The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at ؊196°C and ؊20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (؊196°C and ؊80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at ؊20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.Concentrates of lactic acid bacteria (LAB) are widely used as starters for manufacturing cheese, fermented milk, meat, vegetables, and bread products (19). Freezing and storage at low temperatures (ϽϪ40°C) are commonly applied to preserve the viability of concentrates while maintaining their technological properties upon thawing (acidification activity, production of aroma compounds, and contribution to product texture). However, bacterial resistance to freezing and to frozen storage depends on the strain, culture conditions before freezing, harvesting, formulation (type and concentration of cryoprotectant), freezing conditions, and final storage temperature (9).Freezing is a critical step in the production of LAB concentrates, as it affects both the viability and acidification activity upon thawing (10, 32). However, the mechanisms of freezing damage to bacteria are not well understood. Two damage mechanisms dependent on the cooling rate have usually been proposed. At low freezing rates, cellular damage is principally caused by osmotic stress to cells (so-called "solution effects") (24). The formation of extracellular ice induces high solute concentrations in the extracellular medium, exposing the cells to an increase in ionic concentration, changes in pH, etc., for extended periods of time. At very high cooling rates, it has been assumed that damage is due to the formation of int...