Purification and identification of hydrolyzed peptides from Lates calcarifer by-products and their anti-inflammatory function in lipopolysaccharide-induced RAW 264.7 macrophage cells

Abstract Fish by-product is considered a sustainable source for obtaining natural protein and hydrolysates with functional and biological activity. The present study explored the potential of barramundi, Lates calcarifer by-products as a natural source to generate bioactive peptides with anti-inflammatory properties in LPS-induced RAW264.7 macrophage cells. The results indicated that the highest degree of hydrolysis was achieved at 4% papain at a rate of 26.15 ± 2.67% in 48 hours. The hydrolyzed peptides had a total amino acid content of 51.55%, with essential and non-essential amino acids accounting for 27.28% and 24.27%, respectively. The active peptides were purified with ultrafiltration and Sephadex G-15 column. Eight peptide exhibited anti-inflammatroy properties were identified by using LC-MS/MS. Evaluation of anti-inflammatory peptides using the PreAIP database revealed high anti-inflammatory scores (0.501–0.659) for peptide sequences LKLLLL, KPKLLL, and LQLLL, and moderate scores (0.433–0.436) for peptide sequences LALDIEIATYR and LQLL, while GPVS, FGVS, and AMSP had lower scores (0.261–0.293). Molecular docking simulations revealed that two peptide sequences, LALDIEIATYR and LKLLLL, can effectively bind to Toll-like receptor (TLR4), with leucine playing a major role in receptor binding. The hydrolyzed peptides from barramundi by-products exhibit potential for improving the inflammatory response, as they effectively inhibited the production of proinflammatory cytokines such as IL-6, IL-1β, and TNF-α after LPS stimulation and increased the gene expression levels of anti-inflammatory factors such as IL-10 and IL-12. Therefore, the study suggests that the hydrolyzed peptides from barramundi by-products offer a promising therapeutic strategy for the treatment of inflammatory responses.