Discovery of pea protein-derived DPP-IV inhibitory peptides: Transport across Caco-2 cell monolayers and molecular mechanisms

Dipeptidyl peptidase IV (DPP-IV) inhibitory peptides derived from food proteins have been studied to regulate hyperglycemia, but their effects on transepithelial transport and inhibitory mechanisms remain unclear. In this study, pea protein was ultrasonicated then dually hydrolyzed by Pseudomonas aeruginosa protease (PaproA) and acid protease to exhibit great DPP-IV inhibitory activity (IC₅₀ of 1.34 mg/mL). Novel non-cytotoxic DPP-IV inhibitory peptides were discovered and ranked according to their in vitro activities as follows: YPR > YPHY > YPHYR > WAK > SPR. However, SPR and YPR performed excellent in situ DPP-IV inhibitory behaviors (luminescence of ~1.78 × 10⁵) and permeabilities (P_app of ~1.15 × 10^-7 cm/s) during transepithelial transport mediated by PepT1. Studies on DPP-IV inhibitory kinetics and molecular docking have revealed that tyrosine and arginine at the N- and C-termini of the peptides played key roles in competitive inhibition of DPP-IV through π-π stacking, π-anion interaction and hydrogen bonding, respectively. Overall, ultrasonication-assisted sequential hydrolysis by PaproA and acid protease could be a reliable strategy for releasing DPP-IV inhibitory peptides from pea protein for the treatment of diabetes mellitus.