Adaptive mechanism of Lactobacillus amylolyticus L6 in soymilk environment based on metabolism of nutrients and related gene‐expression profiles

Abstract Lactobacillus amylolyticus L6 isolated from naturally fermented tofu‐whey was characterized as potential probiotics. To give insight into the adaptive mechanism of L. amylolyticus L6 in soymilk, the gene‐expression profiles of this strain and changes of chemical components in fermented soymilk were investigated. The viable counts of L. amylolyticus L6 in soymilk reached 1012 CFU/mL in the stationary phase (10 hr). The main sugars reduced gradually while the acidity value significantly increased from 45.33° to 95.88° during fermentation. About 50 genes involved in sugar metabolization and lactic acid production were highly induced during soymilk fermentation. The concentration of total amino acid increased to 668.38 mg/L in the logarithmic phase, and 45 differentially expressed genes (DEGs) in terms of nitrogen metabolism and biosynthesis of amino acid were detected. Other genes related to lipid metabolism, inorganic ion transport, and stress response were also highly induced. Besides, the concentration of isoflavone aglycones with high bioactivity increased from 14.51 mg/L to 36.09 mg/L during the fermentation, and the expression of 6‐phospho‐β‐glucosidase gene was also synchronously induced. This study revealed the adaptive mechanism of L. amylolyticus L6 in the soymilk‐based ecosystem, which gives the theoretical guidance for the application of this strain in other soybean‐derived products.

Although the stachyose and raffinose in soymilk have been regarded as prebiotics, excessive intake by human body would cause gastric bloating or diarrhea, requiring probiotics to own ability of hydrolyzing soybean oligosaccharides in soymilk with α-galactosidase (Donkor et al., 2007). In addition, soymilk is rich in low-absorptive isoflavone glycosides (occupying approximately 90% of isoflavone content) (Izumi et al., 2000), and the probiotic strain with the ability of converting isoflavone glycosides into high-absorptive aglycones by β-glucosidase were the best choice (Donkor et al., 2007). On the other hand, stachyose and raffinose in soymilk could promote the proliferation of fermentation probiotic strains (Kim et al., 2010;Sarina et al., 2017). In addition, the soymilk could be used as food vehicles of probiotics, protecting bacterial cells from adverse environment such as low pH of gastric acid, bile salt, and various digestive enzymes in the gastrointestinal tract (Zhuang et al., 2009).
Therefore, the selection of probiotic strains suitable for soymilk environment is very important for the production of soybean yoghurt.
Lactobacillus amylolyticus L6 was isolated from naturally fermented tofu-whey, a traditional Chinese tofu-coagulant , and its safety, potential probiotic characteristics, and fermentation properties in tofu-whey have been extensively studied (Fei et al., 2020;. Since L. amylolyticus L6 was one of the dominant bacteria in naturally fermented tofu-whey for a long time, it has evolved the adaptability to nutritional environment in soybean products, which makes L. amylolyticus L6 one of the best candidate probiotic strains for fermenting soymilk. In this study, the changes of nutrient and functional substances in soymilk and gene-expression profiles of L. amylolyticus L6 during fermentation were investigated to reveal the molecular mechanisms of synergistic effect between soymilk and L. amylolyticus L6.

| Strains and cultivation
Lactobacillus amylolyticus L6 (CGMCC NO.9090) was isolated from naturally fermented tofu-whey . This strain was preserved in 15% glycerol at −80°C and cultivated in De Man, Rogosa and Sharpe (MRS) (Guangdong Huankai Microbiology Biotech Inc., Guangzhou, China) plate at 37°C for 36 hr before use. A single colony was then picked and inoculated into 10 ml of MRS broth and incubated for 24 hr.

| Preparation of fermented soymilk
Soymilk was prepared according to the method described by Salma et al. (Elghali et al., 2014) with slight modification. Soybean (100 g) was washed and then soaked in 600 ml of drinking water with 0.5% NaHCO 3 at 26°C for 14 hr. The soaked soybean was ground and heated with 800 ml of drinking water in a soymilk maker (DJ12B-DEF4, Midea, China). The slurry was filtered through a doublelayered cotton cloth and then mixed with drinking water in a ratio of 8:2. Glucose (Sigma Chemical Co., Ltd, Guangzhou, China) with a concentration of 1.5% (w/v) was added to make soymilk. Soymilk was heated at 85°C for 15 min for sterilization and then cooled to 37°C. Subsequently, the soymilk was inoculated with 10% (w/v) L. amylolyticus L6 and incubated at 37°C for 24 hr. The growth curve was plotted according to the viable counts determined at 0 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, and 16 hr during fermentation (Tang et al., 2007). All analyses were performed in triplicate.

| Transcriptomic analysis
The fermented soymilk was sampled at the fermentation time of 4 hr, 7 hr, and 10 hr corresponding to the lag phase, logarithmic phase, and stationary phase, respectively. Three parallel samples were obtained in each sampling point. The quality and integrity of total RNA were assessed by 1% agarose gels and RNA 6000 Nano Assay Kit of the Bioanalyzer 2100 system. Probes were used to purify mRNA from the total RNA of prokaryotic samples. Fragmentation was carried out using divalent cations under hyperthermal temperature in first strand synthesis reaction buffer (5X). Synthesis of first strand complementary DNA (cDNA) was performed with random hexamer primer and Moloney murine leukemia virus (M-MuLV) reverse transcriptase. The second strand was synthesized by DNA Polymerase I and M-MuLV reverse transcriptase. The 3' ends of DNA fragments were adenylated and then ligated to the adaptor with hairpin loop structure for hybridization. The cDNA library fragments with 350-400 bp were selected and purified with AMPure XP system. Polymerase chain reaction (PCR) was carried out with Phusion High-Fidelity DNA polymerase and the PCR products were purified with AMPure XP system. Finally, library quality was evaluated with Agilent 2100 Bioanalyzer system (Cheng et al., 2019). Gene descriptions and annotations were performed in the Genome Database of L. amylolyticus strain L6 in National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih. gov) with GenBank Accession Number of CP020457.1. The annotated genes were then used to predict biochemical pathways. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms were retrieved from the KEGG database (http:// www.kegg.jp/kegg) and gene ontology (GO) database (http://geneo ntolo gy.org), respectively.
Real-time quantitative PCR (RT-qPCR) was applied to verify the accuracy of transcriptomic results. Primers were designed and synthesized according to gene sequences on NCBI (Table S1). The gene-expression levels were calculated via the 2 -△△Ct method (Xu et al., 2015), which was used to compare with the sequencing results of the transcriptome.

| Sugars and organic acid
The determination of sugars, including sucrose, glucose, fructose, galatose, raffinose, and stachyose in fermented soymilk, was performed by high-performance liquid chromatography (HPLC) according to the National Standard of China GB/T 22,221-2008. The samples with a volume of 5 ml were collected at 0 hr, 4 hr (lag phase), 7 hr (log phase), and 10 hr (stationary phase) and then centrifuged at 10,000 r/min for 10 min. The supernatant was filtered through a 0.22µm syringe membrane into HPLC vials for testing. HPLC was carried out on Thermo-Ultimate 3000 equipped with HP-NH2 column (4.6 mm×250 mm, 5 μm) and differential refraction detector RefractoMax 520. Acetonitrile (68%)-deionized water (32%) were used as mobile phase with a flow rate of 1.0 ml/min. The detection wavelength was 280 nm, and the column temperature was set at 35°C. The standard of sucrose, glucose, fructose, galactose, raffinose, and stachyose (Sigma Chemical Co., Ltd, Guangzhou, China) was dissolved in deionized water and transferred to a 10-mL volumetric flask for gradient dilution. The equation parameters of standard curve were used to determine the concentration of sugar in fermented soymilk.
The acidity values of fermented soymilk samples were determined according to a previous description . The distilled water with a volume of 20 ml was added to 10-mL collected samples. Then, 30 ml of mixture was mixed with 0.5 ml of phenolphthalein indicator to titrate the amount of acidity against NaOH solution (0.1 mol/L). All analyses were performed in triplicate.
The content of lactic acid and acetic acid in fermented soymilk was detected by HPLC according to GB/T 5009.157-2003. The samples with a volume of 5 ml were collected at 0 hr, 4 hr (lag phase), 7 hr (log phase), and 10 hr (stationary phase) and then centrifuged at 10,000 r/min for 10 min. The supernatant was filtered through a 0.22µm syringe membrane into HPLC vials for testing. HPLC was performed on Agilent 1100 equipped with Luna C18(2) 100A column (4.6 mm×250 mm, 5 μm) and VWD 3100 ultraviolet detector.
KH 2 PO 4 (95%) with a concentration of 10 mmol/L-methanol (5%) was used as mobile phase with a flow rate of 0.5 ml/min. The detection wavelength was 210 nm, and the column temperature was set at 25°C. The standard of lactic acid and acetic acid (Sigma Chemical Co., Ltd, Guangzhou, China) was dissolved in deionized water and transferred to a 10-mL volumetric flask for gradient dilution. The equation parameters of standard curve were used to determine the concentration of organic acid in fermented soymilk.

| Analysis of isoflavones and amino acids by HPLC
The content of isoflavones in fermented soymilk was determined by HPLC according to our previous report . The samples were collected at 0 hr, 4 hr (lag phase), 7 hr (log phase), and 10 hr (stationary phase) and then centrifuged at 10,000 r/min for 10 min at 4 ℃. The supernatant (4 ml) was transferred to a 10-mL volumetric flask, diluted with methanol to the constant volume, and extracted with sonication for 1 hr. The resulting extracts were filtered through a 0.22µm membrane into HPLC vials for HPLC testing.
The content of amino acid in fermented soymilk was determined by HPLC according to Agilent AdvanceBio AAA method (www. agile nt.com/chem/advan cebioaaa). Briefly, the pretreated samples were derivatized with o-phthalaldehyde (OPA), and the specific operations were carried out according to the method provided by Agilent. Analysis of amino acid by HPLC was carried out on Agilent 1100 equipped with an Agilent AdvanceBio AAA amino acid column (4.6 mm×100 mm, 2.7 μm) under isocratic elution. Na 2 HPO 4 with a concentration of 0.01 mol/L and acetonitrile-methanol solution (acetonitrile:methanol:water 45:45:10) were used as mobile phase A and mobile phase B, respectively, with a flow rate of 1.5 ml/min. The detection wavelength was 338 nm, and the column temperature was set at 40°C. The standard of amino acids (Sigma Chemical Co., Ltd, Guangzhou, China) was dissolved in deionized water and transferred to a 10-mL volumetric flask for gradient dilution. The equation parameters of standard curve were used to determine the concentration of amino acids in fermented soymilk.

| Statistical analysis
Analyses were performed using SPSS (SPSS Inc., Chicago, IL, USA, V23.0.0). One-way analysis of variance (ANOVA) was performed using to compare between groups, which was considered statistically significant at the p <.05 level.

| Growth characteristics of L. amylolyticus L6 in soymilk
The growth curve of L. amylolyticus L6 in soymilk was plotted according to viable counts ( Figure 1). L. amylolyticus L6 started to grow after 2 hr inoculation in soymilk. It needed approximately 4 hr for bacteria to grow from lag phase into the logarithmic phase. Bacteria grew into the stationary phase at the time of 10 hr with a cell concentration of 10 12 CFU/mL. It was reported that Lactobacillus casei Zhang grew from the lag phase into the logarithmic phase at a time of 3 hr and reached stationary phase at 14 hr with a cell concentration of 10 9 CFU/mL . L. amylolyticus L6 need less time than L. casei Zhang to grow into stationary phase, while L6 could produce more bacterial cells in soymilk than L. casei Zhang.
That might be because tofu-whey isolated L. amylolyticus L6 is more adaptable in the soymilk-based ecosystem than koumiss-isolated L.
To provide a guide for industrial applications of L. amylolyticus L6 in fermenting soymilk, 1.5% (w/v) of glucose was added to provide enough carbon source for the growth of L6. The metabolism of carbohydrate to produce organic acid by L.amylolyticus L6 during its fermentation in soymilk is shown in Figure 5. The results indicated that four kinds of sugars reduced significantly (p <.5) during the fermentation and the main carbon sources used for the growth of L.amylolyticus L6 were sucrose and glucose (Table 1 and Figure 5).
In the stationary phase, few genes related to glucose metabolism were induced while many genes involved in sucrose (B1745_04485, B1745_04615, B1745_06775), raffinose, and stachyose utilization were found to be significantly up-regulated (Table 3). Among these sugars, only the content of galactose increased slightly (Table 1), which was due to the partial hydrolysis of raffinose and stachyose by α-galactosidase (B1745_RS08070) ( Table 2). This phenomenon has also been reported in several researches of soybean products Besides, two genes (B1745_05365 and B1745_06945) relevant to ATP production were also significantly up-regulated.
During the fermentation, the acidity of soymilk increased significantly from 45.33° to 95.88° (Table 1). The acidity increment was mainly derived from lactic acid with its content increased from 2.62g/L to 4.65g/L (stationary phase) (p <.05). In addition, the con- showed that the expression of LDH gene (B1745_03165) encoding L-lactate dehydrogenase was significantly increased in the log phase (Table 2). In addition, it was also observed that adhE (B1745_05695) encoding acetaldehyde hydrogenase related to acetic acid production was up-regulated in the log phase. Therefore, high expression of LDH and adhE genes promotes the production of lactic acid and acetic acid which are important for coagulating soymilk.

| Nitrogen metabolism and biosynthesis
Due to the lack of various biosynthetic pathways, especially amino acid synthesis pathways, LAB generally need various nutritional ingredients and therefore they are usually found in nutrient-rich environments, such as vegetables, meat, and milk (Fernández & Zúñiga, 2006). Amino acids as an important nitrogen resource for LAB played important roles in physiological functions such as intracellular pH maintenance, stress resistance, and energy generation (Lei et al., 2018;Slonczewski et al., 2009). As a consequence, the proteolytic enzyme system serves a key role for LAB to grow in protein-rich soymilk. A total of 17 kinds of amino acids were detected in the fermented soymilk, including seven kinds of essential amino acids (EAAs) and 10 kinds of nonessential amino acids (NEAAs) ( Table 4). The content of EAAs decreased gradually along with the fermentation and reached 177.26 mg/L in the stationary phase. But the content of total amino acids and NEAAs increased significantly and reached the highest 668.38 mg/L and 187.40 mg/L in the logarithmic phase respectively. Although the content of total amino acids and NEAAs decreased slightly in the stationary phase, it was still higher than that of unfermented soymilk. The increase of free amino acid content in the soymilk fermented by different lactobacilli and their mixes has been widely F I G U R E 4 Reliability analysis between RNA-sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) reported (Ceh et al., 2020;Song et al., 2008). Besides, the content of glutamate and arginine was higher than those of other amino acids in unfermented and fermented soymilk, accounting for approximately 40% content of total amino acid. This phenomenon has been found in soy powder yoghurt fermented by L. brevis WCP02 and L. plantarum P120 that content of arginine is the highest (reached 380 mg/g), and accounted for almost 50% content of the total amino acid in soy powder yoghurt (Ceh et al., 2020).
Therefore, fermentation of soymilk by L. amylolyticus L6 could promote the hydrolysis of protein into amino acid, improving the nutritional quality and digestibility of soymilk.
The proteolytic system of LAB generally consisted of protease, transport systems of amino acid or peptides, and peptidases . The protein in soymilk was first hydrolyzed by protease into amino acids and peptides, which were then transported to cytoplasm by transport systems. Finally, the translocated peptides were degraded by peptidases (Savijoki et al., 2006). The transport of peptides into the cell is an essential step for LAB multiplying in soymilk (Hagting, 1995). Transcriptomic data showed that genes involved in the transport and hydrolysis of peptide in the cytoplasm also exhibited high expression levels (Tables 2   and 3). The gene cluster oppDFBCA encoding the oligopeptide transport system (Opp) and PepC encoding the aminopeptidase, which have been identified in an operon of Lactococcus lactis (Tynkkynen et al., 1993), were found to be up-regulated in soymilk-grown L. with penultimate proline residue in the fermented soymilk. In the stationary phase, there were only two aminopeptidase genes (pepC, B1745_00910 and B1745_01515) that were significantly upregulated, which might be due to the stagnation of cell growth and proliferation, reducing the requirement for peptide and amino acid.
Meanwhile, the content of asparagine in the fermented soymilk reduced significantly from 21.7 mg/L to 5.48 mg/L, and this phenomenon did not occur in other amino acids (Table 4). The data presented in this study suggested that the growth and proliferation of L. amylolyticus L6 required a large amount of aspartate and asparagine, therefore promoting the uptake of free asparagine from soymilk into cell and simultaneously synthesizing aspartate by inducing the expression of asnA and lysC. In the stationary phase, asnB gene that encoded asparagine synthase (glutamine-hydrolyzing) catalyzing the conversion of aspartate into asparagine with glutamine as the nitro- glnH, B1745_05195; glnH, B1745_05195) was highly induced in the logarithmic phase to transport the high concentration of free glutamate from the soymilk into the cell ( Table 2 and Table 4).
The overexpression of the glutamate transporter operon has also been reported in L. casei Zhang under soymilk environment . Meanwhile, many uncharacterized amino acid permease genes (B1745_03105, B1745_06875, B1745_06870, and B1745_06860) were up-regulated, while two amino acid permease genes (B1745_04680 and B1745_03815) were down-regulated in the logarithmic and stationary phase. Interestingly, two genes livB and brnQ coding for branched-chain amino acid transport system II carrier protein and branched-chain amino acid ABC transporter permease were significantly down-regulated in the stationary phase.
That's because L. amylolyticus L6 could synthesize branched-chain amino acids (leucine, isoleucine, and valine) and did not require the help of their transporters, therefore repressing the expression of corresponding genes.

| Lipid metabolism, inorganic ion transport, and stress response
There are 14 genes involved in fatty acid biosynthesis which were identified in the genome of L. amylolyticus L6, which includes accA the intracellular concentrations of metal ions (Boyaval, 1989 During the fermentation, the pH values and acidity of soymilk in the stationary phase could reach 4.0 and 95.88°, respectively, which would induce the expression of genes in responding to acidity stress. Molecular chaperones have been regarded as a ubiquitous feature of cells, including LAB, in which these proteins cope with stressinduced denaturation of other proteins (Feder & Hofmann, 1999).
Chaperone proteins GroL, DnaK, and GrpE participate actively in the response to stress conditions by preventing the aggregation of stress-denatured proteins (Lemos et al., 2007). Transcriptomic analysis indicated that the expression of genes groEL (B1745_01775), dnaK (B1745_03015), and grpE (B1745_03010) coding for chaperone proteins was highly up-regulated in the stationary phase, while these two genes were not significantly induced in logarithmic phase. The difference was mainly due to a relatively higher pH value in logarithmic phase that is not enough to cause acid stress to L. amylolyticus L6 (Table 1)

| Change of isoflavones in fermented soymilk
Soymilk was rich in isoflavonesi in the form of isoflavone aglycones (10%) and their corresponding glucosidic conjugates (90%) (Rodriguez-Roque et al., 2013). Isoflavones' glucosidic conjugates could be converted into highly bioactive aglycones by β-glucosidase in lactobacilli (Tang et al., 2007;Wei et al., 2007;Xia et al., 2019). Table 5 Transcriptomic data indicated that the expression of bglA gene coding for 6-phosphoβ-glucosidase increased significantly in logarithmic phase, which was consistent with the increasing concentrations of isoflavone aglycones. And 6-phosphoβ-glucosidase that could convert isoflavone glucosides into aglycones has been reported in our previous study .

| CON CLUS ION
This study revealed the chemical component changes and transcriptomic changes of L. amylolyticus L6 in fermented soymilk.

TA B L E 5 Concentration of isoflavones (mg/L) in soymilk fermented with L. amylolyticus L6
Large amount of genes related to carbon metabolism in L. amylolyticus L6 were significantly up-regulated in the logarithmic phase and stationary phase, which allowed this strain to metabolize various sugars in soymilk. Highly expressed α-galactosidase gene could help to reduce the content of raffinose and stachyose that caused flatulence of human body. Meanwhile, the concentration of total amino acid increased significantly in the logarithmic phase for highly induced genes involved in the proteolysis, hydrolysis, and transport of peptide, transport and biosynthesis of amino acid. Highly efficient utilization of carbon and nitrogen sources significantly raised the viable counts of L. amylolyticus L6 in soymilk. High expression of 6-phosphoβ-glucosidase promoted the conversion of isoflavone glycoside into highly bioactive aglycones. Besides, other genes related to lipid metabolism, inorganic ion transport, and stress response were also up-regulated. Further study should be conducted in terms of applying this strain into developing soymilk products and vitro digestion simulation test to testify its production performance.
In conclusion, this study reveals that L. amylolyticus L6 isolated from the soybean-derived environment exhibited excellent adaptability in a soymilk-based ecosystem, which is expected to become the specific probiotic strain used for the fermentation of soybean products.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C A L A PPROVA L
The authors declare that they have no conflict of interest. This article does not contain any studies involving animal's trails performed by any of the authors. Furthermore, this article does not contain any studies involving human participants performed by any of the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original contributions presented in the study are included in the article/Supplementary Material, and further inquiries can be directed to the corresponding authors.