Purification and characterization of antifungal lipopeptide produced by Bacillus velezensis isolated from raw honey

Raw honey contains a diverse microbiota originating from honeybees, plants, and soil. Some gram-positive bacteria isolated from raw honey are known for their ability to produce secondary metabolites that have the potential to be exploited as antimicrobial agents. Currently, there is a high demand for natural, broad-spectrum, and eco-friendly bio-fungicides in the food industry. Naturally occurring antifungal products from food-isolated bacteria are ideal candidates for agricultural applications. To obtain novel antifungals from natural sources, we isolated bacteria from raw clover and orange blossom honey to evaluate their antifungal-producing potential. Two Bacillus velezensis isolates showed strong antifungal activity against food-isolated fungal strains. Antifungal compound production was optimized by adjusting the growth conditions of these bacterial isolates. Extracellular proteinaceous compounds were purified via ammonium sulfate precipitation, solid phase extraction, and RP-HPLC. Antifungal activity of purified products was confirmed by deferred overlay inhibition assay. Mass spectrometry (MS) was performed to determine the molecular weight of the isolated compounds. Whole genome sequencing (WGS) was conducted to predict secondary metabolite gene clusters encoded by the two antifungal-producing strains. Using MS and WGS data, we determined that the main antifungal compound produced by these two Bacillus velezensis isolates was iturin A, a lipopeptide exhibiting broad spectrum antifungal activity.