Introducing Bacillus natto and Propionibacterium shermanii into soymilk fermentation: A promising strategy for quality improvement and bioactive peptide production during in vitro digestion.

Herein, the texture properties, polyphenol contents, and in vitro protein digestion characteristics of soymilk single- or co-fermented by non-typical milk fermenter Bacillus natto (B. natto), Propionibacterium freudenreichii subsp. shermanii (P. shermanii), and traditional milk fermenter were evaluated. Co-fermenting procedure containing B. natto or P. shermanii could raise the amounts of gallic acid, caffeic acid, and GABA when compared to the unfermented soymilk. Co-fermented soymilk has higher in vitro protein digestibility and nutritional protein quality. Through peptidomic analysis, the co-work of P. shermanii and Lactobacillus plantarum (L. plantarum) may release the highest relative percentage of bioactive peptides, while the intervention of B. natto and Streptococcus thermophilus (S. thermophilus) resulted in more differentiated peptides. The multi-functional bioactive peptides were mainly released from glycine-rich protein, ß-conglycinin alpha subunit 1, and ACB domain-containing protein. These findings indicated the potential usage of B. natto/S. thermophilus or P. shermanii/L. plantarum in bio-enhanced soymilk fermentation.

The Combination of Bacillus natto JLCC513 and Ginseng Soluble Dietary Fiber Attenuates Ulcerative Colitis by Modulating the LPS/TLR4/NF-κB Pathway and Gut Microbiota.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that is currently difficult to treat effectively. Both Bacillus natto (BN) and ginseng-soluble dietary fiber (GSDF) are anti-inflammatory and helps sustain the intestinal barrier. In this study, the protective effects and mechanism of the combination of B. natto JLCC513 and ginseng-soluble dietary fiber (BG) in DSS-induced UC mice were investigated. Intervention with BG worked better than taking BN or GSDF separately, as evidenced by improved disease activity index, colon length, and colon injury and significantly reduced the levels of oxidative and inflammatory factors (LPS, ILs, and TNF-α) in UC mice. Further mechanistic study revealed that BG protected the intestinal barrier integrity by maintaining the tight junction proteins (Occludin and Claudin1) and inhibited the LPS/TLR4/NF-κB pathway in UC mice. In addition, BG increased the abundance of beneficial bacteria such as Bacteroides and Turicibacter and reduced the abundance of harmful bacteria such as Allobaculum in the gut microbiota of UC mice. BG also significantly upregulated genes related to linoleic acid metabolism in the gut microbiota. These BG-induced changes in the gut microbiota of mice with UC were significantly correlated with changes in pathological indices. In conclusion, this study demonstrated that BG exerts protective effect against UC by regulating the LPS/TLR4/NF-κB pathway and the structure and metabolic function of gut microbiota. Thus, BG can be potentially used in intestinal health foods to treat UC.

A comparative study on the nutritional composition, protein structure and effects on gut microbiota of 5 fermented soybean products (FSPs).

In this study, we conducted an analysis of the differences in nutrient composition and protein structure among various fermented soybean products and their impacts on the gut microbiota of rats. Conventional physicochemical analysis was employed to analyze the fundamental physicochemical composition of the samples. Additionally, we utilized high-performance liquid chromatography and ELISA techniques to quantify the presence of antinutritional compounds. Fourier infrared spectroscopy was applied to delineate the protein structure, while 16 s rRNA gene sequencing was conducted to evaluate alterations in gut microbiota abundance. Subsequently, KEGG was utilized for metabolic pathway analysis. Our findings revealed that fermented soybean products improved the nutritional profile of soybeans. Notably, Douchi exhibited the highest protein content at 52.18 g/100 g, denoting a 26.58 % increase, whereas natto showed a 24.98 % increase. Douchi and natto demonstrated the most substantial relative amino acid content, comprising 50.86 % and 49.04 % of the total samples, respectively. Moreover, the levels of antinutritional factors markedly decreased post-fermentation. Specifically, the α-helix content in doujiang decreased by 13.87 %, while the random coil content in soybean yogurt surged by 132.39 %. Rats that were fed FSP showcased notable enhancements in gut microbiota and associated metabolic pathways. A strong correlation was observed between nutrient composition, protein structure, and gut microbiota abundance. This study furnishes empirical evidence supporting the heightened nutritional attributes of FSPs.

A case of community-acquired pneumonia caused by Bacillus subtilis subsp. natto in an immunocompetent patient.

A 70-year-old immunocompetent male with a history of insomnia presented with pneumonia and bacteremia caused by Bacillus subtilis. The patient took benzodiazepines and regularly consumed alcohol and natto (fermented soybeans). Initial antibiotic treatment was not effective, and bronchoalveolar lavage was performed. Bronchoalveolar lavage fluid (BALF) analysis revealed an increased lymphocytes fraction, and B. subtilis was detected in the BALF. Whole-genome sequencing confirmed the congruence of the genetic sequences between the strain in the blood culture of the patient, BALF, and strain isolated from the consumed natto, confirming B. subtilis subsp. natto as the causative pathogen of pneumonia and bacteremia. Vancomycin followed by levofloxacin and systemic corticosteroid were used to treat the condition. This case highlights community-acquired pneumonia and bacteremia caused by B. subtilis subsp. natto, particularly in individuals who consume natto.

Fermentation of millet bran with Bacillus natto: enhancement of bioactivity levels and the bioactivity of bran extract.

BACKGROUND: Millet bran (MB), a byproduct of millet production, is rich in functional components but it is underutilized. In recent years, researchers have shown that fermentation can improve the biological activity of cereals and their byproducts. This study used Bacillus natto to ferment millet bran to improve its added value and broaden the application of MB. The bioactive component content, physicochemical properties, and functional activity of millet bran extract (MBE) from fermented millet bran were determined. RESULTS: After fermentation, the soluble dietary fiber (SDF) content increased by 92.0%, the ß-glucan content by 164.4%, the polypeptide content by 111.4%, the polyphenol content by 32.5%, the flavone content by 16.4%, and the total amino acid content by 95.4%. Scanning electron microscopy revealed that the microscopic morphology of MBE changed from complete and dense blocks to loosely porous shapes after fermentation. After fermentation, the solubility, water-holding capacity, and viscosity significantly increased and the particle size decreased. Moreover, the glucose adsorption capacity (2.1 mmol g-1), glucose dialysis retardation index (75.3%), and α-glucosidase inhibitory (71.4%, mixed reversible inhibition) activity of the fermented MBE (FMBE) were greater than those of the unfermented MBE (0.99 mmol g-1, 32.1%, and 35.1%, respectively). The FMBE presented better cholesterol and sodium cholate (SC) adsorption properties and the adsorption was considered inhomogeneous surface adsorption. CONCLUSION: Fermentation increased the bioactive component content and improved the physicochemical properties of MBE, thereby improving its hypoglycemic and hypolipidemic properties. This study not only resolves the problem of millet bran waste but also encourages the development of higher value-added application methods for millet bran. © 2024 Society of Chemical Industry.

Fermentation Technology and Functional Foods.

Food is an integral part of our civilization. It is a cultural phenomenon that, while having evolved, is associated with societal traditions and identity. This work analyzes studies conducted to highlight the health properties of the most common ethnic foods. Although these foods were originally created from the need to preserve perishable produce, presently, we know that the fermentation process makes them nutritionally more complete. The basis of these transformations lies in that vast range of prokaryotic and eukaryotic microorganisms that, similar to small biochemical factories, can transform the initial nutrients into metabolically more active biomolecules through fermentation. Although naturally occurring microbes work together for mutual benefit, environmental conditions enhance or inhibit their development. Starting from a selection of microorganisms naturally present on a substrate, we attempt to select the most suitable species to obtain a fermented food with the best nutritional qualities and the richest in nutraceuticals.

Natto (fermented soybeans)-induced anaphylaxis in a surfer with the possibility of sensitization to poly(γ-glutamic acid) from cutaneous exposure to jellyfish sting: a case report.

BACKGROUND: We report a case of anaphylaxis induced by natto (fermented soybeans) allergy that occurred following dermal sensitization from a jellyfish sting. CASE PRESENTATION: A 49-year-old male presented to the emergency room complaining of an acute onset of erythema with pruritis that appeared while he was surfing. Given that his heart rate dropped to ~ 40 bpm without a decline in blood pressure or oxygen saturation, we suspected anaphylaxis and administered 0.5 mg of adrenaline intramuscularly. Immediately after the muscular adrenaline injection, his heart rate recovered to ~ 60-70 bpm. CONCLUSIONS: The major allergen that induces natto allergy is poly(γ-glutamic acid) (PGA), which is present in its mucilage. Given that PGA is also produced by jellyfish tentacles, it can be inferred that the PGA sensitization occurred via dermal exposure to jellyfish PGA. This is an example of a food allergy induced by animal stings. As PGA is a high-molecular-weight polymer, natto allergy, despite being IgE-mediated, often presents with late-onset anaphylaxis, which typically develops half a day after digestion. PGA has a wide range of applications in pharmaceuticals, cosmetics, and foods. Patients may develop allergic symptoms and experience repeated anaphylaxis with no known cause. Therefore, it is important to obtain a detailed medical history and individually instruct patients suspected of being allergic to PGA to avoid PGA-containing products.

Comparative genomics analysis and transposon mutagenesis provides new insights into high menaquinone-7 biosynthetic potential of Bacillus subtilis natto.

This research combined Whole-Genome sequencing, intraspecific comparative genomics and transposon mutagenesis to investigate the menaquinone-7 (MK-7) synthesis potential in Bacillus subtilis natto. First, Whole-Genome sequencing showed that Bacillus subtilis natto BN-P15-11-1 contains one single circular chromosome in size of 3,982,436 bp with a GC content of 43.85 %, harboring 4,053 predicted coding genes. Next, the comparative genomics analysis among strain BN-P15-11-1 with model Bacillus subtilis 168 and four typical Bacillus subtilis natto strains proves that the closer evolutionary relationship Bacillus subtilis natto BN-P15-11-1 and Bacillus subtilis 168 both exhibit strong biosynthetic potential. To further dig for MK-7 biosynthesis latent capacity of BN-P15-11-1, we constructed a mutant library using transposons and a high throughput screening method using microplates. We obtained a YqgQ deficient high MK-7 yield strain F4 with a yield 3.02 times that of the parent strain. Experiments also showed that the high yield mutants had defects in different transcription and translation regulatory factor genes, indicating that regulatory factor defects may affect the biosynthesis and accumulation of MK-7 by altering the overall metabolic level. The findings of this study will provide more novel insights on the precise identification and rational utilization of the Bacillus subtilis subspecies for biosynthesis latent capacity.

A rare case of Bacillus subtilis variant natto-induced persistent bacteremia with liver and splenic abscesses in an immunocompetent patient.

Bacillus subtilis var. natto, a low-pathogenic bacterium used in the traditional Japanese food "natto" (fermented soybeans), has rarely been reported as a pathogen of infectious diseases in humans. Herein, we report the first case of persistent bacteremia caused by B. subtilis var. natto in an immunocompetent patient without any gastrointestinal involvement. A 53-year-old Japanese woman who had been consuming natto every day was admitted to our hospital with complaints of fever and chills. B. subtilis was isolated from blood cultures collected during the initial visit. Abdominal contrast-enhanced computed tomography (CT) showed multiple low-absorption areas in the liver and spleen. Treatment commenced with vancomycin; however, Bacillus sp. was re-detected in the blood culture on day 4 after treatment initiation. The blood culture on day 8 was negative. Subsequently, the treatment was switched to ampicillin-sulbactam and oral amoxicillin-clavulanic acid, and the patient recovered after 28 days of treatment from the time the blood cultures became negative. Contrast-enhanced CT of the abdomen at the end of treatment showed that the multiple low-absorption areas in the liver and spleen had disappeared. Later, the variant of the bacteria was identified as B. subtilis var. natto by DNA analysis. B. subtilis var. subtilis and B. subtilis var. natto cannot be distinguished using matrix-assisted laser desorption/ionization-time of flight mass spectrometry or 16S rRNA analysis. Biotin auxotrophy of B. subtilis var. natto is used to distinguish between the two variants.

Prevention of high-fat-diet-induced obesity in mice by soluble dietary fiber from fermented and unfermented millet bran.

Obesity-related diabetes, cardiovascular disease, and hypertension pose many risks to human health. Thus, mice on a high-fat diet were gavaged with millet bran (unfermented/fermented) soluble dietary fiber (RSDF/FSDF, 500 mg·kg-1) for 10 weeks in current research, and then evaluated the various biological indicators. These findings revealed that RSDF and FSDF supplements could prevent fat synthesis by inhibiting sterol regulatory element-binding protein-1c gene expression. The RSDF supplements can also accelerate fat catabolism through enhanced the mRNA expression levels of adipose triglyceride lipase and peroxisome proliferator-activated receptor α. FSDF supplements can prevent obesity by decreasing 3-hydroxy-3-methyl-glutaryl-CoA reductase expression and increasing cholesterol 7α-hydroxylase expression. Moreover, FSDF also controls obesity development by lowering total cholesterol and low-density lipoprotein cholesterol levels in the blood, triglyceride, total cholesterol, and bile acid levels in the liver. Notably, FSDF supplements can promote Bacteroides and Prevotella propagation; excretive propionic acid binds to free fatty acid receptor 2/3 and then stimulates intestinal epithelial cells to generate glucagon-like-peptide-1 and peptide YY, which can reduce food and energy intake and ultimately prevent obesity. All evidence suggests that FSDF supplements play a crucial role in preventing obesity.

Fermented natto powder alleviates obesity by regulating LXR pathway and gut microbiota in obesity rats.

AIM: This study aimed to investigate the positive effect of natto powder on obese rats fed with a high-fat diet (HFD). METHODS AND RESULTS: Sprague-Dawley rats were fed with a HFD for 8 weeks continuously and gavaged with natto powder, respectively, for 8 weeks starting from the ninth week. The results showed that natto powder significantly reduced the body weight of rats and maintained the balance of cholesterol metabolism in the body by inhibiting the activity of liver X receptors (LXR) target genes, increasing the active expression of cholesterol 7 alpha-hydroxylase, and reducing the active expression of sterol-regulatory element-binding protein and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Furthermore, natto powder increased the relative abundance of potentially beneficial microbiota in gut and decreased the relative abundance of obesity-related harmful bacteria, and also increased the Bacteroidetes/Firmicutes ratio and improved the composition of gut microbiota. CONCLUSIONS: Natto powder maintains the balance of cholesterol metabolism by inhibiting the LXR pathway and regulating the gut microbiota.

Patterns of the Nutrients and Metabolites in Apostichopus japonicus Fermented by Bacillus natto and Their Ability to Alleviate Acute Alcohol Intoxication.

The aim of this study was to understand the changes in nutrient composition and differences in metabolites in Apostichopus japonicus fermented by Bacillus natto and their function in alleviating acute alcohol intoxication (AAI) through in vivo studies. The results showed no significant difference between the basic components of sea cucumber (SC) and fermented sea cucumber (FSC). The SC proteins were degraded after fermentation, and the amino acid content in FSC was significantly increased. The differentially abundant metabolites of SC and FSC were identified by LC-MS/MS. The contents of amino acid metabolites increased after fermentation, and arachidonic acid metabolism was promoted. The results demonstrated that FSC alleviated AAI by improving the activities of alcohol-metabolizing enzymes and antioxidant enzymes in the liver but did not alleviate the accumulation of triglycerides. Our results will provide beneficial information for the development and application of new products from FSC.

Untargeted metabolomics revealed the effect of soybean metabolites on poly(γ-glutamic acid) production in fermented natto and its metabolic pathway.

BACKGROUND: Natto mucus is mainly composed of poly(γ-glutamic acid) (γ-PGA), which affects the sensory quality of natto and has some effective functional activities. The soybean metabolites that cause different γ-PGA contents in different fermented natto are unclear. RESULTS: In this study, we use untargeted metabolomics to analyze the metabolites of high-production γ-PGA natto and low-production γ-PGA natto and their fermented substrate soybean. A total of 257 main significantly different metabolites with the same trend among the three comparison groups were screened, of which 114 were downregulated and 143 were upregulated. Through the enrichment of metabolic pathways, the metabolic pathways with significant differences were purine metabolism, nucleotide metabolism, fructose and mannose metabolism, anthocyanin biosynthesis, isoflavonoid biosynthesis and the pentose phosphate pathway. CONCLUSION: For 114 downregulated main significantly different metabolites with the same trend among the three comparison groups, Bacillus subtilis (natto) may directly decompose them to synthesize γ-PGA. Adding downregulated substances before fermentation or cultivating soybean varieties with the goal of high production of such substances has a great effect on the production of γ-PGA by natto fermentation. The enrichment analysis results showed the main pathways affecting the production of γ-PGA by Bacillus subtilis (natto) using soybean metabolites, which provides a theoretical basis for the production of γ-PGA by soybean and promotes the diversification of natto products. © 2023 Society of Chemical Industry.

Enhancing menaquinone-7 biosynthesis through strengthening precursor supply and product secretion.

Menaquinone-7 (MK-7) is an important class of vitamin K2 that is essential in human health and can prevent osteoporosis and cardiovascular disease. However, due to the complex synthesis pathway, the synthesis efficiency is low. The main objective of this study was to explore the effect of enhanced supply of precursors in Bacillus natto. Three precursors of pyruvate, shikimic acid, and sodium glutamate were chosen to investigate the effect of enhanced supply of precursors on MK-7 synthesis. Then, the optimal concentrations, different combinations, and different adding times were systematically studied, respectively. Results showed that the combination of shikimic acid and sodium glutamate could boost MK-7 production by 2 times, reaching 50 mg/L of MK-7 titer and 0.52 mg/(L·h) of MK-7 productivity. Furthermore, adding shikimic acid and sodium glutamate initially and feeding pyruvate at 48 h and 72 h increased MK-7 production to 58 mg/L. At the same time, the expression of the three related genes was also significantly upregulated. Subsequently, a new fermentation strategy combining the precursors enhancement and product secretion was proposed to enhance MK-7 yield and MK-7 productivity to 63 mg/L and 0.45 mg/(L·h). This study proposed a new fermentation regulation strategy for the enhancement of vitamin K2 biosynthesis.

Quantitative profiling of supersulfides naturally occurring in dietary meats and beans.

Sulfur is essential in the inception of life and crucial for maintaining human health. This mineral is primarily supplied through the intake of proteins and is used for synthesizing various sulfur-containing biomolecules. Recent research has highlighted the biological significance of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiol and proteins. Ingestion of exogenous sulfur compounds is essential for endogenous supersulfide production. However, the content and composition of supersulfides in foods remain unclear. This study investigated the supersulfide profiles of protein-rich foods, including edible animal meat and beans. Quantification of the supersulfide content revealed that natto, chicken liver, and bean sprouts contained abundant supersulfides. In general, the supersulfide content in beans and their derivatives was higher than that in animal meat. The highest proportion (2.15 %) was detected in natto, a traditional Japanese fermented soybean dish. These results suggest that the abundance of supersulfides, especially in foods like natto and bean sprouts, may contribute to their health-promoting properties. Our findings may have significant biological implications and warrant developing novel dietary intervention for the human health-promoting effects of dietary supersulfides abundantly present in protein-rich foods such as natto and bean sprouts.

Complete genome sequence of Bacillus subtilis subsp. natto NARUSE using PacBio sequencing.

We report the complete genome sequence of Bacillus subtilis subsp. natto NARUSE, which has been traditionally employed for fermenting soybeans in Japan. The genome was sequenced using the PacBio system, yielding a sequence, yielding a sequence length of 4,148,793 nucleotides for the circular chromosome and 62,770 nucleotides for the plasmid.

Natto extract inhibits infection caused by the Aujeszky's disease virus in mice.

Aujeszky's disease virus (ADV), also known as Suid alphaherpesvirus 1, which mainly infects swine, causes life-threatening neurological disorders. This disease is a serious global risk factor for economic losses in the swine industry. The development of new anti-ADV drugs is highly anticipated and required. Natto, a traditional Japanese fermented food made from soybeans, is a well-known health food. In our previous study, we confirmed that natto has the potential to inhibit viral infections by severe acute respiratory syndrome coronavirus 2 and bovine alphaherpesvirus 1 through their putative serine protease(s). In this study, we found that an agent(s) in natto functionally impaired ADV infection in cell culture assays. In addition, ADV treated with natto extract lost viral infectivity in the mice. We conducted an HPLC gel-filtration analysis of natto extract and molecular weight markers and confirmed that Fraction No. 10 had ADV-inactivating ability. Furthermore, the antiviral activity of Fraction No. 10 was inhibited by the serine protease inhibitor 4-(2-Aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF). These results also suggest that Fraction No. 10, adjacent to the 12.5 kDa peak of the marker in natto extract, may inactivate ADV by proteolysis. Our findings provide new avenues of research for the prevention of Aujeszky's disease.

Probiotic properties of Bacillus subtilis DG101 isolated from the traditional Japanese fermented food natto.

Spore-forming probiotic bacteria offer interesting properties as they have an intrinsic high stability, and when consumed, they are able to survive the adverse conditions encountered during the transit thorough the host gastrointestinal (GI) tract. A traditional healthy food, natto, exists in Japan consisting of soy fermented by the spore-forming bacterium Bacillus subtilis natto. The consumption of natto is linked to many beneficial health effects, including the prevention of high blood pressure, osteoporosis, and cardiovascular-associated disease. We hypothesize that the bacterium B. subtilis natto plays a key role in the beneficial effects of natto for humans. Here, we present the isolation of B. subtilis DG101 from natto and its characterization as a novel spore-forming probiotic strain for human consumption. B. subtilis DG101 was non-hemolytic and showed high tolerance to lysozyme, low pH, bile salts, and a strong adherence ability to extracellular matrix proteins (i.e., fibronectin and collagen), demonstrating its potential application for competitive exclusion of pathogens. B. subtilis DG101 forms robust liquid and solid biofilms and expresses several extracellular enzymes with activity against food diet-associated macromolecules (i.e., proteins, lipids, and polysaccharides) that would be important to improve food diet digestion by the host. B. subtilis DG101 was able to grow in the presence of toxic metals (i.e., chromium, cadmium, and arsenic) and decreased their bioavailability, a feature that points to this probiotic as an interesting agent for bioremediation in cases of food and water poisoning with metals. In addition, B. subtilis DG101 was sensitive to antibiotics commonly used to treat infections in medical settings, and at the same time, it showed a potent antimicrobial effect against pathogenic bacteria and fungi. In mammalians (i.e., rats), B. subtilis DG101 colonized the GI tract, and improved the lipid and protein serum homeostasis of animals fed on the base of a normal- or a deficient-diet regime (dietary restriction). In the animal model for longevity studies, Caenorhabditis elegans, B. subtilis DG101 significantly increased the animal lifespan and prevented its age-related behavioral decay. Overall, these results demonstrate that B. subtilis DG101 is the key component of natto with interesting probiotic properties to improve and protect human health.

Bacillus subtilis Bacteremia from Gastrointestinal Perforation after Natto Ingestion, Japan.

We report a case of Bacillus subtilis variant natto bacteremia from a gastrointestinal perforation in a patient who ingested natto. Genotypic methods showed the bacteria in a blood sample and the ingested natto were the same strains. Older or immunocompromised patients could be at risk for bacteremia from ingesting natto.

Microbiology of fermented soy foods in Asia: Can we learn lessons for production of plant cheese analogues?

The food industry is facing the challenge of creating innovative, nutritious, and flavored plant-based products, due to consumer's increasing demand for the health and environmental sustainability. Fermentation as a unique and effective tool plays an important role in the innovation of food products. Traditional fermented soy foods are popular in many Asian and African countries as nutritious, digestible and flavorful daily staples or condiments. They are produced by specific microorganisms with the unique fermentation process in which microorganisms convert the ingredients of whole soybean or soybean curd to flavorful and functional molecules. This review provides an overview on traditional fermented food produced from soy, including douchi, natto, tempeh, and sufu as well as stinky tofu, including the background of these products, the manufacturing process, and the microbial diversity involved in fermentation procedures as well as flavor volatiles that were identified in the final products. The contribution of microbes to the quality of these five fermented soy foods is discussed, with the comparison to the role of cheese ripening microorganisms in cheese flavor formation. This communication aims to summarize the microbiology of fermented soy foods in Asia, evoking innovative ideas for the development of new plant-based fermented foods especially plant-based cheese analogues.