T. Van de Wiele

Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability

Background and aims: Obese and diabetic mice display enhanced intestinal permeability and metabolic endotoxaemia that participate in the occurrence of metabolic disorders. Our recent data support the idea that a selective increase of Bifidobacterium spp. reduces the impact of high-fat diet-induced metabolic endotoxaemia and inflammatory disorders. Here, we hypothesised that prebiotic modulation of gut microbiota lowers intestinal permeability, by a mechanism involving glucagon-like peptide-2 (GLP-2) thereby improving inflammation and metabolic disorders during obesity and diabetes. Methods: Study 1: ob/ob mice (Ob-CT) were treated with either prebiotic (Ob-Pre) or non-prebiotic carbohydrates as control (Ob-Cell). Study 2: Ob-CT and Ob-Pre mice were treated with GLP-2 antagonist or saline. Study 3: Ob-CT mice were treated with a GLP-2 agonist or saline. We assessed changes in the gut microbiota, intestinal permeability, gut peptides, intestinal epithelial tight-junction proteins ZO-1 and occludin (qPCR and immunohistochemistry), hepatic and systemic inflammation. Results: Prebiotic-treated mice exhibited a lower plasma lipopolysaccharide (LPS) and cytokines, and a decreased hepatic expression of inflammatory and oxidative stress markers. This decreased inflammatory tone was associated with a lower intestinal permeability and improved tight-junction integrity compared to controls. Prebiotic increased the endogenous intestinotrophic proglucagon-derived peptide (GLP-2) production whereas the GLP-2 antagonist abolished most of the prebiotic effects. Finally, pharmacological GLP-2 treatment decreased gut permeability, systemic and hepatic inflammatory phenotype associated with obesity to a similar extent as that observed following prebiotic-induced changes in gut microbiota. Conclusion: We found that a selective gut microbiota change controls and increases endogenous GLP-2 production, and consequently improves gut barrier functions by a GLP-2-dependent mechanism, contributing to the improvement of gut barrier functions during obesity and diabetes.

Inulin-type fructans of longer degree of polymerization exert more pronounced in vitro prebiotic effects.

Aims:  We assessed to what extent fructans of different degrees of polymerization (DP) differ in their prebiotic effectiveness towards in vitro microbial communities from the proximal and distal colon.

Bacteria and chocolate: A successful combination for probiotic delivery

In this work, chocolate has been evaluated as a potential protective carrier for oral delivery of a microencapsulated mixture of Lactobacillus helveticus CNCM I-1722 and Bifidobacterium longum CNCM I-3470. A sequential in vitro setup was used to evaluate the protection of the probiotics during passage through the stomach and small intestine, when embedded in dark and milk chocolate or liquid milk. Both chocolates offered superior protection (91% and 80% survival in milk chocolate for L. helveticus and B. longum, respectively compared to 20% and 31% found in milk). To simulate long-term administration, the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) was used. Plate counts, Denaturing Gradient Gel Electrophoresis and quantitative PCR showed that the two probiotics successfully reached the simulated colon compartments. This led to an increase in lactobacilli and bifidobacteria counts and the appearance of additional species in the fingerprints. These data indicate that the coating of the probiotics in chocolate is an excellent solution to protect them from environmental stress conditions and for optimal delivery. The simulation with our gastrointestinal model showed that the formulation of a probiotic strain in a specific food matrix could offer superior protection for the delivery of the bacterium into the colon. The chocolate example could act as a trigger for new research to identify new balanced matrices.

Comparison of five in vitro digestion models to in vivo experimental results: Lead bioaccessibility in the human gastrointestinal tract

This paper presents a multi-laboratory comparison study of in vitro models assessing bioaccessibility of soil-bound lead in the human gastrointestinal tract under simulated fasted and fed conditions. Oral bioavailability data from a previous human in vivo study on the same soil served as a reference point. In general, the bioaccessible lead fraction was significantly (P < 0.05) different between the in vitro methods and ranged for the fasted models from 2% to 33% and for the fed models from 7% to 29%. The in vivo bioavailability data from literature were 26.2 ± 8.1% for fasted conditions, compared to 2.5 ± 1.7% for fed conditions. Under fed conditions, all models returned higher bioaccessibility values than the in vivo bioavailability; whereas three models returned a lower bioaccessibility than bioavailability under fasted conditions. These differences are often due to the methods digestion parameters that need further optimization. An important outcome of this study was the determination that the method for separating the bioaccessible lead from the non-bioaccessible fraction (centrifugation, filtration, ultrafiltration) is crucial for the interpretation of the results. Bioaccessibility values from models that use more stringent separation methods better approximate in vivo bioavailability results, yet at the expense of the level of conservancy. We conclude from this study that more optimization of in vitro digestion models is needed for use in risk assessment. Moreover, attention should be paid to the laboratory separation method since it largely influences what fraction of the contaminant is considered bioaccessible.

Arabinoxylan-oligosaccharides (AXOS) affect the protein/carbohydrate fermentation balance and microbial population dynamics of the Simulator of Human Intestinal Microbial Ecosystem

Arabinoxylan‐oligosaccharides (AXOS) are a recently newly discovered class of candidate prebiotics as – depending on their structure – they are fermented in different regions of gastrointestinal tract. This can have an impact on the protein/carbohydrate fermentation balance in the large intestine and, thus, affect the generation of potentially toxic metabolites in the colon originating from proteolytic activity. In this study, we screened different AXOS preparations for their impact on the in vitro intestinal fermentation activity and microbial community structure. Short‐term fermentation experiments with AXOS with an average degree of polymerization (avDP) of 29 allowed part of the oligosaccharides to reach the distal colon, and decreased the concentration of proteolytic markers, whereas AXOS with lower avDP were primarily fermented in the proximal colon. Additionally, prolonged supplementation of AXOS with avDP 29 to the Simulator of Human Intestinal Microbial Ecosystem (SHIME) reactor decreased levels of the toxic proteolytic markers phenol and p‐cresol in the two distal colon compartments and increased concentrations of beneficial short‐chain fatty acids (SCFA) in all colon vessels (25–48%). Denaturant gradient gel electrophoresis (DGGE) analysis indicated that AXOS supplementation only slightly modified the total microbial community, implying that the observed effects on fermentation markers are mainly caused by changes in fermentation activity. Finally, specific quantitative PCR (qPCR) analysis showed that AXOS supplementation significantly increased the amount of health‐promoting lactobacilli as well as of Bacteroides–Prevotella and Clostridium coccoides–Eubacterium rectale groups. These data allow concluding that AXOS are promising candidates to modulate the microbial metabolism in the distal colon.

In vitro modulation of the human gastrointestinal microbial community by plant-derived polysaccharide-rich dietary supplements

The use of prebiotics is a possible strategy to manage and steer the complex gut microbial community towards a health-promoting composition (Gastrointestinal Resource Management). In this study, the Simulator of the Human Intestinal Microbial Ecosystem was used to investigate the effects of two commercially-available plant polysaccharide supplements on the structure, composition and metabolism of an in vitro cultured colon microbial community. Microbial analyses showed both a bifidogenic (up to +1.3 log cfu/mL) and a lactobacillogenic (up to +0.9 log cfu/mL) effect during treatment with the dietary supplements. Quantitative PCR confirmed that the increase of Bifidobacteria spp. was statistically significant (P<0.05) in all of the colon compartments and showed a significant increase of the bacteroides-prevotella group concentration (+0.6 log cells/ml) in the compartment simulating the proximal colon. Denaturant gradient gel electrophoresis analyses and a relative ecological interpretation, in combination with sugar and short-chain fatty acids quantification, provided evidence of a positive effect of both the tested products. Overall, the treatment period was associated with (i) good and selective fermentability of the polysaccharide supplements along the entire colon; (ii) positive and selective bifidogenic effect; (iii) the possibility of enhancing species belonging to Bacteroidetes, a phylum recently associated with body weight management.

Selected nondigestible carbohydrates and prebiotics support the growth of probiotic fish bacteria mono-cultures in vitro

Aims:  To search for nondigestible but fermentable (NDF) carbohydrates and prebiotics with a potency to promote the growth of selected bacteria in vitro.

Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion.

The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored.

Microbiota and their role in the pathogenesis of oral mucositis

Oral mucositis in patients undergoing cancer therapy is a significant problem. Its prevalence ranges between 20 and 100%, depending on treatment type and protocols and patient-based variables. Mucositis is self-limiting when uncomplicated by infection. Unfortunately, the incidence of developing a local or systemic infection during the course of the treatment is very high. At this stage, it is unclear which role oral microbiota play in the onset, duration, and severity of oral mucositis. Nevertheless, there is growing interest in this underexplored topic, and new studies are being undertaken to unravel their impact on the pathogenesis of mucositis.

A simulated mucus layer protects Lactobacillus reuteri from the inhibitory effects of linoleic acid

Lactobacillus reuteri is a commensal, beneficial gut microbe that colonises the intestinal mucus layer, where it makes close contact with the human host and may significantly affect human health. Here, we investigated the capacity of linoleic acid (LA), the most common polyunsaturated fatty acid (PUFA) in a Western-style diet, to affect L. reuteri ATCC PTA 6475 prevalence and survival in a simulated mucus layer. Short-term (1 h) survival and mucin-agar adhesion assays of a log-phase L. reuteri suspension in intestinal water demonstrated that the simulated mucus layer protected L. reuteri against the inhibitory effects of LA by lowering its contact with the bacterial cell membrane. The protective effect of the simulated mucus layer was further evaluated using a more complex and dynamic model of the colon microbiota (SHIME®), in which L. reuteri survival was monitored during 6 days of daily exposure to LA in the absence (L-SHIME) and presence (M-SHIME) of a simulated mucus layer. After 6 days, luminal L- and M-SHIME L. reuteri plate counts had decreased by 3.1±0.5 and 2.6±0.9 log cfu/ml, respectively. Upon supplementation of 1.0 g/l LA, the decline in the luminal L. reuteri population started earlier than was observed for the control. In contrast, mucin-agar levels of L. reuteri (in the M-SHIME) remained unaffected throughout the experiment even in the presence of high concentrations of LA. Overall, the results of this study indicate the importance of the mucus layer as a protective environment for beneficial gut microbes to escape from stress by high loads of the antimicrobial PUFA LA to the colon, i.e. due to a Western-style diet.