Bioactive potential and chromatographic characterization of body mucus from Portugal coastal fish halobatrachus didactylus

The body mucus in fish provides a stable physical or chemical barrier against invading pathogens. In mucus, antimicrobial peptides are secreted as a response to immune stimulation. Studies have shown antimicrobial activity against multidrug-resistant pathogens and low toxicity to eukaryotic cells. Previously, body mucus from five captive Halobatrachus didactylus individuals was collected. We aim to explore the mucus studying molecules with bioactive potential. Size exclusion high­performance liquid chromatography (SE­HPLC) analyses were performed on the five body mucus samples showing a similar molecular size distribution with a maximum peak of ca. 800 Da. These five mucus samples were pooled to assess the following bioactivities: antioxidant (ABTS and ORAC), antimicrobial (minimal inhibitory concentration), and cytotoxicity (Caco­2 and HaCaT human cell lines). The protein content in the mucus, determined by the bicinchoninic acid methodology, was 16836 ± 1020 µg BSA/mL. The antioxidant activity resulted in 268 ± 11 µmol TE/g mucus protein for ABTS and 306 ± 11 µmol TE/g mucus protein for ORAC. The antibacterial activity was assessed against five pathogenic bacteria: Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, within minimal inhibitory concentrations of 421 to 105 µg mucus protein/mL. Moreover, the mucus showed non­cytotoxic for Caco­2 cells in concentrations between 196 to 25 µg mucus protein/mL, while it showed cytotoxicity for HaCaT cells. In the future, liquid chromatography­tandem mass spectrometry (LC­MS/MS) analysis will be performed to determine the molecules behind these bioactivities, namely antimicrobial peptides.