Selection and study on technological parameters of symbiotic starter cultures Lactobacillus bulgaricus and Streptococcus thermophilus for production of Vietnamese yogurt

In this paper ten symbiotic starter cultures for yogurt production were examined for their coagulation time, titratable acidity, pH at the moment of coagulation. Their maximum rate of acidification was also determined by model of fermentation kinetics. Three starter cultures were selected for production of Vietnamese yogurt. With the selected starter culture, yogurt from natural milk and reconstituted whole milk was obtained. Their coagulation time, acidity, maximum rate of acidification and rate of acidification during storage of product were studied. As a result of this study and mathematical modeling, we concluded that maximum rate of acidification at moment of coagulation and during storage was affected by the type of milk used in yogurt production.


Introduction
The benefits of traditional Bulgarian yogurt on human health have been investigated since many years. The yogurt has immunostimulatory effect due to components in the cell wall of the lactic acid bacteria such as peptidoglycan, teichoic acids, lipoproteins, exopolysaccharides (Takahashi et al. 1993;Akira et al. 2006) and bioactive substances produced from the growth of microorganisms in starter culture (Sanders 1999;Lourens-Hattingh and Viljoen 2001;Zubillaga et al. 2001). Yogurt also has antimicrobial activity due to high concentration of active microorganisms Lactobacillus bulgaricus and Streptococcus thermophilus. These lactic acid bacteria maintain the balance between the bacterial species in the gastrointestinal tract by producing short-chain acids (mainly lactic acid), bacteriocins, (Bhatia et al., 1989), BLIS (Bacteriocin-Like Inhibitory Substances) and other metabolites which inhibit enteropathogens (Davidson et al. 2000;Fang et al. 2001). Consuming yogurt is beneficial in lowering the high level of total serum cholesterol, which is considered a factor for cardiovascular disease (Akalin et al. 1997;Anderson and Gilliland 1999). This effect can be explained by high proteolytic activity of Lb. bulgaricus, which synthesizes low molecular weight bioactive peptides such as αlactotensin (His-Ile-Arg-Leu) from β-Lactoglobulin (Yoshikawa et al. 2000). Furthermore, the high hydrolysis activity of Streptococcus thermophilus and Lactobacillus bulgaricus in yogurt can improve lactose tolerance in human (McBean 1999). Vietnamese dairy industry is developing rapidly in the recent years, although the first industrial dairy products were produced in 1990 (Khoi and Dung 2014). However, there has been still limited research about the Vietnamese yogurt and its production. The Vietnamese yogurt has specific organoleptic characteristics such as sweet taste and viscous consistency. The cause for the sweet taste of Vietnamese yogurt can come from the low acidity of the yogurt and addition of a certain amount of sugar during their production. The starter cultures used to produce Vietnamese yogurt mainly include strains of Lactobacillus bulgaricus and Streptococcus thermophilus.
In Vietnam, a large percentage of milk powder was used to produce yogurt and dairy products due to the shortage of natural cow's milk. Natural cow's milk obtained in Vietnam provides only 30% of domestic demand and the remaining 70% is imported from different countries such as: New Zealand, America, Singapore, and Netherlands. Having reviewed the cited literature, the aim of the present paper was to study on technological parameters of symbiotic starter cultures and the effect of different type of milk using for Vietnamese yogurt production.

Materials and Methods
Combinations starter cultures. Modeling the kinetics of the fermentation process. Models 1 and 2 were used for the kinetics of the fermentation process, as the pH and titratable acidity were presented in a non-dimensional form (Tishin and Fedorov 2016;Bouguettoucha et al. 2011;Gordeev et al. 2017). Identification of the model parameters was performed by the method of least squares.
Model 1: Where: Vmax is maximum rate of acidification, 1/h; рНb -pH in non-dimensional form; рНb0 -initial value of pH in non-dimensional form; KT -titratable acidity in non-dimensional form; qP -rate of post-acidification, 1/h; n -exponent indicator or the change in rate of postacidification over time (characterized for intensity of post-acidification); τ -cultivation time, 1/h.

Results and Discussion
Ten combinations of symbiotic starter cultures with selected strains of Lactobacillus bulgaricus, characterized by antimicrobial activity were obtained. The obtained starter cultures were studied for coagulation time, pH and titratable acidity at moment of coagulation, maximum rate of acidification to select starter cultures for production of Vietnamese yogurt with desired quality. The results of these studies were presented in Figure 1 and Table 2.
According to the presented data in the Figure 1 and  The differences in coagulation time, titratable acidity and pH at the moment of coagulation were explained by the composition and the ratio between the lactic acid bacteria, as well as in the specific acidification ability of the various strains.
To determine the effect of the acidification ability of the starter cultures, the models of kinetics of pH reduction (acidification of milk) were studied and the maximum rate of acidification was determined. The data are presented in Table 3. According to  Yogurt products were obtained from natural cow's milk and reconstituted whole milk using the selected starter cultures. The effect of the type of milk on coagulation time, titratable acidity, pH, maximum rate of acidification, and rate of postacidification during the storage of yogurt was studied. Figure 2 shows the comparative results between the experimental data and the data obtained by modeling of pH kinetics during the formation of yogurt using natural cow's milk using the selected starter cultures. The kinetic parameters for the models were presented in Table 4. As shown in Figure 2, model's data were corresponded to the experimental data with very high accuracy and could be used to predict the dynamics of lactic acid fermentation. According to data in Table 4, the maximum rates of acidification in combinations 2B and 3B respectively were 0.043 1/h and 0.042 1/h, which were slightly lower than the one in combination 6 (0.052 1/h).
The coagulation time of the natural milk with these selected combinations varied in the range of 4h10min to 4h25min. The pH values of combinations 2В, 3В и 6 at the moment of coagulation were respectively 5.50 ± 0.03, 5.53 ± 0.03 и 5.34 ± 0.04. The titratable acidity of 3 combinations were in the range of 70 ± 0.4 to 80 ± 0.4°Т. Another important factor for the production of Vietnamese products is the rate of post-acidification of the starter cultures. The mathematical model for kinetics of post-acidification was studied. The model's data were compared to the experimental data, which is shown in Figure 3.   37), which exhibited the change of intensity of post-acidification process. The slightly higher rate of post-acidification in combination 2B and the high exponent indicator showed the intensification of post-acidification in the early stage of storage of yogurt. For combinations 3B and 6, exponent indicators were similar at 0.87 and 0.77, showing that postacidification process for those combinations was moderate during the storage period. The rate of acidification for combination 6 was determined at 8.10 -4 1/h. From reviewed the cited literature, the main source for the production of Vietnamese yogurt is reconstituted whole milk (3% fat). In this case, yogurt was obtained from reconstituted whole milk using three combinations starter cultures 2B, 3B and 6. During the fermentation process, the rate of acidification, the dynamics of pH and titratable acidity at the moment of coagulation, the coagulation time and the rate of post-acidification were determined. The results are shown in Figure 4. The kinetics of model's parameters is shown in Table 5.
As shown in Figure 4, there's well fit between experimental data and model's data. The coagulation time of reconstituted whole milk with the selected starter cultures was shorter than the one of natural milk. According to Table 5, the higher rate of acidification was recorded -from 0.068 to 0.087 1/h. The shorter coagulation time and higher rate of acidification can be explained by the change in structure and hydration characteristics of protein as a result of evaporating of milk to dryness. The mathematical model of the rate of postacidification and the comparison between the experimental data and model data were determined. The results are shown in Figure 5.
The presented results showed that the titratable acidity in non-dimensional type by the model was very well agreed with values by experiments. The combination 3B was characterized with highest rate of post-acidification (1.40.10 -3 1/h) and highest exponent indicator (1.25). It means for this combination starter culture there's more intensive process of post-acidification than the other two combinations.
For the starter cultures 2B and 6, lower rates of postacidification were observed, respectively at 1.23. 10 -3 1/h and 9.3.10 -4 1/h. The exponent indicators in both combinations are lower than 1 (0.77 and 0.97) which exhibit that the process of postacidification proceeded with lower intensity.

Conclusions
Our examination of symbiotic starter cultures with selected strains of Lactobacillus bulgaricus and Streptococcus thermophilus showed that the combinations starter cultures 2B, 3B and 6 are more suitable for production of Vietnamese yogurt. As a result of mathematical modeling, we concluded that maximum rate of acidification at moment of coagulation and during storage was affected by the type of milk used in yogurt production. From that point of view, it allows us to select the appropriate starter cultures for various type of milk for production of Vietnamese yogurt.