Enzymatic characterization and synergistic effects of protease combinations on DPP-IV inhibitory peptide release from bovine casein

This study focused on the enzymatic characteristics, with regard to primary specificity, secondary specificity, and hydrolysis ability of five proteases (Alcalase, Protamex, papain, Flavourzyme, and ProtexA), towards the release of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from bovine casein. The secondary specificity of the five proteases, especially the release capability for Xaa-Pro-type peptide played a determining role in the release of DPP-IV inhibitory peptides from bovine casein. Moreover, protease combinations, chosen based on their Xaa-Pro-type peptide release abilities, resulted in superior DPP-IV inhibitory activity compared to other combinations. Particularly, the hydrolysate with the highest DPP-IV inhibitory activity (IC50 value of 0.45 ± 0.07 mg/mL) was obtained by the combination hydrolysis of ProtexA and papain, achieving a 74.63% ± 1.62% (peak area) release of Xaa-Pro-type peptide, and a significant improving glucose tolerance in mice (33.42% ± 1.68% reduction of blood glucose AUC compared with control mice). The mechanistic insights revealed that papain efficiently released long Xaa-Pro-type peptides in the early hydrolysis stage, and subsequently, the synergistic effect of papain and ProtexA led to an accelerated and extensive release of short Xaa-Pro-type peptides. These findings provide valuable insight into enzymatic processes for generating DPP-IV inhibiting peptides from casein.