Abstract

Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics in aquaculture, offering broad activity and reduced risk of resistance. Microalgae represent an underexplored source of bioactive peptides with antimicrobial properties. In this study, three green microalgae (Tetradesmus obliquus, Dunaliella salina, and Chlamydomonas reinhardtii) were initially screened for antibacterial potential through enzymatic hydrolysis using pepsin and trypsin. Among the resulting hydrolysates, the trypsin-digested extracts of C. reinhardtii exhibited the strongest inhibitory activity against the salmonid pathogens Vibrio anguillarum and Piscirickettsia salmonis, prompting its selection for further analysis. Protein extracts were fractionated by solid-phase extraction and RPHPLC, and the active fractions were screened for antibacterial activity. The trypsin hydrolysate fraction (Cr_T40) showed the highest antibacterial effect, with minimum inhibitory concentrations (MIC) of 2.3-4.7 mu g center dot mL- 1 and low cytotoxicity toward ASK salmonid cells at bactericidal concentrations, with moderate cytotoxic effects observed at higher concentrations. LC-MS/MS analysis of the active fraction identified ten peptide sequences (9-18 amino acids). Chemically synthesized peptides were evaluated against a panel of Gram-negative and Gram-positive bacteria, including fish pathogens. Three peptides (5323, 5329, 5349) displayed inhibitory activity, with MIC values of 26-150 mu g center dot mL- 1, notably against M. luteus, S. aureus, and V. anguillarum. Synergistic interaction between peptides 5323 and 5329, the former active against V. anguillarum individually, reduced the combined MIC against V. anguillarum to 26.6 mu g center dot mL- 1 (FIC = 0.5), highlighting their combined potential. None of the peptides showed cytotoxic effects at bactericidal concentrations. This study gives new highlights on naturally derived antibacterial peptides from C. reinhardtii. These findings position microalgae as a new source of AMP candidates for sustainable antimicrobial strategies in aquaculture.