Impact of Dietary Isoflavone Supplementation on the Fecal Microbiota and Its Metabolites in Postmenopausal Women.

Isoflavones are metabolized by components of the gut microbiota and can also modulate their composition and/or activity. This study aimed to analyze the modifications of the fecal microbial populations and their metabolites in menopausal women under dietary treatment with soy isoflavones for one month. Based on the level of urinary equol, the women had been stratified previously as equol-producers (n = 3) or as equol non-producers (n = 5). The composition of the fecal microbiota was assessed by high-throughput sequencing of 16S rRNA gene amplicons and the changes in fatty acid excretion in feces were analyzed by gas chromatography. A greater proportion of sequence reads of the genus Slackia was detected after isoflavone supplementation. Sequences of members of the family Lachnospiraceae and the genus Pseudoflavonifractor were significantly increased in samples from equol-producing women. Multivariable analysis showed that, after isoflavone treatment, the fecal microbial communities of equol producers were more like each other. Isoflavone supplementation increased the production of caproic acid, suggesting differential microbial activity, leading to a high fecal excretion of this compound. However, differences between equol producers and non-producers were not scored. These results may contribute to characterizing the modulating effect of isoflavones on the gut microbiota, which could lead to unravelling of their beneficial health effects.

Use of high throughput amplicon sequencing and ethidium monoazide dye to track microbiota changes in an equol-producing menopausal woman receiving a long-term isoflavones treatment.

This work describes the impact of long term consumption of an isoflavone-rich dietary daily supplement on the composition and diversity of the faecal microbiota of a menopausal, equol-producing woman. Sequencing of 16S rDNA amplicons was performed on faecal samples taken at 0, 1, 3 and 6 months of treatment. Additionally, and for comparative purposes, ethidium monoazide (EMA) was used to avoid detection of DNA from dead bacteria. Members of two genera of the family Coriobacteriaceae (Eggerthella and Collinsella) were found in greater proportions at all sampling points during isoflavone supplementation. Different genera of the family Ruminococcaceae (e.g., Ruminococcus and Faecalibacterium), as well as members of the family Lachnospiraceae (Coprococcus) also underwent significant increases. For this last genus a positive correlation with the levels of equol excretion in urine was found. Currently bacterial strains known to be involved in isoflavone metabolism and equol production have been assigned to these taxa. The use of EMA dye allowed us to unravel those bacterial gut linages (e.g., Lachnospiraceae) that were more susceptible to damage. Our study contributes to the identification of microorganisms possibly involved in the production of isoflavone-desirable metabolites (such as equol), which could ultimately be isolated and further used as probiotics by people who cannot naturally benefit from isoflavones.

Fecal Changes Following Introduction of Milk in Infants With Outgrowing Non-IgE Cow's Milk Protein Allergy Are Influenced by Previous Consumption of the Probiotic LGG.

Cow's milk protein allergy (CMPA) is the most common allergy in the first year of life. Non-IgE mediated CMPA is characterized by digestive symptoms and tolerance development before the age of three. Gut microbiota composition in early life has been associated with food allergy. The ingestion of different foods/nutrients may mark different shifts in the microbial colonization of the infant intestine as well as the consumption of probiotics. Aim: To analyze changes in microbiota composition and metabolic and cytokine profiles in fecal samples from infants with non-IgE mediated CMPA after successful milk challenges, tolerance acquisition, and increasing dairy introduction in their diet. Methods: Twelve children with CMPA, aged between 1 and 2 years old, were recruited for the study. Participants were initially consuming hypoallergenic hydrolyzed formulas (four of them supplemented with the probiotic Lactobacillus rhamnosus GG), before being exposed to a standardized oral challenge (SOC) with cow's milk. Fecal samples were collected before, 1 week, and 1 month after performing the SOC. Changes in gut microbiota were determined by high-throughput amplicon sequencing of the 16S rRNA gene. Levels of lactobacilli were also determined by quantitative PCR (qPCR). Microbial metabolites were analyzed by chromatographic methods and fecal cytokines related to the Th1/Th2 balance were determined by immunoassay. Results: Lactic acid bacteria significantly increased in infants who outgrew non-IgE CMPA, after the introduction of milk. Microbial metabolites derived from the fermentation of proteins, such as branched chain fatty acids, and p-cresol, diminished. After the SOC, some cytokines related to inflammation (TNF-α, IFN-γ) increased. Accompanying the introduction of an unrestricted diet, we found significant differences in fecal microbial composition, metabolites, and cytokines between infants who did not consume the probiotic L. rhamnosus GG and those that did. Conclusions: These findings indicate that the introduction of intact milk proteins is followed by modifications in the infant gut environment through changes in immune mediators, microbiota, and its metabolic end-products. Consumption of probiotics during CMPA may contribute to gut homeostasis by fine-tuning these profiles.

Reply: "Letter to the editor Re: Diaz M., et al. Nutrients 2018, 10, 1481".

The objective of this letter of reply is to provide answers to the doubts and critical issues that Martín Martinez and López Liñan [...].

Microbiota and Derived Parameters in Fecal Samples of Infants with Non-IgE Cow's Milk Protein Allergy under a Restricted Diet.

Cow's milk protein allergy (CMPA) is the most common food allergy in infancy. Non-IgE mediated (NIM) forms are little studied and the responsible mechanisms of tolerance acquisition remain obscure. Our aim was to study the intestinal microbiota and related parameters in the fecal samples of infants with NIM-CMPA, to establish potential links between type of formula substitutes, microbiota, and desensitization. Seventeen infants between one and two years old, diagnosed with NIM-CMPA, were recruited. They were all on an exclusion diet for six months, consuming different therapeutic protein hydrolysates. After this period, stool samples were obtained and tolerance development was evaluated by oral challenges. A control group of 10 age-matched healthy infants on an unrestricted diet were included in the study. Microbiota composition, short-chain fatty acids, calprotectin, and transforming growth factor (TGF)-ß1 levels were determined in fecal samples from both groups. Infants with NIM-CMPA that consumed vegetable protein-based formulas presented microbiota colonization patterns different from those fed with an extensively hydrolyzed formula. Differences in microbiota composition and fecal parameters between NIM-CMPA and healthy infants were observed. Non-allergic infants showed a significantly higher proportion of Bacteroides compared to infants with NIM-CMPA. The type of protein hydrolysate was found to determine gut microbiota colonization and influence food allergy resolution in NIM-CMPA cases.

Characterization of four ß-glucosidases acting on isoflavone-glycosides from Bifidobacterium pseudocatenulatum IPLA 36007.

Bifidobacterium pseudocatenulatum IPLA 36007 acts on isoflavone glycosides, releasing their corresponding aglycones. This strain-specific activity might be a key step in making isoflavones bioavailable and harnessing their oestrogenic activity. To investigate the molecular mechanisms involved in this activity, four glycosyl hydrolase-encoding genes in the IPLA 36007 genome (AW18_01575, AW18_09810, AW18_08145, and AW18_08090) were selected, synthesized with heterologous promoter and terminator signals (r-ß-gluA, r-ß-gluB, r-ß-gluD and r-ß-gluE, respectively), cloned into Escherichia coli, overexpressed as His-tagged proteins, and the enzymes purified and characterized. All four enzymes - GluAHis, GluBHis, GluDHis and GluEHis - proved to have ß-glucosidase activity and deglycosylated (although at different rates) the isoflavone glycosides daidzin and genistin, releasing the aglycone moieties daidzein and genistein, respectively. GluDHis and GluEHis were also shown to hydrolyse ß-glucosyl disaccharides such as cellobiose and gentiobiose, while GluAHis and GluBHis did not. Differences in activity were recorded for all four ß-glucosidases at different pHs and temperatures under otherwise similar assay conditions, suggesting they have complementary activities under different environmental conditions. Two of the recombinant genes, r-ß-gluA, and r-ß-gluD, were cloned and expressed in the model lactic acid bacterium Lactococcus lactis, suggesting starter and probiotic organisms could be endowed with ß-glucosidase activity. B. pseudocatenulatum IPLA 36007 contains additional ß-glucosidases to those studied in this work, indicating a high level of redundancy for this enzymatic activity. Knowledge of glycoside-degrading enzymes should facilitate the development of novel, more effective or more selective prebiotics or functional foods for the promotion of bifidobacterial numbers in the human gut. It might also be of interest in the development of novel probiotics with specific health-promoting activities.

Development and Use of a Real-Time Quantitative PCR Method for Detecting and Quantifying Equol-Producing Bacteria in Human Faecal Samples and Slurry Cultures.

This work introduces a novel real-time quantitative PCR (qPCR) protocol for detecting and quantifying equol-producing bacteria. To this end, two sets of primers targeting the dihydrodaidzein reductase (ddr) and tetrahydrodaidzein reductase (tdr) genes, which are involved in the synthesis of equol, were designed. The primers showed high specificity and sensitivity when used to examine DNA from control bacteria, such as Slackia isoflavoniconvertens, Slackia equolifaciens, Asaccharobacter celatus, Adlercreutzia equolifaciens, and Enterorhabdus mucosicola. To demonstrate the validity and reliability of the protocol, it was used to detect and quantify equol-producing bacteria in human faecal samples and their derived slurry cultures. These samples were provided by 18 menopausal women under treatment of menopause symptoms with a soy isoflavone concentrate, among whom three were known to be equol-producers given the prior detection of the molecule in their urine. The tdr gene was detected in the faeces of all these equol-producing women at about 4-5 log10 copies per gram of faeces. In contrast, the ddr gene was only amplified in the faecal samples of two of these three women, suggesting the presence in the non-amplified sample of reductase genes unrelated to those known to be involved in equol formation and used for primer design in this study. When tdr and ddr were present in the same sample, similar copy numbers of the two genes were recorded. However, no significant increase in the copy number of equol-related genes along isoflavone treatment was observed. Surprisingly, positive amplification for both tdr and ddr genes was obtained in faecal samples and derived slurry cultures from two non-equol producing women, suggesting the genes could be non-functional or the daidzein metabolized to other compounds in samples from these two women. This novel qPCR tool provides a technique for monitoring gut microbes that produce equol in humans. Monitoring equol-producing bacteria in the human gut could provide a means of evaluating strategies aimed at increasing the endogenous formation of this bioactive compound.

Effect of Soy Isoflavones on Growth of Representative Bacterial Species from the Human Gut.

The present work aimed to assess the susceptibility of dominant and representative bacterial populations from the human gut to isoflavones and their metabolites. To do so, the minimum inhibitory concentration (MIC) of isoflavone glycosides, isoflavone aglycones, and equol to 37 bacterial strains was determined by broth microdilution. Additionally, for 10 representative strains, growth curves, growth rate (µ), and optical density (OD600 nm) of the cultures at 24 h were also determined. MICs of daidzin, genistin, daidzein, and genistein were >2048 µg mL-1 for all strains assayed, while that of equol ranged from 16 µg mL-1 for Bifidobacterium animalis subsp. animalis to >2048 µg mL-1 for Enterobacteriaceae strains. Changes in growth curves, µ, and final OD were observed among the species in the presence of all tested compounds. Genistein reduced µ of Bacteroides fragilis, Lactococcus lactis subsp. lactis, and Slackia equolifaciens, while both genistein and equol increased that of Lactobacillus rhamnosus and Faecalibacterium prausnitzii. Compared to controls, lower final OD in the presence of aglycones and equol were recorded for some strains but were higher for others. Altogether, the results suggest that isoflavone-derived compounds could modify numbers of key bacterial species in the gut, which might be associated with their beneficial properties.

Bacterial communities and metabolic activity of faecal cultures from equol producer and non-producer menopausal women under treatment with soy isoflavones.

BACKGROUND: Isoflavones are polyphenols with estrogenic activity found mainly in soy and soy-derived products that need to be metabolised in the intestine by the gut bacteria to be fully active. There is little knowledge about isoflavone bioconversion and equol production in the human intestine. In this work, we developed an in vitro anaerobic culture model based on faecal slurries to assess the impact of isoflavone supplementation on the overall intestinal bacterial composition changes and associated metabolic transformations. RESULTS: In the faecal anaerobic batch cultures of this study bioconversion of isoflavones into equol was possible, suggesting the presence of viable equol-producing bacterial taxa within the faeces of menopausal women with an equol producer phenotype. The application of high-throughput DNA sequencing of 16S rRNA gene amplicons revealed the composition of the faecal cultures to be modified by the addition of isoflavones, with enrichment of some bacterial gut members associated with the metabolism of phenolics and/or equol production, such as Collinsella, Faecalibacterium and members of the Clostridium clusters IV and XIVa. In addition, the concentration of short-chain fatty acids (SCFAs) detected in the isoflavone-containing faecal cultures was higher in those inoculated with faecal slurries from equol-producing women. CONCLUSIONS: This study constitutes the first step in the development of a faecal culturing system with isoflavones that would further allow the selection and isolation of intestinal bacterial types able to metabolize these compounds and produce equol in vitro. Although limited by the low number of faecal cultures analysed and the inter-individual bacterial diversity, the in vitro results obtained in this work tend to indicate that soy isoflavones might provide an alternative energy source for the increase of equol-producing taxa and enhancement of SCFAs production. SCFAs and equol are both considered pivotal bacterial metabolites in the triggering of intestinal health-related beneficial effects.

Genetic and biochemical characterization of an oligo-α-1,6-glucosidase from Lactobacillus plantarum.

Although encoded in the genome of many Lactobacillus spp. strains, α-glucosidases have received little attention compared to other glycosyl hydrolases. In this study, a putative oligosaccharide(oligo)-α-1,6-glucosidase-encoding gene (malL) was identified in the genome of Lactobacillus plantarum LL441. malL coded for 572 amino acid residues with a calculated total molecular mass of 66.31kDa. No predicted signal peptide was observed, suggesting this enzyme to be localized within the cytoplasm of the cell. Homology studies of the deduced amino acid sequence in the area of its active sites classified the enzyme as a member of the α-amylase (AmyAC) superfamily of glycosyl hydrolases (GH), family 13 (GH13), subfamily 31 (GH13_31). malL was cloned in Escherichia coli and the coded enzyme overexpressed as a histidine-tagged protein (MalLHis). It was then purified and characterized. MalLHis protein showed strong hydrolytic activity towards 4-nitrophenyl-α-d-glucopyranoside (pNP-α-Glu) but not to other pNP-α-d- or pNP-ß-d-derivatives. When using pNP-α-Glu as a substrate, MalLHis showed similar specific activities between pH5.0 and 6.0, and between 20 and 42°C (optimum 30°C). Among the natural carbohydrates assayed, MalLHis showed specificity towards isomaltose (Vmax and Km values of 40.64µmolmin-1mg-1 and 6.22mM) and much less to isomaltulose (Vmax and Km values of 168.86µmolmin-1mg-1 and 244.52mM). However, under the conditions of the assay, the enzyme showed no transglycosylation activity. Characterization of the entire complement of glycosidases in L. plantarum might reveal how strains of this species could be used in new biotechnological applications or in the development of functional foods.

Profiling of Phenolic Metabolites in Feces from Menopausal Women after Long-Term Isoflavone Supplementation.

Phenolic compounds were screened by UPLC-ESI-MS/MS in the feces of 15 menopausal women before and after long-term isoflavone treatment. In total, 44 compounds were detected. Large intertreatment, interindividual, and intersample variations were observed in terms of the number of compounds and their concentration. Four compounds, the aglycones daidzein and genistein and the daidzein derivatives dihydrodaidzein and O-desmethylangolensin, were associated with isoflavone metabolism; these were identified only after the isoflavone treatment. In addition, 4-ethylcatechol, 3-hydroxyphenylacetic acid, and 3-phenylpropionic acid differed significantly in pre- and postintervention samples, whereas the concentration of 4-hydroxy-5-phenylvaleric acid showed a trend toward increasing over the treatment. The phenolic profiles of equol-producing and -non-producing groups were similar, with the exceptions of 3-hydroxyphenylacetic acid and 3-phenylpropionic acid, which showed higher concentrations in equol-non-producing women. These findings may help to trace isoflavone-derived metabolites in feces during isoflavone interventions and to design new studies to address their biological effects.

A novel UHPLC method for the rapid and simultaneous determination of daidzein, genistein and equol in human urine.

This work reports on a novel method involving reverse-phased ultra-high performance liquid chromatography (UHPLC) plus a spectrophotometric photodiode array/fluorescence (FLR) detection system for determining the concentration of equol and major soy isoflavones (daidzein and genistein) in human urine. The proposed method was validated in terms of its linearity, sensitivity, accuracy (recovery) and precision (intra- and inter-day repeatability). The isoflavone profiles of urine samples from a group of menopausal women following oral soy isoflavone supplementation were determined and compared. Screening for equol-producer status was accomplished with high sensitivity (detection limit of the FLR detector 2.93nM). The method involves a short chromatographic run time compared to conventional HPLC methods while allowing for the simultaneous and reliable quantification of daidzein, genistein and equol in human urine. It also allows for the rapid screening of multiple urine samples when testing for equol production status and checking patient adherence to isoflavone treatment regimens.

Equol status and changes in fecal microbiota in menopausal women receiving long-term treatment for menopause symptoms with a soy-isoflavone concentrate.

The knowledge regarding the intestinal microbial types involved in isoflavone bioavailability and metabolism is still limited. The present work reports the influence of a treatment with isoflavones for 6 months on the fecal bacterial communities of 16 menopausal women, as determined by culturing and culture-independent microbial techniques. Changes in fecal communities were analyzed with respect to the women's equol-producing phenotype. Compared to baseline, at 1 and 3 months the counts for all microbial populations in the feces of equol-producing women had increased strongly. In contrast, among the non-producers, the counts for all microbial populations at 1 month were similar to those at baseline, and decreased significantly by 3 and 6 months. Following isoflavone intake, major bands in the denaturing gradient gel electrophoresis (DGGE) profiles appeared and disappeared, suggesting important changes in majority populations. In some women, increases were seen in the intensity of specific DGGE bands corresponding to microorganisms known to be involved in the metabolism of dietary phytoestrogens (Lactonifactor longoviformis, Faecalibacterium prausnitzii, Bifidobacterium sp., Ruminococcus sp.). Real-Time quantitative PCR revealed that the Clostridium leptum and C. coccoides populations increased in equol producers, while those of bifidobacteria and enterobacteria decreased, and vice versa in the non-producers. Finally, the Atopobium population increased in both groups, but especially in the non-producers at three months. As the main findings of this study, (i) variations in the microbial communities over the 6-month period of isoflavone supplementation were large; (ii) no changes in the fecal microbial populations that were convincingly treatment-specific were seen; and (iii) the production of equol did not appear to be associated with the presence of, or increase in the population of, any of the majority bacterial types analyzed.

The genome of Bifidobacterium pseudocatenulatum IPLA 36007, a human intestinal strain with isoflavone-activation activity.

BACKGROUND: Bifidobacterium species, including Bifidobacterium pseudocatenulatum, are among the dominant microbial populations of the human gastrointestinal tract. They are also major components of many commercial probiotic products. Resident and transient bifidobacteria are thought to have several beneficial health effects. However, our knowledge of how these bacteria interact and communicate with host cells remains poor. This knowledge is essential for scientific support of their purported health benefits and their rational inclusion in functional foods. RESULTS: This work describes the draft genome sequence of Bifidobacterium pseudocatenulatum IPLA 36007, a strain isolated as dominant from the feces of a healthy human. Besides several properties of probiosis, IPLA 36007 exhibited the capability of releasing aglycones from soy isoflavone glycosides. The genome contains 1,851 predicted genes, including 54 genes for tRNAs and fie copies of unique 16S, 23S and 5S rRNA genes. As key attributes of the IPLA 36007 genome we can mention the presence of a lysogenic phage, a cluster encoding type IV fimbriae, and a locus encoding a clustered, regularly interspaced, short, palindromic repeat (CRISPR)-Cas system. Four open reading frames (orfs) encoding ß-glucosidases belonging to the glycosyl hydrolase family 3, which may act on isoflavone glycosides, were encountered. Additionally, one gene was found to code for a glycosyl hydrolase of family 1 that might also have ß-glucosidase activity. CONCLUSION: The availability of the B. pseudocatenulatum IPLA 36007 genome should allow the enzyme system involved in the release of soy isoflavone aglycones from isoflavone glycosides, and the molecular mechanisms underlying the strain's probiotic properties, to be more easily understood.