J. Dairying, Foods & H.S., 30 (4) : 246-251, 2011 AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.ar.arccjour ccjournals.com / indianjournals.com nals.com STUDIES ON COMPOSITION OF PROBIOTIC SOYA-FINGER MILLET MILK BASED YOGHURT Deepika Anand and Rashmi Kapoor Sri Sathya Sai Institute of Higher Learning, Department of Home Science, Anantapur- 515 001, India Received : 28-12-2010 Accepted : 31-10-2011 ABSTRACT Probiotics have received considerable attention over the past few years for their health benefits. Soya bean is a unique food because of its rich proximate and phytochemical content; namely high biological value protein, isoflavones, complex carbohydrates and dietary fiber content. Three products were developed viz., soya milk yoghurt, soya and bovine milk yoghurt and soya and finger millet milk yoghurt. The yoghurts were prepared using cultures isolated from curds. Chemical analysis of the products revealed higher levels of protein, total lipids and total solids in bovine yogurt in comparison to other developed yogurt t samples. Total minerals and phytochemicals viz., total polyphenols and vitamin C contents were found to be higher in soya yogurt. Key words: Probiotics, Yoghurt, Soya bean. INTRODUCTION Role of soya foods and probitoics as functional foods is gaining popularity in the modern society. These products are accepted as a means for self care and complementary to medicine. Probiotics are live microbial dietary supplements that upon sufficient ingestion provide health benefits beyond basic nutrition by modulation of the gut microbial ecosystem (Marteau et al., 2001). It is the isoflavone content of soya bean that has attracted most of the attention of health conscious society. Probiotic enriched soya based foods have gained global recognition as a food source of immense importance due to their unique nutritional and phytochemical profile. Incorporation of probiotic microorganisms in dairy products with regard to health-associated benefits has been reflected in the worldwide markets (Lye et al., 2009). Fermentation of soya milk offers improving its flavor and texture as well as enhancing its beneficial health properties. Soya milk based yogurt offers a considerable appeal for a growing segment of consumers with certain dietary and health concerns. It has advantages over milk yogurt including reduced level of cholesterol and saturated fat as well as low level of lactose (Liu, 1997). The concept of synbiotics has been recently proposed to characteristic health enhancing food supplements used as functional food ingredients in humans. Taking into consideration the importance of soya foods and probiotics in the current functional food context, the present study was undertaken to develop and to analyze the probiotic enriched soya based yoghurts. MATERIALS AND METHODS Isolation of the culture: Lactobacillus culture was isolated from curds and kanji samples using the method described by Uzeh et al., (2006). Help was sought from DFRL, Mysore. From the sample, a 1:10 dilution was subsequently made using peptone water followed by making a 10 fold serial dilution. Exactly 0.1 ml from each dilution was sub cultured, in duplicate, into MRS agar (Merck, Germany) used for isolating LAB. To prevent the growing of yeasts, the media were supplemented with 100 mg/l of cycloheximide before being incubated. These plates were then incubated at 37 C for 48 hours for the growth of lactic acid bacteria. The colonies were counted using colony counter. Stock culture of lactobacillus sp. was kept on agar slant at 4 C and sub cultured from time to time. Two products were prepared viz., soya milk and bovine milk yogurt and soya milk and finger millet yogurt utilizing the above isolated culture.
Vol. 30, No. 4, 2011 247 Washing, and soaking (8 10 hours) of whole soyabean Grinding and filtration Extraction of soya milk Standardization of suitable blend of soya milk and bovine milk Heating at 80 85 C for 20 min Cooling at 37 45 C Inoculation with probiotic culture ( Lactobacillus sp. with 2 % ) Incubating at 42 C for 3 4 hours Cooling, packing and storing at 4 C FIG 1: Preparation of probiotic containing soya milk and bovine milk based yogurt. Probiotic efficiency of pure culture: Identification of bacterial isolates was based on morphological and biochemical characteristics, following the standard methods (Buchanam and Gibsons, 1974). The biochemical characteristics tested were ph tolerance test, temperature tolerance test, bile tolerance and cholesterol reduction test. Soya milk based yoghurt: The ingredients used the yoghurts were soya bean, finger millet and bovine milk. To develop standard soya yogurt and soya and finger millet yogurt, appropriate proportions of soya milk and bovine milk as well as soya milk and finger millet milk (100: 0;
248 J. DAIRYING, FOODS & H.S. TABLE 1: Cholesterol reduction and tolerance to ph, temperature, bile acid of isolated cultures. Sample ph tolerance Temperature Bile acid Cholesterol tolerance ( C) tolerance (%) reduction 3 6 9 30 37 42 0.2 0.4 Curds + + + - + + + + + Beet root kanji + + + - - + + - + TABLE 2: Proximate constituents of probiotic enriched soya based products. Sample Moisture Protein Total lipids Ash Total solids (%) (g/100g) (g/100g) (g/100g) (%) Control 85 ± 0.06 4.62 ± 0.02 3.47 ± 0.09 0.63 ±0.04 13.2 ±0.05 Soya yoghurt 91 ± 0.07 3.25 ± 0.01 1.21 ± 2.34 0.65 ± 0.01 5.89 ±0.01 Soya and bovine milk yoghurt 90 ± 0.06 3.93 ± 0.04 2.34 ± 0.04 0.55 ± 0.02 9.53 ±0.01 Soya and finger millet yoghurt 90 ± 0.08 1.92 ± 0.03 0.691 ±.03 0.61 ± 0.04 7.09 ±0.02 Fcal 60.88 14964.6 20267.7 18.6 127401.1 Sem 15.22 2.61 3.062 0.0037 20.7 CD**(P<0.05) 4.67 1.93 2.09 0.0731 5.44 *Values are mean ± SD of three replicates. **Critical difference. 80: 20; 60: 40; 50: 50; 40: 60; 20: 80 and 0: 100) of each component were prepared. The blends were cultured with pure culture of Lactobacilli spp. to prepare yoghurt. Yogurt samples were analyzed for physicochemical characteristics such as ph, gravity, sugar profile and sensory characteristics. The flow diagram of standard probiotic enriched soya based in outlined in Fig.1. Chemical analysis of the samples The products were analyzed for proximate constituents according to the procedures described by AOAC (1990); Total and acid soluble minerals viz., calcium, iron and phosphorus were analyzed by the methods of Hawk et al., (1957) and Raghuramulu et al., (2003); and phytochemicals viz., polyphenols (Singleton and Rossi, 1995), flavoniods (Jia et al., 1999) and vitamin C (Carawy, 1970) were also determined. Statistical analysis Statistical analysis was done to find out significant difference among the samples and with storage by applying one way ANOVA according to standard methods (Gupta, 1991). RESULTS TS AND DISCUSSIONS Probiotic efficiency of isolated culture:the probiotic efficiency of isolated Lactobacilli culture from curds and kanji was assessed by for their probiotic efficiency bile acid resistance, tolerance to ph, temperature and cholesterol reduction property (Table 1). The cultures were tested for their resistance at varying ph. The isolated culture from curds and kanji samples were studied for survival up to ph 3, 6 and 9. The isolated cultures were also evaluated for their resistance to bile acids added at levels of 0.2 and 0.4 per cent in the media. Cultures from curds exhibited resistance at both the concentration levels of bile acids. However, cultures from kanji could show resistance only at two per cent but not at 4 per cent level of bile acid addition. Isolated cultures from curds revealed survival at 37 and 42ÚC. However, the culture from kanji showed resistance only at 42 C. Both the isolated cultures from curds and kanji samples revealed effective cholesterol reduction at the studied concentration level. Since the pure culture from curds exhibited better effects than kanji. The products were prepared utilizing cultures isolated from curds. Chemical composition Proximate composition: The results of the proximate composition of probiotic enriched soya based products are shown in Table 2. The moisture content of soya based yoghurt ranged from 85 in bovine yoghurt to 91 per cent in soya milk yoghurt. Soya milk yoghurt recorded the highest moisture content (91 per cent) compared with the other yoghurt samples and subsequently the total solid content was the least in the same sample.
Vol. 30, No. 4, 2011 TABLE 3: Carbohydrate analysis of probiotic enriched soya milk based yoghurt. Sample Total carbohydrate Total soluble Reducing Non-reducing (g/ 100g) sugars(g/ 100g) sugars(g/ 100g) (g/ 100g) Control 5.60 ± 0.05 4.50 ± 0.08 3.6 ± 0.07 0.90 ± 0.03 Soya yoghurt 3.22 ± 0.06 0.92 ± 0.05 0.59 ± 0.05 0.33 ± 0.04 Soya and bovine milk yoghurt 3.90 ± 0.05 2.12 ± 0.03 1.92 ± 0.06 0.20 ± 0.07 Soya and finger millet yoghurt 3.02 ± 0.06 1.45 ± 0.03 1.10 ± 0.07 0.35 ± 0.08 Fcal 11.6 6.28 10.7 8.46 Sem 0.07 0.21 1.68 0.87 CD**(P<0.05) 0.12 0.34 0.99 0.34 *Values are mean ±SD of three replicates. ** Critical difference. Total solids content was found to be the highest in bovine yoghurt (13.2 per cent) and the lowest in soya milk yoghurt (5.89 per cent). The analysis for protein content (g/100g) in the prepared yoghurt sample and showed a wide range (1.92 to 4.62). The protein content was found to be the highest in bovine yoghurt (4.62) followed by soya and bovine yoghurt (3.93), soya yoghurt (3.25) and the least in soya and finger millet yoghurt (1.92). The level of proteins in different samples differed significantly (p<0.01). The total lipid content (g/100g) in soya based yoghurt ranged from 0.691 in soya milk and finger millet yoghurt to 3.47 in standard yoghurt. All the samples significantly (p< 0.05) showed lower total lipid content than that of bovine yoghurt. The total ash content (g/100g) of soya based yoghurts was found to range from 0.55 in soya milk and bovine milk yoghurt to 0.65 in soya milk yoghurt. The carbohydrate analysis of probiotic enriched soya milk based yoghurt is presented in Table 3. The carbohydrate content ranged from 3.02 to 5.60 g per cent in soya and finger millet yoghurt and bovine yoghurt, respectively. The highest carbohydrate content was recorded in bovine yoghurt (5.60 per cent). Soya milk yoghurt showed significantly (p<0.05) low carbohydrate content as compared to all other yoghurt samples. The total soluble, reducing and non-reducing sugars observed in the present study ranged from 0.92 to 4.50; 0.59 to 3.60; 0.20 to 0.90 g per cent in bovine and soya milk based yoghurts, respectively. Mineral composition Three important elements analyzed in the developed products were calcium, iron and phosphorous. Table 4 presents the mineral 249 composition of probiotic enriched soya based yoghurts. The observed value for phosphorus (mg/ 100g) ranged from 89 in soya milk and finger millet yoghurt to 98 in soya milk yoghurt. The incorporation of soya bean in yoghurt enhanced the phosphorous availability of the product considerably in comparison to bovine yoghurt (50). The per cent phosphorus extractability was found to range from 55 to 60 in probiotic enriched soya milk based yoghurt in comparison with 54 per cent in bovine yoghurt. The observed value for iron (mg/100g) in the developed products ranged from 0.39 in soya milk and finger millet milk yoghurt to 0.50 in soya milk yoghurt. The available iron values (mg/100g) ranged from 0.21 in soya milk and finger millet milk yoghurt to 0.31 in soya milk yoghurt. The per cent iron extractability was found to range from 53 to 62 per cent in probiotic enriched soya milk based yoghurts. The increased availability could be attributed due to the enhanced microbial activity and partly due to decreased content of phytic acid in the fermented product. The observed value for calcium (mg/100g) in the developed products ranged from 118 in soya milk and finger millet milk yoghurt to 132 in soya milk yoghurt. The incorporation of soya bean in the yoghurts enhanced the native calcium content of the developed products. The available calcium values (mg/100g) ranged from 67 in soya and finger millet milk to 90 in soya milk yoghurt. The per cent calcium extractability as measured by HCl solublization was found to range from 59 to 68 per cent in probiotic enriched soya milk based yoghurts in comparison with 56 per cent in bovine yoghurt.
250 J. DAIRYING, FOODS & H.S. TABLE 4: Mineral analysis of probiotic enriched soya based powders. ple Phosphorus (mg/ 100g) Iron (mg/ 100g) Calcium (mg/ 100g) Total Acid soluble Mineral extractabi lity (%) Total Acid soluble Mineral extractabili ty (%) Total Acid soluble Mineral extractabilit y (%) trol 92 ±0.04 50 ±0.07 54 0.46 ±0.08 0.26 ±0.01 56 120 ±0.06 67±0.09 56 oghurt 98 ±0.09 57 ±0.01 58 0.5 ±0.09 0.31 ±0.12 62 132 ±0.21 90±0.05 68 d bovine oghurt d finger oghurt 95 ±0.03 53 ±0.09 55 0.49 ±0.01 0.29 ±0.04 59 127 ±0.10 75±0.02 59 89 ±0.01 54 ±0.12 60 0.39 ±0.03 0.21 ±0.07 53 118 ±0.05 77±0.04 65 *Values are mean ± SD of three replicates. TABLE 5: Phytochemical screening (mg/100g) of probiotic enriched soya based products. Sample Total polyphenols Total flavanoids Vitamin C Control 0.92 ± 0.06 0.14 ± 0.03 1.02 ± 0.06 Soya yoghurt 56.2 ± 0.09 0.69 ± 0.05 1.29 ± 0.02 Soya and bovine milk yoghurt 26.1 ± 0.01 0.47 ± 0.07 1.14 ± 0.01 Soya and finger millet yoghurt 60.0 ± 0.04 0.84 ± 0.17 1.19 ± 0.09 Fcal 111.4 98.46 242.5 Sem 0.84 0.93 3.17 CD**(P<0.05) 0.56 0.48 1.53 *Values are mean ± SD of three replicates. **Critical difference. Phytochemicals Different phytochemicals analyzed were total polyphenols, flavonoids and vitamin C (Table 5). Analysis revealed a total phenolic content (mg/100g) ranging from 26.1 in soya and bovine milk yoghurt to 60 in soya and finger millet milk yoghurt. The flavonoid content ranged from 0.14 in soya milk yoghurt to 0.84 mg/100gm in soya and finger millet yoghurt. The vitamin C levels were found to be almost similar in the developed products. CONCLUSIONS Yogurt samples prepared from bovine milk; soya milk yogurt and finger millet milk yogurt were analyzed for their chemical characteristics such as proximate constituents, available minerals, and phytochemicals viz., total polyphenols, flavonoids and vitamin C. Chemical analysis of the products revealed higher levels of protein, total lipids and total solids in bovine yogurt in comparison to other yogurt samples. Total minerals and phytochemicals viz., total polyphenols and vitamin C contents were found to be higher in soya yogurt sample in comparison to bovine and other developed yoghurt samples. REFERENCES AOAC. Official Methods of Analysis,16th edn. Washington D.C., 1990. Hawk, Osei, B.L. and Scimmerson (Eds).(1957) Practical Physiological Chemistry. 13th Edn., 1957, 644. Raghuramulu, N., Madhavan, N.K and Kalyanasundaram, S. (Eds).( 2003) A Manual of Laboratory Techniques. National Institute of Nutrition, ICMR. 319-320. Singleton, S. L. and Rossi, J.A. (1995)Colorimetry of total phenolics with phosphomolybidic-phosphotungstic acid reagents (1930). Am. J. Enol Vatic., 16, 144-158.
Vol. 30, No. 4, 2011 251 Marteau, P.R., de Verse, M., Cellier, C.J., and Schrezenmeir, J (2001). Protection from gastrointestinal diseases with the use of probiotics. Am. J. Clin. Nutr., 73: 480s 436s. Lye, H.S., Kuar, C.Y., Eme, J.A., Fung, W.Y. and Liong, M.T. (2009). The improvement of hypertension by probiotics : Effects on cholestrol, diabetis, renin and phytoestrogees. Int. J. Mol. Sci. 10: 3755-3775. Liu, K. (1997). Soyabean chemistry, Technology and utilization. New York: International Thomsoa Publishing, Newyork. Buchanam, R.E. and Gilbbons, N.E. (1974). Bergey s Manual of Determinative Bacteridogy, 8th Ed., The Williams and Wiltons Co., Battimore, M.D. Uzeh, R.E., Uhenhen, R.E. and Rojugbokan, A.K. (2006). Microbiological and nutritional qualities of dietary product : Nono and wara. Nature and Science. 4(3): 37-40. Jia, Z. Tang, M and Wu. J. (1999). Food Chem. 64: 555-559. Gupta, S.K. (1991). Statistical Methods. Sultan Chand and Sons. New Delhi. 234-243.