INTRODUCTION

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Research into the application of high-pressure (HP) processing for food preservation began more than a century ago, when Hite (20) demonstrated that the shelf life of milk, fruit, and vegetables could be extended by HP treatment (20, 21). In contrast to thermal processing, the application of HP to food does not affect its taste, color, or flavor (18, 22). The pressures required to achieve microbial inactivation are high, usually in the range of 300 to 700 MPa, but as the pressure applied is uniform in all directions, the treated product remains intact. Consequently, products such as cheese can be treated with minimal effect on texture and consistency. The first commercial HP-treated products appeared on the market in 1991 in Japan (24), where HP processing is now used commercially for products such as jams, sauces, fruit juices, rice cakes, and desserts. Progress in the design of HP equipment has facilitated commercial semicontinuous HP processing of liquid and solid foods (10).

The mechanisms by which HP-induced inactivation of microorganisms takes place have not yet been fully elucidated, but HP is known to cause morphological, biochemical, and genetic alterations in vegetative microorganisms (9, 22). Generally, yeasts and molds are most sensitive to HP, gram-positive microorganisms are most resistant, possibly due to their cell wall structure, and gram-negative microorganisms are moderately sensitive (9). Bacterial spores are extremely resistant to HP and can survive pressures in excess of 1,000 MPa (9). The stage of growth of microorganisms is also important in determining sensitivity to HP, as log-phase cells are more sensitive than stationary-phase cells (9). Important parameters affecting HP inactivation of microorganisms in food include the constituents and physical conditions within the food, which may induce a protective effect against HP (8). Therefore, a direct extrapolation of data for microbial inactivation by HP obtained with buffer or physiological solutions to predict levels of inactivation in foodstuffs may give misleading results.

HP treatment of microorganisms may result in either no loss of viability, sublethal injury, or cell death, although the last may or may not be accompanied by cell lysis. Sublethal injury may occur at pressures lower than those at which cell death occurs (29). After treating E. coli in phosphate buffer at 270 MPa, Hauben et al. (17) found that 99.58% of the surviving population was comprised of injured cells.

The present study defines the effects of a range of pressures and temperatures on the viability of selected contaminants of Cheddar cheese and demonstrates the potential application of HP for controlling undesirable microorganisms in this product.

	MATERIALS AND METHODS

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Stock cultures. The cultures used in this study were Escherichia coli K-12 (ATCC 29425; American Type Culture Collection, Rockville, Md.), Staphylococcus aureus ATCC 6538 (American Type Culture Collection), and Penicillium roqueforti IMI 297987 (International Mycological Institute, Egham, Surrey, England). These cultures were selected because they are well-defined typed strains which have been used in many previous laboratory investigations, including HP studies (17, 26).

Preparation of cultures for addition to cheese curd. Tryptone yeast phosphate broth was inoculated with E. coli K-12 and incubated at 30°C overnight. S. aureus ATCC 6538 was grown in Trypticase soy broth (Becton Dickinson and Co.) containing 0.6% yeast extract at 37°C overnight. Both cultures (3 ml) were centrifuged at 20,800 × g for 1 min, the supernatants were discarded, and the pellets were washed with maximum-recovery diluent (MRD; Oxoid). The washed pellets were resuspended in 2 ml of MRD. Four milliliters of the P. roqueforti spore suspension was centrifuged at 20,800 × g, and the pellet was resuspended in 4 ml of MRD.

Preparation of Cheddar cheese slurries. Cheddar cheese curd chips prior to being salted were obtained from Wexford Creamery (Wexford, Ireland) and stored at 20°C until required. Starter bacteria, lactobacilli, and coliforms were enumerated as described by Ryan et al. (30) in the cheese curd chips. S. aureus organisms were enumerated on Baird Parker medium (Merck) plus egg yolk tellurite (Merck) after incubation at 37°C for 48 h, E. coli organisms were enumerated on violet red bile agar (VRBA) after incubation at 30°C for 24 h, and yeasts and molds were enumerated on yeast extract-glucose-chloramphenicol (Merck) agar after incubation at 25°C for 5 days.

HP treatment of inoculated Cheddar cheese slurries. Inoculated Cheddar cheese slurry samples were pressurized in an HP rig (Stansted Fluid Power Ltd., Stansted, England), which has a 300-mm-deep chamber with a diameter of 37 mm. The pressurization medium used was 15% (vol/vol) castor oil in ethanol. Pressurization was carried out at pressures of 50, 100, 200, 300, 400, 500, 600, 700, and 800 MPa for 20 min at temperatures of 10, 20, and 30°C on two independent replicate slurry samples. Duplicate unpressurized control slurry samples were maintained at ambient temperature, as there would be little difference in the levels of growth detected following the short incubation time (20 min).

Microbiological assay of the pressurized product. Immediately following decompression, serial dilutions of the cheese slurry samples were prepared. E. coli K-12, S. aureus ATCC 6538, and P. roqueforti IMI 297987 organisms were enumerated as described above. The species-selective media used were determined to be suitable for enumerating individual species in the mixed microbial population and were effective in suppressing the background microflora present in the cheese curd chips. Each replicate cheese slurry sample was plated in duplicate.

Kinetic experiments to determine microbial inactivation by HP. The decimal reduction time (D value) is the time required at a specified pressure to reduce the number of cells by 90%, and the Z value is the increase in pressure (in megapascals) needed to change the D value by 90%.

Strain-to-strain variation in HP sensitivity. Variation in strain sensitivity to HP was examined by subjecting a range of strains of S. aureus, E. coli, and mold spores to HP treatment at 100 to 400 MPa in cheese slurries for 20 min at 20°C as described above. The extra strains of S. aureus and E. coli tested were isolated from commercial-smear-ripened cheeses, and the species were confirmed. Mold strains were isolated either from a commercial-smear-ripened cheese or from Cheddar cheese made at pilot scale. These molds were not confirmed as being P. roqueforti but were typical mold contaminants of cheese.

Effect of HP treatment on log-phase cells. As the inactivation experiments described above used stationary-phase cells, log-phase cells of E. coli and S. aureus were also added to cheese slurries to compare inactivation rates of log- and stationary-phase cells. The cultures were grown to log phase, as determined by optical density measurement, inoculated into the slurries, and HP treated at 100 to 400 MPa for 20 min at 20°C as described previously.

Detection of sublethal injury. In order to determine the level of sublethal injury, a differential plating technique using nonselective agar incorporating salt or glucose was employed. These are injury-selective media, as opposed to the species-selective media described above. Tryticase soy agar yeast extract (TSAYE) and TSAYE with salt incorporated were used for detection of unstressed and stressed E. coli (5% salt) and S. aureus (7% salt) (23). Yeast glucose [YG; yeast extract at 5.0 g/liter and D-(+)-glucose at 20.0 g/liter] agar and YG agar with the D-(+)-glucose level increased to 30% (2) were used to elucidate the stress response in P. roqueforti. Both noninjured and injured cells were able to form colonies on TSAYE and YG agar, whereas only noninjured cells formed colonies in the presence of salt or glucose. To enable the use of nonselective media, the slurries were pasteurized at 63.5°C for 30 min prior to the addition of the individual cultures to inactivate residual starter microflora, whose growth was suppressed on the species-selective media. In these experiments, individual addition of the microorganisms to the slurry was necessitated by the use of the nonselective differential plating media used for injury detection. Two independent replicate slurry samples of E. coli and P. roqueforti were HP treated (20°C for 20 min) at 300, 400, and 500 MPa, and those containing S. aureus were treated at 400, 500, 600, and 700 MPa. Following HP treatment, slurries were incubated at 8°C, at which temperature growth did not occur, and any increase in cell numbers could thus be attributed to recovery from sublethal injury. Following incubation at 8°C for 0, 24, and 72 h, HP-treated and control slurries were sampled and the numbers of injured cells which recovered were determined by using the nonselective differential plating technique. Samples were also plated on the species-selective media used previously to determine the influence of these media on the growth of sublethally injured cells.

Manufacture of cheese. Miniature cheeses were produced by using a modification of the protocol of Shakeel-Ur-Rehman et al. (32). Pasteurized milk (800 ml) was added to six plastic centrifuge bottles (1 liter) and cooled to 31°C. The starter culture (1% inoculum of Lactococcus lactis subsp. cremoris 303) and the three microbial species under study were then added to the milk, which was ripened for 30 min. Following renneting and coagulum formation, the curd was cut and stirred. Curds and whey were then centrifuged at room temperature for 60 min at 5,442 × g in a Mistral 6,000 centrifuge (MSE Scientific Instruments, Sussex, England). The whey was drained, and the curds were held in the centrifuge bottles at 36°C until the pH decreased to 5.2 to 5.3, whereupon cheeses were inverted and recentrifuged at 5,442 × g for 20 min at room temperature. The remaining whey was then drained, and cheeses were brine salted (20% NaCl, 0.05% CaCl₂ · 2H₂O) for 100 min at room temperature, vacuum packed, and stored at 8°C.

Microbial inactivation in buffer systems. The test microorganisms were suspended in 0.06 M phosphate buffer (pH 5.3), and two independent replicates were treated at 100 to 500 MPa for 20 min at 20°C in order to compare inactivation ratios (the log of the number of cells detected following pressurization treatment to the total number of CFU in the control) with those obtained in the model cheese slurry system and in the miniature cheese.

	RESULTS

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Inactivation experiments with cheese slurry. Analyses of the background microflora of the Cheddar cheese curd chips showed that starter bacteria and nonstarter lactic acid bacteria were present at 1.14 × 10⁹ and 2.90 × 10² CFU/g, respectively, while yeasts, molds, coliforms, and S. aureus were not detected.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Inactivation of S. aureus ATCC 6538 (A), E. coli K-12 (B), and P. roqueforti IMI 297987 (C) in Cheddar cheese slurry by HP treatment at 10°C (), 20°C (), and 30°C () for 20 min (No is the total number of CFU in the control, and N is the number of cells detected following pressurization treatments). Each curve shows the mean values obtained from results from two independent replicates, and error bars represent standard deviations.

Sublethal injury and recovery of microorganisms after HP treatment. Sublethal injury and recovery of S. aureus ATCC 6538 (Fig. 2A to C), E. coli K-12 (Fig. 2D to F), and P. roqueforti IMI 297989 (data not shown) after HP treatment at 20°C were investigated using injury-selective media. For control S. aureus samples, to which no pressure was applied, no difference was evident between growth on nonselective and growth on selective media (Fig. 2A). At 400 MPa, a >1-log-unit cycle of kill (indicated by the difference in levels obtained in Fig. 2A and B) and a further 1-log-unit cycle of injury (indicated by the difference in counts obtained on TSAYE and TSAYE plus salt) occurred, and slow recovery of injured cells was seen following incubation for 72 h (Fig. 2B). At 500 MPa there was a 3- to 4-log-unit cycle of kill, accompanied by 3-log-unit cycles of injury, with complete recovery following 72 h of incubation at 8°C (Fig. 2C). At 600 MPa there was a 7-log-unit cycle of kill on TSAYE, from which S. aureus did not recover following 72 h of incubation at 8°C, and on Baird Parker agar there was a 6-log-unit cycle of kill and a 1-log-unit cycle of injury, from which the organism did not recover after 72 h at 8°C (not shown). At 700 MPa the entire culture was killed, as no recovery was evident following incubation at 8°C (not shown). These results suggest that Baird Parker medium supported growth of noninjured and injured cells equally well; thus, all inactivation results obtained in the model cheese slurry system for S. aureus plated on this medium reflect actual kill.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Sublethal injury in Cheddar cheese slurry of S. aureus ATCC 6538 without pressurization (control) (A), at 400 MPa (B), and at 500 MPa (C) plated on Baird Parker agar (), TSAYE (), and TSAYE plus 7% salt () and of E. coli K-12 without pressurization (D), at 300 MPa (E), and at 400 MPa (F) plated on VRBA (), TSAYE (), and TSAYE plus 5% salt () (pressure treatments were carried out for 20 min at 20°C). Each curve shows the mean values obtained from results from two independent replicates, and error bars represent standard deviations.

Kinetics of inactivation. The rates of inactivation of the three test organisms at 20°C in Cheddar cheese slurry were studied (Table 1). S. aureus exhibited a linear rate of inactivation at the three pressures tested. Increasing the pressure from 300 to 400 MPa had a greater effect on inactivation than extending the pressurization time (not shown in Table 1), with the D values decreasing from 38 to 20 min over this pressure range. With the mold species, results obtained at the lower pressure (300 MPa) were very similar to those obtained with S. aureus, in that little effect of increasing pressurization time was evident (not shown), and a D value of 56 min was obtained at this pressure. However, at 350 and 400 MPa the effects of extending the pressurization time became significant and even greater in magnitude than were observed for S. aureus. The D value was reduced to 5 min after pressurization of P. roqueforti at 400 MPa. With E. coli at 300 and 350 MPa, the D values were 22 and 19 min, respectively, although the D value could not be calculated at 400 MPa as the inactivation curve was not linear at this pressure.

Influence of strain variation on inactivation by HP treatment. Pressure treatment at 100 to 200 MPa caused negligible inactivation of any of the three strains of S. aureus, E. coli, or mold species studied (data not shown). Inactivation increased for all strains at 300 MPa, where the greatest degree of interstrain variation was seen for the mold species (2-log-unit cycle difference between strains). However, as the viability of the mold spores was greatly reduced at 400 MPa, the degree of strain variation at this pressure was small (<1-log-unit cycle). The range of pressure sensitivity within strains of S. aureus and E. coli increased to a 2-log-unit cycle difference at 400 MPa.

Effect of HP treatment on log-phase and stationary-phase cells. Both S. aureus ATCC 6538 and E. coli K-12 cells in the log phase of growth were more sensitive to HP treatment in Cheddar cheese slurry than they were in stationary phase (Fig. 3). Log-phase S. aureus cells were more resistant than log-phase E. coli cells.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Inactivation of stationary-phase S. aureus ATCC 6538 () and E. coli K-12 () cells and log-phase S. aureus ATCC 6538 () and E. coli K-12 () cells in Cheddar cheese slurry by HP treatment at 20°C for 20 min (No is the total number of CFU in the control, and N is the number of cells detected following pressurization treatments). Each curve shows the mean values obtained from results from two independent replicates, and error bars represent standard deviations.

Inactivation of microorganisms in cheese by HP treatment. Relative sensitivities of the three microbial species to HP in the miniature cheeses assayed immediately after pressurization were as found for the cheese slurry system, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus (Fig. 4). However, there were substantial differences between media, particularly with E. coli; e.g., organisms in cheese were much more sensitive to HP than they were in the cheese slurry system (Fig. 4B). The same trend was evident for S. aureus and P. roqueforti, although the effect was less dramatic.

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Inactivation of S. aureus ATCC 6538 (A), E. coli K-12 (B), and P. roqueforti IMI 297987 (C) in pH 5.3 buffer (), Cheddar cheese slurry (), and Cheddar cheese () by HP treatment at 20°C for 20 min (No is the total number of CFU in the control, and N is the number of cells detected following pressurization treatments). Each curve shows the mean values obtained from results of at least two independent experiments, and error bars represent standard deviations.

	DISCUSSION

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The purpose of this study was to determine the effect of HP treatment on microbial contaminants in Cheddar cheese and to provide information, not previously available, on the influence of the Cheddar cheese environment on HP inactivation.

HP inactivation was studied in three different systems: cheese slurry, Cheddar cheese, and a buffer system. Cheese slurries are recognized as an acceptable experimental model for cheese conditions (12, 13) and were used to develop a matrix relating microbial inactivation to conditions of pressure and temperature. The data from the matrix were subsequently selectively applied to laboratory-manufactured Cheddar cheese deliberately contaminated with the microorganisms of interest. Inactivation in buffer was studied in order to elucidate the degree of protection provided by the cheese against HP inactivation.

The greater sensitivity of the microorganisms in the Cheddar cheese than in the cheese slurry system may be explained by acid injury to the bacteria during fermentation. Also, some cells may have been in the log phase of growth, as overnight cultures were added directly to the milk, which underwent a 5-h cheese-making process. This circumstance may have resulted in greater inactivation, as the HP sensitivity of log-phase cells is greater than that of stationary-phase cells (9), as demonstrated also in the present study (Fig. 3). It has been shown that water availability can markedly affect the response of microorganisms to HP (28, 31, 33), with microorganisms in dehydrated foods being very pressure resistant (9). It is likely that differences in water availability explain the greater sensitivity of microorganisms to HP inactivation in the buffer system than in cheese.

The importance of the interaction between pressure and temperature (even over the range of moderate temperatures used here) in determining the degree of inactivation of microbial species was evident from the results obtained in the cheese slurry experiment. Adjustment of temperature can significantly affect the pressure necessary to achieve inactivation. Results obtained by others (6, 7, 34, 35) working over a greater range of temperatures indicate an even greater response to pressure-temperature interactions. However, temperatures higher than those used in this study cannot be realistically applied in a commercial Cheddar cheese process.

Yeasts and mold spores have been previously shown to be readily inactivated at 400 MPa (33), while Ogawa et al. (27) reported a >5-log-unit reduction when nine species of yeasts and molds in fruit juice were treated at 350 MPa for 30 min or 400 MPa for 5 min. These results are in agreement with results obtained in this study for P. roqueforti in cheese slurry at temperatures of 20 and 30°C. The pattern of inactivation by HP observed for the mold spores was different from that obtained with the bacterial species. However, it has been demonstrated that eucaryotic microorganisms are generally more sensitive to pressure than procaryotic microorganisms (22). Inactivation of molds was not evident until a pressure of 300 MPa was reached, after which cell numbers decreased rapidly.

Sublethal injury experiments indicated that, at the higher pressures, the injured population was more severely damaged and took longer to recover. Hauben et al. (17) also found that increasing the pressure from 180 to 270 MPa resulted in an increased percentage of sublethally injured cells. Those authors studied the effect of 320 MPa for 15 min at room temperature on E. coli K-12 in buffer at pH 7 and found approximately a 4-log-unit cycle reduction in numbers, of which 2 log units of organisms were sublethally injured.

Previous studies of HP inactivation of microorganisms have reported either a first-order kinetic reaction or a sigmoidal response, indicating the presence of a pressure-resistant subpopulation (11, 19, 22, 33). Generally, S. aureus displays first-order inactivation kinetics (5, 14), as found in this study with Cheddar cheese slurries. E. coli is reported to demonstrate either first-order inactivation kinetics (25, 34) or a two-phase tailing effect (1, 9), depending on the strain and treatment conditions, both of which effects were observed for E. coli in the present study.

Strain-to-strain variation is a significant problem which arises when the extent of HP inactivation is being defined for a particular species. This study demonstrated a strain response to pressure, with values varying by ~2-log-unit cycles for both the mold and bacterial species. Other studies have found greater degrees of variation. Benito et al. (1) found a 5-log-unit cycle difference in pressure sensitivity between stationary-phase cells of strains of E. coli O157 treated at 500 MPa for 20 min in pH 7.0 buffer. Food isolates are generally reported to be more pressure resistant than strains from culture collections (9). In this study the food isolates of S. aureus were more pressure resistant than S. aureus ATCC 6538 pressure treated at 400 MPa at 20°C for 20 min. However, E. coli K-12 and P. roqueforti IMI 297987 spores were more pressure resistant than the food isolates under the conditions studied.

The results from this study underline the importance of taking the suspending medium and the effects of processing into account when assessing the impact of HP on the viability of microorganisms. Application of HP substantially reduces the microbial load in Cheddar cheese, with 400 MPa for 20 min at 20°C being sufficient to reduce the numbers of viable E. coli and P. roqueforti by 7- and 6-log-unit cycles, respectively, and to reduce the levels of S. aureus by 3-log-unit cycles. The technology could be applied to control post pasteurization contaminants in cheese where heat treatment cannot be used. However, the HPs required to inactivate microorganisms combined with the high cost of HP technology are factors currently limiting the use of HP processing in industrial applications. Nevertheless, there is increasing interest in combining HP with other technologies, such as the use of bacteriocins as demonstrated by Morgan et al. (26), which may lead to microbial inactivation at reduced pressures. These results thus favor the development of commercial processes for HP treatment of dairy products such as cheese.