Comparative enzymatic hydrolysis of porcine tracheal cartilage generates ACE/DPP-IV-inhibitory and antioxidant peptides with enhanced bioactivity following simulated gastrointestinal digestion and molecular docking analysis

Porcine tracheal cartilage, an underutilized Type II collagen-rich by-product, was systematically evaluated as a source of angiotensin I-converting enzyme (ACE) inhibitory, dipeptidyl peptidase-IV (DPP-IV) inhibitory, and antioxidant peptides. Neutrase at 4 h outperformed Papain in generating ACE-inhibitory and antioxidant peptides. The <3 kDa ultrafiltration fraction, enriched in small hydrophobic peptides, exhibited the highest bioactivities. LC-MS/MS identified eight sequences (870–1565 Da). Chemical synthesis confirmed LGLGADMFHR as the most potent ACE inhibitor (IC50 = 0.52 mM), QGLPGPGAVAEYT as the lead DPP-IV inhibitor (IC50 = 1.84 mM), and LGFGADRAGPQ as the strongest antioxidant (5.64 mM Trolox equivalents). Molecular docking revealed LGLGADMFHR binding through Zn2+ coordination and hydrogen bonds with Cys352, Glu376, and Asp453. Standardised INFOGEST gastrointestinal digestion progressively enhanced ACE inhibition and antioxidant activities while DPP-IV inhibition declined, establishing porcine tracheal cartilage as a viable source of gastrointestinally stable cardiovascular bioactive peptides.