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A high-density phylogenetic microarray targeting small subunit rRNA (SSU rRNA) sequences of over 1000 microbial phylotypes of the human gastrointestinal tract, the HITChip, was used to assess the impact of faecal inoculum preparation and operation conditions on an in vitro model of the human large intestine (TIM-2). This revealed that propagation of mixed faecal donations for the production of standardized inocula has only a limited effect on the microbiota composition, with slight changes observed mainly within the Firmicutes. Adversely, significant shifts in several major groups of intestinal microbiota were observed after inoculation of the in vitro model. Hierarchical cluster analysis was able to show that samples taken throughout the inoculum preparation grouped with microbiota profiles observed for faecal samples of healthy adults. In contrast, the TIM-2 microbiota was distinct. While members of the Bacteroidetes and some groups within the Bacilli were increased in TIM-2 microbiota, a strong reduction in the relative abundance of other microbial groups, including Bifidobacterium spp., Streptococcus spp., and Clostridium clusters IV and XIVa, was observed. The changes detected with the HITChip could be confirmed using denaturing gradient gel electrophoresis (DGGE) of SSU rRNA amplicons. DOI 10.1099/mic.0.042044-0

The release characteristics of a unique blend of carotenoid beadlets designed to increase bioavailability were tested using the dynamic gastrointestinal model TIM‐1. Individual carotenoid bioaccessibility peaks were observed over approximately 3–4 hr in the order of lutein and zeaxanthin first, followed by lycopene, and then finally α‐ and β‐carotene; when tested as a beadlet blend or when the beadlets were compressed into tablets. Bioaccessibility measurements of 7%–20% were similar to those previously reported in literature and comparable between the two formulations, beadlet blend and tablet formulations. Total recovery of carotenoids from all compartments ranged from 70% to 90% for all carotenoids, except lycopene where almost 50% was unrecoverable after digestion in the TIM system. [ABSTRACT FROM AUTHOR]

Lactulose, a disaccharide of galactose and fructose, used as a laxative or ammonia-lowering drug and as a functional food ingredient, enhances growth of Bifidobacterium and Lactobacillus at clinically relevant dosages. The prebiotic effect of subclinical dosages of Lactulose, however, remains to be elucidated. This study analyses changes in the microbiota and their metabolites after a 5 days Lactulose treatment using the TIM-2 system, a computer-controlled model of the proximal large intestine representing a complex, high density, metabolically active, anaerobic microbiota of human origin. Subclinical dosages of 2-5 g Lactulose were used. While 2 g Lactulose already increased the short-chain fatty acid levels of the intestinal content, 5 g Lactulose were required daily for 5 days in this study to exert the full beneficial prebiotic effect consisting of higher bacterial counts of Bifidobacterium, Lactobacillus, and Anaerostipes, a rise in acetate, butyrate and lactate, as well as a decrease in branched-chain fatty acids, pH (suggested by an increase in NaOH usage), and ammonia. [ABSTRACT FROM AUTHOR]

Oral iron administration in African children can increase the risk for infections. However, it remains unclear to what extent supplementary iron affects the intestinal microbiome. We here explored the impact of iron preparations on microbial growth and metabolism in the well-controlled TNO's in vitro model of the large intestine (TIM-2). The model was inoculated with a human microbiota, without supplementary iron, or with 50 or 250μmol/L ferrous sulfate, 50 or 250μmol/L ferric citrate, or 50μmol/L hemin. High resolution responses of the microbiota were examined by 16S rDNA pyrosequencing, microarray analysis, and metagenomic sequencing. The metabolome was assessed by fatty acid quantification, gas chromatography-mass spectrometry (GC-MS), and 1H-NMR spectroscopy. Cultured intestinal epithelial Caco-2 cells were used to assess fecal water toxicity. Microbiome analysis showed, among others, that supplementary iron induced decreased levels of Bifidobacteriaceae and Lactobacillaceae, while it caused higher levels of Roseburia and Prevotella. Metagenomic analyses showed an enrichment of microbial motility-chemotaxis systems, while the metabolome markedly changed from a saccharolytic to a proteolytic profile in response to iron. Branched chain fatty acids and ammonia levels increased significantly, in particular with ferrous sulfate. Importantly, the metabolite-containing effluent from iron-rich conditions showed increased cytotoxicity to Caco-2 cells. Our explorations indicate that in the absence of host influences, iron induces a more hostile environment characterized by a reduction of microbes that are generally beneficial, and increased levels of bacterial metabolites that can impair the barrier function of a cultured intestinal epithelial monolayer. [ABSTRACT FROM AUTHOR]

A validated in vitro model of the large intestine ( TIM-2), set up with human or pig faeces, was used to evaluate the impact of potentially probiotic Lactobacillus amylovorus DSM 16698, administered alone (i), in the presence of prebiotic galactooligosaccharides ( GOS) (ii), and co-administered with probiotic Bifidobacterium animalis ssp. lactis Bb-12 ( Bb-12) (iii) on GOS degradation, microbial growth ( L. amylovorus, lactobacilli, bifidobacteria and total bacteria) and metabolite production. High performance anion exchange chromatography revealed that GOS degradation was more pronounced in TIM-2 inoculated with pig faeces than with human faeces. Denaturing gradient gel electrophoresis profiling of PCR-amplified 16S r RNA genes detected a more complex Lactobacillus spp. community in pig faecal material than in human faecal inoculum. According to 16S r RNA gene-targeted q PCR, GOS stimulated the growth of lactobacilli and bifidobacteria in faecal material from both materials. The cumulative production of short chain fatty acids and ammonia was higher ( P < 0.05) for pig than for human faeces. However, lactate accumulation was higher ( P < 0.05) in the human model and increased after co-administration with GOS and Bb-12. This study reinforced the notion that differences in microbiota composition between target host organisms need to be considered when animal data are extrapolated to human, as is often done with pre- and probiotic intervention studies. [ABSTRACT FROM AUTHOR]

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]

This study introduces a stable-isotope metabolic approach employing [U-13C]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-13C]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 13C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed 13C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the 12C contents and 13C 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. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The article discusses a study which determined the effect of maize-based fibers on the activity and composition of microbiota in the colon. Results showed that by microbial metabolites analysis, an increase in short-chain fatty acids for all fibers, including different butyrate production levels for each fiber, were identified. All the fibers reduced toxic metabolites and showed increased numbers of bifidobacteria and Lactobacillus species. The study concluded that all fibers displayed prebiotic activity with regard to more beneficial microbes.

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-13C]-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 13C-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 13C-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. [ABSTRACT FROM AUTHOR]

A polymerase chain reaction (PCR)-based method was developed for the identification of isolates of Bifidobacterium at the species level. Using two Bifidobacterium-specific primers directed against the 16S ribosomal gene (Bif164 and Bif662), a PCR product was obtained from the type strains of 12 different bifidobacterial species that have been found in the human alimentary tract. After purification of the PCR products, the DNA was restricted with five different restriction enzymes. The size of the different restriction fragments was used as a fingerprint for the identification of individual bifidobacterial species. The amplified ribosomal DNA restriction analysis method was subsequently used to speciate bifidobacterial isolates from child’s feces and from an in vitro model of the large intestine. [Copyright &y& Elsevier]

Galacto-oligosaccharides (GOS) are considered to be prebiotic, although 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 (13)C-labeled GOS. The normal (control) medium and unlabeled or (13)C-labeled GOS was added to a dynamic, validated, in vitro model of the large-intestine containing an adult-type microbiota. Liquid-chromatography MS was used to measure the incorporation of (13)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 primary members within the complex microbiota that were directly involved in GOS fermentation were shown to be Bifidobacterium longum, B. bifidum, B. catenulatum, Lactobacillus gasseri, and L. salivarius, in line with the prebiotic effect of GOS, although some other species incorporated (13)C 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.

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.