(The) leakage of cellualr materials in lactobacillus lactis induced by freezing and drying

Abstract

Rapid freezing and freeze-drying have been widely used for the storage of bacterial cultures. Substantial degree of cell damage, however, has been accompanied in these methods and the mechanism of cell damage was tried to be explored in this study. When $\mbox{\underline{Lactobacillus}}$ $\mbox{\underline{lactis}}$ cells were frozen for 5 minutes at -196$^\circ$C or further dried for 24 hours under vacuum and then incubated in 1.0 \% phosphate buffer for 90 minutes at 37$^\circ$C, a considerable amount of cell materials were observed to be released in the incubation medium which could be detected by the UV-analysis having an optimum absorption at 260 nm. Among various agents tried, 1.0 \% phosphate solution showed the highest leakage of cell materials during the period of incubation. The UV-absorbing materials from the frozen or freeze-dried lactic acid bacteria cells during incubation in the phosphate solution was found to be dependent on PH and temperature having optimum values of 7.2 and 45$^\circ$C, respectively. Addition of $Mgcl_2$ in the incubation medium at the level of 0.1\% inhibited the leakage of the cell materials markedly. The leaked cell materials were analyzed and found that RNA and protein occupied the major part with negligable amounts of DNA and carbohydrates. And when cells were incubated for a longer period, only RNA continued to release in the incubation medium. It was also found that the addition of cell protecting agents, such as glycerol, dextrose, dimethyl sulfoxide, and 0.1 \% $Mgcl_2$ in the suspending solution of the cells during freezing could prevent the leakage of cell materials during the process of the subsequent incubation. It suggested that the protectants could prevent the damage of permeability barrier which might be related to the death of cells. However, no direct relationship between the increasing of the leaked cell materials and the cell viability could be detected.