Innovative discovery and validation of ACE-inhibitory peptides from eggshell membrane keratin byproducts: An integrated computational and in vitro approach.

To advance high-value utilization of egg-processing byproducts, we systematically characterized eggshell membrane (ESM) keratin as a novel source of angiotensin-converting enzyme (ACE)-inhibitory peptides. LC-MS/MS coupled with in silico analysis identified protein A0A8V0ZQN6 as the predominant ESM keratin with strong ACE-inhibitory potential. Computational simulations nominated five candidate proteases for hydrolysis, and subsequent bioinformatic screening with molecular docking yielded five peptide candidates. Of these, SICR (IC50 = 106.5 μg/mL) and MTKW (IC50 = 55.23 μg/mL) demonstrated significant ACE inhibition in vitro, showed non-cytotoxicity, and retained bioactivity in a human umbilical vein endothelial cell (HUVEC) model. Notably, MTKW restored the viability of angiotensin II (Ang II)-injured HUVECs from 66.05% to 86.8%. Molecular docking further confirmed that both peptides bind stably within the ACE active site, with -CDOCKER energies of 100.623 and 108.573 kcal/mol for SICR and MTKW, respectively, surpassing the reference ligand LPR (91.61 kcal/mol), and forming seven and eight hydrogen bonds. Collectively, these results position SICR and MTKW as promising natural ACE-inhibitory candidates for incorporation into functional foods or nutraceuticals targeting blood pressure regulation. This study validates ESM as a viable yet underutilized food protein resource, establishes an integrated pipeline spanning ESM keratin preparation, protein identification, in silico enzymatic screening, and in vitro peptide validation, and provides a theoretical basis for developing natural ACE inhibitors from eggshell membrane byproducts.