DETERMINATION OF THE SOURCE AND SELECTION OF THE REGIMEN OF EXTRACTION OF TRICHODERMA ATROBRUNNEUM VKPM F-1434 METABOLITES WITH ANTIBIOTIC ACTIVITY AGAINST REPRESENTATIVES OF THE ENTEROBACTERICEAE GROUP

Abstract

Relevance. It is relevant to determine the source and select the mode of extraction of biologically active metabolites of the microbial producer in or-der to further develop the technology for their production. The purpose of the study. Determination of the source and selection of the mode of extraction of Trichoderma atrobrunneum F-1434 metabolites with antibiotic activity. Material and methods. The object of the study is a strain of Trichoderma atrobrunneum VKPM F-1434 synthesizing low molecular weight bacterio-static compounds against Enterobactericeae. The producer was cultured in deep conditions for 5 days, at t – 28 oC, with a sucrose limit of up to 50% of the weight the standard composition of the Chapek medium. The biomass extract and the culture fluid of the producer were purified by centrifugation, the supernatants were examined for antagonism. The active components were isolated by single-stage mixing and settling extraction. The complete-ness of extraction was determined by varying the volume of the extractant and the time of interaction with the culture fluid. The eluents for thin-layer chromatography were determined by the principal component method. The extract was separated by normal-phase TLC on the stationary phase of Sorbifil, varying the ratio of solvents from 10A:0B to 0A:10B in increments of 1.2x20 cm. Preparatively, the fractions were obtained by column gel-penetrating chromatography with fixed phase Sephadex G-15, particle size 40–120 µm. The mobile phase is dimethyl sulfoxide (DMSO). The molecular weight was calculated from the ratio of the Rf fraction to the Rf of the witnesses. Results. The maximum sensitivity of the test culture is shown in an experiment with a culture fluid preparation. The most available extractants for the extraction of metabolites were ethyl acetate and butanol. The greatest value of the completeness of extraction is achieved when equal volumes of ex-tractant and culture fluid interact for 180 minutes. The optimal mobile phases were determined by the principal component method: I – hexane : chlo-roform; II – hexane : acetone; III – hexane : acetone : methanol (10%). Three fractions were isolated, bacteriostatic activity was determined for each, the Rf was calculated and the molecular weights, as well as the coefficients of antibiotic activity, were theoretically calculated. Conclusions. The components of the culture liquid T. atrobrunneum VKPM F-1434, having bacteriostatic activity, can be extracted by stirring for three hours equal volumes of ethyl acetate and culture liquid, and determined in the extract using TLC on silica gel with impregnated methanol in the system of hexane and DMSO – 6:4 eluents.