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It is increasingly evident that digestion can affect the biological activity of cheese by the release of new active peptides from their precursors or, on the contrary, giving rise to fragments without activity. The characterization of the peptidome of a Spanish blue cheese, Valdeon, has been conducted before and after gastrointestinal digestion, and the digests have been compared to those obtained from pasteurized skimmed milk powder (SMP) using a bioinformatics platform. Peptidomic profiling of digests revealed several regions that are especially resistant to digestion (among them β-casein 60-93, 128-140, and 193-209). Some of them correspond to well-conserved regions between species (human, cow, sheep, and goat) and include peptide sequences with reported bioactivity. The great peptide homology found between both digests, cheese and SMP, suggests that the gastrointestinal digestion could bring closer the profile of products with different proteolytic state. Although most of the biologically active peptides found in cheese after digestion were also present in SMP digest, there were some exceptions that can be attributed to the absence of the relevant precursor peptide before digestion.

It is increasingly evident that digestion can affect the biological activity of cheese by the release of new active peptides from their precursors or, on the contrary, giving rise to fragments without activity. The characterization of the peptidome of a Spanish blue cheese, Valdeón, has been conducted before and after gastrointestinal digestion, and the digests have been compared to those obtained from pasteurized skimmed milk powder (SMP) using a bioinformatics platform. Peptidomic profiling of digests revealed several regions that are especially resistant to digestion (among them β-casein 60-93, 128-140, and 193-209). Some of them correspond to well-conserved regions between species (human, cow, sheep, and goat) and include peptide sequences with reported bioactivity. The great peptide homology found between both digests, cheese and SMP, suggests that the gastrointestinal digestion could bring closer the profile of products with different proteolytic state. Although most of the biologically active peptides found in cheese after digestion were also present in SMP digest, there were some exceptions that can be attributed to the absence of the relevant precursor peptide before digestion.

In this report, we present the use of CE-MS as complement to RP separation for the identification of novel angiotensin-converting enzyme-inhibitory (ACEI) peptides from a complex milk protein hydrolysate. As preliminary step, fast protein LC (FPLC) was used to isolate the different casein fractions from raw ovine milk. Enzymatic hydrolysis of these fractions was performed by using proteolytic enzymes of gastrointestinal origin. The most active hydrolysate, corresponding to kappa-casein hydrolyzed with pepsin, chymotrypsin, and trypsin, was fractionated by RP-HPLC and the peptides contained in the active fractions were sequenced by CE coupled to IT-MS (CE-MS). The use of CE-MS allowed the identification of short peptides with ACEI activity included in the scarcely retained fraction obtained by semipreparative RP-HPLC. Among the identified peptides, those with hydrophobic or positively charged residues at the C-terminal tripeptide were chemically synthesized to determine their ACEI activity. This procedure allowed us to identify four novel potent ACEI peptides from kappa-casein with sequences IAK, YQQRPVA, WQVLPNAVPAK, and HPHPHLSF. These active sequences could be obtained by enzymatic hydrolysis either of the individual kappa-casein fraction or the total casein fraction from ovine milk.