Improvement of the resistance of Lactobacillus delbrueckii ssp. bulgaricus to freezing by natural selection.

Lactic acid bacteria are often produced as frozen or freeze-dried cultures that can be used for the direct inoculation of milk in cheese and fermented milk production processes. The objective of this study was to investigate whether the resistance of Lactobacillus delbrueckii ssp. bulgaricus to freezing could be improved by natural selection. Three parallel cultures of strain CFL1 were propagated for 30 cycles in which each cycle involved three serial transfers through milk, one freezing step, and one thawing step. The concentration in viable cells after thawing as well as the acidifying activity of the thawed cultures increased dramatically throughout the experiment. This may be explained by the random appearance of better-adapted mutants that can outcompete the other genotypes. However, after 30 cycles of subcultivation, freezing, and thawing, all the cultures contained subpopulations having different survival rates to freezing. Our results show that serial transfer culture experiments may be used to improve technological properties of lactic acid bacteria. Furthermore, investigation of the mutations that are responsible for an increased cryotolerance may help to define new targets for improving the resistance of lactic acid bacteria to several stresses.