Comparison and Optimization of Different Protein Nitrogen Quantitation and Residual Protein Characterization Methods in Dietary Fiber Preparations.

Proteins are plant cell wall components but they are not included in the definition of dietary fiber. Therefore, dietary fiber preparations have to be corrected for their residual protein contents. This is commonly done by calculating the residual protein concentrations from the nitrogen contents after Kjeldahl digestion. Here, three different methods to determine nitrogen in Kjeldahl digests were compared: conventional titration with hydrochloric acid after steam distillation, a colorimetric assay (24-well microplates and cuvettes), and the determination by using an ammonia electrode. All assays gave similar results but detection using the ammonia electrode was found to be the most time-efficient approach. Also, an amino-acid profiling method, which is not based on commercial kits and which is suitable for routine analysis of dietary fiber preparations, was established. For this purpose, an HPLC-FLD method following amino acid derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was optimized for fiber samples. Although all commonly used dietary fiber preparation methods involve the application of proteases the amino acid profiles of fiber samples from different sources were shown to be quite diverse. Considering the amino acid composition of the residual protein in various dietary fiber preparations, residual protein is probably not only based on structural proteins.

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.

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.

A stable-isotope dilution GC-MS approach for the analysis of DFRC (derivatization followed by reductive cleavage) monomers from low-lignin plant materials.

The derivatization followed by reductive cleavage (DFRC) method is a well-established tool to characterize the lignin composition of plant materials. However, the application of the original procedure, especially the chromatographic determination of the DFRC monomers, is problematic for low-lignin foods. To overcome these problems a modified sample cleanup and a stable-isotope dilution approach were developed and validated. To quantitate the diacetylated DFRC monomers, their corresponding hexadeuterated analogs were synthesized and used as internal standards. By using the selected-ion monitoring mode, matrix-associated interferences can be minimized resulting in higher selectivity and sensitivity. The modified method was applied to four low-lignin samples. Lignin from carrot fibers was classified as guaiacyl-rich whereas the lignins from radish, pear, and asparagus fibers where classified as balanced lignins (guaiacyl/syringyl ratio=1-2).