Methods for microbial inactivation are important in the food industry; however, conventional external heating (CH) reduces food quality. Accordingly, the nonthermal effects of ohmic heating (OH) on Bacillus subtilis spores in a sodium chloride aqueous solution at 101°C (i.e., the boiling point), as well as the effects of electric field intensity and frequency during OH, were investigated. Survival kinetics were compared between OH and external CH. The inactivation effect on B. subtilis was greater for all electric field conditions (5, 10, and 20 V/cm) than for CH. In particular, 20 V/cm showed a significantly higher inactivation effect (P < 0.05) on B. subtilis than those of CH at 8, 10, 12, 14, and 16 min. The survival data were fitted to various primary kinetic models. In the Weibull model and the log-linear model, there were significant differences (P < 0.05) in the rate parameters δ and kmax between OH at 20 V/cm and CH. However, there were no significant differences (P > 0.05) in survival kinetics between 20, 40, and 60 kHz; B. subtilis spores were inactivated more efficiently as the frequency increased. B. subtilis spores were almost completely inactivated at 14 to 16 min for the 60-kHz treatment, but spores were still alive at 20 and 40 kHz for the same treatment times. These results demonstrated that OH inactivates B. subtilis spores more effectively than CH. OH conditions with high electric field intensities and high frequencies resulted in efficient B. subtilis spore inactivation.
The essential goal of microbial inactivation is to secure food safety by reducing spoilage and eliminating pathogenic bacteria. Microbial inactivation is simultaneously required to secure food safety and maintain food quality. Thermal processing is the most common technology for microbial inactivation. However, high-temperature treatments induce the deterioration of food, resulting in changes in flavor, taste, and texture or the loss of nutrients. To overcome the limitations of thermal processing, various nonthermal microbial inactivation techniques, such as high pressure processing, high electric field pluses, and ultrasonication, have been developed. However, without heating foods excessively, most nonthermal processing methods do not completely inactivate bacterial spores and, accordingly, do not sufficiently meet the needs of the food industry (3, 4, 10).
Ohmic heating (OH) is a procedure in which an alternating electric current is applied to foods. OH rapidly and uniformly heats materials (6) and achieves close to 100% energy transfer efficiency because heat is generated and distributed internally in foods (11). OH has the potential to reduce thermal damage and retain the quality of processed foods (7, 16), unlike conventional external heating (CH), owing to the highly efficient temperature increase.
In addition, OH is expected to inactivate bacterial spores not only by thermal effects but also by nonthermal effects via electricity (1, 2, 9, 12, 13). Cho et al. (2) compared the effects of OH and CH on Bacillus subtilis spores at various temperatures and concluded that B. subtilis spores heated in water with 0.1% NaCl at 92.3°C showed a significant reduction in the D-value under OH treatment. However, there was no significant difference in D-values between OH and CH at 88.0°C. Moreover, Cho et al. (2) concluded that the z-value is not significantly different between the two methods. Pereira et al. (9) compared the effects of OH with those of CH on Bacillus licheniformis spores at various temperatures and concluded that B. licheniformis spores heated in cloudberry jam at 70, 75, and 80°C showed significantly reduced D-values under OH treatment. However, there was no significant difference in D-values between OH and CH at 90°C, and the z-values for the two methods were not significantly different. In contrast, the effect of OH was not significantly (P > 0.05) different from that of CH on B. licheniformis spore inactivation (14).
Although OH is expected to have nonthermal effects on bacterial spores and contribute to reductions in the food heating temperature and time, the results of previous studies have been variable. Conflicting results may be explained by unstable or fluctuating electric field stress during OH treatment. It is technically difficult to maintain a steady temperature and apply a continuous electric field simultaneously because the temperature of the heating medium increases as a constant electric current is applied. In previous studies, the electric field has been increased or decreased to maintain a constant temperature (1, 2, 9, 12, 13). In the present study, an electric field was applied constantly by maintaining the heating medium at the boiling point. Electric field stress was constantly applied to bacterial spores by using the procedure examined in the present study. The objective of this study was to clarify the nonthermal effects of OH on the inactivation of B. subtilis spores when a constant electric field is applied. In addition, the effects of the magnitude of the electric fields and electric frequency on B. subtilis spore inactivation were evaluated.
MATERIALS AND METHODS
Bacterial strains. B. subtilis (NBRC13719) was used as a representative bacterial spore because NBRC13719 has the highest thermal resistance strain among B. subtilis strains in our laboratory. A stock culture maintained on nutrient agar (Difco, BD, Sparks, MD) slants at 3°C was inoculated in glucose broth (2 g of beef extract, 3 g of yeast extract, 10 g of peptone, 5 g of dextrose, 5 g of sodium chloride, and 1 liter of deionized water, pH 7.0) and incubated at 37°C for 48 h for the activation of the bacterial cells.
Preparation of spore suspension. To induce the sporulation of B. subtilis cells, an aliquot of activated bacterial cells was inoculated on a nutrient agar plate and then incubated at 37°C for 24 h. The colonies that formed on the nutrient agar were inoculated in 25 ml of Difco sporulation medium (8 g of Bacto nutrient broth [Difco], 10 ml of KCl, 10 ml of MgSO4·7H2O, 1 M NaOH (pH to 7.6), 1 ml of Ca(NO3)2, 1 ml of MnCl2, 1 ml of FeSO4, and 1 liter of deionized water) and incubated at 37°C for 48 h. Greater than 90% of sporulation was obtained, as verified by observations of refractile spores under phase-contrast microscopy. The culture was centrifuged at 3,000 × g for 20 min. Recovered spores were washed with distilled water four times with repeated centrifugation for 10 min at 3,000 × g. Between the second and third centrifugation steps, the suspension was pasteurized at 80°C for 20 min to eliminate vegetative cells. The suspensions were stored at 4°C until use.
OH treatment. The heating unit consisted of titanium square electrodes (length, 110 mm; width, 110 mm; and thickness, 2 mm) in contact with the sample, a Teflon vessel (thickness, 10 mm; height, 110 mm; width, 123 mm; and depth, 55 mm), and a thermocouple (K-type) inserted at the center of the Teflon vessel with a capacity of 300 ml. The distance between the two titanium square electrodes was 30 mm.
For the OH treatment, a 250-ml aliquot of sodium chloride aqueous solution (0.05, 0.2, and 0.6%, wt/vol) was added to the Teflon vessel and heated to the boiling point (101°C) by applying a constant electric field. Once the desired temperature was reached in the salt water, 1 ml of spore suspension (~107 CFU/ml) was added to 250 ml of the heating medium. The electric field intensity (5, 10, and 20 V/cm) and the frequency (20, 40, and 60 kHz) were controlled by the power supply. Every 2 min, a 1-ml aliquot was collected from the heating medium and kept in a plastic tube in an ice bath until the microbiological analysis (<20 min).
In many studies of the inactivation of bacterial spores using OH, sporular liquid in the medium is heated to a targeted temperature, and the temperature is maintained by OH and CH. In contrast, in this study, sporular liquid was added to the heated medium at the targeted temperature to ensure identical temperature histories for the OH and CH treatments.
CH treatment. For the CH treatment, a 250-ml aliquot of sodium chloride aqueous solution (0.05, 0.2, and 0.6%, wt/vol) was heated in a glass beaker by using a heating magnetic stirrer to the boiling point (101°C). After the temperature reached 101°C, 1 ml of spore suspension (~107 CFU/ml) was added to 250 ml of the heated water.
Microbiological analysis. To determine the number of surviving spores at each sampling point (every 2 min) during the heat treatment, appropriate serial dilutions (1:10) were performed with sterile 0.1% peptone water and were plated on duplicate tryptic soy agar plates. The plates were then incubated at 37°C for 24 h.
Survival kinetics analysis. Survival kinetics were analyzed by using the Geeraerd, Valdramidis, and Van Impe inactivation model-fitting tool (GInaFiT, Version 1.6), a freely available add-in for Microsoft Excel (Microsoft Corporation, Redmond, WA) (5). The Weibull model, the log-linear regression model, and the log-linear regression plus shoulder model were used.
The Weibull model is represented by the following formula
where S(t) is the momentary survival ratio and N(t) and N0 are the momentary and initial counts at 101°C, respectively. The initial counts (N0) were viable cell counts at time zero. The parameters δ and p represent the survival rate and kinetics curvature, respectively. The parameter δ is the first time at which a 10-fold reduction in the viable cells is observed, and p is the shape parameter.
The log-linear regression model is described by the following formula
where S(t) is the momentary survival ratio and N(t) and N0 are the momentary and initial counts at 101°C, respectively. The parameter kmax is a first-order inactivation constant. Therefore, the traditional decimal reduction time (D-value) can be calculated from the kmax parameter according to the equation: D = 2.303/kmax.
The log-linear regression with shoulder model is described by the following formula
where kmax is the first-order inactivation constant and Sl is the shoulder length. This model describes the survival curves based on two parameters, i.e., the shoulder length (Sl), defined as the time until exponential inactivation begins and the inactivation rate (kmax), defined as the slope of the exponential portion of the survival curve. Therefore, the D-value can be calculated from the kmax parameter, as mentioned previously.
Statistical analysis. All experiments were repeated three times, and each data point was determined based on duplicate plate counts. Data from three samples subjected to each treatment in each of three independent replicate experiments were analyzed. For the statistical evaluation, significant differences (P < 0.05) in the survival ratio between the OH and CH treatments at each heating time and the parameters for the model fits for OH and CH were analyzed by using the Tukey-Kramer method. The statistical analysis was conducted by using the R statistical environment (Version 3.2.4 for Mac OS; R Foundation for Statistical Computing, Vienna, Austria; http://www.R-project.org).
The temperature was 101°C, and the electric fields were constantly applied during OH (Fig. 1). Electric field conditions for OH were controlled by adjusting the sodium chloride concentration. As the sodium chloride concentration increases, the electric current flows more easily, and the intensity of the electric field declines. The sodium chloride concentration did not affect the inactivation of B. subtilis spores during heating at the boiling point (Fig. 2). Therefore, we concluded that sodium chloride concentrations of 0.05 to 0.6% do not significantly influence the survival kinetics of B. subtilis spores.
Greater inactivation effects on B. subtilis spores were observed for all of the electric field conditions (5, 10, and 20 V/cm) for OH than for external CH (Fig. 3). As the intensity of the electric field increased, a greater, more rapid log reduction in viable cells was observed. In particular, the OH treatment with 20 V/cm showed significantly (P < 0.05) greater log reductions at 8, 10, 12, 14, and 16 min than those obtained for the CH treatments. However, there were no significant differences in log reduction between CH and OH for 20 V/cm at 18 and 20 min.
In the log-linear model, the log-linear plus shoulder model, and the Weibull model, kmax of OH tended to be higher, and δ of OH tended to be lower than those of CH (Table. 1). In the Weibull model and the log-linear model, there were significant differences (P < 0.05) in δ and kmax between OH at 20 V/cm and CH. These results showed that B. subtilis spores were inactivated more efficiently as the electric field intensity increased.
As the frequency increased during OH treatment at 20 V/cm, OH showed a trend toward a faster and greater log reduction in viable B. subtilis spores (Fig. 4). In particular, the OH with 60 kHz completely inactivated B. subtilis spores at 14 to 16 min, while viable spores remained after treatment with OH at 20 and 40 kHz for the same treatment duration. However, there were no statistically significant differences (P > 0.05) in surviving B. subtilis spores (Fig. 4) and the fitted model parameters between frequencies (Table 1). Although there were no significant differences among the frequencies, the time until complete inactivation of B. subtilis spores was apparently frequency dependent.
A few studies have compared the efficiency of bacterial spore inactivation between OH and CH. Cho et al. (2) studied the inactivation kinetics of inactivation of B. subtilis spores by using OH and CH and reported that OH has a greater inactivation effect than CH. Moreover, Cho et al. (2) indicated that bacterial spore inactivation during OH is primarily attributed to the thermal effect, but an additional killing effect occurs via the electric current. In the present study, the electric field and frequency had additional effects because B. subtilis was inactivated not only by OH but also by CH. B. subtilis would be damaged by heat, as well as increases in the electric field and frequency.
The additional effect of the electric field on bacteria may be primarily explained by a related phenomenon: electroporation. High electric fields, such as pulsed electric fields (>10 kV/cm), cause electroporation, and low frequencies (usually 50 to 60 Hz) allow cell walls to build up charges and form pores (15). Park and Kang (8) studied the effects of electric field–induced OH on the inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in buffered peptone water and apple juice at 30 and 60 V/cm. The authors found that bacterial reduction differed significantly between OH and CH treatments. Moreover, propidium iodide values (an index of cell membrane damage) were significantly different between OH and CH treatments. Yoon et al. (17) investigated the effects of OH on the structure and permeability of the cell membrane of Saccharomyces cerevisiae. They found that the amount of exuded protein increased significantly as the electric field increased from 10 to 20 V/cm, while the amount of exuded protein increased when higher frequencies (600 Hz and 6 and 60 kHz) were applied. Thus, vegetative bacterial cells and yeast cells would be damaged by higher electric fields and frequencies similar to electroporation.
However, the responses of bacterial spores to various electric fields and frequencies are unclear. Baysal and Icier (1) studied the effect of electric field (30, 40, and 50 V/cm) on Alicyclobacillus acidoterrestris spores heated by OH. Baysal and Icier (1) reported that although there were significant differences among the electric fields at 70°C, there were no significant differences among the electric fields at 80 and 90°C. Somavat et al. (12) indicated that 10-kHz treatment resulted in comparatively more effective inactivation than 60 Hz for Geobacillus stearothermophilus spores. In contrast, Somavat et al. (13) observed that 60-Hz treatments for Bacillus coagulans spores tended to more efficiently inactivate spores than 10-kHz treatments.
Because a constant electric field was not applied to maintain the temperature to avoid overheating in these previous studies, the effects of electric fields are expected to be variable. In contrast, in the present study, a constant electric field was applied at the boiling point during OH treatment and a higher electric field and frequency increased the nonthermal effect of OH on B. subtilis. These results demonstrated that the continual application of a certain electric field would increases nonthermal effects on bacterial spores. Moreover, the effect of frequency on the inactivation of spores is pronounced under a constant certain electric field.
Although the mechanism by which electric field and frequency influence bacterial spores is unclear, it is common for ion molecules, such as hydrogen ions, to be drawn by electric fields and polar substances, such as water molecules, and vibrate in response to electric fields. These attractive forces and vibrations may cause the changes in cell structure or the leakage of intercellular substances in bacterial spores. The heat resistance of bacterial spores may decline above a certain frequency, which may explain why higher frequencies increase the nonthermal effects of OH on B. subtilis. In the future, it is necessary to investigate the effects of electric field and frequency on other kinds of bacterial spore types by applying a constant electric field.
In conclusion, the results of the present study indicated that nonthermal B. subtilis spore inactivation results from OH when an electric field is constantly applied. The OH conditions with higher electric field intensities and higher frequencies improve the efficiency of B. subtilis spore inactivation and reduce the heating time to inactivate B. subtilis spores. Consequently, although we need to examine the effect of OH on food quality or sensory evaluation or both, the OH treatment would have the potential for reducing thermal treatment time for ensuring food safety.
This study was financially supported by a research grant from the Toyo Institute of Food Technology.