Glutathione Protects Lactobacillus sanfranciscensis against Freeze-Thawing, Freeze-Drying, and Cold Treatment

Chemicals.MRS broth was purchased from Becton Dickinson company (Sparks, MD). GSH, cysteine (Cys), glutathione reductase, NADPH, NADP⁺, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), bovine liver superoxide dismutase (SOD), and ouabain were purchased from Sigma-Aldrich (Steinheim, Germany). An SOD activity detection kit was purchased from Wako Pure Chemicals Industries (Osaka, Japan).

Bacterial strains and cultivation conditions.The type strain of Lactobacillus sanfranciscensis DSM20451 used in this study was purchased from DSMZ (Braunschweig, Germany). Three media were used in this study: chemically defined medium (CDM) (10), MRS broth, and wheat flour hydrolysate (WFH) prepared as reported previously (13). CDM, which lacks GSH, was used to demonstrate whether GSH can play a protective role in L. sanfranciscensis. MRS broth is a complex medium in which L. sanfranciscensis grows better than in CDM. MRS broth was used to verify whether the protective role of GSH is reproducible. The concentration of GSH in MRS broth was determined to be 48.8 ± 0.2 μM. Incubation of strain DSM20451 in this medium did not result in a detectable intracellular GSH concentration, suggesting that the GSH taken up by strain DSM20451 from MRS broth need not be taken into consideration. WFH was used to mimic real sourdough fermentation conditions.

An inoculum was transferred from a −70°C frozen stock to MRS broth supplemented with 5 g/liter maltose and incubated at 30°C statically for 24 h as the preculture. The preculture of strain DSM20451 was used to inoculate CDM, MRS broth, or WFH with or without 1.5 g/liter (4.8 mM) GSH to obtain L. sanfranciscensis cells with intracellular GSH (designated GSH⁺ cells) or without GSH (designated GSH⁻ cells). Meanwhile, considering the possibility that other low-molecular-weight thiol compounds might play a protective role similar to that of GSH, 0.58 g/liter (4.8 mM) Cys was added to CDM, MRS broth, or WFH, and the corresponding cells grown in these media were designated Cys⁺ cells. The inoculum size used was 1% (vol/vol).

Preparation of cell extracts.Bacteria were harvested by centrifugation. Cell pellets were washed twice with ice-cold saline (0.85% NaCl [wt/vol]) and resuspended in an equal volume of phosphate buffer (0.2 M potassium phosphate, 2 mM EDTA, pH 7.0). Three milliliters of the cell suspension was disrupted ultrasonically at 4°C for 40 cycles of 5 s (ACX 400 sonicator at 20 kHz; Sonic and Materials, Newton, MA). Cell debris was removed by centrifugation (10,000 × g for 10 min at 4°C), and the cell-free extracts (CFE) were used for determination of GSH concentration and enzyme activity.

Cold challenge.For cold challenge at 4°C, portions (10 ml) of cultures of L. sanfranciscensis GSH⁺ and GSH⁻ cells grown to middle-late stationary phase (36 h) were centrifuged at 10,000 × g for 5 min. Cell pellets were washed with saline to remove the residual medium and resuspended in fresh MRS broth to exclude the possible effect of starvation. The suspension was divided into 1-ml aliquots and stored at 4°C for cold treatment.

For freezing-thawing cycle (FTC) manipulation, each sample was exposed to five repeated FTCs (frozen at −20°C for 3 days and thawed at 30°C for 1 h). After being given FTCs, the cell suspensions were centrifuged at 10,000 × g for 5 min, washed with saline twice, and prepared for the following experiments.

For freeze-drying, cells were frozen at −80°C for 3 h, followed by desiccation under vacuum (50 mtorr) in a freeze-drier (Martin Christ, Germany) for 30 h. The freeze-dried cells were rehydrated with an equal volume of saline (0.85% NaCl [wt/vol]) for a survival assay.

Dough preparation.The freeze-dried cells were resuspended in 63 ml of sterile tap water and mixed with 90 g of wheat flour until dough formation.

pH and TTA measurement.Sourdough samples (10 g) were homogenized with 90 ml of sterile distilled water using a Stomacher apparatus (Seward, London, United Kingdom). The pH value was recorded, and the acidity was titrated with 0.1 N NaOH to a final pH of 8.5. The total titratable acidity (TTA) was expressed as the volume of 0.1 N NaOH consumed.

Electron microscopy.To prepare samples for transmission electron microscopy (TEM), cells were fixed by adding 2.5% (vol/vol) glutaraldehyde for 30 min. Cell pellets were harvested by centrifugation (5,000 × g for 5 min) and mixed with 1.25% (wt/vol) water agar. The agar was then cut into ca. 1-mm pieces and fixed in phosphate-buffered 2.5% (vol/vol) glutaraldehyde for an additional 30 min. The agar pieces were rinsed with 0.02 M phosphate buffer (pH 6.8) three times and postfixed in phosphate-buffered saline containing 1% (wt/vol) osmium tetroxide for 1 h, followed by rinsing with water and fixing for 1 h in 1% (wt/vol) aqueous uranyl acetate. All fixations were carried out at room temperature. After dehydration in a graded series of ethyl alcohol concentrations and twice in propylene oxide, the agar pieces containing L. sanfranciscensis cells were embedded in Epon 812 (Spi Supplies, New Chester, PA). Thin sections stained with uranyl acetate and lead citrate were examined in a JEM 1200 EXII electron microscope (Jeol, Tokyo, Japan).

Fatty acid extraction and analysis.Extraction of bacterial lipids and preparation of fatty acid methyl esters (FAMEs) were carried out according to the method of Miller and Berger (31). Briefly, cells were collected by centrifugation (7,500 × g at 4°C for 20 min). Cell pellets were washed with cold saline (0.85% NaCl [wt/vol]) twice and then mixed with 1 ml NaOH in a methanol-distilled water solution (3:10:10 [wt/vol/vol]), heated at 100°C for 30 min, and cooled at room temperature. Methylation was carried out with 2 ml methanol-HCl 6 M solution (13:11 [vol/vol]) by heating at 80°C for 10 min. After rapid cooling in an ice bath, FAMEs were extracted with 1.25 ml methyl tertiary butyl ether-hexane (1:1 [vol/vol]) for 10 min and washed with 3 ml 0.33 M NaOH. The organic phase (0.8 ml) was transferred to a 2-ml silyl vial, evaporated under a nitrogen flow, and then adjusted to 10 ml with hexane.

One microliter of the concentrated FAME extract was assayed by chromatography on a 30-m fused-silica capillary polar column (inner diameter 0.22 mm, film thickness 0.25 μm, 70% biscyanopropyl polysiloxane BPX70; SGE, TX) with a model Shimadzu GC-17A chromatograph coupled with a QP-5000 mass spectrometer. The carrier gas was helium at a flow rate of 29.6 ml/min, the column pressure was 63.4 kPa, and the column flow was 0.5 ml/min. The temperatures used were 260°C for the injection port and 280°C for the detector. The temperature program was 100°C isothermally for 1 min followed by 4°C/min to 250°C and then 250°C isothermally for 5 min. FAMEs were identified by their retention times in comparison to those of the standards (C₉ through C₂₀; Supelco, Bellefonte, PA) and their mass spectra versus a spectrum database. The relative amounts of FAMEs were calculated from peak areas. The degree of desaturation (mol% unsaturated fatty acids over mol% saturated fatty acids [U/S ratio]) was assayed. All experiments were carried out in triplicate.

Superoxide dismutase assay.The SOD activity of the CFE was measured by using an assay kit (SOD test; Wako) based on the NBT (nitroblue tetrazolium) method (5). Three thousand units of bovine liver SOD was used as the positive control.

GSH assay.Total glutathione (reduced form, GSH, plus oxidized form, glutathione disulfide [GSSG]) was determined by the enzymatic recycling procedure, as modified from the procedures of Tietze (40), which has been detailed in a previous study (25). Protein concentrations were determined with the Bradford method, using bovine serum albumin as a standard (6).

Na⁺,K⁺-ATPase assay.The Na⁺,K⁺-ATPase assay was carried out according to the method of Baginski et al. (3). Ten-milliliter amounts of cell culture were centrifuged at 10,000 × g for 5 min at 4°C, and cell pellets were washed twice with 10 ml hypotonic solution (1 mM MgCl₂, 0.25 mM EDTA, 0.1% bovine serum albumin, and 1 mM imidazole, pH 7.4) and then incubated with 1 ml assay buffer (130 mM NaCl, 20 mM KCl, 4 mM MgCl₂, 1 mM EGTA, 3 mM sodium azide, 0.1% saponin, and 30 mM imidazole-HCl, pH 7.4) or 10⁻⁴ M ouabain in background assays at 37°C for 20 min. Reactions were started by adding 3 mM ATP, and reaction mixtures incubated at 37°C for an additional 45 min. The release of inorganic phosphate (P_i) by the enzyme was within the linear range during the incubation period. The liberated P_i was measured by absorption at 850 nm. The Na⁺,K⁺-ATPase activity was defined as the difference between the total ATPase activity and the background ATPase activity.

Statistical analysis.Student's t test was employed to investigate statistical differences. Samples with P values of <0.05 were considered to be statistically different.

GSH maintains cellular integrity of L. sanfranciscensis cells upon cold treatment.Considering the practical application for sourdough preparation, L. sanfranciscensis cells were incubated in WFH with or without GSH to prepare the GSH⁺ and GSH⁻ cells. To investigate whether other low-molecular-weight thiol compounds play a similar role, L. sanfranciscensis cells containing cysteine (Cys⁺) were also prepared. Upon freeze-drying treatment, the survival advantage of GSH⁺ cells was remarkable, with survival rates 8.2-fold and 4.3-fold higher than those of the GSH⁻ and Cys⁺ cells, respectively (Fig. 1A). Cys⁺ cells did show some degree of protection against freeze-drying, but the protection was less prominent than that of the GSH⁺ cells. Further investigation showed that the pH of the sourdough containing GSH⁺ cells decreased faster than the pH of sourdough containing GSH⁻ cells, especially in the first 12 h of fermentation (Fig. 1B). Moreover, the TTA of the sourdough containing GSH⁺ cells reached 10.9 after 36 h of fermentation, while the value was only 7.1 for the control (Fig. 1B). Scanning electron microscopy showed that the number of GSH⁺ cells maintaining intact structures was much greater than for GSH⁻ cells (data not shown). All these observations suggested that GSH protects L. sanfranciscensis cells against freeze-drying treatment and improves the subsequent sourdough fermentation performance.

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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).

To test if GSH can protect L. sanfranciscensis against cold stress induced by freeze-thawing, cells were subjected to freeze-thawing cycle (FTC) treatment. In the comparison of the survival of L. sanfranciscensis GSH⁺, GSH⁻, and Cys⁺ cells, the survival rates of Cys⁺ and GSH⁻ cells were comparable (data not shown), suggesting that the protective role of Cys against FTCs was not significant. However, the survival rates of GSH⁺ cells were significantly higher than those of the GSH⁻ cells in both MRS (Fig. 2A) and CDM (Fig. 2B). The surface morphology of L. sanfranciscensis GSH⁺ and GSH⁻ cells was examined by transmission electron microscopy (TEM). TEM with 200,000-fold magnification showed that L. sanfranciscensis GSH⁺ and GSH⁻ cells were both intact before cold treatment (data not shown). After FTC treatment, the integrity of L. sanfranciscensis GSH⁺ cells was maintained as normal (Fig. 3A and C). However, the cell walls of L. sanfranciscensis GSH⁻ cells were significantly thinner (Fig. 3B) than those of GSH⁺ cells (Fig. 3A). Moreover, many L. sanfranciscensis GSH⁻ cells cracked and their cytoplasm effused (Fig. 3D). These results suggest that the presence of GSH in L. sanfranciscensis GSH⁺ cells maintains cell integrity during FTC treatment.

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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).

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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.

GSH protects membrane fatty acids from oxidation upon cold treatment.The survival rates of L. sanfranciscensis GSH⁺, GSH⁻, and Cys⁺ cells treated at 4°C in both MRS and CDM broth were compared (Fig. 4). The results were similar to what has been observed for freeze-drying and FTC treatment, suggesting that the protective role of Cys is not remarkable. In contrast, the survival rates of GSH⁺ cells were significantly higher, thus confirming the protective role of GSH against cold treatment.

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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.

Membrane fluidity affects the resistance of LAB cells to cold damage (15). Since membrane fluidity is correlated with the fatty acid composition of the cell membrane, we investigated the effects of 4°C treatment on the membrane fatty acid composition of L. sanfranciscensis GSH⁺ and GSH⁻ cells. Before cold treatment, seven fatty acids were present in the membrane of L. sanfranciscensis GSH⁺ cells, of which the major fraction was octadec-8-enoate (C_18:1ω8c). In GSH⁻ cells, the composition of membrane fatty acids was similar to that of GSH⁺ cells except for the absence of oleic acid (C_18:1ω9c). After being treated at 4°C for 14 days, the composition of membrane fatty acids changed considerably. Pentadecanoic acid (C_15:0) and heptadecanoic acid (C_17:0) were detected as new components of saturated fatty acids in L. sanfranciscensis GSH⁺ cells (Fig. 5A and B), while the molar percentages of hexadecanoic acid (C_16:0) and octadecanoic acid (C_18:0) were significantly reduced (Fig. 5A). Correspondingly, the average chain length of saturated fatty acids of GSH⁺ cells was not significantly altered (from 16.43 to 16.69). On the other hand, heptadecanoic acid (C_17:0) was detected as a new component of saturated fatty acids of L. sanfranciscensis GSH⁻ cells, and the fractions of the saturated fatty acids all increased in GSH⁻ cells upon cold treatment (Fig. 5A), except for hexadecanoic acid (C_16:0). Correspondingly, the average chain length of saturated fatty acids of GSH⁻ cells increased significantly (from 15.87 to 16.88). Regarding the profile of unsaturated fatty acids, no increase in the molar percentage of unsaturated fatty acids was observed in GSH⁻ cells, whereas the molar percentage of C_18:1ω9c in GSH⁺ cells increased significantly (Fig. 5B). The average chain length of the unsaturated fatty acids of either GSH⁺ cells or GSH⁻ cells did not alter significantly (data not shown).

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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.

Remarkable differences in the U/S ratio, an important parameter reflecting the membrane fluidity of cells (9), were observed between GSH⁺ and GSH⁻ cells (Fig. 6). Generally, both GSH⁺ and GSH⁻ cells showed a tendency to increase their U/S ratio in the initial phase of cold treatment (Fig. 6), whereas GSH⁺ cells exhibited a greater capability to further increase their U/S ratio, which enabled the GSH⁺ cells to maintain a much higher U/S ratio than the GSH⁻ cells after 30 days of cold treatment (Fig. 6). This suggests that besides maintaining the average chain length of saturated fatty acids, GSH⁺ cells might have greater membrane fluidity as indicated by the increased U/S ratio, which might be beneficial to maintain a higher survival rate during cold treatment.

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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.

Effect of GSH on membrane LA (C_18:2) concentration during cold treatment.Since the ratio of the unsaturated fraction of membrane fatty acids of L. sanfranciscensis GSH⁺ cells increased during cold treatment, linoleic acid (C_18:2) (LA) was chosen as a representative unsaturated fatty acid to further investigate the effect of GSH. We determined the molar percentages of LA and found that GSH⁺ cells before cold treatment have a much higher fraction of LA than GSH⁻ cells (Fig. 7), suggesting that the concentration of LA is related to the redox status (33). As shown by the results in Fig. 7, no LA can be detected in GSH⁻ cells after being treated at 4°C for 14 days, whereas GSH⁺ cells retained 50% of their LA. Even after they were treated at 4°C for 30 days, we were able to detect the presence of LA in GSH⁺ cells (Fig. 7). These results suggest that the presence of GSH increased the initial molar percentage of LA, thus enabling the cells to maintain a high level of LA during cold treatment.

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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.

Cold treatment triggers SOD response.The alteration of the degree of saturation of membrane fatty acids suggested a redox change during cold treatment. We detected the superoxide dismutase activities of L. sanfranciscensis GSH⁺ and GSH⁻ cells treated at 30°C (control) and 4°C. The SOD activity of the control cells treated at 30°C increased by 10 to 15% after 7 days of treatment, suggesting that no significant redox disturbance occurred at the normal growth temperature. However, exposure of L. sanfranciscensis GSH⁺ and GSH⁻ cells to 4°C for 7 days led to a significant increase in SOD activities in both cells (Table 1). These results suggest that oxidative stress is a contributory factor for chilling injury, consistent with previously reported studies of several microorganisms (8, 12, 37) and plants (21).

GSH protects Na⁺,K⁺-ATPase against cold stress.The activities of key cell membrane-localized enzymes with important physiological functions were determined to investigate whether GSH plays a protective role. Na⁺,K⁺-ATPase is an integral membrane enzyme which plays a key role in maintaining the electrochemical gradients of Na⁺ and K⁺ and regulating their active transport across the cell membrane (16). In our study, a remarkable decrease in Na⁺,K⁺-ATPase activity was detected in L. sanfranciscensis GSH⁻ cells when treated at 4°C (Fig. 8A). Surprisingly, L. sanfranciscensis GSH⁺ cells showed a strong capability to maintain greater Na⁺,K⁺-ATPase activity, and 75% of the initial Na⁺,K⁺-ATPase activity could be retained after treatment at 4°C for 30 days (Fig. 8A). This implies that the presence of GSH effectively prevents Na⁺,K⁺-ATPase from damage during cold treatment.

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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.

Changes in Na⁺,K⁺-ATPase activity and intracellular GSH during the recultivation process.When cold-treated L. sanfranciscensis GSH⁻ cells were reinoculated into fresh MRS, no increase in Na⁺,K⁺-ATPase activity was observed, suggesting that the decrease in Na⁺,K⁺-ATPase was irreversible (35). When the cold-treated L. sanfranciscensis GSH⁺ cells were reinoculated into fresh MRS broth, the Na⁺,K⁺-ATPase activity increased gradually (Fig. 8B), although the cell number did not increase. Meanwhile, the intracellular GSH concentration showed a significant increase during the recultivation process (Fig. 8B). We therefore hypothesized from the synchronous increase of GSH level and Na⁺,K⁺-ATPase activity that GSH might protect Na⁺,K⁺-ATPase in a reversible way.