Inactivation of Vibrio fischeri by the Application of a Pulsed Electric
Field
Takahisa Ueno1*, Kyohei Asami1, Junko Ninomiya1, Takashi Furukawa2, Takashi Sakugawa3, Sunao Katsuki3
11National Institute of Technology, Oita College, Oita, 870-0165, Japan 2)Kitasato University, Kanazawa, 252-0374, Japan
3)Kumamoto University, Kumamoto, 860-8555, Japan
*E-mail: ueno@oita-ct.ac.jp
The pulsed electric field method, which is cited as a non-thermal physical sterilization method, enables continuous treatment using a simple protocol. Given that this method causes minimal protein denaturation, it can be used to sterilize eggs, fish, and similar protein-rich food items, if its bactericidal strength and energy efficiency can be improved. In this study, the ability of a pulsed electric field to sterilize Vibrio fischeri was examined. When the current applied to V. fischeri was limited, it was impossible to sterilize the bacteria even when the applied voltage and applied time were increased up to 13 kV and 15 min, respectively. Subsequently, when helium flow was used to increase the current to V. fischeri, sterilization was confirmed at an applied voltage of 13 kV for 5 min or more, and ozone water was detected in the bacterial suspension. Here, we show that sterilization could be achieved due to factors besides heat because applying the voltage at 20 °C or lower facilitated sterilization.
1. Introduction
Conventionally, heating is a widely used method to sterilize food, and if the heating process is uniform, highly reliable sterilization can be performed only by controlling the temperature and time. However, this approach causes heat-induced denaturation of food ingredients, as well as changes in flavor and color, warranting a preference for non-thermal physical disinfection methods. Especially when processing food items containing high amounts of heat-susceptible protein, such methods will ensure an uninterrupted food supply because they will enable storage for long durations without the degradation of quality [1-3].
For foods and similar substances, available non-thermal sterilization techniques include methods that use ultraviolet light, electron beams, ultra-high pressure, and disinfectant[4]. Among
them, sterilization by pulsed electric fields has high energy efficiency, and this approach has attracted considerable research interest[5]. In this study, we report the effect of sterilization through
electric current by applying a pulsed electric field to Vibrio fischeri. 2. Experimental equipment and methods 2.1 Target bacterium and culture method
In this experiment, the target organism was V. fischeri (ATCC 49387), which is a bioluminescent marine gram-negative bacterium. We selected this species owing to its bioluminescence, relative ease for artificial culture, and lack of toxicity. Frozen V. fischeri (ATCC 49387-MINI PACK )was thawed at room temperature, and 1 mL of the stock bacterial suspension
was adjusted to a suitable concentration (107 CFU/mL) by diluting and suspending the bacteria in
sterile milli-Q water, after which 10 mL of the bacterial suspension was dispensed into petri dishes. 2.2High-voltage impulse application and bacterial count determination
High-voltage impulses were applied to V. fischeri suspensions using an impulse generator. Fig. 1(a) shows a circuit diagram of the equipment. This equipment uses transformer pressurization. The charging voltage in the primary capacitor is boosted by a transformer and then accumulated in the secondary capacitor, and a high-voltage impulse is applied to the load. The step-up ratio of the primary to secondary windings was 1:46. A thyristor (N1718NS180; Westcode) was used as a semiconductor switch. A function generator was used for thyristor gate signal control. The primary resistance R0and charging capacitance C0were set at 100 Ω and 2.5 μF, respectively.
A schematic diagram of the experimental equipment is shown in Fig. 1(b). A petri dish containing 10 mL of V. fischeri culture medium was placed between a stainless steel needle-array electrode and a flat plate electrode, and a high-voltage impulse was applied. The distance between the electrodes from the needle tip to the applied target was set to 10 mm. At this time, gas (air or helium gas) was flowed into the container at 1 L/min, and then, a fan was used to flow the gas from the container to the outside.
At a constant frequency of 100 Hz, the applied voltages were 5.0 and 13 kV, with application durations of 5 and 15 min each. The number of viable bacteria and luminescence intensity were measured immediately and 4 h and 6 h after voltage application, and the respective values were compared for each elapsed duration. At these timepoints, the temperature of the bacterial suspension was measured by an infrared thermometer (73010; Shinwa Rules); luminescence was measured by a luminometer (Lumat LB 9507; Berthold); and turbidity for bacterial count was measured in a mixture of treated bacterial solution and 3% NaCl solution at a wavelength of 600 nm (OD600) using
a spectrophotometer (AE-450; Elma). In addition, 100 μL of the treated bacterial suspension was plated on nutrient agar (NA medium; Difco), and the number of colonies was counted after 2 days. Three bacterial suspensions subjected to the same conditions were prepared, and the average was used as the result. Five milliliters of the bacterial suspension subjected to 13 kV was collected, and the ozone water concentration was measured using the pack test (DPM2-O3; Kyoritsu-Lab) according to the 4-aminoantipyrine colorimetric method.
Fig. 1(b) Schematic diagram of the experimental equipment 3. Application of a pulsed electric field to V. fischeri 3.1 Application of high-voltage impulse to V. fischeri under current limitation
The effect of sterilization on V. fischeri was confirmed when the current was limited by a plastic petri dish. The gas flowing into the reactor was air. Fig. 2 shows the current waveform at an output voltage of 5 kV. The voltage pulse width was 7.5 μs. The current was a vibration waveform, and the maximum current was 37 mA. The maximum current value increased in direct proportion to the applied voltage, which was 122 mA at 13 kV.
The number of bacteria and the luminescent intensity are shown in Fig. 3. Although the number of bacteria increased slightly under all conditions from immediately after the voltage was applied to after 6 h elapsed, no significant change was observed.
Next, a 1 mL aliquot was taken from the culture suspension to be treated, and the luminescence value was measured with a luminometer. The results are shown in Fig. 4. Comparing the results immediately after the voltage was applied and that after 6 h, we confirmed that the amount of light emission did not decrease substantially and was relatively constant across the studied conditions. Fig. 5 shows the number of colonies; the number of bacteria did not change greatly under any of the studied conditions.
In addition, temperature was measured using an infrared radiation thermometer to investigate the temperature of the bacterial suspension. The temperature ranged from 26 °C and 28 °C, indicating that there were no significant changes. The pack test did not detect ozone water in any condition.
←Gas flow Electrod
Gap: 10 mm Petri dish
-4 -2 0 2 4 -40 -20 0 20 40 -20 -15 -10 -5 0 5 Vout Iout V ou t [k v] Iou t [ m A ] Time [µs]
*Vout: output voltage, Iout: output current
Fig. 2 Output waveforms
Fig. 3 OD600turbidity of V. fischeri Fig. 4 Luminescence of V. fischeri
0 0.05 0.1 0.15 0.2 0.25 0.3 0 4 6 Control 5kV, 5min 5kV, 15min 13kV, 5min 13kV, 15min O D 60 0 [l o g s ca le ] Time [ hour ] 0 2 4 6 8 10 0 4 6 Control 5kV, 5min 5kV, 15min 13kV, 5min 13kV, 15min B io lu m in e s ce n c e [ a. u .] Time [ hour ] 1.0E+05 1.0E+06 1.0E+07 1.0E+08 b e r o f c o lo n y / m l]
3.2 Application of high-voltage impulse to V. fischeri under increased current in a helium atmosphere
The sterilization of V. fischeri by high-voltage pulse was attempted by increasing the current with helium gas. The waveforms of the output voltage and the output current at the time of application are shown in Fig. 6. When the output voltage was 13 kV, the current was 21 A, which was more than 10 times that compared with the current limit.
Fig. 7 shows a graph of time variation for V. fischeri OD600 (turbidity). As in the previous
experiment, the total bacterial count did not change significantly from immediately after the application to 6 h after the application. Fig. 8 shows a graph that summarizes the changes in the amount of light emitted by V. fischeri with time. When the applied voltage was 13 kV, we confirmed that the amount of luminescence was remarkably low immediately after the application for any application time from 5 to 15 min. Fig. 9 shows the number of colonies per milliliter of bacterial suspension. Compared to the untreated sample, the number of colonies did not change significantly at 5 kV. However, colony number greatly decreased at 13 kV, suggesting that V. fischeri was sterilized (order of 107CFU/mL).
Fig. 10 shows the temperature change of the culture suspension when 13 kV was applied for 15 and 5 min. The temperature tended to rise over time and increased to 52 °C when the voltage was applied for 15 min. Although this value markedly exceeded the culture temperature, the temperature at 5 min after application was about 16 °C, much lower than the culture temperature.
*Vout: output voltage, Iout: output current
Fig. 9 Number of V. fischeri colonies under increased current
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 4 6 5kV, 15min 13kV, 5min 13kV, 15min O D 60 0 [l o g s ca le ] Time [ hour ] 0 2 4 6 8 10 0 4 6 5kV, 15min 13kV, 5min 13kV, 15min B io lu m in e sc e n c e [a .u .] Time [ hour ] 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08
Control 5kV, 5min 5kV, 15min 13kV, 5min 13kV, 15min
V . f is he ri n u m b e r o f c o lo n y [C F U / m l] 50 60 15kV, 15min 15kV, 5min ]
Fig. 7 V. fischeri OD600(turbidity) under
increased current
Fig. 8 V. fischeri luminescence under increased current
4. Dicussion
When voltage was applied to V. fischeri using a plastic petri dish, the current was limited because the petri dish acts as an insulator. Even when a maximum voltage of 13 kV was applied for 15 min, the luminescence of V. fischeri did not change with time, indicating that it was not sterilized. The slight increase in turbidity was due to an increase in the number of bacteria, but the amount of light emission did not change significantly because the increase was slight [7].
When helium gas was supplied, the ionization voltage was low and the discharge voltage decreased; hence, glow discharge was generated, and the current applied to the bacterial suspension increased. The bacteria were not killed at a voltage of 5 kV, but when a voltage of 13 kV was applied, perfect sterilization was achieved (an order of 107 CFU/mL) with 5 min application, and the light
emission greatly decreased. When the application time was 15 min, the temperature of the bacterial suspension reached 52 ℃. We surmise that the temperature rose by the current flowing in the bacterial suspension, which was converted into heat. Furthermore, factors besides heat, which were also by-products of the electric field and discharge, were considered to underlie the sterilization, because the bacterial suspension was sterilized even when the temperature was 20 ℃. The by-products include plasma and ozone. Park et al. showed that colonies of Escherichia coli were sterilized by plasma irradiation for 20 s [8]. However, the plasma-irradiated surface temperature was
75–130 °C, which was much higher than the temperature of the bacterial suspension in the present experiment; thus, the plasma effect was considered to be negligible herein.
As regards the ozone water concentration, a study showed that 102–103 CFU/mL
Escherichia coli was sterilized by ozone water at 0.1–0.2 ppm in a treatment time of about 20 -30
min[9-11]. In the present experiment, the ozone water concentration became 0.1 ppm by 13 kV voltage
application for 5 min, yielding sterilization of the order of 107CFU/mL. We found that factors apart
from ozone water were important for the sterilization, because the overall sterilization value was over four orders of magnitude larger than that of the sterilization by only ozone water.
When the current was limited, the effect of the electric field was not observed, but it has been reported that the sterilization rate improves over 103CFU/mL by an electric field due to heat.
This might be because lipid, which is a part of cell membranes, softens by heat[12-15], and similarly,
ozone water has been reported to affect lipid, causing holes in the cell membrane[16-19]. Therefore, it
is considered that sterilization of an order of 106CFU/mL could be achieved by the combined effects
of ozone water and the electric field.
5. Conclusion
In this study, we demonstrated differences in the effect of bacterial sterilization by applying a pulsed electric field to V. fischeri at different voltage and durations. The results are summarized as follows.
(1) The current was restricted when a pulsed electric field was applied by flowing air into the reactor and by using a plastic petri dish. Voltages of 5 and 13 kV were applied for 5 and 15 min, respectively, but it was confirmed that V. fischeri were not sterilized in all cases.
(2) Pulsed electric fields were applied to V. fischeri suspensions without current limitation with helium gas for sterilization. On comparing the turbidity, luminescence, and colony number of V.
voltage of 13 kV. For the 5 min 13 kV application, the temperature was 20 °C and ozone water concentration was 0.1 ppm. Since it is difficult to sterilize bacteria to an order of 107CFU/mL with
only 0.1 ppm ozone water, the electric field is also a factor underlying sterilization. Acknowledgments
This work was supported by JSPS KAKENHI Grant Number 18K04096, Joint Research Center on Pulsed Power Science and Technology.
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