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 10^12 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.

   Keywords: chemical components, Lactobacillus amylolyticus L6, soymilk,
   transcriptome
     __________________________________________________________________

   During fermentation, 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 metabolism 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.

   graphic file with name FSN3-10-1548-g003.jpg

1. INTRODUCTION

   Soybean and its derived food products are important part of the Asian
   diet. These foods are rich in various nutrients, such as protein,
   oligosaccharides (raffinose and stachyose), grease, vitamins, and
   insoluble dietary fiber (Lokuruka, [34]2011). Meanwhile, they were
   reported to have many beneficial functions to consumers, including the
   prevention of cardiovascular disease, osteoporosis, hormone‐related
   cancers, and modulation of immunity and intestinal flora (Ko, [35]2014;
   Yan et al., [36]^2017). As consumers become increasingly interested in
   functional foods, they have higher requirements for the varieties of
   functional soybean products. In recent years, fermentation of soymilk
   by probiotics has become one of the research hotspots because of the
   function‐promoting effects brought about by these microorganisms and
   soymilk (Marazza et al., [37]2013; Wang et al., [38]^2012; Wei
   et al., [39]^2007).

   Probiotics were able to attach to the surface of intestinal mucosa and
   colonize in the intestinal tract, which could allow them to bring
   beneficial effects to human health (Bron et al., [40]2012). For
   example, the probiotics could competitively exclude and inhibit
   pathogens in the intestinal tract (Kholy et al., [41]2014), enhance
   intestinal flora (Gerritsen et al., [42]2011), augment both cellular
   and humoral immunity (Yan & Polk, [43]2011), and relieve inflammation
   and food allergy (Majamaa & Isolauri, [44]1997). Except for the above
   functional characteristics, the probiotics used as soymilk starter were
   required to adapt to a complex nutritional environment of soymilk. In
   general, the minimum number of living probiotics in the final product
   of soybean yoghurt should reach 10^8 CFU/ml (Shah, [45]2000).
   Meanwhile, the pH for coagulating soymilk was 4.5–5.0 (Qiao &
   Li, [46]2007), which required the strong acid‐producing ability of
   probiotics in soymilk. 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., [47]2007). In addition, soymilk is rich in
   low‐absorptive isoflavone glycosides (occupying approximately 90% of
   isoflavone content) (Izumi et al., [48]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., [49]2007). On the other hand, stachyose and raffinose in
   soymilk could promote the proliferation of fermentation probiotic
   strains (Kim et al., [50]2010; Sarina et al., [51]^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., [52]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 (Fei et al., [53]2018),
   and its safety, potential probiotic characteristics, and fermentation
   properties in tofu‐whey have been extensively studied (Fei
   et al., [54]2020; Fei, Li, et al., [55]^2017; Fei, Liu,
   et al., [56]^2017). 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.

2. MATERIALS AND METHODS

2.1. Strains and cultivation

   Lactobacillus amylolyticus L6 (CGMCC NO.9090) was isolated from
   naturally fermented tofu‐whey (Fei et al., [57]2018). 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.

2.2. Preparation of fermented soymilk

   Soymilk was prepared according to the method described by Salma et al.
   (Elghali et al., [58]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 double‐layered 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., [59]2007). All analyses were
   performed in triplicate.

2.3. 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., [60]2019). Gene descriptions and
   annotations were performed in the Genome Database of L. amylolyticus
   strain L6 in National Center for Biotechnology Information (NCBI)
   ([61]https://www.ncbi.nlm.nih.gov) with GenBank Accession Number of
   [62]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 ([63]http://www.kegg.jp/kegg) and gene ontology (GO) database
   ([64]http://geneontology.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 [65]S1). The gene‐expression
   levels were calculated via the 2^—△△Ct method (Xu et al., [66]2015),
   which was used to compare with the sequencing results of the
   transcriptome.

2.4. 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 0.22‐µm 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 (Fei, Liu, et al., [67]2017). 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 0.22‐µm
   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.

2.5. Analysis of isoflavones and amino acids by HPLC

   The content of isoflavones in fermented soymilk was determined by HPLC
   according to our previous report (Fei et al., [68]2018; Fei, Liu,
   et al., [69]^2017). 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 0.22‐µm 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
   ([70]www.agilent.com/chem/advancebioaaa). 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.

2.6. 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.

3. RESULTS AND DISCUSSION

3.1. Growth characteristics of L. amylolyticus L6 in soymilk

   The growth curve of L. amylolyticus L6 in soymilk was plotted according
   to viable counts (Figure [71]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 (Wang et al., [72]2012). 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. casei Zhang (Fei,
   Liu, et al., [73]2017).

FIGURE 1.

   FIGURE 1
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   Growth of L.amylolyticus L6 in soymilk incubated at 37°C

3.2. Gene‐expression profiles of L. amylolyticus L6 during fermentation in
soymilk

   The gene‐expression profiles of L.amylolyticus L6 during growth in the
   soymilk ecosystem were investigated by the RNA‐sequencing (RNA‐seq).
   Our research mainly focused on the comparative transcriptomic analysis
   of different growth phases next to each other. A total of 313
   significantly differentially expressed genes (SDEGs) were identified.
   There were 260 SDEGs in logarithmic phase versus lag phase and 171
   SDEGs identified in the stationary phase versus logarithmic phase
   (FigureS [75]1). The SDEGs of L.amylolyticus L6 in logarithmic phase
   versus lag phase were functionally categorized, indicating that SDEGs
   mainly enriched in biological process (transmembrane transport,
   oxidation and reduction, and translation), cellular component (ribosome
   and membrane), and molecular function (structural constituent of
   ribosome, nucleotide binding, and catalytic activity) (Figure [76]2a).
   In the stationary phase compared to the logarithmic phase, SDEGs mainly
   enriched in molecular function, such as structural constituent of
   ribosome, nucleotide binding, and catalytic activity (Figure [77]2b).
   The pathway enrichment for different growth phases is shown in
   Figure [78]3 according to the KEGG pathway database. As for logarithmic
   phase versus Lag phase group, the pathway of SDEGs enriched in starch
   and sucrose metabolism, fatty acid degradation, ribosome, biosynthesis
   of secondary metabolites, and folate biosynthesis. The pathway of SDEGs
   in stationary versus logarithmic phase group enriched in ribosome,
   starch, and sucrose metabolism, adenosine triphosphate (ATP)‐binding
   cassette (ABC) transporter, phosphotransferase system (PTS), and amino
   acid metabolism.

FIGURE 2.

   FIGURE 2
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   Significantly differentially expressed genes (SDGEs) between different
   growth phase based on gene ontology (GO) analysis. Logarithmic phase
   versus Lag phase (a), Stable phase versus Logarithmic phase(b); BP, CC,
   and FF refer to biological process, cellular component, and molecular
   function, respectively

FIGURE 3.

   FIGURE 3
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   Scatter plot of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   enrichment analysis for different growth phases. Rich factor is the
   ratio of the number of differentially expressed genes (DEGs) annotated
   to the Pathway Term to the number of genes annotated to the Pathway
   entry

   In order to determine the reliability of transcriptomic results,
   expression changes of nine target genes (B1704_03855, B1704_02440,
   B1704_01765, B1704_01760, B1704_00925, B1704_00910, and B1704_06165)
   between lag phase and stationary phase were selected for detection. The
   result indicated a high consistency between platform of RNA‐seq and
   real‐time quantitative polymerase chain reaction (RT–qPCR), proving the
   validity of RNA‐seq data (Figure [81]4).

FIGURE 4.

   FIGURE 4
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   Reliability analysis between RNA‐sequencing (RNA‐seq) and real‐time
   quantitative polymerase chain reaction (RT‐qPCR)

3.3. Carbon metabolism of L. amylolyticus L6

   It has been reported that Lactobacillus amylolyticus could metabolize
   various carbohydrates such as dextrin, fructose, galactose, glucose,
   maltose, mannose, sucrose, melibiose, and raffinose, and in some cases
   salicine, esculin, amygdalin, and starch (Bohak et al., [83]1998; Fei
   et al., [84]^2018). Soymilk mainly contained four different kinds of
   sugars, including glucose, sucrose, raffinose, and stachyose
   (Table [85]1). 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 [86]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 [87]1 and
   Figure [88]5). Many genes related to glucose metabolism were
   significantly up‐regulated in logarithmic phase, such as genes coding
   for PTS β‐glucoside transporter (B1745_01765),
   6‐phospho‐alpha‐glucosidase (B1745_05130), gluconate kinase
   (B1745_01565), and glucose‐6‐phosphate dehydrogenase (B1745_01805)
   (Table [89]2). However, several genes involved in sucrose
   transportation, especially sugar ABC transporters (B1745_06760,
   B1745_06745 and B1745_06745), and galactose metabolism (B1745_05485 and
   B1745_05490) were significantly down‐regulated in logarithmic phase.
   Microbes intend to utilize easily metabolizable carbohydrate and
   inhibit the metabolism of the other carbohydrate by down‐regulating the
   expression of related genes (Luesink et al., [90]1998), the phenomenon
   of which, called carbon catabolite repression (CCR), has been widely
   found in lactic acid bacteria (LAB) (Görke & Stülke, [91]2008; Wang
   et al., [92]^2012). 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 [93]3).
   Among these sugars, only the content of galactose increased slightly
   (Table [94]1), which was due to the partial hydrolysis of raffinose and
   stachyose by α‐galactosidase (B1745_RS08070) (Table [95]2). This
   phenomenon has also been reported in several researches of soybean
   products fermented by LAB (Battistini et al., [96]2018; Elghali
   et al., [97]^2014; Xia et al., [98]^2019). The production of energy for
   L. amylolyticus L6 is mainly through the Embden–Meyerhof–Parnas pathway
   (EMP). The gene (B1745_01805) of glucose 6‐phosphate dehydrogenase that
   is the key regulatory enzyme of the Hexose Monophosphate Pathway (HMP)
   was highly expressed in the log phase, indicating that HMP was also
   indispensable in the glycometabolism of L. amylolyticus L6. Besides,
   two genes (B1745_05365 and B1745_06945) relevant to ATP production were
   also significantly up‐regulated.

TABLE 1.

   Changes of sugar and organic acid in soymilk fermented with L.
   amylolyticus L6
   Unfermented Lag phase Logarithmic phase Stationary phase
   Sugars (g/L) Sucrose 3.75 ± 0.09^a 3.36 ± 0.09^b 2.89 ± 0.10^c
   2.61 ± 0.05^c
   Glucose 8.01 ± 0.15^a 7.65 ± 0.12^ab 7.36 ± 0.10b^c 7.06 ± 0.14^c
   Fructose 0.13 ± 0.01^a 0.11 ± 0.00^b 0.09 ± 0.00^b 0.09 ± 0.00^b
   Raffinose 0.59 ± 0.00^a 0.59 ± 0.01^ab 0.57 ± 0.01^b 0.53 ± 0.05^c
   Stachyose 2.89 ± 0.02^a 2.88 ± 0.02^a 2.88 ± 0.01^a 2.76 ± 0.01^b
   Galactose 0.23 ± 0.01^c 0.35 ± 0.01^b 0.41 ± 0.01^a 0.44 ± 0.02^a
   Organic acids (g/L) Lactic acid 2.62 ± 0.04^d 3.38 ± 0.04^c
   4.12 ± 0.04^b 4.65 ± 0.06^a
   Acetic acid 0.36 ± 0.02^c 0.43 ± 0.03^b 0.57 ± 0.04^a 0.62 ± 0.03^a
   Acidity (°) 45.33 ± 1.14^c 47.64 ± 1.86^c 88.42 ± 2.31^b 95.88 ± 2.65^a
   pH 6.7 ± 0.1^a 6.6 ± 0.1^a 5.5 ± 0.2^b 4.3 ± 0.2^c
   [99]Open in a new tab

   Data are the mean ±standard deviation (n = 3). Means in the same column
   with different superscript letters (a–d) are significantly different
   (p <.05)

FIGURE 5.

   FIGURE 5
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   Schematic representation of the presumptive carbohydrate metabolic
   pathways in L. amylolyticus L6 during fermentation in soymilk. Changes
   in the amounts of the substances (average of three replicates) are
   represented by histograms. Different colored bars represent period:
   unfermented (red), lag phase (yellow), log phase (blue), and stationary
   phase (green)

TABLE 2.

   Genes differentially expressed in the logarithmic phase compared to lag
   phase
   Function group and ORF Gene Description Value of log[2] variable
   expression
   Genes up‐regulated
   Carbohydrate transport and metabolism
   B1745_05615 nagZ beta‐N‐acetylhexosaminidase 3.54
   B1745_05130 glvA 6‐phospho‐alpha‐glucosidase 3.26
   B1745_03165 ldh L‐lactate dehydrogenase 3.18
   B1745_05695 adhE acetaldehyde dehydrogenase /alcohol dehydrogenase 2.67
   B1745_RS08070 galA alpha‐galactosidase 2.32
   B1745_01765 scrA PTS beta‐glucoside transporter subunit EIIBCA 1.98
   B1745_04615 spp HAD family hydrolase 1.93
   B1745_03820 gyaR hypothetical protein 1.80
   B1745_03825 spp sugar‐phosphatase 1.69
   B1745_01805 zwf glucose−6‐phosphate dehydrogenase 1.62
   B1745_06170 gpmB histidine phosphatase family protein 1.58
   B1745_07215 ‐ transcriptional regulator 1.44
   B1745_02045 dacC D‐alanyl‐D‐alanine carboxypeptidase 1.44
   B1745_02005 bcrC phospholipid phosphatase 1.44
   B1745_03140 fumC class II fumarate hydratase 1.43
   B1745_04590 murB UDP‐N‐acetylenolpyruvoylglucosamine reductase 1.38
   B1745_04460 glgC YebC/PmpR family DNA‐binding transcriptional regulator
   1.36
   B1745_04525 spp HAD family hydrolase 1.33
   B1745_03145 frdA flavocytochrome c 1.32
   B1745_02465 galE UDP‐glucose−4‐epimerase 1.30
   B1745_07245 poxL pyruvate oxidase 1.29
   B1745_04860 pgl 3‐carboxymuconate cyclase 1.29
   B1745_06310 rpe ribulose phosphate epimerase 1.22
   B1745_00870 ‐ aldose 1‐epimerase 1.19
   B1745_07130 bdhAB aldo/keto reductase 1.19
   B1745_03235 lysozyme lysin 1.12
   B1745_01565 gntK, idnK gluconate kinase 1.09
   B1745_04605 pta phosphate acetyltransferase 1.09
   B1745_05160 rpiA ribose 5‐phosphate isomerase A 1.03
   B1745_04420 ackA acetate kinase 1.00
   B1745_03855 bglA 6‐phospho‐beta‐glucosidase 1.00
   B1745_05365 zntA copper‐translocating P‐type ATPase 1.09
   B1745_06945 ‐‐ cadmium‐translocating P‐type ATPase 2.16
   Amino acids transport and metabolism
   B1745_06855 asnA aspartate‐‐ammonia ligase, asparagine biosynthetic
   process 4.16
   B1745_02860 pepO peptidase M13 2.44
   B1745_00965 hsp20 heat‐shock protein Hsp20 2.39
   B1745_04695 clpP ATP‐dependent Clp protease proteolytic subunit 2.35
   B1745_04960 clpE Clp protease ClpE 2.25
   B1745_05185 glnP glutamine ABC transporter permease 2.16
   B1745_03105 att amino acid permease 2.14
   B1745_00925 oppB peptide ABC transporter substrate‐binding protein 2.07
   B1745_00550 htpX zinc metalloprotease HtpX 1.97
   B1745_06070 pepT peptidase T 1.89
   B1745_00950 oppF ABC transporter ATP‐binding protein 1.83
   B1745_03200 prmA ribosomal protein L11 methyltransferase 1.81
   B1745_00910 pepC aminopeptidase 1.67
   B1745_01265 clpC ATP‐dependent Clp protease ATP‐binding protein ClpC
   1.63
   B1745_04890 ‐ transcriptional activator, Rgg/GadR/MutR family
   domain‐containing protein 1.60
   B1745_05190 glnM glutamine ABC transporter permease 1.60
   B1745_02545 pepX dipeptidyl‐peptidase 1.56
   B1745_02045 dacD D‐alanyl‐D‐alanine carboxypeptidase 1.44
   B1745_06875 ‐ amino acid permease 1.44
   B1745_00945 oppD peptide ABC transporter ATP‐binding protein 1.43
   B1745_RS07280 lysC aspartate kinase 1.41
   B1745_06870 att amino acid permease 1.40
   B1745_04855 atpA haloacid dehalogenase 1.40
   B1745_00955 pepC aminopeptidase 1.38
   B1745_05195 glnH glutamine ABC transporter substrate‐binding protein
   1.32
   B1745_01775 groEL chaperonin GroEL 1.23
   B1745_06165 ‐ metalloprotease 1.18
   B1745_05200 GlnP glutamine ABC transporter permease 1.08
   B1745_00920 oppA peptide ABC transporter substrate‐binding protein 1.07
   B1745_07210 cth aluminum resistance protein 1.05
   B1745_04725 uvrB excinuclease ABC subunit B 1.02
   B1745_00940 oppC peptide ABC transporter permease 1.01
   B1745_00320 gadC glutamate:gamma‐aminobutyrate antiporter 1.79
   Lipid metabolism, inorganic ion transport and stress response
   B1745_05970 ‐ esterase 1.41
   B1745_00245 ‐ esterase 1.03
   B1745_02830 ‐ acyl‐CoA thioesterase 1.40
   B1745_05670 ‐ biotin carboxylase 1.58
   B1745_00100 mgtC putative Mg^2+ transporter family protein 1.26
   B1745_00845 pot potassium transporter 1.36
   B1745_05305 amt ammonium transporter 1.97
   B1745_06945 ‐ cadmium‐translocating P‐type ATPase 2.16
   Genes down‐regulated
   Carbohydrate transport and metabolism
   B1745_06195 ‐ hypothetical protein −1.03
   B1745_05125 glvB PTS alpha‐glucoside transporter subunit IICB −1.06
   B1745_05025 atoB 3‐ketoacyl‐CoA thiolase −1.08
   B1745_00695 ddl D‐alanine–D‐alanine ligase A −1.27

   B1745_05485

   B1745_05490
   tagA galactosyltransferase

   −1.33

   −1.56
   B1745_06760 Malk sugar ABC transporter ATP‐binding protein −1.36
   B1745_02355 acyP acylphosphatase −1.50
   B1745_06765 pgmB beta‐phosphoglucomutase −1.61
   B1745_06730 gpmB hypothetical protein −2.38
   B1745_06745 ganQ sugar ABC transporter permease −2.58
   B1745_06750 ganP sugar ABC transporter permease −2.79
   Amino acids transport and metabolism
   B1745_01435 rpoA DNA‐directed RNA polymerase subunit alpha −1.02
   B1745_02350 yidC insertase −1.07
   B1745_04680 ‐ amino acid permease −1.14
   B1745_05775 secG preprotein translocase subunit SecG −1.17
   B1745_06880 lepB S26 family signal peptidase −1.19
   B1745_04160 valS valine‐tRNA ligase −1.22
   B1745_01405 secY preprotein translocase subunit SecY −1.38
   B1745_02435 cth aluminum resistance protein −1.74
   B1745_03815 ‐ amino acid permease −2.08
   [101]Open in a new tab

TABLE 3.

   Genes differentially expressed in the stationary phase compared to
   logarithmic phase
   Function group and ORF Gene Description Expression ratio
   Genes up‐regulated
   Carbohydrate transport and metabolism
   B1745_04550 glmS glutamine‐‐fructose−6‐phosphate transaminase
   (isomerizing) 2.34
   B1745_04615 spp HAD family hydrolase 1.91
   B1745_04860 pgl 3‐carboxymuconate cyclase 1.30
   B1745_06170 gpmB histidine phosphatase family protein 1.26
   B1745_04485 spp sugar‐phosphatase 1.20
   B1745_06775 nplT alpha‐glycosidase 1.14
   Amino acid transport and metabolism
   B1745_04550 glmS glutamine‐fructose−6‐phosphate transaminase
   (isomerizing) 2.34
   B1745_00615 adaB cysteine methyltransferase 2.00
   B1745_01515 pepC aminopeptidase 1.55
   B1745_06860 ‐ amino acid permease 1.54
   B1745_00965 hsp20 heat‐shock protein Hsp20 1.52
   B1745_03805 uvrC excinuclease ABC subunit C 1.45
   B1745_06885 clpE ATP‐dependent Clp protease ATP‐binding subunit 1.40
   B1745_04960 clpE Clp protease ClpE 1.33
   B1745_04860 pgl 3‐carboxymuconate cyclase 1.30
   B1745_01540 cysS cysteine‐tRNA ligase 1.29
   B1745_04890 ‐ transcriptional activator, Rgg/GadR/MutR family
   domain‐containing protein 1.23
   B1745_03010 grpE nucleotide exchange factor GrpE 1.23
   B1745_00550 htpX zinc metalloprotease HtpX 1.19
   B1745_04855 atpA haloacid dehalogenase 1.18
   B1745_01260 ‐ histidine kinase 1.16
   B1745_00985 ‐ CPBP family intramembrane metalloprotease 1.15
   B1745_04695 clpP ATP‐dependent Clp protease proteolytic subunit 1.14
   B1745_07110 pcp pyroglutamyl‐peptidase I 1.06
   B1745_06165 ‐ metalloprotease 1.05
   B1745_00910 pepC aminopeptidase 1.03
   Lipid metabolism, inorganic ion transport, and stress response
   B1745_02830 ‐ acyl‐CoA thioesterase 2.30
   B1745_01775 groEL chaperone 1.22
   B1745_03010 grpE nucleotide exchange factor 1.23
   B1745_03015 dnaK molecular chaperone 1.10
   B1745_00745 uspA universal stress protein 1.14
   B1745_01850 trxA thioredoxin 1.20
   Genes down‐regulated
   Carbohydrate transport and metabolism
   B1745_01165 murF UDP‐N‐acetylmuramoyl‐tripeptide‐‐D‐alanyl‐D‐ alanine
   ligase −1.02
   B1745_05730 manY PTS mannose/fructose/sorbose transporter subunit IIC
   −1.04
   B1745_05730 manY PTS mannose/fructose/sorbose transporter subunit IIC
   −1.04
   B1745_05735 manX PTS mannose transporter subunit IIAB −1.15
   B1745_05130 glvA 6‐phospho‐alpha‐glucosidase −1.33
   B1745_07135 nagB glucosamine−6‐phosphate deaminase −2.70
   Amino acid transport and metabolism
   B1745_04680 ‐ amino acid permease −1.01
   B1745_05320 livB branched‐chain amino acid transport system II carrier
   protein −1.02
   B1745_05735 manX PTS mannose transporter subunit IIAB −1.15
   B1745_02915 tsf translation elongation factor Ts −1.17
   B1745_03935 ‐ peptide‐binding protein −1.24
   B1745_02235 thrS threonine‐tRNA ligase −1.41
   B1745_00785 asnB asparagine synthase (glutamine‐hydrolyzing) −1.47
   B1745_00270 brnQ branched‐chain amino acid ABC transporter permease
   −1.69
   Lipid metabolism, inorganic ion transport, and stress response
   B1745_00715 ‐ acetylesterase −1.10
   [102]Open in a new tab

   During the fermentation, the acidity of soymilk increased significantly
   from 45.33° to 95.88° (Table [103]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 content of acetic
   acid also increased slightly. Organic acid production was produced by
   L. amylolyticus L6 through the consumption of sugars in the soymilk
   (Wang et al., [104]2012; Xia et al., [105]^2019). The production of
   acetic acid indicated that this strain was a facultatively
   heterofermentative bacterium. After glucose is converted into pyruvate
   by glycolysis, it can generate lactic acid through the action of
   lactate dehydrogenase under anaerobic conditions (Figure [106]5).
   RNA‐Seq results showed that the expression of LDH gene (B1745_03165)
   encoding L‐lactate dehydrogenase was significantly increased in the log
   phase (Table [107]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.

3.4. 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, [108]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., [109]2018;
   Slonczewski et al., [110]^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 [111]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
   reported (Ceh et al., [112]2020; Song et al., [113]^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., [114]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.

TABLE 4.

   Changes of amino acids in soymilk fermented with L. amylolyticus L6
   Free amino acids (mg/L) Period
   Unfermented Lag phase Log phase Stationary phase
   Essential amino acids
   Lysine 39.68 ± 1.72^a 36.60 ± 1.21^b 35.13 ± 0.28^bc 33.06 ± 0.99^c
   Phenylalanine 29.62 ± 0.79^ab 30.39 ± 0.86^a 28.87 ± 2.03^ab
   26.79 ± 1.39^b
   Methionine 47.04 ± 1.50^a 43.05 ± 1.79^b 43.72 ± 1.09^ab 42.30 ± 1.85^b
   Threonine 8.59 ± 0.21^c 9.31 ± 0.25^ab 9.59 ± 0.25^a 9.01 ± 0.22^bc
   Isoleucine 20.32 ± 1.33 21.28 ± 0.92 20.80 ± 0.53 19.57 ± 2.50
   Leucine 50.91 ± 1.98^a 48.33 ± 1.76^ab 46.65 ± 1.20^b 43.40 ± 2.11^bc
   Valine 5.74 ± 0.33^a 2.19 ± 0.22^d 2.63 ± 0.08^c 3.13 ± 0.08^b
   Total of EAA 201.90 ± 7.95^a 191.14 ± 8.18^b 187.40 ± 4.82^b
   177.26 ± 9.56^c
   Nonessential amino acids
   Asparagine 21.70 ± 0.76^a 14.10 ± 1.14^b 10.64 ± 1.02^c 5.48 ± 0.52^d
   Glutamate 102.04 ± 3.21^b 133.14 ± 2.90^a 133.30 ± 3.15^a
   129.07 ± 0.99^a
   Serine 23.27 ± 0.82^b 26.16 ± 1.63^a 27.98 ± 0.92^a 27.53 ± 0.66^a
   Histidine 4.65 ± 0.41^c 7.03 ± 0.23^a 6.82 ± 0.13^a 6.15 ± 0.25^b
   Glycine 48.10 ± 1.61^d 61.88 ± 1.93^c 76.37 ± 1.00^b 83.97 ± 1.12^a
   Arginine 125.25 ± 2.85^a 121.10 ± 2.56^a 120.45 ± 2.65^ab
   115.86 ± 1.73^b
   Alanine 60.34 ± 1.87^a 51.74 ± 2.00^b 53.91 ± 1.00^b 52.86 ± 0.98^b
   Tyrosine 11.42 ± 0.19^d 16.97 ± 0.24^a 16.23 ± 0.51^b 15.09 ± 0.41^c
   Cysteine 0.79 ± 0.06^d 1.21 ± 0.04^c 1.92 ± 0.09^b 3.18 ± 0.05^a
   Proline 32.66 ± 1.55 32.83 ± 1.84 33.35 ± 0.67 32.69 ± 1.05
   Total of NEAAs 430.23 ± 13.34^c 466.16 ± 14.53^b 480.99 ± 11.14^a
   471.86 ± 7.77^ab
   Total amino acids 632.12 ± 21.28^c 657.30 ± 22.70^ab 668.38 ± 15.97^a
   649.12 ± 17.33^bc
   [115]Open in a new tab

   Data are the mean ±standard deviation (n = 3). Means in the same row
   with different superscript letters (a–d) are significantly different
   (p <.05)

   The proteolytic system of LAB generally consisted of protease,
   transport systems of amino acid or peptides, and peptidases (Wang
   et al., [116]2012). 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., [117]2006). Transcriptomic
   results indicated that the expression of clpP (B1745_04695), clpC
   (B1745_01265), and clpE (B1745_04960) genes encoding subunits of
   caseinolytic protease (Clp) previously identified in Lactobacillus
   plantarum IIA‐1AS (Mega et al., [118]2020) was highly induced in the
   logarithmic and stationary phase. Meanwhile, two genes (B1745_00550 and
   B1745_06165) coding for metalloprotease were also found to be
   up‐regulated in the logarithmic and stationary phase. Besides, the gene
   of another intramembrane metalloprotease showed higher induction levels
   in the stationary phase than in the logarithmic phase. These highly
   expressed protease genes indicated the strong proteolytic ability of L.
   amylolyticus L6 in the fermentation of soymilk.

   The transport of peptides into the cell is an essential step for LAB
   multiplying in soymilk (Hagting, [119]1995). Transcriptomic data showed
   that genes involved in the transport and hydrolysis of peptide in the
   cytoplasm also exhibited high expression levels (Tables [120]2 and
   [121]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., [122]1993), were found to be up‐regulated in soymilk‐grown L.
   amylolyticus L6 cells. Five genes in Opp operon were highly expressed,
   including B1745_00920 (OppA coding for substrate‐binding proteins),
   B1745_00925 and B1745_00940 (OppB and OppC coding for membrane
   proteins), and B1745_00945 (OppD and OppF coding for ATP‐binding
   proteins). Meanwhile, pepC coding for aminopeptidase that could
   hydrolyze oligopeptide into amino acid also exhibited high expression
   levels. Additionally, another five peptidase genes were highly induced
   in the logarithmic phase, which include two peptidases (pepO,
   B1745_02860; pepT, B1745_06070), two aminopeptidases (pepC, B1745_00910
   and B1745_00955), and a dipeptidyl‐peptidase (pepX, B1745_02545).
   Dipeptidyl‐peptidase (pepX, B1745_02545) identified in Lactobacillus
   helveticus (Ojennus et al., [123]2019) and Lactococcus lactis (Nurizzo
   et al., [124]2002) could hydrolyze peptide bonds from the N‐terminus of
   substrates when the penultimate amino acid residue is a proline. The
   highly induced dipeptidyl‐peptidase might suggest the high content of
   oligopeptides 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
   up‐regulated, which might be due to the stagnation of cell growth and
   proliferation, reducing the requirement for peptide and amino acid.

   Genome analysis of L. amylolyticus L6 with KEGG pathways revealed that
   this strain was able to synthesize nine kinds of amino acids, including
   valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe),
   tryptophan (Trp), tyrosine (Tyr), aspartate (Asp), asparagine (Asn),
   and arginine (Arg). asnA (B1745_06855) and lysC (B1745_RS07280) genes
   responsible for the synthesis of aspartate (Asp) showed high induction
   levels in the logarithmic phase (Table [125]2), while other amino acid
   synthetic genes were not up‐regulated. 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 [126]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 nitrogen source was significantly
   down‐regulated, which was due to the feedback repression by a high
   concentration of substrate glutamate inside and outside the cell.

   L. amylolyticus L6 could not synthesize other amino acids, except those
   mentioned above, hence it needed the help of amino acid transporters to
   supplement amino acid. A glutamate transporter operon (glnQHMP, glnP,
   B1745_05185; glnM, B1745_05190; 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 [127]2 and Table [128]4). The overexpression of the glutamate
   transporter operon has also been reported in L. casei Zhang under
   soymilk environment (Wang et al., [129]2012). 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.

3.5. 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
   (acetyl‐CoA (coenzyme A) carboxylase), accB (acetyl‐CoA carboxylase
   biotin carboxyl carrier protein), accC (acetyl‐CoA carboxylase, biotin
   carboxylase subunit), accD (acetyl‐CoA carboxylase carboxyl transferase
   subunit beta), fabD (acyl‐carrier‐protein S‐malonyltransferase), fabF
   (3‐oxoacyl‐[acyl‐carrier‐protein] synthase II), fabG
   (3‐oxoacyl‐acyl‐carrier protein reductase), fabH
   (3‐oxoacyl‐[acyl‐carrier‐protein] synthase III), fabI
   (enoyl‐[acyl‐carrier protein] reductase I), fabZ
   (3‐hydroxyacyl‐[acyl‐carrier‐protein] dehydratase), and mch
   (medium‐chain acyl‐[acyl‐carrier‐protein] hydrolase). Only accC gene
   (B1745_05670) was found to be up‐regulated in the logarithmic phase
   during the growth of L. amylolyticus L6 in soymilk. It was reported
   that soymilk contained 2.64% grease, and the relative content of
   unsaturated fatty acid is more than 80% (Xiangnan et al., [130]2019),
   which inhibited the expression of genes related to fatty acid
   biosynthesis in L. amylolyticus L6. Meanwhile, acyl‐CoA thioesterase
   gene (B1745_02830) that catalyzes the hydrolysis of acyl‐CoAs to the
   free fatty acid and regulates intracellular levels of free fatty acids
   and acyl‐CoAs (Tillander et al., [131]2017) was highly induced during
   its growth in soymilk. Besides, several genes coding for esterase
   (B1745_05970 and B1745_00245) were also up‐regulated in logarithmic
   phase to utilize the grease in soymilk.

   Inorganic ions, especially metal ions, are important for LAB to
   maintain normal functions in the metabolism (Mrvčić et al., [132]2012).
   Generally, membrane transporters play a crucial role in regulating the
   intracellular concentrations of metal ions (Boyaval, [133]1989). The
   expression of five genes, such as mgtC (B1745_00100) coding for Mg^2+
   transporter, pot (B1745_00845) coding for potassium transporter, amt
   (B1745_05305) coding for ammonium transporter, and
   cadmium‐translocating P‐type ATPase gene (B1745_06945), was
   significantly induced in the logarithmic phase, indicating the
   importance of inorganic ions in regulating physiological functions of
   L. amylolyticus L6, such as ion homeostasis, coenzyme factor, and
   electron transport system.

   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 stress‐induced
   denaturation of other proteins (Feder & Hofmann, [134]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., [135]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 [136]1). The
   increased expression level of a universal stress protein (B1745_00745)
   in the stationary phase that was required for resistance to DNA damage
   also engaged in acid tolerance of L. amylolyticus L6. In addition, the
   high transcript level of thioredoxin (trxA, B1745_01850) in the
   stationary phase that acts as an antioxidant by promoting the reduction
   of other proteins through the cysteine thiol–disulfide bond exchange
   was related to stress adaptation in L. amylolyticus L6. The gene highly
   expressed in logarithmic phase was glutamate:gamma‐aminobutyrate
   antiporter (gadC, B1745_00320) that exchanges the intracellular
   γ‐aminobutyric acid (GABA) with extracellular Glu to expel protons in
   the cytoplasm (Dan et al., [137]2012).

3.6. 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., [138]2013). Isoflavones’ glucosidic conjugates
   could be converted into highly bioactive aglycones by β‐glucosidase in
   lactobacilli (Tang et al., [139]2007; Wei et al., [140]^2007; Xia
   et al., [141]^2019). As shown in Table [142]5, most of the isoflavones
   in unfermented soymilk occurred in the form of glucosides with the
   concentration of 285.77 mg/L and the content of aglycones was only
   14.51 mg/L. During the fermentation, the total concentration of
   isoflavone aglycones increased from 14.51 mg/L to 36.09 mg/L, and three
   forms of aglycones’ (daizein, glycitein, and genistein) concentration
   also increased significantly. However, the content of glucosidic
   isoflavones changed irregularly during the fermentation. Compared with
   the unfermented phase, the glucosidic isoflavones (daidzin, glyctin,
   and genistin) exhibited a decreasing tendency in the lag phase (2h),
   and then the content of glucosidic isoflavones increased gradually in
   the logarithmic and stationary phase. A similar phenomenon has been
   reported in the soymilk beverage fermented by Kombucha rich in LAB (Xia
   et al., [143]2019). It is presumed that the fermentation of L.
   amylolyticus L6 could promote the release of free flavonoids from
   binding forms with soluble fibers in the soymilk. Transcriptomic data
   indicated that the expression of bglA gene coding for
   6‐phospho‐β‐glucosidase increased significantly in logarithmic phase,
   which was consistent with the increasing concentrations of isoflavone
   aglycones. And 6‐phospho‐β‐glucosidase that could convert isoflavone
   glucosides into aglycones has been reported in our previous study (Fei,
   Liu, et al., [144]2017).

TABLE 5.

   Concentration of isoflavones (mg/L) in soymilk fermented with L.
   amylolyticus L6
   Isoflavones (mg/L) Period
   Unfermented Lag phase Log phase Stationary phase
   Glycosides
   Daidzin 216.65 ± 3.80^a 154.96 ± 1.92^c 188.45 ± 1.83^b 217.22 ± 2.01^a
   Glyctin 46.02 ± 1.77^b 35.17 ± 0.47^d 43.60 ± 0.19^c 50.73 ± 0.29^a
   Genistin 23.09 ± 0.12^a 11.87 ± 0.63^b 12.10 ± 0.06^b 12.62 ± 0.77^b
   Total 285.77 ± 5.50a 202.01 ± 1.80c 244.15 ± 1.87b 280.57 ± 1.41a
   Aglycones
   Daizein 10.03 ± 0.49^c 9.17 ± 0.48^d 11.89 ± 0.08^b 16.63 ± 0.14^a
   Glycitein ND ND ND 5.61 ± 0.07^a
   Genistein 4.48 ± 1.16^b 5.65 ± 1.87^b 12.11 ± 0.99^a 13.85 ± 1.27^a
   Total 14.51 ± 1.65 ^c 14.82 ± 0.62 ^c 24.00 ± 1.07 ^b 36.09 ± 1.48 ^a
   [145]Open in a new tab

   Data are the mean±standard deviation (n = 3). Means in the same column
   with different superscript letters (a–d) are significantly different
   (p <.05). ND means not detected.

4. CONCLUSION

   This study revealed the chemical component changes and transcriptomic
   changes of L. amylolyticus L6 in fermented soymilk. 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‐phospho‐β‐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.

CONFLICT OF INTEREST

   None declared.

ETHICAL APPROVAL

   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.

Supporting information

   Supplementary Material
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   Supplementary Material
   [147]Click here for additional data file.^ (37.1KB, docx)

ACKNOWLEDGMENTS