Food Control 41 (2014) 1e6

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Whole soybean as probiotic lactic acid bacteria carrier food

in solid-state fermentation
ShanTing Zhang, Yan Shi, ShuLi Zhang, Wei Shang, XueQin Gao, HaiKuan Wang*
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology,
No. 29, 13th Avenue TEDA, Tianjin, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: For the purpose of preparing lactic acid bacteria (LAB) carrier food, the solid-state fermentation of whole
Received 26 October 2013 soybean was performed using Bifidobacterium animalis 937, Lactobacillus casei Zhang and Lactobacillus
Received in revised form plantarum P-8 mixed with Bacillus subtilis natto, respectively. The physicochemical properties, the amino
18 December 2013
nitrogen content and peptide molecular weight distribution of the fermented whole soybean products
Accepted 20 December 2013
were examined during this process. After 48 h of fermentation, the viable counts of the three samples
were 1.41
108 CFU/g (B. animalis 937), 1.74
1010 CFU/g (L. casei Zhang) and 2.19
1010 CFU/g
Keywords:
(L. plantarum P-8), with the pH declined rapidly from 6.32 to 5.78, 5.60 and 5.44 at the early stage of the
Whole soybean
Solid-state fermentation
fermentation and increased to 6.71, 6.47 and 6.60 at the later stage of the fermentation. The fermentation
Lactic acid bacteria caused a sharply increase in the content of the free amino nitrogen from 99.7 mmol/g to 301.9 mmol/g,
Bacillus subtilis natto 390.1 mmol/g and 529.1 mmol/g in the solid fermented soybean products, due to the multiplication of
microorganism and the effect of enzyme system. Furthermore, the levels of soybean peptide with mo-
lecular weight less than 1000 Da increased 30.7%, 71.2% and 81.3% relative to that of the control group at
48 h. The result of the present work implied that whole soybean fermented by LAB can provide the
different probiotics for the host, and there is potential to develope nutritious fermented soybean
products.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction of the microbial enzyme, insoluble macromolecular substances

Soybean is an excellent source for nutrition that include plant
protein, oligosaccharides, VB, VE and mineral substance (Dajanta,
Chukeatirote, & Apichartsrangkoon, 2012). Acceptance of soybean
protein products has increased because of the low cost and high
nutritional quality for human consumption and also as protein
source for animal meals (Frias, Song, Martinez-Villaluenga, Gon-
zalez De Mejia, & Vidal-Valverde, 2008). Soybean is also a crucial
protein source for Asian people (Ojokoh & Wei, 2011). For example,
soybean curd and soymilk are their favorite food (Amadou, Le, Shi,
& Jin, 2011; Feng, Eriksson, & Schnurer, 2005). It has been shown
that the production of soybean have some functional compound
which can reduce the risk of cardiovascular diseases and cancers
(Amadou, 2011). In addition, fermented soybean products such as
black bean sauce, natto and tempeh are people’s daily diet (Juan &
Chou, 2010; Lv, Guo, & Yang, 2009). After fermentation, the nutri-
tion inhibitory factor in soybean was eliminated. Under the action
* Corresponding author. Tel.: þ86 22 6060 1958; fax: þ86 22 6060 2298.
E-mail address: hkwang@aliyun.com (H. Wang).
such as protein, fat and carbohydrate were degraded into poly-
peptides, fatty acids and oligopeptides, which can improve the
nutrition utilization of soybean.
Lactic acid bacteria (LAB) and Bacillus subtilis natto as probiotics
can exert different health effects on the consumers, which leads to
the developing of certain functional foods (Molina, Médici, Font de
Valdez, & Taranto, 2012). LAB are well known to be major benefi-
cial microflora in human intestine, and they have been widely uti-
lized to manufacture fermented soymilk products and other types of
foods (Kim, Lee, & Yoo, 2012). For example, it can reduce the soy flour
immunoreactivity by fermenting with Lactobacillus plantarum by
decreasing the IgE immunoreactivity (Frias, Song, Martinez-
Villaluenga, Gonzalez De Mejia, & Vidal-Valverde, 2008; Nguyen,
Guyot, Icard-Vernière, Rochette, & Loiseau, 2007). Lactobacillus casei
as a significant member of LAB can survive naturally in the intestinal
tract to regulate the gut microorganism and reduce the risk of cancer,
and also be used for fermenting soymilk (John, Nampoothiri, &
Pandey, 2007). In addition, soybean also has natural oligosaccha-
ride, such as raffinose, stachyosea, and sugar, and they have some
unique characteristics to make Bifidobacterium proliferating, to
improve the Bifidobacterium viable count, and to reduce harmful

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.12.026
2 S. Zhang et al. / Food Control 41 (2014) 1e6
bacteria, so as to improve the quality of soybean fermentation
products (Han, Ebert, Zhao, Li, Zhang, & Tian, 2005). Bacillus subtilis is
the most broadly used strain for soybean fermentation, which can
increase antioxidant activity, anti-allergic activity and fibrinolytic
function of the soybean (John, Nampoothiri, & Pandey, 2007; Juan,
Wu, & Chou, 2010; Kwon, Lee, Lee, Chang, & Chang, 2000).
Many studies suggested that LAB was widely used in the
fermentation of soybean derived products, such as sufu (a Chinese
fermented soybean food), soybean flour and soymilk (Georgetti
et al., 2009; Han, Cao, Rombouts, & Nout, 2004; Marazza,
Nazareno, de Giori, & Garro, 2012). However, to our knowledge,
the whole soybean fermented by LAB mixed with B. subtilis natto as
probiotics carrier food was not investigated. Besides, the solid-state
fermentation possesses several biotechnological advantages, such
as higher fermentation productivity, higher end-concentration of
products, higher product stability, lower catabolic repression,
cultivation of microorganisms specialized for water-insoluble
substrates and lower demand on sterility (Holker, Hofer, & Lenz,
2004). After the solid-state fermentation of the whole soybean,
the products can be lyophilized directly without centrifugation. As
a result, the solid-state fermentation of the whole soybean is a
more economical and simple fermentation technology in order to
produce probiotics carrier food.
In our research, the solid-state fermentation of whole soybean
was performed using Bifidobacterium animalis 937, L. casei Zhang
and L. plantarum P-8 mixed with Bacillus subtilis natto, respectively.
The physicochemical properties, the amino nitrogen content and
peptide molecular weight distribution of the fermented whole
soybean products were examined during this process.
2. Material and methods
2.1. Microorganisms
The microorganism, L. casei Zhang and L. plantarum P-8 were
obtained from Key Laboratory of Dairy Biotechnology and Engi-
neering, Ministry of Education, Inner Mongolia Agricultural Uni-
versity (Inner Mongolia, PR China), which had been considered as
new probiotic strains (Bao, Wang, & Zhang, 2012; Bao, Zhang, & Li,
2012; Wang, Zhang, Zhang, Wei, Bao, Zhang, Sun, Postnikoff, Meng,
& Zhang, 2012; Wu, Zhang, Sun, Wu, Yue, Meng, & Zhang, 2011)
isolated form traditional fermented foods in Inner Mongolia of
China. B. animalis 937 was isolated from the feces of a healthy child
in our laboratory and B. subtilis natto was isolated in our laboratory
from a traditional fermented food (natto) collected from Hei-
longjiang, China (Wang, Zhang, Sun, & Dai, 2013).
2.2. Inoculum preparation
For inoculum preparation, L. casei Zhang and L. plantarum P-8
were grown in MRS broth (peptone 10 g/L, yeast extract 5 g/L, beef
extract 10 g/L, sodium acetate anhydrous 5 g/L, ammonium citrate
tribasic 2 g/L, K2HPO4 2 g/L, MgSO4$7H2O 0.1 g/L, MnSO4$H2O
0.05 g/L, tween-80 1 g/L, pH 6.8) at 37  C for 20 h. B. animalis 937
was grown in MRS broth at 37  C for 20 h under anaerobic condi-
tions. B. subtilis natto was grown in the broth containing 1% soy-
bean meal and 2% glucose at 37  C，150 rpm for 20 h. The cells
were then harvested and resuspended in sterilized physiological
saline and adjusted to 106 CFU/mL. This cells suspension was ready
to serve as inoculum for soybean fermentation.
2.3. Soybean fermentation
The soybeans (Northeast soybean, Heilongjiang, PR China) were
cleaned and soaked in the water (pH 6.5) overnight at 4  C and then
the soaking water was discarded. The soybeans were sterilized at
115  C for 25 min and cooled to 24  C and then were inoculated
with 106 CFU/g of L. plantarum P-8, L. casei Zhang and B. animalis
937 mixed with 106 CFU/g of B. subtilis natto, respectively. The
fermentation process was carried out at 37  C for 48 h and the
fermentation of B. animalis 937 mixed with B. subtilis natto was
cultured with anaerobic conditions. The samples were taken for
analysis after 0, 24, 28, 32, 36 and 48 h of the fermentation.
2.4. Microbiological analysis (total viable bacterial count)
Total viable counts were determined during the fermentation.
The fermented soybeans (10 g) were homogenized with 90 ml of
the sterilized physiological saline (0.85%). Serial dilutions were
prepared in sterilized physiological saline and 1 ml of appropriate
dilutions was poured in triplicate plates for total viable count. Total
viable counts of L. plantarum P-8, L. casei Zhang and B. animalis 937
were made using a pour plate method and MRS agar after serial
dilution in maximum recovery diluents. A pre-prepared test sample
(1 ml) of 107, 108, and/or 109 dilution was transferred into a
sterile petri dish, in triplicate, and warm (45  2  C) sterile plate
count MRS agar (15 ml) was mixed with the inoculums. Cultures
were incubated anaerobically at 37  C for 48  2 h. Total Viable
counts of B. subtilis natto was made using spread plate technique.
About 15 ml nutrient agar medium was poured into plates prior to
use. A pre-prepared test sample (0.1 ml) of 106, 107, and/or 108
dilution was transferred to the surface of nutrient agar medium, the
inoculums was spread evenly over the entire surface of the agar by
a rotary twirling motion of the plate under the rod. Cultures were
incubated at 37  C for 24  2 h. The colonies were then counted and
expressed as logarithmic colony forming units per gram (lg CFU/g)
of the sample.
2.5. Physicochemical analysis
2.5.1. The determination of pH
10 g of the fermented soybeans (wet weight) were homogenized
in a blender with 90 ml of distilled water for 30 s and the pH value
of the suspension was measured with an FE20 pH meter (Mettler-
Toledo, Shang Hai, PR China).
2.5.2. The determination of free amino nitrogen
10 g of the fermented soybeans (wet weight) were homogenized
in a blender with 90 ml of distilled water for 30 s. The free amino
nitrogen of the homogenate was extracted at 4  C for 24 h,
centrifuged at 7000 rpm for 15 min and the supernatant was
reserved. The free amino nitrogen was determined according to the
ninhydrin method (Abernathy, Spedding, & Starcher, 2009).
2.5.3. The determination of molecular weight distribution
Fermented soybeans (10 g) were homogenized with 50 ml of
distilled water and incubated at 4  C for 4 h, centrifuged at
4500 rpm for 10 min at 4  C and then the supernatant was passed
through 0.45 mm Millipore filters (Minisart, Sartorious, Germany).
The filtered supernatant was collected, lyophilized and stored
at 20  C until further used.
1.0 g freeze-dried samples was dissolved in 99 ml mobile
phase (50 mM phosphate buffer containing 0.15 M NaCl,
pH ¼ 7.2). The sample was loaded onto GE SuperdexÔ Peptide
10/300 GL column (i.d. 10
300 nm), eluted with mobile phase
at a flow rate of 0.5 ml/min and monitored at 220 nm. A mo-
lecular weight calibration curve was obtained from the following
standards: Cytochrome C (12,500 Da), Trasylol (6512 Da),
Glutathione Disulfide (615 Da), Reduced Glutathione (310 Da)
and Glycine (75 Da).
 S. Zhang et al. / Food Control 41 (2014) 1e6 3

2.6. Statistical analysis

All experiments were performed in triplicate, and the results

were expressed as means  standard deviation. Data were tested
for statistical significance by the Statistical Analysis System soft-
ware (SAS 9.00; SAS Institute Inc. NC, USA).

3. Results and discussion

3.1. Microbial counts during fermentation

Microbiological monitoring of the fermented soybean was co-

inoculated with individual LAB species and B. subtilis natto. As
shown in Figs. 1e3, the population of the LAB increased remarkably
from 24 h to 36 h, and then entered a stationary phase. The addition
of B. subtilis natto resulted in an increase in the number of viable
cells of the LAB. As it was reported, B. subtilis natto can produce
catalase, which exhibit a similar growth-promoting effect on lac-
tobacilli (Hosoi, Ametani, Kiuchi, & Kaminogawa, 2000). After 48 h
of the fermentation, the viable count of the sample fermented by
L. plantarum P-8 mixed with B. subtilis natto was 2.19
1010 CFU/g,
which is higher than that of the samples fermented by B. animalis
937 mixed with B. subtilis natto and L. casei Zhang mixed with
B. subtilis natto with the viable count of 1.41
108 CFU/g and
1.74
1010 CFU/g, respectively. The results indicated that the fer-
mented whole soybean could serve as LAB probiotic carrier food
effectively. Besides, fermentation could decrease soy immunore-
activity (Frias, Song, Martinez-Villaluenga, Gonzalez De Mejia, &
Vidal-Valverde, 2008) and increase fibrinolytic enzyme (nattoki-
nase) activity proportionately (Kim, Hwang, & Lee, 2010).
3.2. The pH values
At the initial stage of the fermentation, the microbiological
monitoring of the fermented samples revealed that the LAB was able
to dominate soybean fermentation and the production of the lactic
acid resulted in the decrease of pH (Fig. 4). The pH values of the
samples fermented by B. animalis 937, L. plantarum P-8 and L. casei
Zhang mixed with B. subtilis natto declined rapidly from 6.32 to 5.78,
5.60 and 5.44, respectively. At the later period of the soybean
fermentation, B. subtilis natto could largely hydrolyze soy proteins
leading to release of amines and ammonia and a rise of pH value. The
Fig. 2. The growth cure of L. casei Zhang and B. subtilis natto in the co-fermentation of
the whole soybean. The samples were taken for analysis after 0, 6, 12, 18, 24, 28, 32, 36
and 48 h. Data were expressed as mean  SD from three independent experiments.
pH of the three samples increased to 6.71, 6.47 and 6.60 at the end of
the fermentation. As compared with other fermented soybean
foods, due to its characteristic of the musty odor and slimy appear-
ance, natto (the sterilized whole soybean was only fermented by
B. subtilis natto) was not so popular and widely consumed, even
though it was well known in Japan (Weng & Chen, 2010). The lowest
pH of the mixture slurries (rice/soybean slurries) fermented by
L. plantarum A6 was 3.79 (Nguyen, Loiseau, Icard-Vernière, Rochette,
Trèche, & Guyot, 2007), which was too sour for the consumer to
accept. The results of our study indicated that the combination of the
LAB and B. subtilis natto in the fermentation could improve the odor
and the slimy appearance of the fermented whole soybean by
maintaining at an appropriate pH value and providing acidic taste.
3.3. Free amino nitrogen
The results of the microbial counts during the fermentation
showed that LAB grew well with B. subtilis natto. The content of the
free amino nitrogen (Fig. 5) sharply increased at the middle and

Fig. 1. The growth cure of L. plantarum P-8 and B. subtilis natto in the co-fermentation Fig. 3. The growth cure of B. animalis 937 and B. subtilis natto in the co-fermentation of
of the whole soybean. The samples were taken for analysis after 0, 6, 12, 18, 24, 28, 32, the whole soybean. The samples were taken for analysis after 0, 6, 12, 18, 24, 28, 32, 36
36 and 48 h. Data were expressed as mean  SD from three independent experiments. and 48 h. Data were expressed as mean  SD from three independent experiments.
4 S. Zhang et al. / Food Control 41 (2014) 1e6
Fig. 4. The effect of LAB and B. subtilis natto on the pH in the solid-state fermentation
of the whole soybean. The samples were taken for analysis after 0, 24, 28, 32, 36 and
48 h. Data were expressed as mean  SD from three independent experiments. d-d
Whole soybean was fermented by B. animalis 937 mixed with B. subtilis natto. dCd
Whole soybean was fermented by L. casei Zhang mixed with B. subtilis natto. d:d
Whole soybean was fermented by L. plantarum P-8 mixed with B. subtilis natto.
later stages of the fermentation with the multiplication of micro-
organism and the effect of enzyme system, and reached 301.9 mmol/
g (B. animal 937), 390.1 mmol/g (L. casei Zhang) and 529.1 mmol/g
(L. plantarum P-8) at the end of the fermentation, increased 202.8%,
291.3% and 430.7% relative to that of the control group (the control
group was not inoculated with LAB and B. subtilis natto). As it was
reported that the soybean meal was fermented by L. plantarum with
the total amino acids increasing significantly (p < 0.05) by Song,
Frías, Martinez-Villaluenga, Vidal-Valdeverde, & de Mejia (2008).
The solid-state fermentation of whole soybean can cause a
significant increase in protein solubility, available amino acid and
in vitro digestibility. It is well known that the hydrolysis of protein
Fig. 5. The effect of LAB and B. subtilis natto on the content of the free amino nitrogen
in the solid-state fermentation of the whole soybean The samples were taken for
analysis after 0, 24, 28, 32, 36 and 48 h. Data were expressed as mean  SD from three
independent experiments. d-d Whole soybean was fermented by B. animalis 937
mixed with B. subtilis natto. dCd Whole soybean was fermented by L. casei Zhang
mixed with B. subtilis natto. d:d Whole soybean was fermented by L. plantarum P-8
mixed with B. subtilis natto.
Fig. 6. The molecular weight distribution of the fermented whole soybean with LAB
and B. subtilis natto after 24 h of the fermentation. Data were expressed as mean  SD
from three independent experiments. d d Control group was not inoculated with
LAB and B. subtilis natto. d d Whole soybean was fermented by B. animalis 937
mixed with B. subtilis natto. d d Whole soybean was fermented by L. casei Zhang
mixed with B. subtilis natto. d d Whole soybean was fermented by L. plantarum P-
8 mixed with B. subtilis natto.
in fermented soybean is greatly dependent upon microbial strain,
substrate and moisture content (Kim, Lee, & Yoo, 2012). Such as, in
the process of the natto, the most nonessential amino acids and
essential amino acids are found to increased after 48 h of fermen-
tation (Weng & Chen, 2010). And the level of total free amino acids
of the soy protein extract hydrolyzed by the enzyme of the LAB also
increases (Aguirre, Garro, & de Gioria, 2008). In our study, the
greatly increase of the content of free amino nitrogen of the whole
soybean fermented by LAB mixed with B. subtilis natto demon-
strated that the enzyme system of LAB and B. subtilis natto could
work well to hydrolyze the soybean protein.
Fig. 7. The molecular weight distribution of the fermented whole soybean with LAB
and B. subtilis natto after 48 h of the fermentation. Data were expressed as mean  SD
from three independent experiments. d d Control group was not inoculated with
LAB and B. subtilis natto. d d Whole soybean was fermented by B. animalis 937
mixed with B. subtilis natto. d d Whole soybean was fermented by L. casei Zhang
mixed with B. subtilis natto. d d Whole soybean was fermented by L. plantarum P-
8 mixed with B. subtilis natto.
 S. Zhang et al. / Food Control 41 (2014) 1e6 5

3.4. Molecular weight (MW) distribution
The size of peptides is known to be a significant factor to assess
the nutritional value of soybean product (Song et al., 2008). Fig. 6
showed that the levels of peptide with MW less than 1000 Da in
the samples fermented by L. casei Zhang mixed with B. subtilis
natto, L. plantarum P-8 mixed with B. subtilis natto for 24 h
increased 5.0% and 12.9%. The sample fermented by B. animalis 937
mixed with B. subtilis natto decreased 4.6% relative to that of the
control group, because the production of proteases was poor at the
initial stage of the fermentation, the protein of the soybean was
only partly hydrolyzed and the most peptides were utilized by
B. animalis. The levels of peptide with MW more than 10,000 Da
(Fig. 6) in the samples decreased 14.5% (B. animalis 937 with
B. subtilis natto), 20.5% (L. plantarum P-8 with B. subtilis natto) and
22.3% (L. casei Zhang with B. subtilis natto) with the hydrolysis of
proteases.
After 48 h of the fermentation, the levels of peptide with MW
less than 1000 Da (Fig. 7) in the three samples fermented by
B. animalis 937, L. casei Zhang and L. plantarum P-8 mixed with
B. subtilis natto increased 30.7%, 71.2%, and 81.3% relative to that of
the control group. And the levels of peptide with MW more than

Fig. 8. Chromatograms of molecular weight calibration curve, control group and the fermented whole soybean. (A) Chromatograms of molecular weight calibration curve: Cy-
tochrome C (12,500 Da), Trasylol (6512 Da), Glutathione Disulfide (615 Da), Reduced Glutathione (310 Da) and Glycine (75 Da). (B) Control group (whole soybean was not fermented
by B. animalis 937 mixed with B. subtilis natto). (C) Whole soybean was fermented by B. animalis 937 mixed with B. subtilis natto. (D) Whole soybean was fermented by L. casei Zhang
mixed with B. subtilis natto. (E) Whole soybean was fermented by L. plantarum P-8 mixed with B. subtilis natto.
6 S. Zhang et al. / Food Control 41 (2014) 1e6
10,000 Da in the three samples decreased 34.3%, 75.2%, and 77.2%,
respectively. And the molecular weight distribution of peptide in
the samples fermented by B. animalis 937, L. casei Zhang and
L. plantarum P-8 mixed with B. subtilis natto was showed in Fig. 8.
As it was reported that the soybean protein meal fermented by
L. plantarum Lp6 increased the amount of low molecular weight
peptides (Amadou, Le, Shi, Gbadamosi, Kamara, & Jin, 2011).
Therefore, the effect of LAB and B. subtilis natto on the levels of
peptide with MW less than 1000 Da of the fermented whole soy-
bean could lead to the development of the nutritious and functional
fermented soybean products.
4. Conclusion
This study presented a series of the fermented whole soybean
foods by using LAB mixed with B. subtilis natto in solid-state
fermentation, which had received recognition as a potential
biotechnological process without causing serious environmental
pollution. Due to the microbial autolysis and enzyme chemistry
reaction, the whole soybean fermented by LAB mixed with
B. subtilis natto could provide different probiotics for the host and
had the capacity to improve nutritional and functional properties.
The results also indicated that the viable count, the content of free
amino nitrogen and the level of peptide with MW less than 1000 Da
in the sample fermented by L. plantarum P-8 mixed with B. subtilis
natto were the highest compare with B. animalis 937 mixed with
B. subtilis natto and L. Casei Zhang mixed with B. subtilis natto. In
conclusion, a novel probiotic LAB carrier food was provided by
using LAB mixed with B. subtilis natto to ferment the whole soybean
in the solid-state fermentation.
Acknowledgments
The authors wish to acknowledge the financial support provided
by the National Natural Science Foundation of China (No.
30900961) and Tianjin Social Science Planning Program (No.
TJGLWT11-08).
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