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Applied and Environmental Microbiology, August 2008, p. 4835-4840, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00571-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Intracellular pH Homeostasis Plays a Role in the Tolerance of Debaryomyces hansenii and Candida zeylanoides to Acidified Nitrite{triangledown}

Henrik Dam Mortensen,1 Tomas Jacobsen,2 Anette Granly Koch,2 and Nils Arneborg1*

Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark,1 Danish Meat Association, Maglegårdsvej 2, 4000 Roskilde, Denmark2

Received 10 March 2008/ Accepted 24 May 2008


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ABSTRACT
 
The effects of acidified-nitrite stress on the growth initiation and intracellular pH (pHi) of individual cells of Debaryomyces hansenii and Candida zeylanoides were investigated. Our results show that 200 µg/ml of nitrite caused pronounced growth inhibition and intracellular acidification of D. hansenii at an external pH (pHex) value of 4.5 but did not at pHex 5.5. These results indicate that nitrous acid as such plays an important role in the antifungal effect of acidified nitrite. Furthermore, both yeast species experienced severe growth inhibition and a pHi decrease at pHex 4.5, suggesting that at least some of the antifungal effects of acidified nitrite may be due to intracellular acidification. For C. zeylanoides, this phenomenon could be explained in part by the uncoupling effect of energy generation from growth. Debaryomyces hansenii was more tolerant to acidified nitrite at pHex 5.5 than C. zeylanoides, as determined by the rate of growth initiation. In combination with the fact that D. hansenii was able to maintain pHi homeostasis at pHex 5.5 but C. zeylanoides was not, our results suggest that the ability to maintain pHi homeostasis plays a role in the acidified-nitrite tolerance of D. hansenii and C. zeylanoides. Possible mechanisms underlying the different abilities of the two yeast species to maintain their pHi homeostasis during acidified-nitrite stress, comprising the intracellular buffer capacity and the plasma membrane ATPase activity, were investigated, but none of these mechanisms could explain the difference.


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INTRODUCTION
 
Nitrite (NO2–) is a well-known antimicrobial agent used especially for the curing of meat (5, 26). The early studies of Tarr, in 1941 (29), and Castellani and Niven, in 1955 (6), on bacteria showed that the antimicrobial effect of nitrite is limited at neutral pH values but is significantly increased by acidification. These early findings have subsequently been confirmed in a variety of bacteria (for examples, see references 2, 7, 8, 27, and 28) and in yeast species (1, 3, 37). The reports covering this issue on yeasts are, however, very few (1, 3, 37), and they comprise only clinical yeast species.

When nitrite is acidified, a complex mixture of nitrogen oxides and nitrous acid are produced (19); i.e., nitrite is protonated under acidic conditions to generate nitrous acid (HNO2; pKa = 3.2), which is unstable and will spontaneously decompose to produce nitric oxide (NO) and nitrogen dioxide (NO2). The antimicrobial effect of acidified nitrite has, as yet, been attributed mainly to the nitrogen oxides causing alteration and/or inactivation of DNA, proteins, and membrane lipids (5, 16, 19, 20).

Furthermore, under acidic conditions, the anionic form of nitrous acid may severely decrease the intracellular ATP content in the yeast Saccharomyces cerevisiae by inhibiting the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (13-15). Hinze and Holzer explain this phenomenon as follows: at low external pH values, more nitrite will be in its undissociated form as nitrous acid and will passively diffuse across the plasma membrane of the cell, and due to the neutral intracellular pH, nitrous acid will be trapped inside the cell in its anionic form (13-15). This mechanism for the antimicrobial effect of nitrous acid resembles very much that of weak organic acids, which cause an intracellular acidification, besides causing an intracellular accumulation of their anionic forms. To counteract this acidification, the cell pumps out protons via the energy-requiring plasma H+-ATPase, thereby leading to the uncoupling of energy generation from growth (4). Interestingly, no reports examining whether the antimicrobial effect of acidified nitrite may be caused by intracellular acidification seem to exist.

Yeasts may cause spoilage of processed meats, such as slimes and discoloration on the surfaces of frankfurters and sausages and gas swelling of packaged, sliced meats (10). Debaryomyces hansenii and Candida zeylanoides are two of the most frequently isolated yeast species from meat products (10), probably due to the fact that they are both tolerant to low temperatures and high salt concentrations (9, 24, 35). At present, it is not known how the growth and physiology of these two yeast species are affected by acidified nitrite. A better understanding of these issues would enable a more focused use of nitrite as an antifungal agent in the meat processing industry.

In this study, we investigated the effect of nitrite at pHex values of 4.5 and 5.5 on the growth (measured as cell area) and pHi of individual cells of D. hansenii and C. zeylanoides isolated from a bacon-manufacturing process (23).


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MATERIALS AND METHODS
 
Composition of media, buffers, and solutions.
Yeast peptone dextrose (YPD) agar (pH 5.6) contained the following per liter: 5 g yeast extract, 10 g peptone, 10 g glucose, and 20 g Bacto agar. Brain heart infusion broth (BHIB) (pH 4.5 and 5.5) contained 37 g BHI (Oxoid) per liter. BHIB-NO2 (pH 4.5 and 5.5) consisted of BHIB plus 300 mg NaNO2 (i.e., 200 mg nitrite) per liter. Solutions and buffers used in this study included cFDA,SE (4.48 mM carboxyfluorescein-diacetate succinimidyl ester [C-1157; Molecular Probes] in water-free dimethyl sulfoxide), a citrate-sodium phosphate buffer (pH 7.0) containing 0.1 M citric acid and 0.2 M Na2HPO4 (CPB), and 0.15 M KCl solutions in Milli-Q water with (KCl-ethanol) and without 70% (vol/vol) ethanol (pH 6.0).

Yeast strains, growth media, and preparation of inocula.
Two yeast species, Debaryomyces hansenii (DMRICC 4862) and Candida zeylanoides (DMRICC 4866), isolated from a bacon-manufacturing process, were used. For the preparation of the inoculation culture of each yeast species, several colonies from YPD agar, kept in a refrigerator at 5°C, were transferred to 10 ml BHIB (pH 5.5) in a 100-ml shake flask and incubated with agitation (120 rpm) at 25°C for 24 h. Subsequently, these cells were transferred to 50 ml BHIB (pH 5.5) in 250-ml shake flasks to an initial concentration of 1.0 x 106 cells/ml (Neubauer improved counting chamber) and incubated with agitation (120 rpm) at 25°C for 22 h (late exponential growth phase).

Fluorescence staining of cells.
Yeast cells were harvested in the late exponential growth phase (approximately 1.0 x 108 cells/ml) by centrifugation at 7,000 rpm for 5 min. Pellets were washed and resuspended in 980 µl CPB. Ten microliters of 1 M glucose was added for activation of the cells, and the cells were then stained by adding 10 µl cFDA,SE and incubated for 25 min at 30°C. The cells were harvested by centrifugation at 7,000 rpm for 5 min and resuspended in 1 ml BHIB (pH 4.5 or 5.5).

Perfusion setup and nitrite stress.
Stained cells were transferred to a perfusion chamber (RC-21B; Warner Instruments) mounted with a concanavalin A (Sigma)-coated bottom coverslip as previously described (36). Subsequently, the chamber was filled with BHIB (pH 4.5 or 5.5), and the cells were left to settle and immobilize on the concanavalin A surface for 10 min. The chamber was then mounted on an inverted epifluorescence microscope (Zeiss Axiovert 135 TV; Carl Zeiss). Two peristaltic pumps were connected to the chamber. The inlet pump (Alitea XV) was calibrated to a flow rate of 0.5 ml/min, whereas the outlet pump ensured removal of the waste. NaNO2 stress was introduced by perfusion with BHIB-NO2 (pH 4.5 or 5.5). Perfusion with BHIB (pH 4.5 or 5.5) was performed as a control. Both media used for perfusion (BHIB and BHIB-NO2) were aerated for 30 min before use. All experiments were conducted at 22°C.

Microscope setup for cell area index (CAI) and pHi determinations.
The same microscope setup as previously described (12) was used. The cells were focused under bright-field illumination with a Zeiss Fluar 40x objective (numerical aperture of 1.3). Stained cells were excited for 3 s at 435 and 490 nm using a monochromator (Monochromator B; TILL Photonics) as the excitation source and a 6% Zeiss gray filter to minimize photobleaching. Fluorescence emission was collected with a cooled charge-coupled-device camera (CoolSnapfx; Photometrics). Perfusion was initiated at time zero, and subsequently, images were acquired at various time intervals. At each acquisition time, a bright-field image for the determination of the cell area and fluorescence (i.e., 435 and 490 nm) images for the determination of pHi were acquired concurrently. The images were stored on a computer for later analysis by using MetaMorph 4.5 (Universal Imaging).

Determination of CAI, pHi, and pH gradient.
Single-cell growth was determined by measuring the CAI, and the pHi was determined for the same cells that were used for CAI measurements by using fluorescence ratio imaging microscopy, as previously described (22). Subsequently, the pH gradient was calculated by subtracting the extracellular pH from the intracellular pH of each cell. BHIB was used as the basis medium, and a minimum of 47 individual cells in each experiment were analyzed.

Determination of intracellular buffer capacity.
The intracellular buffer capacity was determined as previously described (22). Briefly, cells were harvested from the late exponential growth phase and washed twice with 0.15 M KCl (pH 6.0). Subsequently, cell suspensions were divided into 15-mg cell dry weight (CDW) aliquots, and each aliquot was resuspended in 2 ml KCl-ethanol (pH 6.0) and 2 ml 0.15 M KCl (pH 6.0) for permeabilization and nonpermeabilization, respectively, and incubated for 15 min at 30°C. After incubation, the pH values of the permeabilized- and nonpermeabilized-cell suspensions were measured before and after the addition of an acid pulse (50 µl of 20 mM HCl) by using a PHM 95 pH electrode (Radiometer). The pH of a nonpermeabilized-cell suspension was adjusted to match that of its corresponding permeabilized-cell suspension before the addition of the acid pulse. The total and extracellular buffer capacities were calculated as the [H+] pulse added to the suspensions of permeabilized and nonpermeabilized cells, respectively, divided by the observed [H+] difference (calculated from the pH values before and after the acid pulse) per mg CDW. The intracellular buffer capacity was calculated by subtracting the extracellular buffer capacity from the total buffer capacity.

Determination of plasma membrane ATPase activity.
The specific activity of plasma membrane ATPase was expressed in nmol of Pi released min–1 mg protein–1 and determined in crude membrane suspensions basically as previously described (31, 33), but with the following modifications. Membrane suspensions were washed twice with suspension buffer before storage at –80°C to avoid the interference of sugars with the protein detection method. Furthermore, preliminary experiments showed a pH optimum of 7.0 for the plasma membrane ATPase of both yeasts (data not shown). Thus, the assays were conducted at this pH value. Moreover, assays were conducted for 30 min at 30°C. Finally, results from orthovanadate (final concentration of 2 mM) inhibition assays showed that under these conditions, 85% (or more) of the ATPase activity in C. zeylanoides could be attributed to the plasma membrane ATPase, whereas for D. hansenii, this figure was only 50% (or less) (data not shown). We therefore decided to perform an assay with and without orthovanadate concurrently on each sample, and we calculated the plasma membrane ATPase activity by subtracting the value obtained in the orthovanadate inhibition assay from the value obtained in the assay without orthovanadate.

Determination of nitrite in the culture broth.
The concentration of nitrite in the culture broth was determined using the Griess modified reagent (G-4410; Sigma). Samples were mixed with an equal volume of Griess modified reagent and allowed to react for 15 min at room temperature, and the absorbance was read at 540 nm using a microtiter reader (Labsystems Multiskan MCC/340). Nitrite was quantified using a standard curve prepared from NaNO2 salt.

Statistical analysis of data.
Statistical analysis of the buffer capacity and the plasma membrane ATPase activity data was performed using the two-sample t test, assuming unequal variances and considering both sides of the distribution (two-tailed distribution). Probabilities of less than 0.05 were considered significant.


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RESULTS
 
Effect of nitrite on cell growth.
During the experiments, crowding of cells on the glass surface occurred, limiting the analysis to an average CAI increase of approximately 350% (Fig. 1). Thus, our experimental setup detected only the growth initiation of the two yeast species.


Figure 1
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  		FIG. 1. CAIs of Debaryomyces hansenii (A) and Candida zeylanoides (B) during perfusion with BHIB at pHex 4.5 (circles) and 5.5 (triangles) containing 0 µg/ml (open symbols) and 200 µg/ml (filled symbols) nitrite. Perfusion was initiated at t = 0. Cell areas at t = 0 are set to 100%. Values are means of results for at least 47 cells, and vertical bars represent standard errors of the means.

At pHex 5.5, the growth initiation of the D. hansenii cells perfused with BHIB containing 200 µg/ml of nitrite was only scarcely affected compared with that of the control and reached a CAI of approximately 330% after 330 min (Fig. 1A). At pHex 4.5, however, in the BHIB with nitrite, the D. hansenii cells did not initiate growth, and the CAI was still 100% after 360 min of perfusion (Fig. 1A).

The growth initiation of C. zeylanoides cells in BHIB without nitrite was very slightly delayed by decreasing the pHex from 5.5 to 4.5 (Fig. 1B). However, in BHIB with nitrite, C. zeylanoides cells were not able to initiate growth, either at pHex 4.5 or at pHex 5.5, and CAIs of 100% were observed after 360 min of perfusion (Fig. 1B).

Nitrite consumption.
It is well known that D. hansenii is able to utilize nitrite as a nitrogen source (34). However, under the conditions used in this study, i.e., during 360 min of incubation in BHIB with 200 µg/ml of nitrite at pHex 4.5 and 5.5, our D. hansenii strain did not consume any nitrite (data not shown). Similar results were found for our C. zeylanoides strain (data not shown). It should be stressed that these results do not exclude the possibility that our yeast strains could use nitrite as a nitrogen source under other conditions.

Effect of nitrite on pHi.
The pHi values of D. hansenii and C. zeylanoides (Fig. 2) were measured in the same cells used for growth determination (Fig. 1). Cells without an intact plasma membrane pH gradient, i.e., if subtracting the pHex from the pHi yielded a value of <1.0, were considered dead (21), and data from these cells were not included in Fig. 1 and 2. It was possible to determine the pHi values of growing cells of both yeast species only during the first 60 min of perfusion (Fig. 2) due to the loss of fluorescent signal caused by the yeast growth and dilution of the fluorescent probe. In nongrowing cells, however, it was possible to follow the pHi throughout the full duration of the experiments (i.e., 360 min).


Figure 2
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  		FIG. 2. pHi values of Debaryomyces hansenii (A) and Candida zeylanoides (B) during perfusion with BHIB at pHex 4.5 (circles) and 5.5 (triangles) containing 0 µg/ml (open symbols) and 200 µg/ml (filled symbols) nitrite. Perfusion was initiated at t = 0. Values are means of results for at least 47 cells, and vertical bars represent standard errors of the means.

The pHi values of D. hansenii in BHIB without nitrite at pHex 4.5 and 5.5 were very similar and remained rather constant at 7.4 to 7.5 during 60 min of perfusion (Fig. 2A). This was also the case when nitrite was added to the medium at pHex 5.5 (Fig. 2A). However, when the D. hansenii cells were perfused with BHIB containing nitrite at pHex 4.5, a drastic decrease in pHi values from approximately 7.5 to 6.6 was observed within 1 min after the start of perfusion (Fig. 2A). After this initial decrease, the pHi remained constant at approximately 6.6 for the next 60 min (Fig. 2A), and even after 360 min of perfusion, the pHi was only approximately 6.5 (data not shown).

The pHi values of C. zeylanoides in BHIB without nitrite at pHex 4.5 and 5.5 remained constant within the range of 7.2 to 7.3 (Fig. 2B) and, thus, were slightly lower than those of D. hansenii (Fig. 2A). Upon the addition of nitrite at pH 5.5, the pHi of C. zeylanoides decreased from 7.3 to a minimum of 6.9 after 5 min of perfusion, after which it slowly increased to 7.2 after 60 min of perfusion (Fig. 2B). Subsequently, it remained constant, being 7.3 after 360 min of perfusion (data not shown). When nitrite was added at pHex 4.5, the pHi decrease of C. zeylanoides was even more pronounced than that of D. hansenii, dropping from approximately 7.3 to 5.9 within the first 1 min of perfusion (Fig. 2B). Within the following 60 min, the C. zeylanoides cells were apparently able to slightly recover their pHi, reaching a level of 6.0 to 6.1 (Fig. 2B). Subsequently, the pHi remained rather constant, and a pHi of 6.2 was detected after 360 min of perfusion (data not shown).

Effect of nitrite on retention of pH gradient.
A very large proportion (i.e., ≥96%) of both the D. hansenii and C. zeylanoides cells were able to retain a pH gradient during the 360 min of perfusion (Table 1), despite the decrease in average pHi values caused by the addition of a high concentration of nitrite (Fig. 2). These results indicate that although 200 µg/ml nitrite totally inhibited the growth of C. zeylanoides at pHex 4.5 and 5.5, and D. hansenii at pHex 4.5, virtually none of the cells died within the duration of the experiments.


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  		TABLE 1. Proportions of D. hansenii and C. zeylanoides cells with intact pH gradients (gradients of ≥1.0) at different time intervals after the initiation of perfusiona

Effect of nitrite on plasma membrane ATPase activity.
The plasma membrane ATPase activities of D. hansenii, ranging from 41 to 122 nmol Pi min–1 mg protein–1, were significantly lower (P < 0.05; data not shown) than those of C. zeylanoides, ranging from 276 to 498 nmol Pi min–1 mg protein–1 (Table 2). Whereas the low plasma membrane ATPase activity of D. hansenii is consistent with previously reported results (18, 30), the plasma membrane ATPase activity of C. zeylanoides, to our knowledge, has never been reported before. Its level of activity, however, seems to resemble very much that of, e.g., S. cerevisiae under stressed conditions (33).


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  		TABLE 2. Plasma membrane ATPase activity of D. hansenii and C. zeylanoides after 60 min of incubationa

Regardless of the pHex, the plasma membrane ATPase activity of C. zeylanoides after 60 min of exposure to nitrite was approximately 1.7 to 1.8 times higher than that after 60 min without nitrite (Table 2). For D. hansenii, however, the plasma membrane ATPase activities at pH 5.5 did not differ significantly (P > 0.05), whereas at pH 4.5, the plasma membrane ATPase activity of the cells after 60 min of exposure to nitrite was approximately 2.4 times lower than that after 60 min without nitrite (Table 2).

Intracellular buffer capacity.
Usually, the buffer capacity of cells is expressed as the amount of acid pulse needed to titrate a cell suspension 1 pH unit down per mg CDW or protein (17). We, however, added one predetermined acid pulse in all experiments, measured the pH difference, and converted the difference to changes in proton concentration [H+]. Subsequently, we calculated the buffer capacity as [H+] added x {([H+] difference x mg CDW)–1} (Table 3). We found this method to be more robust than the existing methods, since no adjustments or equilibrations of cell suspension pH values were required before the experiments were performed.


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  		TABLE 3. Extracellular, intracellular, and total buffer capacities of D. hansenii and C. zeylanoides

Both the extracellular and total buffer capacities of D. hansenii were significantly higher (P < 0.05) than those of C. zeylanoides, leaving no significant differences (P > 0.05) between the intracellular buffer capacities of the two yeasts (Table 3).


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DISCUSSION
 
According to our results, 200 µg/ml of nitrite is not sufficient to kill the two yeast strains belonging to the species D. hansenii and C. zeylanoides used in this study within 6 h of incubation in BHIB at pHex 4.5 and 5.5 at 22°C. Within this period of time, however, 200 µg/ml of nitrite is sufficient to totally inhibit the growth of D. hansenii at pHex 4.5 and C. zeylanoides at pHex 4.5 and 5.5. In fact, shake flask growth experiments performed in our lab show that the yeast cells are unable to initiate growth within 48 h of incubation under these conditions (data not shown). These results strongly suggest that acidified nitrite will not prevent the growth of D. hansenii in meat products with pH values above 5.5, whereas it may prove to be an effective antifungal agent against C. zeylanoides in these products. It could be stated that our experimental conditions are not comparable to those occurring in meat at cooling temperatures. Additional experiments in our lab, however, show that 100 µg/ml nitrite inhibits growth of C. zeylanoides, but not of D. hansenii, in a heat-treated, sliced-meat sausage model (pH 6.3, 3.2% NaCl aqueous phase and 100 µg/ml nitrite added) packed in a modified atmosphere (0.5% O2, 20% CO2, 79.5% N2) and stored at 8°C (data not shown). These findings indicate that our results obtained in BHIB at 22°C can, in fact, be used to predict yeast growth in processed meat at cooling temperatures. Furthermore, it should be stressed that since the salt tolerance of yeasts has proven to be highly strain dependent (22), tolerance toward acidified nitrite may very well also be strain dependent. This issue, however, remains to be further investigated.

The absolute pHi values (i.e., 7.4 to 7.5) of the nonstressed D. hansenii cells used in this study are comparable to the previously reported results for other D. hansenii strains using a similar experimental setup (22). The absolute pHi values of C. zeylanoides (i.e., 7.2 to 7.3) are, to our knowledge, the first of their kind, and they indicate that the two yeast species maintain rather similar pHi values under nonstressed conditions.

Our results confirm earlier findings for clinical yeasts showing that the antifungal effect of nitrite is increased by external acidification (1, 3, 37). To our knowledge, however, this is the first paper reporting that nitrite causes an intracellular acidification of yeasts. The fact that nitrite causes pronounced growth inhibition and intracellular acidification of D. hansenii at pHex 4.5 but does not at pHex 5.5 strongly indicates that nitrous acid as such plays a crucial role in the antifungal effect of acidified nitrite. Moreover, since both yeasts experience severe growth inhibition and pHi decrease at pHex 4.5, at least some of the antifungal effect of acidified nitrite may be attributed to intracellular acidification.

For C. zeylanoides, the antifungal effect of acidified nitrite due to intracellular acidification may, in part, be explained by the uncoupling of energy generation from growth, as previously described for weak organic acids (4). Since, however, the growth of C. zeylanoides is inhibited to similar degrees by nitrite at pHex 4.5 and 5.5, whereas the intracellular acidification caused by acidified nitrite is much lower at pHex 5.5 than at pHex 4.5, the uncoupling seemingly occurs when the amount of energy for maintenance purposes has exceeded a certain minimum value. This hypothesis is supported by the fact that the plasma membrane ATPase activities of C. zeylanoides are increased by acidified nitrite to similar degrees at the two pHex values. However, it should be stressed that our results do not exclude that the toxic effects of the other acidified nitrite derivatives, i.e., nitrogen oxides and the anionic form of nitrous acid, also contribute to the observed growth inhibition of C. zeylanoides.

For D. hansenii, the growth inhibitory effect of intracellular acidification cannot be explained by the uncoupling mechanism mentioned above, since its plasma membrane ATPase activity is not increased by acidified nitrite. Interestingly, at pHex 4.5, acidified nitrite causes a decrease in the plasma membrane ATPase activity of D. hansenii. These results suggest that other mechanisms, such as those caused by the anionic form of nitrous acid in S. cerevisiae (13-15) and/or by nitrogen oxides (5, 16, 19, 20), may be those primarily responsible for the growth inhibition of D. hansenii. This phenomenon, however, requires further investigation.

Our results clearly show that D. hansenii is more tolerant than C. zeylanoides to acidified nitrite stress at pHex 5.5. We have shown that this phenomenon is not caused by the different abilities of the two yeast species to utilize nitrite as a nitrogen source for growth under these conditions. In fact, within the duration of our experiments (i.e., 360 min), neither of the two yeast species consumed any nitrite. Instead, our results may be explained by the fact that D. hansenii is able to maintain pHi homeostasis at pHex 5.5, whereas C. zeylanoides is not. This hypothesis is sustained by previous observations in the literature showing that pHi homeostasis is important for the proper function of yeast metabolism (25) and that lag phases are prolonged for yeast cells unable to maintain pHi homeostasis (11). Thus, our results strongly suggest that the ability of D. hansenii to maintain pHi homeostasis plays an important role in its ability to tolerate acidified nitrite stress.

One potential mechanism to explain why the D. hansenii cells are able to maintain pHi homeostasis during acidified nitrite stress at pHex 5.5 could be that they have a higher plasma membrane ATPase activity than C. zeylanoides. However, as described above, this does not seem to be the case. Another possible mechanism could be the intracellular buffer capacity, which has been suggested to increase and stabilize the pHi in yeast cells exposed to weak organic-acid stress (32). It may be suggested that the D. hansenii cells have a higher buffer capacity than the C. zeylanoides cells, but our results show that the intracellular buffer capacities of the two species are virtually similar. A third mechanism could be that the plasma membrane compositions of the two yeast species are regulated differently, thereby rendering the plasma membrane of D. hansenii more rigid and less permeable for nitrous acid. This issue, however, remains to be elucidated.

In conclusion, our results clearly show that acidified nitrite causes an intracellular acidification of yeasts. Furthermore, they strongly indicate that the antifungal effect of acidified nitrite is caused at least partly by intracellular acidification. Finally, they suggest that the ability to maintain pHi homeostasis plays a role in the tolerance of D. hansenii and C. zeylanoides to acidified nitrite. This knowledge provides a basis for focusing the use of nitrite as an antifungal agent in the meat processing industry.


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ACKNOWLEDGMENTS
 
This research was financially supported by the Danish Directory for Food, Fishery and Agribusiness (grant 3414-05-01333) and the Danish Meat Association.

The excellent technical assistance of Søs Nielsen is highly appreciated.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. Phone: 45 35333266. Fax: 45 35333214. E-mail: na{at}life.ku.dk Back

{triangledown} Published ahead of print on 6 June 2008. Back


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Applied and Environmental Microbiology, August 2008, p. 4835-4840, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00571-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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