food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 2, 2017 www.scholink.org/ojs/index.php/fsns 100 challenge test improvement: analytical costs and time optimization marco romani1* & chiara romani1 1 merieux nutrisciences, prato, italy * marco romani, e-mail: marco.romani@mxns.com received: october 1, 2017 accepted: october 10, 2017 online published: october 18, 2017 doi:10.22158/fsns.v1n2p100 url: http://dx.doi.org/10.22158/fsns.v1n2p100 abstract the study’s aim was to develop a quantitative risk assessment model of listeria monocytogenes in liver chicken paté. the model was performed using the integrated challenge test (italian journal of food safety, vol. 1 n. 6 2012) with the objective to reduce the analytical cost and time. the challenge test was carried out on 3 different batches stored at 12°c and inoculated with a mix of listeria monocytogenes strains. lactobacillus spp. and listeria monocytogenes plate counts were performed daily on each sample until the stationary phase was reached by both populations. the challenge test results at 12°c were input in the combase dmfit software to determine the growth parameters of listeria monocytogenes and lactic flora which showed mutual interaction. then, using the combase predictor for listeria monocytogenes and the fssp (food spoilage and safety predictor) software for lactic flora, the growth parameters were extrapolated at 4°c and 8°c. the growth parameters of both populations at 4°c, 8°c and 12°c were then used to apply the model in order to predict the maximum daily concentration of listeria monocytogenes. model results were assessed against the results of an additional challenge test conducted with the same strain mix inoculum in 3 different batches stored for 4 days at 4°c, 4 days at 8°c and then 4 days at 12°c. the proposed model represents a reliable quantitative risk evaluation which provides realistic results with limited cost. keywords listeria monocytogenes, challenge test, anti-listerial activity, lactic acid bacteria, predictive microbiology 1. introduction the integrated challenge test was created in response to anses guide (november 2008) to focus on its strengths and overcome its limitations. the first version of the integrated challenge test (italian journal of food safety, vol. 1, no. 6, december 2012) aimed to develop a quantitative risk assessment model of l. monocytogenes starting with experimental data only. this involved high analytical cost and www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 101 published by scholink inc. time. therefore, the following steps consisted in providing a more accessible model without compromising the scientific strictness. the current version of the integrated challenge test starts with experimental results, but uses predictive microbiology to extrapolate that data which, if obtained experimentally, would have greatly increased the cost and time. the model has been developed on a sample of liver chicken paté (rte food) that, for the values of the chemical-physical parameters (ph and aw) and the growth potential (> 0.5 log cfu/g), can support l. monocytogenes growth. this model is applicable also to the cooked meat products in which the lactic flora is predominant (example: sliced cooked ham, sliced mortadella, fresh sauces). 2. method the microbiological population of the product was mainly composed of lactic flora that was subjected to the following tests: agar well diffusion assay (parente et al., 1994) and agar drop test (paparella et al., 1992). the first test aimed to evaluate the antilisterial activity; the second one determined the substances with antilisterial activity. later a challenge test was conducted on three batches (three repetitions for batch). selected l. monocytogenes strains, most meat isolates, were grown at 8°c to post exponential phase, mixed and inoculated in liver paté samples (approximately 1.6 log cfu/g, according to the anses guide, november 2008). the samples were incubated at 12°c and tested daily for l. monocytogenes plate count (uni en iso 11290-02: 2005) and lactic acid bacteria plate count (uni en iso 15214: 1998). microbiological analysis were conducted until the stationary phase of both populations (1 week). the challenge test was conducted at 12°c because the time to reach by both population the stationary phase occurred in a short time (1 week). this allowed to speed the study, reducing costs significantly (at 4°c the lactic flora reaches the stationary phase after 20 days). l. monocytogenes and lactic flora growth data were then put in combase dmfit software in order to determine the growth curves and parameters: lag phase (days), daily growth rate (log/day), beginning of the stationary phase (days) and concentration of the stationary phase (log cfu/g). starting from the average growth parameters at 12°c, growth parameters (lag phase and growth rate) were extrapolated at 4°c and 8°c using two predictive software (combase predictor and fssp) and the formula of baranyi roberts relating to the physiological state of the microorganisms [physiological state = 1/10 (lag phase x growth rate)]. the experimental data at 12°c with those extrapolated at 4°c and 8°c were used to set the model which, taking also in consideration the difference time between l. monoctogenes and lactic flora stationary phase, aimed to define the maximum concentration reached by the pathogen in the liver chicken pate stored 4 days at 4°c, 4 days at 8°c and 4 days at 12°c. the model was then statistical assessed (t-test) against real data coming from a challenge test carried out according to the same time-temperature profile: 4 days at 4°c, 4 days at 8°c and 4 days at 12°c. the t-test was applied to the averages of the experimental and predictive data (95% confidence limit). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 102 published by scholink inc. 3. result agar well diffusion assay showed that the indigenous lactic flora had antilisterial activity against l. monocytogenes strains used for inoculation. agar drop test showed that such activity is supported by organic acids. in table 1 the averages of growth parameters of lactic flora and l. monocytogenes at 4°c, 8°c and 12°c are reported. from table 1 it can be observed that at 12°c l. monocytogenes achieved the stationary phase 0.43 days before lactic flora. this result with the other growth parameters permitted the development of the model. table 1. average values of the growth parameters of l. monocytogenes (l.m.) and of lactic acid bacteria (lab) at the temperatures of 4°c, 8°c, 12°c. the table shows the values used for the development of the model lab lag phase days lab growth rate log/day lab stationary phase log cfu/g lab stationary phase days l.m. lag phase days l.m. growth rate log/day l.m. stationary phase log cfu/g l.m. stationary phase days 4°c 4,78 0,37 / / 5,62 0,15 / / 8°c 1,95 0,92 / / 2,52 0,33 / / 12°c 1,05 1,70 8,96 5,22 1,26 0,66 / 4,79 the statistical comparison (t-test) between l. monocytogenes maximum concentration coming from the experimental challenge test and the predictive model (table 2) showed a slight difference. the model underestimated the reality by little (<0.5 log), suggesting the possibility to correct the average predictive data (3.43 log ufc/g) with the limit of 95% confidence. since the model underestimated the reality, the correction of the predictive data consisted in adding the upper limit of the confidence interval (-0.47 log). table 2. comparison of the average of predictive and experimental data regarding the maximum concentration of l. monocytogenes among the three batches. the lower confidence limit (lcl) and the upper confidence limit (ucl) were also calculated: confidence interval 95% challenge test predictive data log cfu/g experimental data log cfu/g 1° batch, 1° rep. 3,42 3,74 1° batch, 2° rep. 3,33 3,72 1° batch, 3° rep. 3,47 4,15 2° batch, 1° rep. 3,61 3,90 2° batch, 2° rep. 3,54 3,80 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 103 published by scholink inc. 2° batch, 3° rep. 3,46 3,95 3° batch, 1° rep. 3,42 3,52 3° batch, 2° rep. 3,39 3,70 3° batch, 3° rep. 3,21 3,78 average 3,43a 3,74b lower confidence limit (lcl) -0,23 log upper confidence limit (ucl) -0,47 log 4. discussion the study shows that the proposed quantitative risk assessment model is very realistic because taking into consideration the characteristics of the food, as well as the growth parameters of lactic flora and of l. monocytogenes, it is possible to predict the maximum concentration of the pathogen very closely the real data. also, the use of predictive microbiology for the extrapolation of data at temperatures at which the experimental test would be extended too much, gives the integrated challenge test a greater commercial value. food companies can implement the integrated challenge test at an affordable cost which is immediately translated into a competitive advantage. the proposed model, in fact, is reliable and provides an accurate quantitative risk assessment with limited cost as the result of synergy between experimental and predictive data. knowing the concentration of lactic flora and l. monocytogenes at any time of the shelf life, the model allows a determination of the maximum l. monocytogenes concentration. references beaufort, bergis, lardeux, & lombard. (2008, november). technical guidance document on shelf life studies for listeria monocytogenes in ready to eat foods. colombo, s., romani, m., romani, c., & matteini, p. (2012). il challenge test integrato. italian journal of food safety, 1(6). paparella, a., ruocco, g., & barbieri, b. (1992). lattobacilli come inibitori della microflora delle carni fresche. parente, e., brienza, c., moles, m., & ricciardi, a. (1995). a comparison of methods for the measurement of bacteriocin activity. j. microbiol. meth., 22, 95-108. https://doi.org/10.1016/0167-7012(94)00068-i uni en iso 11290-2/a1. 2005. uni en iso 15214:1998. 1998. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 4, no. 2, 2020 www.scholink.org/ojs/index.php/fsns 21 case study hunger in madagascar: a case study lincoln j. fry1* 1 athens institute for education and research (atiner), 974 sw. general patton terrace, port st lucie, florida, 34953, united states received: april 21, 2020 accepted: april 29, 2020 online published: may 21, 2020 doi:10.22158/fsns.v4n2p21 url: http://dx.doi.org/10.22158/fsns.v4n2p21 abstract this paper is a continuation of a larger study which assesses hunger in african countries. the purpose of these papers in this is to look at the scope of hunger in countries and then to identify the factors that predict hunger in each individual country. this is the 5th paper in the series and is concerned with hunger in madagascar, one of africa’s and the world’s hungriest countries. the paper is important for several reasons. one is the fact that it, like all the country level papers, is based on a national probability sample, something the literature stresses is lacking and needed to improve hunger research. a second is that all the papers in the series, including this one, found that the literature’s suggestion that gender and the rural-urban dimension are significant predictors of hunger. these studies have provided a consistent list of significant hunger predictors. employment in the country’s agricultural sector, wealth as measured by asset ownership, education and age were consistent significant predictors, .the surprising findings were related to respondent perception of the role of government in addressing hunger, questions like the way the government was handling whether people had enough to eat or addressing the living standards of the poor. the major policy implication of this and earlier papers is that governments need to reach out to citizens, presenting them with their food related plans and assuring them that the government is doing everything within its power to address hunger in their countries. keywords hunger, madagascar, hunger predictors, respondent perceptions of government actions, national probability sample 1. introduction this paper is a continuation of a larger project where the purpose is to demonstrate that existing survey research provides a means to assess the scope of hunger in african countries. because this study reports on a project that generated national probability samples and utilizes a self-report www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 22 published by scholink inc. measure to assess hunger, the results may be seen to reflect a country’s total hunger level. that is because persons found in each country’s sample might have been respondents that could have been enumerated in formal hunger statistics as well as unreported persons that reflect what is known as hidden hunger. 2. hunger among the various meanings of hunger, one refers to the want or scarcity of food in a country, and it is in that sense that this note addresses hunger. there are formal measures which include those who demonstrate clear cut hunger. in the latest un food and agriculture organization report (2019), the estimate was that 925 million people were hungry worldwide, and that 239 million people in sub-saharan africa were hungry or undernourished. this made africa the continent with the second largest number of hungry people, following asia and the pacific with 578 million. due to the difference in population sizes, sub-saharan africa had the largest proportion of hungry-undernourished people, estimated at 30 percent of the population compared to 16 percent in asia and the pacific. what is known as hidden hunger is a major issue and there are an estimated two billion persons that are affected by a chronic deficiency of essential vitamins and minerals. among this population the signs of malnutrition and hunger are less visible, but it has negative and long-term consequences for long-term health, productivity and cognitive development (mµthayya et al., 2013). 2.1 hunger in sub-saharan africa as clover (2004) has suggested, while the right to food is one of the most consistently acclaimed assertions in international human rights law, yet no other human right has been so frequently and spectacularly violated. her discussion of food insecurity in sub-saharan africa leads to the conclusion that hunger is a multi-faceted issue in africa, and that just growing more food will not eradicate the problem. agriculture is important and clover points out that africa has gone from being a key agricultural commodity exporter into being a net importer; the african continent now receives the most food aid. perhaps the most important point clover made was to suggest hunger will not be eradicated by just throwing money at the problem. 2.2 hunger in madagascar madagascar is one of the poorest and hungriest countries on the planet. the world food program (2015) noted that 92 percent of the population lives on less than two us dollars per day, and concern world wide (2019) listed madagascar as one of four countries that suffer from a level of hunger that is alarming; the country was ranked 114 out of 117 countries in terms of the degree of hunger in the country. climate change is also a major focus of the literature devoted to hunger in madagascar, especially as it affects small holder farmers (harvey et al., 2014). small farmers in madagascar are portrayed as particularly vulnerable to extreme weather events, particularly cyclones, which cause significant crop loses. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 23 published by scholink inc. 2.3 measuring hunger as godecke et al. (2018) have indicated, measuring hunger remains problematic. this does not mean that hunger has been ignored. for example, there has been an effort in the research community to develop hidden hunger measures, indices and maps. these indices and mapping efforts have been productive and are useful here because they highlight the role of several important factors that are addressed in this paper. one of these is fact that african farmers may be hungrier than the rest of the population, and that gender may also be a significant factor, with women hungrier than men. both godecke et al. and muthayya et al. point to the lack of national probability samples as the primary hunger research roadblock in africa. this study does use a national probability sample, with the objective being to identify policy related factors that possibly help alleviate hunger problems at the country level, madagascar in this instance. 3. data the study’s data source is the afrobarometer project, round 6. the project started 1999 with 12 countries in round 1 and by 2016 when the 6th round survey was completed in there were 36 countries included in the survey. the surveys utilize a standardized questionnaire, with new items added to the next administration of the instrument. sampling sized are either 1,200 or 2,400 depending on the country’s population. the procedures used in all the afrobarometer surveys are explained in in bratton, mattes and gymah-boadi (2005). 3.1 measures the study’s questionnaire included what is called the lived poverty index used in the afrobarometer studies which was adopted from mattes (2003). one of the five questions in the index askes “over the past year, how often, if ever, have you or anyone in your family gone without enough food to eat”. fixed responses to this question were: never, just once or twice, several times, many times, always. these responses were coded as follows: never = 1, just once or twice = 2 and many times and always = 3. these categories provide the basis for the ordered logistical analysis presented in the results section. the study’s independent variables were basic socio-demographic variables included in the questionnaire and the significant factors that predicted hunger in the earlier papers. each of these paper looked at hunger and the factors that predicted it at the country level. these countries were burundi (fry, 2017), benin (fry, 2018a), zambia (fry, 2018b) and madagascar (fry, 2020). 4. results this study’s sole analytic procedure was to conduct an ordered logistical regression analysis and the results of that procedure appear in table 1. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 24 published by scholink inc. table 1. the results of madagascar’s ordered logistical analysis variable coefficient standard z p education -.38 .07 -5.33 .000 ag worker .81 .18 4.56 .000 total assets -.28 .07 -3.83 .000 enough to eat -.55 .18 -3.14 .000 age .28 .09 3.16 .000 extra funding prior .44 .16 2.86 .000 living stands-poor -.43 .19 2.30 .000 job status -1.09 -1.94 1.78 .08 urban-rural .25 .14 -1.77 .08 gender .08 .14 -.59 .55 occupation .04 .05 .67 .50 reduce gap-poor -07 .22 -.33 .74 number of observations 1,129 chi square 215.15 probability .000 pseudo r2 .09 table 1 shows that nine independent variables reached statistical significance in madagascar’s ordered logical regression analysis. this was to be expected in that most of the independent variables in the regression equation had been found to be significant predictors of hunger in the previous studies. again, gender and the urban rural dimension were found not to be significant predictors. the ordered logical regression produced a pseudo r square of 09. 5. discussion as mentioned above, what is unusual about this afrobarometer file is that it contains respondent surveys from some of the world’s and africa’s hungriest countries, madagascar is one of those nations, and the fifth country to be included in this series of papers; the others were burundi, benin, tanzania, and senegal. perhaps what is most important about these earlier papers is that the two major factors identified in the literature as hunger predictors were not significant predictors in any of these countries, namely gender and the rural-urban dimension. a consistent set of predictive factors did emerge from this study. as expected, factors like poverty and employment in the agricultural sector were found to be consistent predictors of hunger, but the most striking findings were respondent’s perception of the government’s handling of food related problems, like assuring people have enough to eat, the major implication of this and earlier papers is that governments need to reach out to citizens, presenting their www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 25 published by scholink inc. food related plans and assuring them that the government is doing everything within its power to address hunger in their countries. references bratton, m., mattes, r., & gyimah-boadi, e. (2005). public opinion, democracy, and market reform in africa. cambridge university press. clover, j. (2003). food security in sub-saharan africa: african security review 12 facts. retrieved from http://www.globalcitizen.org/en/content/the-worlds-10 fry, j. l. (2017). is hunger destined to be perpetual in burundi? food science and nutrition studies, 1, 15. fry, j. l. (2018). hidden hunger in benin: the scope and prospectus. j food sci nutr., 1(1), 3-8. fry, j. l. (n.d.). continuities in the hiv/aids policy debate in south africa. african journal of infectious technology, 3(3). global hunger index. (2018). retrieved from http://www.globalhungerindex.org godecke, t., stein, a., & qaim, m. (2018). the global burden of chronic and hidden hunger: trends and determinants. global food security, 17. the world’s ten hungriest countries. (2019). retrieved from http://www.concernusa.org/story/worlds-ten-hungriest-countries top 10 hungriest african countries. (2015). retrieved from http://www.africaranking.com/top-10-hungriest-african-countries-of-2015 un food and agriculture organization report. (2015). the state of food and agriculture. retrieved from http://www.fao.org/publications/sofa http://www/ http://www.africaranking.com/top-10-hungriest-african-countries-of-2015 http://www.fao.org/publications/sofa food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 2, no. 2, 2018 www.scholink.org/ojs/index.php/fsns 25 original paper production and characteristics of a traditional food: molasses (pekmez) lale sariye akan1* 1 department of nutrition and dietetics, ankara yildirim beyazit university, ankara, turkey * lale sariye akan, faculty of health sciences, department of nutrition and dietetics, ankara yildirim beyazit university, 06450, ankara, turkey received: september 10, 2018 accepted: september 25, 2018 online published: october 4, 2018 doi:10.22158/fsns.v2n2p25 url: http://dx.doi.org/10.22158/fsns.v2n2p25 abstract pekmez, which has been produced for a long time in turkey, is one of the popular and traditional turkish foods (tosun & üstün, 2003; celik & surucuoglu, 2005; türkben, 2016). pekmez is produced primarily from grapes (arici et al., 2004; alpaslan & hayta, 2002; sürücüoglu & celik, 2005; batu et al., 2007; dag, 2016; demir, 2014). molasses are usually preferred for breakfast in winter (kusçu & bulantekin, 2016). suitable for juice production, sugar content, acid value and ripening time are suitable for grape molasses production. turkey, approximately 4185.126 tons of grapes are produced per year (tuik, 2012), and approximately 30% of the grapes produced in turkey are used for pekmez, wort and sausage with pekmez production in a year. in this study, some information is given on the history of molasses, production stages, its types, effects on health and the relevant regulations and in terms of product chemical and microbiological characteristics. keywords molasses, pekmez, traditional food 1. introduction our country has suitable climatic and soil conditions due to its viniculture in terms of its geographical position on the world. almost all regions have anatolian lacquerware, and the date of the lacquering of this region extends to 3000 bc (şimşek 2000; didin et al., 2000; çelik & sürücüoğlu, 2005). anatolia, which is the motherland of grape, has been famous for its rich grape varieties since ancient times and almost everywhere in the country is suitable for viniculture (çelik & sürücüoğlu, 2005). in the past years molasses, which is one of the basic nutrients of human beings, has become less productive in the changing world conditions. in our country molasses is made between at the end of september and the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 26 published by scholink inc. beginning of october when the grapes are mature and this time is called molasses time. pekmez is a concentrated and extended shelf-life form of several fruit juices, and it is formed by boiling without the addition of sugar or other food additives (yoğurtçu & kamışlı, 2006; celik & surucuoğlu, 2005; ekin & celikezen, 2015). pekmez is a good energy and carbohydrate source due to its high sugar content (up to 50%-80%) in the form of glucose and fructose; therefore, it easily passes into the blood without digestion (karababa & isikli, 2005; akbulut et al., 2008; karaca, 2009; ekin & celikezen, 2015). according to the turkish standards institute (ts 3792), grape molasses are defined as a product that is produced by the addition of dark-matter substances obtained by vacuum or lightening according to the method of the present invention, without reducing the acidity of fresh or raisin syrup, or by reducing the acidity with calcium carbonate (tetik et al., 2010; türkben et al., 2010). table 1 shows the chemical and microbiological properties of grape molasses according to ts3792 (tse, 1989). table 1. chemical and microbiological properties of grape molasses chemical properties limits 1. water soluble solids minimum % 65 2. sucrose 0 3. total ash max % 2.0 4. the max % of ash that is not soluble in %10 hcl solution 0.3 5. artificial colours 0 6. preservative 0 7. max arsenic (mg/kg) 0.2 8. max copper (mg/kg) 5.0 9. max zinc (mg/kg) 5.0 10. max iron (mg/kg) 15.0 11. max tin (mg/kg) 150.0 12. max lead (mg/kg) 0.3 13. max sum of copper, iron and zinc (mg/kg) 20.0 microbiological properties 1. max number of mesophilic aerobic bacteria (ad / g) 104 2. the number of yeast and mold (ad / g) 103 3. the number of osmophilic yeast (ad / g) 102 2. production of molasses, regulations and analysis methods in the production of traditional molasses, the grapes are filled with nuts and the slaves are removed by chewing with the feet in boats made of wood or concrete. for deacidification, excess lime, white or neaby white molasses soil are used (guldas et al., 2004; toker & hayoğlu, 2004; tetik et al., 2010; www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 27 published by scholink inc. koca, 2014; ekin & çelikezen, 2015). the grape syrup is boiled on a strongly burning stove to provide easy and quick effect of the soil to the syrup, to prevent the action of the yeasts and to speed up the clarification; this is called the curdling of a grape syrup. after the curdling, the syrup is left to rest, after resting the clear part is separated from the sediment and clear syrup is obtained. the syrup is darkened on the open flame in boilers. in the case of increased acidity in the open boiler method, it is stated that the reducing sugars in the medium during the concentration process are decomposed by hmf to formic acid and levulinic acid when the ph level is lowered (i̇zgi, 2011). at the beginning of the molasses cooking process, foams are formed on the surface of the must, called skimmed fat, and they must be taken from the medium with the flat cheeks in order to provide a clear molasses appearance. the syrup which have been cleaned from their skimmed fat are left to boil in their own form for a while to darken. even if there is a sudden foaming on the syrup surface during this time, it is only a temporary foaming which does not boil and is not in the form of skimmed fat. however, in order to avoid caramelization in this excess molasses, it is necessary to constantly mix it (vardin, h. & vardin, b. c., 2004; batu, 2005; koca, 2014). in the modern method, the raisins are first moistened and passed through the mincing machine. the minced raisins are extracted according to the principle of reverse flow. in grapes obtained from fresh grapes, the grapes that have been cleaned are separated from their stems by being passed through the stalk separating machine, pounded, and crushed by passing through the grape crushing mill. in order to obtain syrup, the grapes are passed through a separator to separate prestine and coarse materials. after the seperation process, molasses soil or calcium carbonate (caco3) is added for acidity. the grape syrup is heated to 70˚c to provide easy and quick effect of the soil to the syrup, to prevent the activity of the yeast and to speed up the clarification. clarification is carried out so that the grape syrup can be clarified and the bitter flavors can be removed. the sieving process can be carried out by applying heat, by tannin-gelatin application or enzymatically. after clarification, the syrup is concentrated by vacuum and the desired dry matter value is reached (aydınlık, 2012; koca, 2014). acid removal and clarification of fresh grapes after pressed and raisins after extraction become cloudy. grape juice blurring is caused by organic molecules that give rise to a viscous structure in the product with the crust particles, the fiber, the cell and the cell fragments in various dimensions of the fruit juice, and these suspension particles give stability to the particles (şengül et al., 2007; koca, 2014). the organic molecules in the colloidal dimensions that are responsible for the formation of a stable suspension of turbidimetric particles are: pectic substances, polyphenols, proteins, starch and arabindin these, the pectic substances have a separate prescription due to their protective colloid properties. for this reason, for a successful cleaning process, it is first necessary to break down the pectic substances to galacturonic acids, which are building blocks with pectolytic enzymes (kayışlıoğlu, 2001; batu, 2005; karaca, 2009; aydınlık, 2012; koca, 2014). the obtained juice contains various turbidity materials which cause a large majority to form tartaric acid and cause the syrup to have a blurred appearance as well as the free acidity which leads to the ph being www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 28 published by scholink inc. between 3-4. for this reason, the free acidity of the syrup should be neutralized to the 6-6.5 ph level, so that the syrup can be produced at the desired sweetness level. in the meantime, it is still possible to obtain a clear view of the molasses by removing the various turbidity materials contained in the syrup. in order to accomplish this, a suitable practice is to use a high (over 80%) soil of calcium carbonate content called molasses soil (batu & aktan, 1993; batu et al., 2007; tetik et al., 2010; ekin & çelikezen, 2015). this process takes place by adding the soil to the syrup and heating it for 5-10 minutes to neutralize the free acidity in the environment with caco3 contained in the soil. thus, the precipitate can be easily separated as a result of the reduction of the colloid substances causing the turbidity in the medium, the loss of the isoelectric point due to the change of the ph level of the medium and the calcination of ca ions in the medium as calcium tartrate (zengin, 2006; turhan et al., 2007; akaydın, 2009; koca, 2014; ekin & çelikezen, 2015). 2.1 grape molasses production flow chart figure 1. stages of molasses production as a physical analysis, homemade liquid-solid and fabricated liquid-solid molasses samples were examined for the presence of foreign substances in the molasses. sensory analyzes of the molasses samples were evaluated for appearance, texture, taste and smell. for microbiological analysis, 10 grams of molasses samples were weighed, 90 ml of sterile physiological saline (0.85% nacl solution) was added to them, and the mixture was homogenized for 1 minute and then a dilution series was prepared up to 10-5. as a microbiological analysis, the total number of mesophilic aerobic microorganisms of molasses www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 29 published by scholink inc. samples was measured at 35°c for 48 hours in plate count agar (pca-merck), mold-yeast number at 25oc in potato dextrose agar (pda-merck) 5 days, staphylococcus aureus in baird parker agar (bpabiomerieux) at 37°c for 24 hours and the number of escherichia coli was determined by incubating for 48 hours at 44°c in coli id (biomerieux). determination of microorganism numbers; the most probable number method was used for the coliform group and the bulk sowing method was used for the others. 3. results 3.1 product features the sensory analysis results of the product characteristics of the molasses samples studied are as follows: the samples are suitable for appearance and have no burning odor or foreign matter. its appearance is unique and homogeneous, without sediment and sugar. solid molasses is not fluid and looks bright. microbiological analysis results of four molasses samples are given in table 2. table 2. microbiological analysis results of molasses samples (cfu/g) company total count of mesophilic aerobic bacteria yeast-mold coliform (mpn) e.coli s.aureus a liquid-homemade 5.7x103 1.4x105 4 unidentified <10 solid-homemade 7.8x102 1.0x102 <3 unidentified 2.0x101 b liquid-fabrication 6.5x103 1.4x104 9 unidentified 3.1x101 solid-fabrication 1.2x102 1.7x104 9 unidentified 5.0x101 the total number of mesophilic aerobic microorganisms generally provides information on food quality, not only the safety of food, but also the quality of food, shelf life and post-heat transmission (colak et al., 2007). the total number of mesophilic aerobic microorganisms in molasses samples ranged from 8.0 x 101-6.9 x 104 cfu / g and the number of yeast and mold was 1.0 x 102-1.4 x 105 cfu / g. the presence of coliform group bacteria in foods is considered a sign of poor sanitation conditions, inadequate or incorrect pasteurization practices, re-infection after cooking and pasteurisation (colak et al., 2007). since all of the coliform group bacteria are not of fecal origin, e.coli has been sought as a marker of fecal contamination. e. coli can not be detected in this study. the presence of staphylococcus aureus in foods is considered a sign of personnel hygiene (colak et al., 2007). the number of s. aureus in the molasses samples was <10-5.0 x 101 cfu / g. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 30 published by scholink inc. 4. conclusion as a result, the samples included in the study are scarce to give a general conclusion. however, it is a fact that the molasses has some disadvantages in terms of microbiological properties if it does not cause any problems in terms of sensory properties. problems can be solved if hygiene and sanitation rules are obeyed in production and marketing and the regulations are taken into consideration. adversities are not encountered in large businesses that fit into hygiene rules and are named for packaging. grape production which occupies the most important place in every period of turkish history, grape molasses which is made with grapes, and many other products are required to be made in conformity with the standards and quality. many companies have been selling molasses through the internet, but there is not enough information about their content and product quality. references akaydın, m. (2009). ticari olarak üretilen bazı sıvı ve katı üzüm pekmezlerinin özelliklerinin belirlenmesi. yüksek lisans tezi, gaziosmanpaşa üniversitesi fen bilimleri enstitüsü, tokat. akbulut, m., çoklar, h., & özen, g. (2008). rheological characteristics of juniperus drupacea fruit juice (pekmez) concentrated by boiling. food science and technology international, 14(4), 321-328. https://doi.org/10.1177/1082013208097193 arici, m., gümüs, t., & kara, f. (2004). the fate of ochratoxin a during the pekmez production from mouldy grapes. food control, 15(8), 597-600. https://doi.org/10.1016/j.foodcont.2003.10.001 aydınlık, z. (2012). niğde i̇linde üretilen üzüm pekmezi örneklerinin fenolik madde i̇çeriğinin belirlenmesi. yüksek lisans tezi, niğde üniversitesi fen bilimleri enstitüsü, niğde. batu, a. (2005). production of liquid and white solid pekmez in turkey. journal of food quality, 28(5‐6), 417-427. https://doi.org/10.1111/j.1745-4557.2005.00045.x batu, a., aktan, n. (1993). üzüm pekmezlerinde asit ve ph değerleri üzerinde bir araştırma. gıda ve yem dergisi, 4, 38-43. batu, a., karagöz, d. d., kaya, c., & yıldız, m. (2007). dut ve harnup pekmezlerinin depolanması süresince bazı kalite değerlerinde oluşan değişmeler. gıda teknolojileri elektronik dergisi, 2, 7-16. dag, b., & tarakçi, z. (2016). comparatives of physico-chemical composition, mineral and heavy metal properties of the grape juices, grape pekmez and dried grape products in difference plant. journal of current research in science, 4(3), 147. demir, m. k. (2014). effect of the replacement of sugar with spray dried grape pekmez (pekmez powder) on some properties of cookies. quality assurance and safety of crops & foods, 6(2), 229-235. https://doi.org/10.3920/qas2013.0242 didin, m., kızılaslan, a., & fenercioglu, h. (2000). suitability of some cornelian cherry cultivars for fruit juice. gida, 25(6), 435-441. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 31 published by scholink inc. gökçe, k., & çizmeci, m. (1965). pekmez. tarım bakanlığı ziraat i̇şleri genel müdürlüğü yayınları, ankara. guldas, m., gucbilmez, m., & dokuzlu, c. (2004). haccp model application in the production of canned grapefruit segment. food technology (turkey). i̇brahim, e. (2015). bitlis i̇linde geleneksel olarak üretilen gezo pekmezinin bazı kimyasal özelliklerinin i̇ncelenmesi (master’s thesis). bitlis eren university, bitlis, turkey. i̇zgi, n. (2011). ev yapımı andız pekmezinin bileşimi, reolojik özellikleri, antioksidan ve antimikrobiyal aktivitelerinin belirlenmesi. yüksek lisans tezi, namık kemal üniversitesi fen bilimleri enstitüsü, tekirdağ. karababa, e., & develi isikli, n. (2005). pekmez: a traditional concentrated fruit product. food reviews international, 21(4), 357-366. https://doi.org/10.1080/87559120500222714 karaca, i̇. (2009). pekmez örneklerinde vitamin ve mineral tayini. yüksek lisans tezi, i̇nönü üniversitesi sağlık bilimleri enstitüsü, malatya. kayışoğlu, s. (2001). tekirdağ i̇linde farklı yöntemler ile üretilen üzüm pekmezlerinin bazı özellikleri üzerine depolamanın etkisinin saptanması üzerine bir araştırma. doktora tezi, trakya üniversitesi fen bilimleri enstitüsü, tekirdağ. koca, i̇. (2014). pekmezden üretilen çerezlerin bazı fiziksel ve kimyasal özellikleri. gıda teknolojileri elektronik dergisi, 9(1), 36-39. koch, j., & klesaat, r. (1960). zeitchrift für lebensmitteluntersuhung und troschung 130 band heft 5abgeschlassen, 2, 45. kuşçu, a., & bulantekin, ö. (2016). the effects of production methods and storage on the chemical constituents of apple pekmez. journal of food science and technology, 53(7), 3083-3092. https://doi.org/10.1007/s13197-016-2281-1 sengül, m., fatih ertugay, m., sengül, m., & yüksel, y. (2007). rheological characteristics of carob pekmez. international journal of food properties, 10(1), 39-46. https://doi.org/10.1080/10942910600627996 şimşek, a., (2000). farklı hammaddelerden üretilen pekmezlerin bileşimi üzerine araştırma. yüksek lisans tezi, ankara üniversitesi, fen bilimleri enstitüsü, ankara. tetik, n., turhan, i̇., karhan, m., & öziyci, h. r. (2010). keçiboynuzu pekmezinin karakteristiği ve 5-hidroksimetilfurfural i̇çeriği (i̇ngilizce). gıda dergisi, 35(6). toker, a., hayoğlu, i̇. (2004). şanlıurfa yöresi gün pekmezlerinin üretim tekniği ve bazı fiziksel kimyasal özellikleri. harran üniversitesi. ziraat fakültesi dergisi, 8(2), 67-73. tuik. (2012). tüik 2012 yılı istatistik raporu. meyveler, içecek ve baharat bitkilerin üretim miktarları (seçilmiş ürünlerde). retrieved from http://www.tuik.gov.tr/ turhan, i̇., tetik, n., & karhan, m. (2007). keçiboynuzu pekmezinin bileşimi ve üretim aşamaları. gıda teknolojileri elektronik dergisi, 2, 39-44. 37. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 32 published by scholink inc. türkben, c., suna s., i̇zli g., uylaşer, v., & demir, c. (2016). physical and chemical properties of pekmez (molasses) produced with different grape cultivars. tarım bilimleri dergisi, 22(3), 339-348. https://doi.org/10.1501/tarimbil_0000001392 türkben, c., uylaşer v., & i̇ncedayı, b. (2010). influence of traditional processing on some compounds of rose hip (rosa canina l.) fruits collected from habitat inbursa, turkey. asian journal of chemistry, 22(3): 2309-2318. vardin, h., & vardin, b. c. (2004). kuru üzümden doğal pekmez üretimi. geleneksel gıdalar sempozyumu, van. yoğurtçu, h., & kamışlı, f. (2006). determination of rheological properties of some pekmez samples in turkey. journal of food engineering, 77(4), 1064-1068. https://doi.org/10.1016/j.jfoodeng.2005.08.036 zengin, s. (2006). kahramanmaraş gün pekmezlerinin bazı fiziksel, kimyasal, organoleptik ve mikrobiyolojik özellikleri (pp. 6-7). yüksek lisans tezi, sütçü i̇mam üniversitesi fen bilimleri enstitüsü, kahramanmaraş. kahramanmaraş sütçü i̇mam üniversitesi fen bilimleri enstitüsü. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 2, no. 2, 2018 www.scholink.org/ojs/index.php/fsns 33 original paper production and characteristics of a turkish traditional food in another country (jonkoping-sweden): molasses lale s. akan1* & yahya özdogan1 1 department of nutrition and dietetics, ankara yildirim beyazit university, ankara, turkey * lale sariye akan, faculty of health sciences, department of nutrition and dietetics, ankara yildirim beyazit university, 06450, ankara, turkey received: september 15, 2018 accepted: september 28, 2018 online published: october 4, 2018 doi:10.22158/fsns.v2n2p33 url: http://dx.doi.org/10.22158/fsns.v2n2p33 abstract pekmez, which has been produced for a long time in turkey, is one of the popular and traditional turkish foods (tosun & üstün, 2003; celik & surucuoglu, 2005). although it is not very common, it is being tried in houses and production places in some countries where turks live. pekmez is produced primarily from grapes (alpaslan & hayta, 2002; sürücüoglu & celik, 2005; batu et al., 2007). grapes suitable for fruit juice yield, sugar content, acid value and ripening time are suitable for molasses production.turkey, approximately 4185.126 tons of grapes are produced per year (tuik, 2012), and approximately 30% of the grapes produced in turkey are used for pekmez, wort and sausage with pekmez production in a year. in this study, some information is given on molasses (produced in another country), production stages, effects on health and the relevant regulations and in terms of product chemical and microbiological characteristics. keywords molasses, traditional food, kulu, jonkoping, sweden 1. introduction our country has suitable climatic and soil conditions due to its viniculture in terms of its geographical position on the world. almost all regions have anatolian lacquerware, and the date of the lacquering of this region extends to 3000 bc (şimşek, 2000; didin et al., 2000; çelik & sürücüoğlu, 2005). anatolia, which is the motherland of grape, has been famous for its rich grape varieties since ancient times and almost everywhere in the country is suitable for viniculture (çelik & sürücüoğlu, 2005). in the past years molasses, which is one of the basic nutrients of human beings, has become less productive in the changing world conditions. in our country molasses is made between at the end of september and the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 34 published by scholink inc. beginning of october when the grapes are mature and this time is called molasses time. pekmez is a concentrated and extended shelf-life form of several fruit juices, and it is formed by boiling without the addition of sugar or other food additives (yoğurtçu & kamışlı, 2006; celik & surucuoğlu, 2005; ekin & celikezen, 2015). jonkoping, sweden’s 10th largest city, is very similar to our country in terms of eating habits. the city that is established in the 13th century, is centrally located on the coast of vattern in south of sweden. sweden has many immigrants from turkey, especially from kulu. since 1965 it has been migrating. the total population of kulu, which is a district of konya province, is over 70.000 and this number is doubled in summer with the majority population coming from sweden. kulu families have adopted swedish culture as well as introducing their diets and traditional foods to the swedes. molasses consumption is very high, especially in athletes whose has high energy consumption, so it has started to be produced at many home. this production is done only to meet their own consumption. kulu people coming to turkey for the summer period, for the grape harvest in early october period, take grapes with themselves which are requested to take along sweden. since swedish families have already made wine, vinegar, etc. from grapes, they have quickly adopted this idea together with its taste and energy value. (erişim tarihi: september 12, 2018, https://www.ju.se/en). pekmez is a good energy and carbohydrate source due to its high sugar content (up to 50%-80%) in the form of glucose and fructose; therefore, it easily passes into the blood without digestion (karababa & isikli, 2005; akbulut et al., 2008; karaca, 2009; ekin & celikezen, 2015). according to the turkish standards institute (ts 3792), grape molasses are defined as a product that is produced by the addition of dark-matter substances obtained by vacuum or lightening according to the method of the present invention, without reducing the acidity of fresh or raisin syrup, or by reducing the acidity with calcium carbonate (tetik et al., 2010; turkben et al., 2010). table 1 shows the chemical and microbiological properties of grape molasses according to ts3792 (tse, 1989). table 1. chemical and microbiological properties of grape molasses chemical properties limits 1. water soluble solids minimum % 65 2. sucrose 0 3. total ash max % 2.0 4. the max % of ash that is not soluble in %10 hcl solution 0.3 5. artificial colours 0 6. preservative 0 7. max arsenic (mg/kg) 0.2 8. max copper (mg/kg) 5.0 9. max zinc (mg/kg) 5.0 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 35 published by scholink inc. 10. max iron (mg/kg) 15.0 11. max tin (mg/kg) 150.0 12. max lead (mg/kg) 0.3 13. max sum of copper, iron and zinc (mg/kg) 20.0 microbiological properties 1. max number of mesophilic aerobic bacteria (ad / g) 104 2. the number of yeast and mold (ad / g) 103 3. the number of osmophilic yeast (ad / g) 102 2. production of molasses, regulations and analysis methods in the production of traditional molasses, the grapes are filled with nuts and the slaves are removed by chewing with the feet in boats made of wood or concrete. for deacidification, excess lime, white or neaby white molasses soil are used (guldas et al., 2004; toker & hayoğlu, 2004; tetik et al., 2010; koca, 2014; ekin & çelikezen, 2015). the grape syrup is boiled on a strongly burning stove to provide easy and quick effect of the soil to the syrup, to prevent the action of the yeasts and to speed up the clarification; this is called the curdling of a grape syrup. after the curdling, the syrup is left to rest, after resting the clear part is separated from the sediment and clear syrup is obtained. the syrup is darkened on the open flame in boilers. in the case of increased acidity in the open boiler method, it is stated that the reducing sugars in the medium during the concentration process are decomposed by hmf to formic acid and levulinic acid when the ph level is lowered (i̇zgi, 2011). at the beginning of the molasses cooking process, foams are formed on the surface of the must, called skimmed fat, and they must be taken from the medium with the flat cheeks in order to provide a clear molasses appearance. the syrup which have been cleaned from their skimmed fat are left to boil in their own form for a while to darken. even if there is a sudden foaming on the syrup surface during this time, it is only a temporary foaming which does not boil and is not in the form of skimmed fat. however, in order to avoid caramelization in this excess molasses, it is necessary to constantly mix it (vardin, h. & vardin, b. c., 2004; batu, 2005; koca, 2014). in the modern method, the raisins are first moistened and passed through the mincing machine. the minced raisins are extracted according to the principle of reverse flow. in grapes obtained from fresh grapes, the grapes that have been cleaned are separated from their stems by being passed through the stalk separating machine, pounded, and crushed by passing through the grape crushing mill. in order to obtain syrup, the grapes are passed through a separator to separate prestine and coarse materials. after the seperation process, molasses soil or calcium carbonate (caco3) is added for acidity. the grape syrup is heated to 70˚c to provide easy and quick effect of the soil to the syrup, to prevent the activity of the yeast and to speed up the clarification. clarification is carried out so that the grape syrup can be clarified and the bitter flavors can be removed. the sieving process can be carried out by applying heat, by tannin-gelatin application or enzymatically. after clarification, the syrup is concentrated by vacuum and www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 36 published by scholink inc. the desired dry matter value is reached (aydınlık, 2012; koca, 2014). acid removal and clarification of fresh grapes after pressed and raisins after extraction become cloudy. grape juice blurring is caused by organic molecules that give rise to a viscous structure in the product with the crust particles, the fiber, the cell and the cell fragments in various dimensions of the fruit juice, and these suspension particles give stability to the particles (şengül et al., 2007; koca, 2014). the organic molecules in the colloidal dimensions that are responsible for the formation of a stable suspension of turbidimetric particles are: pectic substances, polyphenols, proteins, starch and arabindin these, the pectic substances have a separate prescription due to their protective colloid properties. for this reason, for a successful cleaning process, it is first necessary to break down the pectic substances to galacturonic acids, which are building blocks with pectolytic enzymes (kayışlıoğlu, 2001; batu, 2005; karaca, 2009; aydınlık, 2012; koca, 2014). the obtained juice contains various turbidity materials which cause a large majority to form tartaric acid and cause the syrup to have a blurred appearance as well as the free acidity which leads to the ph being between 3-4. for this reason, the free acidity of the syrup should be neutralized to the 6-6.5 ph level, so that the syrup can be produced at the desired sweetness level. in the meantime, it is still possible to obtain a clear view of the molasses by removing the various turbidity materials contained in the syrup. in order to accomplish this, a suitable practice is to use a high (over 80%) soil of calcium carbonate content called molasses soil (batu & aktan, 1993; batu et al., 2007; tetik et al 2010; ekin and çelikezen 2015). this process takes place by adding the soil to the syrup and heating it for 5-10 minutes to neutralize the free acidity in the environment with caco3 contained in the soil. thus, the precipitate can be easily separated as a result of the reduction of the colloid substances causing the turbidity in the medium, the loss of the isoelectric point due to the change of the ph level of the medium and the calcination of ca ions in the medium as calcium tartrate (zengin, 2006; turhan et al., 2007; akaydın, 2009; koca, 2014; ekin & çelikezen, 2015). 2.1 grape molasses production flow chart www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 37 published by scholink inc. figure 1. stages of molasses production as a physical analysis, homemade liquid-solid and fabricated liquid-solid molasses samples were examined for the presence of foreign substances in the molasses. fabrikasyon pekmez türkiye’den satın alınmış olup hazır katı ve hazır sıvı pekmezdir. sensory analyzes of the molasses samples were evaluated for appearance, texture, taste and smell. for microbiological analysis, 10 grams of molasses samples were weighed, 90 ml of sterile physiological saline (0.85% nacl solution) was added to them, and the mixture was homogenized for 1 minute and then a dilution series was prepared up to 10-5. as a microbiological analysis, the total number of mesophilic aerobic microorganisms of molasses samples was measured at 35°c for 48 hours in plate count agar (pca-merck), mold-yeast number at 25oc in potato dextrose agar (pda-merck) 5 days, staphylococcus aureus in baird parker agar (bpabiomerieux) at 37°c for 24 hours and the number of escherichia coli was determined by incubating for 48 hours at 44°c in coli id (biomerieux). determination of microorganism numbers; the most probable number method was used for the coliform group and the bulk sowing method was used for the others. 3. results 3.1 product features the sensory analysis results of the product characteristics of the molasses samples studied are as follows: the samples are suitable for appearance and have no burning odor or foreign matter. its appearance is unique and homogeneous, without sediment and sugar. solid molasses is not fluid and looks bright. microbiological analysis results of four molasses samples are given in table 2. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 38 published by scholink inc. table 2. microbiological analysis results of molasses samples (cfu / g). company total count of mesophilic aerobic bacteria yeast-mold coliform (mpn) e.coli s.aureus a liquid-homemade 6.7x102 1.5x105 3 unidentified <10 solid-homemade 4.8x103 1.2x103 <2 unidentified 1.9x101 b liquid-fabrication 6.5x102 1.4x103 9 unidentified 2.1x101 solid-fabrication 1.3x102 1.5x104 9 unidentified 4.0x101 the total number of mesophilic aerobic microorganisms generally provides information on food quality, not only the safety of food, but also the quality of food, shelf life and post-heat transmission (colak et al., 2007). the total number of mesophilic aerobic microorganisms in molasses samples ranged from 8.0 x 101-6.9 x 104 cfu / g and the number of yeast and mold was 1.0 x 102-1.4 x 105 cfu / g. the presence of coliform group bacteria in foods is considered a sign of poor sanitation conditions, inadequate or incorrect pasteurization practices, re-infection after cooking and pasteurisation (colak et al., 2007). since all of the coliform group bacteria are not of fecal origin, e.coli has been sought as a marker of fecal contamination. e. coli can not be detected in this study. the presence of staphylococcus aureus in foods is considered a sign of personnel hygiene (colak et al., 2007). the number of s. aureus in the molasses samples was <10-5.0 x 101 cfu / g. as a result, the samples included in the study are scarce to give a general conclusion. however, it is a fact that the molasses has some disadvantages in terms of microbiological properties if it does not cause any problems in terms of sensory properties. problems can be solved if hygiene and sanitation rules are obeyed in production and marketing and the regulations are taken into consideration. adversities are not encountered in large businesses that fit into hygiene rules and are named for packaging. grape production which occupies the most important place in every period of turkish history, grape molasses which is made with grapes, and many other products are required to be made in conformity with the standards and quality. many companies have been selling molasses through the internet, but there is not enough information about their content and product quality. references akaydın, m. (2009). ticari olarak üretilen bazı sıvı ve katı üzüm pekmezlerinin özelliklerinin belirlenmesi. yüksek lisans tezi, gaziosmanpaşa üniversitesi fen bilimleri enstitüsü, tokat. akbulut, m., çoklar, h., & özen, g. (2008). rheological characteristics of juniperus drupacea fruit juice (pekmez) concentrated by boiling. food science and technology international, 14(4), 321-328. https://doi.org/10.1177/1082013208097193 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 39 published by scholink inc. aydınlık, z. (2012). niğde i̇linde üretilen üzüm pekmezi örneklerinin fenolik madde i̇çeriğinin belirlenmesi. yüksek lisans tezi, niğde üniversitesi fen bilimleri enstitüsü, niğde. batu, a. (2005). production of liquid and white solid pekmez in turkey. journal of food quality, 28(5‐6), 417-427. https://doi.org/10.1111/j.1745-4557.2005.00045.x batu, a., & aktan, n. (1993). üzüm pekmezlerinde asit ve ph değerleri üzerinde bir araştırma. gıda ve yem dergisi, 4, 38-43. batu, a., karagöz, d. d., kaya, c., & yıldız, m. (2007). dut ve harnup pekmezlerinin depolanması süresince bazı kalite değerlerinde oluşan değişmeler. gıda teknolojileri elektronik dergisi, 2, 7-16. didin, m., kızılaslan, a., & fenercioglu, h. (2000). suitability of some cornelian cherry cultivars for fruit juice. gida, 25(6), 435-441. gökçe, k., & çizmeci, m. (1965). pekmez. tarım bakanlığı ziraat i̇şleri genel müdürlüğü yayınları, ankara. guldas, m., gucbilmez, m., & dokuzlu, c. (2004). haccp model application in the production of canned grapefruit segment. food technology (turkey). i̇brahim, e. (2015). bitlis i̇linde geleneksel olarak üretilen gezo pekmezinin bazı kimyasal özelliklerinin i̇ncelenmesi (master’s thesis). bitlis eren university, bitlis, turkey. i̇zgi, n. (2011). ev yapımı andız pekmezinin bileşimi, reolojik özellikleri, antioksidan ve antimikrobiyal aktivitelerinin belirlenmesi. yüksek lisans tezi, namık kemal üniversitesi fen bilimleri enstitüsü, tekirdağ. jönköping university (ju). (n.d.). retrieved september 12, 2018, from https://www.ju.se/en karababa, e., & develi isikli, n. (2005). pekmez: a traditional concentrated fruit product. food reviews international, 21(4), 357-366. https://doi.org/10.1080/87559120500222714 karaca, i̇. (2009). pekmez örneklerinde vitamin ve mineral tayini. yüksek lisans tezi, i̇nönü üniversitesi sağlık bilimleri enstitüsü, malatya. kayışoğlu, s. (2001). tekirdağ i̇linde farklı yöntemler ile üretilen üzüm pekmezlerinin bazı özellikleri üzerine depolamanın etkisinin saptanması üzerine bir araştırma. doktora tezi, trakya üniversitesi fen bilimleri enstitüsü, tekirdağ. koca, i̇. (2014). pekmezden üretilen çerezlerin bazı fiziksel ve kimyasal özellikleri. gıda teknolojileri elektronik dergisi, 9(1), 36-39. koch, j., & klesaat, r., (1960). zeitchrift für lebensmitteluntersuhung und troschung 130 band heft 5abgeschlassen, 2, 45. sengül, m., fatih ertugay, m., sengül, m., & yüksel, y. (2007). rheological characteristics of carob pekmez. international journal of food properties, 10(1), 39-46. https://doi.org/10.1080/10942910600627996 şimşek, a., (2000). farklı hammaddelerden üretilen pekmezlerin bileşimi üzerine araştırma. yüksek lisans tezi, ankara üniversitesi, fen bilimleri enstitüsü, ankara. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 40 published by scholink inc. tetik, n., turhan, i̇., karhan, m., & öziyci, h. r. (2010). keçiboynuzu pekmezinin karakteristiği ve 5-hidroksimetilfurfural i̇çeriği (i̇ngilizce). gıda dergisi, 35(6). toker, a., & hayoğlu, i̇. (2004). şanlıurfa yöresi gün pekmezlerinin üretim tekniği ve bazı fiziksel kimyasal özellikleri. harran üniversitesi. ziraat fakültesi dergisi, 8(2), 67-73. tuik. (2012). tüik 2012 yılı istatistik raporu. meyveler, içecek ve baharat bitkilerin üretim miktarları (seçilmiş ürünlerde). retrieved from http://www.tuik.gov.tr/ turhan, i̇., tetik, n., & karhan, m. (2007). keçiboynuzu pekmezinin bileşimi ve üretim aşamaları. gıda teknolojileri elektronik dergisi, 2, 39-44. 37. türkben, c., uylaşer, v., & i̇ncedayı, b. (2010). influence of traditional processing on some compounds of rose hip (rosa canina l.) fruits collected from habitat inbursa, turkey. asian journal of chemistry, 22(3), 2309-2318. vardin, h., & vardin, b. c. (2004). kuru üzümden doğal pekmez üretimi. geleneksel gıdalar sempozyumu, van. yoğurtçu, h., & kamışlı, f. (2006). determination of rheological properties of some pekmez samples in turkey. journal of food engineering, 77(4), 1064-1068. https://doi.org/10.1016/j.jfoodeng.2005.08.036 zengin, s. (2006). kahramanmaraş gün pekmezlerinin bazı fiziksel, kimyasal, organoleptik ve mikrobiyolojik özellikleri (pp. 6-7). yüksek lisans tezi, sütçü i̇mam üniversitesi fen bilimleri enstitüsü, kahramanmaraş. kahramanmaraş sütçü i̇mam üniversitesi fen bilimleri enstitüsü. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 5, no. 1, 2021 www.scholink.org/ojs/index.php/fsns 1 original paper prevention of kidneys failure in patients with heart diseases at cardiology institut of abidjan anvoh koutoua yves blanchard1,2*, niaba koffi pierre valery3, kouadio larissa adjoua4 & kouadio amani ange beryl1 1 department of science of food and technologies nangui abrogoua university, 02 bp 801 abidjan 02, côte d’ivoire 2 cardiology institute of abidjan, bpv 206 abidjan, côte d’ivoire 3 agro-valorization laboratory, agro forestry department, jean lorougnon guede university, daloa, côte d’ivoire 4 department felix houphouet boigny university, côte d’ivoire * corresponding author, e-mail: akybcr6@gmail.com abstract the prevalence of chronic kidney disease (ckd) is high and it is gradually increasing. arterial hypertension accelerates many forms of renal disease and hastens the progression to esrd. patients with heart diseases with medication have not enough knowledge about the diet increasing kidney failure risks. methods: this descriptive study included 42 selected randomly patients with cardiovascular complications with hypercreatininemia. food frequency questionnaire was used. creatininemia and clearance of creatininemia measurement were done. results: the results of food frequency questionnaire shown that patients with higher serum creatinine are those who drink less than 1,5 liters of water a day and/or those have not a good repartition in water consumption. patients with higher serum creatinine are those who drunk less than 1,5liters of water a day and those (p≤0.5%). then, meals with frying induced more triglyceride production than meals with sauce (p≤0.5). among beverage, sodas and homemade juices were most consumed respectively at 36.6 and 51.3%. these beverages were associated with higher serum creatininemia. after 3 months of nutritional advises, drop in serum creatininemia were observed. conclusion: lowering serum creatinine involved augmentation in water amount consumption and good water consumption establishing. it also recommends sodas and homemade juice reducing sodas should be reduce consumption and promoting water drinking. keywords hypercreatininemia, clearance, water volume, water distribution www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 5, no. 1, 2021 2 published by scholink inc. 1. introduction the prevalence of chronic kidney disease (ckd) is high and it is gradually increasing. estimates of the global burden of disease indicate that diseases of the kidney and urinary tract account for approximately 830,000 deaths and 18,467,000 disability-adjusted life years annually (hostetter, 2004). there are many causes of these diseases. they varied from chronic diseases such as diabetes, cardiovascular diseases, diet and others. indeed, diabetes has become the single most important cause of esrd in the united states and europe (stengel, billon, van dijk, jager, dekker, & simpson, 2003). hypertension and kidney disease are closely related. arterial hypertension accelerates many forms of renal disease and hastens the progression to esrd (luke, 1999). the influence of certain types of nutrients has been widely studied in relation to kidney function. high protein intake may lead to increased intraglomerular pressure and glomerular hyperfiltration. this can cause damage to glomerular structure leading to or aggravating chronic kidney disease (ckd) (gang jee ko, yoshitsugu obi, amanda r. tortoricci, & kamyar kalantar-zadeh, 2017). high protein diet, usually defined as >1.2 grams of dietary protein per kilogram of body weight a day (g/kg/day), is known to induce significant alterations in renal function and kidney health (kalantar-zadeh, moore, & tortorici, 2016). about sugar, authors shown that people who regularly consumed one or more sugar-sweetened soft drinks a day had 58% increased risk of developing ckd compared to those who did not consume this type of beverage (cheungpasitporn, thongprayoon, o'corragain, edmonds, kittanamongkolchai, & erickson, 2014). despite the fact that the influence of certain types of nutrients has been widely studied in relation to kidney function and overall health condition of ckd patients, there are few studies on the impact of specific diet precisely, the impact of water consumption on their survival. recent studies have firmly established the importance of continuous blood pressure reduction to slow the progression of many forms of renal injury, particularly glomerular disease (agodoa et al., 2001; peterson et al., 1995). the primary objective of this study was to examine real causes of hypercreatininemia in our patients under treatment. a second aim was to propose an adequate diet within water consumption to reduce kidney diseases. 2. research design and methods this study was conducted at cardiology institute of abidjan (côte d’ivoire) in 2018. during 4 months, dietary trials were proposed to patients with high creatininemia by modification of their habitual diets when necessary. 2.1 screening phase forty two (42) volunteers with high creatininemia with average aged about 47.5 ± 79.5 years participate to the study. the minimum age was 44 years and the maximum one was 72 years. their creatinine levels varied between 15 and 20 mg/l. these patients with hypercreatininemia were non-smoker and no taking medication known to affect lipid metabolism from the clinical practices. javascript:; www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 5, no. 1, 2021 3 published by scholink inc. 2.2 dietary interventions recommendations were delivered during one-to-one consultation sessions. volunteers were asked about their habitual diets by answering a questionnaire developed on local foods. food frequency questionnaire focused on type of breakfast, lunch and dinner were used. it also focused on type of meal, number of meal and type of beverage. diets mistake were then explained and advices were given to them during 30 to 45 minutes for changing experimenting. the nutritionist, in consultation with each volunteer, drew up meal programs for the study period and patients noted no change from the original program. the nutritionist gave dietary advice to participant in order to avoid stressful diets. they met 2 weeks after the first consultation for the check-up. no portion size was indicated. they had choice among the proposed diet at breakfast and lunch, but they should follow strictly the dinner diet day by day. only men were authorised to add 40 g of bread to vegetable soup on tuesday and friday when required. it was a hypocaloric diet especially in the evening. the diet included low saturated fats, and increased in breads (morning and evening), roots tubers, vegetables and fish. it also had less red meat and more poultry. the participants were also taught to prepare their own meals or not. in addition, fruits consumption was studied and limited to one fruits during the study. 2.3 none dietary interventions although exercising was encouraged during 45 minutes, patients were allowed to choose other types of moderate-intensity physical activity twice or 3 times a week. 2.4 creatininemia and clearance control analyses were led each 2 months and half (75 days). first samples were made at t0. second samples were made 75 days later and this date was mentioned t0 then t1 and t2. plasma levels of creatininemia and clearance of creatininemia were measured using automated procedures in cardiology institute laboratory. 2.5 exclusion criteria patients with weight higher than 100kg and those who aged more than 75 were not included in this study. 2.6 statistical analysis the creatinine and creatinine clearance measurements made at each time (t0, t1 and t2) were compared in relation to the variation in the volume of water and soda consumed daily. the frequency of consumption and the daily distribution of catches in the period concerned. this was achieved by various analysis tests (anova) with one / and / or two classification criteria in order to see if there is a difference in the evolution of the level of each parameter studied (serum creatinine and creatinine clearance). the significance of the difference in the means is determined by comparing the probability p associated with the fischer-senedecor test statistic to the theoretical threshold of α = 0.05. so when p ≤0.05, there www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 5, no. 1, 2021 4 published by scholink inc. is a significant difference between the means. to process all of our data. all statistical tests were carried out using r software (r core team, version 3.6.2 and the graphics were carried out on the one hand with this same software and on the other hand with microsoft excel software. 3. results the results of our study is shown in tables and figures below. table 1. values according to daily water consumption parameters daily water volume pr(>f) less than 1.5 l up than 1.5 l number (percentage) 27 (64.28%) 15 (35.72%) serum creatinine (mg/l) 18.9 ± 3,73 17.06 ± 1.75 0.01808 clearance of creatinine (ml/min) 57.04 ± 13,51 60.32 ± 9.8 0.37043 table 1 shows the results of the water consumption survey on serum creatinine values and serum creatinine clearance. among the 42 volunteers, 27 of them (64%) of the patients consumed less than a liter and a half of water per day compared to 15 others who drank more. people with a summation of less than a liter and a half had the highest values 18.9 ± 3.73 mg / l against 17.06 ± 1.75 mg / l the others. regarding creatinine clearance, it changes in the opposite direction to creatinine. the lowest values 57.04 ml / min were observed in patients with low water consumption. those with water consumption above 1.5 l per day had an average clearance value of 60.32 ± 9.8 ml / min. table 2. values according to daily water consumption plan paramètres matin et soir toute la journée cure d’eau pr (>f) effectif 26 (61.91%) 12 (28.57%) 04 (9.52%) créatininémie (mg/l) 19.31 ± 2,86 17.61 ± 4.17 18.25 ± 2.75 0.15540 clairance de la créatininémie (ml/min) 56.55±9.49 62.02±20.75 57.7±10.92 0.77287 the results of serum creatinine and creatinine clearance according to the distribution of water consumption are presented in table 2. it emerges from this survey that patients with morning and evening water consumption are in the majority (61.91%) with the highest mean creatinine levels around 19.31 ± 2.86 mg / l and a clearance of 56.55 ± 9.49 ml / min. patients with kidney problems with a consumption of water distributed throughout the day represent 28.57% with an average serum creatinine of 17.61 ± 4.17 mg / l. this group presents the critical mean www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 5, no. 1, 2021 5 published by scholink inc. clearances (62.02 ± 20.75 ml / min). patients who consume water only in the evening represent 16.67% with an average serum creatinine of 16.85 ± 2.41 mg / l and an average clearance of 62.02 ± 20.75 ml / min. the number of patients taking water cures is the least (9.52%) with an average serum creatinine of 18.25 ± 2.75 and an average clearance of 57.7 ± 10.92 ml / min. the clearance does not vary with the volume of water consumed by the subjects (p> 0.05), it also does not vary with the distribution of water consumption during the day (p> 0.05). on the other hand, the interaction between the volume of water consumed and its distribution during the day is very significant, so the clearance varies (p <0.05). 27 the subjects who have an usual consumption of water greater than or equal to a liter and half (1.5l) distributed throughout the day have the highest average clearance. table 3. analysis table of variance of serum creatinine and clearance according to the mode of water consumption parameter factors of variation parameters of anova n f-value ddl p amount of water (1) 6.17 1 0.018 creatinine water consumption plan (2) 42 1.86 3 0.155n.s interaction between (1) et (2) 1.46 3 0.243 n.s amount of water (1) 0.82 1 0.370n.s clearance of creatinine water consumption plan (2) 42 0.38 3 0.773 n.s interaction between (1) et (2 ) 8.67 3 0.000206 (*) : p < 0,05 ; (**) : p< 0,01 ; (***) : p< 0,001 ; n.s : non significatif (p > 0,05) figure 1. evolution of serum creatinine www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 5, no. 1, 2021 6 published by scholink inc. figure 1 shows the evolution of serum creatinine during our study. a decrease in serum creatinine is observed. after 2 months of diet, the value drops from 18.31 ± 3.26 mg / l to 12.88 ± 1.99 mg / l, a decrease of 29.65%. during the second period of dietary monitoring, the drop in blood creatinine level is 16.3%, a final value of 10.78 ± 1.52 mg / l. the total decrease is estimated at almost 45.95%. figure 2. variation in creatinine clearance during our work, we observe an increase in the clearance value of creatinine. this constant goes from its initial value of 57.95 ± 12.25 mg / l to 81.24 ± 18.05 mg / l, an increase 40.18%. during the second part of our study, the clearance reached 91.61 ± 24.78, an increase of 12.51%. the total increase is estimated at almost 52.69%. (figure 2) 4. discussion this study analyzed the correlation between water intake and the prevalence of kidney diseases in patients with heart diseases with medication. the research focuses on water amount intake and water plan establishing within other beverages. this study highlights one of the main causes of kidney diseases in patients in cardiology under medication. indeed, the results of the survey showed that 64% of patients with higher serum creatinine were used to drink less than a liter and a half of water a day. studies in humans have shown that numerous factors affect fluid intake availability, ambient temperature, flavor, flavor variety, beverage temperature, proximity of the beverage to the person, and even beverage container have all been shown to impact intake (who, 2004). water consumption could vary with ageing according to some authors (kim, shin, & kim, 2003). for these authors, during ageing, water consumption decreases as a result of reduced metabolic activities. also, some patients unfortunately replace waters by sugar-sweetened beverage (homemade juice and soda) with water consumption reducing. the first mistake by replacing water by these beverages is that these sweetened juice have a higher salt content in the form of sodium. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 5, no. 1, 2021 7 published by scholink inc. the sodium level of these drinks like apple juice sometimes reaches 218.03 ± 0.22% mg/l according to (agbangnan dossa cokou pascal, ggohaïda virginie, bothon fifa théomène diane, kanfon rose estelle, avlessi félicien, wotto dieudonné valentin, & sohounhloue koko codjo dominique1, 2018). these values are widely higher than those contained in running water (3 to 18mg/l). recent studies have firmly established the importance of continuous blood pressure reduction to slow the progression of many forms of renal injury, particularly glomerular disease (agodoa et al., 2001; peterson et al., 1995). indeed, high dietary sodium is an important factor influencing blood pressure, predisposing patients with established ckd to salt-sensitive hypertension and fluid retention (carrero & cozzolino, 2014; luik et al., 2002). renal damage associated with salt intake may be a result of its interaction with aldosterone (lambers heerspink, navis, & ritz, 2012). for these authors, more the blood pressure is higher, more renal damage are important. on one hand, the first finding of the present study is that the amount of daily intake of water around 2liters and half revealed very useful to decrease the risk of renal failure by reduce the serum creatinine. this finding could be explained by the therapeutic benefits of water drinking on heart activity previously shown by several authors (lu et al., 2003). the mechanism(s) by which water has its beneficial effect is unknown. it is known that water evokes a pressor response in patients with autonomic failure (shannon et al.,, 2002) and improves orthostatic responses in the postural tachycardia syndrome (kobayashi, 1957). water drinking also enhances cardiovagal tone in young healthy subjects (routledge, chowdhary, coote, & townend, 2002). some authors advocate that water drinking should be utilized as an adjunct to other methods of treatment for patients with postural syncope and suggest that these patients be encouraged to drink water (claydon, schoeder, lucy, norcliffe, & hainsworth, 2006). our results are supported by the proposals made in korea where the 2010 reference nutritional intake recommends, for men and women to drink 2.1 and 2.6 l / day and 1.8 and 2.1 l / day, respectively (anonymos 2, 2010). our results are also similar to that author who suggests drinking 30 ml of water per kilogram of weight (chernoff, 1994). on the other hand, beside of the amount of water intake, the main finding of our study was the correlation between daily intakes of water plan and clearance of creatinine. our study has shown a significant decrease of clearance of creatinine when the daily consumption was spread over the whole day on 5 intakes. 5. conclusion our study showed that there is now substantial evidence that lower water intake increases the risk of kidney diseases by increasing the serum creatinine level in patients with heart diseases with under medication. dietary interventions have proven effective in reducing the risk of developing kidney by reducing serum creatininemia and increasing clearance of creatinine. the identification of risk factors can prevent or limit disease through lifestyle modifications. these findings reinforce the need to www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 5, no. 1, 2021 8 published by scholink inc. promote the continuing education for healthcare teams involved in the treatment of these patients, sponsoring the prevention and diagnosis of ckd at the early stages. references agbangnan dossa cokou pascal1, ggohaïda virginie, bothon fifa théomène diane, kanfon rose estelle, avlessi félicien, wotto dieudonné valentin, & sohounhloue koko codjo dominique. 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(2003). trends in the incidence of renal replacement therapy for end-stage renal disease in europe, 1990-1999. nephrology dialysis transplantation, 18, 1824-1833. https://doi.org/10.1093/ndt/gfg233 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 2, 2017 www.scholink.org/ojs/index.php/fsns 114 effects of propolis and black seed oil on the shelf life of freshly squeezed pomegranate juice ibrahim kahramanoglu1,2* & serhat usanmaz1 1 department of horticultural production and marketing, european university of lefke, lefke, cyprus 2 alnar narcılık ltd., güzelyurt, cyprus * ibrahim kahramanoglu, e-mail: ibrahimcy84@yahoo.com received: october 23, 2017 accepted: october 30, 2017 online published: november 6, 2017 doi:10.22158/fsns.v1n2p114 url: http://dx.doi.org/10.22158/fsns.v1n2p114 abstract during the last decade, consumers began to pay more attention on the 100% natural, pure and not from concentrate (nfc) fruit juice. however, 100% natural fruit juice has shorter shelf life than the concentrated juice, due to the development of yeast and mould. therefore, present research aimed to study the effects of propolis and black seed oil on the shelf life of freshly squeezed pomegranate juice. according to the results obtained, both propolis and black seed oil have delaying effect on the development of yeast and mould. moreover, it was also found that combination of these treatments with freezing, increases the efficiency of tested natural treatments, as well as the shelf life. keywords fresh juice, shelf life, colony forming unit, ascorbic acid, maturity index 1. introduction pomegranate (punica granatum l.) is predicted to be among the oldest known cultivated fruit crops. result of some scientific studies showed that antioxidant and anti-microbial capacity of pomegranate fruit are high, it decreases blood pressure and it can be used against some illness such as cancer and diabetic (aviram et al., 2000; jurenka, 2008). however, consumption of pomegranate fruit is difficult due to the hassle of aril extraction. on the other hand, pomegranate has lots of low quality products as a result of sunburn and fruit cracking. therefore, using low quality products for juice production is important for both producers and consumers. the demand for 100% natural, pure and not from concentrate (nfc) juice is increasing because of the increase in consumer awareness on the negative effects of synthetic food additives on human health (aijn, 2016). however, development of yeast and mould cause 100% natural pomegranate juice to have shorter shelf life as in many other fruit juice. although, the shelf life of pomegranate juice can be prolonged by using chemical additives i.e. sodium benzoate and potassium sorbate, changes in the consumer preferences has been directed producers to www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 115 published by scholink inc. find alternative natural and healthy methods. in the light of this information, present work aimed to study the effects of propolis and black seed oil on the shelf life of freshly squeezed pomegranate juice. 2. meterials and methods pomegranate fruit samples of present study are belonging to the wonderful culturae, which has been dominating pomegranate trade in the world. this culturae was originated in florida. fruit size of this culturae is big with deep-red fruit color. fruit juice content is high and taste is sweet-tart. harvesting period is between october and november in the northern hemisphere. fruit samples of present study were harvested on october 2015, from a 7-years old pomegranate orchard located in güzelyurt province in cyprus. fruits were harvested by hand at commercial maturity (>17% tss and >1.80 titratable acidity) and immediately after harvest, fruits were transferred to the factory of alnar narcılık ltd. with a ventilated truck. after that, arils were extracted from the fruits by automatic machine and arils were pressed to produce juice. crude propolis was gathered by hand from bağlıköy province in the western part of cyprus. the propolis exudates collected by bees (apis mellifera cypria) were primarily from a mixture of wild and cultivated plant species, including pine (pinus brutia l.), olive (olea europea l.), eucalyptus (eucalyptus globulus l.), citrus (citrus spp.), trifoliums (medicago spp. and trifolium spp.), pimpernel (anagallis arvensis l.), hordeum (hordeum bulbosum l.), field bindweed (convolvulus arvensis l.), chrysanths (chrysanthemum spp.) and locust (acacia spp.). preparation of the propolis extract was done according to the method by krell (1996) with some modifications. the propolis extracts were frozen to -20ºc for 1 month, then cut in small pieces, and ground in a chilled mortar. after that, 10% ethanol extracted propolis was prepared by adding 100 g of the propolis to 900 ml of 70% ethanol and agitating for 1 week. agitating was done with automatic machine by shaking the extract for 1 minute with 60 minutes interval. the mixture was maintained at room temperature during preparation and was subsequently filtered through whatman 1 filter paper. the extracts were kept at 4ºc in dark storage until use. black seed oil is a product of nigella sativa plant which is native to asia. the black seed oil of present study is belonging to the pelmur ltd. with a brand name of biotama. the black seed oil is obtained by the cold-press of black cumin seeds. the purchased black seed oil was 100% pure and was dissolved in ethanol by adding 100 ml of the black seed oil to 900 ml of 70% ethanol and agitating for 1 day. first of all, pomegranate juice was pasteurized for 15 second at 72ºc and it cooled to 4ºc in 4 minutes. immediately after that, pomegranate juice was filled in 250 ml bottles. numbers of main treatments of present study are 3, which are: (1) untreated control, (2) propolis application [1 drop/250 ml] and (3) black seed oil application [1 drop/250 ml]. all treatments were subjected to 2 different factors, these are: (a) shelf life test after filling [storage at 4ºc] (b) shelf life test after 1 year storage at -18ºc [storage at 4ºc]. experiments were set up with 145 samples for each unique treatment. five samples from each unique treatment were subjected to yeast and mould analyzes (colony forming unit-cfu/g) for 2-days www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 116 published by scholink inc. intervals starting from the 5th day (totally 29 measurements). therefore, experiments were continued for 61 days in total. moreover, following tests were conducted for the samples of (i) 0 day after squeezing, (ii) 15 days after squeezing, and (iii) 380 days after squeezing [15 days shelf life after 1 year freezing storage]. the tests were: (1) antioxidant activity (%), (2) total soluble solids (tss), (3) titratable acidity (%), and (4) ascorbic acid content [mg/l]. determination of yeasts and moulds was done as colony forming unit/g. for this 1 ml of each juice was placed on plate surface that contained sabouraud dextrose agar (sda) and distributed by a sterilized swab. plates were incubated for 5 days at 25°c. colonies were counted and expressed as cfu/g. the antioxidant activity of the pomegranate juice was evaluated using the dpph free radical-scavenging method. measurements were carried out according to the modified method of klimczak et al. (2007). a total of 5 ml pomegranate juice was mixed with 5 ml of methyl alcohol (80%) in teflon tubes and then centrifuged (4000 rpm, 10 min, at 4ºc). briefly, 0.1 ml of supernatant was added to 2.46 ml of 1,1-diphenyl-2-picrylhydrazyl radical (dpph; 0.1 mg/l in 80% methyl alcohol) and mixed by vortex. absorbance of the samples was measured at 515 nm using the spectrophotometer after incubating for 10 min in the dark. antioxidant activity was expressed as the percentage decline of the absorbance from control group. total soluble solids content of the fruits were measured by a hand refractometer. titratable acidity (g/100 g of citric acid) of juice samples was determined according to aoac (1990) with wtw ph-meter (weilheim, germany). titratable acidity was determined by titrating 2 ml of fruit juice in 38 ml of distilled water with 0.1 n naoh to an end point of ph 8.1. ascorbic acid determination was performed by following the method of lee and coates (1999) by using the hplc method. the hplc column was maintained at 25ºc and the flow rate was 0.5 ml min-1. a total of 10 𝜇l supernatant was injected into the c18 xterra (waters, 4.6 × 250 mm) column. the photodiode array detector was set at 244 nm, and 2% kh2po4 (ph 2.4) was used as the mobile phase. collected data was summarized by using microsoft excel and figures and simple tables were prepared with the mean and standard deviations. the data of the experiments was subjected to analysis of variance (anova) with main treatments and storage conditions as factors using spss software. mean separations was done by using tukeys (hsd) multiple range test at p ≤ 0.05. significant differences were showed at the tables by using different letters. 3. results and discussions results for the development of yeast and mould at the juice samples which were subjected to different treatments are given in figure 1. it is clear from the figure that yeast and mould development was firstly observed at the control treatment at 15th day. colony forming unit was 72 ± 41 at 15th day and it increased with the increase in the storage duration. according to the result it can be concluded that freshly squeezed pomegranate juice can be stored for 15th days at 4ºc without any additive (with pasteurization at 72ºc for 15 sn). development of yeast and mould had been observed at 19th day of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 117 published by scholink inc. control treatment when samples stored 1 year at -18ºc and then taken out to shelf. these result shows that freezing of freshly squeezed pomegranate juice delays the development of yeast and mould for 4 days. frozen storage is known to have less detrimental effect on the juice. application of both propolis and black seed oil had been found to delay the development of yeast and mould. first measurement of colony forming unit for the application black seed oil and propolis were at 21th and 23th days, respectively. similarly koç et al. (2007) conducted a study about the anti-fungal effects of propolis in 4 different fruit juice (mandarin, orange, apple and white grape). they reported that presence of propolis inhibited the growth of all spoilage yeast at 25ºc. however, that study was conducted for only 48 hours. anti-fungal activity of propolis was also reported by some other scientist (özcan, 1999; oliveira et al., 2006; senka et al., 2011; temiz et al., 2013). on the other hand, hafez (2008) reported that 0.5% black seed oil application on the cucumber had showed protective effect against powdery mildew. in another study, forouzanfar et al. (2014) reported that black seed oil contains thymoquinone which has high anti-microbial activity. they also noted that black seed oil had a strong antibacterial activity against all the strains of l. monocytogenes. in present study, similar with the control treatment, freezing had been found to delay the development of yeast and mould for both the application of propolis and black seed oil. yeast and mould development had firstly observed at 33th day for black seed oil application. this means that freezing helped the black seed oil to increase the shelf life of pomegranate juice for 12 more days. the best result (longer shelf life) had been obtained from propolis application + freezing, where the development yeast and mould was suppressed until 37th day. figure 1. effects of propolis and black seed oil on the yeast and mould development antioxidants protect the body from the harmful effects of free radicals. antioxidants include some vitamins (i.e., vitamins c and e), some minerals and flavonoids, which are found in plants. pomegranate fruit is among the good sources of antioxidants (valko et al., 2007). antioxidant activity www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 118 published by scholink inc. of samples measured as 95.6 ± 0.17 immediately after squeezing. at 15th day it was observed that antioxidant activity of all treatments decreased (table 1). according to the results, antioxidant activity of control treatment was found to be significantly lower than the other treatments. when the samples stored 1 year at -18ºc, it was determined that antioxidant activity continued to decrease. however, at the same time antioxidant activity of control treatment was again found to be lower than the other treatments. table 1. effects of propolis and black seed oil on the antioxidant activity treatments antioxidant activity (%) day 0 day 15 day 15 after 1 year freezing control 95.6 ± 0.17 a 93.3 ± 0.12 b 90.5 ± 0.22 b propolis 95.6 ± 0.17 a 94.4 ± 0.15 a 92.8 ± 0.11 a black seed oil 95.6 ± 0.17 a 94.2 ± 0.04 a 92.5 ± 0.14 a values followed by the same letter or letters within same column are not significantly different at a 5% level (tukeys (hsd) multiple range test). ascorbic acid is among the important components of pomegranate (miguel et al., 2010). however, it is believed that the storage duration of fruit causes a decline in the concentration of ascorbic acid (zarei et al., 2011; kulkarni & aradya, 2005). as stated by these studies, the ascorbic acid content of pomegranate juice showed a considerable decline during storage in present study (table 2). the ascorbic acid in control treatment decreased from 70.3 mg/l to 66.7 mg/l in 15 days of storage at shelf. the ascorbic acid content of other treatments also showed a decline but for both propolis and black seed oil applications, it was found to be higher than the control treatment. when the samples freeze for 1 year, the ascorbic acid content for control, propolis and black seed oil treatments were found to decrease until 18.7, 23.9, 23.3, respectively. table 2. effects of propolis and black seed oil on the ascorbic acid treatments ascorbic acid (mg/l) day 0 day 15 day 15 after 1 year freezing control 70.3 ± 0.45 a 66.7 ± 0.23 c 18.7 ± 0.38 b propolis 70.3 ± 0.45 a 67.8 ± 0.88 a 23.9 ± 0.49 a black seed oil 70.3 ± 0.45 a 67.3 ± 0.11 ab 23.3 ± 0.17 a values followed by the same letter or letters within same column are not significantly different at a 5% level (tukeys (hsd) multiple range test). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 119 published by scholink inc. the ratio (maturity index) between total soluble solids content (tss) and titratable acidity (ta) is the main factor determining pomegranate fruit taste and fruit maturity (cristosto et al., 2000). the maturity index (mi) is related to the taste and flavor of fruit. the mi of present study is determined as 9.7 at the first day of juice production (table 3). table 3. effects of propolis and black seed oil on the maturity index treatments maturity index (tss/ta) day 0 day 15 day 15 after 1 year freezing control 9.7 ± 0.24 a 10.3 ± 0.19 a 23.2 ± 0.6 a propolis 9.7 ± 0.24 a 10.2 ± 0.12 a 18.9 ± 0.5 c black seed oil 9.7 ± 0.24 a 10.3 ± 0.10 a 21.8 ± 0.2 b values followed by the same letter or letters within same column are not significantly different at a 5% level (tukeys (hsd) multiple range test). the maturity index showed slight increase in 15 days of storage at shelf. at that time, no significant difference was determined among the treatments. approximately 2-fold increase had been determined at the samples when they freeze for 1 year and then taken out to the shelf for 15th days. main reason of increase in the maturity index is the considerable decrease in the titratable acidity. at 15th day, significant differences were calculated for the different treatments. the lowest maturity index was determined from the propolis treatment and highest from control. when the maturity index of a food increases, it causes the food to be sweeter. not only for the pomegranate juice, but for all juice types, consumers do not prefer high changes in the taste. therefore, it can be concluded that application of propolis reduces this change in the taste. application of black seed oil has also been found to have significant effect on the maturity index, but lower than the propolis application. 4. conclusions yeast and the mould are the main reason for the spoilage of freshly squeezed pomegranate juice and thus main cause of the decrease in the shelf life. in present study, propolis and black seed oil applications showed good performance in controlling the development of yeast and mould. the efficiency of both treatments showed considerable increase; when they combined with freezing technique. results suggested that the shelf life of freshly squeezed pomegranate juice can be extended to 37 days with the application of propolis (1 drop/250 ml) plus freezing. on the other hand, both propolis and black seed oil applications have been found to protect anti-oxidant activity, ascorbic acid content and maturity index, as compared to control treatment. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 120 published by scholink inc. references aijn. (2016). liquit fruit 2016 market report, eruropean fruit juice association, brussels (p. 43). retrieved from http://www.aijn.org aoac. (1990). official method of analysis of the association of official analytical chemistry (15th ed.). aoac, arlington, va, usa. aviram, m., dornfeld, l., rosenblat, m., volkova, n., kaplan, m., coleman, r., … fuhrman, b. (2000). pomegranate juice consumption reduces oxidative stress, atherogenic modifications to ldl, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein e-deficient mice. the american journal of clinical nutrition, 71, 1062-1076 [abstract only]. cristosto, c. h., mitcham, e. j., & kader, a. a. (2000). pomegranate: recommendations for maintaining postharvest quality. produce facts postharvest research and information centre, university of california, davis, usa. retrieved august 3, 2017, from http://www.postharvest.ucdavis.edu/pffruits/pomegranate/ forouzanfar, f., bazzaz, b. s. f., & hosseinzadeh, h. (2014). black cumin nigella sativa and its constituent (thymoquinone): a review on antimicrobial effects. iranian journal of basic medical science, 17, 929-938. hafez, y. m. (2008). effectieness of the antifungal black seed oil against powdery mildews of cucumber (podosphaera xanthii) and barley (blumeari graminis f.sp. hordei). acta biologica szegediensis, 52(1), 17-25. jurenka, j. (2008). therapeutic applications of pomegranate: a review. alternative medicine review, 13(2), 128-144. klimczak, i., malecka, m., szlachta, m., & gliszczyńska-świglo, a. (2007). effect of storage on the content of polyphenols, vitamin c and the antioxidant activity of orange juices. j. food compos anal, 20, 313-322. https://doi.org/10.1016/j.jfca.2006.02.012 koc, a. n., silici, s., mutlu-sariguzel, f., & sagdic, o. (2007). antifungal activity of propolis in four different fruit juices. food technol. biotechnol., 45(1), 57-61. krell, r. (1996). value-added products from beekeeping. fao agricultural services bulletin (no. 124, pp. 85-89). food and agriculture organization of the united nation, rome. kulkarni, a. p., & aradhya, s. m. (2005). chemical changes and antioxidant activity in pomegranate arils during fruit development. food chemistry, 93, 319-324. https://doi.org/10.1016/j.foodchem.2004.09.029 lee, h. s., & ve coates, g. a. (1999). vitamin c in frozen, fresh squeezed, unpasteurized, polyethylene-bottled orange juice: a storage study. food chemistry, 65(2), 165-168. https://doi.org/10.1016/s0308-8146(98)00180-0 miguel, m. g., nevesa, m. a., & antunes, m. d. (2010). pomegranate (punica granatum l.): a medicinal plant with myriad biological properties. journal of medicinal plants research, 4, 2836-2847. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 121 published by scholink inc. oliveira, a. c. p., shinobu, c. s., longhini, r., franco, s. l., & svidzinski, t. i. e. (2006). antifungal activity of propolis extract agaist yeasts isolated from onychomycosis lesions. mem inst oswaldo cruz, 101(5), 493-497. https://doi.org/10.1590/s0074-02762006000500002 özcan, m. (1999). antifungal properties of propolis. grass y aeites, 50(5), 395-398. https://doi.org/10.3989/gya.1999.v50.i5.685 senka, b., pasic, s., behija, d., & agnesa, c. (2011). antifungal activity of propolis originated from bosnia and herzegovina. veterinaria, 60(3-4), 187-193. temiz, a., mumcu, a. ş., tüylü, a. ö., sorkun, k., & salih, b. (2013). antifungal activity of propolis samples collected from different geographical regions of turkey against two food-related molds, aspergillus versicolor and penicillium aurantiogriseum. gida, 38(3), 135-142. valko, m., leibfritz, d., moncol, j., croninc, m. t. d., mazura, m., & telserd, j. (2007). free radicals and antioxidants in normal physiological functions and human disease. international journal of biochemistry and cell biology, 39(1), 44-84. https://doi.org/10.1016/j.biocel.2006.07.001 zarei, m., azizi, m., & bashir-sadr, z. (2011). evaluation of physicochemical characteristics of pomegranate (punica granatum l.) fruit during ripening. fruits, 66, 121-129. https://doi.org/10.1051/fruits/2011021 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 4, 2019 www.scholink.org/ojs/index.php/fsns 122 original paper hydrothermal treatment to remove tannins in wholegrains sorghum, milled grains and flour maría del rosario acquisgrana1, laura cecilia gómez pamies1 & elisa inés benítez1,2* 1 departamento de ingeniería química, facultad regional resistencia, quitex-universidad tecnológica nacional, french 414, 3500 resistencia, chaco, argentina 2 facultad de ciencias exactas y naturales y agrimensura, iquiba-nea, conicet, universidad nacional del nordeste, av. libertad 5460, 3400 corrientes, corrientes, argentina * elisa inés benítez, departamento de ingeniería química, facultad regional resistencia, quitex-universidad tecnológica nacional, french 414, 3500 resistencia, chaco, argentina received: october 7, 2019 accepted: october 18, 2019 online published: october 30, 2019 doi:10.22158/fsns.v3n4p122 url: http://dx.doi.org/10.22158/fsns.v3n4p122 abstract pigmented sorghum with high content of tannins were studied in this work. tannins bind to proteins and reduce their availability. a hydrothermal treatment was carried out to reduce tannins. a control sample of non-pigmented pericarp variety was used. after the treatment, grains were milled, and a part was separated for wholegrain flour elaboration. several determinations were done after treatment: tannins (t), total antioxidant capacity (tac) and total polyphenols (tpp) content. tpp and tac in wholegrain pigmented sorghum were 3.9 to 12.3 and 2.3 to 3.5 times higher than those of non-pigmented sorghum, respectively. in all sorghum varieties the extractions of tpp decreased with milling. tac in flour increased 3.3 times the initial value for non-pigmented sorghum, whereas for the other sorghum samples it increased slightly from 1.1 to 1.3 times the initial value. in flours there was a noticeable reduction in t, with respect to the wholegrain. it was possible to conclude that the hydrothermal treatment allowed lower levels of tannins than those established in the codex for both wholegrain sorghum and flour. this reduction makes it possible to obtain flour which may be suitable for food processing and the recovery of tannins for other uses. keywords steeping, annealing, milling, sorghum, tannin www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 123 published by scholink inc. 1. introduction sorghum (sorghum bicolor (l.) moench) is the fifth most important cereal crop in the world after wheat, rice, corn and barley (singh et al., 2011), being an excellent source of energy used for both animal and human feed (carvalho teixeira et al., 2016). the world consumption of this cereal is considerable (althwab et al., 2015) and it is probably due to the ability of the grain to grow over extensive agro-ecological zones (girard et al., 2018; taylor et al., 2014). this cereal has nutrients common to all varieties, including several minerals, vitamins and amino acids (althwab et al., 2015). the presence of polyphenol in the grain is typical in all varieties. the antioxidant level of polyphenols in sorghum is higher than in any other cereal analyzed (rao et al., 2018). the presence of polyphenols in the sorghum grain provides natural protection against microorganism and insect attacks (chandrashekar & satyanarayana, 2006). the levels of phenols and antioxidant activity are highest when sorghums have secondary purple/red plant color; a black or dark red, thick pericarp and a pigmented testa (dykes et al., 2005). in pigmented sorghum, condensed tannins, belonging to the group of polyphenols, become important. in this variety, a positive correlation between total phenolic content and proanthocyanidin, flavan-4-ols and 3-deoxyanthocyanidins (condensed tannins) has been reported. in the same study, 55% of polyphenols correspond to proanthocyanidin 18% to flavan-4-ols and 7.5% to 3-deoxyanthocyanidins (dicko et al., 2005). from this previous study, analyzing tannin content in pigmented sorghum through polyphenol determination leads to a good estimation. such hypothesis is evaluated in this new study. condensed tannins have a negative impact on sorghum flours because they reduce the digestibility of many nutrients, which can affect animal productivity and health (awika & rooney, 2004). its main effect on nutritional value is reducing the digestive availability of protein and starch (aguiar moraes et al., 2015). however, there is a tendency to use milled wholegrain (van der kamp and lupton, 2013), because this type of food may be suitable for diets among people with type 2 diabetes, it is proven that polyphenols in sorghum bind to digestive enzymes, specifically alpha-amylase and retard the degradation of starch into glucose, attenuating hyperglycaemia (links et al., 2015). therefore, the hydrothermal treatment proposed in this work would has a double benefit, since on one hand it would reduce the tannins in flours, where they are not desired due to their coloration, astringency and reduction of protein and starch availability (links et al., 2015), and on the other hand their separation by means of a suitable solvent allowing the use of them as an additive in other foods or as nutraceutical, to reduce type 2 diabetes. the production of wholegrain flour consists of grinding the whole grain to take advantage of the nutrients found in the pericarp and fibers thus improving the gastrointestinal tract health and reducing the incidence of chronic diseases (van der kamp & lupton, 2013). the milling of sorghum could cause contact of the pericap polyphenols and proteins from the inside of the grain and reduce protein availability. on the other hand, the codex alimentarius standard states that wholegrain and flour of sorghum cannot contain more than 0.5% and 0.3% tannins, respectively (codex alimentarius www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 124 published by scholink inc. commission (cac), 2018). previous studies proposed water treatment to reduce the content of polyphenols present in the pericarp of the wholegrain, to take advantage of polyphenols for other uses in food, and to obtain flour with better starch and protein availability (acquisgrana et al., 2016). after treatment there is still enough polyphenols retaining their antioxidant capacity. for that reason, the purpose of this work is to quantify the amount of the remaining polyphenols in the grain after treatment, after grain milling and in final flour. however, in this work both determination of tannins and polyphenols was done, because tannins are the real problem in food. the method to quantify the concentration of polyphenols in cereals is not direct, because it is required to extract them from the food matrix and in many cases, extraction is incomplete depending on the solvent used (tufan et al., 2013). for a better comparison between different types of solvents, tannins in flour is done both, with water and a methanolic extraction. it is interesting to quantify the residual antioxidant capacity after extraction, because it is an attractive quality in food and it is associated with the presence of polyphenol, then an important loss of this property with the hydrothermal treatment applied to wholegrain is expected. the cupric ion reducing antioxidant capacity (cuprac) method has been applied to cereals and has proved to be a reliable determination (tufan et al., 2013); therefore, this is the method used in this work. 2. material and methods 2.1 steeping five samples of sorghum with high content of tannin were obtained from the experimental agricultural stationnational institute of agricultural technology (inta), argentina. four of the samples were red or brown sorghum (dk 61t, dow 108, tob 60t and malón– simplified nomenclature: dk, dw, t and m). the other sample was non-pigmented sorghum, named blank sample (b). total tannin concentration for each sample was obtained from extractable tannins during hydrothermal treatment and the second extraction used in this study. total tannin concentrations were summarized in table 1. table 1. total tannin concentration of each wholegrain sorghum sample sample ums mg/kg ums b 1220 ± 21 dk 7577 ± 54 dw 9252 ± 60 m 4443 ± 26 t 9683 ± 51 data are mean values ± standard deviation www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 125 published by scholink inc. sorghum grains (50 g) of each sample were steeped in 100 ml of sodium hypochlorite (naocl) solution, containing 0.5% (v/v) available chorine. the procedure was done at 25ºc during 18 h (acquisgrana et al., 2017). the samples were washed to eliminate the naocl solution and 100 ml of water was added. the preparation was incorporated to a heat bath at 75ºc for 60 min (acquisgrana et al., 2016), a stage called “annealing” (singh et al., 2011). finally, the samples were dried for 12 h at 60ºc. 2.2 milling after annealing and extracting polyphenol, each sample was divided in three samples: unmilled sorghum (ums), milled sorghum (ms) and flour (f). the ms and f samples were milled with a two-roller mill (cibart, argentina) with a separation of 0.5 mm between rollers. ms samples went through the mill once; f samples underwent the same process eight times. f samples were screened through a 500 m mesh (astm 35) to obtain fine flour, according to cac. 2.3 polyphenol extraction a sample of ums, ms and f was weighed, and polyphenols were extracted with double the amount of water. each preparation was incorporated to a heat bath at 75ºc for 120 min. samples were taken every 30 minutes to analyze total polyphenols (tpp) and total antioxidant capacity (tac). 2.4 measures tpp were estimated using the folin-ciocalteu method (singleton et al., 1999) and tannis (t) were estimated using the hclvainillin midific method (price et al., 1978). both methods were expressed in mg catechin/kg of solid matter, ums or f. tac was estimated with the cuprac method (özyürek et al., 2011) and expressed as mmol trolox equivalents (mmol te/kg of solid matter, ums or f) (tufan et al., 2013). measurements were carried out in triplicate. 2.5 statistical analysis mean values were calculated, and the software infostat (2002) was used to analyze variance. tukey test was carry out at the 0.05 significance level. 3. results and discussion 3.1 polyphenol determination in all ums samples, it was observed that even after treatment to reduce the content of polyphenols, concentration was still significant (figure 1). polyphenols were extracted using different solvents: water and methanol. table 2 shows the final concentration of polyphenols and tannins for each sample at the end of the extraction process. total values correspond to extraction at 120 minutes. the same table shows the values for the extraction of tannins in aqueous solution and methanolic solution. for ums (figure 1), it is observed that pigmented sorghum t contains more polyphenols than the rest of the samples (0.40%), while non-pigmented sorghum shows the lowest value (<0.03%). for ums, extractable polyphenols corresponds to those found in the pericarp. in all the ums analyzed a second-order polyphenol extraction kinetics could be obtained, which may be modeled with a quadratic www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 126 published by scholink inc. polynomial similar to those previously obtained by acquisgrana et al. (2016). this fact suggests that the treatment could continue for longer than the expected 60 minutes, from previous results (acquisgrana et al., 2016). sorghum with lower tannin content was obtained with longer extraction time, but as it will be seen in the ms and f samples, it would not be necessary since the final concentrations were adequate according to the codex. on the other hand, an excessive reduction may cause a greater loss of antioxidant capacity, which is not be desirable for food. furthermore, in previous studies it has been observed that the implementation of the proposed treatment greatly improves availability of soluble proteins (acquisgrana et al., 2017). figure 1. polyphenol extraction vs. extraction time for unmilled sorghum (ums) table 2. tpp, t-we and t-me and ac after 120 min of extraction for ums, ms and f samples ums mg/kg ums ms mg/kg ums f mg/kg f tpp b 324 ± 31 377 ± 9 145 ± 47 dk 3119 ± 11 1515 ± 13 1194 ± 21 dw 3156 ± 62 1710 ± 50 983 ± 14 m 1255 ± 41 1008 ± 3 1179 ± 41 t 3977 ± 36 1537 ± 12 1068 ± 39 t-we (water extraction) b 440 ± 22 608 ± 23 180 ± 13 dk 2622 ± 16 3150 ± 38 2787 ± 30 dw 2828± 9 2900 ± 39 2056 ± 43 m 1093 ± 25 2218 ± 26 2181 ± 35 t 3417 ± 55 3471 ± 52 2861 ± 28 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 127 published by scholink inc. t-me (methanolic extraction) b 640 ± 38 284 ± 18 dk 2678 ± 56 2613 ± 32 dw 3524 ± 83 2144 ± 71 m 1790 ± 45 2258 ± 55 t * 3375 ±72 1930 ± 39 ac** mmol te/kg b 1.2 ± 0.2 2.9 ± 0.4 4.0 ± 0.4 dk 4.2 ± 0.4 5.2 ± 0.2 4.7 ± 0.4 dw 3.8 ± 0.1 5.5 ± 0.1 4.2 ± 0.1 m 2.7 ± 0.1 4.4 ± 0.3 3.4 ± 0.3 t 3.5 ± 0.2 6.1 ± 0.3 5.1 ± 0.2 * determination of t-me in ums was not done. data are mean values ± standard deviation ** ac units are mmol te/kg instead of mg/kg figure 2 shows the extraction of polyphenols for ms. it is observed that practically in all samples, the values are stabilized at 90 minutes. the concentrations of polyphenols in ms samples of all pigmented sorghums showed lower values than the ums samples: dk was reduced in 51.4%, dw in 45.8%, m in 19.6% and t in 61.3% in relation to ums. white non-pigmented sorghum presented an increase with respect to ums by 16.4% (table 2). the decrease of polyphenols in all sorghum varieties could be directly linked with their interaction with proteins. when the grain is milled, polyphenols could interact with proteins and could not continue to be extracted, which does not necessarily imply the reduction of the proteins, because they continue in the ground matrix. in the flour samples (f), since they are completely ground and have been separated from the pericarp by sieving, extraction speed is higher than for ums and ms samples, reaching stability within the initial 30 minutes and remaining invariable the rest of the time, therefore only the final value of extraction is indicated in table 2. since most polyphenols have been extracted during the annealing process and the retention of most of the pericarp and germ during sieving, the flours of all the variety present a lower concentration of polyphenols than the ums and ms. extraction stability after 30 minutes at 75ºc could indicate the appropriate time to extract all the polyphenols present in the f samples. previous studies have reported the high tac present in sorghum grains, regardless of their variety (dlamini et al., 2007). this previous study considered pigmented sorghums with and without tannins. however, it is known that the presence of tannins confers the greatest antioxidant capacity in sorghum varieties, due to the presence of proantocyanidin and other condensed tannins (rao et al., 2018). for example, in the case of proanthocyanidin, mean values of 9400 mg/kg for the red variety, against 1300 mg/kg for the white variety have been reported. furthemore, the proanthocyanidin levels were www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 128 published by scholink inc. positively correlated with the total phenolic content. other examples and quantities can be comparated in bröhan et al. (2011). figure 2. polyphenol extraction vs. extraction time for milled sorghum (ms) other previous studies have reported a strong correlation between the content of polyphenols and tannins (dicko et al., 2005). this observation could be verified in the present report work (figure 3), but only in whole sorghum the content of tannins is lower than that of polyphenols, finding the linear adjustment that is reported in table 3, with the setting parameters of equation 1: 𝑇𝑃𝑃 = 𝑎 ∙ 𝑇 (1) table 3. setting parameters for eq. (1) correlation of tannins and polyphenols in ums, ms and f, with water (we) and methanolic (me) extraction t a r2 ums-we 00.91a 0.985 ms-f-we 2.11b 0.914 ms-f-me 2.02b 0.949 mean in same row in different lowercase are significantly different (p<0.05). from the linear adjustments through the origin for ums samples, it is obtained that 91% of the polyphenols correspond to tannins. however, a strong correlation between tannins and polyphenols is possible for ms and f samples, but tannins exceed more than twice the content of polyphenols, obtaining no significant differences between both extractions, with water or methanol. there were also no significant differences between f and ms samples (table 3). probably, no significant differences are observed due to the high temperature used for the different types of solvent. the main difference www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 129 published by scholink inc. between tannins and polyphenols may be the limitation of the folin-ciocalteu method (singleton et al., 1999) which does not allow adequate quantification of condensed tannins. recent studies provide more evidence on this behavior where a similar relationship between tannins and popyphenols was found (adetunji et al., 2015). in all cases the tannins values found are much lower than that established in the cac 0.5% for wholegrains and 0.3% for flours, therefore with the treatment proposed by acquisgrana et al. (2016) it is possible to obtain a flour suitable for human consumption from the varieties of colored sorghum studied and that can be used for the production of gluten-free foods for people with celiac disease. figure 3. correlation between tannins (t) and total polyphenols (tpp) in ums, ms and f samples. full lines represent eq. (1) with constant values in table 2. vertical bars represent the standard deviation in each value 3.2 antioxidants tac determination in table 2 shows that pigmented sorghum contain a greater quantity of antioxidants than non-pigmented sorghum in the ums. in all cases, the tac is the highest in the ums samples, and the pigmented varieties show higher tac values than the non-pigmented sorghum variety. this is a nutritional advantage of the wholegrain of treated pigmented sorghum since it is possible to reduce the content of tannins to suitable levels according to the cac and that preserve an antioxidant capacity greater than the white variety. however, during flour production, the pericarp part containing most of the fiber and probably the tannins, is separated with the sieving process, leaving the flour of pigmented sorghum with an tac similar to the non-pigmented variety (table 2). in the case of flour, it is observed that in all cases the values obtained are lower than those found by tufan et al. (2013) for the aqueous extract of 18.28 mmol te/kg barley, rye 8.64, wheat 4.31 and 7.51 oat, with the same cuprac method. it is interesting to show that the previous cereal did not undergo sieving, and probably in this samples the tac will be reduced. however, the values of tac obtained in the sorghum samples are of the same order of magnitude obtained with the other cereals studies and similar to milling wheat. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 130 published by scholink inc. the reduction in the tac is due to the initial annealing procedure to extract polyphenols. the methodology used in this work and in the previous studies (acquisgrana et al., 2016) can be used to regulate the content of tannins to meet the requirements of the cac and present a residual antioxidant capacity that makes it attractive to obtain products with the selected flour. 4. conclusion from the result obtained, it was possible to conclude that the treatments of steeping and annealing allowed lower levels of tannins than those established in the codex for both ums and f samples. it may also be possible to handle this reduction to improve the antioxidant capacity of flour. this reduction makes it possible to obtain flour, from pigmented varieties, which may be suitable for food processing and the recovery of tannins for other uses. acknowledgments the authors thank the facultad regional resistencia-universidad tecnológica nacional and the consejo nacional de investigaciones científicas y técnicas (conicet) for their financial support, and the estación experimental agropecuaria del inta “las breñas” for their contribution of experimental samples. special thanks to mr. julio osvaldo jimenez from the estación experimental agropecuaria del inta “las breñas”. references acquisgrana, m. r., benítez, e., gomez pamies, l. c., sosa, g. l., peruchena, n. m., & lozano j. e. (2016). total polyphenol extraction from red sorghum grain and effects on the morphological structure of starch granules. international journal of food science and technology, 51, 2151-2156. https://doi.org/10.1111/ijfs.13194 acquisgrana, m. r., gomez pamies, l. c., & benítez, e. i. (2017). uses of sorghum with tannins for food production. in advances in chemistry research, 42, 231-244. aguiar moraes e., da silva marineli r., lenquiste s. a., joy steel c., beserra de menezes c., vieira queiroz v. a., & maróstica júnior m. r. (2015). sorghum flour fractions: correlations among polysaccharides, phenolic compounds, antioxidant activity and glycemic index. food chemistry, 180, 116-123. https://doi.org/10.1016/j.foodchem.2015.02.023 althwab s., carr t. p., weller c. l., dweikat i. m., & schlegel v. 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(2013). direct measurement of total antioxidant capacity of cereals: quencher-cuprac method. talanta, 108, 136-142. https://doi.org/10.1016/j.talanta.2013.02.061 van der kamp, j. w., & lupton, j. (2013). fibre-rich and wholegrain foods (p. 496). cambridge: woodhead publishing limited. https://doi.org/10.1533/9780857095787.1.3 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 2, no. 3, 2018 www.scholink.org/ojs/index.php/fsns 57 original paper effect of live lactobacillus paracasei nfri 7415 on the preference for a lard diet or fish oil diet in rats akie sato1* & noriko komatsuzaki1 1 department of human nutrition, seitoku university chiba, japan * akie sato, department of human nutrition, seitoku university chiba, 271-8555, chiba, japan received: october 20, 2018 accepted: october 31, 2018 online published: november 13, 2018 doi:10.22158/fsns.v2n3p57 url: http://dx.doi.org/10.22158/fsns.v2n3p57 abstract this study examined the effect of live lactobacillus paracasei nfri 7415 on the preference of a lard diet (ld) and a fish-oil diet (fd) in rats. 4-week-old male fischer 344 rats were fed one of four diets; ld, ld + lactic acid bacteria (lld), fd and fd + lactic acid bacteria for 4 weeks (dietary experimental period). the lld and fld groups freely ingested water containing lb. paracasei nfri 7415 (107 cfu/ml). after 4 weeks, all rats were placed on a two-choice diet program in which they self-selected from two food cups, each containing either the ld or the fd for 5 weeks (self-selection period). after the dietary experimental period, there was no significant difference in the final body weight and total food intake among the four groups. the intake of fish-oil and live lb. paracasei nfri 7415 was increasing the fecal lipids excretion, and it effectively reduced plasma total cholesterol concentration (p<0.05). it was indicated that the intake of live lb. paracasei nfri 7415 was no influence on the preference for fat in the dietary experimental period and the self-selection period. keywords lactobacillus paracasei nfri 7415, self-selection, ratio of fish oil diet intake 1. introduction it is estimated that there are approximately 10 million patients with diabetes and pre-diabetes in japan (ministry of health labour and welfare 2016). according to the national nutrition survey in 2016, the meat intake per day for individuals in their twenties is 2.5 times the intake of fish and shellfish. animal products such as meat contain a large quantity of saturated fatty acids and cholesterol. the risk and incidence of obesity, type-2 diabetes, atherosclerotic vascular disease, and coronary heart disease are increasing in rapid proportion to the increased intake of animal fat (walker et al., 2009; ota et al., 2007). meanwhile, n 3 polyunsaturated fatty acids (pufa) such as eicosapentaenoic acid (epa) and www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 58 published by scholink inc. docosahexaenoic acid (dha) contained in fish oil are known to exhibit plasma lipid-lowering, antithrombotic, and other health-related actions (guichardant et al., 2015; komatsuzaki et al., 2010; arai et al., 2009). previously, to investigate why young people disliked fish, sato and colleagues performed experiments using three groups of rats fed different diets: a lard diet (ld), a soybean-oil diet (sd) or a fish-oil diet (fd) (sato et al., 2009). after 8 weeks, all rats were placed on a self-selected regimen and allowed to choose ld or fd for 3 weeks. the results showed that the rats that were initially fed an ld, i.e., rats who had ingested exclusively animal-fat and no n-3 fatty acids consumed a large amount of fd immediately after the start of the self-selection period, demonstrating that rats have the ability to compensate a deficiency of n-3 fatty acids. lactic acid bacteria (lab) have been utilized as a natural health food since ancient times, and the health-promoting effects of lab are well recognized (elmadfa et al., 2010). some labs are used in fermentation; typical examples can be found in the dairy industry for the production of cheese, yogurt, and other fermented milk products (zhao et al., 2015). lactobacillus paracasei nfri 7415, an lab isolated from a traditional japanese fermented fish (funa-sushi), exhibits high  -aminobutyric acid (gaba)-producing ability (komatsuzaki et al., 2005). we previously reported that lb. paracasei nfri 7415 removed cholesterol from the plasma and liver of rats fed an ethanol-containing diet (komatsuzaki & shima, 2012). oral administration of this strain may have the potential to improve intestinal conditions and immune functions in humans (komatsuzaki et al., 2017). our data suggest that this strain may be effectively applied as a probiotic lactobacillus. even though fat has no taste or smell in itself, as a food component fat has a highly compelling taste for which humans show a strong preference (fushiki et al., 2003). similarly in rats, fatty acid has been shown to be preferred over triglyceride (tsuruta et al., 1999). lab have been shown to affect the lipid metabolism in intestinal cells (tomishige et al., 2016). thus, it is speculated that the intake of lab may have an influence on the preference for fat. in order to examine these assumptions, we investigated the preference for fat in rats over an ld/fd self-selection period (5 weeks) following a 4 week dietary treatment period in which they received either ld or fd either with or without lb. paracasei nfri 7415. the body weight, fat tissue weight and serum lipid concentrations of the rats were also examined and discussed in relation to their observed dietary preferences. 2. materials and methods 2.1 animals and diets four-week-old male fischer 344 rats were commercially obtained from charles river, japan (yokohama, japan). they were housed individually in stainless steel cages in a room kept at a constant temperature (23 ± 1°c) and 50% humidity and illuminated in cycles of 12 h light/12 h dark (lights on from 7:00 to 19:00). the rats were given free access to food and ion-exchanged water. they were weighed, and their food intake was measured every other day from 10:00 to 12:00. the studies were www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 59 published by scholink inc. performed in accordance with the animal experimentation guidelines of the laboratory animal care committee of seitoku university. the composition of the experimental diet is shown in tables 1 and 2. the experimental diet was based on the ain-93g diet (reeves et al., 1993). in order to avoid an n-6 fa deficiency, soybean oil (3 g/100 g diet) was added to the four diets. casein, lard, soybean oil, and dietary components were obtained from oriental yeast (tokyo). fish oil was purchased from nihon-suisan (tokyo). the fatty acid composition of the diet is shown in table 2. the n-6/n-3 ratios of the ld and the fd were 9.9 and 0.9, respectively. table 1. composition of the experimental diets 1 ingredient (g/100g) casein 20.0 l-cystine 0.3 cornstarch 49.95 sucrose 10.0 soybean-oil 3.0 fat2 7.0 cellulose 5.0 mineral mixture3 3.5 vitamin mixture3 1.0 choline bitartrate 0.25 tert-butylhydroquinone 0.0014 1 diet components were purchased from oriental yeast. 2 fat: lard (lard diet) or fish oil (fish-oil diet). 3 mineral mixture (g/kg of mix): cahpo4, 500.0; nacl, 74.0; k3c6h5o7⋅h2o, 220.0; k2so4, 52.0; mgo, 24.0; mnso4⋅5h2o, 6.77; feso4⋅7h2o, 4.95; znco3, 1.6; cuco3cu(oh)2h2o, 0.3; klo3, 0.01; naseo3, 0.01; crk(so4)2⋅12h2o, 0.55; naf, 0.06; sucrose, 115.75. vitamin mixture (g/kg of mix): retinol, 4.8; cholecalciferol, 0.4; thiamine, 24.0; riboflavin, 0.6; pantothenic acid, 0.6; pyridoxine, 0.7; cobalamin, 0.01; menadione, 0.05; nicotinic acid, 3.0; d-calcium pantothenic acid, 1.6; folic acid, 0.2; biotin, 0.02; para-aminobenzoic acid, 5.0; inositol, 10.0; glucose, 949.02. table 2. fatty acid composition of the diets (% of total fatty acids) fatty acid lard diet fish oil diet 10:0 0.07 12:0 0.1 0.1 14:0 1.2 6.1 14:1 0.1 0.4 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 60 published by scholink inc. 15:1 0.1 16:0 20.9 16.6 16:1 6.6 17:0 1.8 0.3 17:1 0.3 0.7 18:0 10.1 2.2 18:1 (n-9) 38.9 19.2 18:2 (n-6) 22.5 16.7 18:3 (n-3) 2.3 2.6 20:0 0.3 0.1 20:1 (n-9) 0.5 6.3 20:2 0.3 0.1 20:3 0.1 20:4 (n-6) 0.1 0.5 20:5 (n-3) 10 22:3 0.1 22:5 (n-3) 1.5 22:6 (n-3) 6.2 unknown 0.3 3.0 total n-6 22.8 17.9 total n-3 2.3 20.3 n-6/n-3 9.9 0.9 2.2 preparation of extract a pre-culture of lb.paracasei nfri 7415 was grown to the stationary phase at 37°c for 20 h in de man, rogosa, sharpe (mrs) (difco laboratories, detroit, mi) medium. the medium was separated from cells by centrifugation (5,000 rpm for 10 min at 4°c). the cells were washed with phosphate-buffered saline (pbs; at ph 7.0) containing 8 g of nacl, 0.2 g of kcl, 1.44 g of na2hpo4, and 0.24 g of kh2po4 (per liter), and then the cells were diluted (107 cfu/g) with sterilized water. the cell suspension was used in animal experiments. 2.3 experimental design forty-two 4-week-old male rats were divided into four groups. the groups respectively received the ld (n=10), the ld + lab (lld) (n=11), the fd (n=10), or the fd + lab (fld) (n=11) during the dietary treatment period (figure 1). the rats in the lld and fld groups freely ingested water containing lb. paracasei nfri 7415 (107 cfu/ml). after 4 weeks, half of the rats in each of the four groups were sacrificed. all rats were put on a two-choice diet program in which they self-selected from two food cups, one containing the ld and the other the fd, for 5 weeks (self-selection period). after 5 weeks, all rats were anesthetized. there was no fasting after the feeding period. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 61 published by scholink inc. figure 1. experimental design the blood was centrifuged at 3,000 rpm for 10 min to separate the plasma, which was then stored at -80°c until analysis. liver, perirenal fat tissue and epididymal fat tissue were removed and weighed. feces were collected for 24 h on the day before the end of the dietary treatment period and on the day before the end of the self-selection period. feces were dried in an oven at 105°c for 24 h. 2.4 analytical methods liver lipids were extracted by the methods of folch et al. (1996), plasma triacylglycerol (tg) and total-cholesterol (t-cho) concentrations were measured, and liver extracts were similarly analyzed using test kits (triglyceride e-test wako and cholesterol e-test wako; purchased from wako pure chemical industries, osaka, japan). 2.5 assay of fecal lipids and fecal cholesterol to assay the fecal lipids and cholesterol, 0.1 g of homogenized dry fecal matter was added to 4 ml of concentrated sulfuric acid in test tubes for 30 min at room temperature. diethyl ether was added to reach 25 ml, and the solution was mixed. the diethyl-ether-containing layer was moved to a flask, and the diethyl ether was evaporated. the fecal lipid in the flask was then weighed. the t-cho concentration in fecal matter was determined in the same way as the liver t-cho concentration. fecal bile acids were measured by the procedure described in iwami et al. (2002); 10 mg of the sample was mixed with 0.2 ml of 90% ethanol during vortex mixing, and incubated for 1 h at 65°c. the mixture was subjected to centrifugation at 5,000 rpm for 3 min. the supernatant was transferred to a 1.5 ml tube, and the ethanol was evaporated. then, 0.2 ml of 90% ethanol was added to the precipitate for vortex mixing. the sample was dissolved in 1 ml 90% ethanol and measured using test kits (total bile acid test by enzyme colorimetric method; wako pure chemical industries). 2.6 statistical analysis values were expressed as means ± sd. repeated-measures analysis of variance was used to evaluate the effects of preference group and time on food intake. differences in mean values between groups were tested by scheffe’s multiple-range test. differences were considered significant at p<0.05. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 62 published by scholink inc. 3. results 3.1 food intake, body and organ weight, plasma and liver lipid concentrations after the 4-week dietary treatment period during the dietary treatment period, no significant difference in the food intake, body weight, or perirenal fat and epididymal fat tissue weights were observed among the four groups (table 3). the liver weight of the fld group was lower than those of the ld and lld groups (p<0.05) (table 3). no significant difference was observed in lab intake between the lld and fld group, based on monitoring of the drinking water levels (data not shown). the plasma tg concentration of the fld group was lower than that of the ld group (p<0.05) (table 3). the plasma t-cho concentrations of the fd and fld groups were lower than those of the ld and lld groups (p<0.05) (table 3). no differences were observed in liver tg and t-cho concentration among the four groups. table 3. food intake, body, liver, and fat tissue weights and plasma and liver lipid concentrations after a dietary treatment period of 4 weeks group ld (n=5) lld (n=5) fd (n=5) fld (n=5) food intake (g) 212 ± 21 213 ± 21 213 ± 21 211 ± 21 body weight (g) 194 ± 9 191 ± 17 190 ± 15 191 ± 9 liver weight (g/100g bw) 4.12 ± 0.17b 4.00 ± 0.24b 3.88 ± 0.25ab 3.56 ± 0.13a perirenal fat tissue weight (g/100g bw) 0.76 ± 0.10 0.71 ± 0.05 0.76 ± 0.14 0.73 ± 0.08 epididymis fat tissue weight (g/100g bw) 1.54 ± 0.36 1.45 ± 0.10 1.37 ± 0.15 1.41 ± 0.11 plasma lipid (mg/dl) triacylglycerol 242.5 ± 104.6b 181.0 ± 30.2ab 120.6 ± 94.5ab 54.5 ± 12.7a t-cholesterol 78.3 ± 5.8b 70.0 ± 4.9b 51.6 ± 4.4a 50.6 ± 5.6a liver lipids (mg/g) triacylglycerol 14.4 ± 4.2 14.5 ± 10.9 10.2 ± 3.6 7.0 ± 4.6 t-cholesterol 2.06 ± 0.60 1.01 ± 0.21 1.65 ± 0.47 2.27 ± 0.90 values represent means ± sd. within a row, values not sharing a common superscript letter are significantly different at p<0.05. 3.2 food intake, body and organ weight, plasma and liver lipid concentrations after the self-selection period for 5 weeks after the self-selection period, no significant difference in the food intake, body weight, or perirenal fat and epididymal fat tissue weights were observed among the four groups (table 4). when calculating the ratio of fd intake (fish-oil intake/total intake) over 5 weeks, it became about 30% in each of the four groups, and no significant difference was observed. immediately after the start of the self-selection period (1 week), the ratios of fd intake of the ld and lld groups were higher than that of the fld group (p<0.05) (figure 2). however, the ratio of fd intake after 7 days of self-selection was approximately 30%; there was no significant difference among the four groups. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 63 published by scholink inc. 0 10 20 30 40 50 60 ld lld fd fld r a ti o o f fi sh o il d ie t in ta k e (% ) 1week b a 0 10 20 30 40 50 60 ld lld fd fld r a ti o o f fi sh o il d ie t in ta k e( % ) 5week figure 2. ratio of fish oil diet intake to total intake of groups fed a lard diet or fish oil diet during a self-selection period of 5 weeks after being fed a lard diet or fish oil diet for 4 weeks values are expressed as means ± sd. ld and fd groups: n=5, lld and fld groups: n=6. values not sharing a common superscript letter are significantly different at p<0.05. a b www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 64 published by scholink inc. table 4. food intake, body, liver and fat tissue weights and plasma and liver lipid concentrations after a self-selection period of 5 weeks group ld (n=5) lld (n=6) fd (n=5) fld (n=6) food intake (g) 585 ± 20 598 ± 41 554 ± 46 595 ± 55 lard diet intake (g) 388 ± 53 386 ± 75 433 ± 69 408 ± 49 fish oil diet intake (g) 197 ± 39 209 ± 54 135 ± 43 142 ± 31 ratio of fish oil diet intake (%) 34 ± 7 35 ± 10 24 ± 8 26 ± 5 n-6/n-3 ratio 2.5 2.5 3.0 3.2 body weight (g) 297 ± 12 306 ± 17 303 ± 20 309 ± 23 liver weight (g/100g bw) 3.26 ± 0.17 3.07 ± 0.22 2.36 ± 1.04 3.04 ± 0.15 perirenal fat tissue weight (g/100g bw) 1.25 ± 0.24 1.25 ± 0.18 1.25 ± 0.15 1.31 ± 0.13 epididymis fat tissue weight (g/100g bw) 2.24 ± 0.31 2.16 ± 0.32 2.38 ± 0.26 2.45 ± 0.15 plasma lipid (mg/dl) triacylglycerol 76.1 ± 21.6a 101.7 ± 35.1ab 122.3 ± 13.0b 113.3 ± 15.1ab t-cholesterol 45.7 ± 10.2 38.7 ± 12.4 52.9 ± 9.7 56.7 ± 7.9 liver lipid (mg/g) triacylglycerol 18.7 ± 2.9 16.6 ± 7.1 10.8 ± 6.0 12.5 ± 5.8 t-cholesterol 1.08 ± 0.15 1.24 ± 0.22 1.18 ± 0.17 1.28 ± 0.27 values represent mean ± sd. within a row, values not sharing a common superscript letter are significantly different at p<0.05. after the self-selection period, the plasma tg concentration of the ld group was lower than that of the fd group (p<0.05) (table 4). there were no significant differences in the plasma t-cho and liver lipid concentrations among the four groups. 3.3 fecal weight and fecal lipid concentration after the dietary treatment period and the self-selection period there were no significant differences in fecal weight among the four groups during the dietary treatment period and the self-selection period (tables 5, 6). after the dietary treatment period, the fecal total fat concentration of the ld group was higher than that of the fd group (p<0.05) (table 5). after the self-selection period, the fecal t-cho concentration of the fld group was higher than those of the ld and lld groups (p<0.05) (table 6). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 65 published by scholink inc. table 5. fecal weight and fecal lipids after a dietary treatment period of 4 weeks ld ( n=5) lld ( n=5) fd ( n=5) fld ( n=5) weight (fresh) (g) 2.21 ± 0.68 2.06 ± 0.58 2.13 ± 0.22 2.27 ± 0.36 weight (dry) (g) 2.11 ± 0.44 1.79 ± 0.48 1.81 ± 0.24 1.91 ± 0.32 total fat (mg/g) 50.6 ± 8.95b 46.1 ± 4.5ab 32.0 ± 9.34a 40.2 ± 6.61ab bile acid (mg/g) 8.40 ± 1.62 6.57 ± 0.93 7.99 ± 1.03 7.01 ± 0.76 t-cholesterol (mg/g) 0.35 ± 0.10 0.37 ± 0.10 0.32 ± 0.03 0.37 ± 0.05 values represent mean ± sd. within a row, values not sharing a common superscript letter are significantly different at p<0.05. table 6. fecal weight and fecal lipids after a self-selection period of 5 weeks values represent mean ± sd. 4. discussion no significant differences in total dietary intake, body weight, and perirenal fat tissue weight were observed among the four groups during the treatment period and the self-selection period. therefore, it was suggested that a difference in ingested fats did not affect the appetite of rats. however, the liver weight and plasma tg of the fld group were lower than those of the ld and lld groups after the dietary treatment period (p<0.05) (table 3). previous studies have reported that lb.paracasei nfri 7415 can remove cholesterol from the plasma and liver of rats fed high-fat diets (komatsuzaki et al., 2016). in another report, oral administration of this strain reached the intestinal tract of mice and improved enteric bacterial flora (komatsuzaki et al., 2017). no significant differences in the liver weight and plasma tg were observed among the ld, lld, and fd groups after the dietary treatment period (table 3). some labs metabolized unsaturated fatty acids in the intestinal tract and produced conjugated fatty acid (cla) has effect like probiotics (kishino et al., 2011). n-3 pufas such as γ-lenolenic acid, epa, and dha are contained in fish oil (table 2). it was assumed that these n-3 pufas were metabolized in the intestinal tract of the fd group by lb.paracasei nfri 7415. in order to clarify this assumption, it was necessary to construct an in vitro experiment to produce cla or metabolize n-3 pufa from this strain. after the self-selection period, the plasma tg of the ld group was lower than that of the fd group ld (n=5) lld (n=6) fd (n=5) fld (n=6) weight (fresh)(g) 2.70 ± 0.31 3.10 ± 0.65 2.65 ± 0.69 2.89 ± 0.68 weight (dry) (g) 2.02 ± 0.24 2.42 ± 0.37 2.08 ± 0.49 2.28 ± 0.54 total lipid (mg/g) 77.0 ± 10.3 70.5 ± 15.7 87.2 ± 22.6 88.5 ± 17.9 bile acid (mg/g) 7.42 ± 1.87 7.19 ± 1.60 8.37 ± 1.51 6.61 ± 3.07 t-cholesterol (mg/g) 0.40 ± 0.08 0.39 ± 0.10 0.47 ± 0.31 0.64 ± 0.23 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 66 published by scholink inc. (p<0.05), although no significant differences were found in the ratio of fd intake among the four groups (table 4). the ratios of fd intake in the ld group and fd group were 34 ± 7 (%) and 24 ± 8 (%), respectively (figure 2). many reports have shown that diets enriched with fish-oil reduce the plasma tg concentration (guichardant et al., 2015; arai et al., 2009; ikeda et al., 2001). our results suggested that plasma tg concentrations in the ld group declined as a result of ingesting a large amount of fd in order to counteract the effect of n-3 fatty acids deficiency during the dietary treatment period. live lb.paracasei nfri 7415 has the capacity to accelerate fecal t-cho excretion, and it has been shown to effectively reduce the plasma t-cho concentration (komatsuzaki et al., 2014). arai et al., (2009) reported that fish-oil inhibited body weight gain and exhibited an anti-obesity effect. to investigate the cholesterol excretion effect of fish-oil and this strain, we measured the fecal lipids in the rats. although the fecal total fat concentration of the ld group was higher than that of the fd group (p<0.05) (table 5), there was no significant difference between the lld group and the fld group after the treatment period. the t-test analysis showed that the fecal fat concentration of the fld group tended to be higher in the fd group (p=0.06). caesar et al. (2015) showed that mice fed lard for 11 weeks have increased bacteroides in the intestine, white adipose tissue inflammation, and reduced insulin sensitivity compared with mice fed fish oil. this phenotypic difference between the dietary groups can be partly attributed to differences in microbiota composition. more than 100 trillion intestinal bacteria inhabit the intestinal flora in the mammalian bowels, and more than 100 types of bifidobacteria and anaerobic bacteria form intestinal flora (mitsuoka et al., 1990). over 99% of the bacteria in the gut in the intestinal flora of mammals are anaerobes. among them, streptococcus spp. and bacteroides spp. are known as human carcinogens; further, it has been shown that the number of anaerobic bacteria exceeds that of aerobic bacteria in the feces of cancer patients (shinohara, 1990). in this study, the intake of fish oil and live lb.paracasei nfri 7415 increased the fecal lipids excretion, and it effectively reduced the plasma t-cho concentration. at the same time, it is possible that useful bacteria such as resident lab improve the balance of bacterial flora in the intestines. at one week after the start of the self-selection period, the ratios of the fd intake in the ld group and lld group were 34.4 ± 4.0% and 37.5 ± 15.6%, respectively (figure 2). it was speculated that the ld and lld groups ingested large amounts of the fd to compensate for deficient n-3 pufa in the dietary treatment period. the plasma tg in the ld group exhibited an approximately three-fold decrease, from 242.5 ± 104.6 mg/dl to 76.1 ± 21.6 mg/dl, between the dietary treatment period and the self-selection period (tables 3, 4). on the other hand, the plasma tg concentration in the fd group was 120.6 ± 94.5 mg/dl in the dietary treatment period and 122.3 ± 13.0 mg/dl in the self-selection period; it was nearly unchanged. after the self-selection period, the n-6/n-3 ratios of the ld, lld, fd, and fld groups were 2.5, 2.5, 3, and 3.2, respectively. as described in our previous paper, it was concluded that the proper n-6/n-3 ratio for the effective prevention of arteriosclerosis and cardiac diseases was 3 according to self-selection of the ld and the fd (sato et al., 2009). also in this study, it was suggested that the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 67 published by scholink inc. rats had the ability to purposefully compensate for insufficient levels of essential fatty acids by consuming ld and fd. it was previously reported that intraduodenal injection of lactobacillus johnsonii la1 elevated efferent gastric vagal nerve activity, thereby increasing food intake (horii et al., 2013). in that study, it was suggested that the intake of lab increases the appetite. the lactic acid produced by lab is known to have a sour taste, and a substantial proportion of young children have a preference for extremely sour tastes (liem et al., 2004). however, in humans this preference appears to be related to the willingness to try unknown foods and a preference for intense visual stimuli; it is difficult to attribute this to a physiological need. because humans have preferences derived from food experiences and food culture, they are less strongly dominated by physiological needs than animals. however, it is possible to scientifically clarify preferences because humans and animals have physiological similarities (fushiki, 2003). we previously reported that rats have the ability to compensate for a deficiency of n-3 fatty acids (sato et al., 2009), and similar results were obtained from this study. at the same time, it was indicated that the intake of live lb.paracasei nfri 7415 had no influence on the preference for fat in the dietary treatment period or the self-selection period. 5. conclusion as in our previous study, the rats had the ability to purposefully compensate for insufficient levels of essential fatty acids in the experimental food intake period by consuming ld and fd during the self-selection period. in addition, ingestion of lactic acid bacteria did not affect this ability. the total cholesterol concentration in feces after the self-selection period was significantly higher in the fld group than in the fd group. by ingesting fish oil and lactic acid bacteria, the excretion effect of total cholesterol in feces of lactic acid bacteria was considered to be enhanced. acknowledgments we thank nihon-suisan (tokyo) for providing the fish oil. references arai, t., kim, h. j., chiba, h., & matsumoto, a. 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(2017). effects of live lactobacillus paracasei nfri 7415 on the intestinal immune system and intestinal microflora of mice. int j biotechnol food sci, 5, 56-62. komatsuzaki, n., ebihara, k., honda, m., ueki, y., & shima, j. (2014). effects of lactic acid bacteria isolated from japanese fermented fish (funa-sushi) on fecal cholesterol excretion of mice. j integr stud diet habits, 25, 287-291. https://doi.org/10.1155/2018/6491907 komatsuzaki, n., shima, j., kawamoto, s., momose, h., & kimura, t. (2005). production of -aminobutyric acid (gaba) by lactobacillus paracasei isolated from traditional fermented foods. food microbiol, 22, 497-504. https://doi.org/10.1016/j.fm.2005.01.002 komatsuzaki, n., usui, t., toriumi, e., yamada, y., & nakashima, y. 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(2015). preventive effect of lactobacillus fermentum zhao on activated carbon-induced constipation in mice. j nutr sci vitaminol, 61(2), 131-137. https://doi.org/10.3177/jnsv.61.131 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 1, 2017 www.scholink.org/ojs/index.php/fsns 50 micronutrients as an innovation opportunity for the pharmaceutical industry: a case study of vitamin d in brazil heros teixeira rabelo1, wanise borges gouvea barroso2 & jorge lima magalhães2,3* 1 research and marketing department, gross pharmaceutical laboratory, rio de janeiro, brazil 2 research and development in the pharmaceutical industry, centre for technological innovation/nit-far, oswaldo cruz foundation/fiocruz, ministry of health, brazil 3 global health and tropical medicine (ghtm), instituto de higiene e medicina tropical (ihmt), universidade nova de lisboa (unl), rua da junqueira, lisboa, portugal * jorge lima magalhães, e-mail: jorgemagalhaes@far.fiocruz.br received: april 11, 2017 accepted: april 21, 2017 online published: may 17, 2017 doi:10.22158/fsns.v1n1p50 url: http://dx.doi.org/10.22158/fsns.v1n1p50 abstract this paper aims to demonstrate the micronutrients as an attractive alternative of innovation for pharmaceutical industries with limited resources. a case study of vitamin d as a pharmaceutical supplement is launched in the brazilian market. the results demonstrate that some opportunities of several new products for populations with nutritional deficiency—the so-called micronutrients for hidden hunger, that is any deficiency unnoticed where micronutrients assume metabolic functions in humans. this deficiency unnoticed lead to disease onset or an organism dysfunction. who estimate that more than 2 billion humans are affected by hidden hunger. scientific studies show that these ills cause serious problems for pregnant women, elderly and children. thus, the micronutrients in a normal diet comes up the news opportunities for pharmaceuticals industries as well as vitamin supplements. pharmaceutical industry is the most innovative company in healthcare. launcher a blockbuster as a new product through of radical innovation is so hard and with higher costs. in this sense, the micronutrients are an attractive alternative of innovation for pharmaceutical industries with limited financial resources. so, pharmaceutical supplements appear as an incremental innovation of known chemical substances. keywords micronutrients, vitamin d, public health, pharmaceutical industry competitive intelligence www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 51 published by scholink inc. 1. introduction according to sakar (2007), innovate derives from latin in+novare and its meaning can be expressed as doing something new, renewing or altering something. drucker (2010) points out that innovating is the ability to transform something that already exists into a resource that generates wealth. therefore, innovation is the process that includes the technical activities, design, development, and management that result in the marketing of new (or improved) products, or in the first use of new (or improved) processes. it may be a new marketing method, or a new organizational method in business practices, workplace organization, or external relations (freeman, 2008). organization for economic co-operation and development (oecd) highlights innovation encompasses several scientific, technological, organizational, financial, commercial and marketing activities. technological innovation activities are the set of scientific, technological, organizational, financial and commercial steps, including investments in new knowledge, leading to or attempting to implement new or improved products and processes (oecd, 2005). in this innovation context, the pharmaceutical industry is a global example of this innovative capability. in 2014, according to ims health (2015), innovative medicines accounted 24% of total global drug sales, accounting only 19% of global sales in 2009 (ims institute for healthcare informatics, 2013; healthcare informatics, 2015). in ten years, r & d investment by these companies has reached us$ 1.2 trillion and should continue to grow there about 2.4% pa. until 2020. considering the 100 global companies that have invested more in r & d, it is observed that 22 are pharmaceutical companies, outperforming the automotive and electronics industries (evaluate, 2015). innovations are characterized by as radical, incremental, disruptive or frugal. radical innovations resulted in drugs whose therapeutic indications differed substantially from existing drugs on the market, thus it creating a new class of drugs. already the incremental is an improvement of an already existing drug and whose performance has been improved significantly or the research of that same existing drug are directed to a second therapeutic indication. concerning disruptive innovations, they provoke a rupture in established business models. usually favor the appearance of new products with performance equivalent to the market standard, but lower than the leading product from radical innovation, however, adopts a new approach making it cheaper and more convenient or easier to use. the frugal innovation discovers new business models, reconfigures value chains, and redesigns products to serve users who face extreme affordability constraints in a scalable and sustainable manner. furthermore, it involves either overcoming or tapping institutional voids and resource constraints to create more inclusive markets (vieira, 2006; kotler, 2011). in this context, the area of human health is the one that has demanded the different types of innovation for the improvement of the quality of life of humankind. according to the food and agriculture organization of the united nations (fao), about 800 million people remain chronically undernourished and more than 2 billion suffer from micronutrient deficiencies (essential vitamins and minerals required from diet to sustain cellular functions and molecular mechanisms of virtually all www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 52 published by scholink inc. living things). although very small amounts of these micronutrients are required in the human body, micronutrient-deficiency (mnd) can lead to health impacts as deaths if left untreated. the most common deficiencies in the human body are vitamin a, d, folic acid, iron, iodine and zinc. however, there are several other mnd disorders (bailey, 2015; fao, 2016; west, 2012). unfortunately, there are no biomarkers available for all micronutrients or existing biomarkers are not practicable/feasible for widespread evaluation or use in the clinical environment (bailey, 2015). in this way, brazilian pharmaceutical laboratories have made efforts to launch several concentrations of vitamin d in the market to act in the public health area, specifically mnd. 2. method this is a descriptive study involving data mining in bibliographical references in indexed scientific databases such as scopus, medline (pubmed) and officials portal websites as ims health (http://www.imshealth.com/), food and agriculture organization of the united nations (http://www.fao.org/home/en/) and national health surveillance agency (anvisa-brazilian term, http://www.portal.anvisa.gov.br/). in order to identify the state of the art of the subject in question, searches were carried out in the databases indexed with the following terms: adult, biotin, birth, child, chrome, deficiency, disease, essential, folate, folic acid, food, hidden hunger, infant, innovation, iodine, iron, micronutrient, mineral, nutrient, nutrition, old, vitamin d, pregnant, pregnancy. 3. results and discussions 3.1 the hidden hunger historically, hunger has been one of the greatest woes of humankind. about 30 million die in china between 1958 and 1961 and 4 million in india in 1943. in this sense, continues to plague sub-saharan africa. however, even with this scenario, far fewer people are hungry today than in the past due to the adoption of mechanization, agrochemicals and agricultural techniques to obtain high crop yields during the last 50 years. thus, the average per capita calories consumed in the developing world jumped from around 2,000 kcal to over 2,700 kcal (muthayya et al., 2013; shekhar, 2013). however, there is one type of malnutrition that continues to devastate many populations: hidden hunger which is the invisible deficiency in the diet of essential vitamins and minerals. although supplements and fortified foods are helping to address this problem they are expensive and sometimes impractical. an alternative strategy is to encourage farmers to switch to basic crops especially planted with improved micronutrient levels. so, such biofortified varieties could yield much more than cultivation on micronutrient-poor soils (shekhar, 2013; muthayya, 2013). hidden hunger compromises several stages of the metabolic process, impacting mainly on the immune system and the physical and mental development of the individual. it occurs to economic, geographic and/or educational reasons, the individual has access to a basic diet that is not diversified and is usually deficient in several micronutrients with the number of families affected being even more significant than www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 53 published by scholink inc. those affected by protein-energy malnutrition. it should be noted, pregnant women, infants and children are the populations most vulnerable to micronutrient deficiency and consequently suffer the worst adverse effects (black, 2013; tulchinsky, 2010). hidden hunger is present in every country in the world even the richest. hidden hunger and obesity are not mutually exclusive. the hierarchy of food prices is such that foods with high energy density, composed of refined grains, added sugars, or fats have become the least expensive sources of energy in the concentrated diet. not surprisingly, diets high in sugars and fats are cheaper than prudent diets based on lean meats, fish, fresh vegetables and fruits. the more nutrient-rich foods not only cost more but the price difference between foods that are energy dense versus nutrient-rich continues to grow. thus, populations in developed countries are becoming malnourished even though they are overfed (biesalski, 2013). 3.2 micronutrient supplementation in the 1980s, infant mortality in northern sumatra caused by vitamin a deficiency was reduced by 1/3 of the previous data at the cost of 4 cents per micronutrient capsule. in this sense, other ways of attenuating mnd were implemented such as increasing the frequency of supplementation through fortified foods. thus, the united nations children’s fund (unicef) distributes between 400 and 600 million vitamin a capsules annually (shekhar, 2013). since the mid-twentieth century, food fortification has been accepted and used by food processors whose goal is to enhance nutritional value and correct or prevent nutritional deficiencies (boen et al., 2007). in brazil, the regulation took place by cnnpa resolution 12 of 1978 and minister order (portaria-brazilian term) nº 31 of january 13, 1998. the fortified food is that which in 100 ml or 100 g of the product ready for consumption contain at least 15% of dietary reference intakes (dri) in the case of liquids and 30% of dri in the case of solids. according to the technical regulation of complementary nutrition information (ms, 1978), the food is considered fortified/enriched and declared on the label “high content” or “rich” (vellozo, 2010). according to boonen (2007), vitamin d supplements of at least 800 iu/day may be useful in reducing bone loss and fracture rates in the elderly. for the preservation of bone health, one should consume from 1,000 to 1,200 mg per day of calcium through diet and/or supplements (boonen, 2007). the brazilian society of endocrinology and metabolism (sbem), recommends that people with osteoporosis and increased risk of fractures use vitamin d supplements in daily doses between 1,000 and 2,000 iu required to maintain concentrations of 25 (oh-) d ratio was above 30 ng/ml, with respect to the full benefits of preventing secondary hyperparathyroidism, decreased risk of falls, and improved bone densitometry (sbem, 2014). vitamins are a group of organic compounds necessary for the normal physiological function of living organisms and which cannot be synthesized-produced-in adequate amounts by these organisms and therefore need to be obtained from external sources (lieberman, 1990). scientific knowledge about the importance of vitamins only occurred at the beginning of the 20th century www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 54 published by scholink inc. when beriberi disease was associated with vitamin b1 deficiency and pellagra due to lack of vitamin b3. in the 21st century, all vitamins are produced on an industrial scale in chemical and pharmaceutical companies, either by synthesis, semi-synthetic or by fermentation (philippsborn, 2007). the main source of vitamins is diet, so, the feeding. however, some vitamins are obtained in other ways such as vitamin d which is synthesized by the skin when it is exposed to ultraviolet radiation from sunlight. the main sources of vitamin d are ultraviolet-b (uvb), cod liver oil, mackerel, pink salmon, tuna, milk, sardines, liver steak, egg yolk, cheese and mushroom. figure 1 shows the essential organic compounds and vitamin d nutrients are ergocalciferol (d2) and cholecalciferol (d3) (hendler, 2008; mahan, 2012). figure 1. chemical structure of vitamin d according to lips (2006), a clinical trial in 18 countries involving more than 2,600 women with osteoporosis revealed that 64% of them had vitamin d deficiency. however, vitamin d deficiency may also be associated with increased risk of cancer, autoimmune diseases, hypertension, and infectious diseases (holick, 2008). 3.3 vitamin d in brazil even in tropical countries where the sunlight is constant such as brazil with an average annual temperature of 28°c in the north and 20°c in the south of the country, a large part of the population has mnd. regions with low latitude (10°s), even with tropical humid climate and predominant sunlight, the state of recife, presents women with vitamin d deficiency in postmenopausal. the prevalence of vitamin d deficiency increases significantly with age. it founds in 30% of women between 50 and 60 years of age and in more than 80% in women with 80 years of age (bandeira, 2006). in the state of são paulo, brazil, 250 elderly people with a mean age of 79 years showed 57% with plasma levels of vitamin d below the recommended limit (saraiva, 2005). in brazil, in order to obtain a license to market food supplements, they must meet a series of requirements described in specific legislation managed by anvisa such as shown in figure 2. ergocalciferol cholecalciferol www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 55 published by scholink inc. figure 2. synthesis of brazilian sanitary legislation for micronutrients considering mnd scenario in brazil and the knowledge of the essentiality of the vitamin d for bone formation and maintenance, the national pharmaceutical laboratories have been striving for a disruptive innovation in vitamin d. considered as disruptive innovation on the grounds that these laboratories introduced in the pharmaceutical market a new prescription form for vitamin d. so, they created a new market for vitamin supplements and/or pharmaceutical minerals (vspm). as too much sun exposure is counteracted by the brazilian society of dermatology (sbd), due to the risk of skin cancer, this fact has generated a market environment even more conducive to the incremental and/or disruptive innovations of vitamin d. in may 2010, the pharmaceutical laboratory hypermarcas launched the first vspm of vitamin d isolated in the brazilian market: addera d3®, in the form of drops, registered in anvisa as a specific drug, reaching sales of around us$ 1 million in its first year of launching. subsequently, revenues reached approximately us$ 5 million, us$ 8 million and us$ 13 million in 2011, 2012 and 2013, respectively. in 2011, the multinational pharmaceutical laboratory sanofi with a strong presence around vaccines and medicines r & d, decided to enter in this mnd market launching the depura® containing 200 iu per drop and containing lemon flavor. this fact is a differential in relation to addera d3. depura sales reached about us$ 2.3 million in 2011 and exceeded sales of addera d3 in the following years. sales in 2012 and 2013 were recorded at approximately us$ 8.4 million and us$ 17.2 million, respectively. following this fact, there were new releases of vitamin d on the market, namely:  maxxi d3®, launched in 2011 by the national laboratory myralis with concentration limited to 200 iu and without flavor.  vitersol d®, launched in 2011 by the national laboratory marjan. it was the first vitamin d isolated in the form of soft capsules also in the concentration of 200 iu. sales reached about us$ 3.5 million in 2014. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 56 published by scholink inc.  depura kids®, launched in 2012, sanofi launched the first isolated vitamin d designed for children in the form of raspberry-flavored drops. the sales of this presentation grew discreetly and continuously year after year and in 2015 reached us$ 3.8 million.  supra d®, launched by o kley hertz laboratory. this containing orange flavored in drops. sales totaled about us$ 4 million in 2015.  vitax d3®, launched in 2013 by arese laboratory. presentations in drops and soft capsules with sales of approximately us$ 300,000 in the first year of launch.  desol®, launched by the apsen laboratory. in drops and with a sweet-milk flavor. revenue was approximately us$ 1.5 million from the launch year and in 2015 reached about us$ 2.2 million.  font d® launched in 2013 by the union chemical laboratory in the form of drops. sales reached about us$ 2.2 million in 2015.  maxxi d3 launched in 2013 by myralis laboratory. tutti frutti flavor.  dose d® by the laboratory aché in the year 2013 containing a version apple-green flavor drops. the great diversity of products in the form of drops was justified by the physician’s flexibility to prescribe high doses of vitamin d (above 1000 iu/day), since the medical practices pointed to treatments of 2000 iu/day where the blood tests indicated an adequate level of vitamin d in the individual. in this way, doctors sometimes prescribed up to 5000 iu/day—equivalent to 25 drops of the depura or 200 iu/drop. thus, two competing pharmaceutical laboratories developed presentations in the form of tablets at high dosages (above the limits established by ordinance no. 40 of 1998). therefore, they products were classified as type 2 specific medicine. so, getting a new license to market. now as a medicine and no longer as a supplement. in this new scenario, in 2014, the myralis laboratory launched d-prev®, being coated tablets in the concentrations of 1,000 iu, 2,000 iu, 5,000 iu and 7,000 iu per pharmaceutical unit. the brand’s sales reached approximately us$ 1.5 million in the launch year at us$ 5 million in 2015. following the groundbreaking wave in 2014, the pioneering lab relaunched addera d3 with three coated tablet concentrations: 1,000 iu, 7,000 iu and 50,000 iu. the presentations achieved sales of approximately us$ 10 million and in 2015 reached about us$ 36 million. other vitamin d releases:  doss®, in the year 2014 by biolab laboratory, a product as coated tablets of 1,000 iu.  font-d® by união química laboratory: in the form of soft capsules.  detamax d3® by momenta laboratory: a new version in the form of drops and without flavor.  devera® launched in the year 2014 by takeda laboratory: in the form of drops and honey flavor.  tridevit® by zodiac laboratory: it innovated in the pharmaceutical form and launched the first chewable vitamin d tablet isolated with strawberry flavor.  deforce® launched by zydus laboratory: in chewable form and strawberry flavor.  depura® from sanofi lab: in the year 2015 as 1000 iu. figure 3 shows the evolution of the pure vitamin d market consolidated as the largest micronutrient www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 57 published by scholink inc. market in brazil reaching around us$ 100 million in sales in 2015. figure 3. vitamin d—sales in brazil source: ims health, 2016. given this scenario of micronutrients, the national pharmaceutical medium-sized laboratory gross, developed efforts to the possibility of a better repositioning in the market of its products such as adefort drops. this product, having as active principle vitamin d. it was relaunched with a new concept of marketing—totally reworked. the pharmaceutical laboratory had already license to market in anvisa, but it opted for its market repositioning rather than launching it a new vspm (food). it should be noted that the formulation of adefort drops consists of the association of three fat soluble vitamins (vitamins a, d and e). after the implementation of new packaging and distribution to the medical class the product was consolidated along with another vitamin d existing ones. in 2010, the presentation of adeforte drops totaled 22,451 units. the following year, in 2011, sales grew by 133%, representing 52,421 units. already in the year 2012, a total of 130 thousand units and in 2013 about 170 thousand units sold. considering this scenario, the gross laboratory developed the soft capsule form for the already existing formulation of adeforte (vitamins a, d and e), but registering as vspm of pure vitamin d. but, in this way, the concentrations of the soft capsule formulation were adjusted following the rdi limits established by rdc 269/2005 of anvisa. table 1 shows the comparison between the dosages of adeforte drops (medicament) and adeforte capsules (svmf) formulations according to the amounts recommendations to ingest of each micronutrient. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 58 published by scholink inc. table 1. concentration of adeforte® formulations micronutrient drops soft capsules vitamin a 10.000 ui/day 600 ui/day (100% idr) vitamin d 800 ui/day 200 ui/day (100% idr) vitamin e 10 ui/day 10 ui/day (100% idr) in 2013, the sales of soft capsule form exceeded 13 thousand units which corresponds practically to the presentation in drops. only capsule version reached sales in the order of us$ 350,000, us$ 500,000, us$ 700,000 and us$ 1 million from 2013 to 2016 respectively. this scenario of vitamin success for mnd treatment also occurs by other associations with vitamin d. this fact has been observed by market innovation since the launch of addera d3 from the sanofi laboratory, mainly the associations of calcium with vitamin d where its synergistic effect potentiates the absorption of the two substances in the bone mineralization of the human being. 4. conclusions there is a worldwide concern with the topic of hidden hunger where the micronutrients inserted in the daily food can contribute to the resolution of the problem. nevertheless, governmental regulatory actions for the pharmaceutical industries have assisted in the direction of new treatments through specific dosages for both medicaments and the vspm class, as in the case of brazil, through its sanitary regulatory agency, anvisa. national pharmaceutical brazilian laboratories have intensified their efforts in the introduction of new pharmaceuticals in the market, given that the lack of micronutrients is still a very present reality in society. it should be noted that many of these deficiencies are not caused by nutritional problems. in this sense, efforts should be made for new studies that enable mnd treatment with vitamins and minerals for populations in need. it is noted that small repositioning in the portfolio of pharmaceutical industries may favor a new opportunity in the pharmaceutical market share of a given region. this is especially interesting for laboratories that have limited financial resources for high r & d investments. references bailey, r. l., west, k. p. jr., & black, r. e. 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(2013). hidden hunger: addressing micronutrient deficiencies using improved crop varieties. chemistry & biology 20 issue 11. retrieved june 24, 2016, from http://www.sciencedirect.com/science/article/pii/s107455211300392x tulchinsky, t. h. (2010). micronutrient deficiency conditions: global health issues. public health reviews, 32, 243-255. vellozo, e. p., & fisberg, m. (2010). a contribution of the stronguers food (a contribuição dos alimentos fortificados na prevenção da anemia ferropriva—portuguese version). revista brasileira de hematologia e hemoterapia, são paulo, 32(2), 140-147. vieira, v. m. m., & ohayon, p. (2006). innovation in drugs and medicines (inovação em fármacos e medicamentos: estado-da-arte no brasil e políticas de p&d—portuguese version). revista economia & gestão. belo horizonte: puc minas. west, k. p., stewart, c. p., caballero, b., & black, r. e. (2012). nutrition. in m. h. merson, r. e. black, & a. j. mills (eds.), global health: diseases, programs, systems, and policies (3rd ed., pp. 271-304). burlington: jones & bartlett learning. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 2, 2017 www.scholink.org/ojs/index.php/fsns 61 comparative study of starch characteristics, in-vitro starch digestibility and glycemic index of some starchy foods consumed in nigeria israel olusegun otemuyiwa1*, adedayo muideen sanni1 & emmanuel ayorinde oyewumi1 1 department of chemistry, obafemi awolowo university, ile-ife, osun state, nigeria * israel olusegun otemuyiwa, e-mail: otemuyisegun@oauife.edu.ng received: june 25, 2017 accepted: july 8, 2017 online published: july 29, 2017 doi:10.22158/fsns.v1n2p61 url: http://dx.doi.org/10.22158/fsns.v1n2p61 abstract the study investigated the carbohydrate characteristics and in-vitro starch digestibility of some starchy food consumed in nigeria. ten foods samples (cassava, yam, red and white sorghum, rice, plantain, banana, semovita, noodles and bread) were selected. the content of starch, amylose and sugar were determined by colorimetric method, in vitro rate of starch hydrolysis was evaluated by multi-enzyme digestion method over a period of two hours, the glucose released was estimated by colorimetric method and was compared to the reference food (bread). the result showed that the percentage moisture content and total starch ranged from 9.8 to 15.3% and 236 to 248 mg/g, while amylose, rapidly digestible starch (rds) and resistance starch (rs) ranged from 8.41 to 19.2%, 30.8 to 51% and 7.8 to 37.4%, respectively. the in-vitro digestibility study indicated that the equilibrium concentration (cα), kinetic constant (k ), hydrolysis index (hi) and glycemic index (gi) ranged from 34 to 64.9, and 0.02 to 0.07, 56.6 to 104 and 71 to 96.8, respectively. positive correlations (p < 0.05) exist between rds and gi (r = 0.700) and rs and amylose (r = 0.899) the study revealed that, structure of dietary carbohydrate could greatly influenced the glycemic index, plantain and noodles with low rds and low hydrolysis constant may be beneficial in management of diabetes whereas sorghum, semovita, cassava and bread with high rds, and high gi should be taken sparingly or combined with high protein and low glycemic load foods. keywords glycemic index, resistant starch, amylose, in vitro digestion, nigeria 1. introduction in recent years it has become evident that significant health risks and benefits are associated with dietary food choice. a particular disease has been associated with carbohydrate foods with high glycemic index www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 62 published by scholink inc. and high digestibility rate. about 104 millions of people worldwide are being afflicted with the perfect epidemic known as the diabetes. this figure is increasing daily. diabetes meets all criteria for a public health disorder (seal et al., 2003). nutrition is a significant cornerstone of diabetes care as described in intensive management. the main focus in nutritional management of diabetes is to improve glycemic control by balancing food intake with endogenous and/or exogenous insulin level (heacock et al., 2004). historically, attempt has been made to control the glycemic response to food, particularly carbohydrate-containing foods, including use of very low carbohydrate and starvation diets, artificial sweeteners and pharmacological preparations such as fast acting insulin and inhibitors of carbohydrate absorption (heacock et al., 2004). in nigeria, the diet of the people is predominantly carbohydrate obtained from either root tuber or cereal grains. there are varieties of food that were consumed which complement one another without the empirical knowledge of their digestion rate and optimum intake that will give sufficient nutrient intake. foods like carbohydrates for instance should be monitored carefully. there is the need to consider the rate at which these foods digest and be able to predict their glycemic index in order to prevent glucose induced ailment. one way to classify the glycemic response of various carbohydrate-containing foods is glycemic index (gi). the glycemic index (gi) is an in-vitro measurement based on glycemic response to carbohydrate-containing foods. the index allows ranking of carbohydrate foods on the basis of the rate of digestion and absorption (jenkins et al., 1981; englyst et al., 1992). in-vitro method has also been used to classify foods based on their digestion characteristics similar to the in vivo situation, and to identify slow release of carbohydrate in foods (jenkins et al., 1984). the foods with gi values more than 70%, between 56% and 69% and lower than 55% were classified as high, medium, and low gi foods, respectively (brand-miller et al., 2003). the study carried out using human subject by asinobi et al. (2016) to determine the blood sugar response of some traditional fortified staple meals in nigeria concluded that unripe plantain had the lowest glycemic index value with lowest postprandial glucose response. also fasanmade and anyakudo (2007) concluded that yam based food product should be generously used by diabetes patient because of its low glycemic index. these experiments were carried out under in-vivo conditions as such none of the researcher addressed the nature and characteristics of the starch present in foods analysed. the digestibility of starch in foods may vary widely (björck et al., 1994). hence, a nutritional classification of dietary starch has been proposed, which takes into account both the kinetic component and the completeness of its digestibility, thus comprising rapidly digestible (rds), slowly digestible (sds), and indigestible or resistant fractions (rs) (englyst et al., 1992). the objectives of this study were to carry out in-vitro digestibility studies of some starchy staple diets consumed in nigeria, determine the rate of hydrolysis and the starch content characteristics, and also predict the glycemic index. the study would provide an insight into the basic cause of epidemics associated with elevated glucose induced type 2-diabetes among nigerians. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 63 published by scholink inc. 2. materials and methods 2.1 sample and sample preparation the samples selected for this study are yam flour, cassava flour, unripe plantain flour, unripe banana flour, flour of white and red sorghum, semovita, rice, noodles and bread. the samples were dried and milled using a locally fabricated mill (lawood metals, osogbo, nigeria). the milled samples were sieved using a local sieve (aperture size of 0.6 mm) to remove the coarser fragments. all the samples were milled as one batch, mixed thoroughly and sub-samples randomly taken from different parts of each milled sample, mixed together and stored in the freezer until analyzed. 2.2 analysis of proximate composition the proximate composition of the samples (moisture, ash, crude fibre) were determined by the method of aoac (2000). 2.3 determination of total and reducing sugar content soluble sugar was extracted from 2.0 g sample with 85% ethanol using soxhlet extractor and refluxed for 2 h as described by bambridge et al. (1996) reducing sugar and total sugar were determined from the ethanolic extract by the ferricyanide method (aoac, 1984). glucose was used as a standard and the glucose content of the sample was calculated using a linear equation y = 1.6216 0.001x (r2 = 0.972). 2.4 determination of total starch the total starch content of the samples was determined on the residue obtained after ethanolic extraction of sugar. residue (200 mg) was refluxed with 0.7 m hcl for 2.5 h. the acid hydrolysate was neutralized to ph 7.0 using 5.0 m naoh, pour into 500 ml standard flask and made up to volume with distilled water. the hydrolysate was filtered through a whatman no. 541 filter paper and the starch was determined as the reducing sugar using the ferricyanide method (bainbridge et al., 1996). the glucose content was calculated using a glucose standard linear equation and then converted to starch content using the aoac (1984) equation. 2.5 determination of amylose content amylose content in rice samples were determined based on the iodine-binding procedure as described by thomas et al. (2013). the sample (100 mg) was measured into 100 ml standard flask, 1.0 ml of ethanol (95%) and 9.0 ml of 1.0 m naoh were added, the mixture was heated on a boiling water bath for 10 min to gelatinize the starch. 5.0 ml of the gelatinized starch solution was transferred to a 100 ml standard flask, 1.0 ml of 1.0 m acetic acid and 2.0 ml of iodine solution were added and made up to volume with distilled water. all the contents were thoroughly vortex mixed and allowed to stand for 20 min. the absorbance was measured at 620 nm using a uv-spectrophotometer (model aa-6650, shimadzu co. japan). the amylose content was calculated from the standard curve of potato amylose using the linear equation (r2 = 0.899). 2.6 in-vitro starch hydrolysis the in-vitro starch digestibility was determined by multi-enzyme procedure described by deepa et al. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 64 published by scholink inc. (2010). the sample (250 mg) was gelatinized in 10 ml distilled water on a hot plate. the gelatinized sample was homogenized with 10 ml of hcl-kcl buffer (ph 1.5) using a basic homogenizer (kika labortechnik 725, janke and kukel gmbh & co., stanfen germany) at 9500 rpm for 1 min and the homogenate was then digested with 20 mg of pepsin (sigma; cas 2001/75-6, code 10132561, 666 iu/mg, porcine gastric mucosa) solution (prepared by adding 1.0 g of pepsin/10 ml of hcl-kcl buffer) for 1 h in a shaking water bath at 37oc. the ph of the digestate was adjusted to 6.9 and the volume made to 25 ml using tris-maleate buffer (ph 6.9). then 5.0 ml of α-amylase (2.6 iu in 5 ml buffer ph 6.9) was added to the digestate which was incubated at 37oc in a shaking water bath. one ml of sample aliquots was collected at intervals of 30 min for 180 min, the enzyme activity in the aliquot withdrawn was inactivated by immediately placing the tube in a boiling water bath maintained at 100oc for 5 min and then refrigerated till the end of the incubation period, to these aliquots, 3 ml of 0.4 m sodium acetate buffer (ph 4.75) and 60 µl amyloglucosidase (sigma, no;10105-5gf,70 ui/mg. aspegilius niger) were added and incubated at 60oc for 5 min to hydrolyse the starch to glucose. the glucose released was determined using dinitrosalicylic acid (miller, 1959). the concentration of glucose was calculated from the linear equation of glucose standard (r2 = 0.980) and glucose was converted into starch by multiplying with 0.9. all the experiments were conducted thrice and with triplicate analysis each. the rate of starch digestion was expressed as the percentage of ts hydrolyzed at different times. the digestibility curve for each food sample was fitted into the first-order equation (grandfeidt et al., 1992). )1( kt t ecc    where ct is the percentage of starch hydrolyzed at time t (min), c∞ is the equilibrium starch hydrolysis after 180 min, k is a pseudo-first order rate constant. the parameters, k and cα were estimated for each sample based on the data obtained from starch hydrolysis procedure using microsoft excel software. hydrolysis index (hi) was obtained by dividing the area under the hydrolysis curve of the sample by the corresponding area of a reference food (white bread) expressed as a percentage (grandfeidt et al., 1992). glycemic index (gi) was estimated using the equation of goni et al. (1997). )549.0(71.39 higi  2.7 determination of rapidly digestible, slowly digestible and resistance starch the method of han et al. (2007) was used to estimate the rapidly digestible, slowly digestible and resistance starch. the rapidly digestible starch (rds) is the fraction of starch hydrolyzed within 30 min of incubation, slowly digestible starch (sds) is the fraction hydrolyzed between 30 to 180 min and the fraction that remained un-hydrolyzed after 180 min is regarded as the resistant starch. 2.8 statistical analysis analyses were carried out in triplicate for each determination and the results were expressed as mean www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 65 published by scholink inc. and standard deviation. the data were subjected to analysis of variance, and pearson correlation coefficient and the levels of significant difference was performed. graphpad instat version 3.06 for windows 2003 was used for statistical analysis. 3. results and discussion the results of starch and sugar and chemical characteristics of the starch were presented in table 1. the moisture content of the local foods ranged from 8.2 to 15.3%, the highest value was found in yam flour, these values compares with 12 to 14% predicted as optimum moisture content for storage of flour foods and for obtaining quality product during milling (souilah et al., 2014). the fibre content ranged from 0.48% in rice to 4.9% in noodles. the fibre content of rice is expectedly low compared to others, this is because it rice has been subjected to the process of milling and polishing in which the outer layer (bran) containing fibre has been completely removed. though flours of yam, cassava, plantain and sorghum were sieved during processing but still contain high fibre content which could be adduced to pore size of the sieve. diets with a high content of fiber, have a positive effect on health since their consumption has been related to a decreased incidence of several types of diseases as due to its beneficial effects like increasing the volume of fecal bulk, decreasing the time of intestinal transit, lowering cholesterol and glycemic levels, and stimulating the proliferation of the intestinal flora (dingra et al., 2012; souilah et al., 2014). the total and reducing sugar content (table 1) ranged from 5.8 to 25.6 mg/g and 1.2 to 12.1 mg/g, respectively. the sugar content of banana flour was higher (total and reducing sugar) and this could be attributed to glucose release resulting from the activity of endogenous enzymes during processing of the flour the presence of sugar will help improve taste of the food products. the total starch ranged from 222 to 293 mg/g, plantain recorded the highest starch, the starch was not significantly different (p < 0.05) among banana, yam, sorghum and cassava flours. amylose and amylopectin ranged from 8.41 to 26% and 74 to 92.4%, respectively. sorghum and cassava flour recorded the least values for amylose though according to amylose classification in food (juliano et al., 1981), these samples could be categorise as having intermediated amylose content. the implication of this amylose level is that these foods is expected to be soft and not sticky and will not become hard when cooling. heating of starch in the presence of water will lead to gelatinisation that makes starch more easily digested, however after cooling amylose tend to recrystallise and form retrograded amylose which is inaccessible to enzymatic hydrolysis. amylose and amylopectin are important in determining the structure of a carbohydrate food which may have a profound effect on starch digestibility. the mechanism of how an increased amylose/amylopectin ratio affect glycemic response is that linear amylose chain form a compact structure that limit enzyme accessibility and rate of amylosis (halistrom, 2011). amylopectin on the other hand with its branched structure is less ordered and therefore more easily digested. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 66 published by scholink inc. the structural characteristics of digestible starch showed that rapidly digestible starch (rds), slowly digestible starch (sds) and resistant starch (rs) ranged from 31.8 to 55.2%, 8.9 to 40.2% and 7.8 to 37.4%, respectively. flours of cassava, banana, sorghum and bread were higher in rds while low value was reported for plantain and noodles. also the highest value for rs was found in rice (46.5%) followed by noodles and plantain and low values were found in white sorghum and yam flour though yam had high sds. in foods, rs could corresponds to the physically inaccessible starches entrapped in cellular matrix or are native uncooked granules, the crystallinity of which makes them scarcely susceptible to hydrolysis or retrograded starch (englist et al., 1999). rs has also been shown to have positive effects on colonic health by increasing faecal bulk and by generating short chain fatty acids (scfas) such as butyric acid, which is the main energy source for colonocytes and may therefore, have a protective role in inflammatory bowel diseases and colon cancer (hallstrom et al., 2011). several factors can explain the difference found in the resistant starch quantities, some of which are: interaction of starch with different components present in the food system such as proteins, fats; botanical source of starch; and storage conditions (perera et al., 2010). from the results, it was observed that noodles recorded high value for fibre as well as resistant starch this could be from two sources; through enrichment of the ingredient with soluble dietary fibre or through heat processing of starch that lead to formation of retrograded starch which hinders enzymatic hydrolysis of starch (englist et al., 1992). table 1. total starch, rapidly digested starch (rds), slowly digested starch (sds), resistance starch (rs), amylose and amylopectin content of starchy food (expressed as% of total starch) sample moisture total starch rds % sds % rs % amylose % amylopect in % fiber % total sugar mg/g red sugar mg/g plaintain flour 11.3 ± 0.1 c 239 ± 13 c 31.8 ± 1.0 f 19.2 ± 0.4 f 31.4 ± 0.5 c 17.1 ± 0.3 c 82.9 ± 0.3 c 1.3 ± 0.02 b 10.5 ± 0.5 e 8.45 ± 1.4 b banana flour 9.8 ± 0.2 d 242 ± 0.9 b 53.7 ± 0.4 b 8.9 ± 0.5 g 37.4 ± 1.2 b 17.7 ± 0.3 c 82.3 ± 0.3 c 1.4 ± 0.05 b 24.6 ± 0.6 ab 12.1 ± 0.6 a yam flour 15.3 ± 0.3 a 248 ± 1.0 b 44.3 ± 0.2 d 47.9 ± 1.6 a 7.8 ± 0.1 h 14.5 ± 0.2 d 85.5 ± 0.2 b 3.22 ± 0.8 a 21.1 ± 1.4 b 1.45 ± 0.5 e red sorghu 12.7 ± 1.1 b 246 ± 0.9 b 51.6 ± 0.6 bc 33.3 ± 0.2 c 15.1 ± 0.6 f 14.4 ± 0.3 d 85.6 ± 0.3 b 2.29 ± 0.09 a 15.6 ± 0.26 d 2.6 ± 0.07 d white sorghum 13.1 ± 0.6 b 241 ± 10 c 49.4 ± 0.2 c 40.2 ± 1.5 b 10.4 ± 0.2 gh 8.41 ± 0.7 e 92.4 ± 0.5 a 2.0 ± 0.07 ab 17.1 ± 0.22 c 2.2 ± 0.07 d cassava flour 12.5 ± 1.0 b 248 ± 2.0 b 55.2 ± 1.0 a 22.6 ± 0.1 e 22.2 ± 0.4 e 12.8 ± 1.1 d 87.8 ± 2.0 b 3.6 ± 0.3 a 10.08 ± 0.05 e 1.83 ± 0.05 e rice 8.2 ± 0.3 e 222 ± 0.14 c 38.0 ± 0.3 e 16.5 ± 0.2 f 46.5 ± 0.9 a 26.0 ± 0.8 a 74.0 ± 0.6 e 0.48 ± 0.01 c 10.9 ± 0.02 e 1.17 ± 0.13 e semovita 10.2 ± 1.2 cd 237 ± 8.0 c 43.8 ± 0.3 d 27.9 ± 1.4 d 28.3 ± 1.0 d 17.4 ± 0.2 c 82.6 ± 0.2 c 2.4 ± 0.01 a 25.6 ± 0.03 a 2.71 ± 0.1 d noodles 12.4 ± 0.8 b 236 ± 5.0 c 30.8 ± 0.8 f 35.4 ± 0.8 c 33.8 ± 0.6 c 19.2 ± 0.6 b 80.8 ± 4.0 d 4.9 ± 0.9 a 5.8 ± 0.62 f 5.3 ± 0.007 c bread 10.6 ± 0.3 cd 293 ± 5.0 a 49.0 ± 1.0 c 21.0 ± 1.0 ef 30.0 ± 2.0 c 21.0 ± 0.2 b 79.0 ± 0.8 d 1.2 ± 0.02 b 17.8 ± 0.67 c 6.53 ± 0.5 b note. mean ± sd—mean and standard deviation of triplicate analysis. values in the same column with same superscript are not significantly different p ≤ 0.05. 1 rapidly digestible starch; 2 slowly digestible starch; 3 resistance starch. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 67 published by scholink inc. the results of in-vitro digestibility and the kinetic parameter were presented in figure 1 and table 2, the results indicated that the equilibrium concentration (cα), kinetic constant (k), hydrolysis index (hi) and glycemic index (gi) ranged from 34 to 64.9, and 0.02 to 0.07, 56.6 to 104 and 71 to 96.8, respectively. with the exception of yam flour and noodles that recorded low hydrolysis index, all sample had both high hydrolysis and glycemic index. correlation coefficient (table 3) showed that there is a positive correlation hi and rds (r = 0.700), gi and rds (r = 0.701) whereas fibre was negatively correlated with gi (r = -0.624) and hi (r = -0.628). figure 1. starch hydrolysis curve of some starch foods in nigeria table 2. kinetic parameters of in-vitro starch hydrolysis of starchy foods * white bread—control sample. mean and standard deviation of triplicate analysis. cα-equilibrium concentration, k-rate constant, auc-area under curve, hi-hydrolysis index, gi-glycemic index. values within the same column with different superscripts are significantly different at p ≤ 0.05. sample cα k auc hi(%) gi (%) plaintain flour 44.7 ± 1.0d 0.03 ± 0.001c 6606 ± 12c 73.01 ± 0.9d 79.8 ± 2.1c banana flour 60.0 ± 3.0b 0.07 ± 0.002a 9442 ± 22a 104 ± 10a 96.8 ± 1.1a yam flour 43.7 ± 0.8 d 0.02 ± 0.001d 5911 ± 10g 65.32 ± 0.8e 75.6 ± 1.2d semovita 55.2 ± 0.7c 0.05 ± 0.001b 8800 ± 2.9d 97.2 ± 0.5c 93.0 ± 2.8b white sorghum flour 61.9 ± 0.1ab 0.07 ± 0.001a 9014 ± 23c 99.6 ± 1.4b 94.4 ± 3.0ab red sorghum flour 64.9 ± 2.0a 0.07 ± 0.004a 9113 ± 25b 100 ± 6.0b 94.8 ± 4.0a cassava flour 60.0 ± 0.6b 0.07 ± 0.009a 9403 ± 15a 104 ± 0.7a 96.8 ± 1.2a rice 58.6 ± 0.5c 0.03 ± 0.001c 8603 ± 9.0e 95.1 ± 2.6c 91.9 ± 0.5b noodles 39.0 ± 0.4e 0.02 ± 0.002d 5123 ± 8.5h 56.6 ± 0.8f 71.0 ± 0.8e bread* 54.5 ± 0.3c 0.07 ± 0.001a 9048 ± 12h 100 ± 0.0b 94.6 ± 0.3ab www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 68 published by scholink inc. the kinetic constant k of amylolysis has been proposed as a reliable index of the inherent susceptibility of flour starches to amylase hydrolysis (goni et al., 1997; frei et al., 2003). from the results, the hydrolysis constant of plantain flour, yam flour and noodles is low (k = 0.02), the content of rsd was lower and rs was higher than in bread, this implied that the rate at which they will digest may take a longer time which may not adversely affect the blood sugar. this observation agrees with the report from in-vivo study that yam and unripe plantain had low glycemic index (fasanmade & anyakuro, 2007; asinobi et al., 2016). banana flour unlike plantain recorded high hydrolysis constant (k = 0.07) which is the same as bread, the flour recoded high rds and sugar content which may result from enzymatic degradation (endogenous enzymes ) of starch that led to ripening. sorghum flour (red and while cultivars) also recorded high hydrolysis rate which is higher than bread. the hydrolysis constant in cassava flour also did not differ from that of bread, processing of cassava into flour involve grating, soaking and fermentation to make pulp free of cyanide (a toxic compound that is lethal), during fermentation starch is broken down by enzymes an aerobically to sugar which is rapidly released when it is consumed as food. the glycemic index is even higher than white bread (reference food) from this study, it was discovered that the glycemic index of cassava flour, banana flour, semovita and sorghum flour (red and white cultivars) were higher whereas those of plantain flour, yam flour and noodles were lower compared to bread which was taking as standard high glycemic index food. 4. conclusion the study revealed that starchy foods which are staple diets among nigerians are high glycemic load foods and that structure of dietary carbohydrate could greatly influenced the glycemic index of the foods, therefore, foods like plantain and noodles with low rds and low hydrolysis constant may be beneficial in management of diabetes whereas sorghum, semovita, cassava and bread which contain high content of rapidly digestible starch should be taken sparingly. hence, in order to effectively reduce the high incidence of type 2-diabetes, these foods should be complimented with high protein sources. references aoac. (2000). official methods of analysis, association of official analytical chemists (aoac). washington dc. asinobi, c., uzoagba, h., mba-anyadioha, a., & johnkennedy n. (2016). glycemic index of some traditional fortified staple meals on the postprandial blood glucose responses of nigerian undergraduate students: an open-label study. functional foods in health and disease, 6(7), 414-424. bainbridge, z., tomlins, k., wellings, k., & westby, a. (1996). method of assessing quality charactristics of non grain starch staples part 3, laboratory methods, natural resources www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 69 published by scholink inc. institute chatham, uk. björck, i. m., granfeldt, y., liljeberg, h., tovar, j., & asp, n. g. (1994). food properties affecting the digestion and absorption of carbohydrates. american journal of clinical nutrition, 59, 699s-705s. brand-miller, j., wolever, t. m. s., foster-powell, k., & colagiuri, s. (2003). the new glucose revolution (2 nd ed.). marlowe & company, new york, ny. deepa, g., singh, v., & maidu, k. a. (2010). comparative study on starch digestibility glycemic index and resistance starch of pigmented (“njavara” and “jyothi”) and a non pigment (“ir 64”) rice varieties. journal of food science and technology, 47(6), 644-649. https://doi.org/10.1007/s13197-010-0106-1 dhingra, d., michael, m., rajput, h., & patil, r. t. (2012). dietary fibre in foods: a review. journal of food science and technology, 49(3), 255-266. https://doi.org/10.1007/s13197-011-0365-5 englyst, h., kingman, s., & cummings, j. (1992). classification and measurement of nutritionally important starch fractions. european journal of clinical nutrition, 46, s33-s50. englyst, k. n., englyst, h. n., hudson, g. j., cole, t. j., & cummings, j. h. (1999). rapidly available glucose in foods: an in vitro measurement that reflects the glycemic response. american journal of clinical nutrition, 69, 448-454. fasanmade, a. a., & anyakudo, m. c. (2007). glycemic indices of selected nigerian flour meal products in male type 2 diabetic subjects. diabetologia croatica, 36-42. frei, m., siddhuraju, p., & becker, k. (2003). studies on the in vitro starch digestibility and glycemic index of six different indigenous rice cultivars from the philippines. food chemistry, 83, 395-402. https://doi.org/10.1016/s0308-8146(03)00101-8 goni, i., garcia-alonsa, a., & saura-calixto, f. (1997). a starch hydrolysis procedure to estimate glycemic index. nutrition research, 17, 427-437. https://doi.org/10.1016/s0271-5317(97)00010-9 grandfeidt, h. n., veenstra, j., & hudson, g. j. (1992). measurement of rapidly available glucose (rag) in plant foods: a potential in in-vitro predictor of the glycemic responses. british journal of nutrition, 46, 649-660. hallstrom, e., sestili, e. f., lafiandra, d., bjorck, l., & ostman, e. (2011). a novel wheat variety with elevated content of amylose increases resistant starch formation and may beneficially influence glycaemia in healthy subjects. food and nutrition research, 55, 70-74. https://doi.org/10.3402/fnr.v55i0.7074 han, x. z., ao, z., janaswamy, s., jane, j.-l., chandrasekaran, r., & hamaker, b. r. (2006). development of a low glycemic maize starch: preparation and characterization. biomacromolecules, 7, 1162-1168. https://doi.org/10.1021/bm050991e heacock, p. m., hertzler, s. r., & wolf, b. (2004). the glycemic, insulinemic, and breath hydrogen responses in humans to a food starch esteriwed by 1-octenyl succinic anhydride. nutrition research, 24, 581-692. https://doi.org/10.1016/j.nutres.2003.10.015 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 70 published by scholink inc. jenkins, d. j. a. et al. (1981). glycemic index of foods: a physiological basis for carbohydrate exchange. american journal of clinical nutrition, 34, 362-366. jenkins, d. j. a. et al. (1984). the relationship between glycemic response, digestibility and factors influencing the dietary habits of diabetics. american journal of clinical nutrition, 40, 1175-1191. juliano, b. o., perez c. m., & blackney, a. b. (1981). international cooperative testingon the amylose content of milled rice. starch, 33(5), 157-182. https://doi.org/10.1002/star.19810330504 miller, g. i. (1959). use of dinitrosalicylic acid reagent for determination of reducing sugar. analytical chemistry, 51, 126-129. https://doi.org/10.1021/ac60147a030 perera, a., meda, v., & tyler, r. t. (2010). resistant starch: a review of analytical protocols for determining resistant starch and of factors affecting the resistant starch content of foods. food research international, 43, 1959-1974. https://doi.org/10.1016/j.foodres.2010.06.003 seal, c. j., daly, m. e., thomas, l. c., bal, w., birkett, a. m., jevcoat, r., & mathers, j. c. (2003). postprandial carbohydrate metabolism in healthy subjects and those with type 2 diabetes fed starches with slow and rapid hydrolysis rates determined in-vitro. british journal of nutrition, 90, 863-864. https://doi.org/10.1079/bjn2003972 souilah, r., djabali, d., belhadi, b., mokrane, h., boudries, n., & nadjemi, b. (2014). in vitro starch digestion in sorghum flour from algerian cultivars. food science and nutrition, 2(3), 251-259. https://doi.org/10.1002/fsn3.104 thomas, r., wan-nadiah, w. a., & rajeev, b. (2013). physiochemical properties, proximate composition, and cooking qualities of locally grown and imported rice varieties marketed in penang, malaysia. international food research journal, 20(3), 1345-1351. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 3, 2019 www.scholink.org/ojs/index.php/fsns 96 original paper sugar spectra of syrups produced from different tuber starches via crude enzymes and amyloglucosidase sources okafor d.c.1*, agunwah i.m.1, ezegbe c.c.2, ekeoma c.l.1 & onuegbu n.c.1 1 department of food science and technology, federal university of technology, owerri, imo state, nigeria 2 department of food science and technology, faculty of agriculture, nnamdi azikiwe university awka, anambra state, nigeria * okafor d.c., department of food science and technology, federal university of technology, owerri, p. m. b. 1526 owerri, imo state, nigeria received: november 12, 2018 accepted: november 24, 2018 online published: august 19, 2019 doi:10.22158/fsns.v3n3p96 url: http://dx.doi.org/10.22158/fsns.v3n3p96 abstract syrup production was done via enzyme hydrolysis. enzymes used were crude enzymes from malted sorghum, wheat and millet and exogenous enzyme by name amyloglucosidase (amg) which hydrolyzed cassava (manihot esculenta,), water yam (dioscorea alata) and potato white (ipomoea batatas l) starches. syrup sugars were determined using high performance liquid chromatography (hplc) and the sugar profile found are fructose; glucose, sucrose, maltose, d-xylose, and d-raffinose which manifested as a result of the interaction between starches and enzymes. the sugar fructose was in the range of 17.34 ± 0.651 g/l to 28.16 ± 0.982 g/l, glucose sugar was in the range of 6.09 ± 0.165 g/l to 177.04 ± 1.229 g/l. the highest glucose yield (177.04 ± 1.229 g/l) was observed in cassava starch reaction with the commercial enzyme –amg. sucrose content was in the range of 5.78 ± 0.180 g/l to 21.59 ± 0.536 g/l, maltose (23.71 ± 0.125 g/l to 48.04 ± 0.125 g/l) was the most predominant sugar in all syrups gotten from the starch and crude enzymes interaction. the hydrolysis of starches using different enzyme sources yielded sugar spectra of different sugars concentrations with each starch source predisposed to the natural activity of the enzyme peculiar to their variety or cell structure. d-xylose and d-raffinose were in the range of 0.004-0.225 g/l which is very small in quantity compared to other sugars seen while no d-stachyose was detected. keywords amyloglucosidase, crude, enzymes, hydrolysis, starches, sugars www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 97 published by scholink inc. 1. introduction root and tuber crops are among the most important group of food crops in many tropical african countries. in nigeria for instance, cassava (manihot esculenta) is the most important of these crops in terms of total production, importance and economic value (okoye et al., 2008). cocoyam (colocasia esculenta), which belongs to the araceae family, ranks third after cassava and yam (onyenweaku & okoye, 2007). according to a report by ogunniyi (2008), nigeria is the world’s largest producer of cocoyam, accounting for about 40% of total world output as recorded by the food and agriculture organization in 2007 (fao, 2007). despite this, nigeria and other developing nations are beset by the problem of lack of proper storage facilities for these tubers and as such, a large number of these tubers in the order of millions of tons are destroyed through pest infestation, deterioration, physical damage to the tubers, pilfering etc. (omemu et al., 2005). in order to recover the losses resulting from these wastages, it is important to expand the processing range of these tubers with particular focus on converting them into value-added products. one of the processing methods that can reduce wastage of these tubers is converting them into syrups for multipurpose usages and applications in food and beverage industries, as well as in the pharmaceutical industries. again, with the demand on cane sugar as a result of increasing need by the teeming population and industries who seek to satisfy nutritional and commercial requirements, there is deficient supply of cane sugar as the quantity produced in the country cannot meet up with the increasing demand. hence the need to source for alternative sources of sugar and this is what syrup production provides. however, over the years, commercially produced enzymes have been used for syrup production which not only is expensive and not readily accessed or available in nigeria and other tropical countries but also very difficult to preserve because they are heat labile. hence this paper is geared towards producing syrup using an alternative source of enzyme other than the exogenous source. the main objectives of this paper is to produce sugar syrups by enzyme hydrolysis with exogenous and endogenous enzymes [amyloglucosidase (amg) and crude enzymes from millet, sorghum and wheat respectively] and to determine the sugar spectra present in the syrups produced using high performance liquid chromatography (hplc). the quality and state of this paper is new, different and interesting in that it will help us to ascertain the ability of tropical malted grains such as wheat, millet, and sorghum in hydrolyzing the starches extracted from tropical tubers and roots (cassava, water yam, and potato) and the extent of their hydrolysis as when compared to the action of the exogenous enzyme (amyloglucosidase) hydrolysis on these starches. it will advance the knowledge of crude enzymes in the field of food science and technology. 2. materials and methods 2.1 raw material collection the cassava (manihot exculenta) tuber known as tme 419, yam (dioscorea alata ) tuber commonly known as water yam, and potato white (ipomoea batatas l), white colored sorghum (sorghum vulgare) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 98 published by scholink inc. with varietal name fara-fara, millet (pennisetum glaneum) known as ex borno and wheat (triticum eastivum) ex jos used were gotten from the department of crop science michael okpara university of agriculture umudike, abia state, nigeria. the equipment and some chemicals used for this research were all of analytical standards gotten from the department of food science and technology, federal university of technology owerri, imo state and the international institute of tropical agriculture (iita) ibadan, oyo state, nigeria. the exogenous enzyme amyloglucosidase used was gotten from novo nordisk of denmark. the solvents used for hplc analysis are hplc grade and are gotten from e-merck (darmstadt, germany). other chemicals are also of analytical grade and are gotten from sigma aldrich (steinheim, germany). 2.2 preparation of starch the method described by ige and akintunde (1981) was used. starch extraction was carried out from the fresh cassava tubers. the tubers were washed, peeled and milled into slurry. the slurry was properly stirred and allowed to settle for about 6 hours. after settling of the cassava slurry, a heterogeneous mixture was observed, the top part o of the mixture was a transparent liquid and the bottom part was a white thick liquid which is starch. the supernatant was decanted and the sediment which contains the starch was filtered with muslin cloth and oven-dried at 45-55ºc for 30 minutes to produce the dry starch, which was later milled to produce a fine powder. this procedure was followed in preparing starch from potato roots and water yam tubers 2.3 production of crude enzymes by malting the grains [fara fara (sorghum vulgare, a variety of guinea corn) ex -borno (pennisetum glaneum a variety of millet) and ex -jos (triticum eastivum a variety of wheat)], enzymes were developed in them. these enzymes are crude enzymes. the malting was prepared according to the method described by subramanian et al. (1992). the grains were carefully sorted to separate dirt, stones and other contaminants. the cleaned grains were soaked in water at a grain to water ratio of 1:3 the grains were washed and soaked/steeped separately in portable water for 24 hours and the water were changed every 6 hours interval to undergo air rest of 10 mins. they were spread on a jute bag in a room for the commencement and completion of sprouting at an ambient temperature (35ºc). 250 ml of water for every 500 g of grain was sprinkled every 6hours interval on the grains to enhance sprouting (figure 1). it was germinated for 24 hours before the emergence of grain rootlets after which they were dried in an oven at 45ºc to stop the germination. dry milling was done using a machine and was sieved to a fineness of flour using a 5.3 um aperture size sieve to get the malted cereal which contains the crude enzymes developed during malting of the grains. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 99 published by scholink inc. figure 1. flow diagram for production of malted grains (wheat, millet and sorghum) 2.4 procedure for syrup production using exogenous enzyme application (amyloglucosidase) the method described by osuji and anih (2011) and okafor et al. (2018) was used. the mash water to be used for the syrup production was prepared to a ph of 11 with aid of calcium hydroxide. 20 grams of the starch samples were weighed respectively into clean pots. slurry was made by adding 250 ml of the mash water respectively into the weighed starches. the temperature of the slurries was raised to 45ºc, after which 20 grams of the amylogucosidase enzyme was added to each of the slurries, the slurries were stirred and maintained at that temperature for 20 minutes. temperature was raised to 55ºc, they were stirred and allowed to rest for 10 minutes. iodine tests were carried out by adding 2 drops of iodine to a few drops of the samples on a ceramic tile. the temperature of the slurries was raised to 65ºc and maintained for 1 hour. another iodine tests were carried out. temperatures were further raised to 90-93ºc and maintained for another 1 hour. the slurries were then boiled for 5 minutes, after which iodine tests were carried out. the samples were then cooled to 60ºc by placing them in an ice water bath. the ph of the samples was checked. after hydrolysis, the liquors were boiled for 10 minutes to denature enzymes. the converted slurries were then filtered across a double-layered muslin cloth. the samples were then evaporated and concentrated through evaporation using a water bath, and then packaged. 2.5 procedure for syrup production using crude enzyme application from malted cereals the method described by okafor et al (2018 and 2019) was used. the mash water to be used for the syrup production was prepared to a ph of 11 with aid of calcium hydroxide. 20 grams of the starch samples were weighed respectively into clean pots. slurry was made by adding 250ml of the mash www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 100 published by scholink inc. water respectively into the starches. the temperature of the slurries was raised to 45ºc, after which 3 grams of the malted grains respectively was added to each of the slurries, the slurries were stirred and maintained at that temperature for 20 minutes. temperature was raised to 55oc, they were stirred and allowed to rest for 10 minutes. iodine tests were carried out by adding 2 drops of iodine to a few drops of the samples on a ceramic tile. the temperature of the slurry was raised to 65ºc and maintained for 1 hour. another set of iodine test were carried out. temperatures were further raised to 90-93ºc and maintained for another 1 hour. the slurries were then boiled for 5 minutes, after which iodine tests were carried out. the samples were then cooled to 60ºc by placing them in an ice water bath. the ph of the samples was checked, after hydrolysis, the liquors were boiled for 10 minutes to denature enzymes. the converted slurries were then filtered across a double-layered muslin cloth. the samples were then evaporated and concentrated through evaporation using a water bath, and then packaged. this same procedure was repeated for 6, 9, 12, 15 and 20 grams of the malted cereals respectively on each of the three starches. 2.6 determination of brix level the apparent brix was determined using a portable digital handheld refractometer (vbr32t bellingham and stanley uk brix/atc 0-32%). the digital refractometer was cleaned with a clean wiper and standardized with distilled water at 20ºc until the brix value reads zero. two drops of syrup sample at 20ºc was dropped on the lens (sensitive surface) of the refractometer and measured. the syrups with the highest brix level were chosen for the sugar spectra analysis using high performance liquid chromatography. 2.7 determination of sugar spectra with high perfomance liquid chromatography using refractive index detector (hplc-ri) determination and quantification of specific sugars was performed according to method of zielinski et al. (2014) using hplc with refractive index detector (waters e2695, usa; 2414 ri detector) with sugar analysis 300 x4.6mm column. the analysis was performed at 35ºc with a flow rate of 1 ml min-1using isocratic elution with 75% acetonitrile (acn): 25% water (h2o) mixture as a mobile phase. all samples were centrifuged at 4,000 rpm for 10 min (hermle labnet z323) and the supernatant was filtered through a 0.22 μm pes membrane (sartorius stedim biotech, germany). then the filtrate was diluted 10 times before direct injection into the hplc. the sugars seen and detected were recorded. 2.8 statistical analysis data were expressed as mean ± standard deviation (sd) from two parallel measurements. the analysis of variance (anova) and least significant difference (lsd) were performed with the spss 20.0 software (spss inc.) significance difference was defined at ɵ = 0.05. 3. results and discussion 3.1 brix reading of syrups produced with crude enzymes increase in the brix level of the syrups confirms that there was an interaction between the starches and enzymes. from the refractometer readings at different concentration for each of the crude enzyme used, www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 101 published by scholink inc. it was observed that the potato white starch with millet crude enzyme at all concentrations gave the highest brix level (table 1). all other starch sources (cassava and water yam starches) on the millet crude enzyme gave their highest degree brix at 15 grams concentration. when sorghum crude enzyme was used in the hydrolysis, cassava starch was the only starch that gave a reading of 3.0 0bx at 0gram concentration of the crude enzyme as against all starch sources that gave zero reading at the 0gram concentration of the sorghum crude enzyme. cassava had degree brix level of 3.0 without any enzyme treatment this can be attributed to the variety used and other substances in the multi component matrix in which the starch occurs naturally as the gelatinized starch has lost its crystallinity thereby accelerating its hydrolysis. also, since this variety tme 419 gives higher tonnage yield and higher starch content as opposed to other cassava varieties which are lower in starch content, it is possible that some hydrolysis took place by inherent enzyme in the peel as starch extraction processes took place (tester et al., 2004). cassava, water yam and potato white starches gave their highest brix level of 16.0, 13.5 and 15.0 respectively at the 15 grams concentration of the sorghum crude enzyme treatments (table 1). for wheat enzyme treatments, potato white starch set the pace at all concentrations of the crude enzyme giving its highest degree brix level at 15 grams concentration of the crude enzyme. all starch sources when treated with different crude enzymes gave their best/highest degree brix readings at 15 grams concentration of the crude enzyme (table 1). as a result of the high brix level at this concentration, only syrups produced at the 15 gram concentration of the crude enzyme was subjected to sugar spectra analysis using hplc. table 1. brix reading of syrups produced with crude enzyme application at different concentrations for different starch sources sources of enzymes sources of carbohydrates quantity of crude enzymes applied (grams) 0.0 3.0 6.0 9.0 12.0 15.0 20.0 millet cassava starch 0.0 7.0 9.0 12.0 13.6 15.40 15.30 water yam starch 0.0 9.5 11.3 12.5 13.5 15.0 14.8 potato white starch 0.0 10.5 11.8 13.8 15.5 18.5 18.0 sorghum cassava starch 3.0 9.5 11.9 13.5 14.5 16.0 15.8 water yam starch 0.0 7.4 8.1 10.5 11.7 13.5 13.10 potato white starch 0.0 8.5 9.8 11.5 13.7 15.0 14.8 wheat cassava starch 0.0 6.3 8.5 10.7 12.6 14.7 14.0 water yam starch 0.0 8.5 9.8 10.6 11.3 12.8 12.4 potato white starch 0.0 11.3 12.6 15.0 19.0 25.0 24.8 3.2 sugar spectra of syrups as a result of effect of only starch source interaction for fructose, interaction between the starches showed that, water yam starch gave the highest mean yield of fructose (11.95 g/l) followed by cassava starch (7.55 g/l) while potato white starch gave the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 102 published by scholink inc. least fructose yield (5.78 g/l) irrespective of the enzymes used. for glucose, cassava starch gave the highest mean yield of glucose (48.47 g/l) followed by potato white starch (9.69 g/l) with water yam starch giving the lowest yield (3.80 g/l). for sucrose, potato white starch gave the highest mean yield of sucrose (11.01 g/l), followed by water yam starch (6.41 g/l) with cassava starch given the least yield for sucrose (3.16 g/l). for maltose, potato white starch gave the highest mean yield of maltose (26.09 g/l), followed by water yam starch (20.05 g/l) with cassava starch given the least yield for maltose (17.14 g/l). for d-xylose, potato white starch gave the highest mean yield (0.0082 g/l), followed by water yam starch (0.0079 g/l) with cassava starch given the least mean yield for d-xylose (0.0047 g/l). for d-raffinose, potato white starch gave the highest mean yield (0.1218 g/l), followed by cassava starch (0.0400 g/l), with water yam starch given the lowest mean yield for d-raffinose (0.0198 g/l) shown in table 2. different sugars at variable concentrations indicated interaction that took place as a result of the inherent properties (genetic makeup) of the different starch sources table 2. effect of starch source interaction on sugar spectra in grams per liter (g/l) starch source fructose glucose sucrose maltose d-xylose d-raffinose d-stachyose cs 7.5514 48.4675 3.1639 17.1416 0.0047 0.0400 0.0000 wys 11.9484 3.8013 6.4148 20.0471 0.0079 0.0198 0.0000 pws 5.7823 9.6994 11.0100 26.0931 0.0082 0.1218 0.0000 means with different alphabet superscript along columns are significantly different (p≥0.05) key: cs = cassava starch, wys = water yam starch, pws = potato white starch 3.3 sugar spectra from enzyme interaction only when the enzymes interaction only is considered without paying attention to the starch source, millet enzyme gave the highest total yield of fructose with mean yield of 14.76 g/l, followed by sorghum enzyme (8.76 g/l), wheat (5.14 g/l) and amg enzyme with the lowest mean yield of 5.05 g/l. amg enzyme gave the highest total yield of glucose (59.65 g/l) followed by millet enzyme (10.03 g/l), wheat (6.68 g/l) with sorghum enzyme with the lowest mean yield of 6.27 g/l. the wheat enzyme gave the highest total yield of sucrose (9.24 g/l), followed by millet enzyme (9.20 g/l), amg (5.96 g/l) with sorghum enzyme giving the least yield (3.05 g/l). the wheat enzyme gave the highest total yield of maltose (28.53 g/l), followed by millet enzyme (20.37 g/l), sorghum (18.37 g/l) and amg enzyme gave the lowest mean yield of 17.10 g/l. the amg enzyme gave the highest total yield of d-xylose (0.0093 g/l), followed by wheat enzyme (0.0073 g/l), sorghum (0.0063 g/l) with millet enzyme being the lowest (0.0047 g/l). the millet enzyme gave the highest total mean yield of d-raffinose (0.0830 g/l), followed by wheat enzyme (0.0830 g/l), amg (0.0463) while sorghum enzyme gave the lowest mean yield of 0.0273 g/l (table 3). the ability of this enzyme sources to liberate this sugar can be attributed to their diastatic power resulting from the amount of diastatic enzymes present in the malted grains which varies between the grains (john et al., 1998). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 103 published by scholink inc. table 3. effect of only enzyme interaction on the sugar spectra in grams per liter (g/l) enzyme source fructose glucose sucrose maltose d-xylose d-raffinose d-stachyose amg 5.0552 59.6484 5.9563 17.1053 0.0093 0.0463 0.000 millet 14.7637 10.0264 9.2048 20.3677 0.0047 0.0853 0.000 sorghum 8.7552 6.2730 3.0491 18.3693 0.0063 0.0273 0.000 wheat 5.1354 6.6763 9.2414 28.5333 0.0073 0.0830 0.000 key: amg = amyloglucosidase 3.4 sugar spectra as shown by the interaction of both enzyme and starch sources when the interaction of both enzyme and starch sources are considered together, the sugars spectra are affected. water yam starch and millet gave the highest mean yield of fructose (28.16 g/l), followed by the interactive effect of cassava starch and sorghum with mean yield of 17.34 g/l while potato white starch and wheat gave the lowest yield (10.49 g/l). the interactive effect between cassava starch and amg enzyme gave the highest mean yield of glucose (177.04 g/l), followed by that of potato white starch and wheat enzyme with mean yield of (17.11 g/l), with water yam starch and millet giving the lowest glucose yield of 6.09 g/l. the interactive effect between potato white starch and wheat gave the highest yield of sucrose (21.59 g/l), followed by the interaction of water yam starch and amg enzyme with yield of 13.9 g/l whereas cassava starch and wheat gave the lowest yield of 4.12 g/l. the interaction between potato white starch and wheat enzyme gave the highest yield of maltose (48.04 g/l), followed by the interaction effect between water yam starch and sorghum enzyme yielding (32.30 g/l), with cassava starch and amg enzyme giving the lowest maltose yield (24.56 g/l). the interactive effect between potato white starch and amg enzyme gave the highest yield of d-xylose (0.018 g/l), followed by the interaction of water yam starch and wheat enzyme yielding (0.0127 g/l), with cassava starch on all enzyme sources giving the lowest yield of 0.0047 g/l. the interaction effect between cassava starch and amg enzyme gave the highest mean of d-raffinose yield (0.0920 g/l), followed by the interaction effect between water yam starch and sorghum enzyme with mean yield (0.0390 g/l), while potato white starch and wheat enzyme gave the lowest yield of 0.2250 g/l. for fructose yield, there is no significant difference between cassava starch on wheat and water yam starch on amg enzyme as, likewise no significant difference exists between water yam starch on wheat and potato white starch on sorghum. other starch sources and enzymes are significantly different with respect to yield of fructose sugar. for glucose yield, cassava starch on millet and water yam starch on wheat are not significantly different, also potato white starch on millet and sorghum are not significantly different but all other starch source on all other enzymes are significantly different with regards to the glucose yield. for sucrose yield, water yam starch on millet, sorghum and wheat enzyme are not significantly different. all other starches on all other enzyme sources are significantly different. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 104 published by scholink inc. for maltose yield, all starch sources on all enzyme sources are not significantly different. for d-xylose yield, cassava starch on amg, millet, sorghum, and wheat enzymes are not significantly different, alongside water yam starch on amg enzymes, and potato white starch on millet, sorghum and wheat while water yam starch on millet, sorghum, wheat and potato white starch on amg are significantly different from all other starches on all other enzymes sources but are not significantly different in their yield of d-xylose. for d-raffinose yield, cassava starch on wheat and water yam starch on amg are not significantly different. cassava starch on sorghum and potato white starch on millet, sorghum and wheat are not significantly different. cassava starch on millet and potato white starch on amg are not significantly different. potato white starch on millet and sorghum are not significantly different, but cassava starch on amg and potato white starch on wheat are significantly different from among all the starch sources and enzymes as shown in table 4. the discussions agree with the results of the study conducted by other researchers. in fact, osuji and okafor (2013), okafor et al. (2019) have worked on soymilk and revealed the existence of significant differences among the sugar content of soymilk after different enzyme treatment using hplc. among the sugars identified include glucose, fructose, maltose, raffinose, xylose and stachyose. the presence of stachyose as identified in their work can be attributed to the action of the cell wall degrading enzyme used as opposed to this research where stachyose was not identified because the hydrolysis was mainly as a result of oligosaccharide degrading enzymes. table 4. the sugar spectra of the interactive effect of enzymes and starches (g/l) starch enzyme fructose glucose sucrose maltose d-xylose d-raffinose d-stachyose cs amg 0.1270d ± 0.010 177.0383a ±1.229 3.1607h ± 0.055 24.5643h ± 0.125 0.0047b ± 0.006 0.0920d ± 0.004 0.000 millet 11.1170f ± 1.210 6.9790h ± 0.069 1.3996g ± 0.005 13.5533k ± 0.125 0.0047b ± 0.006 0.0160b ± 0.004 0.000 sorghum 17.3413b ± 0.651 8.9627e ± 0.061 3.9877j ± 0.044 16.5993l ± 0.125 0.0047b ± 0.006 0.0390e ± 0.004 0.000 wheat 1.6203c ± 0.451 0.8900i ± 0.014 4.1077e ± 0.105 13.8493i ± 0.125 0.0047b ± 0.006 0.0130f ± 0.004 0.000 wys amg 14.8217c ± 0.563 1.0910f ± 0.020 13.9087f ± 0.128 23.5833f ± 0.125 0.0047b ± 0.006 0.0140f ± 0.004 0.000 millet 28.1590a ± 0.982 6.0930j ± 0.165 5.7837h ± 0.180 0.5873d ± 0.125 0.0047a ± 0.006 0.0150e ± 0.004 0.000 sorghum 1.5130j ± 0.265 5.9940g ± 0.025 3.9430h ± 0.128 32.3033j ± 0.125 0.0097a ± 0.006 0.0390e ± 0.004 0.000 wheat 3.3000h ± 0.159 2.0270h ± 0.097 2.0237i ± 0.103 23.7143g ± 0.125 0.0127a ± 0.006 0.0110e ± 0.004 0.000 pws amg 0.2170g ± 0.003 0.8160c ± 0.075 0.7998b ± 0.004 3.1683a ± 0.125 0.0187a ± 0.006 0.0330b ± 0.004 0.000 millet 5.0150i ± 0.265 17.1120d±0.389 20.431c ± 0.527 46.9623c ± 0.125 0.0047b±0.006 0.2250a±0.004 0.000 sorghum 7.4113h ± 0.751 3.8623d ± 0.064 1.2167a ± 0.093 6.2053b ± 0.125 0.0047b ± 0.006 0.0040a ±0.0040 0.000 wheat 10.4860d ± 0.854 17.1120b ± 0.389 21.5930d ± 0.536 48.0363e ± 0.125 0.0047b ± 0.006 0.2250c ± 0.004 0.000 lsd 1.098 0.654 0.405 0.218 0.011 0.006 0.000 key: cs = cassava starch, wys = water yam starch, pws = potato white starch, amg = amyloglucosidase. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 105 published by scholink inc. 4. conclusion it is evident that enzyme hydrolysis by means of endogenous enzyme can hydrolyze starches to produce syrups. sources of the starches hydrolyzed determines to a great extent the type of sugars to be produced in a hydrolysis, as the constituents enzymes developed in a particular malted grain and the cell structure of the starch source must be compatible to the intended end product (sugar) in that particular hydrolysis. the diastatic power of the grains developed during malting led to more production of maltose sugar by the crude enzymes. food and beverage industries, institutions and, private investors should harness the opportunity of producing syrups from crude enzymes and choices as to what type of sugar they want to dominate the syrups affect their starch source and enzyme source. this can be achieved by choosing among the starches and enzymes that produce the best result for any particular sugar of interest. for a syrup rich in fructose water yam starch is the ideal starch and malted millet as source of enzyme, for a sucrose rich syrup, potato white starch is the ideal starch and malted wheat as the source of enzyme etc. this paper has established the fact that a particular sugar type can dominate all other sugars in syrup. this paper has provided alternatives on how a particular sugar can be obtained in syrup at different preferred or desired concentrations to meet consumer’s preferences using crude enzymes. references fao. (2007). faostat statistics division of the food and agricultural organization. retrieved from http://www.faostat.fao.org ige, m. t., & akintunde, f. o. (1981). studies on the local techniques of yam flour production. international journal of food science and technology, 16(3), 303-311. https://doi.org/10.1111/j.1365-2621.1981.tb01019.x john, k. m., james, l. c., & peter, j. b. (1998). distatic power and alpha-amylase activity in millet, sorghum and barley grains and malt. j. am. soc. brew. chem, 56(4), 131-135. https://doi.org/10.1094/asbcj-56-0131 ogunnyi, l. t. (2008). profit efficiency among cocoyam producers in osun state, nigeria. international journal of agricultural economics and rural development, 1(1), 38-46. okafor d. c., onuegbu n. c., ibeabuchi j. c., agunwah i. m., kabuo n. o., nweje n. o, … izuakam, j. c. (2019). physio-chemical properties of sugar syrup produced from two varieties of yam (dioscorea dumetorum and dioscorea alata) using exogenous enzymes. research journal of food science and nutrition, 4(1), 12-21. okafor, d. c., ofoedu, c. e., nwakaudu, a. a., & daramola, m. o. (2018). enzymes as additives in starch processingshort review. in m. kuddus (ed.), enzymes in food biotechnology. elsevier sand t books. okoye, b. c., onyenweaku, c. e., & agwu, a. e. (2008). technical efficiency of small-holder cocoyam farmers in anambra state nigeria: implications for agricultural extension policy. journal www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 106 published by scholink inc. of agricultural extension, 12(1), 107-116. https://doi.org/10.4314/jae.v12i1.47032 omemu, a. m., akpan, i., bankole, m. o., & teniola, o. d. (2005). hydrolysis of raw tuber starches by amylase of aspergillus niger amo7 isolated from the soil. african journal of biotechnology, 4(1), 19-25. onyenweaku, c. e., & okoye, b. c. (2007). technical efficiency of small-holder cocoyam farmers in anambra state nigeria: a translog stochastic frontier production function approach. international journal of agriculture and rural development, 9(1), 1-6. https://doi.org/10.4314/ijard.v9i1.2655 osuji, c. m., & anih, p. o. (2011). physical and chemical properties of glucose syrup from different cassava varieties. nig. food j., 29, 83-89. osuji, c. m., & okafor, d. (2013). effects of combined cell-wall degrading enzyme treatment on the total dissolved solids and sugars of soymilk. the annals of the university of dunarea de jos of galati. fascile vi. food technology, galati, 37(1), 50-60. subramanian, v., murty, d. s., rao, n. s., & jambunathan, r. (1992). chemical changes and diastatic activity in grains of sorghum (sorghum bicolor) cultivars during germination. journal of the science of food and agriculture, 58(1), 35-40. https://doi.org/10.1002/jsfa.2740580107 tester, r. f., karkalas, j., & qi, x. (2004). starch structure and digestibility enzyme-substrate relationship. world poultry science journal, 60(2), 186-195. https://doi.org/10.1079/wps200412 zielinski, a. a. f., braga, c. m., demiate, i. m., beltrame, f. l., nogueira, a., & wosiacki, g. (2014). development and optimization of a hplc-ri method for the determination of major sugars in apple juice and evaluation of the effect of the ripening stage. food science and technology (campinas), 34(1), 38-43. https://doi.org/10.1590/s0101-20612014005000003 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 2, 2017 www.scholink.org/ojs/index.php/fsns 87 food quality and nutritional profile of students from a public school in brazil loriana l. teixeira1, estelamar mb teixeira1*, helen mara gomes1 & lucas arantes-pereira1 1 instituto federal de educação, ciência e tecnologia do triângulo mineiro-iftm, uberaba-mg, brazil * estelamar mb teixeira, e-mail: estelamar@iftm.edu.br received: september 13, 2017 accepted: september 26, 2017 online published: october 4, 2017 doi:10.22158/fsns.v1n2p87 url: http://dx.doi.org/10.22158/fsns.v1n2p87 abstract feeding is directly related to nutritional status and alterations as obesity and malnutrition, affecting human body and increasing the risk of morbidity and mortality. this study aimed to evaluate the nutritional status of children and adolescents in a public school located in uberaba mg brazil by comparing macro and micronutrients on the school menu with pnae (brazilian government program) recommendations. a descriptive cross-sectional study including 547 individuals was carried out. nutritional assessment was done based on anthropometric z-score analysis. only the food ingestion made at the school was considered, in order to verify if the meals offered by the school meet the percentage of nutrients recommended by pnae. with regard to the nutritional status, there was a small proportion of students (0.18%) with low weight (-3 ≥ z < -2). there was 15% prevalence of overweight (overweight, obesity, and severe obesity) among students. menus composition revealed that macronutrients, fiber, and energy intake was lower than that recommended by pnae for all age groups studied. the prevalence of a considerable number of overweight students is a warning for interventions to prevent obesity. a small percentage of students classified as low weight represents a nutritional transition trend in recent years. keywords school meals, nutritional status, nutritional education 1. introduction overweight and obesity among school children (aged five to nine years) and adolescents almost tripled in the last 20 years, reaching one-fifth of children and one-third of teens (brazilian institute of geography and statistics, 2010). food consumption is related to obesity, including not only the volume of food intake but also the quality of diet (triches & giugliani, 2005). in this context, public health policies work as a tool for governments around the world to reverse the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 88 published by scholink inc. epidemiological situation (silva, 2010). school health programs aim at development of promotion, prevention, and health care in the school environment, besides monitoring the nutritional status of students (ministry of health, 2009). at school, children can try other foods and new preparations, and change eating habits (fisberg et al., 2000). in this context, the brazilian national school feeding program (pnae) has proposed to meet the nutritional needs of students during their stay in school to contribute to food security and nutrition (ministry of education, 2006). therefore, the present study aimed to evaluate the nutritional status of children and adolescents (aged 7-16 years) in a state school in uberaba mg-brazil; to compare macro and micronutrients in school menu and the recommendations by pnae. 2. method 2.1 population this research is a descriptive cross-sectional study including children and adolescents. data were collected during the school year of 2014 and first half of 2015. the school director was asked to select all students enrolled in the afternoon shift. five hundred and forty-seven (547) students of the state school, of both sexes aged 7 to 16 years were assessed. in this research, only the food ingestion made at the school was considered, in order to verify if the meals offered by the school meet the percentage of nutrients recommended by the pnae. the study was approved by the ethics and research committee of the universidade federal do triângulo mineiro-brazil, register number 877.724 and the inclusion of students in the study occurred only after obtaining the term of consent signed by parents or guardians. 2.2 identification questionnaire a structured questionnaire was used to assess the name, sex, and birth date of the participants according to the school enrollment registers. age was calculated as the difference between anthropometric measurement and birth dates. 2.3 nutritional status to calculate the body mass index (bmi) and characterize the nutritional status, an anthropometric evaluation from measurements of weight (kg) and height (m) was performed. the weight measurements were carried out in a platform electronic scale (filizola), with 300 kg capacity and height measurements were done using an inelastic tape measuring 1.5 m length, fixed to the wall at 1 meter from the floor. for the experiment, individuals dressed light clothes and did not wear shoes. individuals were classified according to body mass index (bmi). body mass index (bmi) was calculated by dividing weight (kg) by height squared (m2). the z score is considered an effective method to study population, since age and gender factors are taken into account (world health organization, 2007). z score is given by the ratio of the difference between the value measured at the individual and the value of the reference population, divided by the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 89 published by scholink inc. standard deviation of the same population (ministry of health, 2011), according to the following equation: populationreferenceofdeviationdards populationreferenceofaveragevalueobserved zscore tan   anthropometric indexes used to assess the nutritional status of students aged 6-10 years were weight for age (w/a), height for age (h/a), and bmi for age (bmi/a) measured as z scores. height for age (h/a) and bmi for age (bmi/a) measured as z scores were used to evaluate the nutritional status of students aged 11-17 years. the classification criterion used was the recommended by who, and adopted by the brazilian ministry of health (ministry of health, 2011), as shown in tables 1 and 2. table 1. nutritional status of children aged 5-10 years for each anthropometric index critical values anthropometric index weight for age height for age bmi for age < z score -3 severely underweight severely stunted severely wasted ≥ z score z -3 < z score -2 underweight wasted wasted ≥ z score -2 e < score -1 adequate weight for age adequate height for age eutrophia ≥z score -1 ≤ z score +1 >z score +1 ≤ z score +2 overweight > z score +2 ≤z score +3 high weight for age obesity > z score +3 severly obesity source: adapted from ministry of health (ministry of health, 2011). table 2. nutritional status of adolescents for each anthropometric index critical values anthropometric index height for age bmi for age < z score -3 severly stunted severly wasted ≥ z score -3 e < z score -2 stunted wasted ≥ z score -2 e < z score -1 adequate height for age eutrophia ≥ z score -1 e ≤ z score +1 > z score +1 e ≤ z score +2 overweight > z score +2 e ≤ z score +3 obesity > z score +3 severe obesity source: adapted from ministry of health (ministry of health, 2011). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 90 published by scholink inc. 2.4 nutrition analysis of school meals weekly menus of the full month of june, 2015 were studied. french bread was served in the morning throughout the research period, together with chocolate milk or mate tea. menus for lunch and afternoon snack were prepared for five weeks, returning to the beginning of the menu after the fifth week. dietwin® software was used to calculate the nutrients and energy consumed by students in school meals, after updating the information about nutrients and portion sizes and registering receipts. for registration of recipes not included in the software, a food composition table developed by the center for studies and research in food-nepa, universidade estadual de campinas-unicamp (unicamp, 2011) was used. energy, macronutrients (carbohydrates, proteins, and lipids), fiber, vitamins a and c, and minerals (calcium, iron, magnesium and zinc) intake was investigated, as suggested by pnae. the analyses on nutrient intake according to students’ needs were carried out using the values set by pnae as a reference. table 3 shows the pnae recommendations for students according to age group. table 3. reference values for energy and nutrients, according to age group, established by the national school feeding program (pnae) to meet 70% of the daily nutritional needs energy and nutrients age group 06 to 10 11 to 15 16 to 18 energy (kcal) 1000 1500 1700 carbohydrate (g) 162.5 243.8 276.3 protein (g) 31.2 46.9 50.0 lipids (g) 25.0 37.5 42.5 fiber (g) 18.7 21.1 22.4 vitamin a (µg) 350.0 490.0 560.0 vitamin c (mg) 26.0 42.0 49.0 ca (mg) 735.0 910.0 910.0 fe (mg) 6.3 7.5 9.1 mg (mg) 131.0 222.0 271.0 zn (mg) 4.7 6.3 7.0 source: national school feeding program-pnae (ministry of education, 2013). 2.5 statistical analysis data to assess the nutritional status were processed in microsoft office excel 2007 software; the dietwin® software was used to calculate macro and micro nutrients in school meals. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 91 published by scholink inc. 3. result 3.1 nutritional status the results for the nutritional status of the students (n = 547), based on the body mass index z scores are shown in table 4. there was a prevalence of 10.9% overweight (≥ 1 z < 2) and 3.2% obesity (≥2 z ≤ 3) among students. a percentile of 0.9% students was classified with severe obesity (z > 3), while 0.18% students were classified with low weight index (≥ -3 z <-2). table 4. distribution of students according to age and nutritional status (based on bmi z scores) age (years) gender n % low weight eutrophia overweight obesity severe obesity ≥ -3 z < -2 ≥ -2 z < 1 ≥ 1 z < 2 ≥ 2 z ≤ 3 z > 3 n % n % n % n % n % < 9 male 20 3.5 0 0.00 18 3.30 1 0.18 1 0.18 0 0.00 female 111 20.3 1 0.18 97 17.7 8 1.50 5 0.90 0 0.00 9 i11 male 28 5.1 0 0.00 26 4.70 1 0.18 0 0.00 1 0.18 female 114 20.8 0 0.00 89 16.2 18 3.30 6 1.10 1 0.18 11 i13 male 36 6.6 0 0.00 35 6.40 1 0.18 0 0.00 0 0.00 female 110 20.1 0 0.00 96 17.5 13 2.40 1 0.18 0 0.00 13 i15 male 34 6.1 0 0.00 24 4.40 7 1.20 3 0.50 0 0.00 female 65 11.9 0 0.00 54 9.90 8 1.40 2 0.36 1 0.18 > 15 male 12 2.2 0 0.00 9 1.60 2 0.36 0 0.00 1 0.18 female 17 3.2 0 0.00 13 2.40 1 0.20 2 0.3 1 0.18 total 547 100 1 0.18 461 84.1 60 10.9 20 3.52 5 0.9 the classification of students according to the height-age index is shown in table 5. the results demonstrated that 2.75% students have low height for age. table 5. distribution of students according to the height-age index for children age (years) gender n % low height for age adequate height for age >= -3 and < -2 >= -2 n % n % < 9 male 20 3.6 1 0.18 19 3.5 female 111 20.3 2 0.36 109 20 9 i11 male 28 5.1 1 0.18 27 5 female 114 20.8 7 1.3 107 19.5 11 i13 male 36 6.6 0 0 36 6.5 female 110 20.1 4 0.73 106 19.3 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 92 published by scholink inc. the classification of students according to weight-age index is shown in table 6. the results showed that 4.06% of female students and 0.50% of male students have high weight for age. table 6. distribution of students according to the weight-age and high weight for age index for children age (years) gender n % adequate weight for age high weight for age >= -2 z < 2 z > 2 n % n % ≤ 9 male 35 17.8 34 17.2 1 0.50 female 162 82.2 154 78.2 8 4.06 total 197 100 188 95.4 9 4.56 3.2 nutritional analysis of school meals table 7 shows the school menus used at the school during the research period. table 7. menus used at the school during the research period meal menu breakfast french bread, chocolate milk french bread, mate tea lunch cooked white rice, cooked carioca beans, polenta bolognese, lettuce salad cooked white rice, pasta with white sauce and ham cooked white rice, feijoada cooked white rice, cooked carioca beans, polenta bolognese cooked white rice, cooked carioca beans, ground beef with potato seasoned rice, chicken mayonnaise cooked white rice, cooked carioca beans, polenta bolognese, cooked cabotia pumpkin cooked white rice, minced beef with carrots, vinaigrette and banana snack cornmeal soup cooked white rice, cooked carioca beans, crumbs banana sweet rice 13 i15 male 34 6.2 0 0 34 6.2 female 65 11.9 0 0 65 11.8 > 15 male 12 2.2 0 0 12 2.2 female 17 3.2 0 0 17 3.2 total 547 100 15 2.75 532 97.2 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 93 published by scholink inc. cooked white rice, chicken salad rice, beans drover avocado, papaya and banana smoothie vegetable soup and noodles cooked white rice, cooked carioca beans, crumbs with ground beef and cabotia pumpkin table 8 shows the average energy, macronutrients, micronutrients, fiber, calcium, iron, magnesium, zinc, and vitamins a and c intake of school meals. the number of calories consumed at breakfast, lunch, and snack was 124.87, 290.48, and 266.62 kcal, respectively. regarding the macronutrients, carbohydrate was the most consumed item, especially at lunch, averaging 50.77 g, while protein intake at breakfast, lunch, and snack was on average 3.85 g, 10.62 g, and 11.6 g, respectively. regarding lipids intake, an increase was observed as the meals were served, from 4.21 g and 5.01 g at breakfast and lunch, respectively, reaching 8.22 g at afternoon snack. a low fiber intake was verified at breakfast, with 0.56 g, which was higher at lunch and snack, being 4.81 g and 4.83 g respectively. a difference in vitamin a intake was also observed among the periods, with values of 32.14 g, 2.64 g, and 9.94 g at breakfast, lunch, and snack, respectively. the vitamin c intake at lunch was 4.98 g, while at snack and breakfast the values were 4.97 and 0.58 g, respectively. regarding the calcium intake, breakfast represented the highest intake, with 72.51 g, followed by 17.95 g at lunch and 52.96 g at snack. the average iron intake was 0.57 g, 1.3 g, and 1.31 g at breakfast, lunch, and snack, respectively. with regard to magnesium, the highest intake was observed at lunch with 38.19 g, followed by 33.18 g and 13.37 g at snack and breakfast, respectively. zinc intake was higher at snack, with 2.37 g, when compared to lunch and breakfast with values of 1.98 g, and 0.42 g. table 8. energy and nutrient intake per school meal nutrient/meal breakfast lunch afternoon snack energy (kcal) 124.87 ± 27.17 290.48 ±116.52 266.62 ± 132.99 carbohydrate (g) 17.96 ± 2.87 50.77 ± 22.93 36.45 ± 21.79 protein (g) 3.85 ± 1.17 10.62 ± 4.35 11.6 ± 9.01 lipids (g) 4.21 ± 1.3 5.01 ± 2.76 8.22 ± 5.14 fiber (g) 0.56 ± 0.06 4.81 ± 2.44 4.83 ± 4.87 vitamin a 32.14 ± 15.00 2.64 ± 5.09 9.94 ± 14.84 vitamin c 0.58 ± 0.37 4.98 ± 5.83 4.97 ± 5.73 ca 72.51 ± 42.91 17.95 ± 8.70 52.96 ± 47.38 fe 0.57 ± 0.08 1.3 ± 0.84 1.31 ± 1.27 mg 13.37 ± 3.42 38.19 ± 30.10 33.18 ± 33.67 zn 0.42 ± 0.15 1.98 ± 1.12 2.37 ± 268 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 94 published by scholink inc. regarding the composition of school meals, the energy, macronutrients, and fiber intake was below the recommended value by pnae, as shown in table 9. the energy value was only 69.19% of the recommended value for the group aged 6-10 years, 45.46% for the group aged 11-15, and 40.11% for students aged 16-18 years. the carbohydrates intake reached 64.72%, 43.14%, and 38.06% of the recommended value for the students aged 6 -10, 11-15, and 16-18 years, respectively. the average protein intake reached 83.5% of the recommended value for the students aged up to 10 years. in contrast, for the students aged 11-15 and 16-18 years, the protein intake was 55.6% and 52.14% of the recommended value, respectively. lipids intake was 69.76% of the recommended value for children aged 6-10 years, and 46.50% for the group aged 11-15 years. for the group aged 16-18 years, the average intake reached 41.03% of the recommendations. fiber intake was 54.5%, 48.3%, and 45.5% of the recommended value by the pnae for the groups aged 6-10, 11-15, and 16-18 years, respectively. table 9. energy and macronutrients composition of school meals when compared to 70% of the daily nutritional needs recommended by pnae according to age group nutrient 6-10 years old 11-15 years old 16-18 years old provided by the school recommended recommended recommended energy (kcal) 1000 1500 1700 681.97 carbohydrate (g) 162.5 243.8 276.3 105.18 protein (g) 31.2 46.9 50 26.07 lipids (g) 25 37.5 42.5 17.44 fiber (g) 18.7 21.1 22.4 10.2 the composition of micronutrients of school meals when compared to 70% of the daily nutritional needs recommended by pnae for full day students is presented in table 10. calcium intake was 19.5% and 16.8% of the recommended value for students aged 6-10 years and over 11 years old, respectively. iron intake was 50.5%, 42.4%, and 35%, for the groups aged 6-10, 11-15, and 16-18 years, respectively. with respect to magnesium intake, percentages of 64.6%, 38.2%, and 31.27% were observed for students aged 6-10, 11-15, and 16-18 years, respectively. the average vitamin c intake was 40.5%, 25.07%, and 21.5% of the recommended value by the pnae for students aged 6-10, 11-15, and 16-18 years, while vitamin a intake reached 12.8%, 9.1%, and 21.49% of the recommended value by the pnae for those age groups, respectively. as regards the average zinc intake for the group aged 6-10 years, the school meal supplied the recommendations proposed by the pnae. in contrast, for those aged 11-15 years and 16-18 years, zinc intake supplied 75.7% and 68.1% of the recommended value, respectively. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 95 published by scholink inc. table 10. micronutrients of school meals when compared to 70% of the daily nutritional needs recommended by pnae nutrient 6-10 years old 11-15 years old 16-18 years old provided by the school recommended recommended recommended calcium (mg) 735 910 910 143.42 iron (mg) 6.3 7.5 9.1 3.18 magnesium (mg) 131 222 271 84.74 vitamin a (µg) 350 490 560 44.72 vitamin c (mg) 26 42 49 10.53 zinc (mg) 4.7 6.3 7 4.77 4. discussion 4.1 nutritional status although only one school has been investigated in the present study, it followed the trend in the literature about the decline in malnutrition and increase in overweight and obesity. studies on obesity and underweight in school-age children and adolescents in brazil, china, united states, and russia have found that low weight increased only in russia. in contrast, brazil, china, and the united states have shown an increase in the prevalence of overweight (wang et al., 2002). pegolo and silva (2008) assessed the nutritional status of schoolchildren aged 7-14 years and found 4% students with low height for age. as for the analysis of bmi, 10.7% students were underweight, while 1.6% was classified as obese. koga (2005) investigated schoolchildren aged 7-10 years, and reported prevalence of 4.5% underweight and 11% overweight. regarding the height for age index, stunting of 2.3% was observed. a study in spain found obesity in 16.8% of pre-school children aged 4 years, especially girls. the increase in chronological age raises obesity rates for 32.6% of 8-year-old girls. a decline in female obesity is observed among teenagers, probably due to negative social connotations associated with excess body fat, since vanity is aroused in adolescence (travé et al., 2012). a study in japan investigated children from primary school to high school, by evaluating weight and height every year. the results showed that approximately 50% of obese children in primary school remained obese at age 17. at high school, 40% of obese continued overweight at age 17, while 70% of the obese adolescents in high school remained obese at age 17 (sugimori et al., 1999). abrantes et al. (2002) studied the prevalence of overweight and obesity in children and adolescents in the southeast and northeast of brazil. the results showed that 11.9% of children in the southeast, and 8.2% in the northeast region were obese. in adolescence, a smaller obesity was observed in the southeast over the northeast with 1.7% and 4.2% respectively. in this research, the prevalence of overweight and obesity was higher among women in both regions. silva et al. (2002) evaluated 211 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 96 published by scholink inc. students aged 10-19 years in recife, brazil, and found prevalence of 6.2% overweight and 5.7% obesity among students. salles et al. (2000) assessed the prevalence of obesity among students aged 10-17 years in florianópolis, brazil, and observed a higher prevalence in public schools (13.1%) when compared in private schools (7.6%). in fortaleza, brazil, a survey assessed the nutritional status of adolescents from public and private schools. the results indicated that the prevalence of overweight and obesity in private schools reached 23.9% students, while in public schools this value was 19.5% (campos et al., 2007). 4.2 nutritional analysis of school meals the results of this study showed that in relation to macronutrients, energy, and fibers, the students consumed lower amounts than recommended by the pnae for all age groups. regarding to micronutrients, only zinc intake for the group aged 6-10 years was adequate. a major limitation of the study was the fact that there is no pattern in food portion sizes. pnae recommendations are stratified by age group however meals at school are served equally for all students who receive the same menu and portion sizes. this attitude suggests a lack of guidance on the school feeding program, which mischaracterizes the dietary guidelines according to the nutritional needs (ministry of education, 2013). in this research, the consumption of foods in out-of-school environments has not been studied to associate the nutritional status and food routine at home. however, the identification of insufficient nutrients and energy intake in school meals suggests that students may have a regular intake at home, since there is no major commitment in height of the students evaluated (2.75%). this study showed a low intake of pnae recommendations for all nutrients (macro and micro), but indicates a high consumption of macronutrients outside the school environment according to the nutritional profile presented by the children. low vitamin a ingestion, with predominance among students in the public school system, and the low consumption of vitamin c were also recorded in other research (albuquerque e monteiro, 2002; maestro, 2002). these data are worrying, since hypovitaminosis a is considered a public health problem in brazil and can cause night blindness, loss of vision and less resistance to infections (ministry of health, 2015). low vitamin c intake is associated with lower iron uptake, especially in vegetable foods (carvalho et al., 2001). the deficiencies in vitamins a and c are possibly due to the insufficient consumption of fruits and vegetables, and it is necessary to encourage the students to consume foods that are sources of these nutrients, prioritizing the regional ones, since they are low cost and high nutritional density. proportionally calcium was the nutrient with the lowest supply, and its intake was below the recommendations, which is also demonstrated in other studies (albano e souza, 2001; albuquerque e monteiro, 2002; garcia et al., 2003), corroborating the results of this study. despite the favorable daily consumption of milk, the low ingestion of calcium can be due to the intake of milk in a very diluted www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 97 published by scholink inc. form, with frequency and fractionation insufficient to reach the recommendation of this mineral in the organism. this improper diet practice may lead to low linear growth, impair the bone mass formation (albano e souza, 2001) and favor the early onset of osteoporosis (carvalho et al., 2001). 5. conclusion the prevalence of 15% overweight (overweight, obesity, and severe obesity) among students is a warning for interventions to prevent obesity. a small group classified as underweight (0.9%) proved to be consistent with the nutritional status in the country, where malnutrition has been replaced by excess weight. in relation to energy and macronutrients intake, there was an insufficient intake in relation to the requirements by the pnae for all age groups of students. it is worth mentioning the low occurrence of fibers in school meals, confirmed by fiber intake of 10.2 g per day, probably due to lower consumption of fruits and vegetables. regarding micronutrients intake, only zinc intake was within the recommended value for the group aged 6-10 years. in general, calcium, iron, vitamin a, vitamin c, magnesium intakes were considered insufficient for all ages, and zinc for the groups aged 11-15 and 16-18 years. references abrantes, m. m., lamounier j. a., & colosimo, e. a. (2002). prevalence of overweight and obesity in children and adolescents in the southeast and northeast regions, rio de janeiro. journal of pediatrics, 78, 335-340. albano, r. d., & souza, s. b. (2001). ingestion of energy and nutrients by adolescents of a public school, rio de janeiro. brazil. j. pediatr, 77, 512-516. albuquerque, m. f. m., & monteiro, a. m. (2002). food intake and nutrient adequacy in late childhood. brazil. j. of nutrition, 15, 291-299. brazilian institute of geography and statistics. (2010). 2010 census data. brazil. retrieved from http://www.cidades.ibge.gov.br/xtras/perfil.php?lang=&codmun=317010 campos, l. a., leite, a. j. m., & almeida, p. c. (2007). prevalence of overweight and obesity in school adolescents in the city of fortaleza. brazilian journal of maternal and child health, 7, 183-190. carvalho, c. m. r. g, nogueira, a. m. t., teles, j. b. m., paz, s. m. r., & souza, r. m. l. (2001). food consumption of adolescents enrolled in a private school in teresina, piauí, brasil. brazil. j. of nutrition, 14, 85-93. fisberg, m., bandeira, c. r. s., bonilha, e. a., halpern, g., & hirschbruch, m. d. (2000). eating habits in adolescence. pediatr. mod, 36, 724-734. garcia, g. c. b., gambardella, a. m. d., & frutuoso, m. f. p. (2003). nutritional status and food consumption of adolescents at a youth center in the city of são paulo. brazil. j. of nutrition, 16, www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 98 published by scholink inc. 41-50. koga, c. r. (2005). nutritional status of schoolchildren aged 7 to 10 years: diagnosis and comparison of methods. faculty of public health, university of são paulo, são paulo, brazil. maestro, v. (2002). food pattern and nutritional status: characterization of schoolchildren from the city of são paulo. university of são paulo, são paulo, brazil. ministry of education. national development fund for education. (2006). resolution fnde/cd/nº 32. establish standards for the implementation of the national school feeding program-pnae. brasília, brazil. retrieved from http://www.ftp.fnde.gov.br/web/resolucoes_2006/res032_ 10082006.pdf ministry of education. national development fund for education. (2013). resolution/cd/fnde nº 26. provides on the attendance of school feeding to students of basic education within the scope of the national school feeding program-pnae. brazil retrieved from https://www.fnde.gov.br/fndelegis/action/urlpublicasaction.php?acao=getatopublico&sgl_tipo= res&num_ato=00000026&seq_ato=000&vlr_ano=2013&sgl_orgao=fnde/mec ministry of health. national health surveillance agency. (2015). food guide for the brazilian population: promoting healthy eating. brasília, brazil. ministry of health. national health surveillance agency. department of basic attention. (2011). guidelines for the collection and analysis of anthropometric data in health services: technical standard of the food and nutrition surveillance system. series g. statistics and health information, brasília, brazil. ministry of health. secretariat of health care. department of basic attention. (2009). health at school. basic attention notebooks, series b basic health texts. brasília, brazil. pegolo, g. e., & silva, m. v. (2008). nutritional status of school children in the public school of peidade-sp, campinas, brasil. food and nutrition security, 15, 76-85. salles, r. k., kazapi, i. a., & di pietro, p. f. (2000). occurrence of obesity in adolescents of the teaching network of the city of florianópolis. in j. e. d. oliveira et al. (eds.), obesity and deficiency anemia in adolescence: symposium. danone institute, são paulo, brazil. silva, g. a. p., balaban, g., nascimento, e. m. m., baracho, j. d. s., & freitas, m. m. (2002). prevalence of overweight and obesity in adolescents of a public school in recife, recife. brazilian journal of maternal and child health, 2, 37-42. silva, j. r. m. (2010). health promotion: healthy eating in the school context. faculty of health sciences, university of brasília, brasília, brazil. sugimori, h., yoshida, k., miyakawa, m., izuno, t., takahashi, e., & nanri, s. (1999). temporal course of the development of obesity in japanese school children: a cohort study based on the keio study. the journal pediatrics, 134, 749-759. https://doi.org/10.1016/s0022-3476(99)70292-1 travé, t. d., olascoaga, j. h., & torres, i. g. (2012). childhood excess body weight in navarra and its www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 99 published by scholink inc. impact on adolescence. med clin (barc), 138, 52-55. triches, r. m., & giugliani, e. r. j. (2005). obesity, feeding practices and nutrition knowledge in schoolchildren, são paulo, brazil. journal of public health, 39, 541-547. unicamp-federal university of campinas. (2011). brazilian food composition table-taco. nucleus of studies and research in food-nepa, campinas, brazil. wang, y., monteiro, c. a., & popkin, b. m. (2002). trend of obesity and underweight in older children e adolescents in the usa, brazil, china and russia. am j clin nutr, 75, 971-977. world health organization. (2007). development of a who growth reference for school-aged children and adolescents, geneva. bulletinofthe who, 85, 660-667. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 2, no. 1, 2018 www.scholink.org/ojs/index.php/fsns 1 original paper knowledge and determinants of fruit and vegetable consumption among adults in hohoe municipality, ghana thomas k. awuni1*, gideon kye-duodu2, charles duodu3, francis b. zotor4 & basma ellahi5 1 ghana health service, municipal health directorate, elmina, ghana 2 department of epidemiology and biostatistics, university of health and allied sciences, ho, ghana 3 ghana health service, volta regional health directorate, ho, ghana 4 department of family and community health, university of health and allied sciences, ho, ghana 5 faculty of health and social care, university of chester, chester, uk * thomas kwasi awuni, ghana health service, municipal health directorate, 852-8102, elmina, ghana received: october 28, 2017 accepted: november 7, 2017 online published: december 4, 2017 doi:10.22158/fsns.v2n1p1 url: http://dx.doi.org/10.22158/fsns.v2n1p1 abstract the world health organization (who) recommends that a person consumes at least 400g of fruit and vegetable (fv) daily to prevent chronic disease risk. we assessed knowledge of current who guidelines and other determinants of fv intake among adults (≥ 18 years, n = 397) in hohoe municipality, ghana. face-to-face interviews using a questionnaire adopted from who risk factor surveillance system were undertaken. knowledge of fv daily servings and determinants of intake were evaluated by descriptive statistics and binary logistic regression. there was a 99.2% response rate with approximately 9% of participants correctly stating the who daily recommended amount (p = .296). most (54%) of respondents’ fv intake was affected by unavailability of desired choice (p = .050). odds of inadequate consumption for persons aware of adequate intake amount was 1.97 (95% ci: 0.64, 6.05, p = .234) higher than persons without awareness. participants with problems accessing their desired choice of fv had 0.59 odds (95% ci: 0.36, 0.95, p = .030) of consuming inadequate amount compared to those with easy access. adequate fv intake depends on availability of consumer prefered choice regardless of knowledge of recommendations. individual home based fv cultivation is relevant for availability of preferred choice and adequate consumption for ncds risk reductions among ghanaians. keywords non-communicable diseases, awareness, africa, recommendations www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 2 published by scholink inc. 1. introduction inadequate fruit and vegetable (fv) consumption is ranked high among behavioural risk factors associated with increasing incidence of global non-communicable diseases (ncds) such as cardiovascular disease, ischaemic heart disease, stroke, and cancers (who, 2016; oyebode, gordon-dseagu, & walke, 2014; vaidya, oli, aryal, karki, & krettek, 2013; boeing et al., 2012; hall, moore, harper, & lynch 2009). other research has also linked the phenomenon to disabilities and mortalities worldwide (who, 2016; hall et al., 2009). notably, in 2000, almost 27 million disability adjusted life years (dalys) reported worldwide was associated with poor fv consumption (hall et al., 2009). more regrettably, in 2013, low fv intake accounted for an estimated 5.2 million global deaths (who, 2016). studies have shown positive associations between increased fv intake and ncds risk prevention (who, 2016; oyebode et al., 2014; boeing et al., 2012). consumers of fv hardly become obese and are less likely to develop chronic diseases (who, 2016; oyebode et al., 2014; boeing et al., 2012). this evidence underpins the world health organization (who) guidance on individual consumption of ≥ 400g daily (≥ 5 servings of 80 g each) of varieties of fv as reported (hall et al., 2009; agudo, 2014). the guideline aims to achieve and maintain adequate fv intake globally for ncds prevention and has since been translated into diverse national health promotion campaigns in different geographical areas (agudo, 2004). notably, in ghana, the ministry of health (moh) promoted fv intake as “medicine” as part of the regenerative health campaign strategy to enhance healthy eating and for averting increasing risk of ncds among residents (moh, 2008). despite this, the moh campaign seems to be silent on who recommendations. there is a lack of data on awareness of the who guidance for maintaining adequate intake in previous studies (amo-adjei & kumi-kyereme, 2015; nti, hagan, bagina, & seglah, 2011; hall et al., 2009). in particular, hall et al.’s global study and similar studies on fv intake in ghana only focused on consumption (amo-adjei & kumi-kyereme, 2015; nti et al., 2011; hall et al., 2009). more importantly, subsequent assessments of interventions on fv consumption showed negligible successes and warrants further research (amo-adjei & kumi-kyereme, 2015; nti et al., 2011; tagoe & dake, 2011; pomerleau, karen, cécile, & mckee, 2005). we therefore aimed to assess knowledge on the who guideline of daily fv intake and determinants of consumption among adults in the hohoe municipality, ghana, to provide evidence for future interventions. 2. materials and methods 2.1 study population and design a cross-sectional study was undertaken in three locations (urban, peri-urban and rural) in the hohoe municipality, volta region of ghana using a multistage sampling method. the municipality is located in the northern part of volta region, and shares its border with the eastern part of the republic of togo. most of the residents are farmers who cultivate cash crops and vegetables and also rear livestock. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 3 published by scholink inc. farming activity is more popular among the rural dwellers whilst trading and other industrial activities are undertaken by most of the urban inhabitants. a total sample size (n) of 397 adults (18 years and above) was estimated using low fv intake population prevalence (p = 38%), margin of error (e = 5%), confidence level desired (zα/2 = 1.96), 95% confidence interval (ci) and published formula n = (zα/2) 2 p (1-p) ÷ е2 (hall et al., 2009; snedecor & cochran, 1989). selected adults are eligible since in ghana they represent a constitutionally acceptable and self-informed decision-making body whose decisions can invariably impact households eating habits. adolescents below 18 years and those whose mental judgement could influence responses including proxy respondents were excluded in this study. 2.1.1 sampling techniques eligible respondents were selected with probability proportionate to sample size in each location: urban (232), sub-urban (117) and rural (48). this ensures that the information gathered represent rural/urban dynamics. researchers grouped the municipality into three strata and one community was randomly selected from each stratum using lottery method where individual units were interviewed. in the chosen community, the investigators located the centre, spun a pen on the ground and followed the direction in which the head of the pen indicated to select the first house where a household was interviewed at random. this method (spin-the-pen) for selecting respondents has been discussed elsewhere (grais, angela, & jean-paul, 2007). subsequently, the investigators visited the next house whose entrance was facing them in that manner until the number of respondents was interviewed. the procedure was repeated throughout in each selected stratum. 2.1.2 ethical approval individual written informed consent was obtained from all participants. the study was approved by ghana health service ethical review committee (ghs-erc, id: 02/04/15). 2.1.3 data collection a face-to-face interview was undertaken between may and june 2015 by trained research assistants using the pre-tested semi-structured questionnaire adapted from who step wise approach to surveillance of non-communicable diseases (ncds) fv module (who, 2015; who, 2008). the tool is reported to be valid and data gathered reflects the usual consumption reflecting positively on respondents’ habitual rather than actual intake (agudo, 2004). the questionnaire was translated from english to the local language (ewe and akan) by an independent bilingual researcher or translator prior to field implementation for ease of understanding by the subjects and used as appropriate. it was back translated by members of the research team following (who, n.d). researchers also used a pictorial show card that presented samples of locally available fv and their amounts, excluding juices, in line with who standard amounts (modified from hall et al., 2009; who, 2008). field pre-testing of the show card was undertaken prior to data collection. the show card supported the interview processes for standard estimation of serving size and number of servings as reported elsewhere (hall et al., 2009; who, 2008; agudo, 2004). this ensured participants understood www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 4 published by scholink inc. how each count was calculated in relation to the responding question and enhanced the content validity of the survey tool. the study examined data on sex, age, marital status, education, occupation and monthly income, and source of information. knowledge of experts’ daily recommendation was measured by asking participants the following questions: 1. in your opinion, are you eating enough fv to be healthy? limited to a yes or no response. 2. what is the who minimum recommended fv servings day/person? responses selected from 1, 2, 3, 4 and ≥ 5 servings respectively. other determinants of fv intake were analysed by perceived barriers/facilitators including access to materials to cultivate, (land, seeds and equipment) quality, availability of preferred choice, initiatives and fv information source. 2.1.4 statistical analysis data was coded, entered and analysed using the statistical package for social sciences (spss) version 20, 2011 (ibm inc.). of the 397 questionnaires, a total of 394 respondents’ completed questionnaires were analysed representing a 99.2% useable response rate due to 0.8% missing data. participants’ knowledge of adequate fv consumption was classified based on their response to who defined criterion (hall et al., 2009; agudo, 2004). dependent variables included fv intake grouped into inadequate serving (< 5 servings) and adequate intake (≥ 5 servings) whereas independent variables were respondents’ background characteristics, knowledge and factors of fv consumption. statistical analysis performed included descriptive and binary logistic regression to establish odds ratio of factors influencing consumption among adequate (≥ 5 servings coded “0”) and inadequate (˂ 5 servings coded “1”) respondents. a chi-square test was used to test for statistical difference for categorical variables and p-value 0.05 was considered significant. 3. results the demographic characteristics for the respondents are provided in table 1. respondents mean age was 35.6 years (standard deviation [sd] ± 12.6y). urban participants represented the majority (58.4%) and almost 52% were within low income group with 33.5% of them being traders. junior high/middle school and tertiary graduates respectively dominated respondents’ educational background. although, the respondents’ marital status, income and educational levels show no statistical significance with fv intake, there was a statistical significant relationship between their location, their occupation and fv consumption. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 5 published by scholink inc. table 1. background characteristic stratified with subjects’ fruit and vegetable intake variables fruit and vegetable daily consumption p-value < 5, n = 108(27.4%) ≥ 5, n = 286(72.6%) total n = 394(%) age (years) mean age 35.6(sd 12.6) 18-29 55 (50.9) 114 (39.9) 169 (42.9) .287 30-39 25 (23.1) 70 (24.5) 95 (24.1) 40-49 17 (15.7) 54 (18.9) 71 (18.0) 50-59 7 (6.5) 31 (10.8) 38 (9.6) 60 plus 4 (3.7) 17 (5.9) 21 (5.3) gender of respondents .820 male 45 (41.7) 124 (43.4) 169 (42.9) female 63 (58.3) 162 (56.6) 225 (57.1) marital status of respondents .748 single 34(31.5) 77(26.9) 111(28.2) married/co-habiting 66(61.1) 185(64.7) 251(63.7) divorced 2(1.9) 9(3.1) 11(2.8) widowed/widower 6(5.6) 15(5.2) 21(5.3) respondents community of residents .001 urban 80 (74.1) 150 (52.4) 230 (58.4) sub-urban 23 (21.3) 93 (32.5) 116 (29.4) rural 5 (4.6) 43 (15.0) 48 (12.2) educational level of respondents .593 none 6 (5.6) 18 (6.3) 24 (6.1) primary 20 (18.5) 38 (13.3) 58 (14.7) junior high school 39 (36.1) 122 (42.7) 161(40.9) senior high school 23 (21.3) 52 (18.2) 75 (19.0) tertiary 20 (18.5) 56 (19.6) 76 (19.3) occupation of respondents .044 artisan 28 (25.9) 64 (22.4) 92 (23.4) farming 6 (5.6) 34 (11.9) 40 (10.2) government/private employee 16 (14.8) 62 (21.7) 78 (19.8) trading 37 (34.3) 95 (33.2) 132 (33.5) unemployed 21 (19.4) 31 (10.8) 52 (13.2) income status of respondents .800 low 145 (50.7) 58 (53.7) 203 (51.5) average 127 (44.4) 44 (40.7) 171(43.4) high 14 (4.9) 6 (5.6) 20 (5.1) sd: standard deviation, n: sample size; % per cent; < less than; ≥ greater than or equal to. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 6 published by scholink inc. table 2 shows there is no significant association between knowledge of recommended daily quantity of fv and its consumption. although the majority (80.2%) of respondents affirmed they consumed adequate fv to remain healthy (p = .667), this did not reflect their knowledge as only 8.9% could indicate correctly the who recommendation (p = .296). about 43% and 39% of the respondents had access to land and seed/equipment respectively for their own fv cultivation. majority (more than 80%) of both adequate and inadequate consumption participants indicated they could easily obtain good quality fv (p = .288). however, a significant association existed between availability of the kind of fv and what kind a participant’s desire to eat at a particular time (season of the year). of 54.3% participants who were challenged with their desired choice of fv, 46.3% consumed below who predefined standard (p = .050). in relation to where knowledge is obtained from the highest proportion (78%) of participants reported they were informed about fv intake via radio (p = .903), compared to television (45.6%) (p = 1.00) and 37.3% by health workers (p = 1.00) though there was no statistically significant association (bonferroni correction due to multiple comparison). table 2. knowledge and factors of fruit and vegetable consumption among respondents variables daily amount of fruit and vegetable consumed p-value < 5, n = 108(27.4%) ≥ 5, n = 86(72.6%) total n = 394 in your opinion, are you eating enough fruit and vegetable to be healthy? .667 yes 84 (77.8) 232 (81.1) 316 (80.2) no 13 (12.0) 26 (9.1) 39 (9.9) don’t know 11 (10.2) 28 (9.8) 39 (9.9) what is the who daily recommended minimum amount of fruit and vegetable per person? .296 1 serving 15(13.9) 33(11.5) 48(12.2) 2-4 servings 16(14.8) 36(12.6) 52(13.2) ≥ 5 servings 5 (4.6) 30(10.4) 35 (8.9) do not know 72(66.7) 187(65.4) 259(65.7) do you think eating a diet high in fruit and vegetable can decrease chances of developing ncds? yes 77 (71.3) 206 (72.0) 283 (71.8) .626 no 19 (17.6) 41 (14.3) 60 (15.2) don’t know. 39 (13.6) 12 (11.1) 51 (12.9) is it easy for you to get good quality fruit and vegetable? .288 yes 87(80.6) 243(85.0) 330(83.8) no 21(19.4) 43(15.0) 64(16.2) do you have problem getting your choice of fruit and vegetable to consume? .050 yes 50(46.3) 164(57.3) 214(54.3) no 58(53.7) 122(42.7) 180(45.7) do you have access to an area to grow your own fruit and vegetable? .066 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 7 published by scholink inc. yes 38 (35.2) 130(45.5) 168(42.6) no 70 (64.8) 156(54.5) 226(57.4) can you easily obtain seeds/equipment you need to grow your own fruit and vegetable in your area? .079 yes 33(30.6) 120(42.0) 153(38.8) no 5(4.6) 13(4.5) 18(4.6) not applicable 70(64.8) 153(53.5) 223(56.6) are there initiatives promoting fruit and vegetable intake? .617 yes 75(69.4) 207(72.4) 282(71.6) no 33(30.6) 79(27.6) 112(28.4) where do you get information on fruit and vegetable intake? radio 50(84.7) 138(75.8) 188 (78.0) .903 television 24(40.6 86(47.3) 110(45.6) 1.000 newspaper 1(1.69) 7(3.8) 8(3.3) 1.000 leaflet 1(1.6) 7(3.8) 8(3.3) 1.000 poster 2(3.4) 15(37.9) 17(7.1) 1.000 health workers 21(35.5) 69(37.9) 90(37.3) 1.000 % per cent; < less than; ≥ greater than or equal to; n: sample size. table 3 illustrates possible influence of availability on fv consumption using binary logistic regression analysis. the odds ratio (or) of consuming inadequate serving of fv was 0.59 (95% ci: 0.36, 0.95, p = .030) among participants with problem getting a desired choice of fv compared with persons without problem of choice. this result was statistically significant. the or of having inadequate servings for persons who are aware of what an adequate serving is, was 1.97 (95% ci: 0.64, 6.05, p = .234). this is higher than persons with no awareness, although the result is not statistically significant. table 3. logistics regression analysis of knowledge and determinants of fruit and vegetable intake < 5 fruit and vegetable servings β s.e. p-value odds ratio 95% ci lower upper is it easy for you to get good quality fruit and vegetable? -.431 0.313 .168 0.650 0.352 1.199 do you have area to grow fruit and vegetable? -.384 0.238 .107 0.681 0.427 1.087 do you get your desired choice of fruit and vegetable? -.531 0.245 .030 0.588 0.364 0.950 can fruit and vegetable intake prevent ncds? -.003 0.260 .990 0.997 0.599 1.658 do you know the recommended amount fruit and vegetable to be consumed daily? .680 0.572 .234 1.974 0.644 6.054 are there initiatives promoting fruit and vegetable intake? .035 0.263 .893 1.036 0.619 1.734 < less than; β: regression coefficient; s.e: standard error of the mean, ci: confidence interval. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 8 published by scholink inc. 4. discussion we established that less than 10% of the respondents are correctly aware of who predefined daily minimum intake amount of fv (p = .296). this finding did not correspond with the majority who indicated they consumed adequate amounts to remain healthy (p = .667). the odds ratio of having inadequate servings to persons aware of adequate servings was rather higher (or > 1) than among persons without awareness. therefore, awareness about who predefined criterion had no positive influence on quantity of fv consumption. indeed, the low level of knowledge found in this study corroborates a previous study in ghana (aitken et al., 2014). in contrast, a review that extensively discussed adequate fv intake and chronic diseases risk reduction benefits fell short of finding out whether people know what adequate consumptions are (boeing et al., 2012). of note, hall and colleagues reported intake below standard globally, including ghana (hall et al., 2009). studies conducted in three zones of ghana involving the study region also documented poor intake (amo-adjei & kumi-kyereme, 2015; nti et al., 2011). however, these studies did not adequately address subjects’ knowledge of health experts’ recommended amount that could possibly trigger individual adequate consumption and this is a clear limitation to assessing population fv consumption (agudo, 2004). certainly, measuring population fv consumption in relation to the quantity consumed to derived health benefits strongly reinforces public health strategies of which our study supports (agudo, 2004). in this study, we affirm that media advertisement serves as one of such platform to facilitate increased fv intake, notwithstanding the result of our study showing no statistical significance. radio and television were two main ways found in this study to have informed segment of ghanaian population about the nutritional benefits of fv as had been reported (nti et al., 2011). adequate health education on healthy lifestyle benefits supposedly influences healthy behaviour changes among african american women (delores, harville, efunbumi, & martin, 2015). a well-informed people likely adopted healthy lifestyles and equally applied such information obtained based on the source (delores, 2015). also, several other reviews of wide ranges of worldwide intervention and local policies on fv intake implemented provide evidence to suggest that intensification of healthy life campaigns may be a determinant of improvement in fv intake, though data revealed just modest increase on the amount consumed (amo-adjei et al., 2015; tagoe et al., 2011; pomerleau et al., 2005). interestingly though, awareness of the who recommendation reflected poorly on quantity consumed, evidence from this study suggest that lack of desired choice of fv and location negatively influenced subjects’ consumption. our result shows association between participants’ consumption at a particular time (season of the year) and availability of choice and their locations. indeed, the greater proportion of the respondents who could easily obtain good quality fv even consumed poorly as a result of not finding what they desired for and this increased disproportionally from among rural to urban respondents (p = .288). in our study, we also established that the odds ratio of consuming inadequate serving for persons who had challenges accessing their desired choice of fv was lower compared with persons without problem of desired choice. possibly, the respondents limited access to land and www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 9 published by scholink inc. materials for cultivating their own fv threatened availability and thus consumption. the decision to consume fv has strong linkages with numerous factors ranging from personal to the environment (amo-adjei et al., 2015; ting, wojciech, sarpong, manjeet, & anna, 2014; marie, minot, & lisa, 2004; pollard, kirk, & cade, 2002). previous studies elucidated individual preference, accessibility and availability (quality and quantity), sensory appeal (taste or texture) and location, all of which our result substantiates (ting et al., 2014; marie et al., 2004; pollard et al., 2002). indeed, we confirmed that those who consumed adequate fv actually got their best choice that they wanted regardless of what health experts have indicated to be the appropriate amount. moreover, although we did not examine ethnicity and cultural preference, which has strong influence, it has been reported in other studies (di noia, monica, cullen, pérez-escamilla, gray, & sikorskii, 2015; marie et al., 2004). our finding therefore advances the understanding of drivers of fv intake and recognition of who guideline and practices. this study employed quantitative terms to defining fv intake and used same to explain respondents’ views about experts’ advice on consumption. this method has been proven valid for evaluating population fv consumption (agudo, 2004). the who system for ncds risk-factor surveillance fv intake assessment model is validated, flexible and easily applicable and reports reliable estimate of usual consumption and therefore our finding from recruiting respondents from three strata enhanced views from diverse background is justified for population comparisons (who, 2008; agudo 2004). however, the study had limitations. fv intake knowledge was measured by self-reporting relying on respondent recall, thus there existed a natural tendency of recall bias, random error and social desirability (oyebode et al., 2014; agudo 2004). additionally, cultural preferences were not assessed and determining the amount of servings pictorially from show card of which the questionnaire did not measure fv juices could lead to imprecise estimation of intake (agudo, 2004). the strength of the study includes the use of field investigators who were trained using appropriate protocols (hall et al., 2009; who, 2008). they also explained the portion size and how it is counted from the show card to the participants before the interview enhancing understanding of reliability of information gathered. 5. conclusion we established poor knowledge on daily fv consumption based on the who guideline and furthermore the few knowledgeable participants tended to consume below the recommended levels. importantly, the absence of desired choice of fv impacted negatively on consumption. adequate fv intake depends on consumer preference and availability regardless of health experts’ advice. this suggests individual home based fv cultivation is relevant for availability of preferred choice and adequate consumption to mitigate risk of ncds among ghanaians. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 10 published by scholink inc. acknowledgements trained research assistants who assisted with data collection led by carl agbitor (nutritionist, ghana health service), for data collection, and the participants who took part in the study. wisdom takrama of the department of epidemiology and biostatistics, university of health and allied sciences, ho, ghana who assisted with data analysis. references agudo, a. (2004). measuring intake of fruit and vegetables. background paper for the joint fao/who workshop on fruit and vegetables for health. kobe, japan. aitken, a. et al. (2014). street food vendors in ghana: fruit and vegetable knowledge and influences on vending practice. proceedings of the nutrition society summer meeting, carbohydrates in health: friends or foes. gasgow, uk. amo-adjei, j., & kumi-kyereme, a. (2015). fruit and vegetable consumption by ecological zone and socioeconomic status in ghana. j biosoc sci., 47(5), 613-631. https://doi.org/10.1017/s002193201400025x boeing, h. et al. (2012). critical review: vegetables and fruit in the prevention of chronic diseases. eur j nutr., 51(6), 637-663. https://doi.org/10.1007/s00394-012-0380-y delores, j., harville, c., efunbumi, o., & martin, m. (2015). health literacy issues surrounding weight management among african american women: a mixed methods study. j hum nutr diet., suppl 2, 41-49. di noia, j., monica, d., cullen, k. w., pérez-escamilla, r., gray, h. l., & sikorskii, a. (2016). differences in fruit and vegetable intake by race/ethnicity and by hispanic origin and nativity among women in the special supplemental nutrition program for women, infants, and children. prev chronic dis., 13, e115. https://doi.org/10.5888/pcd13.160130 grais, r. f., angela, m. c., & jean-paul, g. (2007). don’t spin the pen: two alternative methods for second stage sampling in urban cluster surveys. emerg themes epidemiol., 1, 4-8. https://doi.org/10.1186/1742-7622-4-8 hall, j. n., moore, s., harper, s. b., & lynch, j. w. (2009). global variability in fruit and vegetable consumption. am j prev med., 36(5), 402-409. https://doi.org/10.1016/j.amepre.2009.01.029 marie, t. r., minot, n., & lisa, s. (2004). patterns and determinants of fruit and vegetable consumption in sub-saharan africa. background paper for the joint fao/who workshop on fruit and vegetables for health. kobe, japan. measurement and methods core of the center for aging in diverse communities. (2007). guidelines for translating surveys in cross-cultural research. university of california: san francisco. retrieved march, 2015, from http://www.dgim.ucsf.edu/cadc/cores/measurement/ translationguide.pdf moh. (2008). regenerative health and nutrition: a new paradigm for health promotion. source www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 1, 2018 11 published by scholink inc. manual for the training of trainers. ministry of health, ghana. retrieved april 7, 2015, from http://www.rhnp.gov.gh/admindox/rhn%20source-%20manual.pdf nti, c. a., hagan, j., bagina, f., & seglah, m. (2011). knowledge of nutrition and health benefits and frequency of consumption of fruits and vegetables among ghanaian homemakers. african journal of food science, 5(6), 333-339. oyebode, o., gordon-dseagu, v., walke, a., & jennifer, s. m. (2014). fruit and vegetable consumption and all cause, cancer and cvd mortality: analysis of health survey for england data. j epidemiol community health, 0, 1-7. https://doi.org/10.1136/jech-2013-203500 pollard, j., kirk, s. f. l., & cade, j. e. (2002). factors affecting food choice in relation to fruit and vegetable intake: a review. nutr res rev., 15(2), 373-387. https://doi.org/10.1079/nrr200244 pomerleau, j., karen, l., cécile, k., & martin, m. (2005). interventions designed to increase adult fruit and vegetable intake can be effective: a systematic review of the literature. j nutr., 135(10), 2486-2495. snedecor, g. w., & cochran, w. g. (1989). statistical methods (8th ed.). ames: iowa state press. tagoe, h. a., & dake, f. a. a. (2011). healthy lifestyle behaviour among ghanaian adults in the phase of a health policy change. global health, 7, 7. https://doi.org/10.1186/1744-8603-7-7 ting, m., wojciech, j. f., sarpong, d., manjeet, c., & anna, v. a. (2014). expenditure on fresh vegetables, fresh fruits, and peanut products in urban ghana: does location matter? selected paper prepared for presentation at the southern agricultural economics association annual meeting. dallas, tx. vaidya, a., oli, n., aryal, u. r., karki, d. b., & krettek, a. (2013). disparities in fruit and vegetable intake by socio demographic characteristics in sub-urban nepalese adults: findings from the heart-health associated research and dissemination in the community (hardic) study, bhaktapur, nepal. nepal med coll j., 2(3), 3-11. who step wise approach to non-communicable disease risk factor surveillance (steps). (n.d.). who, geneva. retrieved march 23, 2015, from http://www.who.int/chp/steps/instrument/q-byq_steps_instrument_v3.1.pdf?ua=1 who. (2008). steps surveillance instrument. who. retrieved april 11, 2015, from http://www.who.int/chp/steps/part5.pdf who. (2008). steps surveillance training and practical guide. who. retrieved april 11, 2015, from http://www.who.int/chp/steps/part3.pdf who. (2016). increasing fruit and vegetable intake to reduce the risk of non-communicable diseases: e-library of evidence for nutrition actions (e-lena). retrieved november 19, 2016, from http://www.who.int/elena/titles/fruit_vegetables_ncds/en/.html who. (n.d.). management of substance abuse. retrieved march, 2015, from http://www.who.int/substance_abuse/research_tools/translation/en/ food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 2, no. 2, 2018 www.scholink.org/ojs/index.php/fsns 12 original paper physiological activities of perilla frutescens var. frutescens leaf extracts and storage stability in kneaded noodles yoshiyuki watanabe1*, hiroko kitayama1, mina yamamoto1, yusuke kono1, yasue yamada1 & masato nomura1 1 department of biotechnology and chemistry, faculty of engineering, kindai university, higashihiroshima, japan * yoshiyuki watanabe, department of biotechnology and chemistry, faculty of engineering, kindai university, higashihiroshima, 739-2116, japan received: may 16, 2018 accepted: may 22, 2018 online published: june 4, 2018 doi:10.22158/fsns.v2n2p12 url: http://dx.doi.org/10.22158/fsns.v2n2p12 abstract perilla (perilla frutescens var. frutescens) leaves were fractionated based on their chemical properties, and the physiological activities of the fractions were evaluated. the acidic fraction had high radical scavenging ability, whereas the superoxide dismutase-like activities of all fractions were low. a positive correlation was observed between scavenging activity and polyphenol content. the inhibitory effects of the extracts on -amylase and on -glucosidase activities were low, indicating a weak suppressive effect of the leaf extracts on diabetes. the acidic and phenolic fractions suppressed pancreatic lipase activity and accelerated lipid hydrolysis in adipocytes differentiated from 3t3-l1 cells. flour noodles kneaded with leaf powder were prepared, and storage stability was examined. the functional compounds in the leaves were heat-sensitive in the flour noodles. we fractionated perilla leaves to isolate and identify valuable components to provide functionality to processed food and determined that some conditions, such as storage temperature, must be considered to effectively use the compounds. keywords flour noodle, perilla leaves, physiological activity, storage stability 1. introduction perilla frutescens var. frutescens is an annual labiate that exclusively inhabits east and south asia. perilla seeds have been used for their extracted oil for a long time. perilla seed oil is more than 60% comprised of the n-3 polyunsaturated fatty acid -linolenic acid (ichikawa, 2006). as -linolenic acid has various physiological functions, such as lowering blood lipid concentration and preventing diabetes www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 13 published by scholink inc. and hypertension, much attention has been focused on the edible oils as dietary supplements. on the other hand, perilla leaves are rarely used as food except in some countries and regions for its unique flavor and bitter and harsh taste. some polyphenols are expected to be contained in perilla leaves as in other plants. polyphenols occur naturally in more than 4,000 plant species, where they are synthesized during photosynthesis and play a role eliminating reactive oxygen species. hence, polyphenols are effective for maintaining and promoting good health, and preventing disease (jakobek, 2015; lu et al., 2016). in the present study, perilla leaves were fractionated and the chemical characteristics and physiological activities of extracts of each fraction were evaluated for functional components and applications to processed food. moreover, flour noodles kneaded with powdered perilla leaves were prepared as a representative application, and storage stability was investigated to verify its utility. 2. objective the objective of this study is evaluation for the several physiological activities of components in perilla leaves and examination about effective utilization of the leaves as a food material. 3. method 3.1 materials perilla leaves were supplied by fukutomi bussan shakunagekan (hiroshima, japan). caffeic acid, cholic acid, glycine, tris, 2-morpholinoethane sulfonic acid monohydrate (mes), maleic acid, folin-ciocalteu reagent, dexamethasone, 3-isobutyl-1-methylxanthine (ibm-x), insulin, formalin, lecithin from egg yolk, superoxide dismutase (sod), lipase from porcine pancreas, the sod-test wako, the amylase-test wako, the glucose cii-test wako, and the nefa c-test wako were all purchased from wako pure chemical industries (osaka, japan). (±) -tocopherol, (+)-catechin, glyceryl trioleate, -amylase type vi-b, rat intestinal acetone powder, oil red-o, and fetal bovine serum (fbs) were purchased from sigma-aldrich (st. louis, mo, usa). trolox was obtained from merck-millipore co. (darmstadt, germany). the 1,1-diphenyl-2-picrylhydrazyl (dpph) free radical was purchased from tokyo chemical industry (tokyo, japan). dulbecco’s modified eagle medium (dmem) was purchased from thermofisher scientific inc. (waltham, ma, usa) and boehringer ingelheim gmbh (ingelheim, germany), respectively. 3t3-l1 cells were obtained from the japan human science foundation (tokyo, japan). strong and weak commercial-grade flour products were purchased from nisshin flour milling, inc. (tokyo, japan). n-tris hydroxymethyl methyl-2-aminoethane sulfonic acid (tes) was purchased from dojindo molecular technology, inc. (kumamoto, japan). all other analytical grade chemicals were purchased from either wako pure chemical industries or yoneyama chemical (osaka, japan). 3.2 extraction and fractionation of perilla leaves based on chemical properties fresh perilla leaves (20 g) were added to 300 ml distilled water and heated at 95°c for 10 min. after filtering the mixture and evaporating the filtrate under reduced pressure, the condensate was dried in a desiccator containing silica gel to obtain a hot water extract. the fractionation based on chemical www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 14 published by scholink inc. properties was executed as follows: fresh perilla leaves (100 g) were mixed with 1 l methanol and left in the dark at ambient temperature for 7 days. after filtration and evaporation, the methanol extract was prepared by drying the condensate in a desiccator. in accordance with the fractionation procedure shown in figure 1 (urabe et al., 2003), acidic, alkaline, neutral, and phenolic fractions were obtained in their corresponding organic phases. each fraction was prepared by concentration in a rotary evaporator, freeze-dried (dc400, yamato scientific co., ltd., tokyo, japan), and applied to various assays for evaluating their physiological activities. methanol extract from perilla leaves water phase organic phase (basic fraction) water phase organic phase (phenolic fraction) water phase organic phase (neutral fraction) water phase organic phase (acidic fraction) water phase organic phase water phase organic phase extraction by ethyl acetate and 0.2 ml/l glycinehydrocholoric acid buffer (ph2.5) after adjusting to ph 12, extraction by ethyl acetate extraction by 0.2 mol/l sodium phosphate buffer (ph 7.5) extraction by 0.2 mol/l sodium hydroxidepotassium chloride buffer (ph 13) after adjusting to ph 3, extraction by ethyl acetate after adjusting to ph 3, extraction by ethyl acetate figure 1. fractionation procedure for the perilla leaf methanol extracts based on chemical properties 3.3 measurement of radical scavenging activity and total polyphenol content the radical scavenging activity of the extracts of each perilla leaf fraction was measured according to a previous method (fujita et al., 2006). a dpph radical solution (15 ml of 0.4 mm) was mixed with 15 ml of 0.2 m mes buffer (ph 6.0), and the same volume of 3.4 m ethanol solution. the sample solution was prepared at six concentrations by dissolving the powdered fraction in 13.7 m ethanol solution. then, 600 l of the sample solution and 1.8 ml of the mixture containing the dpph radicals were added to an amber vial. the headspace in the vial was filled with nitrogen gas to suppress oxidation of the substrate during the reaction, and it was tightly sealed. the vial was shaken and incubated for 20 min at 25°c. absorption of the sample was measured at 520 nm using a spectrophotometer (dr4000; hach co., loveland, co, usa), yielding a. a 13.7 m ethanol solution www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 15 published by scholink inc. was used as blank b. the dpph radical scavenging activity was determined using the following formula: 100 b a -1 = [%]activity scavenging radical dpph       -tocopherol and trolox were used to compare the extracts of the perilla leaf fractions. the sod-like activity assay was carried out using the sod-test wako (yamashoji et al., 1979). sod and the extract solution were prepared by dissolving them in 0.1 m phosphate buffer (ph 8.0) at 352.9 unit/ml and 2 mg/ml, respectively. the extract solution (50 l), sod solution (500 l), and coloring reagent solution (500 l) were mixed and incubated at 37°c. after sampling periodically and stopping the reaction, absorbance was measured at 560 nm with the spectrophotometer. the total quantity of polyphenolic compounds in the perilla leaf extracts was determined according to the folin-ciocalteu procedure (kähkönen et al., 1999). folin-ciocalteu reagent (1 ml) was added to 1 ml of the extract solution and allowed to stand for 3 min at room temperature. then, 1 ml of 10% (w/v) sodium carbonate was added, and mixed for 1 h. the mixture was centrifuged at 3,000 rpm for 10 min, and absorbance of the supernatant was measured at 760 nm using the spectrophotometer. caffeic acid was used as the standard to express the total amount of polyphenolic compounds as caffeic acid equivalents/mg extract. 3.4 measurement of inhibitory effects on -amylase, -glucosidase, and pancreatic lipase activities the inhibitory effect of the perilla leaf extracts on -amylase activity was tested by the amylase-test wako (anno et al., 2004; hara & honda, 1990; mcdougall et al., 2005; saito et al., 2007). first, 5.35 unit/ml -amylase solution was prepared in 250 mm phosphate buffer (ph 7.0). starch solution (1 ml) as the substrate and 100 l of each extract were mixed at specific concentrations and incubated at 37°c for 5 min. then, 20 l of -amylase solution was added to the mixture and incubated again for 7.5 min. the absorbance of the mixture was measured at 660 nm in a spectrophotometer, following the addition of 1 ml of coloring reagent solution and distilled water. (+)-catechin was used as the standard for comparison with each extract. inhibition of -glucosidase activity was tested as follows (anno et al., 2004; mcdougall et al., 2005; saito et al., 2007): rat intestinal acetone powder (2 g) was added to 45 ml of 56 mm tris-maleic acid buffer (ph 6.0), and the mixture was homogenized in ice. the supernatant was obtained as a coarse enzyme solution by centrifugation at 3,000 rpm and 4°c for 10 min. a 50 l aliquot of 2% (w/v) maltose, the extract at the given concentrations, the coarse enzyme solution, and the tris-maleic acid buffer were mixed, and the reaction was carried out at 37°c for 1 h. after stopping the reaction by heat shock at 60°c for 10 min, the concentration of glucose that formed was measured using the glucose cii-test wako. the reaction solution (20 µl) was incubated with 3 ml of coloring reagent solution at 37°c for 5 min, and the absorbance of the mixture was measured at 505 nm using the spectrophotometer. a substrate emulsion was prepared by ultrasonic treatment of 80 mg glyceryl trioleate, 10 mg lecithin, www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 16 published by scholink inc. and 5 mg cholic acid in 9 ml of 0.1 m tes buffer (ph 7.0) containing 0.1 m sodium chloride to measure pancreatic lipase activity (edashige et al., 2008; kwon et al., 2003). the porcine pancreas lipase solution was prepared in buffer. the extracted sample (50 l), 10 units of lipase solution (25 l), and the emulsion (125 l) were mixed at 37°c for 30 min. the amount of free fatty acids in the sample solution was determined by the nefa c-test wako. the sample and coloring reagent solutions were mixed at 37°c for 20 min, and absorbance was measured at 550 nm. 3.5 3t3-l1 adipocyte differentiation assay the effect of the perilla leaf extracts on adipocyte differentiation of 3t3-l1 cells was investigated to evaluate suppression of obesity according to previously reported methods with a slight modification (furuyashiki et al., 2004; hayashi et al., 1981; iwashita et al., 2001; kuri-harcuch & green, 1978; sakuramata & kusano, 1998). 3t3-l1 cells were cultured in 96-well plates at 1 × 104 cells/well in dmem containing 20% (w/v) fbs under 5% (v/v) co2 and 37°c. the culture medium was exchanged with dmem plus 0.2 m dexamethasone, 0.5 mm ibm-x, and 0.8 m insulin before the cells were confluent, and the mixture was incubated at 37°c for 2 days to begin adipocyte differentiation. then, differentiation proceeded by continuously exchanging the culture medium with dmem plus 20% (w/v) fbs and 0.8 m insulin every 2-4 days. the concentration of fbs used in the cultures was decreased from 10% to 4% (w/v) over 2 days. this culture solution (150 l) was added to 150 l/well dmem with 5-500 g/ml of each of the perilla leaf extract solutions and 4% (w/v) fbs, and the mixture was incubated at overnight 37°c. adipocyte differentiation of 3t3-l1 cells was confirmed by oil red-o staining (kuri-harcuch & green, 1978). first, 500 mg of oil red-o was dissolved in 100 ml isopropanol. the solution was diluted with distilled water to 60% (v/v) and was left at room temperature for 10 min before filtration. after washing the cells with phosphate buffer, they were fixed for 15 min in buffer with 10% (w/v) formalin. the cells were washed again, and left in 60% (v/v) isopropanol for 1 min. then, the cells were stained for 15 min with the oil red-o solution. the stained cells were observed under a phase-contrast microscope (ix72; olympus co., tokyo, japan). free fatty acid content in the culture was measured using the nefa c-test wako. 3.6 preparation and preservation of flour noodles kneaded with leaf powder two grams of the powder obtained by milling perilla leaves, 50 g of strong and 50 g of weak flour, 200 mg sodium carbonate, and 36 ml distilled water were mixed and fully kneaded to obtain the dough. the dough was rolled out to a ca. 3 mm thickness, and 3 mm × 5 cm noodles were prepared by cutting the dough. the noodles were placed in a plastic container that had a glass vial filled with a saturated potassium carbonate solution to regulate relative humidity at 44%. the container was stored in the dark at 75°c for 5 days. noodles were removed at appropriate intervals and ground with a mortar and pestle. the ground sample (35 g) was added to 84 ml of methanol, and the mixture was stirred for 3 min with a magnetic stirrer. after ultrasonic treatment at 40°c for 10 min, the mixture was centrifuged at 3,000 rpm for 10 min. this treatment was repeated three times, and the three supernatants were mixed. a powdery sample was extracted from the noodles by concentrating and drying the supernatant mixture in www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 17 published by scholink inc. a rotary evaporator. the same storage and extract procedures were applied for the perilla leaf powder, and the extracted sample was used as a reference to the noodle sample in several physiological activity tests. 3.7 statistical analysis all measurements were carried out in triplicate, and mean values were compared by analysis of variance. significant differences were estimated by t-tests at p < 0.05. 4. result and discussion 4.1 antioxidant activities of extracts of the perilla leaf fractions the weights of the powders obtained from the hot water extract, alkaline fraction, acidic fraction, neutral fraction, and phenolic fraction after fractionating the 100 g of fresh perilla leaves were 0.958, 0.254, 0.885, 2.66, and 0.325 g, respectively. the neutral fraction was the largest of all extracted samples. the hot water extract results suggest that the effluent of components from the leaves during branching treatment, which is often used for processing and cooking vegetables. concentration [mg/ml] 0.02 0.040 s c a v e n g in g a c ti v it y [ % ] 100 80 60 40 20 0 total polyphenolic compunds as caffeic acid [mg/mg] 10 102 103 (a) (b) figure 2. the relationship between dpph radical scavenging activity and (a) the concentrations of the () acidic, () alkaline, () neutral, and (◇) phenolic fractions, as well as () the hot water extract from perilla leaves and (b) the total amount of polyphenolic compounds in each extract. the scavenging activities in (b) are plotted for the 50 mg/ml concentration of each extract figure 2 (a) shows the relationship between dpph radical scavenging activity and the concentration of each perilla leaf extract. the 50% scavenging concentration (sc50) for dpph radicals on the hot water extract, acidic fraction, and phenolic fraction were estimated to be 35, 7.7, and 41 g/ml, respectively, from these profiles. the sc50 values for the alkaline and neutral fractions could not be calculated, as the activities did not reach 50% in the tested concentration range. as the values were ≥ 5.6 and 8.3 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 18 published by scholink inc. g/ml for -tocopherol and trolox, respectively, the acidic fraction had high antioxidant ability. on the other hand, the alkaline and neutral fractions hardly exhibited any radical scavenging activity. figure 2 (b) shows the relationships between radical scavenging activity of each 50 g/ml extract and the total amount of polyphenolic compounds in the corresponding extract. a positive correlation was obtained from the plots, suggesting the contribution of polyphenols to the antioxidant activity of the extracts. the high radical scavenging activity and total polyphenolic content in the hot water extract indicates a loss of antioxidant functionality by the leaf branching treatment. figure 3 shows sod-like activity of each perilla leaf extract. the activity of the phenolic fraction was the highest, but it was almost half that of sod. sod-like activities of the extracts were low. time [h] 0 1 2 3 a b s o rb a n c e a t 5 6 0 n m 0 0.1 0.2 0.3 0.4 figure 3. superoxide dismutase (sod)-like activities of the () acidic, () alkaline, () neutral, and (◇) phenolic fractions, () as well as the hot water extract from perilla leaves. black circle, ●, and square, ■, represent control and sod, respectively 4.2 suppressive effects of the extracts of the leaf fractions on diabetes and obesity the diabetes preventive effects of the perilla leaf extracts were evaluated by testing their inhibitory effects on -amylase and -glucosidase activities. figure 4 (a) shows the inhibitory effects of each extract on -amylase activity. the phenolic fraction exhibited the highest inhibitory activity, and the 50% inhibitory concentration (ic50) was 1.7 mg/ml. the ic50 value of catechin was 55 g/ml, but the inhibitory effects of these extracts on -amylase were very low. the inhibitory effects of the extracts on -glucosidase activity are shown in figure 4 (b). the ic50 value of the acidic fraction was 1.8 mg/ml, but the values could not be estimated for other extracts at concentrations < 2.0 mg/ml. therefore, the inhibitory effects of the leaf extracts on -glucosidase activity were very low. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 19 published by scholink inc. in h ib it io n [ % ] 60 40 20 0 0 1 2 concentration [mg/ml] (a) (b) 0 1 2 figure 4. inhibitory effects of the perilla leaf extracts on (a) -amylase and (b) -glucosidase activities. symbols are the same as shown in figure 2 the effects of the perilla leaf extracts on pancreatic lipase inhibitory activity and 3t3-l1 adipocyte differentiation were examined to evaluate the ability to prevent obesity. pancreatic lipase inhibitory activities of the extracts at the 500 g/ml concentration are shown in table 1. the activities were high in the order of the acidic fraction > hot water extract > phenolic > alkaline > neutral fractions. in particular, the activity of the acidic fraction was twice that of the other extracts. however, not all of the values were high. next, the effect of the extracts on adipocyte differentiation was investigated. the 3t3-l1 cells differentiated and oil red-o staining revealed the formation of lipid droplets under a phase-contrast microscope. table 2 shows the amounts of free fatty acids released from the cells into the culture medium after adding the extracts. the free fatty acid concentration released in response to the 500 g/ml neutral fraction was almost equal to that of differentiated control 2 cells with no extract added. the free fatty acid concentrations released in response to the 500 g/ml acidic fraction, alkaline fraction, and hot water extract were 1.8-1.9-fold that of the control. the free fatty acids released in response to 500 g/ml of the phenolic fraction was 2.6 times that of the control. thus, the phenolic fraction seemed to be the most effective for suppressing obesity in the tested samples through hydrolysis of triglycerides in the adipocytes. however, concentrations that exhibited an accelerating effect on lipid hydrolysis were too high for the cells. because the acidic and phenolic fractions suppressed pancreatic lipase activity and accelerated lipid hydrolysis in differentiated adipocytes, respectively, the compounds responsible for these physiological functions must be identified and isolated. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 20 published by scholink inc. table 1. pancreatic lipase inhibitory activities of the extracts from perilla leaves at the concentration of 500 mg/ml extracts inhibition [%] (n=3) hot water extract 17.4 + 3.9 alkaline fraction 5.8 + 1.3 acidic fraction 37.5 + 1.3 neutral fraction 4.9 + 3.8 phenolic fraction 16.0 + 1.0 table 2. effects of the extracts from perilla leaves on the release of free fatty acids from 3t3-l1 cells into the culture added extracts [μg/ml] released free fatty acids [μg/ml] control 1 (not differentiated cell) 0.046 + 0.034 control 2 (differentiated cell) 0.070 + 0.015 hot water extract 5 0.076 + 0.011 50 0.091 + 0.001 500 0.130 + 0.029 alkaline fraction 500 0.125 + 0.004 acidic fraction 5 0.070 + 0.019 50 0.076 + 0.004 500 0.124 + 0.023 neutral fraction 500 0.088 + 0.004 phenolic fraction 5 0.085 + 0.001 50 0.094 + 0.026 500 0.252 + 0.079 4.3 storage stability of physiological components in flour noodles kneaded with perilla whole leaf powder flour noodles kneaded with perilla whole leaf powder were prepared and the stability of the physiological activities after storage was verified to explore opportunities for use in processed food. the noodles were preserved under accelerated test conditions at a temperature of 75°c to evaluate short-term stability. figure 5 shows the dependence of dpph radical scavenging activity on the concentration of the extract from noodles kneaded with leaf powder. the whole leaf extract exhibited significantly higher activity, and activity decreased only slightly after 5 days. the immediate dpph radical scavenging activity after preparing the noodles with the leaf powder was only about 10%. in addition, activity decreased during the preservation and was close to zero. the reason for the large www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 21 published by scholink inc. decrease is unclear. the radical scavenging activity of the leaf extracts was likely due to the polyphenols in the leaves, and the oxidation reaction is generally affected by water activity. therefore, water sorption onto a matrix, such as starch or gluten, in the noodles may have stabilized the polyphenols during preservation. 0 20 40 60 80 0 0.2 0.4 0.6 concentration of extract [mg/ml] s c a v e n g in g a c ti v it y [ % ] (a) (b) 0 0.2 0.4 0.6 figure 5. dependence of dpph radical scavenging activities on the concentrations of () the extract from perilla leaves, () the extract from noodles kneaded with leaf powder, and () the extract from noodles with no kneading (a) before preservation and (b) after preservation for 5 days at 75ºc and 44% relative humidity the relationships between inhibited -glucosidase activity and the concentrations of the extracts of the noodles kneaded with leaf powder are shown in figure 6. the immediate inhibition of the extract from the noodles with the leaf powder was ca. 35% at 2 mg/ml, but the difference from the extract with no lead kneading was small. the difference also remained small after 5 days, indicating that the inhibitory ability of the noodles with leaf powder was low but that the inhibitory compounds in the noodles were relatively hard to degrade under high temperature storage. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 22 published by scholink inc. 0 20 40 60 0 1 2 in h ib it io n [ % ] concentration of extract [mg/ml] 0 1 2 (a) (b) figure 6. dependence of the inhibitory effect on -glucosidase activity on the concentrations of () the perilla leaf extracts, () the noodles kneaded with leaf powder extract, and () the extract from noodles with no kneading (a) before preservation and (b) after preservation for 5 days at 75ºc and 44% relative humidity figure 7 shows the change in the inhibitory effect of the extract from the noodles kneaded with leaf powder on pancreatic lipase activity during preservation at 75°c and 44% relative humidity for 5 days. the inhibition of the noodles with the leaves was no less low, as that of the leaf extract intrinsically was not high. the noodles with no leaves had little inhibitory activity. the inhibition of the noodles with the leaf powder was calculated to be near zero after storage, compared with the result for noodles without kneading. this was similar to the radical scavenging activity results of the extracts. the inhibitory ability of the leaf extracts on pancreatic lipase activity may have also been due to the same polyphenolic compounds as described for radical scavenging activity. 0 5 10 15 20 in h ib it io n [ % ] extract from leaves extract from noodle with leaves day 0 day 5 day 0 day 5 day 0 day 5 extract from noodle without leaves figure 7. inhibitory effect of the perilla leaf extract, the noodles kneaded with the leaf powder extract, and the extract from noodles with no kneading on pancreas lipase activity (a) before preservation and (b) after preservation at 75ºc and 44% relative humidity for 5 days www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 23 published by scholink inc. 5. conclusion perilla leaves had some physiological activities, and fractionation based on the chemical properties of the leaf extracts made it possible to clarify the functions of each fraction. for example, the acidic and phenolic fractions effectively suppressed oxidation and obesity. in addition, some functional compounds were detected in the hot water extract. however, the promising components in the leaves were heat-sensitive in flour noodles. therefore, some conditions, such as storage temperature, must be considered to use these compounds effectively. references anno, t., tamura, k., oono, h., & tomi, h. (2004). maltase, sucrase and -amylase inhibitory activity of morus leaves extract (in japanese). food preservation science, 30(5), 223-229. https://doi.org/10.5891/jafps.30.223 edashige, y., murakami, n., & tsujita, t. (2008). inhibitory effect of pectin from the segment membrane of citrus fruits on lipase activity. journal of nutritional science and vitaminology, 54, 409-415. https://doi.org/10.3177/jnsv.54.409 fujita, a., masumoto, k., kawakami, k., mikami, t., & nomura, m. (2006). anti-oxidation activity of various rice brans. journal of oleo science, 55(11), 585-591. https://doi.org/10.5650/jos.55.585 furuyashiki, t., nagayasu, h., aoki, y., bessho, h., hashimoto, t., kanazawa, k., & ashida, h. (2004). tea catechin suppresses adipocyte differentiation accompanied by down-regulation of ppar 2 and c/ebp in 3t3-l1 cells. bioscience biotechnology and biochemistry, 68(11), 2353-2359. https://doi.org/10.1271/bbb.68.2353 hara, y., & honda, m. (1990). the inhibition of -amylase by tea polyphenols. agricultural and biological chemistry, 54(8), 1939-1945. https://doi.org/10.1080/00021369.1990.10870239 hayashi, i., nixon, t., morikawa, m., & green, h. (1981). adipogenic and anti-adipogenic factors in the pituitary and other organs, proceedings of the national academy of sciences of the united states of america, 78(6), 3969-3972. https://doi.org/10.1073/pnas.78.6.3969 ichikawa, k. (2006). nutritional properties and utilization of perilla seed oil (in japanese). journal of oleo science, 6(5), 257-264. https://doi.org/10.5650/oleoscience.6.257 iwashita, k., yamaki, k., & tsushida, t. (2001). effect of flavonoids on the differentiation of 3t3-l1 adipocytes. food science and technology research, 7(2), 154-160. https://doi.org/10.3136/fstr.7.154 jakobek, l. (2015). interactions of polyphenols with carbohydrates, lipids and proteins. food chemistry, 175, 556-567. https://doi.org/10.1016/j.foodchem.2014.12.013 kähkönen, m. p., hopia, a. i., vuorela, h. j., rauha, j. p., pihlaja, k., kujala, t. s., & heinonen, m. (1999). antioxidant activity of plant extracts containing phenolic compounds. journal of agricultural and food chemistry, 47, 3954-3962. https://doi.org/10.1021/jf990146l kuri-harcuch, w., & green, h. (1978). adipose conversion of 3t3 cells depends on a serum factor. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 24 published by scholink inc. proceedings of the national academy of sciences of the united states of america, 75(12), 6107-6109. https://doi.org/10.1073/pnas.75.12.6107 kwon, c. s., sohn, h. y., kim, s. h., kim, j. h., son, k. h., lee, j. s., … kim, j. s. (2003). anti-obesity effect of dioscorea nipponica makino with lipase-inhibitory activity in rodents. bioscience biotechnology and biochemistry, 67(7), 1451-1456. https://doi.org/10.1271/bbb.67.1451 lu, w., kelly, a. l., & miao, s. (2016). emulsion-based encapsulation and delivery sytems for polyphenols. trends in food science & technology, 47, 1-9. https://doi.org/10.1016/j.tifs.2015.10.015 mcdougall, g. j., shpiro, f., dobson, p., smith, p., blake, a., & stewart, d. (2005). different polyphenolic components of soft fruits inhibit -amylase and -glucosidase. journal of agricultural and food chemistry, 53, 2760-2766. https://doi.org/10.1021/jf0489926 saito, y., nishi, s., koaze, h., hironaka, k., & kojima, m. (2007). antioxidant and inhibitory activity on -amylase and -glucosidase in legume polyphenols (in japanese). nippon shokuhin kagaku kogaku kaishi, 54(12), 563-567. https://doi.org/10.3136/nskkk.54.563 sakuramata, y., & kusano, s. (1998). screening of plant extracts with potential to stimulate lipolysis in 3t3-l1 cells (in japanese). nippon eiyo shokuryo gakkaishi, 51(6), 361-364. https://doi.org/10.4327/jsnfs.51.361 urabe, k., kitao, s., kouyama, k., nadamoto, t., kawamura, m., & nishikawa, y. (2003). growth inhibition of escherichia coli, staphylococcus aureus and bacillus subtilis by wild grasses extracts (in japanese). nippon shokuhin kagaku kogaku kaishi, 50(8), 350-355. https://doi.org/10.3136/nskkk.50.350 yamashoji, s., yoshida, h., & kajimoto, g. (1979). photooxidation of linoleic acid by ultraviolet light and effect of superoxide anion quencher. agricultural and biological chemistry, 43(6), 1249-1254. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 4, 2019 www.scholink.org/ojs/index.php/fsns 107 original paper investigation of processing technology for aged black jujube lin gao1, duanyin gu2, xin sun1 & rentang zhang1* 1 college of food science and engineering, shandong agricultural university, tai’an, people’s republic of china 2 tai’an academy of agriculture sciences, tai’an, china * rentang zhang, college of food science and engineering, shandong agricultural university, tai’an, people’s republic of china received: august 27, 2019 accepted: september 9, 2019 online published: september 23, 2019 doi:10.22158/fsns.v3n4p107 url: http://dx.doi.org/10.22158/fsns.v3n4p107 abstract we investigated how to use jujubes from xinjiang to make black jujube by temperature-controlled wet high-temperature curing. single-factor and orthogonal experimental designs l9(3)3 were used to optimize the color change process of the black jujube. the effect of blackening temperature, blackening time and added water amount on the content of camp, 5-hmf, polyphenols, total acids, reducing sugars and moisture content were investigated and optimized. the results showed that the optimum process conditions of red jujube aging and blackening black include blackening temperature of 75°c, blackening time of 55 h, and water addition amount of 150 ml per 600 g. under the best technological conditions, the black jujube was fragrant, sweet, and delicious, and the content of all functional substances, including camp (0.0137 g/100g), 5-hmf (0.103 g/100g), polyphenols (2.71 g/100g), total acids (17.09 g/kg), reducing sugars (76.7 g/100g), reached high levels, at a moisture content of 26%. keywords red jujube, aging, blackening, black jujube, processing technology 1. introduction jujube fruit, also known as big jujube, dry jujube, old chinese jujube, is the mature fruit of ziziphus jujuba mill. from the rhamnaceae family. jujube is a unique fruit and vegetable resource in china. it is native to china and has been cultivated for over 4000 years (li, fan, ding, & ding, 2007). with an area and output of more than 90% of the world’s total, china is the world’s largest producer of jujube fruits (zhao, zhang, liu, xue, & pan, 2014). red jujube is a characteristic fruit of china which integrates nutrition, health care and medicinal functions. jujube is rich in vitamins, minerals, phytosterols, amino acids, saponins, polyphenols, flavonoids, camp, cgmp, and polysaccharides www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 108 published by scholink inc. (pawlowska et al., 2009). jujube can enhance immunity, help the body resist inflammation, protect the liver, intestines and stomach, ameliorate insomnia and act as an antioxidant (chen et al., 2015; zhang, w. y., zhang, l., & xu, 2016; almansa, hernández, legua, nicolás-almansa, & amorós, 2016; yue et al., 2015). at present, there are many red jujube products on the market, such as dry products, fermented products, preserved jujube, red jujube beverages and so on. however, there are few reports on aging black jujube. ji et al. (2013) have done relevant research on the aged jujube, but they only investigated the changes of composition. there are some conventional jujube products in china, such as jiaozao, wuzao, etc., but they are produced differently from the production process of aging black jujube. the processing method of black jujube is similar to that of black garlic. black jujube is produced by non-enzymatic browning of red jujube at high temperature and high humidity. the color, flavor and nutrition of jujubes are changed after blackening. it has been found that the antioxidant capacity of red jujube is also enhanced after aging and blackening (park et al., 2012). there is a lack of research on processing technology for black jujube. so, the development of low-sugar aged black jujube and related products has broad market prospects. 2. materials and methods 2.1 plant material and reagents jujube fruits (moisture content 15-20%) were obtained from hami (gps e93°32′n42°49′) xinjiang province, china. chemicals, including zinc acetate, potassium ferrocyanide, naoh, phenol, concentrated sulfuric acid, and ethanol were purchased from tianjin kaitong chemical reagent co., ltd (tianjin, china); 5-hmf (98%), camp (98%) and gallic acid (98%) were purchased from shanghai yuanye biotechnology co., ltd (shanghai, china). hplc grade methanol and acetonitrile were purchased from shandong yuwang industrial co., ltd (yucheng, shandong, china). 2.2 the influence of blackening conditions on the content of various index compounds in black jujube 2.2.1 influence of blackening time on the content of various index compounds in black jujube dried jujubes were divided into 6 equal portions, 600 g each, and cleaned. according to the proportion of jujube water mass ratio 1:5, the fruits were rehydrate at room temperature for 1 h, removed and drain for 15 min, and then 400 ml distilled water added to the bag and sealed. then, the red jujube were put in the 80°c oven for blackening. at 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h. cyclic adenosine monophosphate, 5-hydroxymethylfurfural, polyphenols, total acids, reducing sugars, water content and other indicators of each sample were detected respectively. 2.2.2 influence of blackening temperature on the content of various index compounds in black jujube dried jujubes were divided into 6 equal portions, 600 g each, then cleaned. according to the proportion of jujube water mass ratio 1:5, rehydrate at room temperature for 1 h, remove and drain for 15 min, and www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 109 published by scholink inc. then 400 ml of distilled water was added to the bag and sealed. the portions were separately aged for 60h at 65°c, 70°c, 75°c, 80°c, 85°c, and 90°c. cyclic adenosine monophosphate (camp), 5-hydroxymethylfurfural, polyphenols, total acids, reducing sugars, water content and other indicators of each sample were measured respectively. 2.2.3 influence of the amount of added water on the content of various index compounds in black jujube dried jujubes were divided into 6 equal portions, 600 g each, then cleaned. according to the proportion of jujube water mass ratio of 1:5, the fruits were rehydrated at room temperature for 1 h, removed and drained for 15 min. then, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml and 600 ml of distilled water was added to individual bags and sealed, followed by aging at 80°c for 60 h. the contents of camp, 5-hydroxymethylfurfural, polyphenols, total acids, reducing sugars, water and other indicators of each sample were measured respectively. 2.3 orthogonal experimental design for optimizing the blackening conditions on the basis of single factor tests, an orthogonal test was carried out by selecting factors such as blackening time, blackening temperature and amount of added water. an orthogonal experiment [l9(3)3] test design in the blackening mode was used for optimizing the blackening conditions. the key parameters that influenced the contents of key components of black jujube were analyzed, including the time of blackening (a), temperature of blackening (b) and amount of added water (c). every factor had three levels. nine extractions were carried out at blackening times 55, 60 and 65 h, blackening temperatures of 75, 80 and 85°c, and added water amounts of 150, 200, and 250 ml. the factor levels are shown in table 1. table 1. orthogonal design level factor a, blackening time/h b, blackening temperature (°c) c, amount of added water (ml) 1 55 75 150 2 60 80 200 3 65 85 250 2.4 index measurement method 2.4.1 analysis of total acids the total acid content of black jujube was determined using the gb/t 12456-2008 method. black samples (20 g) were broken, dissolved in water at 80°c, and then placed in a boiling water bath for 30 min (shaken 2-3 times). after the solution was cooled, the volume was set to 250 ml. the sample was filtered, and 25 ml of the filtrate was added to 50 ml of water and titrated with 0.1 mol/l sodium hydroxide solution until the ph was 8.3. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 110 published by scholink inc. 2.4.2 analysis of reducing sugars the reducing sugar content of black jujube was determined using the gb 5009.7-2016 method. a sample comprising 2.5 g of jujube paste was dissolved in 50 ml of water and transferred to a 250 ml volumetric flask after stirring on a magnetic stirrer for 10 min. zinc acetate and potassium ferricyanide were added, 5 ml each, to a constant volume, shaken well, and allowed to stand for 30 min. the solution was filtered, the primary filtrate discarded, and the subsequent filtrate saved for later use. aliquots comprising 5.0 ml alkaline cupric tartrate solution and 5.0 ml alkaline cupric tartrate solution were absorbed, then put in a 150 ml conical flask, 10 ml of water and 2~4 glass beads added. the sample filtrate was used for titration, which was heated to boiling within 2 min, and the titration continued at a rate of 1 drop per 2s until the blue color just faded as the end point. 2.4.3 analysis of camp a sample comprising 2 g of black jujube paste was added to 80 ml water, stirred with a magnetic stirrer for 10 min, transferred to a 100-ml volumetric flask, and filled to the mark with water. the sample was liquid incubated at 80°c for ultrasonic extraction for 30 min. after cooling, the liquid was passed through a 0.45 μm pore-size membrane and analyzed by hplc using a sb-c18 column (150 mm x 2.1 mm, 3.5 m; shimadzu, japan). the mobile phase was composed methanol and 50 mm potassium dihydrogen phosphate (10:90, v/v). the flow rate was 1 ml/min. the column temperature was 30°c. the detection wavelength was 254 nm. the injection volume was 10 μl. 2.4.4 analysis of 5-hmf a sample comprising 5 g of black jujube paste was suspended in 10 ml methanol, after which a small amount (approx. 5 ml) of water was added. after magnetic stirring for 10 min, the mixture was transferred to a 50 ml brown volumetric flask and filled to the mark with water. then, the sample solution was ultrasonicated for 30 min and filtered to the sample bottle through a 0.45 μm organics filtering membrane. a total of 10 μl was injected into the hplc column. the analytes were separated on a intertsustain c18 column (250×4.6 mm, 5 μm; shimadzu, japan) at 35°c. the mobile phase was a methanol-water mixture (2:98, v/v). the absorbance wavelength for determination was 282 nm. 2.4.5 analysis of polyphenols this analysis was done based on a previous study[10] with some modifications as follows. 1 g of black jujube paste was dissolved in 30 ml 70% ethanol solution and stirred with a magnetic stirrer for 10 min, after which the volume was adjusted to 50 ml with 70% ethanol. the sample was then ultrasonicated at 66°c for 30 min and filtered, after which 0.2 ml of the sample solution was added to 0.5 ml of 100% folin phenol reagent diluted 1 time, and the mixture was evenly mixed. then, 1.5 ml of na2co3 solution with a mass fraction of 10% was added, and the mixture was mixed evenly with distilled water in a constant volume until 10 ml. the mixture was allowed to react at 75°c for 10 min, after which its absorbance at 760 nm was measured. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 111 published by scholink inc. 2.5 data processing and statistical analysis all experiments were conducted in triplicate. the experimental results were presented as means±se. spss 20.0 software (ibm corp., usa) was used for analysis, and origin 8.0 software (originlab corp., usa)was used for drawing charts. 3. results and discussion 3.1 the influence of blackening conditions on the content of various index compounds in black jujube 3.1.1 effect of blackening time on the biochemical contents of black jujubes table 2. influence of blackening time during the aging process of red jujubes on important biochemicals index blackening time 36h 48h 60h 72h 84h 96h total acids (g/kg) 19.76±0.00b 18.66±0.16a 19.59±0.00b 22.40±0.00c 23.86±0.00e 23.45±0.00d reducing sugars (g/100g) 71.6±0.00b 74.60±0.17d 75.67±0.29e 74.10±0.10c 71.6±0.00b 70.27±0.07a polyphenols (g/100g) 2.16±0.01a 2.30±0.09ab 2.32±0.13ab 2.39±0.38b 2.22±0.38ab 2.30±0.09ab 5-hmf (g/100g) 0.143±0.003a 0.177±0.006b 0.203±0.006c 0.290±0.010d 0.323±0.006e 0.353±0.006f camp g/100g 0.029±0.006e 0.026±0.001d 0.023±0.001c 0.021±0.000b 0.015±0.001a 0.014±0.001a note. in the same line, numbers marked with the same letters in the upper right corner indicate that there is no significant difference between groups (p > 0.05), while those without the same letters indicate that there is significant difference between groups (p < 0.05). figure 1. influence of blackening time on the moisture content of aged black jujubes www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 112 published by scholink inc. the influence of blackening time on the aging indexes of red jujubes is shown in table 2 and figure 1. blackening time was fixed at 36 h, 48 h, 60 h, 72 h, 84 h and 96 h. it can be seen from table 2 that the blackening time has a significant effect on the contents of total acids, reducing sugars, furfural, camp, flavonoids and water in aged jujubes, but has little effect on the polyphenol content. with the extension of blackening time, the total acid content decreased significantly at 36-48 h, but increased significantly after 48 h. the content of reducing sugars increased significantly between 36 and 60 h, but decreased significantly after 60 h. the content of polyphenols did not change significantly with the extension of blackening time. furfural content showed a significant increasing trend with the extension of blackening time (p < 0.05), the while camp content showed a decreasing trend and leveled off after 84 h. as can be seen in figure 1, the water content fluctuated greatly with the extension of blackening time, with the lowest content of 42% observed at 60 h. during the whole process, the total acid content increased, which may be caused by the fact that the reduced ketones generated by the maillard reaction can be lysed to produce acids, resulting in the increase of the total acid content (martins, jongen, & van boekel, 2000; troise, 2018; karbasi & madadlou, 2018). in addition, during the browning reaction, the increase of acids is also related to the production of carboxylic acids. for example, it was reported that carboxylic acids are produced by the oxidation of aldoses (sang, cho, yong, lee, & park, 2014). in addition, due to the presence of amino and carbonyl groups, organic acids may change during the maillard reaction. the increase of reducing sugars may be due to the fact that sucrose was decomposed into monosaccharides or changed into other disaccharides. our analysis showed that the sucrose content decreased continuously during the aging and blackening of jujube, and finally dropped to 0. therefore, sucrose may be converted into other reducing sugars during the blackening process. since reducing sugars are one of the reactants of maillard reaction, they were continuously consumed and their content decreased with the extension of blackening time. the 5-hmf increased with the extension of black time, but the accumulation rate was higher in the early stage and decreased in the later stages, which may be because 5-hmf is one of the sources of melanoid, the final product of the maillard reaction. 5-hmf was gradually consumed in the process of increasing melanoid accumulation in the later stages. camp was consumed in the non-enzymatic browning reaction during aging. the reducing sugar content reached the highest point at 75.67 g/100g after 60 h of blackening, whereby the polyphenols and camp still remained at a relatively high level, so as to better maintain the nutrients and functional components of jujube. in addition, the moisture content first increased with the increase of blackening time, and then decreased to 42% at 60 h, which was suitable for subsequent processing. however, the total acid index maintained an upward trend, and too much acidity was not conducive to subsequent processing. furfural has also had a rising trend, but there is still a controversy surrounding the impact of 5-hmf at present (durling, busk, & hellman, 2009), and how it affects the taste of the final product, especially in terms of bitterness. therefore, blackening jujube for 60 h had the best quality effect. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 113 published by scholink inc. 3.1.2 effect of blackening temperature on the biochemical composition of black jujube table 3. influence of blackening temperature on the aging process of red jujubes index blackening temperature 65°c 70°c 75°c 80°c 85°c 90°c total acids (g/kg) 13.81±0.01a 15.03±0.01b 17.59±0.02c 20.34±0.01d 21.72±0.17e 25.29±0.15f reducing sugars (g/100g) 75.59±0.03e 74.41±0.05d 67.40±0.03b 77.54±0.03f 71.61±0.03c 63.06±0.00a polyphenols (g/100g) 3.64±0.11a 3.50±0.06a 3.43±0.32a 5.47±0.10c 4.46±0.07b 4.25±0.04b 5-hmf (g/100g) 0.043±0.001a 0.097±0.003b 0.171±0.006c 0.256±0.012d 0.418±0.005e 0.698±0.027f camp g/100g 0.034±0.001e 0.027±0.002d 0.021±0.002bc 0.023±0.001c 0.018±0.001b 0.014±0.000a note. in the same line, the numbers marked with the same letters in the upper right corner indicate that there is no significant difference between groups (p > 0.05), while those without the same letters indicate that there is significant difference between groups (p < 0.05). figure 2. influence of blackening temperature on the moisture content of aged black jujubes the influence of different blackening temperatures on camp, furfural, total acid, reducing sugar, polyphenol and moisture indexes was studied, as shown in table 3 and figure 2. the blackening temperature was fixed at 65°c, 70°c, 75°c, 80°c, 85°c and 90°c. as shown in table 3, with the increase of blackening temperature, the content of total acids and furfural showed a significant increasing trend. it is generally believed that 5-hmf is a product of dehydration and decomposition of fructose or glucose under acidic conditions, and it is a common intermediate product of the maillard reaction, ascorbic acid oxidation reaction and caramelization. studies have pointed out that the accumulation of 5-hmf is strongly correlated with the browning rate. however, 5-hmf is produced at high temperatures and is the most important pollutant among heat-induced products, especially in www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 114 published by scholink inc. baked goods (capuano & fogliano, 2011). the reducing sugar content increased significantly with the increase of blackening temperature. at 80°c the content was 77.54 g /100g. the increase of the reducing sugar content may be associated with the decomposition of other sugars. studies have shown that the reducing sugar content is directly proportional to the browning rate in the maillard reaction. in the later stages, with the increase of blackening temperature, the maillard reaction was accelerated, melanoid-like substances accumulated more, and the color of the jujube was deepened (majid, mehdi, & moein, 2019). for the polyphenol content, the rise of temperature was within the range 65-75°c, but the maximum of 5.47 g/100g reached at 80°c was significantly lower. at temperatures higher than 85°c the content stabilized. with the increase of temperature, the increase of polyphenol content may be caused by the formation of maillard reaction products or by the cleavage of the esterified and glycosylated compounds (kavita et al., 2015). overall, camp displayed a reducing trend, but increased somewhat at 80°c, in spite of generally faster decomposition at higher temperatures. as can be seen in figure 2, with the increase of blackening temperature, water content reduced first reduced and then rose again, but was significantly lower at 75°c. this may be because the maillard reaction rate was faster in the early stage and the water consumption was faster. when the temperature exceeded 80℃, the maillard reaction was weakened by the temperature, and the evaporation of water increased with the temperature in the later stage, resulting in the water content of jujube decreasing. therefore, the jujubes blackened at 80°c were optimal, with the functional material content maintaining a high level. 3.1.3 effect of added water amount on the biochemical contents of black jujube table 4. influence of added water amount on the aging process of red jujubes index added water amount 100ml 200ml 300ml 400ml 500ml 600ml total acids (g/kg) 21.69±0.12e 21.94±0.01f 21.15±0.00d 18.77±0.01c 19.26±0.01b 17.15±0.08a reducing sugars (g/100g) 63.78±0.02a 70.47±0.09c 66.92±0.11b 72.68±1.66d 76.10±0.16e 77.27±0.13e polyphenols (g/100g) 3.45±0.11b 4.37±0.04e 3.07±0.05a 3.61±0.03bc 4.15±0.06d 3.63±0.03c 5-hmf (g/100g) 0.337±0.010e 0.314±0.006d 0.281±0.002c 0.264±0.002b 0.260±0.005b 0.245±0.000a camp g/100g 0.017±0.002a 0.018±0.001a 0.019±0.001a 0.020±0.001a 0.027±0.002b 0.026±0.002b note. in the same line, the numbers marked with the same letters in the upper right corner indicate that there is no significant difference between groups (p > 0.05), while those without the same letters indicate that there is significant difference between groups (p < 0.05). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 115 published by scholink inc. figure 3. influence of water addition on the moisture content of aged black jujubes the effects of different amounts of added water on camp, furfural, total acid, reducing sugar, polyphenols and water indexes in the blackening process were studied, as shown in table 4 and figure 3. the amount of added water was fixed at 100 ml, 200 ml, 300 ml, 400 ml, 500 ml and 600 ml. as shown in table 4, with the increase of water content, the total acids and furfural showed a significant downward trend, i.e., the higher the humidity, the lower the content. there was a significant difference of reducing sugars between 100 ml and 400 ml of water added, but it tended to be stable after 500 ml. the polyphenol content fluctuated greatly, and the increase was the most obvious at 200 ml, reaching 4.37 g/100g. there was no significant difference in the content of camp at 100-400 ml and 500-600 ml. as can be seen from figure 3, with the increase of total water content, the water content of the jujube also showed an upward trend. when the amount of added water exceeded 400 ml, the black jujube not only had a large water content, but also had a large amount of residual water in the blackening container, resulting in an incomplete appearance and soft collapse of the black jujube. when 100 ml of water was added, the water content of black jujubes was 28%, and the black jujubes were hard and bitter. to sum up, when the water content was 200 ml, the water content of jujube was appropriate, and cyclic adenosine monophosphate, reducing sugars and polyphenol were all at high levels. therefore, the water content of jujube was the most appropriate with the addition of 200 ml of additional water. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 116 published by scholink inc. 3.2 optimization of the blackening process of red jujube using an orthogonal experiment table 5. the orthogonal experimental design for assessing different blackening conditions level a b c 5-hmf (g/100g） camp (g/100g） total acids (g/kg） reducing sugars (g/100g） polyphenols (g/100g） 1 1 1 1 0.103 0.0137 17.09 76.7 2.71 2 1 2 3 0.234 0.0116 22.48 72.2 3.605 3 1 3 2 0.391 0.0082 24.69 66.3 3.122 4 2 1 2 0.135 0.0150 19.44 72.5 3.42 5 2 2 1 0.277 0.0124 23.22 72.3 3.302 6 2 3 3 0.395 0.0092 24.59 61.3 3.296 7 3 1 3 0.157 0.0158 19.91 76.7 2.799 8 3 2 2 0.325 0.0100 25.13 70.2 3.509 9 3 3 1 0.475 0.0217 24.99 66.2 3.525 5-hmf k1 0.728 0.395 0.855 k2 0.807 0.836 0.851 k3 0.957 1.261 0.786 r 0.199 0.866 0.069 camp k1 0.0335 0.0445 0.0478 k2 0.0366 0.034 0.0332 k3 0.0475 0.0391 0.0366 r 0.014 0.0105 0.0146 total acids k1 64.26 56.44 65.3 k2 67.25 70.83 69.26 k3 70.03 74.27 66.98 r 5.77 17.83 3.96 reducing sugars k1 215.2 225.9 215.2 k2 206.1 214.7 209 k3 213.1 193.8 210.2 r 9.1 32.1 6.2 polyphenols k1 9.437 8.929 9.537 k2 10.018 10.416 10.051 k3 9.833 9.943 9.7 r 0.581 1.487 0.514 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 117 published by scholink inc. table 6. variance analysis table of furfural for orthogonal test design factor sum of squares freedoms mean square error f sig. correction model 0.135a 6 0.023 502.583 0.002 intercept 0.690 1 0.690 15409.588 0.000 a 0.009 2 0.005 100.722 0.010 b 0.125 2 0.063 1395.859 0.001 c 0.001 2 0.001 11.169 0.082 error 8.956e-005 2 4.478e-005 total 0.825 9 corrected total 0.135 8 according to the range and variance analysis shown in tables 5 and 6, the primary and secondary order of each influencing factor is: b blackening temperature > a blackening time > c water addition amount. the blackening temperature and blackening time had a significant effect on furfural, while the addition of water had no significant effect. with furfural as index, an orthogonal test was done to determine the best technology of blackening for a1b1c2, which were found to encompass a blackening time of 55 h, blackening temperature of 75°c, and added water of 200 ml. table 7. anova table of cyclic adenosine monophosphate for orthogonal test design factor sum of squares freedoms mean square error f sig. correction model 9.333e-005a 6 1.556e-005 0.716 0.682 intercept 0.002 1 0.002 70.737 0.014 a 3.605e-005 2 1.802e-005 0.830 0.547 b 1.838e-005 2 9.190e-006 0.423 0.703 c 3.891e-005 2 1.945e-005 0.896 0.528 error 4.345e-005 2 2.172e-005 total 0.002 9 corrected total 0.000 8 according to the range and variance analysis shown in tables 5 and 7, the primary and secondary order of each influencing factor was: c water addition amount > a blackening time >b blackening temperature. the effect of blackening temperature, blackening time and water content on camp was not significant. with camp as index, an orthogonal test was conducted to determine the best technology of blackening for a3b1c1, which was found to encompass a blackening time of 65 h, blackening temperature of 75°c, and added water of 150 ml. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 118 published by scholink inc. table 8. total acid variance analysis table for orthogonal test design factor sum of squares freedoms mean square error f sig. correction model 67.831a 6 11.305 109.511 0.009 intercept 4513.152 1 4513.152 43717.976 0.000 a 5.551 2 2.776 26.887 0.036 b 59.646 2 29.823 288.890 0.003 c 2.634 2 1.317 12.756 0.073 error 0.206 2 0.103 total 4581.190 9 corrected total 68.037 8 according to the range and variance analysis shown in tables 5 and 8, the primary and secondary order of each influencing factor is: b blackening temperature > a blackening time > c water addition. the blackening temperature and blackening time had a significant effect on the total acid content, while the addition of water had no significant effect. with total acids as index, an orthogonal test was conducted to determine the best technology of blackening for a1b1c1, which was found to encompass a blackening time of 55 h, a blackening temperature 75°c, and added water of 150 ml. table 9. anova table of reducing sugars for orthogonal test design factor sum of squares freedoms mean square error f sig. correction model 199.307a 6 33.218 7.759 0.119 intercept 44718.151 1 44718.151 10445.454 0.000 a 15.136 2 7.568 1.768 0.361 b 176.962 2 88.481 20.668 0.046 c 7.209 2 3.604 0.842 0.543 error 8.562 2 4.281 total 44926.020 9 corrected total 207.869 8 according to the range and variance analysis shown in tables 5 and 9, the primary and secondary order of each influencing factor is: b blackening temperature > a blackening time > c water addition. the time of blackening and the amount of water added had no significant effect on reducing sugars, while the temperature of blackening had a significant effect. using separate raw sugars as an index, an orthogonal test was conducted to determine the best technology of blackening for a1b1c1, which was found to encompass a blackening time of 55 h, blackening temperature of 75°c, and water addition of 150 ml. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 119 published by scholink inc. table 10. anova table of orthogonal test design for polyphenols factor sum of squares freedoms mean square error f sig. correction model 0.490a 6 0.082 0.503 0.782 intercept 95.310 1 95.310 588.045 0.002 a 0.059 2 0.029 0.181 0.847 b 0.385 2 0.192 1.187 0.457 c 0.046 2 0.023 0.142 0.876 error 0.324 2 0.162 total 96.123 9 corrected total 0.814 8 according to the range and variance analysis shown in tables 5 and 10, the primary and secondary order of each influencing factor is: b blackening temperature > a blackening time > c water addition. the effect of blackening time, blackening temperature and water content on polyphenols was not significant. with the polyphenol content as index, an orthogonal test was conducted to determine the best technology of blackening for a2b2c2, which encompassed a blackening time of 60 h, blackening temperature of 80°c, and water addition of 200 ml. in conclusion, the three factors of blackening time, blackening temperature and water addition had no significant influence on the camp and polyphenol indexes. therefore, furfural, total acid, reducing sugar indices were used as the basis to determine the optimal processing parameters for a1b1c1, which were a blackening time of 55 h, blackening temperature of 75°c, and water addition of 150 ml. 4. conclusion red jujube is widely planted in china, but there are few reports on the production technology of black jujube. in this paper, an orthogonal l9(3)3 test was used to optimize the production process of black jujube. the results revealed optimum processing parameters as follows: aging blackening time 55 h, blackening temperature 75°c, added water 150 ml. the black jujube produced under the optimal technological conditions had a strong fragrance, sweet and sour taste, and contents of various functional substances reaching (dry matter meter): camp 0.0137 g/100g, 5-hmf 0.103 g/100g, polyphenols 2.71 g/100g, total acids 17.09 g/kg, reducing sugars 76.7 g/100g, and water content 26%. acknowledgments this work was supported by the shandong province key research and development fund (2016gnc113015, 2019gnc106061) and shandong province major application of technological innovation projects. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 120 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(2016). chemical and volatile composition of jujube wines fermented by saccharomyces cerevisiae with and without pulp contact and protease treatment. food science and technology, 36, 204-209. https://doi.org/10.1590/1678-457x.0011 almansa, s., hernández, f., legua, p., nicolás-almansa, m., & amorós, a. (2016). physicochemical and physiological changes during fruit development and on-tree ripening of two spanish jujube cultivars (ziziphus jujuba mill.). journal of the science of food and agriculture, 96, 4098-4105. https://doi.org/10.1002/jsfa.7610 yue, y. et al. (2015). wild jujube polysaccharides protect against experimental inflammatory bowel disease by enabling enhanced in testinal barrier function. food & function, 6, 2568-2577. https://doi.org/10.1039/c5fo00378d ji-eun, k., min-ah, k., jung-seok, k., dong-cheol, p., & sam-pin, l. 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(2019). the maillard reaction products as food-born antioxidant and antibrowning agents in model and real food systems. food chemistry, 644-660. https://doi.org/10.1016/j.foodchem.2018.09.083 kavita, sharmaa. et al. (2015). temperature-dependent studies on the total phenolics, flavonoids, antioxidant activities, and sugar content in six onion varieties. journal of food and drug analysis, 23, 243-252. https://doi.org/10.1016/j.jfda.2014.10.005 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 1, 2019 www.scholink.org/ojs/index.php/fsns 9 original paper proximate and sensory evaluation of different zobo-moringa blends packaged in tea bags ibeabuchi, j.c.1, okafor, d.c.1*, agunwah, i.m.1, agim, o.a.1, nwosu, m.o.1, eluchie, c.n.1 & aneke, e.j.1 1 department of food science and technology, federal university of technology, imo state, nigeria * okafor d.c., department of food science and technology, federal university of technology, p.m.b. 1526 owerri, imo state, nigeria received: november 4, 2018 accepted: november 22, 2018 online published: february 12, 2019 doi:10.22158/fsns.v3n1p9 url: http://dx.doi.org/10.22158/fsns.v3n1p9 abstract different blends of roselle calyx and moringa leaves (90:10%. 80:20%, 70:30%, and 50:50% respectively) were used in processing the novel instant “zobo-moringa mix” by drying, grinding and mixing of zobo calyx, moringa leaves, ginger, cloves and orange flavour. various recipes were formulated using linear programming, after which sensory evaluation was carried out to obtain an acceptable formula. dried moringa leaves were added at different proportion by varying the amount of zobo calyx used. all samples were packaged in teabags. proximate analysis was carried out on the samples. the results of the proximate analysis showed that the proximate composition of the samples was significantly higher (p < 0.05) in protein and it increases as the proportion of moringa increases from 2.325% to 28.05%. for the crude fiber, there was a decrease in its composition from 14.00% to 5.70% as the proportion of moringa increases. it is significantly low in carbohydrate from 59.98% to 29.62% as the proportion of moringa increases. the results of the sensory evaluation of the samples showed that the samples into which moringa were incorporated were generally more accepted than the conventional zobo sample (control). keywords zobo, moringa, mix, sample, blend 1. introduction the major nigerian local beverages are burukutu (sorghum beer), kunnuzaki (millet food drink), pito (fermented alcoholic beverage from sorghum or maize), palmwine, adoyo (ripe pineapple juice and supernatant derived from ogi), ogogoro (distilled palm wine or local gin), nunu (fermented skim milk), www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 10 published by scholink inc. fura da nunu (fermented skim milk with millet dough), zobo (extracts of calyx of hibiscus sabdariffa), wara (cheese whey), etc. zobo drink isa traditional non-alcoholic beverage which is consumed in most part of nigeria, mostly in northern part of nigeria (osuntogun, 2004). the zobo drink is a red liquid drink and taste like fruit punch, served as a fair source of vitamin a, riboflavin, niacin, calcium and iron (qi et al., 2005), and is low in sugar content. it is extracted from the dried reddish purple calyces of the plant hibiscus sabdriffa (scott, 2003). hibiscus sabdariffa (roselle) is an annual herb that is grown in the tropics and it is widely cultivated in nigeria mainly in the northeastern and middle-belt regions (bolade et al., 2009; nwafor & ikenebomeh, 2009; yadong et al., 2005; and omemu et al., 2006). moringa leaf powder is an excellent nutritional supplement and can be added to any dish (fuglie, 2001).the shelf life of any drink is dependent on the packaging material used; the use of teabags proves to be a means of preventing loss of quality of products, inhibitmicrobial contamination, preventing discolouration of the drink, and above all permitting large-scale production and preservation of the beverage for a longer period with maximum retention of nutritive value (vermeiren et al., 1999). the production process of hibiscus sabdariffa (zobo) drink has not been mechanized nor standardized and the shelf life of the drink is less than two days due to microbial attack, making it loose its physico-chemical and organoleptic quality (nwafor & ikenebomeh, 2009; and olawale, 2011). this work is therefore aimed at producing instant “zobo-moringa mix” which can keep longer and can be prepared easily by extraction using hot water. 2. materials 2.1 sample collection the dried zobo calyx, moringa leaves, sweetener, flavours and other ingredient used in this work were bought from the main market (eke-onuwa) in owerri and transported to futo where the production and analysis of the product was carried out. the materials were procured in its wholesome condition and reasonable quantities. 3. methods 3.1 preparation of raw materials for the developed instant zobo-moringa beverage 3.1.1 processing of hibiscus sabdariffa calyxes the hibiscus sabdariffa calyces were obtained and the dirt was sorted out by winnowing. it was mixed and dried in the oven to further reduce the moisture content. the dried calyces were then crushed to a mesh size of 200 micron and sieved. these tea size particles were then stored in an air tight vessel. 3.1.2 processing of moringaoliefera leaves powder leaves were dried at room temperature inside a room protected from light (to prevent the loss of vitamins) and protected from dust and pests (to prevent contamination). dried leaves were made into powder using a burr mills (motor driven). the powder is sifted to remove any remaining stems. it was stored in airtight containers protected from heat, humidity and light. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 11 published by scholink inc. 3.1.3 processing of ginger and cloves the ginger bulbs were dried under sunlight and it was grounded into powder using an attrition mill. the cloves were also grounded into powder using the attrition mill. the entire grounded ingredients were stored in an air tight container, in other to prevent them from losing their volatile components. figure 1. image showing processed ingredients 3.2 recipe formulation linear programming is a mathematical technique used in computer simulations to find the best possible solution in allocating limited resources or ingredient to achieve maximum profit and cost. it can be applied to a wide variety of fields of study and has proved useful in planning, routing, scheduling, assignment and designing, such as transportation or manufacturing industries. this model was used to calculate the formulation needed to obtain the desired product. five recipes were obtained and these were the result: www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 12 published by scholink inc. table 1. formula for instant “zobomix” in teabag derived using linear programming formulations in % ingridient 1 2 3 4 5 roselle calyx 58.80 56.70 54.20 52.50 50.70 ginger bulb 23.80 25.70 26.55 27.75 28.55 cloves 12.85 13.05 14.70 15.20 16.20 orange flavor 4.55 4.55 4.55 4.55 4.55 table 2. samples and their different proportions samples ingredients zm90 zm80 zm70 zm50 zcon zobo 90% 80% 70% 50% 100% moringa 10% 20% 30% 50% ginger 26.12% 26.12% 26.12% 26.12% 26.12% cloves 13.38% 13.38% 13.38% 13.38% 13.38% orange flavor 4.6% 4.6% 4.6% 4.6% 4.6% key; zcon= zobo control, zm90= zobo:moringa 90:10, zm80= zobo:moringa 80:20, zm70= zobo:moringa 70:30, zm50= zobo:moringa 50:50 4. proximate analysis the proximate analysis was carried out on the roselle-moringa blend. they were analyzed chemically according to the official methods of analysis described by association of official analytical chemists (a.o.a.c, 1990). 4.1 determination of moisture content the moisture content was determined by weighing out 2 g of each of the sample into a dry petri dish of a known mass, charged into the oven at temperature of 105oc and heated for 3 hours. the dried samples were then withdrawn from the oven and placed in a desiccator to cool. they were weighed using the analyticalbalance (electronic) and the whole process was repeated until a constant mass was obtained. the difference in mass as percentage (% moisture) was calculated thus: %moisture = where; m1 = mass of dish m2 = mass of dish + sample before drying m3 = mass of dish + sample after drying. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 13 published by scholink inc. 4.2 determination of crude fat a soxhlet extraction unit was setup with a reflux condenser. a small round bottom flask was weighed after washing and drying, and half filled with light petroleum ether (boiling point 40-60oc) and fixed into the unit. two (2) grams of each of the samples were wrapped with a whitman filter paper and gradually lowered into the thimble which was fitted into the cleaned, dried and weighed round bottom flask containing 120 ml of petroleum ether. samples were slowly heated with heating mantle for 5 hours. refluxed petroleum ether was recovered and the flask containing the fat and oil was cooled in the desiccator and reweighed after drying. by difference, the mass of oil extracted was determined and thus expressed as percentage; % crudefat = 4.3 determination of crude protein the keldjhal method as described by aoac (1990) was used. the total nitrogen was determined and 6.25 were used to multiply to obtain the protein. two (2) grams of each of the samples was boiled in 10ml of concentrated h2so4 in the presence of selenium catalyst. boiling was done under a fume cupboard until a clear solution was formed. the digest was transferred into a volumetric flask containing a 100 ml of distilled water and 10ml of it was mixed with equal volumes of 45% naoh solution and was poured into a keldjhal distillate apparatus. on distillation of the mixture; the distillate was collected in a 100ml of 4% boric acid solution containing 3 drops of a mixed indicator (methyl red and bromocresolgreen). a total of 50ml distillate was collected and titrated against 0.02n h2so4 solution. titration was done from green to a deep red end point. a reagent blank was determined as discussed above but without the sample. the protein content was calculated. 4.4 determination of crude fiber two (2) grams of each sample were defatted and boiled in 200 ml of 1.25 h2so4 for 30 minutes. the boiled samples were washed with hot water using a twofold muslin cloth to retain particles. the retained particles were returned to the flask and boiled again in 200 ml of 1.25 naoh solution and was again washed with hot plate and allowed to dry before been transferred to the oven to dry at 105oc to a constant weigh and was subsequently placed in muffle furnace at 550oc for 4hours and finally cooled in a desiccator and reweigh. by difference in mass, the mass of the fiber was determined and was given by; % crudefibre = where; w1 = weight of sample before incineration w2 = weight of sample after incineration w3 = weight of original sample www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 14 published by scholink inc. 4.5 determination of ash content five (5) grams of the sample was put in a crucible, ignited and tarred. the crucible was placed in a drying oven at 100°c for 4 hours and then transferred to a cool muffle furnace as the temperature was increased to 550°c ± 5°c. the temperature was maintained for 8 hours until white ash was obtained. the crucible was placed in a desiccator with the aid of thongs, to cool and then weight was determined. the percentage (%) ash was calculated as thus; % ash= 4.6 determination of carbohydrate the carbohydrate content was determined by the difference method. 100% a + b + c + d + e = % carbohydrate where; a = % moisture b = % ash c = % crude fibre d = % fat e = % crude protein 4.6 sensory evaluation sensory evaluation was carried out using an 18-man panelist to assess the organoleptic attributes of the roselle-moringa blend samples. the organoleptic attributes assessed were; colour, taste, aroma, mouth-feel and general acceptability. the panelists were selected randomly from the staff and students of federal university of technology, owerri. the sensory evaluation was conducted using a 9-point hedonic scale as described by ihekoronye and ngoddy (1985), where scoring scale ranges from 9 = liked extremely to 1 = disliked extremely. 4.7 statistical analysis the results of the proximate analyses and the sensory evaluation were computed and a one-way analysis of variance (anova) and fishers least significant difference (lsd) was used to establish the significance differences among the value at 0.05 level of confidence. the statistical analysis was computed using the program, minitab 16.2.1 (2010). 5. results and discussion 5.1 recipe formulation of instant zobo drink five formulas of instant “zobo mix” (table 4) were selected using linear programming. the result from the sensory evaluation using the ranking-for-preference test showed that formula 3 was the most generally accepted of the lot; with a mean score of 7.994±0.938, the panelists liked the product very much. formula 3 was however similar to formula 2 and formula 4. formula 5 was accepted at a similar level to formula 4. formula 1 was the least accepted of the lot, its mean score of 5.278±1.274 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 15 published by scholink inc. meant the panelists were indifferent to the product. the acceptance level of formula 1 was significantly different from other formulations produced. the sensory analysis showed formula 3 to be the best in terms of taste, aroma, mouthfeel and general acceptance. it lagged behind formula 2, formula 4 and formulae 5 in terms of colour but was still deemed similar to them. formula 1 was the least appreciated of the lot in all criteria with a similarity only in colour and in aroma to any of the products. the tabular representation of the results of the sensory evaluation of the various recipes done by the 18-man panelists is shown in table 3, while the optimum formula obtained from linear programming is given in table 4. table 3. sensory evaluation carried out on the recipes formulations formulations taste colour aroma mouth-feel general acceptabili ty 1 6.000b 7.278a 6.944b 6.444a 6.389b 2 7.000ab 7.556a 7.000ab 6.722a 7.500a 3 7.611a 6.944a 7.889a 7.222a 7.944a 4 7.000ab 7.556a 7.000ab 6.722a 7.500a 5 4.611c 6.389b 6.278b 5.167b 5.278c lsd 1.091 0.836 0.937 0.983 0.904 table 4. ingredients used for the instant “zobo mix” ingredient quantity used (%) roselle calyx 55.40 ginger bulbs 26.12 cloves 13.88 orange flavor 4.60 5.2 proximate result of the zobo-moringa mix the tabular representation of the results of the proximate analysis carried out on the zobo-moringa mix for the different samples is shown in table 3.2. from the result of the proximate analysis (see table 5 for codes) for the fat analysis, it was observed that zcon (6.30 ± 0.02) (control) is significantly different (p < 0.05) from the samples that were mixed (i.e., zm90, zm80, zm70, and zm50). also there was no significant difference (p > 0.05) between zm90 (8.50 ± 0.02) and zm80 (11.40 ± 0.01), while zm70 (14.50 ± 0.01) and zm50 (17.10 ± 0.01) was significantly different (p < 0.05). for protein the zcon (control) (2.325 ± 0.03) ranked lowest while zm50 (28.050 ± 0.21) ranked highest and this is attributed to the high protein content of the dried moringa leaves. also samples with moringa (i.e., zm90, zm80, zm70, and zm50) were significantly different (p < 0.05) from the control (zcon), and this difference increases as the composition of moringa increases. there was no significant difference (p > 0.05) in the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 16 published by scholink inc. moisture content in all samples and this is as a result that the ingredient used was uniformly dried and the samples were prepared from the stock. for the ash, there was significant difference (p < 0.05) amongst the samples. zcon (6.4 ± 0.09) has the least mean score, while zm50 (7.75 ± 0.18) ranks the highest. it was observed that the higher the proportion of the blend the greater the ash content of the sample. for carbohydrate: zcon (59.98±0.61) has the highest mean value than other samples (i.e., zm90, zm80, zm70, and zm50), and these values are significantly different (p < 0.05). for fibre the samples (i.e., zm90, zm80, zm70, and zm50) are all significantly different (p < 0.05) and their mean values are lower than zcon (control). table 5. mean value of proximate analysis carried out on the samples sample s moisture protein fiber cho fat ash zcon 11.00a±0.05 2.32a ±0.03 14.00a±0.04 59.98a±0.61 6.30e±0.02 6.40d ±0.1 zm90 12.28a ±1.25 19.48b ±0.48 8.50b±0.02 44.45b±1.7 8.50d±0.02 6.80c±0.8 zm80 10.75a ±0.25 23.44c ±0.04 7.00b±0.03 40.37c±0.33 11.40c±0.01 10bc±0.1 zm70 11.00a ±0 25.43d ±0.04 6.30bc±0.11 35.37d±0.04 14.50b±0.01 7.40b±0.1 zm50 11.75a ±0.25 28.05e ±0.15 5.70c±0.1 29.62e±0.46 17.10a±0.01 7.75a±0.05 lsd 2.263 0.816 0.248 3.017 0.042 0.335 key; zcon= zobo control, zm90=zobo:moringa 90:10, zm80= zobo:moringa 80:20, zm70= zobo:moringa 70:30 zm50= zobo:moringa 50:50. 5.3 sensory evaluation on the samples (zobo-moringa blend) the results of the sensory evaluation are shown in table 5. there was no significant difference (p < 0.05), in colour among the samples, but there was a significant difference between the four samples and control. the highest value of7.90 was obtained from sample zcon, while the lowest value was obtained from the zm50. generally, the value of the zcon were greater than other samples, this may be as a result green pigmentation of the chlorophyll present in the moringa leaves which makes the colour brighter and sharper. for the aroma, there was a significant difference (p < 0.05), between zm50 and zcon, zm90 and zm50, but there was no significant difference (p < 0.05), between zm70 and zm80, zm70 and zm50, zm70 and zm90, zm80 and zm90, zm80 and zcon. for taste, there was no significant difference (p < 0.05) among the samples, but there was a significant difference between the samples and the control. for the mouth feel, there was no significant difference between sample zm90, zm80 and zm50 but there was a significant difference (p < 0.05), between samples zm50, zm70, zm80, zm90 and zcon; there was also a significant difference between zm80 and zcon. for the general acceptance there was no significant difference among the samples, also there was no significant difference (p < 0.05) between the samples and the control, this may be attributed to the same recipe (ginger, close and flavour) used for the samples, also the packaging in teabags makes the products more acceptable to the panelists. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 17 published by scholink inc. table 6. mean values sensory evaluation of the various samples sample colour aroma taste mouthfeel general zm90 7.80a ±0.87 6.70 b ±0.90 8.6 a ±0.49 8.0 a ±0.78 8.70a ± 0.46 zm80 7.70a ±1.01 6.80ab ±0.60 8.70a ±0.46 7.9 a ±0.54 8.30 a ±0.46 zm70 7.70a ±0.46 7.00ab ±0.89 8.70 a ±046 7.3 b ±0.64 8.60a ± 0.49 zm50 7.00b± 0.63 7.30 a ±0.90 8.70a ±0.46 7.4 a ±0.49 8.70 a ±0.46 zcon 7.90a ±1.04 6.30bc ±0.46 7.90b ±0.54 6.50c ± 0.81 7.60 a ±0.49 lsd 0.449 0.574 0.366 0.640 key; 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(2005). biological characteristics of plants. nutritional and medicinal journal, 1-17. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 4, 2019 www.scholink.org/ojs/index.php/fsns 133 original paper maintain the chili colour of a sea-food dipping sauce in product processing w. puminat1* 1 division of food chemistry and physic, institution of food research and product development, kasetsart university, p.o.box 1043, kasetsart bangkok, 10900 thailand received: october 24, 2019 accepted: november 5, 2019 online published: november 15, 2019 doi:10.22158/fsns.v3n4p133 url: http://dx.doi.org/10.22158/fsns.v3n4p133 abstract products of sea-food dipping sauce were prepared by sugar, water, acid, salt with chili and herbs. in the experiments, products were prepared and designed on a factor model for the study on the program. they were designed as factorial 4×2×4 by management with rbcd on conditions model of blanching, heating and cooling for the study on chili colour. the results of the experiments performed on physicochemical properties of product such as brix 50.0-66.9 ph 3.52-4.29, aw 0.814-0.879, solid content 2.80-5.75 g/100g, chlorophyll a 0.20-1.66, chlorophyll b 1.38-4.13. colour value of products was l*29.99-23.97, a*-2.56-3.0 and b*8.28-1.21. the green value of products was preserved green colour for processing on blanching, short-time heating and all cooling. the color preservation of chili depended on the preparation process. blanching and cooling supported to preserve the colour and decreasing of decay texture. usage chemicals as ca2+ and mg2+ treatment.in processing of products were preserved a green colour more than no chemical treatment. blanching, cooling and short time for cooking were product development in order to preserve the green colour. the stability of the color of product was improved by blanching and cooling including boiling with short time. keywords chilli color, dipping sauce, color preservation 1. introduction chlorophyll is a green plant pigment that is important function of plant physiology as well as the possible health effects. chlorophyll is found in the chloroplasts of plants. there are various types of chlorophyll structures but plants contain chlorophyll a and b. chlorophyll a has blue-green color. chlorophyll b has green-yellow color. chlorophyll has antioxidant and anti-inflammatory properties that prevent chronic diseases such as cancer. a molecular structure of chlorophyll is similar to heme www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 134 published by scholink inc. chlorophyll has magnesium as its central metal ion, and the large organic molecule to which it bonds is known as a porphyrin. heme consists of a porphyrin similar to that in chlorophyll with at its center. heme is bright red, the pigment that characterizes red blood. in the red blood cells of vertebrates, heme is bound to proteins to form hemoglobin. heme is the red pigment of blood in humans and animals whereas in the central atom of heme is an iron (ii) ion. the metal in the center of chlorophyll molecule is magnesium. it makes chlorophyll remains green. the sea salt in cooking composes of sodium or magnesium ions replaces in food it will also have green vegetables are fresh. therefore, the addition of salt to the vegetables boiling are preserved green vegetables, it makes pretty fresh. if we boiled vegetables for a long time the vegetables had been a pale color. in acid conditions, added lemon juice in vegetable for cooking changes color from green to brown. porphyrin structure of chlorophyll replaces with hydrogen ion in reaction and changes to a reddish brown. frozen vegetables should be blanched and decreased temperature with cold water such that green vegetables are fresh longer. to add a little oil in vegetable, it will coat the cell walls of vegetables that are boiled. more flexible and the surface of the vegetables are even more luster. it makes a green shadow more. chili is an ingredient to enhance the color, flavor and aroma in food. it is also used as economic plant for medicinal plant and product food. several types of food products contain chili such as chili dipping sauce, chili sauce and canned food. some are used as a cooking ingredients and main ingredient is chili pepper and paste chili. they are used to season a dish after preparation. the chlorophyll green gives fresh color for various food. colour of chlorophyll has unique characteristics that can degrade through enzymatic and non-enzymatic reactions. the maillard reaction and oxidation make change to dark brown. usage high-temperature processing of the product degraded green colour through several processes. chlorophyll degradation progresses rapidly as the chlorophyll structure changes into its derivative compounds that result in its green color. the discoloration of green chlorophyll change into dark-green, yellow or black. one of the most important qualities is control parameters in process of industry. 2. materials and methods 2.1 processing of a sea food dipping sauce from chili 2.1.1 preparation of chili by chemical treatments for process 5% of chemicals (ca(oh)2, mgo, mgcl2 and cacl2) were prepared by dissolve with distillation water. chili was soaked in chemicals of the following solutions over night or about 5hrs. they were eluted with filtered water 2l for three times. they were drained until dry and packed in cool stores for the next to product preparation. 2.1.2 steps on the preparation of product to boil the mixing of water and sugar until boiling added acid and salt added herbs and chili (blanching or no blanching) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 135 published by scholink inc. to cook by heating and cooling or no cooling 2.1.3 study on temperature and time on the process blanching was a heat treatment by steaming and followed by cooling with low temperature or room temperature. blanching, heating and cooling were important for processed products. there was study on time of blanching as 0, 1 and 3 min for the difference of chili colour before processing of product. in the processing, they were divided into 6 groups as the following: bcc = blanching and cooling with cooling after process heating bcn = blanching and cooling with no cooling after process heating bnc = blanching and no cooling with cooling after process heating bnn = blanching and no cooling with no cooling after process heating nbc = no blanching with cooling after process heating nbn = no blanching with no cooling after process heating 2.1.4 experimental designs for the product of processing in the experiments, products were designed on a model of preparation and factor of conditions for the study on chili colour. they depended on chili groups, bleaching, heating and cooling. the experimental design was factorial 4×2×4 by management with rbcd model as 2 blocks. the first factor was four groups of fixing colour as ca(oh)2, mgo, mgcl2, and cacl2. the second was two types of bleaching and no bleaching. the third was bleaching by with or without cooling and with or without cooling after heating. products of processing were kept on study the quality and property of characteristics. 2.2 physicochemical properties 2.2.1 dry weight of product and preparation of sample solid the sample solid was prepared by filtration and drying. the sample was filtrated with gauze cloth. residue from the filtration was made for drying with vacuum oven at 50°c 7hrs. the drying sample was kept in vial with screw cap for colour measurement. 2.2.2 soluble solid content or degrees brix (obx) the brix scale or degrees brix (obx) was numerically equal to the percent of dissolved solids in the products. the soluble solid content was determined by using a refractometer (abbe refractometer, model). % brix of a solution was performed by refractive index of a solid-containing solution. three determinations of each sample were taken with triplicate readings. 2.2.3 measurement the green colour of chlorophyll drying solid was packed in a transparent bag and mixture sample was taken in the transmission compartment for determination of colour value. all samples were measured a colour by reflectance and transmission with the data colour international measurement model colour tools by transmission compartment. cie value (commission internationale de i'eclaerage) display colour value in cielab system. l*(0 = black and 100 = white), a*(-a* = green and +a* = red) and b*(-b* = blue and +b* = yellow) at d65 10deg (light source illuminant d). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 136 published by scholink inc. 2.2.4 determination of chlorophyll content samples were stirred together and mixed until homogeneous. after that sample was allowed to separate the layers. a transparent layer of 1ml dipping sauce was soaked and extracted with 10 ml acetone in vial with screw cap by vortex mixer for 30 min. mixer solution was centrifuged at 4,500 rpm for 20 min. the upper layer was supernatant for chlorophyll analysis by uv-visible spectrophotometer at absorbance of wavelength 645 and 663 nm. to calculate the amount of chlorophyll a and chlorophyll b by equation below. chlorophyll (a) = [12.7 (od663) -2.69 (od645)] x v /1000 x wt chlorophyll (b) = [22.9 (od645) -4.68 (od663)] x v /1000 x wt (od = absorbance of the sample v= volume of the solution sample wt. = weight of the sample). 2.2.5 determination of β-carotene content samples were soaked and extracted with acetone-hexane mixture (10:1) by vortex mixer. extracts were centrifuged at 4500 rpm 30 min and upper transparency of the sample was determined total carotene content using the established method of uv-visible spectrophotometer at wavelength 445 nm. 2.2.6 measuring the water activity (aw) aw was equilibrated and measured at 25°c by an instrument of the novasina labmaster-aw. the water activity was recorded with sorption behavior of water and bounded water in food water activity was usually controlled by the use of salt or sugar. changing the aw of the finished product indicated the process and changes in ingredients. 2.2.7 ph measurement the ph (sartorius model docu-ph+ meter) was calibrated according to the manufacturer's instructions using buffer standards of ph 7 and ph 4. the ph meter was immersed in the sample. the ph value of each product was measured and recorded the values of the concentration of the acidity. 2.3 statistical analysis statistical analysis of mean and variance in each treatment was taken with duncan's new multiple range test at the significance level of p ≤ 0.05 by spss (statistical package for the social sciences) program. in each treatment was scaned and checked the correlation in order to compare the differences at p ≤ 0.05 and p ≤ 0.01 level (2-tailed). the data were analyzed by statistical program of anova and the average treatments were analyzed by duncan new’s multiple range test at significant level. 3. results and discussions 3.1 characteristics of products and quality of colour products of sea food dipping sauce were prepared in ingredients with proportion by figure1. the products were classified the food processing by ph< 4.5 as low acidity food. the buffer of sodium acetate/acetic was shown ph value as acids and measured in the ph range 3.7-5.6. the acidity of product should be satisfied with preparation at ph range 3.40 -3.75. acid and salt of ingredients kept ph buffer and control the sour taste of product quality. https://global.britannica.com/science/hydrogen-ion www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 137 published by scholink inc. figure 1. the percentage of ingredients for preparation of sea food dipping sauce in the experiments, blanching was done by heating and the temperature cool down so that the chilies were destroyed the enzyme and reduced the number of microorganisms. while it was cooked together with all ingredients for the product, chili color depended on the preparation process. the measured values were claimed to better preserve the quality of green colour. process of blanching, cooling and treatment of chemicals (ca2+, mg2+) were shown the color enhancer. blanching and cooling supported to reduce the decay of chilies and become tender more. the green value of products was increased for process on blanching and all cooling. time of chili blanching with 5 min indicated less green color than 3 min. 3.2 statistical comparisons of the different processing and treatments table 1. influence of blanching and thermal cooking in process on the difference of products and total individual quality sample cielab colour all products of sea food dipping sauce chili dry solid of sea food dipping sauce chili l* a* b* l* a* b* classification of product by heating and cooling bcc 27.31±0.25e* -1.60±0.64f* 5.18±0.91ef* 49.74±6.96ab* 2.41 ±1.04a* 26.82±2.72b* bnc 26.22±0.25e* -1.27±-0.45f* 4.67±0.40ef* 49.09±5.37ab* 2.74±1.02a* 26.77±1.85b* bcn 28.28±1.14e* -1.68±0.69f* 5.65±1.94ef* 48.10±4.41ab* 2.82±0.99a* 26.64± 1.86b* bnn 26.84±1.23e* -1.23±0.46f* 5.24±1.10ef* 49.38±6.23ab* 2.76±0.87a* 27.28±2.81b* nbc 26.51±2.23e* -1.52±0.86f* 5.22±1.54ef* 50.96±4.80ab* 2.32±0.80a* 27.36±1.33b* nbn 27.83±0.74e* -1.41±0.28f* 5.25±1.34ef* 47.69±5.65ab* 2.96 ±1.19a* 25.97±3.16b* classification of product by chemicals treatment www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 138 published by scholink inc. none 24.98±4.93 bd* 2.37±5.16 bc* 5.57±4.09cd* 44.76±3.33a* 3.25±0.73a* 24.78±1.96* mgcl2 26.27±1.40 bd* -0.86±0.44 bc* 4.51±1.86cd* 52.14 ±3.05a* 2.76 ±1.13a* 28.31±2.10* mgo 27.39±1.56 bd* -1.77±0.55 bc* 5.68±1.42cd* 52.25± 4.29 a* 3.29±0.88a* 27.37±2.03* ca(oh)2 27.64±1.11 bd* -1.49±0.45 bc* 5.00±1.13cd* 54.04±4.35 a* 2.60±1.00 a* 27.91±1.65* cacl2 27.35±0.80 bd* -0.98±0.27 bc* 4.51 ±1.01cd* 52.15±2.38 a* 2.18±0.62 a* 27.46±1.18* * mean the values in a row are significantly different at p ≤ 0.05 (2-tailed). abc mean the correlation by regression of the same alphabet are significant at p ≤ 0.05 (2-tailed). def show the correlation of the same alphabet are significant at p ≤ 0.01 (2-tailed). the different letters in the same column indicate a significant difference at the level of p ≤ 0.05. the differences of row l*, a* and b* for blanching, heating and cooling were significant at p ≤ 0.05. average mean of l*, a* and b* for a kind of process by blanching and cooling were significantly different at the level of p ≤ 0.01. all products by processing with heating and cooling of bcc, bcn, bnc, bnn, nbc and nbn had significantly a difference at the 0.01 level (2-tailed). blanching and no blanching process, the pearson’s correlation coefficient (rx) of bcc, bcn, bnc with bnn were highly 0.984, 0.989 and 0.989 at the level of p ≤ 0.01. nbc and nbn were highly the correlation as rx = 0.991 at p ≤ 0.01. but blanching and no blanching process were not the correlation at the level of p ≤ 0.01. . figure 2. colour value and product of classification by processing six groups with heating and cooling the differences between types of chlorophyll was displayed physicochemical properties and colour. chlorophyll a and chlorophyll b were orderly bluish-green and yellow-green. the green value of products was increased for process on blanching and all cooling. average mean of l*, a* and b* for a difference of row by blanching, heating and cooling were significant at p ≤ 0.01. colour of chlorophyll content was determined along prepared treatments in order to assess both the comparative evaluation process and the quality of the green index. the chlorophyll of bcc, bcn, bnc, bnn, nbc and nbn indicated a significant difference at p ≤ 0.05. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 139 published by scholink inc. table 2. comparison of identity and properties of the processed product in each group samples β-carotene ± sd bx ± sd ph ± sd aw ± sd % dry wt. ± sd chlorophyll ± sd type a type b classification of product by time for blanching none 0.20±0.01* 54.20±2.54xw* 3.72±0.17xz* 0.873±-0.008z* 4.01±0.50* 0.84±0.29y* 2.72 ±0.78y* 1min 0.19±0.03* 53.98±1.79xw* 3.75±0.20xz* 0.872z±0.006z* 4.40±0.88* 0.77±0.35y* 2.63±0.55y* 3 min 0.17±0.01* 56.20±2.60xw* 3.72±0.04xz* 0.853z±0.019z* 4.61±-0.5* 0.89±0.21y* 2.27±0.61y* classification of products by processing for chemicals and no chemicals treatment none 55.38±2.49rs* 3.69±0.04mpt* 0.861±0.016q* 4.48±0.82pq* 0.61±0.24m* 2.23±0.63t* mgcl2 56.14±4.76rs* 3.58±0.05mpt* 0.867± 0.013q* 4.11±0.37pq* 0.69±0.17m* 2.31±0.44t* mgo 53.95±2.93rs* 3.72±0.068mpt* 0.873±0.060q* 3.81±0.60pq* 1.19±0.20m* 3.79±0.44t* ca(oh)2 54.30±2.14rs* 4.17±0.070mpt* 0.875±0.002q* 4.06±0.33pq* 1.10±0.28m* 1.88±0.51t* cacl2 53.76±2.45rs* 3.65±0.04mpt* 0.875±0.002q* 4.52±0.48pq* 0.78±0.29m* 3.02±0.40t* * mean the values in a row are significantly different at the 0.05 level (2-tailed). xmpq mean the correlation of the same alphabet are significant at p ≤0.01 (2-tailed). y show the correlation of the same alphabet are significant at p ≤ 0.05. wzrst mean the correlation by regression of the same alphabet are significant at p ≤ 0.01 (2-tailed). all β-carotene measurements were derived using absorbency readings obtained for standard β-carotene. the standard curve for all β-carotene measurements of spectrophotometric readings was at concentrations as 0 0.5 mg. the calculation of β-carotene was followed by the same comparison on samples and standard solution by using a linear equation y = 3.879x 0.001 at r2 = 0.9978. β carotene of sample for blanching with 3 min and 5 min were orderly 0.1845-0.3332 mg/100g and 0.1125-0.2582 mg/100g. there was a statistically significant difference at p ≤ 0.01. the water activity was used as a point of definition for the regulations of determining an acidified food. water activity was usually controlled by changes in ingredients and the use of salt or sugar. 3.3 correlation between physicochemical characteristics and products in the experiments on chili with chemical treatments were shown the effects of ca(oh)2 on change of ph. both mgcl2 and cacl2 were related to increasing content of total solid and all ca2+ion (ca(oh)2, cacl2) were related to a decreasing of degrees brix. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 140 published by scholink inc. figure 3. the significant correlation of aw & brix and ph & brix for product by chili with chemical treatments at the level of p ≤ 0.01 figure 4. display graph of the correlation in each of the following groups by 3d surface plots with distance-weighted least squares of statistica chlorophyll (a) correlated with green colour value ( a*) and the correlation was significant at the level of p ≤ 0.01. all products from chili with chemical treatments, aw and ph were shown a highly significant negative correlation rx = 0.708 at p ≤ 0.01(2-tailed). the correlation of aw and brix were orderly negative by significant at rx = 0.723 by chilies with chemical treatments and rx = 0.619 at p ≤ 0.01(2-tailed) by all chilies with none and chemical treatments. chili with chemical treatments, the correlation ph & wt. and ph & brix were highly negative by significant at rx = 0.774 and were highly positive by significant at rx = 0.768 at p ≤ 0.01. 4. conclusion development and improvement of products of sea-food dipping sauce were a technique of process in product producing. process of blanching, cooling and treatment of chemicals (ca2+, mg2+) were indicated the color enhancer. the green value of products was increased for process control on heating and cooling. a short time of chili blanching indicated to preserve a green color more. the measured www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 141 published by scholink inc. values were claimed to preserve the better quality of green colour. the green colour of product was preserved in product processing on blanching and all extreme cooling. references aoac. (1995). official method of analysis (15th ed.). association of analytical chemists, washington, dc. gross, j. (1991). pigments in vegetables: chlorophylls and carotenoid (p. 351). van nostrand reinhold. newyork. hornero-mendez, d., & minguez-mosquera, m. i. (2001). rapid spectrophotometric determination of red and yellow isochromic carotenoid fractions in paprika and red pepper oleoresins. j agric food chem., 49(8), 3584-3588. https://doi.org/10.1021/jf010400l puminat, w., & teangpook, c. (2017). phytochemicals extraction and nutraceuticals of purple corn. food science and nutrition studies, 2(1), 104-113. https://doi.org/10.22158/fsns.v1n2p104 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 1, 2019 www.scholink.org/ojs/index.php/fsns 23 original paper influence of women age on berries consumption: the role of liking and properties of berries as determinants of their intake mariana lares-michel1, fatima ezzahra housni1*, virginia gabriela aguilera cervantes1, maría del carmen barragán carmona1 & claudia llanes cañedo1 1 research center in food behavior and nutrition, university center of the south, university of guadalajara, av. enrique arreola silva 883, col. centro. zip code 49000. ciudad guzmán, jalisco, méxico * fatima ezzahra housni, research center in food behavior and nutrition, university center of the south, university of guadalajara, av. enrique arreola silva 883, col. centro. zip code 49000. ciudad guzmán, jalisco, méxico received: january 13, 2019 accepted: january 25, 2019 online published: february 27, 2019 doi:10.22158/fsns.v3n1p23 url: http://dx.doi.org/10.22158/fsns.v3n1p23 abstract due to the benefits of berries to health and aging in women and the fact that as women age, they are increasingly concerned about remaining healthy and looking young, the aim of this paper is to determine if the consumption of berries among women is influenced by their age; also, to identify if the reasons why berries are ingested vary with age. this study evaluated berries intake and motives of consumption of 240 adult women from jalisco, mexico, ranging from 18 to 65 years old, divided into three groups: young adult women (yaw, from 18 to 35 years old), middle-aged adult women (maw, from 36 to 55 years old) and older adult women (oaw, from 56 to 65 years old). berries (blueberry, raspberry, strawberry and blackberry) consumption was collected by an adapted food consumption frequency questionnaire validated for mexican population, and intake reasons were assessed by a qualitative section. spearman’s correlations, anova and bonferroni test were done. while berries consumption lowers in the maw group, it increases when women reach the advanced adulthood. yaw consumes berries mainly because of liking and oaw due to its properties. only 60% of oaw consumes berries and frequency of consumption is occasional in all women, so policies to increase berries intake are needed. keywords berries consumption, age, women, anti-aging, liking, antioxidants, berries properties www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 24 published by scholink inc. 1. introduction the potential health benefits of foods are one of the main reasons why consumers prefer to ingest certain type of products over others (jaeger, axten, wohlers, & sun-waterhouse, 2009; roininen, lähteenmäki, & tuorila, 1999; roininen et al., 2001). women, in particular, have been found to engage in more health-promoting behaviors than men and to have a healthier lifestyle and a greater interest in eating a diet that may benefit their long-term health, compared to men. in fact, numerous evidence shows that women’s diets are healthier than men’s (arganini, saba, comitato, virgili, & turrini, 2012; huntley, 2009; prättälä et al., 2006); however, age is an important factor that can alter their food consumption habits, especially when taking into account that two of the main determinants of food choices for women are: disease prevention and the desire to delay the aging (bennett, clarkea, kowalski, & crocker, 2017; fraser, welch, luben, bingham, & day, 2000; holcomb, 1995). the consumption of food rich in antioxidants has increased steadily in the last decades, due to the numerous health-promoting properties attributed to them (huntley, 2009). berries (blueberry, raspberry, strawberry and blackberry) have plenty of attributes. besides being low in calories and a rich source of fiber, ascorbic acid, carotenes and polysaccharides, they are a rich source of bioactive compounds, such as phenolic compounds (including flavonoids such as anthocyanins and procyanidins), tannins and phenolic acids, substances that are considered antioxidants. these bioactive compounds have received considerable attention from researchers. so far, it has been found that they improve the markers of heart disease, help to prevent cancer of the digestive system, reduce the risk of mortality from stroke and improve the immune system (gramza-michałowska, sidor, & kulczynski, 2017; heinonen, 2007; ros et al., 2010). clinical evidence indicates that a diet rich in berries helps to control and prevent chronic diseases. also, consumption of berries improves the postprandial glycemic response, and its long-term intake can improve the lipid profile in plasma, reduce chronic inflammation and benefit cardiovascular health, especially in populations with a basal metabolic profile at higher risk of metabolic syndrome (yang & kortesniemi, 2015). in recent years, there has been an increasing amount of research on the health benefits of berries antioxidants, many of them highlighting the effects on women (huntley, 2009; lehtonen et al., 2011). the studies focused on evaluating the consumption of berries in women have found that a relatively higher intake of these fruits is associated with lower rates of cognitive deterioration in older women (devore, kang, breteler, & grodstein, 2012), a reduction in memory loss (o’connor, 2012), and with a healthy aging in women over 50, which is mainly due to their flavonoids (samieri, sun, townsend, rimm, & grodstein, 2014). berries intake can also help to prevent breast cancer (aiyer, srivani, & gupta, 2011) and to delay aging (willis, shukitt-hale, & joseph, 2009). it has also been found that the consumption of berries is positively related to a lower risk of death from cardiovascular diseases (cardiovascular disease mortality) in post-menopausal women (mink et al., 2007). the numerous attributes of berries, make its consumption on women worth studying, yet identifying how the consumption of these fruits varies according to women’s age, may further help in creating www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 25 published by scholink inc. policies to promote their consumption. due to the above the aim of this paper is to determine if the consumption of berries among women is influenced by their age; also, to identify if the reasons why berries are ingested vary with age. 2. method 2.1 sample a cross-sectional study was undertaken in ciudad guzman, jalisco, mexico, a region producing berries (housni et al., 2018). the sample to be studied were 240 women between 18 and 65 years old, from ciudad guzman, jalisco, mexico. the sample was divided into the following age groups, according to the classification of petry (2002): women from 18 to 35 years old, who are considered as young adult women (yaw); women from 36 to 55 years old, considered as middle-aged adult women (maw) and women from 56 to 65 years old, who are considered as old adult women (oaw). the sociodemographic characteristics of the sample appears in table 1. the average age of yaw was 24.88  4.77, maw was 43.35  5.66 and oaw was 60.48  2.95. the highest monthly salary was that of oaw (363.81  325.90 usd), however, this group obtained the lowest average with respect to school years taken. yaw turned out to be the group with the highest number of school years attended (12.26  3.69). table 1. demographic characteristics of the three age groups young adult women (yaw) middle-aged adult women (maw) older adult women (oaw) age ranges1 from 18 to 35 years old from 36 to 55 years old from 56 years to 65 years old n 113 77 50 % 47.08 32.09 20.83 age mean 24.88 43.35 60.48 sd 4.77 5.66 2.95 monthly income2 mean 307.36 354.30 363.81 sd 288.13 289.59 325.90 min 41.77 52.21 78.32 max 2,088.48 1,566.36 2,088.48 school years completed mean 12.26 11.49 8.66 sd 3.69 4.37 4.81 min 2 1 0 max 19 19 19 1age classification according with petry (2002). 2american dollars (usd). the minimum monthly wage in mexico in 2018 was 138.36 usd, according with service tax administration (2018). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 26 published by scholink inc. 2.2 instrument and data collection an adapted food consumption frequency questionnaire validated for the mexican population was applied (macedo-ojeda et al., 2013), which asked about berries consumption, considering as such: blueberry, raspberry, strawberry and blackberry. the method used was the same as the used by housni et al. (2018), where the consumption of blueberry, raspberry and blackberry was questioned in grams or cups, and strawberry consumption in grams or pieces. the consumption frequency ranges used were those proposed by willet (2013), which were chosen according to the exact and personal frequencies of each participant, which were also captured for further analysis. the amount of consumption was the one consumed each time berries are eaten. subsequently, the reasons why berries are ingested were questioned. the application of surveys was carried out in local markets where berries are sold. the information was obtained by personnel with knowledge in health of the university center of the south, university of guadalajara, who were trained in the application of surveys. 2.3 statistic analysis the data obtained was converted to monthly frequency, considering the daily consumption as a maximum of 30 days per month. the information regarding the amounts of intake was converted to grams, using as reference the weight of the established portions of berries, in the mexican system of equivalent foods [smae] (pérez, palacios, castro, & flores, 2014). per capita daily consumption was obtained, multiplying the frequency of consumption of each berrie, of each woman, by the amount of consumption referred, and dividing the result by 30 days per month. finally, the consumption frequency data of each berrie type was averaged to obtain the average frequency of the four berries evaluated. in the case of the quantities, the consumption information of the four types of berries was added, to obtain the total consumption of all the berries, in grams, every time they are consumed and per capita by day. in addition to the averages and total consumption quantities, the standard deviation of the information was calculated. the consumption reasons were grouped in the two most mentioned. the rest was considered as “other reasons”. frequency of participants was described as well as the percentage they represented for each motive. per capita daily consumption and standard deviation were calculated for each reason. the data was analyzed to evaluate its distribution by means of the shapiro-willk test. spearman’s rank correlations coefficient was applied, to correlate the age and the consumption data of each age group and the reason of consumption according to the age group. size of correlation coefficient was taken from mukaka (2012). a value of p < 0.05 was considered significant. to assess differences in berries intakes and reasons of consumption between age groups, an analysis of variance (anova) test was performed with a post hoc multiple comparison bonferroni test. descriptive statistics and statistical analysis was carried out using stata/se v12. 2.4 etic considerations this research is considered without risk in accordance with the regulation of the general health law on health research (official gazette of the federation [dof], 2014), for which the consent of the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 27 published by scholink inc. participants was granted orally. the data were confidential and the name, address or telephone number of the participants was not requested, as it was irrelevant for this study and for the protection of the subject’s identity. 3. results 3.1 consumption of berries table 2 shows the consumption of berries by age group, including number of women who consume berries, frequency and amount of consumption and daily consumption per capita. it was identified that the percentage of yaw that consumes berries presented a greater tendency to ingest them. while 71.68% of yaw consume berries, only 60% of oaw reported consuming them, nevertheless, there were no statistically significant differences between the groups (p = 0.318). despite the above, oaw were the ones who ingest berries most frequently with 5.6  8.37 times per month. regarding the amounts of consumption per capita, per day, yaw eats an amount of 89.35  120.21 grams of berries, nevertheless, as age increases, consumption decreases to 73.32  96.77 grams per day, but when women reach a stage where they are more than 56 years old, the intake increases to 89.38  146.59 grams. although the per capita daily consumption is similar in yaw and oaw, the ration consumed each time berries are eaten in yaw is considerably higher than the other groups. however, no statistically significant differences were found. spearman’s rank correlations did not show strong correlation between any consumption variables and age, however, a negative correlation was find in maw consumption (rho = -0.226 / p = 0.047*), which confirms that in this period, as age increase consumption decrease. anova test neither showed any significant different between age groups (see table 2). table 2. berries consumption by age group young adult women (yaw) middle-aged adult women (maw) older adult women (oaw) variable anova consumption n 81 50 30 0.318 % 71.68 64.94 60 rho 0.109a -0.226a 0.142a p-value 0.246 0.047* 0.324 monthly frequency of consumption mean 5.18 4.56 5.6 0.559 sd 5.82 6.01 8.37 rho 0.056a 0.020a 0.048a p-value 0.549 0.858 0.737 quantity consumed in grams1 mean 423.95 357.01 332.98 0.517 sd 715.71 617.23 573.95 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 28 published by scholink inc. rho 0.195a -0.102a 0.123a p-value 0.038* 0.377 0.391 daily consumption per capita in grams mean 89.35 73.32 89.38 0.626 sd 120.21 96.77 146.59 rho 0.246a 0.033a 0.132a p-value 0.008* 0.769 0.359 sd = standard deviation; rho = spearman’s rank correlation; p value from spearman’s rank correlation; anova = p value from analysis of variance test between age groups; a value of p <0.05 was considered significant. 1grams consumed each time berries are ingested, according to the frequency of consumption expressed; * = statistically significant differences; a = negligible correlation (0.00 to 0.30 and 0.00 to -0.30), b = low positive correlation (0.30 to 0.50), c = moderate positive correlation (0.50 to 0.70), d = high positive correlation (0.70 to 0.90), e = very high positive correlation (0.90 to 1.00), according with mukaka (2012). although no differences were identified in the anova test, table 3 shows a multiple comparison bonferroni analysis to identify the exact values between each comparison group. the results of this analysis proved that there are no statistical differences between the groups. table 3. multiple comparison bonferroni test in berries consumption by age group young adult women (yaw) middle-aged adult women (maw) consumption middle-aged adult women (maw) p-value 0.014 1.000 older adult women (oaw) p-value -0.060 0.631 -0.075 0.436 monthly frequency of consumption middle-aged adult women (maw) p-value -0.615 1.000 older adult women (oaw) p-value 0.423 1.000 1.038 0.900 quantity consumed in grams middle-aged adult women (maw) p-value -66.936 1.000 older adult women (oaw) p-value -90.990 0.930 -24.054 1.000 daily consumption per capita in grams middle-aged adult women (maw) p-value -15.986 1.000 older adult women (oaw) p-value 0.057 1.000 16.043 1.000 * = statistically significant differences www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 29 published by scholink inc. table 4 shows the consumption frequencies of berries of each age group. the results show that berries consumption frequency of yaw and maw are mainly into a mid-scale (1-3 times a month to once a week) whereas oaw consumption are skewed towards lower frequencies, since 40% of these women never consume berries. in the three age groups, daily consumption, from 1 to 3 times, is represented by less than 8% of responders. table 4. frequency of consumption of berries in women by age group frequency of consumption young adult women (yaw) middle-aged adult women (maw) older adult women (oaw) n % n % n % never 32 28.32 27 35.06 20 40 1-3 time a month 27 23.89 14 18.18 9 18 1 time a week 25 22.12 15 19.48 8 16 2-4 time a week 22 19.47 18 23.38 7 14 5-6 time a week 2 1.78 1 1.30 2 4 1 time a day 3 2.66 2 2.60 3 6 2-3 time a day 2 1.78 0 0 1 2 3.2 reasons of berries consumption the main reasons of consumption for which the women consumers said to ingest berries, were liking and the effects that this fruits bring to their health, including the nutritional properties such as antioxidants, their content in vitamins, fiber, among others. some women expressed within this reason, that their intake was due to the anti-aging effects of these red fruits and its capabilities to prevent diseases such as cancer and diabetes mellitus. the rest of the reasons for consumption expressed were incorporated in “other reasons” and included consumption for availability at home or in the market, because they are necessary for some culinary preparations and because they are given the product because someone in the family works in greenhouses producing berries. as it is shown in table 5, it was found that the principal reason why yaw eats berries is because they like them. more than 50% of women of this age group consume berries because this reason, and only 14.81% eat them for their nutritional, anti-aging or health benefits. as age advances, reasons of consumption changes, since 26% of maw prefer to eat berries because of their proprieties than for their taste. when woman reaches 56 years old, the reasons of consumption changes even more, and from the half of the yaw who prefer to eat berries for their taste, only 33.33% of oaw, consume them for this reason, as the most important motive for consume them are the properties that these foods can bring to their health and nutrition (36.67%), including their content in antioxidant which was a highly mentioned factor among this group of women. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 30 published by scholink inc. spearman’s rank correlation showed a low positive correlation between age and liking berries in yaw. same size correlation was found in other reasons of consumption for yaw. a moderate positive correlation was find in oaw who consumes berries because they like them and this size correlation was also obtained in women of this group who said to eat berries because of their nutritional, health or anti-aging proprieties. in no case the correlation was strong enough. frequencies of participants by reason of consumption did not showed correlation, in fact, a negative correlation was identified in the case of oaw. anova test showed that the grams of daily per capita consumption of berries due to their liking presented statistically significant differences between the groups (0.002*). this means that it is verified that age, besides of modify the reason of consumption also change the amount of consumption. table 5. reasons of consumption of berries in women by age group and their daily consumption per capita in grams young adult women (yaw) middle-aged adult women (maw) older adult women (oaw) reason of consumption anova liking n 41 22 10 0.449 % 50.63 44 33.33 dcpc 24.28 22.43 23.62 0.002* sd 47.21 28.26 30.58 rho 0.396b 0.039a 0.640c p 0.012* 0.864 0.046* health, nutritional or antiaging effects n 12 13 11 0.378 % 14.81 26 36.67 dcpc 38.13 28.85 25.24 0.863 sd 81.56 28.19 32.35 rho -0.139a 0.143a 0.588c p 0.700 0.655 0.044* other reasons n 28 15 9 0.589 % 34.56 30 30 dcpc 38.45 35.64 70.28 0.392 sd 50.42 70.86 121.14 rho 0.425b 0.174a -0.066a p 0.010* 0.289 0.773 scf rho 0.072a 0.269a -0.131a p 0.523 0.059 0.498 dcpc: daily consumption per capita in grams; sd = standard deviation; rho = spearman’s rank www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 31 published by scholink inc. correlation; p value from spearman’s rank correlation; anova = p value from analysis of variance test between age groups by frequency of participants and dcpc in each reason of consumption; a value of p <0.05 was considered significant. scf = spearman correlation’s in frequencies of participants; * = statistically significant differences; a = negligible correlation (0.00 to 0.30 and 0.00 to -0.30), b = low positive correlation (0.30 to 0.50), c = moderate positive correlation (0.50 to 0.70), d = high positive correlation (0.70 to 0.90), e = very high positive correlation (0.90 to 1.00), according with mukaka (2012). table 6 presents a multiple comparison bonferroni test in reasons of berries intake and daily consumption per capita in grams, between the age groups. this was due to the identification of statistically significant differences between women who said they consumed berries due to liking. statistically significant differences were identified between daily per capita consumption of yaw and oaw (0.011*) and of maw and oaw (0.002*) in this intake reason. in the other variables, as in the anova test, no statistically significant differences were identified. table 6. multiple comparison bonferroni test in reasons of berries consumption in women by age group and their daily consumption per capita in grams young adult women (yaw) middle-aged adult women (maw) liking consumption middle-aged adult women (maw) p-value -0.094 0.640 older adult women (oaw) p-value -0.051 1.000 0.042 1.000 daily consumption per capita in grams middle-aged adult women (maw) p-value -39.925 0.741 older adult women (oaw) p-value 135.822 0.011* 175.748 0.002* health, nutritional or antiaging effects consumption middle-aged adult women (maw) p-value 0.083 0.661 older adult women (oaw) p-value 0.083 0.719 0 1.000 daily consumption per capita in grams middle-aged adult women (maw) p-value -6.192 1.000 older adult women (oaw) p-value 17.045 1.000 23.237 1.000 other reasons consumption middle-aged adult women (maw) p-value -0.017 1.000 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 32 published by scholink inc. older adult women (oaw) p-value -0.142 0.932 -0.125 1.000 daily consumption per capita in grams middle-aged adult women (maw) p-value 10.446 1.000 older adult women (oaw) p-value -56.053 0.694 -66.5 0.568 * = statistically significant differences 4. discussion in first instance, it could seem that the consumption of berries is not related to the age of the women, since no correlations were found between the intakes and frequencies of consumption and the age. in addition, having found that the amount of yaw that consume berries has a trend in a greater consumption than oaw, could indicate that age is not an important factor in the consumption of these fruits and that in fact, there are more young women who consume berries in comparison with those who are older. however, interesting patterns of intake were identified. when women is younger consumes around 89 grams by day, but as age increase, consumption decrease more than 16 grams, nonetheless, when women reaches a higher age, above 56 years old, the consumption returns to 89 grams. now, what is interesting in this patterns is the reason why women is eating this fruits. when women is between 18 and 35 years old, liking berries result to be the most import reason for consuming them, however, as age increase, reasons of consumption changes, and get closer to a constantly worry for improve health, prevent chronic disease and delay aging. this coincide with the results of chambers, lobb, butler, & traill (2008) since, like them, it was identified that the older participants (60+ years old in their case) were more likely to make food choices based on health considerations, compared with younger women. likewise, the fact that oldest women consume berries mainly due to their nutritional properties, coincides with studies carried out in places such as germany and united kingdom, where the population refers concern about eating foods that provide health benefits (farruggiaa, crescimannoa, galatia, & tinervia, 2016; huntley, 2009). in this sense, it can be affirmed that in mexico, as the age increases, berries consumption revolve more around the health and nutrition than their taste, but the quantities and frequency of consumption are low, since as it was possible to verify, berries are consumed mainly occasionally in the three age groups studied. however, in spite of the low amounts of consumption, the results showed that statistically the per capita daily consumption of berries is different between the age groups depending of the reason of consumption, especially in the liking reason. thus, although not completely, this study can find similarity to the one referred by gibson, wardle and watts (1998) who mentioned that beliefs and attitudes about the relationship between diet and disease, have an important influence on food consumption. but nevertheless, although the concern for health, nutrition and anti-aging leads oaw to resume the consumption that was observed in the yaw group, www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 33 published by scholink inc. this motivation is not enough to consume berries every day. according to brug et al. (1995), laaksonen et al. (2016) and roininen et al. (1999) the sensory characteristics of food and especially its taste and satisfaction, can be considered as one of the key factors in the acceptance of food. as well as the preference for certain food (awuni, kye-duodu, duodu, zotor, & ellahi, 2018). in short, this pattern was identified in the three age groups, however, it was much more important in yaw and maw, due to in oaw liking berries took second place, preferring to consume them for their nutritional, health or anti-aging properties. in yaw and maw liking berries was more important than their properties, to consume them, same as in the studies of holm and kildevang (1996) and wardle (1993); even though authors such as laaksonen et al. (2016), tang, kälviäinen and tuorila (2001) and viljanen, heiniö, juvonen, kössö and puupponen-pimiä (2014) claim that the chemical components contained in berries (such as phenolic compounds and organic acids) induce an acidity that has a strong impact on their sensory quality, which affects the acceptance of consumption and may have a negative influence on the acceptation. however, according to laaksonen, ahola and sandell (2013) for some, this may be a positive feature to some extent, which could be the case of yaw. as mention, when age increases, the importance given to the flavor of the berries diminishes and they are consumed for their properties. according to pohjanheimo and sandell (2009) and verbeke (2006) consumers who consider health as an important reason for choosing food and in order to obtain a benefit to their health, can accept more bitter flavors, compared to people who consider the comfort, taste and familiarity of food, more important than the benefit that these can provide to their health and that are not willing to choose healthiness over the taste of the product. in this sense, it is confirmed that age is a factor that marks a trend toward the flavors go to second term by preferring to prevent diseases and slow aging through the consumption of berries. contributing to this, it has been reported that older women are more attached to a diet than younger women (lietchy, 2012), regardless of its taste. kaume, gbur, dibrezzo, howard and devareddy (2014) suggest that consuming 45 grams of berries daily, like blackberry for 9 months, can provide certain health benefits, such as prevent bone loss induced by smoking in postmenopausal women, however, the results of the present study are far from this consumption, but why, if per capita daily consumption indicates more than 73 grams in all groups? even though daily consumption per capita of the three age groups analyzed in this study could suggest that this ration is exceeded, it is important to remember that we talk about averages and when frequency of consumption is analyzed as an isolated variable, the daily consumption turn to be occasional, as the frequency of intake does not exceeded 6 times per month, although when consumed, ingested amounts are higher than 300 grams, i.e., greater than 1 cup of 250 grams, according with the smae (pérez et al., 2014). so far, the consumption of berries has shown to provide multiple benefits, especially to old adult women. in this specific age group, berries are of great help delaying the onset of cardiovascular diseases and cancer; help to improve memory and avoid cognitive problems in this period where these www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 34 published by scholink inc. functions decline (huntley, 2009). in accordance with shukitt-hale, lau and joseph (2008) early nutritional interventions with berries may even prevent or delay the onset of diseases such as alzheimer’s disease, because they can reduce oxidative stress and inflammation. as well, berries consumption help control body weight (bertoia et al., 2016), and also, it has been even found that berries prevent and help in the treatment of viral diseases such as influenza (gramza-michałowska et al., 2017). due to this, consumption in oaw should be promoted, because statistically, it turned out to be the age group that consumes less berries (60% of participants in this age range). to this point, in first world countries, such as finland, berries are part of traditional diets (heinonen, 2007), nevertheless, in mexico the consumption of berries is in a gradual process of addition to the diet, due to the important increase that has had its agricultural production (housni el al., 2018). 5. conclusions although it might seem that the consumption of berries is independent of age, this study shows an interesting relationship between them. while the daily amounts of intake are statistically equal in the three groups, descriptive analysis showed that although maw consumption is lower compared to yaw, when women are reaching a stage of older adulthood, consumption returns to the levels observed in the younger ages, but the consumption motives diverge. to be more specific, the main motives switch from the pleasant flavor of the berries to those related to the potential of berries to delay the onset of chronic diseases and to slow down aging. from these findings, we conclude that age is a factor that influences the decisions of berries consumption. also, it was found that age actually modify the daily per capita ingested amounts depending of the reason why are ingested, especially in liking. however, nutritional programs to promote the consumption of these fruits in the category of oaw are needed, since currently, only 60 percent of the group consumes them. when consumption frequency is considered as a single variable, we find that consumption is only occasional; on average, women of all groups consume berries less than 5.6 times a month, that is, less than twice a week. for this reason, future studies evaluating the consumption of berries, or food in general, should consider both the quantity and the frequency of consumption as isolated and joint variables, for example, expressing the data of each one, but also including per capita daily consumption, obtained from these data. in mexico, one of the world’s top producers of berries (housni et al., 2018), the advertising of the attributes of berries and the implementation of nutritional education programs are needed to increase their consumption levels. these will benefit all adult women, and especially those reaching the stage of older adulthood. acknowledgments the authors thank to the professional development program (prodep) and the trained staff of the university center of the south who collaborated with the data collection. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 35 published by scholink inc. funding this work was supported by the professional higher education development program (prodep-academic groups). references aiyer, h., srivani, r., & gupta, r. 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(2015). clinical evidence on potential health benefits of berries. current opinion in food science, 2, 36-42. https://doi.org/10.1016/j.cofs.2015.01.002 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 2, 2017 www.scholink.org/ojs/index.php/fsns 71 comments on the high pressure preservation of human milk s. j. rzoska1*, e. rosiak1,2, m. rutkowska1, a. drozd-rzoska1, a. wesolowska3 & m. k. borszewska-kornacka4 1 institute of high pressure physics polish academy of sciences, warsaw, poland 2 warsaw university of life sciences—sggw, warsaw, poland 3 warsaw medical university, faculty of health science and laboratory of milk and lactation research at the regional bank of human milk, warsaw, poland 4 warsaw medical university, clinic of neonatology and intensive care of the newborn, warsaw, poland * s. j. rzoska, e-mail: sylwester.rzoska@unipress.waw.pl received: august 17, 2017 accepted: august 26, 2017 online published: september 13, 2017 doi:10.22158/fsns.v1n2p71 url: http://dx.doi.org/10.22158/fsns.v1n2p71 abstract the current state of studies on the high pressure preservation of the human milk is briefly presented. it is indicated that reaching (i) the antimicrobial safety, (ii) antiviral safety, and (iii) high nutritional, metabolic and immunological quality, may be difficult for a “classical” single pressure pulse high pressure preservation (hpp) treatment. it is shown that the sudden decompression leads to additional physical processes, which can be important for supporting the hpp technology. additional advantages were reached due to the two-pulse compression, with subsequent values: p = 200 mpa and 400 mpa. tests included the microbiological insight for the two-weeks storage. it is also shown that the decay of the number of microorganisms under the high pressure follows the relation n(t) = n0exp(at)exp(bt2). finally, issues regarding containers for the high pressure preservation of human milk are discussed. keywords human milk, foods preservation, high pressures, thermodynamics, microbiology 1. introduction the human milk is not only the first food for new born infants. it is the natural continuation of the intrauterine nutrition which delivers the energy and enables the development. human milk is essential for avoiding many chronic diseases during the whole life, being the source of constituents important in metabolic processes. for instance, epidemiological data show the protective role of the human milk to prevent acute and chronic diseases and obesity. if the breastfeeding of newborn is not possible, the best solution seems to be banks of human milk (mimouni et al., 2017). nowadays, the microbiological safety in human milk banks is assured via the holder “soft” pasteurization: the thermal treatment at t = www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 72 published by scholink inc. 62.5oc for t = 30 min (kim & unger, 2010). however, any protocol associated with approaching or passing the pasteurization threshold, required for reaching the microbiological safety, is associated with the very notable reduction of key nutritional and bioactive features (peila et al., 2016). the new hope for banks of human milk can be the implementation of innovative methods based on non-thermal physical treatments (peila et al., 2017). the most prominent candidate seems to be the high pressure preservation (hpp), which has already reached a great success in the pro-health foods market. the key features of the hpp technology can be concluded as follows (boziaris, 2014; huang et al., 2016; barba et al., 2017):  microbiological safety even up to 90 days,  taste, flavor and texture of fresh foods,  vitamins content and nutritional characterizations of fresh foods,  bioactivity close to the fresh product and long-time preserved,  the lack of chemical preservatives,  using of salt can be avoided,  in selected cases deactivation of enzymes is possible,  the tool for the creation of new functional foods,  used for already packed foods: the secondary microbiological contamination can be avoided,  applicable for fluid and “solid” products,  isotropic and “almost immediate” action of pressure on the product,  environment-friendly method: no pollutions and it requires up to 10x less energy than the thermal pasteurization. one can expect that these extraordinary advantages of the hpp technology have to occur also for the human milk. however, human milk is much more than simply a food. it is an extremely complex system, being also one of the most important factors shaping the human health for the whole life (mimouni et al., 2017). consequently, possible implementations of the hpp technology for human milk banks require careful optimization and consideration of all possible issues. this report discusses problems of the hpp technology application for the human milk. it focuses on physical issues which have been not clearly addressed so far, including compressing protocols and the convenient packing. 2. method new experimental results presented in this report are based on the application of large volume high pressure processors, working up to p > 600 mpa and in the possible temperature range from -20oc to 120oc. the working volumes of pressure chambers: v = 1 l and v = 2 l they enable “programming” of the form of high pressure pulses (see the description below) and measurement of temperature via thermocouples located within the chamber. both processors were designed and manufactures in the institute of high pressure physics polish academy of sciences (warsaw, poland). samples of human milk were collected via the bank of human milk at warsaw medical university (see below). prior to www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 73 published by scholink inc. the high pressure treatments the human milk samples were place in special container able to 20%-30% decrease of volume without any permanent and non-reversible distortions. all details regarding containers and microbiological tests are given is subsequent paragraphs. 3. results 3.1 emerging optimal parameters of hpp processing of human milk generally, studies of the high pressure based preservation of human milk explore the pattern developed for the food industry: the high compression in the range between 300 and 600 mpa, for few/several minutes, at the near room temperature. the key target in this treatment is the high microbiological safety (huang et al., 2016). this pattern is also applied in studies on the human milk pascalization carried out so far (vazquez-landaverde et al., 2006; viazis, 2008; viazis et al., 2008; permanyer et al., 2010; moltó-puigmartí et al., 2011; lou et al., 2012; delgado et al., 2014; sousa et al., 2014; windyga et al., 2015; sousa et al., 2016; kiełbratowska & kołodziejska, 2017; li et al., 2017). tables 1 and 2 below illustrate some recent results, focusing on trends and the scatter of experimental data. table 1. selected result showing the decrease of the number of key parasitic microorganisms on compressing microorganism n 400 mpa n 500 mpa n 600 mpa t ( o c) ref. staphylococcus aureus 5 log/30 min 21-31 [1] staphylococcus aureus 6-8 log/30 min 21-31 [1] staphylococcus aureus 5 log/15 min 4 [1] staphylococcus aureus 8 log/15 min 50 [1] staphylococcus aureus 2.5 log/15 min 5 log/15 min 4 [2] staphylococcus aureus 2 log/15 min 4 log/15 min 20 [2] staphylococcus aureus 3.5 log/50 min 8.5 log/15 min 50 [2] streptococcus agalactiae 8 log/7 min 21-31 [1] listeria monocytogenes 8 log/2 min 21-31 [1] escherichia coli 8 log/10 min 21-31 [1] gram positive bacteria 5 log 5 log 30 [3] gram negative bacteria 5 log 30 [3] yeast, molds 300-400 30 [3] note. the decay (cfu/ml) is presented in “customary n-log units” reflecting the change in the number of microorganism following the hpp process:  treateadnativen 10log . [1]—(viazis, 2006; viazis et al., 2008), [2]—(windyga et al., 2015), [3]—(mimouni et al., 2017). http://pubs.acs.org/author/vazquez-landaverde%2c+pedro+a http://www.sciencedirect.com/science/article/pii/s0308814610006539 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 74 published by scholink inc. table 2. key metabolic and immunological factors of the human milk after the hpp treatment and after the reference “classical” thermal holder pasteurization factor 0.1 mpa (holder) 400 mpa/duration 600 mpa/duration t ( o c) ref. iga 72% 100%/5 min 69.3% 12 [1] siga 51.2% 107%/30 min 30 [2] lysozome 51.2% 99.6%/6 min 72.7%/6 min 30 [2] α-tocopherol 74.5% 71%/6 min 72.6%/6 min 10 [3] γ-tocopherol 52.7% 83.3%/6 min 56.4%/6 min 10 [3] δ-tocopherol 66.7% 102.9% 75%/6 min 10 [3] saturated fatty acids 99.3% 95.5%/6 min 98.8% 10 [3] polysaturated fatty acids 99.6% 99.2%/6 min 83.7%/6 min 10 [3] monounsaturated fatty acids 99.6% 90.8%/6 min 106.3%/6 min 10 [3] interleukin (6) 25.4% 35%/6 min 102%/6 min 10 [3] interferon 55% 93.8%/6 min 99% 10 [3] tumour necrosis factor 5% 100% 111%/6 min 10 [3] vitamin c 84% 100% 100% 20 [4-7] ascorbic acid 80% 100%/15 min 100% 20 [4-8] lysozome 56% 100%/15 min 20 [5-8] note. prepared basing on recent results from refs.: [1]—(permanyer et al., 2010), [2]—(viazis, 2008), [3]—(delgado, 2014), [4]—(moltó-puigmartí et al., 2011), [5]—(sousa et al., 2014), [6]—(sousa et al., 2016), [7]—(mimouni et al., 2017), [8]—(montserrat et al, 2016). table 3. the analysis of the multi-cycle hp treatment (duration t = 6-10 min) factors holder p = 0.1 mpa n% temp. ( o c) number of cycles p = 325 mpa p = 425 mpa nutritional: energy 101% 101% 99.5% 4 1-4 99% 99.5% 37 1-4 nutritional: lipids 102% 98% 98% 4 1-4 97% 97.6% 37 1-4 nutritional: protein 102.2% 95% 99.2% 4 1-4 92% 101% 37 1-4 immunological: iga 66% 92% 94% 4 1-4 92% 82% 37 1-4 immunological: igg 68% 117% 90% 4 1-4 112.5% 87% 37 1-4 immunological: igm 32% 111% 73.5% 4 1-4 104% 48% 37 1-4 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 75 published by scholink inc. note. prepared using results of ref.: (girard et al., 2016). recently, hpp studies of human milk focused on the impact of the sequence of high pressure pulses (lasting 6-10 minutes), with respect to nutritional, immunological and metabolic factors have been carried out (girard et al., 2016). their results are concluded in table 3. the emerging picture from above results can be concluded as follows:  the post-hpp treatment nutritional, metabolic and immunological features are close to the native human milk, whereas the thermal hpp treatment is notably destructive. it seems that the optimal values of pressure is located for p = 300-400 mpa, i.e., in the region where the deactivation of pathogenic bacteria decreases.  the high pressure pascalization offers much better microbiological safety regarding key pathogenic bacteria and the total bacterial load. this target can be reached for p = 400-500 mpa. worth stressing is the notable scatter of results obtained in different laboratories.  regarding pathogenic viruses the evidence is still limited. notwithstanding, one can expect that at least in some cases the safety is reached for p >> 600 mpa. this range of pressures is highly destructive for nutritional, metabolic and immunological factors of human milk. 3.2 the form of high pressure pulses for the high pressure “cold pasteurization” of foods, products are placed within the pressure chamber in a container capable for the reversible elastic deformation caused by the isotropic shrinking during compressing. the high pressure is applied for the time riset up to the planned stationary value which is kept constant for the time .statt , usually between 3 and 15 minutes. subsequently, the high pressure is released within decayt , down to ambient conditions. pressure is transmitted to the product/sample via the pressurized medium, most often water with a possible addition of glycol or a similar liquid to facilitate the lubrication of elements of the high pressure processor and to avoid freezing during the metabolic: lactoferrin 16% 100% 63% 4 1-4 113% 84% 37 1-4 metabolic: lysozome 71% 97% 94.6% 4 1-4 101% 99% 37 1-4 metabolic: lipase 48.5% 31% 43% 4 1-4 70% 66% 37 1-4 bacterial load decay (“log units”) 1.4-3.4 1.9-3.1 4 4 2.2-3.5 37 4 ~4.4 37 1 ~5.1 37 1+2 ~5.1 37 1+2+3 ~8 (?) 37 1+2+3+4 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 76 published by scholink inc. process. in our studies two hpp processors, designed and build in ihpp pas, with volumes of pressure chambers v = 2l (2r = 100 mm) and v = 1l (diameter 2r = 65 mm) were used. for the first processor (type i) the piston method for high compression was used (yordanov & angelova, 2010). in this case the durations of compressing ( riset ) and decompressing ( decayt ) is obligatory in the range of 1-2 minutes. the pattern of high pressure pulse is shown in figure 1. for the second processor (type ii) the high compression was created due to the external pumping system, supported by the pressure mutiplicator. for the possible profile see figure 2. the associated changes of temperature are also given (red, dashed curve and the right scale). this is related for type i hpp processor with the piston-manostatic principle, volume v = 2 l, diameter 2r = 100 mm. fast increasing/decreasing of pressure is coupled to notable changes of temperature, as shown in figures 1 and 2 (dashed curves in red). this is associated with the adiabatic nature of the process, most often recalled within the ideal gas equation frames: .)(constcnrtpv  , where p, t and v stand for pressure, temperature and volume; n is the number of moles and r denotes the gas constant (stanley, 1972; rzoska et al., 2010). 0 200 400 600 800 1000 0 100 200 300 400 500 600 p re s s u re (m p a ) time (s) -10 -5 0 5 10 15 20 25 30 t ( o c ) figure 1. the form of the pressure vs. time pulse applied for hp processing (solid, black lines, left scale) following this, for adiabatic heating/cooling associated with compressing/decompressing one obtains     1 1   initialfinalinitialfinall pptt , where vp cc is the ratio of specific heats for constant pressure and volume. such process is isentropic (the entropy s = const) and it is associated with the notable change of the internal energy      12 1    initialfinalinitial pprtfu . www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 77 published by scholink inc. 0 200 400 600 800 0 100 200 300 400 500 600 p re s s u re ( m p a ) time (s) -10 0 10 20 30 t (o c ) figure 2. the form of the pressure vs. time pulse applied for hpp processing (solid, black lines, left scale) the associated changes of temperature are also given (red, dashed curve and the right scale). the plot is for the type ii hp processor with the external pumping of pressure, volume v = 1 l, diameter 2r = 65 mm. the temperature sensor was located on the axis of the chamber, 20 mm below its top. it is worth stressing that the adiabatic change of pressure and temperature occurs “immediately” and homogeneously within the whole processed sample/material. ideal adiabatic conditions appears for the perfect thermal isolation from the surrounding or for processes fast enough to reduce the heat exchange from/to surrounding. above relations although “classical” in discussions on the adiabatic heating/cooling assume the ideal gas equation as the reference. its validity is limited to gases since for the ideal gas the phase transition to the liquid state is absent (stanley, 1971). for liquids, a dense and elastic medium, notable repulsive and attractive interactions are significant. consequently, one can explore solely the isentropic nature of the adiabatic compression/decompression the nature of the given phenomenon, namely:     0,0,  ptdspts and then   0,                  dp p s dt t s ptds pp (1)                             p p p t p t s c t tstt psvv ts ps dp dt   1 1 (2) where p is for thermal expansion coefficient, pc denotes the isobaric  is for density (stanley, 1971; rzoska et al., 2010). the solution of the latter equation yields ca. kt 13 13 k temperature change for mpap 400 = 400 mpa pressure change and kt 23 k for mpap 600 = 600 mpa pressure jump, assuming water as the host pressurized medium. notable is the similarity of the above dependence to the 2nd ehrenfest equation (stanley, 1971; rzoska et al., 2010), used for describing the pressure evolution of the phase transition:   pp ctdpdt   , where p and pc are for jumps of the expansion coefficient and the specific heat at the discontinuous phase transition. in the experimental practice for the hp technology www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 78 published by scholink inc. the rapid change of pressure is mostly adiabatic but some heat exchange also can take place. this can lead to the supplementary change of entropy, related to the clausius-clapeyron equation vsdtdp  (stanley, 1972; rzoska et al., 2010). figure 1 shows the most classical form of hpp pressure vs. time pulse. first, the pressure increases (for strise 125 125 s), subsequently it is kept at the processing pressure for .statt (~ 5 min in figure 1). finally, pressure is released for stdecay 125 125 s. this is associated with heating above the base level up to ktrise 3.6 +6.3 k, returning to the base level during the high compression and finally, cooling below the base value by ktdecay 5.21 -8.1 k. both values are below the maximal adiabatic heating/cooling values, what suggests the notable “isothermic contribution” during the rise and decay of pressure. figure 1 is associated with the type i pressure processor, and the temperature measured on the axis of the pressure chamber, 5 cm. from its top. figure 2 presents the pressure vs. time pulse, with the same value of strise 125 125 s and the sudden pressure decay down to the ambient value for stdecay 1.0 0.1 s. the rise of pressure is associated with ktrise 1.4 +4.1 k and the decompression related to ktdecay 5.21 -21.5 k. this result was obtained in type ii pressure processor. it is visible that for the decay one approaches the adiabatic cooling temperature limit. this can be associated with the sudden, very short time decay of pressure. this scenario was realized by opening a valve linked to the pressure chamber. 3.3 containers for the hpp treatment of human milk and fluid foods for the hpp technology, products are placed within an elastic container, made from a plastic (polymer-based) material which does not interact with the product. the container has to ensure the isolation from the pressure-mediated liquid (water, often with lubricated additives as glycol) during the high pressure processing. it also must provide excellent pressure transmission to the product from the pressurized (liquid) medium and the return to the initial state after decompressing (mertens, 1993; balasubramaniam et al., 2016). for fluids the container is most often a bottle, consisting from the main body and the closure cap with a sealing system. for the hpp technology the cap should have the same or higher compressibility than the main body. consequently, the tightness of the closure can increase with rising pressure. a notable problem appears if filling of the bottle is not complete and the “parasitic” air-layer remains. compressing water up to p = 600 mpa results in   %6.14%0  vv relative volume change. for p = 400 mpa one obtains   %5.11%0  vv . air is ca. 8105.1  more compressible than water. all these may cause irreversible distortions or even break of the bottle when compressing. this issue is particularly important for hpp implementations in human milk banks, since the human milk is too precious to allow losses associated with the destruction of containers/bottles to solve the problem one can propose the design of the bottle given figure 3a. it enables an additional anisotropic (longitudinal) shrinkage, what causes that even larger than 20% “parasitic”; air layer can be easily compensated. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 79 published by scholink inc. cap ba bottlebottle cap figure 3. the proposal of containers/bottles for the high pressure preservation (hpp) of human milk or other fluid foods/drinks the solid (thick, blue) arrows indicates the (isotropic) way of compressing. the left bottle (a), made from an elastic plastic, enables hpp processing also in the case if the bottle is only partially filled with the fluid. the dashed (thick, blue) arrows show the additional “anisotropic” shrinkage due to the specific design. all these enable the “safe” compressing of a fluid “product” with a notable air layer inside the bottle. the right part of the figure (b) shows the possibility of making the bottle from an arbitrary material, including the silicate/oxide glass. the pressure is transmitted via increasing the volume of the internal “soft” immersed part (rzoska & drozd-rzoska, 2017). generally, almost non-compressible glass bottles are considered as non-proper for the hpp technology. on the other hand glass-bottles are multi-use, easy-cleaned and free from parasitic interactions with any food, including human milk. figure 1b shows the possible solution of the “glass bottle problem”. the pressure is transmitted to the fluid by means of a properly shaped and “soft” element connected to the cap and immersed in the hpp processed liquid. this element can be also “reversed” and subsequently used as the bottle teat, if made from latex or similar a similar material. 3.4 microbiological tests of the new hpp protocol figure 4 presents the decay of the number (n) of s. aureus bacteria in microbiologically contaminated human milk during such processing at p = 400 mpa for three selected isotherms. such value of pressure seems to be close to the possible compromise between the antimicrobial safety and the preservation of key constituents (tables i and ii). for the applied semi-log scale the linear dependence is the indicator of the simple exponential decay    atntn exp0 , i.e.,     atntn  010 10ln1log of the number of microorganism for the compressing time t. this is the general advised pattern for the microbial deactivation under pressure (balasubramaniam et al., 2016). a glimpse—test of figure 4 suggests that such pattern obeys for t = 4oc and t = 20oc isotherms, but definitively fails for t = 50oc. figure 5 presents results of the distortions sensitive and derivative-based analysis of data from figure 4. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 80 published by scholink inc. in the basic case of the simple exponential decay one obtains:    atntn exp0    adttnd ln (3) such description is related to horizontal lines in figure 5. however, it obeys only for t = 4oc and t = 20oc isotherms, for the limited period of time. results presented in figure 4 indicate the occurance of the more general pattern:      2 0 expexp btatctc     btadttcd 2ln  (4) horizontal lines in fig. 5 indicate the clear “single” exponential decay    attn exp . solid curves are for the “extend” exponential decay      2expexp btattn  : (i) solid lines are for the process faster than the single exponential decay and described by b > 0, (ii) the dot-sloped line is for the process slower than the single exponential decay, described by b < 0 and (iii) for b = 0 the simple exponential case takes place. results are obtained basing on experimental data from figure 4. in eq. (4) the value b = 0 is for the simple exponential decay, b > 0 leads to the decay lesser that the simple exponential one and b < 0 is for the effective decay stronger the simple & basic exponential decay. the latter behavior is characteristic for the whole tested time range at t = 50oc. for t = 4oc and t = 20oc isotherms the notable change of the behavior during compressing takes place. it is visible that for the’ most convenient’ for applications isotherm t = 20oc the decay of the number of microorganisms is relatively mild, namely only 1.14 “log-unit” for t = 10 min compressing and 1.7 “log-unit” for t = 15 min. the above resume of recent studies on the hpp technology for human milk focused on (i) microbiological safety, (ii) nutritional, metabolic and immunological agents, and (iii) anti-virus issues. consequently, the question arises how to overcome the problem of different optimal pressures for these key targets. a possible solution is a new compressing protocol. in ref. (girard et al., 2016) the application of a sequence of up to 4 pressure pulses p = 400 mpa, duration 6-10 minutes each, for instance was tested. results presented above indicate the necessity of the hpp+ application protocols for human milk, beyond the dominated “classical” hpp pattern, i.e., the single high pressure pulse. in this paragraph results of such hpp+ approach using 2 high pressure pulses are presented: (i) compression at p = 200 mpa for  tstat. = 10 min, (ii) for  twait. = 10 min samples were kept at p = 0.1 mpa, and finally (iii) the compression at p = 400 mpa for tstat. = 10 min: i.e., the same total period as in the holder “standard” treatment. such pattern was successfully tested (skąpska et al., 2012; skąpska et al., 2013) in studies on fruits and vegetable juices. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 81 published by scholink inc. 0 5 10 15 10 5 10 6 10 7 10 8 10 9 7 4 o c 20 o c 50 o c n ( cf u / m l ) t (min.) lo g [n (cfu /m l )] 9 8 6 5 p = 400 mpa figure 4. changes of the amount of s. aureus in human milk during p = 400 mpa compressing, for selected temperatures 0 5 10 15 -1.0 -0.8 -0.6 -0.4 -0.2 4 o c 20 o c 50 o c dl nn (t ) / dt t (min.) p = 400 mpa figure 5. the derivative of the amount (n) of s. aureus as the function of compressing time the first moderate-pressure pulse (p = 200 mpa) did not influenced the total amount of bacteria but made them more sensitive to impact of the second “killing” high pressure pulse (p = 400 mpa). it is notable that the destruction of spores was also reported. one can speculate that the first pulse weakens the cohesion within cellular walls. additionally, microbiological consequences of different forms of high pressure pulses shown in figures 1 (type i) and 2 (type ii) were tested. finally, the storage studies, up to 14 days, were also carried out. for testing the impact of type i (figure 1) and type ii (figure 2) advanced hpp+ technology human milk was contaminated by s. aureus up to the reference control level log 7.47 cfu/ml. the mix of three strains of staphylococcus aureus: atcc 25923 commonly used as indicator in high pressure inactivation tests; 4.4. strain isolated from food came from the department of industrial food microbiology university of warmia and mazury in olsztyn; hm01 strain isolated from raw human milk in the department of food hygiene warsaw university of life sciences—sggw. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 82 published by scholink inc. table 4. results of microbiological test when applying different modes of hpp treatments note. pressure day native daydd nnn   : the index d is for the given day and d = 0 stands for the reference onset day. for the reference see results of ref. (windyga et al., 2015) where the single high pressure pulse for p = 400 mpa,  tstat. = 15 min at t = 20oc yielded ca. 2-log decay of s. aureus. three replicates of a 14-day storage cycle of humane milk contaminated with staphylococcus aureus inactivated by high pressure were made. the reference number of s. aureus determined in the control sample was changed significantly during the 9 days of refrigerated storage of milk samples. the number of bacteria recorded on that day was 4.43 log cfu/ml. changing the number of bacteria by more than two orders of magnitude signified the natural aging of the population of the bacteria tested, as confirmed by the 14th day of storage, in which a 3.91 log cfu/ml was found. in 9th day of the storage also in pressure samples (type ii), the significant reduction in the tested microorganisms was recorded: down to a level of 1.79 log cfu/ml. however, the last day of the storage cycle was not labeled s. aureus in the pressurized milk samples. a similar phenomenon took place in the pressure samples in device i. the significant change in the number of bacteria compared to the reference sample was observed on day 4 of storage to level 1.75 log cfu/ml. results presented below clearly show the increasing effectiveness of the proposed double-pulse high pressure pattern of compressing, reached already for the near-room temperature of processing t = 20oc. the obtained result is qualitatively better than for the single pulse for p = 400 mpa processing at the near room temperature. the next notable result is much better reduction of hpp processing for the treatment with the “sudden” decay of pressure (the form of pulse presented in figure 2). the application of the single 10 minutes, p = 600 mpa completely removes pathogens but it is strongly destructive basic functional features of the human milk. taking into account results from table 4 and ref. (windyga et al., 2015) one can conclude the notable advantage of the 2-pulse scenario, with the properly selected values of compressing, supplemented by the sudden decay of the high pressure pulse. storage days number of microorganisms during the storage native nn 10log n 200+400 (ii) mpa relative values ddd nn   0/ n 200+400 (i) mpa relative values ddd nn   0/ 0 7,47 4,88 2.59 3,09 4.38 1 6,96 3,69 3.27/3.78 3,20 3.76/4.27 2 7,17 4,10 3.07/3.37 3,75 3.42/3.72 4 6,71 3,81 2.09/3.66 1,75 4.96/5.72 9 4,43 1,79 2.64/5.60 0 4.43/7.47 14 3,91 0 0 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 83 published by scholink inc. 4. discussion this report discusses selected issues associated with the high pressure “cold pasteurization” of human milk. the analysis of experimental data clearly shows that this non-thermal method may lead to results qualitatively better than obtained for the holder thermal pasteurization, regarding the antimicrobial and antiviral safety as well as the preservation of bioactive nutritional, metabolic and immunological features. however, such completed preservation target seems to be not possible for the single pulse hpp technology. figure 6. the sketch of the denaturation curve in the pressure—temperature plane: prepared following ref. (smeller, 2002). such breakthrough target seems to be possible only for the expanded “hpp+” approach, such as discussed in this report 2-pulses protocol (p = 200 mpa and p = 400 mpa). for presented studies, it has been obtained that results of the treatment can remain valid for the 2-weeks storage. it is worth stressing that the deeper decrease of the number or s. aureus occurred for the sudden decompression of high pressure pulses. one can speculate that this is associated with the short-time, deep adiabatic cooling, the pressure-shock change and the pressure-gradient wave front propagated through the tested sample. the analysis of changes of the number of s. aureus under p = 400 mpa compression showed that the most often used near room temperature (t = 20oc) for the high pressure processing is less effective than the treatment related to t = 4oc and t = 50oc. the decay of the number of microorganisms was particularly strong for the latter. it is also notable that the obtained decay of the number of microorganism is complex:      2 0 expexp btatntn  , i.e.,   2 10log btatctnn  , where the constant 10lnlog 010 nc  . the distortions-sensitive analysis   btadttdn 2 revealed that the pattern and then the key mechanism of the decay of microorganisms can change after few minutes of compressing. it is worth recalling that for the thermal pasteurization the process is related mainly to the denaturation of proteins. one can link this to reaching the activation energy high enough to create new intermolecular links between secondary and ternary structures of proteins, definitively www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 84 published by scholink inc. changing their forms. the resulted denaturation curve extends in the pressure—temperature plane as shown in figure 6. however, under high pressure appears additional: the break of cellular walls, the destruction of cellular organelle or changes in the permeability of cellular walls (rzoska et al., 2015; starzonek et al. 2015). moreover, the contribution of all these mechanism seems to depend on the distance from the denaturation curve in the p-t plane. concluding, it seems that the “revolutionary” expectations for totally safe human milk and with “native” nutritional, metabolic and immunological features, also during, the long term storage in a “home refrigerator” (~ 8oc), are justified. however, this requires rather hpp+ treatment, than a simple implementation of the hpp technology used in the food industry. acknowledgements the authors would like to acknowledge the support of the grant “lactotechnology as a response to the special nutritional needs of prematurely born children” (natl. centre for res. & develop. ncbir, poland: for years 2015-2017). adr was supported by the national science centre (ncn, poland) project, ref. 2016/21/b/st3/02203. references balasubramaniam, v. m., barbosa-cánovas, g. v., & lelieveld, h. l. m. 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(2010). maintenance of breast milk immunoglobulin a after high-pressure processing. j. dairy science, 93, 877-883. https://doi.org/10.3390/nu8080477 rzoska, s. j., & drozd-rzoska, a. (2017). submission to the urząd patentowy rzeczpospolitej polskiej (the patent service of the republic of poland). rzoska, s. j., mazur, v., & drozd-rzoska, a. (2010). metastable systems under pressure. springer verlag, berlin. rzoska, s. j., musiał, f., rutkowska, m., fonberg-broczek, m., sokołowska, b., drozd-rzoska, a., & nowakowska, j. (2015). simultaneous impact of high pressures and pulsed electric field on saccharomyces cerevisiae model system. j. food process. technol., 6, 5. skąpska, s., sokołowska, b., fonberg-broczek, m., niezgoda, a., chotkiewicz, a., dekowska, a., & rzoska, s. j. (2013). factors influencing the inactivation of alicyclobacillus acidoterrestris spores exposed to high hydrostatic pressure in apple juice. high pressure research, 33, 73-82. https://doi.org/10.1080/08957959.2013.772170 http://www.sciencedirect.com/science/article/pii/s0308814610006539 https://link.springer.com/journal/217 https://www.ncbi.nlm.nih.gov/pubmed/?term=permanyer%20m%5bauthor%5d&cauthor=true&cauthor_uid=20172207 https://www.ncbi.nlm.nih.gov/pubmed/?term=castellote%20c%5bauthor%5d&cauthor=true&cauthor_uid=20172207 https://www.ncbi.nlm.nih.gov/pubmed/?term=ram%c3%adrez-santana%20c%5bauthor%5d&cauthor=true&cauthor_uid=20172207 https://www.ncbi.nlm.nih.gov/pubmed/?term=aud%c3%ad%20c%5bauthor%5d&cauthor=true&cauthor_uid=20172207 https://www.ncbi.nlm.nih.gov/pubmed/?term=p%c3%a9rez-cano%20fj%5bauthor%5d&cauthor=true&cauthor_uid=20172207 https://www.ncbi.nlm.nih.gov/pubmed/?term=castell%20m%5bauthor%5d&cauthor=true&cauthor_uid=20172207 https://www.ncbi.nlm.nih.gov/pubmed/?term=franch%20a%5bauthor%5d&cauthor=true&cauthor_uid=20172207 https://www.ncbi.nlm.nih.gov/pubmed/?term=franch%20a%5bauthor%5d&cauthor=true&cauthor_uid=20172207 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 86 published by scholink inc. skąpska, s., sokołowska, b., fonberg-broczek, m., niezoda, k., dekowska, a., & rzoska, s. j. (2012). the combined effect of high pressure and nisin or lysozyme on the inactivation of alicyclobacillus acidoterrestris spores in apple juice. high pressure research, 32, 119-124. https://doi.org/10.1080/08957959.2012.664642 smeller, l. (2002). pressure-temperature phase diagrams of biomolecules. biochimica et biofisica acta, 1595, 11-29. https://doi.org/10.1016/s0167-4838(01)00332-6 sousa, s. g., delgadillo, i., & saraiva, j. a. (2014). effect of thermal pasteurization and high pressure processing on immunoglobin content and lysosome and lactoperoxidase activity in human colostrum. food chemistry, 151, 79-85. https://doi.org/10.1016/j.foodchem.2013.11.024 sousa, s. g., delgadillo, i., & saraiva, j. a. (2016). human milk composition and preservation: evaluation of high-pressure processing as a non-thermal pasteurisation technology. crit. rev. food sci. & nutr., 56, 1043-1060. https://doi.org/10.1080/10408398.2012.753402 stanley, h. e. (1971, 1992). introduction to phase transitions and critical phenomena. pergamon, new york. starzonek, s., rutkowska, m., rzoska, s. j., drozd-rzoska, a., fonberg-broczek, m., sokołowska, b., & martinez-garcia, j. c. (2014). dielectric spectroscopy of pressurized saccharomyces cerevisiae. food biophysics, 362, 1-6. vazquez-landaverde, p. a., torres, j. a., & qian, m. c. (2006). effect of high-pressure-moderate-temperature processing on the volatile profile of milk. j. agric. food chem., 54, 9184-9192. https://doi.org/10.1021/jf061497k viazis, s. (2006). high pressure processing of human milk for improved nutrient retention and microbial safety (phd thesis). north caroline state university. viazis, s., farkas, b. e., & jaykus, l. a. (2008). inactivation of bacterial pathogens in human milk by high-pressure processing. j. food. prot., 71, 109-118. https://doi.org/10.4315/0362-028x-71.1.109 windyga, b., rutkowska, m., sokołowska, b., skąpska, s., wesołowska, a., wilińska, m., ... rzoska, s. j. (2015). inactivation of staphylococcus aureus and native microflora in human milk by high pressure processing. high pressure research, 36, 1-8. yordanov, d. g., & angelova, g. v. (2010). high pressure processing for foods preserving. biotechnol. & biotechnol., 24, 1940-1945. https://doi.org/10.2478/v10133-010-0057-8 http://pubs.acs.org/author/vazquez-landaverde%2c+pedro+a http://pubs.acs.org/author/torres%2c+j+antonio http://pubs.acs.org/author/qian%2c+michael+c https://www.ncbi.nlm.nih.gov/pubmed/?term=viazis%20s%5bauthor%5d&cauthor=true&cauthor_uid=18236670 https://www.ncbi.nlm.nih.gov/pubmed/?term=farkas%20be%5bauthor%5d&cauthor=true&cauthor_uid=18236670 https://www.ncbi.nlm.nih.gov/pubmed/?term=jaykus%20la%5bauthor%5d&cauthor=true&cauthor_uid=18236670 https://www.ncbi.nlm.nih.gov/pubmed/18236670 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 1, 2019 www.scholink.org/ojs/index.php/fsns 1 original paper fermented starch: production testing of process stabilization aton yulianto1, palupi tri widiyanti1*, suparman1 & musa1 1 agency for assessment and application of technology, national laboratory for starch technology, jl. zainal abidin pa 36, bandar lampung, lampung, indonesia * palupi tri widiyanti, agency for assessment and application of technology, national laboratory for starch technology, jl. zainal abidin pa 36, bandar lampung, lampung, indonesia received: december 9, 2018 accepted: december 22, 2018 online published: january 21, 2019 doi:10.22158/fsns.v3n1p1 url: http://dx.doi.org/10.22158/fsns.v3n1p1 abstract tapioca is one of starch product that is widely produced in indonesia. the use of tapioca, especially from modern industry, as an ingredient of various food products (such as fish cake, crackers) is often limited by physicochemical characteristics, for example, swelling power, solubility, and viscosity causing limitation in its use. therefore, tapioca needs to be modified to yield the desired characteristics. in this research, tapioca is modified through fermentation using microorganism. starch modification via fermentation provides new chemical groups or change of shape, size and molecule structure. the fermented starch is made from fresh cassava planted by national laboratory of starch technology (b2tp) in lampung-indonesia and starter of fermentation is lactic acid bacteria (lab). fermentation is carried out for five days at room temperature. fermentation product is dried under the sun. the quality stabilization of fermented starch is observed through its functional characteristics including paste clarity, swelling power, and expanding capability. the research conducting in flask scale in b2tp obtains fermented starch with a relatively stable characteristic, especially in expanding capability, around 3.07-5.26 ml/g. the quality stabilization of fermented starch has to be maintained to preserve the desired product characteristics. keywords fermented starch, tapioca, product quality 1. introduction starch is carbohydrate as food reserve of plants. most of the starch is kept in roots (cassava, sweet potato, potato, etc.), grains (maize, paddy, wheat, etc.), trunk (sago) and fruits. besides, starch is an essential nutrition in daily life, where the human body needs energy almost 80% from carbohydrate. starch is composed by at least three components that are amylose, amylopectin, and intermediate www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 2 published by scholink inc. materials (protein and fat). generally, starch contains 15-30% amylose, 70-85% amylopectin, and 5-10% intermediate materials. the structure and type of intermediate materials for each starch source is different depending on the botanical characteristics of the sources (greenwood & munro, 1979). starch plays an important role in the processed food industries. native starch such as tapioca, starch from maize, sago, and other starchy substances have some obstacles when used as ingredient both in food and non-food industries. when cooked, starch needs a long time (needs quite high energy) and forms a hard and unclear paste. besides, its character is too sticky and cannot resist with acidic treatment. these obstacles cause limitation in the use of native starch in industry. in contrast, the sources and production of starch in indonesia is abundance, consisting of tapioca (cassava starch), starch from other roots, sago, paddy, fruits (e.g., banana) and other starch sources that have not commercially produce yet (koswara, 2006). native starch can be modified so that it has desired characteristics. the modification is meant to change the molecular structure of starch that can be done by chemically, physically, and enzymatic treatment (james et al., 1997). each method produces modified starch with a different character. native starch can be processed as modified starch to have the desired character or as needed (sangseethong et al., 2009). the modification involves physical treatment (heat, pressure), chemically treatment (acid, alkali, oxidation, cross-link, etc.), enzymatic, and biologically treatment (fermentation). tapioca is one of starch product that is widely produced in indonesia. the use of tapioca, especially from modern industry, as an ingredient of various food products (such as fish cake, crackers) is often limited by physicochemical characteristics, for example, swelling power, solubility, and viscosity causing limitation in its use. the processed food industry prefers using tapioca from conventional tapioca manufacturer compared to modern industry. this is an obstacle for the industries that applied dewatering system before changing to use separator and hydrocyclone to improve its production capacity and brightness of its tapioca. they receive complaints from the consumers. therefore, an effort to modify tapioca needs to be done to yield tapioca with desired characteristics. modification of tapioca through fermentation is a suitable method. this method involves microorganism. starch modification via fermentation yields new chemical groups or changes of shape, size and molecular structure. b2tp constructed tapioca pilot plant with the capacity of 5 ton/day in 2015. its process production is a combination from production system in the modern industry and that of in the conventional industry. through the facility, there are two types of product, consisting of native starch and fermented starch. cassava is peeled and washed, then rasped to be cassava mash. next, the mash is extracted to obtain starch suspension. its concentration is concentrated and purified via dewatering process using hydrocyclone. after that, starch concentrate is settled in a pond. for the product of native starch, wet tapioca is then dried either under the sun or by mill dryer. in case of fermented tapioca, the wet tapioca is fermented using starter of lab in a certain incubation period, and then dried under the sun, so that tapioca with specific characteristic and suitably used in the food industry can be achieved. process www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 3 published by scholink inc. production of tapioca is shown in figure 1. the assessment for production of fermented starch in b2tp has been done since 2015. since then, the production parameter has been determined, but the product quality has not been stable yet. therefore, testing for the quality stabilization of fermented starch is needed. this report confirms stabilization of quality of fermented starch through several times production in flask scale. cassava peeling & washing crushing & rasping extraction starch suspension cassava pulp concentration & purification drying fermentation fermented tapioca drying tapioca figure 1. flow diagram of the production process of tapioca and fermented tapioca in tapioca pilot plant b2tp 2. methodology 2.1 materials fresh cassava used is harvested from b2tp plantation in central lampung-indonesia, and the starter of fermentation is lab with a commercial brand of bimo-cf. 2.2 equipment equipment used in cassava starch extraction is cassava peeler, crusher, plastic container, and filter cloth. equipment used for fermentation is stainless steel container and erlenmeyer 5000ml. equipment used for product analysis involves glassware, spectrophotometer, ph meter, porcelain cup, oven, desiccator, waterbath, and scanning electron microscope (sem). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 4 published by scholink inc. 2.3 method 2.3.1 production of fermented starch in flask scale, cassava is peeled and washed, then rasped to be cassava mash. next, the mash is extracted using filter cloth to obtain starch suspension. then, the suspension is settled in a plastic container. for fermentation media, starch suspension is set to have a concentration of 20°be (46.68% w/w) and added 0.1% w/w lab to dry weight of tapioca. media is incubated within five days at room temperature. the wet tapioca is then washed for three times. it is then thinned out and dried under the sun to reach moisture content less than 13%. next, dry starch is milled and sifted to have powder size of 80 mesh. 2.3.2 functional characterization of fermented starch functional characteristics of fermented starch observed are paste clarity 1% (method by stuart et al., 1989), swelling power at 70°c (method by leach et al., 1959), and expanding capability (method by demiate et al., 2000). besides, the profile of starch granule is also provided by sem. 2.3.3 data analysis testing of quality stabilization of fermented starch product is carried out by several repetitive testing. each product is named with a code of pf-(production sequence), for example pf-2, pf-2, etc. data obtained is then analysis to observe the stabilization of production process through quality of fermented starch product that is yielded from a series of production process. 3. results and discussions 3.1 process production of fermented starch cassava starch is wetly extracted to separate the starch from other components. in flask scale, wet extraction of cassava starch requires quite a large amount of water compared to that of an industrial scale. this is important to release starch granule from any binding substance so that pure starch is obtained. process parameter for production of fermented starch in this report is mostly adjusted to that of in tapioca pilot plant b2tp. cassava is peeled to remove the outer brown skin and small part of inner white skin. peeled cassava is then washed under running water to eliminate any dirt and mucus on the root surface so that cyanide acid can be reduced. next, clean cassava is rasped to tear the tissue inside to facilitate starch extraction. the cassava mash is then mixed with water, squeezed, and filtered. starch suspension is obtained and settled. naturally, starch will settled because it is not dissolved in water and has higher density than water. starch fermentation in this report conducted in a media with concentration of 20°be (46.68% w/w) and lab concentration of 0.1% w/w to dry weight of tapioca as used in tapioca pilot plant. the top of fermentation container is tightly closed to maximize the fermentation process due to the anaerobic lab. after five days, wet tapioca is dried under the sun. drying process is evidently impact on the characteristics of fermented starch. vatanasuchart et al. (2005) shows that lactic acid and ultraviolet www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 5 published by scholink inc. (uv) energy with a wave length of 310-330 nm can cause partial depolymerization on amylose structure of sour tapioca so that the resulted expanding power is higher than that of using oven. therefore, when starch paste is heated, water molecule is easier to absorb and then forms hydrogen binding so that viscous paste is faster to be achieved. visually, fermented starch is not significantly different to native tapioca. fermented starch is slightly white compare to tapioca. besides, fermented starch has a unique odor. this comes from the lab that generates nonvolatile odor component, lactic acid, as the main component (onyango et al., 2004). furthermore, starch modification in this fermentation is able to improve the product characteristic, especially expanding capability, so that the easiness in its application may be achieved. the modification is due to hydrolysis by lactic acid generated by lab and heat treatment from sun drying with various wavelengths. the mechanism of starch modification allows structure change of amylose and amylopectin. the amorphous and crystalline structures of those segments contribute to starch characteristic, specifically functional characteristics. the amorphous structure can be dissolved, whereas the crystalline cannot be dissolved. in fermented starch, amorphous structure is damaged by lactic acid. this structure consists of weak bonds and is generally found in intersection of starch structure so that it is easier to break by acid. when damaged by acid, the intersection is broken and then some straight chains are formed in accordance with the amount of the damage. in the production of fermented starch, the needed amount of the damage of amorphous structure is only some parts. so, the easiness of the user application can be obtained by observing the functional characteristics of the product. 3.3 functional characterization of fermented starch data analysis for fermented starch in this report is focused on functional characteristics including swelling power at 70°c, and expanding capability. result of analysis is shown in table 1. table 1. functional characteristics of fermented starch sample paste clarity (%) swelling power (g/g) expanding capability (ml/g) pf-1 33.25 8.59 3.07 pf-2 39.00 12.42 4.32 pf-3 50.85 3.37 5.26 pf-4 50.75 4.84 4.69 3.3.1 paste clarity 1% paste clarity 1% is measured by spectrophotometer. light transmission can be used to directly determine the development of starch granule through light reflection of paste. the obtained transmission identifies the homogeneity of starch granule inside the paste. in this measurement, the more transparent the paste, the higher percentage of transmission is obtained. the value indicates that www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 6 published by scholink inc. amorphous fraction from fermented starch is partly cut and dissolved in water. in other words, functional characteristic of paste clarity 1% is related to the dispersion and retrogradation of starch. according to table 1, the value of paste clarity 1% of fermented starch in this report is around 33.25-50.85%. this quite high value means fermented starch resulting paste that is relatively clear or transparent so that the product is suitable to be used as ingredient of food product with clear color, such as fish cake. 3.3.2 swelling power swelling power identifies ability of starch molecule to retain water in its hydrogen bond. starch swells when heated in water. in the process, water hydrates starch granule in the amorphous area that has weak hydrogen bond among molecules. as a result, some parts of starch molecules, especially amylose, are released from starch granule and dissolved in water. the higher heating temperature, the more starch molecules are released. in fermentation process, the amount of lactic acid will continue to rise along with the length of fermentation time. lactic acid damages the amorphous fraction, not only inside amylose but also amylopectin structure contained in fermented starch so that hydration when heated is higher. this causes starch granule to increasingly swell and result in higher swelling power. the value can be used as parameter to decide the dimension of process equipment when processed/cooked. according to table 1, the value of swelling power of fermented starch in this report is around 3.37-12.4 g/g. the value of swelling power is inversely proportional to viscosity. starch with high value of swelling power has lower viscosity. this is a benefit of fermented starch. the low viscosity of fermented starch is able to yield food product with soft or tender texture (rembulan et al., 2012). 3.3.3 expanding capability expanding capability shows starch ability to expand and raise the dough volume at the same time. the volume changing is expressed in specific volume (ml/g). dough made from fermented starch has low viscosity, so it is faster to expand. in baking process, hydrogen bonds among molecules are weaker causing the more amorphous fractions are cut off and resulting in the higher hydration on starch granule. besides, the drying system of fermented starch is also impact on the value of expanding capability. sun drying provides a higher value of expanding capability than another drying method, such as oven or drying machine. sunlight radiation, especially uv-b with the wave length of 310-330 nm will react to amylose and amylopectin causing partial depolymerization in the linear fragment and amorphous structure while in baking process (vatanasuchart et al., 2005). according to table 1, the value of expanding capability of fermented starch in this report is around 3.07-5.26 ml/g. this value is quite high compared to native starch (1.44-2.52 ml/g). this is another advantage of fermented starch where it can be used as an ingredient for food product that requires expanding dough so that the use of expanding agent (e.g., yeast) can be reduced. based on the three functional characteristics observed, expanding capability from fermented starch in this report has quite stable value. the quality stabilization of product can be maintained by concerning www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 7 published by scholink inc. some points that are suspected impact on the product quality including variety and harvest age of cassava that is varied, and also the period of sun drying. 3.3.4 granule shape of fermented starch fermented starch is a product modification from native starch. in detail observation of the shape of starch granules by sem, some differences are found when compared to the granule of native starch. as shown in figure 2, fermentation causes starch granules to have various shapes, i.e. truncated granules, granules with partly-broken surface, and even moon-like surface. meanwhile, granules of native starch have a shape with smooth surface, but some portions are being irregular. this observation is in line with putri et al. (2011), where some granules of fermented starch are digested during the fermentation process. they find broken granules and also irregular surface of granules as the evidence of fermentation. the granular change represents the structural change of amylose and amylopection due to the existence of lactic acid during fermentation that damages the amorphous fraction. figure 2. shape of starch granule observed by scanning electron microscope (sem): native tapioca (right) and fermented starch (left) 4. conclusion testing of fermented starch in this report yields quite stable product, especially in the functional characteristic of expanding capability around 3.07-5.26 ml/g. the process stabilization of fermented starch production has to be maintained to preserve the desired product characteristics. some factors need to be maintained including variety and harvest age of cassava that is used as raw material, drying period and sunlight intensity, and environmental condition that supports the fermentation process. references demiate, i. m., dupuy, n., huvene, j. p., cereda, m. p., & wosiacki, g. (2000). relationship between baking behavior of modified cassava straches and starch chemical structure determinated by ftir spectroscopy. carbohyd polym, 42, 149-158. https://doi.org/10.1016/s0144-8617(99)00152-6 greenwood, c. t., & munro, d. n. (1979). effects of heat on foodstuffs. in carbohydrates (r. j. priestley, ed.). applied science publ. ltd, london. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 1, 2019 8 published by scholink inc. james, n. be miller, & west lafayette. (1997). starch modification: challenges and prospects, usa, review, 127-131. koswara. (2006). teknologi modifikasi pati. ebook pangan. leach, h. w., mccowen, l. d., & schoch, t. j. (1959). structure of the starch granules. in swelling and solubility patterns of various starches. cereal chemistry, 36, 534-544. onyango, c., bley, t., raddatz, h., & henle, t. (2004). flavour compounds in backslop fermented uji (an east african sour porridge). eur food res technol, 218, 579-583. https://doi.org/10.1007/s00217-003-0870-5 putri, w. d. r., haryadi, marseno, d. w., & cahyanto, m. n. (2011). effect of biodegradation by lactic acid bacteria on physical properties of cassava starch. international food research journal, 18(3), 1149-1154. rembulan, g. d., sunarti, t. c., & meryandini, a. (2012). addition of encapsulated lactic acid bacteria to suppress the growth of pathogenic bacteria during tapioca production. jurnal teknologi dan industri pangan, 26(1), 34-43. sangseethong, k., lertphanich, s., & sriroth, k. (2009). physicochemical properties of oxidized cassava starch prepared under various alakalinity levels. starch/stärke, 61, 92-100. https://doi.org/10.1002/star.200800048 stuart, a. s. c., cladualdoc, m., paul, a. s., & hosenney, r. c. (1989). starch paste clarity. cereal chem, 66, 173-182. vatanasuchart, n., naivikul, o., charoenrein, s., sriroth, k. (2005). moleculer properties of cassava starch modified with different uv irradiations to enhance baking expansion. carbohyd polym, 61, 80-87. https://doi.org/10.1016/j.carbpol.2005.02.012 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 1, 2017 www.scholink.org/ojs/index.php/fsns 1 nutritive profile of sun dried fermented mud fish (clarias anguiliaris) and tiger fish (hydrocynus vittatus) locally known as abil alier sold in markets in south sudan amegovu k. andrew1*, mawadri michael1 & juliana mandha2 1 department of food technology, college of applied and industrial sciences, university of juba, juba, republic of south sudan 2 faculty of public health and management, international health sciences university, kampala, uganda * amegovu k. andrew, e-mail: kiri_andrew@yahoo.com received: january 10, 2017 accepted: january 24, 2017 online published: february 5, 2017 doi:10.22158/fsns.v1n1p1 url: http://dx.doi.org/10.22158/fsns.v1n1p1 abstract fishes are irreplaceable animal food in developing countries as a source of high quality protein and micronutrients. this study was carried out to determine the proximate composition, mineral content and fatty acids of sun-dried fermented mud fish (clarias anguiliaris) and tiger fish (hydrocynus vittatus) sold in local markets of south sudan. international organization for standardization procedures were used to determine proximate composition, atomic absorption spectrophotometer for mineral content and gas chromatography-mass spectrometry for fatty acids. c. anguiliaris had higher concentrations of crude protein (75.2%), crude fat (24.9%) and moisture content (14.3%) than h. vittatus with 65.98%, 7.81%, 8.12% respectively. h. vittatus had more ash content (4.1%) and carbohydrate (3.59%) than c. anguiliaris at 2.7% and 2.12% respectively. palmitic fatty acid was the dominant saturated fatty acid in h. vittatus (21.12%) and c. anguillaris (21.32%). eicosatrienoic acid was 11.21% in h. vittatus and 10.64% in c. anguillaris and was the highest polyunsaturated fatty acid. calcium was the highest mineral followed by magnesium and zinc. lead and mercury were significantly (p < 0.0001) higher in c. anguiliaris (0.104 ± 0.001 g100g-1) than h. vittatus (0.06 ± 0.000 g100g-1). c. anguiliaris and h. vittatus are highly nutritious and rich sources of protein, moisture, lipid, ash and minerals. keywords south sudan, tiger fish, mud fish, nutritive profile www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 2 published by scholink inc. 1. introduction fish, either produced through fish farming/aquaculture activity or caught from wild marine or freshwater stocks, is a primary source of protein and essential nutrients. there is a growing recognition of its nutritional and health-promoting qualities (tahergorabi, matak, & jaczynski, 2014). the presence of essential nutrients (such as iodine, vitamin b12 and d), the long-chain fatty acids (lc-pufa), eicosapentaenoic (epa) and docosahexaenoic (dha) omega-3 fatty acids, protein of high quality (with all the essential amino-acids) and fish is very rich content in calcium, iron, zinc and vitamin a, is well documented (belton & thilsted, 2014; kawarazuka & béné, 2011; salem & eggersdorfer, 2015). fish contributes to 16% of all animal protein consumed worldwide (world bank, 2013). fish is a particularly nutritious food, rich in numerous micronutrients in their in bioavailability form (golden et al., 2016) that are often missing in diets, particularly those of the poor. recent studies have reported high micronutrient deficiencies. globally, 0.9% of children under 5 years and 7.8% of pregnant women have night blindness due to insufficient vitamin a (black et al., 2013). africa has the highest proportion of pregnant women with iron deficiency anemia (20.3%) and zinc deficiency (23.9%) (black et al., 2013). their deficiencies pose risks of infant mortality, cognitive under-development, weak immunity, maternal and perinatal deaths and growth retardation (schaible & kaufmann, 2007). fish is an essential, cheap and often irreplaceable animal food for the poor in developing countries with access to water resources (youn et al., 2014). fish and fish-related products also provide income and livelihoods for numerous communities across the world (food and agriculture organization, 2016) and is the fastest growing food-supply industry in the world (béné, barange, & subasinghe, 2015). fish, in a broad sense, including fisheries and aquaculture, plays a crucial role for food security as a purveyor of food (availability), livelihoods and income (mcclanahan, allison, & cinner, 2015) particularly for some vulnerable and marginalized populations (accessibility) (lynch et al., 2016). consumption of fish is projected to increase by 57% by 2020 in developing countries (world bank, 2013) , however, fish has a short shelf life. various methods food processing techniques such as salting, canning, drying, curing, freezing and fermentation are used for preservation and value addition (hall, 2012). in south sudan, sun-drying and fermentation are the most common traditional methods of fish processing. an estimate of proximate composition gives a good justification for better processing, preservation and provides the nutritional value of fish species. this study was carried out to determine the proximate composition, mineral content and fatty acids of sun-dried fermented mud fish and tiger fish locally known as abil alier and commonly sold in every markets in south sudan. 2. methods 2.1 sample collection fish samples of abil alier were aseptically collected from konyokonyo market randomly and transported to chemiphar food laboratory for analysis. samples (n = 12) were cleaned manually and www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 3 published by scholink inc. homogenized using a mixer grinder. the minced samples were then stored at -40oc for further use. 2.2 proximate composition crude protein, ash (mineral), crude fat, carbohydrate and moisture content were determined in triplicates using standard protocols. standard procedures by the international organization for standardization (iso) were used to determine moisture (iso 1442:1997), fat content (iso 1443:1973), ash content (iso 936:1998) and crude fiber (iso 5498:1981). crude protein was determined according to kjeldahl method (976.05; aoac, 2000) and protein calculated from the nitrogen content multiplied by 6.25. total carbohydrate content was determined using antrone method, using standard protocol d-glucose as standard (dreywood, 1946). 2.3 mineral analysis samples were dried in an oven at 125oc and ashed at 550oc. the samples were then initially digested in 15 ml of hno3 until colorless. distilled water was added to make up 25 ml. minerals including heavy metals were determined using atomic absorption spectrophotometer (aas) (aoac, 2000). analyses were carried out in triplicates and results expressed as dry weight mean values. 2.4 fatty acid analysis total lipids in the fish samples were extracted using a method developed by folch et al. (1957) and the fatty acids were classified using gas chromatography-mass spectrometry (chatzimichalakis, samanidou, & papadoyannis, 2004). each individual constituent was identified and quantified by comparing retention times and peaks with the standards. 2.5 statistical analysis statistical significance was analyzed by unpaired student’s t-test method using graph pad prism. p < 0.05 was considered statistically significant and the results were expressed in mean ± se. 3. results there was a significant difference (p < 0.0001) in the proximate composition of c. anguiliaris and h. vittatus. h. vittatus had higher concentrations of crude protein (75.18%), ash content (4.1%) and carbohydrate (3.59%) than c. anguiliaris at 65.98%, 2.7% and 2.12% respectively. however, c. anguiliaris had more fat (24.9%) and moisture content (14.3%) than h. vittatus at 7.81% and 8.12% respectively (table 1). table 1. proximate composition of c. anguillaris and h. vittatus component content (%) c. anguiliaris h. vittatus moistur 14.3 ± 0.62a 8.12 ± 0.08b protein 65.98 ± 0.01a 75.18 ± 0.04b fat 24.9 ± 0.93a 7.81 ± 0.16b www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 4 published by scholink inc. carbohydrates 2.12 ± 0.00a 3.59 ± 0.01b ash 2.7 ± 0.10a 4.1 ± 0.21b crude fibre 0.062 ± 0.002 0.185 ± 0.006 energy (kcal) 495.97 ± 1.32 378.80 ± 1.782 values are reported as mean ± standard deviation of three replicates. values for pairs with different superscripts across the rows are significantly different (p < 0.05). the mineral composition of c. anguillaris and h. vittatus is presented in the table 2. minerals detected were zinc, magnesium, calcium, lead, mercury, and arsenic. the highest mineral in both fish species was calcium at 467.00 ± 2.735 g100g-1 and 388.51 ± 8.691 g100g-1 in c. anguiliaris and h. vittatus respectively. heavy metals specifically lead and mercury were significantly (p < 0.0001) higher in c. anguiliaris at 0.104 ± 0.001 g100g-1 and 0.06 ± 0.000 g100g-1 respectively than in h. vittatus. table 2. mineral composition c. anguillaris and h. vittatus mineral (ppm) content (g100g -1 of wet sample) c. anguiliaris h. vittatus nutritional minerals ca 467.00 ± 2.735a 388.51 ± 8.691b mg 241.72 ± 0.029 242.65 ± 2.25a zn 6.08 ± 0.340a 4.24 ± 0.175b heavy metals pb 0.104 ± 0.001a 0.096 ± 0.001b hg 0.06 ± 0.000a 0.045 ± 0.005b as 0.014 ± 0.000a 0.014 ± 0.000a values are reported as mean ± standard deviation of three replicates. values for pairs with different superscripts across the rows are significantly different (p < 0.05). more than half of the total fat content in h. vittatus and c. anguillaris constituted of total saturated fatty acids (sfas) at 58.94 ± 0.32% and 60.36 ± 1.99% respectively as shown in table 3. palmitic fatty acid (c16:0) was found to be the dominant sfa in h. vittatus (21.12%) and c. anguillaris (21.32%). monounsaturated fatty acids (mufa) was found to be at 9.27 ± 0.83% and 8.70 ± 0.33% for h. vittatus and c. anguillaris respectively. oleic acid (c18:1 ω9) was the highest mufa in h. vittatus (3.99%) and c. anguillaris (4.08%). the overall amount of pufas found in h. vittatus was 31.79 ± 0.04% and 30.94 ± 0.92% in c. anguillaris. the level of eicosatrienoic acid (ete) (c20:3ὠ3) was found to be 11.21% in h. vittatus and 10.64% in c. anguillaris which was the highest among all pufas, followed by linoleic acid (c18:2) and eicosapentaenoic acid (epa) (c20:5ω3). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 5 published by scholink inc. table 3. fatty acid composition of c. anguillaris and h. vittatus fatty acid % composition of total area h. vittatus c. anguillaris saturated (sfa) 10:0 5.50 6.01 11:0 0.22 0.27 13:0 2.80 3.01 14:0 0.54 0.54 15:0 10.24 10.35 16:0 21.12 21.32 17:0 0.65 0.64 18:0 16.06 16.49 20:0 0.54 0.54 21:0 1.29 1.18 ∑ sfa 58.94 ± 0.32 60.36 ± 1.99 mono-unsaturated (mufa) 14:1 0.54 0.54 15:1 0.22 0.27 16:1 0.54 0.54 17:1 1.40 1.40 18:1 3.99 4.08 20:1 1.08 1.13 22:1 0.32 0.16 24:1 1.19 0.59 ∑ mufa 9.27 ± 0.83 8.70 ± 0.33 polyunsaturated (pufa) 18:2 8.62 8.81 18:3 ω 6 2.05 2.20 18:3 ω 3 2.16 2.26 20:3 ω 6 0.43 0.43 20:3 ω 3 11.21 10.64 20:4 ω6 0.75 0.70 20:5 ω3 6.36 5.91 22:5 ω9 0.32 0.16 ∑ pufa 31.79 ± 0.04 30.94 ± 0.92 ∑ ω-3 19.94 ± 0.61 18.80 ± 0.6 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 6 published by scholink inc. ∑ ω-6 3.23 ± 0.72 3.33 ± 0.07 ∑ ω-9 4.31 ± 0.54 4.24 ± 0. 41 iodine value 23.35 ± 2.19 23.41 ± 0.15 oleic acid(o)/linoleic(i) ratio 0.46 ± 0. 01 0.46 ± 0.00 4. discussion fish is the most efficient converter of feed into high quality food (high level panel of experts on world food security, 2014). fish is a good source of high quality protein with all the essential amino acids and micronutrients (kawarazuka & béné, 2011). however, its composition varies from one fishing ground to another, season to season, the amount and quality of feeds, amount of fish movement, size, sex, age, within and across fish species (jan, shah, manzoor, & ganie, 2012; stansby, 1962) . in the present study, significant differences (p < 0.05) were observed in moisture, fat, protein, carbohydrate and ash content of the sun-dried fermented fish species of c. anguiliaris and h. vittatus. moisture content was found to be higher in c. anguiliaris than h. vittatus. this could be attributed to the duration and temperature of sun-drying as water is lost due to evaporation (eyo, 2001). the fat content and moisture content in fish is said to be inversely related (fao, 1999). this was found to be true for c. anguiliaris while in h. vittatus fat and water content were almost similar at 7.81% and 8.12% respectively. fish feeds, habitat and geographical locations influences the protein and fat content of fish (ahmed et al., 2015). protein content was found to be higher in the h. vittatus than in c. anguiliaris. c. anguiliaris with a protein content of 65.98 ± 0.01 was similar to a previous study by idakwo et al. (2016) in fermented solar tent-dried fish. the time of reproduction may affect protein content because during spawning, protein is transferred from the muscle to the ovaries to meet the energy requirements (jan et al., 2012). in addition, during the growth cycles, microbial metabolism is inhibited which decreases synthesis of proteins (suchitra & sarojinalini, 2012). the measure of mineral content is ash given it’s the inorganic residue that remains after the organic matter has been removed (ogundiran et al., 2014). the ash content observed was higher in h. vittatus than c. anguiliaris. h. vittatus is a predator that swallows small prey fish whole mainly of the families of characidae and cichlide (gerking, 2014). the amount of vitamins and minerals is species-specific and can furthermore vary with season. fish meat is regarded as a valuable source of calcium and phosphorus in particular but also of iron, copper and selenium (fao, 2016). the nutritional minerals observed in c. anguiliaris and h. vittatus fish were calcium, magnesium and zinc. calcium was the highest mineral content as with other similar previous studies (bogard et al., 2015; mahanty et al., 2014). calcium deficiency leads to the development of rickets especially in children (craviari et al., 2008). lead and arsenic concentrations observed in c. anguillaris differed from a study carried out on river okpokwu, nigeria which recorded 0.05 ± 0.01 μg/g as and 0.08 ± 0.03 μg/g pb (biosci, abah, ubwa, onyejefu, & nomor, 2013). the heavy metals in fish species could be due to the agrochemical wastes from pesticides, chemical fertilizers, herbicides, www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 7 published by scholink inc. domestic wastes, and fuels in the water and their bio-accumulation in fish which can affect human health in the long term (harikumar & jisha, 2010). the levels of heavy metals observed in both fish species were below the recommended permissible limits of pb 0.02 mg/g (world health organization, 2006) and as 0.01 mg/kg (butu & iguisi, 2013). fish is the richest source of long chain ὠ pufa that improve human health and nutrition (mahanty et al., 2014). omega 3 and omega 6 fatty acid have been found to reduce hypertension, cancer, coronary heart disease, atherosclerosis, alzheimer’s disease and dementia (davis & kris-etherton, 2003; shahid & miraliakbari, 2004). fatty acid profiles of c. anguiliaris than h. vittatus showed that they both contain more sfa than pufa which was different from mahanty et al. (2014) who reported more pufa than sfa in p. sophore. in conclusion, this study has shown that the mud fish (clarias anguiliaris) and tiger fish (hydrocynus vittatus) are highly nutritious. both fish species are rich sources of protein, moisture, lipids, ash and minerals. more than half of the total fat content in h. vittatus and c. anguillaris constituted of total saturated fatty acids (sfas) and polyunsaturated fatty acids (pufa) were eicosatrienoic acid (ete), linoleic acid, eicosapentaenoic acid (epa). calcium was highest nutritious mineral followed by magnesium and zinc. heavy metals detected were lead, mercury, and arsenic. fish absorb and bio-accumulate minerals from their diets and the water bodies. references ahmed, t., muhammad, s., naqvi, a., abdullah, s., abbas, k., zakir, s., … zia, m. a. (2015). comparative proximate body composition of wild captured and farm cultured cirrhinus mrigala. pakistan journal of agricultural sciences, 52(1), 203-207. aoac. (2000). official method of analysis (17th ed.). gaithersburg: association of official analytical chemists. belton, b., & thilsted, s. h. (2014). fisheries in transition: food and nutrition security implications for the global south. global food security, 3(1), 59-66. http://dx.doi.org/10.1016/j.gfs.2013.10.001 béné, c., barange, m., & subasinghe, r. (2015). feeding 9 billion by 2050—putting fish back on the menu. food security, 7(2), 261-274. http://dx.doi.org/10.1007/s12571-015-0427-z biosci, i. j., abah, j., ubwa, s. t., onyejefu, d. i., & nomor, s. a. (2013). assessment of the levels of some heavy metals in mudfish (clarias anguillaris ) from river okpokwu, apa, benue state, nigeria. international journal of biosciences, 3(4), 142-150. http://dx.doi.org/10.12692/ijb/3.4.142-150 black, r. e., victora, c. g., walker, s. p., bhutta, z. a., christian, p., de onis, m., … uauy, r. (2013). maternal and child undernutrition and overweight in low-income and middle-income countries. the lancet, 382(9890), 427-451. http://dx.doi.org/10.1016/s0140-6736(13)60937-x bogard, j. r., thilsted, s. h., marks, g. c., wahab, m. a., hossain, m. a. r., jakobsen, j., & stangoulis, j. (2015). nutrient composition of important fish species in bangladesh and potential www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 8 published by scholink inc. contribution to recommended nutrient intakes. journal of food composition and analysis, 42, 120-133. http://dx.doi.org/10.1016/j.jfca.2015.03.002 butu, a. w., & iguisi, e. o. (2013). concentration of heavy metals in sediment of river kubanni, zaria, nigeria, compr. j. earth env. sci, 1(2), 10-17. chatzimichalakis, p. m., samanidou, v. f., & papadoyannis, j. 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(2004). omega-3 (n=3) fatty acid in health and disease: www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 10 published by scholink inc. part1-cardiovascular disease and cancer. j med food, 7, 387-401. https://doi.org/10.1089/jmf.2004.7.387 stansby, m. e. (1962). proximate compoistion of fish. fish in nutrition, 55-60. suchitra, t., & sarojinalini, c. (2012). effect of temperature on biochemical and microbiological qualities of ngari. nature science, 10(2), 32-40. tahergorabi, r., matak, k. e., & jaczynski, j. (2014). fish protein isolate: development of functional foods with nutraceutical ingredients. journal of functional foods, 18, 1-11. http://dx.doi.org/10.1016/j.jff.2014.05.006 world bank. (2013). fish to 2030: prospects for fisheries and aquaculture. agriculture and environmental services discussion paper, 3(83177), 102. http://dx.doi.org/83177-glb world health organization. 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(2014). inland capture fishery contributions to global food security and threats to their future. global food security, 3(3-4), 142-148. http://dx.doi.org/10.1016/j.gfs.2014.09.005 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 2, no. 3, 2018 www.scholink.org/ojs/index.php/fsns 86 original paper quality oil for deep frying in foods and changes acidity on various kind of oils after frying puminat w.1* & teangpook c.2 1 department of food chemistry and physic, institution of food research and product development, kasetsart university, bangkok, thailand 2 department of food processing and preservation, institution of food research and product development, kasetsart university, bangkok, thailand * puminat w., department of food chemistry and physic, institution of food research and product development, kasetsart university, 10900, bangkok, thailand received: november 9, 2018 accepted: november 20, 2018 online published: november 29, 2018 doi:10.22158/fsns.v2n3p86 url: http://dx.doi.org/10.22158/fsns.v2n3p86 abstract human eat a lot of highly acidic and oxidant foods. acid chemicals and oxidants in food cause harmful to the health. acid value, free fatty acid and polarities of the fried oil change during cooking for food. statistical data of fried oils estimate and systematize on three recipes of food (doughstick, fish cake and fried chicken). their recipes are on the best of sensory evaluation. in each food recipes are selected from market and evaluated by traditional and popular merchant. in the sampling, the experimental model is planned by trial rbcd with factorial 3 × 2 × 3. the use of three type oil, two level temperature and three sampling collector are treated on experiments with three recipes. they showed that palm olien oil can change a higher ph than soybean oil and rice bran oil by a statistically significant difference at 95% confidence level. deep frying by high temperatures and short time have change a quality oil less than low temperatures and long time. frying in larger quantities and a longer period can change the more acidity and polarities. quality foods for flavor and healthy oil must no more high and low temperature in cooking. keywords acid value (av), acidity, fried oil and food frying 1. introduction cells and tissues of normal body is level ph 7.35. in normal function, the body has been completely absorbed the oxygen for metabolic processes. the body can prevent cancer including the growth of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 87 published by scholink inc. pathogens, bacteria and fungi. if ph level is more acid the normal cells in the body is misbehaving by more oxygen. in normal tissues can hold oxygen up to 20 times of acidic conditions. if blood develops a more acidic condition the body will inevitably deposit the excess acid. as this cycle continues, the dead cells will then turn into acids. these areas will increase in acidity until some cells die. the body obtains and contains an acid into the cell body. blood will equillibrate and maintain the acidity. survivable cell can become the toxic cells. in long time, those cells will be the addition of more acid. some cells will die by the age of it. remaining cells become abnormal cells and hazardous toxic cells. if the growth does not stop cells becomes abnormal cells or cancer cells. cooking oil help penetrate heat into the food and distribute heat for cooking. the correct frying is necessary to keep the enough high heat and no overheat conduction. oil heat conduction helps cook quickly. food is brought in direct with hot oil. frying is a complex process by heat, mass transfer and chemical reactions change. the food surface becomes golden yellow to dark brown in color and develops a pleasant fried food flavor in the frying process, the food is fried in a layer of oil and fried until completion. heat turns the internal moisture of food into vapor. the food loss moisture and develop surface color, firm texture, flavor and aroma. the food surface becomes golden yellow to dark brown in color and develops a pleasant fried food flavor. extremely cooking is brown according to the time in the recipe. cooking food with oil take good texture, taste and more appetizing. food frying makes it palatable and desirable to consumer include texture, fried food flavor. deep frying is cooked using oil as a medium heat. cooking food by frying, oil is absorbed and penetrated in context food. the penetration of fried oil is enough to make the food taste better. if food is adsorbed more oil and became oily it will destroy the taste of food. the frying of food must have an understanding of the nature of the oil and type of fried materials. the frying temperature depends on the thickness and type of food. regulating lies between 175 and 190°c (345-375°f). the heat cooks food extremely quickly. oil for cooking originate in several major sources. ability of resistance the change and oxidation of oil in each method are differential reasons. oil for deep frying, it should be a high smoke point so that high temperature and more long time make change less quality oil. repeated frying with over high heat, the oil will change the physical and chemical properties by moisture in the food and oxygen from the air. new compounds are occurred such as free acid, polar compounds, polymers, carbonyls, ketones, etc. they may accumulate in the body and harm to the health of consumers. chemicals in food are acidic in accrued process of cooking will affect the body by exposure with increasing of acidity. control the ph balance and chemicals are able to resist life-threatening disease and reduce the risk of cancer. 2. materials and methods 2.1 experimental design and random sampling for deep-fried oils management and preparation of food recipes to deep frying (doughstick, fish cake and fried chicken) for study the fried oils. http://en.wikipedia.org/wiki/celsius http://en.wikipedia.org/wiki/fahrenheit www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 88 published by scholink inc. figure 1. raw material of oil and oil for deep frying experimental design use management as trial factorial 3 × 2 × 3 and 2 blocks in experiments by rbcd. three types of fried oil are soybean, rice bran and palm olein with two levels of high (h) and low (l) temperature. oil samples after frying are been sampling by batch at three period in sampling level of fried food weight. 2.2 pick up oils from three food cooking by sampling between deep frying processing doughstick recipes are directly selected from a professional merchant. the best recipe is chosen by preference test and sensory evaluation. standard recipes of dough flour are deep-fried in each oil by two level temperature and period time of frying (180°c 2 minutes and 160°c 4 minutes). frying oil are collected by batch of weight of dough flour at 0.5, 1 and 1.5 kilogram in each interval of sampling. fish cake recipes are selected by finished product with spices from a market. they are sensory for the best of ingredient and favorite recipe. deep-fried oils for fish cake 7.5 kg, are collected by batch at the end of the weight at 1, 5 and 7.5 kg respectively. two levels of temperature and frying time are 150°c for 3 minutes and 130°c for 5 minutes. fried chicken recipes are chosen a testing by one of the most favorite. marinated spicy chicken deep-fried on two levels of temperature and time as 175°c 7 minutes and 155°c 8 minutes. the frying oils of fried chicken 5 kg. are collected by batch at weight 1, 3 and 5 kg respectively. 2.3 solvent extraction on oil after process solvent extraction for optimum, the food analysis with a convenient method of separating are made by extract the oil soluble components and other contaminates. lipid and oil are soluble in three groups of organic solvent. percentage of polar and nonpolar are 0%, 50% and 100%. 2.4 polar compounds (polar cpds) in deep-fried processing polar compounds are formed during deep frying as a function of process time and product exposure. sample is filtered through silica column chromatography and eluted with hexane/diethyl ether. solvent is evaporated and weighted for polar cpds (gutierrez et al., 1988) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 89 published by scholink inc. 2.5 studying acid value and the quality oil in processing of deep-fried the standard analysis is a general method by aoac reference. the chosen process for the more better is diethyl ether and ethanol solvent for oil extraction (kardash & yakov, 2005). this method is the most reliable to improvement and increase efficiently a high solubility. quantitative analysis of acidity is acid value and free fatty acid. sampling the fried oil of cooking process are evaluated on a type of oil, frying temperature, amount of sampling and kind of fried food. sample oil (1-2 g) is weighted in flask with cover lid and poured a solvent mixture 25 ml of ethyl ether : ethanol (1 : 1). soluble mixer is filled 1-.2 drops of phenolpthalien indicator and titrated with 0.01 n potassium hydroxide in alcohol until the endpoint. color of mix soluble change no color to pink. the volume of titration calculate as the acidity and compare against the equivalent gram (gm-e) of potassium hydroxide. acid value and free fatty acid are evaluated on changes an acidity between cooking process. 2.6 color and the color changes of oils measuring color is taken with the data color international measurement model color tools. cie value (commission internationale de i’eclaerage) display the color value in cielab system l*(0 = black and 100 = white), a*(-a* = green and +a* = red) and b*(-b* = blue and +b* = yellow) at d65 10deg (light source illuminant d). 2.7 statistical analysis experimental designs are replicated two treatments in each value. average values of different parameter by statistical analyses are expressed as the mean and standard deviation. evaluation of significant difference among the mean values of analysis are conducted using statistical software as spss 15.0. graph and t-test are performed on statistical analysis package of statistica program at 95%.confidence level. identify differences among samples evaluate by duncan new’s mutiple range test at significant level p < 0.05. 3. results and discussions in the frying process of food is brought in direct with hot oil. the food surface becomes golden yellow to dark brown in color and develops a pleasant fried food flavor. food frying make it palatable and desirable to consumer include texture and fried food flavor. crispyness colour, flavor and texture are product characteristics of thai fried foods. frying is suitable on temperature and time. it make a high quality and identity of food such as appearance, taste and acceptance. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 90 published by scholink inc. figure 2. three food recipes (doughstick, fish cake and fried chicken) of deep frying in three food recipes (doughstick, fish cake and fried chicken) of deep frying by appropriate temperatures and short time have change a quality oil less than overheating and long time. normally fried food should use an adequate temperature. quality food for flavor and healthy oil must no more high and low temperature in cooking. the frying temperature depends on the thickness and type of food. regulating the heat lies between 175°c and 190°c (345°f and 375°f). it cooks food completely. heat of deep-frying is a complex process by mass transfer and chemical reactions change. heat turns the internal moisture of food into vapor. the water vapor change bubbles around the food. the foods loss moisture and develop surface color, firm texture, flavor and aroma. table 1. the parameter of oil after deep-frying for three food recipes parameter food recipes of deep-frying doughstick fish cake fried chicken l 92.50±3.85a – 92.77±5.14a 90.95±3.91a – 91.52±5.21a 88.21±4.04a – 88.77±5.38a a* -1.60±0.20a – -1.54±0.10a -0.55±0.20b – -0.54±0.10a -8.76±0.10a – -8.76±0.10a b* 21.14±2.42a – 22.62±1.48a 27.97±3.21a ± 36.49±2.39a 75.18±8.64a – 78.52±5.15a vicosity (cp.) 61.62±1.74a – 62.04±0.78a 60.16±1.78b – 61.18±0.77a 60.63±1.77a – 60.65±0.76a acid value 0.81±0.01a – 0.83±0.04a 1.05±0.01a – 1.10±0.06a 0.53±0.05a – 0.55±0.02a polar cpds (%) 12.02±1.77b – 12.65±0.78a 11.67±1.71a – 12.17±0.72a 11.62±1.71a – 11.83±0.75a note. a,b are the vertical meanings in different alphabets and have significantly the differences at 0.05 level. table 1 shows the average parameter and standard deviation of fried oil by different temperature in three food recipes of deep-frying. color oil after frying of fried chicken has more dark than fish cake and doughstick.vicosity and polar compounds are the highest for doughstick. fish cake has a high http://en.wikipedia.org/wiki/celsius http://en.wikipedia.org/wiki/celsius http://en.wikipedia.org/wiki/fahrenheit http://en.wikipedia.org/wiki/fahrenheit www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 91 published by scholink inc. changing of ph and the highest amount of acid value in three food recipes. acid value and free fatty acid obtained as a result of the breakdown of the total fried oil. measurement of free acid in the fryer oil indicates the degradation. in the experiments, extraction technique with solvent is chosen one of the best of efficiency in all matrices. conditions are fluctuation in control and comparison of extraction. selection the best solubility of extract help decrease the interference of determining the total acidity in lipid contents of food. normally the free fatty acid content in the fryer oil 0.25% to 0.4% for most snack food products and it allowed to go up > 0.5% in the fryer. if oils contain 2% free fatty acid the oil will begin to oxidize. increasing of acidity during frying can evaluate as destroy oil and the broken bonds of molecule to free acid. table 2. the free fatty acid (ffa) of fried oil after deep frying by different temperature in three food recipes deep-fried of food recipes oil for frying temperature level intervals of sampling doughstick fish cake fried chicken ffa±sd ffa± sd ffa± sd rice h 1 0.2050±0.0021 0.1772±0.0006 0.0552±0.0005 bran oil 2 0.2250±0.0021 0.27875±0.0016 0.1006±0.0002 3 0.2700±0.0028 0.3226±0.0017 0.1230±0.0002 l 1 0.2250±0.0007 0.2370±0.0003 0.0723±0.0001 2 0.2450±0.0007 0.3201±0.0001 0.0945±0.0001 3 0.3500±0.0008 0.3640±0.0001 0.1196±0.0002 soy h 1 0.3250±0.0016 0.3947±0.0012 0.2494±0.0016 bean oil 2 0.3450±0.0013 0.4563±0.0012 0.2356±0.0010 3 0.3750±0.0011 0.4647±0.0007 0.2260±0.0006 l 1 0.3500±0.0016 0.3486±0.0021 0.2376±0.0012 2 0.3650±0.0015 0.4203±0.0013 0.2207±0.0007 3 0.4200±0.0017 0.4802±0.0009 0.1838±0.0001 plam h 1 0.7500±0.0017 0.7865±0.0007 0.3085±0.0036 oil 2 0.7400±0.0014 0.8719±0.0002 0.1819±0.0020 3 0.7000±0.0008 0.9062±0.0001 0.1685±0.0019 l 1 0.8650±0.0009 0.8041±0.0009 0.4963±0.0002 2 0.8700±0.0013 0.8633±0.0006 0.4261±0.0003 3 0.8350±0.0009 0.8995±0.0004 0.3535±0.0002 note. (h,l) mean the level high (h = 180-190°c) and low (l = 150-160°c) of temperature usage deep frying in each food. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 92 published by scholink inc. in a batch fryer, the oil is heated directly with an external heater. the food is fried in a layer of oil and fried until completion. fried products are removed the excess oil through a drain device. frying oils are collected by batch in three intervals of sampling. they have free fatty acid (ffa) ±sd by the kinds of oils in each deep-fried food on table 2. figure 3. changing acid value (av) and free fatty acid (ffa) by sampling the deep fried oil in three food recipes frying oil of doughstick are been sampling with high and low temperature. deep-frying of doughstick must slowly penetrate heat so that crispiness and browning are spread out and change an equability. frying of doughstick at high temperature must use short time in order that fermented flour do not have a burn and hardness. changes the acidity of deep-fried oils are orderly made the descending as doughstick, fish cake and fried chicken. the use of three type oils are deep-fried with foods at high temperature. palm olein oil increase an acidity more than rice bran oil and soybean oil. changing of av and ffa are quickly when cooking deep-fried with more long time. they have range of acid value (max-min) and free fatty acid (ffa±sd) in table. av and ffa of sampling oil in all of foods have significantly difference at =0.01. graph show the acid value in three type of fried oils. frying with the high temperature and short time of them have less the acid value and free fatty acid. deep frying of doughstick is both the more high temperature and the more quantity flour. it is different the ascending values of av and ffa by a statistically significant difference at 95% confidence level. frying oil for doughstick by palm olein oil, soy bean oil and rice bran oil have mean and standard deviation of the acid value and free fatty acid as 1.58 ± 0.22, 0.72 ± 0.22, 0.50 ± 0.11, and 0.79 ± 0.11, 0.36 ±0.11, 0.25 ± 0.05 respectively. av and ffa of palm olein oil are the highest average. fried oil of fish cake change the av and ffa more than doughstick and fried chicken. graph show the acid value (av) and free fatty acid (ffa) in three kinds of fried oil. plam olein oil change the acidity more than soy bean oil and rice bran oil respectively. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 93 published by scholink inc. figure 4. increasing the acid value of three oils at different collected time in deep frying three sampling of frying oil in two level temperature show increasing of acid value by more long time. the deep frying in a great amount of fried foods can made change more acidity. the three sampling of deep frying oil by low and high temperature for all foods increase the acidity respectively. the mean average in each weight period of sampling oil have difference significantly at = 0.05. the maximum level for acid value of edible fat and oil were established by the administration of public health at 0.6 (unit of av = mg koh/1 g oil for refined fat and oil). the oil degradation cause loss a nutritional value and the destruction of essential fatty acid. consuming of the deteriorated oil get acid components and toxic products. normally ratio of alkaline-acid food for consumption should be about four to one, or eighty percent as alkaline foods and twenty percent as acid foods for a healthy body. experts recommend health by balance body with acid and alkaline foods. the evaluation on the acidity of fat and oil should develop and control a quality of cooking oils for consumer health. 4. conclusions fat and oil are a nutrition and a high source of energy in five main foods. they release nine calories per gram and dissolve a necessary vitamins. oils which help distribute heat in cooking of fried food make increase browning, crispy and flavor food. the use of fat and oil for cooking food take effect to healthiness and unhealthiness. acid value was used to assessment the degradation of frying oil. it is often a measure of the break down of triacylglycerols into free fatty acids and other free acid, including new compounds such as free acid, polar compounds, carbonyls, ketones, etc. the amount of free acids and volatiles are related to the quality of fried food. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 94 published by scholink inc. references aoac. (2000). official methods of analysis (17th ed.). the association of official chemists. washington dc. usa. fritch, c. w. (1981). measurements of frying fat deterioration: a brief review. journal of the american oil chemists’ society, 58, 272-274. https://doi.org/10.1007/bf02582355 gutierrez, r., quijano, g., & dobarganes, m. c. (1988). frying of food: principles, changes, new approaches (pp. 141-154). chichester: ellis horwood. kardash, e., & yakov, i. t. (2005). acid value determination in vegetable oils by indirect titration in aqueous—alcohol media. croatica chemica acta, 78(1), 103-199. normand, l., eskin, n. a. m., & przybylski, r. (2000). evaluation of the very-fry pro ffa-75 quick test for measuring free fatty acids in deep—frying oils. journal of food lipids, 7, 63-69. https://doi.org/10.1111/j.1745-4522.2000.tb00161.x saguy, i. s., shaani, a., weinberg, p., & garti, n. (1996). utilization of jojoba oil for deep-fat frying of foods. food science and technology, 29, 573-577. yi-chang, t., moreira, r., & sun, x. (1996). total frying-use time effects on soybean-oil deterioration and on tortilla chip quality. international journal of food science & technology, 31(3), 287-294. https://doi.org/10.1046/j.1365-2621.1996.00338.x food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 1, 2017 www.scholink.org/ojs/index.php/fsns 43 anthocyanin, lutein, polyphenol contents and antioxidant activity of black, red and white pigmented rice varieties saravanan ponnappan1, arun thangavel1 & omprakash sahu1* 1 faculty of chemical and food engineering, bahir dar institute of technology, bahir dar university, ethiopia * omprakash sahu, e-mail: ops0121@gmail.com received: april 10, 2017 accepted: april 22, 2017 online published: may 16, 2017 doi:10.22158/fsns.v1n1p43 url: http://dx.doi.org/10.22158/fsns.v1n1p43 abstract colour rice varieties are rich in antioxidants and functional based properties such as anthocyanin, lutein and phenolic compounds. in this experiment, two of red pigmented (tps-1and tkm-9), one white (glutinous rice) and black pigmented rice varieties are cultivated from india were analysed to determine their antioxidants and nutrition based functional properties. based on the result, the anthocyanin content was very high on black rice than other variety contents up to 244.45 mg/100 g. polyphenol compound were varied significantly within the compared varieties. highest polyphenol compound content (463.05 mg/100 g) was found in the black rice and also showed rich antioxidant properties. obviously, black rice rich source of lutein compound was also higher than other varieties where under the experimental condition. dpph (determination of 2, 20-diphenyl-1picrylhydrazyl radical scavenging ability) scavenging capacity starting from 69.46% to 76.4% ranged to level of remain dpph. keywords antioxidants, anthocyanin, polyphenol compound, dpph 1. introduction rice is the essential cereal crop in developing world countries. rice is utilizing as a staple food for one half of the population in developing countries (bhattacharjee et al., 2002). but most of the developing countries living populations are widely eating white rice, even though they are cultivating enormous rice variety which have been containing various pigments such as black, red and purple color kernels. that kernels contains rice are rich in inevitable antioxidants such as phenolic compounds and lutein (perera & yen, 2007). from the report of previous study have been conducted by lee et al. (2008), anthocyanin playing major role of cholesterol reduction and also inhibitory effects on in vitro allergic reaction in human body www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 44 published by scholink inc. (abdel-aal et al., 2006; yang et al., 2008). peonindin-3-glucoside and cyanindin-3-glucoside are all the major anthocyanin compounds which are present in the extracts of black rice, also these compounds are influencing an inhibitory effect of invasion of cells on different cancer (chen et al., 2006). lamberts and delcour (2008) studied that carotenoids decreases the formation of cancer cells and other heart disease. enormous in vitro and in vivo studies have revealed that ldl modification through the help of oxidation mainly plays onset of exacerbates and atherosclerosis on clinical manifestation (siddiq, 2004). pigmented rice are studied as a major source of antioxidants and other vital functional properties especially lutein and phenolic compounds (frei & becker, 2005). normally, the health benefits of pigmented rice varieties are very high but the production and supply are very less for the demand of population needs (yawadio et al., 2007). zeazanthin and lutein are the essential non provitamin-a which were present in the eye protection against both infection and disease (rose, 1999; tan et al., 2005). with significant health benefits of therapeutic values in rice varieties have been known since from heritage scriptures and experience from the peoples (siddiq, 2004; johnson, 2002). 2. methods 2.1 sample collection to study the sample experimental pigmented rice varieties (figure 1) were selected and purchased from various available markets in india. the paddy of black rice (kavuni rice) was obtained from dry land agriculture research station, kanadukathan, chettinad, india. red rice paddy varieties tps-1 were obtained from regional rice research station, thirupathisaram and tkm-9 obtained from rice research station, thirurkuppam, thiruvallur. glutinous white rice which was sourced from mumbai was purchased from remuki departmental stores, madurai, india. finally, the macerated and grounded ricesample flours were prepared in laboratory. under the following methods were analysed appropriate compounds from different pigmented rice varieties. 2.2 anthocyanins potassium chloride buffer (0.03 mol/l) and sodium acetate buffer were added with 20 µl rice sample extracts from pigmented rice varieties. mixed well and also allow those compounds for 15 min to absorb measurements on spectrophotometer at 500 nm and 700 nm (nicoue et al., 2007). distilled water used for blank purpose. the concentration of anthocyanin content (mg/l) from the extracted samples was calculated with standard formula and also result has expressed as cyanin-3-glucoside equivalents. anthocyanin content = (a x mw x df / ɛ x 1) x1000 where, a = (aλ700) ph 1 (aλ700) ph 4.5; mw = mol. wt. of cyanidin-3-glucoside; df = factor of dilution; ɛ= extinction coefficient (l x cm 1 x mol-1) = 26,900 for cyanidin-3-glucose; www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 45 published by scholink inc. where l (length) = 1. 2.3 polyphenols compounds in test tubes, the extraction of methanol was taken and also prepare the volume up to 3 ml with distillation water. then, 0.5 ml folin-ciocalteau reagent was mixed with methanol solution. about 20 per cent sodium carbonate was added under the volume of 2 ml after three min approximately and mixed well again. absorbent measurement range on spectrophotometer wavelength range at 650 nm against the blank using with vis spectrophotometer. a set of standard solutions of gallic acid prepared using with distilled water (10 µg-100 µg per ml) was treated in the same manner as described earlier and read against a blank. polyphenol compounds was expressed in mg and also it was equivalent to gallic acid per 100g on fwb basis. 2.4 lutein content of selected pigmented rice varieties chemicals: acetonitrile, hexane, methanol, ethanol and dichloromethane were of hplc grade standard. carotenoid extraction: macerated colour rice samples flour were mixed with sodium sulfate (5 g) and 2 mm-rtocopherol in methanol solution. ice-cold acetone was used to prepared colorless carotenoids content extracted solution (volume: 400 ml). prepared crude extract was mixed and shaken with 100ml hexane. three or four times the extraction was repeated finally make the known volume up to 250 ml. the mixed crude hexane was dried with anhydrous sodium sulfate (20 g) and filtered through filter paper (whatman no.1). an aliquot filtered known extracted volume (100 ml) solution was dried under the nitrogen stream and the remaining residue of the hexane solution was redissolved in 1 ml acetonitrile chemical. finally, the prepared samples were analysed by hplc. hplc analysis: sge c-18 (ods) column was used to separate carotenoids through hplc instrument. about 0.1 per cent ammonium acetate with methanol was used as mobile phase for the carotenoids separation. 20 µl measured samples were injected in ods column on hplc equipment and also isocratic condition was continued at 1 ml/min flow rate. λmax values of the compound were determined by the time of retention and standard chromatograms assessed through the help of spd-10 avd detector. in column result where the quantified peak area were related to the standard reference. 2.5 anti-oxidant activity 2.5.1 diphenyl-picryl-hydrazyl (dpph) assay dpph is a purple coloured stable free radical and will form yellow colour when it was reduced as diphenyl-picryl-hydrazine complex. the sample extracts electron donation ability was calculated from purple colour beaching of dpph methanol solution. scavenging antioxidant potential indicates by discoloration. the dpph assay was performed by goupy et al. (1999). 2 ml of 60 µm 2, 2-diphenyl-1 picryl-hydrazyl in methanol (initial absorbance of dpph was 0.62 ± 0.02.) was added to one ml of various concentrations of sample extract. prepared 1 min vortexed mixture and kept in room temperature under the dark condition for 30 minutes. decreased absorbance was measured in spectrophotometer under the wavelength of 517 nm with used methanol as a blank portion. ascorbic www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 46 published by scholink inc. acid was applied as positive control point. calibration curve was diagrammed indicated for using absorbance versus concentration of ascorbic acid and the results were noted that as a mg (vit-c) equivalent per 100 g of sample on fwb basis. figure 1. selected pigmented rice varieties for study 3. results carotenoids are essential chemical compounds of coloured rice varieties. selected rice flour samples were very low levels of β-carotene content were eluted by hplc. but the coloured rice varieties were good source of lutein shown in figure 2. the lutein content found to be maximum in tps-1 (1075 ng/100 g) followed by black rice (280 ng/100 g), glutinous white rice (240 ng/100 g) and tkm-9 (215 ng/100 g) respectively. the different significant ratio (p < 0.05) was noticed in terms of lutein content between the pigmented rice varieties and white rice. the data indicates the presence of maximum level of anthocyanin mainly in black rice (244.45 mg/100 g) while the other pigmented rice varieties such as tps-1 and tkm-9 had anthocyanin levels of 1.39 and 1.25 mg/100 g respectively, with lowest levels recorded in glutinous white rice (0.32 mg/100 g). table 1. lutein, anthocyanin, polyphenols content and antioxidant activity of selected pigmented rice varieties varieties lutein (ng/100 g) anthocyanin (mg/100 g) polyphenols (mg/100 g) antioxidant activity dpph (%) glutinous white rice 240 ± 2.08 0.32 ± 0.01 88.21 ± 0.04 69.46 ± 0.05 black rice 280 ± 1.52 244.45 ± 0.02 463.05 ± 0.07 86.12 ± 0.05 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 47 published by scholink inc. tps-1 1075 ± 4.50 1.39 ± 0.01 340.13 ± 0.08 84.42 ± 0.05 tkm-9 215 ± 1.52 1.25 ± 0.01 208.51 ± 0.06 79.79 ± 0.03 sed 1.2247 0.0053 0.0156 0.0091 cd (0.05) 2.9970** 0.0129** 0.0381** 0.0223** the polyphenol content of pigmented rice varieties was noticed to be minimum in glutinous white rice (88.21 mg/100 g) compared to higher levels recorded in black rice (463.05 mg/100 g), followed by red rice varieties tps-1 (340.13 mg/100 g) and tkm-9 (208.51 mg/100 g). the data pertaining to antioxidant activity of the pigmented rice varieties is presented in table.1. the stable dpph radical is continuously used to test hydrogen donating antioxidants in enormous plant species. it can be inferred, that antioxidant activity of selected pigmented rice varieties was maximum in black rice at 15.81 per cent in terms of dpph value followed by tps-1 (13.42%), tkm-9 (12.68%) and lowest in glutinous rice (10.48%). significant difference in terms of total antioxidant activity was noted between the selected varieties. figure 2. lutein content of selected pigmented rice varieties 4. discussion anthocyanin, lutein and polyphenol compounds were essential antioxidants which were majorly present in pigmented rice varieties. the data results shows presence the level of anthocyanin, polyphenol and other vital antioxidant activities were higher in black rice variety. but the lutein content www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 48 published by scholink inc. was maximum in tps-1 red rice variety. sompong et al. (2011) reported that cyanidin 3-glucoside and peonoidin 3-glucoside as the dominant anthocyanins in black rice varieties with contents ranging from 19.4 to 140.8 mg/100 g dm and 11.1-12.8 mg/100 g dm respectively. red rice varieties to have polyphenol compounds in the range of 79.2 and 691.4 mg fa equivalent/100 g with a mean tpc of 364.8 mg fa equivalent/100 g. the black rice had a higher mean polyphenol compounds of 492.8 mg fa equivalent/100 g than the red ones. also he was evaluated the dpph value of three black rice varieties to range from 16.04 to 30.25 per cent and that of 10 red rice varieties to range from 12.99 to 76.38 per cent. no significant difference between the black and red rice varieties were reported. in conclusion, this experimental study has shown that the pigmented rice varieties were good source of antioxidants and other vital functional properties such as lutein, anthocyanin, and polyphenol compounds. the lutein content found to be maximum in tps-1 (1075 ng/100 g) followed by black rice (280 ng/100 g), glutinous white rice (240 ng/100 g) and tkm-9 (215 ng/100 g) respectively. the different significant ratio (p < 0.05) was noticed in terms of lutein content between the pigmented rice varieties and white rice. maximum level of anthocyanin mainly in black rice (244.45 mg/100 g) while the other pigmented rice varieties such as tps-1 and tkm-9 had anthocyanin levels of 1.39 and 1.25 mg/100 g respectively, with lowest levels recorded in glutinous white rice (0.32 mg/100 g). the polyphenol content of pigmented rice varieties was noticed to be minimum in glutinous white rice (88.21 mg/100 g) compared to higher levels recorded in black rice (463.05 mg/100 g), followed by red rice varieties tps-1 (340.13 mg/100 g) and tkm-9 (208.51 mg/100 g). antioxidant activity of selected pigmented rice varieties was maximum in black rice at 15.81 per cent in terms of dpph value followed by tps-1 (13.42%), tkm-9 (12.68%) and lowest in glutinous rice (10.48%). references abdel-aal, e. s. m., young, j. c., & rabalski, i. (2006). anthocyanin composition in black, blue, pink, purple, and red cereal grains. journal of agricultural and food chemistry, 54, 4696-4704. bhattacharjee, p., singhal, r. s., & kulkarni, p. r. (2002). basmati rice: a review. international journal of food science and technology, 37(1), 1-12. chen, p. n., kuo, w. h., chiang, c. l., chiou, h. l., hsieh, y. s., & chu, s. c. (2006). black rice anthocyanins inhibit cancer cells invasion via repressions of mmps and u-pa expression. chemico-biological interactions, 163(3), 218-229. frei, m., & becker, k. (2005). fatty acids and all-trans-beta-carotene are correlated in differently colored rice landraces. journal of science in food and agriculture, 85, 2380-2384. johnson, j. l., carson, k. g., & jackson, c. l. (2002). characteristics of intact and ruptured atherosclerotic plaques in brachiocephalic arteries of apolipoprotein-e knockout mice. arteriosclerosis thrombosis vascular biology, 22, 788-792. lamberts, l., & delcour, j. a. (2008). carotenoids in raw and parboiled brown and milled rice. journal of agricultural food chemistry, 56(24), 11914-11919. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 49 published by scholink inc. lee, j. c., kim, j. d., hsieh, f. h., & eun, j.-b. (2008). production of black rice cake using ground black rice and medium-grain brown rice. international journal of food science and technology, 43(6), 1078-1082. nicoue, e. e., sylvanin, s., & khaled, b. (2007). anthocyanin in wild blueberries of quebec: extraction and identification. journal of agriculture and food chemistry, 55(15), 5626-5635. perera, c. o., & yen, g. m. (2007). functional properties of carotenoids in human health. international journal of food properties, 10, 201-230. rose, r. (1999). atherosclerosis: an inflammatory disease. the new england journal of medicine, 340, 115-126. siddiq, e. a. (2004). aromatic and medicinal rices; urgency for collection, validation and conservation for sustainable utilization towards value added rice farming. compendium of papers. science-society interface on medicinal and aromatic rices. august organized by m.s.swamination research foundation, chennai and kerala agricultural university. sompong, r., siebenh, l-ehn, s., linsberger-martin, g., & berghofer, e. (2011). physicochemical and antioxidative properties of red and black rice varieties from thailand, china and sri lanka. food chemistry, 124, 132-140. tan, j., baisakh, n., oliva, n., parkhi, v., rai, m., torrizo, l., … datta, s. k. (2005). the screening of rice germplasm, including those transgenic rice lines which accumulate beta-carotene in their polished seeds, for their carotenoid profile. international journal of food science and technology, 40, 563-569. yang, d. s., lee, k. s., jeong, o. y., kim, k. j., & kays, s. j. (2008). characterization of volatile aroma compounds in cooked black rice. journal of agricultural and food chemistry, 56, 235-240. yawadio, r., tanimori, s., & morita, n. (2007). identification of phenolic compounds isolated from pigmented rices and their aldose reductase inhibitory activities. food chemistry, 101(4), 1616-1625. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 3, 2019 www.scholink.org/ojs/index.php/fsns 84 original paper differences in dietary intake of women with standard weight but varying body fat percentages in japan minatsu kobayashi1,2*, mayuko hirata1, eri abe2 & mieko horiguchi3 1 department of food science, faculty of home economics, otsuma women’s university, tokyo, japan 2 institute of human culture studies, otsuma women’s university, tokyo, japan 3 department of domestic science, junior college division, otsuma women’s university, tokyo, japan * minatsu kobayashi, department of food science, faculty of home economics, otsuma women’s university, tokyo, japan; institute of human culture studies, otsuma women’s university, tokyo, japan received: june 13, 2019 accepted: june 24, 2019 online published: july 8, 2019 doi:10.22158/fsns.v3n3p84 url: http://dx.doi.org/10.22158/fsns.v3n3p84 abstract “hidden obese people” have a high body fat percentage (bfp) despite having a normal bmi (18.5 <bmi ≦ 25.0 bfp ≧30) due to an excessive accumulation of visceral fat, which increases their risk of lifestyle-related diseases. we aimed to identify factors that contribute to hidden obesity among japanese female students at the nutritionist training facility university using their anthropomorphic measurements, lifestyle characteristics and nutritional intake. characteristics of participants with hidden obesity physique (18.5 <bmi ≦ 25.0 and bfp >30%) (n = 160) and standard physique (18.5 <bmi ≦ 25.0 and bfp <30%) (n = 376) were compared using student’s t-test or welch’s t-test. the participants with hidden obesity physique have lower intake of energy (p = 0.044) and fat (p = 0.036) and a higher intake of carbohydrates (p = 0.023), cereals (p = 0.009) and sugary beverages (p = 0.020). this study suggests that a reduction in carbohydrate-dense foods is effective in preventing hidden obesity. keywords body fat percentage, bmi, carbohydrate-dense foods, hidden obesity www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 85 published by scholink inc. 1. introduction body mass index (bmi) which is calculated from the height and weight is used as an indicator of obesity worldwide (who expert consultation, 1995). the report of a who expert consultation stated that at bmis lower than the existing who cut-off point for overweight (= 25 kg/m2), a substantial proportion of asian people have a high risk of type 2 diabetes and cardiovascular disease (who expert consultation, 2004). however, an excessive accumulation of body fat is a risk factor for many diseases such as diabetes mellitus, dyslipidaemia of lipid metabolism, hypertension, and myocardial diseases (wulan, westerterp, & plasqui, 2010). therefore, it is important to consider not only body weight but also body fat in assessing for obesity. compared to other developed countries, the percentage of young japanese women who are categorized as underweight (bmi <18.5) is high. the japan national survey of 2017 showed that 20.7% of japanese women in their twenties were underweight (ministry of education, culture, sports, science & technology in japan, 2017). additionally, among persons with a bmi within the normal range, persons with normal body weight with a high body fat percentage (bfp), the so-called ‘hidden obese persons’ are of increasing importance (omori, tanaka, & nakajima, 2016). the ‘hidden obesity’ among young women is thought to be due to a decrease in muscle mass and bone mass, and an increase in body fat volume among persons whose diet is of poor quality due to a strong “desire for thinness” (niibori, hatsushika, takanami, & akedo, 2013). people with a strong “desire to be thin” tend to have a high score on the eating attitude test (eat-26), which is a screening test for eating disorders (uehara & sakakibara, 2015). among young women, there is a risk of consuming a poor diet due to a strong desire to be slender, resulting in anemia, menstrual abnormality and eating disorders (nicholls & viner, 2005). in addition, of the body weight of a young woman also affects her future pregnancies and childbirth. pregnant women who are underweight have a higher risk of giving birth to infants of low weight (suzuki, nomura, takenoshita, ando, & kido, 2016). a mothers’ weight in pregnancy also affects foetal development (kiserud et al., 2018) and predisposes to the infant to an earlier onset of lifestyle-related diseases (fukuoka, 2016). additionally, after menopause, there is an increased risk of osteoporosis and fractures. therefore it is important to institute the appropriate measures to maintain a healthy body weight (gallagher & tella, 2014). in the literature, there are reports that illustrate that the bmi is an indicator of the relationship between adolescent female physique and nutritional status (koike, hardy, & richards, 2016); however, limited reports exist that consider bfp as an indicator of nutrition status (parizkova, 1994). in this study, we assessed the nutritional status of young women using bmi and bfp by classifying them as follows: (bmi <18.5), (18.5 ≦ bmi < 25.0 and bfp <30), (18.5 ≦ bmi < 25.0 and bfp ≧30) and (bmi ≧ 25.0). we then compared the food and nutrient intake of participants with “standard physique” (18.5 ≦ bmi < 25.0 and 20 ≦ bfp <30) to those of participants with “hidden obesity physique” (18.5 ≦ bmi < 25.0 and bfp ≧30) (sasamori, 2002). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 86 published by scholink inc. 2. method 2.1 participants and ethical considerations we measured the height, weight and body fat ratios of 670 female students at the nutritionist training facility university located in tokyo japan between 2012 and 2017. we assessed lifestyle characteristics using questionnaire, dietary intake using a food frequency questionnaire and assessed their food intake, total energy and nutrient intake. informed consent was obtained from all students. this study was approved by the ethics committee of the otsuma women’s university (permission number: 25-0006). 2.2 determination of physique and biochemical indicators height was measured using a metal height gauge ys 101 s (yoshida works co., ltd.). body weight, bfp, body fat mass, muscle mass and basal metabolism were measured using a body composition analyser inbody770 (in body japan co., ltd.). bmi was calculated using the following formula: bmi = weight (kg) / (height (m))2 study participants were classified using bmi and bfp as follows into 4 groups; “bmi <18.5”, “18.5 ≦ bmi < 25.0 and bfp <30”, “18.5 ≦ bmi < 25.0 and bfp ≧ 30” and “bmi ≧ 25”. blood pressure and pulse were measured using digital automatic sphygmomanometer hem-7000 (omron ltd.). osteo sono-assessment index (osi) was measured using ultrasonic bone evaluation device aos-100nw (hitachi aloka medical ltd.). 2.3 assessment of lifestyle characteristics participants were asked about average weekly exercise time which is stronger than the intensity of walking. residence were asked using the following categories: “living alone, living with family, or dormitory”. bowel frequency was asked using the following categories: “once a day or more than once a day, 5-6 times per week, 3-4 times per week, twice per week or less than twice per week”. bowel movement was asked using the following categories: “loose stool, normal stool, hard stool, or repetition of diarrhea and constipation”. sleeping time was asked using the following categories: “less than 5 hour, 6 hour, 7 hour, or more than 8 hour”. satisfaction of daily life was asked using the following categories: “satisfied, anything satisfied, anything is not satisfied, and not satisfied”. menstrual cycle was asked using the following categories: “regularly and irregularly”. 2.4 estimation of food and nutrient intakes information on dietary intake was obtained using a 165-item semi-quantitative food frequency questionnaire (ffq). participants were asked to report their average frequency of consumption and portion size for each item during the past year. nutrient intake was evaluated using a previously described method (sasaki, kobayashi, ishihara, & tsugane, 2003). the ffq was modified as follows. participants were presented with the eight frequency categories that included: none, once/month, 2-3 times/month, once/week, 2-3 times/week, 4-6 times/week, once/day and more than 2 times/day. portion sizes were also described for every food item. coefficients for the categories of relative portion size were presented in the five categories of 0.5, 0.75, 1.0, 1.25 and 1.5. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 87 published by scholink inc. the composition values of the 165 foods on the ffq were multiplied by the frequencies and relative portion sizes for the food items from the ffq. intakes of total energy and 38 nutrients for each food were calculated using a food composition table developed for the ffq based on the standard tables of food composition in japan, 2017 edition (watanabe & kawai, 2018). food and nutrient intake were energy adjusted using the residual method (henriquez-sanchez et al., 2009). estimated nutrient intake was validated using a 3-day dietary record, a modified ffq that gives reasonably valid estimates of energy, and nutrient and food intake (takada, 2017). 2.5 statistical analysis participants were classified into 4 groups, however only two groups were indicated in the analysis: “standard physique” (18.5 ≦ bmi < 25.0 and bfp <30) and “hidden obesity physique” (18.5 ≦ bmi < 25.0 and bfp ≧ 30%). differences in biochemical indicators, food and nutrient intake estimated from ffq by physique status, and exercise time were assessed using the student’s t-test or welch’s t-test. lifestyle characteristics were assessed using the fisher’s exact test. data were analyzed using sas statistical software ver. 9.4. (sas institute inc.). all analyses were considered significant at the 0.05 level. 3. result a total 670 participants were interviewed. they were categorized as follows: lean students (n = 101), fat students (n = 33) and normal bmi (n = 536) who were then classified into standard physique (n = 376) and hidden obesity physique (n = 160) groups. the distribution of participants by physique status is shown in table 1. of the 670 participants, 376 (56.1%) had a standard physique and 160 (23.9%) a hidden obesity physique. biological indicators of participants by physique status are shown in table 2. participants with a standard physique were taller than those with a hidden obesity physique (p=0.033). conversely, when compared to participants with a standard physique, those with hidden obesity physique were heavier and had higher bmi, bfp, body fat mass (p<0.0001) and diastolic blood pressure (p=0.002). lifestyle characteristics of participants by physique status are shown in table 3. there were no statistically significant differences between participants with a standard physique and those with a hidden obesity physique. the frequency of bowel movements did not differ by physique status. there was a higher proportion of participants with hard stool among those with a hidden obesity physique compared to those with a standard physique (p=0.081). although participants with hidden obesity physique spent less time exercise than those with standard physique, this was not statistically significant (p=0.530). energy and nutrient intake for 38 nutrients of participants by physique status are shown in table 4. energy intake was higher (p=0.044) and total fat and saturated fat intake were also higher (p=0.036, p=0.017) among participants with a standard physique compared to participants with a hidden obesity physique. conversely, compared to participants with a standard physique, carbohydrate intake was www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 88 published by scholink inc. higher (p=0.023) and manganese and iodine intake also were higher (p=0.001, p=0.013) among participants with a hidden obese physique. food intake by participant physique status is shown in table 5. the intake of cereals and beverages other than alcohol was higher (p=0.009, p=0.020) among participants with a hidden obese physique compared to that among participants with a standard physique. in this study, drinking-water was not included in their beverages and there were no differences in the estimated intake of drinking-water by physique status (data not shown). conversely, compared to participants with a hidden obesity physique, the intake of confectionery was higher (p=0.047) among participants with a standard physique. table 1. the distribution of the physique of the participant (n=670) frequency % bmi <18.5 101 15.1 18.5 ≦ bmi <25.0 and bfp <30 376 56.1 18.5 ≦ bmi <25.0 and bfp ≧ 30 160 23.9 bmi ≧ 25 33 4.9 table 2. biological indicator of standard group and hidden obese group standard group (n=376) hidden obese group (n=160) p value* mean ± sd mean ± sd height cm 158.8 ± 4.9 157.8 ± 5.1 0.0333 weight kg 51.2 ± 4.6 55.2 ± 4.7 <0.0001 bmi 20.3 ± 1.2 22.1 ± 1.3 <0.0001 body fat percentage (bfp) % 25.8 ± 2.6 32.3 ± 2.0 <0.0001** body fat mass g 13.3 ± 2.3 17.9 ± 2.1 <0.0001 muscle mass g 35.7 ± 3.1 35.2 ± 2.9 0.079 basal metabolism kcal 1182.0 ± 82.2 1184.2 ± 81.3 0.773 systolic blood pressure mmhg 105.5 ± 9.8 106.3 ± 10.3 0.379 diastolic blood pressure mmhg 66.7 ± 7.1 69.2 ± 8.9 0.002** pulse times 72.7 ± 11.2 74.4 ± 10.8 0.093 osteo sono-assessment index osi(×106) 2.9 ± 0.4 2.9 ± 0.3 0.908** hidden obese group; participants with 18.5≦bmi<25.0 and bfp ≧ 30; standard group; participants with 18.5≦bmi<25.0 and 20 ≦ bfp <30. * student t-test; ** welch’s t-test www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 89 published by scholink inc. table 3. lifestyle characteristics of standard group and hidden obese group standard group (n=376) hidden obese group (n=160) p value* exercise time min per 1 week 58.2±127.6 50.9±111.6 0.530** residence alone 18.6 20.0 0.548 with family 77.7 75.6 dormitory 3.7 3.8 bowel frequency ≧ once a day 52.8 45.0 0.331 5-6 times / week 18.1 23.8 3-4 times / week 21.1 23.1 < 2 times / week 8.0 8.1 bowel movement loose stool 8.0 6.9 0.081 normal stool 76.8 68.8 hard stool 11.7 20.0 repetition of diarrhea and constipation 3.5 4.4 sleeping time less than 5 hour 30.4 25.6 0.430 6 hour 55.2 61.3 7 hour 13.1 11.9 more than 8 hour 1.3 1.3 satisfaction of life satisfied 16.3 17.5 0.313 anything satisfied 65.1 57.5 anything is not satisfied 14.4 20.6 not satisfied 4.0 3.8 menstrual cycle regularly 72.5 73.0 0.861 irregularly 25.6 26.4 hidden obese group; participants with 18.5≦bmi<25.0 and bfp ≧ 30; standard group; participants with 18.5≦bmi<25.0 and 20 ≦ bfp <30. * fisher’s exact test; ** student’s t-test www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 90 published by scholink inc. table 4. energy and nutrient intake*** of standard group and hidden obese group standard group (n=376) hidden obese group (n=160) p value* mean ± sd mean ± sd energy (kcal) 1654.8 ± 548.9 1568.3 ± 407.0 0.044** protein (g) 53.3 ± 7.1 52.1 ± 6.6 0.074 total fat (g) 52.1 ± 10.1 50.0 ± 10.4 0.036 sfa (g) 18.0 ± 4.2 17.0 ± 4.4 0.017 mufa (g) 18.2 ± 3.8 17.5 ± 3.9 0.082 pufa (g) 10.6 ± 2.6 10.3 ± 2.6 0.318 cholesterol (mg) 234.1 ± 73.6 234.2 ± 76.4 0.993 carbohydrate (g) 204.3 ± 27.0 210.1 ± 25.8 0.023 total dietary fibers (g) 8.7 ± 2.2 8.4 ± 2.4 0.251 natrium（mg) 1672.9 ± 577.5 1664.6 ± 643.3 0.884 potassium (mg) 1867.3 ± 391.5 1845.5 ± 422.3 0.565 calcium (mg) 492.0 ± 185.8 465.8 ± 175.0 0.129 magnesium (mg) 206.0 ± 37.7 203.1 ± 42.2 0.419 phosphorus (mg) 846.8 ± 146.3 825.5 ± 136.0 0.117 iron (mg) 6.1 ± 1.3 6.1 ± 1.4 0.936 zinc (mg) 6.9 ± 0.8 6.8 ± 0.7 0.861 copper (mg) 0.9 ± 0.2 0.9 ± 0.2 0.743 manganese (mg) 2.8 ± 1.0 3.1 ± 1.3 0.001** iodine (µg) 629.4 ± 202.4 682.3 ± 232.6 0.013** serene (µg) 48.7 ± 11.1 48.4 ± 10.7 0.768 chrome (µg) 4.3 ± 1.7 4.1 ± 1.7 0.302 molybdic (µg) 156.8 ± 53.2 159.1 ± 53.2 0.648 alpha-carotene (µg) 365.2 ± 293.4 398.2 ± 326.5 0.249 beta-carotene (µg) 1674.6 ± 876.7 1727.4 ± 1043.0 0.575** vitamin a (µg) 587.3 ± 413.1 550.1 ± 329.5 0.270** vitamin d (µg) 4.4 ± 1.7 4.3 ± 1.7 0.699 alpha-tocopherol (mg) 5.9 ± 2.1 5.8 ± 2.1 0.640 vitamin k (µg) 179.98 ± 102.2 169.2 ± 110.5 0.276 vitamin b1 (mg) 0.71 ± 0.11 0.69 ± 0.1 0.210 vitamin b2 (mg) 1.1 ± 0.31 1.14 ± 0.30 0.754 niacin (mgne) 11.3 ± 2.3 11.3 ± 2.14 0.767 vitamin b6 (mg) 0.9 ± 0.1 0.9 ± 0.1 0.833 vitamin b12 (µg) 4.6 ± 1.8 4.4 ± 1.7 0.267 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 91 published by scholink inc. folate (µg) 224.6 ± 61.6 229.1 ± 65.6 0.441 pantothenic acid (mg) 5.2 ± 0.9 5.1 ± 0.9 0.294 biotin (µg) 26.5 ± 6.5 26.6 ± 6.2 0.800 vitamin c (mg) 65.0 ± 24.9 68.1 ± 28.5 0.238** salt (g) 4.2 ± 1.5 4.1 ± 1.6 0.815 alcohol (g) 4.0 ± 6.6 3.7 ± 6.8 0.574 hidden obese group; participants with 18.5≦bmi<25.0 and bfp ≧ 30; standard group; participants with 18.5≦bmi<25.0 and bfp <30. * student t-test, ** welch’s t-test, *** each nutrient were adjusted with residual method table 5. food intake*** of standard group and hidden obese group standard group (n=376) hidden obese group (n=160) p value* (g) mean ± sd mean ± sd cereals 223.2 ± 59.6 237.8 ± 58.6 0.009 potatoes and starches 25.2 ± 14.4 24.2 ± 15.2 0.483 sugars 3.0 ± 3.3 2.7 ± 2.8 0.375** beans 39.0 ± 24.8 36.3 ± 28.4 0.283** nuts and seeds 1.6 ± 3.1 1.6 ± 2.8 0.834 vegetables 125.0 ± 58.2 125.7 ± 65.0 0.906 green vegetables 48.5 ± 26.2 46.8 ± 27.4 0.507 white vegetables 71.3 ± 39.6 73.3 ± 41.4 0.590 pickles 5.3 ± 6.0 5.5 ± 7.9 0.807** fruits 80.3 ± 59.7 76.0 ± 58.9 0.448 mushroom 8.3 ± 6.7 8.0 ± 5.8 0.528** seaweed 5.1 ± 4.7 4.6 ± 4.0 0.249** fish 39.0 18.0 37.9 17.0 0.505 meats 71.8 30.1 69.2 30.3 0.358 eggs 23.0 ± 16.6 25.2 ± 16.2 0.167 dairy 169.7 ± 132.7 160.7 ± 111.9 0.423** fats & oils 9.5 ± 3.7 9.3 ± 3.5 0.440 confectionery 76.3 ± 45.0 68.6 ± 38.5 0.047** alcohol beverage 54.2 ± 92.9 54.4 ± 127.9 0.986** beverage other than alcohol 836.6 ± 609.9 1004.4 ± 817.8 0.020** hidden obese group; participants with 18.5≦bmi<25.0 and bfp ≧ 30; standard group；participants with 18.5≦bmi<25.0 and bfp <30. * student t-test; ** welch’s t-test; *** each food were adjusted with residual method. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 92 published by scholink inc. 4. discussion in our study, 24% of participants had hidden obesity, even though their bmis were within the normal range. in a study of young taiwanese, 29.7% of women were diagnosed with hidden obesity (hung, chen, guo, chang, & jan, 2017). there were differences in biological indicators, lifestyle, nutrients and food intake between the standard physique group and hidden obesity physique group. in particular, the hidden obesity group had a higher intake of cereals, beverages and carbohydrates. although it the body fat mass is higher in persons with a hidden obesity physique, there were no differences in muscle mass and bone density between the two groups. in this study, participants with a standard physique spent more time exercise, but the difference in exercise time did not seem to be enough to affect muscle mass or bone density. there were only two persons with a standard physique and three persons with a hidden obesity physique that were diagnosed with hypertension based on a systolic blood pressure of 140 mm hg or more or diastolic blood pressure of 90 mm hg or more. however, the mean of blood pressure was higher among persons with a hidden obesity physique, especially the diastolic blood pressure. in a recent study of college students that explores differences in blood pressure levels across adiposity, it was found that both waist circumference and fat mass percentages acted as mediators between cardiorespiratory fitness and blood pressure (diez-fernandez et al., 2017). in persons with a hidden obesity physique, a higher proportion had hard stools; on the other hand, their dietary fiber intake was low. dietary fiber intake is effective in relieving constipation (suares & ford, 2011; yang, wang, zhou, & xu, 2012). on the other hand, the intake of water or fluid foods relieves hard stools (kira, 2013). although persons with a hidden obesity physique had a higher intake of beverages (other than alcohol), there were no differences in the estimated intake of drinking-water or water amount in foods by physique status. the diet consumed by the hidden obesity group was characterized by a low intake of fat and sfa and a high intake of carbohydrates. in addition, they had a low intake of sweets and a high intake of cereals and sweet beverages other than alcohol. in a study of women in new zealand, the bfp was directly related to the energy density diet pattern, such as red meat, processed meat or deep-fried foods (schrijvers, mcnaughton, beck, & kruger, 2016). because our study participants were young women, particularly among those with a hidden obesity physique, it is possible they reduced the intake of fat and sweets for the purposes of dieting, which may have led to an increase in the intake of cereals and sweet beverages. several randomized controlled trials have reported differences in the effect of low-carbohydrate diets and low-fat diet on weight reduction (tobias et al., 2015). low-fat diet, particularly low sfa diet, has generally been evaluated for its effectiveness in reducing cvd risk (hu & willett, 2002). however, low-carbohydrate diets are associated with not only reduction in body weight, but also a reduction in the levels of total cholesterol and triglyceride and an increase in hdl cholesterol levels (bueno, de melo, de oliveira, & da rocha ataide, 2013). a recent randomized controlled trial reported no differences in the effect of low-carbohydrate diets and low-fat diet on www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 3, 2019 93 published by scholink inc. weight reduction (gardner et al., 2018). although the effect of low-carbohydrate diets and low-fat diet on the weight reduction is controversial, there may be a number of our study participants who had a desire to reduce their weight that selected the low-fat diet. because the hidden obesity group tended to refrain from total fat intake, sfa intake and snack intake, and the hidden obesity group was expected to have a strong desire to lose weight. this study had some limitations. firstly, because participants were students at the nutritionist training facility university, they may have been knowledgeable about the relationship between nutrition and health risks, and this may have had implications on the differences in their eating and lifestyle habits. nevertheless, dietary intake among persons with a hidden obesity physique was characterized by a low intake of fat, a low intake of sfa and a high intake of carbohydrates. secondly, although the ffq used to estimate food and nutrient intake in this study has been validated, the accuracy of the absolute value of the available intake may not be guaranteed due to the type of the dietary survey conducted. however, our results appear to correctly reflect the difference in the average value of the food and nutrient intake level in the hidden obesity group and the standard group. thirdly, we could only compare the daily exercise time of the hidden obesity group to the standard group; the intensity of the exercise and the activity level in the daily life were not made. 5. conclusion in this study, approximately one quarter of young women with standard bmi were reclassified as having a hidden obesity physique based on a body fat ratio of 30% or more. among all participants, those with a hidden obesity physique had a low intake of fat, a low intake of sfa and a high intake of carbohydrates. obesity is not only identified by bmi but also by bfp (hidden obesity), which is related to the risk of diabetes mellitus, abnormality of lipid metabolism, hypertension and myocardial disease. therefore, the results of this study should be utilized in dietary education in order to reduce the number of persons with hidden obesity. acknowledgement we are deeply grateful to all participants who took part in this study, and to hospital staff for their cooperation. references bueno, n. b., de melo, i. s., de oliveira, s. l., & da rocha ataide, t. 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(2004). appropriate body-mass index for asian populations and its implications for policy and intervention strategies. lancet, 363(9403), 157-163. https://doi.org/10.1016/s0140-6736(03)15268-3 wulan, s. n., westerterp, k. r., & plasqui, g. (2010). ethnic differences in body composition and the associated metabolic profile: a comparative study between asians and caucasians. maturitas, 65(4), 315-319. https://doi.org/10.1016/j.maturitas.2009.12.012 yang, j., wang, h. p., zhou, l., & xu, c. f. (2012). effect of dietary fiber on constipation: a meta analysis. world j gastroenterol., 18(48), 7378-7383. https://doi.org/10.3748/wjg.v18.i48.7378 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 2, 2019 www.scholink.org/ojs/index.php/fsns 54 short research article fat content and fatty acids profile in follow-on formulas commercialized in côte d'ivoire andré philippe s. kpaibé1*, yao aya k.a. kouassi1, n’goran jean simon t. yao1,2, alexandre koko n’bra2, sylvain k. dibi3 & michèle aké1,2 1 département de chimie analytique-bromatologie-chimie minérale-chimie générale, ufr sciences pharmaceutiques et biologiques-université felix houphouët-boigny, abidjan, côte d’ivoire 2 laboratoire de nutrition, institut national de santé publique, abidjan, côte d’ivoire 3 laboratoire de la police scientifique, côte d’ivoire * andré philippe s. kpaibé, département de chimie analytique-bromatologie-chimie minérale-chimie générale, ufr sciences pharmaceutiques et biologiques-université felix houphouët-boigny, abidjan, côte d’ivoire received: march 11, 2019 accepted: march 17, 2019 online published: april 6, 2019 doi:10.22158/fsns.v3n2p54 url: http://dx.doi.org/10.22158/fsns.v3n2p54 abstract this study evaluates the follow-on formula for infants. these products are available under several brands in the ivorian market. in order to verify their conformity to the who standards a post-market control by gravimetric method and gas chromatography with mass spectrometry is executed to evaluate the quantity and quality of fat products contained in the milks of brands available in côte d’ivoire. out of the nine brands of milks analyzed, only four of them were close to the values revealed by their manufacturers, whereas the other products had their values below their respective indications. keywords milk, follow-on formulas, fats, control 1. introduction a follow-on formula is food obtained from the natural cow milk with some additional ingredients. these formulas are appropriate to support growth and development of 6 to 12 months children (who, 2001). a follow-on formula is produced using some physical means. so it is preciously packaged to prevent spoilage and contaminations caused all normal conditions of handling, storage and distribution in the country where the product is sold (codexalimentarius fao-who, 1987). who recommends that follow-up formula be proposed to complete or replace human milk to children www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 55 published by scholink inc. of at least 6 months to ensure that all essential nutrients such as, proteins, lipids, carbohydrates, minerals and vitamins cover their nutritional needs (who, 2015, 2018). follow-on formula are becoming increasingly important in africa, particularly in côte d'ivoire. several imported brands are available pharmacies, supermarket and other small places of distribution (pereira, ford, feeley, sweet, badham, & zehner, 2016; champeny et al., 2016). due to strict standards aimed to maintain the quality of these products, follow-on formula should contain fat content and particularly essential fatty acids similar to those of human milk (suthutvoravut et al., 2016; koletzko et al., 2005). as in breastmilk, saturated and unsaturated fatty acids as well as essential fatty acids must be present in these formulas to ensure growth in children (vaysse et al., 2011; bernard & annie, 1995). as part of a post-marketing quality control of formulas, a study was initiated to evaluate total fat content on and fatty acid profile in follow-on formulas commercialized in abidjan (côte d’ivoire). 2. material and method 2.1 samples and chemicals the samples of follow-on formulas were collected in pharmacies of abidjan. nine brands were found and selected. they were numerically labelled from 1 to 9 for the purpose of the study. four boxes per brand were purchased for analysis. all chemicals and solvents were analytical grade from different suppliers. hydrochloric acid, sodium chloride, dichloromethane, hexane, ethanol, methanol, sodium hydroxide, chloroform from sigma aldrich (missouri, usa). 2.2 instrument the analysis performed on agilent model 7890a gas chromatograph coupled to agilent s975c mass spectrometer (agilent, ca, usa) operating in the electron impact (ei) and selected ion monitoring (sim) modes. samples (1 µl) were injected at 250 °c in split mode. a capillary column (30 m×0.25 mm i.d., 0.25 m agilent j&w capillary columns, ca, usa) was used. 2.3 fat content determination the fat in the milk samples was extracted by the folch lipid extraction method (suthutvoravut et al., 2016). for each sample, 5g of formula were weighted and put into a 250 ml flask. then 40 ml of chloroform and 20 ml of methanol were successively added. the mixture was homogenized on a magnetic stirrer at 400 rpm for 30 minutes before being filtered on a whatman filter paper (70 mm, 8 µm). the filtrate was recovered in a 500 ml separatory funnel. thus, 0.73% sodium chloride solution was deposited in the separating funnel at 20% the volume of the recovered organic solvents. the organic phase obtained was placed in the flask and evaporate till it dried on rotary evaporator at 300 rpm for 40 minutes. then the flask was dried in the oven at 104°c for 30 minutes. the fat content was thus weighed after cooling the flask in a desiccator. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 56 published by scholink inc. 2.4 fatty acids profile 2.4.1 preparations formula of the samples were prepared in dichloromethane to obtain concentrated solutions at 1 mg/ml. then 1 ml of solution was vortexed in a 15 ml test tube with 1 ml of 0.5 n methanolic solution of sodium for 30 seconds. the tube was then placed in a water bath at 70°c for 30 minutes of saponification. methylation was carried out by the addition of 1 ml of methanolic solution of hydrochloric acid, before reheating the tube in a water bath at 70°c for 30 min. the fat was extracted by the successive mixture of 4 ml of 9 mg/ml nacl and 4 ml of hexane. the mixture was allowed to settle for 5 minutes before recovering the organic phase in a test tube. the organic solvent was evaporated using a rotary evaporator at 75°c at 60 rpm. then 100 μl of absolute ethanol was added to remove all traces of water and evaporated on the rotary evaporator. a solution of volatile methylated fatty acids was obtained by adding 1.2 ml of hexane before being analyzed by gas chromatography (gc). 2.4.2 analysis conditions gc temperature program was as follows: 150°c for 2 min, followed by temperature ramp at 1,3°c/min to 200°c, then temperature ramp at 40°c/min to 250°c, and hold for 5 min. full scan data was acquired under the following conditions: mass range 50-550 m/z, scan time 1 s, solvent delay 13.5 min. quantitation of the analytes was carried out in the sim mode, once their characteristic masses were selected from their full spectra. the electron energy was 70 ev, and the electron multiplier was operating at 200 to 300 v (kuo & ding, 2004). analyses of each brand of milk were repeated 6 times. 3. results and discussion the analysis in the follow-on formula marketed in abidjan revealed fat content between 5.01 g and 20.82 g per 100 g of milk powder. very low values of standard deviation have been obtained (table 1). table 1. average total fat content of follow-up formula brands designation fat content (g/100g of milk) content reports on packaging (g/100 g of milk) mean standard deviation (sd) 1 16.40 0.22 22 2 21.32 0.70 22 3 15.19 0.35 21.85 4 21.23 0.06 21.70 5 20.82 1.84 23.50 6 15.48 0.36 25.40 7 20.72 0.77 21.10 8 21.68 0.33 21.80 9 5.01 0.23 21.50 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 57 published by scholink inc. among the nine brands of milks analyzed, only four brands showed content were close to the values reported by the manufacturers, whereas the other five brands (labelled 2, 4, 7, 8) presented low values, particularly brand 9. this last result could indicate a non-compliance of the analyzed batch. besides brand 9, these milks were found to be most concentered in fat than breast milk (kent, mitoulas, cregan, ramsay, doherty, & hartmann, 2006). in all samples analyzed, 07 fatty acids were identified which include 04 saturated fatty acids, 02 monounsaturated fatty acids and only 01 polyunsaturated fatty acids (table 2). table 2. fatty acid profile of milk analyzed group fatty acids detected proportion of fatty acids identified in follow-up formula (%) 1 2 3 4 5 6 7 8 9 fatty acids saturated c 12 :0 (lauric acid) 0,49 7,45 4,97 6,95 5,15 7,74 7,61 7,23 3,93 c 14 :0 (myristic acid) 0,63 6,05 3,16 3,83 3,42 3,92 4,16 4,13 2,53 c 16 :0 (palmitic acid) 26,88 27,79 22,46 22,76 25,59 23,37 21,7 19,29 24,53 c 18 :0 (stéaric acid) 3,71 5,40 3,96 3,90 4,40 3,56 3,67 3,89 3,89 mono unsaturated fatty acids c 18 :1n-9 (oleic acid) 46,69 32,12 44,82 43,15 43,22 43,03 47,19 48,57 45,41 c 18 :1n-7 (elaidic acid) 2,11 1,70 2,00 1,85 1,82 1,88 2,12 2,19 2,52 polyunsaturated fatty acids c 18 :2n-6 (linoleic acid) 19,50 19,49 18,62 17,57 16,39 16,49 13,55 14,77 17,18 oleic acid is the fatty acid found in greater proportion in all analyzed brands while elaidic acid is the one that is generally in the lowest proportion except in the sample of brand 1. only one essential fatty acid has been identified, linoleic acid whereas packaging reported the presence of two essential fatty acids: linoleic acid and alph-linolenic acid (hansen, haggard, boelsche, adam, & wiese, 1958). further investigation must be realized as it seems that α linolenic acid content could not be detected with the proposed method while the latter plays an important role in growth and development in infants. setting up a post-marketing control system for follow-on formula is necessary. these regulatory measures would aim to ensure the quality of follow-on formula made available on the market in côte d’ivoire and remove non-compliant batch and or brands. 3. conclusion post-marketing quality control of follow-on formulas for infants marketed in côte d'ivoire constitutes a contribution to the nutrition monitoring of infant’s formulas commercialized. this study evaluated the fat content of these formulas and the fatty acids profile. the results showed fat content close to the one reported on packaging for 4 out of nine analyzed brands. fat content was also higher than those www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 58 published by scholink inc. reported in human milk by literature. only one essential fatty acid was identified in these formulas. further investigation must be pursued as regarding the importance of these fatty acids in growth and development infants. references bernard, d., & annie, r. (1995). acides gras essentiels et prématurité : une triple approche expérimentale. société de biologie., 189, 781-796. champeny, m., pereira, c., sweet, l., khin, m., ndiaye coly, a., sy gueye, n. y., … huffman, s. l. (2016). point-of-sale promotion of breast milk substitutes and commercially produced complementary foods in cambodia, nepal, senegal and tanzania. matern. child. nutr., 12(suppl 2), 126-139. codexalimentarius fao-who. (1987). standard for follow-up formula cxs156. retrieved december 11, 2018, from http://www.fao.org/fao-who-codexalimentarius hansen, a. e., haggard, m. e., boelsche, a. n., adam, d. j. d., & wiese, h. f. (1958). essential fatty acids in infant nutrition iii. clinical manifestations of linoleic acid deficiency. j. nutr., 66, 565-576. kent, j. c., mitoulas, l. r., cregan, m. d., ramsay, d. t., doherty, d. a., & hartmann, p. e. (2006). volume and frequency of breastfeedings and fat content of breast milk throughout the day. pediatrics, 117, e387-e395. koletzko, b., baker, s., cleghorn, g., neto, u. f., gopalan, s., hernell, o., … zong-yi, d. (2005). global standard for the composition of infant formula: recommendations of an espghan coordinated international expert group. j. pediatr. gastroenterol. nutr., 41, 584-599. koletzko, b., bhutta, z. a., cai, w., cruchet, s., el guindi, m., fuchs, g. j., …walker, a. (2013). compositional requirements of follow-up formula for use in infancy: recommendations of an international expert group coordinated by the early nutrition academy. ann. nutr. metab., 62, 44-54. kuo, h.-w., & ding, w.-h. (2004). trace determination of bisphenol a and phytoestrogens in infant formula powders by gas chromatography-mass spectrometry. j. chromatogr. a., 1027, 67-74. pereira, c., ford, r., feeley, a. b., sweet, l., badham, j., & zehner, e. (2016). cross-sectional survey shows that follow-up formula and growing-up milks are labelled similarly to infant formula in four low and middle income countries. matern. child. nutr., 12(suppl 2), 91-105. suthutvoravut, u., abiodun, p. o., chomtho, s., chongviriyaphan, n., cruchet, s., davies, p. s. w., … koletzko, b. (2016). composition of follow-up formula for young children aged 12-36 months: recommendations of an international expert group coordinated by the nutrition association of thailand and the early nutrition academy. ann. nutr. metab., 67, 119-132. vaysse, c., billeaud, c., guesnet, p., couëdelo, l., alessandri, j. m., putet, g., & combe, n. (2011). teneurs en acides gras polyinsaturés essentiels du lait maternel en france : évolution des teneurs www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 59 published by scholink inc. en acides linoléique et alpha-linolénique. médecine nutr., 47, 5-9. who. (2001). the world health organization's infant feeding recommendation. retrieved december 11, 2018, from https://www.who.int/nutrition/topics/infantfeeding_recommendation/fr who. (2015). up to what age can a baby stay well nourished by just being breastfed? retrieved december 11, 2018, from http://www.who.int/features/qa/21/fr who. (2018). clarification on the classification of follow-up formulas for children 6-36 months as breastmilk substitutes. retrieved december 11, 2018, from http://www.who.int/nutrition/publications/infantfeeding/information-note-followup-formula food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 1, 2017 www.scholink.org/ojs/index.php/fsns 23 entrepreneurial skill development through aonla processing in punjab, india manoj kumar mahawar1*, kirti jalgaonkar1, dattatreya kadam2 & prasad chavan1 1 hcp division, icar-central institute of post-harvest engineering and technology abohar, punjab, india 2 asec division, icar-central institute of post-harvest engineering and technology ludhiana, punjab, india * manoj kumar mahawar, e-mail: manojmahawar362@gmail.com received: april 5, 2017 accepted: april 25, 2017 online published: may 5, 2017 doi:10.22158/fsns.v1n1p23 url: http://dx.doi.org/10.22158/fsns.v1n1p23 abstract indian economy is predominantly dependent on agriculture and allied sectors as they are the important source of raw material and demand for many industrial products. as one among the allied sector, food processing sector is nowadays gaining attention amid entrepreneurs across the country. famer’s interest as an entrepreneur is constantly increasing in terms of investment which creates a promising atmosphere for affluent growth of food processing industry. ministry of food processing industries, govt. of india is a wing which is actively engaged in promoting entrepreneurial activities in fruits and vegetables processing sector. however, there exists a definite demand to attract the small and marginal farmers to avail this entrepreneurship facility so that it can help in improving their livelihood and become a source of their family income. this particular case study is a representation of facilities availed by some entrepreneurs in icar-ciphet abohar for aonla processing. such kind of information may become a source of encouragement for the farmers who are having a desire to promote themselves as farmer cum businessman. keywords entrepreneurship, aonla, processing, pilot plant 1. introduction although, india stands second in agricultural production but indian agriculture is severely suffering with unacceptable level of wastages and failed to provide remunerative prices to farmers. this problem could be curtailed if appropriate strategies can be adopted for processing of agricultural commodities (especially fruits and vegetables) at their production catchments. in a study conducted by nanda et al. (2012), the estimated losses are 5.8% to 18% for fruits and 7.5% to 13% for vegetables, respectively. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 24 published by scholink inc. the food processing sector surrounds with plentiful opportunities for the entrepreneurs, nevertheless the industry is forced to revamp the overall value and speed of its innovations. significant progress of entrepreneurship in food processing segment will create work opportunities for rural youth and thereby augmenting the living standard of the people across the country (negi, 2013). indian government under various schemes of mofpi is encouraging the entrepreneurial development programmes (edp) which are intended to provide assistance to an individual in strengthening his entrepreneurial motive and in achieving skills and capabilities required for playing his entrepreneurial role effectively (kumari, 2014). however, due to lack of skill, basic materials and inexperience, it might be difficult for new entrepreneur to launch and manage their enterprises. it is not only needed to motivate them but skilled them to manage their venture (awasthi et al., 2006). processing of horticultural produce into value added products is emphasized by the government of india by providing ample opportunities to the young potential entrepreneurs through edp’s and encourages them to establish their own processing industries. edp helped the trainees in gaining basic knowledge of processing methodology, technological interventions and marketing of the product. hands-on-training experience provides motivation and confidence to the trainees to initiate and manage a business venture. during such programs, trainees are also educated about the opportunities and financial assistance available for food processing units. the information to avail credit facilities from banks/financial institutions and assistance from the developmental organizations to procure equipments/raw material is also provided. proper guidance towards establishment of marketing linkages for the developed products and the possible risk factors in running the units is also explained during the tenure of training (anonymous, 2013). under indian council of agricultural research (icar), central institute of post-harvest engineering & technology (ciphet), ludhiana is established with the vision of ensuring better income to farmers through post-harvest processing interventions. this can be achieved through value addition and by-product utilization of the agricultural produce (cereals, oilseeds, fruit and vegetables). indian gooseberry or aonla (phyllanthus emblica l.) is one of the richest sources of vitamin c and is known for its medicinal values since long time. the crop is most ideal for arid regions of punjab and rajasthan like abohar, sriganganagar, etc. the main cultivated varieties of aonla are kanchan (na-4), banarasi, bansi red, desi, krishna, francis (hathijool), chakaiya, pink tinged, na-6, na-7, na-8, na-9 and na-10 (rakesh et al., 2004). due to its high astringency and low shelf life, the fruit is not popular as table fruit. the other methods of extending shelf life are by processing to murabba, pickle, juice syrup, squash and dehydrated powder (kalra, 1988). as of now, the commercialization of aonla processing is limited and hence the growers are encouraged for entrepreneurship and ultimately towards establishment of small/medium/cottage level industries. but, for confidence boosting of small scale entrepreneurs and beginners to establish the industry on its own, it requires hands on experience so that they can adequately invest for procurement of machineries. the knowledge and practical exposure provided during the training will help in examining the entire facilities required to establish www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 25 published by scholink inc. the plant according to their needs and available infrastructure (kadam et al., 2012). to enhance the processing perspectives of aonla as well as to improve the awareness of processing among the local farmers, horticultural crop processing division, icar-ciphet abohar is committed to organize need-based trainings for a period of 3-4 days. interested participants from punjab and rajasthan attend such programs and gain requisite exposure under the guidance of scientific experts. a pilot plant facility is available at the division which is used for the processing operations to develop various valorized products. this study summarizes the overall evaluation of the processing and value addition of aonla using the existing pilot plant facility at icar-ciphet abohar. 2. materials and methods an edp on “aonla processing and value addition” was organized from 29th february to 2nd march, 2016 at hcp division which was attended by 4 farmers from punjab and rajasthan. for processing operations, aonla fruits with similar maturation characteristics were procured from the farm of icar-ciphet abohar, punjab, india. dust and other impurities were removed by washing the fruits in tap water. the cleaned fruits were sorted and weighed as per the usage. the existing pilot scale facility was used for various processing operations (grading, shredding, pricking, juicing, etc.). 2.1 pilot scale processing plant existing pilot plant contains, fruit washing tank (100 kg/h), fruit holding tank (100 kg/h), aonla grader (4 grades), aonla shredder (300 kg/h), aonla juice extractor (55 lit/h) and batch pasteurizer (30 lit/h). the view of the established pilot plant has been shown in figure 1. figure 1. pilot plant established at agro processing unit 2.2 preparation of aonla juice raw material of good quality aonla were sorted and washed with normal water. cleaned fruits were weighed and crushed in aonla shredder in the batches of 5 kg. aonla shreds were collected from the two outlets while seeds were separated and collected at the outlet provided at bottom. after removal of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 26 published by scholink inc. the seeds, the recovery of the shred was 60% to 65%. the collected shreds were subjected to pressing using hydraulic juice extractor (pressure range: 0-420 kg/cm2) for collection of juice. juice was then pasteurized at 82 ± 4°c for 2 min and then allowed to cool to 55 ± 2°c. potassium metabisulphate (2 g) was added to the juice as a preservative and the juice was packed in plastic bottles and stored in a cooling chamber at 12 ± 2°c. the remaining shreds were dried in tray dryer (60 ± 3ºc) for 6-8 hours and the dried product can be used to prepare mouth freshener, etc. the detailed procedure of juice preparation by means of flow chart is depicted in figure 2. figure 2. process flow chart for aonla juice preparation 2.3 determination of physico-chemical properties sphericity and colour of about 100 aonla fruits were taken and average values are calculated. the obtained juice quality was determined in terms of total soluble solids (tss), titrable acidity and ph. 2.4 sphericity the major, minor and intermediate intercepts of the fruit were measured using digital vernier calliper aonla reception inspection, sorting and weighing washing shredding (separation of aonla seed) hydraulic pressing of shreds (juicing) pasteurization (82°c for 2 min) cooling (55°c) + addition of preservative bottling cold storage www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 27 published by scholink inc. (make mitutoyo with least count 0.01 mm). sphericity of the fruit was computed using following formula (mohsenin, 1970; mahawar et al., 2017): sphericity = (𝑎𝑏𝑐)1/3 𝑎 where, a = major intercept, b = intermediate intercept, c = minor intercept. 2.5 total soluble solids (tss) the amount of sugar and soluble minerals present in fruits determines the tss of any product. tss of the juice was recorded using a digital refractometer (make atago, range 0-85°brix). 2.6 colour colour of aonla fruits as well as aonla juice was recorded using hunterlab colour analyzer (hunterlab msez-4500l, virginia, usa) and respective l, a, b values were obtained. 2.7 titrable acidity (ta) it is a measure of the amount of acid present in a solution and was measured using the method described by (ranganna, 2001). ta (%) = titre × equivalent weight of acid × 100 volume of sample taken × 1000 3. results and discussion 3.1 physicochemical properties diameter of the aonla fruit was in the ranged from 42.37 to 45.71 mm whereas length was varied from 35.35 to 39.28 mm. sphericity of the fruit was varied from 0.92 to 0.95 which was in accordance. weight of 10 aonla fruits was ranged from 225 to 235 gm. the fruits were of almost same colour with l* value ranging from 54.60 to 64.06, a* value ranging from 23.27 to 30.52 and b* value ranging from 23.68 to 33.93. the fruits were light greenish in colour with yellow shadow. pulp of the fresh fruit contains 200 to 900 mg of vitamin c as reported by several other workers (kalra, 1988). chemical properties of the aonla juice were tested to evaluate the quality of juice. total soluble solidity of the juice was in the range of 15.7 to 17.3 whereas acidity and ph were in the range of 1.8 to 2.4 and 3.17 to 4.61, respectively. the average values of the determined properties are tabulated in table 1. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 28 published by scholink inc. figure 3. aonla shredding in operation performed by the entrepreneur table 1. physicochemical properties of aonla major diameter intermediate diameter minor diameter sphericity l a b tss acidity ph 44.79 ± 1.09 44.11 ± 1.61 37.02 ± 1.23 0.93 ± 0.01 58.15 ± 3.35 25.92 ± 2.15 23.82 ± 3.59 16.74 ± 0.61 2.08 ± 0.25 3.85 ± 0.66 note. values given in the table are mean ± standard deviation. 4. economic analysis one of the trained entrepreneurs has used the pilot plant facilities to process his raw material brought from the local area of abohar. he used the facility in the last season for about 15 days on custom-hiring basis and some selected parameters were observed in order to obtain the detailed cost estimation of the overall juice extraction process. the information is tabulated in table 2. table 2. cost analysis of juice preparation specification observation total quantity of aonla processed 7 ton total quantity of juice recovered 2250 litre cost of fresh aonla 13 rs/kg number of working days 15 working hours per day 7-8 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 29 published by scholink inc. juice recovery 32.14% capacity of the shredder 300 kg/h capacity of juicer 55 lit/h electricity consumed for shredding 1 kw/40 kg consumption of electricity for juicing 1 kw/35 kg labour engaged 7 (5 men, 2 women) labour charge 350/day 5. conclusion entrepreneurship development program has prominent role in entrepreneurial skill development, employment generation and it also provides opportunities to the small scale industries holders to improve their livelihood. the technologies developed by icar-ciphet are well accepted and adopted by small and medium entrepreneurs in the nearby surroundings. to get larger impact of the technologies developed, there is urgent need to propagate the technologies in systematic manner, through imparting trainings; establishing faith among entrepreneurs towards viability of projects; establishing business units and making it profitable under the supervision and guidance of production/processing and marketing experts. references anonymous. (2013). nmfpgoi. guidelines for implementation of human resources development scheme during the remainder of 12th plan (2013-2017) under nmfp. arya, r. s. s., & moond, s. k. (2004). processed products of aonla. processed food industry, february, 20-23. awasthi, d., jaggi, r., & padmanand, v. (2006). manual for the entrepreneurs: food processing industry. a report of ministry of food processing industries, government of india, new delhi. goyal, r. k., patil, r. t., kingsly, a. r. p., walia, h., & kumar, p. (2008). status of post harvest technology of aonla in india. a review. american journal of food technology, 3(1), 13-23. kadam, d. m., kaushik, p., & kumar, r. (2012). evaluation of guava products quality. international journal of food science and nutrition engineering, 2(1), 7-11. kalra, c. l. (1988). the chemistry and technology of aonla: a resume. in indian food packer (pp. 67-82). kalra, c. l. (1990). role of blanching in vegetable processing. in indian food packer (pp. 3-15). kumari, m. (2014). a study on entrepreneurship development process in india. indian journal of research, 4(3), 51-53. mahawar, m. k., jalgaonkar, k., kumar, m., meena, v. s., & bhushan, b. (2017). determination of some physical properties of date palm fruits (cv. khadrawy and medjool). acta agrophysica, 24(2), www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 30 published by scholink inc. 217-223. mohsenin, n. n. (1970). physical properties of plant and animal materials. gordon and breach science publishers, new york. nanda, s. k., vishwakarma, r. k., bathla, h. v. l., rai, a., & chandra, p. (2012). harvest and post-harvest losses of major crop and livestock produce in india. indian council for agricultural research. negi. s. (2013). food processing entrepreneurship for rural development: drivers and challenges. in iim, suscon iii third international conference on sustainability: ecology, economy & ethics (pp. 186-197). new delhi: tata mcgraw hill education. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 2, 2017 www.scholink.org/ojs/index.php/fsns 104 phytochemicals extraction and nutraceuticals of purple corn w. puminat1* & c. teangpook2 1 division of food chemistry and physic, institution of food research and product development, kasetsart university, kasetsart, bangkok, thailand 2 division of food processing and preservation, institution of food research and product development, kasetsart university, kasetsart, bangkok, thailand * w. puminat, e-mail: ifrwnp@hotmail.com received: october 19, 2017 accepted: october 25, 2017 online published: november 3, 2017 doi:10.22158/fsns.v1n2p104 url: http://dx.doi.org/10.22158/fsns.v1n2p104 abstract phytochemicals are naturally formed in plant of corn. many kinds of flavonoids are the most important plant pigment. purple corn is extracted with polar and nonpolar solvent and analyzed for flavonoids and antioxidants. the extract residue of purple corn is compared on extraction with various solvent groups. in the designed experiments, conditions of extraction depend on solvent, solvent ratio and evaporation temperature. the residue quantity of purple corn is respectively from extraction with three kinds of solvent such as acetone, chloroform: methanol (3:1) and pentane: hexane (1:1). all residue extract are eluted and with mobile phase and analyzed with diode array detector by hplc. anthocyanin, β-carotene and total tannin of purple corn were taken analysis an average (mean ± sd) in 100 g sample and shown as 198.42 ± 0.33 mg, 175.82 ± 0.17 mg and 273.75 ± 0.33 mg respectively. the efficiency of solvent extraction depends on the polarity of the substances. the extract by acetone can take the highest residue and mixture solvent take a high residue. the different polarities of solvent can make the difference of residue product in the extraction. improving and development on the changes are useful for the best of raw material and products. keywords purple corn, nutraceuticals, extraction and anthocyanin 1. introduction flavonoids are naturally formed in plant. flavonoids are widely distributed in plants, fulfilling many functions. some flavonoids are phenolic substances that act in plant as antioxidants. many kinds of flavonoids are the most important plant pigments. plant pigments include a variety of different kinds of molecule, including porphyrins, carotenoids, anthocyanins and betalains. purple pigments of purple corn have the properties of free radicals and reduce symptoms of cancer tumor types. they enhance the strengthen of body, increase an immunity antibodies and the red blood cells. they reduce the fat in www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 105 published by scholink inc. artery clogs, prevent heart disease and control blood sugar levels. pigments color help anti-aging, the digestive system and deterioration of eye. physical and chemical changes with growth, this is useful in helping to identify compounds in pigments. if we know the chemical properties of substances, we are likely to predict what kind of reaction. we see a chemical change based on certain chemical properties of pigments. these pigments are generally measured by determining the amount and wavelength of light that they absorb. the wavelength and amount of absorbed light can show the identify and content of pigments. all substances have properties that functional and identifying. we can utilize by separating them from one another by extraction with solvents and purity by chromatography. purple corn has a rich composition of phytochemicals such as anthocyanins and phenolic compounds. the phytonutrients are natural compounds or substances found in certain plants which is believed to be beneficial to human health and help prevent various diseases. this class of phytonutrients includes pigments such as carotenoids (beta-carotene, lutein), flavonoids or phenolics, alkaloids, nitrogen-containing compounds and organosulfur compounds (indoles, glucosinolates). the color categories of phytonutrients are red, red purple (and blue), orange, orange-yellow, yellow-green, green and white-green. liquid extraction also known as solvent extraction is a method to separate compounds based on their relative solubilities by preferentially dissolving that substance in a suitable solvent. generally, polar solvents dissolve polar compounds and non-polar solvents dissolve non-polar compounds. strongly polar compounds dissolve only in very polar, while strongly non-polar compounds dissolve only in very non-polar organic solvents. polar and non-polar are not miscible with each other and will quickly separate into two layers even after being shaken well. the type of polarity, dipole moment, and hydrogen bonding of solvent is able to specify the dissolve. 2. materials and methods 2.1 the characteristics and physical properties of purple corn this classification scheme will be based on their shapes and structures for morphological classification. physical characteristics are defining traits or features about general quality. the samples of purple corn are classified and weighted for determining an average of groups. a classification is provided the groups in order to understand the general standard of purple corn. 2.1.1 preparation of purple corn powder and drying fresh purple corn is sliced in size 1-2 mm and crushed to a fine shred. sample is taken drying by vacuum drying at 60°c for 7 hr. the samples are pulverized to a fine powder and stored in seal container for further analysis and the other process. 2.1.2 determination of moisture content fresh purple corn is sliced in size 1-2 mm and dry with the vacuum oven. moisture is determined by drying 1-3 g sample in a vacuum oven (25-100 mmhg) at 60°c for 7 hr. the moisture content of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 106 published by scholink inc. sample is calculated as percentage of the different weight. 2.1.3 measurement the colour of purple corn sample is measured in three times and seven sampling. colour measuring is taken with the data colour international measurement model colour tools. cie value (commission internationale de i’eclaerage) display the colour value in cielab system l*(0 = black and 100 = white), a*(-a* = green and + a* = red) and b*(-b* = blue and + b* = yellow) at d65 10deg (light source illuminant d). 2.2 analysis of nutraceuticals purple corn 2.2.1 determination total anthocyanin by ph-differential method total anthocyanin content is extracted and determined by uv-vis spectroscopy. the extract is added to test tubes containing of buffers ph 1.0 and ph 4.5. they are thoroughly mixed by vortex mixer. transperent sample mixer is measured at different ph with an absorbance at wavelength 530 nm and 700 nm by uv-visible spectrophotometer. total anthocyanin content is calculated and determined as anthocyanin (cyanidin) in milligram/100 gram by the following equation % anthocyanin pigments (mg/l) = a* mw * df * 1000 /  * l; a = (a530nm-a700nm) ph 1 (a530nm-a700nm) ph 4.5. a is a difference of absorbance at ph 1and ph 4.5. mw. is molecular weight (cyanidin-3-glucoside = 449.2 g/mol) and  is the molar absorptivity for cyanidin-3-glucoside ( = 26900 l/mol/cm). l is standard of pathlength1cm and df is dilution factor. 2.2.2 determination pigment content and pigments extract each sample is prepared by 5 g pasted purple corn immersed in 1-butanol 25 ml. they are extracted in an ultrasonic water bath for 2 hrs, and centrifuged at 4,500 rpm for 20 min. the upper layer is supernatant for pigment analysis by uv-visible spectrophotometer. the absorbance at 435 nm show pigment content of purple corn. 2.2.3 determination β-carotene extract by uv-visible spectrophotometer purple corn are extracted with hexane and acetone. sample 1 g is added with 15 ml hexane: acetone (2:3). mixture is soaked and shaken with vortex mixer for 10 minutes. they are allowed to stand by magnesia adsorber and centrifuged at 4500 rpm for 10 minutes. the extract is separated in vial with cap and measured by spectrophotometry at wavelength 436 nm. the determination of β-carotene is compared and calculated with standard solution. the standard curve of β-carotene is prepared at concentrations of 0.001-0.015 mg/ml. the concentrations are calculated using a linear regression of the five points in standard curve. 2.2.4 determination of polyphenol or total phenolic assay total phenolic compounds are quantified as tannin content by using folin ciocalteu’s method. sample is extracted with water and filtered by filter paper. extract is formed a complex compound with folin-ciocalteu reagent. polyphenol or tannin is analyzed by spectrophotometer. blue colour compound is determined an absorbance at wavelength 760 nm. blank is prepared by replacing the reagent on the same way by sample and standard reference. samples are compared with standard calibration of tannin and calculated as total tannin content. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 107 published by scholink inc. 2.2.5 determination of amylose content in purple corn the samples are dissolved with urea and dimethylsulfoxide (udmso) in a water bath at high temperature until changed in gel formulation. contaminated fat is eluted with alcohol and dried in oven. residues without ethanol are dissolved udmso again and adjusted the volume with water. solution is formed the color with iodine in potassium iodide solution and measured the absorbance at 635nm wavelength. absorbance is compared with standard reference and calculated for amylose content. 2.3 extraction and solvent extract in the designed experiments on extraction model, conditions of extraction for the yield depend on solvent, solvent ratio and evaporation temperature. control on the extract ratio is 1:7 wt/ml. sample is approximately weighted in 10 g and mixed with 70 ml solvent extract. it is tightly closed the lid and shaken a mixture. controls on the condition are the same temperature and shaker at speed 180 rpm for 8 hour. the suspension is filtered through filter paper. all of extract liquids are done the evaporation of solvent by buchi syncorne analyst in three stage of temperature at 40°c, 50°c and 60°c. the evaporation is controlled by temperature, vacuum pressure and speed controller at 150 rpm until dryness. residue is blowed with nitrogen gas in order to evaporate the remained solvent. the specific substances are focused on weight of recovery residue. % extracted = total residue * 10 / wt.sample the extract in each experiment can be compared the differences and taken for the other analysis. 2.4 identification of flavonoids and antioxidants by hplc 2.4.1 sample and preparation sample analysis of anthocyanin is determined by hplc (high performance liquid chromatography). residue is weighted and mixed with 25 ml ethanol. it is tightly closed the lid and shaken to dissolve by a vortex mixer. the sample is extracted with ultrasonic agitation for 15 min and shaken with a vortex mixer again. solution is precipitated with centrifuge (minicentrifuge c 1200). the upper layer of solution is followed by filter membrane ф 0.45 μm and injected for 1microl into the hplc. 2.4.2 determining of flavonoids and total flavonoids conditions of analysis in grouping, mobile phase is acetonitrile: 5% formic acid: methanol: water; 75: 10: 10: 5 and elute with column at flow rate 0.9 ml/min. column is ods hypersil particle size 5 μm id 4.6 mm length 250 mm and detect the peak with diode array detector at a wavelength 280 nm. the concentration of cyanidin-3-glucoside standard reference is level 1.5-6 ng for determining as flavonoids and another series. 2.4.3 determining of antioxidants and total antioxidants antioxidant analysis, extracted sample is carried out by hplc analysis with uv detector. mobile phase is acetonitrile: methanol; 90: 10 and elute column at flow rate 0.7 ml/min. both antioxidants and flavonoids analysis detected on the same wavelength and column. the analysis is determined at room temperature and identified the chromatogram in series of epicatechin. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 108 published by scholink inc. 2.5 statistical analysis the data is analysed by statistical program of anova (analysis of variance). statistical analysis of mean and variance in each treatment are taken with duncan’s new multiple range test at the significance 0.0l. 3. results and discussions 3.1 evaluation of physical and chemical properties the physical and chemical characteristics are a defining of traits about qualification. their identities are sometimes taken into consideration to evaluate certain corn. in the experiments, their properties are classified as either extensive or intensive. extensive properties depend on the amount of matter in the sample such as colour, % silk or pollen, average weight per three pods. intensive properties do not depend on the sample size or mass such as pigment, anthocyanin, tannin and amylose. mean and standard deviation (mean ± sd.) of % weight of purple corn are pod 46.16 ± 0.57%, silk or pollen 19.88 ± 2.71%, peel 33.96 ± 2.55% and bulk weight per three pod 1.257 ± 0.015 kg. 3.1.1 moisture content and dry sample an average (mean ± sd.) moisture of fresh corn and silk is about 73.40 ± 1.01% and 24.47 ± 0.40 respectively. drying sample is prepared by heating in vacuum oven. the moisture is removed by evaporation of water. moisture in the sample is measured by the difference of mass before and after drying. the moisture content of the sample is calculated as percentage of the different weight loss. an average moisture of drying powder is about 12.19 ± 0.02%. the samples are pulverized to a fine powder and stored in seal container for further analysis and process. 3.1.2 purple colour of corn and colour value (cielab) the pollen or silk has a high red colour but seed has a high blue colour. colour in the cielab system (l*, a* and b*) of purple corn in seed and silk are different a statistical significance at α = 0.05. the relationships between the association of two variables are shown with pearson’s correlation. table 1 is shown pearson’s correlation of colour and some characteristics of purple corn. in each part of corn pod and colour value in the table are compared the average and a variance on random analysis by statistics at significant level α = 0.05. table 1. the characteristics and colour value of purple corn and its silk purple corn total weight colour value (kg) per 3 pod l* a* b* c kenel and seed 1.257 ± 0.017 18.964 ± 3.924a 2.047 ± 0.746a,b 0.742 ± 0.480a 2.282 ± 0.523b silk 0.250 ± 0.038 20.173 ± 3.49c 3.647 ± 2.052c 0.520 ± 0.585c,d 3.807 ± 1.784d a,c correlation is significant at the 0.05 level (2-tailed) and b,d correlation is significant at the 0.01 level (2-tailed). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 109 published by scholink inc. in table 1, a,c and b,d show the relationships according to duncan’s multiple range test at α = 0.05 and α = 0.01. colour value (cielab) in each column are significantly different at α = 0.05. 3.2 evaluation on nutraceuticals of purple corn anthocyanins are a group of naturally occurring flavonoid compounds, which play an important role in the colour quality of purple corn. in the experiments, the analysis of extract has respectively anthocyanin and pigment an average (mean ± sd) 198.42 ± 0.33 mg/100 g and 204.52 ± 0.30 mg/100 g. tannin and β-carotene of purple corn are taken analysis and shown as fresh, frozen and drying sample in table 2. table 2. some phytonutients of fresh corn, frozen corn and dry corn phytonutrients fresh corn (mean ± sd) frozen corn (mean ± sd) dry corn (mean ± sd) tannin (mg/100 g) 273.75 ± 0.33 259.55 ± 0.48 1274.32 ± 0.49 β-carotene (mg/100 g) 175.82 ± 0.17 156.60 ± 0.02 496.54 ± 0.19 standard cruve of linear equation and r-squared value (tannin and β-carotene) in puple corn are respectively displayed as y = 0.2244x 0.0066: r² = 0.9958 and y = 1.9256x 0.0129: r2 = 0.9899. in the experiments, amylose of fresh purple corn is 121.75 ± 0.32 mg/100g and drying powder average 834.35 ± 0.75 mg/100g. the calibration graph is shown a linearity, with a correlation coefficient of r² = 0.9986. the representative linear of regression equation for standard amylose is y = 0.1966x + 0.0261. 3.3 solvent extraction and residue of purple corn with various solvent extract figure 1. the extract residues of the various solvent polarities www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 110 published by scholink inc. 3.3.1 effect of solvent on the extraction of nutraceuticals in purple corn all substances have properties as functionals and nutraceuticals. the crude extract is identified and analyzed for determination of flavonoids and antioxidants. the polarity of the substances are compared to the different extraction. the residue content is evaluated by comparing with various solvent extract. by separating them by extraction with solvents, difference of nutraceuticals are a process that substances dissolve selectively by one or more of the mixture of solvent. in the experiments, mixture solvent of chloroform and methanol is the best described system for partially polar and nonpolar can extract to high residue. the relationships of residue and solvent polarity in extraction of purple corn show on figure 1. 3.3.2 residue extract and solvent polarities in the extraction of purple corn figure 1 shows the different types of weight (%) of chemical composition by priority of polar extraction. they are found that the extract residue have higher yield than another extract solvent. if the dielectric constant of the extract is divided into three groups the difference of extract residue is from the solvent polarity. it is found that they have statistically a difference at significant level α = 0.05. table 3. capacity and potential of solvent extract for the extraction of purple corn solvent extract focus on functional groups %crude residue (g/ml) acetone pigments 10.682 chloform: methanol; 3:1 the best partially polar and nonpolar 7.693 pentane +hexane; 1:1 fat & oil 7.644 ether + petroleum ether; 1:1 fat & oil 5.635 butanol + ether; 1:1 pigments 5.635 hexane non polar 5.586 ethanol sugar 4.018 chloroform soluble matter 2.205 n-butanol colour 0.763 hexane + acetone; 4:1 fat & oil 0.847 n-butanol colour 0.763 n-heptane unsaturated fat 0.490 in the experiments, the extract by acetone can take the highest residue and mixture solvent take a high residue. the different polarities of solvent can make the difference of residue product in the extraction. the extract residue of purple corn is from extraction with three kinds of solvent such as pentane: hexane (1:1), chloroform: methanol (3:1) and acetone. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 111 published by scholink inc. 3.4 determination of flavonoids and antioxidants by hplc 3.4.1 determination the flavonoid and antioxidant compounds from chemical extract with the different solvent by hplc purple corn is extracted from polar and nonpolar solvent by eleven groups of solvent extraction. they are analysed and detected on flavonoids and antioxidants by hplc. the extract is eluted by mobile phase for flavonoids (acetonitrile: 5% formic acid: methanol: water; 75: 10: 10: 5) and antioxidants (acetonitrile: methanol; 90: 10) scaned with wavelength at 280 nm. the different types of molecule weight by solvent separation are defined with chemical positions. these flavonoids are generally indicated by the identifying wavelength and amount absorbance of light. a qualitative property is one that defines something based on characteristics. the wavelength and amount of absorbed light can determine of flavonoid and antioxidant content. on the table, estimate at peak area of flavonoids and antioxidants are analysed and compared with various solvent extract. table 4. the peak area (min-max) flavonoid and antioxidant by the different eluent moblie phase series of functional groups peak area of epicatichin min-max (mau) total peak area min-max (mau) acn: 5%hcooh: meoh: h2o; 75: 10: 10: 5 flavonoids 2.32-226.21 11.34-1053.50 acn: meoh; 90: 10 antioxidants 8.97-214.07 27.32-1054.42 3.4.2 chromatogram and chemical fingerprints the pattern of the extract components of purple corn are shown some chemicals. by solvent extraction, chemical composition of different types are orderly separated by weight (%) with hplc. simultaneous measurement at the same wavelength is shown that uv detector is the ability to perform spectrophotometric scanning and precise absorbance readings of wavelengths. chemical profile or chromatogram of fingerprint is compared on the different solvent in analysis of flavonoids and antioxidants pattern. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 112 published by scholink inc. figure 2. the difference of mobile phase in flavonoid and antioxidant analysis of crude residue in peak of chromatogram at 280 nm, fingerprints allow to show height and area of peak at the difference of retention time. they are used to identify a determination for flavonoids and antioxidants. the different polarity of the extracting solvent, in each crude residue is constructed and compared on the peak area. scanning and comparing with the profile of reference, the pattern profile is from the characteristics of wavelength by mobile phase. it is indicated some chemical components of flavonoids and antioxidants in purple corn. figure 3. the comparison on flavonoid and antioxidant content by extraction with the difference of solvent extract www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 113 published by scholink inc. on the statistic analysis, figure 3 shows the comparison on the difference of mobile phase in flavonoid analysis. the relationships of peak area base on the different solvent of the extract. they have pearson correlation by 2-tailed level at 0.940 and have significantly a difference by statistic at the 0.01. 4. conclusion purple corn has a rich composition of nutraceuticals and phytochemicals. all substances have properties as functionals that we can use to identify them. by the nature, substance characteristics base on properties of quality and unique identity. improving and development on the changes are useful in helping to the best of raw material and products. references alan, d., mc., & andrew, w. (1997). flavonoids: isoflavonoids and neoflavonoids. iupac compendium of chemical terminology (2nd ed.). oxford: blackwell scientific. aoac. (2000). official methods of analysis. the association of official analytical chemists, arlington virginia. lee, j., rennaker, c., & wrolstad, r. e. (2008). correlation of two anthocyanin quantification method: hplc and spectrophotometric methods. food chemistry, 110, 782-786. https://doi.org/10.1016/j.foodchem.2008.03.010 puminat, w., & teangpook, c. (2014). the acidity enhancement in extraction of anthocyanin and color of rambutan peel. the journal of food technology, 106, 220-226. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 2, 2017 www.scholink.org/ojs/index.php/fsns 122 glycemic and lipid metabolic markers in type 2 diabetes mellitus patients after consuming red pigmented parboiled rice as a staple—a clinical trial upul cosomn1, priyadarshika hettiarachchi2, kamani wanigasuriya3 & rasika perera4* 1 department of allied health sciences, faculty of medical sciences, university of sri jayewardenepura, gangodawila, sri lanka 2 department of physiology, faculty of medical sciences, university of sri jayewardenepura, gangodawila, sri lanka 3 department of medicine, faculty of medical sciences, university of sri jayewardenepura, gangodawila, sri lanka 4 department of biochemistry, faculty of medical sciences, university of sri jayewardenepura, gangodawila, sri lanka * rasika perera, e-mail: rasika@sjp.ac.lk received: november 13, 2017 accepted: november 22, 2017 online published: november 27, 2017 doi:10.22158/fsns.v1n2p122 url: http://dx.doi.org/10.22158/fsns.v1n2p122 abstract red pigmented rice has been proven to have unique properties beneficial to health. these might be further enriched if parboiled. this study investigated the effects of consumption of rppr on glycemic response, lipid profile and bmi in diabetics. for this prospective study patients with diabetes mellitus (aged 40-75 yrs) in a prison (n = 69) were recruited. their usual diet in prison was red pigmented rice. they were served 180 g of rppr for 16 weeks during intervention period. fasting plasma glucose (fpg) and bmi was assessed at 0, 4, 8, 12 weeks and glycated haemoglobin and lipid profile at 0 and 16 weeks. values at 0 weeks were compared with those at 4, 8, 12, 16 weeks after consuming rppr using anova repeated measures. hba1c and lipid profile at 16 weeks were compared with the 0 week value. during consumption of rppr, fpg was significantly reduced at 8 (p = 0.006), 12 (p = 0.002), and 16 weeks (p = 0.005), with a significant reduction of the bmi at 8 (p = 0.028) and 16 weeks (p = 0.003). at the end of 16 weeks of consuming rppr, ldl, total cholesterol (tc) and tc/hdl ratio were significantly reduced compared to 0 weeks (p = 0.001, p = 0.013, p = 0.032, respectively. these results suggest that rppr consumption reduces fpg, ldl, tc,tc/hdl ratio and bmi. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 123 published by scholink inc. keywords glycemic control, lipids, type 2 diabetes mellitus, red pigmented parboiled rice 1. introduction due to increasing obesity rate and westernization of life styles, the prevalence and the incidence of type 2 diabetes mellitus (t2dm) in developing countries are increasing dramatically (jayawardena et al., 2012). rice (oriza sativa) is the staple diet consumed by more than half the world population. although studies have indicated that white rice consumption is associated with increased risk of t2dm, there are only few interventional studies carried out to identify a healthy rice variety (hu et al., 2012). colored rice (black, purple, red, brown) due to its edible pigments deposited in the nutrient containing pericarp of the rice grain is a special entity studied worldwide. colored rice contains a variety of pigments and bioactive substances. this include numerous anti-oxidant molecules such as phenolic acids, flavonoids, anthocyanins, proanthocy-anidins, tocopherols, tocotrienols and y-oryzanol, etc. (nam et al., 2003). as evidenced by epidemiological studies, there might be an association between low incidence of certain chronic diseases amongst rice consumers,which might be due to anti-oxidant compounds in rice (goufo & trindade, 2014). among the antioxidants, anthocyanin, which is the pigment found in red or purple pericarp of rice, is considered a natural colorants of foods. it is reported that there are unique properties of anthocyanin pigment such as antioxidant activity (nam et al., 2006), anti-inflammatory (wang et al., 2007), anticancer (hyun & chung, 2004) and hypoglycemic (takikawa et al., 2010) which makes consuming pigmented rice beneficial for health. pigmented rice is commonly consumed by people in south east asia including sri lanka, thailand, china, etc. (sompong et al., 2011). red pigmented rice is amongst the rice varieties with high activity of antioxidants. anthocyanins: the pigment in red rice comprised of several types cyanidin 3-glucoside, cyanidin 3-galactoside, cyanidin 3-rutinoside, cyanidin 3,5-diglucoside and malvidin 3-galactoside, etc. (goufo & trindade, 2014). numerous recent observational studies have evaluated the hypoglycemic effects of anthocyanin. a study conducted in an obese diabetic rat model has found that, anthocyanin suppresses lipogenesis,reduces hepatic steatosis in hepatocytes and plays a role in inhibiting fatty acid synthesis (guo et al., 2011). quality of the red pigmented rice might be enhanced by parboiling. parboiled rice, obtained after soaking paddy in water, steaming to complete gelatinization and drying, has many advantages over raw rice (figure 1). strengthening of kernel integrity, increased milling recovery, prevention of the loss of nutrients during milling, reduced cooking time and improved shelf life as well as prevention of the proliferation of fungi and insects are some of the reasons for the increasing demand for parboiled rice (hoover, 2010; patindol et al., 2008). it is claimed that parboiling reduces the glycemic index by almost 30% compared to its non parboiled variety due to starch retrogradation, high soluble fiber content and increased physical hardness of the grain for digestion (larsen et al., 2000). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 124 published by scholink inc. figure 1. red pigmented parboiled rice parboiling of red pigmented rice might have several advantages over other types of rice. although no significant differences in macronutrient composition among the red pigmented and red pigmented parboiled rice have been observed there were differences in the glycemic indices and the soluble fibre contents (table 1; hettiarachchi, 2000). due to the different levels of anthocyanic pigment differences in the digestibility has also been observed (perera & jansz, 2000). table 1. composition and glycemic indices of cooked red pigmented and red pigmented parboiled rice nutrient/glycemic index bw 351 bw272-6b bw 351* bw272-6b* carbohydrates (g) 73.5 82.6 78 74 amylose/amylopectine 26.4/47.3 = 0.5 26.4/56.2 = 0.4 29.9/48.1 = 0.6 26.1/47.5 = 0.5 protein (g) 9.2 11.6 6.7 9.6 fat (g) 1.5 0.9 1.1 2.1 total fiber (g) 5.3 2.6 5.7 4.3 soluble fiber (g) 1.7 1.4 1.9 3.1 glycemic index 73 68 56 58 moisture (g) 14.7 13.7 12.2 13.7 macronutrients are calculated on dry basis. * denotes parboiled rice. a preliminary in vitro study with red pigmented parboiled rice has shown that there is alteration in the pigments in parboiling (perera & jansz, 2000). a lower glycemic response in red parboiled rice might be due to protein pigment complex permeating the starch gel causing inhibition of enzyme hydrolysis during parboiling (perera & jansz, 2000). rice pigments and the antioxidant properties have been extensively investigated, yet interventional studies in human testing the physiological effects of red pigmented rice are few. owing to certain characteristics postulated such as, presence of anti-oxidants, anti amylase activity and the ability to improve the soluble fibre content after parboiling and lower glyceamic index, consuming www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 125 published by scholink inc. red pigmented rice might be beneficial in health as well as in diabetics. the effect of consumption of red pigmented parboiled rice on lipid markers and bmi has not been ascertained in diabetic patients. consuming red pigmented parboiled rice might favorably answer an emerging public health problem in the developing world by achieving the expected outcomes of diabetes mellitus in improving the lipid and glucose metabolism markers. thus we hypothesized that red pigmented parboiled rice consumption lowers the glycemic response, improves the lipid profile and lowers the bmi in t2dm patients. the aim of this study was to investigate the effect of red parboiled rice consumption on the glycemic control, lipid profile and bmi in t2dm subjects. 2. method 2.1 ethics statement ethics review committee of the faculty of medical sciences, university of sri jayewardenepura, sri lanka specifically approved this study. permission to conduct the study was obtained from the commissioner of prison, prison head quarters, welikada, sri lanka. informed written consent was obtained from all participants before enrollment to the study and they were given the option of withdrawing from the study at any moment. the study was conducted according to the principles expressed in the declaration of helsinki. 2.2 participants and study design this analytical clinical trial (ctri/2014/06/004666) was conducted at welikada prison in colombo, the capital of sri lanka, where more than 2000 prisoners with long-term imprisonment serve their sentences. the study evaluated the effects of pigmented parboiled rice consumption on blood glucose control, bmi and lipid profiles in t2dm patients for a period of 16 weeks. the list of all registered diabetes patients in welikada prison was obtained from the prison hospital and they were invited for screening through posters displayed at the prison hospital and dispensary. all responded patients were screened according to the inclusion/exclusion criteria and their dietary habits, exercise patterns and presence of complications were assessed by an interviewer administered questionnaire. out of 363 registered t2dm patients, 102, t2dm patients fulfilled the inclusion criteria and were recruited for the study following obtaining informed written consent. t2dm patients between the ages of 40 and 75 yrs, those who were not receiving medication for any disease other than diabetes were included. those who had complications of diabetes, who received insulin therapy, whose medication regimen was altered during the study, those who had elevated cholesterol levels and blood glucose levels were excluded. of an initial diabetic population of 102 only 69 were recruited as those patients who had elevated serum cholesterol (n = 12) and elevated fpg (n = 14) were removed from the study sample and referred for treatment and seven subjects opted out from the study. the usual diabetic diet in the prison comprised of cooked red pigmented rice (180 g), starchy vegetable 50 g, leafy vegetable 50 g www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 126 published by scholink inc. and fish or meat (100 g). in this intervention red pigmented parboiledrice was served instead of red pigmented rice. at 0 weeks, fpg, lipid profiles, hba1c and bmi were assessed. these measurements were considered as baseline measurements. fpg and bmi were determined every 4 weeks from the day of commencement of the study whereas lipid profiles, and hba1c were determined at the end of 16 weeks (figure 2). figure 2. time points at which biochemical parameters and bmi were assessed 2.2.1 activities prisoners generally have a fixed daily routine of physical activities and all of them were following the same schedule of physical activities. 2.2.2 medication patients on antidiabetic medication (sulfonylurea: n = 18, biguanides: n = 21, thiazolidiones: n = 8, and sulfonileurea + biguanides: n = 22) were included in the study. in case of change in medication during the course of the study, those subjects were removed from the study (n = 0). 2.3 data collection venous blood was collected after an overnight fast into edta tubes, sodium fluoride containing tubes and plain tubes for hba1c, fpg and lipid profile respectively. samples were transported immediately to the nawaloka metropolis laboratory, colombo 2, sri lanka and centrifuged at 4000 rpm for 10 minutes. plasma was stored until analysis at 2-80c up to a maximum of 1 week, unless analyzed immediately. 2.4 assessment of bmi and blood pressure weight was measured using a digital weighting scale (chyo mu-150k, japan), to the nearest 0.1 kg while standing with barefoot on the center of the scale-platform, keeping the hands at sides and looking straight. height was measured only at 8 weeks to the nearest 0.1 cm, using stadiometer (no. 265m), fb g, bm i fb g, bm i intervention with red pigmented parboiled rice 16 wks 0 wks bmi fpg hba1c lipid profile 4 wks bmi fpg 8 wks bmi fpg 12 wks bmi fpg 16 wks bmi fpg hba1c lipid fb g, bm i fb g, hb a1c lipi d prof ile bm i www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 127 published by scholink inc. while standing up straight against the backboard, with barefoot, heels together, toes apart, and the head, shoulder blades, buttocks and heels in contact with the backboard with the head kept in the frankfurt’s horizontal plane. bmi was calculated by determining the weight to height squared ratio. as a base line investigation systolic and diastolic blood pressure was determined using the sphigmomanometer. 2.5 analytical methods blood samples for fbg were analyzed by glucose oxidase method which is an adaptation of the hexokinase-glucose-6-pospate dehydrogenase method, in a fully automated biochemistry analyzer (siemens dimension: ct. df 40 usa). hba1c was analyzed by d-10 hba1c program which utilizes the principles of ion-exchange high-performance liquid chromatography (hplc), using fully automated analyzer by immunoturbidimetric method (roche hitachi (902) cobas system) (yasmeen et al., 2011). blood samples for lipid profiles were analyzed by enzymatic method using fully automated siemens dimension ct. df 27 (usa) instrument (flegg, 1973; rautela & liedtke, 1978). 2.6 statistical analyses fbg and, bmi, at 0 weeks were compared with values obtained at 4, 8, 12 and 16 weeks (table 2, figure 2, figure 3), using anova repeated measures, general linear model (glm). hba1c and lipid profiles at 0 weeks were compared with values obtained at 16 weeks (table 2, figure 2, figure 3), using anova repeated measures, glm. the values at 0 weeks was considered as the baseline for rppr intervention. all analyses were performed using spss (version 17.0) software and a p value < 0.05 was taken as statistically significant. 3. results base line characteristics: the mean (sem) age of the study group was 50.72 years (1.08). there were 17 females and 52 males in the study group. at 0 weeks subjects’ mean (sem) of weight, height and bmi were 62.98 kg (0.97), 1.62 m (0.01) and 23.88 kg/m2 (0.29), respectively. at 0 weeks their mean (sem) diastolic blood pressure was 83.18 (0.70) and the mean (sem) systolic blood pressure was 128.19 (1.20). body weight significantly decreased after consumption of rppr compared to 0 weeks. hence bmi was significantly decreased after consumption of rppr for 8 (p = 0.028) and 16 weeks (p = 0.003) compared to 0 weeks. 3.1 glycemic control after the intervention with rppr consumption, fpg was significantly reduced at 8 (p = 0.006), 12 (p = 0.002), and 16weeks (p = 0.005) compared to 0 weeks (table 2, figure 3). although there was a decreasing trend, the hba1c did not show a significant change at 16 weeks compared to the 0 week value (table 2). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 128 published by scholink inc. table 2. assessment of biochemical parameters and bmi of t2dm after consuming red pigmented parboiled rice (n = 69) parameter consumption of red pigmented parboiled rice at 0 weeks 4 weeks 8 weeks 12 weeks 16weeks bmi 23.88  2.39 23.87  2.40 23.82  2.36 23.81  2.30 23.78  2.29 fpg mg/dl 1.12  26.39 1.06  24.25 1.03  18.13 1.02  18.51 1.03  16.95 ldlmg/dl* 1.27  30.11 1.14  27.03 hdlmg/dl* 41.82  10.59 42.47  10.40 tcmg/dl* 1.95  45.02 1.83  37.43 tgmg/dl* 1.63  84.57 1.52  66.16 tc/hdl* 4.93  1.51 4.50  1.27 hba1c (%)* 6.37  1.40 6.31  1.20 note. mean +/-sd. *parameters were assessed at 0 and 16 weeks. figure 3. variations in fpg and bmi during the intervention period 3.2 lipid profile at the end of 16weeks of consuming rppr, ldl (p = 0.001) and tc (p = 0.013) were significantly reduced compared to the 0 weeks value (table 2, figure 4). there was no change in the hdl or tg concentration. however the tc/hdl ratio (p = 0.032) was significantly reduced compared to the 0 weeks value. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 129 published by scholink inc. figure 4. changes in lipid parameters during the intervention period 4. discussion investigations to identify rice varieties which produce a low glycemic response have been carried out worldwide. the chemical composition of red rice has been extensively investigated. anti oxidant and lipid lowering effects of pigmented rice has been reported. however no interventions determining the effect of consuming red pigmented rice were reported on t2dm patients. the present study is the only study to report on glycemic response, lipid profile and bmi on consuming red pigmented parboiled rice in t2dm patients. a reduction in the fasting plasma glucose was seen with rppr consumption at 4 (p = 0.236), 8 (p = 0.006), 12 (p = 0.002), and 16 (0.005) weekscompared to 0weeks. however at the end of 16 weeks the hba1c concentration was unchanged in spite of a decreasing trend. probably consuming rprr for a longer duration, for more than 16 weeks, might reduce the hba1c concentration. in contrast during consumption of pre germinated brown rice for 16 weeks in people with impaired glucose tolerance the hba1c concentration compared to white rice was reduced (bui et al., 2014). in another study comparing pre germinated brown rice with white rice consumption for a similar time duration, a reduction of fructosamine concentration was observed (hsu et al., 2008). this may be because in both studies pregerminated brown rice was compared with white rice whereas the present study compared the basal blood glucose concentration. a study which substituted brown rice for white rice for 16 week did not show conclusive evidence in improvement in the metabolic risk factors such as fpg, hba 1c, hdl, tc and tg (zhang et al., 2011). although the brown rice contain a higher fibre content compared to white rice the 16 weeks duration of consumption of brown rice might be inadequate. similarly even though the fibre content in red pigmented parboiled rice is high to observe a difference in hba1c probably a the duration of consumption should be more than 16 weeks. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 130 published by scholink inc. at the end of 16 weeks after consuming of red pigmented parboiled rice lipid markers such as ldl cholesterol (p = 0.001, total cholesterol (p = 0.013) and tc/hdl ratio (p = 0.032) werereduced compared to the basal level. this might be attributed to the presence of anthocyanin in red pigmented rice. supplementation of anthocyanin for 12 weeksin a double-blind, randomized, placebo-controlled trial in dyslipideamic subjects has shown an improvement in serum ldland hdl-cholesterol concentrations (qin et al., 2009). as proven by many prospective studies it is clear that the risk of diabetes increases with white rice consumption implying red pigmented parboiled rice might be an alternative for white rice (villegas et al., 2007). consuming pre germinated brown rice compared to white rice, for a period of 16 weeks produced a lower ldl and triglyceride level and significantly higher hdl cholesterol level, in vietnamese women with impaired glucose tolerance. however the tc concentration was not reduced (bui et al., 2014). another study in patients with diabetes and individuals with impaired glucose tolerance in taiwan also proved the improvement on levels of serum tcl, tri-acylglycerol (tg) and hdl-cholesterol (hdl-c) with pregerminated brown rice for 16 weeks (hsu et al., 2008). this implies that the changes in the lipid profile occur due to consuming pre germinated brown rice unlike white rice. thus red pigmented parboiled rice can be suggested as another alternative to be recommended to improve the lipid profile in diabetes mellitus in prison. it was demonstrated in diabetic rats that the hypocholesterolemic effects of germinated brown rice were partly mediated through the upregulation of the ldl-r and apo a1 genes (ithnin, 2013). it was shown that the pre germinated brown rice bran contains a y-oryzanol which are effective in lowering hyperlipideamia (sugano & tsuji, 1997). pre-germinated brown rice (pgbr) is obtained by allowing the rice kernels to slightly germinate once soaked in water. although it is a common practice in japan most people might not be familiar in preparing pgbr. although a low glycemic indices were found in, easy-cook long-grain rice and white basmati, long term physiological effects of consuming these varieties were not determined (hsu et al., 2008). improvement of lipid markers after consuming pre-germinated brown rice for 16 weeks has been also attributed tophysical shape of the rice grain causing delayed digestion and high fibre content (hsu et al., 2008). it is reported that the glycemic response in rice: is influenced by the physical texture, chemical structure amylose amylopectine ratio and soluble fibre content, anti amylase activity and antioxidants, etc. (björck & elmståhl, 2003; gunaratne et al., 2013). parboiling of the red pigmented rice has increased in the soluble fibre content, lowered the digestibility and the glycemic index. this might be a added factor which increase its efficacy (hettiarachchi, 2000; hettiarachchi et al., 2001; larsen et al., 2000; perera & jansz, 2000). this prospective study ascertains the efficacy of consuming red pigmented parboiled rice as a staple while maintaining other ingredients in the diet relatively constant. our hypothesis was tested in a group of people having a generally fixed dietary menu throughout the study period. due to presence of a fixed www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 131 published by scholink inc. dietary menu, and organized daily physical activities of prison in-mates, it was assumed that energy expenditure did not vary in the intervention period as before. hence reduction of the bmi at 8 weeks (p = 0.028) and 16 weeks (p = 0.003) compared to basal level might be attributed to consumption of red pigmented parboiled rice. similar to consuming red pigmented parboiled, consuming pre germinated brown rice compared to white rice, for a period of 16 weeks produced a significant reduction in the bmi in vietnamese women with impaired glucose tolerance. thus red pigmented parboiled rice might be prescribed in patients with diabetes mellitus although the previous study data is from individuals withimpaired glucose tolerance. controlling energy expenditure to a scientifically acceptable degree throughout the intervention period was one of the strengths of this study. dietary habits of the prison inmates did not vary except for introducing red pigmented parboiled rice and the fact that assessors and lab technicians were blind on the variety of rice were strengths of this study. during the intervention medications were not changed among the final study population which was taken for analysis. when compared to other intervention studies with rice, the sample size of this study was larger and the total diabetics in the prison were considered and this was strengths of this study. firstly due to the financial and time constrains intervention period was limited to 16 weeks and this would have preferably been longer to see changes in hba1c concentration. secondly due to the practical difficulties groups of prison inmates could not be randomized to an intervention and non intervention group. future randomized clinical trial in diabetics with a longer duration is suggested. to ascertain the actual effect of consuming red pigmented parboiled rice on hba1c the duration would have to be lengthened to more than 16 weeks. in conclusion, this study proves that, consuming red pigmented parboiled rice for 16 weeks can significantly reduce the blood glucose level. furthermore, consuming red pigmented parboiled rice for 16 weeks can produce a significant reduction in the glycemic response, lipid profile and bmi. due to the impact of red pigmented parboiled rice on these markers this variety might be recommended for t2dm patients. considering the unique physiological and physiochemical properties as discussed before, further prospective studies may confirm that red pigmented parboiled rice consumption might even besuitable for the general community. acknowledgement we are grateful to the prison inmates who volunteered and the staff of the prison including the commissioner general of prison for their assistance given in conducting the study in the prison premises. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 132 published by scholink inc. references bjorck, i., & elmastahl, h. l. 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(2010). the impact of heat-moisture treatment on molecular structures and properties of starches isolated from different botanical sources. critical reviews in food science and nutrition, 50, 835-847. https://doi.org/10.1080/10408390903001735 hsu, t.-f., kise, m., wang, m.-f., ito, y., yang, m.-d., aoto, h., … yaamamoto, s. (2008). effects of pre-germinated brown rice on blood glucose and lipid levels in free-living patients with impaired fasting glucose or type 2 diabetes. journal of nutritional science and vitaminology, 54, 163-168. https://doi.org/10.3177/jnsv.54.163 hu, e. a., pan, a., malik, v., & sun, q. (2012). white rice consumption and risk of type 2 diabetes: meta-analysis and systematic review. british medical journal, 344. https://doi.org/10.1136/bmj.e1454 hyun, j. w., & chung, h. s. 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(2000). glycemic index of parboiled rice depends on the severity of processing: study in type 2 diabetic subjects. european journal of clinical nutrition, 54, 380-385. https://doi.org/10.1038/sj.ejcn.1600969 nam, s. h., choi, s. p., kang, m. y., koh, h. j., kozukue, n., & friedman, m. (2006). antioxidative activities of bran extracts from twenty one pigmented rice cultivars. food chemistry, 94, 613-620. https://doi.org/10.1016/j.foodchem.2004.12.010 nam, s., chang, s., & kang, m. (2003). varietal difference in antioxidative activity of ethanolic extracts from colored rice bran. journal of the korean society of agricultural and biotechnology. patindol, j., newton, j., & wang, y. j. (2008). functional properties as affected by laboratory‐scale parboiling of rough rice and brown rice. journal of food science, 73, e370-e377. https://doi.org/10.1111/j.1750-3841.2008.00926.x perera, a., & jansz, e. (2000). preliminary investigations on the red pigment in rice and its effect on glucose release from rice starch. journal of the national science foundation of sri lanka, 28, 185-192. https://doi.org/10.4038/jnsfsr.v28i3.2660 qin y., xia, m., ma, j., hao, y., liu, j., mou, h., cao, l., & ling, w. (2009). anthocyanin supplementation improves serum ldl-and hdl-cholesterol concentrations associated with the inhibition of cholesteryl ester transfer protein in dyslipidemic subjects. the american journal of clinical nutrition, 90, 485-492. https://doi.org/10.3945/ajcn.2009.27814 rautela, g. s., & liedtke, r. j. (1978). automated enzymic measurement of total cholesterol in serum. clinical chemistry, 24, 108-114. sompong, r., siebenhandl-ehn, s., linsberger-matin, g., & berghofer, e. (2011). physicochemical and antioxidative properties of red and black rice varieties from thailand, china and sri lanka. food chemistry, 124, 132-140. https://doi.org/10.1016/j.foodchem.2010.05.115 sugano, m., & tsuji, e. (1997). rice bran oil and cholesterol metabolism. the journal of nutrition, 127, 521s-524s. takikawa, m., inoue, s., horio, f., & tsuda, t. (2010). dietary anthocyanin-rich bilberry extract ameliorates hyperglycemia and insulin sensitivity via activation of amp-activated protein kinase www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 2, 2017 134 published by scholink inc. in diabetic mice. the journal of nutrition, 140, 527-533. https://doi.org/10.3945/jn.109.118216 villegas, r., liu, s., gao, y.-t., yang, g., li, h., zheng, w., & shu, x. o. (2007). prospective study of dietary carbohydrates, glycemic index, glycemic load, and incidence of type 2 diabetes mellitus in middle-aged chinese women. archives of internal medicine, 167, 2310-2316. https://doi.org/10.1001/archinte.167.21.2310 wang, q., han, p., zhang, m., xia, m., zhu, h., ma, j., … ling, w. (2007). supplementation of black rice pigment fraction improves antioxidant and anti-inflammatory status in patients with coronary heart disease. asia pac j clin nutr., 16, 295-301. yasmeen, f., mumtaz, a., adhami, s., & qureshi, s. (2011). comparison of cation exchange hplc and immunoturbidimetric method for determination of hba1c. biomedica, 27, 161-165. zhang, g., pan, a., zong, g., yu, z., wu, h., chen, x., … chen, x. (2011). substituting white rice with brown rice for 16 weeks does not substantially affect metabolic risk factors in middle-aged chinese men and women with diabetes or a high risk for diabetes. the journal of nutrition, 141, 1685-1690. https://doi.org/10.3945/jn.111.142224 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 2, 2019 www.scholink.org/ojs/index.php/fsns 73 original paper selection and characterization criteria of probiotics intended for human use from the past to the future valeria sagheddu1*, elena guidesi1, serena galletti1 & marina elli1 1 aat-advanced analytical technologies, fiorenzuola d’arda, piacenza, italy * valeria sagheddu, aat-advanced analytical technologies, fiorenzuola d’arda, piacenza, italy received: april 28, 2019 accepted: may 10, 2019 online published: may 20, 2019 doi:10.22158/fsns.v3n2p73 url: http://dx.doi.org/10.22158/fsns.v3n2p73 abstract the probiotic product consumption has recently increased with the prevalent intent to promote human and animal wellbeing. the complex selection process dealing with new-isolated probiotic candidates is the first challenge that has to be faced. from the isolation to the launch on the market, information about safety, tolerance to host physiological conditions, adhesion properties, genetics and interaction with the host has to be collected. probiotics must be safe, survive to the exposition to bile salts and to gut transit, adhere to intestinal cells lining and colonize the lumen of the tract. the evaluation process of the possible probiotic health benefits is widely supported by in-vitro assays simulating the in-vivo conditions. the aim of this work is to summarize the classical models usually employed for the probiotic screening by underlying strengths and weaknesses of all models and to present some more recent analysis tools used in the probiotic field. the long term goal in new probiotic candidate selection experiencing these combined essays together would lead to the hypothetical assignment acknowledged as one strain-one function. keywords screening, platform, in-vitro, in-vivo, model 1. introduction international scientific association for probiotics and prebiotics defined probiotics as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (hill et al., 2014). in the recent years the global probiotic market has faced a massive growth, this expansion should be related to a robust and univocal legislation (baldi & arora, 2015). nevertheless, focusing the attention on the european regulatory framework, it has to be noticed the absence of harmonized regulation. the food products directive and regulation (regulation 178/2002/ec; directive www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 74 published by scholink inc. 2000/13/eu) and herbal medicinal products directive (2004/24/ec) are the main laws supervising probiotic products (coppens et al., 2006). furthermore, the european regulatory pressure in the field of probiotics led to a double negative impact by limiting the communication of their beneficial effects to consumers and by adversely impacting the market. after ten years from the enter into force of the regulation on health claims made on food (eu 1924/2006), the market of probiotics shows a renewed interest about the health improving potential of beneficial microbes. the restored enthusiasm is mainly driven by the big pool of information derived from the omics approach for the study of the gut microbiota (baugher & klaenhammer, 2011). unfortunately, the legislative framework seems to not run with the innovation associated with new analytical approaches. actually, of the about 300 calls for health claims submitted to efsa experts’ panel on the food area none has been accepted. the only exception was represented to the use of yoghurt for lactose intolerance (papadimitriou et al., 2015). the implementation of new and comprehensive models, exploiting the newest omics technology could ameliorate the understanding of the probiotics mechanism of action and assessing the beneficial impact on health exerted by the administration of probiotics at a quantifiable level (rebollar et al., 2016). the integration of the traditional in-vitro and ex-vivo screening methods of microorganisms with potential probiotic features are associated with omics techniques with the intent to boost the selection process, to deeper understand their functionality and to examine the opportunity to employ cell fractions instead of viable probiotics in finished products (franzosa et al., 2015; lahtinen, 2012). recent market trends concerning probiotic are interestingly targeted to the zootechnic field. for instance, it has been proposed the probiotic use to produce safe and high-quality poultry meat (park et al., 2016). other application fields could be retrieved in cereals, dairy products, fermented meat, baked food, and dry food productions (competitive market share & forecast, 2016). for the application related to human consumption, the validated approach applied to the selection of probiotics firstly includes the safety assessment intended to verify the absence of pathological and virulence traits (hill et al., 2014). the next step evaluates the potential application of the isolated strain by considering the prophylactic, therapeutic use or its ability to reduce the risk to develop a certain pathological condition. regrettably, the implication of this validation results strictly dependent on the availability of measurable biomarkers related to a certain physiological condition. 2. classical in-vitro approaches for new probiotics screening guidelines for the evaluation of the probiotic use in food have been published several years ago (fao/who, 2002) the principal focus identified was defined as a selection scheme for the conventional assessment of probiotic properties and for the characterization of functional bacterial strains as probiotics in food. despite their time-consuming application, the in-vitro assays are routinely used due to their presumed predictive value to predetermine, the putative association between the isolated probiotic candidate strain and a specific health claim, targeting its potential general or specific application (kumar et al., 2013). the major limitation of these assays is represented by the high www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 75 published by scholink inc. variability in terms of reproducibility even if a certain degree of standardization has been suggested. the natural continuation of these essays would be theoretically represented by in-vivo tests that are laborious, expensive and not feasible for ethical reasons. 3. persistence and survival in the human gut a peculiar required probiotic feature is represented by the ability to survive the passage through the gastrointestinal tract remaining in a viable state and with the sufficient number of cells necessary to exert the probiotic effect. to verify this ability simulated gastric and pancreatic juices (charteris et al., 1998; lavermicocca et al., 2008) and porcine or bovine bile have been extensively used, in fact these assays are easy and cheap if compared to more recent approaches including git simulators. unfortunately, those in-vitro approaches are affected of some biases dealing with the unrealistic conditions simulated during benchtop assays. moreover, potential probiotic candidates are tested against severe environmental parameters not really miming the stomach acidity and lower intestine conditions during food consumption (papadimitriou et al., 2015). 4. safety for the use in human nutrition the joint working group fao/who (2002) underlined the relevance of some safety issues for the use of probiotics in food. the efsa panel of experts suggested the precise determination of the minimal inhibitory concentrations (mics) towards 9 listed antibiotics and also stated the microbiological cutoff values to evaluate a new probiotic candidate as susceptible or resistant to antibiotics and the reference methodology to assess it (efsa; 2012). safety aspects usually assessed by in-vitro tests also include aggregation to platelets, to fibronectin and to fibrinogen, hemolytic activity, the synthesis of certain enzymes and the production of biogenic amines. the major concern related to these essays is based on the fact that the expression levels of the in-vivo traits could significantly vary from the in-vitro conditions inducing to an overor an under-estimation of some vexing features of probiotics (papadimitriou et al., 2015). 5. colonization ability the colonization ability is an indicator of the probiotic cellular features to interact with the host. the adhesion of bacteria to mucus and epithelial cells is an important parameter evaluated with several in-vitro assays; this trait could both represent positive connotations associated with pathogens displacement and negative implications for instance the increasing risk of translocation (sanders et al., 2010). furthermore, caco-2, ht-29 epithelial cell adhesion assays often produce weak results due to the low reproducibility maintenance of the tested conditions. in order to circumvent the problem, resected gut tissue or whole tissue models including the mucus layer have been taken into consideration but reliability bias are still present (vesterlund et al., 2005; van tassell & miller, 2011). to evaluate the ability of a strain to interact with the host, some physical features of probiotics have been considered, www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 76 published by scholink inc. for instance, the hydrophobicity of the cell surface (yadav et al., 2014). unfortunately, the scientific significance of these tests is only considered partially because of the lack of the peculiarity of this trait. the enzymatic activity expression such as gapdh (glyceraldeyde-3-phosphate dehydrogenase) promoting the probiotic attachment to the mucus has been evaluated as a potential screening parameter even if with scarce results (kinoshita et al., 2008). the assay conditions, even if optimized could not reproduce the complexity of the bacterial community structure and the possible interactions among different microorganisms. the adhesion assays are performed on a single and purified strain without considering different microbial species leading to doubts about the results, significance and their possible extensive transposition to the gut microbiota. 6. antimicrobial potential it is known that probiotics could exert competitive inhibition towards pathogens exploiting several mechanisms, for instance the production of metabolites showing antimicrobial activity. this feature has been proven influence other bacteria, viruses and fungi at a large spectrum, taking advantage of different compounds like organic acids or bacteriocins (denkova et al., 2013). several in-vitro models have been experienced, from the agar diffusion test to more advanced systems targeting the release of antimicrobial substances using indicator strains (szweda & szweda, 2016). probiotics exhibit other features as co-aggregation with pathogens and/or by reinforcing the integrity and functions of the gut inner barrier. briefly, probiotic cells bind pathogens causing a sort of clumping effect limiting the pathogen interaction with the surfaces of the host and facilitating the excretion of pathogens trough biological fluids (e.g., saliva, feces) (janković et al., 2012). the gut barrier could exert its function only if the tight junctions integrity is maintained, this condition represents a key issue to partially prevent the bacterial translocation, pathogens included. it has been proved that probiotics may improve the barrier integrity as measured by specific enzymatic activities (putaala et al., 2008). the evaluation of probiotics antimicrobial potential is affected by some biases since the laboratory conditions only partially reproduce the in-vivo situation. similarly to what has been observed for the described above in-vitro models, the level of expression of the real antimicrobial potential of tested probiotic strains is not completely defined. substances as exopolysaccharides (eps), biosurfactants, bacteriocins and organic acids represent the main antimicrobial compounds produced by probiotics (evivie et al., 2017). 7. immunomodulatory actions probiotics could stimulate the secretion of antibodies by host cells and activate cell-mediated immune responses (haghighi et al., 2005). usually, immunomodulatory effects are promoted by m cells from the payer’s patches or dendritic cells (dcs) and the response to the occurrence of bacterial cells is assessed by co-culture of probiotics and immune cells and by the detection and the quantification of cytokines (gad et al., 2011). these co-incubation models are frequently applied to the selection of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 77 published by scholink inc. probiotics showing anti-inflammatory features useful in the management of chronic inflammatory diseases (e.g., ibd) (rutella and locatelli, 2011) or to be employed as adjuvant in the treatment of allergic symptoms (ozdemir, 2010). however, the center of the debate still remains the in-vivo modality of action of probiotics and on the possible solution to translate experimental findings to the concrete evaluation of the probiotic strains efficacy in the modulation of the gastrointestinal and systemic immunity. 8. in-vitro models of cardiovascular diseases another trait that is required from probiotic candidates is represented by the expression of the bile salt hydrolase activity (bsh). this enzymatic activity allows the deconjugation of bile acids and it is pretty easy and quick to obtain qualitative and quantitative results (cani & van hul, 2015; zheng et al., 2013; tomaro-duchesneau et al., 2014). however, these routinely used models are sometimes considered relatively insufficient to estimate the real probiotics contribution to reduce cardiovascular diseases risks associated (papadimitriou et al., 2015), albeit the beneficial effects seem to be highlighted by scientific evidences (ebel et al., 2014). 9. in-vitro anti-cancer models it has been proven that probiotics reinforce natural host body protection functions by improving metabolic and immunological parameters (ashraf & shah, 2014). furthermore, these beneficial bacteria could exert antigenotoxic and antimutagenic activities and promote nitrosamines and heterocyclic amine degradation whose accumulation within the intestinal ecosystem could lead to potential negative effects (duangjitcharoen et al., 2014; faridnia et al., 2010). excluding the ability of withdrawing dangerous metabolites, the data support the probiotics ability of releasing short chain fatty acids (scfa) within the intestinal lumen (table 1). these substances have been proven to exert some anticarcinogenic effects in-vivo (burns & rowland, 2004; castro et al., 2010). nevertheless, despite a supportive background is already available, researchers consider these in-vitro tests as not sufficient to provide consistent probiotic anticancer activity information and, for this reason, the in-vivo validation is compelled (chong, 2014). to easily complete the probiotic profile of beneficial bacteria, a pool of other in-vitro model has been proposed, among them the measurement of b-galactosidase activity to monitor lactose intolerance symptoms (vonk et al., 2012), b-group vitamin production (leblanc et al., 2011), oxalate-degrading features (abratt & reid, 2010) and inhibition of pathogen oral strains responsible for volatile sulfur compounds (vscs) production (lee & baek, 2014). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 78 published by scholink inc. table 1. short chain fatty acids (scfa) produced by probiotics probiotic microorganisms produced scfa reference l. salivarius spp salcinius jcm 1230, l. agilis jcm 1048 propionate/butyrate meimandipour et al., 2010 l. acidophilus crl 1014 acetate/butyrate/propionate sivieri et al., 2013 l. rhamnosus lgg propionate le blanc et al., 2017 l. gasseri pa 16/8 propionate le blanc et al., 2017 b. longum sp 07/3 propionate/acetate le blanc et al., 2017 b bifidum mf 20/5 propionate/acetate le blanc et al., 2017 bifidobacteria acetate/lactate pessione et al., 2012 10. development of innovative screening platforms the rationale underlying standardized in-vitro assays to study several aspects of probiotic effectiveness is driven by the necessity of set reproducible experimental protocols and consequently decrease the percentage of false positive and negative results. in-vivo models should be considered integrative for in-vitro ones with the purpose of choosing the appropriate probiotic for a potential application. several animal models including invertebrates (e.g., insect, worm) and vertebrates (e.g., rodents, dogs, monkeys and swine), have been tested even though the predictivity for the situation in humans seems to be powerless and for this reason, a critical approach should be applied when results are transposed to humans. in the latest years the conventional screening pipeline for probiotics (in-vitro assays—animal model—clinical trial) including mice or rat models has strongly been debated and there was the tendency to directly check the findings from in-vitro evidences on small number human cohorts. however, animal models will certainly be exploited, particularly for studying specific pathological conditions, both induced by chemicals administration or using animal with genetic predispositions, or pathogenic infection conditions. some probiotic positive modulation effects of intestinal inflammation have been successfully studied thanks to animal models. for instance, the co-occurrence of probiotics and carcinogenesis prevention, metabolic disorders control or auto-immunological conditions have been experienced. on the contrary, immune-deficient animals carrying human tissue or genes also known as humanized mice or rat models, are basically used more for pharmaceuticals purpose instead of probiotic validation (papadimitriou et al., 2015). the combination of in-vitro and in-vivo test represents the new frontier, europe coupled the two tests in some in-silico models to examine from a mathematical point of view the interaction between nutrients, epithelial cells, gut communities and host (tan & liong, 2014). these applications are supposed to mimic the complex gut environment processes by proposing predictive functions and taking in consideration the possible interactions between probiotics with multiple factors. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 79 published by scholink inc. the omics approaches with the advent of new molecular methods greatly contributed to the comprehension of the usefulness of probiotic towards hosts, mainly quantifying the expression variation of certain housekeeping genes of probiotic cells in response to specific environmental conditions. the resistance to stressful conditions, modulation of the immune system, adhesion to host tissues, production of antimicrobial or noble nutrient compounds, degradation of prebiotics and quorum sensing are areas of interest investigated with special consideration (papadimitriou et al., 2015). omics technologies are assumed to explicate the complexity of the communication dynamics inside microbiome or between it and host tissues and to provide new insights on the impact of a foreign probiotic strain meeting those structured communities (de melo pereira et al., 2018). 11. conclusions the number of available assays to validate probiotic efficacy in-vitro and in-vivo is rather complex. the major limitations linked to those approaches are that they consider variables explanatory and not easily reproducible. as a consequence, often the efficacy of new-isolated probiotic candidates could be accomplished only by combining several assays. at the state of the art, on the one hand, there is an urging need of defining a clear workflow for the probiotics selection in order to obtain reliable and unquestionable proof of efficacy, and on the other, the design of appropriately designed clinical trials. human studies are often associated with significant costs, consequently probiotics are still screened following the “funnel-like” approach, firstly using in-vitro tests and subsequently by expensive and appropriate validations on selected strains. the objective sought in new probiotic candidate selection would lead to the development of screening platforms, reducing from a large pool of strains to a restricted group intentionally selected for a specific 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(2013). probiotic properties of lactobacillus strains isolated from tibetan kefir grains. plos one, 22;8(7), e69868. https://doi.org/10.1371/journal.pone.0069868 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 4, no. 1, 2020 www.scholink.org/ojs/index.php/fsns 1 original paper acceptability and satiety evaluation of a developed nutritionally balanced school meal manal abdeen ali1*, ibtihag awad mahmoud1, sawsan ahmed balla1, rekha lecomte2, laura dehondt2, eman ahmed abdekareem3 & abdelhalim rahma gubara1 1 nutrition & health center for training and research, school of health sciences, ahfad university for women, sudan 2 tweed research center of nutriset group, france 3 samil industrial co., khartoum, sudan * manal abdeen, ahfad university for women, al arda street, omdurman, sudan received: january 19, 2020 accepted: january 30, 2020 online published: march 9, 2020 doi:10.22158/fsns.v4n1p1 url: http://dx.doi.org/10.22158/fsns.v4n1p1 abstract five nutritionally designed school meals were developed in a form of pastes based on leguminous seeds (lentil, chick pea, and soya) mixed with dairy ingredients. all formulas contain groundnut, and few contained crushed nuts providing inclusions inside the prototypes to test acceptance. the products were fortified with essential minerals and vitamins following the dri guideline for children. developed meals were served as a breakfast meal to ~200 students (6-9 years juniors; 10-14 years seniors) of one of the sudanese basic schools (omdurman city) for 21 days to substitute a traditional breakfast meal boiled pigeon pea colloquially called “balila” served routinely to the students. this paper covers acceptability of favorable products behavioral responses of senior and junior students to four meal/subject interactions coverings temptation to finishing the whole meal, reason(s) for not finishing the whole meal, time needed to finish the meal, and degree of satiety the students feel during teaching hours. almost 95% of the senior students interacted positively with pastes based on lentil or chick pea finish the product as evidenced by the short time (<10 min) to finishing or stop taking the meal in ≤ 10 minutes. all past products provide satiety; senior students judged that lentil based paste(s) as the meal that secures full satiety during school hours. unlike seniors, more than 95% of junior students finished more of the soy-based meal, one third of them took more time to finish lentil-based meal indicate that the results reflected satisfaction by taking less quantities from all products offered. degree of satiety feeling among junior students fluctuated where >90% of them confirmed satisfactory degree of satiety www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 2 published by scholink inc. compared to all products offered. keywords acceptability, satiety, school meal, nutrition 1. introduction nutritional readers need no reminding of the consequences of the spread of malnutrition among population of most developing countries. black et al. (2008) summarized such consequences; whereas fao (2006) highlighted root causes of food insecurity leading to such consequences, and when it comes at the door of vulnerable groups such as children, fao (2009) itself indicated kind of strategies that prevent or reduce under-nutrition and combat child mortality. almost, a decade back, sudan ministry of general education (smge, 2008) launched a school feeding program that reached about 1.2 million children throughout the country with the objective of increasing school attendance, particularly of girls, and relieving short-term hunger while transforming resources to vulnerable households. lack of food in the households is reportedly one of the main reasons for school dropout, according to baseline survey carried out by smge (2008). the child performance in morning schooling is very much related to taking right nutritious food that is rich in energy, protein, carbohydrates and calorific sources, e.g., breakfast items, to help in focus and concentration, a pre-requisite behavior for better learning and hence more opportunities in life. the market for children’s foods is continuously growing, and children have an increasing influence on food purchase decisions. this has led to the use of children in product development programs by food manufacturers, to the development of twin target markets for adults and children in many food categories (jean-xavier, 2001), and to requirements for palatable, healthy and nutritious food for children amidst trends towards convenience foods and demands for responsible nutrition. the sensory properties of any food item are important determinants of its acceptance among consumers, and as a result, the need for sound methodology for sensory testing with children has increased (astm, 1992; guinard, 2001; astm, 2003). the principal objective of this effort was to test the acceptability of nutritionally balanced school meals developed to substitute existing conventional breakfast meal served to needy students at most of the sudanese primary (basic) schools. the test also embraced satiety profile of meals to answer their level of stomach fulfillment during the school hours. 2. methodology based on an earlier preliminary study by ahfad university for women (auw) team, (nhctr, 2017) concerning acceptability and quality of several school meals, only five meals were then selected and developed by samil industrial co., onyx, and tweed research center of nutriset group, based on plant foods (e.g., soybean, chickpea, lentil, peanuts, etc.), and dairy ingredients, in a form of pastes. the meals were additionally fortified with recommended gaps in iron, iodine, zinc, vitamins of a, b9, b12, and c. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 3 published by scholink inc. a total of ~200 school children (6-9 year juniors; 10-14 year seniors) studying at one of omdurman city (sudan) primary schools were selected to assure minimum classes attendance of 180 children throughout the study. recruited students were served ethically (dius, 2007) developed meals in a form of paste-in sachets (100 g each) for 15 school days plus 5 days given to the reference conventional school meal prepared traditionally from boiled salted or sweetened pigeon pea colloquially called “balila” and served routinely to the children as a school meal. using a concise questionnaire consisted of elements relevant to sensorial issues (brace, 2004), the students with the help of the study group, were asked to judge on the five products (meals) guided by multidescriptive sensory evaluation techniques (gordin, 1987; iso, 1988; poste et al., 1991; lawless & heymann, 1998; schutz & cardello, 2001; meilgaard et al., 2007), and facial drawing procedures to express level of satisfaction by one of three choices (happy face that reflects acceptance of product; noexpression face that expresses neither/nor decision; and sad face that expresses rejection of the product) simplified to suit their level of perception (goldstein, 2006). 3. results and discussion table 1 shows distribution and number of school students served school meals by class and age group. the initial week started with serving the conventional school meal (balila) for 5 days as a control, followed by the newly developed products (5 of them; a: soy based product “vitamamba”; b: lentil based product; c: lentil based product + inclusion product; d: chick pea based product; e: chick pea based product + inclusion product) served for consecutive 15 school study days giving 3 alternating days for each product. the number of students receiving breakfast meal daily for 5 days ranged between 38-54 for males and 39-55 for females from the junior class; 6-9 years (3rd grade) giving a total range of 81-109 students of this category. similarly, the number of students from the senior class; 10-14 years (7th grade) receiving the same meal for the same period of time ranged between 35-55 for males, and 41-53 for females giving a total range of 79 to 105 students of this category. table 1 itself shows a total of 1854 meals offered to students from the juniors and 1844 meals offered to students from the seniors by the end of the study. table 1. distribution and number of meals for school students served by class and age group junior students -class 3 (age 6-9) senior students -class 7 (age 10-14) total of meals/gendre total m % f % total m % f % total m % f % week 1 (balila meal) sunday 86 44 51.2 42 48.8 week 1 balila meal sunday 100 55 55 45 45 186 99 53.2 87 46.8 monday 83 44 53.0 39 47.0 monday 105 52 49.5 53 50.5 188 96 51.1 92 48.9 tuesday 82 38 46.3 44 53.7 tuesday 85 40 47.1 45 52.9 167 78 46.7 89 53.3 wednesday 106 51 48.1 55 51.9 wednesday 92 45 48.9 47 51.1 198 96 48.5 102 51.5 thursday 109 54 49.5 55 50.5 thursday 79 35 44.3 44 55.7 188 89 47.3 99 52.7 week 2 paste products a 88 42 47.7 46 52.3 week2 a 91 47 51.6 44 48.4 179 89 49.7 90 50.3 b 81 40 49.4 41 50.6 b 92 47 51.1 45 48.9 173 87 50.3 86 49.7 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 4 published by scholink inc. c 92 49 53.3 43 46.7 c 95 47 49.5 48 50.5 187 96 51.3 91 48.7 d 88 43 48.9 45 51.1 d 93 44 47.3 49 52.7 181 87 48.1 94 51.9 e 94 45 47.9 49 52.1 e 94 45 47.9 49 52.1 188 90 47.9 98 52.1 week 3 paste products a 90 45 50.0 45 50.0 week3 a 90 42 46.7 48 53.3 180 87 48.3 93 51.7 b 95 45 47.4 50 52.6 b 89 45 50.6 44 49.4 184 90 48.9 94 51.1 c 98 49 50.0 49 50.0 c 89 48 53.9 41 46.1 187 97 51.9 90 48.1 d 97 49 50.5 48 49.5 d 92 45 48.9 47 51.1 189 94 49.7 95 50.3 e 98 50 51.0 48 49.0 e 99 51 51.5 48 48.5 197 101 51.3 96 48.7 week 4 paste products a 88 49 55.7 39 44.3 week4 a 93 44 47.3 49 52.7 181 93 51.4 88 48.6 b 91 47 51.6 44 48.4 b 92 48 52.2 44 47.8 183 95 51.9 88 48.1 c 92 45 48.9 47 51.1 c 88 45 51.1 43 48.9 180 90 50.0 90 50.0 d 97 49 50.5 48 49.5 d 89 44 49.4 45 50.6 186 93 50.0 93 50.0 e 99 50 50.5 49 49.5 e 97 47 48.5 50 51.5 196 97 49.5 99 50.5 total intervention 1854 928 50.1 926 49.9 total 1844 916 49.7 928 50.3 3698 1844 1854 average per day (20 school days) 185 92 93 m: male; f: female. a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. figures 1 and 2 (tables 2a and 2b, respectively) show percent contribution by junior female and male students, and senior ones, respectively, receiving balila in first week calculated from total. it was obvious that senior females were more punctual to attending school classes (table 2b; figure 2), the observation that concerned us for the following three weeks assigned for serving the newly developed meals. the negative fluctuation in percent number of junior female and male students (table 3) receiving new meals was tolerable and seems normal with respect to students’ daily attendance. surprisingly, unlike the junior students, the week following the initial balila week witnessed positive fluctuation in number of senior participants (table 4) which would suggest more or less consistency in number of juniors and seniors male and female students who participated to the four week testing program. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 5 published by scholink inc. the increase or decrease in number of students taking the meals during the course of the study did not reflect improvement or decline in school attendance, since the latter phenomenon is associated with certain factors such as in-door or out-door school activities. the second week of serving newly developed products (tables 3 and 4) started to partially reflect actual response of participant’s contribution to taking the new meals. the same second week of feeding the new meals witnessed highest number of meals served (478 meals) with a slight decrease in the final week (467 meals) mainly coming from female students due to certain domestic school reasons beyond acceptability factors. table 3. percent contribution by junior male and female students to taking new products for 3 weeks serving week product a product b product c product d product e male female male female male female male female male female f % f % f % f % f % f % f % f % f % f % week 1 42 47.7 46 52.3 40 49.4 41 50.6 49 53.3 43 46.7 43 48.9 45 51.1 45 47.9 49 52.1 week 2 45 50 45 50 45 47.4 50 52.6 49 50 49 50 49 50.5 48 49.5 50 51.5 48 49 week 3 49 55.7 39 44.3 47 51.6 44 48.4 45 48.9 47 51.1 49 50.5 48 49.5 50 50.5 49 49.5 f= frequency a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. table 4. percent contribution by senior male and female students to taking new products for 3 weeks serving week product a product b product c product d product e male female male female male female male female male female f % f % f % f % f % f % f % f % f % f % week 1 47 51.6 44 48.4 47 51.1 45 48.9 47 49.5 48 50.5 44 47.3 49 52.7 45 47.9 49 52.1 week 2 42 46.7 48 53.3 45 50.6 44 49.4 48 53.9 41 46.1 45 48.9 47 51.1 51 51.5 48 48.5 week 3 44 47.3 49 52.7 48 52.2 44 47.8 45 51.1 43 48.9 44 49.4 45 50.6 47 48.5 50 51.5 f= frequency a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. tables 5 and 6 show response of senior and junior students, respectively; to finishing the whole new meal, in other words finishing the whole sachet content (100 g) of 5 products (a, b, c, d, and e) presented to the students for 3 successive weeks (15 school days). balila meal was taken as in-memory www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 6 published by scholink inc. control against all products presented to the students. the number of the senior students (10-14 years) finishing the whole sachet content (table 5) increased by serving days (weeks 2 and 3) with all products presented, although products b (lentilbased) and d (chick peabased) showed better consumption (> 95% of participants) compared to the rest of the products. soya based product (a) seemed to be least acceptable to senior students by the end of the test. the response of junior students (6-9 years) to finishing the whole sachet content is shown in table 6. surprisingly, juniors seemed to have different opinion on the products by giving product a (soy-based) the lead in finishing whole sachet content by the end of the feeding test period of time (> 95% of participants), followed by products c (lentil/inclusion-based), and b(lentil-based) which registered 93.5% and 93.4% number of participants, respectively, finishing the whole sachet contents. with juniors, product e (chick pea/inclusion-based) was the least tempting to finish it all. table 5. %response of senior students to finishing the whole meal serving week product a product b product c product d product e frequency % frequency % frequency % frequency % frequency % week 1 yes 67 73.6 72 78.3 67 70.5 79 84.9 70 74.5 no 24 26.4 20 21.7 28 29.5 14 15.1 24 25.5 total 91 100 92 100 95 100 93 100 94 100 week 2 yes 74 82.2 84 94.4 78 87.6 85 92.4 93 93.9 no 16 17.8 5 5.6 11 12.4 7 7.6 6 6.1 total 90 100 89 100 89 100 92 100 99 100 week 3 yes 84 90.3 88 95.7 81 92.0 85 95.5 90 92.8 no 9 9.7 4 4.3 7 8.0 4 4.5 7 7.2 total 93 100 92 100 88 100 89 100 97 100 a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. table 6. %response of junior students to finishing the whole meal serving week product a product b product c product d product e frequency % frequency % frequency % frequency % frequency % week 1 yes 68 77.3 69 85 79 86 74 84 79 84 no 20 22.7 12 15 13 14 14 16 15 16 total 88 100 81 100 92 100 88 100 94 100 week 2 yes 80 88.9 82 86 88 90 88 91 90 92 no 10 11.1 13 14 10 10 9 9 8 8 total 90 100 95 100 98 100 97 100 98 100 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 7 published by scholink inc. week 3 yes 84 95.5 85 93 86 93 89 92 90 91 no 4 4.5 6 7 6 7 8 8 9 9 total 88 100 91 100 92 100 97 100 99 100 a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. tables 7 and 8 show responses of senior and junior students, respectively; to reasons for not finishing the whole contents of sachet (whole meal). none of the senior students (table 7) expressed any dislike for finishing 3 products (c: lentil+ inclusion; d: chick pea; & e: chick pea+ inclusion) during the entire 3 weeks testing. about fifth or even less (11-19%) of the senior students expressed dislike as a reason for not finishing soy-based product (product a). palatability of any product decides most of the time length of tolerability and hence decision of continue taking any meal (gregory & kadri, 2015). the junior students’ behaviour in finishing the whole meal is shown in table 8. it seems that the 100g material offered in sachet was too much for most of them since more than two third of them felt they had enough (stomach full) before finishing the sachet content. the latter feeling is slightly different from that full satisfaction feeling expected to reflect lingering satiety during the school hours. product d (chick pea based) seemed to be the favourite for juniors, followed by product b (lentil based). table 7. %response of senior students to reasons for not finishing the whole meal serving week response product a product b product c product d product e frequency % frequency % frequency % frequency % frequency % week 1 dislike 3 12.5 1 5.0 0 0.0 0 0.0 0 0.0 had enough 15 62.5 11 55.0 20 71.4 9 64.3 1 4.2 fully satisfied 6 25.0 8 40.0 8 28.6 5 35.7 23 95.8 total 24 100 20 100 28 100 14 100 24 100 week 2 dislike 3 18.8 0 0.0 0 0.0 0 0.0 0 0.0 had enough 12 75.0 5 100.0 1 9.1 7 100.0 6 100.0 fully satisfied 1 6.3 0 0.0 10 90.9 0 0.0 0 0.0 total 16 100 5 100 11 100 7 100 6 100 week 3 dislike 1 11.1 0 0.0 0 0.0 0 0.0 0 0.0 had enough 6 66.7 3 75.0 7 100.0 4 100.0 7 100.0 fully satisfied 2 22.2 1 25.0 0 0.0 0 0.0 0 0.0 total 9 100 4 100 7 100 4 100 7 100 a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 8 published by scholink inc. table 8. %response of junior students to reasons for not finishing the whole meal serving week response product a product b product c product d product e frequency % frequency % frequency % frequency % frequency % week 1 dislike 2 10 1 8.3 0.0 0.0 0.0 0.0 0.0 0.0 had enough 18 90 9 75.0 12.0 92.3 14.0 100 14.0 93.3 fully satisfied 0 0 2 16.7 1.0 7.7 0.0 0.0 1.0 6.7 total 20 100 12 100 13.0 100 14.0 100 15.0 100 week 2 dislike 1 10 0 0 0.0 0 0.0 0 0.0 0 had enough 7 70 12 92.3 10.0 100.0 9.0 100 7.0 87.5 fully satisfied 2 20 1 7.7 0.0 0.0 0.0 0.0 1.0 12.5 total 10 100 13 100 10.0 100 9.0 100 8.0 100 week 3 dislike 0 0 0 0.0 1.0 16.7 0.0 0.0 0.0 0.0 had enough 3 75 6 100 5.0 83.3 8.0 100 7.0 77.8 fully satisfied 1 25 0 0.0 0.0 0.0 0.0 0.0 2.0 22.2 total 4 100 6 100 6 100 8 100 9 100 a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. tables 9 and 10 show time needed for senior and junior students to finish and/or stop taking more of the meal. it was obvious that more than 90% of the senior students (table 9) did not take much time (≤ 10 minutes) in finishing or stop taking the sachet content of all the 5 products offered to them successively on daily basis. the positive side of this test outcome is that the sachet content (the paste), was easily chewable, non-sticky, easily swallowed, and palatable enough to hasten the eating process. the latter characteristic usually encourages eaters to continue taking more or finishing any stuff offered (barbara & sandra, 2010). table 10 also shows the time taken by junior students to finish or stop taking the sachet content. although more than two third (> 68%) of the junior participants needed 10 minutes only or even less to finish the meal, the other one third is of concern to us for reasons that made them took 15 minutes or more to finish lentil based pastes (b&c). lentil is a conventional legume to all sudanese particularly to school meals planning and preparations (federal ministry of health, 2006). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 9 published by scholink inc. table 9. time needed by senior students to finish and/or stop taking more of the meal serving week finishing time product a product b product c product d product e frequency % frequency % frequency % frequency % frequency % week 1 ≤10 minutes 82 90 86 93 92 97 91 98 90 96 ~15 minutes 7 8 6 7 3 3 2 2 4 4 ˃15 minutes 2 2 0 0 0 0 0 0 0 0 total 91 100 92 100 95 100 93 100 94 100 week 2 ≤10 minutes 86 96 84 94 88 99 90 98 98 99 ~15 minutes 4 4 4 4 1 1 2 2 1 1 ˃15 minutes 0 0 1 1 0 0 0 0 0 0 total 90 100 89 100 89 100 92 100 99 100 week 3 ≤10 minutes 89 96 90 98 88 100 85 96 93 96 ~15 minutes 4 4 2 2 0 0 4 4 4 4 ˃15 minutes 0 0 0 0 0 0 0 0 0 0 total 93 100 92 100 88 100 89 100 97 100 a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. table 10. time needed by junior students to finish and/or stop taking more of the meal serving week finishing time product a product b product c product d product e frequency % frequency % frequency % frequency % frequency % week 1 ≤10 minutes 71 80.7 56 93 67 97 60 98 77 96 ~15 minutes 16 18.2 17 7 19 3 23 2 15 4 ˃15 minutes 1 1.1 8 0 6 0 5 0 2 0 total 88 100 81 100 92 100 88 100 94 100 week 2 ≤10 minutes 86 95.6 77 81.1 84 85.7 79 81.4 75 76.5 ~15 minutes 4 4.4 16 16.8 14 14.3 16 16.5 15 15.3 ˃15 minutes 0 0.0 2 2.1 0 0.0 2 2.1 8 8.2 total 90 100 95 100 98 100 97 100 98 100 week 3 ≤10 minutes 65 73.9 65 71.4 67 72.8 82 84.5 86 86.9 ~15 minutes 22 25.0 25 27.5 25 27.2 15 15.5 13 13.1 ˃15 minutes 1 1.1 1 1.1 0 0.0 0 0 0 0 total 88 100 91 100 92 100 97 100 99 100 a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 10 published by scholink inc. tables 11 and 12 show responses of senior and junior students, respectively; to degree of satiety feeling during class room hours following meal intake. feeling full satiety by senior students (table 11) during the school hours was improved towards the third week of testing the new products. by the end of the feeding test, product b (lentil based) proved to fulfil that degree of satiety (100%) intended to quench hunger until the students finish their lessons and go back home. the other products were by no means less than b in giving that feeling (97.6-98.9% degree of satiety) of full satiety although sachet content of some of them were not fully consumed by seniors (table 5) compared to b product. table 12 shows fluctuation in degree of satiety feeling among junior students during the school hours following meal taking in the first and second weeks of the serving process, with more or less satisfactory degree of satiety offered by all the products by the end of the third week (94.1-98.9%). it then becomes hard to judge on poorness of any of them. table 11. response of senior students to degree of satiety during class room hours following meal taking serving week satiety response product a product b product c product d product e frequency % frequency % frequency % frequency % frequency % week 1 no satiety 1 1.5 1 1.4 0 0.0 1 1.3 0 0.0 not sure 11 16.4 7 9.7 9 13.4 11 13.9 5 7.1 full satiety 55 82.1 64 88.9 58 86.6 67 84.8 65 92.9 total 67 100 72 100 67 100 79 100 70 100 week 2 no satiety 0 0.0 1 1.2 2 2.6 1 1.2 0 0.0 not sure 1 1.4 4 4.8 7 9.0 4 4.7 3 3.2 full satiety 73 98.6 79 94.0 69 88.5 80 94.1 90 96.8 total 74 100 84 100 78 100 85 100 93 100 week 3 no satiety 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 not sure 2 2.4 0 0.0 1 1.2 1 1.2 1 1.1 full satiety 82 97.6 88 100.0 80 98.8 84 98.8 89 98.9 total 84 100 88 100 81 100 85 100 90 100 a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 11 published by scholink inc. table 12. response of senior students to degree of satiety during class room hours following meal taking serving week satiety response product a product b product c product d product e frequency % frequency % frequency % frequency % frequency % week 1 no satiety 0 0 0 0.0 1 1.3 0 0.0 1 1.3 not sure 0 0 5 7.2 2 2.5 10 13.5 10 12.7 full satiety 68 100 64 92.8 76 96.2 64 86.5 68 86.1 total 68 100 69 100 79 100 74 100 79 100 week 2 no satiety 0 0.0 3 3.7 2 2.3 0 0.0 0 0.0 not sure 1 1.3 7 8.5 5 5.7 3 3.4 1 1.1 full satiety 79 98.8 72 87.8 81 92.0 85 96.6 89 98.9 total 80 100 82 100 88 100 88 100 90 100 week 3 no satiety 2 2.4 2 2.4 0 0.0 1 1.1 1 1.1 not sure 2 2.4 3 3.5 2 2.3 0 0.0 1 1.1 full satiety 80 95.2 80 94.1 84 97.7 88 98.9 88 97.8 total 84 100 85 100 86 100 89 100 90 100 a: soy based product vitamamba; b: lentil based product; c: lentil based product + inclusion product; d: chick pea base product; e: chick pea based + inclusion product. in conclusion, senior students (10-14 years) seemed to have appreciated paste meal based on lentil or chick pea as reflected in finishing all the meals in less than 10 minutes and also showed fullsatiety feeling during the daily school hours. one fifth of the seniors expressed dislike for soy based paste as a reason for not finishing the paste. with respect to junior students (6-9 years), they seemed to have different opinions by showing more appreciation to the soy-based paste and taking less time to finish the product, although they expressed fullsatiety feeling with all the products during the daily school hours. references astm. (1992). american society for testing and materials; committee e-18.manual on descriptive analysis for sensory evaluation (r. c. hootman, ed.). philadelphia, pa, mnl. astm. (2003). american society for testing and materials; e2299-03.standard guide for sensory evaluation of products by children. philadelphia, pa. barbara, b., & sandro, d. (2010). nutrition and consumer protection division, fao. proceedings of the international scientific symposium biodiversity and sustainable diets. united against hunger. fao headquarters, rome. black, r. e., allen, h. l., & bhutta, z. a. (2008). maternal and child under nutrition: global and www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 1, 2020 12 published by scholink inc. regional exposures and health consequences. lancet, 371, 243-260. brace, i. (2004). questionnaire design: how to plan, structure and write survey material for effective market research. kogan page, philadelphia, pa. jean-xavier g. (2001). sensory and consumer testing with children, trends in food science and technology, 11 (8) 273-283 dius. (2007). department for innovation, universities and skills rigour, respect and responsibility: a universal ethical code for scientists. fao, (2009). declaration of the world summit on food security, wsfs 2009/2, 16 november 2009. fao, (2006). policy brief, issue 2: food security. rome. federal ministry of health. (2006). policy nutrition policy for sudan and strategy for implementation comprehensive national nutrition policy, strategy or plan. goldstein, e. b. (2006). sensation and perception. wadsworth publishing, florence, ky. gordin, h. h. (1987). intensity variation descriptive methodology: development and application of a new sensory evaluation technique. journal of sensory studies, 2, 187-198. https://doi.org/10.1111/j.1745-459x.1987.tb00416.x gregory, c. a., & kadri, k. (2015). pet food palatability evaluation: a review of standard assay techniques and interpretation of results with a primary focus on limitations, animals (basel) (vol. 5, no. 1, pp. 43-55). guinard, j. x. (2001).sensory and consumer testing with children. trends in food science and technology, 11, 273-283. https://doi.org/10.1016/s0924-2244(01)00015-2 iso. (1988). international organization of standards. iso 8587: methods for sensory analysis of food. lawless, h. t., & heymann, h. (1998). sensory evaluation of food: principles and practices. springer, n.y. meilgaard, m., civille, c. v., & carr, b. t. (2007). sensory evaluation techniques (4th ed.). crc, boca raton, fl. nhctr, nutrition & health center for training & research. (2017). technical report on “the acceptability of school meals developed based on plant sources test no. i”. nhctr, auw, sudan. nhctr, nutrition & health center for training & research. (2018). technical report on “the acceptability of school meals test no. ii”. nhctr, auw, sudan. poste, l. m., mackie, d. a., butler, g., & larmond, e. (1991). laboratory methods for sensory analysis of food. canadian group; publishing centre, ottowa, canada. schutz, h. g., & cardello, a. v. (2001). a labeled affective magnitude (lam) scale for assessing food liking / disliking. journal of sensory studies, 16, 117-159. https://doi.org/10.1111/j.1745-459x.2001.tb00293.x sudan ministry of general education, (2008). baseline survey on basic education on the northern states of sudan. directorate general of educational planning, khartoum, sudan. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4494336/ food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 1, 2017 www.scholink.org/ojs/index.php/fsns 31 evaluation the antimicrobial activity of artemisia and portulaca plant extracts in beef burger marwa al-moghazy1, m. s. ammar2, mohamed m. sief3 & sherif r. mohamed3* 1 dairy science department, food industries and nutrition division, national research centre, dokki, giza, egypt 2 food science and technology department, faculty of agriculture, al-azhar university, cairo, egypt 3 food toxicology and contaminants department, food industries and nutrition division, national research centre, dokki, giza, egypt * sherif r. mohamed, e-mail: sheriframzy4@gmail.com received: april 9, 2017 accepted: april 23, 2017 online published: may 15, 2017 doi:10.22158/fsns.v1n1p31 url: http://dx.doi.org/10.22158/fsns.v1n1p31 abstract medicinal plants contain substances can alternate the traditional chemical preservatives which used for preserving meat products that have negative effects on consumer health. several biological activities have been reported for artemisia and portulaca as antimicrobial agents, so the current study focused on using artemisia and portulaca extracts as antimicrobial agents in beef burger. phytochemical of artemisia and portulaca extracts were analyzed, and both extracts contain alkaloids, flavonoids, phenols, trepenoids and saponin. the results show that artemisia extract was inhibited all tested microorganisms (staphylococcus aureus, pseudomonas aeruginosa, salmonella typhimurium, listeria monocytogenes and e.coli o157:h7) while, portulaca extract affect staphylococcus aureus only. the minimum cidal concentration (mcc) and minimum inhibitory concentration (mic) were carried out for testing microorganisms, since artemisia extract was very effective against staphylococcus aureus followed by e.coli, pseudomonas aeruginosa, salmonella typhimurium and l. monocytogenes. artemisia and portulaca extracts were separately applied in beef burger as antimicrobials at levels 1% and 1.5%. the sensory evaluation of treated beef burger showed no significant differences between control sample and treatments containing portulaca extract while, the addition of artmesia extract had a detrimental effect on taste of beef burger since it causes formation of bitter taste. keywords antimicrobial, artemisia, portulaca, plant extracts, beef burger www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 32 published by scholink inc. 1. introduction nowadays, new preservation techniques are being developed to extend the storage time with maintaining both the natural appearance and safety of fresh meat (patsias et al., 2008; zhou et al., 2010). synthetic preservatives such as nitrite has been widely used for preserving meat products such as sausages and luncheon meats. nitrite is efficient for inhibition of clostridium botulinum (gibson et al., 1984). nitrite may react with secondary amines naturally present in the meat to form n-nitrosamines, which are carcinogenic (iarc, 1998). there is an association between consumption of processed meat and increase risk of different cancers (santarelli et al., 2008; larsson et al., 2006; larsson & wolk, 2012), which cause death (rohrmann et al., 2013). unlike synthetic compounds, natural extracts obtained from plants are rich in phenolic compounds which can enhance the overall quality of food by decreasing lipid oxidation and microbial growth (zhang et al., 2016). artemisia vulgaris is used in asthma emmenagogue, anthelmintic, and stomachic (ambasta, 1994). the essential oil of leaves exhibited significant antimicrobial activity (laxmi & rao, 1991). the leaves have an antibacterial activity on staphylococcus aureus, bacillus typhi, bacillus dysenteriae, streptococci, escherichia coli, bacillus subtilis and pseudomonas. pharmacological studies on portulaca oleracea showed its activities as antibacterial (chan et al., 2015), hepatoprotective (al-sheddi et al., 2015), anti-inflammatory, analgesia (zhou et al., 2015), antioxidant (liu et al., 2015). so the goal of this work is the evaluation of antimicrobial effect for both artemisia and portulaca extracts against microorganisms in vitro and as a preservative agent in beef burger. 2. materials and methods 2.1 materials herbs: artemisia vulgaris and portulaca oleracea were obtained from egyptian herbal market, dokki, giza. microbial strains: three gram negative pathogen bacteria; salmonella typhimurium (atcc 14028), e. coli o157:h7 and pseudomonas aeruginosa and 2 gram positive pathogens; staphylococcus aureus (25923), and listeria monocytogenes (atcc 7644), microbial strains are generously given by microbiology department, faculty of agriculture, cairo university. different microbial strains were preserved at -20oc. ingredients of beef burger: a. beef meat: frozen beef, lean meat was obtained from the local butcher shop in the day before the experiment. the meat was cold stored at 5±1ºc overnight. b. soybean flour: soybean flour was obtained from food technology research institute, agriculture research center, giza, egypt. c. other ingredients: spices, fresh eggs, onion and salt were obtained from the local market. while, sodium tripolyphosphate, and sodium ascorbate were obtained from the adwic laboratory chemicals co., cairo, egypt. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 33 published by scholink inc. 2.2 methods 2.2.1 technological methods preparation of artemisia vulgaris and portulaca oleracea extract: one hundred grams of artemisia vulgaris or portulaca oleracea leaves were added to excessive distilled water: ethanol (2:8 v/v) and incubated at room temperature for 24 h, then the slurry was filtered through filter paper. the water extract was concentrated using a rotary evaporator under reduced pressure and the residues were dissolved in 50 ml of distilled water. beef burger formulation: beef burger patties were processed by the method described by (oroszvári et al., 2005; ou & mittal, 2006) according to the formula showed in table 1: table 1. beef burger formulation main ingredients (%) treatments control artemisia extract portulaca extract 1% 1.5% 1% 1.5% beef meat 62 62 62 62 62 soy flour 12 12 12 12 12 iced water 10 10 10 10 10 fresh eggs 7 7 7 7 7 fresh onion 7 7 7 7 7 salt 1.5 1.5 1.5 1.5 1.5 spices 0.5 0.5 0.5 0.5 0.5 total 100 100 100 100 100 additives (%) sodium tripolyphosphate 0.3 0.3 0.3 0.3 0.3 sodium ascorbate 0.03 0.03 0.03 0.03 0.03 artemisia extract 1 1.5 1 1.5 portulaca extract 1 1.5 1 1.5 2.2.2 analytical methods dpph radical scavenging method: the antioxidant activity of the plant extracts was evaluated using the stable 2, 2-diphenyl-1-picrylhydrazyl radical (dpph) according to a modified method of bandoniene et al. (2002). qualitative phytochemical analysis: phytochemical of plant extracts was carried out by standard methods described by (brain & turner, 1975; evans, 1996) as follows: detection of alkaloids: extracts were dissolved separately in dilute hydrochloric acid and filtered. the filtrates were used to detect the alkaloids by using mayer’s test, since filtrates were treated with mayer’s reagent. formation of a yellow, cream precipitate indicates the presence of alkaloids. detection of saponins: about 0.5 mg of the extract was mixed by agitation with five ml of distilled water. formation of frothing (appearance of creamy miss of small bubbles) shows that the presence of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 34 published by scholink inc. saponins. detection of tannins: a small quantity of extract was mixed with water and heated using water bath. then the mixture was filtered and ferric chloride was added to the filtrate. the formation of dark green colour indicates the presence of tannins. detection of flavonoids: detection of flavonoids was carried out as follows: -lead acetate test: extracts were treated with a few drops of lead acetate solution. the formation of yellow color indicates the presence of flavonoids. -h2so4 test: extracts were treated with a few drops of h2so4. formation of orange color indicates the presence of flavonoids. precipitate indicates that the presence of flavonoids. detection of phenols: detection of phenols was carried out as follows: -ferric chloride test: 10 mg extracts were treated with a few drops of ferric chloride solution. formation of bluish black color indicates the presence of phenol. -lead acetate test: 10 mg extract was treated with a few drops of lead acetate solution. formation of a yellow color precipitate indicates the presence of phenol. 2.2.3 quantitative phytochemical analysis estimation of alkaloids was carried out by using the method of (harborne, 1973) as follows: one gram of extract was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added and the beaker was covered and allowed to stand for 4 h, then the mixture is filtered and concentrated on a water bath to one quarter of the original volume. concentrated nh4oh was added by drop wise to the extract until the precipitation was complete. the whole solution was allowed to settle and the precipitate was collected and washed with dilute nh4oh, then filtered. the residue is the alkaloid, which was dried and weighed. estimation of flavonoids was carried out as follows: one gram of plant sample was repeatedly extracted with 100ml of 80% aqueous methanol at room temperature. the mixture was filtered through a whatman no.1 filter paper into a pre-weighed 250 ml beaker. the filtrate was transferred into a water bath and allowed to evaporate to dryness and weighed (krishnaiah et al., 2009). estimation of total phenols was carried out as follows: the fat free sample was boiled with 50 ml of ether for extraction of phenolic compounds for 15 min. five ml of the extract was pipetted into a 50 ml flask, then 10 ml of distilled water was added. two ml of nh4oh solution and 5 ml of concentrated amyl alcohol were also added. the samples were made up to mark and left to react for 30 min for color development. this was read spectrophotometry at 505 nm. screening of antimicrobial activity: screening of antimicrobial activity was performed using the agar diffusion method. the nutrient agar plate was over layered with approximately 2 ml soft agar inoculated with 105-106 cfu/ml of overnight activated microbial cultures, then wells of 8 mm diameter were holed by cork borer, 60 µl of each tested compounds were injected in every well. negative control was performed using sterile distilled water. plates were incubated for 24 h at 37oc. diameters of inhibition clear zones were measured using graded ruler. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 35 published by scholink inc. 2.2.4 microbiological methods determination of minimum inhibitory concentrations (mic) and minimum cidal concentrations (mcc): determination of minimum cidal concentrations (mcc) of extracts were performed using microplate methods, 100 µl of sterile nutrient broth were loaded in each well of 96 wells plate, then defferent volumes of extract were added to achieve final concentrations ranged from1 to 15% (v/v), then the inoculums of different pathogens were added to have 106 cfu/ml final concentration. after incubation for 24 h at 37oc, bacterial count of each sample was determined using the drop plate method (naghili et al., 2013). drops of 10 µl from each well were pipetted onto surface of solidified nutrient agar, and then the plates were incubated for 24 h at 37oc. minimum inhibitory concentration defined as the concentration which shows no increase or decrease of the initial counting after 24 h of incubation, while minimum cidal concentration (mcc) defined as the less concentration showed no bacterial viability. microbiological assessment of frozen beef burger: microbiological assay of the different beef burger samples was carried out at zero time and after 3 months of frozen storage. total count of bacteria was determined to evaluate the antimicrobial activity of plant extract in a real food system such as beef burger and the ability of this extract to prolong the storage period of beef burger. ten g of each sample was added to 90 ml of sterile pepton water and mixed well for 1 min to homogenize. decimal dilutions in sterilized pepton water were prepared and 1 ml was poured in plate count agar (pca, cm0325, oxoid) to enumerate total bacterial count after incubation for 24 h at 37oc. 2.2.5 statistical analysis one-way analysis of variance (anova) was performed to test for differences between the groups mean. significant differences between the means were determined by duncan’s multiple range test and p<0.05 were regarded as significant (sokal & rohlf, 1995). 3. results table 2 shows the qualitative phytochemical of artemisia and portulaca extracts. from the obtained results it cleared that both extracts contain alkaloids, flavonoids, phenols, trepenoids, saponin while the tannins was positive in artemisia and negative in portulaca extract. table 2. qualitative phytochemical analysis of artemisia and portulaca extracts phytochemicals artemisia extract portulaca extract alkaloids mayer s test + + flavonoids lead acetate test h2so4 test + + + + phenols ferric chloride test + + www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 36 published by scholink inc. lead acetate test + + terpenoids + + saponin + + tannin + note. (+) present; (-) not detected. table 3 shows the quantitative phytochemical of artemisia and portulaca extracts. from the results it cleared that both extracts contained alkaloids, flavonoids, phenol and antioxidant activity at levels 4.25-5.34, 14.34-9.46, 9.47-3.85 w/w, 74%-68% respectively. table 3. quantitative phytochemical analysis of artemisia and portulaca extracts phytochemicals artemisia extract (w/w) portulaca extract (w/w) alkaloids 4.25 5.34 flavonoids 14.34 9.46 phenol 9.47 3.85 antioxidant activity 74% 68% table 4 shows the artemisia and portulaca water extract against pathogenic bacteria. the obtained results showed that artemisia extract has a potent antimicrobial effect against all tested microorganisms, staph aureus, pseudomonas aeruginosa, salmonella typhimurium, listeria monocytogenes and e.coli o157:h7. the maximum effect of the artemisia extract as an antibacterial was against pseudomonas aeruginosa and gave 28 mm inhibition zone diameter followed by listeria monocytogenes, staph aureus, salmonella typhimurium and e.coli o157:h7 since the extract could inhibit the microbial growth and the inhibition zones were 26.25, 26, 24 and 15.4 mm respectively. the portulaca extract was also tested as an antimicrobial against the same microorganisms, but it had a potent effect only against staph aureus while, did not have any effect against pseudomonas aeruginosa, salmonella typhimurium, listeria monocytogenes and e.coli o157:h7. table 4. screening of antimicrobial activity (inhibition zone diameter in mm) of artemisia and portulaca against tested pathogenic bacteria microorganism artemisia extract portulaca extract staph aureus 26 20 pseudomonas aeruginosa 28 nil salmonella typhimurium 24 nil listeria monocytogenes 26.25 nil e.coli o157:h7 15.5 nil table 5 shows the minimum inhibitory concentration (mic) and minimum cidal concentrations www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 37 published by scholink inc. (mcc) of artemisia extract against pathogenic bacteria (portulaca extract was excluded because that it had not antimicrobial activity except against staph aureus only). the minimum cidal concentration (mcc) is the lowest concentration of an antibacterial agent required to kill a particular bacterium. it can be determined from broth dilution minimum inhibitory concentration (mic) tests by subculturing to agar plates that do not contain the test agent. the minimum inhibitory concentration mic and minimum cidal concentration (mcc) were carried out for testing pathogenic bacteria are e. coli, pseudomonas aeruginosa, staph aureus, salmonella typhimurium, l.monocytogenes. the mic was regarded as the lowest concentration of the extracts that prevent the growth of any tested bacterial colony on the medium. from the obtained results in table 4 the artemisia extract was very effective on staph aureus and the mic was appeared at 5 µl/100 µl, while mic for both e.coli and pseudomonas aeruginosa were medium and detected at 7 µl/100 µl, moreover salmonella typhimurium and l.monocytogenes possess the lowest mic which recorded at 9 µl/100 µl. also the minimum cidal concentration mcc was carried out and found that the results related and agreed with the results of mic and the mcc at 6 µl/100 µl was for staph aureus followed by e.coli, pseudomonas aeruginosa and salmonella typhimurium, l.monocytogenes at 8, 10 µl/100 µl respectively. table 5. minimum inhibitory concentration (mic) and minimum cidal concentrations (mcc) of artemisia extract against pathogenic bacteria microorganism mic (µl/100 µl) mcc (µl/100 µl ) e.coli 7 8 staph aureus 7 8 pseudomonas aeruginosa 5 6 salmonella typhimurium 9 10 listeria monocytogenes 9 10 staph aureus pseudomonas aeruginosa www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 38 published by scholink inc. salmonella typhimurium listeria monocytogenes e.coli o157:h7 figure 1. the minimum inhibitory concentration (mic) and minimum cidal concentrations (mcc) of artemisia extract against pathogenic bacteria data in table 6 show changes in the total bacterial count of beef burger during freezing, storage which was determined at zero time and after three months of frozen storage. the results show that portulaca extract had a slight effect on beef burger microbial quality, but the rate of growth was slow which may be due to the polyphenols content of artemisia extract which cause reducing the growth rate and maximum growth population and/or extending the lag-phase of the target microorganism (zhou et al., 2010). the artemisia extract possesses a potent effect as an antimicrobial agent against all tested bacteria opposite the portulaca extract that had only effect against staph aureus, but unfortunately based on the sensory evaluation of beef burger results the artemisia extract has a detrimental effect on the taste of burger patties which impart bitter taste and were unpalatable. so, more researchers are required to remove the bitterness of artemisia extract until we can be used it in beef burger preservation without its affecting the sensory properties. table 6. microbiological changes in total bacterial count (cfu/g) analysis of beef burger samples during frozen storage period storage time treatments control artemisia extract portulaca extract 1% 1.5% 1% 1.5% zero time 2.40 e+06 3.70 e+06 2.10 e+06 2.66 e+06 2.68 e+06 3 months 3.07 e+06 3.85 e+06 2.86 e+06 3.00 e+06 3.40 e+06 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 39 published by scholink inc. table 7 shows that there is no significant differences (p≤0.05) between control and treatments containing portulaca extract in all sensory properties of beef burger, furthermore addition of portulaca extract with 1.5% was enhanced the taste, tenderness, juiciness and overall acceptability of beef burger. on the other hand the addition of artmesia extract had a detrimental effect on taste of beef burger since it impart it bitter taste. also addition of artmesia extract was slightly lower the sensory score of beef burger for tenderness and juiciness but did not affect color and appearance. table 7. effect of addition plant extracts on sensory characteristics of beef burger sensory characteristics treatments control artemisia extract portulaca extract 1% 1.5% 1% 1.5% color 8a 8a 8a 8a 8a taste 8a 1b 1b 8a 9a flavor 8a 1b 1b 8a 8 a tenderness 8ab 7b 7b 8ab 9a juiciness 8ab 7b 7b 8ab 9a appearance 8a 8a 8a 8a 8a overall acceptability 8a 1b 1b 8a 9a * scores ranging from 0-3 = very poor, 4 = poor, 5 = fair, 6-7 = good and 8-10 = very good. ** in rows means have the same superscript are not significantly different. 4. discussion our obtained phytochemical analysis of artemisia and portulaca extracts in agreement with (alireza et al., 2013) who showed that tannins, saponins, alkaloids, amino acids, phenolic compounds, quinines and terpenoids are present in artemisia extract using mass gas-chromatograph. also, okafor (2014) studied the water extract of aerial parts of portulaca and showed that it contain steroids, protein, and alkaloids. the artemisia extract had a potent effect as an antimicrobial against all tested microorganisms while, portulaca extract had a potent effect against staph aureus only. in our respect study (abdul & waheeta, 2010) showed that artemisia nilagirica extracts had a broad spectrum of antibacterial activity against phytopathogens and clinical pathogens except s. aureus, e. faecalis and k. pneumoniae. our results showed that plant extracts contain effective biological compounds such as alkaloids, flavonoids, phenols, tannins and terpenoids, which could be alternate the traditional chemicals to inhibit phytopathogenic bacteria and decrease the negative effects of synthetic drugs. the mic of artemisia extract that gave a positive effect against all tested microorganisms was analyzed and the results was in agreement agreed with (abdul & waheeta, 2010) who studied the effects of various artemisia extracts against bacteria and reported that hexane extract was effective www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 40 published by scholink inc. against all phytopathogens with low mic of 32 μg/ml, while the methanol extract showed higher inhibition activity against escherichia coli, yersinia enterocolitica, salmonella typhi, enterobacter aerogenes, proteus vulgaris, pseudomonas aeruginosa (32 μg/ml), bacillus subtilis (64 μg/ml) and shigella flaxneri (128 μg/ml). the mic analyses of clinical pathogens showed an activity against gram-positive and gram-negative bacteria, which indicate that plant extracts contains several antimicrobials. the phytochemical screening of plant menthol extracts showed that it contain flavonoids, terpenoids, phenols, amino acids, alkaloids and tannins, which have antimicrobial properties (fernandez et al., 1996; mendoza et al., 1997; amaral et al., 1998; cowan, 1999 ; shaheen et al., 2003; amarowicz et al., 2008; chowdhury et al., 2008). artemisia and portulaca extracts were incorporated into the beef burger formula as an antimicrobial agent and both extracts gave good results as antibacterial, but artemisia extract was had superior antimicrobial activity than portulaca extract. the sensory quality of the formulated beef burger was carried out, beef burgers incorporated with portulaca extract were having good sensory quality, while the beef burgers containing artemisia extract were having bad sensory quality (bitter taste), although it showed good microbiological quality, thus further researches are needed to remove the compounds which induce the bitter taste until we can use the artemisia extract as an antimicrobial in meat products. 5. conclusion finally, beef burger can be formulated with incorporation of portulaca extract as antimicrobial without any detrimental effect on its sensory properties, whereas 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(2015). portulaca oleracea l.: a review of phytochemistry and pharmacological effects. biomed. res. int. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 2, no. 3, 2018 www.scholink.org/ojs/index.php/fsns 70 original paper quantity and functionality of protein fractions isolated from 3 ecotypes of indigenous chicken in kenya benard o. oloo1*, mahungu s.1, kahi a.2 & eric amonsou3 1 department of food science and technology, egerton university, egerton, kenya 2 department of animal science and veterinary science, egerton university, egerton, kenya 3 department of biotechnology and food technology, durban university of technology, south africa * benard o. oloo, department of food science and technology, egerton university, p.o box 536-20115, egerton, kenya received: september 19, 2018 accepted: october 5, 2018 online published: november 21, 2018 doi:10.22158/fsns.v2n3p70 url: http://dx.doi.org/10.22158/fsns.v2n3p70 abstract the aim of this study was to evaluate the effect of the cluster ecotype and the part of chicken on nutritional composition, and functionality of sarcoplasmic and myofibrillar proteins that are most relevant to the technological features of chicken meat. over 50 chickens from each ecotype cluster purchased, slaughtered and the meat stored under refrigeration at -20oc and later on transferred in cooler box on ice and flown to south africa, at the durban university of technology. protein fractions were extracted with a cocktail of sodium chloride buffer (50mm nacl, 50mm tris hcl; 75mm dtt and 1mm edta at ph 7) and quantified by bradford method. one dimensional sodium dodecyl polyacrylamide gel electrophoresis (sds page) was applied to separate protein fractions. emulsifying capacity, emulsifying stability, solubility, and in vitro digestibility were determined on the total protein isolates. significant differences in band expressions were recorded for the myofibrillar and the sarcoplasmic proteins. the three ecotypes had high quality proteins with all the limiting and essential amino acids at concentrations higher than fao/who recommended daily allowance for adults and children. distinct protein bands at larger molecular weight proteins >100 kda, corresponding to myosin heavy chain, medium fractions 75 kda and 45 kda and even lower molecular weight fraction <25 kda were present in the chicken breast and the thighs. it concludes that indigenous chicken protein isolates’ nutritional and functional properties are affected by part of chicken and ecotype clusters. keywords indigenous chicken, protein isolate, functionality, sarcoplasmic, myofibrillar www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 71 published by scholink inc. 1. introduction researchers have recently used the microsatellite markers for the genetic evaluation of chicken variability (qu et al., 2006, ngeno et al., 2015). indigenous chicken (ic) in kenya have recently been classified genetically into 3 ecotype clusters based on allele type of lei0258 marker genotype for major histocompatibility complex (mhc) of the variable region (ngeno et al., 2015). limited work if any has been done to evaluate meat quality of ic in kenya according to (ngeno et al., 2015) based on this classification. yet the clusters of indigenous chicken in kenya show distinct morphological, sensorial and other quality characteristics (oloo et al., 2017). this may be attributed to the difference in genetic makeup or the differences in the traditional rearing practices by the separate communities which predominate the areas where these chicken ecotypes are found. the source of this variability and how it impacts the meat quality with regard to the protein isolates’ functionality and expressions is yet to be studied. poultry meat is made up of water, proteins, fat, minerals, and carbohydrates (omana et al., 2010; bender, 1992). these components determine the functional and sensory quality of the meat. chicken is a source of high quality protein because they contain all the essential amino acids needed by human body; they are highly nutritious (friedmann, 1996). protein composition affects overall meat characteristic of appearance, texture, and mouth feel (mudalal et al., 2014). two major types of chicken muscle proteins (myofibrillar and actin) are responsible for muscle texture and water holding capacity (whc) (ooizumi & xiong, 2004). the sarcoplasmic proteins on the other hand, play only a minor role (petracci et al., 2013). the solubility of sarcoplasmic and myofibrillar proteins on the other hand are reported to be correlated to water retention capacity of chicken meat resulting in low cooking losses (li‐chan et al., 1987; warner et al., 1997). lower protein solubility has an influence on the functionality and or defects of chicken proteins such as the pale soft exudative (pse) condition (mudalal et al., 2014). meat proteins are generally categorised in terms of their solubility as; salt, water, or alcohol soluble. chicken protein composition especially of breast; have a crucial impact on processability of meat, its nutritional, and sensory quality (mudalal et al., 2014). the knowledge of protein composition and structure is important to connecting of functionality and applications in various foods. protein composition and structure may vary across different ecotypes or genotypes and this is likely to affect their functionality (arise, 2016). the high molecular weight proteins are known to confer functional properties to the skeletal muscle. myosin, actin, titin and nebulin are the major of the skeletal sarcomere and are most important in this regard. myosin with a (molecular weight) mw of 540 kda and actin of approximate mw 43 kda are most noticeable (clark et al., 2002). the aim of the current study was to evaluate the effect of the cluster ecotype and the part of chicken of indigenous chicken in kenya on the chemical composition, protein properties and functionality with emphasis on sarcoplasmic and myofibrillar proteins that are most relevant to the technological features of chicken meat. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 72 published by scholink inc. 2. method 2.1 chemical composition proximate analysis (moisture, protein, lipid and ash) of breast and thighs of indigenous chicken were estimated in 3 replications for each sample using aoac (1990) official methods. moisture content was evaluated by weighing 5 grams of sample and drying in a conventional oven at 103oc for 5 hours until constant weight was reached. crude protein content was estimated by kjeldhal method while edible fat (intra-muscular fat) was estimated by petroleum ether extraction using soxhlet method. ash content was evaluated by incineration in a muffle furnace at 525oc for 5 hours. 2.2 amino acid composition determination amino acid content of the indigenous chicken fillets were determined on lyophilized, ground and homogenous samples by pico-tag method as described by (bidlingmeyer et al., 1984). a total of 16 amino acids were analysed. 2.3 sds page -analysis frozen chicken breast and thigh muscles were selected to separate the extracted proteins according to their molecular weights by sds-page according to the procedure of (mudalal et al., 2014) with slight modifications. ten (10) grams of samples was mixed with 10 mls cocktail of sample extraction buffer (50 mm nacl2, 50 mm tris-hcl (ph=7.0); ddt (75 mm dtt) and 1 mm edta (ph=7). resulting sarcoplasmic and myofibrillar proteins were mixed with equal volume of standard loading buffer as previously described by (mudalal et al., 2014). samples were then heated at 95oc for 5 minutes in a water bath cooled on ice and then applied to a gel at a volume of 15 ul of both sarcoplasmic and myofibrillar proteins and loaded into the already prepared gels. the small gels were set to run at a constant voltage of 180 v. reservoir buffer was made up of 50 mm tris, 0.384 m glycine, and 0.1% (wt/vol) sds. a mixture of standard proteins (11-190 kda) was used as the molecular weight marker and the gels were stained in coomassie brilliant r-250. 2.4 emulsifying activity and stability emulsifying activity was determined according to the procedure described by (lawal et al., 2007). equal volumes (5 mls) of protein (isolates from chicken) were mixed with equal volumes of pure sunflower oil and homogenized. the emulsifying activity was calculated as the height of the emulsified layer over the overall content of tubes and expressed as a percentage. samples were then heated at 80oc for 30 minutes and the remaining emulsified layer used to calculate emulsifying stability. 2.5 protein solubility to quantify protein solubility, difference in extractability using different ionic strength solutions was exploited. this was modified from the procedure used by (warner et al., 1997). after solubilization of proteins, the supernatant was carefully poured out temporarily to separate the sediment. resultant protein concentration was quantified by bradford with (bovine serum albumin) bsa as the standard (bradford, 1976). for total solubility, a high ionic strength of 1.1 m ki and 0.1 m potassium phosphate buffer of (ph 7.2) was used. the difference in total protein solubility from that of sarcoplasmic protein www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 73 published by scholink inc. solubility was used to determine the myofibrillar protein content (zhang et al., 2009). 2.6 in vitro gi digestion (sangsawad et al., 2017) in vitro digestion of boiled chicken meat was performed according to (garrett et al., 1999) and adopted by (sangsawad et al., 2017), with slight modifications. the boiling of chicken meat was done in six times volume of water in a temperature-controlled furnace at temperature of 95±1°c for 15 minutes; equivalent to well done. cooked samples (2 g dry solid) were homogenized (ika works asia, bhd, malaysia) in 100 mls of deionized water (di) for 1 min. the ph was adjusted to 2.0 ± 0.02 with 1m hcl and pepsin (2.86% of substrate, dry basis) was added, and digestion performed at 37oc for 1 h in a shaker set at 95 rpm speed. subsequently, the ph was adjusted to 7.5 ± 0.02 by adding 5 m naoh. pancreatin enzyme (4.00% of substrate, dry weight basis) was added and the reaction was carried out at 37oc for 2 h in the shaker set at 95 rpms. the enzymatic digestion was terminated by submerging the sample in a 95oc water bath for 10 min and then cooling on ice to room temperature. the digested mixtures were then centrifuged at 10,000 g for 10 min. the peptide content of the supernatant was determined using bradford assay, with bsa as the standard. 2.7 statistical analysis the resulting data was subjected to statistical analysis. a one-way analysis of variance (anova) was employed to determine statistical difference using sas proc glm procedure version 9.3 (sas institute inc, cary, nc). all data was presented as means ± standard deviation. duncan’s multiple range test (p<0.05) was used to identify significant differences among means. 3. results 3.1 chemical composition of indigenous chicken ecotypes in kenya among the ecotypes, no significant difference was reported in moisture content between the thighs and the breasts (p>0.05). in all the ecotypes the thighs recorded higher moisture content than breasts. the mean moisture content reported for the ic ecotypes ranged from 69-72% (table 1.) this compares favourably with the values reported by chepkemoi et al. (2017) on the nutritional quality of five different poultry in kenya. the mean values recorded (breast 71.6% and thigh 71.3%) were lower than those reported by (chumngoen & tan, 2015) who got an average moisture of 74.73% among the taiwan native chicken. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 74 published by scholink inc. table 1. chemical composition of indigenous chicken in kenya from the different ecotypes: values are expressed in (g/100g) of sample ecotype chicken part moisture content fat content protein content ash content kakamega breast 72.53±1.00a 1.75±0.86a 21.21±1.50a 4.43±1.03a thigh 73.32±1.32a 3.59±0.95a 19.02±2.04a 3.88±0.96a naivasha breast 70.40±0.91a 1.64±0.31a 24.08±1.11a 4.61±0.38a thigh 71.69±1.83a 2.95±0.89a 22.99±1.35a 4.23±0.49a taita breast 72.46±0.79a 2.78±1.00a 24.31±0.91a 3.00±0.83a thigh 69.09±4.97a 2.77±0.80a 21.97±1.99a 2.96±0.40a values given as mean ± standard deviation (n=30). a-means and standard deviations within the same column with no common superscript are significantly different at (p>0.05). 3.2 the sds-page of proteins from the kenyan indigenous chicken ecotypes the sds page for kakamega ecotype reveals distinct bands between the myofibrillar and sarcoplasmic proteins. up to twelve (12) bands were noticeable on the sarcoplasmic proteins and a maximum of 10 were reported on the myofiobrillar protein for the breast muscle (figure 1). this confirms the report by (mudalal et al., 2014) that sds-page analysis for meat proteins from normal and white striated fillets showed different patterns for sarcoplasmic and myofibrillar proteins. the myosin heavy chain (mhc) proteins are dominant and prevalent on the myofibrillar proteins on the thighs as well as on the breast muscles. these proteins are shown to be smeared at the wells evidence of their inability to disintegrate into lower molecular weight proteins and thus their separation is hindered (figure 1). the actin (42-45 kda) molecular weight proteins is a distinct poultry muscle protein as is visibly recorded both on the breast and thigh muscles of the kakamega ecotype (figure 1). sarcoplasmic protein fraction separated into at least 12 distinct bands. this was similar for protein fractions either from the breast or thigh. this was also reported by (mudalal et al., 2014) though they stated that only 11 of these bands having molecular weight of between 25 and 90 kda were quantified. a band is observed at the 75 kda mark. not as prominent as the actin band observed at 42-45 kda (figure 1). there is a prominent band at 135 kda which separates into two with one band spotted at the 100 kda mark, representing (c-protein). this separation must have been aided by the application of the dtt and then denaturation of the muscles by boiling for 5 minutes at 95oc (mudalal et al., 2014). they also express very prominent band at the 45 kda (actin). the sarcoplasmic protein from breast muscle had seven fully expressed protein bands (lane 2) compared to only 5 for thigh muscle (lane 4) for kakamega ecotype. the concentration of these specific bands is indicative of the nature of the poultry muscle from those affected by white stripping (mudalal et al., 2014). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 75 published by scholink inc. figure 1. sds-page profile of sarcoplasmic and myofibrillar proteins for kakamega ecotype. diagram (a) breast and (b) thigh. lane 1: marker; lane 2: sarcoplasmic; lane 3: myofibrillar; lane 4: sarcoplasmic; lane 5: myofibrillar figure 2. sds-page profile of taita ecotype. diagram (a)-breast and (b) thigh. lane 1: marker; lane 2: sarcoplasmic (deleted breast); lane 3: myofibrillar; lane 4: sarcoplasmic and lane 5: myofibrillar figure 3. sds-page profile of naivasha ecotype. (a) breast and (b) thigh. lane 1: marker; lane 2: sarcoplasmic; lane 3: myofibrillar; lane 4: sarcoplasmic; lane 5: myofibrillar www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 76 published by scholink inc. 3.3 the characteristic bands of myofibrillar and sarcoplasmic muscles the sds page pattern for all the ecotypes and the parts of chicken demonstrate the presence of sarcoplasmic and myofibrillar proteins patterns (figures 2-4). this is similarly reported by several studies (khiari et al., 2014; fritz et al., 1989; zanetti et al., 2014). the sds-page for the myofibrillar proteins isolated from the three ecotypes show three distinct bands at a molecular weight above the 135 kda as (mhc, c-proteins and the alpha-actin). figure 5 shows the sds page profile of sarcoplasmic proteins from kenyan indigenous chicken displaying some of the most prominent bands identified. figure 4. sds-page profile of myofibrillar protein from kenyan indigenous chicken showing some of the most prominent bands identified. lane 1: marker and lane 2: myofibrillar figure 5. sds-page profile of sarcoplasmic proteins from kenyan indigenous chicken displaying some of the most prominent bands identified. lane 1: marker; lane 2: sarcoplasmic protein fraction 3.4 amino acid composition of the kenyan indigenous chicken ecotypes except for histidine and lysine which were higher in the breasts than in the thigh muscles for all the ecotypes, there were no significant differences reported on the amino acid profile of the breasts and thighs (p<0.05) (table 2). remarkably, all the amino acids tested were higher than those recommended www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 77 published by scholink inc. by who/fao for adults as well as for children (organization & university, 2007) health organization. the values of tryptophan and cysteine were not quantified in this work. table 2. effect of body part (breast and thigh) and ecotype on the amino acid profiles (g/100 g protein) of ic in kenya amino acid chicken parts ecotype fao/who2007 recommendatio ns for adults fao/who2007 recommendatio ns for children breast thigh kakamega naivasha taita his 2.96±0.03a 2.15±0.13b 2.51±0.40 2.46±0.52 2.69±0.30 1.6 1.9 lys 9.02±0.26a 7.76±0.24b 7.95±0.56 8.43±0.75 8.79±0.58 1.6 5.8 met 7.96±0.19 6.77±0.89 6.79±0.84 6.90±1.09 8.42±0.13 1.7 2.7 val 4.33±0.02 3.91±0.22 4.04±0.33 3.99±0.31 4.34±0.01 1.5 3.5 ile 3.91±0.04 3.62±0.17 3.67±0.29 3.69±0.16 3.94±0.02 1.3 2.8 leu 7.25±0.10 6.70±0.41 6.81±0.60 6.73±0.35 7.41±0.11 1.9 6.6 phe 3.42±0.26 3.50±0.27 3.50±0.42 3.25±0.17 3.63±0.38 1.6 6.3 thr 4.20±0.09 4.04±0.23 4.03±0.35 4.01±0.05 4.33±0.16 0.9 3.4 total 43.05 38.45 39.3 39.46 43.55 ser 3.42±0.1 3.46±0.19 3.32±0.12 3.39±0.05 3.62±0.22 arg 5.73±0.06 5.69±0.34 5.64±0.19 5.51±0.23 5.99±0.37 gly 3.89±0.17 4.55±0.37 4.60±0.70 4.20±0.02 3.87±0.27 asp 9.09±0.20 8.62±0.39 8.76±0.72 8.68±0.23 9.13±0.24 glu 14.23±0.27 14.36±0.71 14.05±0.72 13.96±0.08 14.89±0.83 tyr 2.82±0.07 2.79±0.24 2.74±0.22 2.67±0.08 3.02±0.26 als 5.19±0.05 4.93±0.09 5.04±0.15 5.04±0.25 5.11±0.83 pro 2.98±0.07 3.44±0.18 3.42±0.36 3.10±0.06 3.12±0.26 total 47.35 47.74 47.57 46.55 33.86 values given as mean ± standard deviation (n=18). ab means and standard deviations within the same row with no common superscript are significantly different at (p<0.05). 3.5 emulsion capacity and stability of kenyan indigenous chicken ecotypes emulsion capacity and emulsion stability of protein isolates from the breasts and thighs of the kenyan ic ecotypes are presented in table 3. only the kakamega ecotype had a significantly different (p<0.05) emulsion capacity. this difference is also expressed in the stability of the emulsion. the naivasha ecotypes’ emulsion capacity is not significantly different between the breast and the thigh protein isolates. though there is a significant difference in the stability of the emulsions for the naivasha www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 78 published by scholink inc. ecotype. there were no significant differences on the emulsion capacity and emulsion stability of protein isolates from breast and thighs of the taita ecotype (table 3). table 3. emulsion capacity and stability of kenyan indigenous chicken ecotypes ecotype chicken part emulsion capacity (ml oil/g protein) emulsion stability (ml oil/g protein) kakamega breast 22.43±1.38b 41.13±8.87a thigh 30.16±0.61a 41.65±3.81b naivasha breast 31.07±0.51a 40.23±4.93b thigh 28.75±0.98a 44.31±9.02a taita breast 28.04±0.54a 28.13±3.13c thigh 32.11±2.11a 25.54±3.04c values given as mean ± standard deviation (n=18). abc means and standard deviations within the same column with no common superscript are significantly different at (p<0.05). 3.6 protein digestibility and protein solubility the digestibility of the protein isolates shows a significant difference (p<0.05) between the thigh and the breast muscles for taita and naivasha ecotypes (higher digestibility of thigh protein isolates). for kakamega ecotype, the breasts had a higher digestibility. with regard to solubility, only the kakamega ecotype showed a significant difference between the breast and the thigh. the solubility of breast protein isolates for this ecotype was 60.9% compared to 62.98% for the thigh protein isolates (table 4). naivasha and taita ecotypes had no significant difference with regard to protein isolates’ solubility for the breast and the thigh. table 4. protein digestibility and solubility of isolates of kenyan indigenous chicken ecotypes ecotype chicken part protein digestibility (%) solubility (%) kakamega breast 63.50±4.50b 60.90±2.08b thigh 60.00±6.10b 62.98.84±6.89c naivasha breast 66.00±6.10b 65.88±0.63a thigh 75.50±2.50a 66.94±0.06a taita breast 63.90±3.50b 67.32±0.02a thigh 75.50±0.50a 67.22±0.06a values given as mean ± standard deviation (n=18). ab means and standard deviations within the same column with no common superscript are significantly different at (p<0.05). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 79 published by scholink inc. 4. discussion protein content was the main focus of this study. though, no significant differences on crude protein content were noted among the different parts of the ecotypes, the breasts recorded higher values for all the ecotypes than the thigh muscles (p<0.05). the values are also higher than what was reported recently by chepkemoi et al. (2017). these results are supported by the study conducted by sirri et al. (2010) who noted that the breast chicken muscle had higher protein content. the breast and thigh muscle composition has been found to differ in some studies but are not at all significant for the present study. this, is however in contrast to guan et al. (2013) who reported that the genotypes of birds influenced their chemical composition. the rate of growth of chicken also affects their protein content (fanatico et al., 2007; guan et al., 2013). indigenous chicken are all slow growing and hence record high quality protein content ranging from 19.0±2.04 to 24.3±0.91%. khiari et al. (2014) reported protein content of 19.7% among the commercial turkey (a close rival or alternative of chicken meat) purchased from the market. it is reported that protein content determines nutritional quality especially of protein based diets such as chicken. similar crude protein content of native chicken meat were reported by guan et al. (2013). on the contrary, fanatico et al. (2007) reported slightly higher values from leg muscles among five genotypes of slow growing chinese chicken. chumgeon and tan (2015) recorded the higher protein values both for the broiler and taiwan native chicken. with regard to fat content, a different trend was observed as higher values of fat content was recorded for the thighs (3.12%) than for the breasts (2.12%). this value was similar to what was reported by chepkemoi et al. (2017) at an average fat content of 2.23% for indigenous chicken in kenya. these values were however higher than those reported by chumgeon and tan (2015). fat content determines functionality, sensory quality and nutrition of poultry meat (aronal, huda, & ahmad, 2012). the difference in fat content is normally dependent on the feed (guan et al., 2013). the results for this study agree with sirri et al. (2010) and fanatico et al. (2007). with regard to the ash content, the same trend was observed. higher values of ash content were reported on the breasts than on the thigh muscles for all the ecotypes. though the ash content is correlated with the mineral content; chicken meat however, is not critically targeted toward contributing to this. these values of ash content are higher than those reported by chepkemoi et al. (2017). these values were however lower than values reported by khiari et al. (2014). the sds page of the taita ecotype reveals that myofibrillar proteins of both the breast and thighs have distinct and similar band profiles or characteristics. there was less smearing at the top of the gel and this may have been as a result of the denaturation for 5 minutes at 95oc (fritz, swartz, & greaser, 1989). the dominant bands are still the mhc (molecular weight above 135 kda) and actin of 42-45 kda molecular weights. the observation agrees with the report of omana et al. (2010) about the location of this abundant actin band. the myofibrilar proteins isolated from the thighs however show least separation of the heavy myosin chain proteins which indicates a difficulty in their separation or reduction to lower molecular weight proteins. myfibrillar proteins on the other hand had prominent www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 80 published by scholink inc. hmc at the top and some of these were too large as to enter the gel wells for better separation. this may be as a result of the difficulty in reduction of disulfide bonds which results in higher molecular weight species that are transferred out of the gels with greater difficulty than those of lower molecular weight (fritz swartz & greaser, 1989). more bands were expressed in sarcoplasmic proteins than were expressed in the myofibrillar proteins for both the thigh and breast muscle. with regard to the naivasha ecotype, the same trend is observed. more bands totaling 12 are visible for the sarcoplasmic proteins on both the breast and thigh muscles. the myofibrillar proteins expression for breast and thighs are very similar (figure 3). the breast’s myofibrillar protein separates better in to 4 distinct bands whereas the thigh myofibrillar proteins have mainly the bands corresponding to mhc and the c-proteins and alpha-actin. this is supported by the work of omana et al. (2010). at the same time it is evident based on the sizes of the bands that hydrolysis of proteins in this process was greatly reduced and this would suggest intact action of the protein functionality particularly with regard to gel-forming capacity (kristinsson, 2001). the poor separation or mobility observed on the myofibrillar from the thigh may suggest the occurrence of disulfide bonds just before sample applications particularly in the presence of dtt (fritz swartz & greaser, 1989). though they reported that this problem could be eliminated by heating or denaturation for longer than 4 minutes which was done in this study; and the addition of 2-mercaptoethanol yet which was not used in ours study. protein fractions are determined based on their solubility on a chosen ionic strength of buffer in this case 50mm nacl was used. the sds-page patterns revealed the characteristic soluble, regulatory muscles contrasted with the contractile of the abundant myosin and actin. this is in line with the results of zanetti et al. (2009). from these bands, the next prominent band is actin which is very indicative of poultry proteins at molecular weight of 42-45 kda. the slow twitch myosin chains lc1 and lc2 and the fast twitch light chain myosin precede these at molecular weights ranging from 27-16 kda (figure 4). the muscles are part of contractile fibre (mudalal et al., 2014). zannetti et al. (2013), reported that the fast twitch fibres are major components of breast muscles. this study confirms this finding based on the distinct bands of lc1 and lc2 recognized in this study. the sds-page of the sarcoplasmic proteins shows the mhc, c-proteins and the alpha-actin as very distinct bands. the presence of alpha actin is a clear indication of the ability of this protein to be extracted by the nacl. this is supported by the finding of xiong et al. (2000). the next prominent bands visible on the electrophoretograms are located at the 90 kda glucose pyruvate and pyruvate kinase at 68 kda. the lowest molecular weight protein fraction is the phosphoglycerate mutase at 25 kda. in total at least 12 distinct bands are observed for sarcoplasmic protein as opposed to 8-10 (maximum) bands observed for the myofibrillar protein fraction for all the ecotypes. this was irrespective of whether the sds-page was conducted on breast or thigh muscle. the two protein fractions (and myofibrillar) have the highest relevance to nutritional quality and processability (mudalal et al., 2014). there is a clear distinction in profile of protein sub fractions between the myofibrillar and sarcoplasmic proteins. for the myofibrillar proteins, the actin and alpha actin seemed www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 81 published by scholink inc. to be the most extractable proteins (y. xiong, lou, wang, moody, & harmon, 2000). the results of this work also support the findings of zhang et al. (2015) who reported the most intense myofibrillar protein bands as containing the mhc at 200 kda, actin at 43 kda and tropomysin subunit at 35 kda. proteins involved in energy metabolism were identified in the sarcoplasmic proteins (figure 5). notably these were expressed by bands corresponding to the enolase, pyruvate kinase (pk), and the glyceride phosphate dehydrogenase (gap). this confirms the studies of zanetti et al. (2013) who reported an increased expression of five proteins involved in the energy metabolism among these were phosphoglycerate kinase, and beta-enolase. amino acid profiles are most important nutritional (protein quality and digestibility) parameters of protein source foods. the values reported for methionine (at least 6.77 g/100g), and lysine (at least 7.76 g/100g) which are limiting amino acids in cereals and legumes were very high in the chicken. this affirms the assertion that chicken are a good source of high quality protein and confirms that kenyan indigenous chicken under free range are equally a good source of the same. high amounts of lysine, aspartic acid and glutamic acid were found in all the three ecotypes. the same trend was observed as well for the breasts and thighs. this result is in line with the finding of wattanachant et al. (2004); who reported high values for these three amino acids. they also reported very high values of leucine in the breast and thighs of chicken; a result which is supported by our findings. protein quality is closely related to the efficiency of the protein utilization in human digestive system. the protein digestion efficiency further plays an important role in determining the level of protein required for consumption or type of protein source required to satisfy the nutritional requirements of an individual (aronal et al., 2012). this information is vital to the choice of quantity of a given food or food source that is required for digestion. the stability of the emulsions is dependent on the ease of solubility of proteins and this is a function of the protein hydrophobicity. an emulsion refers to of two or more immiscible liquids in which one provides the dispersing phase while the other is dispersed evenly in form of tiny droplets (klemaszewski et al., 1992). proteins are amphoteric and have the unique ability to form a layer that binds to both polar and non-polar liquids hence allowing for the two phases to hold as an emulsion. the emulsion capacity is a measure of the volume of oil that can be solubilized by a given amount of protein (omana et al., 2010). this parameter is useful in qualifying proteins that are best for manufacture of comminuted products such as the frankfurter, bologna and sausages. some studies show that the emulsion capacity of protein isolates may relate to the ph of protein extraction. in the present study, the ph extraction of the isolates was uniform and hence the differences can be attributed to the ecotypes and parts of the chicken. the hydrophobic-hydrophilic balance of proteins also affects the emulsion capacity. the protein solubility has been shown to correlate with process properties such as whc, represented by cooking loss, drip loss or moisture uptake (warner et al., 1997). in another related study, omana and others demonstrated the foam capacity of proteins has an optimal ph of about 11.5 from whence an increase in ph results in a reduced foam expansion (omana et al., 2010). all the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 82 published by scholink inc. extraction of current isolates were done at a ph around neutral where protein solubility is optimal and hence the differences observed in foaming capacity can be ascribed to differences in part of chicken muscle as well as differences in ecotypes. protein solubility is also a factor that determines the quality of meat and is in particular an indicator of different meat defects such as pse and dark, firm and dry (warner et al., 1997). protein solubility according to wang et al. (2017) is a key index of functionality as a direct demonstration of the denaturation and aggregation for proteins. whereas tang et al. (2006) suggested that changes in conformation of proteins could be due to formation of soluble protein aggregates. wang et al. (2017) on the other hand, suggested that the increasing exposure of internal hydrophilic sites could be responsible. the higher solubility observed on the breasts for the kakamega and taita ecotypes could precisely be due to this very reason. as supported by digestibility and texture results, the breasts muscles were better broken down and therefore isolates exposed more hydrophilic sites responsible for the increased solubility. marty, rasale and das (2015) suggested that cavitation including shear and turbulence induced when they used pulsed ultra sound could disrupt the hydrophobic interactions hence molecular association of protein aggregates thus improving solubility. in the present study, the disruptive force was provided by the homogenizer/blender and the disruption must have been greatest in breast muscle than on the thigh muscle. 5. conclusions the expression of proteins from the three kenyan ic ecotypes showed distinct differences between myofibrillar and sarcoplasmic proteins based on sds page but very small differences among the ecotypes and their body parts. with regard to functionality, there are differences in emulsification and digestibility capacity among the three ecotypes and also on the breast and thigh muscles. the ic give high concentration of the glutamic, lysine and lysine amino acids and at levels higher than the set by fao/who values for adults and children. this underscores the intrinsic nutritive value of indigenous chicken in the diet. acknowledgements the authors appreciate the support of egerton university; department of dairy & food science & technology and the department of biotechnology and food technology of durban university of technology (dut), south africa for availing laboratory facilities to the authors during this work. the authors also give a special mention to ms. samantha govender and nyasha mirriam busu and fellow phd and msc. students of the food science laboratory (dut) for laboratory analysis support and to mr. nobert wafula of egerton university for data analysis help. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 3, 2018 83 published by scholink inc. references aronal, a., huda, n., & ahmad, r. 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(2006). pp2a is required for centromeric localization of sgo1 and proper chromosome segregation. dev cell, 10. https://doi.org/10.1016/j.devcel.2006.03.010 warner, r., kauffman, r., & greaser, m. (1997). muscle protein changes post mortem in relation to pork quality traits. meat science, 45, 339-352. https://doi.org/10.1016/s0309-1740(96)00116-7 warriss, p. d., pagazaurtundua, a., & browna, s. n. (2005). relationship between maximum daily temperature and mortality of broiler chickens during transport and lairage. brit poultry sci, 46. https://doi.org/10.1080/00071660500393868 wattanachant, s., benjakul, s., & ledward, d. (2004). composition, color, and texture of thai indigenous and broiler chicken muscles. poultry science, 83, 123-128. https://doi.org/10.1093/ps/83.1.123 xiong, y., lou, x., wang, c., moody, w., & harmon, r. (2000). protein extraction from chicken myofibrils irrigated with various polyphosphate and nacl solutions. journal of food science, 65, 96-100. https://doi.org/10.1111/j.1365-2621.2000.tb15962.x zanetti, e., dalvit, c., molette, c., remignon, h., & cassandro, m. (2009). a proteomic approach to study local chicken breeds characterization. it j animal sci, 8. https://doi.org/10.4081/ijas.2009.s2.174 zanetti, e., masi, a., pivato, m., tolin, s., trentin, a. r., guler, c., … cassandro, m. (2013). a note on protein expression changes in chicken breast muscle in response to time in transit before slaughtering. proteome science, 11, 34. https://doi.org/10.1186/1477-5956-11-34 zhang, l., yue, h. y., zhang, h. j., xu, l., wu, s. g., yan, h. j., … qi, g. h. (2009). transport stress in broilers: i. blood metabolism, glycolytic potential, and meat quality. poult sci, 88. https://doi.org/10.3382/ps.2009-00128 zhang, z., yang, y., tang, x., chen, y., & you, y. (2015). chemical forces and water holding capacity study of heat-induced myofibrillar protein gel as affected by high pressure. food chemistry, 188, 111-118. https://doi.org/10.1016/j.foodchem.2015.04.129 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 1, no. 1, 2017 www.scholink.org/ojs/index.php/fsns 11 is hunger destined to be perpetual in burundi? lincoln j. fry1* 1 academic member, sociology research unit, athens institute for education and research (atiner), athens, greece * lincoln j. fry, e-mail: lincolnfry@bellsouth.net received: february 21, 2017 accepted: march 5, 2017 online published: march 15, 2017 doi:10.22158/fsns.v1n1p11 url: http://dx.doi.org/10.22158/fsns.v1n1p11 abstract hunger is a worldwide problem, and africa is the continent with the world’s highest percentage of hungry persons; burundi is africa’s hungriest country. this paper addresses hunger in burundi and then identifies the factors that predict hunger in that country. burundi is a rural country and its rural population will receive a great deal of attention in this paper, especially because the study looks closely at literature’s suggestion that farmers may be hungrier than the rest of the population, and gender may be a factor. this study is based on a national probability sample of 1,200 burundi respondents included in round 6 of the afrobarometer survey conducted in 2014. the search is for policy related factors that would help alleviate burundi’s hunger problem. to preview the findings, this study did not find any light at the end of the tunnel. the factors that predicted hunger were primarily immutable indicators, education, agriculture as an occupation, and wealth, as measured by assets owned. over 80 percent of the respondents felt the government was not ensuring that people had enough to eat. eighty-seven percent were unemployed, 86 percent were rural residents and 71 percent of the respondents reported some degree of hunger, about one-fourth reported being hungry all of the time. the gender and hunger relationship was significant at the bivariate level, but that relationship disappeared in the ordered logistical analysis. keywords burundi, hunger, rural, agriculture, farmers 1. introduction in 2014, burundi topped the global hunger index for the third year in a row. the country has been described as one of the least developed countries (jenicek & grofova, 2015) and the hungriest, not only in africa, but in the world (africaranking.com, 2015). a landlocked country in the african great lakes region, it is bordered by rwanda, tanzania and the democratic republic of the congo. two civil wars and genocides during the 1970s and again in the 1990s have left this predominantly rural country not only undeveloped and its population of roughly 10.5 million, one of the poorest in the world. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 12 published by scholink inc. against that backdrop, this paper assesses the extent of self-reported hunger among 1,200 burundi respondents and then searches for the factors that predict hunger in that country. even though burundi has been somewhat neglected, the literature devoted to what is commonly called food insecurity will be reviewed. some of the issues raised in the african food insecurities literature will be addressed and are questions central to this paper’s analysis. these include whether rural residents, especially agricultural workers, are hungrier than other burundi respondents. other topics include whether gender differences in hunger are apparent, and are there any implications in this research that add any knowledge about hidden hunger? as the title of the paper suggests, the big question will be is there any light at the end of the tunnel regarding hunger in burundi? 1.1 hunger in the world and africa according to the whes (world hunger education service) world hunger and poverty facts and statistics report (2015), hunger has three meanings. two of those meanings deal with craving or desire for food. the third meaning refers to the want or scarcity of food in a country, and it is in this sense that this paper addresses hunger. there are two classifications of hungry persons that are of interest here. the broadest classification includes those who suffer from what is known as “hidden hunger”. these are an estimated two billion persons that are affected by a chronic deficiency of essential vitamins and minerals. among this population the signs of malnutrition and hunger are less visible, but it has negative and long term consequences, often for long term health, productivity and cognitive development (muthayya et al., 2013). the second classification includes those who demonstrate clear cut hunger; in the latest un food and agriculture organization report (2015), the estimate was that 925 million people were hungry worldwide, and that 239 million people in sub-saharan africa were hungry or undernourished. this made africa the continent with the second largest number of hungry people, following asia and the pacific with 578 million. due to the difference in population sizes, sub-saharan africa actually had the largest proportion of hungry/undernourished people, estimated at 30 percent of the population compared to 16 percent for asia and the pacific. 1.2 food insecurity in sub-saharan africa as clover (2003) has suggested, despite the fact that the right to food is one of the most consistently acclaimed rights in international human rights law, no other human right has been so frequently and spectacularly violated. clover’s discussion of food insecurity in sub-saharan africa leads to the conclusion that hunger is a multi-faceted issue in africa, and that just growing more food will not eradicate the problem. agriculture is important and clover points out that africa has gone from being a key agricultural commodity exporter into being a net importer; the african continent now receives the most food aid. perhaps the most important point clover made was to suggest hunger will not be eradicated by just throwing money at the problem. hunger is a political creation which must be ended by political means, a theme which will be mentioned below and revisited in the discussion section. 1.3 hunger in burundi malnutrition is the 4th leading cause of death in burundi, and the 3rd leading cause of total deaths in the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 13 published by scholink inc. country. the country ranks 6th in the world in this category (worldlifeexpectancy.com, 2016), with over 60% of the country’s population deemed undernourished. according to jenicek and grofova (2015) they classify burundi as the world’s third poorest country, a country that is highly vulnerable to natural disasters which contribute further to nutritional instability. burundi has been afflicted by a wide range of challenges such as land shortage, land degradation, corruption, increased political instability and ethnic civil unrest (especially since 1990), and, hiv/aids. according to fauk et al. (2017), there are 610,000 children in burundi who are orphans. most because of aids, but many have been abandoned by their parents because of their dire economic situation. to make matters worse, even those with intact families are faced with poor access to education, which means that the country’s youth is challenged in the task of bringing about significant development in the near future. burundi is a rural country and its economy is based on agriculture. as jenicek and grofova (2015) noted, as of 2007, 90 percent of burundi’s population lived in rural areas. food crops occupy 85 percent of agricultural land and most crops are produced for the owner’s consumption. there is a shortage of agricultural land and there has been a fourfold increase in population and land holdings have been divided to accommodate the claims of sons for family land. the return of nearly 500.000 refugees has increased the pressure on land ownership. the un started intervening in 1993—first through efforts at peace-keeping, and then through reconstruction projects. today, 42% of burundi’s national income is from foreign aid. 1.4 hunger and farmers, climate change, and gender as sanchez and swaminathan (2005) indicated, roughly half, 50%, of the hungry are found in small holder farming households. another 20% are the landless rural and 10% are pastoralists, fishers and forest dwellers; the remaining 20 percent are urban residents. this paper will look at farmers, in order to determine if they are in fact hungrier than other burundi respondents. there are several issues that emerge from the rural hunger literature. the first is climate change shisanya and mafongoya (2016) who suggested that smallholder subsistence farmers will face severe negative impacts from climate change, with their household food security being seriously affected. this paper examines the extent to which farmers see climate change as an issue the government should address. the final issue addressed here is the way gender affects hunger in burundi, especially female farmers. as abebayo and adekunie (2016) have indicated, the division of labor is becoming blurred. many men have left the land to work in the towns or neighboring countries. also, hiv related diseases and deaths have had a major effect on the agricultural labor force. as a result, women sometimes comprise up to 80% of the adult rural population and are made to take on jobs that were traditionally done by men; farming is one focus in this paper. 1.5 the study: the research question the picture of burundi painted by jenicek and grofova (2015) is grim. they described an impoverished, over-populated country with limited resources that cannot overcome its hunger problem in the near future. they noted that in 2005, the real per capita gnp dropped to $105, which meant that www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 14 published by scholink inc. if that trend persisted, burundi would need 225 years to reduce its poverty by half. against that backdrop, this study looks at hunger in burundi and attempts to identify the factors that are related to hunger in present day burundi. as the title of this paper suggests, the search will be to determine whether there are any rays of hope for the hunger problem in burundi, or is hunger destined to be perpetual. several known rays of hope are currently in the process of development and will be covered below in the discussion section. 2. method the data: this study’s data source is the afrobarometer project. as recently described by fry (2017), it is a collaborative research effort formed in 1999 when three independent research projects merged; there were michigan state university, the institute for democracy in south africa and the center for democratic development. the project’s objectives are as follows: 1) to produce scientifically reliable data on public opinion in sub-saharan africa; 2) to strengthen institutional capacity for survey research in africa; and 3) to broadly disseminate and apply survey results. in 2000, afrobarometer joined other regional barometers to form the global barometer network; the following year, afrobarometer completed the round 1 survey. the project started with 12 countries in round 1, and by 2016 round 6 was completed, in 36 african countries. the project uses a standardized questionnaire, with new questions or country specific questions added by round. the individual country is the unit of analysis and sampling goal is to create national probability samples which represent cross sections of adult citizens, 18 years and older, for each country. sampling sizes are set at either 1,200 or 2,400 respondents, depending upon the country’s population size. the sampling procedures used in all of the afrobarometer surveys are explained in detail in bratton, mattes and gyimah-boadi (2005). 2.1 the dependent variable: hunger the study’s questionnaire included what is called the lived poverty index used in the afrobarometer studies which was adopted from mattes (2003). one of the five questions in the index asked “over the past year, how often, if ever, have you or anyone in your family gone without enough food to eat”. fixed responses to this question were: never, just once or twice, several times, many times, always. these responses were coded as follows: never = 1, just once or twice = 2 and many times and always = 3. these categories provide the basis for the ordered logistical analysis presented in the results section. 2.2 the independent variables the afrobarometer questionnaire does not ask respondents to report their income. as bratton (2008) indicated, this is because many citizens in poor countries operate in informal markets where cash transactions, including income, are unrecorded and difficult to measure. instead, this research used what is called an asset-based wealth index, a summed index created from four questions that ask about household assets. the survey asked respondents: “which of these things do you personally own: a radio? a television? a motor vehicle, car or motorcycle? a cell phone?” responses to these questions www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 15 published by scholink inc. were coded as binary. either (0 = don’t own; 1 = own), and these responses were used to create a summed index for this study. other control variables are listed in table 1 and were measured by a single item, like age, and others were collapsed into fewer categories. race and religion are not included in table 1 because over 99 percent of the respondents were classified as black africans and over 95 percent of the respondents reported that they were christians. education was reduced to four categories from nine by combining no school, informal only and then creating primary, high school. and post secondary categories. respondents were asked a series of work related questions, like their employment status. respondents were also asked to identify the most important problems faced by the country that the government should address. respondents were provided with two hypothetical questions which asked what would be their top and second priorities for additional investment if the country could increase spending. fixed responses were provided, which included education, infrastructure, security, healthcare, agriculture and development, energy supply or none of the above. the responses to these questions are also listed in table 1. note that race and religion are not included in table 1 because over 99 percent of the respondents were classified as black africans and over 95 percent of the respondents reported that they were christians. table 1. social and demographic characteristics of the burundi sample (n = 1,200) variable n (%) gender male 600 (50) female 600 (50) education no formal/informal schooling 456 (38) some/primary school completed 540 (45) some/completed high school 156 (13) postsecondary/college/graduation 46 (4) employment unemployed 1,038 (87) employed part time 38 (3) employed full time 124 (10) residence urban 168 (14) rural 1,032 (86) age 18 through 29 402 (34) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 16 published by scholink inc. 30 through 49 434 (36) 50 and over 364 (30) agricultural worker/occupation yes 818 (70) no 356 (30) asset-based wealth none of these 407 (34) radio 458 (38) radio and tv 241 (20) radio, tv and motor vehicle (car or motorcycle) 93 (8) table 1, shows this burundi sample was relatively young, with 70 percent under the age of 50. forty-five percent of the respondents have some attendance or have completed primary school, while 38 percent have not attended school or have received informal education only. thirteen percent attended some or completed high school and 4 percent of the sample have post-secondary education. only 10 percent of the sample was employed and 87 percent were unemployed. the sample was overwhelmingly rural, 86 percent and 70 percent listed their occupations as in agriculture, farming, forestry or fishing. in terms of the assets they owned, 38 percent indicated they only owned a radio, while 34 percent indicated they did not own any of the assets on the list. eight percent of the sample owned a radio, tv and a vehicle. 3. results the next task in the analysis was to identify the respondents self-reported level of hunger, how often they go with out basic necerssities (food) and perceptions of problems the government should address or where the government should direct funds if money was available. the responses to those items appear in table 2. table 2. self-reported hunger, lack of access to basic necessities (food), and perceptions of governmental priorities and possible investment (n = 1,200) variable n (%) hunger never 348 (29) sometimes 581 (48) always 271 (23) basic necessities (food) about once every two to three months 42 (4) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 17 published by scholink inc. once a month 105 (9) two or three times a month 165 (14) once a week 185 (16) several times a week 455 (38) everyday 158 (13) government ensuring everyone has enough to eat badly 971 (82) well 216 (18) respondent selections of the priorities government should address poverty/food shortage/famine 294 (9) farming and agriculture 266 (8) corruption 233 (7) health 220 (7) crime 215 (7) water supply 207 (6) unemployment 42 (4) management of the economy 88 (3) aids 7 (> 1) votes for top priority for additional government investment (count) agricultural development 649 healthcare 600 education 440 infrastructure 244 security 242 energy supply 182 none of the above 20 table 2 reveals that 71 percent of this burundi sample report some degree of hunger, with 23 percent indicating they are always hungry. thirty-eight percent reported being hungry several times a week. and 13 percent reported being hungry every day. poverty and destitution was the top choice as the priority the government should address, 15 percent, followed by food shortage, 9 percent, and farming and agriculture, 8 percent. agricultural development received the most votes regarding where the government should invest funds if money was available. healthcare was second, followed by education and infrastructure. the next task in the analysis was to cross-tabulate some of the study’s independent variables by hunger. these results appear in table 3. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 18 published by scholink inc. table 3. cross-tabulation hunger and selected independent variables (n = 1,200) hunger level none some a lot total p variable n (%) n (%) n (%) gender male 180 (30) 306 (51) 114 (19) 600 .01 female 168 (28) 275 (46) 157 (26) 600 education no formal/informal only 90 (20) 219 (48) 147 (32) 456 .000 some/primary school completed 150 (28) 283 (52) 107 (20) 540 some/completed high school 72 (46) 71 (45) 15 (9) 158 postsecondary/college/graduation 36 (78) 8 (17) 2 (4) 46 employment unemployed 288 (28) 504 (49) 246 (24) 1,038 .04 employed part time 17 (45) 15 (39) 6 (16) 38 employed full time 43 (35) 62 (50) 19 (15) 124 residence urban 83 (49) 61 (36) 24 (14) 168 .000 rural 265 (26) 520 (50) 247 (24) 1,032 agricultural worker/occupation yes 191 (23) 397 (49) 230 (28) 518 .000 no 145 (41) 175 (49) 36 (10) 356 asset-based wealth none of these 87 (21) 190 (47) 130 (32) 407 .000 radio 124 (27) 229 (50) 105 (23) 458 radio and tv 71 (29) 141 (59) 29 (12) 241 radio, tv and motor vehicle 66 (71) 20 (22) 7 (9) 93 age 18 through 29 118 (29) 196 (49) 88 (22) 402 .71 30 thru 49 134 (31) 203 (47) 97 (22) 434 50 and over 96 (26) 182 (50) 86 (24) 364 government ensuring everyone has enough to eat badly 282 (29) 475 (49) 214 (22) 971 .65 good 64 (30) 99 (46) 53 (24) 216 table 3 shows that most of the variables included in table 3 were statistically significant. the two www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 19 published by scholink inc. exceptions were age and respondent perceptions regarding whether the government was ensuring that people had enough to eat. some other variables included in table 3 were highly significant. these included education, residence, agricultural work as an occupation and the asset based wealth index. all at p = .000. gender at p = .01 and employment status, p = .04, were also significant, but to a lesser degree. the final task in the analysis was to conduct an ordered logistical regression analysis to determine which variables predicted hunger in burundi. an ordered logistical model was appropriate because the study had a categorical dependent variable. the statistical program used for all of the analysis presented in this paper was stata, and long and freese (2006) discuss the use of regression models for categorical dependent variables when using stata. the results of this study’s ordered logistical analysis appear in table 4. table 4. logistic regression with self-reported hunger as the dependent variable variable coefficient standard error z p urban-rural -.13 .21 .61 .54 gender .12 1.31 .19 employment status .06 .19 .59 .55 total assets -.29 .11 -2.63 .01 education -.42 .10 4.27 .00 agriculture worker .48 .15 3.11 .00 age -.10 .08 -1.28 .20 government doing enough .64 .28 2.27 .02 invest agriculture .04 .03 1.50 .13 number of observations = 1,097 lr chi2(12) = 164.29 prob > chi2 = 0.0000 pseudo r2 = 0.07 table 4 shows that 4 variables reached significance in the regression equation. in order of their strength, these were education, agricultural worker as the respondent’s occupation, the asset based wealth indicator, and whether respondents thought the government was ensuring that people had enough to eat. perhaps what is most interesting are those variables that were expected to be significant and were not. these include gender and the rural-urban dimension, which the literature suggested were significant predictors of hunger. perhaps this can be explained by the significance of agriculture as an occupation. another significant variable identified above and not included in the regression analysis was the basic necessity indicator, and that was because the items multicollinearity with hunger. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 20 published by scholink inc. 4. discussion another issue which disappeared in the regression analysis was the need to invest in agriculture, which appears to be a major solution to the world food and hunger crisis found in the recent literature (fan & rosegrant, 2016). the thinking is that increased agricultural growth will play a major role in addressing the world food crisis and the major stumbling block will be the cost. there are grass roots approaches to the hunger problem in burundi noted in the literature and one will be mentioned here. this may be defined as taking advantage of what already exists in the environment. the example is provided by akinnifesi et al. (2006), who noted that among the consequences of most countries in southern africa experiencing acute malnutrition, food insecurity, and poverty among both rural and urban populations is deforestation and loss of biodiversity. it has been recognized what are known as the miombo woodlands are in danger, an area which includes southern burundi. this forest area is known to have over 75 indigenous fruit trees (ifts), which bear edible fruits. these fruits are rich in minerals and vitamins, can be sold for cash income and are an important food source during emergencies. akinnifesi et al. provide an overview of some efforts to domesticate the ifts identified by farmers and users as priority species, which is as an important step to providing opportunities for resource-poor farmers to cultivate and generate income from the sale of fresh and processed products. the approach used involves four basic steps: 1) identification of priority species by communities and other users, 2) participatory selection of superior trees and naming them in situ, 3) propogation and cultivation of trees as fruit orchards, and 4) dissemination and adoption. to this point, over 5000 farmers in four countries are involved in on-farm testing of ifts in the field and homesteads. this example points to the need to create an enabling environment, and demonstrates that policy reforms and market development will be necessary to achieve socioeconomic empowerment of the resource poor farmers in the region through domestication, utilization and commercialization of fruits and other agricultural commodities, which in turn stresses the need for product development research, private sector involvement and strong policy support, in order for other similar projects to have tangible impact. as far as can be determined, this is the only published study that has assessed hunger in burundi through individual level survey methods, yet, the results presented here are consistent with other assessments of hunger in burundi. for example, the borgenproject (2014) reported that the rates of malnutrition have increased and burundi is only one of four nations that has seen an increase in ghi (global hunger index) from 1990 to 2016, indicating a worsening of the food situation in the country. the picture presented here and elsewhere suggest that the prospects for the future of hunger looks dismal for burundi. at first glance, the fact that education was the first predictor to emerge from the regression analysis might seem favorable. in fact, burundi has been identified as the african country least able to retain its top talent (mail guardian, 2015). this comes about because the pursuit of opportunities outside the country is called a feature of working life, and burundi was ranked number one in terms of the country where the best and brightest leave for opportunities in other countries and at www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 21 published by scholink inc. the bottom of the list of african countries able to attract top talent. the country cannot create more farm land, and the remaining forest land must be and will be protected; the example provided by the miombo woodlands makes that case. in conclusion, the answer to the question which generated this paper is that hunger will be or remain perpetual in burundi into the foreseeable future. seventy-one percent of the respondents to this survey reported some degree of hunger, with about one-fourth, 23 percent, reporting that they are hungry all the time. about 87 percent of the respondents in this study were unemployed and 86 percent listed some form of agriculture as their occupation. the surprising finding was that, while significant at the bivariate level, gender was not significant in the regression analysis, suggesting that everyone is hungry in burundi, women and men, and this study suggests there is no apparent improvement on the horizon. references adebayo, s. a., & adekunle, o. a. (2016). socio-economic status of women in group membership in selected areas of kwara state, nigeria. agrosearch, 16(1), 57-64. https://doi.org/10.4314/agrosh.v16i1.5 akinnifesi, f., kwesiga, j., mhango, t., chilanga, a., mkonda, c., & kadzere, k. i. (2006). towards the development of miombo fruit trees as commercial tree crops in southern africa. forests, trees and livelihoods, 16(1), 103-121. https://doi.org/10.1080/14728028.2006.9752548 borgenproject. (2014). malnutrition in burundi. retrieved from https://www.borgenproject.org bratton, m., robert, b. m., & emmanuel, g.-b. (2005). public opinion, democracy, and market reform in africa. cambridge university press. clover, j. (2003). food security in sub-saharan africa: feature. african security review, 12(1), 5-15. https://doi.org/10.1080/10246029.2003.9627566 fan, s., & rosegrant, m. (2016). investing in agriculture to overcome the world food crisis and reduce poverty and hunger. washington, dc: ifpri (international food policy research institute). retrieved from https://www.vtechworks.lib.vt.edu fauk, n., mwakinyali, s., sukma, p., & mwanri, l. (2017). understanding the strategies employed to cope with increased numbers of aids-orphaned children in families in rural settings: a case of mbeya rural district, tanzania. infectious diseases of poverty, 6(1). https://doi.org/10.1186/s40249-016-0233-7 fry, l. (2017). the value of publicly available data sets for social science research and evaluation. international journal of current advanced research, 6(1), 1777-1783. jeníček, v., & grofová, s. (2015). least developed countries-the case of burundi. agric. econ.-czech, 61, 234-247. https://doi.org/10.17221/48/2014-agricecon long, j., & freese, j. (2006). regression models for categorical dependent variables using stata. stata press. mail guardian. (2015). brain drain in africa: burundi least able to retain its top talent, rwanda, kenya www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 1, no. 1, 2017 22 published by scholink inc. top jammeh’s gambia surprises. retrieved from http:://www.mgafrica.com/article.2015-08-28 mattes, r., & gyimah-boadi, e. (2005). public opinion, democracy, and market reform in africa. cambridge: cambridge university press. mattes, r., bratton, m. & davids, y. (2003). poverty, survival, and democracy in southern africa. afrobarometer working paper. muthayya, s., jee, h., sugimoto, j. roos, f., kraemer, h., & black, r. (2013). the global hidden hunger indices and maps: an advocacy tool for action. plos one, 8(6), e67860. https://doi.org/10.1371/journal.pone.0067860 notes, hunger. (2011). world hunger and poverty facts and statistics. washington, dc: world hunger education service. poor people and democratic citizenship in africa. (2008). in a. krishna (ed.), poverty, participation and democracy. new york: cambridge university. sanchez, p. a., & swaminathan, m. s. (2005). hunger in africa: the link between unhealthy people and unhealthy soils. the lancet, 365(9457), 442. https://doi.org/10.1016/s0140-6736(05)70241-5 shisanya, s., & mafongoya, p. (2016). adaptation to climate change and the impacts on household food security among rural farmers in umzinyathi district of kwazulu-natal, south africa. food security, 8(3), 597-608. https://doi.org/10.1007/s12571-016-0569-7 the material and political bases of lived poverty in africa: insights from the afrobarometer. (2008). in barometers of quality of life around the globe (pp. 161-185). springer netherlands. tilman, d., & clark. (2015). food, agriculture & the environment: can we feed the world & save the earth? daedalus, 144(4), 8-23. https://doi.org/10.1162/daed_a_00350 world health rankings: burundi. (2013). retrieved from http://www.worldlife expectancy.com/burundi worldlifeexpectancy, burundi. (n.d.). retrieved from http://www.worldlifeexpectancy.com/burundi-life-expectancy food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 4, 2019 www.scholink.org/ojs/index.php/fsns 160 original paper some physiological responses of clarias gariepinus fed graded levels of cirina forda larvae based diets adewumi, a.a.1*, idowu, e.o.1, edward, j.b.1, iwalaye, o.a.1, fadiya, o.o.2 & obafemi, b.d.2 1 department of zoology and environ. biology, ekiti state university, ado-ekiti, nigeria 2 department of zoology, university of ibadan, nigeria * adewumi a.a., department of zoology and environ. biology, ekiti state university, ado-ekiti, nigeria received: september 25, 2019 accepted: october 8, 2019 online published: november 19, 2019 doi:10.22158/fsns.v3n4p160 url: http://dx.doi.org/10.22158/fsns.v3n4p160 abstract the study examined the growth performance, hematological and digestive enzymes of clarias gariepinus juveniles fed cirina forda meal (cfm) based diets in the laboratory for a period of 10weeks. five iso-nitrogenous (30%) experimental diets were formulated at various levels of cfm inclusion levels of 0% (control), 10%, 20%, 30%, 40% and 50%, designated as diets q, a10, b20, c30, d40 and e50 respectively. fish fed the cfm based diets showed mean weight gain (mwg), specific growth rate (sgr) and protein efficiency ratio (per) comparable to the control diet. there was significant differences in the digestive enzyme activities of the fish as the cfm level in the experimental diets increased. protease and maltase activities significantly increased, with diet c30 recording the highest maltase activity (4.37) while the cellulase and glucanase activities of the fish significantly (p<0.05) reduced. there was no significant difference (p>0.05) between the pcv and rbc of the blood of the fish fed the various diets. highest rbc (2.75 x106 /µl) was obtained in fish fed the control diet while the lowest (2.55 x106 /µl) was recorded in the fish fed diet e50. the white blood cell count and the neutrophils of the fish fed the trial diets were not significantly higher (p>0.05) than those of the fish fed the control diets. from the results of this experiment, it is concluded that up to 50% inclusion level of cirina forda meal in the diet of clarias gariepinus was tolerable for good growth and physiological well-being of the fish. keywords clarias, growth, haematology, digestive enzymes www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 161 published by scholink inc. 1. introduction fish culture is one of the fastest growing sectors of the world’s animal production with an annual increase of about 10% (fao, 2010). the african catfish; clarias gariepinus, is the most sought after species among fish farmers and consumers because it commands good commercial value, not only in nigeria, but all over africa. the growth of aquaculture in nigeria is now largely being boosted by a steady rise in catfish culture. to sustain such high rates of increase in production, a matching increase in fish feed production is imperative. the high cost and fluctuating quality as well as the uncertain availability of fish meal have led to the need to identify alternative protein sources for fish feed formulation. therefore, in order to attain more economically, sustainable, environmentally friendly and viable production, research interest has been directed towards the evaluation and use of non-conventional sources of protein. the edible larvae of cirina forda insect (figure 1) has a wide acceptability as a food source, and also serves as an important item of commerce in such nigerian states like oyo, kwara, kogi, niger and kaduna, where it has become the most important and marketable insect (ande, 1991; fasoranti & ajiboye, 1993). osasona and olaofe (2010) analyzed the insect’s larvae as composed of digestible protein (45.10%) (table 1), fats (18.03%) and small, but significant amount of carbohydrate, minerals, vitamins and polyunsaturated fatty acids. table 2 contains the reports of akinnawo and ketiku (2000) and omotoso on the mineral composition of c. forda. apart from being a widely acceptable food source, a number of factors are known to enhance the availability of the larvae of the insect. such factors include its capability for artificial rearing, the short-lived larval stage, and the high conversion rate (ande, 1991). these factors make it a resourceful replacer for fish meal in animal diets (oyegoke et al., 2006). omotoso (2006), ifie and emeruwa (2011) reported that cirina forda contains less than 0.005% oxalate. omotoso observed that the anti-nutrient composition (mg/100 g) of c. forda larva to include phytic acid (1.02±0.00) and oxalate (4.11±0.05). he however commented that these values are lower than those reported in some proteinous foods. this amount is under the tolerance limit because much higher amounts have been observed in various plant food materials (kalita et al., 2007). vijayakumari et al. (1997) observed that 513 mg of phytic acid was present in 100 g of p. chilensis, a consumable legume, hitherto reported by de lumen et al. (1986), to be very rich in methionine and cysteine. enzymes are protein in nature and comprise biological molecules which are involved in metabolic processes in living organisms. the digestion and absorption of nutrients are mostly dependent on enzyme activities involved in breakdown and assimilation of food (klein et al., 2006). therefore, analysis of enzyme activities is a convenient and reliable technique that can provide comprehensive information relating to digestive physiology and nutritional conditions in the fish (bolasina et al., 2006). digestive enzyme activities in fishes are associated with feeding ecology and composition of diet (fernandez et al., 2011). digestive enzymes activity influence feed utilization by fish, and its understanding is important to optimize diet formulation. feed nutrients must be digested for their www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 162 published by scholink inc. utilization, and pancreatic digestive enzymes have essential roles for the digestion; trypsin and chymotrypsin are the main pancreatic proteases, lipase is the major pancreatic lipolytic enzyme, and amylase is known as the major pancreatic digestive enzyme for carbohydrates (murashita et al., 2015). in general, herbivorous fish species possess greater carbohydrate enzyme activity, while carnivorous fish species exhibit higher proteolytic enzyme activity (hidalgo et al., 1999). the digestion and absorption of nutrients are mostly dependent on enzyme activities involved in breakdown and assimilation of food (klein et al., 2006). the digestion and metabolism of carbohydrates (and other feed ingredients) is dependent on fish species and on the source, inclusion level and treatment of the ingredient (krogdahl et al., 2005; stone, 2003). knowledge of the capacity of a fish to utilize the nutrients in the diet is an essential pre-requisite for appropriate formulation of fish feed (wilson, 1994). blood analysis is a valuable means of evaluating the physiological condition of cultured fish with respect to the effect of diets and other stress factors on fish health. changes in haematology of fish in response to stressing agents are indicators of the stressful stage of fish, producing useful information to curb any unfavourable condition that may affect the fish health (fagbenro et al., 2010). this research is being conducted to evaluate the effects of cirina forda pupae replacement for fish meal in the diet, on the growth, digestive enzymes and haematological parameters of clarias gariepinus. figure 1. dried whole cirna forda pupae www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 163 published by scholink inc. table 1. proximate composition (% dm -1 ) and energy value of cirina forda pupae components percentage (%) this work akinnawo and ketiku (2000) oso and ola-oladimeji (2016) adepoju and daboh (2013) omotoso (2006) moisture 6.02 5.25 10.85 fibre 9.40 7.69 ash content 6.62 7.12 6.49 2.6 10.26 protein 52.34 33.12 45.10 52.6 55.50 fat 17.56 12.24 18.03 16.8 4.68 carbohydrates (by difference) 17.36 38.12 17.44 18.70 energy (kcal) 359.00 458.4 table 2. mineral composition (mg/100g) c. forda pupae minerals omotoso (2006) akinnawo and ketiku (2000) calcium 33.16 7.0 potassium 64.02 2130 magnesium 62.31 32.4 phosphorus 215.54 1090 sodium 45.26 210 iron 5.34 64 zinc 3.81 8.6 manganese 1.14 7.0 copper; cobalt; lead; chromium; nickel not available source: akinnawo o. and ketiku, a.o. (2000) and omotoso (2006). 2. materials and method 2.1 experimental site the experiment was carried out at the aquaculture centre of the department of zoology and environmental biology, ekiti state university, ado-ekiti. 2.2 procurement of the experimental fish and feedstuffs one hundred and eighty juveniles (31.01± 0.23 g) of clarias gariepinus were purchased at adebayo fish hatchery in ado-ekiti, ekiti state. the fish were acclimatized for two weeks being fed with a commercial feed (coppen’s feed). after acclimatization, the fish were randomly distributed into well labeled aquarium tanks in triplicates per treatment. 2.3 the preparation of experimental feed dry cirina forda pupae was purchased from oja-oba (king’s market) in ado-ekiti, ekiti state. the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 164 published by scholink inc. cirina forda was sundried and milled into fine powder meal, tagged cfm. other feed ingredients used for the diet formulation include; fish meal, maize, lysine, methionine, wheat bran, vitamin premix, groundnut oil and salt purchased from afe babalola university, ado-ekiti (abuad), ekiti state, while the groundnut oil and salt were obtained from oja-oba. calculated amounts of each feed ingredient were weighed separately using pearson’s square method of feed formulation. fishmeal was replaced at 0% (control diet), 20%, 40%, 60%, 80% and 100% respectively and the treatments were tagged diets q, a20, b40, c60, d80, and e100 respectively. the various ingredients were properly mixed together and pelleted to a particulate size using a 2mm pellet disc. mixing and pelleting was done at the federal university of technology, akure (futa), in ondo state. the pelleted feeds were sundried for a week and kept inside labeled polythene bags till required. the feed formulation is as shown in table 3. table 3. proximate composition (g 100 -1 dm) of the experimental diets ingredient q a20 b40 c60 d80 e100 fish meal 43.10 38.79 34.48 30.15 25.86 21.55 cirina forda 4.31 8.62 12.93 17.24 21.55 maize 51.40 51.40 51.40 51.40 51.40 51.40 wheat bran 2.50 2.50 2.50 2.50 2.50 2.50 lysine 0.50 0.50 0.50 0.50 0.50 0.50 methonine 0.50 0.50 0.50 0.50 0.50 0.50 groundnut oil 0.50 0.50 0.50 0.50 0.50 0.50 vitamin premix* 1.00 1.00 1.00 1.00 1.00 1.00 total 100 100 100 100 100 100 *contains vit. a 4000000 iu, vit. d: 800000 iu, vit. e: 40000 mg, vit. k: 3800 mg, vit. b1: 1000 mg, vit. b2: 6000 mg, vit. b6: 5000 m, vit. b12: 25 mg, niacin: 6000 mg, patothenic acid: 20000 mg, folic acid: 200 mg, folic acid: 200 mg, biotin: 8 mg, manganese: 300000 mg, iron: 80000 mg, zinc: 20000 mg, cobalt: 80 mg, iodine: 400 mg, selenium: 40 mg and choline: 800000 mg. 2.4 fish sampling three fish samples were taken randomly from each bowl. the initial individual weight and length of these were measured using the weighing scale and calibrated ruler, respectively. triplicate samples of the fish from each bowl were weighed bi-weekly until the experiment was completed. the bi-weekly weighing allowed the adjustment of feeding levels for the subsequent weeks. unconsumed feeds and faecal matters were siphoned off every other day. 2.5 growth performance and nutrient utilization parameters using the weight data and the quantity of feed fed, the growth response and nutrient utilization parameters were determined. mean weight gain (mwg), relative weight and specific growth rate www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 165 published by scholink inc. (sgr), percentage weight gain (pwg), feed conversion ratio (fcr), specific growth rate (sgr) were calculated, using the following formulae; sgr= inw2-inw1 x 100 t-t where, w1=initial weight (gram) at time t w2= final weight (gram) at time t fcr= weight of food consumed per fortnight weight gained by fish per fortnight mwg= mean final body weight (g)-mean initial body weight (g) survival rate (sr) = total fish number harvested total fish number stocked relative weight gained (rwg) rwg = w2-w1 x 100 w1 pwg= wt-w0 x 100 where, w0 = weight at initial wt = weight at time t. 2.6 biochemical analysis the proximate compositions of the various diets (table 4) and the carcass of the flesh of the fish were carried out in the laboratory using the methods of aoac (2006). table 4. the proximate composition (% dm -1 ) of the various diets parameters q a a20 b40 c60 d80 e100 ash 9.51a 9.36a 9.91a 9.21a 8.29ab 7.45ab moisture 12.26a 12.64a 12.03a 11.82a 12.35a 12.65a carbohydrate 35.07a 43.16b 39.81b 37.75b 37.26b 42.55b crude fiber 0.90a 1.20a 0.20b 0.21b 0.47b 0.31b crude protein 40.06a 39.63a 38.77a 41.81a 39.69a 39.75a fat 0.20a 0.20a 1.20ab 2.10b 4.01b 2.30b www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 166 published by scholink inc. table 5. the proximate composition (% dm -1 ) of the carcass of c. gariepinus fed with the various diets parameters q a20 b40 c60 d80 e100 ash 12.77ab 15.67a 13.55a 14.06a 13.77a 13.91a moisture 9.45b 11.98a 12.03a 12.34a 12.73a 11.69a crude protein 73.44a 75.10a 71.78a 72.33a 69.23ab 68.21ab fat 8.26a 7.78a 7.39a 7.55a 6.97a 6.89a 2.7 enzyme analysis 2.7.1 amylase assay amylase activity was assayed by the method of negi and banerjee (2010). 0.5 ml of properly diluted enzyme was added into a tube containing 1.5 ml of 2 % (w/v) of potato starch solution and 1 ml of 0.05 m acetate buffer, ph 5.0. the reaction mixture was incubated at 40°c for 15 min. then, 1 ml of the mixture was transferred to a new tube containing 1 ml of 3,5-dinitrosalicylic acid and kept in boiled water for 10 min. the color density was determined spectrophotometrically at 520 nm. one unit was defined as 1 μmol of glucose released per minute by 1 ml of enzyme. 2.7.2 sucrase assay 0.5 ml of properly diluted enzyme was added into a tube containing 1.5 ml of 2 % (w/v) of sucrose solution and 1 ml of 0.05 m acetate buffer, ph 5.0. the reaction mixture was incubated at 40°c for 15 min. then, 1 ml of the mixture was transferred to a new tube containing 1 ml of 3,5-dinitrosalicylic acid and kept in boiled water for 10 min. the color density was determined spectrophotometrically at 520 nm. 2.7.3 maltase assay 0.5 ml of properly diluted enzyme was added into a tube containing 1.5 ml of 2 % (w/v) of maltose solution and 1 ml of 0.05 m acetate buffer, ph 5.0. the reaction mixture was incubated at 40°c for 15 min. then, 1 ml of the mixture was transferred to a new tube containing 1 ml of 3,5-dinitrosalicylic acid and kept in boiled water for 10 min. the color density was determined spectrophotometrically at 520 nm. 2.7.4 glucanase assay glucanase was assayed by incubating 500 ul of 5.0% laminarin in 50 mm acetate buffer ph 4.8 with 200 ul enzyme solution at 45°c for 30 min and determination of reducing sugars with dnsa (danielson et al., 2010). the amount of reducing sugars was calculated as mmol of glucose per min per ml. 2.7.5 cellulase assay cellulase was measured according to ghose (1987). a 900 ul of 1% cmc solution was added to 100 ul enzyme solution in a test tube. 1.5 ml dns reagent was added and incubated at 50°c in water bath www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 167 published by scholink inc. for 30 min. the absorbance was measured at 540 nm. glucose standard graph was prepared from 0-500ug glucose. one unit of cellulase activity was defined as the amount of enzyme that liberates 1 micromole of reducing sugars equivalent to glucose per minute under the assay conditions. 2.8 haematological analysis blood samples of a set of three fish were collected, at the beginning of the feeding trial (week 0) and at the end of trial (week 10), following the procedure described by stockopf (1993) and joshi et al. (2000a). two ml of the blood sample from each fish was collected by cardiac puncture with 2 ml syringe and needle and put in ethylene-diamine tetra-acetic acid (edta) treated bijou bottles. the blood was stored at -40°c prior to analysis. analysis includes the direct measurement of erythrocyte values: haemoglobin (hb), estimated by cyanomethemglobin method, red blood cells (rbc) and white blood cell (wbc) counted by neubauer’s improved haemocytometer using hyem’s and turks solution as a diluting fluid respectively. 2.9 statistical analysis growth performance, nutrient utilization parameters, haematological parameters and proximate composition data were analyzed using one-way analysis of variance (anova). significant differences among means were determined using duncan’s multiple range test (dmrt) on spss 15.0. 3. results 3.1 growth performance table 5 shows the growth performance and feed utilization of clarias gariepinus fingerlings fed the varying diets. even though the initial weight of the fish used for this trial were not significantly different from one another (p>0.05), the final weight gain of the fish fed diets b20,c30,d40,e50 were significantly lower (p>0.05) than that of the control. the mwg of the fish fed diets a10 and b20 were high, comparable to the control diet, but not significantly higher (p>0.05) than the other experimental diets. the fish fed a10 had the highest mwg (21.50), while the fish fed e50 had the lowest (21.28). fish fed diet c30 had the highest percentage weight gain while the lowest was recorded for the fish fed diet e50. the sgr of fish fed the experimental diets were not significantly different (p>0.05) from one another nor from the control, but the highest value (0.75) was recorded for the fish fed control diet, while the least (0.47) was recorded for the fish fed diets b20 and d40. the fcr of the fish fed the experimental diets were not significantly different (p>0.05) from one another, but significantly lower (p<0.05) than that of fish fed the control diet. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 168 published by scholink inc. table 6. growth performance of clarias gariepinus fed with the experimental diets parameters q a10 b20 c30 d40 e50 initial weight (g) 30.49a 30.42a 30.42a 30.47a 30.50a 30.49a final weight(g) 52.55b 51.92ab 51.89a 51.82a 51.79a 51.77a mean weight gain(g) 21.46ab 21.50ab 21.47ab 21.35a 21.29a 21.28a % weight gain(g) 72.35a 70.68a 70.58a 70.07a 69.80a 69.79a sgr 0.75a 0.50a 0.47a 0.49a 0.47a 0.48a fcr 1.39a 2.34b 2.21b 2.59b 2.52b 2.48b per 1.45a 1.43a 1.39ab 1.42a 1.39ab 1.38ab note. suffixes of different letters indicates statistical significant difference among means and same letters in column indicate no significant difference (p>0.05). per = protein efficiency ratio, fcr = feed conversion ratio, sgr = specific growth rate. 3.2 enzyme activity table 4 shows the enzyme activity in the gut of the experimental fish before and after the experiment. there was no significant difference (p>0.05) between the initial amylase enzyme activity and that of the control. however, the amylase activity of the fish fed the trial diets were significantly (p<0.05) higher than the initial and those of the fish fed the control diet. for cellulase, there was significant difference (p<0.05) between the initial and the control. there was significant reduction (p<0.05) in the cellulase activity of the fish as the cfm level in the experimental diets increased. maltase activity significantly (p<0.05) increased as the fish were fed high level of cfm based diets, with diet c30 recording the highest maltase activity (4.37). the glucanase activity of the fish significantly (p<0.05) reduced in the fish fed the experimental diets when compared with the initial, but when compared with the control, there was significantly higher (p<0.05) glucanase activity of the fish fed the trial diets. when compared with the initial, protease activity of the fish fed the trial diets increased as the fish was fed both the control diet and cfm based diets. the highest protease activity (4.34) was recorded in diet c30. table 7. digestive gut enzyme activities of the experimental fish enzyme initial q a10 b20 c30 d40 e50 amylase 4.18a 3.78a 3.25a 3.72a 4.25b 4.26b 4.28b cellulase 3.17a 0.73b 0.62bc 0.73ab 0.58c 0.69ab 0.65b glucanase 1.79a 0.77bc 1.00b 0.67c 0.52c 0.93b 0.90b maltase 1.22a 1.88ab 3.26b 3.33b 4.37b 2.94bc 3.95b protease 0.59a 2.56b 3.77c 4.02c 4.34c 3.67c 4.17c note. means with the same letters in rows are not significantly different (p>0.05). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 169 published by scholink inc. 3.3 haematological parameters the haematological parameters of c. gariepinus fed the control diet and the various c. forda fortified diets are presented in table 5. the pcv of fish fed the control diet was not significantly higher (p>0.05) than the fish fed the c. forda diets, but diet e50 recorded the least pcv level. there was no definite pattern in the hb of the blood of the variously fed fish but the hb of the fish fed the control diet was significantly higher (p<0.05) than those of the fish fed the experimental diets. there was no significant difference (p>0.05) between the rbc of the blood of the fish fed the control and the experimental diets. however, the highest rbc (2.75 x106 /µl) was obtained in fish fed the control diet and the lowest (2.55 x106 /µl) in the fish fed diet e50. the white blood cell (wbc) count and neutrophils of the fish fed the experimental diets were not significantly different (p>0.05) from those of the fish fed control diets. the lowest wbc (7.81 x106 /µl) was recorded in fish fed the control diet and highest (9.03 x106 /µl) in fish fed diet e50. neutrophils recorded was highest (70.21 x106 /µl) in fish fed diet a50 and the least (56.17 x106 /µl) obtained in diet d10. table 8. haematological parameters of the fish fed the various diets parameters experimental diets q a10 b20 c30 d40 e50 pcv (%) 26.64a 25.33a 24.33a 24.51a 24.44a 24.00b hb (g/100ml) 8.51a 7.37ab 8.01a 8.33a 7.31ab 7.55ab rbc (x10 6 /µl) 2.75a 2.65a 2.61a 2.65a 2.61a 2.55ab wbc (x10 6 /µl) 7.81a 7.69a 8.40ab 8.74ab 8.82ab 9.03ab neutrophil ( x10 6 /µl) 56.33a 56.17a 59.00a 60.33ab 63.13ab 63.27ab mean ± s.e with different superscript are significantly different from each other (p<0.05) hb = haemoglobin; pcv = pack cell volume; wbc = white blood cell; rb = red blood cell. 4. discussion in the present study, fish fed the experimental diets showed increase in weight without an external sign of nutritional deficiency because growth performances of fish fed cirina forda diet, at various levels up to 50% replacement, improved in terms of weight gain, percentage weight gain and specific growth rate. the fish showed good appetite to all the diets as attested to by the increase in body weight. this shows that cirina forda contained some of the necessary growth factors required by clarias gariepinus. this might also be due to good digestibility of the diet. the experimental fish showed great increases in weight, which indicates that the fish was able to convert the feed protein to extra muscles. weight gain and species growth rate are usually considered as the most important measurement of productivity of diets (adesina et al., 2013). fish fed cirina forda meal showed high specific growth rates comparable to the fishmeal based diet. this result agreed with the findings of oyegoke et al. (2006), who reported www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 170 published by scholink inc. that there were no significant differences between the growth performances of broiler chicks fed the compounded cirina forda larvae and those fed the conventional fishmeal. the compounded larval diets contained a crude protein level which is comparable and even higher in quality to that present in the conventional fishmeal. cirina forda larvae, in dried form, had been confirmed to contain 57.96% crude protein (ande, 1991). kodondi et al. (1987) have also analyzed three species of saturniid caterpillars (larvae) prepared by the traditional techniques of smoking and drying and found them to be high in riboflavin and niacin. cirina forda was able to cause higher growth rate compared to that observed in the fishmeal probably because of its high protein content and presence of essential minerals and vitamins like sodium, potassium, zinc and manganese as reported by ande (1991). keshavanath et al. (2002) reported better utilization of protein from low protein-high carbohydrate diets by common carp grown in manured tanks. adepoju and daboh (2013) further reported that the trypsin inhibitor level of c. forda was very low and cannot cause protein malabsorption. the positive growth response of clarias to cirna forda diet could also be attributed to its high gross energy (458.40), crude protein (45.10-55.50), lipid (16.82-18.80) and acceptability by the fish. adepoju and daboh (2013) observed that addition of c. forda at 5, 10, and 15% levels to fermented sorghum and maize flours significantly increased both microand macronutrients of the complementary foods and the nutrient density increased with the inclusion level. high digestive enzyme activity in fish receiving the test diets would have resulted in better utilization of diets, leading to higher growth. high protease activity in the fish fed the control diet and test diets when compared to the initial value, indicates efficient utilization of protein from all the diets. this is also reflected by the per value. however, different researchers have shown different results for protease activity. lopez-lopez et al. (2005) reported that there is no strong correlation between protease activity and dietary crude protein. according to le moullac et al. (1994) and krogdahl et al. (1999) the quantity of protease and amylase enzyme fluctuate with variation in concentration of carbohydrate and protein in fed diets. however, if concentration of these components increases beyond limits, concentration of amylase and protease start to decrease (cara et al., 2003). in this experiment, as also reported by haider et al. (2018), all treatment diets were iso-nitrogenous so there was no significant difference in protease activity. kikuchi (1999) reported that fish usually use less carbohydrate, demanding higher protein levels in the feeds. fish species differ greatly in their ability to digest carbohydrates. de almeida et al. (2006) reported that digestive functions capable of hydrolyzing a greater variety of carbohydrate-containing feedstuffs have been developed in herbivorous and omnivorous fish, in contrast to carnivorous fish. lower amylase levels may be indicative of the limited potential of fish to exploit diets containing high carbohydrate levels. measuring the activity of digestive enzymes is not enough to determine the value of a specific fish feed, as enzymes act in combination with feed composition, thus when associated with metabolic parameters they are a more reliable indicator of the fish nutritional status (lundstedt et al., 2004). digestive enzyme www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 171 published by scholink inc. responses can also be influenced by the feeding period, as changes in protein synthesis and enzyme activity in fishes can be observed after a long feeding period (krogdahl et al., 1994; lópez et al., 1999). an increase in white blood cells (wbc) and lymphocyte count (lymph) is usually associated with microbial infection or the presence of foreign body or antigen in the circulating system (bello, 2013). a measurable increase in white blood cells and neutrophil counts of fish or any animal is a function of immunity and animals’ resistance to some vulnerable illness or disease (akinwande et al., 2004). this increase might indicate that the fish under study had high immunity or resistance to diseases. from the results of this feeding trial, it can be asserted that c. forda can serve as a good source of nutrients in formulating nutrient-rich feed for fish. fish farmers can utilize the advantages of the insect’s availability and nutrient potentials in enhancing the productivity of clarias gariepinus at reduced cost of production. 5. conclusion this feeding trial revealed that up to 50% of cirina forda meal inclusion level in the diet of clarias gariepinus was utilized efficiently for good growth and physiological performances. this indicates, that cirina forda meal if thermo-treated to reduce anti-nutrition factor such tannin, as in the dried form, could replace fishmeal up to 50% in the fish feed composition without physiological distress. this level of inclusion would be significant replacement for the expensive fishmeal in feed formulation, since cirina forda meal is an animal resource with less competition for it’s use. references adepoju, o. t., & daboh, o. o. 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(2006). nutritional quality, functional properties and anti-nutrient composition of the larva of cirna forda (westwood) (lepidoptera: saturniidae). j. of zhejiang univer. science b (biomedine and biotech.), 7, 51-55. https://doi.org/10.1631/jzus.2006.b0051 osasona, a. l., & olaofe, o. (2010). nutritional and functional properties of cirna forda larva from ado-ekiti, nigeria. afri. j. of food sci., 4, 775-777. oso, j. a., & ola-oladimeji, f. a. (2016). preliminary assessment of growth performance and nutrient utilization of clarias gariepinus (burchell 1822) fed cirina forda (westwood 1849) larvae as protein source. int. j. of aquac. and fishery sci., 2(1), 039-042. https://doi.org/10.17352/2455-8400.000019 oyegoke, o. o., akintola, a. j., & fasoranti, j. o. (2006). dietary potentials of the edible larvae of cirina forda (westwood) as a poultry feed. african j. of aquaculture, 21(1), 142-147. stone, d. a. j. (2003). dietary carbohydrate utilization by fish. reviews in fish. sci., 11, 337-369. https://doi.org/10.1080/10641260390260884 stoskopf, m. k. (1993). clinical pathology in fish medicine (p. 55). w.b. saunders company, hartcourt brace jovanourah inc. vijayakumari, k., siddhuraju, p., & janardhanan, k. (1997). effect of domestic processing on the levels of certain antinutrients in prosopis chilensis (molina) stunz. seeds. food chem., 59(3), 367-371. https://doi.org/10.1016/s0308-8146(96)00249-x https://www.sciencedirect.com/science/journal/03088146 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 4, no. 2, 2020 www.scholink.org/ojs/index.php/fsns 1 original paper production and evaluation of nutritional contents of traditional couscous from sprouted wheat fortified with glycine max (l.) merr (soya bean) and cucurbita pepo (pumpkin) seeds raihanatu mb1, falmata as1, bintu bp1, maryam bk2, hadiza ahmed ali1, comfort mb1 & modu s1* 1 department of biochemistry, faculty science, university of maiduguri, nigeria ² department of biological sciences, faculty science, university of maiduguri, nigeria * modu s, department of biochemistry, faculty science, university of maiduguri, nigeria received: june 29, 2019 accepted: july 8, 2019 online published: april 8, 2020 doi:10.22158/fsns.v4n2p1 url: http://dx.doi.org/10.22158/fsns.v4n2p1 abstract the study was carried to process, produce, and evaluate nutritional contents of traditional couscous from sprouted wheat (triticum aestivum), fortified with soya bean (glycine max) and pumpkin (cucurbita pepo) seeds. the composite couscous blends were traditionally produced and compared with commercial couscous. the sprouted wheat couscous blends were blended in different ratios, they include; unprocessed (raw wheat, 100), blend 1 (sprouted wheat mixed with soya bean and pumpkin seeds, 70:20:10), blend 2 (sprouted wheat mixed with soya bean, 60:40) and blend 3 (sprouted wheat mixed with pumpkin seeds, 60:40). traditional wheat couscous blends were fed to experimental albino rats of wister strain weighing between (35 g and 45 g) for a period of 28 days. the nutritional and physiochemical analysis were determined using standard laboratory methods. the statistical package for social sciences (spss), version 20.0 was used to analyze the data collected which were expressed as means ± se. one way analysis of variance (anova) and duncan’s multiple range tests were used to compare the means obtained after each experiment. differences were considered significant at p < 0.05. processing (sprouting) decreases the levels of anti-nutrients, mineral elements and vitamins. supplementation with soya bean and pumpkin seeds increased the nutritional composition of the sprouted wheat couscous blends. results of chemical composition showed that blend 2, recorded high protein (29.95%), fat (8.95%) and low carbohydrate content (49.56%), followed by blend 1 and then blend 3, while commercial couscous crude protein, fat and carbohydrate were 12.53%, 1.42% and 75.10% respectively. there was improved level of in vitro protein digestibility at 1 hour (76.64% to 98.59%) and at 6 hours (96.80% to 99.33%). results of in vivo studies showed that raw wheat www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 2 published by scholink inc. couscous recorded protein quality when compared with spouted wheat couscous blends produced. the biological values of the composite couscous blends range from 95.04% to 95.73% and blend 2, recorded high net protein utilization (98.57%). in terms of sensory evaluation using hedonic method, blend 2 was most acceptable and differ significantly (p < 0.05) with other sprouted wheat couscous blends and commercial couscous. the cost of producing sprouted wheat couscous blends is cheaper than the commercial couscous. the study has therefore, revealed that with proper selection of locally available cereal, it is possible to produce nutritious complementary couscous blends that would be acceptable and nutritionally adequate to meet up the nutritional requirement for both children and adults. it also compares favourably with the commercial couscous in terms of nutrient contents. keywords sprouting, wheat, couscous blends, pumpkin seeds, soya bean and grits 1. introduction couscous is defined as a grits form of either triticum aestivum l. (wheat), pennisetum glaucum l. (millet), sorghum bicolorl.moench (sorghum) or zea mays l. (maize), it usually comes in coarse form which is steam cooked and eaten as a full meal. couscous, is prepared traditionally from mono cereals which lack adequate nutrients for adults and children, there is need for fortification/blending with plant legumes to meet up the nutrients requirement that may be loss during processing and production. couscous is served with vegetables, fish or meat. it can also be absorb in milk and sugar, it is a staple food of north africa, and is very popular in west african countries. wheat (triticum aestivum) is the main raw material for the production of couscous commercially and traditionally. the grains were sprouted to reduce the anti-nutrients, improve protein digestibility and enhances the nutritional value of the couscous being produced. fortification of couscous with glycine max (soya beans) and cucurbita pepo seeds (pumpkin seeds) will further supplement the deficient nutrients that were lost in the process of production. flour is inadequate in terms of some of the essential amino acids (lysine, threonine and methionine). mono cereal (wheat) can be enhanced by the addition of legume flour. amino acids balance of legume flour is good and its protein content is high (lee et al., 1998). today, in many countries including turkey, couscous is made mechanically using extrusion technology and each couscous granule represents an aggregate of several semolina particles (debbouz & donnelly, 1996). a comparison of the characteristics of traditional and commercial wheat couscous has been made by guezlane et al. (1986). these authors found a higher elasticity in the traditionally prepared sample and a lower one in the commercially processed one. common wheat (triticum aestivum l.) is a major cereal crop consumed in many parts of the world, accounting for 30% of the total grain consumption with an annual production of over 660 million tons worldwide (fao, 2010). the origins of are thought to date back more than ten thousand years to the levant region of the near east and ethiopian highlands where remains of the wild progenitors of wheat have been discovered (feldman & kislev, 2007). today, wheat is grown on the most land area of any commercial crop countries currently producing the most www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 3 published by scholink inc. wheat include china, india, european countries, and the united states (usda, 2006). wheat contributes more calories than any other cereal crops (adom & liu, 2002; shewry, 2009). it is nutritious, easy for transportation and storage, and can be processed into different types of food products. wheat is considered as a good source of protein, minerals, b vitamins and dietary fiber although the environmental conditions can affect nutritional composition of wheat grains with its essential coating of bran, indicating that it is a great health-building food (shewry, 2007). soya bean is derived from seed glycine max (l) merr of family –leguminosae or fabacae. soya bean is known as the “golden bean” or “the super legume” of the twentieth century. it represents an excellent source of unsaturated fatty acids, high quality proteins and fibers. soya bean contains very small amounts of saturated fatty acids but do not contain any trans-fatty acids. both omega-6 and omega-3 fatty acids such as linoleic acid (56% total fat) and alpha linolenic acid (7-8% of total fat) are present in soya bean. cooked soya bean are rich in iron, phosphorus, magnesium, vitamin b2 (riboflavin) and folate. it is one of the best vegetarian sources of total protein containing all essential amino acids required in the human diets. common food preparations of soya bean include edamme (whole soybean), soy flour, soymilk, tofu (fermanted soybean paste), soybean oil, soybean lecithin and soy sauce (milland et al., 2014). pumpkin (cucurbita pepo) is a cultivated plant of the genus cucurbita. it yields varieties of winter squash and pumpkin, both a shrubby and creeping plant, ovoid or conical shape, pointed at the apex and with longitudinal grooves, thus resembling a spinning top(1-2). the mature or young fruit and the seeds of c. pepo, as well as to a lesser extent the flowers and young tips of the stems, are eaten in many parts of its native distribution area and in other regions of the world (jeffery, 1986). in nigeria, the different parts of cucurbita pepo are edible: the pulp, seed and leaves. they are used to prepare different types of dishes. the pumpkin seed is valued in regard to nutritional points. several studies have reported the chemical composition and oil characteristics of the pumpkin seed from different origins and varieties (lazos, 1986; stevenson et al., 2007). the four fatty acids presented in significant quantities are palmitic, stearic, oleic, and linoleic acids (stevenson et al., 2007). the pumpkin seed is a good source of potassium, phosphorus, magnesium, and also contains moderately high amounts of other trace minerals (calcium, sodium, manganese, iron, zinc, and copper) and these elements make pumpkin seed valuable for food supplements (lazos, 1986). raw or roasted pumpkin seeds are used as a snack food for human consumption in many cultures all over the world. the kernels of pumpkin seeds have been utilized as flavour enhancers in gravies and soups, and used in cooking, baking and ground meat formulations as a nutrient supplement and a functional agent (tsaknis et al., 1997; el-adawy & taha, 2001). 1.1 objectives of the study the objectives of the study are to: i. produce traditional couscous from sprouted mono cereals fortified with soya bean and pumpkin www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 4 published by scholink inc. seeds. ii. determine proximate, mineral element, vitamin and anti-nutrients compositions of the couscous blends. iii. determine in vitro protein digestibility of the couscous blends. iv. determine endogenous urinary nitrogen and metabolic faecal nitrogen for protein quality. v. carry out sensory evaluation of the couscous blends. 2. materials and methodology 2.1 sources of raw materials the samples (wheat, soybean, pumpkin seeds and commercial couscous) used for this study were obtained at the open market (monday market, and gamboru market). they were authenticated by a seed breeder at the lake chad research institute, maiduguri, borno state, nigeria. 2.2 sample preparation the cereal (wheat) was sprouted for 3 days, while the soya bean was soaked overnight, drained, air dried, roasted, and grounded into coarse form and then steam cooked. the pumpkin seeds was also soaked in water, washed, drained, air dried in an open shed, roasted, grounded into coarse form and then steam cooked. the steam cooked soya bean and the pumpkin seeds were also air dried and packaged. 2.3 sprouting ten kilograms (10 kg) of each cereal grains were sorted out and cleaned with water. the grains were then soaked overnight, and the following morning the cereals were washed with water, drained, and then transferred onto wetted jute bag and placed it in an air tight jar. the various samples of cereal grains were sprouted on wet jute bags for 72 hours at room temperature. the samples were removed from the jute bag, air dried and grinded into fine grits. 2.4 soaking and roasting ten kilograms (10 kg) of soya beans were soaked in tap water for 8 hours and washed with more water, air dried in an open shade for 48 hours and then roasted (soaking and roasting were intended to remove the beany flavour). ten kilograms (10 kg) of pumpkin seeds were also soaked in water for 2 hours, washed, air dried in an open shade and roasted. the soya bean and the pumpkin seeds were then grounded into fine grits; sieved and steam cooked for 20-30 minutes. they were then air dried in an open shade for 48 hours and then packaged. 2.5 production of couscous the cereal grains were sorted out and cleaned, sprouted at room temperature, air dried in an open shade, grinded into fine grits, sieved and then, water, a pinch of salt and small quantity of oil were added into the grits, mixed thoroughly with hand and then place in a steam cooking pot and steam cooked for 20-30minutes at low temperature the result is the “traditional couscous” which is then air dried in an open shade for 48 hours, sieved and finally packaged in a container (fatima, 2013). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 5 published by scholink inc. figure 1. flow chart for production of traditional couscous 2.6 formulation/ blending (fortification) of couscous with soya bean and pumpkin seeds. the cereals were blended based on ratio 70: 20:10, (70 g of the wheat was mixed with 20 g of soya bean and 10 g of pumpkin seeds), 60:40 (60 g of the wheat was mixed with 40 g of soya bean without pumpkin seeds) and 60:40 (60 g of wheat was mixed with 40 g of pumpkin seeds without soya bean) respectively. 2.7 animal experimentation/nutritional studies eighty five (85) albino rats of 35-45 g were obtained from the animal house unit of the department of biochemistry, university of maiduguri. the rats were randomly assigned into four (100, 70:20:10, 60:40 and 60:40) dietary treatment groups of 5 rats per group. the rats in each group were housed together in standard plastic laboratory cages with stainless steel covers and were offered with their respective experimental diets and water ad libitum after one week of acclimatization period to the laboratory environment. the feeding trials lasted for four weeks (28 days). the feed intake was determined as the differential between the quantity of feed served and the quantity of feed left over. the changes in weight were determined by weighing the rats at the commencement of the feeding trial and thereafter on a weekly basis until termination of the experiment. the faecal and urine of the rats were collected on daily basis for 7adys (week four) for determination of percentage nitrogen using kjeldahl method (aoac, 1990). another group of 5 rats with same weight of the initial eighty five rats were fed with protein-free diet (1% or 2%), also their faecal and urine samples were analysed for percentage nitrogen to calculate for endogenous and metabolic nitrogen in biological assay. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 6 published by scholink inc. 2.8 measures of protein quality quality and digestibility of protein are two major determinants whether the dietary protein intake can furnish adequate level of essential amino acids to satisfy their function in the body. protein efficiency ratio (per): per was calculated as described by osborne et al. (1919) per = gain in body weight (g) protein consumed (g) biological value (bv) biological value is then calculated using thomas-mitchell (1924) method as follows; bv = ni− (fn−mfn)−(un−eun) x100 ni−(fn−mfn) where ni = nitrogen intake fn = nitrogen voided through faeces un = nitrogen excreted through urine mfn = metabolic faecal nitrogen eun = endogenous urinary nitrogen apparent digestibility (ad) was calculated using the formular. ad = ni− fn x 100 ni true digestibility (td) the amount of faecal nitrogen excreted when the subject is consuming either a protein –free diet, or a diet with just enough of a highly digestible protein to prevent excessive loss by the body protein. thus, true digestibility can be calculated as follows: td = ni−(fn−mfn) x 100 ni net protein utilization (npu) npu was calculated using a method described by bender and miller (1953). npu = ni−(fn−un) x 100 ni 2.9 proximate analysis the determination of moisture content, ash, crude protein, fat, crude fiber, carbohydrate and energy (kcal) were carried out according to aoac (2002). 2.9.1 moisture content five grams (5.0 g) of samples were weighed into a petri dish and dried in an oven at 1050c. after 2 hours the samples were removed, cooled in a dessicator and weighed. the process of weighing and cooling were repeated several times until a constant weight was obtained. the moisture contents were calculated using the formula. %moisture content = weight of sample after drying x 100 weight of sample before dying www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 7 published by scholink inc. 2.9.2 ash content two grams (2.0 g) of samples were placed in a weighed crucible completely combusted at 550-6000c in a muffle furnace, percentage of ash of the samples were calculated using the formula; % ash = w1−w2 x 100 w where w1 = weight of sample + crucible before ashing w2 = weight of sample + crucible after ashing w = weight of sample 2.9.3 crude protein one gram (1.0 g) of the samples were weighed into a digestion tube and one digestion tablet and 20 ml of conc. h2so4 was added and digested at 4200c using digestion block. the samples were digested, cooled and diluted with 80 ml of distilled water for 4 hours. the samples were then distilled with naoh and boric acid and titrated using 0.1n hcl. the crude protein content of the samples were calculated using the formula. % crude protein = (a−b) x n x f x 6.25 x 100 mg of sample where; a = titrated values b = blank n = normality of acid used f = factor 14.007 6.25 = constant 2.9.4 fat content two grams (2.0 g) of the samples (couscous blends) were weighed and transferred into a fat free extraction thimble plugged tightly with cotton wool. the thimble was placed in the soxhlet extraction chamber and 25 mls of petroleum ether was added until it siphoned over, more ether was also added till the barell of soxhlet chamber was half full with ether. the condenser was detached, heat source was adjusted, flask containing the sample was removed and it content poured into a stock ether bottle. the condenser was detached again to the soxhlet chamber and the flask was also connected to the soxhlet and heat source was readjusted from 300c to 600c and the distillation process was continued until all the ether in the flask have practically dried. the flask containing the sample was then transferred into oven and the samples were dried to a constant weight at a temperature 1050c. the samples were removed and transferred to the dessicator for cooling and weighing immediately for determination of ash content. % fat = w2−w1 x 100 w3 where; w1 = weight of flask w2 = weight of fat + flask w3 = weight of sample www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 8 published by scholink inc. 2.9.5 crude fiber the crude fiber were determined using trichloroacetic acid digestion method by refluxing two grams (2.0 g) of samples (couscous blends) in 100 ml of digestion reagent, for exactly 40minutes, counting from the time boiling commences. the flask was removed from the mantle heater and cooled under a tap. the samples were then filtered using 15 cm of no. 4 white man filter. the samples were then washed six times with hot water and once with petroleum spirit. the filter paper was opened and the residues of the samples were removed using a spatula and transferred to a weighed petri dish and dried at 1050c overnight. the samples were removed, cooled in a dessicator and weighed, then ashed at 6000c. the samples were allowed to cooled and re-weighed again. the percentage crude fibers of the samples were calculated as a difference in weighing multiply by 100. % crude fiber = w2−w3 x 100 w1 w1 = weight of the sample w2 = weight after extraction and drying + crucible w3 = weight of the ash + crucible 2.9.6 total carbohydrate the total carbohydrate content were estimated as the difference between 100 and the total sum of moisture, fat, protein, crude fibre and ash (aoac, 2002). 2.9.7 total energy the total energy or the caloric values were estimated by calculation using the water quantification factors of 4, 9 and 4 kcal/100 g respectively for protein, fat and carbohydrate. 2.10 determination of in vitro protein digestibility in vitro protein digestibility was determined by (nills, 1979). the nitrogen of the undigested samples was determined by kjeldahl method (aoac, 1990). % in vitro protein digestibility = cp1 − cp2 x 100 cp1 where, cp1= total protein of unprocessed couscous cp2 = total protein after digestion with trypsin. 2.11 determination of anti-nutrients contents 2.11.1 determination of tannin contents assay by vanillin-hydrochloric acid. method: quantitative (price et al., 1978). the absorbance of the standard solutions, sample extract and sample blank was read in spectrophotometer at 500 nm exactly 20 minutes after incubation. calculation au cu = astd cstd cu = au x cstd = mg/g astd where au= absorbance of unknown cu= concentration of unknown www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 9 published by scholink inc. astd = absorbance of standard cstd = concentration of standard tannin (% reduction) % reduction = crs – cps x 100 crs where crs = concentration of raw sample cps = concentration of processed sample. 2.11.2 determination of phytic acid phytic acid was determined by a method as described by (davies & reid, 1979). 2.12 determinations of mineral elements atomic absorption spectrophotometer (aas) aa 6800 series shimazocorp was used for the determination of ca, na, k, fe, mg, f and zn. as described by (wittmas et al., 1981). 2.13 determination of vitamin contents vitamin b1, b2, b6, folate and c were determined by a method as described by (angelika et al., 2001). the actual concentration of the vitamins was calculated using bear lambert’s law; a = abc. 2.13.1 determination of vitamin a vitamin a was determined using hplc (angelika et al., 2001). 2.13.2 determination of percentage nitrogen percentage nitrogen for urine and faecal of the test animals was determine as described by aoac (1990). % n was calculated using the formula; %n = (a−b) x n x f x100 mg of sample where; a= ml of acid for titrating the sample b = ml of acid for titrating blank sample n = normality of acid used for titration f = factor (14.007) 2.14 sensory evaluation fifteen panelists evaluated the sensory properties of cooked samples by using nine (9) hedonic scale from 9 (like extremely) to 1 (dislike extremely) for colour, texture, aroma, taste and overall acceptability of the couscous samples. replication was achieved by the five different couscous samples being evaluated by fifteen panelists (penfield & campbell, 1990). 2.15 statistical analysis the statistical package for social sciences (spss), version 20.0 was used to analyze the data collected which were expressed as means ± se. one way analysis of variance (anova) and duncan’s multiple range tests were used to compare the means obtained after each experiment. differences were considered significant at p < 0.05. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 10 published by scholink inc. 3. results table 1 shows the results of proximate nutrient composition of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous. the moisture contents of raw wheat couscous and sprouted wheat couscous are 8.90% and 7.54% respectively. the moisture content of sprouted wheat couscous blends were 7.07%, 8.01% and 6.78% respectively. the commercial couscous moisture content was 9.16%. the ash content of raw wheat couscous and sprouted wheat couscous are 1.89% and 2.38% respectively. sprouted wheat couscous blends ash contents were 2.64%, 3.16% and 2.98% respectively. the commercial couscous ash content was 0.83%. crude protein of raw wheat couscous and sprouted wheat couscous are 12.86% and 5.46%. the sprouted wheat couscous blends protein content were 27.21%, 29.95% and 20.85% respectively. the commercial couscous crude protein was 12.53%. crude fat of raw wheat couscous and sprouted wheat couscous are 1.42% and 2.92% respectively. sprouted wheat couscous blends fat content were 7.48%, 8.95% and 8.35% respectively. the commercial couscous crude fat was 1.42%. crude fiber of the raw wheat couscous and sprouted wheat couscous are 0.52% and 2.68% respectively. sprouted wheat couscous blends crude fiber were 0.27%, 0.31% and 1.65% respectively. the commercial couscous crude fiber was 0.97%. total carbohydrate of raw wheat couscous and sprouted wheat couscous are 74.41% and 79.04% respectively. sprouted wheat couscous blends carbohydrate were 55.33%, 49.56% and 59.40% respectively. commercial couscous carbohydrate was 75.10%. energy (kcal) content of raw wheat couscous and sprouted wheat couscous are 361.89 and 364.54 respectively. the energy (kcal) of sprouted wheat couscous blends were 397.24, 398.54 and 396.11 respectively while the commercial couscous energy content was 363.20 kcal. table 1. chemical composition of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends sample raw sprouted blend 1 blend 2 blend 3 gpc moisture (%) 8.90±0.02e 7.54±0.01c 7.07±0.01b 8.01±0.01d 6.78 ± 0.01a 9.16 ± 0.01f ash (%) 1.89±0.2b 2.38±0.02c 2.64±0.01d 3.16±0.01f 2.98 ± 0.00e 0.83 ± 0.02a protein (%) 12.86±0.01c 5.46±0.02a 27.21±0.01e 29.95±0.01f 20.85± 0.01d 12.53±0.01b fat (%) 1.42±0.01a 2.92±0.02b 7.48±0.01c 8.95±0.02e 8.35 ± 0.01d 1.42 ± 0.01a fiber (%) 0.52±0.01c 2.68±0.01f 0.27±0.01a 0.31±0.01b 1.65 ± 0.01e 0.97 ± 0.01d carbohydrate (%) 74.41±0.04d 79.04± 0.04f 79.04± 0.04f 79.04± 0.04f 79.04± 0.04f 75.10± 0.04e energy (kcal) 361.89±0.16a 364.24±0.21c 397.48±0.07e 398.54±0.15f 396.11±0.04d 363.2± 0.11b values are recorded as mean ± sem, n=4 values on the same row with different superscript are significantly different (p< 0.05) blend 1 (sprouted wheat mixed with soya bean and pumpkin seeds, 70:20:10) blend 2 (sprouted wheat mixed with soya bean, 60:40) blend 3 (sprouted wheat mixed with pumpkin seeds, 60:40) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 11 published by scholink inc. gpc (golden penny couscous) table 2 shows the mineral elements of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous. raw wheat couscous and sprouted wheat couscous had sodium (na) content of 14.81mglg and 17.49 mg/g respectively. sprouted wheat couscous blends recorded sodium values of 20.41 mg/g, 19.83 mg/g and 36.00 mg/g respectively. commercial couscous sodium ion content was 21.23 mg/g. potassium (k) content of raw wheat couscous and sprouted wheat couscous are 2.62 mg/g and 5.91 mg/g respectively. sprouted wheat couscous blends potassium (k) content were 20.81 mg/g, 1.65 mg/g and 1.33 mg/g respectively potassium content of commercial couscous was found to be 2.34 mg/g. calcium (ca) values of raw wheat couscous and sprouted wheat couscous are 2.41 mg/g and 2.54 mg/g respectively. sprouted wheat couscous blends recorded ca values of 4.50 mg/g, 2.93 mg/g and 21.03 mg/g respectively. commercial couscous ca content was 2.62 mg/g. raw wheat couscous and sprouted wheat couscous recorded zinc (zn) values of 1.27 mg/g and 0.42 mg/g respectively. sprouted wheat couscous blends zn values were 0.60 mg/g, 0.53 mg/g and 0.22 mg/g. raw wheat couscous and sprouted wheat couscous recorded magnesium (mg) values of 0.81 mg/g, 0.75 mg/g and 1.41 mg/g respectively. sprouted wheat couscous blends mg values were 1.41 mg/g, 1.01 mg/g and 7.03 mg/g respectively. commercial couscous had a zn value of 0.78 mg/g. the iron (fe) content of raw wheat couscous and sprouted wheat couscous are 0.15 mg/g and 0.55 mg/g respectively. sprouted wheat couscous blends fe content were 0.68 mg/g, 0.44 mg/g and 0.70 mg/g respectively. commercial couscous fe content was found to be 0.71 mg/g. table 2. mineral elements of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous mineral elements (mg/g) raw sprouted blend 1 blend 2 blend 3 gpc na 14.81 ±0.01a 17.49 ±0.00ab 20.41 ±0.01c 19.83 ±0.01bc 86.0 ± 2.00d 21.23 ±0.01c k 2.62 ± 0.01d 5.91 ± 0.00e 20.81 ±0.01f 1.63 ± 0.01b 1.33 ± 0.01a 2.34 ± 0.01c ca 2.41 ± 0.01a 2.54 ± 0.00b 4.50 ± 0.01e 2.93 ± 0.01d 21.03 ±0.00f 2.62 ± 0.01c zn 1.27 ± 0.01f 0.42 ± 0.01b 0.60 ± 0.01e 0.53 ± 0.01c 0.22 ± 0.01a 0.56 ± 0.01d mg 0.81 ± 0.00c 0.75 ± 0.00a 1.41 ± 0.01e 1.01 ± 0.00d 7.03 ± 0.01f 0.78 ± 0.01b fe 0.15 ± 0.00a 0.55 ± 0.01c 0.68 ± 0.01d 0.44 ± 0.01b 0.70 ± 0.01d 0.71 ± 0.01d values are recorded as mean ± sem, n=4 values on the same row with different superscript are significantly different (p< 0.05) blend 1 (sprouted wheat mixed with soya bean and pumpkin seeds, 70:20:10) blend 2 (sprouted wheat mixed with soya bean, 60:40) blend 3 (sprouted wheat mixed with pumpkin seeds, 60:40) gpc (golden penny couscous). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 12 published by scholink inc. table 3 shows vitamins content of raw wheat couscous sprouted wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends and commercial couscous. vitamin a content of raw wheat couscous and sprouted wheat couscous are 13.58 µg/g and 12.99 µg/g respectively. sprouted wheat couscous blends vitamin a content were 7.76 µg/g, 14.59 µg/g and 6.67 µg/g respectively. commercial couscous vitamin a content was found to be 14.23 µg/g. vitamin b1 (thiamine) content of raw wheat couscous and sprouted wheat couscous are 0.13 µg/g and 0. 33 µg/g respectively. sprouted wheat couscous vitamin b1 contents were 2.62 µ g/g, 2.74 µg/g and 2.72 µg/g respectively. commercial couscous vitamin b1 content was 0.23 µg/g. vitamin b2 (riboflavin) content of raw wheat couscous and sprouted wheat couscous are 1.34 µg/g and 0.94 µg/g respectively. sprouted wheat couscous blends vitamin b2 contents were 2.23 µg/g, 2.24 µg/g and 10.82 µg/g respectively. commercial couscous vitamin b2 was found to be 0.64 µg/g. vitamin b6 (pyridoxine) contents of raw wheat couscous and sprouted wheat couscous are 20.23 µg/g and 10.13 µg/g respectively. sprouted wheat couscous blends vitamin b6 contents were 23.53 µg/g, 20.44 µg/g and 28.40 µg/g respectively. commercial couscous vitamin b6 was found to be 0.33 µ g/g. folic acid contents of raw wheat couscous and sprouted wheat couscous are 3.84 µg/g and 6.95 µg/g respectively. sprouted wheat couscous blends folic acid contents were 10.63 µg/g, 28.13 µg/g and 25.50 µg/g respectively. commercial couscous folic acid was found to be 5.54 µg/g. vitamin c content of raw wheat couscous and sprouted wheat couscous are 19.79 µg/g and 13.35 µg/g respectively. sprouted wheat couscous blends vitamin c contents were 19.61 µg/g, 13.39 µg/g and 152.14 µg/g respectively. commercial couscous vitamin c content was found to be 13.37 µg/g. table 3. vitamins contents of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous vitamin (µg/ g) vit. a vit. b1 vit. b2 vit. b6 folic acid vit. c raw wheat 13.58 ± 0.01d 0.13 ± 0.01a 1.34 ± 0.02c 20.23 ± 0.01c 3.84 ± 0.01a 19.79 ± 0.20b sprouted 12.99 ± 0.01c 0.33 ± 0.01c 0.94 ± 0.01b 10.13 ± 0.01b 6.95 ± 0.02c 13.35 ± 0.02a blend 1 7.76 ± 0.01b 2.62 ± 0.02d 2.23 ± 0.01d 23.53 ± 0.01e 10.63 ± 0.01d 19.61 ± 0.22 blend 2 14.57 ± 0.02f 2.74 ± 0.01e 2.24 ± 0.01d 20.44 ± 0.01d 28.13 ± 0.01f 13.39 ± 0.02a blend 3 6.67 ± 0.02a 2.72 ± 0.02e 10.82 ± 0.01e 28.40 ± 0.01f 25.50 ± 0.01e 152.14 ± 0.01c gpc 14.23 ± 0.01e 0.23 ± 0.01b 0.64 ± 0.02a 0.33 ± 0.01a 5.54 ± 0.01b 13.37 ± 0.01a values are recorded as mean ± sem, n=4 values on the same column with different superscript are significantly different (p< 0.05) blend 1 (sprouted wheat mixed with soya bean and pumpkin seeds, 70:20:10 blend 2 (sprouted wheat mixed with soya bean, 60:40) blend 3 (sprouted wheat mixed with pumpkin seeds, 60:40) gpc (golden penny couscous) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 13 published by scholink inc. in vitro protein digestibility of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous are presented in table 4 in vitro protein digestibility of raw wheat couscous at 1 hour and 6 hours are 94.57% and 97.39% respectively. while sprouted wheat couscous in vitro protein digestibility at 1 hour and 6 hours are 98.22% and 89.76% respectively. sprouted wheat couscous blends in vitro protein digestibility at 1 hour were 76.64%, 98.59% and 88.29% and at 6 hours were 96.80%, 99.33% and 97.49% respectively. while commercial couscous in vitro protein digestibility at 1 hour and 6 hours are 97.17% and 97.64% respectively. table 4. in vitro protein digestibility of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous digestibility (%) raw sprouted blend 1 blend 2 blend 3 gpc 1 hour 94.57 ± 0.01d 98.22 ± 0.01c 76.64 ± 0.01a 98.59 ± 0.01f 88.29 ± 0.02b 95.17 ± 0.02e 6 hours 97.39 ± 0.01c 89.76 ± 0.01a 96.80 ± 0.02b 99.33 ± 0.01f 97.49 ± 0.01d 97.64 ± 0.01e values are recorded as mean ± sem, n=4 values on the same row with different superscript are significantly different (p< 0.05) blend 1 (sprouted wheat mixed with soya bean and pumpkin seeds, 70:20:10) blend 2 (sprouted wheat mixed with soya bean, 60:40) blend 3 (sprouted wheat mixed with pumpkin seeds, 60:40) gpc (golden penny couscous) table 5 shows anti-nutrients content of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous. tannin contents of raw wheat couscous and sprouted wheat couscous are 5.33 mg/g and 2.06 mg/g respectively. sprouted wheat couscous blends tannin contents were 4.10 mg/g, 3.27 mg/g and 2.27 mg/g respectively. commercial couscous tannin contents was 0.62 mg/g. phytic acid content of raw wheat couscous and sprouted wheat couscous are 0.34 mg/g and 0.98 mg/g respectively. sprouted wheat couscous blends phytic acid were 0.53 mg/g, 0.55 mg/g and 0.21 mg/g respectively. commercial couscous phytic acid was found to be 0.98mg/g. table 5. antinutrients contents of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous anti-nutrients (mg/g) raw sprouted blend 1 blend 2 blend 3 gpc tannin 5.33±0.01f 2.06±0.01b 4.10±0.1e 3.27±0.01d 2.27±0.01c 0.62±0.02a phytic acid 0.34±0.01b 0.98±0.01d 0.53±0.01c 0.55±0.00c 0.21±0.01a 0.98±0.01d values are recorded as mean ±se, n=4 values on the same row with different superscript are significantly different (p<0.05) blend 1 (sprouted wheat mixed with soya bean and pumpkin seeds (70:20:10) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 14 published by scholink inc. blend 2 (sprouted wheat mixed with soya bean, 60:40) blend 3 (sprouted wheat mixed with pumpkin seeds, 60: 40) gpc (golden penny couscous) table 6 shows protein quality of raw wheat couscous and sprouted wheat couscous blends. raw wheat couscous feed intake and weight gain are 41.79g and 327.54g respectively. sprouted wheat couscous blends feed intake were 39.86g, 38.10g and 40.00g and weight gain were 327.77g, 334.93g and 255.70g respectively. protein efficiency ratio (per) of raw wheat couscous was 7.84% while sprouted wheat couscous blends per were 8.22%, 8.79% and 6.40% respectively. biological value (bv) raw wheat couscous was 97.68% while bv of sprouted wheat couscous blends were 95.73, 95.04% and 95.69% respectively. apparent digestibility (ad) of raw wheat couscous was 96.85% while sprouted wheat couscous blends ad were 93.53%, 91.95% and 93.46% respectively. true digestibility (td) of raw wheat couscous was 99.35% while sprouted what couscous blends td were 97.58%, 96.17% and 97.47% respectively. net protein utilization (npu) of raw wheat couscous was 98.27% while sprouted wheat couscous blends npu were 95.09%, 98.57% and 95.65% respectively. table 6. protein quality of raw wheat couscous and sprouted wheat couscous blends parameter raw wheat blend 1 blend 2 blend 3 feed intake (g) 41.79±0.02c 39.86±0.19b 38.10±0.36a 40.00±0.12b weight gain (g) 327.54±0.02b 327.77±0.02 334.93±0.01c 255.70±0.26a protein efficiency ratio (%) 7.84±0.00b 8.22± 0.04c 8.79±0.01d 6.40±0.00a biological value (%) 97.68±0.01d 95.73±0.01c 95.04±0.01a 95.67±0.01b apparent digestibility (%) 96.85±0.01d 93.53±0.01c 91.95±0.01a 93.46±0.01b true digestibility (%) 99.35±0.01d 97.58±0.01c 96.17±0.02a 97.47±0.02b net protein utilization (%) 98.27±0.01c 95.09±0.01a 98.57±0.01d 95. 56±0.01b values on the same row with different superscript are significantly different (p<0.05) blend 1 (sprouted wheat mixed with soya bean and pumpkin seeds (70:20:10) blend 2 (sprouted wheat mixed with soya bean, 60:40) blend 3 (sprouted wheat mixed with pumpkin seeds, 60: 40) table 7 shows sensory scores of raw wheat couscous, sprouted wheat couscous blends compared with commercial couscous. in terms of all the attributes tested, blend 2 was most acceptable by the panelists and was significantly different (p<0.05) from the data obtained on raw wheat couscous, blend 1, blend 3 and commercial couscous. in all, the samples were accepted with exception of blend1 which recorded lowest values. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 15 published by scholink inc. table 7. sensory scores of raw wheat couscous, sprouted wheat couscous blends compared with commercial couscous sample raw wheat blend 1 blend 2 blend 3 gpc colour 7.92 ± 0.01c 7.59 ± 0.01b 8.34 ± 0.01e 7.48 ± 0.01a 7.98 ± 0.02d texture 8.14 ± 0.01d 5.49 ± 0.02a 7.61 ± 0.01c 6.49 ± 0.02b 8.19 ± 0.01e aroma 7.94 ± 0.01d 7.41 ± 0.01b 7.66 ± 0.01c 6.62 ± 0.02a 8.32 ± 0.01e taste 8.21 ± 0.01d 8.46 ± 0.01e 7.68 ± 0.01c 6.89 ± 0.02a 7.02 ± 0.02b overall acceptability 7.94 ± 0.01d 4.82 ± 0.02a 8.22 ± 0.02e 5.54 ± 0.01b 7.32 ± 0.01c values are recorded as mean ± sem, n=15 values on the same row with different superscript are significantly different (p< 0.05) blend 1 (sprouted wheat mixed with soya bean and pumpkin seeds, 70:20:10) blend 2 (sprouted wheat mixed with soya bean, 60:40) blend 3 (sprouted wheat mixed with pumpkin seeds, 60:40) gpc (golden penny couscous) 4. discussion 4.1 proximate composition of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous table 1 shows that commercial couscous recorded the highest moisture content (9.16%). the ash content of the sprouted wheat couscous blends increased significantly (p<0.05) with addition of soya bean and pumpkin seeds combined and separately. the high protein and fat contents in the composite couscous could have come from the soya bean which is known to contain high protein and fat contents (chike & anita, 2016). dietary fiber of blend 3 (1.65%) was higher than other samples. fiber enhances the gastro intestinal tract (git), aids or helps normal bowel movement thereby reducing constipation problems. the lower content of carbohydrate in the sprouted wheat couscous blends could be due to the soya bean and pumpkin seeds that contributed high protein and low carbohydrate (chike & anita, 2016). the energy (kcal) of the couscous blends range from 361.89 to 398.54. the differences in the caloric value were associated with composition with each of the sprouted wheat couscous blends. the results of mineral elements on raw wheat couscous sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous were shown on table 2 it was observed that sprouting (processing method) have increased the levels of na, k, ca and fe and decreases the levels of zn and mg. fortifications of sprouted wheat couscous with soya bean and pumpkin seeds had improved the mineral composition of the couscous blends produced traditionally. blend 3 was found to have higher levels of na, ca, mg and fe followed by blend 2 and then blend 2. commercial couscous had almost the same trend (values) of mineral elements as in sprouted wheat couscous blends produced traditionally. celeik et al. (2004) reported that addition of soy flour increased nutritional values (protein, www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 16 published by scholink inc. ca, k and fe levels) of couscous and this was agreed with this study. table 3 shows the vitamins (a, b1, b2, b6, folic acid and c) of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous. it was observed that, the vitamin a content of raw wheat couscous (13.58 µg/g) was higher than the sprouted what couscous (12.99 µg/g). sprouted wheat couscous blend 2 had the highest vitamin a (14.23 µg/g). there was significant difference (p<0.05) among the sprouted wheat couscous blends and the commercial couscous. vitamin b1 (thiamine) contents of raw wheat couscous (0.13 µg/g) was lower than the sprouted wheat couscous (0.33 µg/g), the sprouted wheat couscous blends vitamin b1 contents are in close range from 2.62 µg/g and 2.74 µg/g and there was no significant difference between blend 2 and blend 3 vitamin b1 contents while the commercial couscous vitamin b1 (0.23 µg/g) was found to be lower than the sprouted wheat couscous blends vitamin b1 contents. vitamin b2 (riboflavin) contents of raw wheat couscous (1.34 µg/g) was higher than the sprouted wheat couscous (0.94 µg/g), processing method (sprouting) had reduced the vitamin b2 content while on addition of soya bean and pumpkin seeds blend 3 had the highest contents of vitamin b2 while blend 1 and blend 2 are in close range while the commercial couscous vitamin b2 (0.64 µg/g) was lower than the vitamin b2 content of the sprouted wheat couscous blends produced traditionally. vitamin b6 (pyridoxine) content of raw wheat couscous (20.23 µg/g) was higher than the sprouted wheat couscous (10.13 µg/g) fortification of sprouted wheat couscous with soya bean and pumpkin seeds had improved the vitamin b6 content of the sprouted wheat couscous blends, blend 3 had the highest vitamin b6 (28.40 µg/g) followed by blend 1 (23.53µ g/g) and the least was blend 2 (20.44 µg/g), while commercial couscous vitamin b6 (0.33 µg/g) was lower than the sprouted wheat couscous blends produced traditionally. folic acid content of raw wheat couscous (3.84 µg/g) was lower than the sprouted wheat couscous (6.95 µg/g), fortification of sprouted wheat couscous had improved the folic acid contents. blend 2 had the highest folic acid value (28.13 µg/g) followed by blend 3 (25.03 µg/g) and then blend 1 (10.63 µg/g) while commercial couscous folic acid content (5.54 µg/g) was lower than the sprouted wheat couscous blends produced traditionally and there was significant difference (p<0.05) among the sprouted wheat couscous blends and the commercial couscous used for comparison. vitamin c content of raw wheat couscous (19.79 µg/g) was higher than the sprouted wheat couscous (13.35 µg/g) while sprouted wheat couscous blend 3 had the highest vitamin c content (152.14 µg/g) followed by blend 1 (19.61 µg/g) and then blend 2 (13.39 µg/g) and there was no significance difference between blend 2 vitamin c content and that of commercial couscous. results of in vitro protein digestibility of raw wheat couscous, sprouted wheat couscous, sprouted wheat couscous blends compared with commercial couscous are presented on table 4 in vitro protein digestibility of raw wheat couscous at 1 hour (94.57%) and 6 hours (97.39%) and sprouted wheat couscous at 1 hour (98.22%) and at 6 hours (89.76%) respectively. this showed that digestibility of raw wheat couscous increases with increased in time. sprouted wheat couscous digestibility at 1 hour (98.22%) was higher than that of 6 hours (89.76%), the differences in percentage may be as a result of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 17 published by scholink inc. the crude protein contents in the sprouted wheat couscous. sprouted wheat couscous blends in vitro protein digestibility showed a significant difference (p<0.05) among the sprouted wheat couscous blends and also between the sprouted couscous blends and the commercial couscous. blend 2 had highest in vitro protein digestibility at 1 hour (98.59%) and at 6 hours (99.33%), followed by blend 3 at 1 hour (88.29%) and at 6 hours (97.49%) and least was blend 1 at 1 hour (76.64%) and at 6 hours (96.80%), while commercial couscous had in vitro protein digestibility at 1 hour (95.17%) and at 6 hours (97.64%) respectively. raw wheat couscous had high tannin (5.33 mg/g) than the sprouted wheat couscous (2.06 mg/g), this showed that sprouting had significantly reduced the tannin content while phytic acid content of sprouted wheat couscous (0.98 mg/g) was higher than the raw wheat couscous (0.34 mg/g). sprouted wheat couscous blends tannin contents (4.10 mg/g, 3.27 mg/g and 2.27 mg/g) were significantly different (p<0.05), while commercial couscous tannin (0.62 mg/g) and phytic acid (0.98 mg/g) were lower than the values found in sprouted wheat couscous blends. the differences could be as a result of addition of soya bean and pumpkin seeds to the sprouted wheat couscous to complement the nutritional value of the traditional couscous blends produced. feed intake of raw wheat couscous (41.79 g) was higher than the values found in sprouted wheat couscous blends which ranged between 38.10 g to 40.00 g. weight gain of raw wheat couscous (327.54 g) was not significant with the weight gained in blend1 (327.77 g) but there was significant difference (p<0.05) among the sprouted wheat couscous blends. blend 2 had the highest weight gain (334.93 g). per of raw wheat couscous (7.84%) was lower than the per of blend 1 (8.22%) and blend 2 (8.79%) but higher than the per of blend 3 (6.40%). biological value (bv) of raw wheat couscous (97.68%) was higher than the bv of sprouted wheat couscous blends which ranged from 95.04% to 95.73% and are statistically significant (p<0.05). apparent digestibility (ad) of raw wheat couscous (96.85%) was higher than the ad of the sprouted wheat couscous blends which range from 91.95% to 93.53% while true digestibility (td) of raw wheat couscous (99.35%) was also higher than the td of the sprouted wheat couscous blends which range from 96.17% to 97.58%. net protein utilization (npu) of raw wheat couscous (98.27%) was higher than the npu of blend 1 (95.09%) and blend 3 (95.65%) and lower than the npu of blend 2 (98.57%) which are all significantly different (p<0.05). 4.2 sensory evaluation fifteen panelists evaluated the sensory properties of cooked couscous samples by using nine (9) hedonic scale from 9 (like extremely) to 1 (dislike extremely) for colour, texture, aroma, taste and overall acceptability of the couscous samples. replication was achieved by the five different couscous samples being evaluated by fifteen panelists (penfield & campbell, 1990). in terms of all the attributes tested, blend 2 was most acceptable by the panelists and was significantly different (p<0.05) from the data obtained on raw wheat couscous, blend 1, blend 3 and commercial couscous. in all, the samples were accepted with exception of blend1 which recorded lowest values. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 18 published by scholink inc. 5. conclusion this study has shown that complementary couscous of acceptable quality can be produced from composites grits of wheat, soya bean and pumpkin seeds. sprouting significantly reduced the levels of antinutrients, mineral elements and vitamins while supplementation of mono cereals with soya bean and pumpkin seeds have improved the nutritional value of the sprouted couscous blends produced traditionally. the results of proximate composition showed significant increase in ash contents, crude protein, fat content and low carbohydrate contents in all the sprouted mono cereal couscous blends produced traditionally. commercial couscous recorded lowest proximate values compared to sprouted couscous blends. results of mineral elements studied (na, k, ca, zn, mg and fe) showed a significant decrease in some mineral elements, while supplementation of sprouted mono cereal with soya bean and pumpkin seeds have significantly increased levels (na, ca, mg, and fe), with blend 3 of all the sprouted mono cereal recorded higher values of the mineral elements. results of vitamins analyzed (vit. a, vit.b1, vit.b2, vit.b6, folic acid and vit.c) showed a significant difference (p>0.05) among the sprouted mono cereal couscous blends compared with commercial couscous. blend 3 of all the mono cereal couscous blends recorded high levels of vitamins (vit. b2, vit b6, folic acid and vitamin c). commercial couscous vitamin contents were lower or within a close range with the mono cereal couscous blends. in vitro protein digestibility of each of the sprouted mono cereal couscous blends occurred at a range of 76% to 99% at 1 hour and 6 hours respectively. the results of anti-nutrients showed that raw mono cereals contained higher tannin and phytic acid, while sprouting reduced the levels of these anti-nutrients and supplementation with soya bean and pumpkin seeds significantly enhanced the nutritional values of the sprouted mono cereal couscous blends produced. commercial couscous recorded lowest tannin content (0.6 ± 0.02a). biological assay (in vivo studies) showed high protein quality in raw and sprouted mono cereal couscous blends. blend 1 if each of the sprouted mono cereal couscous blends recorded high per, bv, ad, td, and npu. in term of sensory score evaluation provides acceptability therefore, nutritious and acceptable complementary couscous blends can be produced from sprouted mono cereals fortified with soya bean and pumpkin seeds. the cost of producing traditional couscous blends is cheaper than commercial couscous. 5.1 recommendation i. it is therefore recommended that, proper processing method (s) before production and consumption of couscous blends be encourage, as it will reduces the levels of antinutrients and the absorption and utilization of minerals and vitamins will be maximized. ii. fortification of sprouted mono cereals with soya bean and pumpkin seeds be encourage, since it will www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 4, no. 2, 2020 19 published by scholink inc. serve as a food base approach to ameliorate micronutrients deficiency in children and adults. iii. more research should be conducted, as there are limited data available on couscous blends. references adom, k., k., sorrels, m. e., & liu, r. h. 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(2007). oil and tocopherol content and composition of pumpkin seed oil in 12 cultivars. journal of agriculture and food chemistry, 55, 4005-4013. https://doi.org/10.1021/jf0706979 tsaknis, j., lalas, s., & lazos, e. s. (1997). characterization of crude and purified pumpkin seed oil. gras aceit, 48, 276-272. https://doi.org/10.3989/gya.1997.v48.i5.802 usda. (2006). agricultural baseline projection to 2015, february, oce-2006. wittmas, h. e. jnr, allich, a., & aufe demhei del, a. c. (1981). mineral element quantification by graphite furnance atomic absorption spectroscopy. american journal of chemical pathology, 75, 80-85. https://doi.org/10.1093/ajcp/75.1.80 food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 2, 2019 www.scholink.org/ojs/index.php/fsns 60 original paper microbial, ph, titratable acidity, functional and sensory properties of weaning food blends formulated from maize, cowpea, bambaranut and groundnut bintu b.p.1, falmata a.s.1, maryam b.k.1, raihatu m.a.1, chellube z.1, hauwa h.1 & modu s.1* 1 department of biochemistry, biological sciences and chemistry, faculty of science, university of maiduguri, p.m.b 1069, maiduguri, nigeria * modu s., department of biochemistry, biological sciences and chemistry, faculty of science, university of maiduguri, p.m.b 1069, maiduguri, nigeria received: march 1, 2019 accepted: march 18, 2019 online published: april 24, 2019 doi:10.22158/fsns.v3n2p60 url: http://dx.doi.org/10.22158/fsns.v3n2p60 abstract this study investigated the microbial, sensory evaluation and functional properties of cereal/legume complementary weaning food blends using yellow maize, cowpea, bambaranut and groundnut. yellow maize was fermented to produce “akamu”, cowpea, bambaranut and groundnut were roasted. the weaning food blends were formulated as follows: mcbg: 60 (g) yellow maize: 20(g) cowpea: 10(g) bambaranut: 10(g) groundnut, mcb; 60(g) yellow maize: 20(g) cowpea: 20(g) bambaranut, mcg: 60(g) yellow maize: 20(g) bambaranut: 20(g) groundnut and mbg: 60(g) yellow maize: 20(g) bambaranut: 20(g) groundnut. standard laboratory methods were used to determine the parameters. the yellow maize (improved variety), cowpea, bambaranut and groundnut were obtained from lake chad research institute (lcri) maiduguri. data obtained were subjected to analysis of variance (anova) and duncan’s multiple range test was used to separate the means. a decrease in ph with an increase in titratable acidity was observed during the production of “akamu” from yellow maize. the weaning food blend mcbg showed a significant decrease in viscosity, water absorption capacity and bulk density than mcb, mcg and mbg. predominant micro organisms isolated during the production of akamu and the weaning food blends were lactobacillus, saccharomyces cerevisae, and streptococcus lactics. results of the sensory evaluation showed that mcbg had the highest overall acceptability than the three weaning food blends mcb, mcg and mbg. keywords weaning food, ph, cereal/legume blend, fermentation/roasting www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 61 published by scholink inc. 1. introduction children in must developing countries are introduced directly to the regular house diet of cereal foods inadequate complementary food is a major cause for the high incidence of child malnutrition, morbidity and mortality (ijarotimi, 2012) protein malnutrition among infants in low income countries is an important public health problem and can be related to the composition of the complementary foods introduced after the breast feeding period (falmata et al., 2014). according to world bank (2013) malnutrition when it is served can cause premature death, permanent disability and fragility in face of many deadly diseases. among different types of malnutrition protein energy malnutrition affects more than one half of the world’s population especially infants at weaning age (laminu et al., 2014). it has been recognized that high densed with pathogenic microbiological parameters weaning foods is an etiological factor of protein energy malnutrition (saleh, 2015). in many developing countries, traditional weaning foods are prepared mainly from cereals like maize, millet and sorghum, which are usually in protein quantity and quality. this coupled with the high cost and viscous nature of commercial available complementary foods as well as the poor hygiene of food handlers and major constraints in providing children with adequate nutrients (gernah et al., 2012). it is therefore, desirable to study ways and means of developing less costly but nutritious complementary weaning foods using our local available cereals and legumes through simple techniques (elemo et al., 2011). these food staples can be fermented to increase the nutrient content, reduce bulk water absorption capacity, improve their shelf life and be generally acceptable to infants at weaning age. the objective of the study is to formulate a weaning food blends from yellow maize, cowpea, bambaranut and groundnut flours and to assess their ph, titratable acidity, functional microbiological and sensory properties. 2. materials and methods 2.1 materials sources of yellow maize, cowpea, bambaranut and groundnut the yellow maize (improved variety), cowpea, bambara nut and groundnut were obtained and authenticated by a seed breeder/plant taxonomist in the lake chad research institute, and department of biological science, university of maiduguri respectively. source of commercial weaning foods the commercial weaning foods maize based cerelac and wheat based frisogold were purchased from a supermarket in maiduguru, borno state. it is recommended for infants of 6 months and above and it is a product of nestle nigeria plc. 2.2 methods preparation of “akamu” the akamu (ogi) was prepared by the method described by akingbala et al. (1981). one hundred (100 g) of maize (cereal) was cleaned and steeped into 200 ml of distilled water in a 1:2 ratio for 72 hours. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 62 published by scholink inc. at the end of the 72 hours, the top water was decanted and 200 ml of distilled water was added and milled into a slurry. the slurry was then sieved through a nylon cloth to separate the bran. the filtrate was then allowed to settle for 24 hours and the top water decanted. the akamu was sun-dried to a constant weight and was packed into airtight container and stored at 40c until used for weaning food formulation and analysis. preparation of cowpea one hundred (100 g) of the cowpea was cleaned and soaked in distilled water for 5 minutes. the cowpea was dehulled (using a mortar and pestle) and washed to remove the husk. it was then sun-dried to a constant weight roasted and ground into a fine powder as described by theodore et al. (2007). preparation of bambaranut one hundred (100 g) of dry bambara nut was cleaned, roasted and milled into a fine powder after which it was sieved using a sieve as described by theodore et al. (2007). preparation of groundnut one hundred (100 g) of groundnut was cleaned of dirt, roasted and dehulled. the dehulled groundnut was milled as described by davies (2009). formulation of the weaning diets cereal / legume diets were formulated using yellow maize, cowpea, bambara nut and groundnut in the following ratios; 1. 60 parts of yellow maize, 20 parts of cowpea, 10parts of bambaranut and 10 parts of groundnut. i.e., 60:20:10:10-mcbg. 2. 60 parts of yellow maize, 20 parts of cowpea, 20 parts of bambaranut. i.e., 60:20:20-mcb. 3. 60 parts of yellow maize, 20 parts of cowpea, 20 parts of groundnut. i.e., 60:20:20-mcg. 4. 60 parts of yellow maize, 20 parts of bambaranut, 20 parts of groundnut. i.e., 60:20:20-mbg. ph and titratable acidity (ta) during 72 hours of fermentation, ph and titratable acidity were measured. the ph of the supernatant was taken using a ph meter. for the titratable acidity, 10ml of sample was measured into a 50 ml beaker of phenolphthalein indicator was added and titrated against 0.1n naoh (egnan et al., 1981). calculation titratable acidity (g/100) = v x n meq.wt 1000 x v x 100 where v = volume of sodium hydroxide n = normality of sodium hydroxide meq. wt = mill equivalent weight of standard functional properties water absorption capacity (wac) one gramme (1 g) of each diet was weighed into a centrifuge tube and 10 ml of distilled water was added. samples were vortexed for 5 minutes and allowed to stand for 15 minutes at room temperature www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 63 published by scholink inc. before centrifuging (10,000 x c) for 5 minutes excess water was allowed to drain by inverting the tube over absorbent paper. the weight of sample bound to water was determined by difference (lin & humbent, 1974). apparent viscosity viscosity was determined by the methods of bhattachanga et al. (1986). viscosity (av) was determined by placing twenty grams (20 g) of the sample in measuring cylinder of 100 ml of water in a boiling water bath of 750c -800c. the slurry was constantly stirred and until boiling which was continued for five minutes. the slurry was cooled to room temperature 230c-250c and their viscosity was measured with a cannon viscometer. bulk density the bulk density was determined using the method of okezie and bello (1988). ten grams (10 g) of the sample material were placed in a 25 ml graduated cylinder and packed by gentle tapping of the cylinder on a bench top ten times from a height of 5-8 cm. the final volume of the test material was recorded and expressed as g/ml. microbiological analysis microbiological analysis was determined according to the method described by harrigan and mccaine (1976). appropriate dilution of samples was enumerated for counts of bacteria and yeasts using nutrient agar, macconkey agar, sabourraud dextrose agar and blood agar base. inoculated plates was incubated at appropriate time and temperature combinations. colonies of respective microbial types appearing in inoculated plates was counted and expressed as colony forming units (cfu/g). colonies of bacteria and yeasts was isolated and subcultured to obtain pure cultures. media preparation nutrient agar this is a general purpose medium which may be enriched with 10% blood or other biological fluid. it supports the growth of a wide range of microorganisms and contains sufficient nutrients for the organisms. procedure twenty grams (20 g) of nutrient agar (oxoid) was weighed and dissolved in 1.0 l of distilled water in a clean conical flask. it was brought to boil to dissolve completely and then sterilized by autoclaving at 1210c for 15 minutes. it was allowed to cool to 50-550c and them poured aseptically into sterile petri dishes and allowed to set. macconkey agar this is a differential medium for the isolation of coliforms and intestinal pathogens in water, dairy products and biological specimens. procedure fifty grams (52 g) of mcconkey agar was weighed into 1.0 l of distilled water in a clean conical flask. this was brought to boil to dissolve completely and sterilized by autoclaving at 121oc for 15 minutes. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 64 published by scholink inc. it was aseptically poured into sterile petri dishes. the surface of the gel was dried before inoculation. blood agar base forty two grams (42 g) of nutrient agar was dissolved in 1.0 l of distilled water; it was dissolved and sterilized in an autoclave at 121oc for 15 minutes. on cooling 10 ml of blood was added and poured aseptically into sterile petri dishes. sabouraud dextrose agar this is a general purpose medium for the cultivation of yeasts and moulds. sixty five grams (65 g) of sda was suspended in 1.0l of distilled water was boiled to completely dissolve and autoclaved at 1210c for 15 minutes and then cooled and aseptically poured into petri dishes. determination of total viable count after inoculation, the plates was incubated at 370c for 24 hours. the colonies obtained was counted on an electric colony counter (gallen kamp colony counter). isolation and identification a loopful of the sample was smeared over one corner of the solidified medium which was sufficiently dried. a ninchrome wire loop will be sterilized over a spirit lamp then cooled and used to make parallel streaks from the main inoculums. the plates was then incubated at 370c for 24 hours. the colonies was separated from one another based on the difference of colony monopoly. one of the separated colonies was taken using a sterilized wire loop and inoculated in another media then incubated for 24 hours at 370c. colonies was obtained on the medium after 24 hours. sensory evaluation a 10% (w/v) of the weaning food blends were cooked and evaluated by 50 nursing mothers using a nine point hedonic scole (appendix i) as described by land and shepard (1988). the mean scores were analysed by duncan’s multiple range test. statistical analysis all determinations were carried out in triplicates. all data collected were subjected to analysis of variance and duncan multiple range test was used to compare the means using spss 11.0 software. significance was accepted at p≤0.05. 3. results 3.1 ph and titratable acidity the results of ph and titratable acidity (ta) recorded during the fermentation of maize for “akamu” production is presented in table 1. fermentation was found to cause a sharp decrease in ph with time. the change in ph from zero to 72 hours resulted in a ph drop from initial ph of 6.30±005 to 3.00±0.11 at 72 hours. the titratable acidity increased from 0.30±0.05 to 4.73±0.02 during the 72 hours period of maize fermentation. both ph and ta are time dependent and the respective differences are statistically significant (p<0.05). www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 65 published by scholink inc. table 1. ph and titratable acidity (ta) at 72 hour fermentation of maize parameters time (hours) 0 24 48 72 ph 6.30±0.05a 4.65±0.21b 3.31±0.05c 3.00±0.11d titratable acidity (ta) 0.30±0.05d 0.65±0.03a 3.60±0.06c 4.73±0.02b values are recorded as mean ± sd of three determinations. means in the same row with different superscripts are significantly different (p<0.05). 3.2 functional properties of the raw and processed maize, cowpea bambaranut and groundnut and the weaning food blends functional properties of the raw and processed maize, cowpea, bambaranut and groundnut are presented in table 2. the functional properties of the weaning food blends are presented in table 3. apparent viscosity there was a significant (p<0.05) difference in the apparent viscosity of raw and processed cowpea, bambara nut at 30 shear rate. no significant difference (p<0.05) was observed between the raw and processed maize and groundnut at 30 shear rate. signifciant differences (p<0.05) were observed in the apparent visicosity of the weaning food blends mcbg, mcb, mcg and mbg. mcg (1452.70 cps) exhibited the highest viscosity followed by mcb (1442.50 cps) and then mbg (1420.20 cps). the weaning food blend mcbg (1008.60 cps) exhibited the lowest viscosity water absorption capacity the water absorption capacity of the raw and processed, maize, cowpea, bambaranut and groundnut exhibited significant (p<0.05) differences. roasted groundnut had the highest water absorption capacity while fermented maize had the lowest water absorption capacity. the water absorption capcity of the weaning food blends exhibited significiant differences (p<0.05). mbg (1.40 g/ml) is the highest followd by, mcg (1.30 g/ml), mcb (1-10 g/ml) and then mcbg (0.98 g/ml). mbg had the highest water absoption capcity while mcbg had the lowest water absorption capcity. bulk-density there was a significant(p<0.05) difference (p<0.05) in the bulk density of the raw and processed maize, cowpea, bambara nut groundnut and the weaning food blend. raw groundnut had the lowest bulk density, while fermented maize had the highest. the weaning food blend mcbg (1.20 g/ml) had lowest bulk density when compared with mcb (1.50 g/ml), mcg (1.60 g/ml) and mbg (1.80 g/ml) which were significantly higher than mcbg. mcb had the lowest bulk density followed by mbg and then mcg. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 66 published by scholink inc. table 2. functional properties of raw and processes maize, cowpea, bambara nut and groundnut functional properties samples maize cowpea bambara-nut groundnut raw fermented raw roasted raw roasted raw roasted apparent viscosity 30 shear rate 1005.33±0.28a 1005.5±0.10a 999.9±0.10b 1016.0±0.11c 1002.2±0.20e 1007±0.10d 999.7±0.10b 999.2±0.10b water absorption capacity (g/ml) 2.00±0.10b 0.90±0.10g 1.42±0.10c 1.40±0.10d 1.00±0.10f 1.20±0.05h 1.30±0.05e 2.60±0.01a bulk density (g/ml) 1.60±0.10c 1.90±0.05a 1.60±0.10c 1.60±0.05c 1.80±0.01b 1.60±0.05c 0.10±0.05d 1.80±0.10b values are recorded as mean ± sd of three determinations. mean in the same row with different superscripts are significantly (p<0.05) different. table 3. functional properties of the weaning food blends functional properties weaning food blends mcbg mcb mcg mbg apparent viscosity 30 shear rate 1008.70±0.10a 1442.50±0.020c 1452.70±0.07b 1420.20±0.10d water absorption capcity (g/ml) 0.98±0.02d 1.10±0.05c 1.30±0.10b 1.40±0.07a bulk density (g/ml) 1.20±0.02d 1.50±0.06c 1.60±0.10a 1.80±0.03b mcbg—60 parts of yellow maize, 20 parts of cowpea, 10 parts of bambaranut and 10 parts of groundnut mcb—60 parts of yellow maize, 20 parts of cowpea, 20 parts of bambaranut mcg—60 parts of yellow maize, 20 parts of cowpea, 20 parts of groundnut mbg—60 parts of yellow maize, 20 parts of bambaranut, 20 parts of groundnut total bacterial count and microorganisms isolated the total bacterial count during the production of “akamu” is presented in table 4. the total bacterial count at 0 hour was 28 x 103 cfu/ml which then dropped to 20 x 103, 11 x 103, 8 x 103 at 24 hours, 48 hours and 72 hours fermentation of maize respectively. the bacterial count of the slurry after 24 hours was 6 x 103 cfu/ml, 5 x 103 for dried akamu, 1 x 103 cfu/ml for mcbg, 3 x 103 cfu/ml for mcb, 7 x 103 cfu/ml for mcg and 3 x 103 cfu/ml for mbg. the microorganisms isolated from “akamu” production are shown in table 5. lactobacilus, escherichia coli and corynebacteria appeared after 24 hours of maize fermentation. lactobacillus streptococcus lactic, bacillus subtitilitis saccharomyces cerevisae appeared after the grain was milled into a slurry after 24 hours of fermentation. lactobacillus and saccharomyces cerevisae were present after the “akamu” was sun dried to a constant weight ; saccharomyces cerevisae were detected in www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 67 published by scholink inc. mcbg, lactobacillus in mcb, saccharomyces cerevisae and streptococcus lactics in mcg and saccharomyces cerevisae and lactacillus in for mbg. table 4. total bacterial count during production of “akamu” samples total bacterial count (cfu/ml) maize 0 hour 24 hours 48 hours 72 hours steep water 28 x 103 20 x 103 11 x 103 8 x 103 slurry 6 x 103 dried “akamu” 5 x 103 mcbg 1 x 103 mcb 3 x 103 mcg 7 x 103 mbg 3 x 103 table 5. microorganisms isolated during production of “akamu” sample microorganisms isolated maize 24 hours 48 hours 72 hours steep water lactobacillus lactobacillus lactobacillus escherichia coli streptoccus lactic streptococcus lactics corynebacteria e.coli bacteria slurry lactobacillus, streptoccus lactic bacillus substilitis, saccharomyces cerevisae dried “akamu” lactobacillus, saccharomyces cerevisae mcbg saccharomyces cerevisae mcb lactobacillus mcg streptococcus lactics saccharomyces cerevisae mbg saccharomyces cerevisae lactobacillus sensory evalaution of the weaning food blends the sensory evaluation of the weaning food blends is presented in table 6. no significant (p>0.05) differences were observed in the colour, odour, taste and overall acceptability of the formulated weaning food blends mcb, mcg and mbg. however, there was a significant (p<0.05) difference in the color, odour, taste, texture and overall acceptability of mcbg and the three weaning food blends, mcb, mcg and mbg. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 68 published by scholink inc. table 6. sensory evaluation of the weaning food blends parameters weaning food blends mcbg mcb mcg mbg colour 8.3±1.21a 7.6±2.50b 7.5±1.93b 7.0±1.91b odour 8.1±1.17a 7.3±2.48b 7.3±2.00b 7.7±0.98b taste 8.6±2.40a 7.7±2.56b 7.6±1.89b 6.8±2.41b texture 8.2±1.01a 6.9±2.44b 7.0±1.59c 6.9±1.12b overall acceptability 8.00±2.00a 7.8±2.52b 7.6±1.81b 7.1±2.14b 4. discussion 4.1 ph and titratable acidity (ta) fermentation was found to cause a gradual reduction in ph with time. the reduction in ph of maize during fermentation was similar to the result of other workers (elyas et al., 2002; nanson & fields, 2011; sanni et al., 1994). these results also agree with those obtained by gieze (1994), who reproted that as a result of fermentation acidity increased and ph falls and this enhanced the keeping quality of fermented foods, by inhibiting microbial growth and also contributing to the flavour of processed maize. nanson and fields (2011) reported that lactic acid fermentation causes a rapid drop in ph of various food grains. also in a related study, fermented dough is lowered due to the production of organic acids by the microflora; hetero fermentors were reported to convert glucose to equimolar mixture of lactic acid, ethanol and carbon dioxide (singh et al., 2012). titratable acidity increased with time over the entire fermentation period from 0.30±0.05 at 0 hour to 4.73±0.02 at 72 hours of maize fermentation. this finding is in agreement with the work of wakil and kazeem (2012). the increase in acidity is of great significance as it was reported to reduce the incidence of diarrhoea in infants consuming fermented maize porridge (mensah et al., 1990). functional properties apparant viscosity the weaning food blend mcbg had a lower viscosity followed by mcb, mcg and then mbg. decrease in apparant viscosity indicates increase in nutrient density. nkama et al. (2001) made similar observations. the significant reduction in viscosity with fermentation could be due to breakdown of macromolecules such as polysaccharides and polypeptides to smaller units, such as dextrins and peptides respectively by the enzymes mobilized during fermentation (gernah et al., 2012). low viscosity weaning diet with a high nutrient content is a desirable characteristics of weaning food (ariahu et al., 1996). during fermentation of cereals, microbial activity hydrolyses starch granules thereby resulting in reduced viscosity (chavan & kadam, 1989; nout et al., 1998). the action of microbial αand βamylases on the maize modify starch structures and results in low viscosity. this thereby leads to a reduction in dietary bulk which is an important factor in the aetiology www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 69 published by scholink inc. of protein -energy malnutrition (mbata et al., 2009b). water absorption capacity water absorption capacity indicates the volume of water required to form a gruel with suitable consistency for infant feeding (bintu et al., 2015; sodipo & fashakin, 2011). the result showed that the weaning food blend (mcbg) had lowest water absorption capacity followed by mcb, mcg and then mbg. ijarotimi and keshinro (2013) had similar findings. according to ghavidel and mehdi (2011), water absorption capacity gives an indication of the amount of water available for gelatinization. lower absorption capacity is desirable for making thinner gruels. the process of fermentation provides a simple inexpensive means of increasing nutrient density by reducing bulk. fermentation influenced the ability of the weaning food to absorb water (ikujenlola & fashakin, 2005). according to previous reports of barac et al. (2010) and ikujenlola and adurotoye (2014) fermentation activates the inherent amylase enzymes in grains; these enzymes saccharify / dextrinify the starch in the grains to dextrins and maltose which absorbs little water when cooked. weaning food blends with low water absorption capacity tend to have their microbial activities reduced (imtiaz et al., 2011). bulk density the significant reduction in bulk density of the weaning food blend (mcbg) could be attributed to the fact that fermentation tend to soften the seeds thus making milling easier with smaller particle size than the unprocessed grains, hence the reduction in bulk density (iwe, 2003). the significance of this is that the less bulky flours will have higher nutrient density, since more flour can be packaged in the same given volume (gernah et al., 2012). the bulk density is a reflection of the load the flour samples can carry, if allowed to rest directly on one another (wilhelm et al., 2004). values obtained from this study were comparable with the values reported by (singh et al., 2012). total bacterial count and microorganisms isolated fermentation of maize attracted a microflora of bacteria and yeast. lactate bacteria and yeast were predominant in the fermented sample. this could be as a result of a symbiotic relationship between lactate and yeast. it is assumed that the lactic flora provide an acidic condition for growth which yeast provide sufficient growth factors which enhances growth of lactate flora (bintu et al., 2015; ikemefuna, 1998). the microorganisms that were predominant were lactobaciullus and saccharomyces cerevisae. this is in agreement with the findings of abegaz et al. (2002), mbata et al. (2009a), serna-salde and rooney (1995). oyerekua (2011) observed species of sacchoromyces cerevisae in spontaneous lactic acid fermentation in cereals. the decrease of total bacterial counts in the weaning food blend might be due to mixed macro-flora of each cereal and legume that might also indicate a range of enzymes like glycoamylase which degrades starch and yeast and ferments parts of glucose thereby making lactic acid bacteria to co-exist with yeast in proto-cooperative manner. this agrees with the report of nout (2009), amodou et al. (2014) fermentation has also been strongly suggested to have inhibition effects on the groups of microorganisms that can cause spoilage or food poisoning (gernah et al., 2012). sensory evaluation www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 70 published by scholink inc. the result of the sensory evaluation indicates that the colour, odour, taste, texture and overall acceptability of the weaning food blends, mcb, mcg and mbg, showed no significant differences. in the overall acceptability, the weaning food blend mcbg had a highest overall acceptability which indicates like very much followed by mcb, mcg and mbg. this indicates that mcbg was preferred over mcb, mcg and mbg. 5. conclusion fermentation affected the microbiological composition and enhanced microbiological safety of the diet/blends by increasing dominance of lactic acid bacteria and inhibiting growth of pathogenic mciro-organisms and improved the nutrient density by reducing bulk. the sensory evaluation of mcbg was found to be superior in terms of over all acceptability than mcb, mcg and mbg. references akingbala, j. o., rooney, l. w., & faubion, j. m. 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(2012). storage and shelf life prediction of food formulations from malted and fermented maize (zea mays l) of fortified with defatted sesame (sesamun indiucum l). nig. j. nutr. sci., 32(1), 45-54. https://doi.org/10.4314/njns.v32i1.67814 ghavidel, r. a., & mehdi, g. d. (2011). processing and assessment of quality characteristics of composite baby food. world academy of science engineering and technology, 59, 2041-2043. gieze, j. (1994). antimicrobial food safety food technology, 48, 102-110. harrigan, w. f., & mccaine, m. e. (1976). laboratory methods in food and dairy microbiology. academic press london, ny, san francisco. ijarotimi, o. s. (2012). influence of germination and fermentation on chemical composition, protein quality and physical properties of wheat flour (triticumaetivum). j. cereals oilseeds., 3(3), 35-47. ijarotimi, o. s., & keshino o. o. 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(2012). effect of fermentation on physicochemical properties and in vitro starch and protein digestibility of selected cereals. international journal of agricultural and of food science, 2(3), 66-70. sodipo, m. a., & fashakin. j. b. (2011). physico-chemical properties of a complementary diet prepared from germinate maize, cowpea and pigeon pea. journal of food agriculture and environment, 9, 23-25. theodore, i., mbata, m. j., ikeheboneh, i., & ahonkai, s. (2007). nutritional status of maize fermented meal by fortification with bambara nut. ajfand, 7(2), 44-61. wakil, s. m., & kazeem, m. o. (2012). quality assessment of weaning of food produced from fermented cereal legume blend using starter. international journal of food research, 19(4), 1679-1685. wilhelm, r. l., dwayna, a. s., & gerand, h. b. (2004). introduction to problem solving skills in food and processing engineering technology asae. world bank. (2012). infant and young child feeding: world bank nutrition. retrieved january 2013, from http://www.report.siteresources.worldbank.org/nutrition/resources/tool2-chap5.pdf food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 4, 2019 www.scholink.org/ojs/index.php/fsns 142 original paper a synergistic sensitized fluorescent determination of 2,4-dichlorophenoxyacetic acid in vegetable samples based on the derivatives of calix[4]arene xiashi zhu1,2* & qiuyi ren2 1 college of guangling, yangzhou university, yangzhou 225002, pr china 2 college of chemistry & chemical engineering, yangzhou university, yangzhou 225002, pr china * xiashi zhu, college of guangling, yangzhou university; college of chemistry & chemical engineering, yangzhou university, yangzhou 225002, pr china received: october 24, 2019 accepted: november 5, 2019 online published: november 18, 2019 doi:10.22158/fsns.v3n4p142 url: http://dx.doi.org/10.22158/fsns.v3n4p142 abstract a novel fluorescent determination of 2,4-dichlorophenoxyacetic acid (2,4-d) based on the derivatives of calix[4]arene (sax) in β-cyclodextrin(β-cd)/ sodium dodecyl sulfate (sds) synergistic sensitized system was developed. the results were shown that the fluorescence intensity of sax could be quenched by 2,4-d, and the fluorescence quenching (∆f=fsax-f2,4-d -sax) was synergistic sensitized in β-cd/ sds medium. under the conditions of λex/em=332/468 nm and ph 7.0, the linear range for 2,4-d were found to be 0.020-4.00 μg/ml. the mechanism of determination was discussed with quenching type analysis, inclusion interaction and sensitizing effect. this method has been applied for the determination of 2,4-d in vegetable samples with satisfactory results. keywords 2,4-dichlorophenoxyacetic acid, β-cyclodextrin, sds, synergistic sensitization, fluorescence quenching 1. introduction 2,4-dichlorophenoxyacetic acid (2,4-d, figure 1(a)) belongs to the category of benzoic acid pesticides, which has the biological activity of auxin and can be used as plant growth regulator and preservative for vegetable and fruit (jiang, zha, & tie, 2015). nevertheless, 2,4-d residues in agricultural products and environment have great harm to human health due to its carcinogenic, mutagenic and estrogenic activity (garabrant & philbert, 2002). up to now the reported techniques for 2,4-d determination have been performed on lc-ms (jiang, zha, & tie, 2015), fluorescence spectroscopy (wang, yua, & wu, 2016; www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 143 published by scholink inc. boroduleva & eremin, 2016; atta, bera, & chattopadhyay, 2015), high performance liquid chromatography(hplc) (wu, ee, & lee, 2005), capillary electrophoresis (ce) (zhu & lee, 2001) and gas chromatography(gc) (rezazadeh, yamini, seidi, tahmasebi, & rezaei, 2014). although these techniques have good performance, they are complicated, expensive and time-consuming. so it is necessary to establish a rapid, simple and high selectivity method for 2,4-d detection. calixarenes are macrocyclic compounds through a series of phenol connected with the ortho methylene units. in calixarene molecule, the upper edge is composed of para-position substituent of benzene; the lower edge is formed by neatly arranged phenolic hydroxyl group; the middle hydrophobic cavity is composed of benzene rings. schiff base calix[4]arene (sax, figure 1(b)) has been followed with great interests due to the simple structure and high symmetry. the cavity of sax is composed of four benzene rings, meeting the size and stability required for the inclusion interaction. the analytical method based on host-guest chemistry of calix[4]arene derivatives have been reported (ma & zhu, 2012; yang, yan, & zhu, 2014; wang, zhu, & yan, 2013; yang, qin, yan, & zhu, 2015; li, x. y., li, m., & chen, 2011; khan, shah, & ahmed, 2016). but the fluorescence quenching methods using schiff base calix[4]arene derivatives as a fluorescent chemosensor for the determination of 2,4-d seems to be lacking. (a) (b) figure 1. chemical structure of (a)2,4-d and (b)sax the sensitivity of spectral analysis could be improved in suitable medium, such as surfactant (ma & zhu, 2012; yang, yan, & zhu, 2014; wang, zhu, & yan, 2013; yang, qin, yan, & zhu, 2015; li, x. y., li, m., & chen, 2011), β-cyclodextrin (β-cd) (sanchez, rubio, & blanco, 1988; márquez, hernández, & garcía, 1990; zhang, liu, & fan, 2009; sánchez, lopez, & gómez, 1987; zhu, sun, bao, & guo, 2006; sun, zhu, & wu, 2007) room temperature ionic liquid (liu & zhao, 2008; berton & martinis, 2009; martinis & olsina, 2008; zhu & jiang, 2011). in our previous publications, the sensitizing effects of surfactant (wu, ee, & lee, 2005; zhu & lee, 2001; rezazadeh, yamini, seidi, tahmasebi, & rezaei, 2014; ma & zhu, 2012), β-cd and its derivatives (zhu, sun, bao, & guo, 2006; sun, zhu, & wu, 2007), ionic liquids (ils) (zhu & jiang, 2011) on the ultraviolet spectrometry and spectrofluorimetry www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 144 published by scholink inc. were developed. further research showed that mixed medium (such as surfactant/ils, β-cd/surfactant) could synergistically sensitize fluorescence method, which has a better sensitization effect than that single medium (zhu, sun, bao, & guo, 2006; zhu & jiang, 2011; ren & zhu, 2016). in this study, the fluorescence intensity of sax could be quenched by 2,4-d, the fluorescence quenching value (∆f=fsax-f2,4-d-sax) were enhanced in β-cd/sds due to the synergistic sensitization, which has a much better quenching effect than that in single β-cd or sds medium. there was a linear relationship between fluorescence quenching value (∆f) and concentration of 2,4-d, a novel β-cd/sds synergistic sensitized fluorescence quenching method for the determination of 2,4-d was successfully developed. the mechanism of determination was also investigated. the proposed method was applied to analyte 2,4-d in real samples with satisfactory results. 2. experimental reagents and instruments schiff base calix[4]arene was synthesized according to the published methods (bi, sun, & yan, 2012). 0.01% sax (m=1328.7 g/mol, c = 7.5×10-6 mol/l) was prepared in ethanol. 100.0 µg/ml stock solution of 2,4-dichlorophenoxyacetic acid (2,4-d) was prepared by dissolving 0.100g 2,4-d in 100 ml volumetric flask and diluting with anhydrous ethanol to scale. the stock solutions were further diluted with anhydrous ethanol to obtain a standard working solution of 10.0 µg/ml for experiment. 1.0% β-cd solution was prepared by dissolving 1.00g of β-cd in 100.0 ml with distilled water. 1.0% sds solution was prepared by dissolving 1.00g of sds in 100.0 ml volumetric flask with distilled water. and ph=7.0 ch3coonh4 buffer solution was employed. all the fluorescence measurements were performed on a hitachi f-4500 spectrofluorimeter (japan) with excitation and emission slits at 10.0 nm and 5.0 nm, λex=332 nm. the ph was measured on a ph fe20 ph meter (mettler toledo). a uv 2501 spectrophotometer (shimadzu, japan) was used for all absorption spectral recordings and absorbance measurements . 3. experiment method fluorescence measurements. in centrifuge tube (5.0 ml), 2.0 ml 0.01% sax, 1.0 ml ch3coonh4 buffer solution (ph = 7.0), 0.5 ml 1.0% β-cd solution, 0.5 ml 1.0% sds and 0.5 ml reference substance solution of 2,4-d (10.0 μg/ml) were added and then diluted to the scale with distilled water. then fluorescence spectra was recorded in the range of 300650 nm with excitation at 332 nm. quenching type analysis (gong, zhu, & hu, 2007). 2.0 ml 0.01% sax solution, 1.0 ml buffer solution, 0.5 ml 1.0% β-cd, 0.5 ml 1.0% sds and different amount of 10.0 μg/ml 2,4-d solutions were added into 5.0 ml centrifuge tube, then diluted to the scale with distilled water and mixed completely. the fluorescence intensity of sax was measured at different temperature (288 k, 298 k and313 k), respectively. quenching type could be analyzed by stern-volmer eq.(1): www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 145 published by scholink inc. qq ckkc f f 0q 0 11  f0 and f were the fluorescence intensities of sax in the absence and presence of 2,4-d respectively, k was the stern–volmer quenching constant, cq was the concentration of quencher 2,4-d, kq was the quenching rate constant, τ0 was the average lifetime of the sax without 2,4-d. if the quenching type is single static or dynamic quenching, the curve of f0/f versus cq (stern–volmer curve) would be linear within certain concentration. absorption spectrum titrations (fu, zeng, & mu, 2012). the recognition ability of sax and β-cd to 2,4-d can be evaluated through the change of absorption spectrum. the absorption spectrum titrations of 2,4-d with sax and β-cd was made in the range of 200.0-600.0 nm. the absorbance of 2,4-d was measured with nsax: n2,4-d and nβ-cd: n2,4-d. inclusion interaction. the solution of a certain amount of sax, 1.0 ml buffer solution and different amount of 10.0 μg/ml 2,4-d solutions were added into 5.0 ml centrifuge tube, then diluted to the mark with distilled water and mixed thoroughly. the fluorescence intensity was measured at 25℃, then the benes-hildebrand method (vimal, ajay, & narinder, 2008) (double reciprocal plot) was used to calculate the inclusion constant (k) of sax-2,4-d and β-cd-2,4-d assuming a 1:1 inclusion model. the benesi-hildebrand method is a spectroscopic method to determine the inclusion constants of the host guest complexes (including fluorescence spectroscopy and absorption spectroscopy). the equation is as follows (sax as an example):        00 14,2111 saxdsaxkf   where [sax]0 was the concentration of sax, ∆f was the quenching value of fluorescence intensity, α was a constant. thus, the inclusion constant (k) of the 1:1 inclusion complex could been calculated by dividing the intercept by the slope of the double reciprocal plot. determination of critical micelle concentration (cmc). cmc values of the medium were measured by conductivity measurements. the cmc was obtained from the inflection point of the straight lines of before and after micellar concentration range (kumaraguru & santhakumar, 2006; mehta, bhawna, & ram, 2010). determination of fluorescence quantum yield. fluorescence quantum yields of sax were measured using 1.0×10−6 g/ml quinine sulfate as reference substance (zhao & wei, 2006; zhu, gong, & yu, 2008). under the same apparatus conditions, the quantum yield of the sax was calculated. kinetics of the reaction. the kinetics of the reaction could be described by the first order kinetic model, the second kinetic model and the weber maurice diffusion model (azizian & fallah, 2010; wang, wei, & li, 2015). in this study, the first order kinetic fitting of the quenching process was carried out. sample preparation. vegetable samples (green vegetables, chinese cabbage and chrysanthemum coronarium) were purchased from local market. a certain amount of each vegetable sample was cut up www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 146 published by scholink inc. and homogenized. then, 20 g of each sample was weighed and placed in a 50 ml centrifuge tube and 50 ml ethanol was added as well. then tighten up the lid, dipped for 1 hour, shaken thoroughly for 20 min. after centrifugation 10 min (5000 r/min), the test sample solution was prepared after the upper solution was filtered. in order to reduce the fluorescence background, the sample solution was diluted 15 times and then used as an analytical sample (farokhcheh & alizadeh, 2013). 4. results and discussion choice of medium. the effect of different medium on ∆f (∆f = fsax− f2,4-d–sax) was studied. as can be seen in figure 2 that the sequence of ∆f was ∆fβ-cd-sds>∆fsds>∆fβ-cd > ∆fh2o. the fluorescence quenching value in β-cd/ sds synergistic sensitized medium was greater than that in single β-cd or sds medium. so β-cd-sds medium was selected for further experiment. 1 2 3 4 0 100 200 300 400 500 f h 2 o sds -cd -cd-sds figure 2. effect of different medium on fluorescence intensity fluorescence spectra. the fluorescence emission spectrum of sax (present or absent of 2,4-d) in β-cd-sds and h2o medium were shown in figure 3. it can be seen that (1) the fluorescence intensity of sax (fsax) was enhanced in β-cd/sds medium (curves a and c); (2) the fluorescence intensity of sax (fsax or fsax-β-cd-sds) was quenched when 2,4-d was added (curves b and d) and gradually diminished with the increase concentration of 2,4-d (inset figure 3); (3) ∆f= fsax-β-cd-sds – f2,4-d-sax-β-cd-sds was larger than that ∆f’=fsax-f2,4-d-sax with the same concentration of 2,4-d, which was the synergistic sensitizing effect in β-cd/sds. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 147 published by scholink inc. 350 400 450 500 550 600 650 0 500 1000 1500 2000 2500 3000 350 400 450 500 550 600 650 0 500 1000 1500 2000 2500 3000 f nm 1 8 f ' nm f a d f figure 3. fluorescence spectra (a). sax-β-cd-sds (b). 2,4-d-sax-β-cd-sds (c). sax-h2o (d). 2,4-d-sax-h2o inset 1-8: 2,4-d-sax-β-cd-sds, [2,4-d]: (1) 0 µg/ml, (2) 0.02µg/ml, (3) 0.08µg/ml, (4) 0.1µg/ml, (5)0.2µg/ml, (6) 0.4µg/ml, (7) 0.8µg/ml, (8) 1.0 µg/ml effect of ph. the influence of ph on ∆f was investigated. as could be seen in figure 4, ∆f gradually increased with the increase of ph and reached maximum at ph = 7.0, but it diminished at ph > 7.0. the reason may be related to the formation of sax-2,4-d inclusion, which will be discussed in section 3.13.2. so 1.0 ml of ph = 7.0 ch3coonh4 buffer solution was chosen for the further study. 4 5 6 7 8 9 10 0 20 40 60 80 100 ph f(a.u) figure 4. effect of ph on fluorescence quenching value effect of sax amount. the effect of the amount of sax was studied in fig. 5. it was shown that the ∆f was increased and reached a maximum value at a sax (7.5×10-6 mol/l) amount of 2.0 ml, and then decreased (curve 1). this was because that the fsax-β-cd-sds gradually decreased with the increase of sax amount due to the self-quenching of sax at higher concentration (curve 2). thus, 2.0 ml sax (7.5×10-6 mol/l) was selected for the optimized method. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 148 published by scholink inc. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 20 40 60 80 100 120 140 160 180 200 sax/ml f(a.u) 0 500 1000 1500 2000 2500 3000 f 2 1 figure 5. effect of the amount of sax on fluorescence quenching value (left axis) and fluorescence intensity of sax (right axis) (1: fluorescence quenching value, 2: fluorescence intensity of sax) effect of β-cd amount. effect of β-cd was investigated. as is shown in fig. 6, with the increase of β-cd (1.0%) amount, ∆f gradually increased and reached the maximum when the β-cd amount was 0.50 ml, but decreased when β-cd amount was more than 0.50 ml. the change trend of ∆f could be explained from the change of fsax-β-cd-sds and f2,4-d-sax-β-cd-sds with β-cd amount (inset figure 6). (1) fsax-β-cd-sds gradually increased with β-cd amount while f2,4-d-sax-β-cd-sds slowly decreased (vβ-cd = 0-0.50 ml) ( ∆f↑= fsax-β-cd-sds↑ -f2,4-d-sax-β-cd-sds↓); (2) fsax-β-cd-sds gradually decreased, while fcr-sax-m-β-cd-tx-100 remain unchanged ( vβ-cd = 0.50-0.80 ml) (∆f↓= fsax-β-cd-sds↓ -f2,4-d-sax-β-cd-sds). therefore, 0.50 ml 1.0% β-cd was chosen for the following experiments. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 20 40 60 80 100 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 500 1000 1500 2000 2500 3000 3500 2,4-d-sax--cd-sds sax--cd-sds -cd/ml f f(a.u) -cd/ml figure 6. effect of the amount of β-cd on fluorescence quenching value effect of sds amount. effect of sds was studied. as is shown in figure 7, ∆f gradually rose up with the increase of sds (1.0 %) amount and up to the maximum when the sds amount was 0.50 ml, but ∆f decreased when sds amount was greater than 0.50 ml. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 149 published by scholink inc. the change of fsax-β-cd-sds and f2,4-d-sax-β-cd-sds with sds amount (inset figure 7) could declare the change of ∆f with sds. (1) fsax-β-cd-sds obviously increased with sds amount was larger than f2,4-d-sax-β-cd-sds ( vsds = 0-0.30 ml)( ∆f↑= fsax-β-cd-sds↑↑ f2,4-d-sax-β-cd-sds↑); (2) fsax-β-cd-sds continuously increased, while f2,4-d-sax-β-cd-sds gradually decreased ( vsds= 0.30–0.50 ml) (∆f↑= fsax-β-cd-sds↑-f2,4-d-sax-β-cd-sds↓); (3) fsax-β-cd-sds gradually decreased, while f2,4-d-sax-β-cd-sds changed rarely (vsds= 0.50–0.80 ml) (∆f↓= fsax-β-cd-sds↓ -f2,4-d-sax-β-cd-sds). therefore, 0.50 ml 1.0 % sds was chosen for the further study. in conclusion, the ∆f was biggest when mass ratio or molar ratio of β-cd and sds are 1:1 and 1:4, respectively, which illustrated that β-cd and sds showed the synergistic sensitized effect on ∆f. either excess β-cd or sds will weaken the synergistic sensitized effect, resulting the decrease of ∆f. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 20 40 60 80 100 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 500 1000 1500 2000 2500 3000 3500 2,4-d-sax--cd-sds sax--cd-sds sds/ml f sds/ml f(a.u) figure 7. effect of the amount of sds on fluorescence quenching value effect of reaction time. the effect of reaction time on ∆f was tested. it was found from figure 8 that ∆f obviously increased from 10 min to 30 min, then ∆f remained unchanged after 30 min. therefore, 30 min of reaction time was chosen for the following experiments. according to the data ∆f-t, the fluorescence quenching reaction kinetics could be discussed. in this study, the first order kinetic fitting of the quenching process was carried out. as shown in inset fig.8, the curve of lnc2,4-d versus t was linear and r2 was 0.9960. the reaction rate constant was 1.14×10-1 min-1. the results show that the fluorescence quenching process followed the first order kinetic model. the reason may be that the amount of sax is excess in this system, so the rate of fluorescence quenching reaction is only in relation to the concentration of 2,4-d. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 150 published by scholink inc. 0 20 40 60 80 100 0 20 40 60 80 100 5 10 15 20 25 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 t/min ln(ca) f(a.u) t/min first order kinetics model figure 8. effect of reaction time on fluorescence quenching value effect of temperature. the effect of temperature (5-50℃) on ∆f was investigated. as can been seen from figure 9 that ∆f was increased from 5 to 25℃ then it decreased with the increase of temperature. thus, the suitable temperature of 25℃ was chosen for the study. 0 10 20 30 40 50 0 20 40 60 80 100 t/℃ f(a.u) figure 9. effect of temperature on fluorescence quenching value effect of foreign substances. the effect of foreign substrates was discussed for the determination of 1.0 μg/ml 2,4-d. with a relative error of less than ±5%, the majority of these substances showed no remarkable interference in the determination of 2,4-d (table 1). table 1. effect of interfering substances foreign substances foreign substances / 2,4-d(w/w) foreign substances foreign substances / 2,4-d(w/w) k+ 500 al3+ 20 na+ 1200 mn2+ 2 ca2+ 1000 cl1800 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 151 published by scholink inc. mg2+ 300 no3 700 ba2+ 100 so4 2300 zn2+ 200 glucose 500 fe3+ 2 sucrose 500 cu2+ 2 glycine 200 analytical performance. under the optimum conditions, the linear regression equations were determined to be: δf = 10.14 + 2672.1c (μg/ml) in the range of 0.020~0.10 μg/ml, r = 0.9972, the detection limit estimated (s/n = 3) was 0.23 ng/ml; δf = 331.08 + 290.87c (μg/ml) in the range of 0.10μg/ml ~ 4.0 μg/ml, r = 0.9918, the detection limit estimated was 2.8 ng/ml. the relative standard deviation (rsd) was 0.96% (n = 3, c = 1.0μg/ml). sample analysis. the proposed method was successfully applied for the determination the amount of 2,4-d in vegetable samples. the data were listed in table 2. the recovery ratio ranged from 95.0% ~ 105.5% which was satisfactory. table 2. determination results of 2,4-d samples added (μg/g) found（μg/g） recovery (%) chinese cabbage 0.0 nd 0.50 0.53 105.5 1.0 0.95 95.5 2.0 1.9 98.2 green vegetables 0.0 nd 0.50 0.48 95.0 1.0 2.0 95.7 2.0 2.1 102.0 chrysanthemum coronarium 0.0 nd 0.50 0.52 104.9 1.0 0.96 96.5 2.0 2.1 103.4 comparison of different methods. the results obtained from this experiment were compared with those previously reported methods for 2,4-d determination (table 3). the advantages of the proposed method are: easy operation, high sensitivity, low detection limit and high recovery rate. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 152 published by scholink inc. table 3. comparison with previously reported methods methods linearity range lod recovery reference hplc-ms 0.025-1.0 mg/l 0.005 mg/kg 88.3-95.4% (jiang, zha, & tie, 2015) shplc 1.0-500 ng/ml 0.3 ng/ml 105-116 % (wu, ee, & lee, 2005) ce 3.0-500 ng/ml 0.02 ng/ml (zhu & lee, 2001) gc 10-500 ng/ml 5.0 ng/ml (rezazadeh, yamini, seidi, tahmasebi, & rezaei, 2014) fluorescence quenching calix[4]arene methods 0.020-4.0 µg/ml 2.8 ng/ml 95.0-105.5% this method 5. discussion of mechanism in this paper, the discussion of mechanism was included quenching type analysis, inclusion interaction and sensitizing effect. quenching type. quenching types can be divided into static quenching and dynamic quenching. the static quenching is caused by the formation of non or weak fluorescent compound (gong, zhu, & hu, 2007). the dynamic quenching is initiated from the collision of fluorescence substance and quencher, resulting in the decrease of fluorescence intensity and quantum yield. quenching type could be discussed with ksv and kq. with the increasing temperature, ksv would be decreased for static quenching, while ksv would be increased for dynamic quenching (gong, zhu, & hu, 2007). the stern-volmer plots of sax with 2,4-d at different temperature (288 k, 298 k and 313 k) was shown in figure 10. the order of ksv at different temperature were ksv 288k =3.90×104 > ksv 298k =3.45×104 > ksv 313k=3.04×104 l/mol. it demonstrated that the fluorescence quenching mechanism of sax by 2,4-d was a static quenching procedure and a complex was formed between sax and 2,4-d. 0.0 0.5 1.0 1.5 2.0 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 c 2,4-d (g/ml) f0/f a b c figure 10. the stern-volmer curves for the binding of 2,4-d with sax at 288 k (a), 298 k (b) and 313 k (c) www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 153 published by scholink inc. inclusion interaction. from the previous discussion, the type of fluorescence quenching was static quenching, which may be caused by the formation of non or weak fluorescent compound. sax and β-cd have a cavity structure, both of them are likely to form inclusion interaction with 2,4-d. the recognition ability of sax and β-cd to 2,4-d can be evaluated through the change of absorption spectrum. the results of an absorption titration at λ = 281.0 nm (characteristic absorption peak of 2,4-d) (figure 11) was shown that the absorbance of 2,4-d was gradually increased with the increase of sax or β-cd and was unchanged when nsax:n2,4-d = 1:1 or nβ-cd:n2,4-d = 1:1, which implied a 1:1 stoichiometry for binding between sax and 2,4-d or β-cd and 2,4-d. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 n/n2,4-d a 1 2 figure 11. absorption spectrum titrations of 2,4-d with sax and β-cd (1: 2,4-d-sax, 2: 2,4-d-β-cd) the inclusion constant (k) of sax-2,4-d and β-cd-2,4-d could be obtained by benesi-hildebrand method (vimal, ajay, & narinder, 2008). the larger the value of k, the more steady the inclusion complex. as is shown in figure 12, double reciprocal plots of sax-2,4-d and β-cd-2,4-d have good linear relationship, which supports the formation of a 1:1 complex. the calculated inclusion constant (k) was listed in table 4. as can be seen in table 4: (1)k sax-2,4-d >>kβ-cd-2,4-d, which suggested that inclusion complex of sax-2,4-d is more stable than β-cd-2,4-d. sax plays a major role in inclusion interaction with 2,4-d, which bringing about static fluorescence quenching. (2) k sax-2,4-d > k sax-2,4-d-β-cd , the inclusion constant of sax-2,4-d was decreased in presence of β-cd, which is caused by the formation of β-cd-2,4-d complex; (3) k sax-2,4-d, ph 7.0 > k sax-2,4-d, ph 4.5 > k sax-2,4-d, ph 10.0, which indicated that ph had a significant effect on the formation sax-2,4-d and the inclusion complex of sax-2,4-d is more stable at ph 7.0 than in acidic or basic solution; (4) the larger the k, the greater the ∆f, the largest fluorescence quenching value ∆f was at ph=7.0, which is in accordance with the discussion about effect of ph on ∆f in section 3.3. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 154 published by scholink inc. 0 200000 400000 600000 800000 1000000 1200000 0.000 0.002 0.004 0.006 0.008 0.010 0.012 1/c 1/f a 0 200000 400000 600000 800000 1000000 1200000 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 1/f 1/c b 0 1000 2000 3000 4000 5000 0 200 400 600 800 1000 1/c a c figure 12. benesi-hildebrand plot (a: sax-2,4-d, b: sax-2,4-d-β-cd, c: β-cd-2,4-d) table 4. results of inclusion constant system inclusion constant k（l/mol） ph 4.5 ph 7.0 ph 10.0 sax-2,4-d 5.71×104 5.55×1010 2.00×104 sax-2,4-d-β-cd 2.21×109 β-cd-2,4-d 262.47 sensitizing effect. the discussion of sensitizing effect was included the formation of the ordered molecular assembly and fluorescence quantum yield. the formation of the ordered molecular assembly the synergistic sensitized effect of β-cd/sds may be from the formation of the ordered molecular assembly. the interaction of sds and β-cd can change the critical micelle concentration (cmc). by measuring the cmc of a single and mixed medium, it can be determined whether the β-cd/sds has formed a new ordered molecular assembly (zhu & jiang, 2011). the value of cmc can be obtained from the inflection point of the straight lines of before and after micellar concentration range (kumaraguru & www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 155 published by scholink inc. santhakumar, 2006). the results were summarized in figure 13 and table 5. as is shown in figure 13, the cmc of β-cd and sds/β-cd were 3.0-5.0 mmol/l and 5.0-6.0 mmol/l, respectively. the cmc of the mixed medium model could be calculate by the clint model based on the assumption of ideal mixture behavior. as is shown in table 5, the cmc of sds is 8.4 mmol/l (mehta, bhawna, & ram, 2010), the molar ratio of sds and β-cd was 4:1. thus, the cmc of the mixed solution should between 7.8-8.1 mmol/l. there was a difference between the cmc of the mixed medium (β-cd/sds) in determined value and the clint model calculated value. this result illustrated that the β-cd/sds mixed medium formed a new ordered molecular assembly. what’s more, the sensitivity of the determination could be enhanced by the new ordered molecular assembly. 0 2 4 6 8 10 12 14 16 18 0 200 400 600 800 1000 concentration mmol.l-1 th e el ec tr ic c o n d u ci ti vi ty (  s. cm -1 ) a b figure 13. electrical conductivity vs. the concentration of β-cd (a: electrical conductivity vs. the concentration of β-cd, b: electrical conductivity vs. the concentration of sds/β-cd) table 5. cmc of the different medium medium cmc (mmol/l) determination value clint model value sds 8.4a β-cd 3.0-5.0 sds/β-cd (4:1) 5.0-6.0 7.8-8.1b a: the literature (mehta, bhawna, & ram, 2010); b: calculate by clint model fluorescence quantum yield. the fluorescence quantum yield represents the ability of translation of absorption energy to fluorescence. it is one of the basic and significant parameters for fluorescence substances. what’s more, ∆f = fsax – f2,4-d-sax, where ∆f must rise up with the increase of fluorescence quantum yield of sax. the fluorescence quantum yield of sax (y) in the medium of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 156 published by scholink inc. h2o，β-cd，sds and β-cd-sds were listed in table 6. as can be seen in table 6, the order of fluorescence quantum yield of sax in different medium is yβ-cd-sds > ysds > yβ-cd > yh2o. it is in accord with the sensitivities in different medium. the results could be considered with internal and external environment. the fluorescence quantum yield of sax in β-cd-sds medium was increased obviously. it was because that the external environment provided by β-cd/sds mixed medium is more favorable on solubilization than that in single medium (β-cd or sds). moreover, the microenvironment which was formed by the new ordered molecular assembly β-cd/sds could provide the protective environment for the singlet excited state and could reduce the non-radiation of the fluorescent substance sax. in other words, both self fluorescence quenching of sax and fluorescence quenching of external quenchers can be decreased in the medium of β-cd/sds. as a result, the fluorescence quantum yield of sax in the mixed medium is largest due to the synergistic sensitizing effect of β-cd-sds. table 6. the fluorescence quantum yield of 2,4-d medium y h2o 0.011 β-cd 0.038 sds 0.094 β-cd-sds 0.108 in summary, the mechanism of this method was (1) the fluorescence quenching of sax is due to the inclusion interaction of sax-2,4-d; (2)the sensitizing effect is that sds/β-cd medium could form a new ordered molecular assembly, in which fluorescence quantum yield of sax increased obviously, resulting synergistic sensitizing effect on fluorescence quenching value. 6. conclusion in this study, a novel fluorescence quenching method for the determination of 2,4-d has been developed. the fluorescence intensity of sax was quenched due to inclusion interaction between sax and 2,4-d, and the fluorescence quenching value (∆f) was increased in β-cd-sds medium. the proposed method has been applied for the determination of 2,4-d in vegetable samples with satisfactory results. funding the national natural science foundation of china (21375117) and the priority academic program development of jiangsu higher education institutions are acknowledged for funding this research. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 4, 2019 157 published by scholink inc. references atta, s., bera, m., & chattopadhyay, t. 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(2006). effect of β-cyclodextrin-microemulsion on determination of trace bi(ⅲ) by spectrofluormietry. chin. j. appl. chem., 23, 323-328. food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 3, no. 2, 2019 www.scholink.org/ojs/index.php/fsns 39 original paper functional properties of sausage rolls made from cocoyam and wheat flour enriched with soybean flour peter-ikechukwu, a.i.1, ibeabuchi, j.c.1, eluchie, c.n.1, agunwah, i.m.1, aneke, e.j.1, chukwu, m.n.2, ogbuagu, j.c.1 & okafor, d.c.1* 1 department of food science and technology, federal university of technology, owerri p.m.b. 1526 owerri, imo state, nigeria 2 department of food technology, abia state polytechnic, aba, abia state, nigeria * okafor, d.c., department of food science and technology, federal university of technology, owerri p.m.b. 1526 owerri, imo state, nigeria received: november 6, 2018 accepted: november 25, 2018 online published: march 9, 2019 doi:10.22158/fsns.v3n2p39 url: http://dx.doi.org/10.22158/fsns.v3n2p39 abstract functional properties of sausage rolls made from cocoyam and wheat flour enriched with soybean flour was studied. cocoyam cormels and soybean were processed into flour, which were later used to formulate composite flour blends, with wheat flour in the ratio of: 90:10:0 (control 1), 80:10:10, 70:10:20, 60:10:40, 50:10:30, respectively, while 0:10:90 served as control 2. the result of the functional properties showed variation in behavior. there were no significant difference (p≥0.05) in ph, bulk density, swelling index, foam capacity and emulsion capacity of the flour samples while significant difference (p≤0.05) existed in water absorption capacity, oil absorption capacity and wettability. keywords composite flour, amino acids, bulk density, swelling index, oil absorption capacity 1. introduction sausage roll is a british savory snack, popular in commonwealth nations and beyond (http://www.wikipedia.com/what-is-sausage-roll/). the basic composition of a sausage roll is sheets of puff pastry formed into tubes around sausage meat. the pastry is glazed with egg or milk before being baked (http://www.foodnetwork.com/sausage-roll-recipe/html). sausage roll in the modern sense of meat, surrounded by rolled pastry, appears to have been conceived at the beginning of 19th century in france. sausage rolls are popular and widely consumed all over the world by people of all ages. they are traditionally made from wheat, a cereal, which is cultivated in many parts of the world. wheat is www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 40 published by scholink inc. imported by countries with unfavorable climatic conditions (aziah et al., 2009). hence, such importing countries spend a lot of foreign exchange on importation of wheat. ndife et al. (2011) reported that the growth of wheat crops could be affected by natural disasters such as hurricane, flood etc which will in turn affect the yield and price of wheat. ojinnaka and nnorom (2015) also reported that many farmers are beginning to switch from growing wheat to growing “more lucrative” crops (like corn and soy beans), which could be used in the production of bio-fuels. therefore, it is important to develop an adequate substitute for wheat. in the quest for a wheat substitute, flour with improved nutritional quality than wheat would be highly desirable, especially in developing countries where malnutrition is prevalent. composite flour can be defined as a mixture of different ratios of non-wheat flours obtained from roots and tubers, cereals, legumes, etc., with or without the addition of wheat flour (okpala & okoli, 2011). composite flour is considered advantageous in developing countries as it reduces the importation of wheat flour and encourages the use of locally grown crops as flour (hugo et al., 2000). aziah and komathi (2009) reported that there is an increase in the substitution for wheat flour with local raw materials as a result of a growing market for confectioneries. nigeria has not been able to produce wheat in commercial quantities because of climatic and soil conditions. consequently, nearly all the wheat flour used for snacks and other products are imported. however, efforts are being made to partially replace wheat flour with non-wheat flours. this will possibly increase the utilization of indigenous crops cultivated in nigeria as well as contribute to low cost of bakery products (ayo & gaffa, 2002). usually, the aim of producing composite flour is to get a product with improved properties, performances, or improved economies (okpala & okoli, 2011). the nutritional value of cereal flours that are poor in lysine but rich in the sulfur containing amino acids could be improved. this is achieved by the addition of legume flours (eneche, 1999) such as soybeans. moreover, fortification of root and tuber flours with cereal flours can improve their nutritional value (fao, 1990) and baking quality. according to ihekoronye and ngoddy (1985), cereals and legumes are good sources of protein which complement each other with respect to their amino acid profile. soybean (glycine max) is a leguminous plant widely grown for its edible bean which has numerous uses (liu, 2000). it is the only plant source that provides all the essential amino acid (ihekoronye & ngody, 1985), phyosterols, b-vitamins and minerals. soybean is one of the richest and cheapest sources of plant protein that can be used to improve the diet of millions of people (lui, 2000). cocoyam (xanthosoma sagittifolium) is an edible root crop grown in the tropics of which nigeria is a major producer. it belongs to the family araceae (ihekoronye & ngoddy, 1985). cocoyam is regarded as the third most important root crop, after yam and cassava, in west africa (obomeghevie et al., 1998). key (1987) stated that the higher protein and amino acid profile of cocoyam is considered as a nutritional advantage over other tropical root or tuber crops. cocoyam has small size granules with high starch content, which results in high digestibility (howeler et al., 1993; huang et al., 2000). as there is a growing interest in the production of flours from locally available grains that can be used www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 41 published by scholink inc. as substitutes for wheat in baked goods, this study was undertaken to produce sausage rolls of acceptable quality from cocoyam, soybean and wheat flour blends. cocoyam is not utilized properly. it is mostly used as thickener in soup and few indigenous recipes. therefore, its conversion into flour could be used efficiently in baking technology. cocoyam is an indigenous root crop that has not been utilized like other root crops such as cassava and yam. despite the high nutritional value of cocoyam and soybean in relation to other root crops and legumes, lack of knowledge of their uses has limited their adoption, production and processing (osho et al., 2009). to bridge the gap, efforts are being made by research institutes, non-governmental organizations (ngos) and industries to promote the production, processing and utilization of cocoyam in nigeria (osho, 2003). nigeria faces one of the most serious nutritional problems in protein-energy malnutrition. nigeria has not been able to produce wheat in commercial quantity due to climatic and soil conditions (okpala & okoli, 2011). celiac disease is associated with the consumption of gluten which is common in wheat and wheat products. this disease occurs due to interaction of gluten in genetically predisposed individuals (vijay et al., 1992). therefore, the use of cocoyam which is gluten-free to produce food products would be suitable for consumption by individuals allergic to gluten. the main objective of the study was to determine the functional properties of sausage rolls made from cocoyam and wheat flour blends enriched with soybeans. however, the specific objectives of the study were production of cocoyam flour and soybean flour; formulation of composite flour from blends of cocoyam, soybean and wheat and production of sausage rolls from different ratios (90:10:0, 80:10:10, 70:10:20, 60:10:30, 50:10:40, and 0:10:90) of cocoyam, soybean and wheat flour blends respectively 2. materials and methods 2.1 material collection cocoyam (xanthosoma sagittifolium) cultivar, ede uhie, soybeans (glycine max) and wheat flour were purchased from eke-ukwu market, owerri, nigeria. all other reagents used were of analytical grade. 2.2 processing of cocoyam into flour cocoyam comels were processed into flour using the method described by oti and akobundu (2007). cocoyam cornels, cultivar ede uhie were peeled, sliced and washed with water. the slices were blanched at 75°c for 15 minutes in portable water. the blanched slices were oven dried at 60°c for 9 hours and milled to obtain flour which was subsequently sieved to yield flour of fine texture. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 42 published by scholink inc. figure 1. flow diagram of the preparation of cocoyam flour figure 2. flow diagram of preparation of soybean flour www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 43 published by scholink inc. 2.3 preparation of soybean flour soy flour was prepared according to the method described by ndife et al. (2011). the soybeans (1 kg) were thoroughly cleaned to remove dirt and other extraneous materials such as stones and sticks. it was then washed and oven dried. the soybeans were roasted, decorticated, winnowed and milled into fine flour using hammer mill (model eu 5000 d) and sieved through 250 μm aperture sieve. the flour was packed and sealed in polyethylene bags until analyzed. 2.4 formulation of flour blend six blends of composite flour were prepared by mixing cocoyam flour, soybean flour and wheat flour in the percentage ratio of 90:10:0, 80:10:10, 70:10:20, 60:10:30, 50:10:40and 0:10:90 respectively. (90:10:0) and (0:10:90) served as control. 2.5 sausage roll preparation the formulated flour blend samples were used in producing sausage roll respectively. the ingredients used for the production and their right proportions are shown in table 2. sausage rolls were prepared according to the method of nigerian sausage roll preparation (http://www.nigerianfoodtv.com) with some modifications in the recipe. the meat filling was first prepared. the boneless beef was ground and seasonings (salt, nutmeg, mixed spices and stock cube) were thoroughly mixed using a binatone blender (model n0: blg-650). the mixture was set aside. the dry ingredients (flour, baking powder, salt and sugar) were thoroughly mixed in a bowl by hand. the margarine was added in scoops and rubbed into the dry mixture till it formed crumbs. thereafter cold water was added in bits till a non-sticky smooth dough was formed. the dough was kneaded for 5 minutes. the dough was cut out in a rectangular shape, rolled to a flat size. the spiced beef placed on the edges of the dough and folded into a cylindrical shape. whisked egg was brushed on the dough and they were placed on oil-greased baking trays, leaving 25 mm spaces in between and were baked at 170oc for 35 minutes in the baking oven. following baking, the sausage roll was cooled at ambient temperature, packaged in polyethylene chin-chin bags and stored at near ambient temperature (28 ± 2oc) prior to subsequent analysis and sensory evaluation. the procedure is shown in figure 3. table 1. formulation of flour blends blend cocoyam flour, c (%) soybean flour, s (%) wheat flour, w(%) cs0 90 10 0 csw1 80 10 10 csw2 70 10 20 csw3 60 10 30 csw4 50 10 40 0sw5 0 10 90 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 44 published by scholink inc. table 2. ingredients for sausage roll preparation ingredient proportion flour 100g baking powder 2.15g margarine 100g salt 0.76g sugar 1.90g beef 100g nutmeg 0.76g mixed spices 0.76g stock cube 0.76g egg 1 medium size cold water 600ml figure 3. flow diagram of production of cocoyam-soybean-wheat flour sausage roll 2.5.1 evaluation of functional properties the functional properties of the flour composites are determined using the following methods. 2.6 ph ph of the flour was determined according to onwuka (2005). 10 g of the flour was suspended in 100 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 45 published by scholink inc. ml of distilled water and mechanically stirred for 2 hours at room temperature. using ph meter, the ph of the flour was determined and was done in triplicates. 2.7 bulk density using the procedure of onwuka (2005, 2018), about 5 g flour was put into a 10 ml measuring cylinder gently. the bottom of the cylinder was tapped gently on the laboratory bench severally until there was no further change of the sample level to a constant volume. the volume was recorded and the cylinder plus sample was weighed and recorded. triplicates determination were made and average result taken. the bulk density was calculated using the formula below. bulk density = 𝑀𝑎𝑠𝑠 𝑜𝑓 𝐹𝑙𝑜𝑢𝑟 𝑠𝑎𝑚𝑝𝑙𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝐹𝑙𝑜𝑢𝑟 (1) 2.8 determination of water absorption capacity the method of sosulski (1962) was described by abbey and ibeh (1988) and it was adopted. one gram (1 g) of each sample was weighed out into a dry, clean centrifugal tube and both weight noted. 10 ml of distilled water was poured into the tube and properly mixed with the sample to make a suspension. it was then centrifuged at speed of 3500 rpm for 15 minutes. after which supernatant was discarded then the tube and its content re-weighed and noted. the gain in weight is the water absorption capacity of the test sample (ibeabuchi, 2014). wac (g/g) = 𝑒 𝑎 𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (2) 2.9 determination of oil absorption capacity the method of onwuka (2005) was employed. 1g of the flour sample of each blend was weighed separately and placed into clean centrifuge tubes of known weight. 10ml of vegetable oil was mixed with the flour and stirred mechanically. the tubes were centrifuged at 3500 rpm for 15 minutes. the supernatant was decanted and the tubes together with the sample weighed. the amount of oil gain in mass is the oil absorption capacity of the flour samples. the amount of oil absorbed (total free) is multiplied by the density of oil for conversion of grams. the gain in weight is the oil absorption capacity of the sample (ogunbusola et al., 2012). the oil absorption capacity of the test sample was repeated three times for each sample and the average was calculated. oac (g/g) = 𝑒 𝑎 𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (3) 2.10 determination of swelling index the swelling index of the flour samples was determined as reported by njoku and banigo (2006). three grams (3 g) of each flour sample were transferred into a clean dry graduated 50 ml cylinder. the samples were gently leveled and their volumes noted. then, 30 ml of distilled water was added into each of the samples. the cylinder was swirled and allowed to stand for 1hour while changes in volume (swelling) were noted. the swelling index of the test sample was repeated three times for each sample and the average was calculated. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 46 published by scholink inc. swelling index = (4) 2.11 determination of wettability the method of onwuka (2005) was employed. 1 g of the flour sample was added into a 25 ml graduated cylinder with a diameter of 1 cm. a finger was placed over the open-end of the cylinder which was inverted and clamped at a height of 10cm from the surface of a 200 ml beaker containing 500 ml distilled water. the finger was removed and the rest of the sample was allowed to be dumped. the wettability is the time required for the sample to become completely wet. 2.12 determination of emulsion capacity the procedure of onwuka (2005) was adopted. 2 g of the flour sample was mixed with 25 ml of distilled water in a warring blender. a complete dispersion was obtained by blending at 1600 rpm for 30 seconds. subsequently, 25 ml of vegetable oil was gradually introduced into the dispersion and blending action continued for another 30seconds. the dispersion was then transferred into graduated centrifuge at 1600 rpm for 5 minutes. the volume of the oil separated from the sample after centrifuge was read directly from the centrifuge tube. emulsion capacity = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑚𝑢𝑙𝑠 𝑓 𝑒𝑑 𝑙𝑎𝑦𝑒𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑜𝑙𝑒 𝑠𝑜𝑙𝑢 𝑜 𝑒 𝑐𝑒 𝑟 𝑓𝑢 𝑒 𝑢𝑏𝑒 (5) 2.13 determination of foam capacity the method as described by onwuka (2005) was adopted in the determination of foam capacity. one gram of the flour was whipped into 100 ml distilled water and its volume noted. the suspension was blended with a warming blender 1600 rpm for 5 minutes. it was then poured into a 250 ml measuring cylinder, its volume noted and recorded. using abbey and ibeh (1988) formula, foam capacity is expressed percentage increase in volume is as follows: foam capacity = – (6) 3. results and discussion 3.1 functional properties the result of the functional properties of the flour samples are shown in table 3. table 3. mean values of functional properties of flour blends sample ph bd (g/ml) w (second) s i oac (g/g) wac (g/g) fc (g/g) ec (g/g) cs0 6.27±0.11a 0.77±0.00a 76.00±0.07a 1.99±0.01a 2.81±0.01a 3.20±0.01a 1.61±0.11a 2.41±0.01a csw1 6.28±0.01a 0.79±0.02a 65.00±0.07a 2.10±0.01a 2.63±0.01a 3.13±0.07a 1.63±0.02a 2.43±0.01a csw2 6.29±0.58a 0.80±0.12a 64.00±0.01b 2.00±0.00a 2.57±0.00ab 3.10±0.08ab 1.60±0.00a 2.43±0.11a csw3 6.29±0.55a 0.79±0.01a 61.00±0.11c 2.05±0.01a 2.43±0.43bc 2.90±0.33bc 1.62±0.00a 2.44±0.01a www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 47 published by scholink inc. csw4 6.25±0.01a 0.82±0.03a 57.00±0.01d 2.00±0.00a 2.36±0.01cd 2.80±0.57c 1.57±0.02a 2.44±0.01a 0sw 6.10±0.33a 0.80±0.01a 35.00±0.01e 1.66±0.00b 2.24±0.00d 2.71±0.00c 1.60±0.21a 2.45±0.11a lsd 0.3 0.26 0.75 0.26 0.15 0.22 0.23 0.38 mean ± standard deviation of triplicate means with the same superscript within a column are not significantly different (p≤0.05) cs0: cocoyam-soybean flour (90:10) (control 1) csw1: cocoyam-soybean-wheat flour (80:10:10) csw2: cocoyam-soybean-wheat flour (70:10:20) csw3: cocoyam-soybean-wheat flour (60:10:30) csw4: cocoyam-soybean-wheat flour (50:10:40) 0sw: soybean-wheat flour (10:90) (control 2) bd=bulk density w=wettability si=swelling index oac=oil absorption capacity wac=water absorption capacity fc=foam capacity ec=emulsion capacity 3.2 ph ph of the flour samples ranged from 6.10 (0sw: 90% wheat flour; 10% soybean flour) to 6.29 (csw2: 80% cocoyam; 10%-soybean, 10% wheat flour) with csw2 and csw3 (70% cocoyam-10% soybean-20% wheat flour) having the highest ph values. there was significant difference (p≥0.05) among the samples. the ph values of the flour samples indicate that they are low acid food samples and might support the growth and proliferation of microorganisms, especially in slurry form (ogunjobi and ogunwolu, 2010). similar ph values (4.2-5.78) were observed for other cocoyam cultivars (owuamanam et al., 2010). therefore, it is important to involve preservation which includes water activity reduction, use of chemical anti-microbial agent and anti-oxidant in the case of prolong storage of the samples (owuamanam et al., 2010). 3.3 bulk density bulk density is very vital in determining the packaging requirement, material handling and application in wet processing in food industry (karuna et al., 1996). values for bulk density ranged from 0.77 g/ml (cs0) to 0.82 g/ml (csw4). csw4 (50% cocoyam 10% soybean 40% wheat flour) had the highest value while cs0 (90% cocoyam 10% soybean flour) had the least value. the bulk density for the flour samples were not significantly different (p≥0.05). the bulk density of the flour samples is in the same range of 0.69 g/ml and 0.81 g/ml as reported for cocoyam flours by amandikwa (2012). flours with bulk density (>0.7 g/ml) are used as thickeners in food products (akubor & badifu, 2004), therefore, www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 48 published by scholink inc. the cocoyam flour blends in this study could also be suitable as thickeners. 3.4 wettability the flour samples have wettability from 35.00 to 76.00 seconds. wettability was found to be highest 76 seconds in cs0 (90% cocoyam 10% soybean flour) while 0sw (90% wheat flour 10% soybean flour) was the least 35 seconds. significant difference (p≤0.05) existed within the samples. there was an increase in the rate at which the flour sample got wet; as the cocoyam substitution decreased down the trend. the low wettability of the flour samples could be attributed to high temperature treatment using oven, hence making them absorb moisture faster (amandikwa, 2012). flour sample with least time of wettability would perform better in textured comminuted meat and baked products (achinewhu et al., 1998). 3.5 oil absorption capacity oil absorption capacity (oac) ranged from 2.24 g/ml to 2.81 g/ml. cs0 had the highest value (2.81 g/ml) while sw0 had the least value (2.24 g/ml). there was significant difference (p≤0.05) within the samples. oac is the ability of flour to absorb oil. oil gives soft texture and good flavor to food, therefore, the absorption of oil by food product improves texture and mouth feel (aremu et al., 2006). a high oil absorption capacity is valuable in ground meat formulations, meat replacers and extenders, doughnuts, pancakes and soups (onimawo & egbekun, 1998). 3.6 water absorption capacity water absorption capacity (wac) ranged from 2.71 ml/g (0sw) to 3.20 ml/g (cs0). there was significant difference within the flour samples. wac is the ability of a product to associate with water under limiting condition. it has been suggested that flours with high wac as seen in this study, will be very useful in bakery products, as this could prevent staling by reducing moisture loss (obatolu et al., 2007). water and oil absorption capacities are useful to indicate whether flours can be incorporated into aqueous food formulation especially those involving dough handling. niba et al. (2001) also stated that water absorption capacity is important in bulking and consistency of products as well as baking applications. 3.7 swelling index there was no significant difference (p≤0.05) among the flour samples in swelling index. cs0 (control 1) had the highest value (2.10 ml) while 0sw (control 2) had the least (1.66 ml). the swelling index increased with higher substitution with cocoyam flour. according to arathi et al. (2003), the presence of protein bodies around starch granules may restrict granule swelling and hence, reduce the susceptibility to enzymatic attack. generally cocoyam samples shows good swelling index when compared to other root crops like cassava (ojinaka et al., 2009). this is because of the type of granules cocoyam starch has and its highly digestible nature. the extent of swelling with the presence of water depends on the temperature, availability of water, species and starch damage due to thermal and other carbohydrates such as pectin, hemicelluloses and cellulose and protein. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 49 published by scholink inc. 3.8 foaming capacity foaming capacity values ranged from 1.57 to 1.63. csw1 had the highest value while 0sw had the least value. there was no significant difference (p≥0.05) in the foam capacity of the samples. foam formation and stabilization is a surface active function of proteins. 3.9 emulsion capacity the emulsification capacities of the flour samples were not significantly different (p>0.05). the emulsification capacity is within the range of 2.41(cs0) 2.45 (0sw). the emulsion capacity of the flour samples increases as cocoyam substitution decreases. emulsification property plays a significant role in many food systems including meat products, batters and dough and salad dressings. one of the important functions proteins perform in certain food system is emulsification. efficiency of emulsification varies with the type of protein, as concentration, ph, ionic strength, viscosity of the system, temperature and the method of preparation of emulsion capacity is influenced by equipment design, rate of oil addition, salt (type and concentration), sugar and water content (mcwatters et al., 2003). 4. conclusion the result of the functional properties of the composite flour blends showed that they could be suitable for other food formulations. sausage roll sample (csw1), from composite flour (80% cocoyam, 10% soybean and 10% wheat) can be recommended for diabetes, heart disease, obesity and weight management due to its high fiber content. furthermore, this study has created the utilization of cocoyam, so as to add value to it, and help in reducing the dependency on wheat flour. this will go a long way to conserve our economy by saving the huge foreign reserve used for wheat importation. our indigenous tuber crop (cocoyam) will no longer be wasted in terms of excess. again, this technology can simply be used in homes to create variety in meals and prevent monotony in feeding wastes also. the formulation and use of composite flours in baking is gaining popularity in food industries. it is one of the various means of fighting malnutrition resulting from nutrient deficiencies. 4.1 recommendations based on the results obtained and conclusion, the following recommendations are necessary. further studies should be done to improve the color and texture of the sausage roll to make it more appealing and palatable. shelf-life, packaging and storage studies should be done because no preservative was added to the sausage roll and they spoilt within few days of production. farmers of cocoyam will be encouraged as they will be making more sales since a conversion product for their produce has been developed. globally, convenience is the main focus, producing a snack with our local raw material which can compete with the conventional, will be a welcomed development. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 50 published by scholink inc. references abbey, b. w., & ibeh, g. o. 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(1993). tillage systems for root and tuber crops in the tropics. soil and tillage research, 27, 211-240. https://doi.org/10.1016/0167-1987(93)90069-2 http://www.nigerianfoodtv.com https://www.foodnetwork.com/sausage-roll-recipe/html https://www.en.wikipedia.org/wiki/sausage huang, a. s., titchenal, c. a., & meilleur, b. a. (2000). nutrient, composition of taiwan taro corms and breadfruit. j. food comp. anal., 13, 859-864. https://doi.org/10.1006/jfca.2000.0936 hugo, l. f., rooney, l.w., & taylor, j. r. n. (2000). malted sorghum as a functional ingredient in composite bread. cereal science, 79(4), 428-432. https://doi.org/10.1094/cchem.2000.77.4.428 ibeabuchi, j. c. (2014). proximate and functional properties of raw and fermented bottle gourd seeds (lagenaria siceraria). international journal of biotechnology and food science, 2(4), 82-87. ihekoronye, a. i., & ngoddy, p. o. 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(2003). physical and sensory characteristics of sugar cookies containing mixtures of wheat, fanio (digitaria exilis) and cowpea (vigna unguiculata) flours. international journal of food science and technology, 38, 403-410. https://doi.org/10.1046/j.1365-2621.2003.00716.x ndife, j., abdulraheem, l. o., & zakari, u. m. (2011). evaluation of the nutritional and sensory quality of functional breads produced from whole wheat and soybean flour blends. afr. j. food sci., 5, 466-472. niba, l. l., bokanga, m., jackson, f. i., schlimme, d. s., & li, b. w. (2001). physiochemical. properties and starch granular characteristics of flour from various manihot esculenta (cassava) genotypes. j. ed. sc., 67, 1701. njoku, b. a., & banigo, e. o. i. (2006). physio-chemical properties of precooked cassava (manihot esculenta crantz) flour prepared by adaptation of a traditional process. nigerian food journal, 24(1), 98-106. https://doi.org/10.4314/nifoj.v24i1.33639 obatolu, v. a., augustine, o., & iken, j. e. (2007). improvement of home-made maize tortilla with soybean. international journal of food science and technology, 42, 420-426. https://doi.org/10.1111/j.1365-2621.2006.01242.x obomegheive, a. a., obafemi, m. o., & oyibo, s. (1998). processing parameters for cocoyam flour production. in proceeding, 22nd annual conference of nigerian institute of food science and technology (nifst), abeokuta (pp. 131-132). ogunbusola, e. m., fagbemi, t. n., & osundalunsi, o. f. (2012). chemical and functional properties of full fat and defatted white melon (cucumeropsis mannii) seed flours. journal of food sciences and engineering, 2, 691-696. ogunjobi, m. a. k., & ogunwolu, s. o. (2010). physiochemical and sensory properties of cassava flour biscuit supplemented with cashew apple powder. journal of food technology, 8(1), 24-29. https://doi.org/10.3923/jftech.2010.24.29 ojinnaka, m. c., & nnorom, c. c. (2015). quality evaluation of wheat-cocoyam-soybean cookies. nigerian journal of agriculture, food and environment, 11(3), 123-129. ojinnaka, m. c., akobundu, e. n. t., & iwe, m. o. (2009). cocoyam starch modification effects on functional, sensory and cookies qualities. pakistan journal of nutrition, 8, 558-567. https://doi.org/10.3923/pjn.2009.558.567 okpala, l. c., & okoli, e. c. (2011). nutritional evaluation of cookies produced from pigeon pea, cocoyam and sorghum flour blends. african journal of biotechnology, 10(3), 433-438. onimawo, a. i., & egbekun, k. m. (1998). comprehensive food science and nutrition. macmillan www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 52 published by scholink inc. press, ibadan. onwuka, g. i. (2005). food analysis and instrumentation: theory and practice (pp. 63-75). naphtali prints, surulere, lagos. onwuka, g. i. (2018). food analysis and instrumentation: theory and practice (2nd ed., pp. 179-228). naphtali prints, lagos. osho, s. m. (2003). the processing and acceptability of a fortified cassava-based product (garri) with soybean. journal of nutrition and food science, 33(6), 205. osho, s. m., akinleye, s. o., & akanni, k. a. (2009). determinants of soybean production in south western nigeria. journal of life and physical science, 4(2), 113. oti, e., & akobundu, e. n. t. (2007). physical, functional and amylograph pasting properties of cocoyam-soybean crayfish flour blends. nigerian food journal, 25(1), 1616-1625. https://doi.org/10.4314/nifoj.v25i1.33665 owuamanam, c. i. (2007). quality of bread from wheat/cassava composite as affected by strength and steeping duration of cassava in citric acid. natural science, 5, 24-28. owuamanam, c. i., ihediohanma, n. c., & nwanekezi, e. c. (2010). sorption isotherm, particle size, chemical and physical properties of cocoyam corm flours. researcher, 2(8), 11-19. sosulski, f. w. (1962). the centrifuge methods for determining flour absorption in hard-red spring wheat cereal. chemical, 39, 344. appendix plate 1. oven-dry slices of cocoyam (xanthosoma sagittifolium) ede uhie www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 3, no. 2, 2019 53 published by scholink inc. plate 2. cocoyam flour (ede uhie) plate 3. soybean flour plate 4. sausage roll (csw4) (50%cocoyam-10%soybean-40%wheatflour) food science and nutrition studies issn 2573-1661 (print) issn 2573-167x (online) vol. 2, no. 2, 2018 www.scholink.org/ojs/index.php/fsns 41 original paper aberration of metals competing for iron on exposure to lambda cyhalothrin and aflatoxins in dietary fish from selected aquatic sources in kenya faith obegi onyangore1*, julius ochuodho2, odipo osano2 & cornell akwiri omondi3 1 university of kabianga, kenya 2 university of eldoret, eldoret, kenya 3 kenya water towers agency, south rift region-narok, narok, kenya * faith obegi onyangore, university of kabianga, kenya received: september 10, 2018 accepted: october 1, 2018 online published: october 12, 2018 doi:10.22158/fsns.v2n2p41 url: http://dx.doi.org/10.22158/fsns.v2n2p41 abstract excess or deficiency of minerals may seriously disturb biochemical processes and upset internal homeostasis, leading to various diseases and disorders in fish species due to deficiency or excess of micro and macro elements caused by improper nutrition, avitaminosis or poisoning. the specific objectives of the study were to determine the iron levels and aberration of metals competing on exposure to lambda-cyhalothrin and aflatoxins in dietary fish from selected aquatic sources in kenya. the concentration of elements cadmium, zinc, and iron in oreochromis niloticus and clarias gariepinus bred in kenya marine and fisheries research institute at sagana and obtained from river nyando was measured using atomic absorption spectrophotometer. iron availability was lower on treatment with aflatoxin compared to lambda-cyhalothrin with a mean of 3.66 ± 0.84 mg/kg, but on subjection to zinc, competition was 3.82 mg/kg on consideration of zinc competition. the naturally occurring toxins cause micronutrient deprival and therefore relevant stakeholders be keen to prevent contamination from farm to fork. keywords dietary, fish, aberration, metals, competing, aflatoxins, lambda-cyhalothrin 1. introduction fish is a vital source of food for people and a good source of heme iron (fao, 2005). iron, a mineral required by the body to produce red blood cells, occurs in two different forms in the diet heme and www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 42 published by scholink inc. non-heme. heme iron derives from the hemoglobin in animal tissue whereas nonheme iron derives primarily from plant tissues, although a small amount of the iron in animal foods is nonheme iron. the body absorbs heme iron more efficiently than non-heme iron. eating a wide variety of iron-rich foods, and combining foods that contain heme and non-heme iron will help prevent deficiencies. sorption and co-precipitation of metals by fe oxides decreased the bioavailability and toxicity of waterborne metals may increase the dietary supply of metals and lead to toxic effects along the food chain (vuori, 1995). heme iron, found in meat and fish, is more easily absorbed at approximately 15-40% as compared to 1-15% nonheme and is only significantly inhibited by large quantities of dairy products, while fish and meat proteins enhance nonheme iron absorption (onyangore et al.). the food and agricultural organization (fao) estimates that about one billion people worldwide rely on fish as their primary source of animal protein (fao, 2000). it is man’s most important single source of high-quality protein, providing 16% of the animal protein consumed by the world’s population according to the food and agriculture organization (fao) of the united nations (2000). it is a particularly important protein source in regions where livestock is relatively scarce fish supplies <10% of animal protein consumed in north america and europe, but 17% in africa, 26% in asia and 22% in china (fao, 2000). in kenya, annual fish consumption levels in the country are at 3.7 kilograms per person (omondi, 2012). lambda-cyhalothrin is a synthetic pyrethroid insecticide widely used for pest management and public health applications to control insects. lambda-cyhalothrin is categorized as a restricted use pesticide in extension toxicology network for its toxicity to fish (mound, 1998). but its usage for the control of major pest in agriculture is being continued in developing countries. the pesticide exposure causes severe alterations in the tissue biochemistry and histology of fishes (saravanan et al., 2010; susan et al., 2010; velisek et al., 2009). aflatoxin is metabolic byproduct of molds aspergillus flavus and aspergillus parasiticus. it is a toxic compound and the cause of high mortality in livestock, poultry, fish and in some cases of human beings (fotsis et al., 1995; reed et al., 1987). toxicogenic a. flavus produces aflatoxin b1 and b2 whereas a. parasiticus produces aflatoxin g1 and g2. aflatoxin b1 is classified as a group i carcinogen by international agency for research on cancer. effect of aflatoxin on fishes and other animals have been reported by many workers. nunez et al. (1991) reported hepatocellular adenoma and hepatocellular carcinoma in rainbow trout when exposed to aflatoxin b1. caguan et al. (2004) reported a loss of appetite, low survival percent and decreased mean total biomass in tilapia when fed with aflatoxin-contaminated feed. since fishes are important sources of proteins, minerals and lipids for humans and domestic animals, so the health of fishes is very important for human beings. excess or deficiency of minerals may seriously disturb biochemical processes and upset internal homeostasis, leading inconsequence to various diseases. tacon (1992) reported that disorders occurred in organisms of various fish species due to deficiency or excess of micro and macro elements which were caused by improper nutrition, avitaminosis or poisoning. it is therefore important to monitor levels of macro and micro elements in http://www.ncbi.nlm.nih.gov/pmc/articles/pmc1084135/#kve236c8 http://www.ncbi.nlm.nih.gov/pmc/articles/pmc1084135/#kve236c6 http://www.ncbi.nlm.nih.gov/pmc/articles/pmc1084135/#kve236c6 http://www.ncbi.nlm.nih.gov/pmc/articles/pmc1084135/#kve236c8 www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 43 published by scholink inc. fish organisms (brucka-jastrzêbska et al., 2009). the poisoning is majorly caused by toxicants emanating from either feed or water pollution. previous research work in kenya has conducted studies on the effects of toxicants on the other aspects of health, but not taken a step further to conduct research on the influence of essential nutrients like iron. the natural and synthetic toxicants in fish may be causing significant changes in the iron levels due to metabolic disruption, and yet no research has specifically checked for the availability of iron. the knowledge gap on nutrient toxin interaction was addressed as findings from this study, therefore, linked toxicants and availability of iron in fish as a heme food source. results from this study generalized and shared with food scientists, food toxicologists, medical personnel, agriculturalists and the community at large on the magnitude on iron availability in fish on exposure to natural and synthetic toxicants in fish a heme food sources. previous studies have considered the implications of nutrient toxicant interactions but findings from this study encouraged nutritionists to consider the importance of environmental exposures to their study populations and their research questions. furthermore, it encouraged the involvement of nutritionists in the design of high quality, rigorous studies of nutritional assessment and interventions in populations exposed to environmental chemicals. as a growing field, the intersection between nutritional science and toxicology benefited from the expertise of nutritionists. 2. materials and methods 2.1 study design, species, sample size, and setting for fish bred at sagana experimental study design was adopted where manipulation of independent variables to determine their effect on a dependent variable. a completely randomized block design was adopted. for fish from river nyando were collected at three different points (appendix 31). a cross-sectional study design was also adopted where fish samples were collected from river nyando for laboratory analysis. the independent variables were the treatments and the dependent variable being iron levels (kothari, 2004). for fish bred at sagana identification of oreochromis niloticus and clarias gariepinus species was done by the kenya marine and fisheries research institute (kmfri) staff. nine hundred (900) fish, 450 of each species were bred in the lab between january-august 2015. sample size calculation for fish bred at sagana 2 sd2 (zt + zx)2 / d2 sd = standard deviation from pilot study zt = z value from the z table zx = type 1 error of 5% at 80% interval d = effect size, the difference in means (iron in mg) (jaykaran & kantharia, 2013). = 2(1)2 (1.96 + 0.842)2 / 3.5 = 4.48 = 5 fish per tank http://www.ncbi.nlm.nih.gov/pubmed/?term=charan%20j%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=kantharia%20nd%5bauth%5d www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 44 published by scholink inc. fish from river nyando 2 sd2 (zt + zx)2 / d2 = 2(1)2 (1.96 + 0.842)2 / 3.5 = 2(1)2 (1.96 + 0.842)2 / 0.145 = 108.29 = 108 fishes 2.2 sampling techniques for fish bred at sagana, there are about thirty-five species of fish commonly consumed in kenya were written down, and the two fish species were selected randomly. for the fish bred in an aquarium in the lab, all the fish sampled were used for the experiment. the fish species used were oreochromis niloticus and clarias gariepinus and were cultured in the lab for twelve weeks after demineralization of water. the samples were extracted immediately and analyzed for iron levels cadmium and zinc as metals competing with iron, this was after treatment with aflatoxins in feed prepared and lambda-cyhalothrin introduced in water and parameters measured when fish were at various ages. for fish from river nyando, the sampling procedure for fish species collection was the same as in fish bred in sagana. the fish were selected at three different sites along river nyando. for each site fish were collected by means of gill net mesh size. the nets were deployed from early morning and checked two hourly until the required fish quotas were reached. this minimized the amount of time fish spent in the net in order to reduce an imposed stress. only living fish size 10-60 g were selected, kept in a 100 l plastic tank and immediately transported to the laboratory for sample collection and necropsy analysis. a total of 105 fish specimens were collected from river nyando. the samples were extracted immediately and analyzed for iron levels and cadmium and zinc as metals competing with iron. 2.3 laboratory procedures for heavy metal analysis for both fish samples collected from fish bred at kenya marine and fisheries industries (kmfri) sagana and from river nyando, the laboratory sites and procedures were similar. at the laboratory, fish were kept in large holding tanks filled with water from each site to minimize stress. the heavy metal analysis was conducted at kirdi (kenya industrial and research development institute). the sampling containers, preparation and handling of fish samples for analysis, the procedure for the dissection of fish3, sample collection from different parts of the fish, sample collection from different parts of the fish was adopted as per previously published work (icp waters report, 105/2010). all glassware was washed in nitric acid solution and rinsed with distilled water. all reagents used during analysis were of analytical grade. this was digested using the procedure recommended by (chen et al., 2008) as described. an acid mixture of two parts concentrated nitric acid and one part perchloric acid (2:1) was prepared. one gram (1.0 g) dried liver material was weighed and put in a 100 ml digestion tube. 5 ml of the acid mixture was added and the sample was placed on a hot plate. the samples were heated at 600c for 15 minutes. the heat was then increased to 1200c and digested for 75 minutes or until the sample cleared. the tube was removed from the hot plate when the www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 45 published by scholink inc. sample was clear. the sample was then cooled and sufficient distilled water added to bring the solution to 100ml and preserved in the cold room at 40c awaiting analysis by aas. a blank comprising of the reagents in the proportion as for the samples but containing no sample material was prepared likewise. the concentration of elements (fe cd and zn) in muscle and fish liver was measured using atomic absorption spectrophotometer (aas) aanalyst 800 (parkin elmer instrument, 2usa) with an acetylene flame (fe and zn) and argon non-flame (cd), after preparation of calibration standards. the overall recovery rates (mean ± sd) for cd, zn were 103 ± 8.3 and 2.3 and 90 ± 3.5 respectively. the detection limit for fe, cd, and zn was 0.04, 0.02 and 0.10 µg/g respectively. detection of lambda-cyhalothrin was conducted in water from river nyando. sample analysis was done using varian cp 3800 gas chromatograph equipped with an electron capture detector. separation was done using bpx 5 capillary column of dimensions 30 m x 0.25 mm x 0.25 μm film thickness. confirmatory analysis was done using a bpx35 capillary column of dimensions 50 m x 0.25 mm x 0.25 μm film thickness. a temperature program was used starting from 900c (withhold time of 3 minutes), increased to 2150c at 80c/min (withhold time of 25 min), then increased to 2700c at 50c/min (withhold time of 5.37 min), and finally ramped to 2750c at 50c/min (withhold time of 18.63 min). the carrier gas was high purity helium (99.9995%) with white spot nitrogen as the makeup gas. quantification followed external calibration method using high purity pesticide reference standards mixture obtained from ultra scientific usa. quality control and quality assurance were ensured by all sampling, extraction, and analysis being done in triplicate to allow verification detected pops residues. the samples were spiked with the insecticide during extraction and analysis to minimize errors due to detector fluctuations. recovery tests were also carried out using the reference pesticide standards to determine the performance of the methodology. quantification of lch was carried out using high purity organic pollutants. heavy metal assessments for fish both from bred in sagana and river nyando, the iron cutoffs were 20-80 mg/kg, the zinc ranges 20-55 mg/kg, the cadmium ranges 0.05-5 mg/kg were as per the european standards. 3. data management and analysis 3.1 data reporting for both fish obtained from sagana and from river nyando, data included iron levels, zinc levels, cadmium levels. for fish from river nyando, lambda cyhalothrin and aflatoxin were recorded from the water samples (kroglund et al., 2007; rognerud et al., 2002). 3.2 data analysis the independent variables were the treatments aflatoxins and lambda-cyhalothrin. the dependent variables were iron levels and metal levels competing for iron availability, the data was entered into a computer and analyzed using the excel spreadsheets as database and genstat version 12 analysis for means, frequencies and cross-tabulations. for fish obtained from river nyando, spss version 21 was used for data analysis. one way anova was carried out to determine differences in means of iron www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 46 published by scholink inc. levels, zinc levels, and cadmium levels lambda-cyhalothrin, aflatoxins. post hoc hsd (turkey) (was used for post-hoc discrimination between means. the effects of aflatoxins and lambda-cyhalothrin were analyzed using linear regression analysis after performing an outlier analysis. the interaction between lambda-cyhalothrin, aflatoxins, iron levels, zinc and cadmium competing for iron was analyzed by ancova. in all statistical tests, 5% significance level was applied. 3.3 ethical considerations all experiments with fish were conducted in accordance with national and institutional guidelines for protection of animal welfare (prevention of cruelty to animals act, cap 360 of the laws of kenya). authority to conduct research was sought from the fisheries department of kenya and from the various sites, kemfri sagana, and kirdi nairobi. 4. results 4.1 determine iron levels 4.1.1 iron levels in oreochromis niloticus and clarias gariepinus the mean fe in both species was 5.0369 ± 1.443 mg/kg with a maximum level of 7.767 mg/kg. the level of iron of 5.22 mg/kg in oreochromis niloticus was higher than that in clarias gorgeous which was 4.86 mg/kg. table 1. comparison of means of iron on treatment with lambda-cyhalothrin versus aflatoxin in clarias gariepinus and oreochromis niloticus in fish from sagana river nyando, metal concentrations in mg/g dw and n=54 environment species1 sem species2 sem p value 1 sagana 4.86 ± 1.49 0.29 5.22 ± 1.40 0.27 0.367 2 river nyando 17.11 ± 4.47 0.66 25.68 ± 3.52 0.44 0.000 results from table 1 indicate that there were significant differences of treatments on iron levels between species clarias gariepinus and oreochromis niloticus p > 0.05 in fish bred in river nyando and no significant differences in iron levels in species in fish bred in sagana. fish in river nyando had higher iron levels with a mean of 17.11 ± 7.81 mg/kg and 25.68 mg/kg in clarias gariepinus and oreochromis niloticus respectively than in fish bred in river nyando. comparing means on iron levels on analysis of variance in clarias gariepinus and oreochromis niloticus on exposure to aflatoxin and lambda-cyhalothrin in selected environments. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 47 published by scholink inc. table 2. comparing means on iron levels after analysis of variance in clarias gariepinus and oreochromis niloticus on exposure to aflatoxin versus lambda-cyhalothrin in sagana and river nyando condition lambda cyhalothrin s.e.m aflatoxin s.e.m p value sagana 4.78 ± 0.72 0.17 3.66 ± 0.84 0.200 0.01 river nyando 22.09 ± 7.81 0.59 nd nd 0.97 results from this study (table 2) show that the type of treatment had a significant effect (p = 0.01) on iron availability (p < 0.05). however, treatment had no significant effect on iron availability from fish in river nyando. fish from river nyando had higher iron levels than those bred in sagana. 4.2 the significance of the type of treatment table 3. tukey means separation to show the significance of types of treatment on aflatoxin and lambda-cyhalothrin on clarias gariepinus and oreochromis niloticus in cultured fish treatment mean( sagana) s.e.m p value aflatoxin 3.66a 0.20 0.01 control 6.68c 0.13 lambda cyhalothrin 4.78b 0.17 both of the treatments had lower iron levels as compared to the control and lambda-cyhalothrin treatment had higher iron levels as compared to aflatoxin treatment at 3.66 mg/kg (table 3). the treatments influenced the tissues differently, i.e., the treatments were significantly different. ho there is no significant aberration of iron on exposure to lambda-cyhalothrin versus aflatoxins in dietary fish in selected environments in kenya.the null hypothesis was rejected because the treatments influenced the iron levels differently. 4.3 assess metals competing for iron availability 4.3.1 zinc and cadmium availability table 4. summary statistics of zinc in livers of clarias gariepinus and oreochromis niloticus on treatment with lambda-cyhalothrin and aflatoxin in fish bred in sagana size (n) mean (mg/kg) variance sd sem 54 19.85 11.58 3.403 0.4631 the mean zinc levels were 19.85 ± 3.403 mg/kg (table 4). 4.3.2 mean comparisons of zinc and cadmium in levels in the selected environments the zinc levels in fish from river nyando were higher at 34.585mg/kg as opposed to the fish bred in www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 48 published by scholink inc. sagana. results from this study further showed that the type of treatment had a significant difference on zinc levels (p < 0.05) in fish bred in sagana as opposed to from river nyando. there was no significant effect of treatment on the cadmium levels in river nyando (table 5). table 5. comparison of means on analysis of variance in clarias gariepinus and oreochromis niloticus on zinc levels on treatment with lambda-cyhalothrin and aflatoxin in fish bred in sagana and river nyando condition lambda-cyhalothrin s.e.m aflatoxin s.e.m p value sagana 16.60 ± 0.09 0.22 18.72 ± 0.14 0.33 0.01 river nyando 34.585 ± 14.66 1.411 nd nd 0.575 sagana(cd) nd nd nd nd nd river nyando 5.82 ± 1.42 0.136 nd nd 0.429 4.3.3 comparison of means of zinc and cadmium on exposure to lambda-cyhalothrin versus aflatoxins table 6. comparison of means on exposure with lambda-cyhalothrin versus aflatoxin in clarias gariepinus and oreochromis niloticus bred in sagana and from river nyando environment species1 s.e.m species2 sem p value sagana zn 19.78 ± 3.54 0.68 19.91 0.94 ± 3.32 0.64 river nyando 32.76 ± 14.62 2.18 35.88 ± 14.67 1.85 0.278 sagana cd nd nd nd nd river nyando 5.79 ± 1.36 0.20 5.84 ± 1.47 0.18 0.863 there was no significant effect (p > 0.05) in the type of treatment among the species from both environments as presented in table 6. cadmium was not detected in fish that was bred in sagana. table 7. tukey’s comparison of means of zinc on treatment with lambda-cyhalothrin and aflatoxin in clarias gariepinus and oreochromis niloticus in fish bred at sagana treatment means s.e.m p value l-cyhalothrin 16.60a 0.22 0.01 aflatoxin 18.72b 0.33 control 24.22c 0.18 results from this study show that the treatments affected the zinc levels differently. the control had the highest zinc level of 24.22 mg/kg. lambda-cyhalothrin had lower zinc levels compared to aflatoxin of www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 49 published by scholink inc. 18.72 mg/kg (table 7). ho there is no significant aberration of metals competing for iron bioavailability in lambda-cyhalothrin versus aflatoxins compared in dietary fish in selected environments in kenya the hypothesis was rejected because the treatments influenced the metals competing with iron levels significantly. 4.3.4 significance of iron availability on coexistence with zinc on exposure to aflatoxins and lambda-cyhalothrin on conducting an analysis of covariance (ancova), the type of treatment on the zinc and iron interaction was significant (p > 0.05). table 8. comparing the significance of iron availability on competition and without competition with zinc on exposure to aflatoxins and lambda-cyhalothrin treatments in clarias gariepinus and oreochromis niloticus cultured fish treatment means (iron/zinc) df p value means ( iron ) l-cyhalothrin 3.816a 2 0.140 3.659a aflatoxin 5.222b 4.769b control 6.070b 6.681c the means did not differ significantly on exposure when zinc and iron interacted on exposure to the two treatments. the means of iron without competition were (3.659 and 4.769) and got higher on exposure to treatments and on the competition with zinc (3.816, 5.222). however, in the control, the iron levels were higher on interaction with zinc 6.681 mg/g (table 8). the treatment was not significant p < 0.05. 5. discussion fish is a major dietary heme source in our diets and hence should be safe and contain uncompromised iron levels when consumed. exposure of fish to contaminants through feed and environment is likely to cause compromised safety to humans and altered nutritional levels. most insecticides have been known to be highly toxic to nontarget organisms that inhabit natural environments close to agricultural fields. several studies reported that some surface waters and surrounding environments were contaminated with different insecticides (arjmandi et al., 2010; bagheri, 2007; ghassempour et al., 2002; rahiminejad et al., 2009; tarahi tabrizi, 2001). findings from this study conform to the studies that insecticides like lambda-cyhalothrin were detected in river nyando. results from this study show that iron levels were higher in oreochromis spp than in clarias gariepinus when tests were conducted in the cultured fish. the same result was obtained from the fish collected from river nyando showed that the clarias gariepinus had lower levels of iron than www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 50 published by scholink inc. oreochromis niloticus. fish obtained fromriver nyando had higher levels of iron of 22.09 mg as compared to 5.04 mg in from river nyando. fish found in water bodies with high levels of metals accumulate higher amounts of metals than those found in uncontaminated water bodies because they absorb these metals from gills, skin oral consumption of water, food and non-food particles. it is expected that iron levels in c. gariepinus fish caught in the river nyando should have higher values as fish absorbs metals from the sediment, polluted water, and food and thus leads to contamination of the food chain. the low iron levels contrary to other studies could be attributed to toxicity from pesticides in water. metals are not easily biodegradable and consequently can be accumulated in human vital organs. this situation causes varying degrees of illness based on acute and chronic exposure (demirezen et al., 2006; mathenge, 2013). the introduction of these elements into the food chain may affect human health as the excess of iron can cause toxicity (coulate, 1992). further, the iron levels measured by (oyoo‐okoth et al., 2010) in rastrineobola argentea are more or less similar to the c.gariepinus of this study, but r.argentea is herbivorous and thus lower on the food chain than c.gariepinus. it can be expected that c.gariepinus from lake victoria investigated by (oyoo‐okoth et al., 2010), would have had greater levels, due to bio-magnification, as r.argentea are prey to c.gariepinus. the results presented suggest a greater effect of aflatoxin on iron levels in cultured fish livers as compared to lambda-cyhalothrin. since fish is a vital source of proteins and lipids for humans and domestic animals, the health of fish is very important to human beings. fish like other aquatic organisms may be exposed to a great range of insecticides during the course of their life cycle. in fish, different insecticides can be absorbed through gills, skin or alimentary ducts (mahdi banaee, 2012; m banaee et al., 2011). fishes are particularly sensitive to environmental contamination of water. hence, pollutants such as insecticides may significantly damage certain physiological and biochemical processes when they enter into the organs of fishes (m banaee et al., 2011). the differences in iron levels could be attributed to a greater physiological damage the aflatoxin causes as compared to the pyrethroid and hence the effects of aflatoxins on fishes are of great concern at its mode of action is hepatotoxicity. the concern of iron levels is of vital importance more so because fish might look apparently healthy despite accumulating metals to a concentration which substantially exceed maximum values considered safe for human consumption. aflatoxicosis is a major problem related to aquaculture that leads to economic losses and health complications in fish. aflatoxins are well recognized as a cause of liver cancer, but they have additional important toxic effects. in farm and laboratory animals, chronic exposure to aflatoxins compromises immunity and interferes with protein metabolism and multiple micronutrients that are critical to health. these effects have not been widely studied in humans, but the available information indicates that at least some of the effects observed in animals also occur in humans (jonathans et al., 2004). although aflatoxin is known to affect nutritional concentrations of iron in animals (dhanasekaran, 2011) its potential effect on human iron concentrations is not known. www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 51 published by scholink inc. it is, therefore, becoming a matter of concern to environmentalist since the presence of pollutants, particularly; essential metals apparently accumulate in the sediments. in a study that was conducted in lake kanyaboli, metal level in the liver of c.gariepinus was significantly associated with heavy metal concentration in lake sediment indicating that fish liver is an effective indicator of fish exposure to heavy metals (akwiri et al.). in the fish obtained from river nyando, the lambda-cyhalothrin levels were not significant to the iron levels both in the liver. the many available pesticides for use and elevated metals concentrations from effluent has caused an indirect association between pesticides use and iron levels. there was no aflatoxin association in the fish from the river. the iron levels generally on exposure to both lambda-cyhalothrin and aflatoxin were lower than iron levels in other studies. in a study conducted in serbia to ascertain the levels of microelements in freshwater fish, the liver values ranged from 4.6-37.3 mg/kg (brucka-jastrzêbska et al., 2009). in another study conducted in bangladesh to determine iron levels in long-whiskered catfish, iron levels were higher than in the present study with ranges 54.93 ± 4.33 mg/kg. this could be attributed to the interference of the toxicants with normal physiological processes involving iron synthesis. in this study the effects of lambda-cyhalothrin, a synthetic pyrethroid was compared to those of aflatoxin a naturally occurring toxicant on iron levels contained in fish. 5.1 effects of aflatoxins and lambda-cyhalothrin on levels of metals competing for iron bioavailability in fish as a food source in kenya results from this study indicate that there were higher zinc levels in aflatoxin-exposed fish, a metal which competes with iron. in this study, the type of treatment had a significant difference in zinc levels. the treatments affected the zinc levels differently with the treatments having the lower zinc levels compared to the control lambda-cyhalothrin treatment which had a higher level of zinc as compared to aflatoxin treatment. zinc is known to compete with iron during metabolism. further, results from this study indicated that iron levels increased on the association to zinc availability. in the ideal, zinc competition could have seen lower iron levels in fish liver. ewa in a study to determine micro and macro elements in fresh water reported higher and within normal physiological ranges of zinc than in the present study. this study also found out that zinc levels were affected and it is evident that zinc competition was insignificant in the presence of the treatments; this is probably due to the impairment of receptors by the respective treatments. studies also revealed that co-administration of zinc with lambda-cyhalothrin to treat animals retained the level of gsh and the activity sod and gpx at the normal values. catalase, cat, gst activity and lop level were improved, and such alterations were still significant in zinclch-treated rats. the observed normalization trend of gsh, sod, and gpx following zinc treatment could possibly be due to dismutation of o-2 to h2o which is catalyzed by sod. zinc is known to induce the production of metallothionein, which is very rich in cysteine, and is an excellent scavenger of -oh. also, the nadph oxidases are a group of plasma membrane-associated enzymes, which catalyze the production of o-2 from oxygen by using nadph as www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 52 published by scholink inc. the electron donor. zinc is an inhibitor of this enzyme. cytochrome p450 enzymes are essential for the metabolism and detoxification of many xenobiotics (e.g., pesticides). it has been reported that many chemicals (e.g., pesticides, drug) interactions are the result of an alteration of cyp450 metabolism, lch decreased cytochrome p450 activity in lch-treated rat. this may be due to the inhibition of heme synthesis and destruction of cytochrome p450. previous studies showed that many pesticides have been reported to inhibit the activity and alteration in the expression of various cytochrome p450 isoforms (e.g., parathion, methomyl). these changes may increase the sensitivity of cells against reactive endogenous metabolites or other xenobiotics. co-administration of zinc to lch-treated animals improved the activity of cytochrome p450 compared to an lch-treated rat. this change may due to the antioxidant role of zinc and alter the enzyme activities associated with antioxidant defense mechanisms (abbasy et al., 2014). the same reaction is likely to occur in fish. fish from the river had higher zinc levels of 34.56 mg as compared to those bred in sagana. findings to this study suggested that species had no significant influence on metals competing with iron. contrary to findings in this study is that zinc /iron competition is due to species composition and not due to the treatment. the study finding in solomons and jacob’s found that zinc from atlantic oysters which are the most zinc-rich natural food items in the occidental diet, was impervious to the inhibitory effects of a 2:1 ratio of iron to zinc, even when 54 mg of zinc (from oysters) and 100 mg of iron as ferrous sulfate (a total of 154 mg of the metals) were given. the absence of an inhibitory interaction may be explained by the chemical form of zinc in the oyster, a physical-chemical protection against the intrusion of iron or accelerated binding of the iron or oxidation of the iron to its ferric form by the oyster (solomon et al., 2013). cadmium (cd) was not detected in fish under controlled conditions but was detected in the fish from the river nyando. concentrations of elements such cd have been previously reported in water and fish (birungi et al., 2007; oyoo-okoth et al., 2013; wandiga et al., 2002). the findings from this study are consistent with studies conducted by saeed and shaker, 2008 who presented a report about concentrations of fe, zn, cu, mn, cd, and pb in o. niloticus (tilapia ) fish tissues, water and sediments in northern delta lakes. they found that the edible part of o. niloticus from lake edku and manzala contained the highest levels of cd while fish from manzala lake contained the highest level of pb. they reported that nile tilapia caught from these two lakes may pose health hazards for consumers. furthermore, (olowu et al., 2010) determined the concentrations of zn, ni, and fe in tissues of two fish species, tilapia and catfish from two stations in lagos, nigeria. they concluded that both fish species may be considered safe for consumption, but the need for continuous monitoring to prevent bioaccumulation is necessary. results from this study revealed that c garipenus had higher concentrations of zinc and cadmium as compared to zinc. this is consistent with findings from studies conducted in m. furnieri which gave the highest concentrations of pb, zn, and cd by 0.552, 20.535 and 0.090 µg/g, respectively, also m. furnieri accumulated highest concentrations of cd and zn than other species in present study, this may www.scholink.org/ojs/index.php/fsns food science and nutrition studies vol. 2, no. 2, 2018 53 published by scholink inc. be interpreted by the living and feeding habits of m. furnieri (croaker) which is a bottom-dwelling marine species, found in muddy and sandy bottoms in coastal waters. it is considered as a benthic feeder, it feeds on benthic migratory crustaceans and sessile mollusks and occasionally preying on fish. (romeo et al., 1999), reported that levels of metals found in tissues of benthic fish were always higher than those found in pelagic fish as in c. garipenus high concentration of both zn and cd in m. furnieri may be attributed to the relation between them which has been studied in terrestrial and marine mammals. the increase in zn concentration was attributed to compensating the increase in cd concentration due to pollution processes, and this mechanism probably includes the synthesis of metallothioneins (or metallothionein-like proteins), which would bind both cd and zn in a molar ratio of 1:1 (marcovecho, 2004). zinc (zn) is an essential trace element, is relatively nontoxic and is integral to several key functions in human metabolism not only has zn been identified as a component of key enzymes and regulatory proteins, but it was also recently suggested that the preventive effects of zinc may partly be mediated through an increase in cytochrome p450 enzymes. 6. conclusion stored iron in the liver is higher on treatment with lambda-cyhalothrin than with aflatoxin. the latter causes liver damage. the iron levels in both treatments are lower than in the control which signifies impaired iron metabolism. further aflatoxins give a significantly higher level of zinc than lambda-cyhalothrin. on treatment aflatoxins with the facilitation of zinc to compete is better than with lambda-cyhalothrin. this is because aflatoxins disrupt the divalent binding receptors. the fish caught in the wild have the highest levels of toxic metals like cadmium and highest levels of essential elements as compared to cultured fish. many processes and factors in the water body affect these levels. lambda-cyhalothrin, in this particular study a synthetic insecticide has less severe effects on metabolism and iron availability than in the naturally occurring toxin aflatoxin. acknowledgments i would like to acknowledge the university of kabianga for giving time to allow me to conduct the research. i would further like to acknowledge my supervisors prof odipo osano and prof julius ochuodho for tirelessly inputting into this research work. i would also like to acknowledge dr. paul orina of kenya marine, fisheries and research institute for providing research facilities. lastly all my research assistants and my colleagues for their support references akwiri, o. c., raburu, p., okeyo, o., ramesh, f., & onyangore, f. 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