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Lactobacillus sanfranciscensis DSM20451 cells containing glutathione (GSH) displayed significantly higher resistance against cold stress induced by freeze-drying, freeze-thawing, and 4°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.
Lactic acid bacteria (LAB) are considered to be mesophilic and thermophilic from an application point of view (27). Among LAB, Lactobacillus sanfranciscensis is widely used as a sourdough starter due to its outstanding characteristics in nutrition, flavor, and antibacterial properties (11, 14, 24). On a commercial scale, cold storage usually affects cell viability and subsequent fermentation performance (1). Approaches to increasing the cold stress resistance are therefore expected to increase the robustness of L. sanfranciscensis.
A series of changes, e.g., decreases in membrane fluidity and destabilization of secondary structures of RNA and DNA, take place when cells are exposed to abrupt decreases in temperature (9). The cell membrane is considered to be the first barrier that separates cells from their environment and is the primary target for damage induced by environmental stresses. To date, many alterations in the structure and function of the cell membrane have been observed during cold treatment (43). Furthermore, inactivity of membrane-associated enzymes and transporters was demonstrated to be related to membrane damage from undergoing cold stress (2).
Glutathione (γ-Glu-Cys-Gly, the reduced form [GSH]) is the most important nonprotein thiol compound in living organisms. The physiological roles of GSH in living organisms (especially in higher eukaryotic organisms) have been generally recognized as antioxidation, immunity boosting, and detoxification (26). Knowledge about the physiological function of GSH in Gram-positive bacteria is very limited. Recently, GSH was found to play a role against oxidative stress in Lactococcus lactis (21) and L. sanfranciscensis (19) and against acid stress in L. lactis (45). However, it is not yet clear if GSH can protect LAB against cold stress. The objective of this study was to investigate the role of GSH in protecting L. sanfranciscensis against various cold stresses, with the aim of providing a strategy for improving the stability of starter cultures under cold conditions.
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).
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).
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.
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.
The freeze-dried cells were resuspended in 63 ml of sterile tap water and mixed with 90 g of wheat flour until dough formation.
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.
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).
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.
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.
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).
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 (Pi) by the enzyme was within the linear range during the incubation period. The liberated Pi 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.
Student's t test was employed to investigate statistical differences. Samples with P values of <0.05 were considered to be statistically different.
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. (Fig.1A).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. (Fig.1B).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. (Fig.1B).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.
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. (Fig.2A)2A) and CDM (Fig. (Fig.2B).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. (Fig.3B)3B) than those of GSH+ cells (Fig. (Fig.3A).3A). Moreover, many L. sanfranciscensis GSH− cells cracked and their cytoplasm effused (Fig. (Fig.3D).3D). These results suggest that the presence of GSH in L. sanfranciscensis GSH+ cells maintains cell integrity during FTC 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. (Fig.4).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.
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 (C18:1ω8c). In GSH− cells, the composition of membrane fatty acids was similar to that of GSH+ cells except for the absence of oleic acid (C18:1ω9c). After being treated at 4°C for 14 days, the composition of membrane fatty acids changed considerably. Pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) 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 (C16:0) and octadecanoic acid (C18:0) were significantly reduced (Fig. (Fig.5A).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 (C17:0) 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. (Fig.5A),5A), except for hexadecanoic acid (C16:0). 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 C18:1ω9c in GSH+ cells increased significantly (Fig. (Fig.5B).5B). The average chain length of the unsaturated fatty acids of either GSH+ cells or GSH− cells did not alter significantly (data not shown).
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. (Fig.6).6). Generally, both GSH+ and GSH− cells showed a tendency to increase their U/S ratio in the initial phase of cold treatment (Fig. (Fig.6),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. (Fig.6).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.
Since the ratio of the unsaturated fraction of membrane fatty acids of L. sanfranciscensis GSH+ cells increased during cold treatment, linoleic acid (C18:2) (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. (Fig.7),7), suggesting that the concentration of LA is related to the redox status (33). As shown by the results in Fig. Fig.7,7, 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. (Fig.7).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.
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 (Table1).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).
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. (Fig.8A).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. (Fig.8A).8A). This implies that the presence of GSH effectively prevents Na+,K+-ATPase from damage during cold treatment.
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. (Fig.8B),8B), although the cell number did not increase. Meanwhile, the intracellular GSH concentration showed a significant increase during the recultivation process (Fig. (Fig.8B).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.
Cell membranes exposed to subzero temperature may be damaged by the formation of ice crystals (36). Low temperatures above zero (such as 4°C) are used for transport and storage; therefore, maintaining the cell viability of L. sanfranciscensis under low temperatures is essential for their application. Membrane damage is considered one of the most serious deleterious effects caused by cold treatment. In this study, we observed that GSH protects L. sanfranciscensis cells against freeze-drying, freezing-thawing, and 4°C cold treatment and plays an important role in maintaining a firm structure and modulating a favorable composition of the cell membrane. Membrane damage caused by cold stress, such as changes in membrane structure and the activities of membrane-associated protein, can lead to cell death (2, 41). Interestingly, loss of membrane SH groups and subsequent changes in ion homeostasis occurred when the intracellular GSH level decreased rapidly (18). Moreover, GSH might modulate the regulatory protein of the mechanosensitive channel and thus enhance the membrane strength of E. coli (23, 29, 30). We therefore hypothesized that the protective role of GSH in L. sanfranciscensis DSM20451 against cold stress might be related to its function in the cell membrane.
Our data suggest that GSH protects the membrane via two different mechanisms. One possible mechanism is that GSH prevents the oxidation of the cell membrane that is triggered under cold stress. Under 4°C cold challenge, the increased SOD activity at the initial stage can be considered a response to the environmentally induced oxidation (4, 44), which may trigger the oxidation of the C-C double bonds of membrane unsaturated fatty acids. So far, the most widely recognized adaptation of the cell membrane at low temperatures is the desaturation of lipid acyl chains (38). Phospholipids with unsaturated fatty acids have lower melting points and are more flexible than phospholipids with saturated acyl chains (32). The higher U/S ratio of L. sanfranciscensis GSH+ cells showed the adaptation of the cell membrane upon cold challenge. Moreover, during 4°C processing, GSH seems to be beneficial in keeping a significantly higher level of linoleic acid (C18:2) in the membrane, which might increase the freeze tolerance of L. sanfranciscensis cells, as previously observed in Saccharomyces cerevisiae (39).
Another possible mechanism is that GSH protects the cell membrane against cold-induced oxidation through protection of Na+,K+-ATPase. It has been reported that the oxidation of SH groups on membrane surface proteins, such as Na+,K+-ATPase, results in increased permeability (17). In addition, altered composition of the membrane lipid fractions may influence the activity of membrane-associated proteins and subsequently lead to growth arrest and cell death (43). Our data show that GSH prevents the decrease of Na+,K+-ATPase activity upon cold treatment. Previous reports suggested that GSH might be required for the protection of membrane SH groups (18), and the depletion of GSH results in increased susceptibility of the [Na+]/[K+] pump to oxidative damage (34). More interestingly, the intracellular GSH decreased during cold challenge but then increased gradually in the “lag phase” of the recultivation (Fig. (Fig.8);8); this “increased” GSH can neither be synthesized by L. sanfranciscensis cells themselves nor determined by the total glutathione assay from redox form changes. Although it has been reported that damage to Na+,K+-ATPase is irreversible (35), our data suggest that Na+,K+-ATPase might be reversible in GSH+ cells during the recultivation process after cold treatment.
The remarkable level of linoleic acid (C18:2) in L. sanfranciscensis GSH+ cells is another exciting finding that confirms the indirect protection by GSH of Na+,K+-ATPase. As an integrated membrane protein, the activity of Na+,K+-ATPase affects the membrane status and is also affected by several physical membrane properties, including membrane thickness (20), phospholipid composition (42), fatty acyl chain length (28), and membrane fluidity (22). A significant positive correlation was found between the level of Na+,K+-ATPase activity and the total fraction of linoleic acid (C18:2) in the membrane lipid composition (7). We therefore propose that GSH might protect Na+,K+-ATPase indirectly by reducing the oxidation of membrane fatty acids, including linoleic acid (C18:2), thereby improving membrane fluidity.
The investigation described here have provided direct evidence that GSH maintains the integrity and fluidity of L. sanfranciscensis cell membranes during cold treatments. Moreover, the findings about the intracellular status, such as the redox changes under cold stress, inspire us to consider the protective role of GSH in the cross-stress effect, which is little known at present, and provide a new model to investigate the mechanism of stress resistance. Based on the role of GSH during freeze-drying and other cold treatments, the new recognition of its protection against cold stress suggests that selecting GSH-accumulating starters is an effective way to improve sourdough fermentation efficiency.
This work was supported by grants from the National Basic Research Program of China (973) (grants 2007CB714306 and 2007CB707803), the Key Program of National Natural Science Foundation of China (grant 20836003), the National Natural Science Foundation of China (grants 30870040 and 30900013), and the Knowledge Innovation Program of the Chinese Academy of Sciences (grant KSCX2-YW-G-005). Y.L. is supported by the Hundreds of Talents Program of the Chinese Academy of Sciences, and J.C. is supported by the National Science Fund for Distinguished Young Scholars of China (grant no. 20625619).
Published ahead of print on 5 March 2010.