A linear gradient of acetonitrile (20–100%) at a flow rate of 1 mL min−1 was used. Solvent A was deionized water +0.1% trifluoroacetic acid (TFA) and solvent B was acetonitrile +0.1% TFA. Absorbance was monitored at 215 nm. Temperature stability was evaluated by incubating the purified compounds at various temperatures

from 30 to 100 °C for 30 min or at 121 °C for 20 min. Residual anti-Candida activity was determined by disk diffusion assay against C. albicans. The effect of pH was determined using a pH range from 2 to 10 adjusted with diluted HCl or NaOH. After incubation for 2 h at room temperature learn more and neutralization to pH 7, the residual activity was measured. Resistance to proteases was tested by incubating the purified compounds with proteinase K, trypsin, α-chymotrypsin and lipase A at 1 : 10 or 1 : 5 (w/w) ratios, as described previously (Tabbene et al., 2009). HPLC-purified fractions were subjected to TLC using n-butanol–methanol–water (39 : 10 : 20, v/v/v) selleck chemicals as the mobile phase. The bioassay was performed using C. albicans ATCC 10231. TLC plates

were sprayed with water for the detection of hydrophilic compounds. Spraying with ninhydrin or 4,4′-Bis(dimethylamino)diphenylmethane (TDM) allowed the detection of compounds with free amino groups or with peptide bonds, respectively (Yu et al., 2002) and spraying with Pauly reagent allowed the detection of tyrosine-containing peptides (Jutisz, 1960). Lipopeptide compounds were hydrolyzed in sealed tubes with 6 N HCl at 150 °C for 8 h. After total hydrolysis, liposoluble moieties were extracted with chloroform (Besson et al., 1976), analyzed by TLC

on a silica gel 60 plate in chloroform–methanol–water (65 : 25 : 4) and revealed Fenbendazole with ninhydrin as described previously (Russell, 1960). HPLC-purified fractions were analyzed using matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF/MS). Samples dissolved in methanol (1 μL) were mixed with 1 μL of matrix (α-cyano-4-hydroxycinnamic acid) at 5 mg mL−1 in 50 : 50 H2O : CH3CN containing 0.1% TFA. Spectra were acquired using a prOTOF 2000 system (Perkin Elmer) operating in positive reflectron mode with an accelerating voltage of 16 kV. The MIC of the purified compounds against human isolates of the pathogen C. albicans was determined by the microbroth dilution assay. One million cells mL−1 of C. albicans were tested for their sensitivity to twofold increasing concentrations of the compounds (from 1.95 to 1000 μg mL−1). Amphotericin B was used as a positive control. After incubation at 28 °C for 24 h, yeast growth was determined by measuring the OD600 nm with a microplate reader (Bioteck, ELx 800). The MIC was defined as the lowest concentration of the compounds inhibiting the yeast growth. MFC was determined from the same experiments by removing 10 μL from wells displaying no yeast growth after 48 h of incubation. Aliquots were spread onto Sabouraud dextrose agar plates, incubated at 28 °C for 24 h and counted.