Identification and screening of ACE-inhibitory peptides from Camellia oleifera seed protein hydrolysates: Integrating in silico predictions with in vitro validation

In this study, researchers investigated the optimal enzyme for the hydrolysis of Camellia oleifera seed protein (CP), utilising a range of proteases, including alkaline protease, neutral protease, compound protease, and trypsin. The hydrolysate obtained from alkaline protease after 6 h showed the highest Angiotensin-converting enzyme (ACE) inhibitory activity, reaching 57.94% ± 1.58%. Two peptides—EFWDSR and FWDSQ—were identified using ultrafiltration, Sephadex G-15 gel chromatography, LC-MS/MS analysis, and in silico screening. Following solid-phase synthesis, both peptides exhibited ACE inhibitory activity, with IC 50 values recorded at 0.114 mg/mL and 0.238 mg/mL. Kinetic analysis showed EFWDSR was a non-competitive inhibitor and FWDSQ a mixed-competitive inhibitor.Secondary structure analysis showed that EFWDSR primarily consisted of β -sheet and α -helix structures, lacking β -turns or random coils. In contrast, FWDSQ was mainly composed of β -sheet and random coil structures, with a small amount of β -turns and no α -helix. Molecular docking studies indicated that both peptides formed hydrogen bonds with ACE. Stability tests demonstrated that EFWDSR remained structurally stable between 4 and 70 °C under neutral pH conditions, whereas FWDSQ was stable between 4 and 50 °C in neutral and mildly acidic environments. Both peptides maintained their stability during simulated gastrointestinal digestion in vitro . These results suggest that EFWDSR and FWDSQ derived from Camellia oleifera seed have potential applications as functional food components or antihypertensive agents.