Lyophilization of microorganisms standardized by the number of viable cells in low concentration: Development of a drying mode

INTRODUCTION. Modern approaches to microbiological testing in the pharmaceutical industry require the use of microbial test strains standardized by the number of viable cells. The most convenient form for storage and transportation is lyophilized state, which ensures long-term preservation of the viability of microorganisms. Various drying modes for microorganisms in high concentrations (10⁷–10¹² CFU/mL) are described in the scientific literature. However, such modes cannot be directly applied while working with standardized by the number of viable cells containing 10³ CFU/mL. Selection of the optimal lyophilization mode allows solving the problem of preserving the viability of microbial cells in a low concentration.

AIM. Development of a drying mode using a chamber-type apparatus that ensures the survival of microbial test strains standardized by the number of viable cells at a concentration of 10³ CFU/ml.

MATERIALS AND METHODS. Microbial test strains Salmonella enterica subsp. enterica serovar Abony NCTC 6017, Staphylococcus aureus АТСС 6538 and 6538P, Alcaligenes faecalis 415, Pseudomonas aeruginosa ATCC 9027, Yersinia enterocolitica ATCC 9610, Escherichia coli ATCC 25922, Micrococcus luteus ATCC 10240 and Martin Christ Epsilon 2-4 LSCplus lyophilizer as well as sucrose-gelatin protective medium were used in the work.

RESULTS. The experiments established the optimal parameters for lyophilic drying of microorganisms at low concentrations (10³ CFU/mL) in chamber-type devices: freezing to minus 25 °C, primary drying at a shelf temperature of minus 35 °C and a vacuum of 0.4 mbar for 8 h, and final drying for 4 h at a temperature of 30 °C and a residual pressure of 0.001 mbar. When using this mode, the survival rate of test strains at low concentrations ranged from 22 to 100% depending on the type of microorganism. The quality of the samples obtained was assessed using the parameter loss of drying which varied from 0.8 to 2.1%.

CONCLUSIONS. The proposed drying mode allows preserving the number of viable cells of microorganisms standardized at low concentrations using chamber-type lyophilic equipment. Freezing below the eutectic temperature may reduce survival of microbial cells after drying, as shown for A. faecalis strain 415. The survival of a set of eight microorganisms at low concentrations after lyophilization has been assessed for the first time in Russia.

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