Fermented soy whey induced changes on intestinal microbiota and metabolic influence in mice

Wei Hn*, Xuhui Zhung Qin Liu, Bo Sun Hijing Mio Xiolin Zhng*

a Academy of State Administration of Grain, Beijing 100037, China

b Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing 100191, China

c COFCO Nutrition and Health Institute, Beijing 102209, China

Keywords:

Soy whey

Microbiota

Short chain fatty acids

Lactobacillus casei

Akkermansia muciniphila

A B S T R A C T

Soy whey (SW) is generated as process waste while preparing soy protein isolates (SPI), and causing severe environmental pollution.Therefore, its value-added utilization is of prime importance for transforming and upgrading traditional industry.This study aims to utilize SW as a substrate for the growth of probiotics and produce a SW based synbiotics.By a series of trials, the effect of the dietary supplementation with this fermented SW (FSW) was analyzed on ICR mice’s body weight, metabolites, and intestinal microbiota in 4 weeks.The results showed that, when SW was concentrated 15 times, the count of viable Lactobacillus casei reached 3.4 × 109 CFU/mL by liquid fermentation method, which was the highest viable cell count among all test strains.In this FSW, the protein, amino acid, total dietary fibre, soluble dietary fibre, and oligosaccharide were 2.10%, 1.63%, 0.52%, 0.51% and 0.79%, respectively.Compared to two control group, the total yields of the short chain fatty acids (SCFAs) were significantly improved (75%–125% at average), while the SCFAs structure was also significantly changed (especially acetic acid and butyrate) in the faeces of mice fed FSW.Meanwhile, FSW dietary addition was associated with the diversity and richness of the intestinal microbiota.Obviously, with mice’s body weight loss, Firmicutes/Bacteroides ratio reduced accordingly (< 1.21), and the abundance of Akkermansia muciniphila was significantly increased (the maximum amount was about 0.013%).In summary, our results indicated that the dietary supplementation of FSW affected mice’s intestinal microbiota and metabolism and improved their health profile.

1.Introduction

Soy whey (SW) is an abundant source of waste in the soy protein isolates (SPI) preparation process.At present,the industrial SW is generally directly discharged, but this discharged waste can seriously contaminate the environment due to its high biological oxygen consumption index and overnutrition characteristics [1].It is estimated that the 30–35 m3SW are generated if one ton of SPI was produced in the alkalisoluble-acid-precipitation process.

However, there are the high nutritional values in SW.On a dry basis, SW contains 3.6%–4.4% nitrogen (50% is protein nitrogen)and 25%–35% soluble sugars, such as sucrose, raffinose, stachyose,galactans, hemicelluloses and isoflavone aglycones and so on [2].The most of these components are essential for the growth and fermentation of microbes, including some fastidious microbes and lactic acid bacteria (LAB).Recently many studies have addressed the feasibility of microbial fermentation by using an industrial SW-based medium.Roopashri et al.[3]evaluated a laboratory-formulated SW-based medium for enhancing the optimized phytase activity inSaccharomyces cerevisiaeMTCC 5421 and theα-D-galactosidase and antibacterial activity inLactobacillus plantarumMTCC 5422.Singh and Banerjee [4]utilized immobilizedAspergillus nigercells for the production of citric acid from SW.Mitra et al.[5]reported that SW, the alternative and inexpensive substrate, was used to cultureLactococcus lactissubsp.lactisfor nisin production, up to 619 mg/L.Usually, the fermentation method is a valuable technique for utilization of agro-industrial by-products to produce the value added products, which may lead to higher yields and productivities of valuable compounds, such as enzymes, organic acids and many other bioactive compounds.In fact, some wild strains of bacteria also can be screened in SW.So, there is more significance for studying the fermented soy whey (FSW) than SW.

Generally, the synbiotics include probiotics and prebiotics.Probiotics are defined as live microorganisms that confer a health benefit to the host when administered in a sufficient amount [6].Among them, LAB is one of the most important members, which can survive by utilizing oligosaccharides or dietary fibres and plays a beneficial rolein vivo[7].Prebiotics are a substrate that is selectively utilized by host microorganisms conferring a health benefit [8],and many of which also come from soybean and its by-products.In contrast to gaining the single cell protein from yeasts, the few reports have focused on the condensed SW utilization for the production of the highly concentrated LAB.Therefore, we hypothesize that SW’s carbon and nitrogen sources can be fairly used by LAB and served for its reproduction, and the fermented product can be developed as a synbiotic.In addition, several common and legal strains of probiotics,includingLactobacillus fermentum,Streptococcus thermophilus,Lactobacillus buchneri,Lactobacillus rhamnosus,Pediococcus pentosaceus,L.lactissubsp.lactis,Lactobacillus delbrueckiisubsp.lactis,Lactobacillus acidophilus,Pediococcus acidilactici,Lactobacillus casei,Lactobacillus delbrueckiisubsp.bulgaricusandLactobacillus reuteri, were studied in this study.

The aim of this study is to research whether the growth and proliferation of LAB is enhanced in the condensed SW and screen the most suitable strain for growth in test stains.Then the growth,microbiota and metabolism parameters of mice are analyzed in response to dietary treatment with FSW.Furthermore, the results can be discussed and evaluated whether FSW can act as a synbiotic that has a positive effect on animal health.

2.Materials and methods

2.1 Microorganisms and culture conditions

A total of 12strains were purchased and obtained from China General Microbiological Culture Collection Centre (CGMCC) and China Centre of Industrial Culture Collection (CICC), which were the common commercial probiotics (Table 1).These strains were activated in MRS broth medium (10.0 g peptone, 8.0 g beef extract,4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium citrate, 5.0 g sodium acetate, 0.2 g magnesium sulphate, 0.04 g manganese sulfate, 1.0 mL tween-80 and 1 000 mL water) at 37 °C for 24 h.

Table 1Colony count of the strains’ fermentation in SW.

SW was used as the fermentation medium, which was obtained from Yu Wang Co., Ltd.(Shandong, China).The fermentation process was as follows: first, SW was concentrated from 3 to 30 times by heating and negative pressure; second, SW was sterilized at 100 °C for 30 min; third, the pH was adjusted to 6.5–6.8; at last,the fermentation was conducted in a 100-L fermentor (containing the concentrated-SW with 2% (V/m) seed) at 37 °C for 24 h.

2.2 Sample processing and detection

The sample was the FSW.The microbe was identified by 16S rRNA gene sequencing in the sample [9].Subsequently, the FSW’ s main components, including aerobic bacterial count, protein, amino acids, total dietary fibre, soluble dietary fibre and oligosaccharide,were analyzed.The aerobic bacterial count was analyzed by using colony-count method[10].The protein was analyzed by using the Kjeldahl method of GB 5009.5–2010 [11].Amino acids were analyzed by the amino acid analyser and using the method of GB/T 5009.124–2003 [12].Total dietary fibre and soluble dietary fibre were analyzed by using the method of GB 5009.88–2014 [13],whose process need to the function of the enzymes, the organic extraction, and the high temperature and so on.Oligosaccharide was analyzed by the high performance liquid chromatography and using the method of GB/T 22491–2008 [14].

2.3 Mice feeding

The female ICR mice were purchased from Beijing Ding-Guo-Chang-Sheng Biotechnology Co., Ltd.and maintained in a specific pathogen-free environment.The animals were housed in the units of 4 mice per cage in a controlled environment with free access to food and water.After 7 days, the mice were divided into 8 groups of 12 and fed the following diets: group C1 (control-1): basal diet; group C2(control-2): basal diet + 0.5 mL probiotics by gavages (2.0 × 109CFU/mLLactobacillus caseiCGMCC 1.2435); group G1: basal diet +1 mL condensed SW by gavages; group Z1: basal diet + 0.5 mL condensed SW by gavages; group D1: basal diet + 0.25 mL condensed SW by gavages; group G2: basal diet + 1 mL FSW by gavages(2.0 × 109CFU/mL); group Z2: basal diet + 0.5 mL FSW by gavages (1.0 × 1010CFU/mL); group D2: basal diet + 0.25 mL FSW by gavages (5 × 108CFU/mL).The feeding period was 4 weeks[15,16].Probiotics were produced into the live lyophilized powder(3.0 × 1011CFU/g), and diluted and used by the sterile water.The condensed SW is concentrated 15 times.All experimental protocols in this study were approved by the China Agricultural University Animal Care and Use Committee.

2.4 Analysis of SCFAs

Short chain fatty acids (SCFAs) analysis was performed as reported by Zubaidahet al.[17]with modifications, which was measured by gas chromatography (GC, Agilent 6820) analysis.The GC was equipped with a capillary column (30 m × 0.250 mm ×0.25 μm; DB-FFAP; Agilent Technologies, Inc.).N2was supplied as the carrier gas.The initial oven temperature was 40 °C and increased to 200 °C at 10 °C/min and finally held at 200 °C for 3 min.The injection temperature was 270 °C.The injected sample volume for GC analysis was 1 μL, and the run time for each analysis was 20.0 min.

2.5 The analysis of microbial diversity

Bacterial DNA from faeces’ samples was extracted using a DNA kit (Omega Bio-tek Inc., Norcross, GA, USA) according to the manufacturer’s instructions.A total of 467 bp of the V3–V4 region of the bacteria 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) using primers 338F: 5’-ACTCCTACGGGAGGCAGCAG-3’ and 806R:5’-GGACTACHVGGGTWTCTAAT-3’ [18-20].The PCR reaction system (20 μL) consisted of 4 μL of 5 × FastPfubuffer, 2 μL of 2.5 mmol dNTPs, 0.8 μL of each primer (5 μmol), 0.4 μL of FastPfupolymerase, 10 ng of template DNA and UV-sterilized water.The PCR drugs were purchased from TransGen Biotech Co., Ltd.(Beijing, China).The PCR products were sent to Majorbio Bio-Pharm Technology Co., Ltd.(Shanghai, China) for sequencing by an Illumina MiSeq platform.The raw fastq files were multiplexed and qualityfiltered using QIIME (version 1.17).Operational taxonomic units(OTUs) were clustered with a 97% similarity cut off using UPARSE(version 7.1, http://drive5.com/uparse/).Chimerical sequences were identified and removed using UCHIME.The taxonomy of each 16S rRNA gene sequence was analysed by RDP Classifier (http://rdp.cme.msu.edu/) using a confidence threshold of 97% [21-23].

2.6 Statistical analysis

The data was presented as the mean ± standard deviation.Duncan’s test and Student’st-test were used to identify the differences among groups and between two groups, respectively, and were performed using SPSS16.0 software.

3.Results and discussion

3.1 Selection of strains

The result of 12 strains’ growth in SW was shown in Table 1.Thestrains includedL.fermentum,S.thermophilus,L.buchneri,L.rhamnosus,P.pentosaceus,L.lactissubsp.lactis,L.delbrueckiisubsp.lactis,L.acidophilus,P.acidilactici,L.casei,L.delbrueckiisupsp.bulgaricusandL.reuteri.Among them, the colony count ofLactobacillus caseiwas the highest after incubated for 24 h at 37 °C, account for 4.0 × 108CFU/mL(8.60 (lg (CFU/g)).As shown in Fig.1, SW was concentrated 3, 15 and 30 times.When SW was concentrated 15 times and fermented at 37 °C for 24 h, the colony count ofLactobacillus caseireached 3.4 × 109CFU/mL (9.53 (lg (CFU/g)); besides, pH decreased to 4.90.By 16S rRNA genes sequencing and comparing, the similarity was 100% betweenLactobacillus caseiand the microbes from FSW.

Fig.1 Colony count and pH of L.casei (CGMCC1.2435) in different concentrated SW.

3.2 Components’ detection of SW and FSW

The main nutritional components of SW and FSW were analyzed,as shown in Table 2.Compared to SW, the contents of several nutritional components (including protein, amino acid, total dietary fibre, soluble dietary fibre and oligosaccharide) significantly changed in FSW (P<0.05).The contents of protein, amino acid, total dietary fibre and soluble dietary fibre were increased by 4.9%, 2.8%, 2.8% and 2.6%, respectively;while the content of oligosaccharide was decreased by 18.5% in FSW,which may be the result of the microbes’ utilization.

Table 2Nutrient composition of SW before and after fermentation.

3.3 Analysis of SCFAs in vivo

The yields of the total SCFAs in the intestinal tract of mice were shown in Figs.2, 1S–3S and Table 1S.In Fig.2A, the change of the total SCFAs in different feeding days was shown.The yields of SCFAs were not significantly different at 0 day and 14 days (P> 0.05,(3.1 ± 0.5) and (3.0 ± 0.8) mg/g), but that of SCFAs were significantly increased at 28 days (P< 0.05, (3.6 ± 1.0) mg/g).In Fig.2B, the yields of the total SCFAs were compared in different feeding groups,including group C1, C2, NF (i.e.G1 + Z1 + D1) and F (i.e.G2 +Z2 + D2).At 0 day and 14 days, there were no significant fluctuations among the yields of SCFAs of the 4 groups (P> 0.05); instead, the yields were significantly higher in the group NF and F at 28 days,compared with the group C1 and C2 (P< 0.05).Fig.2C showed the yield of the total SCFAs in different feeding groups (C1, C2, G1,Z1, D1, G2, Z2 and D2) at the 28 days.According to the descending of the quantity of SCFAs, the following information was obtained:G2 = D2 ((4.3 ± 0.3) and (4.2 ± 0.0) mg/g) > D1 ((4.0 ± 0.2) mg/g)> Z1 = Z2 ((3.5 ± 0.3) mg/g) > G1 ((2.7 ± 0.3) mg/g) > C2 = C1((1.9 ± 0.3) and (2.0 ± 0.1) mg/g) (P< 0.05).In conclusion, the treatment with FSW enhanced significantly the overall levels of SCFAs in the intestinal tract.Interestingly, the yields of the total SCFAs in group D1 were not necessarily lower than that in other group.

Fig.2 Yields of the total SCFAs in mice’s intestinal tract.(A) Comparison of the yields of SCFAs in different feeding times (0, 14 and 28 days).(B) Comparison of the yields of SCFAs in different feeding groups C1, C2, NF (G1 + Z1 + D1) and F (G2 + Z2 + D2) at the same feeding time.(C) Comparison of the yields of SCFAs in different feeding groups (C1, C2, G1, Z1, D1, G2, Z2 and D2) at the 28 days.* indicate a significant difference (P < 0.05) between the values; and the red line represents the average value.

In addition, the composition and levels of SCFAs was also variable in the different feeding group, including acetic acid,propionic acid, butyrate, isobutyric acid, valerate and isovaleric acid(Table 3).Compared to 2 control groups (group C1 and C2), the yields of acetic acid and butyric acid were significantly higher in groups with FSW addition.However, the propionic acid, valerate and the branched chain fatty acids (isobutyric acid and isovaleric acid)had almost no regular changes among the 8 groups.

Table 3Composition and yields of SCFAs.

3.4 Changes of Lee’s index

In Fig.3, Lee’s index at 28 days was showed, which was directly related to weight changes.As the results showed, Lee’s index had a significant different between the groups added FSW and other groups(except the group added low-dose SW).Compared to group C1, Lee’s index was decreased by 12.1%, 11.4% and 12.4% in group G2, Z2 and D2, respectively (P< 0.05).Compared to group C2, Lee’s index was decreased by 9.2%, 8.3% and 9.3% in group G2, Z2 and D2,respectively (P<0.05).

Fig.3 Lee’s index.The different number of * indicate a significant difference(P < 0.05) between the values, the same number of * indicate no significant difference (P > 0.05).

3.5 Analysis of bacterial microbiota

In Figs.4-5 and 4S-7S, it was shown that the different dietary supplementation caused a dramatic change of mice’s gut microbiota at phylum and genus level.

At phylum level, the ratio of Firmicutes to Bacteroides was obviously lower in hosts with FSW and SW addition than hosts of two control groups (Fig.4A).

Fig.4 Changes of the micorobiota in mice’s intestinal tract.(A) The ratio of Firmicutes/Bacteroides.The values in the figure represent the ratio of each group.(B) PCA on genus level.(B1, B3, B5) The horizontal and vertical coordinates represent two selected principal components.The scale of the horizontal and vertical axes is relative distance, and has no practical significance.(B2, B4, B6) The box diagram represents the discrete distribution of different groups of samples on the PC1 axis.(C) Community heatmap on genus level at 0 day.The abscissa is the sample name and the ordinate is the species name.The proportion of species is represented by a certain color gradient.The value represented by the color gradient is on the right.(D) Community heatmap on genus level at 28 days.The abscissa is the sample name and the ordinate is the species name.The proportion of species is represented by a certain color gradient.

Fig.4(Continued)

At genus level, the principal component analysis (PCA) showed that the microbiota of mice fed FSW (group G2, Z2 and D2) were significantly different from those of mice fed a basal diet (group C1) (Fig.4B).From 0 day to 28 days, the relative abundance of some major resident genera had a big change between the different groups (Figs.4C and 4D).For example, the relative abundance ofBifidobacterium,Lactobacillus,Butyricicoccus,Parabacteroides,Lachnospiraceae andA.muciniphilaincreased after FSW gavages,while that ofStreptococcusdecreased to undetectable levels.Especially, the abundance ofA.muciniphilaincreased by 42.31, 1.83 and 20.51 times from 0 day to 28 days in groups D2, Z2, and G2,respectively.Instead there was an insignificant increase or decrease for the abundance ofA.muciniphilain other groups (Fig.5).

Fig.5 Relative abundance of Akkermansia.

4.Discussion

SW, a by-product generated from SPI production, is regarded as a nutritious and safe waste.In general, SW is disposed of into the“sewage” after generated, which pollutes seriously the groundwater.In fact, if SW is treated by the sensible drying mode and processing method, the obtained SW powder will be edible.It has been explored that the valorisation of SW for the value-add or value creation in addition to eliminating the environmental pollution.Nowadays,the problem-solving strategy can be broadly classified into two categories, which are physical and microbiological/enzymatic; the latter include the production of functional ingredients by using microorganisms and enzymes (like prebiotic and citric acid),propagation of microorganisms (such as probiotics) as well as biofuel production [24].In this study, the cultivation feasibility of 12 probiotic stains was investigated by using the condensed SW as the basal medium.The results indicated that the LAB presented a good culture performance in SW, this may be due to the condensed SW contains enough oligosaccharides and its appropriate C/N ratio.Especially forL.casei, the biomass reached 3.4 × 109CFU/mL after optimization in the heterotrophic mode, which was approximately 1–300 times higher than that of other probiotics.Therefore, SW is highly likely to be useful in the production of highly active LAB.To our knowledge, this is the first report of the production ofL.caseiby using SW as a medium.

Following people’s increasing demand for the food functionality,the concept of “microbiota directed foods” (MDF) was put forward.The food composed of a variety of ingredients including one or more prebiotic components capable of being metabolized by microbes alone, and/or nutrients whose transformation by microbes makes them available to the host for direct use or for further biotransformation, as well as components that do not require microbial metabolism for their effects on the host [25].After fermentation under a certain condition,the original ingredients of SW could not be exhausted.So, the enriched LAB and the original soybean-based prebiotics constituted a potential symbiotic, FSW.It should be focused on whether FSW affects human or animal’s microbiota, and whether the specific members and expressed functions of the microbiota can be identified that are essential contributors to the health status of humans or animal at different stages of life.

Trillions of microorganisms reside in the gastrointestinal tract of animals, and the majority maintain a symbiotic relationship with their host, playing a critical part in biological processes such as nutrient utilization, resistance against infections, maturation of the immune system and host metabolism [26,27].Undoubtedly, the different diets can cause changes for the intestinal microbiota [28].Many studies also showed that the ratio of Firmicutes to Bacteroides was correlated with the obesity of host.In the intestinal tract of food-borne obese hosts, the abundance of Firmicutes increased and that of Bacteroides decreased, while the ratio of Firmicutes to Bacteroides was significantly lower in healthy hosts [29,30].By FSW dietary supplement, Firmicutes/Bacteroidesratio reduced,with mice’s body mass lost accordingly.The results suggested that FSW may be beneficial to improving overweight and controlling the food-borne obesity.

A.muciniphila, the only cultivated representative of the Verrucomicrobia, is an intestinal bacterium that was isolated from a human fecal sample.Due to its specialization in mucin degradation,it becomes a key organism at the mucosal interface between the lumen and host cells, and is closely related to the multiple diseases and metabolic disorders, including inflammatory bowel disease,autism, alcoholic liver, colitis, and type 1 and type 2 diabetes [31-37].The presence of this bacterium inversely correlates with body mass in rodents and humans [38].For example, the concentration of the gram-negative family Enterobacteriaceae was significantly higher(P= 0.036), whereas levels ofDesulfovibrioandA.muciniphilawere significantly lower (P= 0.027 andP= 0.030, respectively) in the obese or overweight children, Instead,A.muciniphila-like bacteria andDesulfovibriowere more abundant in children with normal body mass [33].Also, many factors affect the quantity ofA.muciniphila.The combined effect ofLactobacillus caseiSY13 and lactulose had a significant effect on promoting the abundance ofA.muciniphilain the intestinal tract of mice [39].In our trials, the results indicated that the continuous FSW addition could significantly increase the abundance ofA.muciniphilain the mice’s intestine.

Many studies have revealed a close association between the disturbance of the gut microbial community and the structure and levels of SCFAs [40].SCFAs have a direct role in the regulation of T cells in both the gut and peripheral tissues and can accelerate the extrathymic peripheral differentiation of anti-inflammatory T regulatory cells [41].Acetic acid and propionic acid have an important role in providing energy for cells, while butyric acid can regenerate intestine mucosa cells [42].According to our results, compared to the two control groups, the yield of the total SCFAs, the acetic acid and butyric were significantly improved in the faeces of mice fed FSW after a certain feeding time.

5.Conclusion

In summary, the present findings indicated that SW was a good media-material for the growth and proliferation ofL.casei.FSW dietary supplement affected the intestinal microbiota and metabolism and improved their health profile, which may be developed as a symbiotic product.Therefore, this fermentation treatment can become an effective method for the valorisation of SW.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Appendix A.Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://doi.org/10.1016/j.fshw.2021.07.005.