Alcalase-derived egg white hydrolysates exhibit ACE inhibition In silico and gut microbiota modulation In vivo

This study investigated the dual ACE-inhibitory and gut microbiota-modulating potential of egg white hydrolysate (EWH), obtained through Alcalase enzymatic hydrolysis. LC-ESI-MS/MS analysis of the most bioactive fraction (F2), which exhibited strong antioxidant and antibacterial activities, identified six putative bioactive peptides: VLLPDEVSGL, MANKGPAYGM, AAAAGLNPGLM, GIIQHEL, MAGFVPLLLL, and NVLQPSSVDSQ. Molecular docking revealed that EWH-2 (MANKGPAYGM) and EWH-6 (NVLQPSSVDSQ) exhibited the strongest binding to ACE, with Gibbs free energies (ΔG) of − 14.4 and − 13.8 kcal/mol and dissociation constants (Kd) of 2.7 × 10⁻¹¹ M and 8 × 10⁻¹¹ M, respectively. These interactions involved the S1, S2, and zinc-binding motifs via hydrophobic interactions and hydrogen bonds. These findings were supported by 100 ns molecular dynamics simulations, confirming stable ACE–peptide complexes with particularly favorable binding for EWH-2. In vivo administration of EWH to male rats for 14 days ( n = 6 per group) significantly increased gut microbial alpha diversity and reshaped microbial community composition. EWH treatment enriched genera such as Prevotella , Paraprevotella , Sutterella , Butyricimonas , and Barnesiella , which are associated with short-chain fatty acid production and metabolic health. Collectively, these findings demonstrate that Alcalase-derived EWH exhibits dual ACE-inhibitory and gut microbiota-modulating activities, suggesting potential benefits for blood pressure regulation and metabolic health.