Molecular and Functional Characterization of Black Soldier Fly Derived Peptide Against Food-Relevant Bacteria

Microbial contamination remains a major challenge in food systems, creating demand for stable natural antimicrobials. This study investigates the inducible antibacterial response of Hermetia illucens and characterized the black soldier fly defensin-like peptide DLP4. Larvae were challenged with Staphylococcus aureus or Escherichia coli, and hemolymph activity, antimicrobial-peptide gene expression, C18 solid-phase extraction fractions, and LC-MS/MS profiles were evaluated. Synthetic mature DLP4 was further assessed for antibacterial spectrum, physicochemical stability, and structural properties. Hemolymph antibacterial activity varied with treatment and time, while DLP4 and CLP1 showed distinct transcriptional responses. Antibacterial activity was concentrated in early fractions, with F3 showing the greatest activity. LC-MS/MS detected a conserved DLP-family motif and a cysteine-rich defensin-like peptide in F3, whereas the complete 40-amino-acid sequence of synthetic DLP4 was confirmed. DLP4 showed strong, strain-dependent activity against Gram-positive bacteria, including Bacillus subtilis, Listeria ivanovii, and Staphylococcus spp., while no MIC endpoint was detected for the tested E. coli strains. DLP4 retained substantial activity after heat, ultraviolet, storage, pH, NaCl, and most metal-ion treatments, although Fe³⁺ reduced activity. These findings support DLP4 as a stable, Gram-positive-active antimicrobial candidate for further evaluation in food systems.