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Metabolite production and antioxidant released during colonic fermentation of naturally occurring dietary fiber (DF) from two European diets (Mediterranean and Scandinavian) were determined. With this aim, DF and associated components were isolated from both whole diets, as well as from cereals and fruits and vegetables comprising the diets. DF was used as substrate for colonic fermentation in a dynamic in vitro model of the colon, samples were collected, and fermentation metabolites were analyzed. Statistical differences between samples were observed in the concentrations of short-chain fatty acids and ammonia and in the ratio acetate/propionate/butyrate. Whole grain cereal DF generated a larger amount of propionate than refined flour cereal DF. Fruit and vegetable DF generated higher amounts of butyrate than cereal DF. Most antioxidant compounds were released from DF during in vitro colonic fermentation. It is concluded that different sources of DF may play a specific role in health maintenance mediated by metabolites produced during colonic fermentation.

This study introduces a stable-isotope metabolic approach employing [U-(13)C]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-(13)C]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a (13)C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed (13)C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the (12)C contents and (13)C labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.

Galacto-oligosaccharides (GOS) are considered to be prebiotic, altliough the contribution of specific members of the microbiota to GOS fermentation and the exact microbial metabolites that are produced upon GOS fermentation are largely unknown. We aimed to determine this using uniformly 13C-labeled GOS. The normal (control) medium and unlabeled or 13C-labeled GOS was added to a dynamic, validated, in vitro model of the large-intestine containing an aduit-type microbiota. Liquid-chromatography MS was used to measure the incorporation of C label into metabolites. 16S-rRNA stable isotope probing coupled to a phylogenetic micro-array was used to determine label incorporation in microbial biomass. The priman/ members within the complex microbiota that were directly involved in GOS fermentation were shown to be Bifidobacterium longum, B. bifidum, B. catenulatum, Lactobacillus gasseh, and L. salivarius, in line with the prebiotic effect of GOS, although some other species incorporated label also. GOS fermentation led to an increase in acetate (+49%) and lactate (+23%) compared with the control. Total organic acid production was 8.50 and 7.52 mmol/g of carbohydrate fed for the GOS and control experiments, respectively. At the same time, the cumulative production of putrefactive metabolites (branched-chain fatty acids and ammonia) was reduced by 55%. Cross-feeding of metabolites from primary GOS fermenters to other members of the microbiota was observed. Our findings support a prebiotic role for GOS and its potential to act as a synbiotic in combination with certain probiotic strains. [ABSTRACT FROM AUTHOR]

Carbohydrates, including starches, are an important energy source for humans, and are known for their interactions with the microbiota in the digestive tract. Largely, those interactions are thought to promote human health. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP), we identified starch-fermenting bacteria under human colon-like conditions. To the microbiota of the TIM-2 in vitro model of the human colon 7.4 g l−1 of [U-13C]-starch was added. RNA extracted from lumen samples after 0 (control), 2, 4 and 8 h was subjected to density-gradient ultracentrifugation. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting and phylogenetic analyses of the labelled and unlabelled 16S rRNA suggested populations related to Ruminococcus bromii, Prevotella spp. and Eubacterium rectale to be involved in starch metabolism. Additionally, 16S rRNA related to that of Bifidobacterium adolescentis was abundant in all analysed fractions. While this might be due to the enrichment of high-GC RNA in high-density fractions, it could also indicate an active role in starch fermentation. Comparison of the T-RFLP fingerprints of experiments performed with labelled and unlabelled starch revealed Ruminococcus bromii as the primary degrader in starch fermentation in the studied model, as it was found to solely predominate in the labelled fractions. LC-MS analyses of the lumen and dialysate samples showed that, for both experiments, starch fermentation primarily yielded acetate, butyrate and propionate. Integration of molecular and metabolite data suggests metabolic cross-feeding in the system, where populations related to Ruminococcus bromii are the primary starch degrader, while those related to Prevotella spp., Bifidobacterium adolescentis and Eubacterium rectale might be further involved in the trophic chain. [ABSTRACT FROM AUTHOR]