Proteomics analysis of enzyme systems and pathway changes during the moromi fermentation of soy sauce mash.

BACKGROUND: During the brewing of soy sauce, the conversion of multiple substances is driven by various microorganisms and their secreted enzyme systems. Soy sauce mash is an important source of enzyme systems during moromi fermentation, but the changes of enzyme systems in soy sauce mash during moromi fermentation are poorly understood. In order to explore the predominant enzyme systems existing during moromi fermentation and to explain the characteristics of the enzyme system changes, an enzymatic activities assay and 4D-label-free proteomics analysis were conducted on soy sauce mash at different stages of fermentation. RESULTS: The activities of hydrolytic enzymes in soy sauce mash decreased continuously throughout the fermentation process, while most of the characteristic physicochemical substances in soy sauce mash supernatant had already accumulated at the early stage of fermentation. Four hydrolytic enzymes were found to be positively correlated with important physicochemical indexes by principal component analysis and Pearson correlation analysis. The proteomics analysis revealed three highly upregulated enzymes and two enzymes that were present in important metabolic pathways throughout the fermentation process. Furthermore, it was found that Aspergillus oryzae was able to accumulate various nutrients in the soy sauce mash by downregulating most of its metabolic pathways. CONCLUSION: Enzymes present with excellent properties during the moromi fermentation period could be obtained from these results. Meanwhile, the characterization of the metabolic pathways of microorganisms during the moromi fermentation period was revealed. The results provide a basis for more scientific and purposeful improvement of moromi fermentation in the future. © 2024 Society of Chemical Industry.

Relationship Between the Frequency of Bowel Movements and Fecal Bacteroides in Japanese Women.

The intestinal microbiota is involved in many diseases, such as constipation, obesity, and inflammatory bowel disease. To determine the associations between the gut microbiome and the frequency of bowel movements, we performed cross-sectional correlation analysis at the baseline and longitudinal correlation analysis after the intervention. Forty-three women were enrolled in this study. All participants ingested soymilk-okara powder (15 g) daily for 12 weeks. They recorded the ingested okara powder amounts and their frequency of bowel movements during the entire 12 weeks of the intervention. The fecal microbiota percentages were measured at baseline and after 12 weeks of intervention. Two women who did not completely record the frequency of bowel movements were excluded. In the cross-sectional correlation analysis at the baseline, there was a significant positive correlation between the relative abundance of the Bacteroides genus in the feces and the frequency of bowel movements per week (R = 0.429, p = 0.005) and a significant negative correlation between the relative abundance of Clostridium cluster XI in the feces and the frequency of bowel movements per week (R = -0.315, p = 0.045). Moreover, in the longitudinal correlation analysis, the difference in the relative abundance of Bacteroides genus in feces between the baseline and after the intervention significantly correlated with the changes in the frequency of bowel movements per week (R = 0.492, p = 0.001). Therefore, it was suggested that there was a relationship between the gut relative abundance of the Bacteroides genus and the frequency of bowel movements.

Ratio of Histamine-Producing/Non-Histamine-Producing Subgroups of Tetragenococcus halophilus Determines the Histamine Accumulation during Spontaneous Fermentation of Soy Sauce.

Strain specificity (within-species variation) of microorganisms occurs widely in nature. It might affect microbiome construction and function in a complex microbial environment. Tetragenococcus halophilus, a halophilic bacterium that generally is used in high salt food fermentation, consists of two histamine-producing and non-histamine-producing subgroups. It is unclear whether and how the strain specificity of histamine-producing capacity influences the microbial community function during food fermentation. Here, based on systematic bioinformatic analysis, histamine production dynamic analysis, clone library construction analysis, and cultivation-based identification, we identified that T. halophilus is the focal histamine-producing microorganism during soy sauce fermentation. Furthermore, we discovered that a larger number and ratio of histamine-producing subgroups of T. halophilus significantly contributed more histamine production. We were able to artificially decrease the ratio of histamine-producing to non-histamine-producing subgroups of T. halophilus in complex soy sauce microbiota and realized the reduction of histamine by 34%. This study emphasizes the significance of strain specificity in regulating microbiome function. This study investigated how strain specificity influenced microbial community function and developed an efficient technique for histamine control. IMPORTANCE Inhibiting the production of microbiological hazards under the assumption of stable and high-quality fermentation is a critical and time-consuming task for the food fermentation industry. For spontaneously fermented food, it can be realized theoretically by finding and controlling the focal hazard-producing microorganism in complex microbiota. This work used histamine control in soy sauce as a model and developed a system-level approach to identify and regulate the focal hazard-producing microorganism. We discovered that the strain specificity of focal hazard-producing microorganisms had an important impact on hazard accumulation. Microorganisms frequently exhibit strain specificity. Strain specificity is receiving increasing interest since it determines not only microbial robustness but also microbial community assembly and microbiome function. This study creatively explored how the strain specificity of microorganisms influenced microbiome function. In addition, we believe that this work provides an excellent model for microbiological hazard control which can promote future work in other systems.

Correlation between bacterial diversity and flavor substances in Longgang soy sauce.

Longgang soy sauce is one of the traditional fermented condiments in China, but its bacterial community succession and its unique flavor development during the fermentation process are not well-investigated. This study evaluated the bacterial diversity, flavor changes, and their correlation during the fermentation of Longgang soy sauce. The results showed that Weissella was the dominant bacterial genus in the fermentation stage of sauce fermented grains. In the first 31 days of the moromi fermentation stage, a variety of bacterial genera such as Weissella, Halomonas, Bacteroides, Pseudomonas, and Tetragenococcus were the dominant bacteria. Our results showed that these bacteria have a significantly positive correlation with phenylethyl alcohol, ethyl acetate, and 3-methyl-1-butanol. As the fermentation progressed, a flora structure with Halomonas as the main bacterial genus was formed. This genus exhibited a significantly positive and positive correlation with 1-octanol, ethyl palmitate, heptanol, and 2-nonanol, which are the unique flavor components of Longgang soy sauce.

Comparisons of Physicochemical Properties, Bacterial Diversities, Isoflavone Profiles and Antioxidant Activities on Household and Commercial doenjang.

In this study, the physicochemical properties (pH, acidity, salinity, and soluble protein), bacterial diversities, isoflavone contents, and antioxidant activities of doenjang (fermented soy paste), household doenjang (HDJ), and commercial doenjang (CDJ), were assessed and compared. The values of pH 5.14-5.94 and acidity 1.36-3.03%, indicated a similar level in all doenjang. The salinity was high in CDJ at 12.8-14.6%, and the protein contents (25.69-37.54 mg/g) were generally high in HDJ. Forty-three species were identified from the HDJ and CDJ. The main species were verified to be Bacillus amyloliquefaciens (B. amyloliquefaciens), B. amyloliquefaciens subsp. plantarum, Bacillus licheniformis, Bacillus sp. and Bacillus subtilis. Comparing the ratios of isoflavone types, the HDJ has an aglycone ratio of >80%, and 3HDJ indicates a ratio of isoflavone to aglycone of 100%. In the CDJ, except 4CDJ, glycosides account for a high proportion of more than 50%. The results of antioxidant activities and DNA protection effects were variedly confirmed regardless of HDJs and CDJs. Through these results, it is judged that HDJs have a variety of bacterial species compared to CDJs, and these are biologically active and converted from glycoside to aglycone. Bacterial distribution and isoflavone contents could be used as basic data.

A case of Bacillus subtilis var. natto bacteremia caused by ingestion of natto during COVID-19 treatment in a maintenance hemodialysis patient with multiple myeloma.

A 70-year-old woman, who started on hemodialysis 7 months before for end-stage renal disease due to diabetic nephropathy and was diagnosed with symptomatic multiple myeloma 1 month before, was admitted to our hospital with critical coronavirus disease 2019 and treated with long-term immunosuppressive therapy such as steroids and tocilizumab. During treatment, Bacillus subtilis was detected in the blood cultures. We could not exclude the association of natto (fermented soybeans) with B. subtilis var. natto, which the patient had been eating every day from 8 days after admission. She was prohibited from eating natto and treated with vancomycin. Later, B. subtilis detected in the blood culture was identified as B. subtilis var. natto, which was identical with those contained in the natto that the patient consumed daily using a next-generation sequencer. Gut dysbiosis due to old age, malignant tumor, diabetes mellitus, end-stage renal disease, and intestinal inflammation caused by severe acute respiratory syndrome coronavirus 2 increased intestinal permeability and the risk of bacterial translocation, causing B. subtilis var. natto bacteremia. Therefore, careful consideration might be given to the intake of fermented foods containing live bacteria in patients with severe immunocompromised conditions.

α-Glucosidase Inhibitory Activity of Fermented Okara Broth Started with the Strain Bacillus amyloliquefaciens SY07.

In this work, a new strain of Bacillus amyloliquefaciens SY07 isolated from a traditional fermented soybean food was reported to possess remarkable α-glucosidase inhibitor-producing ability. Different culture media were applied for the proliferation of B. amyloliquefaciens SY07, and it was found that fermented okara broth presented the highest α-glucosidase inhibitory activity, while Luria-Bertani medium showed a negative effect. The extract from fermented okara broth acted in a dose-dependent manner to inhibit α-glucosidase activity, with an IC50 value of 0.454 mg/mL, and main inhibitors in the fermentation extract presented a reversible, uncompetitive pattern according to Lineweaver-Burk plots. Moreover, 1-deoxynojirimycin, a recognized α-glucosidase inhibitor, was found in the extract. Results indicated that B. amyloliquefaciens SY07 could utilize okara, a by-product from the soy processing industry, to generate α-glucosidase inhibitors effectively, and be regarded as a novel excellent microbial candidate for safe, economical production of potential functional foods or ingredients with hypoglycemic effect.

Metagenomic insights into the bacteria responsible for producing biogenic amines in sufu.

Harmful levels of biogenic amines (BAs) are frequently identified in sufu. The microorganisms and mechanisms responsible for BA production in sufu, however, are not well documented. In this study, sufu samples were randomly obtained from various regions of China. Putrescine, tyramine, and histamine were quantitated as the most abundant BAs. According to the metagenome sequencing, the abundances and diversities of genes encoding the critical enzymes in BA production were acquired. The results showed that genes encoding arginine-, ornithine-, tryptophan-, and histidine decarboxylases were the predominant amino acid decarboxylase genes. Furthermore, 34 metagenome-assembled genomes (MAGs) were generated, of which 23 encoded at least one gene involved in BA production. Genetic analysis of MAGs indicated genera affiliated with Enterococcus, Lactobacillus-related, and Lactococcus were the major histamine-synthesizing bacteria, and tyrosine may be utilized by Bacillus, Chryseobacterium, Kurthia, Lysinibacillus, Macrococcus, and Streptococcus to product tyramine. The critical species involved in two putrescine-producing pathways were also explored. In the ornithine decarboxylase pathway, Lactobacillus-related and Veillonella were predicted to be the main performers, whereas Sphingobacterium and unclassified Flavobacteriaceae were the dominant executors in the agmatine deiminase pathway. The present study not only explained the BAs formation mechanism in sufu but also identified specific bacteria used to control BAs in fermented soybean products.

Cheonggukjang-Specific Component 1,3-Diphenyl-2-Propanone as a Novel PPARα/γ Dual Agonist: An In Vitro and In Silico Study.

BACKGROUND: Cheonggukjang is a traditional fermented soybean paste that is mostly consumed in Korea. However, the biological activities of Cheonggukjang specific compounds have not been studied. Thus, we aimed to discover a novel dual agonist for PPARα/γ from dietary sources such as Cheonggukjang specific volatile compounds and explore the potential role of PPARα/γ dual agonists using in vitro and in silico tools. METHODS: A total of 35 compounds were selected from non-fermented and fermented soybean products cultured with Bacillus subtilis, namely Cheonggukjang, for analysis by in vitro and in silico studies. RESULTS: Molecular docking results showed that 1,3-diphenyl-2-propanone (DPP) had the lowest docking score for activating PPARα (1K7L) and PPARγ (3DZY) with non-toxic effects. Moreover, DPP significantly increased the transcriptional activities of both PPARα and PPARγ and highly activated its expression in Ac2F liver cells, in vitro. Here, we demonstrated for the first time that DPP can act as a dual agonist of PPARα/γ using in vitro and in silico tools. CONCLUSIONS: The Cheonggukjang-specific compound DPP could be a novel PPARα/γ dual agonist and it is warranted to determine the therapeutic potential of PPARα/γ activation by dietary intervention and/or supplementation in the treatment of metabolic disorders without causing any adverse effects.

Effect of Tempeh on Gut Microbiota and Anti-Stress Activity in Zebrafish.

Rhizopus oryzae is a fungus used to ferment tempeh in Indonesia and is generally recognized as safe (GRAS) for human consumption by the USA FDA. We previously assessed the effect of a tempeh extract on cortisol levels in zebrafish but did not include behavioral studies. Here, we measured the GABA content in three strains of Rhizopus oryzae, two isolated by us (MHU 001 and MHU 002) and one purchased. We then investigated the effect of tempeh on cortisol and the gut microbiota in a zebrafish experimental model. GABA concentration was the highest in MHU 002 (9.712 ± 0.404 g kg-1) followed by our MHU 001 strain and the purchased one. The fish were divided into one control group fed a normal diet and three experimental groups fed soybean tempeh fermented with one of the three strains of Rhizopus oryzae. After two weeks, individual fish were subjected to unpredicted chronic stress using the novel tank diving test and the tank light-dark test. Next-generation sequencing was used to analyze gut microbial communities and RT-PCR to analyze the expression of BDNF (brain-derived neurotrophic factor) gene and of other genes involved in serotonin signaling/metabolism in gut and brain. Tempeh-fed zebrafish exhibited increased exploratory behavior (less stress) in both tank tests. They also had significantly reduced gut Proteobacteria (include E. coli) (51.90% vs. 84.97%) and significantly increased gut Actinobacteria (include Bifidobacterium spp.) (1.80% vs. 0.79%). The content of Bifidobacteriumadolescentis, a "psychobiotic", increased ten-fold from 0.04% to 0.45%. Tempeh also increases BDNF levels in zebrafish brain. Rhizopus oryzae MHU 001 greatly improved the anti-stress effect of tempeh and microbiota composition in zebrafish gut.

Characteristics of Bacterial Strains with Desirable Flavor Compounds from Korean Traditional Fermented Soybean Paste (Doenjang).

To identify and analyze the characteristics of the microorganisms involved in the formation of the desirable flavor of Doenjang, a total of 179 strains were isolated from ninety-four Doenjang collected from six regions in South Korea, and fourteen strains were selected through a sensory evaluation of the aroma of each culture. The enzyme activities of amylase, protease and lipase was shown in the various strains. Bacillus sp.-K3, Bacillus sp.-K4 and Bacillus amyloliquefaciens-J2 showed relatively high protease activity, at 317.1 U, 317.3 U and 319.5 U, respectively. The Bacillus sp.-K1 showed the highest lipase activity at 2453.6 U. In the case of amylase, Bacillus subtilis-H6 showed the highest activity at 4105.5 U. The results of the PCA showed that Bacillus subtilis-H2, Bacillus subtilis-H3, and Bacillus sp.-K2 were closely related to the production of 3-hydroxy-2-butanone (23.51%~43.37%), and that Bacillus subtilis-H5 and Bacillus amyloliquefaciens-J2 were significantly associated with the production of phenethyl alcohol (0.39% and 0.37%). The production of peptides was observed to vary among the Bacillus cultures such as Val-Val-Pro-Pro-Phe-Leu and Pro-Ala-Glu-Val-Leu-Asp-Ile. These peptides are precursors of related volatile flavor compounds created in Doenjang via the enzymatic or non-enzymatic route; it is expected that these strains could be used to enhance the flavor of Doenjang.

A metabolomics approach to evaluate post-fermentation enhancement of daidzein and genistein in a green okara extract.

BACKGROUND: Okara is a major agri-industrial by-product of the tofu and soymilk industries. Employing food-wastes as substrates for the green production of natural functional compounds is a recent trend that addresses the dual concepts of sustainable production and a zero-waste ecosystem. RESULTS: Extracts of unfermented okara and okara fermented with Rhizopus oligosporus were obtained using ethanol as extraction solvent, coupled with ultrasound sonication for enhanced extraction. Fermented extracts yielded significantly better results for total phenolic content (TPC) and total flavonoid content (TFC) than unfermented extracts. A qualitative liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis revealed a shift from glucoside forms to respective aglycone forms of the detected isoflavones, post-fermentation. Since the aglycone forms have been associated with numerous health benefits, a quantitative high-performance liquid chromatography (HPLC) analysis was performed. Fermented okara extracts had daidzein and genistein concentrations of 11.782 ± 0.325 µg mL-1 and 10.125 ± 1.028 µg mL-1 , as opposed to that of 6.7 ± 2.42 µg mL-1 and 4.55 ± 0.316 µg mL-1 in raw okara extracts, respectively. Lastly, the detected isoflavones were mapped to their metabolic pathways, to understand the biochemical reactions triggered during the fermentation process. CONCLUSION: Fermented okara may be implemented as a sustainable solution for production of natural bioactive isoflavonoids genistein and daidzein. © 2021 Society of Chemical Industry.

The fermentation properties and microbial diversity of soy sauce fermented by germinated soybean.

BACKGROUND: The quality of commercial soy sauce is variable at present. Technical work is needed to improve the quality and flavor of soy sauce, especially in China. Material is a factor for influencing soy sauce characters in fermentation. RESULTS: Germinated soybean sauce (fermented by germinated soybean) had a gamma aminobutyric acid (GABA) concentration of 6.83 µg mL-1 , whereas a control (a soy sauce fermented by soybean) had a GABA concentration of less than 2.42 µg mL-1 . Germinated soybean sauce also contained significantly higher levels of isoflavones, total polyphenol, and amino acid nitrogen than the control soy sauce. Microbial diversity results showed that Bacillus was the dominant bacteria in germinated soy sauce compared with the control. Aldehydes, alcohols, esters, and phenols were the major flavor components of germinated soybean sauce. CONCLUSION: A soy sauce with high levels of GABA, isoflavones, and total polyphenol was developed using germinated soybean. Stenotrophomonas, the typical pathogen found in the control, was reduced dramatically and replaced by Bacillus during fermentation with the germinated soybean. The germinated soybean sauce exhibited a better aroma and taste than the control. Soy sauce fermented by soybeans that germinated for 48 h exhibited greater advantages than soy sauce that germinated for 24 and 72 h. © 2020 Society of Chemical Industry.

Effects of the extract from peanut meal fermented with Bacillus natto and Monascus on lipid metabolism and intestinal barrier function of hyperlipidemic mice.

BACKGROUND: Hyperlipidemia is one of the metabolic disorders that poses a great threat to human health. This study is aimed at investigating the potential hypolipidemic properties of extract from peanut meal fermented with Bacillus natto and Monascus in mice fed with a high-fat diet. Herein, 60 male C57BL/6J mice were randomly divided into six groups: four control groups, comprised of a normal group, a model (M) group, a positive control group (atorvastatin 10 mg kg-1 ), and a nonfermented peanut meal extract group (150 mg kg-1 ), and two experimental groups, comprised of a fermented peanut meal extract low-dose group (50 mg kg-1 ) and a fermented peanut meal extract high-dose group (FH, 150 mg kg-1 ). RESULTS: Body weight (P = 0.001) and levels of serum total cholesterol (P = 0.007), triacylglycerol (P = 0.040), low-density lipoprotein cholesterol (P < 0.001), and leptin (P < 0.001) were remarkably decreased in the FH group, whereas the serum high-density lipoprotein cholesterol levels were increased (P < 0.001) by 78.3% compared with the M group. Ileum tissue stained with hematoxylin and eosin showed that the ileal villus detachments in mice were improved, and the villus height was increased by supplementation with extract from fermented peanut meal. Moreover, the expressions of intestinal ZO-1 (P = 0.003) and occludin (P = 0.013) were elevated in the FH group, compared with the M group. CONCLUSION: Extract of peanut meal fermented by B. natto and Monascus can effectively improve hyperlipidemia caused by a high-fat diet in mice, via regulating leptin and blood lipid levels, and protect the intestinal mucosal barrier, which provides evidence for its anti-hyperlipidemia effects and is a research basis for potential industrial development. © 2020 Society of Chemical Industry.

Identification of SFL1 as a positive regulator for flor formation in Zygosaccharomyces rouxii.

Some wild Zygosaccharomyces rouxii impair the quality of soy sauce through the generation of unpleasant odors induced by the formation of flor. Flor formation in Z. rouxii depends on the expression of the FLO11D gene, which is a homolog of the FLO11 gene that encodes a cell surface protein in Saccharomyces cerevisiae. FLO11 expression in S. cerevisiae is regulated by multiple pathways. To investigate the regulation of FLO11D expression in Z. rouxii, we created 13 gene knockout mutants (STE12, TEC1, HOG1, MSS11, FLO8, MSN1, MSN2/4, SKO1, TUP1, CYC8, YAK1, MIG1, and SFL1) related to those pathways and examined whether these mutants form flor. Unexpectedly, SFL1 knockout mutant could only form a very weak flor due to decreased FLO11D expression, suggesting that SFL1 acts as a potential activator of flor formation through FLO11D expression. This result is in contrast to S. cerevisiae SFL1, which acts as a repressor of FLO11 expression.

Linoleic acid, α-linolenic acid, and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus.

Antibacterial activities against Staphylococcus aureus and Bacillus subtilis were found in an ethanol fraction of tempe, an Indonesian fermented soybean produced using Rhizopus oligosporus. The ethanol fraction contained free fatty acids, monoglycerides, and fatty acid ethyl esters. Among these substances, linoleic acid and α-linolenic acid exhibited antibacterial activities against S. aureus and B. subtilis, whereas 1-monolinolenin and 2-monolinolenin exhibited antibacterial activity against B. subtilis. The other free fatty acids, 1-monoolein, monolinoleins, ethyl linoleate, and ethyl linolenate did not exhibit bactericidal activities. These results revealed that R. oligosporus produced the long-chain polyunsaturated fatty acids and monolinolenins as antibacterial substances against the Gram-positive bacteria during the fungal growth and fermentation of heat-processed soybean.

Identification of the putative N-acetylglucosaminidase CseA associated with daughter cell separation in Tetragenococcus halophilus.

The lactic acid bacterium Tetragenococcus halophilus, which is used as a starter to brew soy sauce, comprises both cluster-forming strains and dispersed strains. The cluster-forming strains are industrially useful for obtaining clear soy sauce, because the cell clusters are trapped by filter cloth when the soy sauce mash is pressed. However, the molecular mechanism underlying cell cluster formation is unknown. Whole genome sequence analysis and subsequent target sequence analysis revealed that the cluster-forming strains commonly have functional defects in N-acetylglucosaminidase CseA, a peptidoglycan hydrolase. CseA is a multimodular protein that harbors a GH73 domain and six peptidoglycan-binding LysM domains. Recombinant CseA hydrolyzed peptidoglycan and promoted cell separation. Functional analysis of truncated CseA derivatives revealed that the LysM domains play an important role in efficient peptidoglycan degradation and cell separation. Taken together, the results of this study identify CseA as a factor that greatly affects the cluster formation in T. halophilus.

Dynamics of microbial communities and metabolites in ganjang, a traditional Korean fermented soy sauce, during fermentation.

Six different ganjang batches were prepared and the microbial communities in the ganjang samples and raw materials (meju and solar salts), as well as the metabolites generated during fermentation were analyzed. The varying amounts of raw materials used differentially affected the microbial communities. Halophilic or halotolerant microbes derived from solar salts were abundant during middle or late fermentation. By contrast, non-halophilic microbes derived from meju were abundant mainly during early fermentation. Debaryomyces, Tetragenococcus, and Staphylococcus were abundant in all ganjang batches, which suggested that these may be the most common microbes involved in ganjang fermentation. The salt concentrations did not affect the abundance of Debaryomyces, which was abundant in all ganjang batches. Tetragenococcus was abundant in low salt ganjang, whereas Staphylococcus was abundant in high salt ganjang. Metabolite analysis revealed that carbohydrate concentrations were high in ganjang prepared using high amounts of meju. The level of lactate, which may be largely produced by Tetragenococcus, in low salt ganjang was high because of high microbial activity. The amino acid concentrations of the ganjang batches were mainly associated with meju quantity, but not salt concentration. These results indicated that the production of amino acids may be associated with indigenous proteases in meju, but not microbial activities during ganjang fermentation.

Exploring core functional microbiota related with flavor compounds involved in the fermentation of a natural fermented plain sufu (Chinese fermented soybean curd).

This study aimed to explore the core functional microbiotas related to flavor compounds involving in a naturally fermented soybean curd (plain sufu). Properties such as physicochemical parameters, flavor compounds (17 free amino acids, 21 fatty acids, and 14 aroma volatiles) and microbiota profiles were investigated, and their correlations were explored at 8 stages during production. Results from principal component analysis, multiple factor analysis, and partial least squares-discrimination analysis showed that these properties varied significantly in the eight stages. Furthermore, based on Pearson correlation coefficients and Variable importance for predictive components values between the microbiota profiles and flavor compounds, nine bacterial (Bacillus, Enterobacter, Lactobacillus, Sphingobacterium, Stenotrophomonas, Tetragenococcus, Trabulsiella, Unclassified, and Weissella) and six fungal (Alternaria, Sterigmatomyces, Actinomucor, Fusarium, Debaryomyces, Candida) genera were identified as core functional microbiotas significantly affecting the production of flavor compounds during the natural production. Overall, this study provided a comprehensive description of the dynamic changes of physicochemical parameters, flavor compounds, and microbiota profiles throughout the natural production of plain sufu. The similarities and variations among different stages, as well as correlation between flavor compounds and microbiotas would help to understand the mechanism of plain sufu production, and further to enhance the quality control of plain sufu.

High-throughput sequencing approach to characterize dynamic changes of the fungal and bacterial communities during the production of sufu, a traditional Chinese fermented soybean food.

Red sufu is a traditional food produced by the fermentation of soybean. In this study, sufu samples were periodically collected during the whole fermentation to investigate the dynamic changes of fungal and bacterial communities using high-throughput sequencing technology. The overall process can be divided into pre- and post-fermentation. During post-fermentation, the pH value showed a gradual decrease over time while the amino nitrogen content increased. Trichosporon, Actinomucor and Cryptococcus were the main genera in pre-fermentation while Monascus and Aspergillus were dominant in post-fermentation. This huge shift in fungal composition was caused by process procedure of pouring dressing mixture. However, the bacterial composition was not greatly changed after pouring dressing mixture, the Acinetobacter and Enterobacter were the predominant genera throughout the whole process. Furthermore, Bacillus species were first detected after adding dressing mixture, but declined abruptly to a very low level (0.07%) by the end of the fermentation. Our work demonstrates the dynamic changes of physicochemical properties and microbial composition in every fermentation stage, the knowledge of which could potentially serve as a foundation for improving the safety and quality of sufu in the future.