Extraction and Identification of Lipopeptide

The endophytic bacteria B. siamensis strain NKIT9, with the most promising antagonistic activity against all the test pathogenic fungi, was further explored for the production of antifungal lipopeptides. The lipopeptide extraction method involved acid precipitation and solvent extraction, as described by Romano et al. (2011). Briefly, extraction of lipopeptide from a cell-free supernatant was done by precipitation method at pH 2 using 6N HCl and incubated at 4°C overnight and then centrifuged at 12,000 rpm for 15 min at 4°C. The pellet was dissolved in a solvent mixture of Chloroform: Methanol (2:1, v/v) followed by centrifugation for at 12000 rpm for 15 min at 4°C to remove any undissolved extract. The lipopeptides present in the supernatant were filtered and concentrated to dryness by rotary evaporation. Waters ACQUITY Ultra Performance Liquid Chromatography (UPLC) H-class system with the BEH C18 column was employed for lipopeptide profiling. The lipopeptide extract (10 mg) was dissolved in 10 mL of HPLC grade ethanol and filtered through 0.22 μm PVDF membrane syringe filter (Durapore, Merk), and a 10 μL aliquot of the sample was injected into the column and the column was eluted at a flow rate of 0.3 ml/min with 100% methanol at 0 min and gradually increased the polarity to acetonitrile: methanol (40:60) for 4 min which was further retained for 22 min followed by 100% methanol for 25 min. The lipopeptides were detected by mass spectroscopy using positive ionization electrospray (ESI+) Synapt G2-Si High Definition Mass Spectrometer (HDMS) coupled with the UPLC system.