Effects of probiotics and antibiotics on the intestinal homeostasis in a computer controlled model of the large intestine.

BACKGROUND: Antibiotic associated diarrhea and Clostridium difficile infection are frequent complications of broad spectrum antibiotic therapy. Probiotic bacteria are used as therapeutic and preventive agents in these disorders, but the exact functional mechanisms and the mode of action are poorly understood. The effects of clindamycin and the probiotic mixture VSL#3 (containing the 8 bacterial strains Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus delbrueckii subsp. Bulgaricus) consecutively or in combination were investigated and compared to controls without therapy using a standardized human fecal microbiota in a computer-controlled in vitro model of large intestine. Microbial metabolites (short chain fatty acids, lactate, branched chain fatty acids, and ammonia) and the intestinal microbiota were analyzed. RESULTS: Compared to controls and combination therapy, short chain fatty acids and lactate, but also ammonia and branched chain fatty acids, were increased under probiotic therapy. The metabolic pattern under combined therapy with antibiotics and probiotics had the most beneficial and consistent effect on intestinal metabolic profiles. The intestinal microbiota showed a decrease in several indigenous bacterial groups under antibiotic therapy, there was no significant recovery of these groups when the antibiotic therapy was followed by administration of probiotics. Simultaneous application of anti- and probiotics had a stabilizing effect on the intestinal microbiota with increased bifidobacteria and lactobacilli. CONCLUSIONS: Administration of VSL#3 parallel with the clindamycin therapy had a beneficial and stabilizing effect on the intestinal metabolic homeostasis by decreasing toxic metabolites and protecting the endogenic microbiota from destruction. Probiotics could be a reasonable strategy in prevention of antibiotic associated disturbances of the intestinal homeostasis and disorders.

Isolation of bifidobacteria for blood group secretor status targeted personalised nutrition.

BACKGROUND: Currently, there is a constant need to find microbial products for maintaining or even improving host microbiota balance that could be targeted to a selected consumer group. Blood group secretor status, determining the ABO status, could be used to stratify the consumer group. OBJECTIVE: We have applied a validated upper intestinal tract model (TIM-1) and culturing methods to screen potential probiotic bacteria from faeces of blood secretor and non-secretor individuals. DESIGN: Faecal samples from healthy volunteers were pooled to age- and sex-matched secretor and non-secretor pools. Faecal pools were run through separate TIM-1 simulations, and bacteria were cultivated from samples taken at different stages of simulations for characterisation. RESULTS: Microbes in secretor pool survived the transit through TIM-1 system better than microbes of non-secretor pool, especially bifidobacteria and anaerobes were highly affected. The differences in numbers of bifidobacteria and lactobacilli isolates after plate cultivations and further the number of distinct RAPD-genotypes was clearly lower in non-secretor pool than in secretor pool. CONCLUSIONS: In the present study, we showed that microbiota of secretor and non-secretor individuals tolerate gastrointestinal conditions differently and that a combination of gastrointestinal simulations and cultivation methods proved to be a promising tool for isolating potentially probiotic bacteria.

Survival of Mycobacterium bovis BCG oral vaccine during transit through a dynamic in vitro model simulating the upper gastrointestinal tract of badgers.

In developing an oral bait BCG vaccine against tuberculosis in badgers we wanted to understand the conditions of the gastrointestinal tract and their impact on vaccine viability. Conditions mimicking stomach and small-intestine caused substantial reduction in BCG viability. We performed in vivo experiments using a telemetric pH monitoring system and used the data to parameterise a dynamic in vitro system (TIM-1) of the stomach and small intestine. Some BCG died in the stomach compartment and through the duodenum and jejunum compartments. BCG survival in the stomach was greatest when bait was absent but by the time BCG reached the jejunum, BCG viability was not significantly affected by the presence of bait. Our data suggest that from a starting quantity of 2.85 ± 0.45 x 108 colony-forming units of BCG around 2 log10 may be killed before delivery to the intestinal lymphoid tissue. There are economic arguments for reducing the dose of BCG to vaccinate badgers orally. Our findings imply this could be achieved if we can protect BCG from the harsh environment of the stomach and duodenum. TIM-1 is a valuable, non-animal model with which to evaluate and optimise formulations to maximise BCG survival in the gastrointestinal tract.

Diet drives quick changes in the metabolic activity and composition of human gut microbiota in a validated in vitro gut model.

The aim of this study was to screen how rapidly the human gut microbiota responds to diet in an in vitro model of the proximal colon (TIM-2 system). Two experimental diets were provided to the gut bacteria: a high carbohydrate and a high protein diet. The metabolic response and the composition of the microbiota were compared to a control diet simulating an average western meal. Short-chain and branched-chain fatty acids (SCFA and BCFA, respectively) production, in addition to changes in the community composition (profiling), were measured. The activity of the microbiota reflected differences between diets, exhibiting a trade-off between saccharolytic and proteolytic fermentation when compared to the control. Diversity analysis revealed a phylum-specific response depending on the diet tested. Most changes in the microbiome composition occurred during the first 24 h of the experiment. The outcome of this study elucidates the fact that human gut bacteria quickly respond to changes in diet. In addition, it confirms that variations in the concentration of carbohydrates and proteins modify the activity and composition of the microbiota, and these changes can potentially have an impact on the health of the host.

Evaluation of an optimal preparation of human standardized fecal inocula for in vitro fermentation studies.

This study investigated the optimal preservation approach to prepare human feces as inoculum for in vitro fermentations as an alternative to the use of fresh feces. The four treatments studied were: Treatment 1) fresh feces resuspended in dialysate solution+glycerol; Treatment 2) fresh feces resuspended in dialysate solution+glycerol and then stored at -80°C; Treatment 3) fecal sample frozen with 1.5 g glycerol; and Treatment 4) fecal sample frozen. All the treatments contained 8.75 g of feces, 3.5 ml dialysate and 4.9 ml glycerol when inoculated in TIM-2 in vitro system. Treatment 1 (fresh fecal preparation) was used as a reference. The effects were evaluated in terms of i) metabolic activity and ii) composition of the microbiota using fermentation experiments in the TIM-2 in vitro system. In all treatments, high levels of acetate were produced followed by n-butyrate and propionate. However, the metabolic activity of the bacteria, in terms of short-chain fatty acid production, was affected by the different treatments. Microbiota composition was analyzed using the IS-pro profiling technique. Diversity in Actinobacteria, Firmicutes, Fusobacteria and Verrucomicrobia and Proteobacteria groups seemed to be preserved in all treatments whereas it was observed to decline in the Bacteroidetes group. Preparing a human fecal inoculum resuspended in dialysate solution with glycerol and then stored at -80°C showed high similarities to the results obtained with fresh feces, and is proposed as the optimal way to freeze fecal material as an alternative to fresh feces for in vitro fermentation studies.

Development and validation of a new in vitro assay for selection of probiotic bacteria that express immune-stimulating properties in chickens in vivo.

Oral administration of immunoprobiotic bacteria may support animal health. Species specificity of such microorganisms requires appropriate selection. An in vitro assay for the selection of immunoprobiotic lactic acid bacteria was developed in chicken. The assay allowed testing of large numbers of individual strains. Immune stimulation in vitro correlated well with the in vivo situation in two experiments and no false negative results occurred. Therefore this assay is an appropriate selection tool for immunomodulating properties of lactic acid bacteria in chicken.

Immunological differences between layer- and broiler-type chickens.

In commercial poultry husbandry, alternatives for the use of antibiotics and vaccines are under investigation, which preferably have to be applicable for both layer- and broiler-type chickens. There are indications that the defense mechanisms vary between layer- and broiler-type chickens. Therefore, the difference in immune response between layer- and broiler-type chickens of the same age was investigated, using TNP-KLH (trinitrophenyl-conjugated keyhole limpet hemocyanin) as antigen without adjuvant. First different routes of immunization (intravenously, intramuscular, subcutaneous and ocular) were examined to find out which immunization route gives the highest antibody titers. The intravenous immunization route resulted in higher TNP-specific antibody responses than the other immunization routes tested and therefore this immunization route was used in both following experiments. In order to investigate the optimal dose of antigen needed for immunization, a dose-response curve in broiler- and layer-type chickens was completed. The humoral immune response was measured in serum by a TNP-specific ELISA and the in vitro cellular immune response by an antigen-specific lymphocyte proliferation assay. The antibody response of layer- and broiler-type chickens appeared to differ, not only in optimal dose and response, but also in kinetics of the response itself. Broiler chickens generated higher IgM anti-TNP titers whereas layer-type chickens generated higher IgG anti-TNP titers. This specific antibody response in broiler-type chickens did not last as long as in layer-type chickens. The TNP-specific cellular immune response was detectable in layer-type chickens, but not in broilers. Both types generate a non-specific cellular immune response, although this response in broilers is lower than in layer-type chickens. From these results, we conclude that broilers primarily respond to TNP-KLH with a high IgM antibody response whereas layer-type chickens respond with a high IgG response. In addition, the cellular response of layer-type chickens is much higher than the response of broilers. The results suggest that broilers are specialized in the production of a strong short-term humoral response and layer-type chickens in a long-term humoral response in combination with a strong cellular response, which is in conformity with their life expectancy.

To pool or not to pool? Impact of the use of individual and pooled fecal samples for in vitro fermentation studies.

This study investigated the stability and the activity of the microbiota from a single and a pool of donors in the TNO in vitro model of the colon (TIM-2 system). Our findings demonstrate the suitability of the preparation of a pool of fecal sample to be used for fermentation experiments.

The bioaccessibility of eicosapentaenoic acid was higher from phospholipid food products than from mono- and triacylglycerol food products in a dynamic gastrointestinal model.

The bioaccessibility of eicosapentaenoic acid (EPA) in the forms of monoacylglycerol (EPA-MAG), triacylglycerol (EPA-TAG), and phospholipid (EPA-PL) during gastrointestinal passage was compared in this study using a dynamic gastrointestinal model (TIM system). The TIM system simulated the average upper gastrointestinal tract conditions of healthy human adults after intake of a meal (fed state conditions). In this study, the three EPA-rich oils were separately homogenized with full fat milk to obtain oil-in-water emulsions. Plain yogurt was added into the mixture at an emulsion/yogurt ratio of 4:1 (w/w) as the food matrix of the test products. The results show that the test meals containing EPA-PL left the stomach compartment most efficiently in comparison with the gastric emptying of EPA-MAG and EPA-TAG. The PLs also showed a significantly (P < 0.05) higher bioaccessibility of EPA (75-80%) in comparison with MAG (30%) and TAG (38%). The better gastric emptying of EPA-PL was likely related to the more stable emulsion of EPA-PL in the test meal. EPA-PL was delivered within the meal matrix into the duodenum instead of floating on the top of the test meal matrix. EPA-MAG had the highest amount of EPA that did not leave the stomach (68% of the test meal). The results from this work indicate that EPA-PL is a more effective form of EPA for a higher lipid bioaccessibility than MAG and TAG under the test conditions.

Digestibility of transglutaminase cross-linked caseinate versus native caseinate in an in vitro multicompartmental model simulating young child and adult gastrointestinal conditions.

Aim of this study was to investigate the digestion of transglutaminase cross-linked caseinate (XLC) versus native caseinate (NC) in solution and in cheese spread under digestive conditions for adults and children mimicked in a gastrointestinal model. Samples were collected for gel electrophoresis and nitrogen analysis. The results showed no relevant differences between XLC and NC for total and α-amino nitrogen in digested fraction under adult and child conditions. However, the rate of digestion was depending on the food matrix. Gel electrophoresis showed the gastric breakdown of XLC without formation of pepsin resistant peptides larger than 4 kDa. NC was slowly digested in the stomach with formation of pepsin resistant fragments and was still detectable in the stomach after 90 min. In the small intestine the proteins were rapidly digested. XLC was digested to small peptides, while NC was resistant against pepsin digestion under gastric conditions of adults and children.

Some phenolic compounds increase the nitric oxide level in endothelial cells in vitro.

The vasorelaxing properties of chocolate and wine might relate to the presence of phenolic compounds. One of the potential mechanisms involved is stimulation of endothelial nitric oxide (NO) production, as NO is a major regulator of vasodilatation. This study aimed to develop an in vitro assay using the hybrid human endothelial cell line EA.hy926 to rapidly screen phenolic compounds for their NO-stimulating potential. The assay was optimized, and a selection of 33 phenolics, namely, procyanidins, monomeric flavan-3-ols, flavonols, a flavone, a flavanone, a chalcone, a stilbene, and phenolic acids, was tested for their ability to enhance endothelial NO level. Resveratrol, a well-known enhancer of NO level, was included as a positive control. Of the 33 phenolics tested, only resveratrol (285% increase in NO level), quercetin (110% increase), epicatechingallate (ECg) (85% increase), and epigallocatechingallate (EGCg) (60% increase) were significant (P