Koen Venema

The Effect of Various Inulins and Clostridium difficile on the Metabolic Activity of the Human Colonic Microbiota in vitro

The influence of inulins with different average degree of polymerization (ranging from 3 to 25) on the metabolic activity of the human colonic microbiota with or without the addition of Clostridium difficile was investigated in vitro. The in vitro system used was a dynamic, computer-controlled model that simulates the conditions of the proximal part of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products. The addition of inulin stimulated the formation of the total amount of short-chain fatty acids acetate, propionate and butyrate up to 50%, and lactate >10-fold for short-chain inulin, while the formation of ammonia and the branched-chain fatty acids iso-butyrate and iso-valerate was suppressed. Ammonia formation was suppressed by about 30% and that of iso-butyrate and iso-valerate was almost completely suppressed. These effects became much more pronounced when C. difficile was present in the system. The introduction of C. difficile caused a stimulation of the production of the protein fermentative metabolites ammonia, branched-chain fatty acids and the phenolic compounds indole, phenol and p-cresol. This stimulatory effect of C. difficile was almost completely prevented by the addition of inulins. Thus, these results indicate a potential of inulins to shift the metabolic activity of the human colonic microbiota towards the production of less potentially toxic metabolites, both under normal conditions and under conditions with a disturbed microbiota (with a high level of C. difficile).

The metabolic activity of fecal microbiota from healthy individuals and patients with inflammatory bowel disease.

The hypothesis was studied that intestinal microbial metabolites play a role in the pathogenesis of inflammatory bowel disease. For that purpose, an in vitro model of the colon was inoculated with fresh feces of six healthy individuals and eight inflammatory bowel disease patients. Samples were taken from the model over time to analyze metabolites from both saccharolytic and proteolytic fermentation. Microbiotas from inflammatory bowel disease patients produced significantly more short-chain fatty acids and ammonia than microbiotas from healthy individuals. Furthermore, the branched-chain fatty acid production was 25% higher after inoculation with microbiotas from patients than after inoculation with microbiotas from healthy individuals. Phenolic compounds were produced by all microbiotas, with large interindividual variation. The production of (potentially toxic) metabolites may play a role in the onset or chronicity of inflammatory bowel disease, because they were produced in higher amounts by microbiotas from these patients than by microbiotas from healthy individuals.

The Effect of Lactulose on the Composition of the Intestinal Microbiota and Short-chain Fatty Acid Production in Human Volunteers and a Computer- controlled Model of the Proximal Large Intestine

The objective of this study was to compare the in vivo effect of lactulose on faecal parameters with the effect in a dynamic, computer-controlled in vitro model of the proximal large intestine (TIM-2). Faecal samples from 10 human volunteers collected before (non-adapted) and after 1 week of treatment (10 g/day) with lactulose (lactulose-adapted) were investigated. Parameters were compared immediately in the faecal samples, and after incubation in the in vitro model of the large intestine. After an adaptation period of the faecal microbiota in the in vitro model of the proximal colon, lactulose (10 g/day) was fed to the microbiota over a 48-h period. Samples taken from the model were investigated for microbiota composition and metabolite production (short-chain fatty acids (SCFAs) and lactate). No changes in the faecal parameters pH, dry weight or SCFA ratio were observed in the in vivo samples. However, the results show a major change in the ratio of SCFAs produced in the in vitro model, with a drastic reduction of butyrate production on lactulose. This was clear in the non-adapted microbiota by the observed arrest in butyrate production 24 h after the start of lactulose feeding. However, in the adapted microbiota butyrate production was already low from the start of the experiment. In fact, only the microbiota of one of the 10 individuals still produced significant amounts of butyrate after lactulose adaptation, the concentration in the other samples was extremely low. Similarly, in the in vitro model lactate production of the non-adapted microbiota started after approximately 24 h, whereas the adapted microbiota produced lactate from the start. In faecal (in vivo) samples no changes in microbiota composition were obvious, except for a significant increase in Bifidobacterium counts after lactulose feeding. With classic plating techniques, the in vitro samples showed an increase in Lactobacillus and Enterococcus species. With denaturing gradient gel electrophoresis, a clear change in banding pattern was observed, indicating a shift in microbiota composition. When the major bands that appeared after lactulose feeding in the in vitro model were excised and sequenced, the sequences showed homology to Lactobacillus and Enterococcus species. This is in agreement with the classic plating technique as well as with the observed increase in lactate production. Sampling in vivo at ‘the site where it all happens’ (the proximal colon) is difficult and inconvenient. We conclude that the in vitro model for the proximal colon reflects much better the fermentation of lactulose, in both metabolite production and changes in microbiota composition, than do faecal samples from an in vivo experiment. Therefore, the in vitro model is an excellent tool with which to study bioconversion of functional food components and/or drugs.

Immunomodulatory effects of potential probiotics in a mouse peanut sensitization model

Peanut allergy accounts for the majority of severe food-related allergic reactions and there is a need for new prevention and treatment strategies. Probiotics may be considered for treatment on the basis of their immunomodulating properties. Cytokine profiles of probiotic strains were determined by in vitro co-culture with human PBMCs. Three strains were selected to investigate their prophylactic potential in a peanut sensitization model by analysing peanut-specific antibodies, mast cell degranulation and ex vivo cytokine production by splenocytes. The probiotic strains induced highly variable cytokine profiles in PBMCs. L.xa0salivarius HMI001, L.xa0casei Shirota (LCS) and L.xa0plantarum WCFS1 were selected for further investigation owing to their distinct cytokine patterns. Prophylactic treatment with both HMI001 and LCS attenuated the Th2 phenotype (reduced mast cell responses and ex vivo IL-4 and/or IL-5 production). In contrast, WCFS1 augmented the Th2 phenotype (increased mast cell and antibody responses and ex vivo IL-4 production). In vitro PBMC screening was useful in selecting strains with anti-inflammatory and Th1 skewing properties. In case of HMI001 (high IL-10/IL-12 ratio) and LCS (high interferon-γ and IL-12), partial protection was seen in a mouse peanut allergy model. Strikingly, certain strains may worsen the allergic reaction as shown in the case of WCFS1.

Dynamic System of the Gastrointestinal Tract to Study Nutritional Quality

Abstract A goal of improvement of crob breeding and food processing is to increase the nutritional quality, which is related to the availability of nutrients. This paper describes a dynamic, computer-controlled system that mimics to a high degree the successive kinetic conditions in the stomach and intestines. The system has been validated in comparison to human and animal studies for a broad variety of applications, and showed a high extrapolation value. It is used successfully for the assessment of nutritional quality and the development of novel and functional foods.