Response surface methodology for optimizing fermentation conditions of goat yogurt with Bifidobacterium bifidum and Lactobacillus casei

Chen, et al.: Optimization of probiotic goat yogurt 548 Emir. J. Food Agric ● Vol 28 ● Issue 8 ● 2016 2009), and with the improvement of awareness of people for health care, adding probiotics in yogurt production will be a good application (Tharmaraj and Shah, 2000). In recent years, there is a trend in the making products of probiotics with the combinations of two or more probiotics strains and it was used to increase the health value of each strain (Collado et al., 2007). With goat milk as raw material, choosing five kinds of lactic acid bacteria to develop a function goat milk containing γaminobutyric acid (GABA) and angiotensin-converting enzyme (ACE) inhibitory peptides, which could improve the function of the human body, reduce blood pressure, relieve the heart and brain vascular disease and nervous disease (Minervini et al., 2009). Besides, probiotics can treat diseases like lactose intolerance, food allergy, acute gastroenteritis, crohn’s disease, microbial community structure changes, cancer preventive, et al. (Marco et al., 2006; Million and Raoult., 2012; Unno et al., 2015; Desrouillères et al., 2015). The influence of variation of parameters such as fermentation temperature, inoculum size and initial cocci/rods ratio on the fermentation process of yogurt was studied by using response surface methodology, and the method was useful for a better fermentation process which considering optimal combinations of the factors (Torriani et al., 1996). Fermentation temperature could affect the growth of yogurt bacteria and flavor of yogurt (Radke-Mitchell and Sandine, 1986; Guler-Akın and Akın, 2007). Inoculum size could affect a normal acidification process (Burgain, et al. 2013) and to ensure final viable counts of bacteria to be a desired level at the end of fermentation (Chen, et al., 2015). In our previous work, the single factor and orthogonal experiment have been conducted to optimize the fermentation conditions of goat milk fermented by S. thermophilus and L. bulgaricus (Chen et al., 2010). Effect of fermentation temperature, strain ratio and inoculum size on goat yogurt fermented by Bifidobacterium bifidum and Lactobacillus casei was also studied by the single factor experiments (Shu et al., 2015a,b). The aim of the present study is to optimize fermentation conditions of goat yogurt containing B. bifidum and L. casei (BC-goat yogurt) through response surface methodology, and improve the viable counts in fermentation broth and then provide a theoretical basis for quality control in the goat milk productions. MATERIALS AND METHODS Strain and culture preparation Starter bacteria of L. casei (LC), B. bifidum (BB), S. thermophilus and L. bulgaricus were obtained from the School of Food and Biological Engineering, Shaanxi University of Science and Technology. MRS (for LC, BB and L. bulgaricus) and M17 (for S. thermophilus) and TJA were purchased from Qingdao Haibo Biological Technology Co., Ltd. Skimmed milk was purchased from a local retail store. All reagents used in the experiment were of analytical grade dissolved with distilled water and formulated into various concentrations. L. bulgaricus and S. thermophilus freeze-dried power were inoculated with MRS and M17 medium, and then cultivated at 37oC for 24 h. Repeated the experiments several times until bacteria viability is stable judged by microscopic, and then 3-5% bacteria which had been fully activated was inoculated with sterile skim goat milk in anaerobic tube, mixed and cultivated at 42oC (for L. bulgaricus and S. thermophilus) and 37oC (for L. casei and B. bifidum), respectively. After that, 3%~5% skimmed milk was inoculated with sterile whole goat milk in the flask after solidification, mixed and cultivated in the incubators, which can be used for the production of goat milk. Viable bacterial and probiotics counts determination Top agar method and plate coating method were used to determine viable bacterial counts, among them, the viable counts were determined by modified Tomato Juice Medium(TJA), MRS agar containing 0.06% bile salt or 0.1% LiCl was used to determine viable bacterial counts of L. acidophilus and L. casei in fermentation goat milk (Shu et al., 2011). Selective counting was used to determine the number of probiotics, and finally get viable bacteria and probiotics counts in goat yogurt products can reached more than 109 cfu/mL, 106 cfu/mL, respectively. Sensory evaluation The sensory evaluation of the product was carried out with an internal panel consisting of 5 assessors (aged 28-45 years). Subject persons were selected for their sensory ability and trained for descriptive analysis according to the standard flavor profile guidelines set by ISO 6564:1985. The sensory properties of the product were surface appearance, taste, smell, structural state, whey precipitation and other sensory properties, and scoring standard was shown in Table 1. Response surface optimization of fermentation conditions Based on the determined key factors, a 3-variable and 3-level design method was selected to build response surface models (Box and Behnken, 1960). The coded variables and their respective level are given in Table 2.The design was employed to find the optimal fermentation conditions of goat milk by fitting a polynomial model through response surface methodology (RSM). This methodology is applied to determine the maximum response value and evaluation Chen, et al.: Optimization of probiotic goat yogurt Emir. J. Food Agric ● Vol 28 ● Issue 8 ● 2016 549 of the main effects, interaction effects, and quadratic effects. Validation of the model To optimize fermentation conditions of goat milk by using response surface method, and carry out further fermentation experiments which was based on optimized parameters, the effective of model was verified by making comparison of model predictive value and experimental values. Statistical analysis Box-Behnken experiments were carried out by DesignExpert 8.0.6 statistical software and regression analysis of the experimental data to estimate the response of the independent variables. The quality of the fit of the secondorder model equations was expressed by the coefficient of determination (R2) and p <0.05 was considered statistically significant for all analysis. RESULTS AND DISCUSSION Experimental design and results of Box-Behnken In order to optimize fermentation conditions of skimmed goat milk fermentation temperature (A), the strain ratio (B), and inoculum size (C), response surface methodology (RSM) which has been demonstrated to allow evaluation of the effects of multiple parameters on response variables (Pinho et al., 2011)was used. Among them, the strain ratio is about (BB: LC:(LB: ST)), L.bulgaricus and S.thermophilus are used as basic culture medium. The corresponding Box-Behnken design and the results are listed in Table 3. The viable counts of B.bifidum and L.casei were represented by Y1 (×10 6 cfu/mL), Y2 (×10 7 cfu/mL), respectively. The number of total bacteria was represented by Y3 (×10 9 cfu/mL) and sensory value was represented by Y4. Regression analysis A quadratic model could predict the response at any point, even the data not included in the design. Multiple regression equation correlating the response function with the independent variables could be established using the data provided by Box-Behnken Design. And the multivariate quadratic regression model which was used to determine the individual effects and mutual interaction effects of candidate variables can be got as follows: Y1= 1 1 3 1 . 1 7 6 0 5 3 A 2 6 . 0 1 B + 8 . 7 6 C + 0 . 7 1 A B 0.16AC+0.65BC+0.80A2-1.37B2-0.17C2 (1) Y2= 4 5 2 . 2 3 + 2 3 . 9 3 A + 9 . 8 8 B 5 . 7 8 C 0.21AB+0.21AC+0.28BC-0.32A2-0.98B2-0.23 C2 (2) Y3= 80.63-3.73A-1.21B-1.39C+0.02AB+0.006AC+0.063 BC+0.047A2+0.07B2+0.08C2 (3) Table 1: The sense evaluation standard of goat yogurt Project Bad Common Good Very good Color (1 point) Gray or atypical color (0~0.25) Color is uneven, Pale yellow/light gray (0.25~0.50) Color is uniform basically, creamy/milky (0.50~0.75) Color is uniform, milky (0.75~1.00) Smell (3points) Lack of flavor (0~0.75) Flavor is slightly, slight goaty flavor (0.75~1.50) Pure yogurt flavor, slight goaty flavor (1.50~2.25) Fragrance/pure yogurt flavor, no criticism (2.25~3.00) Taste (3 points) Corruption/moldy (0~0.75) Sour and sweet taste are too strong or weak (0.75~1.50) Sweet and sour moderate, little astringency (1.50~2.25) Sweet and sour moderate, no criticism (2.25~3.00) Structural state (3 points) Adverse curd, bubbles, whey precipitation serious (0~0.75) Curd uneven, not strong, whey separation (0.75~1.50) Curd is good, state is uniform and fine, little whey precipitation (1.50~2.25) No bubbles , no whey precipitation (2.25~3.00) Table 2: Factors and levels for optimizing fermentation conditions of BC‐goat yogurt Independent variables Level