No abstract

An Escherichia coli library comprising 8,424 strains incorporating gene fragments of the equol-producing bacterium Slackia sp. strain NATTS was constructed and screened for E. coli strains having daidzein-and dihydrodaidzein (DHD)-metabolizing activity. We obtained 3 clones that functioned to convert daidzein to DHD and 2 clones that converted DHD to equol. We then sequenced the gene fragments inserted into plasmids contained by these 5 clones. All of the gene fragments were contiguous, encoding three open reading frames (ORF-1, -2, and -3). Analysis of E. coli strains containing an expression vector incorporating one of the orf-1, -2, or -3 genes revealed that (i) the protein encoded by orf-1 was involved in the conversion of cis/trans-tetrahydrodaidzein (cis/trans-THD) to equol, (ii) the protein encoded by orf-2 was involved in the conversion of DHD to cis/trans-THD, and (iii) the protein encoded by orf-3 was involved in the conversion of daidzein to DHD. ORF-1 had a primary amino acid structure similar to that of succinate dehydrogenase. ORF-2 was presumed to be an enzyme belonging to the short-chain dehydrogenase/reductase superfamily. ORF-3 was predicted to have 42% identity to the daidzein reductase of Lactococcus strain 20-92 and belonged to the NADH:flavin oxidoreductase family. These findings showed that the daidzein-to-equol conversion reaction in the Slackia sp. NATTS strain proceeds by the action of these three enzymes.S oybean isoflavones and their derivatives have been reported to prevent sex hormone-dependent diseases, such as prostate cancer, breast cancer, menopausal disorders, premenstrual syndrome, and osteoporosis (3,9,10,16,21,30). The isoflavone equol is expected to prevent hormone-dependent diseases, such as prostate cancer, because of its ability to bind to dihydrotestosterone and its high capacity to bind to estrogen receptor ␤; moreover, it is the most potent antioxidant of all the isoflavones (1, 2, 5, 17, 23).To date, several bacteria capable of producing equol have been isolated from human or animal feces (18-20, 29, 31). Many of these strains are suggested to first metabolize daidzein as a substrate to dihydrodaidzein (DHD) and to then metabolize DHD to equol. Recently, daidzein reductase, which converts daidzein to DHD, has been purified from the equol-producing Lactococcus strain 20-92 (25). On the other hand, it has been suggested that, in the Eggerthella strain Julong 732, DHD is converted to equol by the production of cis/trans-tetrahydrodaidzein (cis/trans-THD) as an intermediate metabolite (13,14). These studies have therefore suggested that daidzein is converted to equol via DHD and cis/ trans-THD. However, the details of the enzymes involved in the production of equol from daidzein, and of the genes encoding them, remain largely unknown.We have recently isolated Slackia sp. strain NATTS, which has potent daidzein-to-equol conversion ability, from healthy human feces (26). This strain has a more potent daidzein-equol conversion activity than the other equol-producing strains...

Several kinds of carbohydrates such as sorbose, adonitol, and melezitose were found to enhance equol production from daidzein in an in vitro human fecal culture. Sorbose, one of the most effective carbohydrates, was used as a carbohydrate source for isolating the NATTS strain, which was a Gram-positive, non-spore-forming rod bacterium with high ability to convert daidzein to equol isolated from the 7th maintenance culture. The strain was found to belong to the genus Slackia family Coriobacteriaceae by 16S rRNA sequence-based analysis, and the prevalence of the Slackia sp. in Japanese adults was examined by reverse transcription-quantitative PCR (RT-qPCR), which was found to be 40% at a mean population level of 10(6) cells per gram of feces.

Several clinical studies have shown that isoflavones and Lactobacillus casei Shirota (LcS) have beneficial effects on skin condition and the gut microbiota, respectively. Thus, we investigated the effects of consecutive intake of fermented soymilk (FSM) with LcS on skin condition and the gut microbiota, as well as isoflavone bioavailability, in a randomised, double-blind, placebo-controlled trial as a pilot study. Sixty healthy premenopausal Japanese women received FSM containing a moderate level of isoflavone aglycones and a probiotic LcS, or soymilk (SM) containing neither of them, twice a day for 8 weeks. Skin condition was assessed by a subjective questionnaire for face and morphological analysis of the stratum corneum on the inner forearm. Faecal microbiota and urinary isoflavone were analysed by 16S rRNA gene amplicon sequencing and high-performance liquid chromatography tandem mass spectrometry, respectively. Both the FSM and SM groups had improved skin condition as assessed from scores of overall satisfaction, dryness, moisture, elasticity, coarseness, pigmentation and/or stratum corneum morphology, as well as significantly increased levels of urinary isoflavones during the intake period compared with the pre-intake period, although there were no significant differences between the two groups. There was a significant positive correlation between urinary isoflavone levels and skin questionnaire scores. In contrast, the relative abundance levels of Lactobacillaceae significantly increased and those of Bifidobacteriaceae tended to increase during the intake period compared with the pre-intake period. For the after-intake period they only decreased significantly in the FSM group. The levels of Enterobacteriaceae and Porphyromonadaceae significantly decreased during the intake period in the FSM group. These findings suggest that daily intake of FSM, as well as SM, provides health benefits that improve skin condition via increased levels of isoflavone absorption in the body, and that only FSM beneficially modifies the gut microbiota in premenopausal healthy women.

Our study findings suggest that high serum levels of genistein, daidzein, and glycitein are significantly associated with a decreased risk of prostate cancer among Japanese men.