大豆素代谢产物——雌马酚研究进展

雌马酚是大豆异黄酮的主要组份之一大豆素（Dai）的代谢产物,人群中约有30％～50％能将大豆素转化为雌马酚,影响因素目前尚不完全清楚,最重要的因素是肠道菌群。雌马酚较其原型具有更为有效的生物学作用,因此受到普遍关注。研究和开发雌马酚的生物活性,在多种常见慢性病的预防与控制方面具有重要的理论和实际意义。     

肠道微生物代谢产物—S-雌马酚与 人类健康关系研究进展

大豆异黄酮是一种年销量很大、市场普及率较高的保健品。具有抗氧化、抗肿瘤等功能,对延缓女性衰老和改善更年期综合征有很好效果。但研究表明,大豆异黄酮的保健功能主要归功于其肠道代谢产物S-雌马酚。S-雌马酚是豆类食品在肠道内经特定微生物代谢后产生的一种高度稳定小分子,其与雌激素的结构和功能高度相似,能与β-雌激素(ER-β)受体结合。具有防治更年期综合征、心血管疾病和多种雌素依赖性癌症的功能。到目前为止,雌马酚的功能及其与人类健康之间的关系还有待进一步研究。本文结合国内外最新研究进展,着重介绍雌马酚的生理功能及其与各类疾病的研究进展,展望雌马酚在各类疾病防治中可能发挥的重要作用。     

微生物转化影响不同内源雌激素水平大鼠粪便雌马酚含量和菌群结构

雌马酚(Equol)是肠道中特定细菌转化大豆异黄酮的产物,与其前体大豆苷元(Daidzein)相比,雌马酚具有更强的生物学活性。【目的】研究口服雌马酚产生菌对大鼠转化大豆苷元能力的可能促进作用及内源雌激素对大鼠肠道菌群的可能影响。【方法】使用平均体重为211±9g的卵巢摘除和假手术雌性大鼠各30只,分别随机分为5组,并灌胃蒸馏水、雌二醇、大豆苷元、雌马酚和大豆苷元+雌马酚产生菌ZX7。【结果】从灌胃第2天开始,接受大豆苷元后大鼠粪样中始终具有较高水平的雌马酚,显著高于对照和雌二醇组(P<0.01);灌胃大豆苷元+雌马酚产生菌ZX7的大鼠和直接灌胃雌马酚的大鼠在粪样雌马酚含量上十分接近;DGGE图谱的PCA分析显示,卵巢摘除大鼠和假手术大鼠粪便菌群存在明显差异;大鼠粪便拟杆菌门细菌数量与粪样中雌马酚水平显著正相关。【结论】大鼠肠道固有菌群中可能存在能够将大豆苷元转化为雌马酚的细菌,利用外源菌株改变大鼠雌马酚产生能力具有一定的可行性,不同的内源雌激素水平可能影响大鼠肠道菌群结构,拟杆菌门细菌可能在大豆苷元的生物转化过程中起着十分重要的作用。     

雌马酚的生物活性和临床应用

雌马酚是大豆异黄酮(SI)的主要组分之一——大豆素(Dai)的代谢产物。雌马酚较其原型具有更为有效的生物学作用,虽其作用及机制还存在争议,但很多研究表明雌马酚的生物学作用以及在适用人群显然都优于SI,并受到普遍关注。研究和开发雌马酚的生物活性,在多种常见慢性病的预防与控制中有重要的理论和实际意义。     

产S-雌马酚梭菌C1三代全基因组测序及功能基因筛选鉴定

【目的】挖掘产S-雌马酚梭菌C1转化大豆苷元产生S-雌马酚的功能基因,为梭菌C1的S-雌马酚转化机制研究提供参考,并为利用合成生物学方法生产S-雌马酚提供新基因资源。【方法】利用GridION测序平台,对梭菌C1进行第三代全基因组测序、基因组组装和功能注释等分析,从C1菌全基因组中筛选和鉴定参与S-雌马酚生物转化的功能基因。【结果】C1全基因组大小为3 035 113 bp,预测编码3 166个基因,包含53个tRNA、15个rRNA、4个ncRNA和1个基因岛。通过生物信息学分析,发现C1-07020基因编码蛋白与已报道的Lactococcus sp.20-92大豆苷元还原酶具有44.8%的氨基酸序列相似性和相同的3个功能保守结构域,体外蛋白功能验证表明,C1-07020具有大豆苷元还原酶功能。此外,C1菌中没有发现与已知产S-雌马酚菌相似的功能基因簇或大豆苷元还原酶以外的其他功能基因。【结论】在C1中鉴定到一个新的产S-雌马酚功能基因,并发现了C1可能具备特殊的产S-雌马酚机制,实验所获基础数据可为进一步挖掘产S-雌马酚新功能基因、了解S-雌马酚的生成机制及体外产S-雌马酚基因资源...     

重组酿酒酵母催化二氢大豆苷元生产雌马酚

雌马酚是大豆异黄酮的代谢产物,是一种天然的选择性雌激素受体调节剂,稳定性和生物学活性高。为实现雌马酚的微生物合成,采用模块途径工程策略,构建编码雌马酚合成关键酶基因 orf-1、orf-2和orf-3 的表达载体,成功用于转化酿酒酵母BY4741,得到工程菌株。结果表明,工程菌株有效表达了外源基因,并可将大豆异黄酮代谢中间体二氢大豆苷元转化为雌马酚。为构建从头合成雌马酚的微生物细胞工厂提供了重要科学参考。     

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。     

肠道微生物代谢产物雌马酚的生成及其主要影响因素北大核心CSCD

雌马酚是大豆苷原的微生物代谢产物,被认为是大豆异黄酮发挥生理作用的关键,但仅有33%-50%的人群能够产生雌马酚。最新研究表明氢气在雌马酚生成过程中起着非常重要的作用。目前此类研究尚处于起步阶段,本文总结了以往的研究结果,对雌马酚的生成、生物学作用及影响雌马酚生成的因素、氢气与雌马酚之间的关系作了较为详细的介绍。     

一株转化大豆苷元为S-雌马酚菌株的筛选和鉴定

【目的】筛选一株可转化大豆苷元为S-雌马酚的微生物菌株,并对该菌株进行鉴定。【方法】在厌氧条件下采用抗生素抑制非目标菌生长并结合稀释涂平板法进行菌株分离,分离可转化大豆苷元生成S-雌马酚的肠道细菌,并对产物进行结构鉴定。之后通过16S rDNA序列分析,构建该菌系统进化树,结合菌体形态及菌落特征,确立该菌系统发育学地位。【结果】从大鼠肠道内筛选分离到一株可以将大豆苷元转化为S-雌马酚的革兰氏阴性兼性厌氧菌株LH-52(JN861767),16S rDNA序列测序结果 BLAST比对表明该菌株与奇异变形杆菌(Proteus mirabilis)相似度达到了99%,结合形态特征和生理生化实验结果鉴定该菌为奇异变形杆菌。根据HPLC保留时间、质谱、核磁共振等波谱数据分析确定产物为S-雌马酚。【结论】菌株P.mirabilis LH-52为首次筛选到的可转化大豆苷元为S-雌马酚的兼性厌氧菌,相对于文献报道的严格厌氧菌更适合于工业化生产。     

人粪样中雌马酚含量调查及其与菌群结构相关性研究

目的 调查人粪样中大豆素和雌马酚的含量及其与年龄和性别的关系;了解人粪样中雌马酚含量高低与菌群结构的关系.方法 采用高效液相色谱(HPLC)对来自杭州的125份粪样进行大豆素和雌马酚含量检测,并使用生物统计学软件SPSS进行统计学分析;使用PCR-DGGE对粪样中雌马酚含量的高低与菌群结构的关系进行初步研究.结果 HPLC检测结果表明,尽管粪样中雌马酚含量的高低与性别关系不大,但却与年龄大小存在很大的相关性,41 ～50岁年龄组的粪样中雌马酚含量明显高于其他年龄组.PCR-DGGE结果表明,粪样中雌马酚含量的高低与菌群结构无明显相关性.结论 人粪样中雌马酚含量的高低与年龄大小有很强的相关性.     

Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions

Only about one third of humans possess a microbiota capable of transforming the dietary isoflavone daidzein into equol. Little is known about the dietary and physiological factors determining this ecological feature. In this study, the in vitro metabolism of daidzein by faecal samples from four human individuals was investigated. One culture produced the metabolites dihydrodaidzein and O-desmethylangolensin, another produced dihydrodaidzein and equol. From the latter, a stable and transferable mixed culture transforming daidzein into equol was obtained. Molecular fingerprinting analysis (denaturing gradient gel electrophoresis) showed the presence of four bacterial species of which only the first three strains could be brought into pure culture. These strains were identified as Lactobacillus mucosae EPI2, Enterococcus faecium EPI1 and Finegoldia magna EPI3, and did not produce equol in pure culture. The fourth species was tentatively identified as Veillonella sp strain EP. It was found that hydrogen gas in particular, but also butyrate and propionate, which are all colonic fermentation products from poorly digestible carbohydrates, stimulated equol production by the mixed culture. However, when fructo-oligosaccharides were added, equol production was inhibited. Furthermore, the equol-producing capacity of the isolated culture was maintained upon its addition to a faecal culture originating from a non-equol-producing individual.     

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.     

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.     

Isolation and identification of equol-producing bacterial strains from cultures of pig faeces.

Transformation of daidzein to equol was compared during fermentation of three growth media inoculated with faeces from Erhualian piglets, but equol was produced from only one medium, M1. Two equol-producing strains (D1 and D2) were subsequently isolated using medium M1. Both strains were identified as Eubacterium sp., on the basis of morphological and physiological characteristics, and 16S rRNA gene sequence analysis showed that strains D1 and D2 were most closely related to previously characterized daidzein-metabolizing bacteria isolated from human faecal and rumen samples, respectively. This suggests that the ability to metabolize daidzein can be found among bacteria present within the mammalian intestine. The results provided the first account of conversion of daidzein directly to equol by bacterial species from farm animals. These strains may be of importance to the improvement of animal performance, and the use of medium M1 could provide a simple way to isolate bacterial strains capable of transforming daidzein into equol.     

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.     

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.     

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.     

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.     

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.