Short chain fatty acids (SCFA) originate from the bacterial fermentation of dietary fibre in the gastrointestinal tract. They are hypothesised to play a key role in microbiota–gut–brain crosstalk and the effect of individual SCFAs or mixtures thereof has been broadly studied. However, studies using fermentation products to evaluate the effect of microbiota-targeted interventions, such as prebiotics, probiotics, or diet, are sparse, particularly in humans. In addition, the complexity of these physiological processes translates as a challenge for their simulation in vitro. In this work, fermentation products of prebiotic-enriched media by bacteria present in primary human faecal samples were tested using an epithelium model based on a Caco-2/HT29-MTX co-culture. The prebiotics raftilose and fructo-oligosaccharides (FOS) were tested and the experimental conditions (contact time and minimal dilution) optimised to avoid cytotoxicity. None of the conditions tested compromised the intestinal epithelium integrity as verified by the TEER and the expression of the tight junction-specific protein – occludin. In addition, none of the fermentation products caused an inflammatory response as determinedby the expression of inflammatory genes by qRT-PCR. The products of fermentation of media enriched with FOS showed a moderate protective effect against the formation of reactive oxygen species. This work provides an important basis for the development of in vitro models using a simple approach to evaluate host-gut microbiota interactions, using co-cultures of intestinal cell lines and products of in vitro fermentations by primary human gut microbiota.

Short chain fatty acids (SCFA) originate from the bacterial fermentation of dietary fibre in the gastrointestinal tract. They are hypothesised to play a key role in microbiota–gut–brain crosstalk and the effect of individual SCFAs or mixtures thereof has been broadly studied. However, studies using fermentation products to evaluate the effect of microbiota-targeted interventions, such as prebiotics, probiotics, or diet, are sparse, particularly in humans. In addition, the complexity of these physiological processes translates as a challenge for their simulation in vitro. In this work, fermentation products of prebiotic-enriched media by bacteria present in primary human faecal samples were tested using an epithelium model based on a Caco-2/HT29-MTX co-culture. The prebiotics raftilose and fructo-oligosaccharides (FOS) were tested and the experimental conditions (contact time and minimal dilution) optimised to avoid cytotoxicity. None of the conditions tested compromised the intestinal epithelium integrity as verified by the TEER and the expression of the tight junction-specific protein – occludin. In addition, none of the fermentation products caused an inflammatory response as determinedby the expression of inflammatory genes by qRT-PCR. The products of fermentation of media enriched with FOS showed a moderate protective effect against the formation of reactive oxygen species. This work provides an important basis for the development of in vitro models using a simple approach to evaluate host-gut microbiota interactions, using co-cultures of intestinal cell lines and products of in vitro fermentations by primary human gut microbiota.