Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects

Abstract

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 degrees C and -20 degrees 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 degrees C and -80 degrees 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 degrees 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.

FIG. 1.

Freezing protocols (FP 1, FP 2, and FP 3).

FIG. 2.

Acidification activities of L. delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol, as determined by the Cinac system, before (t_mc) and after slow freezing (FP 1) (t_mf) and after 1 month of frozen storage (t_ms) at −20°C.

FIG. 3.

Cryo-SEM of L. delbrueckii subsp. bulgaricus CFL1 suspended in glycerol and cooled by different processing conditions, i.e., FP 3 (a and b), FP 1 (c and d), and FP 2 (e and f).

FIG. 4.

Cryo-SEM of L. delbrueckii subsp. bulgaricus CFL1 cooled with glycerol. Details of the interface between the freeze-concentrated matrix and ice for samples cooled either by FP 2 (a) or by FP 3 (b) are shown.

FIG. 5.

Freeze substitution electron microscopy of L. delbrueckii subsp. bulgaricus CFL1 either suspended in glycerol and cooled by different processing conditions following freezing at different rates to −196°C (FP 3 [a], FP 1 [b], and FP 2 [c]) or suspended in distilled water and directly immersed in liquid nitrogen (d).

FIG. 6.

Cryo-SEM (a and b) and freeze substitution electron microscopy (c and d) of L. delbrueckii subsp. bulgaricus CFL1 suspended in glycerol, cooled by FP 1, held at −20°C for 24 h, and then reimmersed in liquid nitrogen.

FIG. 7.

Schematic of the glycerol concentrations encountered by cells following FP 1, FP 2, and FP 3. The glycerol concentrations and T_g2′ values are from Table 2. Dotted lines indicate less certain glycerol concentration values reached following FP 2 and FP 3.