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

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 µg/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.


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 taken 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.

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 In order to obtain an appropriate growth phase of cells, 100 l 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 10 8 /10 9 cfu/ml. Antimicrobials were added to the final concentration of 400 µg/ml (lysozyme) or 100 and 400 µg/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

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. cycle was obtained (Fig. 3). In samples pressurized in the 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).  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

Antibacterial effect of high pressure and nisin
Nisin at concentration of 100 µg/ml and 400 µg/ml,  Subsequently, the cell density in the present study was 10 8 /10 9 cfu/ml while in that conducted by Stegg et al. (36) amounted to 10 5 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 (10 6 cfu/ml) than at high cell density (10 9 cfu/ml). 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 work the 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.