Bioscience of Microbiota, Food and Health最新文献

Natto is a traditional Japanese fermented soy product high in γ-polyglutamic acid (γ-PGA), whose beneficial effects have been reported. We prepared high-γ-PGA natto and compared the dietary influence on liver lipids and cecal microbiota in mice fed a diet containing it or a standard diet. The mice were served a 30% high-γ-PGA natto diet (PGA group) or standard diet (Con group) for 28 days. Liver lipids, fecal lipids, and fecal bile acids were quantified. Cecal microbiota were analyzed by PCR amplification of the V3 and V4 regions of 16S rRNA genes and sequenced using a MiSeq System. Additionally, the cecal short-chain fatty acid profile was assessed. The results revealed that the liver lipid and triglyceride contents were significantly lower (p<0.01) and amounts of bile acids and lipids in the feces were significantly higher in the PGA group than in the Con group. The cecal butyric acid concentration was observed to be significantly higher in the PGA group than in the Con group. Principal component analysis of the cecal microbiota revealed that the PGA and Con groups were distinct. The ratio of Firmicutes/Bacteroidetes was found to be significantly low in the PGA mice. The results revealed a significantly higher relative abundance of Lachnospiraceae (p<0.05) and significantly lower relative abundance of Coriobacteriaceae (p<0.01) in the PGA group. Analysis of the correlation between bacterial abundance and liver lipids, cecal short-chain fatty acids, fecal lipids, and fecal bile acids suggested that intestinal microbiota can be categorized into different types based on lipid metabolism. Hepatic lipid accumulation typically facilitates the onset of nonalcoholic fatty liver disease (NAFLD). Our findings suggest that high-γ-PGA natto is a beneficial dietary component for the prevention of NAFLD.

We investigated bacteria that have a nutritional symbiotic relationship with respect to milk oligosaccharides in gut microbiota of suckling rats, with specific reference to sialyllactose (SL) degrading Enterococcus gallinarum. Our next generation sequencing analysis of the colonic contents of 12-day-old suckling rats revealed that almost half of the bacteria in the microbiota belonged to the Lactobacillaceae family. Major Lactobacillus species in the contents were identified as L. johnsonii, L. murinus, and L. reuteri. We then monitored changes in numbers of the above Lactobacillus species, E. gallinarum, and the bacteria belonging to the family Enterobacteriaceae (i.e., enterobacteria) in the colonic contents of infant rats at 7, 12, 21, 28, and 35 days of age by using real-time PCR assays targeting these bacterial groups. The 7-day-old infant rats had a gut microbiota in which enterobacteria were predominant. Such dominance was replaced by L. johnsonii and the concomitant E. gallinarum markedly increased in those of 12 and 21 days of ages. During this period, the number of enterobacteria declined dramatically, but that of L. reuteri surged dramatically. Our separate in vitro experiment showed that supplementation of culture media with SL promoted the growth of L. johnsonii and E. gallinarum, with marked production of lactic acid. These findings revealed possible milk oligosaccharide-mediated cross-feeding between E. gallinarum and L. johnsonii, with the former degrading SL to release lactose to be utilized by the latter.

The purpose of this study was to establish reference ranges for gut microbial indices by collecting real-world Japanese microbiome data from a Mykinso cohort. Although several large cohort studies have focused on the human gut microbiome, large cohort studies of the gut microbiome from Japanese populations are scarce, especially from healthy or non-diseased individuals. We collected stool samples and original survey lifestyle information from 5,843 Japanese individuals through the Mykinso gut microbiome testing service. From the obtained 16S rRNA sequence data derived from stool samples, the ratio and distribution of each taxon were analyzed. The relationship between different epidemiological attributes and gut microbial indicators were statistically analyzed. The qualitative and quantitative indicators of these common gut microbiota were confirmed to be strongly correlated with age, sex, constipation/diarrhea, and history of lifestyle-related diseases. Therefore, we set up a healthy sub-cohort that controlled for these attribute factors and defined reference ranges from the distribution of gut microbial index in that population. Taken together, these results show that the gut microbiota of Japanese people had high beta-diversity, with no single "typical" gut microbiota type. We believe that the reference ranges for the gut microbial indices obtained in this study can be new reference values for determining the balance and health of the gut microbiota of an individual. In the future, it is necessary to clarify the clinical validity of these reference values by comparing them with a clinical disease cohort.

Bacterial RNA has recently emerged as an immune-stimulating factor during viral infection. The immune response in an organism is directly related to the progression of virus infections. Lactic acid bacteria in particular have anticancer, bioprotective, and antiallergic effects by modulating immunity. Here, we aimed to demonstrate the effect of bacterial RNA on in vitro production of IL-12, a proinflammatory cytokine, and on in vivo activity against influenza A virus (IFV) infection. Oral administration of heat-killed Enterococcus faecalis KH2 (KH2) or Lactobacillus plantarum SNK12 (SNK) in IFV-infected mice suppressed viral replication and stimulated production of virus-specific antibodies. However, ribonuclease-treated KH2 or SNK abrogated the effect, reducing IL-12 production in vitro and anti-IFV effects in vivo. Taken together, KH2 or SNK showed antiviral effects in vivo when administered orally, and the RNAs of KH2 and SNK play a part in these effects, despite the phylogenetic differences between the bacteria.

A reporter assay system is an essential tool for investigating gene expression mechanisms. In the case of bifidobacteria, several convenient and sensitive reporter systems have been developed. Here, we developed a new reporter system for bifidobacteria using the chloramphenicol acetyltransferase gene (cat) from Staphylococcus aureus. This enzyme stoichiometrically produced free CoA-SH, which was analyzed quantitatively with Ellman's test using 2-nitrobenzoic acid (DTNB). The 2-nitro-5-thiobenzoate (TNB^2-) produced showed a strong yellowish color with maximum absorbance at 412 nm. We also constructed a new pBCMAT plasmid series for CAT assays in bifidobacteria to evaluate promoters and terminators. Analyses using promoters from Bifidobacterium longum NCC2705 indicated that the CAT assay using these promoters is quantitative, has a wide measurement range, and is stable. In addition, this assay was useful for several bifidobacterial species, including B. longum, Bifidobacterium breve, and Bifidobacterium adolescentis. Compared with evoglow-Bs2, a fluorescent protein used under anaerobic conditions, the CAT assay showed about 0.25% background activity. In analyses using this CAT assay, we identified 11 promoters and 12 terminators of B. longum NCC2705. The genes encoding ribosomal proteins, elongation factors, and transfer RNAs possessed strong promoters, and terminators that include strong stem-loops and poly-U tails structures tended to show high activities. Although the abovementioned promoters made stronger contributions to expression activities than the terminators, the maximum fold difference in the activities among the tested terminators was approximately 17-fold. Modification of the -10 box and 5'-UTR in the promoters and the structure around the stem-loop in the terminators affected expression levels. These results suggest that the CAT assay is useful for various analyses of bifidobacterial gene expression.

Due to the high prevalence of vascular obstructive diseases, discovering potent, safe, and affordable fibrinolytic agents is of importance. There is particular interest concerning the use of functional foods that have a fibrinolytic activity, such as natto, a Japanese fermented soy-based product made with Bacillus subtilis (natto) strain BEST195. We recently isolated another bacterial strain from natto commercialized in Indonesia, B. subtilis G8, which has proven to exert fibrinolytic activity. Herein, a further characterization of B. subtilis G8 was assessed through a comparison with commercialized nattokinase, the major fibrinolytic enzyme of B. subtilis, by utilizing various in vitro fibrinolytic assays, namely whole blood clot lysis, euglobulin clot lysis, the fibrin plate method, and zymography. Both nattokinase and B. subtilis G8 were able to dissolve both whole blood and euglobulin clots. Furthermore, both nattokinase and B. subtilis G8 were able to lyse blood clots, presumably due to their ability to directly lyse fibrin. Finally, a crude extract of B. subtilis G8 displayed six zymogram bands of approximately 42.0, 35.5, 30.8, 26.7, 20.0, and 13.7 kDa, with the strongest activity observed at 20.0 kDa. This indicates that B. subtilis G8 contained several fibrinolytic enzymes, which might have comprised nattokinase and other fibrinolytic enzymes. In summary, we demonstrated that a crude extract of B. subtilis G8 has potent fibrinolytic activity and that the activity was mediated by various fibrinolytic enzymes.

The fecal microbiota and short-chain fatty acids (SCFAs) play important roles in the human body. This study examined how hyperbaric conditions affect the fecal microbiota and fecal SCFAs. Fecal samples were obtained from 12 divers at three points during deep-diving training (before the diving training, at 2.1 MPa, and after decompression). At 2.1 MPa, the changes in the frequency of Clostridium cluster IV and fecal iso-valerate levels were positively correlated, and the changes in the frequencies of Bacteroides and Clostridium subcluster XIVa were inversely correlated. After decompression, positive correlations were detected between the changes in the frequency of Bifidobacterium and fecal n-valerate levels and between the changes in the fecal levels of iso-butyrate and iso-valerate. On the other hand, inverse correlations were detected between the changes in the frequency of Clostridium cluster IX and fecal iso-butyrate levels, between the changes in the frequency of Clostridium cluster IX and fecal iso-valerate levels, and between the changes in the frequencies of Bacteroides and Clostridium cluster IV plus subcluster XIVa. During the study period, the changes in fecal iso-butyrate and iso-valerate levels were positively correlated, and inverse correlations were seen between the changes in the frequency of Clostridium cluster IV and fecal propionate levels and between the changes in the frequencies of Prevotella and Clostridium subcluster XIVa. These findings suggest that hyperbaric conditions affect the fecal microbiota and fecal SCFA levels and that intestinal conditions reversibly deteriorate under hyperbaric conditions.

Adherence of probiotics to dietary fibers present in the intestinal tract may affect adhesion to intestinal epithelial cells. The properties of the adhesion of bifidobacteria to mucin or epithelial cells have been well studied; however, adhesion of bifidobacteria to dietary fiber has not been investigated. The adhesion ratio of six Bifidobacterium strains to cellulose and chitin was examined; among the strains, Bifidobacterium animalis subsp. lactis JCM 10602 showed high adherence to both cellulose and chitin, and two strains showed high adherence to only chitin. The ratios of adhesion of B. animalis to cellulose and chitin were positively and negatively correlated with ionic strength, respectively. These data suggest that hydrophobic and electrostatic interactions are involved in the adhesion to cellulose and chitin, respectively. The adhesion ratios of the cells in the late logarithmic phase to cellulose and chitin decreased by approximately 40% and 70% of the cells in the early logarithmic phase, respectively. Furthermore, the adhesion ratio to cellulose decreased with increasing bile concentration regardless of the culture phase of the cells. On the other hand, the adhesion ratio to chitin of cells in the early logarithmic phase decreased with increasing bile concentration; however, that of cells in the late logarithmic phase increased slightly, suggesting that adhesins differ depending on the culture phase. Our results indicated the importance of considering adhesion to both dietary fibers and the intestinal mucosa when using bifidobacteria as probiotics.

Chronic constipation is a functional disorder that decreases a patient's quality of life (QOL). Because dysbiosis has been associated with constipation, we aimed to investigate the efficacy of Bifidobacterium bifidum G9-1 (BBG9-1) in improving QOL in patients with constipation. This was a prospective, single-center, non-blinded, single-arm feasibility trial. A total of 31 patients with constipation and decreased QOL received BBG9-1 treatment for 8 weeks, followed by a 2-week washout period. The primary endpoint was change in the overall Japanese version of the patient assessment of constipation of QOL (JPAC-QOL) score after probiotic administration relative to that at baseline. Secondary endpoints included changes in gut microbiota, stool consistency, frequency of bowel movement, degree of straining, sensation of incomplete evacuation, and frequency of rescue drug use. The overall JPAC-QOL scores and frequency of bowel movement significantly improved after BBG9-1 administration from those at baseline (p<0.01 and p<0.01, respectively). There were no statistically significant changes in other clinical symptoms. Subset analysis revealed that patients with initial Bristol Stool Form Scale stool types of <4 had improvements in stool consistency, a significant increase in the frequency of bowel movements, and a significant alleviation in the degree of straining, following BBG9-1 administration. At the genus and species levels, Sarcina and Sarcina maxima were significantly increased. Functional analysis showed that butanoate metabolism increased significantly, whereas methane metabolism decreased significantly. We concluded that BBG9-1 is safe and improves QOL in patients with constipation. The underlying improvements may be due to changes in stool consistency.

Sarcopenia causes functional disorders and decreases the quality of life. Thus, it has attracted substantial attention in the aging modern world. Dysbiosis of the intestinal microbiota is associated with sarcopenia; however, it remains unclear whether prebiotics change the microbiota composition and result in the subsequent recovery of muscle atrophy in elderly patients with sarcopenia. This study aimed to assess the effects of prebiotics in super-elderly patients with sarcopenia. We analyzed the effects of 1-kestose on the changes in the intestinal microbiota and body composition using a next-generation sequencer and a multi-frequency bioimpedance analysis device. The Bifidobacterium longum population was significantly increased in the intestine after 1-kestose administration. In addition, in all six patients after 12 weeks of 1-kestose administration, the skeletal muscle mass index was greater, and the body fat percentage was lower. This is the first study to show that administration of a prebiotic increased the population of B. longum in the intestinal microbiota and caused recovery of muscle atrophy in super-elderly patients with sarcopenia.