A marine Bacillus strain produced biosurfactant during its growth in a defined glucose-containing medium. An efficient method for separation and purification of biosurfactant isoforms was developed and optimized in high-performance liquid chromatography (HPLC) by manipulating solvent gradient program and flow rates. Starting with an initial run time of 60 min, the final optimized method had a significantly reduced run time of 20 min. By using this method, all the surface-active isoforms (fractions A-D) were eluted within 12 min of elution with much shortened retention time of each component. The purity levels of the isoforms were enhanced using the optimized method as evident from their lower CMC values. Among the four surface-active fractions, antimicrobial action was solely displayed by HPLC fraction A. FTIR analysis revealed all the HPLC fractions to be lipopeptide in nature and MALDI-ToF mass spectral analysis showed that these belonged to the fengycin family containing C(15), C(16), and C(17) fengycins.
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