Identification of pigeon crop milk protein-derived candidate peptides with potential anti-inflammatory and antioxidant activities: Integrated proteomics, in silico screening, and in vitro validation.

China's pigeon industry is large in scale, but insufficient deep processing and lack of high value-added products restrict industrial upgrading. Due to its high protein content and unique composition, pigeon crop milk (PCM) holds potential for generating functional bioactive peptides via enzymatic hydrolysis. This study aims to preliminarily explore the anti-inflammatory and antioxidant activities of PCM protein-derived candidate peptides and the underlying mechanisms. PCM contains a high content of hydrophobic amino acids. Annexin A1 isoform p35, protein-glutamine gamma-glutamyltransferase 4, and thioredoxin-dependent peroxiredoxin were identified as key proteins in PCM by proteomics. Three candidates PCM protein peptides, Pro-Trp-Asn-Phe (PWNF), Gln-Ala-Trp-Phe (QAWF), and Gly-Gln-Pro-Phe (GQPF) were obtained by virtual enzymatic hydrolysis and bioinformatics. These peptides alleviated LPS-induced inflammatory responses in RAW264.7 cells, scavenged free radicals, and protected RAW264.7 cells against oxidative stress. Computational predictions suggested that the dual activities of these peptides might be related to their potential interactions with targets in the PI3K-Akt and TNF-associated inflammatory and oxidative stress pathways. In addition, the presence and relative position of Trp and Phe residues appeared to be associated with the observed differences in activity among these peptides. These findings provide a scientific basis for the future development of PCM protein-derived candidate peptide-based functional foods and support the transformation of the pigeon industry toward high-value-added products.