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The cell-free culture supernatant was precipitated by adjusting pH to 2��0 with 6?mol?l?1 HCl and stored at 4��C overnight. The precipitates were collected by centrifugation at 8000?g for 20?min after the liquid was decanted, and the resulting precipitates were extracted with methanol. After centrifugation and discarding the insoluble substance, the methanol soluble antibiotics were obtained by evaporating the methanol. For isolating the lipopeptide homologues, the HPLC Waters 600 system (Water 996 PDAD; Waters Corp., Milford, MA) was used. The mobile phases were acetonitrile http://www.selleck.cn/products/ON-01910.html and 10?mmol?l?1 ammonium acetate (3?:?4, v/v). Lipopeptide antibiotics were dissolved in the mobile phase and obtained a final concentration http://www.selleckchem.com/products/abc294640.html of 1?mg?ml?1. After filtering through a 0��22?��m filter (PALL; Gelman Laboratory, New York, NY, USA), the lipopeptide homologues were eluted with a Diamonsil C18 column (5?��m, 250?mm?��?4��6?mm; Dikma, Beijing, China). The flow rate was 1?ml?min?1 and the elution was detected at 214?nm. The eluted fractions were collected and used for bioassay and ESI mass spectrometry (ESI-MS) analysis. ESI mass spectrometry analysis was carried out on a LCQ Advantage instrument of Thermo Finnigan (Thermo Electron Corporation, San Jose, CA, USA). Samples were injected with a syringe. The electrospray source was operated at a capillary voltage of 32?V, a spray voltage of 5?kV and a capillary temperature of 320��C. Bacillus subtilis JA and the mutant strain were cultured in 3.5-l fermentor (Bioengineering, Wald, Switzerland). A 6% inoculum of each seed culture was incubated into the fermentor containing 2-l medium with an agitator speed of 300?rev?min?1 at 30��C. Antifoam was added http://www.selleckchem.com/products/loxo-101.html aseptically to the culture if necessary. The lipopeptide production and biomass were measured every 6?h during the fermentation process. The experiment was repeated three times. For the low temperature of plasma glow, helium was chosen as working gas in this study. The helium gas flowing rate was maintained at 21��5?l?min?1. Figure?2 shows the glow of APPJ operated by helium. The temperature of emitting plasma glow was about 40�C50��C and the power consumption was about 20�C30?W. Figure?3 shows the relationship between the survival rate of B.?subtilis JA and the plasma treatment time. The survival rate of B.?subtilis JA decreased sharply in the first 5?s, and then dropped slightly. The survival strains were selected and their antagonistic activities were tested primarily in the screening plate as described in ��Materials and methods��. The mutation efficiency (ME) of B.?subtilis JA was calculated by the equation: , (A, inhibition zone of the mutant strain; B, inhibition zone of the original strain). The ME more than 5% or