The effect of lyophilization and cryoprotection on survival of Lactiplantibacillus plantarum strains

Lyophilization has been recognized for many years as an effective method for storing collection cultures, bacterial starter cultures, and probiotics. Cryoprotectors are used to protect bacterial cells from stress during freezing and drying, and to increase their survival. The purpose of our study was to evaluate an effect of three freezing regimes before freeze-drying at temperatures of –20, –30, and –55 °C and solutions of two cryoprotectors, sucrose and trehalose, on the survival and preservation of the metabolic activity of mesophilic lactobacilli. The cultures were grown in 10 % skimmed milk, and in experimental versions, a 20 % cryoprotector solution was added to the culture in a 4:1 ratio. To determine an effect of freeze-drying conditions on bacterial survival and metabolic activity, complete two-factor, three-level experiments were developed and conducted. Two strains of the species Lactiplantibacillus plantarum from the VNIIMS collection, which are used in bacterial starter cultures for cheeses, were studied as representative microorganisms of lactobacilli. The results of assessing the viability of bacteria after lyophilization showed that both cryoprotectors increased the survival of cultures under all freezing conditions, but the best survival rates were obtained using trehalose. Strain-dependent effectiveness of lyophilization of the studied strains was established: in strain Lactiplantibacillus plantarum 28, the maximum survival was at a freezing temperature of –30 °C, in strain Lactiplantibacillus plantarum 37 — at a temperature of –55 °C with the addition of cryoprotector trehalose to both strains. Pre-freezing at –20 °C, also with trehalose, was the best method for preserving the metabolic activity of both cultures. It was shown that the greatest effectiveness of sucrose and trehalose as lyoprotectants during freeze-drying for both bacterial strains was found within the same pre-freezing temperature range of –30 °C. The identified strain-dependent effectiveness of lyophilization should be considered when selecting drying regimens for various collection cultures intended for long-term storage and requires an individual approach for each culture. The obtained results can also be used in freeze-drying of bacterial mass for the production of single-species bacterial starter cultures.