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Antibacterial and Antioxidant Activities Of View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Journal of Drug Delivery and Therapeutics (JDDT) Kekuda et al Journal of Drug Delivery & Therapeutics; 2013, 3(4), 47-53 47 Available online at http://jddtonline.info RESEARCH ARTICLE ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF STREPTOMYCES SPECIES SRDP-H03 ISOLATED FROM SOIL OF HOSUDI, KARNATAKA, INDIA Rakesh K.N 1, Syed Junaid 1, Dileep N 1, *Prashith Kekuda T.R 1,2 1 Department of Microbiology, S.R.N.M.N College of Applied Sciences, NES Campus, Balraj Urs Road, Shivamogga-577201, Karnataka, India 2 P.G Department of Studies and Research in Microbiology, Sahyadri Science College (Autonomous), Shivamogga-577203, Karnataka, India * Corresponding author Email: [email protected] ABSTRACT In the present study, we report antibacterial and antioxidant activity of ethyl acetate extract of Streptomyces species SRDP- H03 isolated from rhizosphere soil of Hosudi, Karnataka, India. The isolate SRDP-H03 was assigned to the genus Streptomyces based on the cultural and microscopic characteristics. The ethyl acetate extract of the isolate SRDP-H03 showed marked inhibition of Gram positive bacteria than Gram negative bacteria. The extract was found to possess dose dependent DPPH free radical scavenging and Ferric reducing activity. UV spectral data of ethyl acetate extract showed strong absorption maxima (λmax) at 267 and 340nm. The isolate can be a potential candidate for the development of novel therapeutic agents active against pathogens and free radicals. Further studies on genomic characterization of isolate and isolation and structure determination of the bioactive compounds are under progress. Key words: Hosudi, Streptomyces, Cross streak, Agar well diffusion, DPPH, Ferric reducing INTRODUCTION Microorganisms are attractive sources of bioactive in soil. They have been recognized as prolific source of compounds with pharmaceutical and agricultural bioactive microbial metabolites as they produced 75% of importance. Actinomycetes, belonging to the order biologically active compounds 2,3,5,6. Actinomycetales, are Gram positive, filamentous Hosudi (Latitude 13.9222; Longitude 75.6437) is a small eubacteria with high G+C content. They are involved in village in Shivamogga (taluk), Shivamogga (district), important processes in a wide range of habitats. They are Karnataka (State), India. It is located 10km away from the responsible for the production of volatile compounds like Shivamogga city. No microbial studies on the soil of this geosmin responsible for characteristic earthy odor. They village have been carried out earlier. Hence, in the present can degrade a variety of compounds such as study, we report antioxidant and antimicrobial activity of lignocelluloses and many other polymers occurring in soil solvent extract of Streptomyces species SRDP-H03 and litter, and a range of xenobiotic compounds. In isolated from a rhizosphere soil collected in Hosudi. addition, due of their metabolic diversity, actinomycetes are important source of lytic enzymes, antibiotics and other MATERIALS AND METHODS bioactive metabolites such as plant growth promoters, herbicides, insecticides, antitumor agents etc. These Collection of soil sample actinomycetes have been considered as biotechnologically The soil sample was collected at Hosudi during the month and industrially valuable prokaryotes since they have of October 2012. The soil was collected in a sterile plastic produced a large number of compounds of pharmaceutical bag from a depth of 15cm, immediately brought to the and agricultural importance 1-4. laboratory and dried at 40oC under aseptic conditions 7. Rhizosphere is a unique biological niche with a diverse Isolation of actinomycetes microflora comprising bacteria, fungi, protozoa and algae. The dried soil sample was subjected to serial dilution (up It is defined as the soil adjacent to and influenced by plant -5 root exudates. The microbial community in rhizosphere is to 10 ) followed by plating on Starch casein agar (SCA) nutritionally favoured by input of organic materials amended with antibiotic Fluconazole (to prevent fungal contamination). The plates were incubated aerobically at derived from the plant roots and root exudates. o Actinomycetes are important rhizosphere inhabitants of 30 C for 10 days. The colonies of actinomycetes were many plants, where they enhance plant growth and protect selected on the basis of typical morphology. The isolates were subcultured on sterile SCA slants and maintained in the plant roots against attack by phytopathogens. Most soil 7 actinomycetes are saprophytic. Among the various refrigerator . actinomycetes genera, Streptomyces is the best recognized Primary screening of the actinomycetes isolates and well-studied genus of actinomycetes in terms of number and the bioactive metabolites being produced. The In order to screen antibacterial activity of actinomycetes Streptomyces species are aerobic, spore formers with DNA isolates, we employed Cross streak method. Briefly, the actinomycetes isolates were streaked at the centre of SCA rich in GC content (69-73 %). They form extensive o branching substrate, aerial mycelia and widely distributed plates followed by incubation at 30 C for 5 days. After 5 days, the test bacteria were inoculated perpendicular to the © 2011, JDDT. All Rights Reserved ISSN: 2250-1177 CODEN (USA): JDDTAO Kekuda et al Journal of Drug Delivery & Therapeutics; 2013, 3(4), 47-53 48 growth of the actinomycetes isolates, incubated for 24 spectrum of the extract was determined in the UV region hours at 37oC and the extent of inhibition was observed. (200-400nm) using a UV-visible spectrophotometer The absence of growth or a less dense growth of test (Shimadzu UV 2554) 12. bacteria near the actinomycetes isolates was considered Total phenolic content of ethyl acetate extract of isolate positive for production and secretion of antibacterial 8 SRDP-H03 metabolite by the isolates . One isolate, SRDP-H03, showed marked inhibition of test bacteria and the isolate The total phenolic content of ethyl acetate extract was was selected for further studies. determined by employing Folin-Ciocalteu Reagent (FCR) method. Here, a dilute concentration of the extract (0.5 Cultural characteristics of isolate SRDP-H03 mL) was mixed with 0.5 ml of diluted Folin-Ciocalteu The isolate SRDP-H03 was grown on various media reagent (1:1) and 4 ml of sodium carbonate (1 M). The namely SCA, Inorganic salt-starch agar (ISSA), tubes were allowed to stand for 15 minutes and the Actinomycetes isolation agar (AIA) and Tryptone yeast absorbance was read colorimetrically at 765 nm. A extract agar (TYEA) and Yeast extract-Malt extract agar standard curve was plotted using different concentrations (YEMEA). The color of substrate and aerial mycelium and of Gallic acid (standard, 0-1000 μg/mL) and the total production of diffusible pigments were observed. phenolic content of extract was estimated as μg Gallic acid equivalents (GAE)/mg of extract 13. Microscopic characteristic of isolate SRDP-H03 Antibacterial activity of ethyl acetate extract of isolate The characteristic spore arrangement in the isolate SRDP- SRDP-H03 H03 was studied by employing cover slip method. Here, thin blocks of SCA were cut, placed on a sterile glass slide Agar well diffusion method was performed to screen and the culture of SRDP-H03 was inoculated on the entire antibacterial activity of ethyl acetate extract of SRDP-H03 surface of the agar block. A sterile cover slip was placed against three Gram positive bacteria viz., Staphylococcus on the inoculated surface; the slide was placed in a sterile aureus NCIM- 2079, Bacillus cereus NCIM-2016 and moist chamber and incubated until good growth was Bacillus subtilis NCIM-2699 and five Gram negative obtained. The cover slip was then removed, placed on a bacteria viz., Escherichia coli NCIM-2685, Vibrio drop of dilute crystal violet stain taken on a clean glass cholerae MTCC-3905, Shigella flexneri NCIM-4924, slide and observed under oil immersion objective in order Klebsiella pneumoniae NCIM- 2957 and Pseudomonas to observe arrangement of spores 7. aeruginosa NCIM-2242. The test bacteria were inoculated into sterile Nutrient broth (HiMedia, Mumbai) tubes and Staining and biochemical characteristics of isolate o incubated for 24 hours at 37 C. Using sterile swabs, the SRDP-H03 broth cultures of test bacteria were swabbed on sterile The isolate SRDP-H03 was subjected for staining Nutrient agar (HiMedia, Mumbai) plates followed by techniques namely Gram’s and Acid-fast staining and punching wells of 6mm diameter using sterile cork borer. biochemical tests namely starch hydrolysis, gelatin 100µl of ethyl acetate extract (5mg/ml of 10% DMSO), liquefaction, casein hydrolysis, catalse test, oxidase test, standard (Streptomycin, 1mg/ml) and DMSO (10%) were citrate test, cellulose hydrolysis, hydrogen sulfide (H2S) transferred into labeled wells and the plates were incubated production test and sugar fermentation tests 9,10. at 37oC for 24 hours. The plates were observed for zone of inhibition formed, if any, and measured using a ruler 7. The Fermentation experiment was carried in replicate and the average value Mass cultivation of the isolate SRDP-H03 was carried out was recorded. in sterile Starch casein broth (SCB). The spore suspension Antioxidant activity of ethyl acetate extract of isolate of well sporulated culture of SRDP-H03 was inoculated SRDP-H03 into flasks containing sterile Starch casein broth and the flasks were
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