FERMENTED SOY MILK USING LACTIC ACID BACTERIA

Le Ngoc Tiet1 and Cao Ngoc Diep2

1Padagosycho

University of Dong Thap, Dong Thap province, 2Biotechnology R&D Institute, Can Tho University, Can Tho City,
Vietnam

INTRODUCTION
Milk is one of foods for human nutrition and yogurt is a type of fermented cow milk by lactic acid
bacteria, Lactobacillus delbrueckii sp. bulgaricus and Streptococcus salivarius sp. thermophilus.
Yogurt production was invented probably by accident by Balkan tribes thousands of years ago,
yogurt remained mainly a food of Eastern Europe until the 1900s and it is used as a foodstuff
through Europe. Soy yogurt is yogurt prepared using soy milk, yogurt bacteria, mainly
Lactobacillus bulgaricus and Streptococcus thermophilus and sometimes additional sugar, like
fructose, glucose, honey or raw sugar. It is one of the best alternatives to dairy yogurt for vegans, as
the bacteria of store soy yogurt are usually no more grown on a soy milk base. In yogurt from cow
milk, the sugars are fermented by the bacteria into lactic acid, which causes the characteristics of
curd; The acid lowers to pH of the yogurt to about 4.0 4.5, the bacteria produce lactase from
lactose in dairy milk, however soy milk does not contain lactose but other sugars as starchyose and
raffinose acting as probiotic for growth of Bifidobacterium in large intestine. The aim of this study
was to apply lactic acid bacteria fermenting soy milk to soy-yogurt.
MATERIALS AND METHODS
Isolation of lactic acid bacteria
Two samples of yogurt from VINAMILK and YOMOST, one home-made yogurt, and two tofuextract samples (acidic extract collected during production of tofu) were used to isolate
Lactobacillus bulgaricus using MRS medium (De Man et al., 1960) and Streptoccocus
thermophilus using M17 medium (Terzaghi and Sandine, 1973). These isolates were purified for
several times on LB agar medium and stored in LB-agar medium in glass tubes (5 ml).
Selection of optimal starter
A combination of Lactobacillus bulgaricus and Streptoccocus thermophilus isolates was conducted
to produce starter for soy-yogurt production. An experiment was conducted with 25 combinations
of five Lactobacillus bulgaricus and five Streptoccocus thermophilus isolates. The isolates growing
in MRS medium for Lactobacillus bulgaricus and in M17 medium for Streptoccocus thermophilus
approxinately have the initial count of 108 cells/ml. The experimental design was two-factor
completely randomized design. The first factor was 25 combinations of bacteria (60ml) and the
second factor was three fermentation intervals (4, 5 and 6 incubation hours). Experimental
temperature was 45oC. The experimental results were the average of three replications on medium
of 50% soy milk and 50% cow milk (Vinamilk). The raw materials containing soy milk and cow
milk were pasteurized. Lactic acid bacteria was used at a level of 5%. pH, total acid concentration
and count of lactic acid bacteria were recorded.
Soy-yogurt processing
First, experiment of a completely randomized two-factor design were performed. The first factor
was the combinations of soy milk, cow milk, and lactose, namely 100% soy milk, 97% soy milk
and 3% lactose, 90% soy milk and 10% cow milk, and 80% soy milk and 20% cow milk. These
combinations were supplemented with 4% sucrose and 3% glucose. The second factor was the
incubation intervals ranging from 4 to 10 hours. Incubation temperature was 45oC. Experiments
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were performed in triplicate. The samples were pasteurized and then the starter at level of 5% was
added. Viscosity, taste, pH, total acid concentration and count of lactic acid bacteria were recorded
as a function of incubation duration.
The second experiment was conducted as described above with 15% starter and two incubation
durations (4 and 5 hours).
Homogeneity of variance and analysis of variance (ANOVA) at P < 0.01 were conducted using
MSTAC software. All values are means of three replicates and they were presented in tables,
figures with LSD.01.
RESULTS AND DISCUSSION
Isolation of lactic acid bacteria
Five Lactobacillus bulgaricus isolates and five Streptococcus thermophilus isolates from three
yogurt samples and two acid-water of tofu-extract samples were isolated and selected in the
appropriate medium. Lactobacillus bulgaricus isolates have sizes of 3.6 x 0.9 m, rod shade (Figure
1), while Streptococcus thermophilus isolates have cocci shade, size of 1.2 m (Figure 2).

Figure 1. Lactobacillus bulgaricus

Figure 2. Streptococcus thermophilus

Selection of optimal starter

The pH of samples combining Streptococcus thermophilus from acid-water 1 and Lactobacillus
bulgaricus isolates were smaller than 4.5. Total acid concentration of these samples were from 66.4 to
73.3%. The count of Lactobacillus bulgaricus varied from 4.511 to 7.926 log10/g starter, while that of
Streptococcus thermophilus was from 5.367 to 8.022. However, C4 combination composed of
Lactobacillus bulgaricus 3 (original from commercial yogurt) and Streptococcus thermophilus 4
(isolated from acid-water tofu-extract) had a pH of 4.267, total acid concentration of 73.3%, and high
count of lactic acid bacteria (Lactobacillus bulgaricus 7.530 log10/g, Streptococcus thermophilus
7.098 log10/g). This combination was selected for further study on soy-yogurt processing.
Soy-yogurt processing
With the starter level of 5%, the experimental data (Table 1) showed that pH values of the samples
decreased to levels below 4.5 after 10 hour incubation. Among these, treatment 2 (97% soy milk
plus 3% lactose), treatment 3 (90% soy milk plus 10% cow milk) and treatment 4 (80% soy milk
plus 20% cow milk) showed pH values below 4.2 and they significantly differ from treatment 1
[100% soy milk]. The total acid concentration of the four samples was over 60%, especially
treatment 3 had the highest total acid concentration.

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Table 1. pH, total acid concentration and count of lactic acid bacteria of mixture of soy milk and cow
milk after incubation (starter level of 5%)

8 hours
Treatment

10 hours

Lac
bulga.

Strep.
thermo.

(log10/g)

(log10/g)

Total
acid
(%)

Lac
bulga.

Strep.
thermo.

(log10/g)

(log10/g)

pH

Total
acid (%)

5.02

41.16

8.179

8.531

4.27

62.97

8.812

8.922

5.25

37.72

8.009

8.641

4.17

62.06

8.184

8.747

4.81

52.84

8.195

8.750

4.18

68.53

8.322

8.912

4.87

51.96

7.991

8.734

4.21

65.46

8.761

8.821

LSD.01

0.034

0.392

0.116

0.034

0.034

0.392

0.116

0.034

C.V (%)

0.48

0.92

0.49

0.38

0.48

0.92

0.49

0.38

pH

Treatment 1 - 100% soy milk, treatment 2 - 97% soy milk plus 3% lactose, treatment 3 - 90% soy milk plus 10% cow
milk, treatment 4 - 80% soy milk plus 20% cow milk

With the starter level of 15%, the pH values of the samples decreased to levels below 4.5. The total
acid concentration increased to levels above 60% after 4 to 5 hous incubation 45oC. Especially, the
count of L. bulgaricus and S. thermophilus were increasing over values of 7 and 9 log10,
respectively (Table 2). Sample of treatment 3 [90% soy milk plus 10% cow milk) had pH below
4.3, high total acid concentration, count of lactic acid bacteria after 4 hours. On the other hand,
viscosity and taste of soy-yogurt in this treatment were the same as cow-yogurt.
Table 2. pH, total acid concentration and count of lactic acid bacteria of mixture of soy milk and cow
milk after incubation (starter level of 15%)

4 hours
Treatment
pH

5 hours

Total
acid
(%)

Lac
bulga.

Strep.
thermo.

(log10/g)

(log10/g)

pH

Total
acid
(%)

Lac
bulga.

Strep.
thermo.

(log10/g)

(log10/g)

4.37

61.15

7.846

8.888

4.24

62.07

7.766

9.075

4.25

56.42

8.118

9.041

4.21

57.94

8.642

9.226

4.25

54.78

8.332

9.443

4.23

61.09

8.394

9.449

4.32

54.74

7.997

8.729

4.28

61.72

8.308

9.113

LSD.01

0.015

0.079

0.23

0.05

0.015

0.079

0.23

0.05

C.V (%)

0.43

0.09

1.70

0.31

0.43

0.09

1.70

0.31

Treatment 1 - 100% soy milk, treatment 2 - 97% soy milk plus 3% lactose, treatment 3 - 90% soy milk plus 10% cow
milk, treatment 4 - 80% soy milk plus 20% cow milk

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According to Shurleff and Aoyagi (2004), Li Yu-ying and Grandvoinmet suggested the use of lactic
acid bacteria to ferment soy milk to soy-yogurt in 1912. Lactobacillus bulgaricus and Streptococcus
thermophilus catalased the metabolism of lactose to glucose and galactose and then to lactic acid by
EMP pathway (Ray, 2000). Soy-yogurt production by fermenting soy milk is similar to production
of yogurt from cow milk, however, soy milk does not contain lactose but other sugars like glucose
and sucrose which were supplemented to mixture of soy milk and cow milk. These sugars were
fermented by the lactic acid bacteria producing lactic acid, in the end forming curd. Soy milk
contains starchyose and raffinose which act as probiotics for the growth of Bifidobacterium in the
large intestine. Soy-yogurt containing living bacteria and periodic ingredients which are called
symbiotics is a good food to improve our health as it is free of cholesterol (USDA Nutrient
Database for Standard Reference).
CONCLUSION
Two isolates of Lactobacillus bulgaricus and Streptococcus thermophilus were selected to produce
starter. This starter with low cost can be used for soy-yogurt production at home scale. It is easy to
make soy-yogurt at home and it can replace dairy yogurt for diet people.
REFERENCES
Man, J.C, M. Rogosa and M.E. Shape. 1960. A medium for the lactobacilli. J. Appl. Bacteriol. 23:130-135.
Ray, B. 2000. Microbiology of yogurt fermentation in Fundamental Food, CRC Press, Washington, D.C.
pp:169-173.
Terzaghi, S and W.E. Sandine. 1975. Improved medium for lactic Streptococci and their bacteriophages.
American Society for Microbiology 29:807-813.

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