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Applied and Environmental Microbiology, September 2002, p. 4399-4406, Vol. 68, No. 9
0099-2240/02/$04.00+0 DOI: 10.1128/AEM.68.9.4399-4406.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Lehrstuhl für Technische Mikrobiologie,1 Lehrstuhl für Fluidmechanik und Prozessautomation, TU München, 85350 Freising, Germany2
Received 28 January 2002/ Accepted 20 June 2002
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ABSTRACT |
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INTRODUCTION |
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pH) will collapse or approach zero, resulting in loss of cell viability (18). The pH value of buffered solutions is influenced by pressure, because the dissociation constants (pKa values) of water and weak acids or weak bases depend on the absolute value of pressure. Because the pKas of phosphate and carboxylic acids are decreased by pressure, a decrease in pH is observed during pressure application in most biological systems. Such a decrease in pH may promote protein denaturation and inactivation of microorganisms in food (9). Furthermore, hydrostatic pressure induces a phase transition of the cytoplasmic membrane from the physiological liquid-crystalline to the gel phase and thus increases the membrane permeability and induces leakage of sodium and calcium ions (25). Although the irreversible, pressure-induced denaturation of proteins in aqueous solution generally requires pressures greater 300 MPa, pressures as low as 200 MPa have been shown to inactivate the membrane-bound transporters of F0F1 ATPase and HorA, a multidrug resistance (MDR) transporter of the ATP-binding cassette family, in Lactobacillus plantarum (29, 30).
Wouters et al. (30) investigated the effect of treatment at 250 MPa on the regulation of the pHin and the activity of F0F1 ATPase in Lactobacillus plantarum cells grown at pHs 5.0 and 7.0. They found that cells grown at pH 5.0 were more resistant to pressures than cells grown at pH 7.0. This difference in resistance may be explained by a higher F0F1 ATPase activity, a better ability to maintain a transmembrane pH gradient, or a higher acid efflux of the cells grown at pH 5.0. After pressure treatment, the ATPase activity was decreased, the ability to maintain a pH was reduced, and the acid efflux was impaired as the cells lost viability. However, the pHin had already decreased before the number of CFU was reduced (30).
Abe et al. (1, 2) described the effect of hydrostatic pressure on the cell growth and on the vacuolar acidification of Saccharomyces cerevisiae. The pHin was measured during high-hydrostatic-pressure treatment using pH-sensitive fluorescent probes. Application of hydrostatic pressure caused a delay or cessation of cell growth and promoted the acidification of vacuoles in a manner dependent on the magnitude of the pressure applied (up to 60 MPa). Pressure-induced vacuole acidification is caused by the production of carbon dioxide. The ionization of H2CO3 (HCO3- plus H+) is facilitated by elevated pressure because the reaction is accompanied by a decrease in volume. Consequently, a large number of protons is produced in the cytoplasm, which becomes more acidic at elevated pressures. Abe et al. proposed that the yeast vacuole takes up protons from the cytoplasm to maintain a favorable cytoplasmic pH under high-pressure conditions, thereby functioning as a proton sequestrant (1, 2).
One of the main difficulties in understanding pressure inactivation is that the effects on cells are usually analyzed after decompression. For this reason, the aim of the present work was to investigate and to measure in situ the effects of both pHex and high pressure on the pHin and viability of Lactococcus lactis and Lactobacillus plantarum. Furthermore, the ability of the treated cells to restore the pH was determined. To perform pHin measurements, a fluorescent method using 5 (and 6)-carboxyfluorescein succimidyl ester (cFSE) (7, 8) was adapted to Lactococcus lactis and Lactobacillus plantarum.
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MATERIALS AND METHODS |
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Milk buffer.
The milk buffer was chosen to contain the same amounts of minerals and lactose as whey from rennet casein; the buffer contained the following compounds (in grams per liter): KCl, 1.10; MgSO4·7H2O, 0.71; Na2HPO4·2H2O, 1.87; CaSO4·2H2O, 1.00; CaCl2·2H2O, 0.99; citric acid, 2.00; and lactose, 52.00. The pH was adjusted to 6.5, 6.0, 5.0, and 4.0 with KOH (1 M).
Bacterial strain and culture conditions.
Lactococcus lactis subsp. cremoris MG1363 was grown at 30°C in M17 broth supplemented with 1% glucose (GM17 broth). Lactobacillus plantarum TMW1.460 was grown at 30°C in MRS medium.
Labeling of cells with cFSE for pHin determination.
The fluorescence method developed by Breeuwer et al. (8) was adapted to Lactococcus lactis subsp. cremoris MG1363 and Lactobacillus plantarum. Harvested cells were washed and resuspended in 50 mM HEPES buffer, pH 8.0. Subsequently, the cells were incubated for 15 min at 30°C in the presence of 10.0 µM cFDASE, washed, and resuspended in 50 mM potassium phosphate buffer, pH 7.0. To eliminate nonconjugated cFSE, glucose (final concentration, 10 mM) was added and the cells were incubated for an additional 30 min at 30°C. The cells were then washed twice, resuspended in corresponding milk buffer, and placed on ice until required.
Offline measurement of pHin at ambient pressure.
Stained cells were placed in a 2-ml cuvette (Sarstedt, Nümbrecht, Germany) and placed in the cuvette holder of a spectrometer (luminescence spectrometer model LS-50B; Perkin-Elmer, Überlingen, Germany). Fluorescence intensities were measured at excitation wavelengths of 485 and 410 nm by rapidly alternating the monochromator between both wavelengths. The emission wavelength was 520 nm, and the excitation and emission slit widths were 5 and 4 nm, respectively. The ratios of intensities at 485 nm to those at 410 nm were corrected for the background of the buffer. The incubation temperature was 30°C. Calibration curves were determined in buffers with pH values ranging from 4.0 to 8.0. Buffers were prepared from citric acid at pHs 4.0 and 5.0 (50 mM); MES at pHs 5.5, 6.0, and 6.5 (50 mM); and HEPES at pHs 7.0, 7.5, and 8.0 (50 mM). The pH was adjusted with either NaOH or HCl. The pHin and pHex were equilibrated by addition of valinomycin (1 µM) and nigericin (1 µM), and the ratios were determined as described previously. Calibration curves were established for experiments performed on a single day.
Pressurization of cell suspensions.
The cells of an overnight culture were harvested by centrifugation (15 min at 5500 x g), washed, and resuspended in milk buffer to about 109 CFU·ml-1. The cells were suspended in 2-ml portions in sterile plastic micro test tubes, sealed with silicon stoppers, and stored on ice until they were pressurized. The pressure chamber was heated or cooled to a desired level prior to pressurization with a thermostat jacket connected to a water bath. The pressure level, time, and temperature of pressurization were controlled by a computer program. The compression-to-decompression rate was 200 MPa min-1, the temperature was 20°C, and the temperature rise due to compression was 6°C or less. Samples were energized with 10 mM glucose and placed in the pressure chamber 5 min prior to treatment to equilibrate the sample temperature. Cells were exposed to a pressure of 200 or 300 MPa for various time intervals (0 to 120 min). Following the release of pressure the samples were stored on ice for determination of viable cell counts.
Enumeration of viable cells.
The cell suspensions from each vial were serially diluted with saline immediately after the pressurization treatment and were surface plated on GM17 or MRS agar. The plates were incubated for 24 h at 30°C. Selective agars were obtained with the addition of 3% NaCl to GM17 agar and 4% to MRS agar. Data presented are means ± standard deviations obtained from two to three independent experiments.
In situ measurement of pHin.
Fluorescence under hydrostatic pressures was measured in a pressure chamber equipped with a cylindrical sapphire window (10 x 8 mm) as described previously (11). Two milliliters of stained cells was placed in the pressure chamber and energized with 10 mM glucose. The lid was closed, and the device was connected with an optical fiber to a spectrometer (Perkin-Elmer luminescence spectrometer LS-50B). Fluorescence intensities were measured at excitation wavelengths of 485 and 410 nm by rapidly alternating the monochromator between both wavelengths. The emission wavelength was 520, and the excitation and emission slit widths were 15 nm to compensate for the loss of fluorescence intensity caused by the optical fiber. The incubation temperature was 20°C, and 5 min was allowed to pass prior to high-pressure treatment to equilibrate the temperature.
Calibration curves were determined in buffers with pH values ranging from 4.0 to 8.0. Buffers were prepared from milk buffers of pH 4.0, 5.0, and 6.0, and 50 mM HEPES buffer at pHs 7.0 and 8.0. The pH was adjusted with either NaOH or HCl for HEPES buffers and with KOH for the milk buffers. The pHin and pHex were equilibrated by addition of valinomycin (1 µM) and nigericin (1 µM), and the fluorescence was determined as described above. Since the signal-to-noise ratio during measurements in the pressure chamber decreased compared to the measurement in the cuvette, the fluorescence intensities were measured over a period of 5 min at either 0.1, 200, or 300 MPa and the means were calculated for each buffer. A calibration curve was established for each culture stained and pressure treated on a single day.
Reversibility test.
After the pressure treatment at pH 6.5, 1 ml of the pressure-treated samples was placed in a cuvette and the fluorescence intensities were determined as described above. After 3 min of equilibration, glucose was added to a final concentration of 10 mM and the changes in pHin were monitored over up to 30 min. The pH of treated cells (pHin versus pHex) after 30 min of incubation was calculated.
Computation of the pH values during pressure treatment.
Various approaches have been proposed to calculate changes in pKa values of water and weak acids (16, 19). In this communication the following relation described by El'yanov and Hamman (10) was used:
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Vm is the molal volume change between the associated and dissociated forms of the buffering acid in solution, R is the universal gas constant (8,3145 J/kmol), T is the absolute temperature, and b is a universal constant (9.2 Pa-1). El'yanov and Hamman showed that this equation fits very well to experimental data obtained with various buffers and pressures of up to 120 MPa. This equation was found empirically and is in good agreement with the electrostatic theory of Born for the interactions between ions in solutions (6).
The application of equation 1 to mixtures of buffer salts exploits the balance of H3O+ ions. The pH value at ambient pressure, e.g., that measured with a pH glass electrode, must be known. Furthermore, the dissociation constant of water (pKw) and pKa and V values at ambient pressure for all reactions must be available from data in the literature. It is sufficient to know the sum of the concentrations at ambient pressure (c0) of all components from one species, e.g.,
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Equation 1 can be used to calculate the pKw and pKa values at high pressure. For a starting pHi (the subscript i is for iteration) the concentrations of all components are calculated based on the law of mass action. Based on the change of these concentrations, the number of H3O+ ions formed or consumed by a reaction is calculated. Equation 2 shows an example for calculation of H3O+ for phosphoric acid and its salts.
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 | (3) |
H3OpH+).
If equation 4 (with pHi instead of pH) yields a figure lower than 0, one has to start the calculation with a smaller value of pHi and vice versa.
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RESULTS |
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pH across the cell membrane was maintained at around 2.0 whereas under alkaline conditions it was dissipated.
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pH, pressure application had only minor effects on the fluorescence of cFDASE located intracellularly, indicating its suitability as a probe for pHin during high-pressure treatments. However, because the pKa value of cFDASE is 6.5, the fluorescence ratios determined at pHs 4.0 and 5.0 almost coincided. The baseline noise of the ratio data collection was caused mainly by the rapid alteration of the emission monochromator between the two wavelengths. This mode of data collection was nevertheless preferred over a mere intensity measurement because differences in fluorescence intensity between individual samples and pressure ramps do not affect the fluorescence ratios. Generally, the experimental error for in situ pHin determinations was less than 0.5 pH units in the pH range of 5.0 to 8.0 and less than 1 pH unit for values below pH 5.0.
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At 300 MPa of pressure, the curves showing inactivation of Lactococcus lactis in milk buffer with different pH values were sigmoid and asymmetric when they were plotted in a logarithmic scale, as previously described for Lactobacillus plantarum (data not shown and reference 28). After 8 min of pressure holding time, a reduction of cell counts by 2 log units was observed at pH 6.5 whereas a reduction of >5 log units was observed at pH 4.0. After 20 min of pressure holding time, a reduction of cell counts by 6 log units was achieved independent of the buffer pH. As opposed to pH effects on viable cell counts, the pH did not affect the numbers of sublethally injured cells.
Lactobacillus plantarum showed a greater resistance than Lactococcus lactis to pressure inactivation at 300 MPa. Treatments at pHs 4.0 and 5.0 reduced cell counts by 4 to 5 log units within 20 min, whereas treatment at pH 6.0 required 120 min to achieve a 5-log reduction, and at pH 6.5 the reduction was only 3 log units after 120 min. Sublethal injury of 90 to 99.9% of the population could be observed during pressure treatments at pHs 6.0 and 6.5; however, almost no sublethal injury was observed after treatments at pHs 4.0 and 5.0, indicating that injured cells were not able to survive.
Effect of high-pressure on the pHin of Lactococcus lactis.
The intracellular pH was determined by measuring the 485-nm/410-nm-wavelength fluorescence ratio of cFDSE-labeled cells during pressure treatments. The ratios of fluorescence intensities of Lactococcus lactis during pressure treatment at 300 MPa are shown in Fig. 3. The fluorescence ratios are compared to the viable cell counts. Based on the calibration samples, the pHin values at ambient pressure and at high pressure were calculated and are indicated in the graphs. The pHin measured at normal pressure corresponded within experimental error to those values shown in Fig. 1. Pressure treatment at 300 MPa and pH values ranging from 3.5 to 5.5 resulted in a drop in the pHin of Lactococcus lactis cells (Fig. 3), which indicates that the regulation of the pHin was impaired in these cells. The pHin approached the respective values of pHex during the first 90 s of treatment, corresponding to the compression of the samples to 300 MPa. However, a compression-decompression cycle had no apparent effect on cell viability at any pH. During the first 4 min of pressure holding time, greater than 90% of the cells were inactivated at any pH, yet the values for the pHin remained constant during the pressure holding time.
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pH was not fully dissipated after the ramp-up time but only after 1 min of pressure holding time. Thereafter, the values of pHin corresponded to the values of pHex. Despite the fast drop of pHin during treatments at 200 MPa, a reduction in cell viability was not observed during 6 min of pressure holding time at any pH (data not shown).
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pH and to reduce the capacity of the cells to restore a pH gradient upon pressure treatment.
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pH after pressure treatments at 300 MPa (Table 2). The results of the reversibility test did not show an increase of the pHin after pressure release. Even mild treatments, e.g., compression to 300 MPa and decompression without pressure holding time, caused an irreversible reduction of the pHin. |
DISCUSSION |
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pH upon pressure release was monitored.
The cFSE method for pH determination is based on the internal conjugation of cFSE in the cytoplasm of cells, followed by the elimination of free probe by an incubation in the presence of a fermentable sugar. Efflux of fluorescent probe during high-pressure treatment, that may permeabilize the cells envelope were avoided because the succinimidyl group of cFSE is conjugated with the aliphatic amines of intracellular proteins (14). The useful range of fluorescent pH indicators is determined by their pKas; cFSE has a pKa of 6.5, compatible for working in the range expected for the pHins of our microorganisms. For lower pHin, the cFSE may be replaced with 5 (and 6)-carboxy-2',7'-dichlorofluorescein succinimidyl ester, which has a pKa of 3.9 in buffer and a pKa of 4.9 when it is incorporated in bacterial cells (7, 24). The effects of cFSE incorporation on the cells prior to high-pressure treatments were negligible, since the viable counts of cells stained with cFSE were not different from those observed with unstained cells. Furthermore, the pHex did not affect either the cell viability or the pHin during storage of cells for up to 3 h at ambient pressure. The values for the pHin at atmospheric pressure of Lactococcus lactis and Lactobacillus plantarum at pHex values ranging from 4.0 to 8.0 determined in this work were comparable to those reported previously for Lactococcus lactis and Lactobacillus plantarum (4, 8, 24, 30). In both species investigated in this work, the pHin decreased with decreasing buffer pH, resulting in pH values between 1.9 and 2.1 pH units at pH 4.0.
It is well established that a reduction in the pH of the suspending medium causes a progressive increase in the sensitivity of bacteria to pressure (3, 13, 17, 18, 22, 23). For example, pressurization at 345MPa and a pH values of 4.5 increased the viability loss of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella enteritidis by an additional 1.2 to 3.9 log cycles compared to pressurization at pH 6.5 (3). The data on pressure inactivation kinetics at acidic and near neutral pH values presented in this study are consistent with these literature data. Both Lactococcus lactis and Lactobacillus plantarum were less pressure sensitive at pH 6.5 than at pH 4.0. However, independent of the buffer pH, a fraction of about 1 in 106 cells withstood pressure treatment for up to 120 min. For either strain, a greater degree of sublethal injury was observed at pH values of 6.5 and 6.0 compared to pressure treatment at pH 4.0 and 5.0. This indicates that at a pH value near neutral, sublethally injured cells survive and recover eventually upon transfer to a rich medium. At acidic pH values, sublethally injured cells are inactivated during pressure treatment.
Wouters et al. (30) reported a decrease on pHin of Lactobacillus plantarum after high-pressure treatment. Our results confirmed and extended these observations by pH measurements during pressure treatment, which allow the distinction between reversible and irreversible effects of pressure treatment on pH homeostasis. Compression of the samples resulted in a fast and reversible decrease of the pHin. At 300 MPa, the pHin was equal to the external pH with completion of the pressure up ramp, prior to a reduction of viable cell counts. The observation that the decrease of pHin occurred virtually in equilibrium with the pressure build up suggest that both the membrane permeability and the activity of proton pumps were affected by high pressure treatment.
Membrane and membrane-bound enzymes are a major target for the pressure inactivation of microorganisms (26). Under high-pressure the membrane lipids undergo a phase transitions from the liquid crystalline phase to the gel phase. This phase transition enhances the permeability of the membrane to ions, and inflicts cell injury because some transport proteins only function in a liquid-crystalline membrane (5). The sensitivity of bacteria is markedly affected by membrane properties (27, 29).
In Lactobacillus plantarum, pressure treatment caused partial inactivation of the F0F1 ATPase such that the ability of cells to maintain a pH was reduced, and the acid efflux mechanism was impaired (30). Direct denaturation, dissociation of the quaternary structure, or the dislocation of the enzyme from the membrane may account for this effect. Ulmer et al. (28, 29) reported that inactivation of transport proteins is an early event during pressurization of microorganisms. Recent works at our laboratory with L. lactis confirmed the pressure inactivation of the membrane-bound multidrug transporter LmrP in Lactococcus lactis before a reduction on the CFU was observed (unpublished).
Remarkable differences in barotolerance pH homeostasis during pressure treatment were observed between Lactococcus lactis and Lactobacillus plantarum. The minimum pH permissive for growth of Lactococcus lactis is 4.0, whereas Lactobacillus plantarum is a highly acid tolerant beer spoiling organism growing at pH values of 3.5 or lower (12). In Lactococcus lactis, pH homeostasis was irreversibly disturbed after 4 min of pressure holding time at 300 MPa and after 40 min at 200 MPa. The loss of viability related not to the decrease of the pHin but to the loss of ability to restore the pH after pressure treatment. Lactobacillus plantarum was more pressure tolerant that Lactococcus lactis at any pH value. The transmembrane proton potential was not fully dissipated by treatments at 200 MPa. The sensitivity of Lactobacillus plantarum to treatments at 300 MPa was affected to a greater extend by low pH conditions than that of Lactococcus lactis. This observation may relate to the inability of Lactobacillus plantarum to restore a pH upon pressurization at 300 MPa even after 0 min pressure holding time. Our data thus support the hypothesis that pressure treatment restricts the pH range tolerated by bacteria as a consequence of the irreversible inhibition of ATPase-dependent transfer of protons and cations (23). Pagán et al. (20, 21) reported a sensitization of E. coli cells to acid by a pressure treatment. Because the lowering of the pHin upon pressure treatment did not explain the death of pressure damaged cells under acidic conditions, the authors suggested that this acid sensitivity is caused by the loss of protective or repair functions rather than to the loss a transmembrane pH per se. Garcia-Graells et al. (13) reported a secondary inactivation of E. coli during storage in fruit juices at low pH after a primary pressure treatment inflicting sublethal injury to a large proportion of cells. This observation was confirmed for pressure resistant strains of E. coli O157 and Listeria monocytogenes (15). Survival and growth of lactic acid bacteria in beer was prevented by pressure treatments that resulted in a reduction of 50 to 99% of viable cell counts only but fully eliminated hop resistance of lactobacilli (12, 29).
The observation that the ability of microorganisms to survive in harsh environments is eliminated by pressure treatment allows the distinction between primary and secondary effects of high pressure treatments. The reversible or irreversible loss of concentration gradients across the cytoplasmic membrane may be considered as a primary effect of pressure treatment. Cell death may be considered a secondary event depending not primarily on pressure or pressure holding time, but on the environmental conditions during and after pressure treatment. This concept may prove useful to study the relevance of membrane properties in the bacterial response to environmental stress, and is especially relevant for the design of high-pressure processes for food preservation.
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ACKNOWLEDGMENTS |
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We thank our students Micha Hoffmann, Lydia Tänzer, and Michaela Hampel for their contributions to this work.
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FOOTNOTES |
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) in the presence of 10 mM glucose at different values of pHex. The assays were performed at 30°C and 0.1 MPa pressure. The buffers were prepared from citric acid (50 mM), MES (50 mM), and HEPES (50 mM). The pH was adjusted with either NaOH or HCl. Symbols represent means ± standard deviations of results from two independent experiments.
