Effect of lysozyme or nisin on survival of some bacteria treated with high pressure at subzero temperature

INDUSTRIAL MICROBIOLOGY

Effect of lysozyme or nisin on survival of some bacteria treated with high pressure at subzero temperature

Edyta Malinowska-Pańczyk; Ilona Kołodziejska^*

Department of Food Chemistry, Technology and Biotechnology, Chemical Faculty, Gdansk University of Technology G. Narutowicza 11/12, 80-952 Gdańsk, Poland

ABSTRACT

The aim of this work was to examine the inactivation of some Gram-positive and Gram-negative bacteria exposed to the pressure of 193 MPa at –20 °C in the presence of lysozyme or nisin at concentration of 400 mg/ml. The highest effect of pressure at subzero temperature and lysozyme was found with pressure sensitive Pseudomonas fluorescens; viable cells of this strain were not detected in 1 ml of sample after combined treatment. The action of pressure at subzero temperature and lysozyme or nisin against Escherichia coli led to synergistic reduction by 0.7 or 1.6 log cycles, respectively, while it was practically insignificant for two Staphylococcus aureus strains. Viability loss of E. coli and S. aureus occurred during storage for 20 h of the samples at 37 and 5 °C, which were previously pressurized with lysozyme or nisin. The synergistic effect of pressure and nisin at pH 5 against E. coli cells just after the pressure treatment was lower than that at pH 7, however, the extent of the lethal effect after storage was higher.

Key words: Inactivation of microorganisms, High pressure, Subzero temperature, Lysozyme, Nisin

Introduction

High-pressure technique is commercially used for preservation of, mainly, acid food products. However, food with pH close to neutral is a better medium for growth of microorganisms that survived the pressure treatment. Moreover, frequently in the pressure treated population of vegetative bacteria survivor tails can occur (35). It is known that Gram-negative bacteria and cells in the exponential growth phase are more sensitive to pressure treatment than Gram-positive bacteria and cells being in the stationary phase of growth (3, 35). Moreover, species very resistant to high pressure exist even among Gram-negative bacteria. Variations in pressure resistance were also observed among strains belonging to the same species of food-born pathogens and spoilage bacteria (1, 21, 28, 29). It should be also take into consideration that barotolerant mutants can be formed in the pressure process (14). Furthermore, microorganisms are usually more resistant to pressure treatment in food systems than in a buffer (4, 8, 29, 34). For these reasons pressure above the moderate range should be used in cold pasteurization of food. This could limit the practical applications of this technique due to changes in some components of food, especially proteins and because of the higher operating costs with the increase of pressure dose. One of the possibilities for achieving the increase in inactivation of bacteria in low-acidity foods is using pressure in combination with antimicrobial factors. Lysozyme, lactoperoxidase, lactoferrin, lactoferricin, nisin and pediocin are known to enhance the effectiveness of high pressure in inactivation of some bacteria (8, 9, 10, 17, 18, 19, 20, 22, 24, 27). The synergistic effect is a result of sublethal injury of cells that makes them more sensitive to antimicrobial substances (14, 17, 18, 20). Moreover, the high pressure induces outer membrane permeabilization in Gram-negative bacteria. It sensitizes the cells to action of bacteriocins and lysozyme (14, 22, 23, 27). The temperatures used in experiments conducted by the cited authors were in the range of 20-50 °C. However, Steeg et al. (36) observed the enhanced synergistic inactivation of some bacteria when high pressure in the presence of nisin was used at temperature 10 °C. This effect of nisin can probably deepen during further reduction of temperature. Moreover, the limited available data showed higher level of inactivation of microorganisms under high pressure at subzero temperature than in a certain range of temperature above 0 °C (12, 15, 26, 31, 32, 33).

Therefore, it is not excluded that such conditions may enhance the synergistic effect of combined action of high pressure and nisin against microorganisms. Furthermore, it would be worth also to check if lysozyme exerts any antimicrobial activity under pressure at such low temperature as –20 °C. It is known that this polypeptide can act on bacteria according to lytic and non-lytic mechanisms, depending on their type and the environmental conditions (6, 25). Therefore, the aim of this work was to examine the inactivation of some Gram-positive and Gram-negative bacteria exposed to pressure of about 193 MPa at –20 °C in unfrozen system in the presence of lysozyme and nisin followed by prolonged storage at 5 and 37 °C. Such data were not found in the available literature.

Materials and methods

Preparation of lysozyme and nisin solutions

Hen egg white lysozyme (69 480 U/mg proteins, Fluka, Switzerland) dissolved in potassium phosphate buffer (10 mM, pH 7.0) at concentration of 2 mg/ml and nisin (Fluka, Switzerland) dissolved in 0.01 M HCl at concentration of 2 mg/ml, prepared directly before experiments, were used.

Cultures and growth conditions

The following bacterial strains were used: Pseudomonas fluorescens WSRO121 from Collection of Dairy Cultures of Department of Microbiology, University of Warmia and Mazury, Olsztyn, Poland; Escherichia coli K-12 PCM2560 (NCTC10538) and Staphylococcus aureus PCM2054 (ATCC25923) from Polish Collection of Microorganisms, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, Wrocław, Poland; Staphylococcus aureus DSM2569 from German Collection of Microorganisms and Cell Cultures and Listeria innocua III₁ (isolated from fish) kindly provided by Department of Food Microbiology, University of Agriculture, Szczecin, Poland.

Inocula of strains were prepared by inoculating 100 ml of tripticase soy broth supplemented with 0.6% yeast extract (TSBYE; purchased from BTL Sp. z o.o., Łódź, Poland) with 100 μl of liquid culture (at stationary phase of growth) and incubating at 37 °C (E. coli, S. aureus, L. innocua) or 28 °C (P. fluorescens) for 24 h with shaking. Under these conditions the cells were in the stationary phase.

In order to obtain an appropriate growth phase of cells, 100 μl of the stationary-phase culture were inoculated into 100 ml of fresh TSBYE and incubated with shaking at optimal temperature of growth for particular microorganisms. The bacteria growth was measured by determination of optical density at 660 nm for 48 h. The middle exponential and stationary phase were determined from the obtained curves of growth.

Preparation of cell suspensions

The cells in a stationary or exponential phase of growth were centrifuged at 1300xg for 20 min at 5 °C, and the pellets were resuspended in phosphate-buffered saline at pH 7.0 or in some experiments in Mc Ilvaine's buffer at pH 5.0 to give viable counts of about 10⁸/10⁹ cfu/ml. Antimicrobials were added to the final concentration of 400 mg/ml (lysozyme) or 100 and 400 mg/ml (nisin).

Pressure treatment

The pressure was generated similarly as described by Hayakawa et al. (15). The procedure was based on the phenomenon that in a sealed vessel filled with water and kept at subzero temperatures, increasing volume of forming ice I generates internal pressure. According to Bridgman (2) high pressure reduces the freezing and melting points of water to a minimum of –22 °C at 207.5 MPa. Therefore, above this temperature, the sample placed in a sealed vessel is affected by the pressure in unfrozen state.

The equipment used to generate pressure during the experiments was designed and constructed by Edward Dunajski in our Department (Fig. 1).

Cell suspensions (3 ml of each) were placed in sterile glass test tubes (with volume of 3 ml), and sealed with a stopper. At this moment a small excess of liquid was pushed out from the tubes without leaving any bubbles of air inside. Due to the special design the stopper holds the tightness of the tube and it is able to move. This allows to expose the sample to the generated pressure.

The tube containing cell suspension and then a metal spring were placed in a cylindrical metal vessel filled with distilled water. The vessel was closed without leaving any bubbles of air inside. It was immersed in four stages during 40 min (4x10 min) with the closed side down, in a temperature-controlled bath containing a mixture of ethanol, propylene glycol and distilled water (1:1:1, v/v) as a coolant (Fig. 1). So initially, only the bottom part of the vessel was immersed in the cooling bath. The role of the spring was to keep the tube, at this moment and later, when the whole pressure vessel was in the cooling mixture, at upper -nonfrozen zone - of the pressure vessel. The time during which the assumed temperature was achieved has been determined in preliminary experiments using a thermocouple. As high pressure lowers the freezing point of water, thus the material should be in the unfrozen state up to about –20 °C. This fact confirmed the results of experiments in which hydrogels were kept at –20 °C in the pressure vessel and simultaneously at the same temperature under atmospheric pressure. The damage of structure of the latter hydrogel caused by water crystallization was visible. Such changes did not appear when hydrogel was cooled in the sealed vessel.

After pressure treatment at –20 °C the vessel was stuck out from cooling bath in half of its length and warmed in the upper part to 15 °C measured with a thermocouple. Then the vessel was taken out and placed for a few minutes in a water bath at 20 °C. The total time of decompression did not exceed 10 min. The samples were stored in an ice bath prior to determination of viable counts. Unpressurized samples were used as controls. In some experiments the samples were incubated for 2 or 20 h with or without lysozyme/nisin added after the pressure treatment.

Enumeration of viable cells

Pressure-treated samples and untreated control cell suspensions were serially diluted with buffered saline (pH 7.0). Dilutions were plated on tripticase soy agar supplemented with 0.6 % yeast extract (TSAYE; purchased from BTL Sp. z o.o., Łódź, Poland)) and plates were incubated for 48 h at 37 °C (E. coli, S. aureus, L. innocua) or 28 °C (P. fluorescens).

The data presented in the tables and figures are mean values obtained from three independent experiments. The bars on the figures indicate the mean standard deviations for the data points. The results were evaluated statistically by analysis of variance (one-way procedure) or Students t-test using the program Statgraphics, Statistical Graphic Corporation.

Results and Discussion

Antibacterial effect of high pressure and lysozyme

The pressure that can be achieved at –20 °C in the closed vessel amounts to about 193 MPa. The tested bacteria differed in sensitivity to pressure mainly in the exponential phase of growth and the most pressure resistant were S. aureus strains (Fig. 2). The number of viable cells from the stationary phase of growth decreased under these conditions no more than by one log cycle, with exception of P. fluorescens. The pressure reduction in the viability of this strain was about 4 log cycles. The large pressure sensitivity of P. fluorescens was also evidenced by Gervilla et al. (11). Therefore, in the next experiments only bacterial cells in the stationary phase of growth were used.

As expected, lysozyme at ambient pressure was not effective against Gram-negative bacteria: E. coli and P. fluorescens (Table 1). Moreover, the viability loss in the presence of lysozyme in the case of tested Gram-positive L. innocua and two strains of S. aureus did not exceed 0.5 log cycle. As reported by Pellegrini et al. (30) lysozyme at atmospheric pressure did not attack S. aureus ATCC 25923 and S. epidermidis ATCC 1228 cells.It is known that modification of the peptidoglycan of some Gram-positive bacteria results in resistance to its degradation by lysozyme (5).

When combined pressure 193 MPa at –20 °C and lysozyme were used the extra reduction of E. coli of 0.7 log cycle was obtained (Fig. 3). In samples pressurized in the presence of lysozyme the viability of E. coli cells did not decrease during incubation for 2 h at 37 °C (Fig. 3 B and C). Moreover, there were no significant differences in the number of viable cells in the pressurized samples without lysozyme and in the samples incubated for 2 h at 37 °C with lysozyme added after pressure treatment (Fig. 3 A and D). These data suggest that some of the observed antimicrobial impact of lysozyme did not take place after decompression but during pressurization at subzero temperature. Lysozyme probably does not show enzyme activity at –20 °C; thus a non-lytic mechanism of lysozyme action under these conditions could not be excluded. The most likely non-lytic mechanism of antimicrobial activity of lysozyme is based on disruption of bacterial membrane (6).

According to some authors (13, 22, 23, 24) the sensitization of Gram-negative bacteria to lysozyme or nisin under high pressure is a result of a transient permeabilization of the outer membrane. It means that bacteria show sensitivity to the antimicrobial substances only during pressurization. The results of the present study show also such an effect when the samples after pressure treatment were incubated for a short time, up to 2 h at 37 °C. During that time the level of bacteria inactivation did not increase (Fig 3). In the experiments of other authors with lysozyme added after pressure treatment the time of incubation did not exceed 180 min at 25 ºC (13, 25). However, data presented in Table 2 indicate that the reduction of E. coli cells took place when storage time of samples pressurized in the presence of lysozyme was elongated to 20 h. The number of viable cells of E. coli K-12 at 37 and 5 °C was, respectively, 1.9 or 2.7 log cycles lower than that for samples pressurized and stored in the absence of lysozyme. It was also found that although there was no significant reduction in the number viable cells after 24 h of storage at 5 °C of pressurized sample without lysozyme, sublethal injury of the cells was observed. The difference between the number of E. coli cells isolated from TSAYE and MacConkey selective medium increased from 0.1 log cycle (unstored pressurized sample) to about 3 log cycles after storage at 5 °C (unpublished data). Furthermore, analysis of the data presented in Table 2 suggests that all sublethal injured cells remain sensitive to lysozyme.

The synergistic effect of high pressure and lysozyme was not observed in experiments with L. innocua and two strains of S. aureus. During storage for 24 h at 5 and 37 °C the number of viable cells of S. aureus PCM2054 pressurized in the presence of lysozyme decreased but only additive effect was evidenced (Table 2).

The highest effect of pressure 193 MPa at –20 °C and lysozyme was found with pressure sensitive P. fluorescens; viable cells of this strain were not detected in 1 ml of sample after combined treatment. Moreover, the results of the experiment with lysozyme added after pressure treatment of bacteria and immediately plated on agar medium showed that the level of inactivation of the tested bacteria was the same as that for the sample pressurized without lysozyme (Fig 3D' and A). These results again confirmed that lysozyme exerted antimicrobial activity during pressure treatment at low temperature. In addition, during storage of such samples for 2 h at 37 °C further inactivation of bacteria was observed reaching the same level as in the case of bacteria pressurized in the presence of lysozyme. It indicates a large extent of sublethal damage of P. fluorescens cells.

Antibacterial effect of high pressure and nisin

Nisin at concentration of 100 mg/ml and 400 mg/ml, pressure 193 MPa at –20 °C used separately or combined did not exert any bactericidal effect on S. aureus PCM2054 cells just after pressure treatment. Otherwise, E. coli was more sensitive to combined action of high pressure and nisin (Table 3). Synergistic reduction of these bacteria after pressure treatment in samples containing nisin at concentration of 400 mg/ml amounted to 1.7 log cycles and was higher than in the case of lysozyme. According to Masschalck et al. (23) various compounds characterize different bactericidal spectra under pressure. However, in the present study less significant antimicrobial effect of high pressure at subzero temperature and lysozyme than pressure and nisin can result also from low enzymatic activity of lysozyme. Nisin working in different way than lysozyme can be more effective in inactivation of bacteria under these conditions.

The results obtained by Steeg et al. (36) showed that bactericidal effect of combined pressure and nisin increased when treatment was conducted at temperature below 15 °C. The synergistic reduction of E. coli NCTC 9001 treated with pressure of 200 MPa in the presence of nisin was above 3 log cycles at 10 °C while at 40 °C only 0.3 log cycle. The results of the present study show lower synergistic effect (Table 3), although temperature was lower than in the experiments carried out by Steeg et al. (36). These differences can result from higher sensitivity to high pressure of E. coli NCTC 9001 than of the strain E. coli NCTC 10538 used in our study. Masschalck et al. (23) reported that the sensitization of bacteria by high pressure to antimicrobial compounds varies even among strains belonging to the same species. Subsequently, the cell density in the present study was 10⁸/10⁹ cfu/ml while in that conducted by Stegg et al. (36) amounted to 10⁵ cfu/ml. As found Garcia-Graells et al (9) E. coli was more sensitive to antimicrobial compounds like lactoperoxidase system and high pressure at low cell density (10⁶ cfu/ml) than at high cell density (10⁹ cfu/ml).

Then, similarly as in experiments with lysozyme, there was the viability loss of E. coli and S. aureus during storage of the samples at 5 °C, which were previously pressurized with nisin; the numbers of viable cells were decreased by 0.4 (E. coli) and 1.2 log cycles (S. aureus) as compared with those just after pressure treatment. However, the inactivation of E. coli pressurized with nisin after 24 h storage was lower than that obtained in the case of lysozyme. On the other hand, taking into account higher antimicrobial effectiveness of nisin during pressurization than of lysozyme, the total reduction of E. coli was similar for both antimicrobial substances.

Gänzle et al. (7), reported that under ambient pressure the sensitivity of E. coli to nisin increased in acidic conditions. However, Steeg et al. (36) showed that antimicrobial effect of nisin and high pressure on E. coli NCTC 9001 cells was much lower in samples at reduced pH. According to the authors, the culture obtained at pH 7 was treated by pressure at pH 4.5 and the shock induced by pH drop probably reduced the transmembrane potential and in consequence the antimicrobial efficiency of nisin. In the present workthe antimicrobial effect of pressure and nisin at pH 5 against E. coli cells just after pressure treatment was also much lower than that at pH 7 (Table 3 and 4). However, the extent of the lethal effect during incubation of the sample after pressure treatment was higher. It can result from higher stability of nisin at acidic pH than at neutral pH (16, 25). In the case of S. aureus the higher synergistic effect of pressure and nisin was obtained at pH 5 as compared with that of pH 7 and differences deepened during storage of the sample after pressure treatment at 5 °C (Table 3 and 4). S. aureus, in comparison to E. coli,was also more sensitive to acidic conditions and nisin during storage under ambient pressure. It is known that enteric bacteria are tolerant towards adverse environmental conditions such as low pH or other factors (7) and Gram-negative bacteria are more resistant to nisin than Gram-positive ones (3).

Conclusions

The data suggest that lysozyme exerts antimicrobial effect during pressurization at subzero temperature what points to non-lytic mechanisms of its action under these conditions. Further studies are needed to explain this phenomenon.

Pressurization of bacteria at subzero temperature in buffer solution leads to sublethal injury of bacterial cells during subsequent prolonged storage of the samples. In the consequence, these cells become sensitive to antimicrobial substance such as nisin and lysozyme.

Furthermore, the maximal pressure of about 200 MPa that is possible to achieve in the sealed vessel at subzero temperature combined with nisin or lysozyme and refrigerated storage increases synergistically reduction of bacteria. However, such conditions can be insufficient to complete inactivation of vegetative microorganisms, especially in food system. Therefore, taking into account also the economical aspect of using high pressure, it would be worth searching for some other, more effective antimicrobials to extend the shelf-life and enhance the safety of food.

Acknowledgements

This research project was financed by the national research budget in the years 2003‑2006.

Submitted: June 25, 2008; Returned to authors for corrections: October 12, 2008; Approved: July 14, 2009.

• 1. Alpas, H.; Kalchayanand, N.; Bozoglu, F.; Sikes, A.; Dunne, C.P.; Ray, B. (1999). Variation in resistance to hydrostatic pressure among strains of food-borne pathogens. Appl. Environ. Microbiol. 65, 4248-4251.
• 2. Bridgman, P.W. (1912). Water in the liquid and five solid forms under pressure. Proc. Amer. Acad. Arts. Sci XLVII (1912), 439-558.
• 3. Cheftel, J.C. (1995). High pressure, microbial inactivation and food preservation. Food Sci. Technol. Int. 1, 75-90.
• 4. Cheftel, J.C.; Culioli, J. (1997). Effect of high pressure on meat: a reviev. Meat Sci. 46, 211-236.
• 5. Clarke, A.J.; Dupont, C. (1992). O-acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis. Can. J. Microbiol. 38, 85-91.
• 6. During, K.; Porsch, P.; Mahn, A.; Brinkmann O.; Gieffers, W. (1999). The non-enzymatic microbial activity of lysozymes. FEBS Lett. 449, 93-100.
• 7. Gänzle, M.G.; Weber, S.; Hammes, W.P. (1999). Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins. Int. J. Food Microbiol. 46, 207-217.
• 8. García-Graells, C.; Masschalck, B.; Michiels, C.W. (1999). Inactivation of Escherichia coli in milk by high hydrostatic pressure treatment in combination with antimicrobial peptides. J. Food Protec. 62, 12481254.
• 9. García-Graells, C.; Van Opstal, I.; Vanmuysen, A.C.M.; Michiels, C.W. (2003). The lactoperoxidase system increases efficacy of high pressure inactivation of foodborne bacteria. Int. J. Food Microbiol. 81, 211-221.
• 10. García-Graells, C.; Valckx, C.; Michiels, C.W. (2000). Inactivation of Escherichia coli and Listeria innocua in milk by combined treatment with high hydrostatic pressure and the lactoperoxidase system. Appl. Environ. Microbiol. 66, 4173-4179.
• 11. Gervilla, R.; Felipe, X.; Ferragut, V.; Guamis, B. (1997). Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk. J. Dairy. Sci 80, 2297-2303.
• 12. Hashizume, C.; Kimura, K.; Hayashi, R. (1995). Kinetic analysis of yeast inactivation by high pressure treatment at low temperatures. Biosci. Biotechnol. Biochem 59, 1455-1458.
• 13. Hauben, K.J.A.; Bartlett, D.H.; Carine, C.; Soontjens, F.; Cornelis, K.; Wuytack, E.Y.; Michiels, Ch.W. (1997). Escherichia coli mutants resistant to inactivation by high hydrostatic pressure. Appl. Environ. Microbiol. 63, 945-950.
• 14. Hauben, K.J.A.; Wuytack, E.Y.; Soontjens, C.C.F.; Michiels, Ch. (1996). High-pressure transient sensitization of Escherichia coli to lysozyme and nisin by disruption of outer-membrane permeability. J. Food Protec. 59, 350-355.
• 15. Hayakawa, K.; Ueno, Y.; Kawamura, S.; Kato, T.; Hayashi, R. (1998). Microorganism inactivation using high pressure generation in sealed vessels under sub-zero temperature. Appl. Microbiol. Biotechnol. 50, 415-418.
• 16. Hurst, A. (1981). Nisin. Adv. Appl. Microbiol. 27, 85-123.
• 17. Kalchayanand, N.; Frethem, C.; Dunne, P.; Sikes, A.; Ray, B. (2002). Hydrostatic pressure and bacteriocin-triggered cell wall lysis of Leuconostoc mesenteroides. Innov. Food Sci. Emerg. Technol. 3, 33-40.
• 18. Kalchayanand, N.; Sikes, A.; Dunne, C.P.; Ray, B. (1998). Factors influencing death and injury of foodborne pathogens by hydrostatic pressure-pasteurization. Food Microbiol. 15, 207-214.
• 19. Kalchayanand, N.; Sikes, T.; Dunne, C.P.; Ray, B. (1994). Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins. Appl. Environ. Microbiol. 60, 4174-4177.
• 20. Kalchayanand, N. Sikes, T.; Dunne, C.P.; Ray, B. (1998). Interaction of hydrostatic pressure, time and temperature of pressurization and pediocin AcH on inactivation of foodborne bacteria. J. Food Protec. 61, 425-431.
• 21. Linton, M.; McClements, J.M.J.; Patterson, M.F. (2001). Inactivation of pathogenic Escherichia coli in skimmed milk using high hydrostatic pressure. Innov. Food Sci. Emerg. Technol. 2, 99-104.
• 22. Masschalck, B.; García-Graells, C.; Van Haver, E.; Michiels, C.W. (2000). Inactivation of high pressure resistant Escherichia coli by lysozyme and nisin under high pressure. Innov. Food Sci. Emerg. Technol. 1, 39-47.
• 23. Masschalck, B.; Van Houdt, R. Michiels, Ch. (2001). High pressure increases bactericidal activity and spectrum of lactoferrrin, lactoferricin and nisin. Int. J. Food Microbiol. 64, 325-332.
• 24. Masschalck, B.; Van Houdt, R.; Van Haver, E.; Michiels, Ch. (2001). Inactivation of gram-negative bacteria by lysozyme, denaturated lysozyme and lysozymederived peptides under high hydrostatic pressure. Appl. Environ. Microbiol. 67, 339344.
• 25. Moreno, I.; Lerayer, A.L.S.; Baldini, V.L.S.; de F. Leităo, M.F. (2000). Characterization of bacteriocins produced by Lactococcus lactis strains. Braz. J. Microbiol. 31, 184-192.
• 26. Moussa, M.; Perrier-Cornet, J.M.; Gervais, P. (2006). Synergistic and antagonistic effects of combined subzero temperature and high pressure on inactivation of Escherichia coli  Appl. Environ. Microbiol72, 150-156.
• 27. Nakimbugwe, D.; Masschalck, B.; Atanassova, M.; Zewdie-Bosuner, A.; Michiels, C.W. (2006). Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure. Int. J. Food Microbiol. 108, 355-363.
• 28. Pagan, R.; Mackey, B. (2000). Relationship between membrane damage and cell death in pressure treated Escherichia coli cells: differences between exponential- and stationary-phase cells and variation among strains. Appl. Environ. Microbiol. 66, 2829-2834.
• 29. Patterson, M.F.; Quinn, M.; Simpson, R.; Gilmour, A. (1995). Sensitivity of vegetative pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and foods. J. Food Protec. 58, 524-529.
• 30. Pellegrini, A.; Thomas, U.; von Fellenberg, R.; Wild, P. (1992). Bactericidal activities of lysozyme and aprotinin against gram-negative and gram-positive bacteria related to their basic character. J. Appl. Bacteriol. 72, 180-187.
• 31. Perrier-Cornet, J.M.; Tapin, S.; Gaeta, S.; Gervais, P. (2005). High pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures. J. Biotech. 115, 405-412.
• 32. Ponce, E.; Pla, R.; Mor-Mur, M.; Gervilla, R.; Guamis, B. (1998). Inactivation of Listeria innocua inoculated in liquid whole egg by high hydrostatic pressure. J. Food Protec. 61, 119-122.
• 33. Reyns, K.M.F.A.; Sootjens, C.C.F.; Cornelis, K.; Weemaes, C.A.; Hendrickx E.; Michiels, C.W. (2000). Kinetic analysis and modeling of combined high-pressure-temperature inactivation of the yeast Zygosaccharomyces bailii Int. J. Food Microbiol. 56, 199-210.
• 34. Simpson, R.K.; Gilmour A. (1997). The resistance of Listeria monocytogenes to high hydrostatic pressure in foods. Food Microbiol. 14, 567-573.
• 35. Smelt, J.P.P.M. (1998). Recent advances in the microbiology of high pressure processing. Trends Food Sci. Technol. 9, 152-158.
• 36. Steeg ter, P.F.; Hellemons, J.C.; Kok, A.E. (1999). Synergistic actions of nisin, sublethal ultrahigh pressure and reduced temperature on bacteria and yeast. Appl. Environ. Microbiol. 65, 4148-4154.

Publication Dates

• Publication in this collection
  06 Oct 2009
• Date of issue
  Dec 2009

History

• Accepted
  14 July 2009
• Received
  25 June 2008