RAPID IDENTIFICATION OF ANTIMICROBIAL LIPOPETIDES IN BACILLUS SUBTILIS TR50 CELL-FREE SUPERNATANT BY HPLC-HIGH RESOLUTION-ORBITRAP(TM)-BASED MASS SPECTROMETRY

Bacillus subtilis TR50, a gram-positive endospore-forming bacteria isolated from a cured sausage, proved to be source of several antimicrobial compounds against food-borne pathogenic bacteria (Caputo et al., 2011). The wide chemical variability shown by these metabolites and their related antimicrobial efficacy depend on both the specific strain producer and on nutritional/environmental growth conditions (Baruzzi et al., 2011;Dhouha and Ellouze-Chaabouni, 2011). The difficulty for reliably identifying the antimicrobial compounds released by strains hampers their application in many fields such as controlling food microbial spoilage or fighting the emergence of antibiotic resistance to human pathogens. In general, identification of these antimicrobial compounds such as bacteriocins and lipopeptides in culture filtrates is accomplished by MALDI-TOF analysis or filtrate fractionation followed by HPLC separation and ESI -MS/MS analysis (Pabel et al., 2003; Kim et al., 2004; Caputo et al., 2011). In this work we describe a LC/MS method for the rapid screening of antimicrobial compounds produced by Bacillus strain TR50. By using HPLC separation and High Resolution MS analysis on a benchtop Orbitrap(TM)-based mass spectrometer equipped with a collision chamber we identified sixteen compounds belonging to three family of lipopeptides in the cell-free supernatant directly. This approach is herein described and proposed as a high throughput tool for the rapid identification of antimicrobial and antifungal compounds in microbial cultures.