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In- Vitro Antibacterial Activity of Stereum Ostrea a Wood Decaying Macro Fungus

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In- Vitro Antibacterial Activity of Stereum Ostrea a Wood Decaying Macro Fungus Journal of Microbiology Research and Reviews Vol. 2(2): 12-18, February, 2014 ISSN: 2350-1510 www.resjournals.org/JMR In- Vitro Antibacterial Activity of Stereum Ostrea a Wood Decaying Macro Fungus Amiya Kumar Prusty1*, Laxmikumari Samad2, Abhijita Rout2 and Apramita Patra2 1 Institute of Pharmacy and Technology, Salipur, Cuttack, Odisha-754202 2Department of Botany, College of Basic Science and Humanities, OUAT, Bhubaneswar Email for Correspondence: [email protected] Abstract The increasing prevalence of multidrug resistant bacterial strains has prompted the need for antibacterial controls other than the existing antibiotics. In the present study, a wood decaying macro fungi Stereum ostrea was assessed in-vitro for its ability to inhibit the growth of different bacterial strains. The antibacterial activities of acetone, ethanol and aqueous extracts of fruiting body of Stereum ostrea were evaluated against the bacterial strains Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The minimum inhibitory concentration of acetone extract against the test microorganisms was lesser than that of ethanol and aqueous extracts. From the minimum inhibitory concentration data it could be concluded that acetone extract was most active as antimicrobial against the test microorganisms followed by ethanol and aqueous extracts. The acetone extract, ethanol extract and aqueous extract of Stereum ostrea were having maximum zone of inhibition of 19.17mm, 12.67mm and 10.17mm respectively against Bacillus subtilis, and minimum zone of inhibition of 11.33mm, 8.50mm and 7.33mm respectively against Pseudomonas aeruginosa. The study concluded that the different extracts of fruiting body of Stereum ostrea contain potential compounds that inhibit growth of both gram positive and gram negative bacteria. Keywords: macro fungi, Stereum ostrea, In-vitro antimicrobial activity, Minimum inhibitory concentration. INTRODUCTION Infectious diseases remain one of the major threats to human health. With the emergence and increase of microbial organisms resistant to multiple antibiotics, and the continuing emphasis on health care costs, more and more research are carried out to develop new, effective antimicrobial reagents free of microbial resistance and of low cost. Such problems and needs have led to resurgence in the use of natural products that may be linked to broad spectrum activity and far lower propensity to induce microbial resistance than the existing antibiotics and also cheaper (Jun et al., 2007). It had been known since Greek and Roman period that macro fungi were used as food and medicine. Therefore macro fungi may be explored as a source of new and useful bioactive compounds (Anke et al., 1980). Macro fungi need antibacterial and antifungal compounds to survive in their natural environment (Kim et al., 1999). Therefore, antimicrobial compounds could be isolated from many macro fungi species and could be of benefit for humans. As a matter of fact, macro fungi produce a large number of metabolites that show antibacterial, antifungal, antiviral, antitumor, hypoglycemic, antiallergic, immunomodulating,anti-inflammatory, hypolipidemic, and hepatoprotective activities (Hatvani, 2001). This was succeeded by the isolation and identification of pleuromutilin. That compound had served for the development of first commercial antibiotic of Basidiomycete origin (Mustafa and Fatma, 2006). Stereum ostrea a macrofungi belongs to family Stereaceae, and division Basidiomycota. It is inedible due to its 13 tough, leathery texture and is often called as ‘False turkey tail’ as it mimics Trametes versicolor. Like the ‘True turkey tail’, Stereum ostrea has somewhat fuzzy cap that displays zones of brown and reddish brown colors. The Stereum ostrea is distinguished by its relatively large size and it tends to develop individual, sliced funnel-shaped fruit bodies, rather than laterally fused flat ones. It is saprophytic as it is found on the dead hard woods, growing in dense overlapping clusters and widely distributed in various parts of the world. This fungus has long been used in folk remedies even without any knowledge of which compounds are responsible for its activity. The ethnobotanical uses of this mushroom to heal both plant and human diseases have been accumulated but scientific evidences are not yet well known (Praveen et al., 2012). At present there is little information available on the antimicrobial activity of these species but some study is here focused:-Recently, some new compounds such as a sesquiterpene, three aromatic compounds and a known compound methyl 2, 4-dihydroxy-6-methylbenzoate was isolated from a culture broth of the fungus Stereum sp. The novel sesquiterpene was determined to be stereumone and the three new aromatic compounds were elucidated together with the known compound. The combination of these compounds showed evident nematocidal activity against nematode Panagrellus redivivus (Li et al., 2006). The main focus of present study lies in the investigation of a white-rot fungus Stereum ostrea isolated from wood logs and their inhibitory activity against selected Gram-positive and Gram-negative bacteria. MATERIALS AND METHODS Chemicals used The chemicals used like ethanol, methanol, agar, peptone, beef extract, sodium chloride, sodium hydroxide, gentamycin sulphate, etc were of analytical grade. Microbial strains used Following four microbial strains were used for the antimicrobial activity studies. Gram + ve: Bacillus subtilis (MTCC 1789) and Staphylococcus aureus (MTCC 187) Gram – ve: Escherichia coli(MTCC 1591) and Pseudomonas aeruginosa (MTCC 779) [MTCC: Microbial type culture collection] The microbial Strains were obtained from Institute Of microbial technology, Chandigarh, India in lyophilized form. The lyophilized microorganisms were cultured in suitable medium at 37ºC overnight for rejuvenation and preserved in slant culture under refrigeration for future use. METHODS Collection of Stereum ostrea The mushroom Stereum ostrea was collected from the Bateswar village of Salipur, district Cuttack, Odisha during the morning hour of First week of April 2013. The mushroom was authenticated by the taxonomist of Department of Botany, College of basic science and humanities, OUAT, Bhubaneswar and a specimen sample was preserved in the museum of Institute of Pharmacy and Technology, Salipur, Cuttack. After separation from the wood log, the mushroom was washed properly in sterile water to remove any dirt or other unwanted materials adhered to it, shade dried for fifteen days followed by oven dry at 50ºC for 24hours. After complete drying it was preserved in a dry container for future use. Preparation of different extracts of the fruit body of Stereum ostrea The dried fruit body of collected mushroom was grounded to powder by a mechanical grinder. The powdered materials (10 gm) were taken in the thimble of soxhlet extractor packed properly and covered with glass wool. 200ml of acetone and few porcelain pieces were added to the extraction chamber. The condenser was attached with a water source at the lower end and removed from the upper out let; water supply to the condenser was continuous till completion of extraction process. The soxhlet apparatus was fixed properly and heated using a heating mental at 50ºC till liquid present in the siphon was colourless. The soxhlet apparatus was dismantled. The extract present in the boiling flask was separated. The powder was removed from the thimble; air dried and once again packed in the thimble for 14 Table 1. Zone of inhibition of Gentamycin sulphate (50 µg /ml) against different bacteria. Zone of inhibition in mm No of Obs. B. subtilis S. aureus E. coli P. aeruginosa 1 25 22 29 26 2 24 21 30 26 3 25 20 27 25 4 24 21 28 24 5 24 22 29 24 6 25 20 29 25 Mean (mm)+/-SD 24.50+/-0.55 21.00+/-0.89 28.67+/-1.03 25.00+/-0.89 extraction using 200ml ethanol as solvent. After completion of extraction, the procedure was repeated using 200ml distilled water. The extracts were filtered, and the solvents were completely evaporated to dryness at 40ºC using a rotary vacuum evaporator and stored in freezer for future use with proper labeling (Prusty et al., 2008). Determination of Minimum Inhibitory Concentration An agar dilution assay was performed for determining minimum inhibitory concentration (MIC) of the three extracts. Tubes of 15 ml molten agar were prepared and maintained at 50ºC. A single concentration of extract was added to the agar in each test tube to obtain a range of final concentrations of 10, 20, 30, 40 and 50µg/ml. Different concentration of extracts were added to molten agar and poured to pre-sterilized petriplates and allowed to solidify. The test microorganisms were diluted in 0.1% (w/v) peptone water to get a concentration of 106 CFU/ml determined by UV-VIS spectrophotometer. The test microbial culture was added to the extract added petriplates and spreaded by a spreader. A control plate, without added extract, was prepared and inoculated to ensure adequate growth of the test microorganism. The plates were incubated at 37ºC for overnight. The MIC was determined as the lowest concentration that completely inhibited growth of microorganism after incubation (Ahmed et al., 2007). Assay for antibacterial activity The antibacterial activity of the extracts was determined by paper disc method (Norrel and Messley, 1997). The extracts were diluted to 50µg/ml with sterilized distilled water. The sterile filter paper (Whatman filter paper) discs of 6mm diameter were soaked
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