Bacteriocin-like substance Bacillus circulans and way of its production

The development of antibiotic resistance is a key problem for a health care service. An approach to solve it relies on limited and reasonable application of antibiotics as well as on utilization of some other substances with more efficient mechanisms of antimicrobial action. Low-molecular-weight peptides called bacteriocins kill cells by making pores in cell walls irrespective of their antibiotic resistance. In this context, of particular interest are some representatives of the genus of Bacillus, especially of species circulans-polymyxa, producing bacteriocins and wide-spectrum activity bacteriocin-like substances. However, production of the substances in sufficient amounts for practical application is still doubtful. Objectives of the research were to study factors that might influence production of the bacteriocin-like substance Сk2th by newly isolated strain B. circulans, and to select an appropriate procedure for isolation of the substance from fermentats to assess then its properties.The strain was proliferated and subcultured on nutrient agar at 30°С for two days. An appropriate composition of nutrient broth for submerged cultivation of the strain was selected using 750 ml flasks containing 100 ml of nutrient media. Bactericidalactivity of the substance was determined by placing samples of desired volumes in Petri dishes containing freshly seeded lawns of test strains of Gram-positive and Gram-negative microorganisms. The activity was expressed in arbitrary units (AU) measured for 1ml or 1mg of the sample depending on the level of dilution. Such methods as salt precipitation (ammonium sulfate), solid carrier sorption (silica gel, carbon, aluminum oxide, aerosil) and organic solvent two-phase separation (dichlormethane, chloroform, toluene) were tested for isolation of active substances from fermentats. Molecular identification of the bacteriocin-like substance was performed by SDS PAGE followed by 5% glutaraldehyde fixation and coomassie staining. Before biological testing the gel was washed, placed in a Petri dish and overlaid with melted agar containing test cells. After incubation zones of inhibition of the growth of the test strain were fixed against markers. The dependence between the yield of B. circulans Сk2th and a type of nitrogen and carbon source in the medium and time of submerged cultivation was determined. The method of two-phase separation in the presence of dichlomethane was found to be the most effective one to isolate the product from the culture fluid. A 5-6kDa peptide was shown to be responsible for antimicrobial action of the bacteriocin-like substance, making it quite different from all known antibiotics.

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