udedibie (1977) in other legumes. osman (2007) further reported that germination significantly decreased the tia activities in d. lablab by 19.3%. this result was similar to those observed on other legumes like soybean (collins and sanders, 1976), lentil (frias et al., 1995; vidal-valverde et al., 1994), great northern bean (sathe et al., 1983), faba bean (rahma et al., 1987) and chick bean (savage and thompson, 1989). apart from the trypsin inhibitor activity, different treatment methods caused significant decrease in phytic acid (pa) level. the phytic acid content of the raw lablab bean is lower than that reported by deka and sarkar, (1990) but higher than those reported by al-othman (1999). osman (2007) reported that roasting caused greater reduction (60.69%) on phytic acid followed by autoclaving (52.29%), germination (48.94%) and cooking (44.85%), while soaking showed the lowest reduction (22.19%). reduction in the phytic acid content during soaking, cooking or germination has been reported by many investigators (chau and cheung, 1997; aloso et al., 1998; alonso et al., 2000; desphande and sheryan, 1983; vidal-vilverde et al., 1994; sievwright and shipe, 1986) for chinese legumes, pea, faba bean, dry bean, lentil and black bean, respectively. the decrease in phytic acid content by soaking, cooking of presoaked bean or germination may be due to leaching out of this compound in water (osman, 2007). roasting and autoclaving has been reported to decrease phytic acid in dry bean (tabekhia and luh, 1980), chick pea and black gram (duhan et al., 1989), cowpea (akinyele, 1989) and black bean (sievewright and shipe, 1986). vijakumari et al. (1995) reported that cooking or autoclaving of d. lablab seeds reduced the tannin contents by 70 and 60%, respectively. germination significantly increased in vitro protein digestibility of d. lablab seeds to 92.27% whereas, roasting and autoclaving significantly decreased it to 85.28 and 86.97%, respectively. similar results were obtained in d. lablab by shastry and john (1991) and sorghum by bhis et al. (1988) and romo-parade et al. (1985). vijakumari et al. (1995) reported that soaking, cooking or autoclaving of d. lablab seeds increased the in vitro protein digestibility values by 3 and 13%, respectively. osman (2007) concluded his report that soaking, cooking of presoaked beans and germination are good potential methods for improving the nutritional value of lablab beans by reducing the antinutritional factors such as trypsin inhibitors and phytic acid and this enhances the utilization of the lablab beans. ogundipe et al. (2003) reported that cooking had significant effects on the utilization of lablab purpureus beans by pullet chicks. cooking lablab seeds for about 30 min gave the best results in terms of final weight, weight gain, feed consumption and feedgain ratio. omeje (1999) also reported that cooking of legume seeds for about 30 min resulted in the destruction of the anti-nutritional factors such as trypsin inhibitors, haemagglutinins, phytic acids, lectins and goitrogens, thereby improving the nutrient availability for better performance of the bird fed such diets. amaefule and obioha (2001) reported better performance in broilers fed pigeon pea seed meal based diets as compared to diets containing the raw meal. kaankuka et al. (2000) also reported that cooking soybean seeds for about 30 min improved the performance of weaned pigs better than cooking for lesser time periods. ogundipe (1980) reported that moist cooking of soybeans gave better performance in broilers than other processing methods. in order to utilize beans effectively as human foods, it is essential to inactivate or remove these anti-nutritional factors (osman, 2007). this also applies to animal feeds. generally, adequate heat processing inactivates the trypsin and chymotrypsin (dipitero and liener, 1989; osman et al., 2002). heat stable compounds in cereals and legumes such as tannins and hydrates are easily removed after germination (reddy et al., 1985) and fermentation (osman, 2004). a better understanding of the effects of different traditional processing methods on nutritive value may lead to wider use of legumes in the food industry (osman, 2007). omoruyi et al. (2007) reported the antinutritional factors, zinc, iron and calcium in some caribbean tuber crops and the effect of boiling or roasting on them. boiling reduced the phytic acid to zinc molar ratio for yellow yam and cocoyam. boiling or roasting reduced the levels of cyanoglucosides in sweet potato, yellow yam and cocoyam. roasting greatly lowered the level of trypsin inhibitor activity compared to boiling. omoruyi et al. (2007) concluded that boiling and roasting were effective in lowering the levels of anti-nutritional factors. ikemefuna et al. (1991) reported the effects of soaking, sprouting, cooking and fermentation on some nutrient composition and anti-nutrients factors of sorghum (guinesia) seeds. they reported that a combination of cooking and fermentation improved the nutrient quality and drastically reduced the antinutritional factors to safe levels much greater than any of the processing methods tested. ikemefuna et al. (1991) also reported that soaking and fermentation decreased the tannins content. these processes produce enzymes that break down complexes to release free tannins. the free tannins leached out. fermentation reduced cyanide in soaked seeds. cooking and fermentation reduced cyanide in soaked seeds to safe levels. the hcn is soluble in soaking water as such was leached out in the atmosphere. they further reported that cooking and fermentation synergistically reduced tannins. cooking and fermentation broke down tanninenzyme and protein-tannin complexes and released free tannins which subsequently leached out the products. ikemefuna et al. (1991) concluded that soaking, sprouting, cooking and fermentation appeared to have beneficial effects as methods of processing. combinations of cooking and fermentation improved the nutrient quality and reduced the antinutritional factors inherent in sprouted cereal products to safe levels much greater than any of the other processing methods tested. chavan et al. (1981) reported a ten-fold increase in some reducing sugars and disappearance of galactooligosaccharides during sprouting. panasuik and bills (1984) observed that the endogenous autolytic enzymes responsible for hydrolysis of cyanogenic glycosides may remain partially active, even in the dry products. they could be fully activated on re-hydration, as in fermentation. soaked seeds had slight decrease in starch. sprouting for 36 and 96 h caused the greatest decrease. they reported that the decreases may be due to (a) reduced tannins by amylolytic enzymes and (b) microbial enzyme hydrolysis (ogunsua, 1980; osuntokun, 1981). fermentation also influenced the levels of reducing sugars in sprouted seeds. fermentation caused increases in lipids for both soaked and sprouted seeds. the increases were more than twofold in seeds soaked for 48-96 h. the cause of the increases might be due to increased activity of lipolytic enzymes which produced more free fatty acids. these imparted their flavour to the products. a similar observation was reported by kazanas and fields (1981). conclusion factors that determine the nutritive value of foods and feedstuffs are very complex. all available information, both qualitative and quantitative must be used in making judgments about the feed value of particular plant specie. the fact that a plant or part of a plant is eaten by animals is only an indication of acceptability. too much emphasis is usually put on the analyses of crude protein and fibre as indicators of feed value. more importance should be given to the presence of secondary plant compounds such as tannins and hydrolysable phenolics, which may interfere with the level of protein and fibre contents which are used as indicators of high nutritional value. references abeke fo, ogundipe so, oladele sb, sekoni aa, dafwang ii, adeyinka ia, oni, oo, nwagu bi (2003). effect of cooking duration of lablab (lablab purpureus) on organ weights and blood parameters of pullet chicks. proc 28 th 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(family: zingiberaceae) is an important medicinal plant among the seven species of alpinia that occur in bangladesh, india, myanmar, indonesia, thailand, new guinea and the bismark archipelago (chopra et al., 1986; mangaly and sabu, 1992). it is a perennial herb with non-tuberous pungent rootstock. its rhizomes showed antinociceptive activities (arambewela et al., 2004). the economically important part is rhizome, which is a major constituent of many formulations of indigenous system of medicine for relieving throat inflammation, stimulating digestion, purifying blood, improving voice and marinating youthful vigour. the rhizome extract of a. calcarata is used as an expectorant in the treatment of bronchitis and asthma for purifying blood, stimulating digestion and improving voice (kirtikar and basu, 1935). drugs prepared from the rhizomes of a. calcarata are used in treatment of rheumatism, bronchial catarrh and asthma. *corresponding author. e-mail: nazrul119@yahoo.com. tel: +88-031-681761. fax: +88-01552404192. abbreviations: gc-ms, gas chromatography-mass spectrometry; nist, institute of standards and technology; bcsir, bangladesh council of scientific and industrial research. it is also used to stimulate digestion, purify the blood, prevent bad breath, and improve the voice and also to treat inflammation (jayaweera, 1982; sharma and singh, 1980). in sri lanka, a. calcarata is commonly prescribed by ayurvedic physicians along with other plant materials in the treatment of arthritis. in addition, anti-inflammatory activity has also been reported (sharma and singh, 1980). rout et al. (2005) reported that the leaf oil contain β-pinene (16.8-29.1%), 1,8-cineole (21.9-24.7%), camphor (4.9-8.0%), while the root oil had camphene (9.0-12.3%), 1,8-cineole (15.1-15.5%), and α-fenchyl acetate (39.1-45.2%) as the major constituents. the analysis of a. calcarata has revealed the presence of protocatechinic acid, quercetin, 4-o-methyl-syringic acid, vanillic acid methyl cinnamate and several terpenes and diterpenes as constituents (merh et al., 1986). 1,8cineole had been found to be the major constituent in the oil (kong et al., 2000; tewari et al., 1999; rath et al., 1994; merh et al., 1986). chowdhury et al. (2003) reported that the rhizome oil constituents from bangladesh containing α-fenchyl acetate (51.4%) and 1,8-cineole (15.1%) as major constituents. 1,8-cineole is used as an antiseptic (0-25%) in dentifrices and, mixed with zinc oxide, as a temporary dental filling. it is also used in dentistry as a softening agent to adapt gutta perch fillings and cones to cavities and root canals. due to its pleasant spicy aroma and taste, 1,8-cineole is used in flavorings, fragrances, and cosmetics. it is also an 2 table 1. constituents of alpinia calcarata leaf essential oil. s/n name of compound % 1 ℘-cadinene 0.96 2 ℘-muurolene 0.54 3 5-nonanol, 5-methyl 2.08 4 benzene 0.32 5 β -humulene 1.63 6 borneol 1.71 7 bornyl acetate 1.34 8 β -pinene 6.39 9 camphene 1.50 10 camphor 21.40 11 carotol 6.53 12 caryophyllene oxide 1.48 13 daucol 1.18 14 1,8-cineole 28.48 15 isolimonene 2.78 16 limonene 2.26 17 longipinocarvone 1.24 18 α-phellandrene 0.42 19 α-pinene 1.35 20 methyl cinnamate 13.35 21 myrtenol 0.83 22 o-cymene 0.74 23 pinocarvone 0.72 ingredient in many brands of mouthwash and cough suppressant. 1,8-cineole has been demonstrated to be capable of reducing inflammation and pain. so the present study deals with the investigation of the chemical components in oils obtained from various parts of a. calcarata grown in bangladesh for medicinal as well as commercial exploitation. materials and methods plant material fresh leaves of a. calcarata were collected from the plants grown in the campus of bangladesh council of scientific and industrial research (bcsir) laboratory, chittagong during june 2007. onevoucher specimen (y-112) was deposited in the herbarium of bcsir laboratory, chittagong. extraction of essential oil the experiment was conducted when the plants were flowering. samples of different plant parts (leaf, stem sheath and roots) were harvested from healthy, well-grown, two-year-old plants. freshly harvested samples (500 g each) in triplicate were subjected to hydrodistillation using a modified clevenger-type glass apparatus for 4 h for isolation of oils separately from different parts. the oil samples were stored at 0°c in air-tight containers after drying them over anhydrous sodium sulfate for gas chromatography-mass spectrometry (gc-ms) analysis. gc-ms analysis the essential oil from rhizomes of a. calcarata was analyzed by gc-ms electron impact ionization (ei) method on gc-17a gas chromatograph (shimadzu) coupled to a gc-ms qp 5050a mass spectrometer (shimadzu); fused silica capillary column (30 m x 0. 25 mm; 0.25 m film thickness), coated with db-5 (j and w); column temperature 100°c (2 min) to 250°c at the rate of 3°c/min; carrier gas, helium at constant pressure of 90 kpa. acquisition parameters full scan; scan range 40-350 amu. identification of the compounds compound identification was done by comparing the national institute of standards and technology (nist) library data of the peaks with those reported in literature, mass spectra of the peaks with literature data. percentage composition was computed from gc peak areas on db-5 column without applying correction factors. results and discussion the oil yields from different plant parts were: leaf 0.14%, stem sheath 0.06%, root 0.08% and 23, 13 and 6 compounds, respectively (tables 1, 2 and 3). there were similarities and differences among the oils. 1,8-cineole 3 table 2. constituents of alpinia calcarata stem sheath essential oil. s/n name of compound % 1 1,8-cineole 11.78 2 l-camphor 8.76 3 borneol 3.33 4 4-terpinenol 1.96 5 terpinyl acetate 15.09 6 fenchyl acetate 19.16 7 γ-cadinene 2.94 8 methyl cinnamate 6.53 9 β-elemene 1.49 10 cis-p-mentha-2,8-dien-ol 1.97 11 carotol 16.77 12 daucol 3.13 13 longifolene-i2 7.09 table 3. constituents of alpinia calcarata root essential oil. s/n name of compound % 1 1,8-cineole 8.14 2 borneol 11.44 3 fenchyl acetate 51.34 4 thymol methyl ether 7.49 5 carotol 10.85 6 juniper camphor 10.73 and carotol were the common components of all parts oils. the leaf oil was rich in 1,8-cineole (28.48%), camphor (21.40%), methyl cinnamate (13.35%), carotol (6.53%), β–pinene (6.39%), isolimonene (2.78%), limonene (2.26%) and 5-nonanol, 5-methyl (2.08%). the stem sheath oil was rich in fenchyl acetate (19.16%), carotol (16.77%), terpinyl acetate (15.09%), 1,8-cineole (11.78%) and l-camphor (8.76%). the root oil was rich in fenchyl acetate (51.34%), borneol (11.44%), carotol (10.85%), juniper camphor (10.73%) and 1,8-cineole (8.14%). 1,8-cineole is the common compound of all the reported oils in the world including ours (kong et al., 2000; tewari et al., 1999; rath et al., 1994; merh et al., 1986). variations in compositions of oils isolated from different plant parts of alpinia breviligulata and alpinia chinensis were reported from vietnam (dung et al., 1994, 1995). α-fenchyl acetate was found to be one of the major constituents in the root oil of these two species (dung et al., 1994, 1995) and rhizome oil of a. calcarata (tewari et al., 1999). conclusion it may be concluded that a. calcarata, growing widely in bangladesh, may be utilized as a source for the isolation of natural 1,8-cineole and fenchyl acetate, respectively for medicinal and commercial use. references arambewela lsr, arawwawala ldam, ratnasooriya wd (2004). antinociceptive activities of aqueous and ethanolic extracts of alpinia calcarata rhizomes in rats. j. ethnopharmacol., 95(2-3): 311-316. chopra rn, nayar sl, chopra ic (1986). glossary of indian medicinal plants, publication and information directorate, council of scientific and industrial research (csir), new delhi, india. p. 13. chowdhury ju, yusuf m, husain mm (2003). composition of the rhizome oil of alpinia calcarata. indian perfumer, 47(4): 355-377. dung nx, rang dd, chinn td, leclercq ra (1994). volatile constituents of the root, rhizome, leaf, stem and flower oils of alpinia chinensis from vietnam. eighth asian symposium on medicinal plants, spices and natural products, 12-16 june, melaka, malaysia, abst. p94 (1994) curr. res. med. arom. plants, 17(1): 92, a94-101016. dung nx, chinn td, rang dd, leclercq pa (1995). constituents of alpinia breviligulata from vietnam. eighth asian symposium on medicinal plants, spices and natural products, 12-16 june, melaka, malaysia, abstract cp, 3a curr. res. med. arom. plants, 17(1): 92, a94-10-1015. jayaweera dm (1982). medicinal plants used in ceylon. national science council of sri lanka, colombo sri lanka, 5: 213. kirtikar ke, basu bd (1935). indian medicinal plants, iv, 2nd edn., m/s. bishen singh mahendra pal singh, new dehi. p. 16. kong ly, qin mj, niwa m (2000). diterpenoids from the rhizomes of alpinia calcarata. j. nat. prod., 63: 939-942. mangaly jk, sabu m (1992). a taxonomic revision of south indian 4 alpinia roxb. (zingiberaceae). rheedea, 2: 38-51. merh ps, daniel m, sabnis sd (1986). chemistry and taxonomy of some members of the zingiberaceae. curr. sci., 55: 835-839. rath sp, sahoo sb, sviniva sc (1994). analysis of cultivated alpinia calcarata. indian j. natural prod., 10: 12-13. rout pk, sahoo s, rath sp, rao yr (2005). analysis of the leaf, rhizome and root oils of two accessions of alpinia calcarata rosc. cultivated at bhubaneswar. j. essen. oil res., 17(4): 398-400. sharma ak, singh rh (1980). screening of anti-inflammatory activity of certain indigenous drugs on carrageenin induced bind paw oedema in rats. bull. med. res., 11: 262-271. tewari a, pant ak, mathela cs, mengi n, kohl e, bestmann hj (1999). volatile constituents of alpinia calcarata. j. essen. oil res. 11: 739741. 1 in ternationa l scholars journa ls african journal of food science research issn: 2375-0723 vol. 10 (3), pp. 001-006, march, 2022. available online at www.internationalscholarsjournals.org © international scholarsjournals author(s) retain the copyright of this article. full length research paper an exploratory investigation of individual and schoollevel determinants for effective nutrition education in primary school in n’djamena, chad kimassoum d.1,2*, alexis h.1, tidjani a.1,2, kanabet z1 and c. frazzoli3 1 noodleschad, n'djamena, chad. 2 laboratoire de recherche en sciences des aliments et nutrition (larsan), university of n'djamena, chad. 3 dept cardiovascular and endocrine-metabolic diseases, and ageing. istituto superiore di sanita', rome, italy. accepted 23 january, 2022 abstract background. malnutrition is a serious public health problem in chad, with prevalence increasing over the years. most eating habits are formed early in life, with beliefs and tacit knowledge directing eating behaviors. school nutrition education can effectively improve nutrition skill if related social determinants are understood. this study investigated factors for effective school nutrition education in n’djamena, chad. methods. a total of 73 children (girls and boys, age 9-13) were stimulated with a nutrition education initiative in two schools in n’djamena. the nutrition education focused on food groups and their importance, food hygiene, and general rules for a balanced diet based on local foods. results. socioeconomic and sociocultural factors affected nutrition education. girls were more proficient than boys, probably due to the role they generally have in dealing with family eating. results confirmed the children’s developmental stages related to nutrition, and showed the significant impact a nutrition intervention can have on the nutritional skills of chadian children. conclusion. formative research is needed to design school nutrition education programmes in chad, where limited studies have been conducted so far. keywords: nutrition security, diet, hygiene, nutrition education, children, chad. introduction severe acute malnutrition (sam) in children under 5 years is one of the leading causes of morbidity and mortality in chad. more than half of the regions/provinces of chad are in a malnutrition situation according to studies (smart, 2021). sam includes stunting (height for age), wasting (weight for height), underweight (weight for age) and micronutrient deficiencies (i.e. inadequate/imbalanced intake of essential nutrients, such as vitamins and minerals) (roberfroid et al., 2015). sam resulted as associated with diarrhoea, fever, vomiting, and type of complementary meal. *corresponding author e-mail: kimassoumd@gmail.com at household level, it is associated with undernourished caretaker, caretaker's handwashing habits, absence of toilet, and low household food diversity (dodos et al., 2018). poor knowledge regarding causes, such as unbalanced nutrients intake, poor hygiene and sanitation, and consequences of malnutrition results in reduced risk perception and disbelief or disregard of nutritional education. nutritional awareness and skills will contribute breaking the intergenerational cycle of malnutrition and protect people against the risks of the worldwide nutrition transition (frazzoliand mantovani, 2020). most eating habits are formed early in life, therefore it is important that children receive nutrition education in primary school. context-specific scientific literature on nutrition education 2 in chad is very limited. this study began investigating individual and school-level determinants for effective nutrition education in school in n’djamena. indeed, formative research allows to describe the target group, understand the dynamics among factors, and design a context-specific road to effective and structural school nutrition education in chad. matherials and methods two primary schools in n’djamena, namely the georges washington school and the notre dame des apôtres school, were selected for this study. primary schools in n'djamena are mostly private, such as the georges washington school. the school notre dame des apôtres is supported by the chadian state instead, and is a semi-state school. a nutrition education session was held at the notre dame des apôtres school (on january, 2020) with 27 children aged 10 to 13 years old (average 11 years old). the second education session (on february, 2020) was held at the george washington school, n’djamena, with 46 children aged 9 to 12 years old (average 10 years old). in both schools, all children, in the middle course classroom, participated on voluntary basis. a large classroom has been made available in the two schools to the team to bring the children together. the nutrition education session focused on food groups and their importance, food hygiene, and general rules (food pyramid, food selection, dietary diversity) for a balanced and diversified diet based on local foods (supinya et al., 2012). the nutrition education targeted the needs of the urban community in chad. in particular, n’djamena urban community is characterized by low consumption of fruit and vegetable. the education session was based on reviewed training materials developed by the government led programme in chad. the nutrition education was provided by members of the ngo noodles chad trained in nutrition, education and health. questionnaires (preand post-) were used to assess the effectiveness of the lesson. learning was ranked low (score 0-3), medium (score 3.5-6), or good (score 6.5-10). the comparison between nutrition skill score of groups of children in schools was taken as indicator of socioeconomic status. the comparison between nutrition skills of boys and girls was observed as indicator of socio-cultural factors. results the overall mean final score was 6.16 for the group of children with mean age 11 (school notre dame des apotres) and 6.73 for mean age 10 (george washington school), thus indicating age 10 as more benefiting from the course. the overall mean background score was 3.72 at semi-state school notre dame des apotres and 4.17 at private george washington school. figure 1 shows the learning rate before (in blue) and after (in red) the nutrition education session at the school notre dame des apotres. before education 42% of children score low, 53% medium and 3% good. after the nutrition education session, just 3% of children score low, 46% medium and 50% good. we noted a marked improvement in the knowledge of basic nutrition by schoolchildren. figure 2 reports the learning rate before (in blue) and after (in red) the nutritional education session at the school george washington. before the nutrition education session, 43% of children score low, 34% medium, and 21% good; after the nutrition education session just 6% of children score low, 23% medium, and 69.5% good. before the nutrition education session, schoolchildren had limited basic knowledge on nutrition, with some higher knowledge in girls. in figure 3, we can see the comparison of learning score before and after the nutrition education session between boys and girls at the school notre dame des apotres. before education overall score of boys and girls was respectively 3.4 and 3.8. after education, their score was 5.8 and 6.3 respectively. figure 4 shows the comparison of learning score before and after the nutrition education session between boys and girls at the george washington school. overall score of boys and girls was 3.5 and 5.1 respectively before education session. this score was 6.6 for boys and 7.0 for girls after education. the final overall score at notre dame des apotres and george washington schools (respectively 6.2 and 6.7) indicates a huge improvement respect to background score (3.7 and 4.2 respectively) (figures 3,4) and therefore the significant impact a nutrition intervention can have on the nutritional skills of chadian children. girls were both more proficient (final score) and more prepared (background score) than boys in this nutritional education. after the nutrition education, the average score of the children from school notre dame des apotres by sex is 6.34 for girls (background 3.84) and 5.75 for boys (background 3.43). at george washington school the average score by sex was 6.97 for girls (background 5.05) and 6.66 for boys (background 3.50). discussion in the present study, we approach the chadian nutrition education scenario in terms of social determinants. social determinants of health refer to a specific group of social and economic factors within the broader determinants of health (marmot, 2015). these relate to an individual's place in society and include factors like education, environment, social and community context, healthy food, economic stability, and health care. for instance, according to ahinkorah et al. (2020), determinants of under-five mortality in chad are education, sex, and birth 3 figure 1. nutrition education before and after teaching at the notre dame des apotres school, n’djamena, chad. figure 2. nutrition education before and after teaching at the george washington school, n’djamena, chad. rank. socio-economic and proximate factors do impact on health disparities in chad, including nutrition and related skill. children were more proficient at george washington school (private school) than in semi-state school, this socioeconomic factor possibly being explained by higher education grade and/or health seeking behaviour of the mother (figure 1). in both schools we found that girls have both better background knowledge and ability to improve in nutrition and knowledge of local foods than boys (figure 3 and 4). this could be due to the roles that girls generally have in dealing with family eating. in fact, it has been found that effectiveness of nutrition intervention on school-age children increases with interventions on preschoolers’ abilities to understand concepts related to edibleness, nutrition, and digestion based on their food and eating experience (schultz anddanford, 2016; nicholson et al. 2018). boys also go to school, and are more likely to go further on in their studies. boys usually help their dads in their business, e.g. sales at the market, bicycle repair business, convenience store or desk job. in a study carried out by the asnutri organization in 2019 at the college savoir partage in n'djamena, children in the 5th class had no knowledge of basic nutrition before the nutrition education (65% of them had a poor score, 30% a low score, and only 5% had a medium score). the nutrition education resulted in 60% children with good score, 25% with medium score and 5% 0 2 4 6 8 10 background final test sc o re 8 boys 19 girls 0 2 4 6 8 10 sc o re 26 boys 20 girls backgroud final test https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate&term=schultz+cm&cauthor_id=27590502 https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate&term=danford+cm&cauthor_id=27590502 4 figure 3. proficiency in nutrition before and after the training education (27 children) at the notre dame des apôtresschool. figure 4. proficiency in nutrition before and after the training education (46 children) at george washington school. with low score (asnutri, 2019). the present initiative of nutrition education confirmed these trends, with 50% and 69.5% good score, 46% and 23% medium score, 3% and 6% low score at the george washington school and notre dame des apotres school, respectively (figure 1). indeed, school-age children can identify factors that influence their decisions about eating, as well as barriers to being healthy (decosta et al., 2017). nutrition education is more likely to be effective when it focuses on behavior and action rather than only knowledge e.g. school gardening, cooking, and accessibility to fruits and vegetables (kyereet al., 2020). 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 girls boys sc o re background score final score notre dame des apotres figure 5. the attention to hygiene. a remainder to regularly wash your hands on the wall at the georges washington school. https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate&term=decosta+p&cauthor_id=28286164 https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate&term=kyere+p&cauthor_id=32646534 5 research is needed for understanding food taboo, beliefs and tacit knowledge that may differ from declarative knowledge. these are more influential in directing eating behaviors than declarative knowledge or knowing facts about food, nutrition, the body, or health (schultz and danford, 2016). inadequate diet but also hygiene and other factors are behind micronutrient deficiency, thus motivating combined interventions (fankam et al., 2022). the planning of equitable interventions to face malnutrition requires the understanding of both inequalities, such as access to drinking water (lemoine and tounian, 2020; ntouda et al., 2013), and differences, such as cultural rooted habits (frazzoli et al., 2016) in the mondo and mao districts, kanem region of chad, where acute malnutrition is prevalent, intestinal parasitic infection is widespread and attributable to water quality. interventions aiming at improving water quality at household level or at promoting handwashing with soap and clean water storage container significantly reduce diarrhea incidence (altmann et al., 2018). intestinal parasites impair the absorption and digestion of nutrients, thus motivating environmental prevention of parasite cycles, improved sanitation, hygiene educationn (figure 5), and eventually community deworming (rivero et al., 2018). conclusion this study confirmed some of the determinants in nutrition education at individual and school level. further efforts in the analysis of health disparities often associated to others factors areneeded to address malnutrition in chad. results showed the significant impact of nutrition education on the nutritional skills of children, and how girls were more proficient than boysin nutrition education. as closing remarks, we can state that nutritionalknowledge, attitude and practices of children in chadmust beaddressed in terms of foodpyramid and diversification, nutrition security, hygiene and safety. training materials for nutrition education of children as well as educational messages targeted to local food culture and needs should be developed. acknowledgement authors are grateful to the schoolcommunitiesthataccepted to take part in this initiative. this study, conducted bynoodleschad,wassupportedby the university of tuscia, viterbo, italyand the pan-african ngo noodles (nutrition &foodsafety and wholesomeness. prevention, education and research network, www.noodlesonlus.org). references ahinkorah bo, aa seidu, e budu, ek armah-ansah, e agbaglo, c adu (2020). proximate, intermediate, and distal predictors of under-five mortality in chad: analysis of the 2014-2015 chad demographic and health survey data. bmc public health.20(1):1873. altmann m, altare c, van der spek n, barbiche jc, dodos j, bechir m, ait aissa m, kolsteren p (2018). effectiveness of a household water, sanitation and hygiene package on an outpatient program for severe acute malnutrition: a pragmatic cluster-randomized controlled trial in chad. am j trop med hyg.98(4):1005-1012. asnutri (2019). education nutritionnelle éffectuée au collège savoir partage par l’association pour la bonne nutrition au tchad. rapportde 2019, pp.8-10. decostap, p møller, m bomfrøst, a olsen (2017). changing children' seating behaviour. a review of experimental research. appetite.113:327-357. dodos j, altare c, bechir m, myatt m, pedro b, bellet f, lapegue j, peeters j, altmann m (2018). individual and household risk factors of severe acute malnutrition among under-five children in mao, chad: a matched case-control study. arch public health. 76:35. fankam py, pouokam gb, saha foudjo bu, mazzanti f, sando jt, samuel c, kamda a, fokou e, frazzoli c (2022). combined education course on nutrition, hand washing and dental care in primary schools in yaoundé, cameroon. journal of global health reportsts:e2022019. frazzoli c, a mantovani (2020). toxicological risk factors in the burden of malnutrition: the case of nutrition (and risk) transition in sub-saharan africa. food and chemical toxicology. 111789. frazzoli c, gb pouokam, a mantovani, oe orisakwe (2016). health risks from lost awareness of cultural behaviours rooted in traditional medicine: an insight in geophagy and mineral intake. science of the total environment. 566-567: 1465-147. kyere p, veerman jl, lee p, stewart de (2020). effectiveness of school-based nutrition interventions in sub-saharan africa: a systematic review. public health nutr.23(14):2626-2636. lemoine a, p tounian (2020). childhood anemia and iron deficiency in sub-saharan africa – risk factors and prevention: a review. arch pediatr.27(8):490-496. marmot m (2015). the health gap: the challenge of an unequal world. lancet. 386, 10011, 2442-2444. nicholson js; jm barton; al simons (2018). ability to categorize food predicts hypothetical food choices in head start preschoolers. j nutr educ behav.50:238– 246. ntouda j, f sikodf, m ibrahim, i abba (2013). access to https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate&term=schultz+cm&cauthor_id=27590502 https://pubmed.ncbi.nlm.nih.gov/?sort=pubdate&term=danford+cm&cauthor_id=27590502 http://www.noodlesonlus.org/ 6 drinking water and health of populations in subsaharanafrica. c r biol. 336(5-6):305-9. rivero m.r., de angelo c., nuñez p., salas m., liang s (2018). intestinal parasitism and nutritional status among indigenous children from the argentinian atlantic forest: determinants of enteroparasites infections in minority populations. acta trop. 187:248-256. roberfroid d, huybregts l, lachat c, vrijens f, kolsteren p, guesdon b (2015). inconsistent diagnosis of acute malnutrition by weight-for-height and mid-upper arm circumference: contributors in 16 cross-sectional surveys from south sudan, the philippines, chad, and bangladesh. nutr j. 14:86. schultzcm, danford cm (2016). children's knowledge of eating: an integrative review of the literature. appetite.107:534-548. smart (2021).enquêtenationale de nutrition, rapport final republique du tchad, ministère de la sante publique. pp.161. supinya i,i (2012). tridsanun saetae, and boonying manaboriboon. the effectiveness of school-based nutritional education program among obese adolescents: a randomized controlled study. international journal of pediatrics. id 608920, 5 pp. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (3), pp. 001-004, march, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper physico-chemical properties of chakiya variety of amla (emblica officinalis) and effect of different dehydration methods on quality of powder poonam mishra1*, vijeyta srivastava1, deepmala verma1, o. p. chauhan2 and g. k. rai1 1 centre of food technology, university of allahabad, allahabad, india. 2 defence food research laboratory, siddarthanagar, mysore, india. accepted 09 october, 2019 in present study a comparative study of wild variety and cultivated variety of amla were carried out; simultaneously effect of different drying techniques on physicochemical properties of amla powder were also studied. the fruits of wild variety were found smaller than the cultivated variety. in case of wild variety, the total phenolic contents were found to be 32.32 g/100 g of gallic acid equivalent (dwb), whereas chakiya variety had 24.50 g/100 g of gallic acid equivalent (dwb). the fruits of chakiya variety were used to prepare the powder by different techniques like freeze drying, sun drying, spray drying, hot air drying and vacuum drying. powder yield varied with type of drying method as sun drying (10.11%), tunnel drying (8.78%) and vacuum drying (12.48%), spray drying (4.90%) and freeze drying (2.23%). significant differences (p<0.05) in chemical composition of amla powder were observed when prepared by different techniques. the freeze dried powder had the highest ascorbic acid content followed by spray dried powder. the lowest concentration of ascorbic acid was found in sun dried powder. freeze dried samples showed maximum mineral contents in terms of calcium (79.6 mg/100 g), phosphorus (12.38 mg/100 g) and iron (88.03 mg/100 g). key words: amla, dehydration methods, quality, polyphenols. introduction amla (emblica officinalis, family euphorbiacea) is a native of india, ceylon, malaya and china. the fruit is used as a major constituent in several ayurvedic prepa-rations such as chyavanprash and rasayana which promotes health and longevity (rajkumar et al., 2001). amla is one of the richest sources of vitamin c known. in addition to this, potent antioxidant, several active tannoid principles (emblicannin a, emblicannin b, punigluconin and pedunculagin) have been identified which appear to account for its health benefits (rastogi 1993; rao et al., 1985). amla has been reported to posses expectorant, purgative, spasmolytic, antibacterial, hypoglycemic (jamwal et al., 1959; jayshri and jolly, 1993), heaptoprotective and hypolipidemic (thakur and *corresponding author. e mail: mpoonam1@rediffmail.com. tel: 09415081554. mandal, 2004) activity. the aqueous extract has been reported to have anti-pyretic laxative and tonic properties and also showed antibacterial activity (vinaygamoothy, 1982). the ascorbic acid content of fresh amla fruit can range up to 950/100 gm which is said to to highest among all fruits next only to barbados cherry (shankar, 1969). several value added products have been reported from amla. amla was used to prepare ready-to-serve beverage (deka et al., 2001), candy, powder (tripathi et al., 1988), sauce (chauhan et al., 2005) etc. the quality of fi-nal product depends on the nature of the raw material used for its preparation. there are several varieties of amla grown throughout different parts of india. however, these varieties are yet to be researched in terms of physico-chemical properties for value addition (kalra, 1988). in addition, storage conditions also play important role in postharvest quality of amla fruits. chakiya is an important variety of amla which is grown table 1. physical properties of fruits of wild and chakiya variety of amla (n=15). particular chakiya variety wild variety moisture, crude fat and mineral content the moisture and crude fat content of the samples were determined as per aoac (1997) procedures; whereas minerals (ca, p and fe) were estimated as per the aoac (1990) procedures. average length, cm average diameter, cm average weight, g no. of segments per fruit juice yield, % 3.0 a 2.72 b 3.8 a 3.0 b 28.88 a 17.66 b 6 a 6 a 41.9 b 48.3 a total phenolic content total polyphenols were estimated as per procedure described by jayprakash et al. (2001) where 250 mg sample was taken in 10 ml of methanol and water (70:30 v/v) solution in a graduated test tube and heated on water bath at 70 o c for 10 min. the sample was brought to room temperature, centrifuged at 3500 rpm for 10 min. values in same row with different superscripts differ significantly (p < 0.05). in the eastern regions of india (kumar and khurdiya, 2002). however, little work has been carried out with respect to physicochemical characterization of this variety and powder preparation from the fruits of this particular variety. therefore, present work was undertaken to study the physicochemical attributes of the fruits of chakiya varieties as well as a wild variety and preparation of powder. effect of different drying methods such as sun drying, tunnel drying, spray drying, freeze drying and vacuum drying were also studied with respect to the quality of amla powder. materials and methods raw material and preparation of amla powder amla of chakiya variety (cultivated) was procured from local market while wild variety of was obtained from forest of panna (madhya pradesh, india). the amla fruits were cleaned and cut into pieces and immediately pressed to obtain juice using a small laboratory manual press. for the preparation of spray dried powder, juice was evaporated up to 50% using rotary evaporator. maltodextrin (5%, w/v of initial juice) was added to the concentrated juice and stirred for 5 min using mechanical stirrer followed by spray drying (model lu 228 advance, labultima, mumbai, india) at 200°c inlet and 150°c outlet temperature and 40% aspiration speed to dry the sample. for freeze drying 200 ml of juice was subjected to freezing at -35°c for 3 h followed by freeze dehydration in a freeze dryer (model alpha 14, martin christ, germany) at 60°c for 16 h. sun dried powder was prepared from grated amla dried under sun followed by grinding in a mixer grinder and filtration using muslin cloth. the powder was also prepared by drying the grated amla in a vacuum oven (kc instruments, lucknow, india) at 50°c and in a tunnel drier (b. sen berry and co. india) at 70°c followed by grinding and filtration as described earlier. analytical methods physical properties average fruit weight, length, diameter and number of segments per fruit were measured taking 15 samples of both the varieties. the juice yield was calculated by pressing the fruit segments after removal of seeds and the data were converted on percentage basis. the supernatant (0.2 ml) was made up to 10 ml with distilled water. this solution was diluted 10 fold. sample solution (5 ml) was mixed with saturated sodium carbonate (0.5 ml) and folinciocalteaue reagent (0.2 ml) and made up to 10 ml with distilled water. the absorbance was read at 765 nm after 60 min by uv visible double beam spec-trophotometer (model evolution 600, thermo electron, us). ascorbic acid sample solution equivalent to 0.2 mg ascorbic acid/ml was prepared in water containing 3% (w/v) metaphosphoric acid. it was titrated against standard 2, 6 dichlorophenol indophenol (2,6 dcip) solution of 0.5 mg/ml concentration until the pink color developed completely. the operation was repeated with a blank (indian pharmocopia). tristimulus colour tristimulus color in terms of hunter l, a, b values was measured using x-rite spectrophotometer (usa) using d-65 illuminant and 10 o observer. ‘l’ value represents lightness, ‘a’ value shows redness-greenness and ‘b’ value indicates blueness-yellowness of the samples. statistical analysis the data obtained were analyzed statistically for analysis of variance (anova) using completely randomized design with least significant difference (lsd) at p<0.05 using co.stat 6.303, cohort software (usa). results and discussion physical properties of wild and cultivated chakiya variety of amla fruits is given in table 1. the fruits of wild variety were found smaller than the cultivated variety. there was significant difference (p < 0.05) in weight of the fruits of both the varieties. an average weight of the fruits of the wild variety of amla was 17.66 g, whereas cultivated variety showed an average weight of 28.88 g. the fruits of both the varieties have six segments. juice yield was found to be significantly (p< 0.05) higher in wild variety (48.3%) as compared to chakiya variety (41.9%). in case of wild variety, the total phenolic contents were found to be 32.32 g/100 g of gallic acid equivalent (dwb), whereas, chakiya variety had 24.50 g/100 g of gallic acid table 2. chemical properties of fruits of wild and chakiya variety of amla (n=3). particular chakiya variety wild variety ascorbic acid (g/100 g) 463.0 ± 6.83 b 560.0 ± 9.42 a tss ( 0 brix) 9.3 ± 0.23 b 13.1 ± 0.105 a total phenols in residue (g/100g, dwb) 4.0 ± 0.23 b 12.1 ± 0.35 a total phenols in juice (mg/100 gm, fwb) 174.0 ± 4.7 a 176.6 ± 6.18 a total phenols in fresh amla (g/100g, dwb) 24.5 ± 1.11 b 32.3 ±1.35 a values in same row with different superscripts differ significantly (p < 0.05). table 3. mineral content and some chemical properties of amla powder (n=3). drying method ca p fe (mg/100gm) ascorbic acid moisture (%) fat freeze dried 79.6 ± 0.72 a 12.38 ± 0.32 a 88.03 ± 0.75 a 5432.75 ± 16.11 a 6.78 ± 0.03 a 0.24 ± 0.005 d sun dried 70.3 ± 0.72 b 7.90 ± 0.24 c 54.20 ± 0.72 b 3006.00 ± 14.40 e 5.76 ± 0.19 c 0.39 ± 0.009 c vacuum dried 64.0 ± 0.27 c 9.16 ± 0.83 b 53.87 ± 0.66 b 3339.88 ±15.14 d 6.53 ± 0.12 b 0.42 ± 0.094 b spray dried 55.0 ± 0.94 d 5.10 ± 0.25 d 49.26 ± 0.91 c 4058.44 ±11.32 b 5.05 ± 0.91 e 0.23 ± 0.021 e tunnel dried 50.1 ± 1.23 e 7.85 ± 0.43 c 53.67 ± 0.97 b 3401.00 ±13.31 c 5.59 ± 0.16 d 0.59 ± 0.089 a values in same column with different superscripts differ significantly (p < 0.05). equivalent (dwb) (table 2). the total phenolic contents were also found high in the juice and residue (left after extraction of juice) of wild variety of amla than the chakiya variety. the juices had total phenolic concentrations in the range of 174.0 and 176.6 mg/100 g of juice (fwb) in chakiya and wild variety, respectively. the phenolic contents in juices are low compared to fruits due to loss of phenolics during squeezing process. another reason may be that polyphenols are involved in specific physicochemical interactions with the solid part of the fruits, especially the cell wall material (ranaurd et al., 2001). this was reconfirmed by the polyphenolic estimation of residue of juice. ascorbic acid content was also found to be high in wild variety of amla compared to the cultivated variety (table 2). kaur and kapoor (2002) reported more than 70% antioxidant activities as percentage inhibition of oxidation in amla fruits which was correlated positively with total phenols. the total soluble so-lids in juice prepared from cultivated variety (9.3 o brix) of amla were also lower than that of wild variety (13.1 o brix). effect of drying methods on the quality of powder the fruits of chakiya variety were used to prepare the powder. powder yield varied with type of drying method as sun drying (10.11%), tunnel drying (8.78%) and vacuum drying (12.48%), spray drying (4.90%) and freeze drying (2.23%). significant differences (p<0.05) in chemical composition of amla powder were observed when prepared by different techniques. the total ascorbic acid content ranged from 3.00 to 5.43 g/100 g of powder. the freeze dried powder had the highest ascorbic acid content followed by spray dried powder. the lowest concentration of ascorbic acid was found in sun dried powder (table 3). when amla fruits were cut and dried the oxidation process already took place and this may be the reason of low ascorbic acid content in sun dried powder. the rate of oxidation was high during sun drying, hence the sun dried sample showed lowest amount of ascorbic acid than other samples. moisture content varied from 5.05 to 6.78% whereas fat content varied from 0.23 to 0.59% in the powder prepared by using different dehydra-tion methods. freeze dried samples showed maximum mineral contents in terms of calcium (79.6 mg/100 g), phosphorus (12.38 mg/100 g) and iron (88.03 mg/100 g). tristimulus colour values of the powder on hunter scale in terms of l, a, b values are given in table 4. significant differences (p < 0.05) were observed in l, a, b values of the samples. freeze dried sample was found lighter in color as compared to other powder samples, whereas the sun dried sample was found to be darkest in color which can be observed by higher l value in freeze dried (61.64 and lower l value in sun dried (45.78) samples. higher redness was observed in sun dried sample followed by tunnel, vacuum, freeze and spray dried samples. where-as, b value was maximum in tunnel dried sample and mi-nimum in spray dried sample. on the basis of l, a, b co-lour values, the freeze dried sample was considered bet-ter in terms of colour values followed by spray dried one. conclusion amla (var. chakiya) can be used for development of value added products. this variety had a lower content of table 4. tristimulus colour profile of amla powder (n=3). drying method l a b freeze dried 61.64 a -0.16 d 15.02 b sun dried 45.78 e 2.49 a 14.37 c vacuum dried 46.18 d 1.58 c 12.77 d spray dried 53.84 c -0.30 e 7.88 e tunnel dried 59.17 b 1.67 b 15.15 a values in same column with different superscripts differ significantly (p < 0.05). phenolic content, total soluble solids and ascorbic acid as compared to wild variety. however, in terms of average weight, length and diameter the fruit of this variety was found better than wild variety. the powder developed from this variety showed better retention of nutrients and colour in the case of freeze dried samples followed by spray dried ones. references aoac (1990). officials methods of analysis of association of analytic chemists, washington dc. aoac (1997). officials methods of analysis of association of analytic chemists, washington dc. chauhan op, srivastava s, pandey p, rai gk (2005). a study on the development of aonla blended sauce. beverage food world. 32 (5): 31-32. deka bc, sethi v, prasad r, batra pk (2001). application of mixtures methodology for beverages from mixed fruit juice/pulp. j. food sci. technol., 38(6): 615-618. jamwal ks, sharma ip, chopra l (1959). pharmacological investigations on the fruits of emblica officinalis. j. sci. ind. res. 18 c: 180-181. jayprakash gk, singh rp, sakariah kk (2001). antioxidant activity of grape seed extracts on peroxidation models in vitro. food chem., 73:285-290. jayshri s, jolly ci (1993). phytochemical antibacterial and pharmacological investigations on monordica chiranlia and emblica officinalis. ind. j. pharm. sci. 1: 6-13. kalra cl (1988).the chemistry and technology of amla (phyllanthus emblica) a resume. indian food packer. 42(4): 67-82. kaur c, kapoor hc (2002). anti-oxidant activity and total phenolic content of some asian vegetables. international j. food sci. technol., 37(2): 153-161. kumar js, khurdiya ds (2002). studies on juice extraction of aonla (emblica officinalis gaertn.) cv. áchakaiya'. j. food sci. technol.,39(5): 515-516. rajkumar nv, theres m, kuttan r (2001). emblica officinalis fruits afford protection against experimental gastric ulcers in rats. pharmaceut. biol., 39 (5):375-380. ranaurd cmgc, baron a, guyot s, drilleau jf (2001). interactions between apple cell walls and native apple polyphenols: quantification and some consequences. int. j. biol. macromol., 29: 115-125. rao ts, kumari kk, netaji b, subhokta pk (1985). ayurveda siddha. j res. 6:213-224. rastogi rp (1993). compendium of indian medicinal plants, cdri, lucknow and id, new delhi 1: 530. shankar g (1969). aonla for your daily requirement of vitamin c. indian hort. 13: 9-15. thakur cp, mandal k (1984). effect of emblica officinalis on cholesterol induced atherosclerosis in rabbits. ind. j. med. res.,79: 142-146. thakur cp, mandal k (1984). effect of emblica officinalis on cholesterol induced atherosclerosis in rabbits. ind. j. med. res. 79:142-146. tripathi vk, singh mb, singh s (1988). studies on comparative compositional changes in different preserved products of amla (embilica officinalis gaertn) var. banarasi. indian food packer., 42(4): 60-66. vinayagamoothy t (1982). antibacterial activity of some medicinal plants of srilanka ceylon. j. sci. biol., 11: 50-55. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 9 (8), pp. 001-005, august, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper correlation and heritability estimates of maize agronomic traits for yield improvement and striga asiatica (l.) kuntze tolerance s. a. olakojo1* and g. olaoye2 1 institute of agricultural research and training, obafemi awolowo university, p. m. b. 5029, moor plantation, ibadan, nigeria. 2 department of agronomy, university of ilorin, ilorin, nigeria. accepted 19 may, 2021 a study was carried out to estimate percent heritability and correlation coefficients of desirable maize agronomic characters under artificial infestation with striga lutea. forty five f1 hybrid maize of varied striga tolerance and their ten parents inbred were evaluated along with two check entries. this was sited in three locations representing different agro ecologies of south western nigeria in 1999 cropping season. the results showed high and moderate heritability estimates of 94.0 and 40.0 for striga emergence count and striga syndrome rating respectively, while low heritability estimates of 13.0, 11.32, 16.0, 5.70 and 16.22% were obtained for tolerance index, days to tasseling, days silking, kernel rows/cob and grain yield respectively. genotypic correlation coefficients of striga emergence count with maize agronomic traits were positive and significant, while phenotypic correlation between striga count and other maize agronomic traits were also positively and significantly correlated, except striga count which was negatively correlated with striga syndrome rating. on the other hand, maize ear aspect alone contributed 19.4% to grain yield, while inherent yield loss of 23.0% was estimated if striga is left uncontrolled. parameter estimates revealed that all striga and maize agronomic traits were positively associated with grain yield except plant and ear heights under striga infestation. although, implications of these estimates have been discussed, this study affirmed that genotypic and phenotypic correlation coefficients as well as heritability estimates were found suitable as models for yield improvement and selection for s. lutea tolerant genotypes. key words: heritability estimates, correlation coefficient, striga tolerance rating, grain yield. introduction variance component and heritability estimates have been extensively used by plant breeders in selection of promising genotypes, and, in prediction of percentage heritability of desirable traits fanous et al. (1977), morakinyo (1996) and anderson et al. (1991). these models have been used in crop yield improvement, adaptation, better nutritional quality, disease/pest resistance, as well as nitrogen use efficiency in crops. for example, ruming et al. (1998) reported the existence of significant genetic variability for resistance to maize weevil under *corresponding author. e-mail: saolakojo@yahoo.co.uk. artificial infestation with sitophilus zeamaize. when they evaluated crosses between exotic and adapted maize varieties in 1995 and 1996 for remained kernel weight (rkw), the broad-sense heritability estimate obtained for rkw was 0.27 and 0.30 in 1995 and 1996 respectively. similarly, lane et al. (1995) identified three sources of resistant gene in wild relatives of pear millet to striga asiatica. in all pennisetum setosum and sorghum vericolor were resistant to striga. from this resistance pattern, 15% of diploperemis population were resistant to striga hermonthica, while 70% were resistant to s. asiatica. verkleij et al. (1996) on the other hand carried out comparative studies on resistance of cereals and wild grasses to s. hermonthica and s. asiatica. their result indicated that wild grasses had higher resistance level to striga than the crop hosts. this probably suggests that their resistance gene could be transferred to the domesticated crop species. kaman (1996), in kenya reported resistance of some maize varieties to infestation with s. asiatica. he observed that maize ear height and grain yield differed significantly among cultivars, while striga emergence count was generally low in resistant varieties but did not differ significantly. the least striga emergence count was recorded in hybrid txi × ap4 with a mean yield of 1.8 t/ha. the use of heritability estimate was also stressed by morakinyo (1996), especially in enhancing early generation selection of progeny when the estimate is considerably high. obilana et al. (1981), earlier summarized procedures in heritability estimates into three, namely, regression of offspring on parent, variance component estimate, and, recurrent selection experiment. whichever, method a breeder decides to use does not really matter, rather, the predictive ability of the estimate in selecting genotypes for advancement makes it highly relevant in selection procedure. in the same vein, chiang and smith (1967) reported 62% heritability estimate on plot means basis for sorghum head length, while lothrop et al. (1985) reported heritability estimates of 41 and 43% for 1000-seed weight in sorghum. lukhele (1981) also used heritability estimate to predict yield component in sorghum. from these reports, they all affirmed the suitability of these estimates in prediction and selection of promising crop genotypes during crop improvement programmes. strictly speaking, when a breeder decides to use regression, recurrent or variance component methods in estimating heritability, mention can not, but be made of correlation and regression responses of the estimated parameters especially yield and disease/pest resistance. perhaps the reason why many workers including mossad and joppa (1987) and moli et al. (1974) reported the importance of correlation and regression analyses in selecting resistant crop species of various production stresses. the objectives of this study, therefore, were to: (1) estimate percentage heritability of striga and maize agronomic parameters with a view to determining desirable genotypes. (2) to determine percentage contribution of some maize agronomic traits to grain yield under s. lutea infestation. materials and methods twenty-four s. hermonthica resistant maize inbred lines were evaluated in 1995 and 1996, out of which ten s .lutea tolerant inbreds were selected for half-diallel crosses to generate 45 f1 hybrids. the ten parent inbreds, 45 f1 hybrids and two check entries were later evaluated in temidire, eruwa and ilora representing three distinct agro ecologies of south western nigeria namely: derived savanna, southern guinea savanna and forestsavanna transition ecology in 1999 cropping season. the three chosen locations were s. asiatica endemic areas where farmers have abandoned the plots due to high striga infestation land in temidire and eruwa locations were mechanically prepared by two regimes of ploughing, one harrowing and ridging, while ilora location was manually prepared with hoes due to undulating soil topography. planting hills were inoculated with 44000 germinable seeds of s.lutea of previous year 14 days before planting so that striga inoculum can re condition itself to the new environment as early as possible using the methods described by barner et al. (1997).the f1 hybrids, their parent inbreds, and, two checks were planted on the hills to avoid possible erosion of the striga inoculums. three maize seeds were planted on a hill and were later thinned to two, ten days after panting. spacing of 75 × 50 cm was used so as to obtain 55,333 population densities per hectare. low fertilizer dosage (50 kg/ha) n.p.k. 20-10-10 was applied by broadcast before ridging during land preparation to minimize the likelihood of nitrogen (n) suppressing striga emergence. constant removal of other weeds except striga seedlings were carried out to enhance full striga emergence. the trial was replicated thrice using a randomized complete bock design (rcbd) with infested and corresponding uninfested plots. data were collected on striga parameters such as plant stand, striga emergence count 10 weeks after planting, striga syndrome rating using scale 1 to 9 as described by kim (1994) where 1 = normal plant growth with no visible symptom 9 = complete scourching of leaf causing premature death or collapse of host plant with no ear formation and striga tolerance index (yield of infested/yield of uninfested maize). other parameters included maize plant and ear aspects using a rating of 1 to 5 (where 1 = excellent and 5 = poor), plant and ear heights (cm), days to 50% tasseling and silking, kernels/row and kernel rows/cob as well as grain yield (t/ha) under artificial and natural striga infestations. data were statistically analysed using sas (sas institute,1992) soft ware package to compute genetic variances, phenotypic and genotypic correlations, stepwise multiple regression and heritability estimates on plot mean basis for both striga and maize agronomic parameters. results, discussion and conclusion the results from the heritability estimates are presented in table1. striga emergence count, striga rating and striga tolerant index recorded lower error variances. the magnitudes of genotypic variances were for most characters lower than those of error and genotype × location variances. although heritability estimates of 94.0% was obtained for striga emergence count, moderate heritability estimate of 40.0% was obtained for striga ratings as against low heritability estimates of 13.0, 11.32, 16.0, 5.70 and 16.22% respectively for striga tolerance index, day to tasseling and silking, kernel rows/ear as well as grain yield (table1). this suggests that while striga emergence count and rating were highly heritable and can be selected at early generation stage, others might not be as easy as one expects. for example, striga syndrome rating in the present study has heritability estimate of 40%, hence can be easily selected. phenotypic and genotypic correlation coefficients of striga and maize agronomic characters are presented in table 2. at the phenotypic level, striga count was negative table 1. variance component and heritability estimate (on plot mean basis) of striga tolerance parameters and maize agronomic traits in s. lutea infested condition. parameter e δ 2 δ 2 gl δ 2 g h 2 b striga emergence count 11.21 -10.74 7.58 94.00 striga rating 0.75 0.13 0.57 40.00 striga tolerance index 10.3 0.15 0.15 13.00 days to 50% tasseling 6.09 1.99 1.03 11.32 days to 50% silking 9.18 3.10 2.37 16.00 no of kernel row/ cob 2.93 0.05 0.18 5.70 maize grain yield 0.54 0.08 0.12 16.22 e δ 2 variance due to error, δ 2 gl variance genotype × location, δ 2 g variance due to genotype. table 2. phenotypic and genotypic (rp and rg) correlation coefficients between striga related and maize agronomic characters, under s.lutea infestation. parameter striga count striga rating plant height ear height days to silking days to tasseling kernel rows/ cob grain yield striga count 0.91 0.30* 0.17 0.30* 0.40** 0.85** ++ striga rating -0.30* 0.36* -0.72** 0.09 0.50** ++ ++ plant height 0.048 0.025 0.05 0.13 0.112 0.076 0.16 ear height -0.74** 0.30* 0.001 0.23 -0.22 -0.57** 0.23 days to silking 0.06 0.09 0.015 0.14 0.74** -0.53** ++ days to tasseling 0.07 0.60** 0.001 0.06 -0.05 0.77** ++ kernel rows/cob -0.20 -0.34* 0.051 0.20 -0.41** 0.06 ++ grain yield. -0.57** 0.33* 0.114 -0.35** 0.075 0.133 0.18 upper diagonal= genotypic (rg) correlation coefficients. lower diagonal= phenotypic (rp) correlation coefficients. *, ** sig. at 0.005 and 0.01.++, genotypic value > 1.0. and significantly correlated with striga rating (rp=0.30)*, ear height (rp=-0.74*) and maize grain yield (rp=-0.57)* indicating that the two traits are probably controlled by different genes. this was similar to evidence of kim (1994) where he affirmed that striga count and rating in s. hermonthica were been controlled by independent striga syndrome rating was however positively count was positively correlated with striga genes and significantly correlated with ear height (rp = 0.31)*, days to silking (rp =0.60)* and grain yield (rp = 0.33)*. phenotypic correlation of ear height and grain yield was however negatively significantly correlated suggesting that the two traits were not closely associated, and, therefore may not be jointly selected. similarly, days to silking and kernel rows/cob was negative and significantly correlated (rp = -0.47)*. genotypic correlation coefficients shows that striga rating, plant height, days to silking, days to tasseling and kernel rows/cob with coefficients of 0.91*, 0.30*, 0.30*, 0.44** and 0.85** respectively. striga rating on the other hand was positively correlated with plant height(rg = 0.36)* and day to tasseling (rg=0.50) ** as compared to ear height which was negative, and significantly correlated with striga rating (rg= -0.72) **. aurelio et al. (1965) table 3. stepwise multiple regression analysis of striga related parameters on maize grain yield under both infestation conditions. parameter partial r 2 cumulative r 2 f {prob.>f} striga infested condition ear aspect 0.1914 0.1914 99.63** 0.0001 striga count 0.0063 0.2239 3.4180* 0.005 tolerance index 0.0031 0.2271 1.6842 0.198 striga rating 0.0015 0.2285 0.7859 0.37 striga uninfested condition ear aspect 0.167 0.167 84.80** 0.0001 tolerance index 0.130 0.300 77.94** 0.0001 striga count 0.002 0.320 1.01 0.317 table 4. stepwise multiple regression of maize agronomic traits on yield under s. lutea infestation. character partial r 2 cumulative r 2 f {prob.>f} plant height 0.083 0.083 38.24** 0.001 days to silking 0.017 0.100 790 6.0052 days to tasseling 0.021 0.121 10.00** 0.0017 striga rating 0.009 0.130 4.16* 0.042 ear height 0.12 14.2 5.90* 0.015 similarly reported high heritability estimate for agronomic characters in sunflower. he further affirmed the possibility of its early generation selection for desirable agronomic traits. kim (1994) therefore recommended the use of striga rating and tolerance index as the most suitable parameters for assessing the tolerance level of maize plants. days to tasseling was positively correlated with days to silking (rg=0.74**). days to silking on the contrary was negatively and significantly correlated with kernel rows/cob (rg = -0.53**) (table 2). days to tasseling was however positive and significantly correlated with kernel rows/cob (rg = 0.77**), suggesting that kernel row/cob is probably a function of maturity date. some correlation coefficients were greater than unity as found in some traits when correlated with yield related characters. the results of stepwise multiple regression analyses for striga related parameters are presented in table 3. total yield reduction caused by striga related parameters was 23.0%. reda and kebebe (1994) had earlier reported a negative correlation between striga count and fresh maize weight indicating an adverse effect of striga count on maize yield. maize ear height had the highest yield contribution of 19.14%. other parameters jointly contributed less than 4% to grain yield. this shows that while striga must be controlled on maize plots to prevent inherent yield loss of about 23%, selection in favour of good ear aspect may bring about 19.14% yield advantage. similarly, ear aspect and tolerance index recorded yield contributions of 16.7 and 13.0% respectively, striga count and rating on the other hand slightly reduced yield by 0.2 and 0.1% under natural striga infestation. this further suggests that breeding for higher maize yield in striga endemic ecologies should focus more on selection for ear aspect. table 4 presents the results of stepwise multiple regression analysis of maize agronomic characters under s. lutea infestation. these characters jointly contributed 14.2% to maize grain yield, out of which plant height alone contributed 8.3% as compared to ear height, day to silking and tasseling and striga tolerant rating that contributed 1.2, 1.7, 2.1 and 0.9% respectively to grain yield. these contributions though very small, were statistically significant except days to silking. parameters estimates under both infestation conditions are presented in table 5. these estimates determine the significant levels of association of each trait to grain yield. all striga and maize agronomic parameters were positively associated with grain yield under artificial infestation. similarly, all maize agronomic traits were positively associated with grain yields under natural infestation. striga related parameters on the other hand, were negatively associated with grain yield except striga rating (table 5). this probably suggests the need to concentrate on selection for yield related characters such as higher numbers of maize rows/ear, numbers of kernel per row, ear aspect and yield in t/ha rather than selecting table 5. partial r 2 and parameter estimates of striga and maize agronomic characters under both striga infestation conditions. variable striga infested condition striga uninfested condition partial r 2 parameter estimate partial r 2 parameter estimate ear aspect 0.191** 0.414 0.16** 0.42 striga count 0.22* 0.034 0.31** -0.72 tolerance index 0.22* 0.056 0.11** ns striga rating 0.23** 0.0362 0.08** 0.012 plant height 0.08* -0.0042 0.008 -0.67 days to silking 0.01* 0.067 0.020** 0.108 days to tasseling 0.12* 0.060 0.032** 0.033 kernels/row 0.13** 0.019 0.012** 0.0066 ear height 0.14** -0.006 0.003* 0.021 kernel rows /cob n.s n.s n.s n.s p< 0.15. only for s .lutea tolerant genotypes per se for striga endemic areas. this study affirmed that genotypic and phenotypic correlation coefficients as well as heritability estimates were found suitable as models for yield improvement and selection for s. lutea tolerant genotypes. references aurelio l, kinman m, smith jd (1965). heritability of self-compatibility in sunflower helianthus annus. crop sci. 5: 529-532. barner dk, wilson md, award ae, cadwell kf, mohan rdr, kim sk (1997). sa striga research method –mannual 2 nd edn. pp 1-15. chiang ms, smith jd (1967). diallel analysis of the inheritance of quantitative characters in grain sorghum 11. gene action the number effective factor and inheritability. can. j. gene cytol., 9: 823-830. reda f, kebebe y (1994). studies on striga host range and resistance screen in sorghum. afr. crop sci. j. 2(3): 301-307. kaman gm (1996). evolution of maize varieties for striga asiatica tolerance in coastal kenya. striga newslett., no. 6, p. 13. kim sk (1994). genetics of maize tolerance of striga hermonthica. crop sci. 34: 900-907. lane ja, child dv, moore thm, baily ja (1995). wild relatives of cereals as new sources of resistance to striga. paper presented at third regional working group meeting mombassa kenya: striga newslett., no 6, p. 12. lothrop je, atkins re, smith os (1985). variability for yield and yield components in iapir grain sorghum, random-mating population 1. mean variance components and heritability. crop sci., 235-240. lukhele pe (1981). estimation of genetic variability in sorghum population (sorghum bicolor l. moench) msc. thesis ahmadu bello university zaria. moli rh, stuber cw (1974). quantitative geneticsempirical results relevant to plant breeding. adv. agron., 26: 277-319. morakinyo ja (1996). heritability, correlation and expected responses to selection of some yield components in grain sorghum, sorghum bicolor l moench. niger. j. gent., 11: 48-54. mossad mg, joppa lr (1987). study of the host-wheat plant resistance to cereal aphids and antifeeding effect of ecyoel on green bug. cereal res. commum. 16(1): 65-71. obilana at, fakorede mab (1981). heriability: a treaties. samaru j. agric. res., 1: 72-82. ruming li, manjit s, kang o, moren j, pollak m (1998). genetic variability in exotic x adapted maize germplasm for resistance to maize weevil. plant genetic resource newsletter no 114: 22-25. sas institute (1992). sas technical report p229, sas/srat soft ware. changes and enhancement release cary new york, 607: 289359. verkleij ja, kuiper ce, peterse ah (1996). comparative studies on the resistance of cereals and wild grasses to striga species. striga newslett. january, 31(6): 13. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 10 (2), pp. 001-010, february, 2022. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review a review of shelf-life extension studies of nigerian indigenous fresh fruits and vegetables in the nigerian stored products research institute ubani, ozioma ndubunma* and okonkwo, ego ulu nigeria stored products research institute, 3 km asa dam road, p. m. b. 1489, ilorin, kwara state, nigeria. accepted 23 august, 2021 the nigerian stored products research institute has been conducting research in technology development suitable for post-harvest handling of nigerian indigenous fruits and vegetables for extension of the shelf-life of these commodities, retention of nutrients and aesthetics of fresh produce in storage, enhancement of the nutritional status and earning capacity of both the rural and urban poor through proper handling, storage, marketing and consumption of fruits and vegetables; and production of the best quality fruits/vegetables for local and export markets over three decades. the research work carried out and the technologies developed for handling of nigerian indigenous fruits/vegetables postharvest for the farm, market and domestic levels of storage are discussed subsequently. these indigenous fruits and vegetables are prone to chill injury when kept at low temperatures. however, storage structures developed by the nigerian stored products research institute over the years, such as the evaporative coolers and cooler baskets which could record some temperature drop from the ambient, have been used in extending the shelf-life of fresh fruits and vegetables. packing (wrapping) materials have also showed promise in extending shelf life of nigerian indigenous fresh produce. the season and time of harvest also play an important role in storage of fruits and vegetables. garden eggs have very short shelf-life of 7 days during the dry harmattan months in nigeria. fresh fluted pumpkin telfairia occidentalis ‘ugu’ leaves can only be kept for 6 days at 29.1°c and 64.5%rh beyond which physiological breakdown of tissues lead to deterioration and total loss. fresh pawpaw (carica papaya l.) fruits, individually wrapped with waxed paper (tixo) kept at ambient conditions in plastic crate, have a shelf-life of 17 days as compared to fresh pawpaw fruits in evaporative coolers for 7 days. in hot dry weather of the northern parts of nigeria, oranges stored in evaporative cooler (ec) metal-in-brick wall have a shelf-life of 10 weeks. adoption of these technologies will play a very vital role in post-harvest loss reduction in fresh fruits and vegetables along the food chain, increase income and improve nutrition for all stakeholders. the interventions to maintain food quality and safety for reduction of postharvest losses in nigerian indigenous fruits and vegetables through research activities in nspri for three decades are the focus of this review paper. key words: postharvest, technologies, fruits, vegetables, shelf-life. introduction the nigerian stored products research institute (nspri) is one of the national agricultural research institutes in nigeria. the nspri carries out researches on postharvest technology and storage of agricultural products and has five out-stations covering different *corresponding author. e-mail: oziubani@yahoo.com, egoulu@yahoo.com. tel: + 234 706 229 3055. agro-ecological zones of nigeria including two sea ports. at inception, it was known as the west african stored products research unit (waspru) covering the british colonies of west africa under the supervision of the federal ministry of trade under the colonial masters who were mainly concerned with export cash crops (cocoa, palm produce, groundnuts and rubber). on nigeria’s attainment of independence in 1960, the waspru became the present nigerian stored products research institute and the only postharvest institute in nigeria and 2 sub-saharan africa. the institute, in line with nigeria national agricultural policies, conducts researches in bulk storage problems of local foods/crops, both durables (grain legumes, oil seeds and beverage crops, example, cocoa and kola and perishables (roots, tubers, fruits/vegetables and animal products as fish and meat). the main research topics at present in the nspri are the following: i. researches to improve and maintain the quality of nigerian food crops; ii. special studies on food contaminants, pests, pesticides/residues, and other anti-nutrients in stored food products; iii. researches to retain/enhance the nutrients and the aesthetics of fresh produce in storage; iv. researches aimed at improving fruit and vegetable quality for local and export markets; v. extension of research findings through publications, training of stakeholders in various postharvest aspects of different crops. research perspective food preservation is more than engineering and its proper understanding must involve the relationship between engineering, nutritional, biochemical, microbiological, entomological and economic aspects of preservation. only the amalgam of these can be considered to encompass food preservation as a whole. for this reason, the nspri has a multi-disciplinary approach as clearly evidenced by the above listed research topics. under tropical and sub-tropical conditions, most crops deteriorate rapidly after harvest ceasing to be marketable. spoilage can either be complete, as following the development of pathogenic microorganisms or partially, when a significant decrease of nutritional value and/or development of toxic byproducts occurs. the nutritional loss of fresh fruits and vegetables is mainly caused by a fast and extended oxidation of health compounds (example, vitamins, amino acids, etc.). in addition to the pre-harvest losses the ones occurring after harvest contribute significantly to world food shortages especially in developing countries including nigeria where storage facilities are limited. most food preservation processes are only effective by employing a low temperature chain from the field to the fork. low temperature storage with high humidity and high concentration of solutes delay the loss of nutrients and obstruct microbial growth. delays between the harvest and utilization of crops are inevitable and the highly perishable crops (roots, tubers, fruits and vegetables) are prone to quality loss during the intervening period. the world’s major food preservation process is still fresh storage of these crops which is a great challenge in the hot and humid tropical conditions especially where a cold chain policy is difficult or even impossible to become accustomed. food quality and safety the major disadvantages of food produce storage and distribution are the limits it imposes on shelf-life, the difficulty in predicting storage life of different perishable crop varieties. there is lack of control over distribution conditions which occurs in the nigerian situation especially in the local markets. the perishables are often packed and transported under circumstances that accentuate their rapid deterioration from the farm gate to the market. postharvest losses in these produce are high and reach the consumers in conditions that are inimical to food quality and safety. appropriate preservation technologies for the highly perishable crops can reduce postharvest losses and fill the needs of the developing countries which can least afford the postharvest losses that occur in these countries. the interventions to maintain food quality and safety for reduction of postharvest losses in nigerian indigenous fruits and vegetables through research activities in nspri for three decades are the focus of this review paper. nigerian indigenous fruits and vegetables there is a great diversity of fruits and vegetables in the tropical and sub-tropical regions of the world. in nigeria, there is abundance of seasonal fruits which replace others as soon as these are out of season. in the humid southern nigeria for example, the star apple (chrsophyllum albidum) replaces citrus (genus) which are off season from the end of january. mangoes, cashews, and avocado pears are in season after the star apple. many nigerian indigenous fruits and vegetables are native to different ecological zones of the country. however, some of these species have been introduced to other parts of the country. the fluted pumpkin (telfairia occidentalis) which in the past was cultivated exclusively in eastern nigeria is now a national vegetable and grown in all parts of nigeria because of its nutritional importance. most nigerian indigenous vegetables are cultivated all year round especially as dry season crops with irrigation, while others which grow in the wild for example gnetum africanum (ukazi) and piper guinenses (uziza) are cultivated under the agro forestry farming system. fruits and vegetables are important in both human and animal nutrition. these crops provide nutrients such as minerals, vitamins and dietary fibre which are essential for animal and human health. some of these fruits are also rich sources of antioxidants which play a role in 3 cancer prevention from environmental pollutants such as polycyclic aromatic hydrocarbons (pahs) emitted from fossil fuels which are now of great concern globally. fruits and vegetables are cultivated in nigeria for economic purposes both in the rural and urban environments. some of the perennial fruits trees such as citrus, avocado pear, guava and others are planted as ornamental trees in homesteads. the annuals such as tomatoes, leaf vegetables, garden eggs and okra may be cultivated either as seasonal or irrigated crops. farm and market surveys (nspri port harcourt, unpublished data) showed that the prices of these commodities are more than doubled during their off-season period. the producers hardly benefit from these price increases because of the lopsided marketing system where the collectors (middlemen) sell to retailers. in nigeria, economic losses by farmers are in part related to crop deterioration along the food chain, especially by inadequate storage and transportation to distant markets where fruits and vegetables would yield better economic returns. this is particularly during the peak season of fruits and vegetables when there is glut in local markets and crops are sold at very low prices. the nigerian stored products research institute has been conducting research in technology development suitable for postharvest handling of nigerian indigenous fruits and vegetables for over three decades. the general objectives of postharvest research for fruits and vegetables in the institute are: i. extension of the shelf-life these commodities; ii. retention of nutrients and aesthetics of fresh produce in storage; iii. enhancement of the nutritional status and earning capacity of both the rural and urban poor through proper handling, storage, marketing and consumption of fruits and vegetables; iv. production of best quality fruits/vegetables for local and export markets. for over three decades, the nspri has been conducting researches in postharvest handling and technology with the aim to extend the shelf-life of nigerian indigenous fruits and vegetables and to disseminate results of the most promising storage techniques for each fruit species of the different ecological zones of nigeria. here follows a review of some of these researches and the benefits of the results to the economic outcome of nigerian agriculture. research work carried out and technologies developed for handling nigerian indigenous fruits/vegetables postharvest for the farm, market and domestic levels of storage are discussed subsequently. each fruit/vegetable react differently to the same environmental conditions, hence, their shelf-life differs under these conditions. fruit/vegetable post-harvest research activities focus on development of the most suitable storage technique for each fruit species in the different ecological zones of nigeria. extension of shelf-life of various nigerian indigenous fruits and vegetables garden eggs (solanum aethiopicum l.) garden eggs are annuals fruit that are cultivated in all the agro-ecological zones of nigeria and also widely distributed in africa. there are many varieties of garden eggs, each variety is peculiar to the locality where it is cultivated. the fruits are among the most appealing vegetables to sight as they have a wide range of colours, from white, cream, yellow, green, lime, orange, pink, red, purple and dusty black. the fruits can be stripped and multicoloured. garden eggs have both nutritional and medicinal values. it is a fruit of choice by diabetic patients. the fruits are claimed to alleviate liver ailments (lawande and chavan, 1998) and has antioxidant properties (a. ade, nspri, ‘ilorin’, personal communication). garden eggs deteriorate within two to three days after harvest especially when the stalk and calyx are removed and fruits are exposed to warm temperatures. the problem of wastage of fruits during the peak season is enormous especially in transporting to the marketing outlets as well as holding at the retail points in both urban and rural markets. studies on shelf-life extension of garden eggs at various times and seasons of the year using a novel fruit/vegetable cooler basket was carried out by the nspri ‘ilorin’ in the central zone of nigeria. the objective of the experiments was to determine the most effective storage conditions for the fruits that will be beneficial to the stakeholders (farmers, traders and consumers). storage of garden eggs 1 the cooler baskets (cb) were constructed with plastic baskets (each having a capacity for 15 kg of fresh fruits) with lid overlaid with tight polystyrene foam and covered with polypropylene material. the baskets were conditioned for storage before use by dipping both lid and basket in clean water and draining for about 1 h. freshly harvested garden eggs purchased in a local fruit market in ilorin metropolis were sorted, washed, drained thoroughly and stored in the cooler baskets. the control fruits were stored in open plastic crates. both the test and the control batches of fruits were on shelves in a ventilated fruit/vegetable shade. the temperature and relative humidity (rh) of the shade (ambient) and those of the storage cooler baskets and crates were recorded daily. the baskets were moistened on the outside whenever drying was observed. fruits were examined for 4 colour, texture and weight changes and signs of deterioration. fruit flavor and acceptability were assessed by palatability test after termination of storage. the average ambient temperature and rh recorded during the storage period in october 2005 in the fruit/vegetable shade was 25°c and 42% rh. the cooler basket recorded average temperature of 22°c and 65% rh. the garden eggs had a shelf-life of 7 days without spoilage in the cooler baskets. the control fruits were shrivelled and changed colour within the same period. palatability test showed that the fruits in the baskets had an off-flavour due to moisture absorption from the storage atmosphere of the baskets which also resulted in slight weight increase of fruits during storage (ubani and suleiman, 2008). storage of garden eggs 2 the fruits were purchased from the fruit market in ilorin in august 2007. they were sorted into small, medium and large sizes and prepared as in experiment 1 earlier for storage. the three sizes of garden eggs were stored in the cooler baskets (test) and open plastic crates (control) kept in a ventilated room in nspri’s on-station fruit/vegetable processing centre ilorin. daily room temperature and relative humidity and those of the storage structures were recorded. fruits were examined for colour, texture and weight changes during the storage period. the garden eggs storage was terminated on the 9th day. the average room temperature was 26.5°c and 61% rh. the cooler baskets recorded an average of 28.5°c during the storage of fruits and the crates 28.3°c. the temperature build-up in the storage structures resulted in fruit spoilage due to rotting in the cooler baskets and mouldiness in the open crates. fruit loss in the cooler baskets was 25.5, 19.7 and 26% for the small, medium and large sizes of fruit respectively on the 7th day of storage. the control had 18.67, 9.33 and 4% spoilage for small, medium and large sizes of fruits within the same storage period (ubani et al., 2008). storage of garden eggs 3 garden eggs were harvested and purchased from a farm in the lower niger river basin at ejigba, kogi state nigeria, about 150 km from ilorin on 30th august 2007. the fruits were sorted into mixed sizes and prepared for storage with the cooler baskets and plastic crates in the fruit/vegetable shade. the average ambient temperature was 27.8°c and 70.6% rh at this period. the cooler baskets (cb) recorded 27.2°c and open crates 29°c temperature, respectively. fruits were stored for 16 days and had 57 and 47% spoilage in cooler baskets and open plastic crates, respectively. deterioration of fruits was highest on the 8th and 11th days in storage. on the 7th day, there was only 10% loss of fruits due to rotting in the cooler baskets (ubani et al., 2008). storage of garden eggs 4 fruits were purchased in the off-season during the harmattan (a very dry, hot and dusty period between mid november and early march) season in february 2008 from a local fruit market in ilorin. fruits were sorted and prepared for storage in the cooler baskets and the control in open plastic crates. the average ambient temperature 36.6°c and 16.6% rh at this period in the fruit/vegetable shade. the storage atmosphere in the cooler baskets was 28.6°c on the average. the fruits were stored for only 5 days and only 43% of the fruits were recovered fresh and wholesome. there was 13% weight loss of the fruits due to shriveling over the five day period. there was no rotting or decay of fruits both in the test and control batches. the control fruits recorded 100% loss due to dehydration after the five days (ubani et al., 2008). these trials in the storage of garden eggs show that the surrounding environment of the cooler baskets holding the fruits, time and season of the year are important factors in the shelf-life extension of the fruits. the shelf-life of garden eggs in the cooler baskets is 7 days in a well ventilated environment such as the fruit/vegetable shade under ambient conditions of 27 o c temperature and 70% rh. the cooler baskets (cb) normally record some temperature drop from the ambient in this environment. at high temperatures (37 o c) and low (16% rh), garden eggs have a shelf-life of 4 days in the cooler baskets; beyond this time shriveling due to extensive moisture loss is the major cause of fruit spoilage and loss. storage of pawpaw fruits (carica papaya l.) pawpaw fruit is of nutritional, therapeutic and economic importance in the tropics. the fruits are good a sources of -carotene, vitamin c, minerals especially potassium and magnesium, soluble fibre and carbohydrates. the water content is high in ripe fruits. the unripe fruit is a source of papain used in food and pharmaceutical industries. the pawpaw fruit is highly perishable and 100% loss may be recorded for the fruit due to improper harvesting, handling and storage postharvest. the fruit is usually harvested at the breaker pawpaw fruits (solo variety) from an orchard in the national horticultural research institute (nihort) idi-ishin ibada, nigeria were harvested at the breaker stage of ripening and transported to the laboratory in nspri ‘ibadan’. the fruits were wiped with wet tissue paper to remove surface dirt, 5 table 1. nutrient content of fresh pawpaw fruits stored at ambient condition in wrapping materials. sample treatment vitamin c (mg/100 g) titratable acidity (ml 0.1 n naoh) total sugars (mg/g) initial ripe 42.8 0.46 11.72 unripe 21.4 0.92 11.32 terminal newspaper wrap (ripe) 62.0 0.85 10.60 waxed paper 67.0 control, no wrap (ripe) 60.0 60.0 11.34 source: ubani on, nspri ‘ibadan’, 1989. and stored in nspri’s evaporative coolers (ec) each of 5 kg capacity. the control fruits were kept in open fruit crates on the laboratory bench. fruits were examined every two days for ripening (colour change) and signs of deterioration (localized softening, mouldiness, pitting and rotting). mechanical damage during harvesting and transportation of the fruit is a major cause of losses during storage of pawpaw fruit. shelf-life of fresh pawpaw fruits in evaporative coolers the fruits in the evaporative coolers ripened in 7 days and the control in 6 days. the fruits had a shelf-life of 23 days and 50% loss in the ec due to rotting at the termination of storage. the control fruits had a shelf-life of 9 days with 50% loss due to spoilage within the storage period (ubani, 1991). storability of individually wrapped pawpaw fruits at ambient conditions freshly harvested pawpaw fruits (solo variety) at the breaker stage of ripening were cleaned and wrapped individually with waxed paper (tixo) and news paper in 6 replicates. the control fruits were not wrapped. the three treatments were kept in plastic fruit crates on the laboratory bench at ambient conditions. the fruits had a shelf-life of 17 days. the control and the test fruits commenced ripening within 3 days. the control fruits and those wrapped with newspaper ripened over five days unevenly with unattractive yellow colour. fruits wrapped with waxed paper ripened over 8 days with an attractive yellow colour. the percentage losses due to spoilage after storage were 50% for control, 17% for both the newspaper and waxed paper wrap treatments. the fruits were analysed initially and finally for vitamin c, titratable acidity and total sugar as shown in table 1. there was appreciable increase in vitamin c content of the fruits during storage with slight decrease in total sugars. there were no weight changes in the fruits during and after storage. the wholesome fruits from all treatment were organolleptically acceptable in a palatability test. there was no off -flavor of fruits. however one of the fruits wrapped in waxed paper was mouldy from the stalk end to the seeds. the result show that ripening could be delayed in pawpaw fruits when wrapped in waxed paper and fruits can be stored for more than two weeks under ambient conditions. processing and storage of unripe pawpaw slices production of unripe pawpaw slices is an indigenous technology for preservation of pawpaw fruits among the tivs of benue state in nigeria. the dried fruit slices are used to prepare soups and sauces. mature unripe fruits are usually harvested during the peak production season between november and february. fruits are wiped with wet cloth to remove surface dirt, peeled and sliced (0.5 cm thickness). slices are sun-dried, packed in black polythene or polypropylene bags for storage and marketing by the processors during the off-season. these commercial samples have been stored hermetically for 30 months (okonkwo et al., 2009). the indigenous processing method was modified and improved upon in the laboratory by the application of the hazard analysis, and critical control points (haccp) system. 1.5 cm slices were either sun-dried in aluminum trays on a platform or mechanically in a multipurpose dryer at 70°c for 11 and 24 h continuously. the dry samples were packed in thick gauge food grade polyethylene films and stored in a hermetic container (plastic drum with sealed lid) for 18 months. moisture content of the dry slices was in the range 9.6 to 10.3%. moisture re-absorption (weight increase), changes in colour, texture and aroma were not observed in the stored slices during and at the termination of the experiment. there were no significant changes in the 6 proximate content of the slices during and after storage. the thicker slices were organolleptically more preferred than the thinner slices (jagtiani, 1980; okonkwo et al., 2009). production of pawpaw slices have potentials for reduction of postharvest losses of the fruits in the tropics with reference to nigeria. storage of oranges [citrus sinensis (l.) osbeck] citrus fruits are among the most important fruit crops in the tropical and subtropical regions. oranges are a component of the diet of many people globally because of their supply of vitamin c, which is essential in human nutrition. citrus fruits are non-climacteric fruits and have a relatively long postharvest life compared to other fruits. oranges are seasonal in nigeria with the peak production season and availability during the dry season from late october to late january. the problem of postharvest loss of the fruits at farm, market and domestic levels has been of great concern to all stakeholders. wastage occurs during harvesting and transportation of the fruits for the fresh market due to mechanical damage, which in turn gives rise to pathological damages of the fruits. oranges have storage problems which include blue and green mould rot at high temperatures and chill injury which leads to pitting at temperatures below 2 to 3°c. weight loss due to dehydration in citrus during storage is also a contributing factor in postharvest losses. this problem has been minimized by waxing, seal packaging and storage in sub-ambient conditions (ben-yehoshua, 1979; bayindirli et al., 1995). several trials on weight loss reduction in oranges have been carried out in nspri. simple storage techniques applied for preservation of oranges are briefly discussed. fungicidal wax treatment and sealed packaging of oranges for storage freshly harvested ‘agege’ sweet oranges were coated with a proprietary wax emulsion containing the fungicide benlate, sealed in perforated and un-perforated polyethylene films. storage of fruits was at ambient conditions and sub-ambient condition in the refrigerator. the control had no treatment both in ambient and subambient conditions. they were monitored for weight changes, vitamin c content and deteriorations for 12 weeks in storage. waxing and sealed packaging in perforated polyethylene films with storage at ambient conditions and sealed packaging in perforated polyethylene films without wax treatment at sub-ambient conditions were effective techniques in the extension of shelf-high of the oranges. the vitamin c content of the fruits did not change significantly (57 to 59 mg/100 g) during the storage periods (opadokun and ubani, 1983; opadokun and akalusi, 1987). wrapping of oranges with different materials during storage `agege i` variety of sweet oranges was harvested from nihort ‘ibadan’ in early january. fruit was harvested and transported with care to avoid mechanical damage. fruits were cleaned with soft tissue to remove surface dirt, wrapped with materials in five treatment groups as follows: waxed paper, waxed envelopes, news paper, green banana leaves and control. the control was stored in an open plastic crate. all treatments were at ambient conditions of 25 ± 3°c and 68 ± 5% rh. fruits were monitored weekly for weight changes, and signs of deterioration, vitamin c, total sugars and titratable acidity contents. terminal samples were assessed organolleptically for acceptability with a hedonic scale of 1 to 5 for texture, peel colour and flavour. weight loss was highest in the newspaper wrapped and control fruits which also had dry rot, peel shrinkage and ring granulation at the end of 7 weeks of storage. the waxed paper wraps and banana leaves were effective in reducing weight loss and peel drying (ubani, 2006). storage of oranges in evaporative cooler oranges were stored in the ec (with capacity for 300 kg of fresh fruit) for 6 weeks with control fruits at ambient conditions. the temperature drop from the ambient was 3°c and 55% rh on the average. the rate of weight loss of the oranges in the ec was reduced. the control fruits lost weight rapidly resulting in fresh weight loss of 31.1% after 1 month in storage and actually lost appeal on the 14th day in storage. fruits in the ec had weight loss of 4.2% and were in good and acceptable after 6 weeks storage (babarinsa et al., 1986). storage of mangoes (mangifera indica l.) mangoes are climacteric fruits which are highly perishable and can only be kept under ambient conditions after harvest for a few days when nature and green. ripe fruits deteriorate rapidly and in the open markets attract flies because of the fruit acid responsible for the characteristic mango flavour. the mango season is very short usually april to june in nigeria when late maturing varieties are available. despite the brief seasonality of the fruits, there is much post harvest loss at the farm level due to difficulty in transportation of fresh fruits to distant or even local markets. fruit loss is also 7 table 2. nutrient contents in the pulp and peel of ’julie’ mangoes during storage. day sample/treatment vitamin c mg/100 g soluble sugars (mg/g) pulp peel p+p 0 (initial) unripe fruit 17.9 40.5 37.5 14.3 a 17.7 66.4 72.0 b 22.1 39.9 160.0 10 c 15.3 25.5 114.0 d 26.6 44.2 110.0 e 18.05 42.4 108.0 f 17.3 20.4 18.05 66.0 40 e 25.9 74.6 23.5 154.0 f 18.8 25.1 112.0 source: ubani on, nspri ‘ibadan’ (1989). much at the market level due to improper handling and storage of fruits before disposal. mangoes are also lost at the domestic level when fruits cannot be held at ambient conditions for more than one or two days after ripening depending on the variety. reduction of post harvest loss of mangoes in south-west nigeria has been approached using different storage methods. production of mango slices, lather and juice have been experimented on as techniques for post harvest loss reduction of mangoes in southwest nigeria (william et al., 2000; babatola and babalola, stakeholders’ meeting on the processing of mango to slices for local and export market, abuja, 2006). however, mango consumers have greater preference for the fresh fruits. storage of fresh mangoes in wrapping materials the mango variety ‘julie’ harvested mature, unripe, firm and wholesome from nihort orchard ibadan. they were prepared for storage in three treatment groups. fruits were cleaned with soft tissue paper to remove surface dirt and randomly selected for storage in: (a) woven basket lined and covered with green banana leaves (as practiced by farmers); (b) waxed-paperlined perforated cartons (made of fibreboards); (c) cartons with fruits individually wrapped in waxedpaper; (d) open plastic fruit crates (control). all treatment groups were kept at ambient conditions on the laboratory bench. a second batch of fresh fruits was stored in sub-ambient conditions (e) in perforated polyethylene films and (f) individually wrapped in waxedpaper kept in perforated high density polyethylene films. storage was in the fruit/vegetable compartment of the refrigerator at 12°c. the physicochemical parameters including weight, colour, firmness, vitamin c and soluble sugars were measured for the fruits before and after the storage period. fruits were examined every two days for ripening, weight changes and signs of deterioration. the storage of fruits at ambient conditions was 10 and 40 days in the sub-ambient condition. after storage, the fruits in the woven basket (a) ripened with unattractive yellow colour with green patches. weight loss was 30.5% of the initial weight and 33% spoilage was recorded for this treatment. the fruits in treatment (b) were fairly soft and not well ripened after storage. there was 67% spoilage and 64.2% loss in weight of fruits in this treatment. fruits in treatment (c) were very firm and ripened with an attractive yellow colour after storage. weight loss was 10.3 with 16% spoilage of fruits. the control fruits (d) did not ripe well. these had unattractive yellow colour with green patches. the fruits were shriveled and soft at the stem end. weight loss was 65% and spoilage 16% in this treatment. treatments e and f in sub-ambient conditions were stored for 40 days. there was no loss in weight or spoilage in fruits in these treatments after 10 days. the individually wrapped fruits in f were firm with attractive yellow colour at this time. there was progressive spoilage of the fruits beyond this period in treatments e and f till the experiment was terminated after 40 days storage. there was 80% loss of fruits in both treatments after 40 days. there was also pitting and softening of fruits. the fruits in the waxed paper inside the polythene film had 35.2% loss in weight and those in the polythene film only 7.4% weight loss. the biochemical parameter (nutrients) monitored are shown in table 2. fungi isolate from the spoiled fruits were all soil fungi namely geotrichum albidum cladosporium, fulvum, fusarium maniliformis, rhizopus 8 table 3. varietal differences in storage of fresh tomato fruits. percentage* storage losses after 7 weeks tomato variety pathological damage (%) weight loss (%) wholesome fruit (%) euromech 30 18 52 early stone 38 40 12 roma v. f. 46 32 22 piacenza 74 22 4 *mean values source: okoye et al. (1986). arrhizus. storage of fresh tomatoes tomatoes are consumed globally. in nigeria, the main production season is during the dry months of the year in the northern part of the country where the bulk of tomatoes consumed in nigeria are produced. in the southwest, the main production season is in the wet months between july and september. the fruits are soft berries and need special postharvest handing to minimize mechanical damage during transportation to the marketing outlets. at the retail and domestic levels, there is need to keep the fruits wholesome for consumers as the major problems after harvest are mould infestation of the damaged fruits. studies were carried out on the shelflife of fresh tomato varieties at ambient conditions. shelf-life of four tomato varieties at ambient conditions tomato varieties (euromech, roma v.f., early stone and piacenza), cultivated under irrigation at the institute of agricultural research farm samaru, kaduna state were harvested and transported to nspri ‘ibadan’ during the execution of the nationally coordinated project on fruits and vegetable (north↔south transportation of fruits and vegetables). fruits were transported in plastic craters, sorted and stored in the same containers on the laboratory bench at 23.5 to 24.8°c temperature and 66.8 to 78.8% rh ambient conditions. fruits were examined weekly for physical changes and analyzed initially and terminally for titratable acidity and total sugars. the tomatoes were stored for 7 weeks and the effect of varietal differences is shown on table 3. the biochemical parameters were similar and did not vary from the initial values after the storage period. total sugars wraped between 1.08 to 3.43 mg/100 g. acidity was 37.7 to 47.1 ml 0.1 n naoh/100 g. roma vf was more acidic than the other varieties. euromech had the longest shelf-life in good quality followed by roma v.f., early stone and piacenza varieties. the piacenza variety was unacceptable after the 3rd week of storage. after the 5th week in storage, the seeds of the early stone and roma varieties started sprouting internally even when the skin was still apparently wholesome. the above study showed that euromech has potentials for fresh storage over a long period even at ambient conditions (okoye et al., 1986). shelf-life of tomatoes (early stone variety) in the fruit and vegetable cooler basket fresh tomatoes were bought from a local fruit market in llorin metropolis during the harmattan season in february 2008. the fruits were sorted, washed and drained in a fruit crate. the fruits were then stored in the cooler baskets in a fruit/vegetable shade and the control fruit in open fruit crate in the shade. fruits were examined daily for signs of spoilage and monitored for weight changes. the average ambient temperature was 37 ± 2°c and 16% rh in the fruit/vegetable shade. the temperature in the cooler baskets was 27 ± 2°c. the fruits had storage life of only 9 days in the cooler baskets and there was 20.4% loss of fruits due to fungal infection. the wholesome fruits did not record weight loss during storage. the control fruits showed signs of skin shrinkage and weight loss during storage (ubani et al., 2008). storage of okra (abelmoschus esculentus (l.) moench) okra is widely cultivated and consumed in nigeria as well as other african countries. it is the second most important vegetable in the market especially in west africa after tomatoes. fruits are harvested at a very early stage of 3 to 4 days from flowering when they are still tender or when they are close to their maximum size and not yet fibrous (6 to 7 days) when the tips can snap. okra is rich in minerals especially calcium and also vitamins. it is used as a source of gums. shelf-life of fresh okra in cooler baskets freshly harvested okra fruits were purchased from a local 9 market in ilorin. the fruits were sorted and only wholesome fruits were stored in the cooler baskets. the cooler baskets were washed in clean water and allowed to drain and further dried on the inside with a clean napkin to reduce the okra contact with surface water. fruits were weighed in batches before storage. the control fruits were stored in open plastic crate and covered with fresh mango leaves to simulate the practice of the women traders. the storage containers were kept in a fruit/vegetable shade. the ambient temperature, relative humidity and the temperature in the storage containers were recorded daily. fruits were examined every two days for signs of deterioration, colour changes, wilting and rotting. spoilt fruits were sorted and removed from the batches and the weight changes of the fruits recorded. the fruits were stored for 14 days. the average ambient temperature was 32.2°c and 44% rh. the temperature of cooler baskets during storage of the fruits was 29.10°c. after the 7th day of storage, there was 37.5% loss in fruits due to spoilage and the control had 70% loss. at the 14th day, the fruits lost 75.9% of the original weight in the cooler basket and the whole some fruits remained tender. the control fruits lost 90% of their initial weight, changed colour with loss of the mucilage (ubani et al., 2010). storage of leaf vegetables leaf vegetables are highly perishable and under the hot tropical conditions cannot keep fresh for more than 24 h. they are essential part of the diet providing micronutrients and dietary fibre. the poor post-harvest handling and storage practices of leaf vegetables predispose them to huge losses after harvest. good postharvest practices will improve nutrition and economic returns for stakeholders. storage of fresh fluted pumpkin leaves (t. occidentalis hook. f.) fluted pumpkin (ugu) is cultivated in the south-eastern nigeria and is a popular leaf vegetable among many communities in south-eastern nigeria and particularly among the igbos. this vegetable is no longer restricted to south-eastern nigeria, but cultivated and eaten in all the agro-ecological zones of nigeria and has become a ‘national’ vegetable. fluted pumpkin leaves are recommended for malnourished children, pregnant women and adults with nutritional problems in nigerian hospitals. the shoots are harvested when they are up to 50 cm long. traditionally, the tips of first shoots after germination are cut and discarded for new shoots to form. shoots are pruned during harvesting. the plants are damaged when leaves are hand-picked and this hinders development of side shoots. farmers tie the harvested long shoots in bundles which are heaped under the shade of a tree or covered with green leaves before transportation to the markets. in the markets, the bundles are heaped in stalls and this often results in heat build-up in the bundles, leading to loss of chlorophyll and decay (ubani et al., 1997). fluted pumpkin cultivation and marketing is currently a thriving business in nigeria among farmers and traders. improved post-harvest handling and storage of this important vegetable is of importance to the stakeholders including the consumers. fluted pumpkin shoots were harvested from a farm at a village near asa dam in ilorin in the cool hours of the morning and transported in a polypropylene bag to nspri. the vines were cut to length of 55 cm to include the tip of the shoots with very young leaves. the shoots were portioned into 1.5 kg batched and stored in the cooler baskets conditions for storage of the vegetables. the control batch was stored in an open plastic crate. both the test and control samples were kept in a well ventilated fruit and vegetable shade. the ambient and storage structural temperature and relative humidity were monitored during the storage period. the vegetables were examined daily for signs of deterioration. the average ambient temperature was 29 ± 1°c and 64 ± 5% rh during the storage period. the storage atmosphere temperature was 27 ± 2°c and 70 ± 5% rh in the cooler baskets. the fluted pumpkin leaves and remained fresh for 6 days in the cooler baskets and there were no weight changes. the tips of the shoots having the very young leaves showed signs of pathological spoilage on the second day in the baskets and tips were removed from the shoots. the control batch recorded weight loss due to wilting of the leaves. on the 7 th day of storage, there was massive deterioration in terms of leaf decay and chlorophyll loss in the test samples. both the control and test samples had 100% loss on the 7th day in storage (ubani et al., 2010). conclusions the results of the studies on handling and storage of various nigerian indigenous fresh fruits and vegetables show that several factors contribute to shelf-life extension of these commodities in the nigerian environment. the nature of the crop and varietal differences is an important factor in storage of fruits and vegetables. this was evident in the studies with different tomato varieties produced in northern nigeria. the season and time of harvest also play an important role in storage of fruits and vegetables. garden eggs have very short shelf-life during the dry harmattan months in nigeria. fresh fluted pumpkin leaves can only be kept for 6 days beyond which physiological breakdown of tissues 10 lead to deterioration and total loss. these indigenous fruits and vegetables are prone to chill injury when kept at low temperatures. however, storage structures developed by the nigerian stored products research institute over the years such as the evaporative coolers and cooler baskets which could record some temperature drop from the ambient have been used in extending the shelf-life of fresh fruits and vegetables. packing (wrapping) materials also have shown promise in extending shelf life of nigerian indigenous fresh produce. adoption of these technologies will play a very vital role in postharvest loss reduction in fresh fruits and vegetables along the food chain, increase income and improve nutrition for all stakeholders. references babarinsa fa, nwangwa sc (1986). construction and assessment of two evaporative coolers for storage of fruits and vegetables. rep. nig. stored prod. res. inst. tech. report, 3: 45-61. bayindirli lg, sumnu g, kamadan k (1995). effects of semper fresh and jonfresh fruit coating on post storage quality of satsuma mandarins. j. food process. preserv., 19: 399-407. ben-yehoshua s (1979). individual seal packaging of fruits in plastic film. a new postharvest technique. hortsci., 20: 32-37. jagtiani dj (1980). fruit preservation. vikas, delhi, india, pp. 325-328. lawande ke, chavan jk (1998). egg plant: in: salunkle dk, kaddan ss (eds) handbook of vegetable science and technology production, composition, storage and processing. marcel dekker inc. new york, pp. 332-343. okonkwo eu, ubani on, nwaubani si, otitodun og, omoju pk (2009). the potentials in the indigenous technology for the production of mature unripe pawpaw (carica papaya l.) fruit slices in nigeria. j. res. agric., 6(4): 31-34. okoye wi, onwuzulu oc, ubani on (1986). storability of four tomato and five onion varieties. rep. nig. stored prod. res. inst. tech. report, 4: 57-61. opadokun js, akalusi do (1987). moisture decay and colour changes in individual sealed packaged oranges in comparison with waxed oranges during ambient storage. nig. stored prod. res. inst. ann. tech. report, 9: 69-72. opadokun js, ubani on (1983). shelf-life of chemically treated agege sweet oranges. nig. stored prod. res. inst. ann. tech. report, 9: 6973. ubani on (1989). storage of fresh mango fruits in different wrapping materials. paper presented in nigerian stored products research inhouse review meeting held at nspri headquarters, ilorin, kwara state, nigeria, 17th-21st april, 1989. ubani on (1991). shelf-life of fresh pawpaw fruits in evaporative coolers. paper presented in nigerian stored products research inhouse review meeting held at nspri ibadan, oyo state, nigeria, 1st-3rd july, 1991. ubani on (2006). packaging materials effect on quality of oranges (citrus sinensis osbeck) during ambient conditions storage in ibadan south-west, nigeria. j. agric. res. pol., 1: 71-73. ubani on, okonkwo ego u, ade a (2010). shelf-life of four tomato varieties at ambient conditions. paper presented in nigerian stored products research in-house review meeting held at nspri headquarters, ilorin, kwara state, nigeria, 22nd-24th june, 2010. ubani on, okonkwo ego u, ade a, saanu f, atibioke o, nwaehujor iu, adeiza a, omoju pk, umama jt, ogunjirin co (2008). storage of indigenous fruits and vegetables in nspri’s novel foam insulated fruit and vegetable basket for garden egg, mangoes, okra, telfaria occidentalis,amaranthus and tomatoes. paper presented in nigerian stored products research in-house review meeting held at nspri headquarters, ilorin, kwara state, nigeria, 26th-28th august, 2008. ubani on, pessu po, adindu mn (1997). report on thematic survey on handling and preservation of fresh fruits and vegetables in southeastern states of nigeria. ubani on, suleiman a (2008). shelf-life of fresh garden eggs (solanum aethiopicum) in two types of vegetable baskets. int. j. biosci., 3: 9193. williams jo, babarinsa fa, bello i (2000). dried mango chips, ready to eat for all seasons. postharv. news, 1: 2. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 7 (12), pp. 001-007, december, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper essential elements content in core vegetables grown and consumed in ghana by instrumental neutron activation analysis d. k. adotey1,2, y. serfor-armah1,2, j. r. fianko1,2 and p.o. yeboah 1 1 department of nuclear sciences and applications, graduate school of nuclear and allied sciences, university of ghana, p.o. box ae 1, atomic-accra, ghana. 2 department of chemistry; national nuclear research institute; ghana atomic energy commission; p.o. box lg 80, legon-accra, ghana. accepted 10 june 2019 food is the primary source of essential nutrients for man. vegetables are essential part of the ghanaian diet; therefore the nutritional status of vegetables is of importance. vegetables that constitute an esse-ntial part of the ghanaian diet have been analyzed for essential elements content (ca, mg, k, co, br, mn, and na) using instrumental neutron activation analysis (inaa). the purpose of the study was to design a nutrient database of the core vegetables grown and consumed in ghana and in addition to ascertain the content of the essential elements in the vegetables. the accuracy of the method was verified by analysis of a compositionally appropriate reference material, iaea -359 (cabbage). the results of the study revealed the presence of br, ca, co, k, mg, mn, and na in all the five vegetables studied. ca, mg, and k were present in the g/kg range in all five vegetables. the content of na, mn, co and br was in the mg/kg range in all five vegetables investigated. key words: vegetables, instrumental neutron activation analysis, elemental concentration, ghana. introduction essential elements reach the human body through foods, ambient air, drinks, and occupational exposures. human growth and metabolism rely on a balanced diet, which is composed of proteins, lipids, carbohydrates, vitamins and inorganic micronutrients. although constituting a small fraction of the whole diet, inorganic micronutrients play an important role in various metabolic processes and their deficiency or excess may disturb the normal biochemical function of the body (akhter et al., 2004). the essential elements in the body function as constituents of bones and teeth, as soluble salts which help to control the com-position of body fluids and cells and serve as essential adjuncts to many enzymes and other functional proteins (dashti et al., 2004). the elements considered essential are the major elements (sodium, magnesium, phosphorus, chloride, potassium, and calcium) and the minor elements (chromium, manganese, iron, cobalt, copper, zinc, selenium, molybdenum and iodine). in addition there are the newer trace *corresponding author. e-mail: kadotey@yahoo.com. elements, which are possibly essential; these are lithium, boron, fluorine, silicon, vanadium, nickel arsenic tin and lead (crews, 1998). the daily allowances or maximum tolerable intakes of trace elements entering humans through foodstuffs cre-ate the need for accurate and reliable determination of microgram and nanogram quantities of these elements in various foodstuffs (dermelj et al., 1996). core foods are the foods most commonly consumed and consumed in the largest quantities by a population (pennington, 2000). many people do not rely on food to supply the trace metal nutrients they consume. this is because they believe that much of the food available in the marketplace has been depleted of nutrients. others do so because they are convinced that the maintenance of good health and the need to ward off diseases require a higher intake of nutrients than can be met by consuming ordinary food. food supplements are consumed to make up for this perceived shortfall. this state of affairs calls for the analysis of foodstuffs for their elemental concentrations, data acquired from such a study will help allay the fears of the public. the concentration of essential elements in foodstuffs of one region may vary from the other since food supplies are affected by various agricultural practices, type of soil, type of fertilizer and chemicals used, type of pesticides and herbicides sprayed. besides these factors, intake of inorganic elements by the populace of any region may also depend on the geographical location of the area, climatic conditions, eating habits and socio-economic status (waheed et al., 2002). two methods are generally considered applicable for estimating dietary exposure to essential trace elements: one is a biological monitoring programme that measures substances in body fluids and or tissue; the other is food monitoring programme (coancher and mes, 1993). three different approaches can be applied to monitor exposure to essential micronutrient through food: selective analysis of individual foodstuffs, duplicate portion study and total diet study, which is also known as the market basket study (who, 1985; nasreddine and parent-massin, 2002). though selective analysis of individual foodstuffs is labour intensive, it offers a number of advantages over the other methods. it prevents the 'dilution effect', which is the lack of detection of an analyte that may occur when the primary food source is mixed with other foods and analyzed as a food group composite. the analysis of individual core foods allows identification of the source of a nutrient. if a nutient is detected in a food group, it is not possible to determine which food contain the component without additional testing. in addition, the selective analysis of core foods allows for the development of a food composition database derived from analytical data. data from analyses are more accurate than those taken from food composition tables where the values may be outdated or based on mere estimation (pennington, 2000) the essential elements in vegetables are present in trace and ultra-trace quantities. hence, an analytical technique with sufficient sensitivity is required for the accurate determination of the essential elements in food samples. the major techniques employed are flame atomic absorption spectrometry (faas), graphite furnace atomic absorption spectrometry (gfaas), inductively coupled plasma atomic emission spectrometry (icp-aes), and inductively coupled plasma mass spectrometry (icp-ms). besides those mentioned above, other techniques such as differential pulse cathode stripping voltamperometry (dpcsv) and neutron activation analysis (naa) have also been shown as excellent tools for trace and ultratrace analysis (inam and somer, 2000; szefer and nriagu, 2007). neutron activation analysis is a less frequently used technique in the determination of essential elements in food samples because of the necessity of accessing a nuclear reactor. despite this, naa possesses a number of advantages and possibilities. among the advantages is lack of necessity of chemical destruction (in the case of instrumental neutron activation analysis), appropriate accuracy, and possibility of simultaneous quantification of many elements in a small amount of sample (žukowska and biziuk, 2008). vegetable is an important and vital part of ghanaian diet. in this work, five vegetables from various regions in ghana have been studied for seven essential trace elements using multi-element, non-destructive and highly sensitive technique of instrumental neutron activation analysis (inaa). the selection of vegetables was based on importance in the ghanaian diet and high consumption among the entire ghanaian population. data acquired from this study, will help create adequate and reliable nutritional information, as well as create awareness on the nutritional value of these vegetables materials and methods sample preparation five core vegetables were randomly purchased from large municipal markets in the greater accra region of ghana. the quantity of vegetables purchased was normally a few kilograms from which sub-samples for analysis were taken. the vegetables purchased were: tomato (lycopersicom esculentum), pepper (aframomum latifolium), garden eggs (limnanthes sulphurea), onion (allium cepa l.), carrot (daucus carota l.). samples were placed into clean polyethylene containers and transported to the laboratory. at the laboratory, samples were thoroughly washed with ordinary water and then with doubly distilled water. from each market about ten sub-samples of each vegetable were selected and sliced into pieces using a carbon steel knife on a plastic slicing board. the sliced sub-samples were homogenized by thorough mixing. in the case of onions they were first peeled before been sliced into pieces. each sub-sample was further homogenized in a home-styled blender with stainless steel blades. aliquots of each homogenized sample were frozen at -20°c and lyophilized (christ gamma 1-16) at 30°c and 0.370 mbar. the lyophilized samples were milled in the vibratory disc mill (retsch rs 100). the lyophilized homogenates obtained were stored in closed polyethylene bottles with screw caps and kept at 20°c until analysis in order to prevent degradation of the sample. drying by lyophilization was used because it ensures that the initial sample texture is preserved and in addition facilitates subsequent milling of samples (hoenig, 2001). elemental analysis preparation of samples for neutron activation aliquots (about 200.00 mg) of the lyophilized homogenate were weighed into clean polyethylene foils, wrapped with forceps and the foil heat-sealed. six replicates of each sample were prepared. three replicate samples of compositionally appropriate reference material, international atomic energy agency, iaea-359 (cabbage) were also prepared. the reference material was used as a comparator standard for gamma spectrum evaluation using the relative method of standardization for neutron activation analysis; and in addition to check the accuracy of the analytical method used. neutron activation of samples for medium-lived and long-lived radioisotopes the reference material was ‘sandwiched’ between two aliquots of the same lyophilized homogenate and stacked together as close as possible. this was to ensure that samples and standards were table 1. nuclear data used for determination of elemental concentrations (filby et al., 1970). element reaction half-life energy (kev) k 41 k (n, ) 42 k 12.36h 1524.7 na 23 na (n, ) 24 na 15.02h 1368.6; 2754.1 br 81 br (n, ) 82 br 35.3h 554.3; 776.5 mg 26 mg (n, ) 27 mg 9.45min 1014.4 mn 55 mn (n, ) 56 mn 2.58h 846.7; 1810.7; 2112 co 51 co (n, ) 52 co 3.76min 1434.1 ca 48 ca (n, ) 49 ca 8.7min 3084.4 cl 37 cl (n, ) 38 cl 37.3min 1642.4; 2167.5 activated under identical and same conditions as possible (any variation can remarkably affect the precision). the stacked samples and standard were then placed in the same polyethylene irradiation vial and irradiated for 1 and 6 h with thermal neutrons at a neutron flux of 5x 10¹¹ neutrons cm -2 s -1 , at the inner irradiation sites of the ghana research reactor -1 facility, situated at the ghana atomic energy commission. the ghana research reactor-1 is a 30 kw tank-inpool miniature neutron source reactor (china institute of atomic energy). it uses 90.2% enriched uranium (u235)– aluminium alloy as fuel. it is cooled and moderated with light water and beryllium acts as reflectors (akaho and nyarko, 2002). measurement of induced activity in sample and standard followed immediately they were ejected from the reactor. at the end of the irradiation, samples and standards were allowed to ‘cool’ to enable nonanalyte radioisotopes induced together with radioisotopes of interest and which half -life are shorter than that of the radioisotopes of interest to decay. in addition, to allow the activities induced in the samples to reach acceptable levels for handling (human health). neutron activation of samples for short-lived radioisotopes short-lived radioisotopes were induced in the samples and standards by 60 s irradiation with thermal neutrons in the pneumatic sample transfer facility of the research reactor. samples and standards were stacked together as close as possible and placed in the same polyethylene irradiation capsule. measurement of induced activity in sample and standard followed immediately was ejected from the reactor. samples were measured first followed by standards. -activity measurement of samples and standards analysis of induced radioisotopes of interest was performed on a pc-based gammaray spectroscopy system. the spectroscopy system consists of a canberra high purity germanium (hpge) ntype coaxial detector (model gr 2518-7500sl) with a resolution (fwhm) of 1.8 kev relative to the 1332.5 kev -energy line of 60 co; a relative efficiency of 25%; and a peak-to-compton ratio of 55. samples and standards were placed on the high purity germanium (hpge) -ray detectors and the -activity of the induced radioisotopes measured at the same position and distance from detector. the measurement time for short-lived, medium-lived, and long-lived radioisotopes depended on the activity of the induced radionuclide. samples were measured first followed by standards. a plexiglass source support was mounted on the detector in order to ensure easy and reproducible source positioning. evaluation of peak area of -spectrum the peak area of the -spectrum for samples and standards were evaluated using the hpge semiconductor detector spectroscopy accumulation software, ortec maestro-32. the areas under the peaks were integrated and converted into concentration using the relative method of standardization for neutron activation analysis. the nuclear reactions, halflives and -energy of the radionuclides have been summarized in table 1. results and discussion five core vegetables consisting of tomato, garden egg, onion, pepper and carrot were analyzed for essential elements of nutritional significance using instrumental neutron activation analysis (inaa). the concentrations were calculated on dry weight basis and each concentration is an average of six individual determinations. the irradiation scheme employed prevented any serious elemental interference. elemental concentrations in vegetables the results for the analysis of the essential elements in the five vegetables, and the ranges are presented on tables 2 and 3. calcium content in the vegetables the concentration of calcium in tomato was found to vary from 0.002 to 0.24 g/kg with a mean of 0.17 g/kg. the concentration of calcium in garden egg varied from 0.01 to 0.16 g/kg with a mean of 0.10 g/kg. the content of calcium in onion was found to be 0.60 g/kg and varied from 0.44 to 0.88 g/kg. for pepper, the calcium content was in the range of 0.09 to 0.84 with a mean of 0.26 g/kg. calcium in carrots was found to vary from 0.26 to 0.53 g/kg with a mean of 0.37 g/kg. the results from the study show high calcium content in onion (0.60 g kg -1 ); the mean content in garden egg was the lowest (0.10 g/kg). however, some of the concentrations of calcium in garden eggs overlapped with the contents in other vegetables. carrots and pepper contains appreciable levels of calcium. even though plant foods like vegetables can be excellent sources of several minerals, the mineral content of plant food can vary dramatically depending on the minerals in the soil where the plants are cultivated. the maturity level of a vegetable can also influence its mineral content. these factors could have accounted for the wide variation in the content of calcium in the vegetables. other soil factors which may have influenced the variations are ph and salinity. calcium acts as the main structural element of bones and teeth in humans. calcium is also essential for the formation of fibrinogen which is vital for blood clotting. low ca intake causes deficiency in the body leading to table 2. na, br, co and mn contents (mg kg -1 ) in the vegetables. content of essential elements in vegetables (mg kg -1 ) mineral tomato garden egg onion pepper carrot na 323.8± 22.5 46.8 ± 1.9 379.9 ± 9.1 57.3 ± 3.3 981.1 ± 26.2 (44.20 – 1400) (24.49 83.30) (53.70 – 715.40) (25.80 92.60) (104 – 2500) mn 37.02 ± 2.58 21.50 ± 1.34 90.14 ± 4.01 19.07 ± 1.73 36.12 ± 2.55 (27.23 65.10) (12.50 27.32) (43.74 169.70) (16.10 22.50) (29.63 44.14) br 23.47 ± 1.91 36.78 ± 2.06 89.65 ± 4.14 14.89 ± 1.34 17.85 ± 2.21 (15.26 37-93) (22.40 47.50) (70.20 142.60) (7.56 29.10) (12.97 29.70) co 5.36 ± 0.28 5.65 ± 0.35 4.96 ± 0.36 4.36 ± 0.30 7.19 ± 0.37 (5.08 6.30) (4.68 7.00) (4.32 6.43) (3.62 6.10) (6.40 8.10) data are expressed as mean ± standard deviation on a dry weight basis (range). table 3. ca, mg, and k contents (g kg -1 ) in the vegetables. essential elements content (g kg -1 ) in the vegetables mineral tomato garden egg onion pepper carrot ca 0.17 ± 0.01 0.10 ± 0.01 0.60 ± 0.02 0.26 ± 0.01 0.37 ± 0.01 (0.01 0.16) (0.44 0.88) (0.09 0.84) (0.26 0.53) (0.002 -0.24) mg 0.16 ± 0.02 0.15 ± 0.01 0.59 ± 0.03 0.13 ± 0.01 0.16 ± 0.02 (0.14 0.16) (0.53 0.77) (0.09 0.21) (0.04 0.30) (0.10 0.25) k 2.84 ± 0.06 3.79 ± 0.07 2.25 ± 0.07 1.22 ± 0.03 1.75 ± 0.05 (1.44 10.70) (1.74 2.94) (0.75 2.01) (0.74 3.07) (0.28 4.29) data are expressed as mean ± standard deviation on a dry weight basis (range). osteoporosis. calcium deficiency may also cause rickets in children. the functions of calcium in the human system demand sufficient intake of this mineral. the study revealed that onions, pepper, carrots grown in ghana contain appreciably high content of calcium. though the calcium content in tomato and garden egg are relatively low, these vegetables are good sources of calcium. analysis of induced radioisotopes of interest was performed on a pc-based gamma-ray spectroscopy system. the spectroscopy system consists of a canberra high purity germanium (hpge) n-type coaxial detector (model gr 25187500sl) with a resolution (fwhm) of 1.8 kev relative to the 1332.5 kev -energy line of 60 co; a relative efficiency of 25%; and a peak-to-compton ratio of 55. samples and standards were placed on the high purity germanium (hpge) -ray detectors and the -activity of the induced radioisotopes measured at the same position and distance from detector. the measurement time for short-lived, medium-lived, and long-lived radioisotopes depended on the activity of the induced radionuclide. samples were measured first followed by standards. a plexiglass source support was mounted on the detector in order to ensure easy and reproducible source positioning. evaluation of peak area of -spectrum the peak area of the -spectrum for samples and standards were evaluated using the hpge semiconductor detector -spectroscopy accumulation software, ortec maestro-32. the areas under the peaks were integrated and converted into concentration using the relative method of standardization for neutron activation analysis. the nuclear reactions, half-lives and -energy of the radionuclides have been summarized in table 1. results and discussion five core vegetables consisting of tomato, garden egg, onion, pepper and carrot were analyzed for essential elements of nutritional significance using instrumental neutron activation analysis (inaa). the concentrations were calculated on dry weight basis and each concentration is an average of six individual determinations. the irradiation scheme employed prevented any serious elemental interference. elemental concentrations in vegetables the results for the analysis of the essential elements in the five vegetables, and the ranges are presented on tables 2 and 3. calcium content in the vegetables the concentration of calcium in tomato was found to vary from 0.002 to 0.24 g/kg with a mean of 0.17 g/kg. the concentration of calcium in garden egg varied from 0.01 to 0.16 g/kg with a mean of 0.10 g/kg. the content of calcium in onion was found to be 0.60 g/kg and varied from 0.44 to 0.88 g/kg. for pepper, the calcium content was in the range of 0.09 to 0.84 with a mean of 0.26 g/kg. calcium in carrots was found to vary from 0.26 to 0.53 g/kg with a mean of 0.37 g/kg. the results from the study show high calcium content in onion (0.60 g kg -1 ); the mean content in garden egg was the lowest (0.10 g/kg). however, some of the concentrations of calcium in garden eggs overlapped with the contents in other vegetables. carrots and pepper contains appreciable levels of calcium. even though plant foods like vegetables can be excellent sources of several minerals, the mineral content of plant food can vary dramatically depending on the minerals in the soil where the plants are cultivated. the maturity level of a vegetable can also influence its mineral content. these factors could have accounted for the wide variation in the content of calcium in the vegetables. other soil factors which may have influenced the variations are ph and salinity. calcium acts as the main structural element of bones and teeth in humans. calcium is also essential for the formation of fibrinogen which is vital for blood clotting. low ca intake causes deficiency in the body leading to osteoporosis. calcium deficiency may also cause rickets in children. the functions of calcium in the human system demand sufficient intake of this mineral. the study revealed that onions, pepper, carrots grown in ghana contain appreciably high content of calcium. though the calcium content in tomato and garden egg are relatively low, these vegetables are good sources of calcium. magnesium content in the vegetables magnesium is an essential element known to be vital for the activity of a number of human enzymes particularly those that are involved with the oxidative phosphorylation. its chief function in the body includes bone mineralization, building of proteins, and transmission of nerve impulse and maintenance of teeth. magnesium deficiency results in renal failure, acute diarrhea and protein–caloric malnutrition. these deficiencies may then cause irritability, muscular tremor, hallucination and depression. mean magnesium contents in the vegetables were found to be nearly the same: 0.16 g/kg in tomato, 0.15 g/kg in garden egg, 0.13 g/kg in pepper and 0.16 g/kg in carrots. the content of 0.59 g/kg found in onion varied sharply from the content in the other vegetables. the similar content of magnesium in tomato, pepper, garden egg and carrot could be that these vegetables were grown in the same geographical area and therefore there is no marked variation in the mineral composition of the soil in which the vegetables were grown. the high content of magnesium in onion may be due to high uptake of magnesium from the soil by onion, assuming it was also grown in the same geographical area as the other vegetables were grown. the onion might also have been grown in a different geographical area with high magnesium content in the soil. it was not possible to establish where the vegetables were grown as the traders were either not certain about which geographical area the vegetables were grown, or gave conflicting prediction as to the exact geographical areas the vegetables were grown. in addition, the conditions which the vegetables were subjected to before being carted to the marketing centers could not be established. these factors may have accounted for the differences in levels of the essential elements in the vegetables. sodium content in the vegetables sodium content in the vegetables from the markets were in the ranges of 44.20–1400 for tomato, 24.49-83.30 for garden egg, 53.70–715.40 for onion, 25.80-92.60 for pepper and 104 – 2500 for carrots. all values are in mg kg -1 . there was no significant variation in the sodium contents in garden egg and onion. this could be attributed to the sodium content in the soils in which the garden egg and onion were grown, even though they might not have come from the same geographical location. variation in the sodium content in onion and tomato were significant. the variations in the content of sodium in onion, carrots and tomato may be attributed to factors such as the preferential uptake of sodium by these vegetables, which can also be influenced by the age of the plant. ambient climatic conditions and mineral composition of the soil in which the vegetables were grown as well as the type of fertilizer and pesticides sprayed might have influenced the concentration of the elements obtained. na is a major physiological element and a primary extra – cellular cation in humans. na together with cl and k are electrolytes that maintain normal fluid balance inside and outside cells and a proper balance of acid and bases in the body. deficiency of this element may result in muscle cramp and hypertension. potassium content in the vegetables potassium concentrations in all the five vegetables studied were very high. potassium is a macronutrient and table 4. concentrations ( g g -1 ) of some analyzed elements in certified reference material. element present study a reported precision (%) accuracy (%) ca 19200 ± 265 (5) 18500 ± 510 1.38 3.78 k 31600 ± 344 (4) 32500 ± 690 1.09 -2.77 mg 2130 ± 17 (6) 2160 ± 50 0.80 -1.39 mn 32.4 ± 0.3 (6) 31.9 ± 0.6 0.93 1.57 na 590 ±9(4) 580 ± 13 1.53 1.72 a average ± standard deviation (number of determinations). a major intracellular cation in the human body. its deficiency causes nerve irritability, cardiac and mental disorder, muscular weakness and paralysis. potassium also facilitates the transmission of nerve impulses. the concentration of potassium in tomato was found to vary from 0.28 to 4.29 g/kg with a mean of 2.84 g/kg. the concentration of potassium in garden egg varied from 1.44 to 10.70 g/kg with a mean of 3.79 g/kg. the content of potassium in onion was found to be 2.25 g/kg and varied from 1.74 to 2.94 g/kg. for pepper, the potassium content was in the range of 0.75 to 2.01 g/kg with a mean of 1.22 g/kg. potassium in carrots was found to vary from 0.74 to 3.07 g/kg with a mean of 1.75 g/kg. the results from the study show high potassium content in garden egg (3.79 g kg -1 ); the mean content in pepper was the lowest (1.22 g/kg). however, some of the contents of potassium in garden eggs overlapped with the contents in other vegetables. the marked variation in the concentration of potassium in the vegetables might be due to the levels of potassium in the soils in which the vegetables were cultivated. in addition, the nutrient levels in soils vary geographically and the vegetables might have been brought to the market centre from different geographical locations. the high potassium content in garden egg may be attributed to the preferential uptake of potassium from the soil by the garden egg plant. in addition, the content may have been influenced by genetic diversity. amendment of the soil by the application of top dressings and agrochemicals could also have had a significant influence on the potassium content in the soil, thereby affecting the content in the vegetables. manganese content in the vegetables manganese content in the vegetables from the markets was in the ranges of 27.23-65.10 for tomato, 12.50-27.32 for garden egg, 43.74-169.70 for onion, 16.10-22.50 for pepper, and 29.6344.14 for carrots. all values are in mg kg -1 . there was no significant variation in the manganese content in the vegetables. the content of manganese recorded may have been influenced by the insignificant variation in the content of manganese in the soils in which the vegetables were grown. the uptake of manganese from the soil by these vegetables could have been the same or nearly the same. manganese is a key cofactor in reactions of key importance in the human body. it is involved in energy metabolism and urea formation. manganese is a component of the antioxidant enzyme superoxide dismutase. bromine and cobalt content in the vegetables the results for the cobalt content in the vegetables did not show any significant variation. cobalt content in the vegetables from the markets were in the ranges of 5.086.30 for tomato, 4.68-7.00 for garden egg, 4.32-6.43 for onion, 3.62-6.10 for pepper, and 6.40-8.10 for carrots. all values are in mg kg -1 . this may have been influenced by the insignificant variation in the cobalt content in the soil, irrespective of the geographical area. the content of bromine in the vegetables did not also show any significant variation. however, the bromine content in onion was slightly higher than the content in other vegetables. this may be due to preferential uptake of bromine from the soil by the plant. bromine in onion varied from 70.20 to 142.60 mg/kg, with a mean of 89.65 mg/kg. the bromine content in tomato was found to vary from 15.26 to 37.93 mg/kg; for garden egg, it varied from 22.40 to 47.50 mg/kg; in pepper it varied from 7.56 to 29.10 mg/kg; and a variation of 12.97 to 29.70 mg/kg in carrot. the reason for the insignificant variation may also be due minimal variation in the bromine content in the soil irrespective of the geographical area. quality assessment the precision and accuracy of the method was evaluated by repeated analyses of a compositionally appropriate certified reference material, iaea -359 (cabbage) under the same experimental conditions. the detailed results of the assessment are presented in table 4. the precision of the measurement, in terms of relative standard deviation is 1.38, 1.09, 0.80, 0.93, and 1.53% for ca, k, mg, mn, and na, respectively. the accuracy of the measurements, in terms of the relative deviation from the iaea certified values are 3.78% for ca; -2.77% for k; -1.39% for mg; 1.57% for mn; and, 1.72% for na. there is generally good agreement between the iaea certified values and the values obtained in the present study. the equations used for the calculation of precision and accuracy are: rsd  ( ) *100 (1) x where, rsd is the relative standard deviation; is the standard deviation; and, is the arithmetic mean of 8 x independent determinations. rd  (x  xc ) xc (2) where, rd is the relative deviation; x is the measured value in the present study and xc is the nist certified value. comparison of essential trace element content with literature data the mean calcium content in the ghanaian vegetables was remarkably high ranging from 0.10 g/kg (limnanthes sulphurea) to 0.60 g/kg (allium cepa l). comparison of these values with literature data for traditional south african leafy vegetables shows that the calcium content in the leafy vegetables were significantly higher, >1000 mg/100 g for all species of south african leafy vegetables analyzed except galinsoga parviflora (162 mg/100 g) and ceratotheca triloba (705 mg/100 g) (odhav et al., 2007). literature data for south african leafy vegetables show substantial quantities of sodium, 1460 mg/100 g (oxygonum sinuatum). centella asiatica contained the lowest amount of 16 mg/100 g (odhav et al., 2007). the sodium content in the ghanaian vegetables was remarkably low, ranging from 46.8 mg/kg (limnanthes sulphurea) to 981.1 mg/kg (daucus carota l). the magnesium content in the ghanaian vegetables studied ranges from 0.13 g/kg (aframomum latifolium) to 0.59 g/kg (allium cepa l). the values from this study were slightly lower than literature values for the traditional south african leafy vegetables, 193 mg/100 g (wahlenbergia. undulata) to 1409 mg/100 g (justicia flava) (odhav et al., 2007). manganese content in the ghanaian vegetables ranges from 19.07 mg/kg (aframomum latifolium) to 90.14 mg/kg (allium cepa l). comparison of these values with literature data for traditional south african leafy vegetables shows that the manganese content in the leafy vegetables were significantly higher, ranging from 2 mg/100 g (physalis. viscosa) to 82 mg/100 g (amaranthus. dubius) (odhav et al., 2007). conclusion vegetables that constitute an essential part of ghanaian diet were analyzed for elements of nutritional significance using inaa. generally, the results established that ca, mg, and k were present in high concentration in all the five vegetables studied, while br, co, mn, and na were at appreciably levels. the results of this study indicate that the vegetables studied are well endowed with essential nutrients required for human consumption. the data should be used to promote the nutritional values of these vegetables among the ghanaian population. in addition, the data generated will be used to build a nutrient database for vegetables grown and consumed in ghana. from the study, tomatoes, garden eggs, onions, pepper and carrots are good sources of k, mg, ca, na, br, co and mn. references akaho ehk, nyarko 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waheed s, zaidi jh, ahmad s, saleem m (2002). instrumental neutron activation analysis of 23 individual food articles from a high altitude region part 1. determination of 14 essential minor and trace elements. j. radioanaly. nuclear chem. 254: 597-605. world health organization (who) (1985). guidelines for the study of dietary intake of chemical contents. who offset publication. 87:2030. žukowska j, biziuk m (2008). methodological evaluation of method for dietary heavy metal intake. j. food sci. 72(2): r21-r29. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (5), pp. 001-007, may, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper gravitropism of loblolly pine (pinus taeda ) radicles after chemical sterilization of seeds dexian he1 and david b. south2 1college of agriculture, henan agricultural university, zhengzhou, henan 450002, china; 2 school of forestry and wildlife sciences and alabama agricultural experiment station, auburn university, al 36849 5418, u.s.a. tel: 334-844 1022, fax:334 – 844 1084, accepted 10 april, 2020 certain types of chemicals can affect the gravitropism of roots. in a laboratory study, intact loblolly pine (pinus taeda l.) radicles (emerged from h2o2treated seeds) exhibited positive gravitropism 8 h after horizontal placement in sterile conditions. the growth angle decreased from almost horizontal (85) to 21 within one week after treatment (90 is horizontal and 0 is vertical). when seeds were treated with hgcl2, radicles under sterile growing conditions expressed gravitropism 6.9 h after horizontal alignment. growth angle changed from 91 to 64 over a 10-day period. cubic and quadratic functions were used to model growth angle as a function of time. under similar experimental conditions, radicles from hgcl2-treated seeds showed a greater degree of gravitropism than those from h2o2-treated seeds (as indicated by the growth angle). these results indicate that the gravitropism can occur in sterile environments and that the type of chemicals used to sterilize seeds might affect the rate of geotropic response. key words: loblolly pine, radicle, geotropic growth, microorganism, h2o2, hgcl2, seed sterilization. introduction certain chemicals can affect root growth of plants and some will alter root gravitropism. chlorsulfuron and metsulfuron-methyl caused severe ultrastructural alterations and injuries of root caps in pea (pisum sativum l.) and maize (zea mays l.), and affected root gravitropism (fayez et al., 1995). tetrazolium altered gravitropism of primary roots of barley (hordeum vulgare l.), oat (avena sativa l.), rape (brassica napus l.), sheep fescue (festuca ovina l.), and wheat (triticum aestivum l.) (steiner and fuchs, 1987). hydrogen peroxide (h2o2) and mercuric chloride (hgcl2) are sometimes used to decontaminate the surface of seeds (gyimah, 1977; somade, 1998; sharma et al., 2004), but it is not known if these chemicals affect gravitropism of conifer radicles. soaking eucalyptus seeds in h2o2 can sometimes damage radicles and can result in abnormal germination *corresponding author. e-mail: southdb@auburn.edu. abbreviations: h= hour; min= minute; s=standard deviation (donald and lundquist, 1988). when treating pine seeds, concentrations of h2o2 typically ranged from 3% to 30% and exposure times varied from 0.25 to 48 h (barnett, 1976; mason and arsdel, 1978; penafiel, 1982; graham and linderman, 1983). recommended treatment times for loblolly, longleaf (pinus palustris mill.), shortleaf (pinus echinata mill.), and slash (pinus elliottii engelm.) pine varied by species (0.5, 1, 1, 0.25, and 1 h, respectively)(barnett, 1976). hgcl2 has been used to treat a wide range of seeds including groundnut (arachis hypogaea l.), maize, rice (oryza sativa l.), sorghum [sorghum bicolor (l.) moench.], soybean [glycine max (l.) merr.], sunflower (helianthus annuus l.), and wheat (sweet and bolton, 1979) . gilmour and vanner (1973) pointed out that hgcl2 could be used as a fungicide on monterey pine (pinus radiata d. don) seedlings. however, only a few research-ers have sterilized pine seeds with hgcl2 (tang, 2000; han et al., 2003). in one study, some loblolly pine radicles (emerged from seeds that had been treated with hgcl2) did not exhibit gravitropism (personal communication: table1 . experimental conditions involving gravitopism of loblolly pine radicles in sterilized petri dishes in 2001. test chemical temperature conditions start date end date i h2o2 24.6c, measured at 08:00 under a fluorescent lamp on the lab table jun 18 jul 16 and 17:00 ii h2o2 30c, and then 25.5c, incubator (constant light) first, and then under jul 9 jul 31 measured at 17:00 a fluorescent lamp on the lab table iii hgcl2 30c incubator (constant light) aug 22 sep 19 iv h2o2 30c, and then 22.7c, incubator (constant light) first, and then under aug 31 oct 11 hgcl2 measured at 17:00 a fluorescent lamp on the lab table v h2o2 27c incubator (constant light) sep 18 oct 20 hgcl2 dr. walt kelley, auburn university). to date, there are no reports demonstrating that gravitropism of pine radicals are affected by chemical sterilization treatments. therefore, the objectives of this study were: (1) to test the null hypothesis that after chemical treatment, young radicles of loblolly pine do not express gravitropism under sterile conditions, and (2) the expression of gravitropism is the same for both hgcl2 and h2o2. materials and methods all seeds were sterilized with either h2o2 or hgcl2 (table 1). unsterilized seeds were not used for comparison since growth of microorganisms in dextrose agar can hinder root orientation and can interfere with measurement of the growth angle. trials were carried out in the southern forest nursery management cooperative lab and forest biology lab, school of forestry and wildlife sciences, auburn university. experiment i loblolly pine seeds were sterilized in 9% h2o2 for 50-60 min, and rinsed twice for 5 min in sterile distilled water. six seeds were placed on each plastic dish (10015-mm)(fisher scientific co., llc, suwanee, ga 30024) containing potato dextrose agar (difco laboratories, detroit, mi 482327058). thirty-three petri dishes (each containing 6 pine seeds) were placed under a fluorescent lamp at 24.6c. after 10 days, 8 petri dishes with no microbial contamination were selected. seeds with radicles approximately 5 mm long were re-arranged so that 3 seeds per dish had radicles pointing down. gravity stimulus was introduced when radicles reached 1-1.5 cm long. each dish was placed on its edge so the gravity stimulus was perpendicular to the growth direction of the radicle tip. using a lamp and a piece of plotting paper, measurements were made on the position of the growing radicle point. coordinates were recorded every 2 h for the first 24 h after initiation of gravity stimulus and daily for another 5 days. experiment ii pine seeds were sterilized as in experiment i. eighteen petri dishes (each containing 6-7 loblolly pine seeds) were placed under a constant light regime in a low temperature illuminated incubator (model 818 of precision, winchester, va) set for 30c. after 10 days, 6 dishes with no microbial contamination were selected and 3-4 germinated seeds per dish were re-arranged. growing condi tions were similar to that described as in experiment i (placed under a fluorescent lamp at 25.5c). observations were made every 4 h in the first 28 h, twice a day for the following 4 days, and then once a day for additional 7 days. experiment iii pine seeds were sterilized in 0.1% hgcl2 for 3.5 min and rinsed twice for 5 min. treated seeds were placed in 100x15-mm plastic petri dishes containing potato dextrose agar. thirty sterilized seeds were placed in three 100x15-mm plastic petri dishes (10 seeds per dish) containing potato dextrose agar under a continuous temperature regime (30c) with light in the low temperature illuminated incubator. observations for microorganism colonies and germination, and measurements on the position of the growing radicle point were made twice a day during the first 5 days, and then daily until 28 days after treatment. experiments iv and v the same procedure was employed in both experiments iv and v. fifty seeds were sterilized in 0.1% hgcl2 for 3.5 min and 50 seeds in 9% h2o2 for 60 min, respectively, and both rinsed twice for 5 min. hgcl 2-treated seeds and h2o2-treated seeds were placed in 12 dishes per treatment (8-9 seeds per dish). dishes were placed into an incubator at 30c and 27c for experiments iv and v, respectively. clean seeds were transferred to a new dish. at the start of the trial, 46 clean germinated seeds per dish were rearranged. the petri dish was rotated 90 clockwise as soon as radicles reached 2.7 cm (s=1.55 cm) and 4.2 cm (s=0.81 cm) for experiments iv and v, respectively. dishes in experiment iv were placed on their edges on a lab table at room temperature (22.7c) while those in experiment v remained in the incubator. observations on the radicle growing status were made daily for 15 days for the experiment iv, while for experiment v observations were made every 2 h in the first 12 h and then once a day for 10 days. with the aid of a lamp and plotting paper, coordinates were recorded for the origin where a radicle penetrates the agar and for the radicle growing point at each observational stage. growth angle was defined as the angle formed by a vertical line through the origin (where the radicle penetrates into the agar) and a line through the origin and the growing point of the radicle at each observational time (figure 1). to make it easier to compare gravitropic measurements, any angle was recorded as a positive angle. the smaller the angle, the greater the gravitropism. radicle length was defined as the distance from the origin to the growing point at a certain time. radicle increment was the difference between 2 growing points at each observational period. growth rate figure 1. drawings of the orientation of the seed and radicle at day 0 and day 4. the growth angle is defined as the angle formed by the baseline vector and the plant organ vector. both vectors pass through the origin (i.e. the position of the radicle tip at the beginning of the experiment). figure 2. growth of loblolly pine radicles under sterile growing conditions 96 and 168 h after the dish was rotated 90 degrees (to point the radicle tip horizontally). was defined as radicle increment per unit of time. data were analyzed by statistical package for the social sciences 10.0 (spss 10.0), sigmaplot 5.0, and excel 2000. student’s t-test was used to test for treatment differences. results and discussion normal gravitropism obtained under sterile conditions intact loblolly pine radicles responded to gravity under sterile growing conditions (figure 2) . they exhibited normal gravitropism 8.3 h (s=6.48 h) after the gravity stimulus was introduced (figure 3). average growth angle decreased from 84.5 to 21.2 one week after gravity stimulus introduction. change in growth angle slowed 72 h after treatment, which suggested that most of the response occurred within the first 3 days. regression analyses revealed a close relation of growth angle to time. based upon calculation of sse (sum of squares for error) and comparison of predicted figures with figures formed by the observed data, cubic and quadratic models were deemed to be appropriate (table 2). intact radicles grew normally under sterile growing conditions. radicle length increased linearly with time, from 0.52 cm to 2.64 cm at the last observational time (figure 4). growth rate remained steady at 0.2-0.3 cm per day. loblolly pine radicles in hgcl2 treatments exhibited gravitropism 4-11 h ( x =6.9 h, s=2.42 h) after the petri dish was rotated. there was a continuous decrease in growth angle over time (figures 5, 6). the angle was 91 (s=9) at the beginning of experiment v and 64 (s=16) after 10 days (figure 5). based upon calculation of sse and comparisons of predicted with observed data, cubic and quadratic models were considered the most appropriate ( table 2). radicle length increased from 1.5 to 2.3 cm in experiment v (figure 7). however, growth rate decreased 24 h after treatment, and 9 days later it was 0.08 cm per day. g ro w th a n g le ( d e g re e s ) 90 85 80 75 70 65 0 2 4 6 8 10 12 14 16 18 20 22 24 hours after rotation g ro w th a n g le ( d e g re e s ) 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 days after rotation figure 3. growth angle of intact loblolly pine radicles during the first week in experiment i (s=7 at end of study). table 2. models for relating growth angle (y) of loblolly pine radicles from hgcl2-treated seeds in experiments iv and v, and from h2o2-treated seeds to days after initiation of study (x). model equation r 2 p-value cubic y=121.066545-0.123114x-0.000895x 2 +0.000002x 3 0.9328 0.0001 hgcl2-treated seeds in quadratic y=125.679240-0.307143x+0.000425x 2 0.9139 0.0001 experiment iv hgcl2-treated seeds in the cubic y=91.596503-0.345424x+0.001954x 2 -0.000004x 3 0.9976 0.0001 experiment v quadratic y=89.516993-0.207752x+0.000450x 2 0.9686 0.0001 h2o2-treated seeds cubic y=95.414266-0.002788x-0.000649x 2 +0.000001x 3 0.9952 0.0001 quadratic y=96.090979-0.047589x-0.001600x 2 0.9906 0.0001 y – growth angle (). x – time after gravity stimulus introduction (d). e = 2.718282. radicles from mercuric chloride treated seeds showed a greater expression of gravitropism than those from hydrogen peroxide treated seeds under the similar experimental conditions. loblolly pine radicles from h2o2-treated seeds started geotropic growth 5 h after dish rotation. radicle growth angle changed from about 96 to 77 over time. cubic and quadratic models were the most appropriate models for the data observed (table 2). radicle length increased from 2.77 to 3.74 cm over the 10-day period (figure 6) while growth rate increased from about 0.02 to 0.11 cm per day. r a d ic le l e n g th ( c m ) 3 2.5 2 1.5 1 0.5 0 0 1 2 3 4 5 6 7 days after rotation figure 4. length of intact loblolly pine radicles during the first week in experiment i (s=1.9 mm at end of study). g ro w th a n g le ( d e g re e s ) 100 95 90 85 h2o2 80 75 hgcl2 70 65 60 0 1 2 3 4 5 6 7 8 9 10 days after rotation figure 5. loblolly pine radicle growth angle over time in different sterilizing treatments in experiment v (s=5 at end of study). treatments were significantly different (=0.05) for days 1 to 10. based upon comparison of growth angle and radicle curvature development, radicles from hgcl2-treated seeds showed greater expression of gravitropism than those from h2o2-treated seeds under the similar conditions in the experiments (figure 5) . eight h after treatment, the difference in growth angle between the two treatments was significant at the 5% level. from 1 to 6 days after treatment, differences were significant at the 1% level. mercuric chloride was a more effective sterilant and did not have a negative influence on loblolly pine gravitropism. although the applied concentration was lower and treatment time shorter, hgcl2 was more effective than h2o2 for sterilizing loblolly pine seeds. both in experiments iii and v, all 80 seeds were free from fungi, compared to 24% fungal contamination with h 2o 2 in experiment v. in experiment iv, only 2 out of 50 seeds treated with hgcl2 were contaminated with fungi. as for bacterium-infested seeds, there was 16%-27% contamination in the hgcl2 treatment but contamination in the h2o2 treatment was greater than 80%. while the data indicated that hgcl2 did not have a negative influence on loblolly pine gravitropism, 3 out of g ro w th a n g le ( d e g re e s ) 130 120 110 100 90 80 70 60 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 days after rotation figure 6. growth angle of loblolly pine radicles from hgcl2-treated seeds during the first 2 weeks in experiment iv (s=13 at end of study). r a d ic le l e n g th ( c m ) 4 3.5 3 2.5 2 1.5 1 0.5 0 0 1 2 3 4 5 6 7 8 9 10 days after rotation figure 7. loblolly pine radicle length over time in different sterilizing treatments (experiment v) (s=2 mm at end of study). treatments were significantly different (=0.05) for days 1 to 10. the 22 germinated seeds either grew in a twisted manner or were tipless. heavy metals such as cu and zn caused problems with radicle growth in jack pine (pinus banksiana lamb.)(govindaraju and dancik, 1987) . however, it remains unknown if either the cation (hg 2+ ) or the anion (cl ) is harmful to pinus when used at higher rates. future research it is known that vigorous radicle growth only occurs for a short period of time after germination. rate of growth declines as the radicle ages. satisfactory gravitropism observations can be achieved only within a certain time period. it is recommended that a loblolly pine radicle gravitropism tests be completed within 15 days after germination. in order for seedlings to grow uniformly, only a few germinated seeds (with their radicles approximately 0.5 cm long) should be placed in each petri dish. if seedlings with relatively long radicles are selected, radicle growth will be reduced after transfer to the dish. conclusion these studies confirmed that gravitropism of loblolly pine radicles does occur after seeds have been treated with either hgcl2 or h2o 2. however, the chemicals selected in the seed treatment can affect the rate and magnitude of gravitropism expression. researchers who treat pine seeds with chemicals should note that some chemicals can affect the expression of gravitropism. acknowledgement we express our appreciation to the international forest seed company (odenville, al) for providing pine seeds. references barnett jp (1976). sterilizing southern pine seeds with hydrogen peroxide. tree planters’ notes 27: 17-19. fayez ka, gerken i, kristen u (1995). ultrastructural responses of root caps to the herbicides chlorsulfuron and metsulfuron methyl. in: baluska f, ciamporova m, gasparikova o and barlow pw (eds) structure and function of roots—proceedings of the fourth international symposium, slovakia. 1993. kluwer academic publishers, netherlands. pp. 277-284. gilmour jw, vanner al (1973). radiata pine (pinus radiata). terminal crook disease; colletotrichum acutatum f. sp. pinea. new zeal. forest serv. reprint. no. 668, p.1. govindaraju dr, dancik bp (1987). allozyme heterozygosity and homeostasis in germinating seeds of jack pine. heredity 59: 279283. gyimah a (1977). effect of hydrogen peroxide on the germination of grand fir, lodgepole pine and douglas fir seeds. tech. bull. for. prod. res. inst. ghana 1: 28-32. graham jh, linderman rg (1983). pathogenic seedborne fusarium oxysporum from douglas-fir. plant dis. 67: 323-325. han zm, hong yd, zhao bg (2003). a study on pathogenicity of bacteria carried by pine wood nematodes. j. phytopathol. 151: 683689. mason gn, asdel ep (1978). fungi associated with pinus taeda seed development. plant dis. rep. 62: 864-867. penafiel sr (1982). effects of three pasture plant extracts on germination of benguet pine (pinus kesiya). sri lanka for. 15: 154156. sharma ad, thakur m, rana m, sing k (2004). effect of plant growth hormones and abiotic stresses on germination, growth and phosphatase activities in sorghum bicolor (l.) moench seeds. afr. j. biotechnol. 3: 308-312. somade af, adegeye ao, oduwaiye ea, obiaga pc (1998). pretreatment effects on seed germination of nauclea diderrichii (de wild and th. dur.) merril. nig. j. for. 28: 79-81. steiner am, fuchs h (1987). germination and tetrazolium testing in seeds treated with herbicides and pesticides. [german]. seed sci. technol. 15: 707-716. sweet hc, bolton we (1979). the surface decontamination of seeds to produce axenic seedlings. am. j. bot. 66: 692-698 tang w (2000). peroxidase activity of desiccation-tolerant loblolly pine somatic embryos. in vitro cell dev. biol. plant 36: 488-491. in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (2), pp. 001-006, february, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper investigation of the potential of fortified instant matooke flour in rehabilitation of malnourished children (part 1) florence isabirye muranga1,2*, miriam kanyago, fabian nabugoomu3 and james m. ntambi4 1 department of food science and technology, makerere university, p. o. box 7062 kampala, uganda. 2 presidential initiative on banana industrial development (pibid), p. o. box 32747 kampala, uganda. 3 faculty of science and technology, uganda christian university, p.o. box 4 mukono, uganda. 4 department of biochemistry and nutritional sciences, university of wisconsin-madison, wisconsin usa. accepted 21 may, 2019 the aim of this study was to optimize levels of incorporation of soybean and sesame flours into instant tooke (cooking banana) flour (itf) porridge with respect to sensory quality. the flours were prepared using conventional dehydration procedures. a mixture design was developed for incorporating sesame and soybean flours into matooke flour. from the design thirteen porridge formulations were developed on which sensory evaluation was determined using 9-point hedonic scale. sensory parameters determined were taste color, flavor, consistency, mouth feel and overall acceptability. based on sensory evaluation, one optimal formulation was identified. nutritional quality attributes of the optimum formulation were determined. viscosity was determined using the rapid visco-analyser. the optimal formulation was 64.90, 16.34 and 18.76 % matooke, soybean and sesame respectively. the results indicated that incorporation of soybean and sesame into itf porridge significantly (p<0.001) enhanced its sensory quality attributes. energy density and protein content of the optimum formulation was 502.12 kcal and 11.1% respectively. incorporation of sesame and soybean flours into itf resulted in reduction in viscosity. the study suggested that fortification of itf with soybean and sesame enhances sensory and nutritional quality. key words: matooke, fortification, optimization. introduction bananas are an important staple food for more than 70% of uganda’s population. cooking bananas also known as ‘matooke’ (east african highland cooking bananas) are grown by 72% of farmers on an average plot size of one hectare, which is 12.3% of the world total (karugaba and kimaru, 1999). the high production levels not with-standing, nutrition and health reports in uganda have indicated high levels of malnutrition and hence high include low energy density of weaning foods and lack of ready to prepare formulations, which lead to infrequent mortality rates *corresponding author. e-mail: director@pibid.org, fmuranga@agric.mak.ac.ug. tel: +256772363271, +256312265789, +256414371050. fax: +256414340413. among children less than five years. the underlying factors for malnutrition have been identified to feeding due to the heavy workload of the caregivers especially among the rural poor (kikafunda, 1996). however, recent advances in processing of matooke have succeeded in reducing the relative bulk of matooke starch by pre-gelatinization (muranga, 1998). pre-gelatinization can be done either by cooking the matooke before drying or by extrusion cooking. studies on weanling mice showed that both techniques were equally effective in enhancing bioavailability of the matooke starch (muranga et al., 2007). muranga (1998) reported that matooke has high starch content (dry basis 81.8 85.7%) and subsequently has a high potential as a calorie resource base. energy sufficiency in the diet is critical for a good overall nutritional status at the weaning table 1. design for the different levels of incorporation of sesame and soybean flours into itf. code treatment itf % soybean % sesame % g 12 80.56 19.44 0.00 e 7 50.33 19.44 30.22 f 10 91.67 2.78 5.56 j 13 60.67 14.58 24.75 i 11 75.22 0.00 24.78 b 2 58.56 25.00 16.44 c 3 61.44 5.56 33.00 g 8 80.56 19.44 0.00 d 4 71.00 12.50 16.50 e 5 50.33 19.44 30.22 a 1 81.33 6.25 12.42 f 6 91.67 2.78 5.56 h 9 66.78 25.00 8.22 stage and the carbohydrate requirement in the diet is adequately covered by the pregelatinised matooke starch (muranga et al; 2007). additionally, matooke flour when mixed with soybean in substantial ratios, results in increase in the protein level and the products have good consumer quality attributes as well as high protein efficiency ratio (per) because of the latter’s good lysine levels (katebarirwe, 2005). though deficient in the sulphur containing amino acids, (cysteine and methionine), soybeans’ chemical score in comparison to egg protein is high (70%). in compensation for the limiting amino acids, sesame (sesamum indicum) also known locally as simsim can be used (fao, 1989). sesame contains 988 mg per 100 g of food, of methionine and cystein combined, in contrast to soybean 422 mg per 100 g and eggs at 717 mg per 100 g (fao, 1989). matooke, sesame and soybean are available locally at an affordable cost and can therefore be used in combination as part of a child’s diet to ensure good nutrition. besides, pilot studies revealed relatively high protein efficiency ratios (per), when the cooked matooke flour fortified with a soybean/sesame composite was fed to weanling rats (muranga et al., 2003a). methods preparation of itf, soybean and sesame flours the itf was prepared according to patent number ug/p/04/ 00010. the soybean and sesame flours were prepared according to methodology outlined by muranga and mutambuka (2003). fortification of itf and sensory evaluation thirteen (13) design points were generated using design expert software version 6.0 computer package stat-ease, inc., mn, usa; 2003. based on previous studies, the levels of incorporation of sesame flour were varied between 0 33, while soybean was varied between 0 25 and the rest being itf as shown in table 1. the flour mixtures were then used to prepare test porridges for sensory evaluation. the test porridges were then prepared by dissolving 100g of composite flour in 300 ml of cold water after which boiling water was added in varying quantities. 700 ml of boiling water was added to mixture a, g and f, 600 ml to mixture h, i and d, and 500 ml to porridge b, c, j and e. the porridges were boiled for five minutes with stirring, after which 10 g of sugar were added. the porridges were then evaluated by a panel of 13 panelists with respect to sensory indicator parameters using the nine point hedonic scale. the panel consisted of final year students of masters of science in food science and technology, masters of science in applied human nutrition and bachelors of science in food science and technology, all of makerere university. the above individuals were given basic training before commencing the evaluation. the sensory evaluation data used to determine the optimal levels for incorporating soybean and sesame flours into matooke flour using stat-ease design expert. optimization was done with respect to mouth feel, taste and overall acceptability by fitting data from the sensory experiment to a quadratic surface response model as shown in table 2. itf physiochemical properties the viscosity of the porridges was analyzed using the flocken programme of the rapid visco analyser (rva flocken thermocline version 2.0 newport scientific scientific pty, ltd, nsw australia). proximate analysis was done on the optimum formulation derived, to get the energy, protein, fat, iron and zinc contents, using standard aoac (1999) and the vitamin a content using the colorimetric method (underwood and stekel, 1984). data analysis the data was entered into excel spread sheet in preparation for analysis. the sensory evaluation data was analyzed by reml and the viscosity data was analyzed by one way anova using the gen stat 5 release 3.2 (pc/windows nt) statistical package for means table 2. optimization models with respect to taste, mouth feel and overall acceptability. response second order polynomials r 2 significance taste y = 0.052539x1 + 0.11544x2 + 7.31243e-003x3 0.1442 0.0221 mouth feel y = 0.070183 x1 0.035189x2 +0.040823x3 0.1468 0.0204 overall y = 0.11495x1 – 41.23019 x2 + 9.72199 x3 + 0.68827 x1x2 – 0.16649 x1 x3 + 0.52249 0.2109 0.2938 acceptability x2x3 – 4.00042e-003 x1 x2 x3 + 2.92411e-003 x1x2(x1x2) – 7.07623e-003 x1 x3(x1 x3) – 5.50244e-003 x2 x3(x2 – x3) 1 duplication code figure 1. sensory characteristics of the formulations. key: levels of incorporation of matooke/soybean/sesame respectively; a – 81.33/6.25/12.42; b – 58.56/25.00/16.44, c 61.44/5.56/33.00; d – 71.00/12.50/16.50; e – 50.33/19.44/30.33; f – 91.67/2.78/5.56; g – 80.56/19.44/0.00; h – 66.78/ 25.00/8.22; i 75.22/0.00/24.78; j – 60.67/14.58/24.75. means, standard errors of differences and least significant differences. results sensory evaluation: six sensory characteristics namely color, taste, flavor, mouth feel, consistency and overall acceptability were analyzed. the results are presented in figure 1. porridge (i) containing 0.00, 24.78 and 75.22% soybean, sesame and matooke respectively had the lowest scores for color because no soybean was added to it to give the yellow color, while porridge (b) with 25.00, 16.44 and 58.56% soybean, sesame and matooke respectively had the highest score due to the high soybean content. porridge (a) containing 6.25, 12.42 and 81.33% soybean, sesame and matooke respectively had f j 600 g (r v u ) 450 a v is c o s it y h 300 i d k 150 b j c e 0 newport scientific pty ltd 0 3 6 9 12 15 time (min) figure 2. viscoamylograph for matooke/soybean/sesame formulations. key: levels of incorporation of matooke/soybean/sesame respectively. a – 81.33/6.25/12.42; b – 58.56/25.00/16.44, c 61.44/5.56/33.00; d – 71.00/12.50/16.50; e – 50.33/19.44/30.33; f – 91.67/2.78/5.56; g – 80.56/19.44/0.00; h – 66.78/ 25.00/8.22; i 75.22/0.00/24.78; j – 60.67/14.58/24.75; k 65.00/16.00/19.00. the lowest score for taste and flavor because of the high sesame content vis-à-vis the soybean. porridge (e) containing 19.44 and 30.22% soybean and sesame respectively had the lowest score for mouthfeel due to the high sesame content which was not finely ground, while porridge (c) containing 5.56, 33.00 and 61.44% soybean, sesame and matooke respectively had the lowest score for consistency also due to the large particle size of the sesame which was also in a high quantity. porridge (f) containing 2.78, 5.56 and 91.67% soybean, sesame and matooke respectively had the lowest value for overall acceptability while porridge (b) with 25.00, 16.44 and 58.56% soybean, sesame and matooke respectively had the highest score for overall acceptability. porridge (b) containing 25.00, 16.44 and 58.56% soybean, sesame and matooke respectively were the best liked porridge. there was a significant (p < 0.001) enhancement in the sensory characteristics in all the porridges except for color in porridge (i). sensory evaluation of the formulations optimization of the sesame and soybean into the matooke flour: a numerical optimization was done using the design expert of the stat-ease statistical package to come up with the optimal product, using the sensory evaluation data. the most optimum combination had a ratio of 64.9: 16.34: 18.76 for the itf/ soybean/ sesame mixture. physiochemical properties of matooke flour viscosity of the formulations: the matooke/soybean/sesame formulations were analyzed for viscosity and the results are as shown in figure 2. porridge f and g had the highest peak trough, setback and final viscosities due to the low levels of incorporation of the soybean and sesame, while porridge e had the lowest due to the high levels of incorporation of the soybean and sesame. porridge b, c and j had similar trends for the peak, trough, setback and final viscosity, as they both had almost no peak and were level throughout. porridge h, i and d had intermediate peak trough, setback and final viscosities. the decline in peak viscosity was rapid for porridge f, g and a, but was gradual for porridge h, i and d. porridge k, the optimum formulation had a lower peak viscosity, a level trough and setback and a gradual increase to the final viscosity, indicating its stability. proximate analysis: the average composition of moisture, protein, iron, zinc vitamin a and energy of the optimized sample were analyzed. the crude protein content was 11.1%, energy content 502.12 kcal/100g, iron content 0.003%, zinc content 0.009% and vitamin a content was 185 µg/100g. discussion sensory evaluation porridge (b) was liked best; this could be attributed to the addition of soybean and sesame up to 25 and 16% respectively. this gave the porridge a good color, flavor, mouth-feel, consistency and taste. the same percentage of incorporation of soybean was also reported in a previous study (katebarirwe, 2005). there was a significant (p < 0.001) enhancement in the sensory characteristics in all the porridges except for color in porridge (i). this could be attributed to the lack of soybean in this mixture. soybean has a yellow color which is attributed to carotenoids, also present in the commonly available foods (lee, 1983). fennema (1986) reported an apparent increase in yellowness in blanched products, which he attributed to inactivation of lipoxygenase, an enzyme that catalyses oxidative decomposition of carotenoids. soybean has a characteristic beany flavor, which has been attributed to formation of volatile compounds by lipoxygenase activity (kwang et al., 2000). addition of soybean beyond 25% makes the beany flavor more pronounced, hence reducing its acceptability (katebarirwe, 2005). however, addition of sesame helped to mask the beany flavor and subsequently the general enhancement in acceptability as previously recorded. viscosity porridge f and g had the highest peak, setback and final viscosity due to the low levels of soybean and sesame incorporated into the formulations. peak viscosity denotes maximum peak of pasting of native starch and the beginning of the cooking stage, and is said to be indicative of the ease of swelling by the starch granules with a low peak value reflecting restricted swelling (muranga, 1998). solubilised or totally gelatinized starch has virtually no peak viscosity at normal starch/water ratio in the pasting cycle. setback viscosity is a measure of the setback that occurs on cooling the hot paste while the final viscosity gives a measure of the stability of the cooked paste under simulated use conditions (muranga, 1998). according to mazurs et al. (1957), exhibition of restricted swelling is true for modified or cross-linked starches, which often have minimal setback on cooling. addition of sesame and soybean into the matooke flour significantly (p < 0.001) decreased the final and set back viscosity values for all the porridges except porridge e, which did not peak at all. reduction of the peak viscosity can be attributed to solubilization of starch (50%) by the protein supplement in soybean and sesame on the one hand and to the interaction between the starch and the lipids (freidman, 1995 and collar, 2002). a low viscosity is important for weaning foods because it allows incorporation of more solids in a given porridge volume leading to an increase in energy and protein density. proximate analysis addition of soybean and sesame increased the protein content of the optimum formulation. soybean and sesame have been reported to be rich in protein (osho, 1991 and salunkhe et al., 1992), whereas cooking banana is a poor source of protein (simmonds, 1959; morton, 1987 and inibap, 2000). however, the protein content of the optimum formulation was lower (11.1%) than that shown by muranga and bukuusuba (2003) who quoted it to be 13.17%. this was due to the differences in the conversion factors used. the conversion factors used were 5.7, 5.71 and 5.3 for matooke, soybean and sesame respectively, giving an average factor of 5.57 (watt and merrill, 1975), whereas muranga and bukuusuba (2003) used the traditional (6.2) conversion factor. this gives a more accurate picture of the actual protein content of the product. addition of soybean and sesame also increased the energy content of the formulation. this is due to their high protein, carbohydrate and fat contents (harrison and tukamuhabwa, 1992 and salunkhe et al., 1992). the gross energy was also higher (502.12 kcal/100 g) compared to 459 kcal/100 g reported by muranga and bukuusuba (2003). this was due to the higher sesame content of 19% compared to muranga and bukuusuba (2003) who had a sesame content of 16.5%. the optimum product therefore, had a higher nutrition content compared to the product developed by muranga and bukuusuba (2003). the iron and zinc were in trace amounts while the beta-carotene content was 185 µg/100 g. fungo et al., 2006 reported similar results for iron, zinc and beta-carotene in fresh matooke. morton (1987) and inibap (2000) reported that matooke contains 225 µg of betacarotene and 0.9 mg iron. salunkhe et al. (1992) reported that sesame contains 10.5 mg/100 g iron and 30 iu of vitamin a, while osho (1991) reported soybean to contain 110 ppm iron and 50 ppm zinc. the major limitation to the study was time and monetary resources, hence it was not possible to train an expert panel, therefore we had to use the students in the department of food science and technology as panelists. it would also have been useful to determine proteinenergy ratio (per) using animal studies if it was not for financial constraints. summary incorporation of soybean and sesame into matooke flour porridge significantly (p< 0.05) enhanced its sensory quality attributes and improved on its nutritional quality by lowering its viscosity, thereby improving its protein content and energy density. the most optimum combination was 64.9: 16.34: 18.76 for the matooke/ soybean/ sesame respectively. this formulation could be useful in preventing and treating malnutrition in children. from the results, an optimized fortified itf formulation has a high potential as a weaning food due to its high energy and protein content. additionally, it reduces the cost of preparation as it takes a short time to prepare and cook, giving the mother enough time to engage in other economic activities. this formulation therefore has the potential of reducing the high malnutrition rates currently plaguing the country. acknowledgements this research was possible through funding from makerere university through the african institute for capacity development (aicad) grant. we acknowledge prof. kakitahi, j.t. of institute of public health medical school makerere uiniversity and dr. kiboneka, e.k. of mwanamugimu nutrition unit mulago hospital for their input in this study. we also acknowledge the department of food science and technology, makerere university, mwanamugimu nutritional unit, mulago hospital for infrastructural support and the presidential initiative on banana industrial development (pibid) for providing the final infrastructure for writing and publication of this work. references aoac (1999). official methods of analysis of aoac international. patricia cunniff (ed) 16 th edition vol 2 aoac international suite 5000 481 north frederick avenue gaithersburg, maryland. collar c (2002). significance of viscosity profile of pasted and gelled formulated wheat dough on bread staling. eur. food res. technol., 216:505-513 fao (1989). utilization of tropical foods: tropical oil seeds. food and agriculture organization of the united nations, rome pp. 22-26. fennema or (1986). food chemistry, 3 rd ed mercel dekker, inc. new york, pp. 493-562. freidman rb (1995). interaction of starches in food. in: anilkumar gg (ed). ingredient interactions: effects on food quality. maecel dekker, inc ny. isbn 0-8247-9347-1. pp. 185-187. harrison wd, tukamuhabwa p (1992). nutritional value of soybean: in tukamuhabwa, p, ebong c (eds). soybean production and utilization in uganda. proceedings of the 1 st workshop on soybean production and utilization held at mukono district farm institute (2 nd 6 th march, 1992). unpublished data, kampala, uganda, pp. 45-48. inibap (2000). networking banana and plantain: inibap annual report 1999. international network for the improvement of banana and plantain. montpellier, france. pp. 28-31. karugaba a, kimaru g (1999). banana production in uganda. an essential food and cash crop. relma technical handbook no. 18. regional land management unit, relma/sida, icraf house, gigiri p. o. box 63403, nairobi, kenya, pp. 1-2. katebarirwe jg (2005). determination of optimum ratios of incorporation of soybean into banana based weaning foods. msc. dissertation, makerere university, kampala, uganda. pp. 59-62. kikafunda-kakuramatsi j (1996). dietary risk factors for childhood malnutrition in uganda. a phd thesis, university of reading. united kingdom, pp.10-15. kwang geun l, mitchell ae, shibamoto tj (2000). determination of antioxidant properties of aroma extracts from various 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(2003). investigation of potential of low bulk matooke preparations as vehicle foods for malnutrition interventions ii, poster presentation at the workshop for the launching of ibmu project. 1 st october. 2003. muranga fi, sampath h, marlette ja, ntambi jm (2007). impact of processing technique on the apparent bioavailability of cooking banana (matooke) afri j. biochem. res. 5: 072-077. osho sm (1991). soybean as food in nigeria. training manual for a national workshop on soybean processing and utilization for women groups in nigeria. 20 – 24 may, 1991. patent number ug/p/04/00010. pp. 4(7) – 12. salunkhe dk, chavan jk, adsule rn, kadam ss (1992). world’s oil seeds; chemistry, technology and utilisation. van nostrand new york. 11: 373. simmonds nw (1959). bananas. longman. great britain pp. 308-309 underwood ba, stekel a (1984). measuring impact using laboratory methodologies in: sahn de, lockwood r, scrimshaw ns (eds) methods for the evaluation of the impact of food and nutrition programmes. report of a workshop on the evaluation of food and nutrition programmes http://www.unu.ed/unupress/unubooks/80473e /80373e08.htm accessed 26 april 2005 watt bk, merrill al (1975). composition of foods, agriculture handbook no. 8. united states department of agriculture, washington, d.c. p. 161. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 10 (2), pp. 001-005, february, 2022. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review kolanut production, processing and marketing in the south-eastern states of nigeria e. u. asogwa*, a. h. otuonye, f. c. mokwunye, k. a. oluyole, t. c. n. ndubuaku and e. o. uwagboe kola programme research, cocoa research institute of nigeria, ibadan, nigeria. accepted 11 july, 2021 kolanut is an important economic crop in the south eastern region of nigeria. however, there were no contiguous kola plantations in the south east, rather each homestead has about 2 to 15 kola trees scattered around the backyard. the land tenure system was observed to be the major constraint to kola production in large scale in the south east. other limiting factors were poor nutrient status of the south eastern soils, which have been heavily leached; the long gestation period of kola tree; the characteristic low yield of the crop; various pests, diseases and weeds (epiphytes) ravaging the plant. the non-inclusion of kolanut in the list of graded items for exportation by the federal government of nigeria also contributes to its low production and marketing. traditional methods of production, processing, storage and marketing peculiar to the south east are discussed. key words: kolanut, production, processing, management, marketing. introduction kola (a member of the family sterculiaceae) is mostly produced in africa and is cultivated to a large degree in nigeria, but also in ghana, ivory coast, brazil and the west indian islands (eijnatten, 1973; opeke, 1987; oludemokun, 1983). annual production from these countries alone is in excess of 250,000 tons, while the world production is about 300,000 tons (brickell et al., 2002). two species cola nitida (vent) schott and endl and cola acuminata (p. beav) schott and endl., are of major economic importance (oladokun, 1982). c. nitida, which is referred to as “the true kola of commerce” has featured in the internal trade of west africa for a number of centuries (nzekwu, 1961; eijnatten, 1969). kola nuts are a common sight in nigerian markets, cities and villages. they are often sold by street vendors at bus and train depots. many nigerians consume kola nuts regularly, even daily, for the medicinal, stimulating and sustaining properties. kola is an important economic cash crop to a significant proportion of nigerian population who are involved in kola farming, trading and industrial utilization. however, nigeria accounts for about 70% of the total world production of kolanuts (quarco, 1973; jacob, 1973; *corresponding author. e-mail: ucheasogwa1@yahoo.com. pala, 1976; oluokun and oladokun, 1999). the kolanut is used as a masticatory and stimulant in the tropics and has social and traditional significance as it features in many traditional ceremonies in nigeria. it also has industrial usage in pharmaceuticals, production of soft drinks, wines and in confectionaries (beaties, 1970; oguntuga, 1975). the kolanut pod husk, which is a by-product from processing the nut, is widely used for animal feeding because of its high nutritive quality. according to babatunde and hamzat (2005), broilers fed with kolanut pod husk meal diets had an outstanding growth performance. the main objective of this paper therefore, is to study the extent of kola production in the south eastern states with respect to their cultivation, processing, storage and management problems (pests, diseases, weeds etc) associated with it. trend in kola production in south eastern region in the south eastern region there are no contiguous kola plantations, rather each homestead has about 2-15 kola trees around the family house. the kola tree to the ibos is a sacred plant; hence it is planted around the house where it will be in constant interaction with the people and the ancestral spirits of their loved ones buried around the compound. the plants are either planted in orchards or 2 scattered around the backyard of each compound. the major production states`in the south east zone are abia, anambra, ebonyi, enugu and imo states, where kolanut trade predominates. in nigeria, there is a common saying that “kola is produced in the west by yorubas, consumed in the north by hausas and worshiped in the east by ibos”. the south easterners are more interested in c. acuminata, which features prominently in most traditional ceremonies and social functions. but recently due to the high cost of c. acuminata (referred to as ibo kola) and low supply to equate demand, c. nitida and solanium marvelum are gradually gaining ground as supplements for c. acuminata in social functions. there is a very high demand for c. acuminata in the region as each family consumes an average of six nuts a day depending on the number of visitors to a family. however, during ceremonies such as marriages, weddings, coronation, installation of high chiefs and traditional rulers, the consumption rate rises sharply as many nuts will be needed to entertain the guests. the excess of what is consumed locally is reserved for the markets in the northern nigeria (kano, sokoto, boronu, kebbi, jalingo and maiduguri etc). the non-establishment of kola plantations in the south eastern region has been attributed to the densely populated land area and the pattern of land tenure system that exists in these areas. even where land is available, the long gestation period of kola tree and floral incompatibility problem that drastically reduces yield have discouraged the farmers the more. kola production projection for the south east the national proposal for the development of the nigeria kola industry had it that a total of 540,000 acres (216,000 ha) of kola plantations will be established with proven materials during the period of 1971 to 1985 (fda, 1973). of these plantings, it is expected that 120,000 acres (48,000 ha) will represent replanting while 229,000 acres (91,600 ha) will be new plantings on land not previously under kola. the phased project was aimed at increasing the annual production level to 200,000 metric tons by 1985 and beyond (fda, 1973). the projected new plantings for the east central and south east states was a total of 35, 600 acres (14, 240 ha) and 62, 500 acres (25, 000 ha) respectively. it was assumed that the level of production of existing plantings would be more or less constant throughout the period covered by the report and beyond. this assumption according to fda (1973) was based on the following reasons: (i) kola trees attain peak production at 15 to 20 years from planting. (ii) 290,000 acres (116,000 ha) or 91% of existing plantings are over 20 years old, which means they have at least attained peak production. (iii) the estimated productive life of kola trees is 70 to 100 years. (iv) the rehabilitation programme will involve the selective felling and replacement of unproductive or low producing trees, so that total orchard production will be more or less unaffected. kolanut harvesting harvesting of kolanut in kola growing areas of the south eastern states is normally done by plucking ripe pod from the trees with the aid of go-to-hell, which is usually tied to a long bamboo stick. the farmers also pick the pods that naturally fall to the ground. kolanut processing and storage cola acuminata the processing of c. acuminata starts by careful examination and sorting out pods infested with weevils, diseases and other deformities, from the healthy pods. the seed coat or testa of the nuts from these healthy pods are removed by soaking the nuts in clean water for 24 h to enhance rottening, after which the nuts are skinned and rinsed in fresh water. the rinsed nuts are collected in wide flat baskets through which excess water drains off before they are kept inside the room where they are maintained under ambient room temperature for a period of three days to cure. defective/infested nuts are picked out during this curing process that usually involves considerable sweating to reduce the moisture content of the nuts. the nuts are then graded into sizes for proper storage in big sized baskets. the storage baskets are first lined up with thin transparent nylon sheet followed by a layer of newbouldia laevis leaves placed with the ventral (upper) surface facing downwards in the basket thereby exposing the dorsal (back) surface of the leaves to the nuts. the nuts are carefully placed inside, layer by layer and after each layer parkia biglobosa leaves is spread out evenly on top of the nuts. n. laevis leaves are used to cover up the last layer of the nuts, before finally sealing up the whole thing with the first layer of polythene sheet. the baskets are then stored under normal room temperature and relative humidity. the n. laevis leaves and the polythene sheets keep the nuts in an air tight condition and prevent desiccation of the nuts, while the p. biglobosa leaves was believed to brighten the nuts, thereby giving them a bright coloration which makes them attractive. the nuts are inspected every eight days during the first few months of storage. the top leaves are removed and 3 the nuts spread out gently on a mat. kolanut is noted for its characteristic quick shoot emergence without necessarily coming in contact with any growth medium. each nut is carefully examined and any emerged shoot is removed with a pin and coated with a film of palm oil to slow down the emergence rate and prevent the splitting of the nuts. any defective or infested nuts are sorted out during the inspection period. this periodical inspection also helps to prevent the over heating of the nuts. during inspection, the top leaves that have dried and shrunk, are changed. after three months, the inspection period can be extended to between 2 to 3 weeks depending on the quality and condition of the nuts at the last inspection. this cultural method of sorting out deformed and infested nuts during storage ensures maintenance of whole nuts and reduces or eliminates completely chemical usage in storage. the deformed and infested nuts are sometimes processed and sold out immediately, while the wholesome nuts are stored for over 14 months provided processing and handling are carried out with care. cola nitida rottening of the testa is achieved by keeping the extracted fresh nuts on bare ground (with occasional moistening) and covering with jute bags for 3 to 4 days. the testa of the fresh nuts wret (that is softens) and turn black. the premature nut also matures in this process. alternatively the testa or seed coat of the fresh nuts are soaked in water for 24 h after which they are skinned, rinsed and collected in baskets to drain off. defective and infested nuts are sorted out before curing the nuts in flat baskets for 3 days. the nuts are transferred to baskets lined with polythene sheet, followed by sheets of paper (old newspapers) and a layer of fresh leaves of tectonia grandis, or marantochola spp or terminalia catapa (indian almond), which are replaced during weekly inspection of the nuts. alternatively the nuts may be stored in jute bags lined with thin transparent polythene sheet, which is in turn lined with a layer of paper and fresh leaves. after two months, the frequency of inspection may be reduced to between 2 to 3 weeks intervals. kola plantation management problems kola farmers in the south eastern region are faced with the problem of pests, diseases, parasitic weeds (epiphytes) and other production problems, which have adversely affected their rate of production. pests few pests associated with kola production in the south eastern nigeria are: (a) ants: some ants like taylor ants oecophylla longinoda latl. (hymenoptera: formicidae) are observed to weave kola leaves to build nest and scale insect are sometimes harbored in these nests for protection against natural enemies. these ants are both agents of dissemination and sources of inoculum for some pathogens (olunloyo, 1979). high rate of ant incidence in most cases constitutes great nuisance as they bite the farmers and thus disturb normal cultural work on the farm. (b) weevils: the kola weevils balanogastris kolae and sophrorhinus spp (coleoptera: curculionidae) are the most destructive field to store pests of kolanuts in west africa (daramola, 1973; 1978). their destructive activities also extend to the south eastern nigeria, where the beetle reduces the shelf life of kolanuts. the curculionids initiate their attack mainly from the field to storage and are therefore referred to as field to store pests. in nigeria, high levels of infestation have been reported both in the plantations and in storage depending on the sanitary condition of the farm at the time of harvest (daramola, 1973). the high significant level of weevil damage on stored kolanuts has been attributed to the favourable storage conditions, which encourage continuous development of various instar stages of the kola weevil within field infested nuts (daramola, 1973; ivbijaro, 1977; ojo, 1979). (c) stem borers: the kola trees were attacked by some stem borers: phosphorous virescens and phosphorous gabonator. (coleoptera: cerambycidae). trees of various ages were attacked, resulting to severe stunting and malformation on young trees, which often lead to the death of the plant. this makes the establishment of kola plantation very difficult and expensive especially in areas of severe attack by phosphorous spp (squire and iwenjora, 1963). attack on older trees by phosphorous spp does not often result in the death of the trees, but as they are riddled with borers, they barely fruit. they remain living reservoirs for the pests and thus constitute a menace to the neighboring trees (squire and iwenjora, 1963). (d) termites: termites (isoptera: termitidae) attack the kola roots and trunks. the termites tunnel into roots and stems of kola trees of various ages and introduce the rot pathogen into the plant. this result into destruction of the trees by degradation and general weakening of the stem (eijnatten, 1965; libby, 1968). diseases the kola trees are hosts to many field and post harvest 4 diseases such as brown spot, brown blight, brown root rot and tip blight diseases. the brown spot disease manifests itself through chlorotic spots, which latter becomes dark brown with chlorotic surroundings. the causal agent is pestalotia spp. the heavily infected leaves become dry and latter fall off (adebayo, 1966). the casual organism for brown blight is botryodiplodia theobromae. it forms small spots of irregular brown discolouration on the leaves, which later become enlarged and cause the leaves to assume a blight appearance. the brown root rot caused by armillaria mellea and fomes noxius causes extensive root decay of kola trees in the field (odebode and olunloyo, 1989). the tip blight disease caused by phomopsis spp results in the blackening of young kola leaves tips and their subsequent death at a very tender stage (odebode and olunloyo, 1989). the major fungal causing mould and rots of the nuts are botrytis spp; b. theobromae; paecilomyces variotii; mucor spp and fusarium spp, which are all known to be favoured by high relative humidity (adebayo, 1966; olunloyo, 1979; agbeniyi and fawole, 1999; agbeniyi et al., 2000). parasitic weeds / epiphytes the kola trees were noted to be attacked by some parasitic weeds and epiphytes. the two mistletoes identified with kola trees are phyragmanthera incana and tapinanthus bangwensis. during the survey mistletoes were found on a number of kola trees and in some cases they had virtually taken over the foliage of the trees thereby reducing the photosynthetic activities of these trees. the mistletoes are usually transmitted by birds, which feed on the ripe attractive reddish berries. room (1970, 1971) reported that the birds normally feed on the succulent pericarp of the berries and then drop their sticky seeds on the branches of kola trees where they latter germinate and grow into full mistletoe plant. the epiphyte platycerium bifurcatum is also found on most of the kola tree trunks in this region. other problems the farmers in the south eastern states are faced with the problem of sterility and incompatibility in kola, as most of their kola trees had not fruited at all or did not fruit heavily in the past few years. this coincided with the earlier observation of okoloko and jacob (1971) that many kola grooves in nigeria contained both productive and unproductive trees. most of the unproductive trees showed total sterility while some were partially sterile. the varying degree of sterility can be attributed to incompatibility, unavailability of hermaphrodite flowers, lack of effective pollinating agents and interspecific hybrid sterility of c. nitida x c. acuminata (okoloko and jacob, 1971; jacob, 1973). the long gestation period of the plant has also discouraged farmers in this region from planting kola trees. other factor militating against heavy yield from this tree crop includes the poor nutrient status of the soils of south eastern states which have been heavily leached and are deficient in nitrogen, potassium and phosphorous (n.p.k). this problem however, could be corrected by applying 131 kg n; 21 kg p and 139 kg k per hectare to compensate for nutrient removed from the soil (ayodele, 1988). also, there is the problem of lack of appropriate pollinator of the kola tree flowers. kolanut marketing kolanut has for some hundreds of years served as an important article of trade in nigeria and other parts of africa (nzekwu, 1961). the marketing of kolanut in nigeria dated back to ancient times especially in the eras of the ghana, the shanghai, the benin and the kanem borno empires (webster et al., 1967). kolanut is produced in the southern part of nigeria and largely marketed and consumed in the northern part of nigeria (akinbode, 1982). most of the retailers/farmers sell their products in smaller units such as cups and bowls. there is no standard local price or grading for kolanut in nigeria, however information on market transaction, especially with reference to price determination, measurements, sizes of nuts as well as quality of nuts/grading are based on mutual knowledge and understanding of the buyers and sellers. c. nitida is the only kolanut of inter-regional and international trade. kolanut is exported in substantial quantity to other african countries as well as to europe and north america, which generate the necessary foreign exchange earnings to nigerian government (akinbode, 1982). it was estimated that the internal kolanut market in nigeria worth about thirty million naira (n30,000.000.00), while in 1970, kolanut export fetched usd $157,500 to nigerian government (pala, 1976). the white and bright coloured nuts attract more premium than the red and dull coloured nuts. at ariam, in ikwano local government area of abia state, kolanut is wealth, as it appeared to be at the centre of the business transactions (olukayode, 2001). the situation is also the same in most rural markets in the south eastern states. conclusion kola is yet to be granted a full export status by the federal government of nigeria, unlike cocoa, coffee, cashew, palm kernel, palm oil and others, which enjoy favorable market prices in the international markets. the official enlistment of kola as one of nigerians export crops will awaken the interest of farmers in these areas 5 and encourage the establishment of kola farms. the result from the present study has elucidated the numerous problems encountered in kolanut production in the south eastern states of nigeria. there is therefore, the need for government and research institutions with mandate on this crop, to address these problems and make their findings available to kola farmers in form of bulletin, hand bills, farmers guide, or by the use of electronic media as well as extension research liaison service (erls). this will sensitize farmers on improved agricultural techniques of production, processing, storage and marketing of the crop. it will also enhance the establishment and maintenance of kola farms, thereby improving the quantity and quality of kolanuts that will be produced in these areas. acknowledgements the authors hereby acknowledge the assistance of the various staff of the agricultural development programmes and the federal office of statistics across the south eastern states. references adebayo aa (1966). kola diseases. ann. rept. cocoa research institute of nigeria, pp. 127-130. agbeniyi so, fawole b (1999). effects of curing and pre-storage die treatments on mould of kolanuts. eur. j. food res. technol., 208: 447-449. agbeniyi so, otuoye ah, adedeji ar (2000). mycoflora associated with post harvest processing stages of kolanuts (cola nitida vent schott and endlicher). j. food technol. afr., 5(4): 129-131. akinbode a (1982). kolanut production and trade in nigeria. nigerian institute of social and economic research (niser), ibadan. p. 97. ayodele ea (1988). soil supporting cocoa, coffee, kola, cashew and tea in nigeria: in progress in tree crop research in nigeria, 2nd edition a crin commemorative book, pp. 33-34. babatunde bb, hamzat ra (2005). effects of feeding graded levels of kolanut husk meal on the performance of cockrels. nig. j. anim. prod., 32(1): 61-66. beatie gb (1970). soft drink flavours: their history and characteristics. cola or “kola” flavours. flavour ind., 1(6): 390-394. brickell c, cole t, marc-cathey h (2002). the american horticultural society encyclopedia of plants and flowers. d.k. publishing, new york. p. 720. daramola am (1973). the bionomics of kola weevils, sophrorhinus spp (coleoptera: curculionidae). phd. thesis university of ibadan, nigeria, p. 325. daramola am (1978). insect pests of cola in nigeria. res. bull. crin, ibadan, 3: 33. eijnatten clmv (1965). diseases and pests of kola (a literature review) memo no 7. crin, ibadan. eijnatten clmv (1969). kola: its botany and cultivation. communication no. 59. roy. trop. inst. amsterdam, p. 100. eijnatten clmv (1973). kola: a review of the literature. trop. abstr., 28(8): 1-10. federal department of agriculture lagos (fda) (1973). national agricultural development committee report of the study-group on cocoa, kola, coffee and cashew, pp. 63-120. ivbijaro mf (1977). gamma-bhc residues in kola nuts cola nitida and control of the kola nuts weevil balanogastris kolae desbr. india exp. biol., 15(12): 1236-1238. jacob vj (1973). yeild characteristics, incompatibility and sterility studies in cola nitida (vent) schoot and endlicher. p hd. thesis university of ibadan. libby jl (1968). insect pests of nigeria crops. college of agric and life sciences, university wisconsin. res. bull., p. 269. nzekwu o (1961). kolanut. nig. mag. (71): 298-305. odebode ac, olunloyo oa (1989). important diseases of kola. in: progress in tree crop research, a commemorative book to mark the 25th anniversary of cocoa research institute of nigeria, ibadan, nigeria, pp. 111-114. oguntuga dba (1975). chemical composition and potential commercial uses of kola nuts, cola nitida (vent) schoot and endlicher. ghana j. agric sci., 8: 121-125. ojo a (1979). identification of insect pests of kola. annual report, cocoa research institute of nigeria, ibadan, nigeria, pp. 29-30. okoloko ee, jacob vj (1971). kola breeding in nigeria. in progress in tree crops (kola, cocoa, coffee) research in nigeria. crin commemorative book, pp. 23-33. oladokun mao (1982). morpho-physiological aspect of germination, rooting and seedling growth in kola. phd thesis. university of ibadan nigeria, p. 230. oludemokun aa (1983). processing, storage and utilization of kolanuts, cola nitida and cola acuminata. trop. sci., 24: 111-117. olukayode o (2001). agro care. the guardian on sunday, february 11, 2001, p. 52. olunloyo oa (1979). fungi associated with stored kola nuts. nig. j. agric. sci., 1(1): 51-59. oluokun ja, oladokun ea (1999). the effects of graded levels of brewers spent grains and kolanut pod meal in the performance characteristics and carcass quality of rabbits. nig. j. anim. prod., 26: 71-77. opeke lk (1987). tropical tree crops. woye and sons (nig) ltd., ilorin, p. 327. pala ao (1976). african women in rural development: research trends and priorities. in: olc paper (usa), no. 12 / american council on education, washington, d.c. overseas liaison committee, p. 39. quarco t (1973). a handbook on kola. crin ibadan, p. 99. room fm (1970). the mistletoe.t bangwensis and cocao in west africa. proc. 3rd int. cocoa res. conf. accra ghana, pp. 522-527. room fm (1971). some physiological aspect of the relation between cocoa (theobroma cacao) and mistletoe. theobroma bangwensis. (engl. and k krause) ann. bot., 35: 69-174. squire fa, iwenjora fo (1963). on the kola tree borer in nigeria. phosphorous virescens memo. department of agriculture, nigeria. 49: 17-19. webster jb, boahen aa, idowu ho (1967). the growth of african civilization: the revolutionary years – west african science 1800. longman’s group limited, london, uk, pp. 60-82. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 12 (3), pp. 001-007, march, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper predation patterns in heterobranchus longifilis larvae: an analysis of their first exogenous feeding p. o. ajah institute of oceanography, university of calabar, calabar, cross rivers state, nigeria. e-mail: ajapaulo@yahoo.com. accepted 4 october, 2023 two-day-old post hatch heterobranchus longifilis larvae (4.06 ± 0.1 mm, 2.75 ± 0.06 mg and 0.35 ± 0.03 mm average standard length, weight and mouth width, respectively) were introduced into glasswares and exposed to a wide range of live zooplankton (prey size range: 42 to 876 µm) at a density of 2,944 individual/larva/day for 14 days. the 180 guts examined showed that food intake changed markedly with age and size. larger preys were preferred as larvae increased in age and size. chilodonella uncinata and lepadella ovalis (size range: 42 125 µm) dominated the larval gut from day 2 4 post hatch. brachionus quadridentatus, alona monachanta and chydorus sphaericus (225 346 µm) were predominant at days 5 and 6, while asplanchna priodonta, bosmina longirostris and daphnia ambigua (485 876 µm) dominated from day 7 14 post hatch. results from another 146 stomachs batch revealed that 2 to 4 day old post hatch fry preyed on larger prey (225 876 µm) at disproportionate mouth-width to prey ratio from 1: 1.2 to 2 when solely introduced and adequate. initial recognition of prey by 2 4 day old larvae was longer but thereafter feeding became more voracious and intermittent with shorter resting periods compared with older larvae. active feeding in h. longifilis larvae decreased with age while the length of feeding and rest period increased with age. key words: prey selection, behaviour, heterobranchus longiflis, exogenous. introduction finetuning of larval nutrition is important at the transition from endogenous to exogenous feeding in order to avoid high mortalities usually encountered during this first critical stage in larval life. kimpe and micha (1974) and horvat et al. (1978) earlier identified this period as the next critical phase after spawning. natural food is a prerequisite for early larval rearing of african catfish, clarias gariepinus and h. longifilis (msiska, 1981; verreth and denbieman, 1987; villegas, 1990; fermin and bolivar, 1991; kerdchuen and legendre, 1994; xi et al., 2002; hagiwara et al., 2007; ajah, 2008). zooplankton always served as an important first source of natural food during intensive larval rearing of atlantic salmon (reinersten et al., 1984; holm, 1985a and b; holm, 1986), carp (jana and chakrabarti, 1993), two flat fish pleuronectes platessa and limanda limanda (bels and davenport, 1996), american shad (johnson and dropkins, 1996). h. longifilis (kerdchuen and legendre, 1994; ajah, 1998; ajah, 2008) brook charr (mclaughlin et al., 2000; mclaughlin, 2001), endangered fish golden bubblebee goodeid, allotoca dugesi (domínguezdomínguez et al., 2002), artic charr (benhaïm et al., 2003) and whitefish (charal) (chirostoma riojai) larvae (morales-ventura et al., 2004). survival during early fish larval stages depends largely upon the availability of appropriate prey. the paper seeks to elucidate the particular type and size of live food organism fish larvae/frys/fingerlings can swallow at any given stage of growth and development. these findings can be used to improve growth and performance of juveniles in rearing tanks. materials and methods prey selection and larvae nutrition a total of 1,840 two-day old h. longifilis val. larvae (average individual weight of 2.75 ± 0.06 mg, 0.4 ± 0.03 mm mouth width and 4.06 ± 0.06 mm standard length) raised in the hatchery complex of the institute of oceanography, university of calabar, calabar, 2 table 1. prey composition per aquarium per day (± s. d. of density in aquarium). species (size in µm) culture tanks density (org/l) density (org/aquarium/day) composition (%) c. uncinata (40.3 × 23.1) 102 326,400 27.72 l. ovalis (93.6 × 70.3) 94 300,800 25.54 b. quadridentatus (432.9 × 128.7) 44 140,800 11.96 a. priodonta (685 × 356) 36 115,200 9.78 a. monachanta (392 × 231) 25 80,000 6.79 c. sphaericus (476 × 351) 24 76,800 6.52 b. longirostris (523 × 482) 18 57,600 4.89 d. ambigua (688 × 541) 15 48,000 4.08 e. gracilis (1240 × 300) 10 32,000 2.72 total 368 1,177600 ± 108958.055 100 source: ajah 1995. n.b.the nutritional values of most live foods used in this study have been well researched and compiled by hepher (1990). consequently, analyses of their nutrient contents were not embarked upon. nigeria were used for this research. the first 1,200 larvae were reared in three 100 l rectangular glass aquaria (25 x 40 × 100 cm, respectively, for w × h × l) with netted inlet and outlet water pipes of 96 mm diameter. each aquarium received 400 two-day old larvae at 50 larvae/l. flows through system were maintained throughout the experiment. aeration at 4.5 ± 0.6 mgo2l -1 was supplied via an airblower of 1.5 horsepower (model: siemens). the water level was kept at the 10 cm mark making room for 8 l of water per aquaria and the water flow rate averaged 45 ± 7.6 mls/s. feeding was at 1,177,600 ± 108,958 mixed zooplankton/aquarium/day drawn from their monoculture tanks (three 10 m 3 circular tanks, 4 oblong 1 m 3 concrete tanks, 3 fibre glass tanks of 0.6 m 3 each and six 40 l glass aquaria). the larvae were fed with 2,944 ± 256 prey/larva/day given in two installments. these monocultures were developed in both the laboratory and semi-controlled outdoor tanks (ajah, 1995) with mono species compositions as in table 1. the zooplankton species compositions were standardized for three years (1992 1995) to ensure stability in composition (ajah, 1995). same results will be gotten at any time/year given same conditions and culture methods so long, there is no change due to cyclomorphosis and the culture organism’s size remains the same. behavioural pattern during feeding for closer observation of feeding behaviour a total of 40 h. longifilis larvae of various ages’ vis-à-vis 2 4, 5 6, 7 10 and 11 14 days old were stocked at 10 larvae per 2 l erlenmeyer flask. the flasks were placed in an illuminated culture cabinet lined with silver material (aluminum foil) (ajah, 1995). a total of 29,440-mixed live zooplankton was put into each of the four flasks containing larvae of a particular age. duration of feeding was monitored using a stopwatch. a hand lens was used in observing the opening of larval mouth during feeding. further confirmations of feeding was by timely removal of all the larvae and dissecting immediately for gut examination. exposure to exclusively larger prey six aerated 2 l erlenmeyer flasks kept in the culture cabinet were stocked with each of 2 days old h. longifilis larvae at 10 larvae/flask giving a total of 60 larvae/l is a total of 24 larvae. mono species cultures of eudiaptomus gracilis (1240 × 300 µm), b. longirostris (523 × 482 µm), physocypria inflata or a. priodonta (685 × 356 m) were separately administered in two installments per day into the first four flasks to ascertain their acceptance as exclusively mono species prey. the remaining two flasks received a mixture of larger prey at same densities and feeding regime as above. zooplankton counts were usually done using sedgwick rafter (model: ajah 001). predator-prey ratio the predator: prey ratio was calculated by dividing the mouth width of the larvae by the length of the prey. gut analysis sampling was carried out everyday for 14 days on larvae fed on mixed live foods. a total of 180 randomly sampled larvae guts comprising 10, 15, 15, 40, 35, 10, 10, 8, 8, 5, 8, 8 and 8 each for days 2 to 14-post hatch/feeding, respectively, were examined. again, twenty randomly selected guts were examined from each of the 100 larvae exposed to mono species cultures in 4 flasks. twenty six guts were examined from the two-mixed zooplankton flasks, composed of 2 -14 day old larvae exposed to mixed prey. a total of forty guts were each examined when fed on the small and large prey. ten guts were analyzed at day 2 and 15 each at days 3 and 4 with small prey. for the larger prey size 10 guts each were analyzed at days 7 and 8 while 8 guts each were analyzed from days 9 -14 except day 11 that only 5 guts were examined. all examinations commenced few seconds when active feeding terminated. the fish caught were immediately dissected and the contents introduced into plastic containers. the contents were slightly diluted with borehole water. two drops of 4% formaldehyde solution was added to demobilize the organisms in the gut and latter examined after sedimentation using counting chamber (model: ajah 001) with a light microscope at ×100 magnification. the dominance, occurrence and numerical methods were adopted. the dominance method indicated the number of stomachs where each food item occurred as the dominant/main food item expressed as a percentage of the total food items. the numerical method gave the relative abundance of a particular food item in the stomach while the percentage occurrence indicated the number of stomachs each item was found over the total number of stomach examined. 3 table 2. the sized ranges of food organisms consumed in percent at various larval ages and guts examined at each stage are represented. days 2 4 days 5 6 days 7 14 small prey size: 42 125 µm large prey size: 225 345 µm larger prey size: 485 876 µm composition (%) composition (%) composition (%) c. uncinata (85) l. ovalis (15) larval size: 4.06 6.0 mm s.l mouth width 0.4 0.6 mm total guts examined 40 b. quadridentatus (39.99) a. monachanta (30) c. sphaericus (30) copepoda (0.01) larval size: 6.1 8.0 mm s.l mouth width 0.6 0.8 mm total guts examined 40 a. priodonta (28) b. longirostris (24) d. ambigua (47.5) copepoda 0.5% larval size: 8.1 15 mm s.l mouth width 0.8 1.5 mm total guts examined 73 results prey selection and larval nutrition when h. longifilis larvae were exposed to adequate quantities of properly mixed zooplankton species as shown in table 1, gut anaylsisi revealed that within the first 3½ days post hatching, results indicated that initial concentration of feeding was on smaller food organismsciliates like c. uncinata and rotifer (l. ovalis), with chilodonella being more consumed (85%) than lepadella (15%) (table 2). by the eve of the 4th day, they have started shifting to large rotifersb. quadridentatus (40%). a few stomachs containing copepod had not more than one copepod each. towards the end of the 6th day, further consumption of the largest group of zooplankton types present in the mixture was observed. most stomachs predominantly comprised rotifera. priodonta (28%), cladocerad. ambigua (48%), b. longirostris (24%) while up to two copepods was observed in only two of the guts examined. behavioural pattern during feeding when the larval feeding behaviour was monitored, using 2 l erlenmeyer flasks, the following findings were made: it took the younger larvae (2 4 days old post hatch) longer time (average of 1½ min) to notice/sense their prey organisms but thereafter, fed more voraciously and incessantly, allowing shorter resting intervals (5 10 s) inbetween meals. active feeding lasted for average of 30 s. quite terrific wagging of the tail and body movements as well as very rapid movements was observed during this period. older fish larvae (5 6 days old) recognized their prey within 60 s of introduction. they fed for 5 7 min on the average and rested for 10 15 s. fish larvae of 7 14 days old post hatch with average standard length 13 ± 0.2 mm jumped on their prey with the fastest rapidity (5 10 s) following introduction of prey. duration of feeding ranged from 22.5 31 min and period of rests increased to 2 min. at this stage, traces of real competition were observed. larvae upon the slightest confrontation by the other changed location and continued feeding. predator-prey ratio based on the findings in table 2, predator: prey mouth width (mw) ratio of 2 days old larvae (mw = 0.4 mm) fed large prey was 1: 0.5 to 1: 0.86. when larger prey, like a. priodonta, b. longirostris, d. ambigua and e. gracilis were introduced, the ratio increased from 1: 1.2 to 1: 2.2. difficulty in swallowing this larger prey increased with the presence of appendages as in e. gracilis, although the high population densities of prey created room for a sizeable number to be caught though quite often by chance. the ostracods were rather evasive. they aggregated at the edge of the aquarium until pursued out by the predators. as soon as they are scattered, their chances of being preyed upon increased. however, this evasion was not possible when round bottom 2 l erlenmeyer glass flasks were used. it was much easier to lay hold on the cladocerans than either the copepoda or ostracoda. a. priodonta besides its very large size, its rotator/folding habit and the speed at which it does this, made it more difficult to catch. exposure to exclusively larger prey the introduction of larvae during its most active stage (2 4 days old post hatch, figure 1) into a pool of only larger zooplankton species such as cladoceran (b. longirostris, d. ambigua) copepoda (e. gracilis), ostracoda (p. inflata etc.) and rotifera (a. priodonta) left the fish larvae with no other choice than to prey on these larger prey. examination of the 26 guts via the numerical method for 2 days post hatch indicated the presence of the following prey per gut within ten minutes of feeding: 4 16 14 14 12 10 10 day 2 7 day 3 8 6 6 5 5 day 4 4 3 2.5 3 3 2 1 0.1 0 a a a a d r d r e e o o f p c ac ti e o o op d r r la st c c o figure 1. average prey composition per h. longifilis gut upon exposure to only large prey. the numbers of samplings were: 12, 8 and 6, respectively, for days 2, 3 and 4 post hatch. average of 7 copepoda (4 cyclopoids, 2 harparticoids and 1 nauplius), 1 cladoceran, 6 ostracoda and 3 rotifera (figure 1) . the 3rd day post hatch witnessed 5 copepoda, 10 cladoceran, 5 ostracoda and zero rotifera per gut while 3, 14, 2 and 0, respectively, were observed during day 4-post hatch. discussion prey selection according to visser (1982), (1) preference for the more popular prey increases when the total prey density increases, (2) preference for the relatively scarce prey increases when the total prey density increases. diet shift from zooplankton to benthic invertebrates was not due to reduced zooplankton availability, but was instead related to changes in gill raker structure (qin and fast, 1997). milinski (1982), feeding three-spined sticklebacks with daphnia showed that more successful competitors concentrated on large prey, whereas the poorer competitors fed as generalists but not unselectively. holm (1987) did not notice any significant size selection in salmon when bosmina was the main prey item. knight (unpublished data) advised that large food granules should be fed at the early fingerling stage but an admixture of the smallest granules is advisable if a tank still contains very small fish fry. an admixture of the various prey sizes in adequate proportions gave preference to the smaller prey as starter diet by h. longifilis larvae. holm (1987) stated that depending on the fish size, salmon selected different food types on days 40. upon exposure of h. longifilis larvae to adequate quantities of properly mixed zooplankton species within the first 3½ days post hatching, initial concentration of feeding was on smaller food organisms-ciliates like c. uncinata and rotifer (l. ovalis), with chilodonella being more consumed (85%) than lepadella (15%). between days five and six there is a complete shift to large rotifers like brachionus (40%) and cladocerans like alona (30%) and chydorus (30%). by the end of the first week to that of the second week basically, larger cladocerans prey sizes such as daphnia (48%) and bosmina (24%) and asplanchna (28%), a rotifer plus a few copepods (0.5%) were consumed. hurst and conover (2001) found out that the diets of youngof-theyear striped bass (morone saxatilis) captured in winter were dominated by gammarid amphipods and shrimp species with generally higher consumption in early winter than late winter. behavioural pattern during feeding fish larvae naturally prefer smaller prey organisms at first few days of posthatch unless exposed to solely large prey from the start. smaller prey is preferred to larger prey by small larvae when the prey is dense and mixed. the first recognition of the prey by younger larvae (2 4 days old) is usually longer but soon after begins more voracious and intermittent feeding with shorter resting periods. larger fish juveniles recognize and capture its 5 prey within a much shorter time, spend longer time during feeding and resting. the incessant feeding tendency coupled with shorter resting periods could be attributed to higher metabolic rates often noticed among younger organisms. it took the very young larvae (2 4 days old) longer time to detect the presence of food due to incomplete development of the gustatory and chemoreceptor organs but become much more voracious thereafter. the much older (7 14 days old) were more sporadic in capturing their prey, fed for longer time intervals and also rested for longer time than the younger (5 6 days old) and the youngest (2 4 days old). clearly, multiple levels of selection and signaling are operating visual and size selection, free amino acid feeding signals, but also further signals such as spit out signals. holm and walther (1988) indicated that both the high spitout rate of depleted daphnids and the positive response to daphnia extract explained the importance of taste during the early feeding of atlantic salmon. predator-prey ratio based on the findings in table 2, predator: prey mouth width (mw) ratio of 2 days old larvae (mw = 0.4 mm) fed large prey was 1: 0.5 to 1: 0.86. when larger prey, like a. priodonta, b. longirostris, d. ambigua and e. gracilis were introduced, the ratio increased from 1: 1.2 to 1: 2.2. difficulty in swallowing this larger prey increased with the presence of appendages as in e. gracilis, although the high population densities of prey created room for a sizeable number to be caught though quite often by chance. the ostracods were rather evasive. they aggregated at the edge of the aquarium until pursued out by the predators. as soon as they are scattered, their chances of being preyed upon increased. however, this evasion was not possible when round bottom 2 l erlenmeyer glass flasks were used. it was much easier to lay hold on the cladocerans than either the copepoda or ostracoda. a. priodonta besides its very large size, its rotator/folding habit and the speed at which it does this, made it more difficult to catch.. exposure to exclusively larger prey h. longifilis larvae do go for excessively large food organisms (a. priodonta, b. longirostris, d. ambigua etc.) in comparison to its mouth width provided such is in slow motion, attractive and abundant. while larval shad mainly consumed copepods (37.7%) and cladocerans (37.4%), juvenile shad ate chironomids (43.1%) and ostracods (28.4%) both thus, exhibiting diet variation in diet composition and feeding periodicity (johnson and dropkins, 1996). brodeur (1998) while working on juvenile pollock (theragra chalcogramma) observed general selection of larger prey sizes relative to what was available. consequently, as predator size increased, choice of prey skewed towards the bigger prey. brodeur (1998) observed a general increase in prey width with increasing predator size while qin and fast (1997) observed that at both the laboratory and field trials, snakehead diets changed as fish size increased. ware (1973) has shown that motion of prey increases the reactive distance of trout to their food. aloo and dadzie (1995) noticed a switch to crayfish and fish at comparatively smaller length-class while absence of zooplankton from the diet might be due to the small sample size of the juvenile bass. ajah (1998) found out that the larvae fed on copepod alone from days 4 post hatch did not grow since they were unable to capture their evasive prey. however, such evasiveness was very pronounced due to paucity of prey organisms (480 individual copepod/larva/day). when the concentration of this supposedly evasive prey (holm, 1985b; ajah, 1998) was increased in this experiment during days 2 4 post hatch to 2,944 individual copepod/larva/day, larvae were able to capture up to seven copepods within 10 min of feeding and then grew. storebakken (1985) reported that atlantic salmon in nature select food items according to high occurrence and not, for example, in accordance with the nutritional appropriateness such as the protein: lipid ratio found to be optimal in aquaculture. holm and moller (1984) showed that food selection in salmon yearlings was influenced by occurrence, high occurrence allowed size selection. visser (1982), working with three-spined sticklebacks, found that preference for the preferred prey (daphnia) increases when the total prey density increases. morales-ventura et al. (2004) stated that survival during early fish larval stages depends largely upon the availability of appropriate prey. larval behaviour and prey capture mclaughlin et al. (2000) examined the prey capture success of recently emerged brook charr (salvelinus fontinalis) foraging in shallow, clear and stillwater pools, and found that fewer than 42% of attacks ended with ingestion either because of difficulty distinguishing suitable prey from unsuitable items or because of difficulty capturing evasive prey. probabilities of capture upon attack and ingestion upon capture depended upon where attacks were directed in the water column, the 6 fish's level of activity at the time of attack, its fork length and the sampling date. in general, success was higher for larger, sedentary fish attacking prey in the lower portion of the water column than for smaller, active fish attacking prey at the water surface. bels and davenport (1996) made a comparison of food capture and ingestion in juveniles of two flatfish species, p. platessa and l. limanda and found relatively minor differences between species, but plaice captured and transported food more quickly than dab. the low water depth reduced the energy loss by the larvae in search of food and enhanced the aggregation of the diet (ajah, 1997). vivekanandan (1976) earlier reported that the depth of the water column influences the food intake of air-breathing fishes. the frequency of items attacked was only a moderate predictor of the frequency of prey ingested. poor capture success is an important aspect of the early life history of brook charr in particular and probably of young salmonines in general. the ostracods were rather evasive. they aggregated at the edge of the aquarium until pursued out by the predators. as soon as they are scattered, their chances of being preyed upon increased. however, this evasion was not possible when round bottom 2 l erlenmeyer glass flasks were used. it was much easier to lay hold on the cladocerans than either the copepoda or ostracoda. a. priodonta besides its very large size, its rotator/folding habit and the speed at which it does this, made it more difficult to catch. job and bellwood (1996) indicated that visual acuity of premnas biaculeatus larvae improved substantially between early feeding (days 3 post-hatch) and the presettlement (days 10 post-hatch) larvae. the prey capture success with rotifers ranged from 96% at 3 days posthatch to 100% at 10 days post-hatch. h. longifilis fish larvae recognized their prey faster with age and fed and rested longer with age. at older stage, traces of real competition were observed. larvae upon the slightest confrontation by the other changed location and continued feeding. domínguez-domínguez et al. (2002), larval feeding behaviour of the endangered fish, a. dugesi on three zooplanktonic preys, viz. b. calyciflorus (pallas), moina macrocopa (goulden) and d. pulex (leydig) showed that capture success (capture/attack) ranged from 0.80 0.98 with brachionus, 0.72 0.94 with moina and 0.17 0.46 with daphnia. prey preference experiments were conducted using b. calyciflorus, m. macrocopa and d. pulex at a fixed ratio of 5: 2: 2 ind. ml -1 , respectively and revealed a positive selection for rotifers and moina, but avoidance of daphnia. the abundance of food at all times eliminated all forms of competition and/or cannibalism. each fish larva rose from rest to find more than enough to feed upon. some were lured to feed by the body movements/feeding habits of others. feeding was not continuous, since each larva, fry or fingerling needed periods of rest, which increased as larval size increased. live zooplankton is preferred to dry feed (holm, 1987; ajah, 1997, 2008; hagiwara et al., 2 007). benhaïm et al. (2003) young arctic charr, s. alpinus, shortly after the onset of exogenous feeding, large fish (0·11 0·14 g) were more active and fed mainly from the water surface. small fish (0·06 0·09 g) moved less and made fewer foraging attempts. mclaughlin (2001), behavioural diversification in brook charr (s. fontinalis mitchill) foraging in still-water pools along the sides of streams exhibit conspicuous variation in foraging behaviour. some charr are sedentary and eat crustaceans from the lower portion of the water column. others are mobile and eat insects from the upper portion of the water column. in conclusion, h. longifilis larvae select food when exposed to a wide range of properly mixed prey and generally will prefer the smaller as first exogenous food. where only large preys are available, it can still commence preying on such up to a disproportionately predator: prey mouth-width ratio of 1: 1.2 and above, provided the prey was introduced early enough and in large amount. thus, h. longifilis larvae could be start-fed with large food organisms like copepods, ostracods and cladocerans provided that preys are introduced in large quantities from days 2 4 post hatch being the most active stage. at the end of days 4, fish larval activity and motivation by hunger slightly reduces and consequently attraction to food reduces if not fed within the period. the younger larvae do not easily recognize its prey but as soon as prey presence is established, feeding is more voracious, incessant with very short resting periods in comparison to older larvae. acknowledgement the author is grateful to the european economic community through whose auspices this research was carried out. references ajah po (1995). identification and mass production of zooplankton species suitable for early feeding of african clariid catfish. phd thesis. university of calabar, nigeria, p. 164. ajah po (1997). effects of live foods, artificial feed and their combinations on the growth and survival of 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aquac. 63: 251-267. villegas ct (1990). the effects on growth and survival of feeding water fleas (moina macrocopa straus) and rotifers (brachionus plicatilis ) to milk (chanos chanos, fosskal) fry. israeli j. aquacult. bamidgeh 42: 10-17. visser m (1982). prey selection by the three-spined sticklebacks (gasterostes aculleatus l.) decologia 55: 395-402. vivekanandan e (1976). effects of feeding on the swimming activity and growth of ophiocephalus striatus. j. fish. biol. 8: 289-292. ware dm (1973). risks of epibenthic prey to predation by rainbow trout (salmo gairdneri) j. fish. res. board canada 30: 787-797. welcomme rl (1979). fisheries ecology of flood plain rivers. longman, london p. 317. xi yli, liu gyi, jin hj (2002). population growth, body size, and egg size of two different strains of brachionus calyciflorus pallas (rotifera) fed algae. j. freshwater ecol. 17(2): 185-190. 1 full length research paper creation of bread and biscuits using fermented and cooked pearl millet flour rahul1* and prasad2 1 indian institute of information technology, tiruchirappalli, tn, india 2 rajiv gandhi national institute of youth development, sriperumbudur, tn, india accepted 6 january, 2025 pearl millet that ferments on its own is discovered to harbor probiotic microorganisms. bakery goods serve as a medium for incorporating various nutrient-dense components. by enhancing its amino acid profiles, fortifying wheat flour with non-wheat proteins improves the quality of the protein. in the cooked fermented pearl millet, the antinutrient phytic acid decreased from 858.4 mg/100 g in the raw millet to 380.3 mg/100 g. after the pearl millet was cooked and fermented, the level of tannin did not decrease from the raw millet. in place of refined wheat flour, the cooked fermented pearl millet was used to make bread and biscuits. the bread that was made with 10, 15, and 20% cooked fermented pearl millet flour had good physical and textural qualities and was on par with bread from the market. cookies made with 50% cooked fermented pearl millet flour demonstrated acceptable acceptance, as did cookies made with 50% and 100% wheat flour replacement. key words: cooked fermented pearl millet, anti nutrients, fortification, bakery products. introduction one of the most significant crops in semi-arid regions of africa and india is pearl millet (pennisetum glaucum). the pearl millet crop's capacity to withstand heat and drought allows it to adapt widely to local conditions. it is therefore commonly grown in tropical parts of the world, such as asia and africa. a staple food grain for millions of people, pearl millet is currently the sixth most important cereal grain in the world. it is farmed widely in africa, asia, india, and the near east. in terms of both output and area, pearl millet is most abundant in india. food fermentation has been a common technique for enhancing food's flavor, texture, and palatability. because of the presence of antinutritional agents as phytic acid, polyphenols, and tannins, pearl millet has a high nutrient content but a low bioavailability. one method that has been shown to lessen these antinutrients is fermentation. the action of the enzymes that microorganisms produce is what causes the modifications related to the fermentation process. substrates based on carbohydrates are frequently fermented by lactic acid bacteria (lab). the acidity of foods fermented with lactic acid is often lower than ph 4.5. the majority of harmful microbes present in food cannot endure at this low ph; hence, it has been discovered that fermenting food with lactic acid lowers the possibility of harmful microbes growing there. because they favorably modify the balance of intestinal microflora, prevent the growth of bad bacteria, encourage healthy digestion, strengthen the immune system, and raise resistance to infection, probiotics are considered helpful bacteria. some strains of the lactobacillus and bifidobacteria genera are examples of probiotics that have been used in probiotic products. materials and methods fermentation of cooked pearl millet by adding water in different ratios of 1:2, 1:3, and 1:4, the fully cooked pearl millet was given the opportunity to ferment spontaneously. in the selective medium man, rogosa, and sharpe author(s) retain the copyright of this article. african journal of food science research issn 2375-0723 vol. 13 (1), pp. 001-006, january, 2025. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 (mrs), the growth and survival of probiotic bacteria (lactobacillus sp.) are continually counted using the plate count method every 6 hours until fermentation lasts for 48 hours. the process parameters for cooked fermented probiotic pearl millet are displayed in table 1. to promote quicker probiotic bacterial proliferation, the cooked fermented probiotic millet water was incubated for 48 hours at 37°c in a bod incubator (genuine equipment mfrs, coimbatore). probiotic bacterial survival was observed throughout the course of several days of storage for the fermented cooked pearl millet, which was kept at both room temperature and in a refrigerator. preparation of value added products the cooked fermented pearl millet flour is used for the preparation of bakery products like bread and cookies. the details of process parameter for bread and cookies are highlighted in the table 2. preparation of bread the bread is made using refined wheat flour and cooked fermented flour in varying proportions. the composite bread is high in fiber thanks to the fiberrich pearl millet flour. the increased proportion of cooked fermented pearl millet flour reduces the bread's rising volume since pearl millet flour contains very little gluten. therefore, in the creation of valueadded composite bread, the proportion of cooked fermented pearl millet flour to refined wheat flour was restricted to 10, 15, 20, and 25%. preparation of cookies the cooked fermented pearl millet flour was combined with the wheat flour in a 1:1 ratio to make cookies, but it was also mixed separately. specific volume of bread by dividing volume (cc) by weight (g), the specific volume of bread was determined using the aacc method 10-05.01 (aacc, 2000). rapeseed displacement was used to measure the loaf volume as soon as it was taken out of the oven and weighed. loaves were put in a known-volume container, then rapeseeds were poured into it until it was full. the loaf volume was defined as the volume of seeds that the loaf displaced. loaf specific volume (lsv), was calculated according to the following formula: l.s.v = loaf volume (cc)/loaf weight (g) (1) physical properties of cookies cookies diameter (d) and thickness (t) were determined using a vernier caliper while weight of cookies was determined by using electronic analytical weighing balance. spread was calculated using the formula given by akubor (2004): spread ratio = diameter/thickness (2) texture analysis insti the quality and acceptance of fermented pearl millet and value-added goods made from pearl millet are greatly influenced by the product's texture. using a texture analyzer, the textural characteristics of valueadded bread and cookies were identified. firmness in terms of hardness, bourne (1982) defined firmness as the highest force measured during the initial compression stroke. the force (in grams, kilograms, or newtons) needed to compress the product by a predetermined distance (measured at 25% compression of 25 mm) is the definition of firmness according to this approach (aacc, 1983). the sample was compressed to a depth of 10 mm in order to perform firmness tests in the texture analyzer using a compression plate with a diameter of 75 mm (p/75). the compression plate was attached to the movable bar using a probe adapter. prior to starting the studies, the probe was calibrated to make sure there was enough room between the heavy-duty platform and the compression plate. determination of firmness of composite bread one bread slice at a time was subjected to firmness testing, and the graph was then used to obtain the necessary values. the sample was compressed to a predetermined distance of 10 mm by moving the probe. after the probe made contact with the sample, the greatest force needed to pierce the chicken piece from the spent layer was noted and contrasted between the samples. the aacc (74-09) standard method is used to measure the hardness of the bread. ta-xt2 configuration, strain: 40%; pre-test speed: 1.0 mm/s; test speed: 1.7 mm/s; post-test speed: 10.0 mm/s; mode: measure force in compression; option: return to start; type of trigger: auto: 5 g; rate of data acquisition: 250 pps; probe: aacc 36 mm cylinder probe with a 5 kg load cell and a radius of p/36r the cylinder's edges are "rounded" to eliminate sharpness from the probe's perimeter, which lessens the probe's propensity to cut the sample when it penetrates. the bread loaf is automatically cut into uniform slices that are 12.5 mm thick. the test was conducted using the '% strain' data in order to calibrate the probe. the probe was lowered such that it was near the test surface in order to do this. the menu bar's t.a. was selected, followed by calibrate probe to define the approximate 30-mm distance that the probe should return to following the recommended sample compression. before starting the testing, the auto height box in the 'run a test' window was checked. the test was started once the sample was positioned in the middle of the cylinder probe, away from any uneven or non-representative crumb patches. the probe compresses the sample until it reaches 40% of the product height once the trigger force is reached. after that, it leaves the sample and goes back to where it was before. the outcome is a texture expert surpasses graphs. the graph and result window are then used to 3 make the observation. determination of hardness and resistance of composite cookies to bend one bread slice at a time was subjected to firmness testing, and the graph was then used to obtain the necessary values. the sample was compressed to a predetermined distance of 10 mm by moving the probe. after the probe made contact with the sample, the greatest force needed to pierce the chicken piece from the spent layer was noted and contrasted between the samples. the aacc (74-09) standard method is used to measure the hardness of the bread. ta-xt2 configuration, strain: 40%; pre-test speed: 1.0 mm/s; test speed: 1.7 mm/s; post-test speed: 10.0 mm/s; mode: measure force in compression; option: return to start; type of trigger: auto: 5 g; rate of data acquisition: 250 pps; probe: aacc 36 mm cylinder probe with a 5 kg load cell and a radius of p/36r the cylinder's edges are "rounded" to eliminate sharpness from the probe's perimeter, which lessens the probe's propensity to cut the sample when it penetrates. the bread loaf is automatically cut into uniform slices that are 12.5 mm thick. the test was conducted using the '% strain' data in order to calibrate the probe. the probe was lowered such that it was near the test surface in order to do this. the menu bar's t.a. was selected, followed by calibrate probe to define the approximate 30mm distance that the probe should return to following the recommended sample compression. before starting the testing, the auto height box in the 'run a test' window was checked. the test was started once the sample was positioned in the middle of the cylinder probe, away from any uneven or nonrepresentative crumb patches. the probe compresses the sample until it reaches 40% of the product height once the trigger force is reached. after that, it leaves the sample and goes back to where it was before. the outcome is a texture expert surpasses graphs. the graph and result window are then used to make the observation. the "hardness" of the sample was defined as the highest force necessary to break the cookies. the distance at the point of break represents the sample's resistance to bending, which is related to its "fracturability"; a sample that breaks at a relatively short distance is said to have a high fracturability. pre-test speed: 1.0 mm/s; test speed: 3.0 mm/s; posttest speed: 10.0 mm/s; distance: 5 mm; trigger force: auto 50 g; data acquisition rate: 500 pps; ta-xt2 settings, mode: measure force in compression; option: return to start; probe: a heavy-duty platform (hdp/90) with a 3 point bending rig (hdp/3pb) and a 5 kg load cell the rig base plate's two adjustable supports are positioned appropriately apart to support the 40 mm sample. this disparity should be recorded and maintained constant for comparison's sake. after that, the base plate is fastened to the sturdy platform. the sturdy platform was moved and secured so that the upper blade was equally spaced from the two lower supports. just before testing, the sample was taken out of storage and positioned in the middle of the supports. the force was observed to increase once the trigger force was reached until the biscuit broke and fell into two pieces. this was found to be the strongest force and is known as the sample's "hardness." the sample's resistance to bending, or "fracturability," was determined by the distance at the point of break. the graph window provides the outcome. sensory evaluation the 50 semitrainned sensory panelists were given the composite bread with 10, 15, 20, and 25% cooked fermented pearl millet flour added to refined wheat flour at room temperature and humidity levels, while the bread made with only refined wheat flour was used as a control. the bread's color, flavor, chewiness, and general acceptance were all assessed. a similar process was used for the composite cookies made with 50% wheat flour and cooked fermented pearl millet flour, as well as cookies made without addition of wheat flour. after every sensory test, panelists were told to rinse their mouths or sip water to clear their palates. a hedonic scale questionnaire with nine points—1 for extremely dislike, 2 for dislike very much, 3 for dislike moderately, 4 for dislike slightly, 5 for neither like nor dislike, 6 for like slightly, 7 for like moderately, 8 for like very much, and 9 for extremely like—was given to semitrained panelists to rate the composite bread. measures of overall acceptability, softness, flavor, mouthfeel, crumb grain, odor, and general look were all assessed for composite bread. results and discussion the studies on composite bread specific volume of composite bread table 3 shows the impact of cooked fermented pearl millet (cfpm) flour on a particular volume of composite bread. as the quantity of cfpm flour substitution increased, the specific volume of bread reduced dramatically. compared to bread made with pure refined wheat flour, bread made using composite flours had a smaller volume. the maximum specific volume of bread, 5.93 cc/g, was acquired from control loaf, but the bread with the lowest specific volume (5.04 and 5.27 cc/g, respectively) was made using flour that contained 25% cfpm flour. while the particular volume of commercially available bread loaf in the market is 6 cc/g, this conclusion was consistent with that of aluko and olugbemi (1989), who observed lower quantities associated with composite as opposed to 100% wheat. this is explained by the dough having less gluten network and, as a result, a weaker cell wall structure, which limits the dough's capacity to rise. the precise volumes of substitution at the 10, 15, and 20% levels, however, did not differ substantially from one another. 4 textural properties of composite bread when cooked fermented pearl millet (cfpm) flour was added to refined wheat flour in proportions of 10, 15, 20, and 25%, the composite bread's hardness, cohesion, springiness, gumminess, and chewiness were measured. the textural characteristics of composite bread are shown in table 4. the table shown that as the percentage concentration of cfpm flour grows, so does the composite bread's hardness. the bread that had a 25% cfpm flour to refined wheat flour incorporation had a higher value of 21.15 n. conversely, the remaining percentage of incor poration has a lower hardness value. the bread's hardness value. less variation is seen from the control sample, whose hardness was 11.48 n, and the 10% bread's hardness of 12.22 n, when 10% cfpm flour is added. despite whatever percentage of integration, there was no discernible variation in the cohesion and springiness ratings. when compared to the manufactured composite bread, the control showed greater cohesion and springiness values of 0.71 and 0.9, respectively. the composite bread with 25% cfpm flour and refined wheat flour demonstrated the lowest values of cohesiveness and springiness. the gumminess and chewiness of bread made with refined wheat flour are 8.19 and 7.41 n, respectively, and they were observed to rise as the percentage of cfpm flour was increased. the 25% cfpm flour composite bread demonstrated higher gumminess and chewiness values of 12.9 and 10.45 n, respectively. according to abdelghafor et al. (2011), pan bread cooked with whole or decorticated sorghum-wheat composite flour had higher hardness, gumminess, and chewiness and decreased cohesion, springiness, and resilience when the amount of sorghum flour was increased. impact of adding cooked fermented pearl millet flours on the color of bread crumb and bread made with refined wheat flour the colour values l (light-dark), a (red-green), and b (yellow-blue) of the bread and crumb samples of blended table 5 provides the flour. the findings show that l-values changed dramatically from white to gray, a values changed from green to red, and b values changed from blue to yellow as the percentage of cfpm flour substitution rose. overall, the l values of the bread and bread crumb samples that were made with cfpm flour showed a substantial rise in grayish hue, falling from 72.54 to 62.47 and from 64.54 to 55.47, respectively. bread made with 25% cfpm flour had the highest a-value (-0.13), whereas bread made with 100% refined wheat flour had the lowest value (-0.75), as seen by higher intensities of green hue. conversely, bread prepared with 25% cfpm flour had the highest b value (12.67), while bread made with 100% refined wheat flour had the lowest value (9.51). the color of the bread crumb was more clearly affected by the substitution of cfpm flour than the color of the bread itself. compared to the samples manufactured with additional cfpm flour, the bread crumb samples created with 100% refined wheat had higher a and b values and lower l values. however, torres et al. (1993) found that the color of tortilla flour was not significantly (p<0.05) affected by the addition of decorticated pearl millet flour. however, the look of tortillas made using sorghum flour was hampered by undesired black dots. additionally, morad et al. (1984) investigated how the millet variety affected the baking qualities of balady bread, cookies, and ordinary bread in the united states. they discovered that the hue at thirty percent, the values of pocket bread made with whole wheat flour were comparable to those created with brown and yellow millet. the color of the bread crumb may rival that of whole wheat bread, particularly in samples made with brown sorghum. the study's findings show that cfpm flour affected the color of the crumb. this might be because the fibers and pigments lessened the green component of the crumb's hue and slightly changed it from red to gray. sensory evaluation of composite breads a hedonic scale questionnaire with nine points—1 for extremely dislike, 2 for dislike very much, 3 for dislike moderately, 4 for dislike slightly, 5 for neither like nor dislike, 6 for like slightly, 7 for like moderately, 8 for like very much, and 9 for extremely like—was given to semitrained panelists to rate the composite bread. measures of overall acceptability, softness, flavor, mouthfeel, crumb grain, odor, and general look were all assessed for composite bread. table 6 displays the sensory characteristics of both the 100% wheat bread and composite breads formed from mixes of wheat and cooked fermented wheat flours. the blend of cfpm flour showed substantial differences in all sensory categories, including general look, color, flavor, softness, mouth feel, and overall acceptability. table 6 indicates that there was a significant difference in overall acceptability between refined wheat flour and a higher amount of cfpm flour. composite loaves prepared with 5 and 10% substituted cfpm flour had colors that were comparable to the control, which was 100% refined wheat flour; samples were noticeably darker at greater substitution levels. as the amount of cfpm flour rose, the mouth feel score dramatically decreased. with mean scores of 7.59 and 7.5, it was discovered that the mouth feel scores of 10% and 15% composite bread were comparable. the composite bread's sensory evaluation score when cfpm flour was substituted for 25% of the refined wheat flour in terms of sensory qualities, flour falls between 5.22 and 6. these findings concur with those of summer and nielsen (1976), who found that adding 25% millet flour to the bread recipe darkened the color of the loaf on the inside as well as the outside. overall bread quality at the various cooked fermented pearl millet flour substitution levels, ranging from 10% to 20%, was determined to be satisfactory in this investigation. however, as the amount of cfpm flour substituted dropped, acceptance rose. these findings concur with kyomugisha's (2002) research. 5 in terms of sensory qualities, flour falls between 5.22 and 6. these findings concur with those of summer and nielsen (1976), who found that adding 25% millet flour to the bread recipe darkened the color of the loaf on the inside as well as the outside. overall bread quality at the various cooked fermented pearl millet flour substitution levels, ranging from 10% to 20%, was determined to be satisfactory in this investigation. however, as the amount of cfpm flour substituted dropped, acceptance rose. these findings concur with kyomugisha's (2002) research. studies on composite cookies spread ratio of composite cookies the spread ratio difference between the composite cookies made with 50% and 100% cooked fermented flour and wheat flour is displayed in table 7. the control group consisted of cookies made with whole wheat flour. the weight of the composite cookies decreased as the replacement ratio of cooked fermented pearl millet flour with whole wheat flour increased, causing the thickness to grow dramatically. comparing the composite cookies to the control, the diameter grew. the whole wheat flour cookies are 30.80 mm in thickness and 40 mm in diameter. while the cookies made with cooked fermented pearl millet flour decreased to 6.45 g, the control group showed a higher mass of 7.22 g. this decrease in mass could be the result of fermentation reducing the flour's volume. chinma and gernah (2007) found a similar outcome with composite cookies made with flour from soy beans, cassava, and mango. textural properties of composite cookies textural characteristics such as chewiness, hardness, gumminess, and fracturability or breaking strength (the ability of the cookies to withstand bending) were observed for the composite cookies when 50 and 100% (cfpm) flour were mixed with refined wheat flour. as more cooked fermented pearl millet flour was added, all of the textural qualities—hardness, gumminess, chewiness, and fraturability—were enhanced. half of the cooked fermented pearl millet flour was substituted in the composite cookies. shown a minor departure from the control in terms of textural characteristics, however the 100% value displays greater values of 28.9 n hardness, 15.22 n gumminess, 13.68 n gumminess, and 27.94 n fraturability. the samples did not differ significantly in terms of fracturability or hardness. for the control sample, a lower hardness value of 19.24 n was noted. the findings imply that the value of textural qualities is increased when the amount of cooked fermented pearl millet flour is incorporated. these findings concur with the conclusion reached by chinma and gernah (2007). colour difference in composite cookies the color of the cookies made with cooked fermented pearl millet differed from that of the control. the color values of whiteness (l), redness (a), and yellowness (b) for crust colors are displayed in table 6. when comparing the cooked fermented pearl millet flour samples to the control, the l value decreased. it was verified that the cookies made with cfpm flour had a higher browning index (bi) and were redder and darker (a-values) than the control samples. the findings demonstrated that the composite cookie samples with increased levels of cfpm flour from 50 to 100% had higher avalues (redness). these findings are in line with those of kenny et al. (2000), barron and espinoza (1993), ahmed (1999), and eissa et al. (2007). additionally, the data showed that the composite cookies' b value decreased as the amount of cfpm flour substituted increased. the cookies made with 100% cooked fermented pearl millet flour had a higher b value. the information in table 6 indicates that the composite cookies' color was darker than that of the control cookies, which were made with wheat flour. sensory evaluation of composite cookies table 7 shows how the sensory qualities of composite cookies are affected when 50 and 100 percent cooked fermented pearl millet flour is added. comparing the composite cookies to the control sample, the sensory characteristics score decreased significantly as the amount of cooked fermented pearl millet flour substituted increase. 6 cookies made with 100% cfpm flour had a less than ideal flavor. therefore, the cookie taste score was 5.11, and the 50% taste score was 7.99, which does not significantly differ from the control level of 8.2. the information about the cookies' color and look, which varies from 8.2 to 9 on the 9-point hedonic scale, was not statistically significant and is shown in table 7. the results shown in table 7 show that as the amount of cooked fermented pearl millet flour substituted with wheat flour increased, the overall acceptability declined. these findings are consistent with those of takumil et al. (2006), who discovered that millet seeds taste bitter when compared to cookies made with wheat flour. conclusion this bakery goods were also made with the cooked fermented pearl millet (cfpm). due to the lower fiber content, regular intake of bakery products, which are often manufactured with refined or whole wheat flour, might cause constipation and colon cancer in users. pearl millet is used in baking when cooked fermented pearl millet flour is substituted for refined whole wheat flour. there is no discernible difference in the physicochemical and textural characteristics of the bread made with 10% cfpm flour and wheat flour compared to the commercially available bread, which has a hardness of 11.48 n and 12.22 n for 10% cfpm. the morphological and textural characteristics of the 25% integration were unacceptable. because cooked fermented pearl millet flour is not glutinous, its rising level decreases when bread is being made. as a result, only 25% of refined wheat flour could be replaced with cooked fermented pearl millet (cfpm) flour. both a 50% and a 100% substitution of cooked fermented pearl millet flour for wheat flour were used to make the composite cookies. compared to cookies made with 100% cfpm flour, those made using 50% cfpm flour substitute have superior color and texture qualities. compared to the composite cookies made with 50% cfpm flour and refined wheat flour, the cookies made with 100% cfpm had a darker color. as the percentage of cfpm flour grew, so did the hardness and fracturability. despite being made with 100% cfpm flour, cookies made with 50% cfpm flour demonstrated a satisfactory sensory score and acceptability. because of their high fiber content and lack of gluten, cookies made with 100% cfpm flour had increased hardness and fracturability of 28.9 and 27.9 n, respectively. its higher phenol and tannin content was the cause of its darker color. 10% of cfpm flour combined with bread's refined wheat flour is appropriate, while 50% of cfpm flour combined with cookie flour is safe to eat.. references acgih abdelghafor rf, mustafa ai, ibrahim amh, krishnan pg (2011). quality of bread from composite flour of sorghum and hard white winter wheat. adv. j. food sci. technol. 3(1):9-15. akubor pi (2004). chemical composition, functional properties and baking potential of african breadfruit kernel and wheat flour blends . int. j. food sci. technol. 39(2):223-229. aluko re, olugbemi lb (1989). sorghum as a raw material in the baking industry. paper presented at the symposium on the current status and potential of industrial uses of sorghum in nigeria. kano, nigeria. 4-6 december. american association of cereal chemists (aacc) (1983). methods 44-15a moisture air-oven methods. american association of cereal chemists (aacc) (2000). method 1005.01 guidelines for measurement of volume by rapeseed displacement. barron jm, espinoza a (1993). fortification of maize tortilla with alkali treated chickpea flour. int. j. sci. technol. 28(5):505-511. bourne mc (1982). principles of objective texture measurement. in: food texture and viscosity concept and measurement. academic press, new york, usa. 44-117. chinma ce, gernah di (2007). physicochemical and sensory properties of cookies production from cassava/ soyabean/mango composite flours. j. food technol. 5(3):256-260. eissa ha, hussein as, mostafa be (2007). rheological properties and quality evaluation of egyptian balady bread and biscuits supplemented with flours of ungerminated and germinated legume seeds or mushroom. pol. j. food nutr. sci. 57:487-496. kenny s, karina w, catherine s, elke a (2000). incorporation of dairy ingredients into wheat bread: effects on dough rheology and bread quality. eur. food res. technol. 210:391-396. kyomugisha f (2002). production and characterization of bread made from epuripur sorghum. uganda. retrieved from: http://www.gasat international.org/conferences/g11mauritius/proceedings/proceeding %208.pdf. morad mm, doherty ca, rooney lw (1984). effect of sorghum variety on baking properties of u.s. conventional bread, egyptian pita "balady" bread and cookies. j. food sci. 49:1070-1074. summer ak, nielsen ma (1976). sorghum and the millets: their composition and nutritive value. academic press, london. 3:439448. takumil a, muraki e, oshima y, nakano y, shono j, hoshino s, tsuge n, narukami t, tsunoda n, chiba h, kasono k (2006). therapeutic effects of frb on diet-induced metabolic disorders in rats. lipids health dis. 160(1): 546. (http://www.lipidworld.com/content/11/1/58). http://www.lipidworld.com/content/11/1/58) sensory evaluation of composite breads studies on composite cookies spread ratio of composite cookies textural properties of composite cookies colour difference in composite cookies sensory evaluation of composite cookies in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 8 (2), pp. 001-009, february, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper investigation of the potential of fortified instant matooke flour (itf) in rehabilitation of malnourished children (part 2) florence i. muranga1,2*, miriam kanyago1,2, fabian nabugoomu3 and james m. ntambi4 1 department of food science and technology, makerere university, p. o. box 7062 kampala, uganda. 2 presidential initiative on banana industrial development (pibid), p. o. box 32747 kampala, uganda. 3 faculty of science and technology, uganda christian university, p. o. box 4 mukono, uganda. 4 department of biochemistry and nutritional sciences, university of wisconsin-madison, wisconsin usa. accepted 12 september, 2019 this study was done to establish potential of instant tooke (cooking banana) flour (itf) as a vehicle food for malnutrition intervention. hypotheses generated were: (1) there is a significant difference in response with respect to weight gain between malnourished subjects rehabilitated using fortified ift and mwanamugimu dietary regime (mdr); (2) there is a significant difference in response with respect to serum albumin, ferritin and retinol levels between fortified itf and mdr. to test the hypotheses, 100 malnourished children admitted at mwanamugimu nutrition unit in uganda, were randomly allocated to a test (ift diet from part i of study) or control (mdr diet) group. growth rates and food intake were measured daily, while blood samples were withdrawn during recruitment and before discharge for testing serum albumin, ferritin and retinol levels. means were compared using spss version 11. the test and control diets were adequate in protein but relatively lower for energy against rda. protein and energy intake in the test group was significantly (p < 0.05) higher for porridge, while protein intake in the control group was significantly (p < 0.05) higher for kitoobero. weight gain in the test was significantly (p < 0.05) higher than the control group. however, both groups showed no significant difference in the bio-chemical indicators. the superiority of fortified itf formulation over the mdr was proved with respect to weight gain but not for the biochemical indicators. hypothesis one was accepted but two was rejected. key words: instant tooke flour (itf), matooke, malnourished, rehabilitation introduction bananas are an important staple food for more than 70% of uganda’s population. cooking bananas also known as ‘matooke’ (east african highland cooking bananas) are grown by 72% of farmers on an average garden size of one hectare, which is 12.3% of the world total (karugaba and kimaru, 1999). the high levels of production and *corresponding author. e-mail: director@pibid.org. tel: +256772363271, +256312265789, +256414371050. fax: +256414340413. consumption of matooke notwithstanding, there are still high levels of malnutrition in uganda, with 50% of children fewer than five years in rural areas either stunted or underweight (ubos and orc macro, 2006). malnutrition is a condition resulting from an inadequate (under-nutrition) or excessive intake (over -nutrition) or impaired utilization of one or more nutrients in the body. there are two types of under nutrition, macro and micronutrient malnutrition. macro nutrient malnutrition in children occurs as protein energy malnutrition (pem) presenting clinically as kwashiorkor, marasmus or marasmickwashiorkor (waterloo, 1993). symptoms of kwashiorkor may include oedema, sparse brown hair, rashes, misery and apathy, while those of marasmas include retardation, under weight, absence of subcutaneous fat and wasting of muscles (cameron et al., 1983; latham, 1989). marasmic-kwashiorkor is a combination of kwashiorkor and marasmas symptoms (cameron et al., 1983). it has been observed that pem is often accompanied by vitamin and mineral deficiencies especially vitamin a and iron deficiencies. the underlying factors for malnutrition have been identified to include low energy density of weaning foods and lack of ready to prepare formulations, which lead to infrequent feeding due to the heavy workload of the caregivers especially among the rural poor (kikafunda, 1996). in response to the need for energy and protein dense foods, the national children’s rehabilitation centre (mwanamugimu) at mulago, the main referral hospital in kampala, uganda, developed a series of multi-mix recipes also called kitoobero from locally available foods. the recipes incorporate local starchy staples mainly matooke or rice. the mwanamugimu has developed a standard dietary regime which is code named mwanamugimu dietary regime (mdr) comprising a total of seven meals: one from porridge (with maize cereal as energy base) lunch and supper from kitoobero and in between four meals of a high energy milk (hem) with energy enhancement from oil. the porridge and hem diets are sweetened and supplemented by a complex mineral vitamin (cmv). the process of preparing maize porridge and kitoobero are, however, laborious and time consuming, therefore their rate of adoption by the caregivers, after discharge is limited. advances in processing of matooke have succeeded in reducing the relative bulk of matooke starch by pregelatinization (muranga, 1998 and muranga et al., 2007). muranga (1998) reported that matooke has high starch content ( db : dry basis 81.8 85.7%) and subsequently has a high potential as a calorie resource base. muranga et al. (2007) comprehensively reported on the effect of processing techniques on the bioavailability of matooke starch. further in the same study using mice, muranga et al. (2007) demonstrated the significance of available carbohydrate in the diet at the weanling stage of mice. the authors subsequently concluded that solubilisation of matooke starch through heat processing improved its bioavailability and enhanced availability of other nutrients in the diet. the pregelatinised cooked form of matooke has since been branded as instant tooke flour (itf). additionally, pilot studies revealed relatively high protein efficiency ratios (per), when itf fortified with soybean and sesame was fed to weanling rats (muranga et al., 2003). the above studies recommended testing of the pregelatinised flours in combination with high protein mixes in malnourished children in a clinical setting prior to adoption for application as a weaning food. in our first paper we established the optimal level of fortification of itf with soy and sesame as 65:16:19. the objective of this study therefore was to establish whether the pregel form of matooke, as itf brand, fortified at the above ratio with soy and sesame and micronutrients can serve as a vehicle food for malnutrition intervention using human subjects. two hypotheses were thereby tested in this part of the study namely: (1) there is a significant difference in response with respect to weight gain between subjects rehabilitated using fortified ift and mwanamugimu dietary regime (mdr). (2) there is a significant difference in response with respect to serum albumin, ferritin and retinol levels between fortified itf and mdr. methods study subjects a baseline of the age, sex, heights and weights was taken before commencing the study. table 1 shows the general characteristics of the study population. the study was carried out at the national children’s rehabilitation centre (mwanamugimu nutrition unit) in mulago hospital, kampala uganda, where 100 severely malnourished children aged 6 59 months were randomly allocated to either a test or control group of equal size using an open labeled randomized trial design. sample size was determined using a formula reported by kirkwood, (1988). generally age range was between 6-59 months of which 50% had kwashiorkor, 31% marasmus and 19% marasmic kwashiorkor. 55% were otherwise free from secondary infection while the rest suffered malaria or other secondary infections during the study. a blood sample was also taken from subjects during recruitment and before discharge. the test group was fed on fortified instant tooke flour (itf) diet, while the control group was fed on the mwanamugimu dietary regime (mdr). both diets were fortified with a therapeutic complex mineral vitamin (cmv) manufactured by nutriset malaunay, france. the diets consisted of three definite menu categories, porridge or uji, high energy milk (hem) and kitoobero (main menu) for both the test and control groups. the test porridge consisted of itf supplemented with soy and sesame (65:16:19), sugar and milk and fortified with cmv. the control was similar in formulation but had the itf composite replaced by maize flour. the high energy milk (hem) diet basically consisted of milk, sugar and cmv with the energy enrichment coming from ift for the test diet and from oil for the control. the kitoobero test diet (a multi-mix recipe cooked together in one pan) consisted of itf-soy-sesame formulation, salt and water while the control consisted of a triple mix of rice, fish powder and groundnut paste. the test and control groups were fed daily on their respective diet regime to provide calories ranging from 150 kcal to 220 kcal per day, with 150 kcal being the minimum. the calorie (energy) requirement given was calculated basing on the body weight of the subject, and this increased as the weight and appetite of the patient increased. according to who (2003), a vigorous approach is required to achieve very high intakes and rapid weight gain of >10 g gain/kg/d. the formula used in determining the quantity of feeds per meal for each subject is as shown below. each subject was given seven meals per day. amount of food served/meal = 150 kcal x body weight 7 (1) the feeding regime therefore required administration of porridge at 6 am, hem four times at 9 am, 3 pm , 9 pm and 12 midnight) where as kitoobero diets were administered for both lunch and supper at 12 noon and 6 pm respectively. meal times were observed by the table 1. general characteristics of the study population. characteristic test group (%) control group (%) overall (%) sex of child male 28 20 48 female 22 30 52 age (months) 6-12 13 14 27 13–24 34 25 59 25–36 4 7 11 37–59 0 3 3 malnutrition type kwashiorkor 23 27 50 marasmus 18 13 31 marasmic-kwashiorkor 10 9 19 secondary infection malaria 22 17 39 diarrhea 3 2 5 septic wound 0 1 1 no infection 24 31 55 occurrence of infection week 1 12 5 17 week 2 17 17 34 week 3 1 4 5 length of hospital stay 1 week 15 17 32 2 weeks 21 24 45 3 weeks 14 9 23 outcome discharged (blood withdrawn) 43 47 90 runaway 6 0 6 discharged (blood not withdrawn) 1 3 4 study nurse or investigator to avoid sharing of meals. every meal given to the subjects was weighed before feeding and the leftover food was also weighed to determine the actual intake of food by difference. the actual food intake by difference was calculated by subtracting the leftover food from the food served to each child and was used to calculate the protein (p) and energy (e) intakes per meal follows: protein intake per meal = food intake x protein content per diet (2) the energy intake was also calculated as in equation 2 while table 2 shows a summary of the calculations. the adequacy of the meal was benchmarked against the recommended daily allowance (rda) for the ages of the children used in the study. the adequacy was compared with respect to the percentage increase in protein and energy requirements over the standard rda. the increase was calculated as shown thus: % increase in rda for protein = actual protein intake (g/day) rda of age group for protein x 100 protein rda of age group (3) percentage increase in rda for energy was also calculated as in equation (3) but replacing energy for protein. the subjects were fed for a period ranging between 1 and 3 weeks depending on how long they took to attain the median weight or at least 85% of the median weight. this was determined by the initial weight, and secondary infections suffered, such as diarrhea and malaria. the discharge criteria included meeting the weight gain requirement as stated above and being on the study for at least two weeks (if the child had met the weight requirement and had been on the study for two weeks, they were discharged to avoid congestion due to limited space in the ward). however, the ability of the caretaker to adopt and maintain behavior change table 2. calculation of the energy and protein contents of the diets. group diet energy (kcal) calculation of energy intake for each child protein (g) calculation of protein intake for each child test porridge (itf + sesame +soybean + milk + sugar) 898.1 intake*898.1/800 26.3 intake*26.3/800 kitoobero (itf + sesame + soybean) 1004.2 intake*1004.2/700 22.2 intake*22.2/700 high energy milk (hem) 1002.02 intake*1002.02/1000 43.09 intake*43.09/1000 (milk + itf + sugar) control porridge (maize flour + milk + sugar) 837.0 intake*837/1000 28. 5 intake*28. 5/1000 kitoobero (rice + g/nuts + fish powder) 1178.4 intake*1178.4/780 39.78 intake*39.78/780 high energy milk (hem) (milk + oil + sugar) 1006.8 intake*1006.8/1000 38 intake*38/1000 250 test control 200 in ta k e 150 f o o d 100 50 0 porridge intake kitoobero intake hem intake meals served figure 1. comparison in food intake per meal between the test and control group. learning how to feed the child) was taken into consideration in order to avoid a relapse of malnutrition. serum albumin, ferritin and retinol analyses the serum albumin content was determined using bromo cresol purple (bcp) method a who standard method while, ferritin con-tent was determined using a photometric method by a procedure as reported by tietz (1987). retinol content was determined using colorimetric method based on carr-price reactions using trichoro acetic acid as reported by underwood and stekel (1984). data analyses all the data was analyzed using spss version 11. the analysis for each response variable was based on a three factor analysis of variance model in which the groups were one of the factors. for each type of meal there was a three factor anova which included group (test versus control), age and time (length of stay on study). the response variables included protein and energy intake, weight gain, serum albumin, ferritin and retinol levels. results food intake figure 1 shows the comparison in food intake per meal between the test and control groups. the tests here for itf include itf -soy-sesame vs. maize flour in porridge, itf vs. oil in hem, and itf-soy-sesame vs. rice and groundnut in the kitoobero diets. there was no significant differrence (p > 0.05) between the test and the control groups in the intake of porridge, kitoobero 12 test control 10 (g ) 8 in ta k e 6 p ro te in 4 2 0 porridge kitoobero hem figure 2. comparison in protein intake per meal between the test and control group. and hem. however, in all cases, the control group took in more food compared to the test group. protein intake figure 2 shows the comparison in protein intake per meal between the test and control groups. there was a significant difference (p<0.05) between the test and control groups in the protein e n e rg y i n ta k e ( k c a l) 350 test 300 250 200 150 100 50 0 porridge control kitoobero hem figure 3. comparison in energy intake per meal between the test and control groups. table 3. comparison of adequacy of test (itf) and control (mdr) diets for protein and energy with recommended dietary intake (rda) for the respective subjects in the study. age gp protein (g/day) energy (kcal/day) test gp increase in rda (%) control gp increase in rda (%) test gp increase in rda (%) control gp increase in rda (%) 6-11 16.41 49.18 16.98 54.36 596 -13.96 535.86 -22.64 12-23 17.15 31.92 20.2 55.38 616.14 -39.94 630.79 -38.51 24-35 19.16 47.38 21.07 62 684.54 -33.28 659.99 -35.67 36-59 30.31 78.29 960.02 -34.01 intake for porridge. there was indeed a significant difference (p < 0.05) between control and test groups in the protein intake for kitoobero but no significant difference (p > 0.05) in the protein intake for hem. energy intake figure 3 shows the comparison in energy intake per meal between the test and control groups. there was a significant difference (p < 0.05) between the test and control groups in the energy intake for porridge but no significant difference (p > 0.05) in the energy intake for kitoobero and hem. the test porridge was therefore better than the control porridge with respect to energy, which credits the ift formulation, but the energy intake for kitoobero and hem for the two groups was comparable. table 3 shows the relative adequacy of the food intake for the different subject groups against standard recommended daily allowance for normal subjects (national academies press, 2005). the adequacy was compared with respect to the percentage increase in protein and energy requirements over the standard rda. there was no significant (p > 0.05) difference between the test and the control groups within the different age groups. except for age 12 23 months the food intake for both the test and control diets was test control w e ig h t g a in ( g /k g b o d y w e ig h t/ d a y figure 4. comparison in weight gain between the test and control groups over study period. adequate for protein since it was close or well above the 50% recommended increase for subjects under rehabilitation. energy intake was however, low for both the test and control groups since all the percentages were negative and therefore far below the 10% recommended energy increase for such subjects. given the overall adequacy of the protein as per table 2 against the rda values for the respective age groups, focus of the test is therefore on the performance of the energy sources in the test vs. the control diet. weight gain figure 4 shows the comparison in weight gain between test and control group over the period of study. there was a significant difference (p < 0.05) in the weight gain between the test and control groups over the period of study. both the test and control groups had a progressive gain in weight in the first two weeks. however, there was a slight decrease in weight gain for the control group in the third week. the difference in weight gain between the test and control group was particularly significant in the third week as it shot up for the test group but decreased for the control group. figure 5 shows the comparison in weight gain between the test and control groups by age groups over the period of study. there was no significant difference (p > 0.05) in weight gain by age group over the period of study between the test and control groups. however, the test group in the 6 11 months and the 24 – 35 months age groups, had a higher weight gain compared to the control group in week 1, 2 and 3. the 12 23 months age group had a comparable weight gain for both groups in week 1, in week 2, the control group had a higher weight gain, while in week 3, the test group had a higher weight gain. the test group also had a higher weight gain in the 36 59 months age group in week 1 and 2. blood serum levels serum albumin: there was an increase in the serum albumin levels for both groups. the test group had an increase from 31.095 36.137 g/l while the control group had an increase from 27.953 34.148 g/l. however, there was no significant (p < 0.05) difference between the test and control groups in increase in the serum albumin levels over the period of study, implying that the two groups did not respond differently from each other. serum ferritin: the test group had an increase in the serum ferritin level from 13.447 16.056 µmol/l while the control group instead had a drop in the serum ferritin level from 15.169 14.704 µmol/l. however, there was no significant difference (p > 0.05) between the test and control groups over the period of study implying that the two groups did not respond differently from each other. serum retinol: the test group had an increase in the test control figure 5. comparison in weight gain between the test and control groups by age groups over study period without outliers. key: 1 = 6-11 months, 2 = 12-23 months, 3 = 24 – 35 months and 4 = 36 – 59 months. serum retinol level from 5.197 7.421 g/dl, while the control group had a slight drop in serum retinol level from 6.320 6.299 g/dl. however, there was no significant difference (p < 0.05) between the test and control group over the period of study, implying that the two groups did not respond differently from each other over the period of study. discussion the data revealed the relative energy inadequacy of both the test and control diets. further the fact that the food intake for the test group though not statistically significant was less compared to the control group is unfortunate for the instant formulation since it is indicative of a significant barrier to energy emanating from inherent bulk in the itf. in muranga et al. (2009) the end viscosity of the itf-soysesame optimal formulation was set at 191 rvu compared to that 423rvu for pure itf which was approximately 50% reduction in viscosity due to incorporation of the protein mixes. therefore whereas pre gelatinisation of the matooke served the reduction of the bulk to a certain extent further reduction in bulk requires to be effected to enable children in this disadvantaged health state to consume enough amounts to meet their high energy requirements. to this end muranga et al. (2007) proposed extrusion cooking, which yields an instant flour that is virtually soluble in warm water. the above observations are however, consistent with a previous study reported by brown et al. (1995) and bennet et al. (1999) where poor food intake was attributed to the low energy density of the control diet (brown et al., 1995; bennet et al., 1999). generally lower glycaemic and the correspondingly high satiety responses are associated with high amylose starches (fao, 1988) . matooke starch, however, is a low amylose starch, but has been associated with heat mediated transformations that could affect the endotherm values (gelatinization transition temperature) and therefore the pregelatinisation process (muranga, 1998) particularly given that the method of gelatinization was a slow steam operation. that both diet regimes met the rdas for protein is a fact that can be in part attributable to the milk supplementation in both diet regimes but particularly in the control. the differences in the protein intake between the test and control group for the kitoobero meals underscores the significance of animal protein in the diet. the control group consumed animal protein from the dried silver fish also known as ‘mukene’, whereas the test group only consumed plant protein (soybean and sesame) as shown in tables 2 and 3. on the contrary lack of significant differences in the protein and energy intake for hem after substitution of oil for itf is significant for the itf as a vehicle for protein/energy supplementation since oil is a more expensive ingredient. that diets for both the test and control groups did not meet the recommended daily allowance (rda) for energy is consequent to the carbohydrate bulk barrier in the control mdr from the maize porridge and the rice kitoobero. the reason for the ift regime is already high lighted above under nutrient adequacy. the differences observed between the test and control in energy intake with respect to porridge is therefore attributed to the relatively higher energy density and the low viscosity of the ift (kanyago et al., 2009). capdevila et al. (1998) reported volume intake and energy density to be one of main factors influencing energy intake. it is, however, important to note that the rda is calculated for a normal population and the study population was an abnormal one for which management was being done in order to restore them to normalcy. the calculation for energy requirements per meal was based on the weight of the individual subjects and this increased as the weight of the subject increased. the short fall in energy intake notwithstanding, our results served to demonstrate that the ift formulation was a superior to the mdr as vehicle for malnutrition intervention which is evidence of a superior bioavailability of energy from the ift formulation in contrast to the control. muranga et. al. (2007) indeed reported on this availability after testing growth responses of mice on matooke flour formulations. indeed the progressive gain in weight for the test group was commensurate with other studies conducted to rehabilitate severely malnourished children. these studies generally report a higher weight gain for the children receiving high-energy-density porridge compared to those receiving low-energy-density porridge (donmen et al., 1996; bennet et al., 1999). catch-up growth represents accelerated tissue gain beyond that expected for chronological age; it occurs after a period of slowing or cessation of growth. protein and energy may influence both normal and catch-up growth (tanner, 1989). the test diet regime was therefore able to sustain both the normal and catch growth for the subjects throughout the period of study. this is consistent with what was reported by uauy and alvear (1991), that weight loss is usually fully reversible, since weight gain is mainly affected by energy supply in relation with energy expenditure. nonetheless, the excellent performance in weight gain for the test group in only in the third week is indicative of a requirement for a more available source of carbohydrate at the initial rehabilitation phase in malnutrition similar to that in the weaning stage of animals as reported by muranga et al. (2007). the control diet was able to some extent in the first week to serve a lower catch up growth but in the second week it fell under negative energy balance and hence negative catch up growth which is unfortunate given the intensity of labour and good will associated with its administration to date. the change in blood serum levels for albumin, ferritin and retinol for both the test and control groups was not significant. albumin is a hepatically synthesized protein frequently used to estimate overall nutrition status (marinella and markert, 1998), while serum ferritin and retinol are used to determine iron and vitamin a status respectively. serum albumin for both groups was within the normal ranges at the end of the study. the normal levels range from 35 50 g/l (latham, 1997) . the initial serum ferritin levels for both groups were within the normal range, which is between 9.0 30.0 mol/l (tiez, 1987). this could be attributed to infection, inflammation, liver disease, and states of increased rbc turnover (e.g. ineffective erythropoiesis and hemolysis), which can falsely elevate these levels (lanzkowsky, 2000). also there were instances of hemolysis due to resistance from the study subjects during withdrawal blood samples. despite the high reliability of low concentrations of ferritin in plasma or serum as an indicator of iron deficiency, concentrations in the normal range in iron deficiency are not uncommon (garibay et al., 2001). the same author also reported iron deficient subjects to be having plasma ferritin concentrations within the normal range. the drop in serum ferritin level for the control group is not easy to explain and is an area for further study. the serum retinol results for both groups were below the normal levels. blood-serum retinol concentrations of below 20 g/dl signify marginal deficiency and that below 10 g/dl signify definite deficiency (lindblad et al., 1998; saskia and dary, 2002). the control group also had a drop in the serum retinol levels like in the serum ferritin. this also is an area for further study. serum retinol concentration reflects an individual’s vitamin a status, particularly when the body’s reserves of vitamin a are limited, because serum retinol concentration is homeostatically controlled and will not drop until body stores are significantly compromised (olson, 1994) . however, serum retinol concentration is also affected by factors that affect release of holorbp from the liver, infection, protein status, adequacy of other nutrients and organ disease. in general, these factors lower serum retinol concentration (olson, 1994). overall the results of the biochemical indicators in this study showed that they were a less reliable benchmark for monitoring rehabilitation of malnourished children. the major limitation to this study was time and financial constraints. however, the cost effectiveness issues are being covered in the follow-up study. however, the itf preparation is a simple technology in which the government of uganda has invested to ensure effective technology transfer to all matooke growing communities in the country. summary our results demonstrated that there is a significant difference in response with respect to weight gain between subjects rehabilitated using fortified itf and mwanamugimu dietary regime (mdr) so hypothesis one was accepted. further we also evidenced that there was no significant difference in response with respect to serum albumin, ferritin and retinol levels between subjects rehabilitated on fortified itf and those on mdr and so hypothesis two was not accepted. the results further justified the significance of energy density over protein intake in protein energy malnutrition rehabilitation. we therefore recommend exploring technology for further solubilisation of matooke flours in order to optimize energy density. acknowledgement this research was possible through funding from makerere university through the african institute for capacity development (aicad) grant. we acknowledge prof. kakitahi, j.t. of institute of public health medical school makerere uiniversity and dr. kiboneka, e.k. of mwanamugimu nutrition unit mulago hospital for their input in this study. we also acknowledge the department of food science and technology, makerere university, mwanamugimu nutritional unit, mulago hospital for infrastructural support and the presidential initiative on banana industrial development (pibid) for providing the final infrastructure for writing and publication of this work. references bennett va, morales e, gonzález j, peerson jm., romaña gl and brown kh (1999). effects of dietary viscosity and energy density on total daily energy consumption by young peruvian children. am. j. clin. nutr,. 70: 285-291. brown kh, grinan sm, perez f, peerson jm, ganoza l and stern js (1995). effects of dietary energy density and feeding frequency on total daily energy intakes of recovering malnourished children. am. j clin. nutr., 62:13-18. cameron m, hofvander y. (1983). manual for feeding of infants and young children. 3 rd ed. oxford university press, pp. 51-66. capdevila f, vizmanos b, and henneberg cm (1998). implications of the weaning pattern on macronutrient intake, food volume and energy density in non-breastfed infants during the first year of life. . am. col. nutr., 17; 3:256–262 fao (1998) carbohydrate in human nutritionfao food and nutrition paper66. report of a joint fao/who expert consultation, rome 1418 april 1997, isbn 92-5-104114-8. garibay e, torres is, nelson se, ziegler ee, rogers rr, janghorbani m, fomon sj (2001). iron absorption during recovery from malnutrition. am. col. nutr. 20(4): 286-292. kanyago m, muranga fi, nabugoomu f, ntambi jm (2009). optimal level of fortification of instant matooke flour porridge and its nutritional quality attributes. accepted may, 2009 for publication in the african j. food sci. karugaba a, kimaru g (1999). banana production in uganda. an essential food and cash crop. relma technical handbook no. 18. regional land management unit, relma/sida, icraf house, nairobi, kenya. pp. 1-2. kikafunda-kakuramatsi j (1996). dietary risk factors for childhood malnutrition in uganda. phd thesis, university of reading, reading, uk. pp10-15. kirkwood br(1988) essentials of medical statistics, blackwell scientific publications. lanzkowsky p (2000). classification and diagnosis of anemia during childhood. in: lanzkowsky p (ed.) manual of pediatric hematology and oncology, 3 rd ed. academic press, san diego, new york, london, pp. 1-12. latham mc (1997). human nutrition in the developing world. food and nutrition series no. 29 food and agriculture organization of the united nations. http://www.fao.org/docrep/w0073e/w0073e05. htm#p3172_360093. accessed 2005 march 17. lindblad bs, patel m, hamadeh m, helmy n, ahamad i, dawodu a, zaman s (1998). age and sex are important factors in determining normal retinol levels. j. trop. ped., 44:2: 96-99. muranga fi (1998). composition and physicochemical characteristics of starches of different banana varieties. phd thesis, makerere university kampala, uganda. pp. 23-36. muranga fi, nampala p, bukusuba j (2003). investigation of potential of low bulk matooke preparations as vehicle foods for malnutrition interventions ii, poster presentation at the workshop for the launching of ibmu project. 1 st oct., 2003. muranga fi, sampath h, marlette ja, ntambi jm (2007). impact of processing technique on the apparent bioavailability of cooking banana (matooke) afri. j. biochem. res 1(5): 072-077. marinella ma, markert rj (1998). admission serum albumin level and length of hospitalisation in elderly patients. south. med. j., 91; 9: 851854. national academies press (2005). dietary reference intakes for energy, carbohydrates, fiber, fat, fatty acids, cholesterol, protein, and amino acids. a report of the panel on macronutrients, subcommittee on upper reference levels of nutrients and interpretation and uses of dietary reference intakes, and the standing committee on the scientific evaluation of dietary reference intakes. pp 168-191 olson ja (1994). vitamin a, retinoids and carotenoids. in shils et al (eds) modern nutrition in health and disease 8th ed. lea & febiger philadelphia, pa. pp. 287-307. saskia dp, dary o (2002). biochemical indicators of vitamin a deficiency in: serum retinol and serum retinol binding protein supplement: proceedings of the xx international vitamin a consultative group meeting. the american society for nutritional sciences j. nutr., 132: 2895s-2901s. tanner jm (1981). catch up growth in man. br. med. bull 37: 233-238. tietz wn (1987). fundamentals of clinical chemistry 3 rd edition. w. b. saunders company philadelphia, london, toronto, mexico city, rio de janeiro, sidney, tokyo, hongkong. p. 958. uauy r, alvear j (1991). effects of protein-energy interactions on growth in: protein-energy interactions nestle foundation i/d/e/c/g steering committee http://www.unu.edu/unupress/food2/uid07e/ uid07e00.htm. accessed 26 april 2005 ubos, orc macro (2006). uganda demographic and health survey 2006. calverton maryland usa: ubos and orc macro, pp 21-23. waterloo (1993). relationship of gain in height to gain in weight. eur. j. clin. nutr., 48. who (2003). guidelines for the inpatient treatment of severely malnourished children. searo technical publication no. 24. p. 48. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 11 (3), pp. 001-004, march, 2023. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of some bacterial isolates as germination stimulants of striga hermonthica o. o. babalola1* , d. k. berner2 and n. a. amusa3 1 department of microbiology, olabisi onabanjo university, ago-iwoye, ogun state, nigeria 2 plant health management division, international institute of tropical agriculture, ibadan, nigeria. present address: united states department of agriculture, agricultural research service, foreign disease-weed science research unit, frederick, md 21702, usa. 3 department of plant science and applied zoology, olabisi onabanjo university, ago-iwoye, ogun state, nigeria accepted 09 december, 2022 to investigate the role of pseudomonas sp., klebsiella oxytoca and enterobacter sakazakii in striga hermonthica seed germination, we have used two varieties of sorghum to test over time for the selected bacteria, using gr-24, a synthetic strigol analogue as a standard and water as a check. sorghum seeds coated with a mixture of bacterial cells (5 x 10 7 cfu ml -1 ) from 24 h old cultures were planted in pasteurized potted soil, which was infested with 0.05 g (about 3000) viable s. hermonthica seed per pot 14 d before sowing. results have indicated that the bacterial isolates could stimulate s. hermonthica germination in the laboratory and in the screen-house. in vitro, only pseudomonas sp. 4mks8 gave significant stimulation of s. hermonthica seed at 5% level of probability as compared to the water check. in the screen-house, the four bacterial isolates stimulated significant germination of s. hermonthica. key words: striga hermonthica, pseudomonas sp., klebsiella oxytoca, enterobacter sakazakii, germination stimulant, sorghum. introduction in the sub-saharan africa, it is a common occurrence for the plants to be attacked by one pathogen or another. one of such pathogens attacking plant is striga, a genus of obligate root-parasitic flowering plant belonging to the family scrophulariaceae. striga species constitutes a big constraint to sustainable development and poverty alleviation. the main graminaceous crops attacked by striga hermonthica include maize, sorghum, pearl millet, finger millet, upland rice and sugar cane (berner et al., 1995). in addition to the conditions necessary for the germination of any seed, striga requires the presence of a stimulant. today many farmers lack an understanding of its biology and parasitic nature and so cannot manage striga in the field. researchers have come to understand that no single methodology will be completely effective in eliminating striga infestations in farmer’s fields. meanwhile research efforts by scientists employ multidisciplinary approach in the combat of striga. microorganisms are in *corresponding author. e-mail: olubukola_babalola@yahoo.com. tel: +2348058871359. creasingly being considered as control agents for striga (babalola et al., 2002; babalola et al., 2004) . among the microorganisms colonising the root surface are bacteria of the genus pseudomonas (babalola, 2002) of which fluorescent pseudomonads are of special importance (vancura, 1980). the composition of the microbial association on the root surface can be modified by the introduction of bacterial cells on the surface of seed, roots or tubers. plant growth-promoting rhizosphere bacteria possess a number of physiological properties, which are important to colonisation of the surface of plant roots and enhancing the growth and health of plants (vancura and kunc, 1987). several methods are available to control striga including cultural practices, application of fertilizers, herbicides, and germination stimulants, and planting resistant crop varieties. however, none of these methods alone provides acceptable levels of control (abbasher et al., 1996) . therefore, the objective of this study was to increase suicidal germination of striga by using bacteria inoculants in mixed cropping of cowpea with sorghum. 2 materials and methods isolation of rhizosphere bacteria root samples of potted maize and sorghum were shaken vigorously by hand to remove loosely adhering soil. bacteria from the region of soil neighbouring the root and influenced by it (exorhizosphere, klyuchnikov and kozhevin, 1991) and intercellular spaces accessible to bacteria (endorhizosphere) were obtained from 1 g firmly adhering soil or macerated roots (mawdsley and burns, 1994). 1 g of firmly adhering soil was shaken in 100 ml of 0.01 m phosphate buffer solution (pbs) solution (0.88% (w/v) nacl, 2.9 mm kh2po4, 7.1 mm k2hpo4, ph 7.2) for 2 h. on a rotary shaker (orbit shaker bath, u.s. patent no: des. 288.600, u.s.a) at 200 rpm. surface sterilisation effected by immersion in 70% ethanol for 30 s. and in 4% solution of sodium hypochlorite (naocl) for 3 min. was carried out on the root sample to ensure that the isolates to be cultured were from the endorhizosphere. these were transferred to the laminar flow hood (envirco, environmental air control, inc.). subsequently, serial dilutions (up to 10 –8 ) were prepared, beginning with a 1 ml aliquot of the stock suspension. one ml of the suspensions from the dilution levels 10 -3 to 10 -8 was dispensed in separate 9 cm diameter sterilised petri-dishes containing king’s medium b (kb) in triplicate. the plates were swirled gently in a clockwise motion to mix the suspension over the agar. after incubation for 24 h at 28 o c, representative types of bacterial colonies were further purified on kb and stored in 35% glycerol at -80 o c. one hundred and eighty isolates of rhizosphere bacteria were screened in preliminary laboratory tests for in vitro stimulation of striga. hermonthica. four potential isolates, (isolates in which s. hermonthica germination was observed) were further tested in the screen-house by seed coating. s. hermonthica germination test striga seed viability was carried out according to the method of eplee and norris, (1987). variability in infection, which may be due to differences in age and quality of inoculum, hosts, growth conditions, and levels of infestation was minimised by using the same seed stock of s. hermonthica, bida 97. these seed were collected from the same site (bida, latitude 9 0 05’, longitude 6 0 01’) and host (sorghum, cultivar unknown) in the same year (1997), hereafter referred to as s. hermonthica bd 97. besides, the seeds were subjected to uniform conditioning. the s. hermonthica seeds were characterised by a high germinability response to the synthetic strigol analogue, gr-24. all glassware and forceps were sterilised prior to pre-germination tests. the parasite seed were surfacedisinfected for 5 min. in a 1% naocl. floating seeds were discarded. the seeds were then thoroughly rinsed in three changes of sterile de-ionised water inside a funnel lined with whatmann no. 2 filter paper (whatman ® whatman int. ltd, maidstone, england), airdried over a clean surface, and stored in glass vials at 28 o c. to condition, the dried s. hermonthica seeds were sprinkled on 3 mm diameter glass-fiber (gf/c, whatman ® whatman int. ltd, maidstone, england) discs (30 40 seed/disc) that were placed on moistened filter paper in petri-dishes. these were placed in an incubator (gallenkamp, uk) in the dark at 28 o c for 14 d. after incubation, the glass-fiber discs with the conditioned s. hermonthica seeds were removed from the petri-dishes and placed in clean 9 cm diameter petri-dishes. the discs were placed around a 2 cm diameter sterilised film canister filled with tempered potato dextrose agar (pda) (difco) medium, centred in the petri-dish, on freshly moistened filter paper. the discs were arranged in four radii forming a “cross” radiating from the film canister. each line had four discs, with the first disk in each line touching the central canister. pure inoculums of the bacteria to be tested were aseptically streaked on the pda. in negative controls, 300 µl of sterile deionised water was pipetted over uninoculated pda as a substitute for ba cterial inoculum. in the positive control, one parts per million (1 ppm) gr-24 was used. these were then incubated at 28 o c for 3 d and the percentage germination of s. hermonthica seeds on each disk was counted. average percentage germination for each line of disks in each dish was calculated. screen-house experiments plantings were done in screen-house potted soil infested with s. hermonthica. soil sterilisation was done at 100 0 c for 4 h in lindig (boiler) machine (model ‘’sf’’ burner, mp1192, r.w. beckett corp. elyria, ohio, u.s.a). for the infested soil, s. hermonthica was added at a concentration of 500 seeds per 4 kg potted soil. the experimental site was at the international institute of tropical agriculture (iita) ibadan in oyo state, latitude 7 0 43’n longitude 3 0 9’e nigeria. seeds of susceptible sorghum (varieties ck60b and mokwa local) obtained from collections maintained at iita were treated with bacterial isolates by pelleting with a mixture of bacterial cells from 24 h. old culture (5 x 10 7 cfu ml -1 that was suspended in 1% methylcellulose (mc)). mc is effective as a bacterial preservative and does not significantly influence seed germination (suslow and schroth, 1981). finely ground vermiculite, capable of passing through 300 mm mesh sieve was wrapped in aluminum foil and sterilised in the autoclave at 121 o c for 15 min. the vermiculite added to freshly inoculated wet seed in a beaker and mixed quickly for 2 min. until seed were evenly coated. the coated seed appeared as off-white and were allowed to dry overnight by spreading on a clean surface prior to sowing in sterilised potted soil infested with s. hermonthica bd 97. the sorghum seeds were sown immediately; and where this could not be done, the seeds were stored for not more than 3 weeks at low temperatures (4 o c) in a refrigerator. seed, which were pelleted with a mixture, which did not contain bacterial cells (mc + powdered vermiculite), served as control. statistical analysis means of percentage germination were calculated for each petridish (average of four lines) of each bacterium in each test. the data were analyzed using the univariate procedure of sas (statistical analysis system, sas institute, cary, nc) to generate raw means and standard error. test of the null hypothesis that the germination stimulated by each isolate equaled zero was done by employing the univariate procedure. analysis of variance for each isolate was done by the general linear model procedure of sas. experiments were repeated four times with four replicates each. results isolation of rhizosphere bacteria forty isolates of bacteria were obtained from the rhizosphere of maize variety 8338-1 and 140 from rhizosphere of two cultivars of sorghum (ck60b and mokwa local) grown in the screenhouse at iita ibadan. isolations were made from the exorhizosphere and endorhizosphere at biweekly intervals by destructive sampling. the bacterial isolates were evaluated for capability to stimulate germination of s. hermonthica bd 97. in a series of laboratory bioassays, a fluctuating (irregular) germination trend was observed in the s. hermonthica seeds as caused by the bacterial isolates. some of the isolates were tentatively identified as fluorescent pseudomonads based on their water-soluble, 3 table 1. germination of s. hermonthica seeds from a population of sorghum plants from bida, nigeria, stimulated by selected rhizosphere bacteria. bacteria % germination of s. hermonthica seeds x mean y pr>t dish 1 dish 2 dish 3 dish 4 pseudomonas 10mkr4 1.751 0.001 0.001 0.001 0.4378 0.7591 z k. oxytoca 10mkr7 2.351 3.701 0.001 3.381 2.3586 0.1066 pseudomonas 4mks8 10.611 0.001 0.001 1.591 3.0510 0.0398 ent. sakazakii 8mr5 1.211 0.001 2.061 0.001 0.8185 0.5675 gr-24 72.801 57.201 68.101 61.891 64.9985 0.0001 water 0.001 0.001 0.001 0.001 0.0010 1.0000 std error 1.41 lsd 5% 2.59 prob. of f 0.0001 coef. of variation 37.25 n = 16 for each day gr-24 = +ve control (strigol-analogues, a synthetic germination stimulant) water = -ve control x means based on four replications; each conducted on a separate dish; values presented are arcsine transformed. y germination least squares means for mean percent germination z probability of a greater t value, under the null hypothesis that the mean equaled zero, based on percentage germination data. greenish-yellow fluorescent pigment when examined under the ultraviolet light. the standard identification system of ‘appareils et procedes d’identification’ (api) technique (api systems, biomerieux, sa, france) was used for biochemical tests. in in vitro experiment, isolates, enterobacter sakazakii 8mr5, pseudomonas sp. 4mks8 and 10mkr4, klebsiella oxytoca 10mkr7 were found to be promising rhizosphere bacteria that could stimulate the germination of s. hermonthica (table 1). pseudomonas sp. 4mks8 stimulated the germination of seed of s. hermonthica by 3%. the percentage germination of s. hermonthica seed induced by all the isolates (table 1) was weak. consequently, they were tagged low-level germination isolates. k. oxytoca 10mkr7 stimulated the germination of s. hermonthica seed to a limited degree, while the strigol analogue gr-24 induced effective germination in all replications with an average of 65% germination at a concentration of 1 ppm. germination of s. hermonthica in water (control) was 0% in all tests. it was observed that germination percentages obtained with gr-24 solution vary from test to test despite the standard procedure used. screen-house experiments the incidence of striga plants over 9 week duration was recorded (table 2) . striga emergence was significantly stimulated over the control (water check). throughout the entire experimental period, s. hermonthica emergence on the control pots was used as the standard for comparison. percentage striga emergence over the control pots showed that seed treatment with pseudomonas sp. 10mkr4 gave the highest number of emerged striga. this happened to be the least stimulant producer in the laboratory experiment followed by pseudomonas sp. 4mks8. laboratory findings of pseudomonas sp. 4mks8 and e.sakazakii 8mr5 agreed with their screen-house findings. discussion this study demonstrates that there are some potential in certain rhizosphere bacteria to induce germination of s. hermonthica. this is in accordance with the observations of babalola and berner (2004) on length of the afterripening period for s. hermonthica. the fact that the seeds were germinable, indicated that the synthetic strigol, gr24, induced effective germination of s. hermonthica seeds. the low stimulation of s. hermonthica by the bacterial isolates may be explained by the diffusion rates of the stimulant produced by the isolates. however, from the data, it was clear that gr 24 is more effective than all the bacterial isolates in s. hermonthica seed germination stimulatory activity. that notwithstanding, the need for indigenous microbes in the integrated control method can not be overemphasized. the isolates tested could be low stimulant producers, such that, even statistically only stimulant from pseudomonas sp. 4mks8 is significant in the laboratory trial, but it is also possible that the seeds of the strain of s. hermonthica bd 97 do not respond to the isolates. germination stimulant which are specific for s. hermonthica strains have been reported by king and zummo (1977) and bebawi (1981). the isolates so far obtained were identified as low stimulant producers (babalola et 4 table 2. stimulation of s. hermonthica as influenced by different rhizosphere bacteria applied by pelleted treatment on susceptible host plant sorghum (var. mokwa local). selected bacterial isolate used average number of highest striga count highest striga count as % in seed treatment emerged striga per pot x per pot of count in water check uninoculated control 8.2 14.1 100.00 pseudomonas 10mkr4 15.0 30.3 214.89 k. oxytoca 10mkr7 13.9 25.5 180.85 pseudomonas 4mks8 13.4 28.0 198.58 e. sakazakii 8mr5 12.6 27.4 194.33 lsd (0.05) 3.68 cv (%) 36.3 x the soil was infested with 0.05 g striga (about 3,000 germinable seeds out of about 10,000 total number of seeds) seed pot 1 before sowing of sorghum seeds. et al., 2006). when stimulant activity was tested, in both laboratory and pot experiments, the percentage germination effected by the stimulatory action of the bacteria was much lower than could be recommended for s. hermonthica control without any other control method. the purpose of this development was to determine the bacterial stimulants for s. hermonthica, to release germination stimulants into the rhizosphere, and thereby further inducing the germination of s. hermonthica and consequently depleting the striga seed bank when applied during trap cropping. these organisms that stimulate s. hermonthica seed germination may probably explain striga decline in those sites for which there is no plausible explanation. acknowledgement babalola o. o. had the visiting fellowship of the international institute of tropical agriculture (iita), ibadan, nigeria. references abbasher aa, hess de, sauerborn j, kroschel j (1996). biological control of striga. paper presented at the icrisat sector review for striga control in sorghum and pearl millet, 27-28 may 1996, 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this article. full length research paper survival of bifidobacterium pseudocatenulatum g4 during the storage of fermented peanut milk (pm) and skim milk (sm) products barka mohammed kabeir1, abdul manap yazid1, muhammad nazrul hakim2, ali khahatan1, anis shaborin3 and shuhaimi mustafa4* 1 department of food service and management, university putra malaysia, 43400, selangor, malaysia. 2 department of biomedicine, faculty of medicine and health sciences, university putra malaysia, 43400, selangor, malaysia. 3 department of food science, faculty of food science and technology university putra malaysia, 43400, selangor, malaysia. 4 halal products research institute, university putra malaysia, 43400, selangor, malaysia. accepted 08 march, 2019 survivability of bifidobacterium pseudocatenulatum g4 in fermented peanut milk (pm) and skim milk (sm) directly or supplemented with fructooligosacharide (fos) and human grade yeast extract was evaluated under refrigeration and 25°c for a period of 2 weeks. during the study period, the development of lactic acid and short chain fatty acids (acetic, propionic and butyric) were also examined. the fermented products were prepared utilizing inoculum of 0.67% and anaerobic incubation under control conditions using 1 l volume bioreactor to final ph less than 5. initial mean viable number of strain g4 ranged between 7.12 8.35 cfu/ml fermented pm and 7.28 8.39 cfu/ml fermented sm based products. the viable strain g4 in fermented samples held at 25°c for 2 weeks decreased to a level of < 7 log cfu/ml due to the ph reductions, which were explained by increases in lactic, acetic, propionic and butyric acids. this final viability of the fermented products is below the requirement to claim probiotic effects upon their consumptions, while at refrigeration (4°c) storage, the viability maintained in fermented products was higher exceeding the mean recommended level of 7 log cfu viable probiotic per ml product generally. therefore, fermented pm products especially supplemented with fos containing strain g4 could maintain probiotics effects under refrigeration for a period of 2 weeks. key words: bifidobacterium, storage, survival, organic acids, peanut milk. introduction bifidobacterium is the predominant species of human colonic and faecal microbiota. it has been extensively introduced as probiotics in the food industry and pharmaceutical applications (guarner and malagelada, 2003). probiotic bacterium is defined as living microbial supplements, which modify intestinal microbiota balance to improve health beyond inherent basic nutrition (fullar, 1989; schrezenmeir and de vrese as, 2001). the positive health effects emanating from consumption of probiotics at recommended dose of 10 10 live cell *corresponding author. e-mail: shuhaimi@biotech.upm.edu. my. tel.: 603-8946 8402. live cell per include, improving lactose digestion, strengthening antimicrobial activity against pathogens, shortening the duration of diarrhoea, contributing positively to nutrition, stimulating the immune system and preventing and curing intestinal diseases (marteau et al., 2002). these claimed benefits have recently stimulated researches in isolation and screening of new strains for probiotics purposes (prasad et al., 1999; fooks and gibson, 2002; frece et al., 2005). however, examination carried out by adikhari et al. (2003) and temmerman et al. (2003) have revealed poor presence or complete absence of bifidobacterium in commercially claimed yogurt preparations. at present, maintaining viability of bifidobacterium until the products are consumed is essential to ensure the delivery of live microorganisms to consumers. it is recommended that probiotics food should contain at least 6 8 log cfu a live probiotic/ml of the product at the time of consumption (schuler-malyoth et al., 1968). in this experiment, alternative medium of peanut milk was evaluated for survivability maintenance of b. pseudoocatenulatum g4 during the storage of fermented products, compared with that on conventional skim milk (sm) based. further subsequent organic acids profiles of fermented products under storage were also elucidated. peanut milk (pm) could be a potential delivery means for probiotic strain g4. it is characterized as a source of high vegetable proteins, low in cholesterol, thus has attracted much attention from consumers. because it is also extremely rich in minerals and essential fatty acids, such as linoleic and oleic, which are highly valuable in human nutrition (diarra et al., 2005). b. pseudocatenulatum g4 previously has been screened for probiotic purposes (shuhaimi et al., 2001; shuhaimi et al., 2002). the strain found to survive the harsh gut environments (wong et al., 2006), safe in in vitro application to mice (kabeir et al., 2008a; 2008b), and able to utilize prebiotics in growth medium particular, fructooligosaccharides (fos) (shuhaimi et al., 2009). materials and methods preparation of fermentation mediums peanut beans were cleaned, washed with distilled water and soaked for 8 h at 45°c using a temperature controlled water bath. the beans were then hand hulled and blended with 1 l stainless steel warning commercial blender at medium speed for 3 min. the obtained slurry was filtered through doublelayered cheesecloth to prepare the peanut milk (pm), which was used directly or supplemented with fructooligosacharide (fos) and human grade yeast extract (biospringer, maissonc-alfort cedex, france) to a final concentration of 10% w/v. the resultant peanut milk was autoclaved for 15 min at 121°c prior to fermentation. a 10% reconstituted skim milk (sm) was prepared using skim milk (nzmp skim milk powder, auckland, new zealand) and the slurry was then sterilized as was done for pm. starter culture and bifidobacterium inoculum b. pseudocatenulatum g4 was obtained from the stock culture collection of biotechnology and functional food laboratory (faculty of food science and technology, upm, malaysia) . the strain was maintained at -20°c in 20% (v/v) glycerol and skim milk supplemented with yeast extract. a working culture was prepared by activation of the frozen strain. it was grown in skim milk (10%, w/v) supplemented with yeast extract (0.05%), incubated under anaerobic conditions using anaerocult a gaspac system (merck, darmstadt, germany) for 48 h. active cells were then transferred twice at 1% inoculum rate (v/v) in skim milk yeast extract medium followed by anaerobic incubation at 37 °c for 24 h prior to use. batch cultivation conditions fermentation was carried out in batches using a 4 vessels bioreactor with a temperature controlled water bath (jeio tech desk top, korea) and an electronic stirrer (gas-col ltd, usa). the media used for fermentation were pm and sm. they were placed in clean vessels and autoclaved for 15 min at 121°c. the contents were then mixed to equilibrate the temperature to 37°c. inoculations were next carried out with 0.67% (v/v) bifidobacterium strain g4 starter culture. the initial ph was set approximately at 6.5 using human grade sodium bicarbonate and the fermentation was run to a final ph less than 5. oxygen free nitrogen was used during fermentation to create the anaerobic environment. during the fermentation, the growth media were homogenized by stirring at speed of 200 rpm/min. shelf life study on fermented products fermented samples with high viable counts of strain g4 were collected for storage evaluation. the collected samples were stored at 4°c (refrigeration) and 25°c for a period of 2 weeks. during storage, at weekly interval samples were collected to determine the viable population, ph and individual organic acids profile including, lactic, acetic, propionic, and butyric acids. microbiological analysis samples were collected aseptically at initial (0 day) and weekly post incubation up to the second. growth of bifidobacterium was enumerated in colony forming units (cfu) per ml using the plate count method. de man rogosa sharpe (mrs) agar containing 0.05% l-cysteine was used for enumeration. organic acid determination 1 ml of liquid fermented medium was centrifuged at 10000 rpm for 15 min. the supernatant was filtered through a 0.22 µm membrane filter and stored (-20°c) for analysis of organic acids and total soluble solids. profiles of organic acids were analyzed by highperformance liquid chromatography [(hplc) shimadzu lc-10as liquid chromatography, japan] with a shimadzu spd-10av uv-vis detector. an organic column, packed with 9 µm of polystyrene divinylbenzene ion exchange resin (aminex hpx-87h, 300 mm x 7.8 mm, biorad laboratories, usa) and maintained at 65°c was used. the uv detector was set at 220 nm and the mobile phase was 0.009 n sulphuric acid with a flow rate of 0.7 ml/min. statistical analysis one-way anova was performed to examine significant differences between normally distributed data. tukey’s-test was used to perform multiple comparisons between means within each specific growth medium. for abnormally distributed data randomization test was performed and the data were analyzed by kruskal-wallis test. probability level of less than 0.05 was considered significant (p < 0.05). all data were analyzed using minitab statistical software (2006). results and discussion survivability of b. pseudocatenulatum g4 during the storage of fermented pm and sm the viable number of strain g4 of fermented products at the beginning of the storage was in range of 7.12 8.39 table 1. survival of b. pseudocatenulatum g4 (log cfu/ml) during the storage of fermented peanut and skim milk with different supplements a . time b pm pm + fos pm + fos +ye sm sm + fos tpy initial 7.12 ± 0.07 a 8.35 ± 0.36 a 8.12 ± 0.43 a 7.28 ± 0.29 a 8.39 ± 0.27 a 8.25 ± 0.23 a 4 w1 7.20 ± 0.11 a 8.11 ± 0.25 a 8.02 ± 0.15 a 7.24 ± 0.29 a 8.08 ± 0.68 a 8.06 ± 0.43 a 4 w2 6.60 ± 0.30 a 7.77 ± 0.32 a 7.20 ± 0.08 b 7.14 ± 0.26 a 7.29 ± 0.13 a 7.63 ± 0.69 a 25w1 6.93 ± 0.47 a 6.68 ± 0.65 b 6.74 ± 0.96 b 7.05 ± 0.24 a 6.68 ± 0.51 b 7.76 ± 0.68 a 25w2 5.87 ± 0.55 b 6.15 ± 0.27 b 5.10 ± 0.43 c 5.51 ± 0.43 b 5.56 ± 0.31 b 5.60 ± 0.61 b a values are mean ± std of triplicate independent runs. different superscript letters in the same column indicate significant (p < 0.05) differences between means. pm = peanut milk, fos = fructooligosaccharide (0.67% w/v), ye = yeast extract (0.5% w/v), sm = reconstituted skim milk, tpy= trypticase pytone yeast extract. b 4 w1= storage at 4°c for a week, 4 w2= storage at 4°c for two weeks, 25 w1= storage at 25°c for a week, 25 w2= storage at 25°c for two weeks. log cfu/ml fermented pm and 7.28 8.39 log cfu/ml fermented sm products. the strain g4 was maintained high in fermented pm and sm in the first week of refrigeration storage (table 1). however, the viable number decreased slightly in the second week of the storage, with significant (p < 0.05) reduction in pm+fos+ye fermented product. fortunately, the viability of the strain in fermented products was still at a level complying with the standard population requirement for a probiotic product to benefit the host, which equivalent to 6 8 log cfu/ml products. this results for strain g4 is consistence with previous report on b. longum bb536 survivability under refrigeration storage of fermented sudanese medida beverage, where the viability of the strain was not affected for a period of 2 weeks under refrigeration (kabeir et al., 2004). therefore, the temperature of refrigeration is important to preserve fermented products with high viable number of bifidobacteria in different fermented carriers, such as, fermented skim milk, malted rice and peanut milk. generally, refrigeration storage was recommended to maintain the survivability of bifidobacteria in traditional fermented beverages (mcmaster et al., 2005). however, akalin et al. (2004) noted a significant reduction on b. longum bb46 in yogurt after only 1 week refrigeration. this indicates that the viability of bifidobacterium in fermented products was dependent on the carrier type and ph of the fermented products during storage. the statement of vinderola et al. (2000) is in support to this point, where the ph of 4.5 and below was found to jeopardize the viability of probiotics in yogurt stored at lower temperature of 5°c. when the fermented pm and sm products stored at 25°c for a period of 2 weeks, significant (p < 0.05) reductions in viable population of strain g4 was found (table 1). it was also found that the viable populations of bifido-bacterium and lactobacillus tent to decline in fermented soymilk held at 25°c, due to acids accumulation and low tolerance of some probiotics to the acidic environment (wang et al., 2002a; rasic and kurman, 1983). at this high storage temperature of 25°c, bacteria will continue activity, thus accumulation of more metabolic products could occur. in fact, the low buffer capacity was postulated as the main factor inherited to reduce viability of a microorganism in fermented food with high level of carbohydrate and low protein profile (edwards, 2000). in general, most bifidobacteria strains are sensitive to low ph, resulting from accumulations of acids during fermentation process, thus they are likely to encounter viability reduction during storage (gomes and malceta, 1999). the main metabolic products of carbohydrate fermentation by probiotics activity are organic acids substantiated by a drop in ph of the surrounding environment. this statement was approved by the study of mcmaster et al. (2005), who noted a great loss in viability of bifidobacterium due to acidic injury, which justified by its lower survivability in fermented milk than in control without fermentation (ouwehand and salminen, 1998). ph decline and acid accumulation during the storage of pm and sm fermented with b. pseudocatenulatum g4. the ph of strain g4 fermented pm and sm products at the beginning of storage was in range of 4.2667 4.973 (table 2). the reduction of ph was higher in fermented samples kept at 25°c compared to refrigeration at 4°c due to induced metabolic activity of the strain by heat (storage temperature of 25°c). ph reduction of pm and sm fermented products was not significantly (p < 0.05) different as compared to the initial level in each specific fermented product. calculated result from table 2 showed that the reduction in ph of the fermented pm and sm products during storage under refrigeration for 2 weeks were 0.47, 0.17, 0.26, 0.39, 0.19 and 0.16 for fermented pm, pm+fos, pm+fos+yeast extract, sm, sm+fos and tpy, respectively. while in the same fermented products, the storage temperature of 25°c caused respective ph reduction of 0.751, 0.613, 0.5433, 0.788, 0.747 and 0.2867 ph. the results presented in tables 3, 4, 5 and 6 show that accumulation of organic acids in strain g4 fermented products during the storage were higher in samples kept at 25°c than in samples stored at refrigeration (4°c). the table 2. the ph of fermented peanut and skim milk with bifidobacterium pseudocatenulatum g4 during the storage a . time b pm pm + fos pm + fos +ye sm sm + fos tpy initial 4.97 ± 0.37 4.80 ± 0.27 4.64 ± 0.12 4.99 ± 0.36 4.76 ± 0.23 4.27 ± 0.12 4 w1 4.80 ± 0.61 4.71 ± 0.29 4.63 ± 0.48 4.63 ± 0.45 4.54 ± 0.42 4.16 ± 0.06 4 w2 4.50 ± 0.20 4.63 ± 0.31 4.38 ± 0.33 4.60 ± 0.19 4.57 ± 0.37 4.11 ± 0.10 25 w1 4.54 ± 0.51 4.39 ± 0.12 4.31 ± 0.32 4.52 ± 0.37 4.57 ± 0.26 4.00 ± 0.27 25 w2 4.22 ± 0.16 4.19 ± 0.14 4.37 ± 0.06 4.21 ± 0.21 4.03 ± 0.21 3.98 ± 0.26 a values are mean ± std of triplicate independent runs. there were no significant (p < 0.05) differences between means in column of each treatment. pm = peanut milk, fos = fructooligosaccharide, ye = yeast extract, sm = reconstituted skim milk, tpy= trypticase phytone yeast extract. b 4 w1= storage at 4°c for a week, 4 w2= storage at 4°c for two weeks. 25 w1= storage at 25°c for a week, 25 w2= storage at 25°c for two weeks. table 3. lactic acid profile (mmol/ ml) during the storage of peanut and skim milk fermented with b. pseudocatenulatum g4. time b pm pm + fos pm + fos +ye sm sm + fos tpy initial 7.21 ± 2.23 a 9.66 ± 1.29 a 7.90 ± 0.25 a 6.83 ± 0.56 a 10.77 ± 0.16 a 12.30 ± 3.29 a 4 w1 7.38 ± 1.85 a 10.35 ± 0.71 a 9.16 ± 0.52 a 6.79 ± 0.42 a 10.38 ± 0.66 a 12.36 ± 2.53 a 4 w2 7.74 ± 2.09 a 10.61 ± 0.93 a 9.27 ± 0.43 a 7.87 ± 0.42 a 10.80 ± 0.95 a 12.38 ± 2.43 a 25w1 8.85 ± 2.91 a 11.25 ± 1.40 a 9.80 ± 0.66 b 8.12 ± 0.33 b 10.70 ± 3.25 a 12.41 ± 1.79 a 25w2 9.11 ± 2.47 a 11.67 ± 1.49 a 10.34 ± 1.08 b 8.18 ± 0.41 b 11.66 ± 4.16 a 13.01 ± 2.31 a a values are mean ± std of triplicate independent runs. different superscript letters in the same column indicate significant (p < 0.05) differences between means. pm = peanut milk, fos = fructooligosaccharide, ye = yeast extract, sm = reconstituted skim milk, tpy= trypticase phytone yeast extract. b 4 w1= storage at 4°c for a week, 4 w2= storage at 4°c for two weeks, 25w1= storage at 25°c for a week, 25w2= storage at 25°c for two weeks. table 4. acetic acid profile (mmol/ ml) during the storage of fermented peanut and skim milk with b. pseudocatenulatum g4. time b pm pm + fos pm + fos +ye sm sm + fos tpy initial 6.86 ± 0.92 12.74 ± 0.41 7.27 ± 2.15 6.92 ± 1.16 13.40 ± 0.50 15.99 ± 2.44 4 w1 7.28 ± 0.71 13.36 ± 1.28 7.71 ± 1.18 7.25 ± 0.48 13.76 ± 0.95 16.21 ± 1.84 4 w2 7.65 ± 0.56 13.66 ± 1.55 8.09 ± 1.21 7.32 ± 0.59 13.62 ± 0.37 16.89 ± 1.38 25w1 8.20 ± 0.15 13.56 ± 2.95 7.47 ± 1.33 6.87 ± 0.25 14.04 ± 0.41 17.03 ± 2.77 25w2 7.77 ± 0.53 13.73 ± 1.27 8.48 ± 2.31 7.52 ± 0.19 14.64 ± 0.39 17.19 ± 1.43 a values are mean ± std of triplicate independent runs. there were no significant (p < 0.05) differences between means in column of each treatment. pm = peanut milk, fos = fructooligosaccharide, ye = yeast extract, sm = reconstituted skim milk, tpy= trypticase phytone yeast extract. b 4 w1= storage at 4°c for a week, 4 w2= storage at 4°c for two weeks. 25w1= storage at 25°c for a week, 25w2= storage at 25°c for two weeks. accumulation of lactic acid during the storage was higher in fermented pm than in sm fermented products (table 3). moreover, tpy was the highest lactic profile after the two weeks storage of strain g4 fermented products under refrigeration (4°c). at similar storage, the lactic profile in other fermented products was higher in sm+fos, pm+fos, pm+fos+yeast extract, sm, and pm in descending order, due to fos and yeast extract supplementations. furthermore, at 25°c storage temperature of the fermented products, lactic accumulation was in a similar trend of refrigeration storage except for fermented pm, where the increase was higher than in fermented sm (table 3). however, the increase of lactic at the end of the storage table 5. propionic acid profile (mmol/ ml) during the storage of fermented peanut and skim milk with b. pseudocatenulatum g4. time b pm pm + fos pm + fos +ye sm sm + fos tpy initial 4.33 ± 0.87 6.86 ± 1.37 8.89 ± 1.47 4.34 ± 0.91 8.81 ± 1.02 36.41 ± 1.44 4 w1 5.13 ± 1.56 7.24 ± 2.10 9.03 ± 2.31 4.63 ± 1.11 8.96 ± 0.79 36.71 ± 5.16 4 w2 5.27 ± 1.60 7.60 ± 2.09 9.33 ± 2.24 4.80 ± 0.68 9.11 ± 0.61 37.26 ± 6.56 25w1 4.66 ± 1.70 6.71 ± 2.17 9.43 ± 2.32 4.68 ± 0.92 8.98 ± 1.00 37.88 ± 8.65 25w2 5.46 ± 2.37 7.54 ± 1.88 9.82 ± 2.79 4.92 ± 1.47 9.43 ± 0.98 37.72 ± 9.02 a values are mean ± std of triplicate independent runs. there were no significant (p < 0.05) differences between means in column of each treatment. pm = peanut milk, fos = fructooligosaccharide, ye = yeast extract, sm = reconstituted skim milk, tpy= trypticase phytone yeast extract. b 4 w1= storage at 4°c for a week, 4 w2= storage at 4°c for two weeks. 25 w1= storage at 25°c for a week, 25 w2= storage at 25°c for two weeks. table 6. butyric acid profile (mmol/ ml) during the storage of fermented peanut and skim milk with b. pseudocatenulatum g4. time b pm pm +fos pm +fos +ye sm sm +fos tpy initial 2.70 ± 0.41 1.80 ± 1.44 1.45 ± 0.98 0.48 ± 0.10 1.22 ± 0.92 1.96 ± 0.41 4 w1 3.02 ± 0.46 2.05 ± 1.33 1.57 ± 1.18 0.58 ± 0.17 1.38 ± 1.02 1.98 ± 0.41 4 w2 3.34 ± 0.48 2.24 ± 1.39 1.63 ± 1.21 0.64 ± 0.12 1.45 ± 1.02 2.05 ± 0.46 25w1 3.04 ± 0.44 2.28 ± 1.40 1.67 ± 1.23 0.65 ± 0.17 1.54 ± 1.06 2.14 ± 0.53 25w2 3.45 ± 0.51 2.41 ± 1.24 1.75 ± 1.28 0.70 ± 0.08 1.52 ± 0.91 2.22 ± 0.51 a values are mean ± std of triplicate independent runs. there were no significant (p < 0.05) differences between means in column of each treatment. pm = peanut milk, fos = fructooligosaccharide, ye = yeast extract, sm = reconstituted skim milk, tpy= trypticase phytone yeast extract. b 4 w1= storage at 4°c for a week, 4 w2= storage at 4°c for two weeks, 25 w1= storage at 25°c for a week, 25 w2= storage at 25°c for two weeks was only significant (p < 0.05) in fermented pm+fos+ye and sm products, as compared to its level at the beginning of the storage. acetic acid profile slightly increased in the first week under refrigeration of pm and sm fermented products, and then further increased in the second week (table 4) of the storage. the value of increase during the storage was 0.79, 0.92, 0.82, 0.40, 0.22 and 0.9 mmol/ml in fermented pm, pm+fos, pm+fos+ye, sm, sm+fos and tpy, respectively. a t 25°c storage of similar fermented products for a period of 2 week, acetic acid increased by 0.91, 0.99, 1.11, 0.60, 1.24, and 1.20 mmol/ml of product, respectively. however, increase of acetic in fermented pm and sm fermented products at the end of the storage period did not differ significantly (p < 0.05) than the initial level at the beginning of the storage. on the other hand, there was evident of slight increase in profiles of propionic and butyric during the storage of strain g4 fermented products under refrigeration and at 25°c (tables 5 and 6). propionic acid in strain g4 fermented products slightly increased in the first week of the storage and then the amount approximately doubled in the second week except for sm fermented sample (table 5). the level of propionic in fermented products at the end of the storage periods did not differ significantly (p < 0.05) than the initial level at the beginning of the storage. the highest butyric accumulated sample under refrigeration was pm, followed by fermented pm+fos, sm+fos, pm+fos+ye, sm, and tpy in descending orders (table 6). nevertheless, at 25°c storage, sm was the lowest butyric content. in fact, initial butyric acid concentration is also higher in pm and the lower in sm products. conclusion in conclusion, the development of a new delivery medium for probiotic bifidobacterium is encouraged. that was due to both limitation and unavailability of the conventional carrier of the dairy based or high prices of probiotic dairy products in today’s food market. the developed medium such as, pm based is cheap could contributes positively to deliver b. pseudocatenulatum g4, besides confer additional nutritional value to the diet. moreover, under refrigeration storage the viability of the strain g4 in fermented pm and sm products was maintained high at a favorable level (> 7 log cfu/ml) for consumer’s to maintain optimum health. on the other hand, the lower accumulation of propionic and butyric in fermented products is favored, their high profiles during the storage might affect the acceptability and preference of the final fermented products. therefore, the findings of this evaluation have elucidated the potential of using fermented pm with fos for delivery of strain g4. acknowledgment we duly acknowledge the financial support 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from probiotic product. j. food microbiol. 81: 1-10. vinderola cg, bailo n, reinheimer ja (2000). survival of probiotic microflora in argentinean yoghurts during refrigeration storage. food res. int. 33: 97102. wang y, yu r, chou c (2002). growth and survival of bifidobacteria and lactic acid bacteria during the fermentation and storage of cultured soymilk drinks. food microbiol. 19: 501508. wong s, wendy yk, kabeir bm, shuhaimi m, rosfarizan m, yazid am (2006). survival of bifidobacterium pseudocatenulatum strains isolated from beast-fed infants to simulated gastric ph environment. malaysian appl. biol. 35: 57 62. 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 9 (3), pp. 001-008, march, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper identity and genetic diversity of pectobacterium spp., causal agents of potato soft rot in zanjan, iran e. tavasoli1, a. r. marefat2 and n. hassanzadeh1* 1 department of plant pathology, faculty of agriculture and natural resources, science and research branch, islamic azad university, tehran, iran. 2 department of plant pathology, faculty of agriculture, zanjan university, zanjan, iran. accepted 09 october, 2020 zanjan province is one of the areas of potato production in iran with more than 6917 ha of cultivation. one of the most important factors to limit potato cultivation is soft rot bacteria belonging to the family enterobacteriaceae. during the years 2007 to 2008, different potato fields and the most important potato storages in zanjan province were surveyed. phenotypic identification of the strains was performed using recommended biochemical and physiological tests. genetic diversity was determined by box-pcr and eric-pcr. also all of the strains were identified with specific primers (y1, y2 and eca1f, eca2r). the strains belonged to "carotovora" group and identified as pectobacterium carotovorum subsp. carotovorum and pectobacterium atrosepticum. although there were some atypical characteristics among the strains, but a very close relationship were found between dna fingerprints and geographical origins of isolates. key words: pectobacterium, soft rot, black-leg, box-pcr, eric-pcr introduction potato is the fifth most important food crop worldwide. likewise, iran is one of the most important potato producing countries in asia and oceania. zanjan province, located in north west of iran, is one of the areas of potato production in iran with more than 6917 ha of cultivation. the soft rot erwinias in the enterobacteriaceae are economically important, because they cause serious damage worldwide on a wide variety of plants (perombelon and kelman, 1980). pectobacterium carotovorum subsp. carotovorum and dickeya chrysanthemi have a wide host range, infecting crops mainly in tropical and subtropical regions, whereas p. atrosepticum is restricted almost exclusively to potato in temperate regions, causing soft rot of tubers and blackleg of stems (perombelon, 1992). different studies have been done for recognition of these bacteria on potato in some parts of iran, which were based on physiological and biochemical tests as well as the *corresponding author. e-mail: hasanzadehr@yahoo.com. comparison of protein electrophoretic patterns that in most cases did not lead to a distinct group differentiation (bahar and danesh, 1986; zohoor et al., 1998). one of the tools in studying the genetic diversity of bacteria including potato soft rot pectobacteria is rep-pcr (rademaker and de bruijn, 1997; xiu et al., 2006). to perform this, many potato soft rot bacteria were isolated from both farms and stores of zanjan province and their genetic diversity were estimated. materials and methods sample handling and isolate collection during the years 2007 to 2008, different potato fields and the most important potato storages in zanjan province (abhar, ijrood, khodabandeh and khoramdareh) were surveyed and sufficient samples were collected from plants suffering from soft rot disease. nutrient agar, king’s b agar and eosin methylene blue agar media were used to isolate the suspected bacteria (schaad et al., 2001). the type strain of erwinia carotovora 5702 was obtained from the international collection of micro-organisms from plants (icmp), auckland, new zealand. 2 biochemical and physiological tests all strains were tested for gram reaction using the koh test, potato soft rot, oxidase reaction, catalase reaction, gelatin hydrolysis, production of gas from glucose, production of reducing substances from sucrose, growth in 5 and 6% nacl, ability to grow at 37ºc and phosphatase (schaad et al., 2001), fermentative metabolism (hugh and leifson, 1953), lecithinase (fahy and hayward, 1983), production of indole (dye, 1968), and sensitivity to erythromycin (psallidas, 1993). additional biochemical tests were starch hydrolysis, esculin hydrolysis, nitrate reduction, arginine dihydrolase, action on litmus milk, urease production, h2s production from cysteine (schaad et al., 2001), tween-80 hydrolysis (misaghi and grogan, 1969), casein hydrolysis (fahy and hayward, 1983), 3ketolactose production (cowan, 1974), methyl red reaction and acetoin production (dye, 1968) and acid production from carbohydrates utilized as a source of carbon (schaad et al., 2001). preparation of dna and rep-pcr genomic fingerprinting genomic dna was extracted using the whole cell alkaline lysis method (rademaker and de bruijn, 1997). the dna was stored at 20°c until it was required. all strains were subjected to box-pcr using the box a1r primer (5´-cta cgg caa ggc gac gct gac g-3´) (martin et al., 1992). eric-pcr was performed for 10 selected strains from different groups of box-pcr by using the eric 1r primer (5´-tgt aag ctc ctg ggg att cac-3´) and eric 2 primer (5´-aag taa gtg act ggg gtg agc g-3´) (versalovic et al., 1991). the method developed by rademaker and de bruijn (1997) was performed with slight modification in cycling conditions. the pcr master mix (25 µl) containing: 1 µl dna, 0.3 µl taq dna polymerase (0.1 u), 2.5 µl 10 × pcr buffer, 0.5 µl dntps (25 mm), 1 µl mgcl2 (25 mm), 1.1 µl of each primer (10 pmol). dna amplification was performed on a thermocycler (corbett, germany) under the following conditions: 4 min at 94ºc for initial denaturation, 34 cycles of 40 s at 94ºc, 40 s at 50ºc and 1 min at 72ºc, followed by a final elongation step of 10 min at 72ºc. pcr products (6 µl) were separated by gel electrophoresis in 1.5% agarose gels in tbe buffer. following staining with ethidium bromide, the gels were viewed and photographed under uv transilluminator. a 1-kb marker (fermentas, germany) was included on every gel. captured photographs were subjected to the gel-pro® analyzer (media cybernetics, md, usa) computer program. bands were scored in binary form, 1 and 0, indicating the presence and absence of a band, respectively. a similarity matrix was obtained using the jaccard coefficient and the software package ntsys-pc (version 2.02k, applied biostatistics, inc., ny, usa). to determine the relationship among the strains, cluster analysis was performed with upgma (unweighted pair-group method, using arithmetic averages) in the sahn program of the ntsys-pc software. dendrograms were generated for the box and eric binary matrixes individually. identification and detection with specific primers primers y1 (5´tta ccg gac gcc gag ctg tgg cgt-3') and y2 (5'-cag gaa gat gtc gtt atc gcg agt-3') selected from the pectate lyase-encoding pel gene sequences of e. carotovora (darrasse et al., 1994), were used. a specific pcr assay for p. atrosepticum was performed using primers eca1f (5'-cgg cat cat aaa aac acg-3') and eca2r (5'-gca cac ttc atc cag cga-3') following the protocol of de boer and ward (1995), with slight modifications. pcr master mix (25 µl) with y1 and y2 primers containing: 2.5 µl dna, 0.1 u taq dna polymerase, 2.5 µl 10 × pcr buffer, 2 µl dntps (1 mmol), 1.5 µl mgcl2 (25 mmol), 0.1 µmol of each primer. dna amplification was performed on a icycler (bio-rad) under the following conditions: 5 min at 94ºc for initial denaturation, 34 cycles of 30 s at 94ºc, 45 s at 55ºc and 45 s at 72ºc, followed by a final elongation step of 7 min at 72ºc. pcr reaction mix in final reaction volumes of 25 µl was prepared containing 2.5 µl dna, 0.5 u taq dna polymerase, 2.5 µl 10 × pcr buffer, 100 µm each of dntps, 2 mm mgcl2, 0.5 µm of each primer. dna amplification was performed on a icycler (bio-rad) under the following conditions: 5 min at 95ºc for initial denaturation, 40 cycles of 30 s at 94ºc, 45 s at 62ºc and 45 s at 72ºc, followed by a final elongation step of 8 min at 72ºc. pcr products (6 µl) were separated by gel electrophoresis in 1.5% agarose gels in tbe buffer. following staining with ethidium bromide, the gels were viewed and photographed under uv transilluminator. results biochemical and physiological tests on the basis of biochemical profiles, 67 and 2 strains were identified as p. carotovorum and p. atrosepticum, respectively. all of them were gram negative, facultatively anaerobic, oxidase negative, catalase positive, nonfluorescent on king´s b (kb) medium and showed soft rot symptoms on slices of potato. all strains grew at 37ºc and in 5% nacl. gelatin hydrolysis and lecithinase were negative. some of them produced gas from glucose (22.3%), indole (59.2%) and phosphatase (41.8%). 10.4% of strains showed sensitivity to erythromycin. 3% were able reduce substances from sucrose and 94% were able to tolerate 6% nacl. thus these strains were identified as the "carotovora" group belonging to the family enterobacteriaceae (tables 1 and 2). results of complementary tests on 22 strains (selected by results of box-pcr), showed that no strains produced urease, 3ketolactose and acetoin, also no strains were able to hydrolyse tween-80. they hydrolyzed starch (22.7%), casein (27.3%) and esculin (77.3%). litmus milk reaction was acid (86.4%) and arginine dihydrolase was positive (72.7%). all strains could reduce nitrate and produced h2s from cysteine. 50% of strains were positive in methyl red reaction. all of them produced acid from arabitol, arabinose, trehalose, rhamnose, ribose, cellobiose, glucose, fructose, sucrose or lactose, but none produce acid from insulin. in addition they could produce acid from α-methyl-d-glucoside and raffinose (81.8%), maltose (86.4%), sorbitol and myo-inositol (77.3%), melibiose and palatinose (68.2%). all of them utilized citrate and 86.4% utilized malonate. no strains utilized d-tartrate. results showed a high variability among strains. results also revealed that p. carotovorum was the main pathogen and d. chrysanthemi was absent from the strains tested. rep-pcr genomic fingerprinting pcr using the box primer gave genomic fingerprints with 22 bands, ranging from about 250 to 3000 bp. results revealed two main clusters among the pathogen strains and their similarity value, based on upgma, was 3 table 1. bacterial strains used in this study. strain location of origon year isolated z1, z2, z3, z4 kheirabad 2007 z5, z6, z7, z8, z9 khoramdareh 2007 z10, z11, z12, z13 abhar 2007 z14, z15, z16, z17, z18 gonbad soltanieh 2007 z19, z20, z21, z22, z23, z24 khodabandeh 2007 i1, i2, i3, i4, i5, i6, i7, i8, i9, i10, i11, i12, i13, i14, i15, i16, i17, i18, i19, i20, i21 ijrood 2007 e2, e15, e26, e29, e24, e25, e32, e34, e35, e41, e40, e37 ijrood 2008 b5, d8, d9, d10, d20, d22, d25, d34, d17, d24 zanjan 2008 table 2. phenotypic characteristics of the strains of pectobacterium isolated from potato in comparison with standard isolate. results test number of strains positive or positive p. carotovorum negative/number of strains tested strains (%) 5702 fermentative growth +(67/67) 100 + gram reaction -(67/67) 0 potato soft rot +(67/67) 100 + oxidase -(67/67) 0 catalase +(67/67) 100 + lecithinase -(67/67) 0 gelatin hydrolysis -(67/67) 0 + gas from glucose +(52/67) 77.6 reducing substances from sucrose -(65/67) 3 growth in 5% nacl +(67/67) 100 + growth in 6% nacl +(63/67) 94 + growth at 37ºc +(67/67) 100 + production of indole +(39/67) 58.2 sensivity to erythromycin -(60/67) 10.4 + phosphatase -(39/67) 41.8 + starch hydrolysis -(17/22) 22.8 tween-80 hydrolysis -(22/22) 0 casein hydrolysis -(16/22) 27.3 + urease production -(22/22) 0 methyl red reaction +(11/22) 50 + acetoin production -(22/22) 0 esculin hydrolysis +(17/22) 77.3 + 3-ketolactose production -(22/22) 0 nitrate reduction +(22/22) 100 + arginine dihydrolase +(16/22) 72.8 h2s from cysteine +(22/22) 100 + action on litmus milk: acid reaction (19/22) 86.4 + alkaline reaction (3/22) 13.6 acid production from: glucose +(22/22) 100 + fructose +(22/22) 100 + sucrose +(22/22) 100 + 4 table 2. contd. α-methyl-d-glucoside +(18/22) 81.8 + sorbitol +(17/22) 77.3 + melibiose +(15/22) 68.2 + raffinose +(18/22) 81.8 + arabitol +(22/22) 100 + lactose +(22/22) 100 + trehalose +(22/22) 100 + rhamnose +(22/22) 100 + maltose +(19/22) 86.4 + palatinose +(15/22) 68.2 ribose +(22/22) 100 + inulin -(22/22) 0 cellobiose +(22/22) 100 + myo-inositol +(17/22) 77.3 + arabinose +(22/22) 100 + utilization of: citrate +(22/22) 100 + d-tartrate -(22/22) 0 malonate +(19/22) 86.4 + +, positive reaction; -, negative reaction. m b5 e2 e15 e24 e25 e26 e29 e32 e34 e35 e37 e40 e41 tt c m 3000 bp 2000 1500 1000 750 500 250 z1 z2 z3 z4 z5 z6 z7 z8 z9 z10 z11 z12 z13 z14 z15 z16 m figure 1. genomic dna fingerprinting patterns from strains of pectobacterium isolated from potato, generated by box-pcr. m: marker (1-kb); c: control (without dna); tt: p. carotovorum 5702. about 51.5%. the first group was divided into two subgroups at 65.5% similarity. the second group was also divided into two subgroups at 75.5% similarity. this protocol revealed 18 genotypes within the pathogen (figures 1 and 2). based on box-pcr results, 10 strains selected from these groups, were further analyzed with 5 z17 tt z18 z21 z22 z4 z7 z8 z10 d8 d9 d10 d20 i8 z19 d17 z20 i3 z23 i9 z1 z2 z3 z5 z9 z11 z12 d34 d22 d25 z13 z16 z14 z6 z24 i13 z15 d24 i7 i1 e34 e25 e2 i16 i14 i11 i6 i4 b5 e32 i12 i10 i5 e35 i2 e24 i15 i17 e29 e15 i20 i18 e26 i21 i19 e40 e37 e41 0.50 0.60 0.70 0.80 0.90 1.00 coefficient figure 2. similarity dendrogram of pectobacterium strains based on box-pcr. eric-pcr using the eric 1r primer and eric 2 primer. genomic fingerprints in eric-pcr showed 28 bands, ranging from about 250 to 3500 bp including 9 genotypes. based on upgma, the similarity value bet-ween the two main clusters was about 46.5%. the first group divided into two subgroups at 50% similarity and the second group showed one genotype (figures 3 and 4). identification and detection with specific primers all strains from the zanjan province yielded a 434 bp dna fragment in pcr with the y1 and y2 primers (figure 5) and only two strains (e2 and d24) yielded a 690 bp dna fragment in pcr with the eca1f and eca2r primers (figure 6). therefore all of the studied strains were identified as pectobacterium carotovorum and only two strains as p. atrosepticum. discussion in this research, the characteristics of pectolytic erwinias of potato in the zanjan province and their genetic diversity were determined both biochemically and by the 6 m d8 d20 d22 d34 b5 e2 e15 e29 e24 e35 tt c m 3000 bp 2000 1500 1000 750 500 250 figure 3. genomic dna fingerprinting patterns from representative strains of pectobacterium isolated from potato, generated by eric-pcr. m: marker (1-kb); c: control (without dna), and tt: p. carotovorum 5702 d8 d20 b5 e2 e15 e29 e24 e35 tt d22 d34 0.40 0.55 0.70 0.85 1.00 coefficient figure 4. similarity dendrogram of pectobacterium strains based on eric-pcr. 7 c tt z18 d17 z14 d24 e34 e2 e32 i18 i21 e41 m 434 bp figure 5. amplification of a specific 434 bp band in 10 strains generated using y1 and y2 primers. c: water control; m: marker (100 bp); tt: p. carotovorum 5702. c e15 e40 z2 z15 d22 d24 e2 m 690 bp figure 6. amplification of a specific 690 bp band in 7 strains, generated using eca1f and eca2r primers. c: water control; m: marker (100 bp). rep-pcr technique. the specific primers were used for the recognition of these pathogens. there was a considerable variation in phenotypic traits and gene electrophoretic clusters among the strains. in this case, 97% of strains were similar to p. carotovorum and only 3% were similar to p. atrosepticum. properties of a few strains did not correspond with defined characteristics of authentic species and subspecies of pectobacterium. these findings agree with louw's justification on rep-pcr and its high efficiency towards discrimination of pathogen population diversity (louws et al., 1994). in this study, some correlations between biochemical characteristic and box-pcr based genetic fingerprints among some of the strains were observed. thus strains related to a defined genetic group with the same genotype, showed the same phenotype characteristics and vice versa. an exceptional example is the d24 strain, which was phenotypically different from other strains showed 85% similarity in genotype to those of the same group. comparing the results of genetic fingerprint box-pcr and eric-pcr, some similarities and differences were observed in their classification. in both cases, strains were placed in two main fingerprinting groups. in none of the current classifications has complete similarity between the standard strain and the examined one been reported. thus there was no complete correspondence between the box-pcr results and eric-pcr and even those strains which showed the same genotype in boxpcr showed different genotypes in eric-pcr. geographically, the group created by box-pcr was closely related to their sample collection areas. because of this, most of strains of a single area were placed in one of the main groups. in eric-pcr, although only a limited number of strains were compared, these correlations were defined. linkage between rep-pcr results and the geographic origin of bacterial strains has been recognized in various studies (scortichini et al., 2001; mkandawire et al., 2004). louws et al. (1994) believe that one of the important reasons for this phenomenon is that the selection for one geographically suitable area can have influence on the genetic map of bacterium and also dispersion of these repetitive units in the genome of bacterium. this work supports this idea and in some cases artifacts, these relations were observed. no doubt rep-pcr is a reliable tool for epidemiological studies of diseases and one can use the information as a device for detection of pathogens. despite this, more recent work with issr-pcr of bacteria such as clavibacter michiganensis subsp. michiganesisis proved the greater sensitivity, specificity and reliability of this technique as another helpful informative tool in epidemiological studies (baysal et al., 2011). examining strains in zanjan province, with special primers of y1/y2, specialized for p. carotovorum 8 (darrasse et al., 1994) and eca1f, eca2r, specialized for p. atrosepticum (de boer and ward, 1995), the main pathogen of potato soft rot in the zanjan province is p. carotovorum and in some cases p. atrosepticum. failing to isolate d. chrysanthemi is not strange in the province, since this bacterium was reported in delimit potato cultivation areas of the hamedan province. besides this, the weather of zanjan province is cold enough to limit the growth of the bacterium (agrios, 1988). references agrios gn (1988). plant pathology. 3 rd ed. academic press, new york. p. 803. bahar m, danesh d (1986). occurrence of potato blackleg in isfahan. proceedings 8 th plant protection congress of iran. p. 105. baysal ö, mercati f, ikten h, yıldız rç, carimi f, aysany, teixeira da silva ja (2011). clavibacter michiganensis subsp. michiganesis: tracking strains using their genetic differentiations by issr markers in southern turkey. physiol. mol. plant pathol., 75: 113-119. cowan st (1974). cowan and steel, manual for the identific of medical bacteria, 2 nd . combridge university press, cambridge. darrasse a, priou s, kotoujansky a, bertheau y (1994). pcr and restriction fragment length polymorphism of a pel gene as a tool to identify erwinia carotovora in relation to potato diseases. app. environ. microbiol., 60: 1437-1443. de boer sh, ward lj (1995). pcr detection of erwinia carotovora subsp. atroseptica associated with potato tissue. phytopathol., 85: 854-858. dye dw (1968). a taxonomic study of the genus erwinia. i. the `amylovora' group. new zealand j. sci., 11: 590-607. fahy pc, hayward ac (1983). media and methods for isolation and diagnostic test. in: fahy, p. c., persley, g. j. 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of cocos nucifera stem bark and citrulus lanatus seeds buraimoh* and monye bayero university, kano, nigeria accepted 12 february, 2025 a phytochemical, proximate, nutritional, and anti-nutrient composition analysis was performed on the stem bark of citrulus lanatus and cocos nucifera seeds. the outcomes of the two plants' analyses were contrasted. the findings show that both plants have significant levels of phenol, flavonoids, terpenoids, tannins, and saponnins. compared to the amount found in the stem bark of c. nucifera, the content of alkaloids and steroids in the c. lanatus seed was substantially (p<0.05) higher. in terms of ash and fiber, the proximate fractions of c. lanatus seeds were generally low, and the stem bark of c. nucifera showed low percentages of protein, ash, lipids, and fiber. the protein and fat content of c. lanatus seeds was found to be substantially (p<0.05) higher than that of c. nucifera stem bark. the most prevalent microelement was iron, which ranged from 3.66 µg/g in c. nucifera stem bark to 4.089 µg/g in c. lanatus seed. zinc came next; magnesium levels in both plants were highly traced. the results of the examination of c. lanatus seeds and c. nucifera stem bark revealed that while both plants had low concentrations of vitamins b1, b2, and b3, they were rich in certain vitamins, including a, e, and c. low levels of phytate, oxalate, hemaglutinin, and trypsin inhibitor were found in c. lanatus seed and c. nucifera stem bark, according to anti-nutrient analysis. these levels ranged from 0.677 mg/100 g to 2.370 mg/100 g, 0.082 mg/100 g to 0.97 mg/100 g, 0.549% to 0.690%, and 0.456 mg/100 g to 0.550 mg/100 g, respectively. according to this study, c. lanatus seeds and c. nucifera stem bark are good sources of biologically significant phytochemicals, which means they may one day be used to make effective medications to treat a variety of illnesses. key words: phytochemicals; proximate; nutrients; anti-nutrients. introduction it has long been recognized that plants and their derivatives have biological activity and play a significant role in global health. plants were the source of 30% of all contemporary medications (riaz et al., 2010; omoboyowa et al., 2013). approximately 80% of the world's population primarily receives their medical care from plants, according to the world health organization (omoboyowa et al., 2013). particularly in africa, there is increasing interest in using plants for therapeutic purposes (adeniyi et al., 2012). watermelon, or citrulus lanatus, has a considerable amount of citrulline, and blood plasma levels showed an increased concentration after consuming several kilograms of the fruit (mendal et al., 2005). its solitary brown seed contains a lot of oleic and arachidonic acid, two types of fat and oil that are useful to industry and are used in soapmaking and cooking (collins et al., 2007). citruline, which can be converted to arginine, is abundant in watermelon flesh. this amino acid contributes to the immunological and cardiovascular systems and is a substrate for the generation of nitric oxide (collins et al., 2007). the coconut palm, or cocos nucifera, belongs to the arecaceae family of palms. it is widespread in the tropic and subtropical regions and is renowned for its high degree of adaptability due to the numerous applications for its different sections. the coconut water contains sugar, dietary fiber, proteins, antioxidants, vitamins, and minerals, while the seed supplies oil for cooking, frying, and producing margarine (ravi, 2009).in terms of medicine, young coconut juice possesses estrogenic-like properties, the n-hexane fraction of coconut peel includes new anticancer chemicals, it can be used as an intravenous hydration fluid, and the tea made from husk fiber is used to treat severe inflammatory disorders (sarian, 2010). in order to highlight their pharmacological roles in traditional medicine, the current study was designed to author(s) retain the copyright of this article. african journal of food science research issn 2375-0723 vol. 13 (1), pp. 001-008, february, 2025. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 assess the secondary metabolites, proximate, nutrients, and anti-nutrient composition of cocos nucifera stem bark, which is frequently used in alternative herbal medicine in nigeria, and citrulus lanatus seeds, which are frequently discarded after consumption of the fruits. materials and methods plant material the fresh watermelon (citrulus lanatus) fruits utilized in this study were bought from eke market in the nigerian state of ebonyi's afikpo north local government area. a sterile knife was used to extract the seeds from the pulp, and then a mechanical blender was used to carefully grind the seeds into a coase form after they had been washed and allowed to air dry. for this study, stem bark from coconut palms (cocos nucifera) was gathered from ogbu edda, located in the afrikpo south local government area of ebonyi state, nigeria. using a clean cutlass, it was gathered, allowed to air dry for three weeks at room temperature, and then ground into a coarse consistency using a mechanical blender.. preparation of fat free sample two grams (2 g) of the samples were defatted with 100 ml of diethyl ether using a soxhlet apparatus for 2 hours. phytochemical analysis phytochemical analysis chemical tests were carried out on the samples for the quantitative determination of phytochemical constituents. alkaloid determination gravimetric analysis was used to determine the alkaloid concentration. after weighing five grams of the sample into a 250 ml beaker, 200 ml of 20% acetic acid in ethanol was added, and the mixture was left to stand for four hours. after filtering, the extract was concentrated to a quarter of its initial volume in a water bath. concentrated hydroxide of ammonium was added to the extract drop by drop until the precipitation was finished. according to obadoni and ochuko (2001), the entire solution was left to settle before the precipitate was filtered through whatman filter paper no. 4 (125 mm) and weighed. saponin determination obadoni and ochuko's (2001) approach was used to determine the saponin content. 200 milliliters of 20% ethanol were used to scatter 20 grams (20 g) of each powdered sample. for four hours, the suspension was cooked to roughly 55 degrees celsius over a hot water bath while being constantly stirred. after filtering the mixture, 200 milliliters of 20% ethanol were used to remove the residue once more. over a water bath at roughly 90 degrees celsius, the combined extracts were reduced to 40 milliliters. after transferring the concentrate into a 250 ml separator funnel, 20 ml of diethyl ether was added, and the mixture was violently agitated. the ether layer was thrown away, but the aqueous layer was retrieved. the purifying procedure was carried out once more. n-butanol (60 ml) was added. ten milliliters of 5% aqueous sodium chloride were used twice to wash the nbutanol and extract combination. in a water bath, the leftover solution was heated to roughly 90 degrees celsius. the samples were dried at 100 degrees celsius in an oven until their weight remained consistent. the proportion of saponin was determined (obadoni and ochuko, 2001). tannin determination tannin content was calculated using the van-burden and robinson (1981) method. a 100 ml plastic bottle was filled with 500 miligrams of the sample. in a mechanical shaker, 50 milliliters of distilled water were added and agitated for one hour. this was adjusted to the proper level after being filtered into a 50 ml volumetric flask. next, 5 ml of the filtrate was pipetted into a tube and combined with 0.008m potassium ferrocyanide and 3 ml of 0.1m fecl3 in 0.1 n hcl. within ten minutes, the absorbance was measured at a wavelength of 120 nm in a spectrophotometer. the same wavelength was used to develop and read the color of a blank sample. tannic acid was used to create a standard that was measured at 100 parts per million (van-burden and robinson, 1981). flavonoid determination boham and kocipai's (1994) approach was used to determine the flavonoid content. at room temperature, 300 milliliters of methanol:water (80:20) were used to repeatedly extract 10 grams of the powdered materials. whatman filter paper no. 42 (125 mm) was used to filter the entire solution. following that, the filtrate was moved into a crucible, dried out over a water bath, and weighed (boham and kocipai, 1994). determination of total phenols obadoni and ochuko's (2001) method was used to determine total phenols. the fat-free sample was boiled for 15 minutes with 50 milliliters of ether in order to extract the phenolic component. ten milliliters of distilled water were added after five milliliters of the extract had been pipetted into a fifty milliliter volumetric flask. additionally, 5 ml of concentrated amyl alcohol and 2 ml of ammonium hydroxide solution were added. after being prepared to mark, the samples were allowed to react for 30 minutes in order to acquire color. a spectrophotometer set to 505 nm wavelengths was used to measure the solution's absorbance (obadoni and ochuko, 2001). cyanogenic glycoside determination 3 the alkaline picrate approach of onwuka (2005) was the technique employed. in a conical flask, 5 g of samples a and b were combined with 50 ml of distilled water and left to stand overnight. four milliliters of alkaline picrate were added to one milliliter of the sample filtrate in a corked test tube, and the mixture was then incubated for five minutes in a water bath. before the cyanide standard curve was prepared, the absorbance of the samples was measured at 490 mm, as was that of a blank that contained 1 ml of distilled water and 4 ml of alkaline picrate solution. however, none of the corked test tubes containing samples a and b showed any change in color, which is a sign that there was no cyanide present in the sample; instead, the color changed from yellow to reddish brown after 5 minutes of incubation in a water bath (railes, 1992). proximate composition analysis this was carried out according to the method of aoac (1990) moisture content determination a dried weighted crucible was filled with two grams of each sample. the samples were heated for three hours at 1050c in a moisture extraction oven. after being placed in desiccators to cool, the dry samples were weighed again. the process was reported until constant weight was obtained the difference in weight was calculated as a percentage of the original sample percentage moisture = w2 – w1 x 100 w2 – w3 1 where w1 = initial weight of empty dish w2 = weight of dish + un-dried sample w3 = weight of dish + dried sample ash content determination two grams of each sample were placed in a crucible, heated for three hours at 1000c in a moisture extraction oven, and then moved to a muffle furnace at 5500c until it was white and carbon-free. following its removal from the furnace, the sample was promptly reweighed after being allowed to cool to ambient temperature in a desiccator. the weight of the residual ash was then calculated as ash content percentage ash = weight of ash weight of original of sample x 100 1 crude protein determination the a.o.a.c. (1990) micro kjeldahl method was applied. in a heating tube, two grams of each sample were combined with ten milliliters of concentrated h2so4. the tube was filled with one table of selenium catalysts, and the mixture was heated within a fume closet. distilled water was used to hold the digest. an identical volume of 45% naoh was combined with a 10-millimeter chunk of the digest. mixture then transferred onto a kjeldahl distillation device. three drops of methyl red indicator were added to a 4% boric acid solution after the mixture was distilled. additionally, 50 milliliters of distillate were gathered and titrated. a duplicate of the sample was obtained, and the average value was calculated. the crude protein content was estimated by multiplying the nitrogen concentration by 6.25. this is given as percentage nitrogen = (100 x n x 14 x vf) t 100 x va where n = normality of the titrate (0.1n) vf = total volume of the digest = 100ml t = titre value va = aliquot volume distilled crude fiber determination 200ml of 1.25% h2so4 was mixed with two grams (2g) of sample and one gram of asbestos, and the mixture was heated for half an hour. after that, the contents and solution were transferred into a buchner funnel that was covered with muslin material and fastened with an elastic band. after letting it filter, the residue was placed in 200 milliliters of boiling naoh, where it simmered for 30 minutes before being moved to a buchner funnel and filtered. after that, it was given two alcohol washes. petroleum ether was used to wash the material three times. after that, the residue was placed in a dry, clean crucible and dried in the moisture extraction oven until it reached a consistent weight. after drying, the crucible was taken out, let to cool, and weighed. then, difference of weight (i.e. loss in ignition) is recorded as crucible fiber and expressed in percentage crude fiber, = w1 – w2 x 100 w3 1 where w1 = weight of sample before incineration w2 = weight of sample after incineration w3 = weight of original sample fat content determination 200ml of 1.25% h2so4 was mixed with two grams (2g) of sample and one gram of asbestos, and the mixture was heated for half an hour. after that, the contents and solution were transferred into a buchner funnel that was covered with muslin material and fastened with an elastic band. after letting it filter, the residue was placed in 200 milliliters of boiling naoh, where it simmered for 30 minutes before being moved to a buchner funnel and filtered. after that, it was given two alcohol washes. petroleum ether was used to wash the material three times. after that, the residue was placed in a dry, clean crucible and dried in the moisture extraction oven until it reached a consistent weight. after drying, the crucible was taken out, let to cool, and weighed. the percentage oil content is percentage fat = w2 – w1 x 100 4 furnace. the resulting ash was heated gradually on a hot plate until brown fumes vanished after being dissolved in 5 milliliters of hno3/hcl/h2o (1:2:3). five milliliters of deionized water were added to the leftover material in each crucible, and the mixture was heated until it became colorless. after passing through whatman no. 42 filter paper, the mineral solution in each crucible was poured into a 100 ml volumetric flask, and the volume was adjusted with deionized water to reach the desired level. using an atomic absorption spectrophotometer, this solution was utilized for elemental analysis. each element's concentration in the sample was determined using a 10 cmlong cell and expressed as a percentage of dry matter. the digest's phosphorus content was ascertained colorimetrically using the technique outlined by nahapetian and bassiri (1975). four milliliters of demineralized water, three milliliters of 0.75m h2so4, 0.4 milliliters of 10% (nh4)6mo7o24.4h2o, and 0.4 milliliters of 2% (w/v) ascorbic acid were added to 0.5 milliliters of the diluted digest and mixed. after letting the solution remain for 20 minutes, absorbance measurements at 660 nm were taken. the extract's phosphorous content was ascertained. where w1 = weight of the empty extraction flask w2 = weight of the flask and oil extracted w3 = weight of the sample carbohydrate content determination the nitrogen free method described by a.o.a.c (1990) was used. the carbohydrate is calculated as weight by difference between 100 and the summation of other proximate parameters as nitrogen free extract (nfe) percentage carbohydrate (nfe) = 100 – (m + p + f1 + a +f2) where m = moisture p = protein f1 = fat a = ash f2 = crude fiber. nutrients analysis mineral determination the shahidi et al. (1999) method was used to determine the principal elements, which include calcium, phosphorus, sodium, potassium, magnesium, and trace elements (iron and zinc). two grams of each ground plant sample were weighed and put through dry ashing after being sieved through a 2 mm rubber sieve in a well-cleaned porcelain crucible at 550°c in a muffle vitamin analysis determination of ascorbic acid (vitamin c) the baraket et al. (1973) method was used to determine the vitamin c concentration. five grams of the sample were mixed with 100 milliliters of edta/tca (2:1) extracting solution in an extraction tube, and the mixture was shaken for 30 minutes. this was placed in a centrifuge tube and spun at 3000 rpm for 20 minutes. the extracting solution was then transferred into a 100 ml volumetric flask and added until the 100 ml threshold was reached. 20 milliliters of the extract were pipetted into the volumetric flask, and then 1% starch indicator was added. a 20% cuso4 solution was used to titrate them in order to determine a dark end point (baraket et al., 1973). determination of vitamin a twenty milliliters of petroleum ether were used to macerate one gram (1g) of the sample after it had been weighed. after drying it off, 0.2 ml of chloroform acetic anhydride and 2 ml of tca chloroform were added, and the absorbance at 620 nm was recorded. next, the standard curve was used to extrapolate the vitamin a concentration. determination of vitamin e one gram (1g) of the sample was weighed and macerated with 20mls of ethanol. one milliliter (1ml) of 0.2% ferric chloride in ethanol was added, then 1ml of 0.5% α,αdipyridyl was also added, it was diluted to 5mls with distilled water and absorbance was measured at 520nm. then concentration of vitamin e was extrapolated from the standard curve. determination of niacin the niacin content was calculated using okwu and josiah's (2006) methodology. fifty milliliters of 1 n sulfuric acid were added to five grams of the sample, and it was agitated for half an hour. after adding three drops of 0.1n ammonia solution, the sample was filtered. five milliliters of potassium cyanide were added to a 50 milliliter volumetric flask containing 10 milliliters of the filtrate. five milliliters of 0.02 n h2so4 were used to acidify this, and the absorbance was measured at 470 nm using a spectrophotometer (okwu and josiah, 2006). determination of riboflavin this content of riboflavin was calculated using okwu and josiah's (2006) technique. 100 milliliters of a 50% ethanol solution were used to extract five grams of the material, and it was agitated for one hour. ten milliliters of the extract were pipetted into a 50 milliliter volumetric flask after this was filtered into a 100 milliliter flask. after adding 10 milliliters of 5% potassium permanganate and 10 milliliters of 30% h2o2, the mixture was let to stand over a hot water bath for approximately half an hour. 40% sodium sulfate 5 was applied in 2 milliliters. deionized water was used to bring this up to the 50 ml level, and a spectrophotometer was used to detect the absorbance at 510 nm (okwu and josiah, 2006). determination of thiamine the thiamine content was ascertained using okwu and josiah's (2006) methodology. 50 milliliters of ethanolic sodium hydroxide were used to homogenize five grams of the material. after filtering, it was placed in a 100 ml flask. after pipetting 10 milliliters of the filtrate, 10 milliliters of potassium dichromate were added to develop the color, and the reading was taken at 360 nanometers. the color also formed and read at the same wavelength after a blank was made (okwu and josiah, 2006). anti-nutrients analysis oxalate determination the titration method was used to ascertain the samples' oxalate content. in a 250 ml volumetric flask suspended in 190 ml of distilled water, 2 g of samples a and b were added. each sample received 10 milliliters of 6mhcl solution, and the suspension was digested for one hour at 100 degrees celsius. after cooling, the samples were added until the flask reached the 250 ml threshold. after the samples were filtered, a duplicate portion of 125 milliliters of the filtrate was metered out and put into a beaker. four drops of methyl red indicator were then added, and then concentrated nh4oh solution was added drop by drop until the solution became yellow instead of pink. after heating each piece to 90ºc, it was cooled and filtered to get rid of the ferrous ion-containing precipitate. after heating each filtrate to 90ºc once again, 10 ml of a 5% cacl2 solution was added to each sample while being constantly stirred. the samples were cooled and then left overnight. after that, the solutions were centrifuged for five minutes at 2500 rpm. the precipitates were fully dissolved in 10 milliliters of 20% h2so4 after the supernatant was decanted. two grams of each sample were digested, yielding 200 milliliters of total filtrates. after heating 125 ml aliquots of the filtrate almost to boiling, it was titrated against 0.05 m standardized kmno4 solutions to produce a pink color that lasted for 30 seconds. each sample's oxalate content was determined (munro and bassiro, 2000). phytate determination using the method outlined by lucas and markaka (1975), the phytate content of each sample was ascertained by measuring its phytic acid. two grams of each sample must be weighed and placed into a 250 ml conical flask. the samples were soaked in 100 ml of 2% concentrated hydrochloric acid for three hours before being filtered using double-layered filter paper. a 250 ml beaker was filled with 50 ml of each sample filtrate, and 107 ml of distilled water was added to provide or enhance the appropriate acidity. each sample solution was titrated with a standard iron chloride solution containing 0.00195 g iron/ml after 10 ml of a 0.3% ammonium thiocyanate solution was added as an indicator. the end point was indicated by a brownishyellow coloration that lasted for five minutes. the phytic acid % was computed (russel, 1980). trypsin inhibitor determination 50 milliliters of 0.5 m nacl solution were used to disseminate one gram of each of samples a and b. after 30 minutes of stirring at room temperature, the mixture was centrifuged for 5 minutes at 1500 rpm. the filtrates were utilized for the assay after the supernatants were filtered. ten milliliters of each sample's substrate were mixed with two milliliters of the standard trypsin solution. using 10 ml of the same substrate as a blank, the mixture's absorbance was measured at 410 nm (prokopet and unlenbruck, 2002). alkaloid determination: 50 ml of 10% acetic acid in ethanol was mixed with 5 g of each sample, agitated, and left to stand for 4 hours before being filtered. a quarter of the filtrate's initial volume was evaporated. to precipitate the alkaloid, concentrated nh4oh was applied dropwise to each sample. weighed filter paper was used to remove the precipitate, and 1% nh4oh solution was used for washing. in each instance, the precipitates were reweighed after being dried for 30 minutes at 60ºc (griffiths, 2000). hemaglutinin determination twenty milliliters of 0.9% nacl were added to two grams of each sample, and the mixture was rapidly agitated for one minute. after letting the supernatants rest for an hour, the samples were centrifuged for ten minutes at 2000 rpm, and the suspension was filtered. each sample's supernatants were gathered and turned into a crude agglutinating extract. 6 table 1. phytochemical composition of methanol extract of citrulus lanatus (watermelon) seed extract phytochemical composition citrulus lanatus (mg/100g) (watermelon) seed cocos nucifera (coconut stem bark (mg/100g) palm) saponins 1.553 ± 0.0071 0.23 ± 0.04 tannins 0.536 ± 0.0057 6.73 ± 0.03 alkaloids 33.795 ± 0.035 3.42 ± 0.03 flavonoids 2.310 ± 0.014 3.59 ± 0.01 phenol 1.371 ± 0.042 0.21 ± 0.01 cyanogenic glycoside 0.003 ± 0.00 0.002 ± 0.00 data represented in mean ± sem table 2. proximate composition of methanol extract of citrulus lanatus (watermelon) seed extract proximate compounds citrulus lanatus (%) (watermelon) seed cocos nucifera (coconut stem bark (%) palm) protein 27.535 ± 0.064 1.84 ± 0.00 ash 4.138 ± 0.015 3.12 ± 0.00 fats 48.06 ± 0.127 7.12 ± 0.00 fibre 3.932 ± 0.078 1.64 ± 0.00 moisture 6.933 ± 0.076 12.25 ± 0.01 carbohydrate 9.407 ± 0.203 74.04 ± 0.04 data represented in mean ± sem statistical analysis each independent replication was subjected to three analytical evaluations for each parameter. each treatment yielded three independent replicates (n = 3). the results are shown in tables as means ± standard error of mean (sem). the t-test was used to analyze the data (p < 0.05). results the phytochemical components of citrulus lanatus seeds and the stem bark of cocos nucifera are quantitatively determined and summarized in table 1. the findings show that both plants have significant levels of phenol, flavonoids, tannins, and saponnins. when compared to the amount found in the stem bark of c. nucifera, the concentration of alkaloids in the c. lanatus seed was substantially (p<0.05) higher. cyanogenic glycosides had virtually little effect on either plant. table 2 displays the findings of the proximate analysis of the stem bark and seeds of c. nucifera and c. lanatus. in terms of ash and fiber, the proximate fractions of c. lanatus seeds were generally low, and the stem bark of c. nucifera showed low percentages of protein, ash, lipids, and fiber. the percentage moisture and carbohydrate composition in c. nucifera were found to be significantly (p<0.05) higher than the concentration of these proximate compounds in c. lanatus, while the protein and fat composition of c. lanatus was found to be significantly (p<0.05) higher than the compositions present in c. nucifera (table 2). table 3 displays the mineral and nutrient contents of both plants. the most prevalent microelement was iron, which ranged from 3.66 µg/g in c. nucifera stem bark to 4.089 µg/g in c. lanatus seed. after that, zinc was found in c. lanatus seeds at 1.082 µg/g and in c. nucifera stem bark at 3.50 µg/g. magnesium levels on both plants were quite low. vitamins a, e, and c were found to be abundant in both plants, according to the results of an investigation of c. lanatus seeds and c. nucifera stem bark. however, thiamine, riboflavin, and niacin were found to be low in both species. the anti-nutrient analysis of c. lanatus seed and c. nucifera stem bark revealed low level of phytate, oxalate, hemaglutinin and trypsin inhibitor ranging from 0.677 mg/100g to 2.370 mg/100g, 0.082 mg/100 g to 0.97 mg/100 g, 0.549 % to 0.690% and 0.456 mg/100 g to 0.550 mg/100 g respectively (table 4). discussion it is well recognized that the bioactive substances found in these plants have both physiological and therapeutic effects (sofowora, 1993; adeniyi et al., 2012). both plants' analgesic, antispasmodic, and antibacterial properties were demonstrated by the presence of alkaloids (stray, 1998; okwu & okwu, 2004). flavonoids have high anticancer properties and are powerful watersoluble antioxidants and free radical scavengers that stop oxidative cell damage (salah et al., 1995; del-rio et al., 1997). additionally, flavonoids are known to possess antiviral, anti-allergic, and anti-inflammatory qualities (adeniyi et al., 2012). according to okwu (2004) and adeniyi et al. (2012), they can reduce the risk of adenovirus, parainfluenza virus, herpes simplex virus, and osteoporosis, as well as allergies. 7 table 3. nutrients composition of methanol extract of citrulus lanatus (watermelon) seed extract nutrient compounds citrulus lanatus (watermelon) seed cocos nucifera (coconut palm) stem bark zinc (µg/g) 1.082 ± 0.0085 3.50 ± 0.01 iron (µg/g) 4.089 ± 0.037 3.66 ± 0.00 magnesium (%) 0.023 ± 0.016 0.18 ± 0.00 vitamin a (mg/100g) 34.21 ± 0.156 49.50 ± 0.01 vitamin e (mg/100g) 20.62 ± 0.212 30.45 ± 0.04 vitamin c (mg/100g) 22.095 ± 0.092 20.90 ± 0.09 thiamine (mg/100g) 0.115 ± 0.0071 0.09 ± 0.14 riboflavin (mg/100g) 0.135 ± 0.035 0.64 ± 0.04 niacin (mg/100g) 1.332 ± 0.013 0.65 ± 0.06 data represented in mean ± sem table 4. anti-nutrients composition of methanol extract of citrulus lanatus (watermelon) seed extract anti-nutrient compounds citrulus lanatus (watermelon) seed cocos nucifera (coconut palm) stem bark phytate (mg/100g) 0.677 ± 0.0057 2.37 ± 0.00 oxalate (mg/100g) 0.082 ± 0.0042 0.97 ± 0.00 hemaglutin (%) 0.549 ± 0.0085 0.69 ± 0.01 trypsin inhibitor (mg/100g) 0.456 ± 0.0085 0.55 ± 0.00 data represented in mean ± sem additionally, flavonoids can stop atherosclerosis, a condition where fat deposits inside the arterial wall. this type of deposition causes the arteries to narrow, which prevents blood from reaching our body's essential organs, such as the heart and brain. thus, this illness raises the risk of stroke and heart attack. flavonoids reduce the incidence of coronary heart disease by reducing atherosclerosis (adeniyi et al., 2012). excessive tannin content damages the intended track and reduces digestibility, which lowers protein quality (sam et al., 2012). according to dutta (2003), tea's flavor is attributed to its tannins. because of their astringent qualities, they are used to treat skin eruptions and for other medical uses. the ability of plants to precipitate and coagulate red blood cells is shown by the presence of saponin. some of the features include hemolytic activity, bitterness, cholesterol binding qualities, and the production of foams in aqueous solutions (sodipo et al., 2000; okwu, 2004; adeniyi et al., 2012). it was discovered that the amount of cyanogenic glycoside was trace. because cyanide is a powerful cytochrome oxidase inhibitor that interferes with the aerobic respiratory system, it is essential to know the amount of cyanogenic glycosides in food (aina et al., 2012). essential bodily processes like acid-base and water balance depend on minerals. according to onwordi et al. (2009), iron is a crucial component of hemoglobin. thus, this is most likely the reason why some people utilize these herbs to increase their hemoglobin levels, particularly throughout the healing process (adeniyi et al., 2012). according to okaka and okaka (2001), zinc is necessary for the body to assist the pancreas make insulin, enable insulin to function more efficiently, and shield insulin receptors on cells. consequently, the zinc content of the plants under study may indicate that the plants can be useful in the treatment of diabetes, which is brought on by insulin failure. the presence of magnesium ions in the plants under study may help manage type 2 diabetes as they are known hormone activators in the condition. vitamins a, e, and c (ascorbic acids) are all abundant in these plants (table 4). according to okwu (2004), natural ascorbic acid is essential for bodily functions. ascorbic acid deficiency disrupts the body's natural production of intercellular materials, such as collagen, bone matrix, and dentine of teeth. the pathogenic alteration brought on by this flaw is the capillaries' endothelial wall becoming weaker as a result of a decrease in intercellular chemicals (adeniyi et al., 2012). thus, ascorbic acid and proper connective tissue metabolism may be linked to the clinical signs of scurvy bleeding from the mucous membranes of the mouth and gastrointestinal system, anemia, and joint pain (hunt et al., 1980; okwu, 2004). this ascorbic acid activity also explains why normal wound healing is necessary (adeniyi et al., 2012). since vitamins a, c, and e are antioxidants that can scavenge free radicals produced in the body, the ascorbic acid found in both plants makes them suitable for use in herbal medicine to control oxidative stress and prevent oxidative damage to organs and tissues. one significant way that plants store phosphorus is through phytotic acid, a hexaphosphate derivative of inositol. an anti-nutrient called phytotate prevents minerals from the food from being absorbed. according to sam et al. (2012), it produces a deficit of calcium and zinc in humans, which leads to rickets, anemia, and osteomalacia. phytate levels in c. nucifera stem bark and c. lanatus seeds were found to be 2.37 and 0.677 mg/100 g, respectively. the presence of the enzyme phytase, which breaks down phytic acid in plants, may be the cause of the low phytic acid levels (aina et al., 2012). because they decrease calcium absorption and promote the formation of renal calcium, oxalates are considered unfavorable dietary components (fagboya, 1990). citrulus lanatus seeds and the stem bark of cocos nucifera have low anti-nutrient content, which is a sign of a healthy diet and a potential source of medicine. conclusion according to this study, c. lanatus seeds and c. nucifera stem bark are good suppliers of biologically significant phytochemicals, which means they may be used as herbs to treat certain conditions. the plants can be a beneficial dietary supplement, according to the trace levels of antinutrient chemicals. additionally, these plants provide a significant amount of easily accessible proximate compounds, vitamins, and minerals that can be taken to supplement the limited or unavailable nutrient sources. reference adeniyi sa, orjiekwe cl, ehiagbonare je, arimah bd(2012). evaluation of 8 chemical composition of the leaves of ocimum gratissium and vernonia amygdalina. int. j. biol. chem. sci., 6(3): 1316-1323 aina vo, sambo b, zakari a, haruna ms, umar h, akinboboye rm, mohammed a(2012). determination of nutritional and anti-nutrient content of vitis vinifera (grapes) grown in bomo (area c) zaria, nigeria. advance journal of food science and technology, 4(6): 445 448. aoac. 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pp: 55-71. stray f(1998). the natural guide to medicinal herbs and plants. tiger books international: london; 12-16. van-burden tp, robinson wc(1981). formation of complexes between protein and tannin acid. j. agric food chem., 1: 77-82. weigert p(1991). metal loads of food of vegetable origin including mushrooms. in metals and their compounds in the environment: occurrence, analysis and biological relevance. merian e (ed). weinheim, vch; pp: 458-468. http://www.financialexpress.com/ http://www.mb.com.ph/ materials and methods plant material preparation of fat free sample phytochemical analysis phytochemical analysis alkaloid determination saponin determination tannin determination flavonoid determination determination of total phenols cyanogenic glycoside determination proximate composition analysis moisture content determination crude protein determination crude fiber determination fat content determination carbohydrate content determination vitamin analysis determination of vitamin a determination of vitamin e determination of niacin determination of riboflavin determination of thiamine anti-nutrients analysis oxalate determination phytate determination trypsin inhibitor determination hemaglutinin determination statistical analysis results discussion conclusion 1 in ternationa l scholars journa ls african journal of food science research issn 2375-0723 vol. 12 (2), pp. 001-008, february, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper nutritional analysis of homemade infant gruels made from malted cereals, soybeans and groundnuts in north-western nigeria k. m. anigo1*, d. a. ameh1, s. ibrahim1 and s. s. danbauchi2 1 department of biochemistry, ahmadu bello university, zaria, kaduna state, nigeria. 2 department of medicine, ahmadu bello university, zaria, kaduna state, nigeria. accepted 5 november, 2023 prevalence of malnutrition in children in north-western nigeria is high due to poor complementary feeding practices. an attempt was made to formulate low-cost, nutritive complementary foods using malted maize, millet and sorghum with groundnut and soyabean. nutrient qualities of gruel of the formulations thus prepared were evaluated. the ash content ranged from 1.03 ± 0.01 to 2.54 ± 0.35 g/100 g with the crude protein ranging from 6.37 ± 0.23 to 7.88 ± 0.28 g/100 g. levels of lysine (4.80 to 6.87 g/100 g protein) and methionine (1.25 to 3.33 g/100 g protein) in most formulated complementary foods were higher than the international reference values of 4.2 and 2.2 for lysine and methionine, respectively. percentage invitro protein digestibility of the formulations (75 to 82%) is comparable to that of the commonly used complementary foods (72 to 82%) in the zone. zinc content ranged from 74.15 to 107.85 ppm and iron content was between 177.10 and 476.64 ppm. phytates (0.01 to 0.34 mg/100 g), tannins (16 to 37 mg/100 g) and free cyanide (0.16 to 0.99 mg/100 g). levels of microbial contamination in the formulated were lower when compared to that reported in commonly used complementary foods in north western nigeria. there was no presence of samonella and shigella in all the samples while also no staphylococcus spp and escherichia coli in some of the samples. sensory evaluation found the formulated complementary foods preparations to have good acceptability. there is improvement in the nutrient quality of the formulated complementary foods with good acceptability comparable to that commonly used in the zone. key words: complementary foods, malted cereals, soyabean, groundnut, formulated blends, nutrient composition. introduction adoption of recommended breastfeeding and complementary feeding practices and access to the appropriate quality and quantity of foods are essential components of optimal nutrition for infants and young children (lutter and rivera, 2003). complementary feeding period is the time when malnutrition starts in many infants contributing significantly to the high prevalence of malnutrition in children under 5 years of age worldwide (daelmans and saadeh, 2003). many factors contribute to the vulnerability of children during the complementary feeding period. the complementary foods are often of *corresponding author. e-mail: mkanigo@yahoo.com. tel: +2348073197086 low nutritional quality and given in insufficient amounts. when given too early or too frequently, they displace breast milk (villapando, 2000; who, 2002). data available on the regional prevalence of diarrhea, under nutrition and under 5 mortality in nigeria showed a strong interaction among these three factors, with each of them far more prevalent in northern than southern part of nigeria (unicef, 2001). prevalence of malnutrition in children in north western nigeria is high due to early introduction of complementary foods and high levels of microbial contamination (anigo et al., 2007, 2008). poor feeding practices and shortfall in food intake are the most important direct factors responsible for malnutrition and illness amongst children in nigeria (solomon, 2005). the high cost of fortified nutritious proprietary complementary foods is always beyond the reach of most nigerian 2 table 1. formulated complementary food blends. code blend ratio (w/w %) mgbsg malted guinea corn: boiled soybeans: roasted groundnut 60:20:20 mgrsg malted guinea corn: roasted soybeans: roasted groundnut 60:20:20 mlbsg malted millet: boiled soybeans: roasted groundnut 60:20:20 mlrsg malted millet: roasted soybeans: roasted groundnut 60:20:20 mmbsg malted maize: boiled soybeans: roasted groundnut 60:20:20 mmrsg malted maize: roasted soybeans: roasted groundnut 60:20:20 families; hence many depend on inadequately processed traditional foods consisting mainly of unsupplemented cereal porridges made from maize, sorghum and millet (nnam, 2002). the global strategy for infant and young child feeding states that, infants should be exclusively breastfed for the first six months of life to achieve optimal growth, development and health, and thereafter, receive nutritionally adequate and safe complementary foods while breastfeeding continues for up to two years or beyond (who, 2002). improved complementary feeding and breastfeeding practices with reduced morbidity are essential to achieving the millennium development goals (mdgs) for child survival and prevention of malnutrition (lutter, 2003). complementary feeding improvement should be of highest priority for nutrition of infant and young children because of its crucial role in preventing mortality and enhancing children development (lutter and dewey, 2003). over 70% of dietary protein in developing countries is supplied by cereals that are relatively poor sources of protein (glover, 1976). formulation and development of nutritious weaning foods from local and readily available raw materials has received a lot of attention in many developing countries (plahar and annan, 1994). several blends using legumes singly or in combination have been evaluated (ekpenyong et al., 1977; plahar et al., 1983; sefa-dede, 1984) . legumes are largely replacing milk and other sources of animal protein, which are expensive and not readily available, as suitable substitutes for highquality protein (plahar and annan, 1994). therefore, appropriate processing and blending of locally available food commodities are been carried out by a number of researchers in nigeria (solomon, 2005) table 1. the present study is a contribution to addressing the prevalence of malnutrition in children in north western nigeria through formulation of more nutritious complementary foods using locally available components. materials and methods cereal grain (guinea corn, sorghum bicolor; maize, zea mays and millet, pennisetum glaucum), groundnuts and soyabean used for this work were purchased from samaru market in zaria, kaduna state. all the grains and legumes samples were manually cleaned by removing manually the ones that were mouldy or broken. the maize, sorghum soyabean groundnut millet cleaning cleaning cleaning soaking soaking roasting (18 h, 25-30°c) (12 h, 25-30°c) germination washing separation of husks (48 h) separation draining of sprouts boiling (2 h, 100°c) sun drying (36 h) separation of husks washing draining sun drying (36°c) blending grinding packing figure 1. flow chart for preparation of formulated complementary food blends. grains were malted as described by kulkarni et al. (1991) for cereals modified with no addition of sodium benzoate as shown in figure 1. the grains were watered twice daily and non-germinated ones removed. 3 table 2. proximate composition of formulated complementary foods (g/100 g dry matter). proximate composition mgrsg mgbsg mlrsg mlbsg mmrsg mmbsg dry matter 14.39 ± 1.43 ab 13.29 ± 0.01 b 13.59 ± 0.01 b 11.05 ± 0.01 c 13.26 ± 0.71 b 15.68 ± 0.08 a ash 2.28 ± 0.19 a 1.11 ± 0.04 c 2.69 ± 0.96 a 1.30 ± 0.09 b 2.54 ± 0.35 a 1.03 ± 0.01 d crude fat 1.09 ± 0.04 d 1.24 ± 0.02 c 2.06 ± 0.16 ab 1.61 ± 0.40 bc 1.05 ± 0.17 d 1.99 ± 0.28 ab crude protein 7.88 ± 0.28 a 6.76 ± 0.61 b 6.37 ± 0.23 c 7.25 ± 0.05 b 7.20 ± 0.41 a 7.44 ± 0.22 a crude fibre 1.21 ± 0.01 c 1.02 ± 0.02 d 1.74 ± 0.02 a 0.99 ± 0.03 d 0.87 ± 0.01 e 1.54 ± 0.10 b total carbohydrate 88.75 ± 0.28 c 90.89 ± 0.57 a 88.88 ± 1.04 bc 89.84 ± 0.51 b 89.21 ± 0.58 bc 89.54 ± 0.10 b values in the same row with different superscripts are significantly different (p < 0.05). mgrsg = malted guinea corn/roasted groundnut/roasted soybeans. mgbsg = malted guinea corn/roasted groundnut/boiled soybeans. mlrsg = malted millet/roasted groundnut/roasted soybeans. mlbsg = malted millet/roasted groundnut/boiled soybeans. mmrsg = malted maize/roasted groundnut/roasted soybeans. mmbsg = malted maize/roasted groundnut/boiled soybeans. groundnut and a part of soyabean were roasted separately while another batch of soyabean was steeped, washed with tap water and boiled (figure 1) . each formulated blend milled using hammer mill and were mixed thoroughly mistral homogenizer into smooth homogenous powder and then stored in airtight containers at room temperature (25 30 o c) until used. ready-to-eat gruel of each formulated complementary foods was made by mixing flour in cold water at concentrations of 20% and then poured into boiling water (200 ml) with the addition of 20 g of flour, stirred and allowed to remain heated for 15 min to form thick gruel, after which samples were taken for analyses. chemical analyses samples and standard solutions were prepared according to the procedures of the aoac (1990).the proximate composition of the formulated complementary food samples were determined as described by aoac (1990). total carbohydrate was determined by difference. amino acid profile was determined using the method described by spackman et al. (1958). samples were digested in a solution by the acid digestion method (allen, 1974). mineral element (na, k, ca, mg, p, mn, fe and zn) concentrations were determined using atomic absorption spectrophotometer (hitachi, model 18080). phosphorus was determined spectrophotometrically by the vanadomolybdate method (aoac, 1990). phytic acid was determined by method of wheeler and ferrel (1971), tannin by the vanillin-hcl (earp et al., 1981) and cyanide content by method of ikediobi et al. (1980). in-vitro protein digestibility determined as described by mertz et al. (1984). microbiological and sensory analysis all microbiological analyses were carried out based on procedures recommended in the international commission on microbiological specification for foods (1996). appropriate serial dilutions of the formulated complementary foods were carried out and 0.1 ml of the selected dilution was spread on triplicate plates using sterile glass spreader. this technique was used for the enumeration of total aerobic count, escherichia coli counts, staphylococcal counts, salmonella and shigella counts, and fungi and molds counts on nutrient agar, eosin methylene agar, salmonella and shigella agar, and sabourand glucose agar. media used were prepared according to the manufacturers instructions and all cultures were incubated at 37°c for 24 h. sensory evaluation of the ready-to-eat formulated complementtary foods was carried out on the taste, appearance, aroma, texture, colour and overall acceptability by a 14 semi-trained adult panelists which includes mothers with children age 6 to 24 months and postgraduate students in ahmadu bello university, zaria using a 9-piont hedonic scale (williams, 1982) which range between 1 (dislike extremely) and 9 (like extremely). the range method of statistical analysis was applied for the test of the significance to find the preferences. statistical analyses results were expressed as mean ± standard deviation. the difference between groups of each parameter was determined using the t-test and statistical significance was claimed at p < 0.05. results table 2 showed result of proximate analyses of the formulated complementary foods. mmbsg (15.68 ± 0.08%) had significantly higher (p < 0.05) dry matter contents which is comparable to mgrsg (14.39 ± 1.43%). mmbsg had significantly (p < 0.05) lower ash content (1.03 ± 0.01%) while comparable higher values of crude protein were recorded for mgrsg (7.88 ± 0.28%), mmbsg (7.44 ± 0.22%) and mmrsg (7.20 ± 0.41%). crude fat contents of mgrsg (1.09 ± 0.04%) and mmrsg (1.05 ± 0.17%) were significantly (p < 0.05) lower while mgbsg (90.89 ± 0.57%) significantly higher (p < 0.05) in total carbohydrate content. pattern of variation of the non-essential and essential amino acids concentrations indicates that mlrsg was higher compared to the other formulated complementary foods (table 3) . table 4 showed no significant difference (p > 0.05) in the in-vitro protein digestibility of all the formulated complementary foods and commonly used plain cereal pap (kdlp, kbgp) as control. mineral element concentrations showed that mlrsg which had significantly (p < 0.05) higher ash content (table 2), recorded significantly (p < 0.05) higher with comparable values for some of the mineral elements especially k, ca and p (table 5). the result also indicates that sodium, manganese and iron contents were significantly (p < 0.05) higher in mgbsg while significantly (p < 0.05) lower values of calcium and phosphorus contents were observed in mgrsg and mmbsg respectively. there were significant differences (p < 0.05) in magnesium content of mgbsg (146.91 mg/100 g) and mmbsg 4 table 3. amino acids composition of formulated complementary foods (g/100g protein). amino acids mgbsg mgrsg mlbsg mlrsg mmbsg mmrsg aspartate 9.25 6.51 10.03 9.81 8.17 7.21 serine 2.75 2.48 2.18 3.08 2.86 2.00 glutamate 14.30 13.11 15.19 18.85 12.52 11.15 proline 1.91 2.54 2.86 3.61 2.97 1.80 glycine 3.89 4.03 4.49 7.36 3.65 3.59 alanine 3.98 2.50 3.97 5.67 4.45 3.29 cystine 1.19 1.12 1.38 1.65 1.37 1.09 tyrosine 3.22 2.89 3.22 3.54 3.22 3.06 lysine 5.14 4.80 5.74 6.87 6.01 5.26 histidine 2.38 2.16 2.63 3.38 2.32 2.56 arginine 5.45 4.50 5.61 6.63 6.13 4.96 threonine 2.77 3.02 3.02 3.24 2.49 2.20 valine 3.39 2.73 3.25 4.15 2.64 3.00 methionine 1.25 2.48 2.68 3.33 1.87 1.34 isoleucine 3.35 3.28 3.56 4.04 3.04 2.50 leucine 5.72 6.03 6.97 7.19 5.65 4.99 phenylalanine 4.23 4.39 4.56 5.41 4.05 3.59 mgrsg = malted guinea corn/roasted groundnut/roasted soybeans. mgbsg = malted guinea corn/roasted groundnut/boiled soybeans. mlrsg = malted millet/roasted groundnut/roasted soybeans. mlbsg = malted millet/roasted groundnut/boiled soybeans. mmrsg = malted maize/roasted groundnut/roasted soybeans. mmbsg = malted maize/roasted groundnut/boiled soybeans. table 4. percentage in-vitro protein digestibility of formulated complementary foods. complementary foods % in-vitro digestibility mgrsg 82.12 ± 6.35 a mgbsg 79.23 ± 3.10 a mlrsg 75.27 ± 2.70 a mlbsg 80.68 ± 6.40 a mmrsg 76.88 ± 2.16 a mmbsg 77.24 ± 3.83 a kbgp 82.17 ± 3.12 a kdlp 72.51 ± 6.98 a values with different superscripts are significantly different (p<0.05). kbgp = guinea corn pap. kdlp = millet pap. mgrsg = malted guinea corn/roasted groundnut/roasted soybeans. mgbsg = malted guinea corn/roasted groundnut/boiled soybeans. mlrsg = malted millet/roasted groundnut/roasted soybeans. mlbsg = malted millet/roasted groundnut/boiled soybeans. mmrsg = malted maize/roasted groundnut/roasted soybeans. mmbsg = malted maize/roasted groundnut/boiled soybeans. (107.56 mg/100 g) and also in the zinc content of mgrsg (7.42 mg/100 g) and mmrsg (10.79 mg/100 g). levels of antinutrient (table 6) in the formulated complementary foods revealed that mgrsg (0.34 ± 0.02 mg/100 g) and mlbsg (37.42 ± 9.46 mg/100 g) had significantly higher (p < 0.05) phytate and tannin concentrations respectively. significantly (p < 0.05) higher free cyanide contents were recorded in mgbsg (0.84 ± 0.13 mg/100 g) and mlbsg (0.99 ± 0.13 mg/100 g). presented in table 7 are the mean counts of organisms in the formulated complementary foods with significantly (p < 0.05) higher total aerobic count of 5 table 5. mineral element concentration in formulated complementary foods (mg/100 g dry matter). complementary foods na k ca mg p mn fe zn mgbsg 209.72 a 381.32 ab 47.66 a 146.91 a 219.98 a 3.58 a 47.66 a 7.88 ab mgrsg 170.03 b 434.95 a 27.68 b 123.52 ab 193.90 a 1.98 c 23.73 bc 7.42 b mlbsg 139.86 bc 319.68 b 47.95 a 108.18 ab 184.4 a 2.50 bc 23.98 bc 9.16 ab mlrsg 159.39 bc 432.90 a 47.23 a 122.93 ab 215.69 a 1.97 c 17.71 c 8.20 ab mmbsg 135.08 c 278.11 ab 47.68 a 107.56 b 148.98 b 2.98 ab 19.87 bc 7.45 ab mmrsg 167.23 b 437.99 a 39.82 a 114.43 ab 172.28 a 1.99 c 19.91 bc 10.79 a sem 14.79 45.56 7.10 13.59 15.73 0.21 3.25 1.31 values in the same column with different superscripts are significantly different (p < 0.05). mgrsg = malted guinea corn/roasted groundnut/roasted soybeans. mgbsg = malted guinea corn/roasted groundnut/boiled soybeans. mlrsg = malted millet/roasted groundnut/roasted soybeans. mlbsg = malted millet/roasted groundnut/boiled soybeans. mmrsg = malted maize/roasted groundnut/roasted soybeans. mmbsg = malted maize/roasted groundnut/boiled soybeans. table 6. antinutritional factors in formulated complementary foods (mg/100 g). antinutrient mgrsg mgbsg mlrsg mlbsg mmrsg mmbsg phytates 0.34 ± 0.02 a 0.12 ± 0.02 b 0.03 ± 0.00 c 0.03 ± 0.01 c 0.01 ± 0.00 c 0.01 ± 0.00 c tannins 15.57 ± 0.00 b 15.57 ± 0.00 b 15.57 ± 0.00 b 37.42 ± 9.46 a 15.57 ± 0.00 a 15.57 ± 8.99 a total cyanide 1.06 ± 0.20 b 1.85 ± 0.50 a 1.81 ± 0.35 a 1.13 ± 0.13 b 0.22 ± 0.06 d 0.55 ± 0.03 c free cyanide 0.37 ± 0.12 bc 0.84 ± 0.13 a 0.40 ± 0.05 c 0.99 ± 0.13 a 0.16 ± 0.05 b 0.50 ± 0.02 b bound cyanide 0.69 ± 0.29 b 1.01 ± 0.28 a 1.41 ± 0.32 a 0.14 ± 0.03 c 0.06 ± 0.04 c 0.08 ± 0.01 c values in the same row with different superscripts are significantly different (p < 0.05). mgrsg = malted guinea corn/roasted groundnut/roasted soybeans. mgbsg = malted guinea corn/roasted groundnut/boiled soybeans. mlrsg = malted millet/roasted groundnut/roasted soybeans mlbsg = malted millet/roasted groundnut/boiled soybeans. mmrsg = malted maize/roasted groundnut/roasted soybeans. mmbsg = malted maize/roasted groundnut/boiled soybeans. organism obtained in mgrsg (450.00 ± 45.00 cfu/g) and mlbsg (371.00 ± 90.00 cfu/g) while no growth of e.coli in mgrsg, mlrsg and mlbsg. mmrsg (39.33 ± 5.61 cfu/g) had significantly higher (p < 0.05) staphylococcal counts while significantly higher (p < 0.05) fungi counts were obtained in mgrsg (8.33 ± 3.21 cfu/g) and mmbsg (8.50 ± 0.71 cfu/g). all the formulated complementary foods did not record any growth for salmonella and shigella. sensory evaluation of the formulated complementary foods showed no significant difference (p > 0.05) between the colour, odour and taste compared with commonly used plain cereal pap (table 8). overall acceptability rating for the formulated complementary foods was equal (p > 0.05) to the plain cereal pap commonly used in north western nigeria. discussion lack of nutrient-dense complementary foods is one of the common factors accounting for decline in satisfactory growth pattern in children (lartey et al., 1999). martins et al. (1992) reported that appropriate number of feeding by children depends on the energy density of the local foods and the usual amount consumed at each feedings. the dry matter contents (table 2) of the formulated complementary foods when compared with 6.37 to 9.99% reported in plain pap commonly used as complementary foods in north western nigeria (anigo et al., 2009) is an improvement in the nutrient density of the complementary foods which may lead to improved nutrient intake which means more nutrients for same quantity taken and may contribute to solving the problem of malnutrition in the zone. fao (2001) reported that staple foods such as millet, maize and sorghum are high in starch hence absorbed a lot of water during cooking which make them bulky hence infants need to consume large quantities to get enough energy and nutrients but it is difficult because they have small stomach but the problem is solved if families feed children with weaning foods prepared from germinated cereal flour and enrich bulky foods. malting reduces viscosity of the foods and hence a child can eat more at a time (ikujenlola and fashakin, 2005). the crude protein values obtained (table 2) in the formulated complementary foods were lower thanthe 13 to 14 g rda (guthrie, 1989) recommended for infants up to one year, however, it may still be able to meet 50% of the rda but indicates undernutrition which may still lead to nutritional deficiency hence the need to improve on the nutrient density. however, concentrations of crude protein in the formulated complementary foods are higher than 1.38 to3.15 g/100 g reported (anigo et al., 2009) in commonly used complementary foods in northwestern nigeria. the lysine, leucine and phenylalanine 6 table 7. mean count of organisms (cfu/g) in formulated complementary foods. complementary foods total aerobic count ×10 5 e. coli count staphylococcal samonella and shigella ×10 3 fungi count mgrsg 450.00 ± 45.00 a ng 6.00 ± 4.24 c ng 8.33 ± 3.21 a mgbsg 7.00 ± 0.71 d 5.50 ± 0.49 a 4.33 ± 1.15 c ng 2.00 ± 0.00 c mlrsg 160.50 ± 81.32 b ng 15.00 ± 1.51 b ng 4.00 ± 1.00 b mlbsg 371.00 ± 90.10 a ng ng ng 1.50 ± 0.71c mmrsg 2.50 ± 0.12 e 7.00 ± 1.66 a 39.33 ± 5.61 a ng 2.50 ± 0.71 bc mmbsg 12.50 ± 3.54 c 3.00 ± 0.41 b ng ng 8.50 ± 0.71 a values in the same column with different superscripts are significantly different (p < 0.05). mgrsg = malted guinea corn/roasted groundnut/roasted soybeans. mgbsg = malted guinea corn/roasted groundnut/boiled soybeans. mlrsg = malted millet/roasted groundnut/roasted soybeans. mlbsg = malted millet/roasted groundnut/boiled soybeans mmrsg = malted maize/roasted groundnut/roasted soybeans. mmbsg = malted maize/roasted groundnut/boiled soybeans ng = no growth. table 8. sensory evaluation of formulated complementary foods using the hedonic scale blend colour odour taste texture overall acceptability mgrsg 6.50 ± 0.76 a 5.71 ± 1.48 a 6.83 ± 1.07 a 6.57 ± 0.90 ab 6.43 ± 1.29 ab mgbsg 5.67 ± 1.49 a 5.00 ± 1.31 a 4.86 ± 1.73 a 4.43 ± 1.99 b 5.67 ± 0.94 b mlrsg 6.29 ± 1.91 a 6.29 ± 1.28 a 6.43 ± 1.40 a 6.14 ± 1.12 ab 6.43 ± 1.18 ab mlbsg 6.71 ± 1.39 a 4.14 ± 1.88 a 5.71 ± 1.75 a 6.71 ± 1.39 ab 6.29 ± 0.70 ab mmrsg 7.50 ± 1.38 a 6.00 ± 1.51 a 6.57 ± 1.50 a 6.29 ± 1.83 ab 6.57 ± 1.84 ab mmbsg 6.71 ± 1.16 a 6.00 ± 0.93 a 5.43 ± 1.99 a 6.71 ± 1.83 ab 6.57 ± 0.73 ab gcp 8.00 ± 1.41 a 7.14 ± 1.12 a 7.29 ± 0.80 a 7.29 ± 1.03 ab 7.57 ± 0.73 a lps 7.67 ± 1.24 a 7.00 ± 0.29 a 6.57 ± 1.99 a 7.57 ± 1.40 ab 6.86 ± 2.10 ab gmp 8.14 ± 1.36 a 6.29 ± 1.48 a 6.14 ± 1.64 a 7.33 ± 0.75 a 6.71 ± 1.58 ab hedonic scale: 1 = dislike extremely 2 = dislike very much 3 = dislike moderately 4 = dislike slightly 5 = neither like/dislike 6 = like slightly 7 = like moderately 8 = like very much 9 = like extremely. values in the same column with different superscripts are significantly different (p < 0.05). mgrsg = malted guinea corn/roasted groundnut/roasted soybeans. mgbsg = malted guinea corn/roasted groundnut/boiled soybeans. mlrsg = malted millet/roasted groundnut/roasted soybeans. mlbsg = malted millet/roasted groundnut/boiled soybeans. mmrsg = malted maize/roasted groundnut/roasted soybeans. mmbsg = malted maize/roasted groundnut/boiled soybeans gcp = guinea corn pap, lps = millet pap, gmp = guinea corn/millet pap. 7 values (table 3) for all and methionine values for some of the formulated complementary foods were higher than the international reference values for infants (fao/who, 1998). percentage in-vitro protein digestibility of formulated complementary foods was comparable to those of the commonly used complementary foods in northwestern nigeria (table 4). protein quality is a measure of the efficiency of utilization of proteins by the body which depends on the amino acid composition, digestibility of the proteins and the biological availability of its amino acids for the synthesis of tissue proteins (ikujenlola and fashakin, 2005). there was improvement in concentrations of mineral elements in the formulated complementary foods (table 5) compare to the commonly used complementary foods in the zone and values obtained for all the mineral elements were higher than rda for infant up to one year except for calcium and potassium but may also contribute to the overall daily intake of mineral elements. according to fao/who (2001) minerals such as iron and zinc are low in cereals but the addition of legumes can improve the iron content. the body depends on a regular zinc supply provided by the daily diets and improvement of zinc in the diet as obtained in the formulated complementary foods may help decrease the prevalence of stunting in the zone as linear growth was reported (fao/who, 2001) to be affected by zinc supply. bioavailability of nutrients in the formulated complementary foods (table 6) compared to 0.04 to 011 mg/100 g phytates and 0.05 to 0.74 mg/100 g free cyanide reported (anigo et al., 2009) in commonly used plain pap may also be high due to low levels of phytates which were less than 1% reported to interfere with mineral availability (erdman, 1979) and free cyanide below 1 mg/100 g safe level reported by fao/who (1998). phytates strongly inhibit iron absorption in a dosedependent fashion, though small amounts of phytates have a marked effect (gillooly, 1983). germination has been reported (traore et al., 2004) to reduce the concentration of antinutritional factors like phytates in malted grains hence improves its nutritional quality (marero et al., 1991). the levels of microbial contamination in the formulated complementary foods were lower (table 7) when compared to the reported (anigo et al., 2007) unacceptable levels of salmonella and shigella, and predominance of staphylococcus sp. and fungi detected in most of the commonly used complementary foods in northwestern nigeria. although, satisfactory results were obtained for salmonella and shigella in all and staphylococcus spp and e. coli in some of the formulated complementary foods samples, most of them gave unsatisfactory results for total aerobic count. presence of some organisms in the formulated complementary foods though prepared under laboratory conditions, indicate a level of contamination with pathogens which may have be present prior to processing due to the source of raw materials used. sensory quality of some of the formulated complementary foods compare to the commonly used complementary foods in the zone was both liked moderately on the hedonic scale (table 8). a number of organoleptic features, such as flavour, aroma, appearance and texture, may affect infant’s intake of transitional foods (birch, 1998) which may results in increased consumption (hofvander and underwood, 1987) . feeding infants with improved complementary foods as that formulated in this study for children in the zone may cause improvement in their growth (lartey et al., 1999). conclusion there is improvement in the nutrient quality of the formulated complementary foods with good 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climates. the study looked into how conventional processing techniques affected the nutritional and anti-nutritional qualities of soybeans. the following processing methods were applied to the soybeans: soaking for 24 hours, sun drying and milling (sample b); soaking for 12 hours, de-hulling, sun drying and milling (sample c); sprouting for 120 hours, sun drying and milling (sample d); and sun drying and milling, which served as the control (sample a). the proximate composition of soybeans revealed that samples' protein contents ranged from 23.98 to 28.44%, with samples b, c, and d having the highest protein contents. crude fiber ranged from 4.68 to 6.58%, and the fat content ranged from 20.51 to 26.20%. the control's moisture content, which varied from 7.23 to 10.92%, significantly decreased. the samples' anti-nutritional characteristics revealed that the phytic acid content ranged from 5.45 to 8.05%, with samples b, c, and d showing a considerable decrease. while the protease inhibitor ranged from 4.91 to 7.09%, with a notable decline in samples b, c, and d, the tannin concentration varied from 19.23 to 25.23 mg/100g. according to the study, traditional processing techniques can both greatly enhance the nutritional qualities of soybeans and dramatically decrease their anti-nutritional qualities, which increases the nutrients' bioavailability. key words: de-hulling, glycine max, milling, proximate, sprouting, sun drying. introduction education legumes are a good source of protein, carbohydrates, dietary fiber, and minerals. only a small percentage of the known species of legumes are widely used and promoted. the soybean (us) or soya bean (uk) is an east asian legume plant that is commonly grown for its edible bean, which has a variety of uses. the plant is classified as an oilseed rather than a pulse by the un food and agricultural organization (fao, 2008). soybeans are the only protein that can be fed to pigs and poultry. although soybeans are low in sulfurcontaining amino acids like cysteic and methionine (potter and hotchkiss, 1995), their protein quality is comparable to that of animal protein sources like meat and milk, which have enough lysine to make up for cereal's lysine deficiency (fabiyi and hamidu, 2011). to increase the nutritional content of soybeans and other legumes, a range of processing methods can be employed, including as toasting, frying, extrusion, salt treatment, fermentation, germination pressure heating, cooking, soaking, and urea treatment (akande & fabiyi, 2010). about 40–45% protein and 18–22% oil are found in soybeans, which are also rich in vitamins and minerals (goyal et al., 2012). phytic acid, saponins, and trypsin inhibitors are some of the anti-nutritional components found in raw soybeans that lower the nutritional value of grain legumes and can have negative effects on both people and animals when ingested in excess (mikic et al., 2009; sharma et al., 2011). trypsin inhibitors decrease digestibility by preventing the breakdown of dietary protein and the absorption of amino acids. they can inhibit either trypsin or chymotrypsin (roy et al., 2010). these anti-nutrients should be removed from legumes in order to enhance their nutritional value and organoleptic attractiveness for potential human consumption. furthermore, by boosting the bioavailability of vitamins, amino acids, and protein digestibility, processing methods can raise the nutritional value of soybeans (prodanov et al., 2004). most farmers author(s) retain the copyright of this article. african journal of food science research issn 2375-0723 vol. 13 (1), pp. 001-005, march, 2025. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 have found it very challenging to treat the seeds in a way that eliminates anti-nutritional components (okagbare and akpodiete, 2006). given this, the purpose of this study is to evaluate the effects of soybean seed processing methods on the nutritional and anti-nutritional properties of soybeans. materials and methods source of materials soybeans (glycine max) were obtained from a local market in ado-ekiti, nigeria. the beans were sorted and cleaned from extraneous materials. chemicals and reagents mercuric oxide of reagent grade, eagle scientific limited, england, produced paraffin wax, a 40% sodium hydroxide solution, a 4% sodium sulphate solution, a boric acid indicator solution, methyl red, and 0.2% bromocresyl; b.d.h. limited, england, produced petroleum ether, antifoam (like silicone or octyl alcohol), and 95% (v/v) ethyl alcohol. reagent grade potassium sulfate or anhydrous sodium sulfate 98% sulfuric acid, nitrogenfree. all of the other reagents were analytical grade. production of soybeans flour after the soybeans were washed and sorted, four different processing methods were applied to produce soybean flour. soybeans that have been sun-dried and crushed into flour make up sample a, the control, while soybeans that have been soaked in water at 12 °c for 24 hours make up sample b. in sample d, the soybeans sprouted for 120 hours before being dried and crushed into flour; in sample c, the soybeans were soaked for 12 hours, then dehulled, sun-dried, and milled into flour. for the purpose of sprouting, the soybean seeds were immersed in water for a full day. the seeds were able to germinate after being spread out on the jute bags and covered with the same material. water was sprayed on the jute bags twice a day until the seeds began to grow. the sprouting seeds were crushed into flour after being exposed to the sun for four days. determination of proximate composition crude protein, crude fat, ash, fiber, and moisture content were all examined using the aoac (2005) method. the total carbohydrate content was determined by subtracting the percentages of crude protein, fat, ash, and crude fiber from 100%. determination of anti-nutritional properties tannin was determined gravimetrically using the makkar and goodchild (1996) method, while phytate was calculated using the young and greaves (1990) method. the ladd and butler (1972) approach was used to determine the protease inhibitor. statistical analysis the results from the experiment were statistically analyzed using spss version 20. the duncan multiple range test (dmrt) was employed to separate the means when the findings showed substantial differences. results and discussion impact of conventional processing techniques on the soybean proximate composition the results of the study on how traditional processing methods affect the proximate composition of soybeans are shown in table 1. crude protein contents (%) for samples b, c, d, and the control (sample a) were 23.98, 24.67, 25.07, and 28.44, respectively. the statistics showed that sample a had the lowest protein level and sample d had the highest protein content when compared to the other samples. sprouting may have increased the crude protein's bioavailability in the soybeans, resulting in sample d's higher protein level. the significant rise in crude protein in sprouted soybeans could be due to intricate metabolic processes that occur during hydration and sprouting. enzymes break down the protein component during these processes, producing simple molecules that are used to make new compounds. the total crude protein constituent improved as a result of the sprouting enzymes' higher hydrolytic activity and the elimination of starch (ramadan, 2012). the higher crude protein concentration in relation to sprouting is especially noteworthy from a nutritional standpoint since it would enhance absorption and digestion. however, the results showed that there is no appreciable change in the levels of protein in samples b and c, even if soaking and dehulling significantly increases the protein content when compared to sample a. crude fat percentages for samples a, b, c, and d were 26.20, 23.35, 22.43, and 20.51, respectively. the findings showed that crude fat greatly increased in sample a and significantly decreased in sample d (ragab et al., 2010). the dissociation of lipid complexes and a metabolic process may be the reason of this, but it also indicates that sample a will be more vulnerable to rancidity than the other samples. 3 table 1. proximate composition of processed soybeans samples (%) parameters/ samples a b c d moisture content 7.23c + 0.27 8.43bc+ 0.07 9.14b + 0.63 10.92a + 0.29 ash content 4.76c + 0.90 5.27b + 0.68 3.83d + 0.39 6.75a + 0.60 crude fiber 6.58a + 0.38 5.10b + 0.30 5.15b+ 0.17a 4.68c + 0.78 fat content 26.20a + 0.75 23.35b + 0.83 22.43b + 0.90 20.51a + 0.69 crude protein 23.98c + 0.97 24.67b + 0.10 25.07b + 0.63 28.44a + 2.50 carbohydrate 38.48c + 0.68 41.61b + 0.35 43.52a + 0.50b 39.62c + 0.71 values are means of triplicates values along the same row followed by different superscripts are significantly different (p<0.05). where: sample a: control, soybeans dried and milled sample b: soybeans soaked for 24h, dried and milled into flour sample c: soybeans soaked for 12h, de-hulled, dried and milled into flour sample d: soybeans sprouted for 120h, dried and milled into flour. the moisture contents (%) of samples a, b, c, and d were 7.91, 8.43, 9.14, and 10.92, in that order. despite the fact that samples a and b and b and c do not differ much, the results showed that sample d's moisture content has significantly increased while sample a's has drastically dropped. the results were in line with those of ramadan (2012), who discovered that raw soybeans (giza 21 and giza 35) had respective moisture contents of 6.15 and 7.32. therefore, compared to the other samples, sample d would be more susceptible to microbial deterioration. the matching ash (%) contents of samples a, b, c, and d were 3.83, 4.76, 5.27, and 6.75, respectively. the results showed that all of the samples varied significantly. sample d's ash concentration has considerably increased, whilst sample c's is noticeably lower. the substantial effect of sprouting on the bioavailability of soybean minerals is responsible for this, whereas sample b's notable decline might have resulted from de-hulling, since most of the pericarp that constitutes the ash was discovered to have been de-hulled. this, however, is in line with ramadan (2012), who discovered that soybeans (giza 21 and giza 35) had ash contents of 5.80 and 6.80, respectively. the carbohydrate (%) contents of samples a, b, c, and d were 38.48, 41.61, 43.52, and 39.62, respectively. the results showed that sample c had a significantly larger carbohydrate content than sample a, despite the fact that there is no discernible difference between samples a and d. the impact of conventional processing methods on soybean flour's antinutritional properties the results of the investigation into how traditional processing methods affect the anti-nutritional properties of soybeans are shown in table 2. the percentages of phytic acid in samples a, b, c, and d are 8.05, 6.27, 5.58, and 5.45, respectively. the results showed that there were substantial differences among all the samples. despite being high in sample a, phytic acid dramatically decreased in sample d, while it also decreased significantly in samples b and c. the impact of phytic acid reduction in soybeans may be due to leaching, dehulling, and sprouting. odumodu (2010) reported higher quantities of phytotic acid in fermented soybeans than this study did. the phytic acid content of soybean seeds and products ranged from 1% to 1.5% of dry mass (mikic et al., 2009). additionally, osman (2007) observed that soaking, boiling, and germination all reduced the phytic acid content. values are means of triplicates values along the same row followed by different superscripts are significantly different (p<0.05) the tannin concentrations of samples a, b, c, and d were 25.23, 21.55, 19.23, and 20.13 mg/100g, respectively. the results showed that there was a considerable variation in the tannin concentration of the samples. the tannin content of samples b, c, and d significantly decreased. this could be explained by the fact that the tannin concentration was significantly affected by soaking, dehulling, and sprouting. additionally, the results demonstrated that there was no significant difference in the tannin content between samples c and d, suggesting that the dehulling and sprouting of soybeans had a significant reducing effect. protease inhibitors in samples a, b, c, and d are 7.09, 6.03, 5.72, and 4.91%, respectively. the results showed a significant difference between all samples. the significant reduction of the protease inhibitor in samples b, c, and d may be attributed in large part to soaking, dehulling, and sprouting. 4 table 2. anti-nutritional properties of processed soybeans samples parameter a b c d phytic acid (%) 8.05a + 0.87 6.27b + 0.48 5.58c + 0.32 5.45d + 0.72 tannin (mg/100g) 25.23a + 0.28 21.55b + 0.97 19.23c + 0.20 20.13c + 0.18 protease inhibitor (%) 7.09a + 0.81 6.03b + 0.26 5.72c + 0.30 4.91d + 0.39 5 conclusion studies have shown how traditional processing methods affect the nutritional and anti-nutritional properties of soybeans. the findings showed that conventional processing significantly increased soybeans' nutritious content while reducing their anti-nutritional traits. soybeans' protein content rose sharply as a result of being soaked, dehulled, and sprouted, but their fat content sharply dropped. additionally, there was a significant drop in phytic acid due to sprouting, which inhibits the absorption of nutrients. soybeans' lipid content was significantly reduced by the soaking, dehulling, and sprouting processes, which may have had a negative effect on rancidity. references akande ke, fabiyi ef( 2010). effect of processing methods on some anti-nutritional factors in legume seeds for poultry feeding. international journal of poultry science 9: 996-1001. 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seeds produced in sudan for poultry feeding. int. j. poult. sci., 9: 198-204. roy f, boye ji, simpson bk(2010). bioactive proteins and peptides in pulse crops: pea, chickpea and lentil. food research international 43: 432-442. sharma s, kaur m, goyal r, gill bs(2011). physical characteristics and nutritional composition of some new soybean (glycine max (l.) merrill) genotypes, journal of food science and technology. doi 10.1007/s13197-011-05177. young sm, js greaves(1990). influence of variety and treatment on phytin content of wheat. food res., 5: 103-105 materials and methods source of materials chemicals and reagents production of soybeans flour determination of proximate composition determination of anti-nutritional properties statistical analysis results and discussion impact of conventional processing techniques on the soybean proximate composition values are means of triplicates the impact of conventional processing methods on soybean flour's antinutritional properties values are means of triplicates (1) conclusion