Formación y biodisponibilidad de péptidos alimentarios con efecto sobre la salud durante la digestión gastrointestinal de proteínas lácteas

Resumen

In this thesis several aspects related to the production and subsequent modification of the peptide sequences within food matrices have been studied, taking into account the influence of technological treatments, and the modifications once they are ingested, including gastrointestinal digestion, absorption and metabolism. Liquid chromatography coupled to tandem mass spectrometry with different analyzers (ion trap, Q-TOF, MALDI TOF/TOF) has been used to perform sequencing and quantification of peptides within different dairy matrices (fermented milk, cheese, gastrointestinal digests) or biological samples (plasma). Special attention has been paid to the ß-casein region 126-140, which is a well conserved domain among different species and hosts several active sequences, such as LHLPLP, f(133-138) , and HLPLP, f(134-138). The production of active compounds and the impact of the packaging conditions on peptides have been also evaluated in this thesis. An alternative was provided to produce active hydrolysates, containing the antihypertensive peptides from ß-casein, f(134-138), HLPLP, and f(133-138), LHLPLP, by casein fermentation using two foodgrade yeast strains of Debaryomyces hansenii. In addition, the peptide profile of two cheeses packed using different technologies, vacuum and modified atmosphere, was studied during the shelf life of the product. No marked qualitative differences could be found in the peptide profile regarding the packaging technique and storage time, although the relative abundance of the peptides was different in both packaging systems. Once the production of active compounds and the impact of the packaging conditions on peptides have been evaluated, the peptidome generated through digestion under different conditions of several dairy matrices was studied. Skimmed milk powder (SMP) and a highly proteolysed blue cheese were subjected to static gastrointestinal digestion in order to assess the influence of the proteolytic state of the matrix in the release of peptides. Several regions of ß-casein were shown to be resistant to digestion i.e. 60-93, 128-140 and 193-209, in both matrices. The differences found among cheese and milk were attributed to the initial state of proteolysis of cheese, which hosted several precursor fragments for bioactive peptides, i.e. LHLPLP, before digestion, in contrast to SMP. On the other hand, the impact of heat treatment on the release of peptides during gastric digestion of heated and unheated milk has been studied using a dynamic digester, which represents next step after static models to better reproduce physiological conditions. The heat treatment elicited an increase in the resistance of casein fraction to digestion by pepsin, in contrast to ß-Lg, which became more susceptible. Some resistant domains from ß- casein (76-93, 126-140 and 190-209) were identified regardless the heat treatment, where the active sequences LHLPLP and HLPLP are located. Later on, an antihypertensive sequence included in the resistant domain of ß-casein 126-140, i.e. HLPLP, f(134-138), was demonstrated for the first time to be absorbed into blood circulation in rats after its oral administration. The kinetic parameters of HLPLP after oral and intravenous administration were determined, being the effective absorption around 5.18 %, based on the quantification of the intact penta-peptide in plasma. However, it was found that several derived fragments, HLPL ß-casein f(134-137) and LPLP ß-casein f(135-138), were rapidly formed in plasma after both types of administration, and therefore, the absorption of the peptide is probably higher. The degradation of the penta-peptide by the action of plasma peptidases was confirmed by in vitro incubation of HLPLP in plasma, where several fragments i.e. HLPL, LPLP and HLP were released, and the antihypertensive activity of these fragments was demonstrated in a SHR model. Subsequently, the implication of opioid receptors in the mechanism of action of two antihypertensive peptides from ¿s1-casein (RYLGY, f(90-94) and AYFYPEL, f(143- 149)) and of a casein hydrolysate, containing these, was shown by the coadministration of naloxone. The antihypertensive activity of YFYPEL, ¿s1-casein f(144-149) also present in the hydrolysate, was demonstrated for the first time, but its mechanism of action did not seem to involve opioid receptors. The application and optimisation of peptidomic techniques has permitted to characterise the peptide fraction within dairy matrices and to evaluate the influence of the technological treatments, the changes during digestion, absorption, metabolism, and ultimately estimate the in vivo bioavailability of an active sequence.