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NMR in Biomedicine 07/2009; 23(1):2 - 12. · 3.21 Impact Factor
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ABSTRACT: 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.
NMR in Biomedicine 07/2009; 23(1):2-12. · 3.21 Impact Factor
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ABSTRACT: 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-(13)C]-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.
Environmental Microbiology 01/2009; 11(4):914-26. · 5.84 Impact Factor
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ABSTRACT: The importance of the human large intestine for nutrition, health, and disease, is becoming increasingly realized. There are numerous indications of a distinct role for the gut in such important issues as immune disorders and obesity-linked diseases. Research on this long-neglected organ, which is colonized by a myriad of bacteria, is a rapidly growing field that is currently providing fascinating new insights into the processes going on in the colon, and their relevance for the human host. This review aims to give an overview of studies dealing with the physiology of the intestinal microbiota as it functions within and in interaction with the host, with a special focus on approaches involving stable isotopes. We have included general aspects of gut microbial life as well as aspects specifically relating to genomic, proteomic, and metabolomic studies. A special emphasis is further laid on reviewing relevant methods and applications of stable isotope-aided metabolic flux analysis (MFA). We argue that linking MFA with the '-omics' technologies using innovative modeling approaches is the way to go to establish a truly integrative and interdisciplinary approach. Systems biology thus actualized will provide key insights into the metabolic regulations involved in microbe-host mutualism and their relevance for health and disease.
Advances in Microbial Physiology 02/2008; 53:73-168. · 9.88 Impact Factor
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ABSTRACT: 16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-(13)C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct (13)C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the (13)C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.
FEMS Microbiology Ecology 05/2007; 60(1):126-35. · 3.41 Impact Factor
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ABSTRACT: Molecular tools have revealed wide microbial diversity in the human alimentary tract. Most intestinal microorganisms have not been cultured and the in situ functions of distinct groups of the intestinal microbiota are largely unknown but pivotal to understanding the role of these microorganisms in health and disease. Promising strategies to gain more insight into the functionality of the complex microbial communities in the human alimentary tract, including fermentation processes in the colon, are discussed. These research approaches could provide a basis for the definition of a healthy gut based on key properties of microbial functionality. This will also enable the development of direct nutritional strategies for intestinal disease prevention and health promotion.
Trends in Microbiology 03/2006; 14(2):86-91. · 7.91 Impact Factor
