SHARMA et al: PARTIALLY PURIFIED BACTERIOCIN OF BACILLUS SP AND ITS APPLICATIONS 881 Journal of Scientific & Industrial Research Vol. 68, October 2009, pp. 881-886 *Author for correspondence E-mail: NiveditaShaarma@yahoo.co.in Characterization of a partially purified bacteriocin of Bacillus sp MTCC 43 isolated from Rhizosphere of radish (Raphanus sativus) & its application as a potential food biopreservative N Sharma 1 *, G Kapoor 1 , N Gautam 1 and B Neopaney 2 1 Microbiology Research Lab, Department of Basic Sciences, University of Horticulture & Forestry, Nauni, Solan 173 230, India 2 Department of Environment & Scientific Technology, Government of Himachal Pradesh, Shimla, India Received 21 November 2008; revised 10 June 2009; accepted This study presents bacteriocin, produced from Bacillus sp. MTCC 43 isolated from rhizosphere of root crop radish, Raphanus sativus, as inhibitor against serious food pathogens (Staphylococcus aureus and Aeromonas hydrophila). Bacteriocin of Bacillus sp. MTCC 43, partially purified by salt saturation method, exhibits very high activity (2.66 x 10 5 AU/ml). Viability of pathogens decreased drastically (up to 70%) within first 10 h of adding bacteriocin. Bacteriocin showed high thermostability (up to 100°C) for 10 min, expressed wide pH tolerance (4.0-10.0), and found sensitive to proteolytic enzyme trypsin. Keywords: Bacteriocin, Bacillus sp. MTCC 43, Food pathogens, Raphanus sativus Introduction Biopreservation controls growth of pathogenic and spoilage causing organisms, thus enhancing shelf life of food products. Some bacteria produce bacteriocins (proteins or protein complexes), which have great potential as natural food biopreservatives.Bacteriocins possess bactericidal activity 1 against bacterial species, of which Gram positive bacteria of genus Bacillus and lactic acid bacteria (LAB) are main bacteriocin producing species 2-4 . This study partially purifies and characterizes bacteriocin, produced from Bacillus sp. MTCC 43 isolated from rhizosphere of radish (Raphanus sativus), as a potential food biopreservative. Experimental Bacillus sp. MTCC 43 was isolated from rhizo- sphere of radish (Raphanus sativus) from local farm of University of Horticulture & Forestry, Nauni, Solan, India. Isolation was done by dilution series method (10 -2 -10 -8 ) on nutrient agar plates at 37°C for 74 h 5 and identification was confirmed at Indian Institute of Microbial Technology, Chandigarh, India. Composition of nutrient agar (pH 6.8) is as follows: beef extract 3.0 g, peptone 5.0 g, NaCl 8.0 g, and agar 10 g/1000 ml. Culture (10 ml) of Bacillus sp. MTCC 43 (10 -6 dilution) was added into nutrient broth (90 ml) and was incubated at 37°C for 72 h at 150 rpm in order to obtain bacterial culture of 1.0 OD. Ammonium Sulfate Precipitation For partial purification, ammonium sulfate precipita- tion method was chosen 6 . Bacterial culture (1.0 OD) was saturated with different concentrations of ammonium sulfate (20, 40, 50, 70 and 80%) subsequently with constant stirring. At 80% saturation level, preparation was kept at room temperature (30°C) for 12 h. Centrifugation of supernatant was carried out at 20,000g at 4°C for 1 h. Pellet so obtained was dissolved in 100 ml of Tris-HCI Buffer (0.1 M, pH 7.0). Cell free extract of Bacillus sp. was dialyzed. After 24 h, dialyzed bacteriocin suspension was carefully removed from dialysis bags and was centrifuged at 10,000 g for 10 min at 4°C. Calculation of activity Units (Arbitrary Units – AU ml) of Partially Purified Bacteriocin (PPB) Serial Two-Fold Dilution Method Activity units of PPB of Bacillus sp. were calcu- lated by Serial Two-Fold Dilution Method 7 . PPB was di- luted in saline water (10 -2 , 10 -4 ……10 -10 ). Each dilution was used to estimate AU by Well Diffusion Method (WDA) and dilution corresponding to smallest detectable