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Nisin, an antimicrobial protein produced by Lactococcus lactis subsp. lactis, is the only bacteriocin that is approved by the World Health Organization to be used as a food preservative today (1, 7). Its primary target is the cytoplasmic membrane of vegetative cells. Nisin interacts via electrostatic interactions with the phospholipids and increases the permeability of the membrane by pore formation, resulting in a rapid efflux of small molecules (3, 16). The efflux of cellular constituents results in a complete collapse of the proton motive force and cellular death (4, 5). The practical application of nisin is limited because its inhibition spectrum is restricted to gram-positive bacteria only (9) and its activity can be influenced by pH or food ingredients like fat particles (18). However, by combining nisin with other mild preservatives like essential oils (14) or nonthermal pasteurization techniques, these restrictions could be overcome.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Determination of critical field strength (kV/cm) (a) and critical treatment time (b) for B. cereus F46.26.90. (a) The total treatment time given was 1,000 µs. The data points represent the means of two independent measurements. (b) PEF treatment at a fixed field strength of 16.7 kV/cm. The data points represent the averages of three measurements.

Pulsed-electric field (PEF) treatment is a nonthermal pasteurization technique which inactivates microorganisms (15, 20) by irreversible structural changes in the membrane, resulting in pore formation and loss of the selective permeability properties of the membrane (6, 13, 17). The extent of the permeability increase depends on the strength and duration of the electric field pulse (2, 6, 11, 19, 20). An imposed electric field causes polarization and subsequently accumulation of free charges at both sides of the cell surface, leading to an increased transmembrane potential difference and a reduction of the membrane thickness which finally results in pore formation (2, 19). Since nisin and PEF both act on the membrane, an additive effect might be expected.

Bacillus cereus IFR-NL94-25, obtained from the Institute of Food Research (Norwich, United Kingdom) was grown at 20°C in brain heart infusion broth (Oxoid) containing 0.5% (wt/vol) glucose. Cells were harvested in the exponential growth phase, washed, and resuspended in 5 mM potassium-HEPES buffer (pH 7.0) to an optical density at 660 nm of 0.2 (tube diameter = 9 mm). Cell suspensions were checked for spores by microscopy before harvesting and subsequently by analysis of surviving spores after a standard heat treatment (80°C, 5 min).

The PEF system used here was a custom-built batch system applying single square-shaped pulses. The treatment chamber (800 µl) was formed by two stainless steel cylinders with a diameter of 12.6 mm which were tightly fitted into a Plexiglas tube with the same inner diameter. The electrode distance was fixed to 6 mm. In all cases, the temperature during treatment was kept below 30°C to rule out thermal effects. This was secured by spreading the pulses in time and by the large contact surface of the electrodes, which provides substantial cooling. Vegetative cells of B. cereus were subjected to increasing field strengths to determine the experimental parameters of the PEF treatment (Fig. 1a and b). Only after passing a critical field strength, a linear correlation between the log viable count of B. cereus and the field strength was observed (Fig. 1a). At high field strengths the reduction saturates (this can be explained by nonideal behavior of field lines in the chamber near the metal-Perspex-liquid interface), resulting in edge effect. This effect limits the resolution of this experiment to 4 log cycles. A field strength of 16.7 kV/cm was chosen to determine the critical treatment time (Fig. 1b), defined as the product of the pulse duration and the number of pulses (11). The reduction in log viable count was linear with the treatment time. At treatment times higher than 300 µs, the reduction saturates, again caused by the limited resolution of the experimental design. To impose a mild PEF treatment, a field strength of 16.7 kV/cm with a treatment time of 100 µs was chosen.

The effect of nisin (Nisaplin; Aplin and Barrett Ltd., Wilts, United Kingdom) was determined by exposing B. cereus cells to different concentrations of nisin for 11.5 min. A stock solution was made in 50% ethanol and filter sterilized (0.22 µm; Costar), and the concentration of nisin was calculated on the basis of the nisin content. A low dose of 0.06 µg of nisin per ml was chosen for further experiments (Fig. 2), since this concentration caused a small reduction of only 1 log unit.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Influence of nisin concentration on the viability of B. cereus F26.46.90. The treatment time was 11.5 min. The data points represent the means of an average of four measurements.

To determine the combined effect, vegetative cells of B. cereus were subjected to low doses of nisin and mild PEF treatment separately and in combination for 11.5 min. The PEF treatment was spread over a 10-min time interval, since nisin was found to be most active during the first 10 min of exposure (14). The PEF treatment caused a reduction in viable count of 1.2 log units (Fig. 3). However, when the two mild treatments were combined and applied simultaneously, a remarkable increase in the reduction of the viable count was observed. This reduction of 3.8 log units is 1.8 log units larger than the sum of the reductions obtained with the single treatments. This clearly demonstrates that PEF treatment acts synergistically with nisin in reducing the viable count of vegetative cells of B. cereus. This inactivation has been determined as a function of time (Fig. 4). In the presence of 0.06 µg of nisin per ml, only a marginal reduction of the viable count of B. cereus cells was observed. The inactivation kinetics associated with the combined treatment followed a linear pattern. These results clearly show that PEF is able to enhance the bactericidal action of nisin. The mechanism of synergy is not yet fully understood. The PEF treatment can be regarded as an additional stress and possibly facilitates the incorporation of nisin into the cytoplasmic membrane, resulting in more or larger pores or pores with a longer lifetime. This is supported by the observations of Kalchayanand et al. (10), who found that electroporation or ultrahigh pressure caused sublethal injury, and Ho et al. (8), who found that the critical field strength required for cell lysis was reduced by inducing additional stress, like sodium chloride or osmotic pressure, to the cell membrane. The synergism found between nisin and PEF opens new possibilities for applying the hurdle concept (12) as a preservation method.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Effects of PEF treatment alone, nisin alone, and the combined treatment of PEF and nisin on the viability of B. cereus F46.26.90. The data points represent the means of duplicate measurements.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Kinetics of combined action of nisin and PEF treatment on the viability of B. cereus F.46.26.90 monitored in time. , PEF treatment (16.7 kV/cm, 100-µs duration); , nisin treatment (0.06 µg/ml); and , the combined treatment. The data points represent the means of duplicate measurements.