Internship Guideline
Microorganisms as a Source of Antioxidant
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- 4. Methods of extraction, isolation and characterization of bioactive secondary metabolites 4.1 Extraction methods of bioactive secondary metabolites
- High Performance Liquid Chromatography (HPLC)
| 3.4.1 Microorganisms as a Source of Antioxidant
A. Actinomycetes The actinomycetes are gram-positive, aerobic, filamentous and spore-forming bacteria, with a foremost reputation in producing chemically different metabolites bearing a broad spectrum of biological activities, including antifungal, antibacterial and insecticidal activities. Mycothiol (MSH), a principal ‘sugar’ thiol present in the cell wall of actinomycetes that serves as the glutathione (GSH) analog. Actinomycetes lack the enzymes for GSH biosynthesis, but as asubstitute, they utilize alternative low molecular weight thiols (LMW), such as bacillithiol (BSH) and MSH, ergothioneine (ESH). MSH maintains the intracellular redox homeostasis, allowing the appropriate working of many biological processes, counting enzyme activation, DNA synthesis, and cell-cycle regulation. MSH acts as an electron donor/acceptor and also assists as a cofactor in detoxifying free radicals, xenobiotics and alkylating agents. Unlike GSH, MSH has two sugar component viz N- glucosamine and inositol along with cysteine component as an alternative of the two amino acids, glycine, and glutamic acid. 4. Methods of extraction, isolation and characterization of bioactive secondary metabolites 4.1 Extraction methods of bioactive secondary metabolites Secondary metabolites were extracted using solvent extraction method. Chloroform and methanol were used as solvents for the secondary metabolite extraction. The intracellular and extracellular metabolites were extracted as per standard protocol.20 Intracellular secondary metabolites extraction was carried out by dissolving pallet of bacterial broth culture into 0.5 ml of methanol and further after mixing centrifuged at 10,000 rpm for 10 minutes and upper layer was used for further work. Extracellular secondary metabolites extraction was carried out by dissolving bacterial broth culture into 0.5ml chloroform and further after mixing centrifuged at 10,000 rpm for 10 minutes and bottom layer was used for further work. The extraction of metabolites was based on the polarityofsolvent (Demain AL and Fang A., 2000). 4.1.1 High Performance Liquid Chromatography (HPLC) After the removal of cell mass, supernatant was analyzed by spectrophotometer at 265nm. This was carried out for HPLC analysis. 10 μl of sample was injected to C18 column (250mm X 4.6mm X 5mm). The flow rate was 0.50 ml/min. Sample was analyzed at 220 nm wavelength. In order to purify the active fraction (methanol: chloroform 20: 80 extract); chloroform was added gradually (dropwise) until formation of the first precipitate. The precipitate was separated by centrifugation. Each fraction was tested in inhibition test (Singh V., et al., 2016 ). Ammonium sulphate was also used for the extraction. Ammonium sulfate was grind in a glass mortar to be a fine powder that easily to dissolve. This powder was added gradually to the antagonistic solution. The solution was shacked using a vortex. The formed precipitate was separated by centrifugation. The amount of ammonium sulfate was noted for each precipitate and calculated as saturation percent. Each precipitate was dissolved in water and re-precipitated again by ammonium sulfate as a purification step (Berdy J., 2005). Each precipitate was dissolved in methanol and centrifuged to remove any residue of ammonium sulfate. This step was repeated several times until there moving all amounts of ammonium sulfate. Each precipitate was used for the inhibition test and analyzed by HPLC. Download 230.5 Kb. Share with your friends:
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