2.4. Lipopeptides quantification by HPLC analysis

For lipopeptides production, the isolated organism was grown aerobically in Landy medium ²⁰. Culture was carried out for 72 h at 30°C with shaking at 160 rpm. Culture was then centrifuged at 15 000 × g for 15 min.

For their quantification, lipopeptides were purified from culture supernatant by SPE using C18 Maxi clean cartridges (Extract–Clean SPE 500 mg, Alltech Deerfield, IL) used according to the recommendations of the supplier. Lipopeptides were eluted with methanol (100%). The extract was then characterized by HPLC (600 s, Waters, USA) using a C18 column (5 μm, 250 mm × 4.6 mm, 218 TP, VYDAC). Each family of lipopeptides was separately analyzed with the ACN‐water‐TFA solvent system (40:60:0.5 v/v/v for iturin, 80:20:0.5 v/v/v for surfactin, and a gradient from 45/55/0.1 to 55/45/0.1 for fengycin) and a flow rate of 0.6 mL/min. Samples (20 μL) were injected and compared to the appropriate standards (purified iturins, fengycins, and surfactins provided by ProBioGEM laboratory). The retention time and second derivatives of UV‐visible spectra (Waters PDA 996 photodiode array detector; Millenium Software) of each peak were used to identify the eluted molecules.