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Food Control 41 (2014) 1e6

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Food Control

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Whole soybean as probiotic lactic acid bacteria carrier foodin 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

Article history:Received 26 October 2013Received in revised form18 December 2013Accepted 20 December 2013

Keywords:Whole soybeanSolid-state fermentationLactic acid bacteriaBacillus subtilis natto

* Corresponding author. Tel.: þ86 22 6060 1958; faE-mail address: hkwang@aliyun.com (H. Wang).

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.12.026

a b s t r a c t

For the purpose of preparing lactic acid bacteria (LAB) carrier food, the solid-state fermentation of wholesoybean was performed using Bifidobacterium animalis 937, Lactobacillus casei Zhang and Lactobacillusplantarum P-8 mixed with Bacillus subtilis natto, respectively. The physicochemical properties, the aminonitrogen content and peptide molecular weight distribution of the fermented whole soybean productswere examined during this process. After 48 h of fermentation, the viable counts of the three sampleswere 1.41 � 108 CFU/g (B. animalis 937), 1.74 � 1010 CFU/g (L. casei Zhang) and 2.19 � 1010 CFU/g(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 thefermentation and increased to 6.71, 6.47 and 6.60 at the later stage of the fermentation. The fermentationcaused a sharply increase in the content of the free amino nitrogen from 99.7 mmol/g to 301.9 mmol/g,390.1 mmol/g and 529.1 mmol/g in the solid fermented soybean products, due to the multiplication ofmicroorganism 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 at48 h. The result of the present work implied that whole soybean fermented by LAB can provide thedifferent probiotics for the host, and there is potential to develope nutritious fermented soybeanproducts.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Soybean is an excellent source for nutrition that include plantprotein, oligosaccharides, VB, VE and mineral substance (Dajanta,Chukeatirote, & Apichartsrangkoon, 2012). Acceptance of soybeanprotein products has increased because of the low cost and highnutritional quality for human consumption and also as proteinsource for animal meals (Frias, Song, Martinez-Villaluenga, Gon-zalez De Mejia, & Vidal-Valverde, 2008). Soybean is also a crucialprotein 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 shownthat the production of soybean have some functional compoundwhich can reduce the risk of cardiovascular diseases and cancers(Amadou, 2011). In addition, fermented soybean products such asblack 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

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of the microbial enzyme, insoluble macromolecular substancessuch as protein, fat and carbohydrate were degraded into poly-peptides, fatty acids and oligopeptides, which can improve thenutrition utilization of soybean.

Lactic acid bacteria (LAB) and Bacillus subtilis natto as probioticscan exert different health effects on the consumers, which leads tothe developing of certain functional foods (Molina, Médici, Font deValdez, & Taranto, 2012). LAB are well known to be major benefi-cial microflora in human intestine, and they have been widely uti-lized tomanufacture fermented soymilk products and other types offoods (Kim, Lee, &Yoo, 2012). For example, it can reduce the soyflourimmunoreactivity by fermenting with Lactobacillus plantarum bydecreasing the IgE immunoreactivity (Frias, Song, Martinez-Villaluenga, Gonzalez De Mejia, & Vidal-Valverde, 2008; Nguyen,Guyot, Icard-Vernière, Rochette, & Loiseau, 2007). Lactobacillus caseias a significantmember of LAB can survive naturally in the intestinaltract to regulate thegutmicroorganismandreduce the riskof 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 someunique characteristics to make Bifidobacterium proliferating, toimprove the Bifidobacterium viable count, and to reduce harmful

S. Zhang et al. / Food Control 41 (2014) 1e62

bacteria, so as to improve the quality of soybean fermentationproducts (Han, Ebert, Zhao, Li, Zhang,&Tian, 2005).Bacillus subtilis isthe most broadly used strain for soybean fermentation, which canincrease antioxidant activity, anti-allergic activity and fibrinolyticfunction 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 thefermentation of soybean derived products, such as sufu (a Chinesefermented soybean food), soybean flour and soymilk (Georgettiet al., 2009; Han, Cao, Rombouts, & Nout, 2004; Marazza,Nazareno, de Giori, & Garro, 2012). However, to our knowledge,the whole soybean fermented by LABmixed with B. subtilis natto asprobiotics carrier food was not investigated. Besides, the solid-statefermentation possesses several biotechnological advantages, suchas higher fermentation productivity, higher end-concentration ofproducts, higher product stability, lower catabolic repression,cultivation of microorganisms specialized for water-insolublesubstrates 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. Asa result, the solid-state fermentation of the whole soybean is amore economical and simple fermentation technology in order toproduce probiotics carrier food.

In our research, the solid-state fermentation of whole soybeanwas performed using Bifidobacterium animalis 937, L. casei Zhangand L. plantarum P-8 mixed with Bacillus subtilis natto, respectively.The physicochemical properties, the amino nitrogen content andpeptide molecular weight distribution of the fermented wholesoybean products were examined during this process.

2. Material and methods

2.1. Microorganisms

The microorganism, L. casei Zhang and L. plantarum P-8 wereobtained 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 asnew 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 ofChina. B. animalis 937 was isolated from the feces of a healthy childin our laboratory and B. subtilis natto was isolated in our laboratoryfrom 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-8were grown in MRS broth (peptone 10 g/L, yeast extract 5 g/L, beefextract 10 g/L, sodium acetate anhydrous 5 g/L, ammonium citratetribasic 2 g/L, K2HPO4 2 g/L, MgSO4$7H2O 0.1 g/L, MnSO4$H2O0.05 g/L, tween-80 1 g/L, pH 6.8) at 37 �C for 20 h. B. animalis 937was 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 cellswere then harvested and resuspended in sterilized physiologicalsaline and adjusted to 106 CFU/mL. This cells suspension was readyto serve as inoculum for soybean fermentation.

2.3. Soybean fermentation

The soybeans (Northeast soybean, Heilongjiang, PR China) werecleaned and soaked in the water (pH 6.5) overnight at 4 �C and then

the soaking water was discarded. The soybeans were sterilized at115 �C for 25 min and cooled to 24 �C and then were inoculatedwith 106 CFU/g of L. plantarum P-8, L. casei Zhang and B. animalis937 mixed with 106 CFU/g of B. subtilis natto, respectively. Thefermentation process was carried out at 37 �C for 48 h and thefermentation of B. animalis 937 mixed with B. subtilis natto wascultured with anaerobic conditions. The samples were taken foranalysis 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 ofthe sterilized physiological saline (0.85%). Serial dilutions wereprepared in sterilized physiological saline and 1 ml of appropriatedilutions was poured in triplicate plates for total viable count. Totalviable counts of L. plantarum P-8, L. casei Zhang and B. animalis 937were made using a pour plate method and MRS agar after serialdilution inmaximum recovery diluents. A pre-prepared test sample(1 ml) of 10�7, 10�8, and/or 10�9 dilution was transferred into asterile petri dish, in triplicate, and warm (45 � 2 �C) sterile platecount MRS agar (15 ml) was mixed with the inoculums. Cultureswere incubated anaerobically at 37 �C for 48 � 2 h. Total Viablecounts of B. subtilis natto was made using spread plate technique.About 15 ml nutrient agar medium was poured into plates prior touse. A pre-prepared test sample (0.1 ml) of 10�6, 10�7, and/or 10�8

dilutionwas transferred to the surface of nutrient agar medium, theinoculums was spread evenly over the entire surface of the agar bya rotary twirling motion of the plate under the rod. Cultures wereincubated at 37 �C for 24� 2 h. The colonies were then counted andexpressed as logarithmic colony forming units per gram (lg CFU/g)of the sample.

2.5. Physicochemical analysis

2.5.1. The determination of pH10 g of the fermented soybeans (wet weight) were homogenized

in a blender with 90 ml of distilled water for 30 s and the pH valueof the suspension was measured with an FE20 pH meter (Mettler-Toledo, Shang Hai, PR China).

2.5.2. The determination of free amino nitrogen10 g of the fermented soybeans (wet weight) were homogenized

in a blender with 90 ml of distilled water for 30 s. The free aminonitrogen of the homogenate was extracted at 4 �C for 24 h,centrifuged at 7000 rpm for 15 min and the supernatant wasreserved. The free amino nitrogenwas determined according to theninhydrin method (Abernathy, Spedding, & Starcher, 2009).

2.5.3. The determination of molecular weight distributionFermented soybeans (10 g) were homogenized with 50 ml of

distilled water and incubated at 4 �C for 4 h, centrifuged at4500 rpm for 10 min at 4 �C and then the supernatant was passedthrough 0.45 mm Millipore filters (Minisart, Sartorious, Germany).The filtered supernatant was collected, lyophilized and storedat �20 �C until further used.

1.0 g freeze-dried samples was dissolved in 99 ml mobilephase (50 mM phosphate buffer containing 0.15 M NaCl,pH ¼ 7.2). The sample was loaded onto GE Superdex� Peptide10/300 GL column (i.d. 10 � 300 nm), eluted with mobile phaseat a flow rate of 0.5 ml/min and monitored at 220 nm. A mo-lecular weight calibration curve was obtained from the followingstandards: Cytochrome C (12,500 Da), Trasylol (6512 Da),Glutathione Disulfide (615 Da), Reduced Glutathione (310 Da)and Glycine (75 Da).

Fig. 2. The growth cure of L. casei Zhang and B. subtilis natto in the co-fermentation ofthe whole soybean. The samples were taken for analysis after 0, 6, 12, 18, 24, 28, 32, 36and 48 h. Data were expressed as mean � SD from three independent experiments.

S. Zhang et al. / Food Control 41 (2014) 1e6 3

2.6. Statistical analysis

All experiments were performed in triplicate, and the resultswere expressed as means � standard deviation. Data were testedfor 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. Asshown in Figs. 1e3, the population of the LAB increased remarkablyfrom 24 h to 36 h, and then entered a stationary phase. The additionof B. subtilis natto resulted in an increase in the number of viablecells of the LAB. As it was reported, B. subtilis natto can producecatalase, which exhibit a similar growth-promoting effect on lac-tobacilli (Hosoi, Ametani, Kiuchi, & Kaminogawa, 2000). After 48 hof the fermentation, the viable count of the sample fermented byL. 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. animalis937 mixed with B. subtilis natto and L. casei Zhang mixed withB. subtilis natto with the viable count of 1.41 � 108 CFU/g and1.74 � 1010 CFU/g, respectively. The results indicated that the fer-mented whole soybean could serve as LAB probiotic carrier foodeffectively. 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 microbiologicalmonitoringof the fermented samples revealed that the LABwas ableto dominate soybean fermentation and the production of the lacticacid resulted in the decrease of pH (Fig. 4). The pH values of thesamples fermented by B. animalis 937, L. plantarum P-8 and L. caseiZhangmixedwith B. subtilis nattodeclined rapidly from6.32 to 5.78,5.60 and 5.44, respectively. At the later period of the soybeanfermentation, B. subtilis natto could largely hydrolyze soy proteinsleading to release of amines and ammonia and a rise of pHvalue. The

Fig. 1. The growth cure of L. plantarum P-8 and B. subtilis natto in the co-fermentationof 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 ofthe fermentation. As compared with other fermented soybeanfoods, due to its characteristic of the musty odor and slimy appear-ance, natto (the sterilized whole soybean was only fermented byB. subtilis natto) was not so popular and widely consumed, eventhough it waswell known in Japan (Weng & Chen, 2010). The lowestpH of the mixture slurries (rice/soybean slurries) fermented byL. plantarumA6was 3.79 (Nguyen, Loiseau, Icard-Vernière, Rochette,Trèche, & Guyot, 2007), which was too sour for the consumer toaccept. The results of our study indicated that the combinationof theLAB and B. subtilis natto in the fermentation could improve the odorand the slimy appearance of the fermented whole soybean bymaintaining at an appropriate pH value and providing acidic taste.

3.3. Free amino nitrogen

The results of the microbial counts during the fermentationshowed that LAB grew well with B. subtilis natto. The content of thefree amino nitrogen (Fig. 5) sharply increased at the middle and

Fig. 3. The growth cure of B. animalis 937 and B. subtilis natto in the co-fermentation ofthe whole soybean. The samples were taken for analysis after 0, 6, 12, 18, 24, 28, 32, 36and 48 h. Data were expressed as mean � SD from three independent experiments.

Fig. 4. The effect of LAB and B. subtilis natto on the pH in the solid-state fermentationof the whole soybean. The samples were taken for analysis after 0, 24, 28, 32, 36 and48 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 LABand B. subtilis natto after 24 h of the fermentation. Data were expressed as mean � SDfrom three independent experiments. d d Control group was not inoculated withLAB and B. subtilis natto. d d Whole soybean was fermented by B. animalis 937mixed with B. subtilis natto. d d Whole soybean was fermented by L. casei Zhangmixed 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) 1e64

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 controlgroup was not inoculated with LAB and B. subtilis natto). As it wasreported that the soybeanmeal was fermented by L. plantarumwiththe 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 asignificant increase in protein solubility, available amino acid andin 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 nitrogenin the solid-state fermentation of the whole soybean The samples were taken foranalysis after 0, 24, 28, 32, 36 and 48 h. Data were expressed as mean � SD from threeindependent experiments. d-d Whole soybean was fermented by B. animalis 937mixed with B. subtilis natto. dCd Whole soybean was fermented by L. casei Zhangmixed with B. subtilis natto. d:d Whole soybean was fermented by L. plantarum P-8mixed with B. subtilis natto.

in fermented soybean is greatly dependent upon microbial strain,substrate and moisture content (Kim, Lee, & Yoo, 2012). Such as, inthe process of the natto, the most nonessential amino acids andessential amino acids are found to increased after 48 h of fermen-tation (Weng & Chen, 2010). And the level of total free amino acidsof the soy protein extract hydrolyzed by the enzyme of the LAB alsoincreases (Aguirre, Garro, & de Gioria, 2008). In our study, thegreatly increase of the content of free amino nitrogen of the wholesoybean fermented by LAB mixed with B. subtilis natto demon-strated that the enzyme system of LAB and B. subtilis natto couldwork well to hydrolyze the soybean protein.

Fig. 7. The molecular weight distribution of the fermented whole soybean with LABand B. subtilis natto after 48 h of the fermentation. Data were expressed as mean � SDfrom three independent experiments. d d Control group was not inoculated withLAB and B. subtilis natto. d d Whole soybean was fermented by B. animalis 937mixed with B. subtilis natto. d d Whole soybean was fermented by L. casei Zhangmixed 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 assessthe nutritional value of soybean product (Song et al., 2008). Fig. 6showed that the levels of peptide with MW less than 1000 Da inthe samples fermented by L. casei Zhang mixed with B. subtilisnatto, L. plantarum P-8 mixed with B. subtilis natto for 24 hincreased 5.0% and 12.9%. The sample fermented by B. animalis 937mixed with B. subtilis natto decreased 4.6% relative to that of thecontrol group, because the production of proteases was poor at theinitial stage of the fermentation, the protein of the soybean was

Fig. 8. Chromatograms of molecular weight calibration curve, control group and the fermtochrome C (12,500 Da), Trasylol (6512 Da), Glutathione Disulfide (615 Da), Reduced Glutathiby B. animalis 937 mixed with B. subtilis natto). (C) Whole soybeanwas fermented by B. animamixed with B. subtilis natto. (E) Whole soybean was fermented by L. plantarum P-8 mixed

only partly hydrolyzed and the most peptides were utilized byB. animalis. The levels of peptide with MW more than 10,000 Da(Fig. 6) in the samples decreased 14.5% (B. animalis 937 withB. subtilis natto), 20.5% (L. plantarum P-8 with B. subtilis natto) and22.3% (L. casei Zhang with B. subtilis natto) with the hydrolysis ofproteases.

After 48 h of the fermentation, the levels of peptide with MWless than 1000 Da (Fig. 7) in the three samples fermented byB. animalis 937, L. casei Zhang and L. plantarum P-8 mixed withB. subtilis natto increased 30.7%, 71.2%, and 81.3% relative to that ofthe control group. And the levels of peptide with MW more than

ented whole soybean. (A) Chromatograms of molecular weight calibration curve: Cy-one (310 Da) and Glycine (75 Da). (B) Control group (whole soybeanwas not fermentedlis 937 mixed with B. subtilis natto. (D) Whole soybeanwas fermented by L. casei Zhangwith B. subtilis natto.

S. Zhang et al. / Food Control 41 (2014) 1e66

10,000 Da in the three samples decreased 34.3%, 75.2%, and 77.2%,respectively. And the molecular weight distribution of peptide inthe samples fermented by B. animalis 937, L. casei Zhang andL. plantarum P-8 mixed with B. subtilis natto was showed in Fig. 8.As it was reported that the soybean protein meal fermented byL. plantarum Lp6 increased the amount of low molecular weightpeptides (Amadou, Le, Shi, Gbadamosi, Kamara, & Jin, 2011).Therefore, the effect of LAB and B. subtilis natto on the levels ofpeptide with MW less than 1000 Da of the fermented whole soy-bean could lead to the development of the nutritious and functionalfermented soybean products.

4. Conclusion

This study presented a series of the fermented whole soybeanfoods by using LAB mixed with B. subtilis natto in solid-statefermentation, which had received recognition as a potentialbiotechnological process without causing serious environmentalpollution. Due to the microbial autolysis and enzyme chemistryreaction, the whole soybean fermented by LAB mixed withB. subtilis natto could provide different probiotics for the host andhad the capacity to improve nutritional and functional properties.The results also indicated that the viable count, the content of freeamino nitrogen and the level of peptidewithMW less than 1000 Dain the sample fermented by L. plantarum P-8 mixed with B. subtilisnatto were the highest compare with B. animalis 937 mixed withB. subtilis natto and L. Casei Zhang mixed with B. subtilis natto. Inconclusion, a novel probiotic LAB carrier food was provided byusing LABmixedwith B. subtilis natto to ferment thewhole soybeanin the solid-state fermentation.

Acknowledgments

The authors wish to acknowledge the financial support providedby the National Natural Science Foundation of China (No.30900961) and Tianjin Social Science Planning Program (No.TJGLWT11-08).

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