Identification of DPP-IV Inhibitory Peptides from Eggshell Membranes: Structure–Activity Relationships, Digestive Stability, and Molecular Mechanisms

Eggshell membrane (ESM), a protein-rich byproduct of the egg processing industry, contains latent bioactive sequences with physiological relevance, but remains largely underutilized. To evaluate its potential as a novel source of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides, ESM was enzymatically hydrolyzed using five proteases, among which alkaline proteases exhibited the most effective hydrolysis. The resulting hydrolysates were systematically assessed for DPP-IV inhibitory activity, amino acid composition, and peptide sequence characteristics. Sequence-activity analysis revealed that peptides with N-terminal hydrophobic residues and Pro or Ala at the second position demonstrated enhanced inhibitory potency, indicating a strong correlation between the peptide structure and bioactivity. Subsequently, three novel peptides (APSPWE, LFPVTPN, and GPPHFLPF) were identified via liquid-chromatography tandem mass spectrometry (LC-MS/MS) and molecular docking. Among them, APSPWE exhibited the most potent inhibitory activity (IC50 = 246 μM) and showed significant in situ inhibition in Caco-2 cells. Network pharmacology analysis further revealed that APSPWE may modulate multiple glucose-regulatory pathways, including tumor necrosis factor (TNF), IL-17, advanced glycation end-products-receptor for AGE (AGE-RAGE), and insulin signaling. These findings highlight ESM as a promising source of DPP-IV inhibitory peptides and offer mechanistic insights into their structure–function relationship.