BCM7 homologues from ovine and buffalo β-casein: Non-detection in the pH 4.6-soluble blue cheese fractions and transient formation during ex vivo whole-blood proteolysis

This study investigated whether β-casomorphin-7 (BCM-7) homologues derived from ovine and Mediterranean buffalo β-casein are detectable during blue cheese ripening and whether synthetic BCM-9 precursors can generate BCM-7 homologues during ex vivo incubation with whole human blood. Peptidomic analysis of pH 4.6-soluble fractions from ovine and buffalo blue cheeses revealed extensive proteolysis within the β-casein residues 60–70, but no intact BCM-7 homologues were detected with the applied workflow. In contrast, synthetic ovine and buffalo BCM-9 precursors (YPFTGPIPN and YPFPGPIPK, respectively), added directly to EDTA-treated whole blood at a final concentration of 3 nmol/mL, generated transient BCM-7 homologues that underwent rapid further degradation. Gastrointestinal digestion, intestinal transport, and in vivo exposure were not examined. These results demonstrate peptide biochemical feasibility under the specified ex vivo conditions rather than dietary absorption or physiologically relevant circulating concentrations. These findings distinguish non-detection of BCM-7 homologues in the analyzed cheese fractions from their conditional formation in whole blood whether suitable precursors reach the circulatory system.