Evaluation of protein hydrolysis in ripened sheep and goat cheeses during in vitro digestion

Cheese protein hydrolysis and the release of peptides and amino groups during gastrointestinal digestion may be influenced by milk origin, coagulant type, and microbial community composition. Artichoke flower extract (AF), rich in aspartic proteases, may contribute to protein hydrolysis during cheese ripening. This study characterized goat (G) and sheep (S) ripened cheeses produced with AF alone or associated with half-dose bovine rennet (AF+BR) regarding to their microbiota (16S rRNA gene sequencing) and evaluated their protein hydrolysis during simulated gastrointestinal digestion. Cheeses were produced, ripened for 21 days and subsequently subjected to metataxonomic assays and to in vitro gastrointestinal digestion according to the INFOGEST 2.0 protocol, with modifications. Protein content was determined by the micro-Kjeldahl method, and protein hydrolysis was assessed using the o-phthalaldehyde (OPA) assay with L-leucine as the standard. The 16S rRNA gene sequencing assay showed that microbiota of all samples were dominated by Lactoccocus genus, followed by Leuconostoc genus (except for S-AF). Moreover, G-AF showed measurable abundance of Carnobacterium genus (3.3%), although this genus was not inoculated as a starter. Protein hydrolysis differed among the cheese treatments, as indicated by the concentration of free amino groups released during gastrointestinal digestion. G-AF showed the lowest OPA value (39.37 ± 9.57 µmol/mL, p<0.05), significantly lower than G-AF+CB (111.36 ± 1.58 µmol/mL), S-AF (105.44 ± 4.29 µmol/mL), and S-AF+CB (96.64 ± 12.21 µmol/mL), which did not differ significantly from each other. Together, these results suggest that, despite the inoculation of the same starter cultures, factors such as milk source, residual microbiota after pasteurization, interaction between indigenous microorganisms and starter cultures, and the proteolytic profile of the coagulant likely influenced primary and secondary proteolysis in cheese. These differences may, in turn, explain the lower release of amino groups during the digestion of G-AF compared with the other samples. 

Acknowledgments: The authors thank CAPES (Funding Code 001) for financial support.