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°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...

Cell inactivation and membrane damage after long‐term treatments at sub‐zero temperature in the supercooled and frozen states

“…Table IV shows that survival of E. coli after 71 days at À808C (71%) was significantly greater than at À108C (30%). This observation corroborates those of Fonseca et al (2006) who reported that the viability of L. bulgaricus cells was greater when stored at À808C than at À208C. This effect may be caused by the occurrence of vitrification at À808C (Fonseca et al, 2006).…”

“…The response of E. coli cells would suggest that the greater viability of cells in frozen compared with supercooled conditions is associated with the higher concentration of glycerol within the unfrozen fraction. It has been shown that the viscosity of the residual unfrozen solution to which all cells are exposed during freezing in the presence of glycerol increases rapidly (Fonseca et al, 2006). Hence, diffusion is limited because of high viscosity.…”

Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus

“…It was found that increasing the methanol concentration or using the glycerol as a buffer was very effective for reducing cell membrane damage. Previous studies confirmed that both methanol and glycerol were effective cryoprotectants for L. bulgaricus and had the ability to enhance the cell viability against freezing damage (Carvalho et al, 2004;Fonseca et al, 2006). Especially, glycerol as a permeable compound could protect freeze-dried lactic acid bacteria through inhibiting excess dehydration, reducing salt toxicity, and preventing ice crystals formation (Huang et al, 2006).…”

Evaluation of three methods for preservation of Azotobacter: freeze-drying, cryopreservation, and immobilization in dry polymers

“…~10 and 30,000 °C min -1 , respectively). Fonseca et al (2006) also reported that high cooling rates significantly improve survival rates for lactic acid bacteria. It is worth noting that although freeze-drying usually causes more damage to cells than cryopreservation, we observed a minor loss of viability using this first method (Table 2, Table 3).…”

“…Leslie et al (1995) showed how some carbohydrates protect membranes during the rehydration process, decreasing the rate of transition in membranes from gel to the liquid crystalline phase. Other agents as glycerol or DMSO reduce the eutectic point of water preventing the formation of ice crystals (Fonseca et al 2006). Therefore, it is critical to select the appropriate protective agent for improving the storage conditions for Azotobacter cells.…”