Gut bacterial metabolism of the soy isoflavone daidzein: exploring the relevance to human health

The indigenous intestinal microflora are involved in a variety of processes within the human body, and are important for maintaining host health. As such, interindividual differences in the ability to harbor certain intestinal bacteria might be associated with interindividual differences in health and/or disease susceptibility. In the last decade there has been considerable interest in phytoestrogen intakes in relation to human health. Daidzein, an isoflavone phytoestrogen found in soy, is metabolized to equol and O-desmethylangolensin (O-DMA) by intestinal bacteria. The specific bacterium/bacteria responsible for equol and O-DMA production in humans have yet to be identified definitively, but in vitro and animal studies have suggested that equol and O-DMA are more biologically active than their precursor daidzein. Interestingly, substantial interindividual differences in daidzein metabolism exist; following soy or daidzein consumption, approximately 30%-50% of the human population produce equol, and approximately 80%-90% produce O-DMA. Observational and intervention studies in humans have suggested that the ability to produce equol and O-DMA may be associated with reduced risk of certain diseases including breast and prostate cancers. However, relatively few studies have been conducted to date. In this review, we discuss the available evidence for a relationship between daidzeinmetabolizing phenotypes and human health, and suggest potential mechanisms for some of the reported relationships.     

Serum steroid hormones, sex hormone-binding globulin concentrations, and urinary hydroxylated estrogen metabolites in post-menopausal women in relation to daidzein-metabolizing phenotypes

Equol and O-desmethylangolensin (O-DMA) are products of bacterial metabolism of daidzein, an isoflavone in soybeans; thus, the presence or absence of equol and/or O-DMA in urine is a marker of particular intestinal bacteria profiles. Plasma hormone concentrations may be lower in pre-menopausal women who harbor the bacteria capable of producing equol (equol producers) compared to women who do not (equol non-producers). We evaluated concentrations of serum hormones, sex hormone-binding globulin (SHBG), and urinary 2-hydroxyestrone (2-OH E₁) and 16-hydroxyestrone (16-OH E₁) in relation to equol-producer and O-DMA-producer phenotypes in 89 post-menopausal women. Follicle stimulating hormone (FSH) was 23% greater in O-DMA-producers compared to non-producers (P=0.04). No significant differences in serum estrogens, androgens, metabolic hormones, or SHBG were observed in relation to either daidzein-metabolizing phenotype. Compared with non-producers within each phenotype, age-adjusted 2-OH E₁:16-OH E₁ was 27% greater (P=0.06) in equol-producers and 9% greater (P>0.10) in O-DMA-producers, and 2-OH E₁ concentrations were 24% greater in equol producers (P=0.07) and 42% greater in O-DMA producers (P=0.02). No significant differences in 16-OH E₁ were observed in relation to either phenotype. These results suggest that interindividual variability in intestinal bacteria may be related to differences in products of hormone metabolism in post-menopausal women.     

Metabolism of isoflavones, lignans and prenylflavonoids by intestinal bacteria: producer phenotyping and relation with intestinal community

Many studies have investigated the importance of the intestinal bacterial activation of individual phytoestrogens. However, human nutrition contains different phytoestrogens and the final exposure depends on the microbial potential to activate all different groups within each individual. In this work, interindividual variations in the bacterial activation of the different phytoestrogens were assessed. Incubation of feces from 100 individuals using SoyLife EXTRA, LinumLife EXTRA and isoxanthohumol suggested that individuals could be separated into high, moderate and low O-desmethylangolensin (O-DMA), equol, enterodiol (END), enterolactone (ENL) or 8-prenylnaringenin producers, but that the metabolism of isoflavones, lignans and prenylflavonoids follows separate, independent pathways. However, O-DMA and equol production correlated negatively, whereas a positive correlation was found between END and ENL production. In addition, END production correlated negatively with Clostridium coccoides-Eubacterium rectale counts. Furthermore, O-DMA production was correlated with the abundance of methanogens, whereas equol production correlated with sulfate-reducing bacteria, indicating that the metabolic fate of daidzein may be related to intestinal H(2) metabolism.     

The role of diet in the metabolism of daidzein by human faecal microbiota sampled from Italian volunteers

The intestinal microbial transformation of daidzein into equol is subject to a wide inter-individual variability. The aim of this study was to investigate in vitro this transformation and to evaluate possible correlations between individual diet and equol production. The transformation of daidzein was investigated in anaerobic batch cultures inoculated with mixed fecal bacteria from 90 volunteers. The daidzein metabolism was monitored by liquid chromatography-mass spectrometry, and a chiral column was used to distinguish equol and dihydrodaidzein enantiomers. The obtained results show that daidzein was unchanged (≈27%) or degraded to equol (≈28%), O-desmethylangolensin (≈12%) or dihydrodaidzein (≈31%). Furthermore, some subjects (≈2%) are able to produce both equol and O-desmethylangolensin. Bacteria represent sub-dominant populations (10⁵–10⁹ cell/g wet faeces) in “slow” equol producers, while higher counts of equol-producing microorganisms (10¹⁰–10¹¹ cell/g wet faeces) were found in “quick” equol producers. The in vitro test to evaluate equol-producing status is quick and not invasive, and the obtained results are comparable with those reported in vivo. Indeed, the only enantiomer present in the batch cultures containing equol was the S-form. No significant correlations between equol production, BMI, age and sex were found. It seems that the equol-producer group consumed less fibre, vegetables and cereals, and more lipids from animal sources.     

172名上海地区成人饮食及尿雌马酚和代谢产物的调查研究

目的确定上海地区成人雌马酚代谢表型及雌马酚的生理范围;把握由于大豆异黄酮负荷而产生的雌马酚产生者比例;调查雌马酚表型和食物摄取频率及有关激素间的关系。方法应用现状调查方法,筛选出172名居住在上海市区健康成年男女。填写问卷获得研究对象日常饮食频率,检测研究对象血清获得血液激素浓度,采用HPLC法分析负荷大豆异黄酮前后尿中雌马酚等大豆异黄酮24 h排泄量,统计产雌马酚者比例及其与摄食频率和激素的关系。结果负荷前雌马酚生理范围0~33.74μmol/24 h,产雌马酚者比例为30.2%,负荷大豆异黄酮后比例提高至53.5%。产雌马酚者与非产雌马酚者之间日常食品摄取频率的差别无统计学意义(P>0.05)。产Eq者血中游离雌二醇的浓度较非产Eq者低(P<0.05)。结论在通常膳食条件下,约有1/3上海成人尿液中能检测到雌马酚,但负荷大豆异黄酮后,约有1/2能产生雌马酚。     

Dietary isoflavones differentially induce gene expression changes in lymphocytes from postmenopausal women who form equol as compared with those who do not

Human and animal studies suggest that dietary soy isoflavones reduce cancer risk, ameliorate postmenopausal syndrome and decrease bone resorption in postmenopausal women. The capacity to form the metabolite equol from daidzein is suggested as an important modulator of response to isoflavones; this capacity depends on gut colonization with appropriate bacteria. We administered a dietary supplement containing high-dose purified soy isoflavones (genistein, 558 mg/day; daidzein, 296 mg/day; and glycitein, 44 mg/day) to 30 postmenopausal women for 84 days and collected peripheral lymphocytes at timed intervals. Using microarray analysis, we determined whether changes in gene expression associated with this treatment support existing hypotheses as to isoflavones' mechanisms of action. Expression of a large number of genes was altered by isoflavone treatment, including induction of genes associated with cyclic adenosine 3',5'-monophosphate (cAMP) signaling and cell differentiation and decreased expression of genes associated with cyclin-dependent kinase activity and cell division. We report that isoflavone treatment in subjects who have the capacity to produce equol differentially affects gene expression as compared with nonproducers, supporting the plausibility of the importance of equol production. In general, isoflavones had a stronger effect on some putative estrogen-responsive genes in equol producers than in nonproducers. Our study suggests that, in humans, isoflavone changes are related to increased cell differentiation, increased cAMP signaling and G-protein-coupled protein metabolism and increased steroid hormone receptor activity and have some estrogen agonist effects; equol-production status is likely to be an important modulator of responses to isoflavones.     

Equol,an Isoflavone Metabolite,Regulates Cancer Cell Viability and Protein Synthesis Initiation via c-Myc and eIF4G

Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via up-regulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER−) metastatic breast cancer cells, equol induced elevated levels of eIF4G, which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNAs translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of c-Myc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant down-regulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer-promoting effects of equol via up-regulation of eIF4GI and selective initiation of translation on mRNAs that utilize non-canonical initiation, including certain oncogenes.     

Urinary equol excretion in relation to 2-hydroxyestrone and 16alpha-hydroxyestrone concentrations: an observational study of young to middle-aged women

Approximately one-third to one-half of individuals harbor the colonic bacteria that are capable of metabolizing the soy isoflavone daidzein to equol. Results of prior studies suggest beneficial effects of producing equol in relation to breast cancer risk, potentially through effects on endogenous hormones. High urinary excretion of 2-hydroxyestrone (2-OH E(1)) relative to 16alpha-hydroxyestrone (16alpha-OH E(1)) has been associated with a reduced risk of breast cancer. In this pilot study we examined associations between urinary excretion of equol and 2-OH E(1), 16alpha-OH E(1), and their ratio, and investigated whether excretion of these estrogen metabolites differed between two samples collected 48h apart. Isoflavones (genistein, daidzein, O-desmethylangolensin (ODMA), and equol) were measured in two overnight urines from 126 women. Excretion of 2-OH E(1) and 16alpha-OH E(1) were measured in the first overnight urine from all 126 women and in the second overnight urine from 30 of these women; there were no significant differences between samples collected 48h apart in excretion of 2-OH E(1) or 16alpha-OH E(1) (P=0.75 and 0.17, respectively). Among all women, correlations between total isoflavone excretion (sum of genistein, daidzein, ODMA, and equol) and estrogen metabolites were non-significant (P>0.05). Among women with detectable levels of equol, total isoflavone excretion was significantly positively correlated with 16alpha-OH E(1) (r=0.32, P=0.02), but was not correlated with 2-OH E(1) or 2-OH E(1):16alpha-OH E(1) ratio (r=0.21, P=0.14, and r=-0.05, P=0.70, respectively). Equol excretion (adjusted for other isoflavone excretion) was significantly positively correlated with 2-OH E(1):16alpha-OH E(1) ratio (r=0.38, P=0.005), but was not correlated with 2-OH E(1) or 16alpha-OH E(1) (r=0.15, P=0.29, and r=-0.17, P=0.24, respectively). The finding that equol excretion, but not total isoflavone excretion, correlated positively with the 2-OH E(1):16alpha-OH E(1) ratio suggests that the colonic bacterial profile associated with equol production may be involved in estrogen metabolism, and may therefore possibly influence breast cancer risk.     

Epidemiological profiles between equol producers and nonproducers: a genomewide association study of the equol-producing phenotype

Equol is a daidzein (a phytoestrogen isoflavone) metabolite of gut bacteria, and the ability to produce equol varies between individuals and reduces the risks of several diseases. We tested the effects of equol production on health in Koreans and identified the genetic factors that determine the equol-producing phenotype. In 1391 subjects, the equol-producing phenotype was determined, based on measurements of serum equol concentrations. The anthropometric and blood biochemical measurements between equol producers and nonproducers were analyzed by LC-MS/MS. Genetic factors were identified in a genomewide association study (GWAS), and the interaction between genetic factors and the equol-producing phenotype was examined. We observed that 70.1 % of the study population produced equol. Blood pressure was significantly lower in equol producers (beta ± SE = −1.35 ± 0.67, p = 0.045). In our genomewide association study, we identified 5 single-nucleotide polymorphisms (p < 1 × 10⁻⁵) in HACE1. The most significant SNP was rs6927608, and individuals with a minor allele of rs6927608 did not produce equol (odds ratio = 0.57 (95 % CI 0.45–0.72), p value = 2.5 × 10⁻⁶). Notably, the interaction between equol production and the rs6927608 HACE1 SNP was significantly associated with systolic blood pressure (p value = 1.3 × 10⁴). Equol production is linked to blood pressure, and HACE1, identified in our (GWAS), might be a determinant of the equol-producing phenotype.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0292-8) contains supplementary material, which is available to authorized users.     

Fecal Bacterial Community Changes Associated with Isoflavone Metabolites in Postmenopausal Women after Soy Bar Consumption

Soy isoflavones and their metabolism by intestinal microbiota have gained attention because of potential health benefits, such as the alleviation of estrogen/hormone-related conditions in postmenopausal women, associated with some of these compounds. However, overall changes in gut bacterial community structure and composition in response to addition of soy isoflavones to diets and their association with excreted isoflavone metabolites in postmenopausal women has not been studied. The aim of this study was to determine fecal bacterial community changes in 17 postmenopausal women after a week of diet supplementation with soy bars containing isoflavones, and to determine correlations between microbial community changes and excreted isoflavone metabolites. Using DGGE profiles of PCR amplified 16S rRNA genes (V3 region) to compare microbial communities in fecal samples collected one week before and one week during soy supplementation revealed significant differences (ANOSIM p<0.03) before and after soy supplementation in all subjects. However, between subjects comparisons showed high inter-individual variation that resulted in clustering of profiles by subjects. Urinary excretion of isoflavone (daidzein) metabolites indicated four subjects were equol producers and all subjects produced O-desmethylangolensin (ODMA). Comparison of relative proportions of 16S rRNA genes from 454 pyrosequencing of the last fecal samples of each treatment session revealed significant increases in average proportions of Bifidobacterium after soy consumption, and Bifidobacterium and Eubacterium were significantly greater in equol vs non-S-(-)equol producers. This is the first in vivo study using pyrosequencing to characterize significant differences in fecal community structure and composition in postmenopausal women after a week of soy diet-supplementation, and relate these changes to differences in soy isoflavones and isoflavone metabolites.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00244907","term_id":"NCT00244907"}}NCT00244907     

Evaluation of inter‐individual differences in gut bacterial isoflavone bioactivation in humans by PCR‐based targeting of genes involved in equol formation

Aim

To identify human subjects harbouring intestinal bacteria that bioactivate daidzein to equol using a targeted PCR‐based approach.

Methods and Results

In a pilot study including 17 human subjects, equol formation was determined in faecal slurries. In parallel, faecal DNA was amplified by PCR using degenerate primers that target highly conserved regions of dihydrodaidzein reductase and tetrahydrodaidzein reductase genes. PCR products of the expected size were observed for six of the eight subjects identified as equol producers. Analysis of clone libraries revealed the amplification of sequences exclusively related to Adlercreutzia equolifaciens in four of the subjects tested positive for equol formation, whereas in three of the equol producers, only sequences related to Slackia isoflavoniconvertens were observed. No amplicons were obtained for one equol‐forming subject, thus suggesting the presence of nontargeted alternative genes. Amplicons were only sporadically observed in the nonequol producers.

Conclusion

The majority of human subjects who produced equol were also detected with the developed PCR‐based approach.

Significance and Impact of the Study

The obtained results shed light on the distribution and the diversity of known equol‐forming bacterial species in the study group and indicate the presence of as yet unknown equol‐forming bacteria.     

Microbial equol production attenuates colonic methanogenesis and sulphidogenesis in vitro

Hydrogen gas produced during colonic fermentation is excreted in breath and flatus, or removed by hydrogen-consuming bacteria such as methanogens and sulphate-reducing bacteria. However, recent research has shown that H₂ is also consumed by equol-producing bacteria during the reduction of daidzein into equol. In this study, the interactions between methanogens, sulphate-reducing, and equol-producing bacteria were investigated under in vitro simulated intestinal conditions. In the presence of daidzein, the equol-producing bacterial consortium EPC4 gave rise to equol production in cultures of Methanobrevibacter smithii or Desulfovibrio sp. as well as in faecal samples with methanogenic or sulphate-reducing abilities. Moreover, this supplementation significantly (P < 0.001) decreased the methanogenesis and sulphidogenesis. The attenuation did not occur in the absence of a daidzein source. Additionally, there was no influence of soy germ powder, daidzein or equol as such, excluding a possible inhibition by these compounds. Finally, a stronger decrease was observed with increasing amounts of EPC4 and a constant equol production, suggesting that the observed effect was only partly caused by the action of daidzein as a hydrogen sink. These findings are of relevance since abdominal discomfort such as bloating and flatulence, are related to colonic gas production, whereas equol has potential health benefits.     

Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta

Equol is a metabolite produced in vivo from the soy phytoestrogen daidzein by the action of gut microflora. It is known to be estrogenic, so human exposure to equol could have significant biological effects. Equol is a chiral molecule that can exist as the enantiomers R-equol and S-equol. To study the biological activity of racemic (+/-)-equol, as well as that of its pure enantiomers, we developed an efficient and convenient method to prepare (+/-)-equol from available isoflavanoid precursors. Furthermore, we optimized a method to separate the enantiomers of equol by chiral HPLC, and we studied for the first time, the activities of the enantiomers on the two estrogen receptors, ERalpha and ERbeta. In binding assays, S-equol has a high binding affinity, preferential for ERbeta (K(i)[ERbeta]=16 nM; beta/alpha=13 fold), that is comparable to that of genistein (K(i)[ERbeta]=6.7 nM; beta/alpha=16), whereas R-equol binds more weakly and with a preference for ERalpha (K(i)[ERalpha]=50 nM; beta/alpha=0.29). All equol isomers have higher affinity for both ERs than does the biosynthetic precursor daidzein. The availability and the in vitro characterization of the equol enantiomers should enable their biological effects to be studied in detail.     

Segregation analysis of phenotypic components of learning disabilities. I. Nonword memory and digit span

Dyslexia is a common and complex disorder with evidence for a genetic component. Multiple loci (i.e., quantitative-trait loci [QTLs]) are likely to be involved, but the number is unknown. Diagnosis is complicated by the lack of a standard protocol, and many diagnostic measures have been proposed as understanding of the component processes has evolved. One or more genes may, in turn, influence these measures. To date, little work has been done to evaluate the mode of inheritance of individual component-as opposed to composite-phenotypes, beyond family or twin correlation studies that initially demonstrate evidence for a genetic basis of such components. Here we use two approaches to segregation analysis in 102 nuclear families to estimate genetic models for component phenotypes associated with dyslexia: digit span and a nonword-repetition task. Both measures are related to phonological skills, one of the key component processes in dyslexia. We use oligogenic-trait segregation analysis to estimate the number of QTLs contributing to each phenotype, and we use complex segregation analysis to identify the most parsimonious inheritance models. We provide evidence in support of both a major-gene mode of inheritance for the nonword-repetition task, with approximately 2.4 contributing QTLs, and for a genetic basis of digit span, with approximately 1.9 contributing QTLs. Results obtained by reciprocal adjustment of measures suggest that genes contributing to digit span may contribute to the nonword-repetition score but that there are additional QTLs involved in nonword repetition. Our study adds to existing studies of the genetic basis of composite phenotypes related to dyslexia, by providing evidence for major-gene modes of inheritance of these single-measure component phenotypes.     

Patterns of recent natural selection on genetic loci associated with sexually differentiated human body size and shape phenotypes

Levels of sex differences for human body size and shape phenotypes are hypothesized to have adaptively reduced following the agricultural transition as part of an evolutionary response to relatively more equal divisions of labor and new technology adoption. In this study, we tested this hypothesis by studying genetic variants associated with five sexually differentiated human phenotypes: height, body mass, hip circumference, body fat percentage, and waist circumference. We first analyzed genome-wide association (GWAS) results for UK Biobank individuals (~194,000 females and ~167,000 males) to identify a total of 114,199 single nucleotide polymorphisms (SNPs) significantly associated with at least one of the studied phenotypes in females, males, or both sexes (P<5x10^-8). From these loci we then identified 3,016 SNPs (2.6%) with significant differences in the strength of association between the female- and male-specific GWAS results at a low false-discovery rate (FDR<0.001). Genes with known roles in sexual differentiation are significantly enriched for co-localization with one or more of these SNPs versus SNPs associated with the phenotypes generally but not with sex differences (2.73-fold enrichment; permutation test; P = 0.0041). We also confirmed that the identified variants are disproportionately associated with greater phenotype effect sizes in the sex with the stronger association value. We then used the singleton density score statistic, which quantifies recent (within the last ~3,000 years; post-agriculture adoption in Britain) changes in the frequencies of alleles underlying polygenic traits, to identify a signature of recent positive selection on alleles associated with greater body fat percentage in females (permutation test; P = 0.0038; FDR = 0.0380), directionally opposite to that predicted by the sex differences reduction hypothesis. Otherwise, we found no evidence of positive selection for sex difference-associated alleles for any other trait. Overall, our results challenge the longstanding hypothesis that sex differences adaptively decreased following subsistence transitions from hunting and gathering to agriculture.     

雌马酚产生细菌及其雌马酚合成代谢机制

大豆食品中通常富含染料木素和大豆苷元等异黄酮素,人和动物肠道中的某些细菌具有将异黄酮素代谢转化为S-雌马酚的能力。到目前为止,S-雌马酚被认为是一种具有潜在健康调节作用的化合物。啮齿类动物均具备产雌马酚的能力,但不同人群之间存在差异,产雌马酚细菌是否存在可能是造成这种差异的重要原因;不同产雌马酚细菌的代谢机制可能不同,并影响机体最终产雌马酚的能力。本文对已知的各种产雌马酚细菌及其细菌的雌马酚合成机制进行综述,以期为进一步了解雌马酚产生个体差异、雌马酚代谢转化效率、体外雌马酚的发酵生产,以及临床产雌马酚细菌的应用等提供理论参考。     

Growth performance, carcass characteristics and bioavailability of isoflavones in pigs fed soy bean based diets

A growth trial with 38 weaners (castrated male pigs) was designed to compare the growth performance and carcass quality of pigs fed diets containing either soy bean meal or soy protein concentrate in a pair-feeding design. Soy bean meal (SBM) and soy protein concentrate (SPC) differed in isoflavone (daidzein plus genistein) content (782 microg/g in SBM and 125 microg/g in SPC, respectively). During the experiment, all pigs were fed four-phases-diets characterized by decreasing protein concentrations with increasing age (weaner I, weaner II, grower, finisher diets). Rations of control and experimental groups were isoenergetic, isonitrogenous, and isoaminogen. The weanling pigs with an initial live weight of 8.4 +/- 1.1 kg were allotted to flat deck boxes. During the growing/finishing period (days 70-170 of age), the pigs were housed in single boxes. Both, the weaning and the grower/finishing performances (daily body weight gain, feed intake, feed conversion ratio) were similar in both groups. No differences were found between the groups in carcass composition (percentages of cuts, tissues, and protein/fat), and meat quality of pigs. Moreover, the IGF-1R mRNA expression in longissimus muscle was not influenced by the kind of soy product. However, circulating levels of isoflavones were clearly different between pigs fed SBM (genistein 239 +/- 44; daidzein 162 +/- 42; equol 12 +/- 4 ng/ml plasma) and animals fed SPC (genistein 22 +/- 9 and daidzein 8 +/- 3, and equol 10 +/- 3 ng/ml plasma). The results confirm the expected differences in the bioavailability of soy isoflavones, yet, there were no significant differences in performance of pigs fed either soy bean meal or soy protein concentrate.     

Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase,an Enzyme Involved in the Metabolism of Daidzein,from Equol-Producing Lactococcus Strain 20-92

Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.Isoflavones are flavonoids present in various plants and are known to be abundant in soybeans and legumes. These compounds have been called phytoestrogens because their chemical structure is similar to that of the female sex hormone, estrogen. Isoflavones have an ability to bind to estrogen receptors and show protection against or improvement in several sex hormone-dependent diseases, such as breast cancer, prostate cancer, menopausal disorder, lower bone density, and hypertension, due to their weak agonistic or antagonistic effects (1, 19, 27).Daidzein is one of the main soy isoflavonoids produced from daidzin by the glucosidase of intestinal bacteria (17). Equol is a metabolite produced from daidzein by the enterobacterial microflora (5). Recently, equol has attracted a great deal of attention because its estrogenic activity is more potent than that of other isoflavonoids, including daidzein (27). It is well known that individual variation exists in the ability of these enteric microflora to produce equol and that less than half the human population is capable of producing equol after ingesting soy isoflavones (3). Therefore, to increase the production of equol in the enteric environment of each individual, the development of probiotics using safe bacteria which have the ability to produce equol from daidzein is ongoing.Lactococcus strain 20-92 (Lactococcus 20-92; 30a) is an equol-producing lactic acid bacterium isolated from the feces of healthy humans by Uchiyama et al. (30). This bacterium is spherical and Gram positive and is a strain of L. garvieae. The application of Lactococcus 20-92 in probiotics is advantageous because L. garvieae is not pathogenic or toxic to humans.To date, other bacterial strains that are capable of transforming daidzein to dihydrodaidzein or equol have been isolated (9, 21, 22, 23, 29, 32, 36, 37). Daidzein is thought to be metabolized by human intestinal bacteria to equol or to O-desmethylangolensin via dihydrodaidzein and tetrahydrodaidzein (14, 15, 22, 32); however, neither the enzymes involved in the metabolism of daidzein to equol nor even the metabolic pathway has been clarified fully for equol-producing bacteria.In this study, we purified an enzyme from Lactococcus 20-92 that assisted in the conversion of daidzein to dihydrodaidzein. Furthermore, we cloned the L-DZNR gene and expressed the active recombinant enzyme in E. coli.