Pure isolates were spot inoculated on actinomycetes isolation agar medium (Hi-Media,
Mumbai) and plates were incubated at 30 °C for six days followed by inversion for 40 min over chloroform in fumehood. Colonies were then covered with a 0.6% agar layer of nutrient Akt inhibitor agar medium (for bacteria), previously seeded with two Gram positive (Bacillus subtilis and Staphylococcus aureus) and two Gram negative strains (Escherichia coli and Serretia sp.) to evaluate antimicrobial activity. The 16SrRNA gene was amplified with primers forward (5′-GAGTTTGATCC TGGCTCA-3′) and reverse (5′-ACGGCTACCTTGTTACGACTT-3′). Amplified PCR product was sequenced and nucleotide sequence was matched using BLAST program. Phylogenetic tree was constructed using neighbor-joining method [13]. Sequence
of the isolate was submitted to GenBank (Accession ID: JQ964039). Seed culture media for submerged fermentation with following composition (g/l) was used: soybean meal 30, glucose 10, glycerol 10, (NH4)2HPO4 1, (NH4)2SO4 3.5, CaCO3 5.10% of inoculum was added in 100 ml production media with composition: (g/l): sucrose 35, yeast extract 15.0, NaCl 4, KH2PO4 3, K2HPO4 2 and MnSO4 1. Inoculated cultures were grown in a rotary shaker at 200 rpm at 30 °C for seven days. Biomass was separated by centrifugation and filter sterilized supernatant was used for extracellular antimicrobial activity. 100 μl of supernatant of each isolate was administrated in each well. Plates were incubated at 37 °C and zone of inhibition was measured after 24 h of incubation. Optimization of carbon and nitrogen sources Epacadostat ic50 i.e. glucose, starch, lactose, sucrose, galactose, fructose, maltose and xylose were added as individual carbon sources in production media at
1% concentration. Casein, yeast extract, peptone, soya bean meal, NH4Cl, NH4NO3, NaNO3 and urea were provided separately as a nitrogen sources into the production medium. Biomass was separated from growth medium by centrifugation at 4000 rpm HSP90 for 10 min. Crude antimicrobial compound produced in culture was extracted through manual shaking with equal volume of chloroform or ethyl acetate or methanol in a separating funnel. The filtered supernatant was extracted by chloroform in ratio of 1:1 (v/v). The yellow colored residual crude active compound was purified by thin layer chromatography (TLC) in a running solvent system of methanol and chloroform. Two fractions with different Rf values recovered from TLC plates were dissolved in 10% Dimethylsulfoxide (DMSO) and bioassayed against the test microorganisms. Purification of this crude compound was carried out in column chromatography technique on silica gel (MerckLtd. India) using chloroform-methanol (Rankem Ltd. India) gradient (11:3) as running solvent system. Extract were collected and characterized by FTIR and HPLC analysis.
Related posts:
- Synergism against clinical isolates and positive isolates could a
- 25 mm diameter and 0 25 μm film thickness The column oven temper
- The results of MBC-11 on survival of mice inoculated with KAS-6/1-MIP1 a number
- Recent re ports showed that many extracts or compounds from pure
- 2kHz) Figure 6 (A) 2H-spectrum of pure DMPD-d54 in H2O at 298K a