Preparation of an ACE Inhibitory Peptides from Defatted Hairtail (Trichiurus Lepturus) Meat by Compound Enzymatic Hydrolysis: Isolation, Purification, Identification, and Molecular Docking Analysis

This study aimed to screen angiotensin I-converting enzyme (ACE) inhibitory peptides from defatted hairtail (Trichiurus lepturus) meat, using the ACE inhibition rate as the primary evaluation criterion. Among five commonly used proteases, alkaline protease and trypsin were selected and further screened based on the inhibitory activity of their respective hydrolysates. Subsequently, single-factor experiments and response surface methodology (RSM) were used to optimize the compound enzymatic hydrolysis conditions involving alkaline protease and trypsin. The resulting hydrolysate was then fractionated and purified using ultrafiltration followed by Sephadex G-25 gel filtration. Moreover, the amino acid sequences of the peptides in the most active fraction were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Finally, potential bioactive peptides were further screened through bioinformatics analysis, and their interaction mechanisms with ACE were investigated using molecular docking. Based on the Box-Behnken experimental design, the optimal hydrolysis conditions were identified as follows: a temperature of 50 °C, a hydrolysis time of 4.5 h, pH9.0, an alkaline protease-to-trypsin ratio of 2:1, a total enzyme dosage of 3100 U/g, and a solid-to-liquid ratio of 1:150 (g/mL). Under these optimized conditions, the enzymatic hydrolysate exhibited an ACE inhibition rate of 77.48%±1.03%, which was approximately 25% higher than the inhibition rates obtained using alkaline protease or trypsin alone. Following separation and purification, five peptides including VELGV (516.30 Da), SVAPL (486.29 Da), SLAGLP (557.33 Da), TDIGVAGI (745.41 Da), and LGGGPI (513.30 Da) were identified from the active fraction (No. F2). Among these, SLAGLP and LGGGPI which had PeptideRanker scores above 0.5, were selected for molecular docking analysis. The results indicated that both peptides could specifically interact with the active site of ACE through hydrogen bonding and coordination with Zn²⁺. Overall, this study provides a feasible and practical strategies for the value-added processing of hairtail and establishes a scientific basis for the development of functional food-derived bioactive peptides.