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Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy industry, requires further documentation of its physiological status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library were designed to positively select activated genes. Second, recombinant clones were introduced into complementary models mimicking the human gut, the Netherlands Organization for Applied Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine, the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human feces as a colon model. All inserts of activated clones displayed a promoter activity that differed from one digestive condition to another. Our results also showed that S. thermophilus adapted its metabolism to stressful conditions found in the gastric and colonic competitive environment and modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses, a first stage in the identification of gut resistance markers and a key step in probiotic selection.

Fecal microbiota transplantation (FMT) is an innovative therapy already used in humans to treat Clostridioides difficile infections associated with massive use of antibiotics. Clinical studies are obviously the gold standard to evaluate FMT efficiency but remain limited by regulatory, ethics, and cost constraints. In the present study, an in vitro model of the human colon reproducing medically relevant perturbation of the colonic ecosystem by antibiotherapy was used to compare the efficiency of traditional FMT enema formulations and a new oral capsule in restoring gut microbiota composition and activity. Loss of microbial diversity, shift in bacterial populations, and sharp decrease in fermentation activities induced in vivo by antibiotherapy were efficiently reproduced in the in vitro model, while capturing inter-individual variability of gut microbiome. Oral capsule was as efficient as enema to decrease the number of disturbed days and bacterial load had no effect on enema performance. This study shows the relevance of human colon models as an alternative approach to in vivo assays during preclinical studies for evaluating FMT efficiency. The potential of this in vitro approach could be extended to FMT testing in the management of many digestive or extra-intestinal pathologies where gut microbial dysbiosis has been evidenced such as inflammatory bowel diseases, obesity or cancers.

Due to obvious ethical and technical reasons, it remains very difficult to evaluate the survival and expression of virulence genes of food-borne pathogens, such as Shiga toxin-producing Escherichia coli (STEC) in the human gastrointestinal tract. Here, we describe the use of the dynamic TNO (Toegepast Natuurwetenschappelijk Onderzoek) gastrointestinal model (TIM-1) as a powerful in vitro tool to obtain the kinetics of STEC survival by plate counting, the regulation of major virulence genes by RT-qPCR, and the production of Shiga toxins by ELISA, in the human stomach and small intestine. The gut model was adapted in order that in vitro digestions were performed both under adult and child digestive conditions, specific at risk populations for STEC infections.

Benzo[ a ]pyrene (B[ a ]P) is a ubiquitous, persistent, and carcinogenic pollutant that belongs to the large family of polycyclic aromatic hydrocarbons. Population exposure primarily occurs via contaminated food products, which introduces the pollutant to the digestive tract. Although the metabolism of B[ a ]P by host cells is well known, its impacts on the human gut microbiota, which plays a key role in health and disease, remain unexplored. We performed an in vitro assay using 16S barcoding, metatranscriptomics and volatile metabolomics to study the impact of B[ a ]P on two distinct human fecal microbiota. B[ a ]P exposure did not induce a significant change in the microbial structure; however, it altered the microbial volatolome in a dose-dependent manner. The transcript levels related to several metabolic pathways, such as vitamin and cofactor metabolism, cell wall compound metabolism, DNA repair and replication systems, and aromatic compound metabolism, were upregulated, whereas the transcript levels related to the glycolysis-gluconeogenesis pathway and bacterial chemotaxis toward simple carbohydrates were downregulated. These primary findings show that food pollutants, such as B[ a ]P, alter human gut microbiota activity. The observed shift in the volatolome demonstrates that B[ a ]P induces a specific deviation in the microbial metabolism.

The purpose of this work was to study the performance of microspheres of soy protein isolate (SPI), zein, or SPI-zein complex as vehicles of nutraceutical delivery under fasting and prandial conditions in an artificial digestive system (TIM-1). Riboflavin availability for absorption from the small intestine compartments reached 90% of the total load within 4 h, most of it (65-80%) turning up in the jejunum dialysis fluid, suggesting that this segment is the main site of absorption, regardless of the nature of the microspheres. However, the riboflavin concentrations and the availability for absorption profiles depended on microsphere formulation. Release from pure SPI and zein microspheres in the stomach compartment occurred within 15 min. The availability for absorption from both the jejunum and ileum compartment followed first-order kinetics, indicating that the limiting step in nutrient uptake with these two formulations is absorption by passive diffusion. SPI-zein complex microspheres provided sustained release of riboflavin over 4 h and a near-zero-order nutrient availability for absorption profile in both fasting and prandial states. Suspending SPI-zein complex microspheres in yogurt significantly delayed nutrient release, which would increase the likelihood of gastric-sensitive nutrients passing intact into the intestine for absorption. SPI-zein complex microspheres thus show potential for use as nutraceutical delivery vehicles in the creation of novel functional foods.

To exert their health effect, phytochemicals such as carotenoids and vitamin E have to be bioavailable. We investigated the digestive stability and intestinal absorption of lycopene and alpha-tocopherol from a whole food containing red tomatoes and sunflower oil using, for the first time, the dynamic gastrointestinal system TNO gastrointestinal tract model (TIM) coupled with Caco-2 cells. Digestive samples were added to Caco-2 cells after appropriate ultracentrifugation, filtration, and dilution. alpha-Tocopherol was stable during digestion in the TIM, whereas a 25% loss was observed for lycopene. The absorption of both compounds was curvilinear, bidirectional, and concentration-dependent. The percentages of alpha-tocopherol absorbed, but not that of lycopene, were lower with digestas compared to those with pure compounds, suggesting competition for absorption with other components of the test meal. According to in vivo data, a lower bioavailability was found for lycopene compared to that for alpha-tocopherol. These results support the usefulness of this in vitro approach for estimating the bioavailability of active compounds from food.