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.

Soy protein hydrolysates induce menaquinone-7 biosynthesis by enhancing the biofilm formation of Bacillus subtilis natto.

Menaquinone-7 (MK-7) is a form of vitamin K2 with health-beneficial effects. A novel fermentation strategy based on combining soy protein hydrolysates (SPHs) with biofilm-based fermentation was investigated to enhance menaquinone-7 (MK-7) biosynthesis by Bacillus subtilis natto. Results showed the SPHs increased MK-7 yield by 199.4% in two-stage aeration fermentation as compared to the SP-based medium in submerged fermentation, which was related to the formation of robust biofilm with wrinkles and the enhancement of cell viability. Moreover, there was a significant correlation between key genes related to MK-7 and biofilm synthesis, and the quorum sensing (QS) related genes, Spo0A and SinR, were downregulated by 0.64-fold and 0.39-fold respectively, which promoted biofilm matrix synthesis. Meanwhile, SPHs also enhanced the MK-7 precursor, isoprene side chain, supply, and MK-7 assembly efficiency. Improved fermentation performances of bacterial cells during fermentation were attributed to abundant oligopeptides (Mw < 1 kDa) and moderate amino acids, particularly Arg, Asp, and Phe in SPHs. All these results revealed that SPHs were a potential and superior nitrogen source for MK-7 production by Bacillus subtilis natto.

Effects of Bacillus subtilis natto JLCC513 on gut microbiota and intestinal barrier function in obese rats.

AIMS: This study aimed to investigate the effects of Bacillus subtilis natto JLCC513 (JLCC513) on gut microbiota, inflammation and intestinal barrier function in high-fat-diet (HFD) rats. METHODS AND RESULTS: Sprague-Dawley (SD) rats were fed HFD for 16 weeks, and treated with JLCC513 in 9th week. The oral administration of JLCC513 decreased body weight and reduced the inflammation level in HFD rats. Pathologically, JLCC513 prevented the detachment of ileal villus and increased the villus height in rats. Mechanistically, western blot analysis showed that the protein levels of tight junction (TJ) proteins involved in intestinal barrier function, including zonula occludens-1 (ZO-1), occludin and claudin-1, were increased after JLCC513 treatment. Meanwhile, JLCC513 treatment also decreased the protein levels of toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB) and NOD-like receptor protein 3 (NLRP3), indicating inhibition of the TLR4/NF-κB/NLRP3 pathway. Furthermore, faecal analysis showed that JLCC513 increased the abundance of Lactobacillus and Oscillospira and the ratio of Firmicutes/Bacteroidetes (F/B), and decreased the levels of Blautia and C_Clostridium. CONCLUSIONS: JLCC513 alleviated intestinal barrier dysfunction by inhibiting TLR4/NF-κB/NLRP3 pathway and regulating gut microbiota disorders. SIGNIFICANCE AND IMPACT OF STUDY: Our study might provide new treatment strategies for obesity and metabolic diseases.

Study on the mechanism of production of γ-PGA and nattokinase in Bacillus subtilis natto based on RNA-seq analysis.

Poly-γ-glutamic acid (γ-PGA) and nattokinase (NK) are the main substances produced by Bacillus subtilis natto in solid-state fermentation and have wide application prospects. We found that our strains had higher activity of nattokinase when soybeans were used as substrate to increase the yield of γ-PGA. Commercial production of γ-PGA and nattokinase requires an understanding of the mechanism of co-production. Here, we obtained the maximum γ-PGA yield (358.5 g/kg, w/w) and highest activity of NK during fermentation and analyzed the transcriptome of Bacillus subtilis natto during co-production of γ-PGA and NK. By comparing changes in expression of genes encoding key enzymes and the metabolic pathways associated with the products in genetic engineering, the mechanism of co-production of γ-PGA and nattokinase can be summarized based on RNA-seq analysis. This study firstly provides new insights into the mechanism of co-production of γ-PGA and nattokinase by Bacillus subtilis natto and reveals potential molecular targets to promote the co-production of γ-PGA and nattokinase.

Inhibition of nattokinase against the production of poly (γ-glutamic Acid) in Bacillus subtilis natto.

OBJECTIVE: To study the effect of nattokinse (NK) on the synthesis of poly(γ-glutamic acid) (γ-PGA) in Bacillus subtilis natto. RESULTS: γ-PGA yield significantly decreased as NK was added in the original medium. With the increment of NK dosage, the yield decreased increasingly, but biomass increased instead of decreasing. The fact that cell density triggers the synthesis of γ-PGA is a controversial issue. γ-PGA yield and biomass closely correlate with addition time of NK. The later the addition of NK, the more γ-PGA yield decreased but the more biomass increased. It is concluded that cell hunger is a key factor to trigger the transmission of the cell density signal, and NK may inhibit γ-PGA synthesis by alleviating cell hunger. Besides, NK may reduce γ-PGA yield by degrading extracellular γ-PGA molecules. The study of adding L-glutamate of 0-20 g/L to the original medium showed that low concentration of L-glutamate (less than 5 g/L) could promote the synthesis of NK and γ-PGA, and thus NK may inhibit γ-PGA synthesis through strengthening substrate competition. CONCLUSIONS: NK mainly inhibits γ-PGA synthesis in Bacillus subtilis natto through alleviating cell starvation and strengthening substrate competition, and reduces γ-PGA yield through degrading extracellular γ-PGA molecules.

The effect of aeration and mixing in developing a dairy-based functional food rich in menaquinone-7.

Vitamin menaquinone-7 (MK-7) supplementation improves bone health and reduces the incidence of osteoporosis. Despite the recent developments in MK-7 fermentation using Bacillus subtilis natto, low fermentation yields, as well as complicated downstream processing steps, are still the main reasons for the expensive final product. To overcome these issues, developing a fermented dairy-based product rich in MK-7 by avoiding costly downstream steps and optimising the fermentation operating conditions to enhance the MK-7 concentration would be an alternative approach. The present study, therefore, aims to evaluate the role of agitation and aeration as the key operating conditions on MK-7 production by Bacillus subtilis natto using a milk media. The agitation and aeration rates of 525 RPM and 5 VVM were found to be the optimum levels leading to the production of 3.54 mg/L of MK-7. Further, the sensory evaluation was performed to compare the sensory properties of the freeze-dried fermented samples with non-fermented milk samples. The results illustrated that the fermented samples had a significant saltiness with intense aroma resulting in the less acceptability of them by the panellists.

Improvement of menaquinone-7 production by Bacillus subtilis natto in a novel residue-free medium by increasing the redox potential.

Bacillus subtilis natto is a GRAS bacterium. Nattokinase, with fibrinolytic and antithrombotic activities, is one of the major products of this organism. It is being gradually recognized that B. subtilis natto can also be used as a biosynthetic strain for vitamin K2, which has phenomenal benefits, such as effects in the prevention of cardiovascular diseases and osteoporosis along with antitumor effects. Knocking out of the aprN gene by homologous recombination could improve the redox potential and slightly increase the concentration of MK-7. By detecting the change in redox potential during the growth of B. subtilis natto, a good oxygen supply and state of the cell membrane were found to be beneficial to vitamin K2 synthesis. A two-step RSM was used to optimize the operation parameters and substrate concentration in the new residue-free fermentation culture. The optimal conditions for the residue-free medium and control were determined. The optimum concentrations of soybean flour, corn flour, and peptone were 78.9, 72.4, and 24.8 g/L, respectively. The optimum rotational speed and volume of the culture medium using a shaking flask were 117 rpm and 10%, respectively. The state and composition of the cell membranes were more stable when engineered bacteria were cultured in this residue-free fermentation medium. Finally, the concentration of MK-7 increased by 37% to 18.9 mg/L, and the fermentation time was shortened by 24 h.

Comparison of soybean cultivars for enhancement of the polyamine contents in the fermented soybean natto using Bacillus subtilis (natto).

Polyamines have beneficial properties to prevent aging-associated diseases. Raw soybean has relatively high polyamine contents; and the fermented soybean natto is a good source of polyamines. However, detailed information of diversity of polyamine content in raw soybean is lacking. The objectives of this study were to evaluate differences of polyamines among raw soybeans and select the high polyamine-containing cultivar for natto production. Polyamine contents were measured chromatographically in 16 samples of soybean, which showed high variation among soybeans as follows: 93-861 nmol/g putrescine, 1055-2306 nmol/g spermidine, and 177-578 nmol/g spermine. We then confirmed the high correlations of polyamine contents between raw soybean and natto (r = 0.96, 0.95, and 0.94 for putrescine, spermidine, and spermine, respectively). Furthermore, comparison of the polyamine contents among 9 Japanese cultivars showed that 'Nakasen-nari' has the highest polyamine contents, suggesting its suitability for enhancement of polyamine contents of natto.

Effect of Bacillus subtilis Natto on Meat Quality and Skatole Content in TOPIGS Pigs.

This study investigated the effect of Bacillus subtilis (B. subtilis) natto on meat quality and skatole in TOPIGS pigs. Sixty TOPIGS pigs were randomly assigned to 3 groups (including 5 pens per group, with 4 pigs in each pen) and fed with basic diet (control group), basic diet plus 0.1% B. subtilis natto (B group), and basic diet plus 0.1% B. subtilis natto plus 0.1% B. coagulans (BB group), respectively. All pigs were sacrificed at 100 kg. Growth performance, meat quality, serum parameters and oxidation status in the three groups were assessed and compared. Most parameters regarding growth performance and meat quality were not significantly different among the three groups. However, compared with the control group, meat pH24, fat and feces skatole and the content of Escherichia coli (E. Coli), Clostridium, NH3-N were significantly reduced in the B and BB groups, while serum total cholesterol, high density lipoprotein, the levels of liver P450, CYP2A6, and CYP2E1, total antioxidant capability (T-AOC) and glutathione peroxidase and Lactobacilli in feces were significantly increased in the B and BB groups. Further, the combined supplementation of B. subtilis natto and B. coagulans showed more significant effects on the parameters above compared with B. subtilis, and Clostridium, and NH3-N. Our results indicate that the supplementation of pig feed with B. subtilis natto significantly improves meat quality and flavor, while its combination with B. coagulans enhanced these effects.

RNA-Seq reveals transcriptomic interactions of Bacillus subtilis natto and Bifidobacterium animalis subsp. lactis in whole soybean solid-state co-fermentation.

Bifidobacteria are anaerobes and are difficult to culture in conventional fermentation system. It was observed that Bacillus subtilis natto enhanced growth of Bifidobacterium animalis subsp. lactis v9 by about 3-fold in a whole soybean solid-state co-fermentation, in a non-anaerobic condition. For the purpose of understanding the metabolic interactions between Bif. animalis subsp. lactis v9 and Ba. subtilis natto, the transcriptome of Bif. animalis subsp. lactis v9 and Ba. subtilis natto was analyzed in single and mixed cultures using RNA-Seq. Compared with the single culture, 459 genes of Bif. animalis subsp. lactis v9 were up regulated and 21 were down regulated in the mixed culture with Ba. subtilis natto, with more than 2-fold difference. Predictive metagenomic analyses suggested that Ba. subtilis natto up regulated transport functions, complex carbohydrates and amino acid metabolism, DNA repair, oxydative stress-related functions, and cell growth of Bif. animalis subsp. lactis v9. In the mixed culture with Bif. animalis subsp. lactis v9, only 3 transcripts of Ba. subtilis natto were over-expressed and 3115 were under-expressed with more than 2-fold difference. The highest down-regulated genes were those involved in carbohydrate and amino acid metabolism. The data presented here demonstrated a parasitic-like interaction regulated at the transcription level, between Ba. subtilis natto and Bif. animalis subsp. lactis in the mixed culture. The over-expression of genes involved in substrate uptake and metabolism in Bif. animalis subsp. lactis in the mixed culture nevertheless, led to its higher cell concentration in the nutrient rich whole soybean medium.