Abstract
This study aimed to effectively identify anti-inflammatory peptides in Jinhua ham, a dry-cured meat product made from the hind legs of pigs by curing and fermenting processes, and elucidate their anti-inflammatory mechanism. The investigation involved a combination of chromatographic purification, in silico screening, and in vitro validation. The first peak of JHP (JHP-P1) was purified using two-part exchange chromatography, in which 3350 peptides were identified by nano-HPLC-MS/MS, among which QLEELKR and EAEERADIAESQVNKLR showed significant anti-inflammatory potential (prediction scores: 0.759 and 0.841). In molecular docking and in vitro RAW264.7 cell experiments, these peptides displayed a strong affinity for Toll-like receptor 4-myeloid differentiation-2 (TLR4-MD-2), specifically binding around Arg 380, Lys 475, His 401, Gln 423, Asp 426, etc. This binding inhibited TLR4 expression and prevented trimer formation about TLR4-MD-2 and lipopolysaccharide (LPS), strongly inhibiting the inflammatory cascade. JHP suppressed LPS-induced cytokine overproduction and partially inhibited the phosphorylation of proteins in the MAPK/NF-κB pathway. These results demonstrated that combining in silico methods (activity prediction and molecular docking) is an effective strategy for screening anti-inflammatory peptides. This study provided a theoretical basis for identifying more anti-inflammatory peptides and applying them in functional foods.