Sustainable valorisation of sardine by-products through enzymatic hydrolysis: nutritional and bioactive characterization of protein hydrolysates

Purpose: Transforming seafood by-products into valuable ingredients presents a sustainable approach to advancing food innovation. This study aimed to characterize sardine protein hydrolysates (SPH), obtained by enzymatic hydrolysis, and evaluate their nutritional profile and multifunctional bioactivities, including the effect of enrichment with rosemary extract. Methods: Sardine by-products were hydrolyzed with alcalase, and the resulting SPH were analyzed for proximate composition, amino acid profile, peptide size distribution, lipid composition, and mineral content. Bioactivity was assessed through in vitro cell-based assays in Caco-2, RAW 264.7, and 3T3-L1 cells, testing cytotoxicity, oxidative stress modulation, lipolysis inhibition, and TNF-α expression. Results: SPH contained 57.1% protein, abundant essential amino acids, peptides < 3 kDa, and a lipid fraction enriched in EPA and DHA. Minerals such as sodium, potassium, phosphorus, and calcium were present at significant levels. At 2.5% (m/v), SPH was non-cytotoxic, reduced oxidative stress, increased lipolysis, and suppressed TNF-α expression, with rosemary extract further enhancing these bioactivities. However, higher concentrations (5–10 mg/mL) induced cytotoxic effects in macrophages and adipocytes. Conclusion: SPH are nutritionally rich and display multifunctional bioactivities, particularly when combined with rosemary extract. These findings support their potential as sustainable protein-derived ingredients for incorporation into functional food formulations.