Angiotensin I-converting enzyme (ACE) plays an important role in the regulation of blood pressure. Screening peptides with ACE inhibitory activity from food source proteins has become a research hotspot. Spirulina is the earliest algae to be eaten with the highest protein content in food. In this study, ACE inhibitory peptides were prepared by hydrolyzed Spirulina protein with alkaline protease, neutral protease, trypsin, and pepsin. Alkaline protease was selected as the best protease according to hydrolysis degree, DPPH scavenging activity, and ACE inhibitory activity. Two new ACE inhibitory peptides were found from the hydrolyzed fractions using peptidomics, and computational simulation: His-Ile-Ile-Ala-Arg-Pro-His (HIIARPH, IC50=461.5 μmol/L) and Leu-Arg-Leu-Lys-Glu (LRLKE, IC50=155.8 μmol/L). The enzyme kinetics of two new peptides are shown as non-competitive inhibition mode. The results of molecular docking showed that HIIARPH and LRLKE could form hydrogen bonds with S1 and S2 pockets of ACE to play an inhibitory role. Moreover, LRLKE could bind with S3 pockets and Zn2+, which contributed to its better inhibitory activity. Stability study showed that LRLKE had good thermal stability and maintains high activity under acidic or mildly alkaline conditions. Cytotoxic study showed that HIIARPH and LRLKE had no significant influence on HUVEC cells viability under the 1 mmol/L concentration. In summary, this study provides a method for rapid screening of ACE inhibitory peptides and provides theoretical reference for the development of Spirulina-derived antihypertensive peptides.
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