Digestión gastrointestinal de proteínas alimentarias y mecanismos de acción de péptidos con efecto sobre la salud digestiva / gastrointestinal digestion of food proteins and mechanisms of action of peptides on digestive health

In this Thesis several aspects related to the physiological effect of bioactive peptides on the digestive tract have been studied by a double approach. It has been investigated not only the modifications that food proteins and peptides undergo during the gastrointestinal digestion, but also the mechanisms of action involved in the biological function that bioactive peptides might exert due to their contact with digestive cells and receptors. Peptide lunasin, casein and whey proteins, and milk peptides were analyzed. Initially, the behavior of peptide lunasin under digestive conditions simulating the transit through the gastrointestinal tract in the absence or presence of soybean Bowman-Birk isoinhibitor 1 (IBB1), in both active and inactive states, was evaluated. IBB1 isoinhibitor exerted a protective effect on lunasin degradation during digestion. Protection against the action of pepsin was due to the presence of IBB1 and its higher size in comparison with that of peptide lunasin, independently of activity. However, an IBB1 dose-dependent protective effect was found at intestinal level, related to its inhibitory activity of pancreatic enzymes trypsin and chymotrypsin. The peptide profiles of gastric and gastrointestinal digests were characterized. It was demonstrated the notable resistance during the digestive process of some domains of peptide lunasin, especially regions 1SKWQHQQDSC10, 11RKQLQGVN18, 19LTPCEKHIME28 and 29KIQGRGDDDDDDDDD43. The transepithelial transport of these four fragments and the precursor lunasin was evaluated using Caco-2 cell monolayers. While some regions of these peptides were susceptible to epithelial brush-border peptidases, a marked resistance was found for others, particularly for fragments 1SKWQHQQDSC10 and 29KIQGRGDDDDDDDDD43. The transepithelial transport of lunasin and fragment 11RKQLQGVN18 was mediated through diffusion via the paracellular pathway. Additionally, lunasin, as well as its gastrointestinal digests in presence and absence of IBB1, and some lunasin-derived fragments identified in these digests were evaluated for their effect against the viability of gastric cancer AGS and colorectal cancer HT-29 and Caco-2 cells. It is highlighted that the fragment 1SKWQHQQDSC10 was the main responsible for the demonstrated inhibitory effect of lunasin on cellular viability, particularly in HT-29 cells. The chemopreventive mechanisms of action of lunasin against the proliferation of bulk colorectal cancer HCT-116 cells and the expansion of their tumorsphere-derived cancer stem-like subpopulation were evaluated. Peptide lunasin inhibited the viability of bulk tumor cells, as well as the tumorsphere-forming capacity of HCT-116 cells. The inhibitory activity was mediated by inducing apoptosis and arresting cell cycle at G1 phase. These effects were associated to a stimulatory effect on molecular marker caspase-3 linked to a cleavage on PARP signal, and a modest activation of p21 protein expression. Upon chemical-induced oxidative stress, lunasin-treated liver HepG2 cells showed an increased cellular viability. This protective effect was mediated through raising intracellular glutathione levels, and decreasing oxygen reactive species production and glutathione peroxidase and catalase activities. In addition, lunasin protected proteins from oxidative damage and inhibited caspase-3 mediated apoptosis. In the context of gastrointestinal mucus strengthening by food protein compounds, novel peptides derived from αs1-casein 144YFYPE148 and 144YFYPEL149 and, to a lower extent, 144YFY146 and 143AYFYPEL149 were found, for the first time, to exert an opioid agonist activity. By molecular dynamics simulations of peptides binding with the μ-opioid receptor, it was proved that the carboxi-terminal proline residue from peptide 144YFYP147 affected to its interaction with opioid receptor and activity. On intestinal goblet cells, it was demonstrated that peptide 144YFYPEL149 was the minimum fragment able to stimulate expression of MUC5AC, the main secreted mucin gene in HT29-MTX cells. Later on, the protective effect of a peptic casein hydrolyzate containing these opioid peptides was studied in the rat intestinal mucus layer. This hydrolyzate stimulated the gene expression of mucins Muc2 and Muc3 in ileum and colon, and the fecal mucin secretion after its oral administration during two and eight weeks, enhancing in vivo the intestinal mucus barrier. Finally, protein degradation and peptide formation in digests obtained after in vivo and in vitro gastrointestinal digestion of casein and whey protein were analyzed. With this purpose, the peptide profile of jejunal digests of five human volunteers was characterized and compared with peptides generated during in vitro gastrointestinal digestion of milk proteins following a standardized and internationally harmonized static digestion protocol. Whereas protein degradation through the gastrointestinal tract was observed, some protein domains showed resistance against the action of digestive enzymes. In vivo and in vitro protein degradation and digests peptidome were similar. Spearman correlation coefficients between in vivo and in vitro digests were within the range of that obtained between the different human volunteers of the study. Therefore, this in vitro method represented a suitable model to physiologically simulate the gastrointestinal digestion, at least in the case of milk proteins. Altogether, these results demonstrated the key role that digestive tract can play on the bioactivity of ingested compounds. Moreover, the results have allowed increasing the knowledge on the mechanisms of action involved in the beneficial effects of peptide lunasin, milk proteins, and derived peptides on digestive health.