Bosea rubneri sp. nov. Isolated from Organically Grown Allium cepa.

Strain ZW T0_25T was isolated from an onion sample (Allium cepa var. Hytech F1) within a storage trial and proofed to be a novel, aerobic, Gram-stain negative, rod-shaped bacterial strain. Analyses of the 16S rRNA gene sequence and of the whole draft genome sequences, i.e., digital DNA-DNA hybridization (dDDH), Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) showed that this strain represents a new species of the genus Bosea. The genome size of strain ZW T0_25T is 6.19 Mbp, and the GC content is 66.9%. As whole cell sugars, rhamnose, ribose and glucose were identified. Ubiquinone Q-10 is the major respiratory quinone with 97.8%. Polar lipids in strain ZW T0_25T are composed of one phosphatidylethanolamine, one phosphatidylglycerol, one aminophospholipid, two aminolipids, one glycolipid and two phospholipids whereas the fatty acid profile predominantly consists of C18:1 w7c (63.3%), C16:1 w7c (19.5%) and C16:0 (7.1%). Phenotypic traits were tested in the wet lab as well as predicted in silico from genome data. Therefore, according to this polyphasic approach, the new name Bosea rubneri sp. nov. with the type strain ZW T0_25T (= DSM 116094 T = LMG 33093 T) is proposed.

Controversy on health-based guidance values for bisphenol A-the need of criteria for studies that serve as a basis for risk assessment.

Since 2006, the responsible regulatory bodies have proposed five health-based guidance values (HBGV) for bisphenol A (BPA) that differ by a factor of 250,000. This range of HBGVs covers a considerable part of the range from highly toxic to relatively non-toxic substances. As such heterogeneity of regulatory opinions is a challenge not only for scientific risk assessment but also for all stakeholders, the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) analyzed the reasons for the current discrepancy and used this example to suggest improvements for the process of HBGV recommendations. A key aspect for deriving a HBGV is the selection of appropriate studies that allow the identification of a point of departure (PoD) for risk assessment. In the case of BPA, the HBGV derived in the 2023 EFSA assessment was based on a study that reported an increase of Th17 cells in mice with a benchmark dose lower bound (BMDL40) of 0.53 µg/kg bw/day. However, this study does not comply with several criteria that are important for scientific risk assessment: (1) the selected end-point, Th17 cell frequency in the spleen of mice, is insufficiently understood with respect to health outcomes. (2) It is unclear, by which mechanism BPA may cause an increase in Th17 cell frequency. (3) It is unknown, if an increase of Th17 cell frequency in rodents is comparably observed in humans. (4) Toxicokinetics were not addressed. (5) Neither the raw data nor the experimental protocols are available. A further particularly important criterion (6) is independent data confirmation which is not available in the present case. Previous studies using other readouts did not observe immune-related adverse effects such as inflammation, even at doses orders of magnitude higher than in the Th17 cell-based study. The SKLM not only provides here key criteria for the use of such studies, but also suggests that the use of such a "checklist" requires a careful and comprehensive scientific judgement of each item. It is concluded that the Th17 cell-based study data do not represent an adequate basis for risk assessment of BPA.

Commentary of the SKLM to the EFSA opinion on risk assessment of N-nitrosamines in food.

Dietary exposure to N-nitrosamines has recently been assessed by the European Food Safety Authority (EFSA) to result in margins of exposure that are conceived to indicate concern with respect to human health risk. However, evidence from more than half a century of international research shows that N-nitroso compounds (NOC) can also be formed endogenously. In this commentary of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG), the complex metabolic and physiological biokinetics network of nitrate, nitrite and reactive nitrogen species is discussed with emphasis on its influence on endogenous NOC formation. Pioneering approaches to monitor endogenous NOC have been based on steady-state levels of N-nitrosodimethylamine (NDMA) in human blood and on DNA adduct levels in blood cells. Further NOC have not been considered yet to a comparable extent, although their generation from endogenous or exogenous precursors is to be expected. The evidence available to date indicates that endogenous NDMA exposure could exceed dietary exposure by about 2-3 orders of magnitude. These findings require consolidation by refined toxicokinetics and DNA adduct monitoring data to achieve a credible and comprehensive human health risk assessment.

Effects of pectin methyl-esterification on intestinal microbiota and its immunomodulatory properties in naive mice.

Pectins are dietary fibers that are attributed with several beneficial immunomodulatory effects. Depending on the degree of esterification (DE), pectins can be classified as high methoxyl pectin (HMP) or low methoxyl pectin (LMP). The aim of this study was to investigate the effects of pectin methyl-esterification on intestinal microbiota and its immunomodulatory properties in naive mice. Supplementation of the diet with LMP or HMP induced changes in the composition of the intestinal microbiota in mice toward Bacteroides, which was mainly promoted by HMP. Metabolome analysis of stool samples from pectin-fed mice showed a different effect of the two types of pectin on the levels of short-chain fatty acids and bile acids, which was consistent with highly efficient in vivo fermentation of LMP. Analysis of serum antibody levels showed a significant increase in IgG and IgA levels by both pectins, while FACS analysis revealed a decrease of infiltrating inflammatory cells in the intestinal lamina propria by HMP. Our study revealed that the structural properties of the investigated pectins determine fermentability, effects on microbial composition, metabolite production, and modulation of immune responses. Consumption of HMP preferentially altered the gut microbiota and suppressed pro-inflammatory immune responses, suggesting a beneficial role in inflammatory diseases.

A Systematic Review and Comprehensive Evaluation of Human Intervention Studies to Unravel the Bioavailability of Hydroxycinnamic Acids.

Significance: Hydroxycinnamic acids (HCAs) are the main phenolic acids in the western diet. Harmonizing the available information on the absorption, distribution, metabolism, and excretion (ADME) of HCAs is fundamental to unraveling the compounds responsible for their health effects. This work systematically assessed pharmacokinetics, including urinary recovery, and bioavailability of HCAs and their metabolites, based on literature reports. Recent Advances: Forty-seven intervention studies with coffee, berries, herbs, cereals, tomato, orange, grape products, and pure compounds, as well as other sources yielding HCA metabolites, were included. Up to 105 HCA metabolites were collected, mainly acyl-quinic and C6-C3 cinnamic acids. C6-C3 cinnamic acids, such as caffeic and ferulic acid, reached the highest blood concentrations (maximum plasma concentration [Cmax] = 423 nM), with time to reach Cmax (Tmax) values ranging from 2.7 to 4.2 h. These compounds were excreted in urine in higher amounts than their phenylpropanoic acid derivatives (4% and 1% of intake, respectively), but both in a lower percentage than hydroxybenzene catabolites (11%). Data accounted for 16 and 18 main urinary and blood HCA metabolites, which were moderately bioavailable in humans (collectively 25%). Critical Issues: A relevant variability emerged. It was not possible to unequivocally assess the bioavailability of HCAs from each ingested source, and data from some plant based-foods were absent or inconsistent. Future Directions: A comprehensive study investigating the ADME of HCAs derived from their most important dietary sources is urgently required. Eight key metabolites were identified and reached interesting plasma Cmax concentrations and urinary recoveries, opening up new perspectives to evaluate their bioactivity at physiological concentrations. Antioxid. Redox Signal. 40, 510-541.

Acetaldehyde as a Food Flavoring Substance: Aspects of Risk Assessment.

The Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) has reviewed the currently available data in order to assess the health risks associated with the use of acetaldehyde as a flavoring substance in foods. Acetaldehyde is genotoxic in vitro. Following oral intake of ethanol or inhalation exposure to acetaldehyde, systemic genotoxic effects of acetaldehyde in vivo cannot be ruled out (induction of DNA adducts and micronuclei). At present, the key question of whether acetaldehyde is genotoxic and mutagenic in vivo after oral exposure cannot be answered conclusively. There is also insufficient data on human exposure. Consequently, it is currently not possible to reliably assess the health risk associated with the use of acetaldehyde as a flavoring substance. However, considering the genotoxic potential of acetaldehyde as well as numerous data gaps that need to be filled to allow a comprehensive risk assessment, the SKLM considers that the use of acetaldehyde as a flavoring may pose a safety concern. For reasons of precautionary consumer protection, the SKLM recommends that the scientific base for approval of the intentional addition of acetaldehyde to foods as a flavoring substance should be reassessed.

Microbial Metabolism of the Soy Isoflavones Daidzein and Genistein in Postmenopausal Women: Human Intervention Study Reveals New Metabotypes.

BACKGROUND: Soy isoflavones belong to the group of phytoestrogens and are associated with beneficial health effects but are also discussed to have adverse effects. Isoflavones are intensively metabolized by the gut microbiota leading to metabolites with altered estrogenic potency. The population is classified into different isoflavone metabotypes based on individual metabolite profiles. So far, this classification was based on the capacity to metabolize daidzein and did not reflect genistein metabolism. We investigated the microbial metabolite profile of isoflavones considering daidzein and genistein. METHODS: Isoflavones and metabolites were quantified in the urine of postmenopausal women receiving a soy isoflavone extract for 12 weeks. Based on these data, women were clustered in different isoflavone metabotypes. Further, the estrogenic potency of these metabotypes was estimated. RESULTS: Based on the excreted urinary amounts of isoflavones and metabolites, the metabolite profiles could be calculated, resulting in 5 metabotypes applying a hierarchical cluster analysis. The metabotypes differed in part strongly regarding their metabolite profile and their estimated estrogenic potency.

Rathayibacter rubneri sp. nov. isolated from Allium cepa var. Rijnsburger, an onion landrace.

The novel, aerobic, Gram-stain-positive, rod-shaped bacterial strain, ZW T2_19T, was isolated from an onion sample (Allium cepa var. Rijnsburger). Analyses of the 16S rRNA gene sequence revealed that ZW T2_19T represented a member of the genus Rathayibacter but may represent a novel species of this genus. Analyses of the whole draft genome sequences, i.e. digital DNA-DNA hybridisation (dDDH) and average nucleotide identity (ANI) of ZW T2_19T and all type strains of species of the genus Rathayibacter confirmed that ZW T2_19T represents a novel species of the genus Rathayibacter. The genome size of ZW T2_19T is 4.01 Mbp and the DNA G+C content is 71.8 mol%. Glucose, mannose, rhamnose and ribose were detected as whole-cell sugars of ZW T2_19T. The major respiratory quinone of ZW T2_19T is menaquinone MK-10, at 78.9 %. The detected peptidoglycan type in ZW T2_19T is a variant of type B2γ with {Gly} [l-diaminobutyric acid (l-DAB)/l-homoserine (l-Hse)] d-Glu-l-DAB. Polar lipids in ZW T2_19T consisted of one diphosphatidylglycerol, one phosphatidylglycerol, seven glycolipids, one phospholipid and one lipid. The fatty acid profile of ZW T2_19T predominantly consisted of anteiso-C15 : 0 (53 %), iso-C16 : 0 (21 %) and anteiso-C17 : 0 (18 %). In addition, API 20NE, API 50CH, API Coryne, API ZYM, antibiotic susceptibility, haemolysis and growth at different temperatures and with different supplements was investigated. On the basis of the results obtained using this polyphasic approach, including molecular, phenotypic and biochemical analyses, we propose the novel species Rathayibacter rubneri with the type and only strain ZW T2_19T (= DSM 114294T = LMG 32700T).

Pyrrolizidine alkaloids in borage (Borago officinalis): Comprehensive profiling and development of a validated LC-MS/MS method for quantification.

Pyrrolizidine alkaloids (PA) from borage (Borago officinalis) consumed as herb and tea, may pose a food safety risk. Therefore, the European Union (EU) set maximum levels of PA in borage, among other foodstuffs, which are applicable since July 1st, 2022. Here, a comprehensive LC-MS/MS based profiling of PA and their N-oxides (PANO) in B. officinalis leaves is presented. Based on these results a targeted, quantitative LC-MS/MS method for the determination of individual PA/PANO present in borage was developed. Chromatographic separation was achieved for all PA/PANO detected in B. officinalis. An easy and fast extraction procedure was developed using a design of experiments approach (DOE). The most efficient extraction was achieved using 0.2% formic acid in 10% methanol at a temperature of 47.5 °C for 60 min. The final method was validated and showed good overall accuracy (recoveries 85-121%) and precision (RDS ≤11%). The method was applied to B. officinalis leave material, demonstrating its suitability for the intended purpose. In these borage samples, the acetylated forms, which are not regulated by EU, were among the quantitatively most relevant PA.

Time-dependent fermentation of different structural units of commercial pectins with intestinal bacteria.

Many of the proposed health-related properties of pectins are based on their fermentability in the large intestine, but detailed structure-related studies on pectin fermentation have not been reported so far. Here, pectin fermentation kinetics were studied with a focus on structurally different pectic polymers. Therefore, six commercial pectins from citrus, apple, and sugar beet were chemically characterized and fermented in in vitro fermentation assays with human fecal samples over different periods of time (0 h, 4 h, 24 h, 48 h). Structure elucidation of intermediate cleavage products showed differences in fermentation speed and/or fermentation rate among the pectins, but the order in which specific structural pectic elements were fermented was comparable across all pectins. Neutral side chains of rhamnogalacturonan type I were fermented first (between 0 and 4 h), followed by homogalacturonan units (between 0 and 24 h) and, at last, the rhamnogalacturonan type I backbone (between 4 and 48 h). This indicates that fermentation of different pectic structural units might take place in different sections of the colon, potentially affecting their nutritional properties. For the formation of different short-chain fatty acids, mainly acetate, propionate, and butyrate, and the influence on microbiota, there was no time-dependent correlation regarding the pectic subunits. However, an increase of members of the bacterial genera Faecalibacterium, Lachnoclostridium, and Lachnospira was observed for all pectins.

Evaluation of the genotoxic potential of acrylamide: Arguments for the derivation of a tolerable daily intake (TDI value).

This opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) presents arguments for an updated risk assessment of diet-related exposure to acrylamide (AA), based on a critical review of scientific evidence relevant to low dose exposure. The SKLM arrives at the conclusion that as long as an appropriate exposure limit for AA is not exceeded, genotoxic effects resulting in carcinogenicity are unlikely to occur. Based on the totality of the evidence, the SKLM considers it scientifically justified to derive a tolerable daily intake (TDI) as a health-based guidance value.

Chemical Stability and Bioactivity of tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone in in vitro test systems.

Danshen Si Wu is a Traditional Chinese Medicine used for menopausal complains. Beside tanshinone IIA (Tan IIA), Danshen also contains tanshinone I (Tan I), cryptotanshinone (CT) and dihydrotanshinone (DT). The aim of this study was to compare the biological activity of these tanshinones and to determine their cytotoxicity and genotoxicity. Purities and stabilities of the substances were analyzed by LC-DAD and LC-MS analyses. DT and CT concentrations decreased rapidly in dimethylsulfoxide and were converted to Tan I and Tan IIA, respectively. In aqueous solution concentration of all tanshinones decreased after 24 h. Tan I and Tan IIA showed dose-dependent bioactivity mediated by ERα and ERß. No cytotoxic and genotoxic effects for Tan I and Tan IIA were detected. In a yeast transactivation assay Tan I and Tan IIA showed antiandrogenic activity. A significant anabolic activity in C2C12 cells could be detected for Tan I and Tan IIA. In conclusion our data provide evidence that Tan I and Tan IIA are the most relevant bioactive tanshinones in Danshen. Our finding that all tanshinones display a certain instability in aqueous solutions is relevant when discussing their potential therapeutic benefits in humans.

Revisiting the bioavailability of flavan-3-ols in humans: A systematic review and comprehensive data analysis.

This systematic review summarizes findings from human studies investigating the different routes of absorption, metabolism, distribution and excretion (ADME) of dietary flavan-3-ols and their circulating metabolites in healthy subjects. Literature searches were performed in PubMed, Scopus and the Web of Science. Human intervention studies using single and/or multiple intake of flavan-3-ols from food, extracts, and pure compounds were included. Forty-nine human intervention studies met inclusion criteria. Up to 180 metabolites were quantified from blood and urine samples following intake of flavan-3-ols, mainly as phase 2 conjugates of microbial catabolites (n = 97), with phenyl-γ-valerolactones being the most representative ones (n = 34). Phase 2 conjugates of monomers and phenyl-γ-valerolactones, the main compounds in both plasma and urine, reached two peak plasma concentrations (Cmax) of 260 and 88 nmol/L at 1.8 and 5.3 h (Tmax) after flavan-3-ol intake. They contributed to the bioavailability of flavan-3-ols for over 20%. Mean bioavailability for flavan-3-ols was moderate (31 ± 23%, n bioavailability values = 20), and it seems to be scarcely affected by the amount of ingested compounds. While intra- and inter-source differences in flavan-3-ol bioavailability emerged, mean flavan-3-ol bioavailability was 82% (n = 1) and 63% (n = 2) after (-)-epicatechin and nut (hazelnuts, almonds) intake, respectively, followed by 25% after consumption of tea (n = 7), cocoa (n = 5), apples (n = 3) and grape (n = 2). This highlights the need to better clarify the metabolic yield with which monomer flavan-3-ols and proanthocyanidins are metabolized in humans. This work clarified in a comprehensive way for the first time the ADME of a (poly)phenol family, highlighting the pool of circulating compounds that might be determinants of the putative beneficial effects linked to flavan-3-ol intake. Lastly, methodological inputs for implementing well-designed human and experimental model studies were provided.

Dynamics and determinants of human plasma bile acid profiles during dietary challenges.

In recent years, bile acids (BA) have received great interest due to their pleiotropic biological activity and the presence of plasma membrane-bound and nuclear receptors. Moreover, BA in blood have been identified by metabolite screening approaches as biomarkers that are associated with various diseases and even with a human longevity phenotype. With the growing interest in the microbiota contribution to the health-disease trajectory, BA that undergo deconjugation and other modifications by bacteria in the large intestine have become a prime target as a microbiome diversity modifier. We here profiled BA by a quantitative and a semiquantitative approach in 15 healthy and phenotypically very similar young individuals for over a 36-h fasting period, an oral glucose tolerance test (OGTT), and an oral lipid tolerance test (OLTT). We demonstrate a remarkable heterogeneity of the responses and describe the different dynamics of the plasma changes that likely originate from different routes by which BA enters the peripheral blood, and that may represent a direct secretion from the liver into the blood and a route that reaches the blood as a spill-over after passing from the gallbladder through the intestine and the portal system. We discuss the finding that an individual transport process involved in the passage of BA could be a critical determinant in the kinetics of plasma appearance and the overall phenotypic variability found.

Plasma Metabolic Signatures of Healthy Overweight Subjects Challenged With an Oral Glucose Tolerance Test.

Insulin secretion following ingestion of a carbohydrate load affects a multitude of metabolic pathways that simultaneously change direction and quantity of interorgan fluxes of sugars, lipids and amino acids. In the present study, we aimed at identifying markers associated with differential responses to an OGTT a population of healthy adults. By use of three metabolite profiling platforms, we assessed these postprandial responses of a total of 202 metabolites in plasma of 72 healthy volunteers undergoing comprehensive phenotyping and of which half enrolled into a weight-loss program over a three-month period. A standard oral glucose tolerance test (OGTT) served as dietary challenge test to identify changes in postprandial metabolite profiles. Despite classified as healthy according to WHO criteria, two discrete clusters (A and B) were identified based on the postprandial glucose profiles with a balanced distribution of volunteers based on gender and other measures. Cluster A individuals displayed 26% higher postprandial glucose levels, delayed glucose clearance and increased fasting plasma concentrations of more than 20 known biomarkers of insulin resistance and diabetes previously identified in large cohort studies. The volunteers identified by canonical postprandial responses that form cluster A may be called pre-pre-diabetics and defined as "at risk" for development of insulin resistance. Moreover, postprandial changes in selected fatty acids and complex lipids, bile acids, amino acids, acylcarnitines and sugars like mannose revealed marked differences in the responses seen in cluster A and cluster B individuals that sustained over the entire challenge test period of 240 min. Almost all metabolites, including glucose and insulin, returned to baseline values at the end of the test (at 240 min), except a variety of amino acids and here those that have been linked to diabetes development. Analysis of the corresponding metabolite profile in a fasting blood sample may therefore allow for early identification of these subjects at risk for insulin resistance without the need to undergo an OGTT.

Salivary nitrate/nitrite and acetaldehyde in humans: potential combination effects in the upper gastrointestinal tract and possible consequences for the in vivo formation of N-nitroso compounds-a hypothesis.

Subsequent to the dietary uptake of nitrate/nitrite in combination with acetaldehyde/ethanol, combination effects resulting from the sustained endogenous exposure to nitrite and acetaldehyde may be expected. This may imply locoregional effects in the upper gastrointestinal tract as well as systemic effects, such as a potential influence on endogenous formation of N-nitroso compounds (NOC). Salivary concentrations of the individual components nitrate and nitrite and acetaldehyde are known to rise after ingestion, absorption and systemic distribution, thereby reflecting their respective plasma kinetics and parallel secretion through the salivary glands as well as the microbial/enzymatic metabolism in the oral cavity. Salivary excretion may also occur with certain drug molecules and food constituents and their metabolites. Therefore, putative combination effects in the oral cavity and the upper digestive tract may occur, but this has remained largely unexplored up to now. In this Guest Editorial, published evidence on exposure levels and biokinetics of nitrate/nitrite/NOx, NOC and acetaldehyde in the organism is reviewed and knowledge gaps concerning combination effects are identified. Research is suggested to be initiated to study the related unresolved issues.

Formation of B- and M-group aflatoxins and precursors by Aspergillus flavus on maize and its implication for food safety.

Aflatoxins count to the most toxic known mycotoxins and are a threat to food safety especially in regions with a warm and humid climate. Contaminated food reaches consumers globally due to international trade, leading to stringent regulatory limits of aflatoxins in food. While the formation of aflatoxin (AF) B1 by the filamentous fungus Aspergillus flavus is well investigated, less is known about the formation kinetics of its precursors and further aflatoxins. In this study, autoclaved maize kernels were inoculated with A. flavus and incubated at 25 °C for up to 10 days. Aflatoxins and precursors were analyzed by a validated UHPLC-MS method. Additional to AFB1 and AFB2, AFM1 and AFM2 were detected, confirming the ability of the formation of M-group aflatoxins on cereals by A. flavus. The measured relative levels of AFB2, AFM1, and AFM2 on maize compared to the level of AFB1 (mean of days 5, 7, and 10 of incubation) were 3.3%, 1.5%, and 0.2%, respectively. The occurrence and kinetics of the measured aflatoxins and their precursors sterigmatocystin, O-methylsterigmatocystin, 11-hydroxy-O-methylsterigmatocystin, aspertoxin, and 11-hydroxyaspertoxin (group 1) as well as of dihydrosterigmatocystin and dihydro-O-methylsterigmatocystin (group 2) supported the so far postulated biosynthetic pathway. Remarkable high levels of O-methylsterigmatocystin and aspertoxin (17.4% and 4.9% compared to AFB1) were found, raising the question about the toxicological relevance of these intermediates. In conclusion, based on the study results, the monitoring of O-methylsterigmatocystin and aspertoxin as well as M-group aflatoxins in food is recommended.

Metabolism of glyphosate by the human fecal microbiota.

Glyphosate is the most frequently used herbicide worldwide and its application is under discussion due to health concerns. As humans may be exposed to glyphosate, the present study investigated the metabolism of glyphosate by the human fecal microbiota in vitro. Human fecal samples were collected from 15 different volunteers and fecal suspensions were prepared. The human fecal suspension samples were incubated with glyphosate under strictly anaerobic conditions and glyphosate degradation was investigated. Neither a degradation of glyphosate, nor a formation of AMPA (aminomethylphosphonic acid), the known microbial metabolite in soil, was detected. In conclusion, the microbiota of human fecal suspensions did not metabolize glyphosate under the conditions used in our study which hints at the assumption that transformation of glyphosate by the gut microbiota seems to be negligible in humans.

Metabolism of Daidzein and Genistein by Gut Bacteria of the Class Coriobacteriia.

The intake of isoflavones is presumed to be associated with health benefits in humans, but also potential adverse effects of isoflavones are controversially discussed. Isoflavones can be metabolized by gut bacteria leading to modulation of the bioactivity, such as estrogenic effects. Especially bacterial strains of the Eggerthellaceae, a well-known bacterial family of the human gut microbiota, are able to convert the isoflavone daidzein into equol. In addition, metabolization of genistein is also described for strains of the Eggerthellaceae. The aim of this study was to identify and investigate gut bacterial strains of the family Eggerthellaceae as well as the narrowly related family Coriobacteriaceae which are able to metabolize daidzein and genistein. This study provides a comprehensive, polyphasic approach comprising in silico analysis of the equol gene cluster, detection of genes associated with the daidzein, and genistein metabolism via PCR and fermentation of these isoflavones. The in silico search for protein sequences that are associated with daidzein metabolism identified sequences with high similarity values in already well-known equol-producing strains. Furthermore, protein sequences that are presumed to be associated with daidzein and genistein metabolism were detected in the two type strains 'Hugonella massiliensis' and Senegalimassilia faecalis which were not yet described to metabolize these isoflavones. An alignment of these protein sequences showed that the equol gene cluster is highly conserved. In addition, PCR amplification supported the presence of genes associated with daidzein and genistein metabolism. Furthermore, the metabolism of daidzein and genistein was investigated in fermentations of pure bacterial cultures under strictly anaerobic conditions and proofed the metabolism of daidzein and genistein by the strains 'Hugonella massiliensis' DSM 101782T and Senegalimassilia faecalis KGMB04484T.