In vitro evaluation and in silico characterization of ACE-inhibitory peptides released from an α-lactalbumin hydrolysate after simulated gastrointestinal digestion

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Angiotensin-converting enzyme (ACE) inhibition is an important strategy for hypertension management, motivating the search for natural food-derived inhibitors. This study evaluated the ACE-inhibitory potential of peptides released from an α-lactalbumin (α-La) hydrolysate produced with Alcalase and subjected to simulated gastrointestinal digestion. The α-La hydrolysate (HA) exhibited strong ACE-inhibitory activity (IC₅₀ = 0.29 ± 0.01 mg mL⁻¹), which moderately decreased after simulated gastrointestinal digestion (digested HA, DHA; IC₅₀ = 0.49 ± 0.01 mg mL⁻¹). LC–MS/MS analysis of selected DHA fractions identified three α-La-derived peptide sequences: IWCKDDQNPH (P1), KFLDDDLTDDIM (P2), and DKFLDDDLTDDIM (P3). Following chemical synthesis and in vitro evaluation, only P1 showed inhibitory activity (IC₅₀ = 3.91 ± 0.20 mg mL⁻¹), likely associated with its favorable amino acid composition, including aromatic and basic residues related to ACE inhibition. In silico analysis revealed that P1 established specific interactions with key catalytic residues of ACE, showing an interaction pattern resembling that of angiotensin II. These findings highlight α-La hydrolysates as a potential source of food-derived ACE-inhibitory peptides and support the combined use of in vitro and in silico approaches for peptide characterization.