LC/ESI-MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti-adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisationmass spectrometry (ESI-MS) and ESI-MS/MS direct infusions: two sets of molecular ionspeciesbelonging to the fengycin andsurfactin familieswere revealedandtheir structures defined, interpreting their product ion spectra. The LC/ESI-MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI-MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI-MS/MS analysis of fraction I showed the presence of C₁₃, C₁₄ and C₁₅ surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na]⁺ at m/z 1030, 1044 and 1058. LC/ESI-MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H]⁺ at m/z 1478 and 1506 corresponding to C₁₇ fengycin A and C₁₇ fengycin B, respectively. Other homologues (C₁₄ to C₁₆) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, althoughwith the same nominalmass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed.