Activation of proestrogens from hops by human intestinal bacteria: conversion of isoxanthohumol into the potent phytoestrogen 8-prenylnaringenin

Hop, an essential ingredient in beer, contains different prenylflavonoids, among which 8-prenylnaringenin (8-PN) would be the most potent phytoestrogen currently known. Therefore, hop extracts are now marketed to relief menopausal complaints but efficiency varies greatly among individuals. As variability in biological activity was also noted for other phytoestrogens and related with variable intestinal bacterial metabolism, the goal of this work was to investigate whether intestinal bacteria also determine the final activity of hop phytoestrogens by combining batch incubations with SHIME, rat and human studies. Batch incubations learned that 8-PN is stable towards microbial degradation but that isoxanthohumol (IX), another hop prenylflavonoid, can be activated into 8-PN by bacterial O-demethylation. Although IX shows no estrogenic activity, it is present in hop products in 10- to 30-fold higher concentrations than 8-PN, showing that intestinal IX activation may significantly increase 8-PN exposure. SHIME experiments showed up to 80% IX conversion, mainly in the distal colon. Experiments with 51 fecal samples indicated significant interindividual variations, with high, moderate and low 8-PN producers. An experiment with germ-free and human microbiota associated rats and a human intervention trial confirmed the importance of variable intestinal IX activation and provided a definitive explanation for the variable biological activity of hop products. As the final activity of all phytoestrogens depends on variable intestinal metabolism, 100 fecal samples were incubated with precursors from all classes. This learned that the activation of different phytoestrogen classes occurs through separate pathways, with within-class correlations between different metabolites. Moreover, the production of specific metabolites was related to certain intestinal conditions. As variable IX activation implies variable 8-PN exposure, technological applications were investigated to balance the 8-PN exposure between individuals. To do this, an efficiently 8-PN producing Eubacterium limosum strain was selected. Its application as probiotic to increase intestinal 8-PN production was shown in vitro and in the rat model. In conclusion, it was demonstrated for the first time that the exposure to the potent phytoestrogen 8-PN mainly depends on the product IX concentrations and the variable intestinal metabolic potential to activate IX. Moreover, strategies were developed to balance the 8-PN exposure in all individuals

Activation of proestrogens from hops (Humulus lupulus L.) by intestinal microbiota; Conversion of isoxanthohumol into 8-prenylnaringenin

Hop, an essential ingredient in most beers, contains a number of prenylflavonoids, among which 8-prenylnaringenin (8-PN) would be the most potent phytoestrogen currently known. Although a number of health effects are attributed to these compounds, only a few reports are available about the bioavailability of prenylflavonoids and the transformation potency of the intestinal microbial community. To test these transformations, four fecal samples were incubated with xanthohumol, isoxanthohumol (IX), and 8-PN. Upon incubation with IX, present in strong ales up to 4 mg/L, 36% was converted into 8-PN in one fecal sample and the estrogenic properties of the sample drastically increased. In an experiment with 12 fecal cultures, this conversion was observed in one-third of the samples, indicating the importance of interindividual variability in the intestinal microbial community. Eubacterium limosum was identified to be capable of this conversion (O-demethylation) of IX into 8-PN, and after strain selection, a conversion efficiency of 90% was achieved. Finally, strain supplementation to a nonconverting fecal sample led to rapid and high 8-PN production at only 1% (v/v) addition. Up to now, the concentration of 8-PN in beer was considered too low to affect human health. However, these results show that the activity of the intestinal microbial community could more than 10-fold increase the exposure concentration. Because prenylflavonoids are present in many beers with IX being the major constituent, the results raise the question whether moderate beer consumption might contribute to increased in vivo levels of 8-PN and even influence human health

A yeast fermentate improves gastrointestinal discomfort and constipation by modulation of the gut microbiome : results from a randomized double-blind placebo-controlled pilot trial

Background: Constipation and symptoms of gastrointestinal discomfort such as bloating are common among otherwise healthy individuals, but with significant impact on quality of life. Despite the recognized contribution of the gut microbiome to this pathology, little is known about which group(s) of microorganism(s) are playing a role. A previous study performed in vitro suggests that EpiCor (R) fermentate has prebiotic-like properties, being able to favorably modulate the composition of the gut microbiome. Therefore, the aim of this study was to investigate the effects of EpiCor fermentate in a population with symptoms of gastrointestinal discomfort and reduced bowel movements and to evaluate its effect at the level of the gut microbiome. Methods: This pilot study was performed according to a randomized, double-blind, placebo-controlled parallel design. Eighty subjects with symptoms of gastrointestinal discomfort and constipation were allocated to one of two trial arms (placebo or EpiCor fermentate). Randomization was done in a stratified manner according to symptom severity, resulting in two subgroups of patients: severe and moderate. Daily records of gastrointestinal symptoms were assessed on a 5-point scale, and also stool frequency and consistency were documented during a 2-week run-in and a 6-week intervention phases. Averages over two-week intervals were calculated. Constipation-associated quality of life and general perceived stress were assessed at baseline and after 3 and 6 weeks of intervention. Fecal samples were also collected at these same time points. Results: EpiCor fermentate led to a significant improvement of symptoms such as bloating/distension (p = 0.033 and p = 0.024 after 2 and 4 weeks of intervention, respectively), feeling of fullness (p = 0.004 and p = 0.023 after 2 and 4 weeks of intervention, respectively) and general daily scores (p = 0.046 after 2 weeks of intervention) in the moderate subgroup. A significant improvement in stool consistency was observed for the total population (p = 0.023 after 2 weeks of intervention) as well as for the severe subgroup (p = 0.046 after 2 weeks of intervention), and a nearly significant increase in stool frequency was detected for the total cohort (p = 0.083 and p = 0.090 after 2 and 4 weeks of intervention, respectively). These effects were accompanied by an improvement in constipation-associated quality of life and general perceived stress, particularly in the moderate subgroup. Members of the families Bacteroidaceae and Prevotellaceae, two groups of bacteria that have been previously reported to be deficient in constipated patients, were found to increase with EpiCor fermentate in the severe subgroup. In the moderate subgroup, a significant increase in Akkermansia muciniphila was observed. Conclusions: Despite the relatively low dose administered (500 mg/day), particularly when comparing to the high recommended doses for prebiotic fibers, EpiCor fermentate was able to modulate the composition of the gut microbiome, resulting in improvement of constipation-associated symptoms. Conversely, the reported increase in bowel movements may have altered the gut microbial community by increasing those groups of bacteria that are better adapted to a faster gastrointestinal transit time

Exploration of isoxanthohumol bioconversion from spent hops into 8-prenylnaringenin using resting cells of Eubacterium limosum

Hops is an almost unique source of the potent phytoestrogen 8-prenylnaringenin (8-PN). As hops contain only low levels of 8-PN, synthesis may be more attractive than extraction. A strain of the Gram-positive Eubacterium limosum was isolated previously for 8-PN production from more abundant precursor isoxanthohumol (IX) from hops. In this study, spent hops, an industrial side stream from the beer industry, was identified as interesting source of IX. Yet, hop-derived compounds are well-known antibacterial agents and the traces of a large variety of different compounds in spent hops interfered with growth and IX conversion. Critical factors to finally enable bacterial 8-PN production from spent hops, using a food and feed grade medium, were evaluated in this research. The use of bacterial resting cells and complex medium at a pH of 7.8-8 best fulfilled the requirements for 8-PN production and generated a solid basis for development of an economic process

Arabinoxylans, inulin and Lactobacillus reuteri 1063 repress the adherent-invasive Escherichia coli from mucus in a musosa-comprising gut model

The microbiota that colonises the intestinal mucus may particularly affect human health given its proximity to the epithelium. For instance, the presence of the adherent-invasive Escherichia coli (AIEC) in this mucosal microbiota has been correlated with Crohn's disease. Using short-term screening assays and a novel long-term dynamic gut model, which comprises a simulated mucosal environment (M-SHIME), we investigated how (potential) pro-and prebiotics may repress colonisation of AIEC from mucus. Despite that during the short-term screening assays, some of the investigated Lactobacillus strains adhered strongly to mucins, none of them competed with AIEC for mucin-adhesion. In contrast, AIEC survival and growth during co-culture batch incubations was decreased by Lactobacillus rhamnosus GG and L. reuteri 1063, which correlated with (undissociated) lactic acid and reuterin levels. Regarding the prebiotics, long-chain arabinoxylans (LC-AX) lowered the initial mucin-adhesion of AIEC, while both inulin (IN) and galacto-oligosaccharides (GOS) limited AIEC survival and growth during batch incubations. L. reuteri 1063, LC-AX and IN were thus retained for a long-term study with the M-SHIME. All treatments repressed AIEC from mucus without affecting AIEC numbers in the luminal content. As a possible explanation, L. reuteri 1063 treatment increased lactobacilli levels in mucus, while LC-AX and IN additionally increased mucosal bifidobacteria levels, thus leading to antimicrobial effects against AIEC in mucus. Overall, this study shows that pro-and prebiotics can beneficially modulate the in vitro mucosal microbiota, thus limiting occurrence of opportunistic pathogens among those mucosal microbes which may directly interact with the host given their proximity to the epithelium

In vitro bioconversion of polyphenols from black tea and red wine/grape juice by human intestinal microbiota displays strong interindividual variability

Dietary polyphenols in tea and wine have been associated with beneficial health effects. After ingestion, most polyphenols are metabolized by the colonic microbiota. The current study aimed at exploring the interindividual variation of gut microbial polyphenol bioconversion from 10 healthy human subjects. In vitro fecal batch fermentations simulating conditions in the distal colon were performed using polyphenols from black tea and a mixture of red wine and grape juice. Microbial bioconversion was monitored by NMR- and GC-MS-based profiling of diverse metabolites and phenolics. The complex polyphenol mixtures were degraded to a limited number of key metabolites. Each subject displayed a specific metabolite profile differing in composition and time courses as well as levels of these metabolites. Moreover, clear differences depending on the polyphenol sources were observed. In conclusion, varying metabolite pathways among individuals result in different metabolome profiles and therefore related health effects are hypothesized to differ between subjects