Abstract: In this study, pea protein hydrolysate was prepared by enzymatic hydrolysis, followed by isolation and purification. Its basic physicochemical properties were analyzed, revealing a protein content of (89.43 ± 3.03)%, a peptide content of (75.72 ± 1.65)%, and a high proportion (90.39%) of components with a molecular mass of < 1 000 Da. Three core peptide sequences were identified using high performance liquid chromatography-mass spectrometry (HPLC-MS), including Val-Ala (VA), Phe-Pro-Trp (FPW), and Trp-Pro-Phe (WPF). Molecular docking was performed to analyze the targeted binding of these peptides to dipeptidyl peptidase-IV (DPP-IV). The results showed that both FPW and WPF could bind to the S2 active pocket of DPP-IV through docking, and their interaction sites were highly similar to those of the positive control Ile-Pro-Ile (IPI). Under the LibDOCK docking mode, both FPW and WPF achieved higher scores than IPI, while the score of VA was lower than that of IPI. Furthermore, a 100 ns molecular dynamics simulation was conducted on the complex of the highest scored peptide (FPW) and DPP-IV. It was found that the complex maintained stable root mean square deviation (RMSD), radius of gyration (Rg), and number of hydrogen bonds, accompanied by a binding free energy (ΔGbind) of (–43.53 ± 0.23) kJ/mol. This indicates a stable binding conformation and strong affinity between FPW and DPP-IV. In vitro assays confirmed the DPP-IV inhibitory activity of FPW, WPF, and VA in a dose-dependent manner. At a concentration of 20 mg/mL, the inhibition rates of FPW, WPF, and VA reached (99.03 ± 0.89)%, (98.54 ± 0.60)%, and (94.14 ± 1.05)%, respectively. In summary, this study provides a theoretical basis for the development of blood glucose-lowering functional foods based on pea protein hydrolysate.

Key words: pea protein hydrolysate; dipeptidyl peptidase-IV inhibitory peptides; peptide identification; molecular docking; molecular dynamics simulation