The Human Fecal Microbiota Metabolizes Foodborne Heterocyclic Aromatic Amines by Reuterin Conjugation and Further Transformations.

SCOPE: Heterocyclic aromatic amines (HAAs) are process-induced food contaminants with high mutagenic and/or carcinogenic potential. Although the human gut microbiota is known to affect the metabolism of dietary constituents, its impact on HAA metabolism and toxicity has been little studied. Here, the glycerol-dependent metabolism of seven foodborne HAAs (AαC, Trp-P-1, harman, norharman, PhIP, MeIQx, and MeIQ) by the human fecal microbiota is investigated. METHODS AND RESULTS: As analyzed by HPLC-DAD/FLD, the extent of conversion is strongly dependent on glycerol supplementation and HAA structure. AαC (60-100%) and the 2-aminoimidazoazarenes (up to 58%) are especially prone to microbial conversion. Based on high-resolution MS and/or NMR spectroscopy data, 70 fecal metabolites are identified in total, mainly formed by chemical reactions with one or two molecules of microbially derived reuterin. Moreover, it has been demonstrated that the human fecal microbiota can further transform reuterin adducts by reduction and/or hydroxylation reactions. Upon isolation, some reuterin-induced HAA metabolites appear to be partially unstable, complicating structural identification. CONCLUSION: The formation of microbial metabolites needs to be incorporated into risk assessment considerations for HAAs in human health. In this study, several HAA metabolites, mainly reuterin-dependent, are identified in vitro, providing the basis for future human studies investigating microbial HAA metabolism.

Metabolization of the Advanced Glycation End Product N-ε-Carboxymethyllysine (CML) by Different Probiotic E. coli Strains.

N-ε-Carboxymethyllysine (CML) is formed during glycation reactions (synonym, Maillard reaction). CML is degraded by the human colonic microbiota, but nothing is known about the formation of particular metabolites. In the present study, six probiotic E. coli strains were incubated with CML in the presence or absence of oxygen in either minimal or nutrient-rich medium. CML was degraded by all strains only in the presence of oxygen. HPLC-MS/MS was applied for identification of metabolites of CML. For the first time, three bacterial metabolites of CML have been identified, namely N-carboxymethylcadaverine (CM-CAD), N-carboxymethylaminopentanoic acid (CM-APA), and the N-carboxymethyl-Δ1-piperideinium ion. During 48 h of incubation of CML with five different E. coli strains in minimal medium in the presence of oxygen, 37-66% of CML was degraded, while CM-CAD (1.5-8.4% of the initial CML dose) and CM-APA (0.04-0.11% of the initial CML dose) were formed linearly. Formation of the metabolites is enhanced when dipeptide-bound CML is applied, indicating that transport phenomena may play an important role in the "handling" of the compound by microorganisms.

Yeast Metabolites of Glycated Amino Acids in Beer.

Glycation reactions (Maillard reactions) during the malting and brewing processes are important for the development of the characteristic color and flavor of beer. Recently, free and protein-bound Maillard reaction products (MRPs) such as pyrraline, formyline, and maltosine were found in beer. Furthermore, these amino acid derivatives are metabolized by Saccharomyces cerevisiae via the Ehrlich pathway. In this study, a method was developed for quantitation of individual Ehrlich intermediates derived from pyrraline, formyline, and maltosine. Following synthesis of the corresponding reference material, the MRP-derived new Ehrlich alcohols pyrralinol (up to 207 µg/L), formylinol (up to 50 µg/L), and maltosinol (up to 6.9 µg/L) were quantitated for the first time in commercial beer samples by reverse phase high performance liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode. This is equivalent to ca. 20-40% of the concentrations of the parent glycated amino acids. The metabolites were almost absent from alcohol-free beers and malt-based beverages. Two previously unknown valine-derived pyrrole derivatives were characterized and qualitatively identified in beer. The metabolites investigated represent new process-induced alkaloids that may influence brewing yeast performance due to structural similarities to quorum sensing and metal-binding molecules.

Metabolism of Foodborne Heterocyclic Aromatic Amines by Lactobacillus reuteri DSM 20016.

The heterocyclic aromatic amine (HAA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is converted into 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) via a chemical reaction with 3-hydroxypropionaldehyde or acrolein derived from glycerol by reuterin producing gut bacteria. Because it is unknown whether this reaction also applies to other HAAs, seven foodborne HAAs (2-amino-9H-pyrido[2,3-b]indole (AαC), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethyl-3H-imidazo[4,5-f]quinoxaline (MeIQx), 9H-pyrido[3,4-b]indole (norharman), and 1-methyl-9H-pyrido[3,4-b]indole (harman)) were anaerobically incubated with Lactobacillus reuteri DSM 20016 in the presence of glycerol. The extent of conversion, as analyzed by HPLC-DAD/FLD, was dependent on both the studied HAAs and the glucose/glycerol ratio, indicating reuterin to be involved in HAA metabolism. Based on HRMS analyses, PhIP-M1-type metabolites were detected for AαC, Trp-P-1, IQ, MeIQ, MeIQx, harman, and norharman. In the case of AαC, this was confirmed by metabolite isolation (AαC-M8, 2,3,4,10-tetrahydro-1H-indolo[2,3-b][1,8]naphthyridin-2-ol) and one- (1H) and two-dimensional (HSQC, HMBC, COSY, DOSY) NMR spectroscopy. In addition, based on HRMS and/or NMR spectroscopy, a new type of HAA metabolite, resulting from the reaction with two molecules of 3-hydroxypropionaldehyde or acrolein, is hypothesized for AαC, Trp-P-1, IQ, MeIQ, and MeIQx.

Transformation of Free and Dipeptide-Bound Glycated Amino Acids by Two Strains of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae transforms branched-chain and aromatic amino acids into higher alcohols in the Ehrlich pathway. During microbiological culturing and industrial fermentations, this yeast is confronted with amino acids modified by reducing sugars in the Maillard reaction (glycation). In order to gain some preliminary insight into the physiological "handling" of glycated amino acids by yeasts, individual Maillard reaction products (MRPs: fructosyllysine, carboxymethyllysine, pyrraline, formyline, maltosine, methylglyoxal-derived hydroimidazolone) were administered to two strains of S. cerevisiae in a rich medium. Only formyline was converted into the corresponding α-hydroxy acid, to a small extent (10 %). Dipeptide-bound pyrraline and maltosine were removed from the medium with concomitant emergence of several metabolites. Pyrraline was mainly converted into the corresponding Ehrlich alcohol (20-60 %) and maltosine into the corresponding α-hydroxy acid (40-60 %). Five specific metabolites of glycated amino acids were synthesized and characterized. We show for the first time that S. cerevisiae can use glycated amino acids as a nitrogen source and transform them into new metabolites, provided that the substances can be transported across the cell membrane.