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 IC₅₀ 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.