High poly-γ-glutamic acid-containing natto improves lipid metabolism and alters intestinal microbiota in mice fed a high-fat diet.

Several beneficial effects of poly-γ-glutamic acid (γ-PGA) have been reported. To test whether natto, a fermented soy food rich in γ-PGA, can improve intestinal microbiota content and lipid metabolism in a high-fat diet, we compared the intestinal microbiota content, plasma, liver, and fecal contents, and changes in gene expression in the livers and large intestines of a group of mice fed a high-fat diet supplemented with cooked soybeans (SC group) and a group fed a high-fat diet supplemented with natto (NA group) for 42 days; high-fat diet-fed mice were used as a control (Con group). Hepatic lipid levels were significantly lower, the fecal bile acid and lipid levels were significantly greater, and the Bacteroidetes/Firmicutes ratio was significantly higher in the SC and NA groups as compared to Con group. Additionally, plasma glucose and triglyceride levels, the expression of liver fatty acid synthase, and the relative abundance of Lactobacillaceae was significantly higher in the NA group than in the Con group. Although both natto and cooked soybeans impacted the metabolic response to a high-fat diet, the addition of natto had a greater effect on glucose and lipid metabolism. γ-PGA may play an important role in natto functionality.

Effects of a high-γ-polyglutamic acid-containing natto diet on liver lipids and cecal microbiota of adult female mice.

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.

Plasma quercetin metabolites are affected by intestinal microbiota of human microbiota-associated mice fed with a quercetin-containing diet.

Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice has been reported. Recent research has revealed that several intestinal bacteria metabolize quercetin. We hypothesize that the difference in composition of intestinal microbiota affects quercetin absorption from the intestine. Germ-free BALB/cA female mice (18 weeks old) were randomly divided into four groups and orally administered with fecal suspension from four human individuals (HF1, HF2, HF3, HF4) to produce the human microbiota-associated mice. All mice were fed the 0.05% quercetin-containing pelleted diet for four weeks. Significant differences were observed in plasma total cholesterol and cecal microbiota among the four groups. Plasma quercetin concentration was significantly higher in the HF3 group than in the HF1 group. The plasma isorhamnetin/quercetin ratio showed significant negative correlation with visceral fat levels (r = -0.544, p = 0.013). Positive correlation was observed between the Log10 Enterobacteriaceae count and the plasma quercetin metabolites. Principal component analysis showed that all groups were distributed in different regions by using the correlation diagram with the second and third principal component. This study indicates that intestinal microbiota of human microbiota-associated mice inoculated with different fecal suspensions react to dietary quercetin in different ways.

Quercetin metabolism by fecal microbiota from healthy elderly human subjects.

Quercetin is a polyphenol found in food that has numerous health benefits. This study investigated the relationship between quercetin metabolism, gut microbiota composition, and dietary intake in elderly Japanese subjects. A food frequency questionnaire was used to assess dietary intake during the week prior to stool sample collection. Fecal suspensions from 56 subjects were anaerobically incubated with quercetin and fecal microbiota composition was analyzed by next-generation sequencing. Inter-individual variations in quercetin concentration and fecal microbiota composition at family level suggested differences in microbial quercetin metabolism. The abundance of Sutterellaceae (r = -0.292) and Oscillospiraceae (r = -0.334) was negatively correlated whereas that of Fusobacteriaceae (r = 0.361) and Enterobacteriaceae (r = 0.321) was positively correlated with quercetin concentration. Niacin (r = -0.313), vitamin B6 (r = -0.297), vitamin B12 (r = -0.266), vitamin D (r = -0.301), and ratio of animal protein to total protein (r = -0.27) were also negatively correlated with quercetin concentration. Bacterial abundance was positively or negatively related to intake of food components. This is the first report describing the relationship between fecal quercetin metabolism, human microbiota, and dietary intake in the elderly.

Effects of an equol-producing bacterium isolated from human faeces on isoflavone and lignan metabolism in mice.

BACKGROUND: Equol is a metabolite of daidzein that is produced by intestinal microbiota. The oestrogenic activity of equol is stronger than daidzein. Equol-producing bacteria are believed to play an important role in the gut. The rod-shaped and Gram-positive anaerobic equol-producing intestinal bacterium Slackia TM-30 was isolated from healthy human faeces and its effects on urinary phyto-oestrogen, plasma and faecal lipids were assessed in adult mice. RESULTS: The urinary amounts of equol in urine were significantly higher in mice receiving the equol-producing bacterium TM-30 (BAC) group than in the control (CO) group (P < 0.05). However, no significant differences were observed between the urinary amounts of daidzein, dihydrodaidzein, enterodiol, and enterolactone between the BAC and CO groups. No significant differences in the plasma lipids were observed between the two groups. The lipid content (% dry weight) in the faeces sampled on the final day of the experiment tended to be higher in the BAC group than in the CO group (P = 0.07). CONCLUSION: Administration of equol-producing bacterium TM-30 affected the urinary amounts of phyto-oestrogens and the faecal lipid contents of mice. The equol-producing bacterium TM-30 likely influences the metabolism of phyto-oestrogen via changes in the gastrointestinal environment. © 2015 Society of Chemical Industry.

Relationships among fecal daidzein metabolites, dietary habit and BMI in healthy volunteers: a preliminary study.

To investigate the relationships among fecal isoflavone metabolism, dietary habit and Body Mass Index (BMI), 15 healthy men and 15 healthy women were recruited and provided stool samples for analysis of ex vivo anaerobic incubation of fecal suspension with daidzein. A negative correlation was observed between BMI and the dihydrodaidzein (DHD) production in men, and between BMI and the equol production in women. There was a positive correlation between intake of soluble dietary fiber and the DHD production in men. The results suggest that dietary habits and BMI are related to the metabolic activity of isoflavonoids by fecal intestinal microbiota.

Xylitol affects the intestinal microbiota and metabolism of daidzein in adult male mice.

This study examined the effects of xylitol on mouse intestinal microbiota and urinary isoflavonoids. Xylitol is classified as a sugar alcohol and used as a food additive. The intestinal microbiota seems to play an important role in isoflavone metabolism. Xylitol feeding appears to affect the gut microbiota. We hypothesized that dietary xylitol changes intestinal microbiota and, therefore, the metabolism of isoflavonoids in mice. Male mice were randomly divided into two groups: those fed a 0.05% daidzein with 5% xylitol diet (XD group) and those fed a 0.05% daidzein-containing control diet (CD group) for 28 days. Plasma total cholesterol concentrations were significantly lower in the XD group than in the CD group (p < 0.05). Urinary amounts of equol were significantly higher in the XD group than in the CD group (p < 0.05). The fecal lipid contents (% dry weight) were significantly greater in the XD group than in the CD group (p < 0.01). The cecal microbiota differed between the two dietary groups. The occupation ratios of Bacteroides were significantly greater in the CD than in the XD group (p < 0.05). This study suggests that xylitol has the potential to affect the metabolism of daidzein by altering the metabolic activity of the intestinal microbiota and/or gut environment. Given that equol affects bone health, dietary xylitol plus isoflavonoids may exert a favorable effect on bone health.

Effects of rice bran oil on the intestinal microbiota and metabolism of isoflavones in adult mice.

This study examined the effects of rice bran oil (RBO) on mouse intestinal microbiota and urinary isoflavonoids. Dietary RBO affects intestinal cholesterol absorption. Intestinal microbiota seem to play an important role in isoflavone metabolism. We hypothesized that dietary RBO changes the metabolism of isoflavonoids and intestinal microbiota in mice. Male mice were randomly divided into two groups: those fed a 0.05% daidzein with 10% RBO diet (RO group) and those fed a 0.05% daidzein with 10% lard control diet (LO group) for 30 days. Urinary amounts of daidzein and dihydrodaidzein were significantly lower in the RO group than in the LO group. The ratio of equol/daidzein was significantly higher in the RO group (p < 0.01) than in the LO group. The amount of fecal bile acids was significantly greater in the RO group than in the LO group. The composition of cecal microbiota differed between the RO and LO groups. The occupation ratios of Lactobacillales were significantly higher in the RO group (p < 0.05). Significant positive correlation (r = 0.591) was observed between the occupation ratios of Lactobacillales and fecal bile acid content of two dietary groups. This study suggests that dietary rice bran oil has the potential to affect the metabolism of daidzein by altering the metabolic activity of intestinal microbiota.

Effect of Dietary l-arabinose on the Intestinal Microbiota and Metabolism of Dietary Daidzein in Adult Mice.

This study examined the effects of L-arabinose on mouse intestinal microbiota and urinary isoflavonoids. Male mice were randomly divided into two groups: those fed a 0.05% daidzein-2.5% L-arabinose diet (AR group) and those fed a 0.05% daidzein control diet (CO group) for 28 days. The amounts of daidzein detected in urine were significantly lower in the AR group than in the CO group. The ratio of equol/daidzein was significantly higher in the AR group (p<0.01) than in the CO group. The composition of caecal flora differed between the AR and CO groups. The occupation ratios of Prevotella and Lactobacillales were significantly lower in the AR group. This study suggests that dietary L-arabinose has the potential to affect the metabolism of equol from daidzein by altering the metabolic activity of intestinal microbiota.

Effects of new dietary fiber from Japanese Apricot (Prunus mume Sieb. et Zucc.) on gut function and intestinal microflora in adult mice.

Much attention has been focused recently on functional foods. Ume, the Japanese name for the apricot of Prunus mume Sieb. et Zucc., is an example of a Japanese traditional functional food. There are, however, few reports on the effects of fiber from this fruit on bowel function. With this objective, we prepared ume fiber to test the hypothesis that it can change gut function and intestinal flora in mice. Mice were fed an ume fiber (UF) or cellulose (CF) diet (control) for 40 days. The fecal weight, fecal lipids, plasma lipids and cecal composition of the microflora were analyzed. The amount of feces was significantly greater in the UF group than in the CF group (p < 0.01). The fecal lipids content (% DW) of the feces sampled on the final day of the experiment were significantly greater in the UF group than in the CF group (p < 0.01). Plasma non-esterified fatty acids (NEFA) concentrations tended to be lower in the UF compared to the CF group (p = 0.058). Occupation ratios of Bacteroides and Clostridium cluster IV were significantly greater in the cecal flora of the UF group. Our results suggest that ume fiber possesses the fecal lipid excretion effects and feces bulking effects.

Lactobacillus rhamnosus JCM 2771: impact on metabolism of isoflavonoids in the fecal flora from a male equol producer.

Many beneficial effects of probiotics have been reported; however, few have focussed on the effects of Lactobacillus, a probiotic, on the bioconversion of isoflavonoids. We hypothesized that Lactobacillus rhamnosus will modify the metabolism of isoflavone. In an in vitro incubation, L. rhamnosus JCM 2771 produced daidzein from daidzin along with genistein. However, daidzin and genistein were not detected in the incubation solution of daidzein with L. rhamnosus. In the fecal suspension from a male equol producer with daidzein, equol was detected in the presence of a low or high concentration of L. rhamnosus. In the fecal incubation with daidzin, the equol concentration increased with an increasing concentration of L. rhamnosus JCM 2771. L. rhamnosus affected the equol production in the in vitro incubation of daidzein with fecal flora from a male equol producer. We demonstrated for the first time that L. rhamnosus JCM 2771 could produce genistein from daidzin and affect the equol production of fecal flora from a male equol producer in vitro.

Dihydrodaidzein-producing Clostridium-like intestinal bacterium, strain TM-40, affects in vitro metabolism of daidzein by fecal microbiota of human male equol producer and non-producers.

Much attention has been focused on the biological effects of equol, a metabolite of daidzein produced by intestinal microbiota. However, little is known about the role of isoflavone metabolizing bacteria in the intestinal microbiota. Recently, we isolated a dihydrodaidzein (DHD)-producing Clostridium-like bacterium, strain TM-40, from human feces. We investigated the effects of strain TM-40 on in vitro daidzein metabolism by human fecal microbiota from a male equol producer and two male equol non-producers. In the fecal suspension from the male equol non-producer and DHD producer, DHD was detected in the in vitro fecal incubation of daidzein after addition of TM-40. The DHD concentration increased as the concentration of strain TM-40 increased. In the fecal suspension from the equol producer, the fecal equol production was increased by the addition of strain TM-40. The occupation ratios of Bifidobacterium and Lactobacillales were higher in the equol non-producers than in the equol producer. Adding isoflavone-metabolizing bacteria to the fecal microbiota should facilitate the estimation of the metabolism of isoflavonoids by fecal microbiota. Studies on the interactions among equol-producing microbiota and DHD-producing bacteria might lead to clarification of some of the mechanisms regulating the production of equol by fecal microbiota.

Dietary cholesterol lowers plasma and cecal equol concentrations in mice.

This study examined the effects of cholesterol on mouse intestinal microflora and on isoflavonoids in the cecum and plasma. Dietary cholesterol affects bile acid metabolism and bile acids can influence the intestinal microorganisms. Intestinal microflora appear to play an important role in isoflavone metabolism. We hypothesized that dietary cholesterol changes the metabolism of isoflavonoids and intestinal microorganisms in mice. Male mice were randomly divided into two groups, which were fed a cholesterol-daidzein (CDA) or daidzein (DA) diet (control diet) for 60d. Plasma equol and cecal equol concentrations were significantly higher in the DA group (control group) than in the CDA group. However, plasma cholesterol concentrations were significantly higher in the CDA group compared to the DA group. The composition of cecal microorganisms differed between the two dietary groups. The occupation ratios of Clostridium cluster XI, Clostridium subcluster XIVa, and Lactobacillales were significantly higher in the CDA group. The occupation ratio of Bifidobacterium was significantly lower in the CDA group. This study suggests that dietary cholesterol has the potential to affect the metabolism of equol from daidzein by altering the metabolic activity of the intestinal microorganisms and gut physiological function.

Lactobacillus collinoides JCM1123(T): effects on mouse plasma cholesterol and isoflavonoids in the caecum.

The effects of Lactobacillus collinoides JCM1123(T) on plasma cholesterol levels, isoflavonoids in the caecum and faecal flora were assessed in adult mice. L. collinoides JCM1123(T) altered the equol production status in in vitro incubation of daidzein with faecal flora of mice. In in vivo investigation, mice were fed an AIN-93M purified diet for 13 days, and then randomly divided into two groups of seven animals each. All mice were fed an AIN-93M diet for 6 days; then the diet was replaced with a 0.05% daidzein diet, the mice received a 0.05% daidzein diet for 4 days. Two groups of mice were administered either L. collinoides JCM1123(T) (the experimental group) or a physiological saline solution (the control group) daily for 10 days and dissection was performed on the following day. The total plasma cholesterol concentration was significantly higher in the control group than in the experimental group. The amount of daidzein present in the caecum was significantly higher in the control group than in the experimental group. Significantly higher numbers of lactobacilli were observed in the experimental group than in the control group. Our data suggest that the administration of L. collinoides is likely to influence the metabolism of isoflavonoids and endogenous cholesterol via changes in the gastrointestinal environment.

Vitamin K deficiency of germfree mice caused by feeding standard purified diet sterilized by gamma-irradiation.

Germfree mice died when they were fed a purified diet of AIN-76 formula sterilized by gamma-irradiation. Vitamin K deficiency was suspected and this study was performed to confirm the cause of the death. Germfree mice were fed purified diets of AIN-76 or AIN-93M formula, which were pelleted and sterilized by gamma-irradiation at a dose of 50 kGy. One half of the mice fed the AIN-76 diet died within two weeks and the surviving animals were also in poor health, while 91% of mice fed the AIN-93M diet survived. No hemorrhage was observed grossly in any organs of the surviving animals. Histologically, degeneration with inflammatory cell infiltration was observed as well as hemorrhage and fibrosis in the heart muscles of mice fed the AIN-76 diet. No microscopic lesions were observed in the other organs. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were extremely prolonged when mice were fed the AIN-76 diet. The animals totally recovered when they were intragastrically administered 1 microg/day of vitamin K(3) from the third day of feeding of the AIN-76 diet, except for PT and APTT which were still slightly longer than in mice fed the AIN-93M diet. The concentration of vitamin K(3) supplied in the AIN-76 diet decreased to an undetectable level after gamma-irradiation, while the AIN-93M diet contained 240 microg/kg of vitamin K(1). These results indicate that the deaths of the germfree mice fed the gamma-irradiated AIN-76 diet were caused by vitamin K deficiency. Vitamin K deficiency may cause fatal degeneration of cardiac muscle cells.

Isolation of an isoflavone-metabolizing, Clostridium-like bacterium, strain TM-40, from human faeces.

Recently, the biological effects of isoflavones have attracted much attention. Intestinal microbiota plays an important role in the metabolism and bioavailability of isoflavones. However, few reports have discussed intestinal bacteria that metabolize daidzein into dihydrodaidzein. In this study, we isolated the dihydrodaidzein-producing intestinal bacterium TM-40 from a healthy boy's faeces. The bacteria from faecal samples were incubated with daidzein. Among all tested bacteria, one strain (strain TM-40) produced dihydrodaidzein both from daidzein and daidzin. However, in our experimental conditions, strain TM-40 did not produce equol from daidzein. The 16S rRNA partial sequence of strain TM-40 (AB249652) exhibited a 93% similarity to that of Coprobacillus catenaformis (AB030218). This strain seems to be a new species.

Lactobacillus gasseri: effects on mouse intestinal flora enzyme activity and isoflavonoids in the caecum and plasma.

The effects of Lactobacillus gasseri JCM 1131(T) on isoflavonoid levels within the caecum and plasma were assessed in adult mice. Male 5-week-old mice were fed an AIN 93M diet for 30 d. Two groups of mice were administered either L. gasseri JCM 1131(T) (the LGI group) or physiological saline solution (the control (CI) group) daily for 5 d before dissection. The plasma daidzein concentration was significantly higher in the LGI group, however, their plasma equol concentration was significantly less than in the CI group. The total amount of equol present as aglycone in the caecum was significantly greater in the CI group, but there was no significant difference in the total daidzein present as caecal aglycone. In an in vitro incubation of daidzein with the faecal flora of mice, the equol concentration was significantly higher in the CI group. The numbers of lactobacilli present were significantly higher in the LGI group. The present data suggest that the administration of L. gasseri is likely to influence the effect of isoflavonoids on the host via changes in the gastrointestinal environment.

Effects of soy oligosaccharides on plasma and cecal isoflavones, and cecal enzyme activities in mice.

This paper studies the effects of soy oligosaccharides on plasma and cecal isoflavones and floral enzyme activities in adult mice. The isoflavones used in this experiment were daidzein and genistein. Male 6-wk-old mice were fed a soy oligosaccharide-isoflavone (SOI) diet or cellulose-isoflavone (CEI) diet for 10 d. Plasma and cecal isoflavones were analyzed by HPLC, and cecal bacterial beta-glucosidase and beta-glucuronidase activities were also measured. The plasma genistein concentration was significantly higher in the SOI diet group than in the CEI diet group, but no significant difference was observed in plasma daidzein concentration between the two dietary groups. The total amount of cecal isoflavones existing as aglycone were significantly greater in the SOI diet group than in the CEI diet group, and both cecal beta-glucosidase and beta-glucuronidase activities were significantly higher in the SOI diet group than in the CEI diet group. These results indicate that soy oligosaccharides have an impact on the metabolic activity of intestinal microflora, plasma concentrations of isoflavones, and amount of isoflavones in the intestine. The present study suggests that ingestion of soy oligosaccharides may influence the effect of dietary isoflavones on the host.

Effects of rice starch-isoflavone diet or potato starch-isoflavone diet on plasma isoflavone, plasma lipids, cecal enzyme activity, and composition of fecal microflora in adult mice.

The effects of rice starch-isoflavone diet or potato starch-isoflavone diet on plasma concentration of isoflavones, plasma lipids, cecal enzyme activity, and intestinal microflora were studied. Male 15-wk-old mice were fed a rice-starch-based or potato-starch-based diet supplemented with isoflavones for 4 wk, and plasma samples, cecal contents, and feces were collected individually. Plasma equol concentration was significantly higher in the potato-isoflavone diet group than in the rice-isoflavone diet group, but no significant difference was observed in plasma daidzein or genistein concentrations. Plasma total cholesterol concentration was higher in the potato-isoflavone diet group, but no significant difference was observed in plasma triglyceride concentration. Both cecal beta-glucuronidase and beta-glucosidase activities were significantly higher in the rice-isoflavone diet group. The number of bifidobacteria was significantly higher in the potato-isoflavone diet group. These results indicate that different types of starches have different influences on plasma isoflavone and suggest that the influences might be through the change of host physiology and/or the metabolism and composition of intestinal microflora.