Glutathione protects Lactobacillus sanfranciscensis against freeze-thawing, freeze-drying, and cold treatment

Abstract

Lactobacillus sanfranciscensis DSM20451 cells containing glutathione (GSH) displayed significantly higher resistance against cold stress induced by freeze-drying, freeze-thawing, and 4 degrees C cold treatment than those without GSH. Cells containing GSH were capable of maintaining their membrane structure intact when exposed to freeze-thawing. In addition, cells containing GSH showed a higher proportion of unsaturated fatty acids in cell membranes upon long-term cold treatment. Subsequent studies revealed that the protective role of GSH against cryodamage of the cell membrane is partly due to preventing peroxidation of membrane fatty acids and protecting Na(+),K(+)-ATPase. Intracellular accumulation of GSH enhanced the survival and the biotechnological performance of L. sanfranciscensis, suggesting that the robustness of starters for sourdough fermentation can be improved by selecting GSH-accumulating strains. Moreover, the results of this study may represent a further example of mechanisms for stress responses in lactic acid bacteria.

FIG. 1.

Bacterial survival and other sourdough characteristics after freeze-drying treatment. Cells precultured in WFH for 36 h were chosen for freeze-drying, with initial cell counts of (9.1 ± 0.2) × 10⁸ CFU/ml. (A) Survivals of cells after freeze-drying treatment. (B) pH (solid lines) and TTA (dashed lines) values during subsequent fermentation of sourdoughs prepared with GSH⁺ and GSH⁻ cells. Error bars indicate standard deviations (n = 3).

FIG. 2.

Bacterial survival and intracellular GSH concentration during FTCs in MRS (A) and CDM (B). Cells cultivated in MRS for 36 h (middle-late stationary phase) were harvested and subjected to FTCs. The initial cell counts for L. sanfranciscensis cells harvested from the precultures in MRS and CDM were (8.0 ± 0.2) × 10⁸ CFU/ml and (5.6 ± 0.1) × 10⁷ CFU/ml, respectively. Solid lines, survival rates of L. sanfranciscensis GSH⁺ cells (•) and GSH⁻ cells (○); dashed lines, intracellular GSH concentrations of L. sanfranciscensis GSH⁺ cells (▴). Error bars indicate standard deviations (n = 3).

FIG. 3.

Transmission electron microscopy of L. sanfranciscensis cells. (A and C) L. sanfranciscensis GSH⁺ cells. (B and D) L. sanfranciscensis GSH⁻ cells. All cells were resuspended in MRS and subjected to five freezing-thawing cycles (FTCs). Delimitation by a white arrow and an opposing black arrow represents the cell wall.

FIG. 4.

Survival of L. sanfranciscensis cells upon cold treatment at 4°C. The initial cell counts of L. sanfranciscensis cells harvested from the precultures in MRS (A) and CDM (B) were (8.2 ± 0.3) × 10⁸ CFU/ml and (5.7 ± 0.2) × 10⁷ CFU/ml, respectively. Error bars indicate standard deviations (n = 3). d, days.

FIG. 5.

Alterations in the distribution of L. sanfranciscensis membrane saturated fatty acids (A) and unsaturated fatty acids (B) upon cold treatment at 4°C for 14 days. Cells were precultured in MRS. Error bars indicate standard deviations (n = 3). Statistically significant differences (P < 0.05) were determined by Student's t test and are indicated with asterisks. d, days; w, ω.

FIG. 6.

Profile of the U/S ratios of L. sanfranciscensis GSH⁺ cells (•) and GSH⁻ cells (○) upon cold treatment at 4°C. Error bars indicate standard deviations (n = 3). d, days.

FIG. 7.

Linoleic acid (C_18:2) in the fatty acid composition of L. sanfranciscensis cells upon cold treatment at 4°C. Gray bars and white bars indicate the fraction of C_18:2 in the fatty acid composition of L. sanfranciscensis GSH⁺ and GSH⁻ cells, respectively. Error bars indicate standard deviations (n = 3). d, days.

FIG. 8.

Time course of Na⁺,K⁺-ATPase activity upon cold treatment at 4°C (A) and time course of Na⁺,K⁺-ATPase activity and intracellular GSH concentration during the recultivation process of GSH⁺ cells which were treated at 4°C for 30 days (B). The initial cell counts of L. sanfranciscensis cells were (8.0 ± 0.2) × 10⁸ CFU/ml. (A) Gray bars and white bars represent GSH⁺ and GSH⁻ cells, respectively. (B) Na⁺,K⁺-ATPase activity (▴) and intracellular GSH concentrations (•). Error bars indicate standard deviations (n = 3). Statistically significant differences (P < 0.05) were determined by Student's t test and are indicated with asterisks. d, days.