VIABILITY OF PROBIOTIC STRAINS OF MICROORGANISMS AND THEIR ADHESION TO HUMAN ERYTHROCYTES AFTER IMMOBILIZATION IN ALGINATE GEL AND STORAGE AT DIFFERENT LOW TEMPERATURES
Authors: Vysekantsev I.P. , Petrov I.V. , Buriak I.A. , Martsenyuk V.P. 
Published: January 2021

The research was focused on the development of technologies for long-term storage of probiotics immobilized in gel carriers at various low temperatures. The aim of the research was to investigate the viability and adhesive activity of probiotic microorganisms after immobilization in an alginate gel and storage at various low temperatures for 24 months (observation period). Probiotic strains of the microorganisms Escherichia coli M–17 (E. coli M–17), Lactobacillus acidophilus IMB B-2637 (L. acidophilus), Saccharomyces cerevisiae IMB Y-505 (S. cerevisiae) were immobilized in 1% alginate gel either without additives or supplemented with lactose, sucrose, skimmed milk and protective SML medium. The samples were frozen at uncontrolled cooling rates in refrigerators with temperatures of –20, –40, –75 °C. When then frozen down to –196 °C, the samples were cooled to –40 °C at the rate of 1 deg / min, and then immersed in liquid nitrogen. The samples were stored at the indicated temperatures for 24 months (observation period). During storage, part of the granules was thawed in a water bath and dissolved in 4% EDTA solution. The viability of microorganisms was determined by the ability of microbial cells to form colonies on agar media. Adhesive activity was determined by the ability to adhere to human erythrocytes 0(I) group, Rh (+). When cooled down to –20, –40, –75, –196 °C and after subsequent storage at –196 °C, immobilized microorganisms in all varieties of gel did not die. In most samples cells died between 1 and 3 months of storage at –20, –40, –75 °C. After 24 months, it was found that the most resistant to storage at these temperatures were the bacteria L. acidophilus, and the most sensitive were the yeast S. cerevisiae. The viability of all microorganisms immobilized in 1% gel without additives was minimal. The addition of disaccharides of lactose, sucrose, SML medium and milk to the alginate gel increased the cryoprotective properties of the alginate gel. The highest viability of immobilized microorganisms was observed in the gel with SML medium. As the storage temperature decreased from –20 to –75 °C, the number of viable microbial cells in all gel samples increased. Immobilization in gel carriers and storage for 24 months did not affect the adhesive properties of probiotics E. coli M–17, L. acidophilus, S. cerevisiae. The changes in the viability of microbial cells are associated with the severity of damaging physicochemical factors that accompany the processes of water crystallization at temperatures of –20, –40, –75 °C. The protective effect of additives in the composition of the alginate gel is analyzed. The viability of microorganisms immobilized in gel granules during long-term storage at different low temperatures is influenced by the species cryoresistance of microorganisms, temperature regimens and storage duration, the composition of the gel carrier. The processes of immobilization and storage at low temperatures did not change the adhesive activity of immobilized microorganisms.