epidermidis (MTCC435) and P. aeruginosa (ATCC27853) in a microtiter plate assay in triplicates. To examine the bacterial growth or killing rate in the presence of different fractions, bacterial cells were grown in 100 μl of Mueller-Hinton
Anlotinib ic50 broth (MHB, HiMedia, India) supplemented with fixed concentration (10 μg/ml) of each fraction, at 37°C. Growth or killing rates were determined by measuring OD at 600 nm. The OD values were converted into concentration of cells measured in CFU per millilitre (1.0 OD corresponded to 2.16 × 108 CFU/ml). The MIC of selected biosurfactant/selleck lipopeptide was evaluated for strains S. aureus (MTCC1430), M. luteus (MTCC106) and S. marcescens (MTCC 97) along with P. aeruginosa and S. epidermidis by using a microtiter plate dilution assay in triplicates as described earlier . Test strains were grown to logarithmic phase (between 0.3-0.4 OD) under optimal conditions. The lowest concentration inhibiting the growth of test strain without showing any increase in absorption up to 48 h of incubation was considered as MIC. MALDI-TOF-MS and sequencing The purified and active lipopeptides were analysed for molecular mass and MS/MS sequencing by using a Voyager time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA, USA). For MS/MS sequencing, the
HDAC inhibitor lactone ring present in lipopeptide was cleaved by incubating each peptide with 10% NaOH in methanol at room temperature for 16 h. The cleaved peptide obtained was lyophilized and again extracted with methanol, and allowed for
mass spectrometry analysis. Spectra were recorded in the post-source decay (PSD) ion mode as an average of 100 laser shots with a grid voltage of 75%. The reflector voltage was reduced in 25% steps and guide wire was reduced 0.02–0.01% with an extraction delay time of 100 ns. Fatty acid analysis by GC-MS To analyze the fatty acid content associated with the lipopeptides, the peptides (5 mg of each) were incubated with 0.5 ml of 6 M HCl at 90°C for 18 h in sealed tubes for acid hydrolysis. The fatty acids were extracted with ether, treated with 0.95 ml methanol and 0.05 ml of 98% H2SO4 at 65°C for 6 h. Finally, fatty acid methyl esters were obtained with n-hexane extraction selleck screening library and analyzed on GC-MS with a Clarus 500 GC (PerkinElmer, USA). The carrier gas used was helium with a flow rate of 1.0 ml/min. The column temperature was maintained at 120°C for 3 min and thereafter gradually increased (8°C/min) to 260°C. Statistical analysis The statistical significance of the experimental results was determined using one-way ANOVA followed by Dunnett’s test. Values of p<0.05 were considered statistically significant. Prism version 5.0 was used for all statistical analyses. The results are presented as the mean of triplicates (n=3) ± SD.