Preparation of Crude Extracts of Lipopeptide Compounds and Separation and Purification of Organic Solvents

For lipopeptide production, liquid chromatography and mass spectrometry characterization were used. Bacillus amyloliquefaciens YN201732 was streaked on Luria-bertani (LB) plates and cultured at 37°C for 24 hours. After growing, a single colony was picked and inoculated into Landy liquid medium at 37°C, 160 rpm, cultured for 48 hours with shaking to prepare 2 L of fermentation broth (Branda et al., 2001). The fermented suspension was centrifuged to remove bacterial cells (50 mL centrifuge tube, 4°C, 10000 rpm, 20 minutes), and the supernatant was adjusted to pH 2.0 with 6 mol/L HCl and left at 4°C overnight. The precipitate was collected by centrifugation at 10,000 rpm for 10 minutes. The precipitate was further washed three times with sterile deionized water, 5 mL of deionized water was also added to adjust pH to 7.0 with 1 mol/L NaOH. After freeze-drying, the powder was extracted with three to four times the amount of 95% methanol under the assistance of ultrasonic for 3 hours. After centrifugation, the precipitate was collected and extracted twice, and the extracted solution was mixed together. The pooled extraction solution was completely evaporated under pressure using a rotary vacuum evaporator to obtain a crude extract of the lipopeptide. Then the lipopeptide crude extract was sequentially extracted with petroleum ether, ethyl acetate, n-butanol, and water. After the crude extract was dissolved in 100 mL H₂O, it was extracted with 1: 1 (V: V) petroleum ether for 3 hours. During this period, it was shaken every 10 minutes and extracted 3 times. Organic layer combined extracts were concentrated and weighed with a rotary evaporator. Aqueous phase was sequentially extracted with ethyl acetate and n-butanol in the same way, and the remaining aqueous phase was directly concentrated and weighed to obtain secondary extracts of four different polar solvents. The four separated components were concentrated and dried and then dissolved in a certain amount of methanol. The solvents were volatilized and secondary extraction product was dissolved with equal amounts of methanol respectively, and four extracted fractions were collected. Four fractions were filtered through a 0.22-μM-pore-size hydrophilic membrane, which was used for the determination of antifungal activity.