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The resistance to freezing and frozen storage of Streptococcus thermophilus was related to the fatty acid composition of the cell membrane. The effects of four experimental factors were investigated on the fatty acid concentrations and on the recovery of acidification activity of S. thermophilus stored at -20 degrees C by using a complete experimental design: incorporating oleic acid in the culture medium, fermentation pH, addition of glycerol as cryoprotective agent and duration of storage. The acidification activity decreased during the freezing and the frozen storage of S. thermophilus. The storage time slightly enhanced the unsaturated fatty acid concentrations. The addition of glycerol did not modify the fatty acid composition but increased the resistance to frozen storage. The addition of oleic acid and the decrease of the fermentation pH enhanced the ratio unsaturated:saturated fatty acids and improved the recovery of the acidification activity. These results indicate that the resistance to frozen storage was closely related to the membrane fatty acid composition. We interpreted this as an adaptation of S. thermophilus to the addition of oleic acid and the unfavorable growth conditions that corresponded to a low fermentation pH.

We have developed a method to quantify the resistance to freezing and frozen storage of lactic acid starters, based on measuring the time necessary to reach the maximum acidification rate in milk (tm) using the Cinac system. Depending on the operating conditions, tm increased during the freezing step and storage. The loss of acidification activity during freezing was quantified by the difference (Δtm) between the tm values of the concentrated cell suspension before and after freezing. During storage at −20 °C, linear relationships between tm and the storage time were established. Their slope, k, allowed the quantitation of the decrease in acidification activity during 9–14 weeks of frozen storage. The method was applied to determine the resistance to freezing and frozen storage of four strains of lactic acid bacteria and to quantify the cryoprotective effect of glycerol.

The effects of 3 fermentation temperatures (30, 37, and 42 degrees C) and 3 fermentation pH (4.5, 5, and 6) on the cryotolerance of Lactobacillus acidophilus RD758 were studied in relation to their fatty acid composition. Cryotolerance was defined as the ability of the cells to recover their acidification activity after freezing and frozen storage at -20 degrees C. Better cryotolerance was obtained in cells grown at 30 degrees C or at pH 5; these cells showed no loss in acidification activity during freezing and a low rate of loss in acidification activity during frozen storage. On the other hand, cells grown at 42 degrees C or at pH 4.5 displayed poor cryotolerance. The membrane fatty acid composition was analyzed and related to the cryotolerance using principal component analysis. The improved cryotolerance observed during the freezing step was associated with a high ratio of unsaturated to saturated fatty acids, a low C18:0 content, and high C16:0 and cyclic C19:0 relative concentrations. High resistance during frozen storage was related to a high cycC19:0 concentration. Finally, the low cryotolerance observed after fermentation at pH 4.5 was explained by a low C18:2 content.

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