Purification, Identification, and Functional Validation of Anti-Inflammatory Peptides from Protein Hydrolysates of Asian Seabass, Lates Calcarifer by-Product.

Fish processing by-products are increasingly recognized as sustainable raw materials for generating bioactive protein hydrolysates. In the present study, hydrolysates derived from Asian seabass (Lates calcarifer) by-products were produced and their anti-inflammatory potential was investigated in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. The results showed that the hydrolysates were non-cytotoxic at concentrations up to 0.5 mg/mL and significantly reduced nitric oxide (NO) production. Fractionation by ultrafiltration indicated that the 10–30 kDa fraction possessed the greatest inhibitory effect on NO release. Further purification using gel filtration chromatography concentrated the activity in Fraction F2, which effectively suppressed NO generation, decreased the expression of pro-inflammatory cytokines (TNF-α, IL6, and IL1β), and regulated anti-inflammatory mediators. Peptide identification by LC–MS/MS combined with in silico prediction (PreAIP) yielded five peptides with high anti-inflammatory potential. Molecular docking with the TLR4/MD-2 complex (PDB: 2Z64) revealed that two peptides, LALDIEIATYR (LR-11) and LKLLLL (LL-6), exhibited favorable and stable binding interactions. Synthetic peptide assays further confirmed that both LR-11 and LL-6 significantly attenuated LPS-induced inflammatory responses, with LR-11 demonstrating stronger activity. In addition, structure-activity relationship evaluation of LR-11 analogs suggested that its biological function was closely associated with preservation of the original amino acid sequence and balanced physicochemical properties. Overall, these findings highlight Asian seabass by-products as a valuable and sustainable source of anti-inflammatory peptides and identify LR-11 as a promising candidate for future applications in nutraceutical and therapeutic development.