HEALTH-PROMOTING COMPOUNDS IN FOOD AND FEED: AN IN VITRO APPROACH TO STUDY DIETARY BIOACTIVES

The main aim of this thesis was to study the functional properties of health-promoting compounds in vitro with an emphasis on milk proteins, vitamin E and micronutrients. The health-promoting effects of intact whey and casein proteins were evaluated after in vitro SGD. Permeate (absorbed fraction) and retentate (intestinal fraction) were obtained and used to study their health-effects in vitro. Soya protein was include as non-animal protein in all the experiments performed. Following SGD, the milk proteins exhibited antioxidant activity, ACE-inhibitory activity and tropho-functional properties at the intestinal cell level. The whey protein permeate exhibited a higher ACE-inhibitory activity compared with the casein and soya protein permeate. SGD increased the ACE-inhibitory activity of whey protein and the antioxidant activity of all the protein tested. At specific concentrations, casein, whey and soya proteins were able to modulate intestinal cell viability and the production of intestinal mucus. Moreover, the proliferation of Lactobacillus casei was increased by specific concentrations of whey and casein proteins. Modulations of mucus production and probiotic bacteria growth were observed, and casein was the primary protein that was able to stimulate MUC5AC gene expression and promote Lactobacillus casei growth. Altogether, the analyses of goblet cell proliferation and prebiotic bacterial growth may represent complementary approaches to study the bioactivities and the functions of food proteins in the gut. Additionally, the comparison of the effect of the two major intact milk proteins performed in this thesis could provide valuable information regarding which is more efficacious in improving health. α-tocopherol has been demonstrated to play an important role in reducing oxidative stress at cellular level, in different in vitro models. Food toxicants, as OTA, have been found to be able of disrupting the cell monolayer and damaging DNA, which leads to cell death. OTA reduced tight junctions protein localization in cell membranes and influenced cell-cell interactions. The immunofluorescence analyses revealed changes in the patterns of occludin and Zo1 proteins in the presence of OTA, compared with control cells. OTA cytotoxic effects were counteracted by the presence of α-tocopherol. The pre-treatment with α-tocopherol blocked the loss of occludin protein in the tight junctions of kidney cells treated with OTA. α-tocopherol supplementation has demonstrated to counteract short-term OTA toxicity at different cellular levels, supporting the defensive role of this compound in the cell membrane. Finally, the in vitro roles of different micronutrients, specifically choline/methionine and different Zn formulations, were determined. Due to their physiological health-promoting effects, both choline/methionine and Zn are commonly used in animal nutrition. The results of this thesis confirmed that under condition of stress, choline and methionine have important roles in enhancing cell viability and counteracting oxidative stress. Specific concentrations of different Zn sources maintained the viability of human and swine intestinal cells, which underlines the beneficial role of Zn in human and swine intestinal epithelia. Overall, these results contribute to the identification of the roles of different dietary health-promoting compounds in human and animal target tissues by making in vitro models an essential tool. However, further in vivo experiments are necessary to extend these in vitro results and to clarify the contributions of health-promoting components in animal feed and human formulations. The improved knowledge related to milk protein bioactives, antioxidants and micronutrients represents a crucial subject for future systematic efforts to improve food and feed quality.