Establishment of in vitro gastrointestinal models platform for human food and animal feed ingredients development

Globally, the human diet significantly relies on animal-derived products. The agri-food industry needs to optimize animal growth and performance to meet consumers demands, while reducing costs, ensuring product quality, and addressing sustainability and health concerns. Enhancing diets, through feed/food fortification with bioactive ingredients targeting gut microbiota is a promising strategy to promote the well-being and animal’s performance. Agro-industrial byproducts are potential sources of these bioactive ingredients, however, their effects and potential application on animal/human diets must be carefully evaluated. The main goal of this doctoral research was to create a platform of in vitro models that simulate the human and animal gastrointestinal tract (GIT), the GIOTA platform. Thus, models were implemented to assess and validate the impact of feed/food supplemented with bioactive ingredients on the gut microbiota, before resorting to in vivo trials. This supports the development of new products and promotes the inclusion of more sustainable ingredients, such as some of the byproducts tested in these models. The experimental work focused on 3 types of GIT: Human, Poultry and Bovine, In the first part, the preservation of human fecal inoculum was optimized, and the metabolic profile of colonic fermentations was compared between fresh and frozen inoculum. Storage at -20 ºC with a 30% (v/v) glycerol solution allowed maintaining the viability of the inoculum for 90 days without affecting colonic fermentations. Furthermore, with the in vitro model already implemented, the impact of food matrices on microbiota modulation was evaluated. Skim milk (SKM) supplemented at 1% (w/v) by different well-known functional ingredients that modulate the gut microbiota were tested. The combination of SKM with fructo-oligosaccharides proved to be most beneficial for consumer’s health, promoting Lactobacillus, Bifidobacterium and Clostridium cluster IV, increasing the production of short-chain fatty acids, and reducing ammonia production. In second part, a poultry GIT in vitro model, specifically chickens, was designed to predict the effects of feed formulations on their microbiota. Two preservation methods of cecal inoculum were studied: (1) 5 % (v/v) dimethyl sulfoxide (DMSO) at -80 ºC and (2) 30% (v/v) glycerol at -20 ºC. Fresh and frozen inoculums were used in this model, and their fermentation profile were compared. The DMSO preservation method proved to be the most appropriate for cecal inoculum preservation for 90 days. Additionally, to validate the results of the in vitro model, a broiler chicken in vivo assay and the chicken in vitro model were performed, comparing the effect of incorporating in feed 1% (w/w) sugarcane bagasse lignin, a byproduct of the sugarcane industry. The tested supplementation, in the in vivo assay, had a positive impact on broiler chicken cecum, increasing the acetate and butyrate concentrations and reducing the presence of Enterobacteriaceae. The GIT model showed a similar trend in microbiota modulation of the in vivo assay, but still requires adjustments in inoculum preparation. In third part, to assess the impact of diet on the bovine intestinal microbiota’s, 2 in vitro models were developed, one for adult cow (ruminant) and another for calf (monogastric). Preservation studies of rumen and fecal inoculum from adult cows were also performed. In the adult cow model, ruminal and hindgut fermentations were performed with fresh and frozen inoculum, while in the calf model only hindgut fermentations were performed. Preservation at -80 ºC was the condition that least affected inoculum’s bacterial viability and did not affect their fermentative capacity. Simultaneously, a calf in vivo assay and a calf model were conducted and compared on the evaluation of the effect of supplementing 1% (w/w) autolyzed spent yeast (AY) from farnesene production on milk replacer (MR). The AY supplementation, in the in vivo assay, had a positive impact on calf’s hindgut microbiota, increasing the presence of Bifidobacterium. However, this result was not observed in the model, which requires adjustments in the methodology associated with the digestion of liquid feed matrices. Scientific research and innovation play a crucial role in addressing modern challenges in the agri-food industry. The developed models serve as valuable and sustainable tools in evaluating the impact and validating the potential of new products of new products in the animal and human feeding industries.