Enzymic Transformation of Isoflavone Phytoestrogens in Soymilk by β-Glucosidase-Producing Bifidobacteria

ABSTRACT: Five strains of bifidobacteria were screened for β-glucosidase activity using p-nitrophenyl-β-D-glucopyranoside as the substrate, and selected strains were used to ferment soymilk. Enumeration of viable bifidobacteria and quantification of isoflavones using HPLC were performed at 0, 12, 24, 36, and 48 h of incubation. Four strains produced β-glucosidase. B. pseudolongum and B. longum -a displayed the best growth in soymilk, with an increase of 1.3 log₁₀ CFU/mL after 12 h. B. animalis, B. longum -a, and B. pseudolongum caused hydrolysis of isoflavone malonyl-, acetyl- and β-glucosides to form aglycones, and transformed daidzein to equol in soymilk. Fermentation of soymilk with Bifidobacterium sp. resulted in a significant increase (p < 0.05) in the concentration of aglycones.     

Effects of clover-grass silages and concentrate supplementation on the content of phytoestrogens in dairy cow milk

A 2 × 2 factorial continuous experiment was conducted with 28 Norwegian Red dairy cows in early lactation to compare milk content of phytoestrogens when feeding ad libitum white clover (WCS) or red clover (RCS) grass silages prepared from the second and third cut without and with 10 kg/d supplementation of a standard concentrate. The cows were offered either RCS or WCS for 88 d (period 1) and thereafter a mixed red clover-white clover-grass silage for 48 d (period 2). Total dry matter intake and milk yield were not affected by forage type but increased with concentrate supplementation. Intake of isoflavones was several times greater in RCS than in WCS, whereas intake of lignans was greater in WCS. Concentrate supplementation reduced the intake of most phytoestrogens. Compared with WCS, RCS diets yielded milk with a greater content of flavonoids, whereas milk from WCS diets had greater contents of the mammalian lignans enterodiol and enterolactone. The content of the isoflavan equol was particularly high in RCS diets. There was no apparent carryover effect of clover type on milk phytoestrogen content because there was no difference in content between the silage treatments 3 wk after the cows were transferred to the same silage diet (period 2). Concentrate supplementation reduced the milk contents of the flavonoids equol, biochanin A, and daidzein and increased the content of mammalian lignans. The effects of silage type and concentrate supplementation on milk contents of the individual phytoestrogens were related to the intake of the compound or its precursor, except for the effect of concentrate on mammalian lignans, for which the intake of the known precursors was also reduced. Overall, this study shows that feeding cows with silage containing red clover increases the milk content of flavonoids at both low and high concentrate supplementation levels, and decreases the content of nonflavonoids such as mammalian lignans, when compared with silage containing white clover. The increased content of phytoestrogens in milk may be important when the health benefits of milk are studied.     

From Gut to Hormones: Unraveling the Role of Gut Microbiota in (Phyto)Estrogen Modulation in Health and Disease

The human gut microbiota regulates estrogen metabolism through the “estrobolome,” the collection of bacterial genes that encode enzymes like β-glucuronidases and β-glucosidases. These enzymes deconjugate and reactivate estrogen, influencing circulating levels. The estrobolome mediates the enterohepatic circulation and bioavailability of estrogen. Alterations in gut microbiota composition and estrobolome function have been associated with estrogen-related diseases like breast cancer, enometrial cancer, and polycystic ovarian syndrome (PCOS). This is likely due to dysregulated estrogen signaling partly contributed by the microbial impacts on estrogen metabolism. Dietary phytoestrogens also undergo bacterial metabolism into active metabolites like equol, which binds estrogen receptors and exhibits higher estrogenic potency than its precursor daidzein. However, the ability to produce equol varies across populations, depending on the presence of specific gut microbes. Characterizing the estrobolome and equol-producing genes across populations can provide microbiome-based biomarkers. Further research is needed to investigate specific components of the estrobolome, phytoestrogen-microbiota interactions, and mechanisms linking dysbiosis to estrogen-related pathology. However, current evidence suggests that the gut microbiota is an integral regulator of estrogen status with clinical relevance to women's health and hormonal disorders.     

Determination of in vitro isoflavone degradation in rumen fluid

The aim of this study was to determine the degradation of dietary isoflavones in rumen fluid under 2 feeding regimens. The experiments were performed in vitro using a rumen fluid buffer system. The rumen fluid was taken from cows fed either a hay diet or a concentrate-rich diet (the diet consisted of 34.6% maize silage, 17.6% haylage, 12.8% alfalfa hay, and 35.0% supplemental mixture on a dry matter basis). As a source of isoflavones, 40% soybean extract (Biomedica, Prague, Czech Republic) at levels of 5, 25, 50, and 75 mg per 40 mL of rumen fluid was used. Samples of soybean extract were incubated in triplicate at 39°C for 0, 3.0, 6.0, 12.0, and 24.0 h in incubation solution. The metabolism of daidzein and genistein was faster under concentrate-rich diet conditions. In general, production of equol started after 3 to 6 h of incubation and reached the highest rate after approximately 12 h of incubation regardless of the type of diet or concentration of extract. In most of the experiments, production of equol continued after 24 h of incubation. Generally, equol production was greater under the hay diet conditions. Furthermore, experiments with higher amounts of added soybean extract revealed possible inhibitory effects of high levels of isoflavones on the rumen microflora.     

高效液相色谱法测定六种大豆异黄酮及雌马酚的含量

针对豆制品中同时测定大豆异黄酮和雌马酚方法存在的问题,优化并建立了同时测定6种大豆异黄酮和雌马酚的高效液相检测程序,并测定了几种市售豆乳中大豆异黄酮的含量。采用乙腈/甲醇（v/v,5/2）和水为流动相、流速1.0mL/min、检测波长254nm,6种大豆异黄酮和雌马酚均得到良好的分离,相关系数R2分别为0.9998、0.9989、0.9997、0.9986、0.9998、0.9995、0.9999,线性关系良好。该方法准确、灵敏,适合于豆制品中大豆异黄酮及雌马酚的检测。     

测定雌马酚的紫外法建立与应用

分别以雌马酚标准品和食用大豆异黄酮人的尿液为样品,建立一种测定雌马酚的紫外分析方法。考察方法的特性及测定结果可靠性,其结果为:检出限5×10-10g/mL,定量限15×10-10g/mL。应用于人尿液雌马酚的测定,精密度4.45%,回收率100.25%,结果可靠。新建立的紫外法达到了定量分析的要求,同时具有操作简单、快速、测定成本低的优点,适合大批量样品的测定。     

The Oxidation of Equol by Tyrosinase Produces a Unique Di-ortho-Quinone: Possible Implications for Melanocyte Toxicity

Equol (7-hydroxy-3-(4′-hydroxyphenyl)-chroman, EQ), one of the major intestinally derived metabolites of daidzein, the principal isoflavane found in soybeans and most soy foods, has recently attracted increased interest as a health-beneficial compound for estrogen-dependent diseases. However, based on its structure with two p-substituted phenols, this study aimed to examine whether EQ is a substrate for tyrosinase and whether it produces o-quinone metabolites that are highly cytotoxic to melanocyte. First, the tyrosinase-catalyzed oxidation of EQ was performed, which yielded three EQ-quinones. They were identified after being reduced to their corresponding catechols with NaBH₄ or L-ascorbic acid. The binding of the EQ-quinones to N-acetyl-L-cysteine (NAC), glutathione (GSH), and bovine serum albumin via their cysteine residues was then examined. NAC and GSH afforded two mono-adducts and one di-adduct, which were identified by NMR and MS analysis. It was also found that EQ was oxidized to EQ-di-quinone in cells expressing human tyrosinase. Finally, it was confirmed that the EQ-oligomer, the EQ oxidation product, exerted potent pro-oxidant activity by oxidizing GSH to the oxidized GSSG and concomitantly producing H₂O₂. These results suggest that EQ-quinones could be cytotoxic to melanocytes due to their binding to cellular proteins.     

Evaluation of Equol Function on Anti- or Prooxidant Status in vivo

ABSTRACT:  The present study was performed to investigate the effects of equol on oxidative stress and the antioxidant defense system in the livers of mice. Mice were orally administered equol at either 5 or 25 mg/kg body weight/day for 1, 3, or 7 wk. Equol administration significantly inhibited biomarkers of oxidative stress (thiobarbituric acid-reactive substances value, carbonyl content, and serum 8-OH-dG) at all doses and for all durations of administration, and this phenomenon was most pronounced at 3 wk. Moreover, catalase and total superoxide dismutase (SOD) activities and their mRNA expression were significantly increased by equol. Although equol increased the glutathione peroxidase (GSH-px) activity in mice treated with equol for 1 wk, long-term administration of equol (7 wk) caused a decrease in the ratio of reduced/oxidized glutathione (GSH/GSSG) and the activities of GSH-px and glutathione reductase (GR). Taken together, these results suggest that equol may act as an antioxidant through an inhibition of oxidative stress and stimulation of catalase and SOD, but can also cause prooxidant effects such as reduction of the GSH/GSSG ratio, depending on the treatment period.