CRYOPRESERVATION OF PROBIOTIC STRAINS OF MICROORGANISMS IMMOBILIZED IN GEL GRANULES WITH ADDITIONAL COATING

Petrov I. V., Ananina G. E., Onasenko O. S., Stepanyuk L. V., Nardid E. O.

MICROBIOLOGY

Scentific article

Abstract. This paper presents the results of the research devoted to the development of technologies for long-term storage of gel-immobilized probiotic microorganisms at low temperatures. The aim of this work was to study the effect of coating gel granules with an additional layer of alginate gel on the viability of immobilized probiotic microorganisms’ strains after rapid cooling to -196 °C and their long-term storage at this temperature. Objects and research methods. Probiotic strains of bacteria Bifidobacterium bifidum 1 (B. bifidum), Escherichia coli M-17 (E. coli M-17) and yeast Saccharomyces boulardii CNCM I-745 (S. boulardii) were immobilized in granules of 1% alginate by ionotropic gelation. A part of the granules was coated with additional layer of 2% alginate gel. The granules from another part were combined into conglomerates of 10 pieces. These conglomerates were coated with 2% alginate gel. The granules and conglomerates were placed in cryovials and frozen by immersion in liquid nitrogen. The samples were thawed in a water bath at 37 ° C. Granules and conglomerates were dissolved in 4% EDTA solution. The viability of microorganisms was assessed by colony formation on agar plates or in semi-liquid media. Research results and their discussion. Coating of the granules with a layer of 2% alginate gel, as well as the formation of conglomerates of granules coated with a layer of 2% alginate, did not cause changes in the viability of immobilized bacteria after their rapid cooling to -196 °С. In E. coli M-17 samples, the number of viable bacteria per 1 ml of gel decreased by 0.31-0.42 lg. In B. bifidum samples, it decreased by 1.2-1.3 lg. In the samples of immobilized S. boulardii yeast, both coating the granules with 2% alginate gel and the formation of conglomerates of granules and their subsequent coating with 2% alginate gel had a protective effect. The number of viable cells per ml of gel in granules without additional coating decreased by 1.22 lg, while in coated granules it reduced by 0.7 lg and in the conglomerates the viability decreased by 0.6 lg. It can be presumed that coating with 2% alginate gel reduces the cooling rate in the gel granules. Accordingly, this reduces the severity of intracellular crystallization and recrystallization processes during cooling-heating. The storage process at the temperature of -196 °C does not cause additional death of immobilized microbial cells.

probiotics, immobilization, gel carriers, cryopreservation

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«Bulletin of problems biology and medicine» Issue 1 (159), 2021 year, 189-192 pages, index UDK 57.086.13:615:832.9:57.083

10.29254/2077-4214-2021-1-159-189-192