Urinary O-desmethylangolensin (ODMA) concentrations provide a functional gut microbiome marker of dietary isoflavone daidzein metabolism to ODMA. Individuals who do not have gut microbial environments that produce ODMA have less favourable cardiometabolic and cancer risk profiles. Urinary metabolomics profiles were evaluated in relation to ODMA metabotypes within and between individuals over time. Secondary analysis of data was conducted from the BEAN2 trial, which was a cross-over study of premenopausal women consuming 6 months on a high and a low soya diet, each separated by a 1-month washout period. In all of the 672 samples in the study, sixty-six of the eighty-four women had the same ODMA metabotype at seven or all eight time points. Two or four urine samples per woman were selected based on temporal metabotypes in order to compare within and across individuals. Metabolomics assays for primary metabolism and biogenic amines were conducted in sixty urine samples from twenty women. Partial least-squares discriminant analysis was used to compare metabolomics profiles. For the same ODMA metabotype across different time points, no profile differences were detected. For changes in metabotype within individuals and across individuals with different metabotypes, distinct metabolomes emerged. Influential metabolites (variables importance in projection score > 2) included several phenolic compounds, carnitine and derivatives, fatty acid and amino acid metabolites and some medications. Based on the distinct metabolomes of producers v. non-producers, the ODMA metabotype may be a marker of gut microbiome functionality broadly involved in nutrient and bioactive metabolism and should be evaluated for relevance to precision nutrition initiatives.

Flaxseed is a rich source of α-linolenic acid and phytoestrogens, mainly lignans, whose metabolites (enterodiol and enterolactone) can affect estrogen functions. The present study evaluated the influence of dietary flaxseed supplementation on reproductive performance and egg characteristics (fatty acids, cholesterol, lignans and isoflavones) of 40 Hy-Line hens (20/group) fed for 23 weeks a control diet or the same diet supplemented with 10% of extruded flaxseed. The flaxseed diet had approximately three times the content of lignans (2608.54 ng/g) as the control diet, mainly secoisolariciresinol diglucoside (1534.24 v. 494.72 ng/g). When compared with the control group, hens fed flaxseed showed a similar deposition rate (72.0% v. 73.9%) and egg yield. Furthermore, there was no effect of flaxseed on the main chemical composition of the egg and on its cholesterol content. Estradiol was higher in the plasma of the control group (1419.00 v. 1077.01 pg/ml) probably due to the effect of flaxseed on phytoestrogen metabolites. The plasma lignans were higher in hens fed flaxseed, whereas isoflavones were lower, mainly due to the lower equol value (50.52 v. 71.01 ng/ml). A similar trend was shown in eggs: the flaxseed group had higher level of enterodiol and enterolactone, whereas the equol was lower (198.31 v. 142.02 ng/g yolk). Secoisolariciresinol was the main lignan in eggs of the flaxseed group and its concentration was three times higher then control eggs. Flaxseed also improved the n-3 long-chain polyunsaturated fatty acids of eggs (3.25 v. 0.92 mg/g egg), mainly DHA, however, its oxidative status (thiobarbituric reactive substances) was negatively affected. In conclusion, 10% dietary flaxseed did not affect the productive performance of hens or the yolk cholesterol concentration, whereas the lignans and n-3 polyunsaturated fatty acid content of eggs improved. Further details on the competition between the different dietary phytoestrogens and their metabolites (estrogen, equol, enterodiol and enterolactone) should be investigated.

Recent studies suggest that the ability to produce equol, a metabolite of the soya isoflavone daidzein, is beneficial to coronary health. Equol, generated by bacterial action on isoflavones in the human gut, is biologically more potent than dietary sources of isoflavones. Not all humans are equol producers. We investigated whether equol-producing status is favourably associated with risk factors for CHD following an intervention by dietary soya isoflavones. We systematically reviewed randomised controlled trials (RCT) that evaluated the effect of soya isoflavones on risk factors for CHD and that reported equol-producing status. We searched PubMed, EMBASE, Ovid Medline and the Cochrane Central Register for Controlled Trials published up to April 2015 and hand-searched bibliographies to identify the RCT. Characteristics of participants and outcomes measurements were extracted and qualitatively analysed. From a total of 1671 studies, we identified forty-two articles that satisfied our search criteria. The effects of equol on risk factors for CHD were mainly based on secondary analyses in these studies, thus with inadequate statistical power. Although fourteen out of the forty-two studies found that equol production after a soya isoflavone intervention significantly improved a range of risk factors including cholesterol and other lipids, inflammation and blood pressure variables, these results need further verification by sufficiently powered studies. The other twenty-eight studies primarily reported null results. RCT of equol, which has recently become available as a dietary supplement, on CHD and its risk factors are awaited.

A wide range of health benefits have been ascribed to soya intake including a lowered risk of osteoporosis, heart disease, breast cancer, and menopausal symptoms. Because it is a hormonally active diet, however, soya can also be endocrine disrupting, suggesting that intake has the potential to cause adverse health effects in certain circumstances, particularly when exposure occurs during development. Consequently, the question of whether or not soya phyto-oestrogens are beneficial or harmful to human health is neither straightforward nor universally applicable to all groups. Possible benefits and risks depend on age, health status, and even the presence or absence of specific gut microflora. As global consumption increases, greater awareness and consideration of the endocrine-disrupting properties of soya by nutrition specialists and other health practitioners is needed. Consumption by infants and small children is of particular concern because their hormone-sensitive organs, including the brain and reproductive system, are still undergoing sexual differentiation and maturation. Thus, their susceptibility to the endocrine-disrupting activities of soya phyto-oestrogens may be especially high. As oestrogen receptor partial agonists with molecular and cellular properties similar to anthropogenic endocrine disruptors such as bisphenol A, the soya phyto-oestrogens provide an interesting model for how attitudes about what is ‘synthetic’ v. what is ‘natural,’ shapes understanding and perception of what it means for a compound to be endocrine disrupting and/or potentially harmful. This review describes the endocrine-disrupting properties of soya phyto-oestrogens with a focus on neuroendocrine development and behaviour.

The study aimed to determine the effects of red clover (Trifolium pratense) silage with high phyto-oestrogen content on ewe performance during their first breeding season. Red clover silage containing formononetin, biochanin A, genistein, and daidzein was fed to 10 nulliparous ewes of the prolific Finnish Landrace breed before, during and after the breeding season, for a total of 5 months. A control group of 10 ewes was fed with grass silage. The mean numbers of foetuses per pregnancy were 2.1±0.7 and 2.2±0.8 for the red clover and control groups, respectively. The total mass of the uterus with its contents was significantly greater in ewes of the red clover group compared with those of the control group. This difference was mainly explained by the greater volume of foetal fluids. Serum progesterone concentration in the red clover group was significantly lower over the entire period analysed than in the control group. In conclusion, the fecundity of the ewes was not reduced by red clover feed with high phyto-oestrogen concentrations. The volume of foetal fluids increased that could increase the risk for vaginal prolapse before the term.

Equol is a metabolite of the isoflavone daidzein (Dz) and is produced by the bacterial microflora in the distal intestine and colon. Some epidemiological studies have reported an association between increased equol production and intakes of green tea or dietary fibre, which are ingredients of the standard Japanese diet. We examined the effects of a diet supplemented with Dz and tea catechin or dietary fibre on equol production and bone mineral density in ovariectomised (OVX) mice. Female mice of the ddY strain were either sham operated or OVX. OVX mice were fed a control diet, a 0·1 % Dz-supplemented diet or a 0·1 % Dz diet supplemented with one of the food components commonly consumed in the Japanese diet. The mice were given 1 % tea catechin (w/w) as part of the diet in Expt 1 or 5 % polydextrose (PD) and 5 % raffinose (Raf) (w/w) as part of the diet in Expt 2. Catechin reduced serum equol levels and attenuated the beneficial effect of Dz on femoral bone loss. The soluble dietary fibres PD and Raf stimulated equol production, and enhanced the bone-protective effects of Dz on femoral bone. These results suggest that dietary fibre, in particular, PD, may alter the bioavailability of isoflavones and prevent osteopenia in OVX mice.

This study investigated the effects of in ovo administration of equol (Eq) on post-hatch growth and hepatic lipid metabolism in broiler chickens. Fertilized eggs (146 eggs/group) were injected with 0 μg (control, Con), 20 μg (low dose, L) and 100 μg (high dose, H) Eq in the albumen on the 7th day of incubation. Except a trend increase in the weight of total fat (P = 0.09), Eq had no effect on growth or liver weight in broilers at 49 days of age. Males presented higher liver and BWs and lower total fat and relative liver weights than females (P < 0.01). However, there were no significant effects of Eq or Eq–gender interactions on growth performance or tissues weight (P > 0.05). With respect to lipid parameters in the serum, the results showed that female broilers presented higher triacyglycerol (TG) and low-density lipoprotein cholesterol concentrations than males, whereas there was no gender difference in serum total cholesterol (TC) or high-density lipoprotein cholesterol (HDLC) concentration (P > 0.05). Eq administration significantly decreased serum TG and TC but increased HDLC concentrations in serum of broilers at 49 days of age (P < 0.05), whereas there were no interactions between gender and Eq (P > 0.05). To elucidate the mechanism behind the significant changes of serum TG and TC levels, the expression of genes involved in lipid metabolism in the liver was investigated in female chickens using reverse transcription-PCR. Carnitine palmitoyl transferase I (CPTI) messenger RNA (mRNA) was significantly upregulated by 20 and 100 μg Eq (P < 0.05). High-dose Eq significantly decreased fatty acid synthase (FAS) and enhanced cholesterol-7alpha-hydroxylase (CYP7A1) mRNA levels in the liver (P < 0.05). Eq had no significant effects on acetyl-CoA carboxylase, sterol regulatory element binding protein-1c, malic enzyme, low-density lipoprotein receptor or 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in the liver (P > 0.05). These results in female broilers suggest that Eq decreased blood TG by upregulating CPTI and downregulating FAS mRNA expression in the liver, and that high serum cholesterol levels stimulated CYP7A1 gene transcription in the liver.

Previous studies have suggested that the daidzein metabolite equol rather than daidzein itself contributes to the beneficial effect of soya foods in the prevention of CVD. The aim of the present study is to examine the proportion of equol excretion in Chinese adults and compare plasma lipids and carotid artery intima–media thickness (IMT) between equol excretors and non-excretors, and to evaluate the effect of soya isoflavone intakes on serum lipids and IMT in either equol excretors or non-excretors. Subjects (n 572; women n 362, men n 210) were recruited for the present study. An overnight urine sample was provided by each subject on their usual diet to quantify urinary concentrations of daidzein and equol. Far-wall IMT was determined by B-mode ultrasound in the right carotid at two sites, carotid bulb (CB-IMT) and common carotid artery (CCA-IMT), and fasting serum lipids were measured. Habitual dietary intakes were estimated with a FFQ, and soya isoflavone intake derived from the FFQ was assessed. Of the 572 subjects, the proportion of equol excretors on their usual diet was 25·0 % (n 143). Compared with non-excretors, equol excretors showed significantly lower serum TAG ( − 38·2 (95 % CI − 70·4, − 5·9) %, P = 0·012) and CCA-IMT ( − 4·9 (95 % CI − 9·7, − 0·3) %, P = 0·033). Equol excretors with higher daily isoflavone intakes ( − 5·4 mg/d) had significantly lower IMT ( − 16·2 %, P = 0·035) and tended to have higher HDL-cholesterol (P = 0·055) than did those with lower daily isoflavone intakes (1·5 mg/d), while no association was observed between soya isoflavone intakes and serum lipids or IMT in non-excretors. In conclusion, the benefits of soya isoflavones in preventing CVD may be apparent among equol excretors only.

S-Equol is a metabolite resulting from the conversion of daidzein, a soya phyto-oestrogen, by the gut microflora. The potential protective effects of equol in breast cancer are still under debate. Consequently, we investigated the effects of equol on DNA methylation of breast cancer susceptibility genes (BRCA1 and BRCA2) and oncosuppressors in breast cancer cell lines (MDA-MB-231 and MCF-7) and in a dystrophic breast cell line (MCF-10a) following exposure to S-equol (2 μm) for 3 weeks. We demonstrated by quantitative analysis of methylated alleles a significant decrease in the methylation of the cytosine phosphate guanine (CpG) islands in the promoters of BRCA1 and BRCA2 after the S-equol treatment in MCF-7 and MDA-MB-231 cells and a trend in MCF-10a cells. We also showed that S-equol increases BRCA1 and BRCA2 protein expression in the nuclei and the cytoplasm in MCF-7, MDA-MB-231 and MCF-10a cell lines by immunohistochemistry. The increase in BRCA1 and BRCA2 proteins was also found after Western blotting in the studied cell lines. In summary, we demonstrated the demethylating effect of S-equol on the CpG islands inside the promoters of BRCA1 and BRCA2 genes, resulting in an increase in the level of expressed oncosuppressors in breast cancer cell lines.

Equol (EQ) is a metabolite produced by gut bacteria through the chemical reduction of the soya isoflavone daidzein (DE), but only by 30–60 % of the population. EQ is believed to provide benefits derived from soya intake and its production is widely viewed as a relatively stable phenomenon. In a randomised, cross-over intervention with soya foods, seventy-nine pre-menopausal women were challenged with a high-soya and a low-soya diet each for 6 months, separated by a 1-month washout period. Overnight urine was collected at three time points during each diet period and analysed for DE and EQ by liquid chromatography tandem MS. Remaining an EQ producer (EP) or non-producer (NP) or changing towards an EP or NP was assessed using an EQ:DE ratio of ≥ 0·018 combined with a DE threshold of ≥ 2 nmol/mg creatinine as a cut-off point. We observed 19 and 24 % EP during the low-soya and high-soya diet periods, respectively, and found that 6–11 % of our subjects changed EQ status ‘within’ each study period (on an average of 1·2 times), while 16 % changed ‘between’ the two diet periods. The present finding challenges the widely held conviction that EQ production within an individual remains stable over time. The precise factors contributing to changes in EQ status, however, remain elusive and warrant further investigation.

In order to investigate the long-term effects of equol (Eq) on growth and meat quality in broilers, 0 μg (control, Con), 20 μg (low dose, L) and 100 μg (high dose, H) Eq, respectively, were injected into fertile eggs (146 eggs per group) on 7 days of embryos. After hatch, chickens were fed under the same conditions and slaughtered at 49 days of age for sample collection and analysis. The results showed that body weight and composition were marginally affected by Eq administration (P > 0.05). Compared with their male counterparts, the meat quality of female broilers was affected greatly after Eq administration. The redness (a*) of meat color in the L and H groups of female broilers was significantly decreased by 24.10% and 21.50% (P < 0.01), respectively; cooking loss decreased by 12.11% and 16.82%, respectively, in the L and H groups (P < 0.01); 24 h and 48 h drip loss was significantly decreased by 60.27% and 45.72% (P < 0.05), respectively, in the H group. However, for male broilers, only cooking loss was significantly decreased by high dosage of Eq treatment (P < 0.05). The antioxidative status was analyzed for discovering further the mechanism behind the improvement of the water-holding capacity caused by Eq in female broilers. The activity of glutathione peroxidase (GSHPx) in plasma was greatly increased by 15.94% in the L group (P < 0.01), whereas the total superoxide dismutase activity (T-SOD) and the content of malondialdehyde in plasma were not changed (P > 0.05). The T-SOD activity in the breast muscle of the L and H groups were significantly improved by 23.14% and 18.82% (P < 0.05), respectively. GSHPx in the breast muscle of the H group showed a tendency to increase (P = 0.06 < 0.1). These results indicate that Eq injection in ovo does not affect the growth of broilers, but significantly improves the water-holding capacity of the muscle, especially in female broilers, which is related to the improvement of antioxidative status.

Previous studies have suggested that daidzein's metabolites, equol and O-desmethylangolensin (O-DMA), rather than daidzein itself may contribute to the beneficial effects of soya foods in the prevention of CVD. The present study aims to assess the proportion of equol and O-DMA producers, and to compare differences in anthropometric factors, serum lipids, glucose and uric acid between producers and non-producers in Chinese adults aged 20–69 years. For the present cross-sectional study, 202 subjects (100 women and 102 men) were recruited. Twenty-four-hour urinary daidzein and its metabolites were determined in these subjects while on their usual diet and again after a 3-d isoflavone challenge. Fasting serum lipids, glucose and uric acid were examined on their usual diet. Three days of 24 h dietary recalls were used to assess dietary intakes. Of the 202 subjects, 27 (13·4 %) and 27 (13·4 %) excreted equol and O-DMA on their usual diet, and 101 (50 %) and 94 (46·5 %) produced equol and O-DMA after a load of 80 mg/d isoflavones. Equol producers showed lower serum uric acid ( − 10·2 %, P = 0·001), TAG ( − 29·5 %, P = 0·007) and waist:hip ratio ( − 2·6 %, P = 0·032), and tended to have higher HDL cholesterol (6·3 %, P = 0·069) compared with equol non-producers. There were no significant differences in serum lipids, glucose and uric acid between O-DMA producers and non-producers. In conclusion, equol phenotypes might influence cardiovascular risk.

The intake of isoflavones and the resulting equol contents of both plasma and milk of the same red clover-fed cows are reported for the first time in cyclic change-over design study. Cows were fed four different red clover silages and two timothy–meadow fescue silages as controls. The red clover silages contained daidzein, formononetin, biochanin A and genistein, whereas the timothy–meadow fescue silages contained no isoflavones. We found a strong association (y = 0·071x+2·75, R 2 0·71) between the formononetin intake (x) and equol concentration (y) in the plasma, while the formononetin intake and milk equol concentration were weakly associated (y = 0·0035x+0·358, R 2 0·20). This means that a small part of the total formononetin in the silage is secreted into milk as equol. The mean equol contents in plasma and milk of cows fed red clover silage diets were in the range of 4·6–8·4 mg/l and 458–643 μg/l, respectively, while the respective values for the control diets were in the range of 0·8–1·5 mg/l and 171–287 μg/l. We showed that shorter growing periods of red clover resulted in higher silage formononetin contents and plasma and milk equol contents, suggesting that the equol content of milk can be manipulated by varying the harvesting strategy of red clover. We conclude that milk equol is derived from the formononetin of red clover silage and that milk from red clover-fed cows can be considered as a source of equol in human nutrition.

Equol, a derivative of daidzein produced by enterobacteria, has greater activity as a phyto-oestrogen compared with daidzein. Difructose anhydride III (DFAIII) is a newly manufactured non-digestible disaccharide with unique fermentation properties. The present study evaluated the prebiotic effects of DFAIII on equol production and on plasma cholesterol concentrations related to the changes in equol production. We compared plasma equol concentrations at 10.00 and 18.00 hours and faecal isoflavone excretion in three groups of seven rats (male Wistar-ST strain, 6 weeks old) fed a basal diet or a DFAIII or fructooligosaccharide (15/g diet) diet containing 1g soya isoflavone/g diet for 20d. Equol concentrations in the DFAIII group were higher than in the control and fructooligosaccharides groups, especially in the later phase of the light period (18.00 hours) throughout the experiment. Daizein and genistein concentrations did not change between the diet groups. The faecal ratios of equol:daidzein were very high in all groups, but the ratios were higher in the DFAIII group than the control and fructooligosaccharide groups on day 3, and this tendency continued throughout the experiment. On day 20, the plasma total cholesterol concentration was lowest in the DFAIII group. Additionally, the cholesterol concentrations were inversely correlated to plasma equol concentration in all the rats. In conclusion, DFAIII efficiently enhanced plasma equol concentrations, which may be associated with an increase in equol production and a decrease in equol degradation by enterobacteria. Higher plasma equol concentrations may contribute to the hypocholesterolaemic effect of DFAIII feeding.

Epidemiological studies indicate that Asian women have a lower incidence of breast cancer compared with their counterparts in the West, and soya consumption has been suggested as a contributory factor. Clinical and animal studies have revealed that cyclooxygenase-2 (COX-2) expression is associated with a risk of breast cancer. In the present study, we investigated the effect of soya isoflavones on the expression of COX-2 in the breast cell line MCF-7. Genistein, daidzein and equol were found to inhibit COX-2 expression induced by phorbol 12-myristate 13-acetate (PMA). Similar findings were observed in the COX-2 protein analysis. In order to study transcriptional control, a fragment of the 5′-flanking region of the hCOX-2 gene was amplified and inserted into a firefly luciferase reporter plasmid. The reporter assay indicated that the transactivation of the hCOX-2 promoter was induced by PMA, and activity was inhibited with the co-administration of genistein, daidzein or equol. An activator protein-1 (AP-1)/cyclic AMP response element binding protein (CREB) binding site (−59/−53) was identified in hCOX-2 promoter, and this could be critical in PMA-induced COX-2 expression. Truncation reporter plasmids with (−70/−36) and without (−51/−36) AP-1/CREB were constructed for subsequent analysis. The results revealed that the hCOX-2 promoter transactivation suppressed by isoflavone could be dependent on AP-1/CREB binding. Nonetheless, this study illustrated that the soya isoflavones reduced COX-2 expression, which could be important in the post-initiation events of breast carcinogenesis.

Particular intestinal bacteria are capable of metabolizing the soya isoflavone daidzein to equol and/or O-desmethylangolensin (O-DMA), and the presence of these metabolites in urine after soya consumption are markers of particular intestinal bacteria profiles. Prevalences of equol producers and O-DMA producers are approximately 30–50 % and 80–90 %, respectively, and limited observations have suggested that these daidzein-metabolizing phenotypes are stable within individuals over time. Characterizing stability of these phenotypes is important to understand their potential as markers of long-term exposure to particular intestinal bacteria and their associations with disease risk. We evaluated concordance within an individual for the equol-producer and O-DMA-producer phenotypes measured at two time points (T1, T2), 1–3 years apart. Phenotypes were ascertained by analysing equol and O-DMA using GC-MS in a spot urine sample collected after 3 d soya (source of daidzein) supplementation. In ninety-two individuals without recent (within 3 months before phenotyping) or current antibiotics use, 41 % were equol producers at T1 and 45 % were equol producers at T2, and 90 % were O-DMA producers at T1 and 95 % were O-DMA producers at T2. The percentage agreement for the equol-producer phenotype was 82 and for the O-DMA-producer phenotype was 89. These results indicate that these phenotypes are stable in most individuals over time, suggesting that they provide a useful biomarker for evaluating disease risk associated with harbouring particular intestinal bacteria responsible for, or associated with, the metabolism of the soya isoflavone daidzein.

The effects of Lactobacillus gasseri JCM 1131T 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 1131T (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 daidzin 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.

The urinary excretion of soya isoflavones and gut microflora metabolites was investigated in infants and children who had been fed soya-based infant formulas in early infancy. These infants and children were compared with cows'-milk formula-fed controls, to determine at what age gut microflora metabolism of daidzein to equol and/or O-desmethylangolensin (O-DMA) was established, and whether exposure to isoflavones in early infancy influences their metabolism at a later stage of development. Sixty infants and children (aged 4 months–7 years) participated in the study; thirty in each of the soya and control groups. There were four age groups. These were: 4–6 months (seven in the soya group and seven in the control group); 7–12 months (seven in the soya group and nine in the control group); 1–3 years (six in the soya group and eight in the control group); 3–7 years (ten in the soya group and six in the control group). Urine samples were collected to measure isoflavonoids by MS, and faecal samples were collected to measure gut-health-related bacterial composition, by fluorescent in situ hybridisation with oligonucleotide probes, and metabolic activity. A soya challenge (typically a soya yoghurt alternative product containing 4·8g soya protein and on average 22mg total isoflavones) was given to control-group infants (>6 months) and children, and also to soya-group children that were no longer consuming soya, to determine their ability to produce equol and/or O-DMA. Urinary genistein, daidzein and glycitein were detected in all infants (4–6 months) fed soya-based infant formula; O-DMA was detected in 75% of infants but equol was detected in only 25%. In the controls (4–6 months), urinary isoflavonoids were very low or not detected. In the older age groups (7 months–7 years), O-DMA was found in the urine samples of 75% of the soya group and 50% of the controls, after the soya challenge. Equol excretion was detected in 19% of the soya-group infants and children, and in only 5% of the controls. However, in the oldest (3–7 years) children, the proportion excreting O-DMA and equol was similar in both groups. Faecal bacterial numbers for bifidobacteria (P<0·001), bacteroides and clostridia (P<0·05) were significantly lower for the soya group compared with the control group. There appears to be no lasting effect of early-life isoflavone exposure on isoflavone metabolism.