Effect of Temperature and Inoculum Size on Fermentation of Goat Yogurt Containing Lactobacillus acidophilus and Bifidobactrium bifidum

The objective of this study was to investigate the effect of Incubation temperature (35, 37, 39, 41 and 43°C, respectively) and inoculum size (1, 3, 5, 7 and 9%, respectively) on the acidity, pH and viable counts in fermented goat milk containing L. acidophilus and Bifidobactrium bifidum by single factor test, respectively. The results showed that the optimum incubation temperature was 37°C, the acidity, pH, the viable counts of Bifidobactrium bifidum, L. acidophilus and the total viable counts were 81.8°T, 4.55, 3.60×10 7 , 3.8×10 7 and 1.67×10 9 cfu/mL, respectively. The optimum inoculum size was 5%, the acidity, pH, the viable counts of Bifidobactrium bifidum, L. acidophilus and the total viable counts were 98.2°T, 4.51, 5.2×10 7 , 7.7×10 7 and 1.89 ×10 9 cfu/mL, respectively.


Probiotics
have been defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host (FAO/ WHO, 2006).Bifidobacteria and Lactobacilli, which can be considered the most common probiotics (Farnworth et al., 2007;Lourens-Hattingh and Viljoen, 2001), are associated with maintaining optimum microbial balance in the digestive tract with a number of well documented health benefits, including enhancement of the immune system (Lourens-Hattingh and Viljoen, 2001), reduction of lactose intolerance (Kim and Gilliland, 1983) reduction of serum cholesterol level and possible anti carcinogenic properties (Rasic, 2003).Thus, probiotics have been extensively incorporated into dairy foods over the last decade and yogurts containing Lactobacillus acidophilus and/or Bifidobacteria species are widely marketed (Shah, 2000).
L. acidophilus is a kind of important beneficial bacteria; it can regulate intestinal flora balance, enhance immunity, lower cholesterol, relieve the lactose intolerance etc and plays an important role in human health and normal physiological function (Jack et al., 1995;Kashket, 1985).Bifidobacteria can reduce the incidence of colon cancer, anti-tumor, enhance the immune function etc., it is also a beneficial probiotic for human.
In our previous study, the process of fermentation set-style type goat yogurts was optimized by S. thermophilus and L. bulgaricus (Chen et al., 2010), the effect of inoculum and temperature on the fermentation of goat yogurt by L. bulgaricus and S. thermophilus (Shu et al., 2014) was investigated, the effect of the total inoculum size containing L. acidophilus or L. casei on the fermentation of goat milk was studied on the basis of S. thermophilus and L. bulgaricus as starter cultures (Chen et al., 2015).The purpose of this study was to study the effect of incubation temperature and inoculum size on the goat yogurt fermented by L. acidophilus and Bifidobactrium on the basis of S. thermophilus and L. bulgaricus as common starter cultures for developing AB-goat yogurt.

MATERIALS AND METHODS
Microorganism: L. acidophilus, Bifidobactrium bifidum, S. thermophilus and L. bulgaricus were provided from School of Food and Biological Engineering, Shaanxi university of science and technology, they inoculated three successive times with MRS (for L. acidophilus and L. bulgaricus), MRS with 0.5% Cysteine Hydrochloride (for Bifidobactrium bifidum) and M17 (for S. thermophilus) to obtain fresh culture.
Analysis method: Plate coating method was used to determine the viable counts.the total viable counts were determinate by modified Tomato Juice medium, determination of L. acidophilus by MRS agar containing 0.06% bile salt and determination of B. bifidum by MRS agar containing 0.10% LiCl (Chen et al., 2011;Shu et al., 2011).The process was as follows: the agar medium packed in 250 mL flask was sterilized and 15-20 mL poured onto the plates in a clean bench after cooling to 60°C, 0.1 mL aliquot  bacteria dilutions were coated on it after coagulation and then inoculated anaerobic 2-3 day at 37°C, plates containing 30-300 cfu/mL colonies were counted and the results expressed as colony-forming units per mL of sample.
The variation of pH was evaluated using a pHmeter (pHS-3c) at room temperature.Acidity of culture media was determined by sodium hydroxide titration and expressed by Jill Nieer degrees (°T).The sensory evaluation of samples including color, smell, taste, texture were organoleptically assessed by five panelists, who was trained on the basis of normal sensory acuity and consistency.

RESULTS AND DISCUSSION
Effect of temperature on the goat milk fermented by B. bifidum, L. acidophilus and common starter cultures: First, goat milk was heated at 90°C, cooled to 45°C after 10 min, then the starter culture of 7% inoculum size was inoculated.The ratio of B. bifidum, L. acidophilus and common starter cultures was 4:1:1.Inocubation temperature was 35, 37, 39, 41 and 43°C, respectively.The acidity, pH value, viable counts of L. acidophilus and Bifidobactrium bifidum, total viable bacteria were determined every other 1.5 h, then gave a sensory evaluation after 12 h.The result was shown in Fig. 1 and Table 1.
Figure 1a showed viable counts of Bifidobactrium bifidum increased rapidly within 3 h at 35 and 37°C, then increased slowly, reached a peak at 4.5 h, showed a gradual decrease after 4.5 h.The viable counts of B. bifidum grew slow within 4.5 h at 39 and 43°C and then tended to be stable.The viable counts of B. bifidum at 41°C grew slow at the initial stage of fermentation, reached the maximum value at 3 h and then tended to be stable.Among then, the viable counts of B. bifidum at 37°C reached the maximum, 3.60×10 7 cfu/mL, the viable counts of B. bifidum at 41°C was the lowest, 2.00×10 7 cfu/mL.
Figure 1b showed the variation of L. acidophilus viable counts.It had a fast growth within 4.5 h at 35°C and 37°C, reached a maximum at 4.5 h, the viable counts increased slowly in the whole fermentation process at 39, 41 and 43°C, respectively then reached a peak at 4.5 h.It can be suggested that the viable counts of 37°C reached the maximum, 3.80×10 7 cfu/mL, the viable counts of 39°C was the lowest, 1.80×10 7 cfu/mL.
Based on the Fig. 1c, it can be observed that each temperature of the total viable counts of goat yogurt had a fast growth within 3 h.The total viable counts presented a slow increase after 3 h at 37, 39 and 41°C, respectively and then reached the maximum at 4.5 h, while the total viable counts tended to be stable after 3 h at 35 and 43°C.With the temperature of 39°C, the total viable counts presented the highest number, 1.72×10 9 cfu/mL, followed by 37°C, 1.67×10 9 cfu/mL.When the fermentation temperature was 43°C, the total viable counts presented the lowest counting, 1.32×10 9 cfu/mL.
Figure 1d and e showed each temperature of acidity of goat yogurt increased rapidly within 3 h, then began to slow down.The pH of goat yogurt decreased rapidly within 3 h and then began to slow down.The acidity and pH of 37°C were 81.8°T and 4.55 at 4.5 h.It can be observed that the optimum temperature on fermentation of L. acidophilus and B. bifidum was 37°C.
Table 1 presents the sensory evaluation of AB-goat yogurt; it can be observed that the incubation temperature had no obvious influence on the color and texture of goat yogurt.The score of smell and taste is rowing with the increase of the temperature before 41°C, after then, the score decreased with the increase of the temperature.Among then, the goat yogurt tasted a little sweet, no sour and had slight goaty flavor at 35°C.The sour and sweet of goat yogurt were moderate and had a good coagulation at 37, 39, 41 and 43°C, respectively but there is a little goaty flavor at 37°C.

Effect of inoculum size on the goat milk fermented by B. bifidum, L. acidophilus and common starter cultures:
Goat milk was sterilized at 90°C for 10 min, cooled to 45°C, then the mixed liquid starter cultures of different inoculum size (1, 3, 5, 7 and 9%, respectively) were inoculated in the fresh goat milk, the ratio of B. bifidum, L. acidophilus and common starter cultures was 2:1:1, then gave a constant temperature fermentation at 39°C.The acidity, pH value, viable counts of L. acidophilus and B. bifidum, total viable bacteria were determined every other 1.5 h and then there was a sensory evaluation after 12 h.the result was shown in Fig. 2 and Table 2.
Figure 2a showed the viable counts of B. bifidum in goat yogurt had an upward trend within 4.5 h, reached a maximum at 4.5 h, then tended to be stable under different inoculum size.Among then, the viable counts of B. bifidum at 5% inoculum size reached the maximum, 5.20×10 7 cfu/mL, while the viable counts of B. bifidum at 1% inoculum size was the lowest, 2.30×10 7 cfu/mL.
From Fig. 2b, the viable counts of L. acidophilus increased slowly in the whole fermentation process at the 1% inoculum size.Viable counts of L. acidophilus of 3 and 5% increased fast within 4.5 h then began to decrease.The viable counts of L. acidophilus at 7 and 9% inoculum size had a rapidly growth at the initial stage of fermentation, but began to slow down after 3 h, reached the maximum at 4.5 h.Among then, the viable counts of L. acidophilus at 9% inoculum size presented the highest number, 9.90×10 7 cfu/mL, followed by 7 and 5% inoculum size, they were 8.50×10 7 and 7.70×10 7 cfu/mL, respectively.Figure 2c showed the total viable counts at 1, 3 and 9%, respectively inoculum size in goat yogurt had a rapid growth at the beginning of fermentation, then tended to be stable after 3 h, the total viable counts of 5 and 7% inoculum size increased fast at first, then started to slow down, reached the maximum at 4.5 h.Among then, the total viable counts at 5% inoculum size presented the highest number, 1.89×10 9 cfu/mL, the total viable counts at 9% inoculum size was the minimum, 1.37×10 9 cfu/mL.
From Fig. 2d and e, the acidity of goat yogurt of each inoculum size had a dramatically increased trend in the process of fermentation, while the pH had a decreased trend.Among then, the acidity and pH of 5, 7 and 9%, were 98.2, 103.6 and 108°T, 4.51, 4.44 and 4.39, respectively.Table 2 presented the sensory evaluation of ABgoat yogurt.It can be suggested that the sour of goat yogurt tasted a little pale and had slight goaty flavor in 1% inoculum size, the sweet and sour of goat yogurt were moderate and there was not goaty flavor in 3, 5% inoculum size.Seven, 9% inoculum size of goat yogurt had a little acid and slight astringent.Over all, the proper inoculum size of AB-goat yogurt was 5%, the time of coagulation was 4.5 h.

CONCLUSION
Based on the experiments, the incubation temperature and inoculation had significant effects on acidity, pH value, viable counts of L. Acidophilus, B. bifidum and the total viable counts in the AB-goat yogurt.It can be suggested that the optimum temperature on fermentation of L. acidophilus and B. bifidum was 37°C.The acidity, pH, the viable counts of L. acidophilus and B. bifidum and the total viable counts were 81.8°T, 4.45, 3.8×10 7 , 3.60×10 7 and 1.67×10 9 cfu/mL respectively, the score of sensory evaluation was 7.36.The optimum inoculum size of AB-goat yogurt was 5%, the acidity, pH, viable counts of B. bifidum, L. acidophilus viable counts and total viable counts were 98.2°T, 4.51, 5.2×10 7 and 1.89×10 9 cfu/mL, respectively and the score of sensory evaluation was 7.91.

Fig. 1 :
Fig. 1: Effect of bacteria proportion on viable counts of B. bifidum, L. acidophilus, total viable bacteria, pH and acidity in ABgoat yogurt

Fig. 2 :
Fig. 2: Effect of inoculum size on viable counts of B. bifidum and L. acidophilus, total viable bacteria, pH and acidity in AB-goat yogurt

Table 1 :
The sensory evaluation of AB-goat yogurt under different temperature

Table 2 :
The sensory evaluation of AB-goat yogurt under different inoculum size