Production of probiotic soy yogurt

Available online at www.scholarsresearchlibrary.com Annals of Biological Research, 2012, 3 (6):2750-2754 (http://scholarsresearchlibrary.com/archive.html) ISSN 0976-1233 CODEN (USA): ABRNBW Production of probiotic soy yogurt Atefeh Ghorbani a, Rezvan Pourahmad a*, Masoud Fallahpour b, Mahnaz Mazaheri Assadi b a Department of Food Science and Technology, Varamin Branch, Islamic Azad University, Varamin, Iran b Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran _ ABSTRACT Soy yogurt is a fermented product that obtained from inoculation of yogurt starter to soymilk. The aim of this study was to investigate the effect of kind of probiotic culture (Bifidobacterium lactis B-12, Bifidobacterium lactis B-94, mixture of Bifidobacterium lactis B-12 and Bifidobacterium lactis B-94) and incubation temperature ( C and C) on microbiological, physicochemical and sensory attributes of soymilk yogurt. ph, titratable acidity, redox potential, viscosity, viability of probiotic bacteria and sensory characteristics of produced samples were analyzed. The sample made using B.lactis B-12 and incubated at C had the highest score of sensory analysis. This sample was stored at 4 C for three weeks. Physicochemical characteristics and viability of probiotic bacteria during 21- days of refrigerated storage were evaluated. During refrigerated storage, acidity, redox potential and viscosity increased, while ph and syneresis decreased. Viability of Bifidobacterium lactis B-12 until the 14 st day increased and then decreased. Key words: Bifidobacterium lactis, incubation temperature, probiotic soy yogurt, refrigerated storage _ INTRODUCTION Soybean is a rich source of protein and economical protein food. Soy-based foods may have some health benefits to consumers due to their hypolipidemic, anticholesterolemic and antiatherogenic properties as well as due to reduced allergenicity [1]. It also contains isoflavones, which can reduce the risk of most hormone-associated health disorders [2]. Soymilk obtained by aqueous extraction from whole soybean, is a well-known food product that is growing in popularity in many areas of the world [3]. However, consumption of soymilk is undesired due to the presence of unpleasant off-flavors carried over from soy beans [4-5]. Moreover, soymilk contains flatulence causing oligosaccharides including raffinose and stachyose [5]. The problems of soymilk can be improved by lactic fermentation, so production of fermented soymilks such as soymilk yogurt is important. Probiotic bacteria are defined as live microorganisms that when administered in enough amounts confer health benefits on the host [6]. Fermented foods that have potential probiotic properties are produced worldwide from a variety of food substrates [7]. Lactic acid bacteria especially lactobacilli and bifidobacteria are the most common bacteria considered as potential probiotics [8]. The aim of this study was to evaluate the effect of kind of probiotic culture and incubation temperature on microbiological, physicochemical and sensory attributes of soymilk yogurt. MATERIALS AND METHODS Starter cultures The DVS pouches of commercial lyophilized cultures including YC-X11 yogurt bacteria (mixed culture of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) and pure strains of Bifidobacterium lactis 2750

B-12 were supplied by Chr-Hansen (Denmark). Bifidobacterium lactis B-94 was supplied by DSM (Australia). The cultures were maintained according to manufacturer s instructions, at -18 C until used. Soymilk The UHT soymilk was used in this study. It was supplied by Maxsoy Company (Karaj, Iran). Preparation of probiotic soy yogurt Soymilk samples were heated at 85 C for 30 min, then cooled down to - C for inoculation. The samples of soymilk yogurt were made by adding an inoculum consisting of Bifidobacterium lactis B-12, Bifidobacterium lactis B-94, mixture of Bifidobacterium lactis B-12 and Bifidobacterium lactis B-94 to sterilized soymilk. The count of bifidobacteria was 10 7 cfu/ml. Then the samples were inoculated with yogurt starter culture (10 7 cfu/ml). The ratio of yogurt bacteria to bifidobacteria was 1:5. All inoculated soymilk samples were incubated at C and C. Fermentation was stopped at ph 4.5 4.7. Then the samples were kept at 4 C. At the second part of experiments, the sample with the most general acceptability was selected. Physicochemical characteristics and viability of probiotic bacteria in this sample were evaluated during 21-days of refrigerated storage. Physicochemical analysis ph and redox potential of the samples were measured using a ph meter (MA235, HANNA, Milan, Italy). Titratable acidity was determined by AOAC method [9]. Viscosity was measured by Brookfield viscometer that used shear rate 6 and spindle 4 [10]. Syneresis was determined by centrifuging yogurt at 350 G for 30 min and it was expressed as volume of separated whey per 100 ml of yogurt [11]. Microbiological analysis MRS-bile agar (MRS agar from Merck, Germany and bile from Sigma, USA) was used for the selective enumeration of probiotic bacteria in the presence of yogurt bacteria. The plates were incubated anaerobically at C for at least 72 h [12]. Sensory evaluation All samples were evaluated 1 day after production by 9 trained panelists. The sensory attributes were appearance, aroma, taste and texture. The acceptability values were scored on 5 (very good), 4 (good), 3 (moderate), 2 (bad) and 1 (very bad). Statistical analysis Experiments were performed in triplicate. The data were analyzed using One-way Analysis of Variance (ANOVA) and Duncan test by SPSS 18.0. RESULTS AND DISCUSSION Physicochemical analysis Table 1 presents the values of physicochemical parameters in the soymilk yogurt samples. The kind of probiotic culture and incubation temperature significantly affect physicochemical characteristics of these samples (p<0.05). Fermentation was stopped at ph 4.5-4.7 in all the samples. However, the samples reached the mentioned ph (4.5-4.7) in different periods. The samples made using the mixture of B.lactis B-12 and B.lactis B-94 and incubated at C reached ph 4.5-4.7 in the shortest fermentation time (360 min), while the samples produced by B.lactis B-94 and incubated at C had the longest fermentation period (510 min). It shows that incubation at lower temperature with B.lactis B-94 leads to increase in fermentation time due to the lack of proteolytic activity of B.lactis B-94 so it cannot break protein and form free amino acids and small peptides. Fermentation resulted in decreasing the ph value, whereas the titratable acidity increased. The highest final titratable acidity observed in the samples containing B.lactis B-12 and the lowest final titratable acidity was related to the samples made using B.lactis B-94 that is due to the lack of acid tolerance. The acid production in yogurt depends on the growing of microorganisms and their ability for fermentation of the carbohydrates in soymilk. The major carbohydrates present in soymilk are sucrose, raffinose and stachyose, whereas in milk it is lactose [13]. Also, Liu reported that lactic acid bacteria grow well in soymilk but produce less organic acids [14]. The concentration of lactic acid was higher than that of acetic acid. Acetic acid is an undesirable end-product in fermented soymilk due to its vinegary flavor; therefore, the high production of lactic acid over acetic acid by lactobacilli is desirable [15]. Our findings showed that the lowest viscosity observed in the samples made using the B.lactis B-94. It confirmed the claim of manufacturer Company that this strain did not have any effect on texture and can use it without limitation in any product and when the sample was incubated at C, yogurt bacteria prevail over probiotics. On 2751

the other hand probiotics especially bifidobacteria are not able to produce sufficient lactic acid while this component has basic role in increase of texture and taste [16]. Table 1: Physicochemical properties of soymilk yogurt samples (values are means ± SD) T( C) probiotic culture B12 B94 B12+B94 B12 B94 B12+B94 Fermentation time (min) 450 510 420 390 420 360 ph drop rate (min) Physicochemical Properties Acidity Redox potential increase increase rate rate (MV/min) ( D/min) Final Titratable Acidity( D) Viscosity (CP) 0.004±0.0001 b 0.11±0.004 c 0.21±0.005 de 67.500±0.9 a 15.433±0. b 0.003±0.0005 c 0.09±0.003 d 0.2±0.01 e 63.900±0.9 c 12.800±0.7 c 0.004±0.0002 b 0.11±0.004 c 0.24±0.015 bc 65.700±0.9 b 15.933±0.32 b 0.004±0.0003 ab 0.12±0.004 b 0.26±0.011 ab 65.700±0.9 b 21.767±0.55 a 0.004±0.0002 b 0.12±0.004 b 0.23±0.011 cd 67.500±0.9 a 13.0±0.2 c 0.005±0.0001 a 0.14±0.005 a 0.28±0.015 a 66.600±0.9 ab 16.100±0.36 b Microbiological analysis Table 2 shows the counts of probiotic bacteria of the samples. The highest count of probiotic bacteria (8.869±0.14 log cfu/ml) was observed in the sample made using the B.lactis B-12 and incubated at C; in contrast, the sample made using the B.lactis B-94 and incubated at C exhibited the lowest viability(8.100±0.17 log cfu/ml). The kind of probiotic culture significantly affected the Bifidobacterium count (p<0.01).the results demonstrated that the samples produced using B. lactis B-12 had the highest bacterial counts, whereas the lowest counts of Bifidobacterium were recorded in the samples made using B. lactis B-94. As indicated from Table 2, incubation at C resulted in greater viability for all probiotic culture compared to C. This was in agreement with the related previous reports [17-19]. In mixed probiotic cultures, particularly when yogurt starters are co-cultured with probiotic cultures, fermentation temperature significantly affects viability of probiotics. The optimum growth temperature of bifidobacteria and yogurt bacteria is within ranges of - C and 42-45 C, respectively. Therefore, employing higher incubation temperatures (44 C in comparison with - C) leads to significantly lower viability of probiotics due to domination of yogurt bacteria over probiotics [19-20]. Table 2: Microbial counts of soymilk yogurt samples (values are means ± SD) Microbial populations (log cfu/ml) T( C) probiotic culture Initial population (log cfu/ml) Final population (log cfu/ml) B12 8.602 8.869±0.14 a B94 8.602 8.259±0.24 b B12+B94 8.602 8.800±0.03 a B12 8.602 8.861±0.07 a B94 8.602 8.100±0.17 b B12+B94 8.602 8.593±0.11 a Sensory analysis Table 3 demonstrates the sensory analysis results for the samples. The score of appearance, aroma, taste and texture of the samples were not different significantly (p>0.05). Our findings showed that the highest score of appearance observed in the samples made using the B. lactis B-12 and incubated at C, similarly the sample made using B.lactis B-12 and incubated at C. The lowest value was related to the sample made using the mixture of B. lactis B-12 and B. lactis B-94 and incubated at C. The samples made using B. lactis B-12 and incubated at C had the highest aroma score, while the lowest values were remarked in the sample containing mixture of B. lactis B-12 and B. lactis B-94 and incubated at C, similarly the sample made using mixture of B. lactis B-12 and B.lactis B-94 and incubated at C. The highest score of taste and texture observed in the samples made using B. lactis B-12 and incubated at C. The lowest value was related to the sample made using the mixture of B.lactis B- 12 and B.lactis B-94 and incubated at C. The incubation temperature significantly affect the taste and texture of the samples (p<0.05). The kind of probiotic culture significantly affected the appearance, aroma and taste of the samples (p<0.01). The results for certain 2752

attributes showed that the highest overall acceptability was related to the sample made using B. lactis B-12 and incubated at C. Therefore, this sample was stored at 4 C for 21 days. Table 3: Sensory properties of soymilk yogurt samples (values are means ± SD) Sensory attributes T( C) probiotic culture Appearance Aroma Taste Texture B12 4.78±0.441 a 4.67±0.500 a 4.11±0.60 ab 4.67±0.70 ab B94 4.67±0.500 a 4.00±0.707 b 3.67±0.500 b 4.56±0.52 ab B12+B94 3.67±0.707 b 3.44±0.527 c 3.56±0.52 b 4.33±0.500 b B12 4.78±0.441 a 4.89±0.333 a 4.67±0.500 a 5.00±0.000 a B94 4.44±0.726 a 4.56±0.527 a 3.89±0.78 b 4.56±0.52 ab B12+B94 3.89±0.601 b 3.44±0.527 c 3.89±0.78 b 4.89±0.33 a Physicochemical changes during refrigerated storage Table 4 shows 21-day refrigerated storage significantly (p<0.01) affected the ph drop, acidity and redox potential increase of the best sample (made using B. lactis B-12 and incubated at C). The initial ph (day 1) is 4.7 and the final ph is 4.55. This decline in ph was presumably due to continued fermentation by the lactic acid bacteria during refrigerated storage. Physicochemical changes occurred more rapidly at the first 7 days of storage in comparison to final 7 days, it could be referred to more activity of yogurt bacteria and higher amounts of nutritional composition of soy milk that produces lactic acid and acetic acid but generally the trend of changes is very slow due to increase dry matter contents of soy yogurt that has buffering effect. Significant differences in the syneresis and viscosity of the sample during 21-day refrigerated storage were found (p<0.01). Rapid ph drop forms irregular and disharmonic protein structure therefore soy protein hydrophobic links set surface of gel structure and increase syneresis. But refrigerated storage is sufficient time for rearrangement of yogurt gel structure and increase of water activity that leads to decrease syneresis and increase viscosity [21]. Survival of B.lactis B-12 during refrigerated storage Table 4 shows the viability of Bifidobacterium in sample made using the B. lactis B-12 and incubated at C during 21 days of cold storage. The viable probiotic counts increased until day 14 during cold storage, but it decresead from day 14 to 21. According to table 4, the greatest and the lowest viability of B. lactis B-12 were observed in 14 st day (8.95±0.01 cfu/ml) and first day (8.84±0.01 cfu/ml), respectively. In general, the viability of B. lactis B-12 increased during cold storage, and the sample contained more than 10 8 cfu/ml viable probiotic on day 21. Donkor reported that fermented soymilk with L. casei L26 and B. lactis B94 and stored at 4 C for 28 day showed an increased trend of bacterial growth, even though there was a general decline in microbial populations from day 21 to day 28 [6]. Table 4: Physicochemical properties and viable probiotic counts of soymilk yogurt during refrigerated storage (values are means ± SD) Storage time (day) ph Acidity ( D) Redox potential (MV) Viscosity (CP) Syneresis(%) Viable counts (log cfu/ml) 1 4.70±0.0 a 66±1.0 c 139±1.0 d 21.76±0.01 d 16.66±0.01 a 8.84±0.01 d 7 4.62±0.01 b 70±1.0 b 144±1.0 c 22.06±0.01 c 15.62±0.01 b 8.91±0.01 c 14 4.58±0.01 c 71±1.0 ab 147±1.0 b 22.56±0.01 b 14.39±0.01 c 8.95±0.01 a 21 4.55±0.01 d 72±./0 a 149±1.0 a 23.41±0.01 a 12.61±0.01 d 8.93±0.01 b CONCLUSION Results of this study revealed that we can use bifidobacteria to produce probiotic soy yogurt. The kind of probiotic culture and incubation temperature affected ph, redox potential, final titratable acidity and viscosity of the samples. The samples were incubated at C had more viscosity and also the highest viscosity was observed in sample with B. lactis B-12. The kind of probiotic culture significantly affected the viability of probiotic bacteria (p<0.01). The greatest viability of probiotics was observed in the sample with B. lactis B-12 and incubated at C and the lowest viability of probiotic bacteria obtained in sample with B. lactis B-94 and incubated at C. The incubation temperature significantly affected the texture and the kind of probiotic culture significantly affected the appearance, aroma and taste of the samples. The results showed that the highest sensory characteristics were related to sample with B. lactis B-12 and incubated at C. This sample which was set in the second grade of viability of probiotics was selected as the best sample. Viscosity of this sample increased during 21-day cold storage and its syneresis decreased. Viability of B. lactis B-12 increased until the 14 st day and then decreased. 2753

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