Optimization of pea protein hydrolysate preparation and purification of antioxidant peptides based on an in silico analytical approach

Isolation and purification of peptides by enzymatic hydrolysis may have a significant effect on their antioxidant activity. In the present study, antioxidant peptides were isolated and purified from hydrolysates by digestion of the pea protein with alcalase. Electron paramagnetic resonance (EPR) spectroscopy utilizing the 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used to optimize the preparative protocol. The obtained peptides were identified by nano-LC-ESI-MS/MS and their biological activity was evaluated by the Peptide Ranker method. Our results showed that the second fraction (F1-2) purified from <1 kDa hydrolysate by Sephadex G-25 gel had good antioxidant activity, along with a DPPH radical scavenging rate of 37.94 ± 1.24% and a hydroxyl (OH) radical scavenging rate of 28.43 ± 1.54% (P < 0.05). In addition, the biological activity of the identified peptide sequences was estimated, and three peptides with scores higher than 0.5 were obtained, corresponding to the sequences YSSPIHIW (0.74), ADLYNPR (0.65), and HYDSEAILF (0.53).