Extraction, detection and purification of antifungal antibiotic from antagonistic bacterium S. marcescens strain ETR17

Bacterial strain ETR17 was inoculated into semi-solid pigment producing media [58] by pour plate method and incubated at 30°C for 8 d. The total content (180 mL) was crushed in a blender and extracted with 250 mL of 80% aqueous acetone for 24 hours in an orbital shaker [59]. Agar was removed by centrifugation at 15,000 rpm for 20 min at 10°C and the supernatant containing antibiotics was condensed at 40°C in a rotary vacuum evaporator (Eyela CCA-1110, Japan). The aqueous concentrate was filtered through cellulose acetate filter paper (Sartorius, pore size 0.2 μm) and 20 mL portions of the filtrate were extracted twice with 2.5 volumes of diethyl ether. The organic phase containing antibiotics were evaporated to dryness in vacuo at 30°C and the residue was re-extracted with 30 mL of acetone, and finally condensed under vacuum to obtain a red pasty mass [38].

The crude extract (2.5 g) was dissolved in 10 mL methanol, mixed and dried over silica gel (mesh 60–120) and was subjected to silica gel column chromatography. Elution with 100% petroleum ether and petroleum ether -ethyl acetate (95% to 50%) yielded a total of eleven fractions (F1 to F11) of 100 mL each. Each fraction was vacuum concentrated and tested for antifungal activity. The active fractions F3, F4, F5, F10 and F11 were monitored by thin layer chromatography (TLC). Samples were loaded on TLC sheets pre-coated with Silica Gel 60 F254 and co-chromatographed with standard antibiotics pyrrolnitrin, prodigiosin and phenazine (Sigma-Aldrich). The sheets were developed in benzene:acetic acid (9:1) and viewed under UV (254 nm) light. The antibiotics present in the bioactive fractions were further purified by preparative TLC. For this, large volumes (1 mL) of the bioactive fractions were spotted as before on glass-backed preparative TLC plates (prepared manually by coating with silica gel G. The sheets were developed similarly in benzene: acetic acid (9:1) and the zone corresponding to the R_f value obtained on analytical plates were scrapped from the TLC plate, suspended in methanol and centrifuged. The supernatant was dried in vacuo and dissolved in methanol. Partially purified compound in methanol was scanned between 200 to 700 nm on a dual beam Varian Cary 50 Bio UV-Visible spectrophotometer (Varian, Australia) along with standard prodigiosin and pyrrolnitrin. Thus two antibiotics were detected by analytical TLC. High performance liquid chromatographic analysis of the antifungal metabolites purified by TLC was performed in Shimadzu SPD-20A, Japan. Fractions which appeared to contain same antibiotics were combined, dissolved in methanol (50μg per mL) and 20 μl was injected into C18 Reverse Phase column (250 x 4.6mm size and 4μm particle size) (Phenomenex, USA). The pump used was LC-20AD (Shimadzu, Japan). The eluent flow rate was adjusted to 1 mL min^-1 and analyzed isocratically in 100% methanol. Standard antibiotics were used at a concentration of 10 μg mL^-1. Pyrrolnitrin was detected at 225 nm using a D2 detector (Prominence, Shimadzu, Japan) and prodigiosin was detected at 536 nm using tungsten (W) detector (Prominence, Shimadzu, Japan).

Presence of pyrrolnitrin was further confirmed by LC-ESI-MS analysis using a gradient elution program with solvent A (methanol) and solvent D (ammonium acetate buffer, pH 6.5): 50% solvent A and 50% solvent D from 0 to 10 min; 70% solvent A, 30% solvent D at 10 min and 80% solvent A, 20% solvent D till 30 min at a flow rate of 0.8 mL min^-1 and 254 nm. A 20 μl sample was injected into the column without any dilution. The column used was Thermo ODS-2 (250 x 4.6 mm size and 5 μm particle size) (Thermo, India). The electrospray ionization mass spectra were recorded on a Thermo LCQ Advantage Max (Thermo, India) with the following specifications: Source voltage 5.3V, source current 80.0 μA, capillary voltage 3.0 V, tube lens offset 5.0 V and capillary temperature of 300°C.