Enzymatic hydrolysis of buffalo casein enhances DPP-4 inhibition: Structural modifications and bioactive peptide identification

Abstract

Dipeptidyl peptidase-4 (DPP-4), the enzyme responsible for the rapid degradation of incretin hormones, plays a pivotal role in blood glucose regulation, and its inhibition serves as an effective strategy for maintaining glucose homeostasis. The aim of this study was to investigate the effect of enzymatic hydrolysis on the structure of buffalo casein and its DPP-4 inhibitory activity. Results demonstrated that Flavorzyme effectively hydrolyzed buffalo casein, as evidenced by scanning electron microscopy and electrophoretic analysis, with the degree of hydrolysis reaching its maximum value (20.05 ± 0.14%) after 3 h. The results of circular dichroism spectra, as well as endogenous and exogenous fluorescence spectra, indicated marked alterations in the secondary and tertiary structures of buffalo casein following enzymatic hydrolysis. Additionally, the DPP-4 inhibitory effect of buffalo casein was found to increase with longer hydrolysis times. The hydrolysate obtained after 3 h of hydrolysis demonstrated the highest level of inhibition, with a half-maximal inhibitory concentration (IC₅₀) value of 1.04 mg/mL. The DPP-4 inhibitory peptide YPFPGPIPN, with an IC₅₀ value of 0.88 mg/mL, was identified in the 1 to 3 kDa fraction of the 3-h hydrolysate. This peptide interacted with the active site of DPP-4 via hydrogen bonds, hydrophobic interactions, salt bridges, and π-cation interactions. This study offers a novel scientific foundation for the development of functional antidiabetic foods derived from buffalo casein.