Bioprospecting Bioactive Peptides in Halobatrachus didactylus Body Mucus: From In Silico Insights to Essential In Vitro Validation

Fish body mucus plays a protective role, especially in Halobatrachus didactylus, which inhabits intertidal zones vulnerable to anthropogenic contaminants. In silico predicted bioactive peptides were identified in its body mucus, namely, EDNSELGQETPTLR (HdKTLR), DPPNPKNL (HdKNL), PAPPPPPP (HdPPP), VYPFPGPLPN (HdVLPN), and PFPGPLPN (HdLPN). These peptides were studied in vitro for bioactivities and aggregation behavior under different ionic strengths and pH values. Size exclusion chromatography revealed significant peptide aggregation at 344 mM and 700 mM ionic strengths at pH 7.0, decreasing at pH 3.0 and pH 5.0. Although none exhibited antimicrobial properties, they inhibited Pseudomonas aeruginosa biofilm formation. Notably, HdVLPN demonstrated potential antioxidant activity (ORAC: 1.560 mu mol TE/mu mol of peptide; ABTS: 1.755 mu mol TE/mu mol of peptide) as well as HdLPN (ORAC: 0.195 mu mol TE/mu mol of peptide; ABTS: 0.128 mu mol TE/mu mol of peptide). Antioxidant activity decreased at pH 5.0 and pH 3.0. Interactions between the peptides and mucus synergistically enhanced antioxidant effects. HdVLPN and HdLPN were non-toxic to Caco-2 and HaCaT cells at 100 mu g of peptide/mL. HdPPP showed potential antihypertensive and antidiabetic effects, with IC50 values of 557 mu g of peptide/mL for ACE inhibition and 1700 mu g of peptide/mL for alpha-glucosidase inhibition. This study highlights the importance of validating peptide bioactivities in vitro, considering their native environment (mucus), and bioprospecting novel bioactive molecules while promoting species conservation.