The electrospray source was operated at a capillary voltage of 46 V, a source voltage of 4.5 kV and a capillary temperature of 300 °C. The collision-induced dissociation (CID) experiments were also performed on this instrument. Helium was used as the collision gas and the collision energy was set at 25%. The MICs of the purified antibiotics against bacteria were determined using a microbroth dilution method in 96-multiwell

microtiter plates (McVay & Rolfe, 2000). Mueller–Hinton broth was used for the growth of indicator bacteria. The final concentrations of the antibiotics in the wells ranged from 0.20 to 100 μg mL−1. MICs were measured after incubation at 35 °C for 18 h. The MICs against fungi were determined using the agar microdilution test as described previously, with some modification (Lu et al., 2009). PDA was used as a substitute for Sabouraud dextrose agar. Commercial polymyxin B was used as a control. All the tests were performed, with three Selleckchem BI2536 replicates. Morphologically, strain B69 was characterized to be a rod-shaped, gram-negative, motile, spore-forming bacterium. This strain could grow at 15–45 °C or pH 6.0–8.5, and it could tolerate up to 2% NaCl in a nutrient broth. Strain B69 was positive for catalase, nitrate reduction, the methyl red test, starch hydrolyzation, casein decomposition, gelatin liquefaction,

esculin hydrolysis and the arginine dihydrolase test, but negative for oxidase, the Voges–Proskauer test, indole production and H2S formation. The same results were obtained when the reference strain P. elgii SD17 was tested. All these characteristics supported the identification PD0325901 price of this strain as a member of the genus Paenibacillus, and it was closely related to P. elgii. Furthermore, the 16S rRNA gene sequence analysis demonstrated that strain B69 shared the highest sequence similarity to that of P. elgii

SD17T (99.4%). Therefore, it was concluded that strain B69 belonged to P. elgii, and it was designated as P. elgii B69. Although the antimicrobial activity of CFS was unaffected by heat treatment at 40 and 60 °C for 2 h, its activity was significantly reduced after 2 h at 80 or 100 °C. pH variation between 1.0 and 8.0 had no effect, but the ID-8 inhibitory activity was reduced at pH 9.0 and 10.0, and was totally lost at pH 11.0 and 12.0. The results revealed that bioactive compounds of P. elgii B69 were most stable at an acidic or a neutral pH and they became progressively inactivated at an alkaline pH. The enzyme degradation assay showed that the antimicrobial activity of CFS was not affected by any of the enzymes tested. The active compounds were isolated from the n-butanol extract using MCI GEL CHP20P column chromatography and HPLC methods. In the HPLC profile, two antimicrobial compounds eluted at retention times of 30.22 and 32.08 min were isolated and designated as Pelgipeptins A and B, respectively. From a total of 10 L of culture, approximately 15 mg of Pelgipeptin A and 21 mg of Pelgipeptin B were obtained.