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Effect of brine and temperature in sterilization using nanosecond pulsed electric field for packaged fresh foods Koki Saito, Kazuya Hoki, Yasushi Minamitani Yamagata University Yonezawa, Japan [email protected] Abstract—We have investigated sterilization of packaging foods. In sterilization methods of the packaging foods, there is pulsed discharge sterilization and high frequency pulsed electric field sterilization. The pulsed discharge sterilization is suitable for solid fresh foods. The high frequency pulsed electric field sterilization is suitable for liquid fresh foods. However, many liquid fresh foods are difficult to sterilize by the pulsed electric field for having high electrical conductivity like containing salt. In this study, sterilizing properties of the brine of the high conductivity by nanosecond pulsed electric field have been investigated. Target microorganism was Saccharomyces cerevisiae that is detected generally in foods. In experiments, the salinity and temperature of the brine with S. cerevisiae was changed as parameters. As experimental results, by applying the pulsed electric field in the sample temperature of 50 °C, processing time to reduce the number of S. cerevisiae two digits has been about one-fifth of that of the room temperature. In the evaluation of the sterilization ratio by difference of the salinity, the sterilization ratio at the salinity of 3 % has been more than that of 0.3 %. Keywords—Nanosecond pulse electric field, Temperature, high conductivity, sterilization, packaged fresh foods, yeast I. INTRODUCTION For us, foods are important matter concerning the survival. However, the incidents that threaten the safety of the foods occur frequently, e.g. a food poisoning. Sterilization of the foods is necessity from a standpoint of the safety of the foods. Currently, many of the food are on the market in the packaging state in order to keep the freshness. The packaging prevents drying and oxidation of the foods. In addition, There are effects that the adhesion of bacteria after the packaging is also prevented, the extension of the expiration date, prevention of food poisoning. However, this effect can’t be expected when the bacteria adheres prior to packaging. If possible sterilization after packaging, this worry is eliminated. But now, sterilization possible method after the packaging is the only heat sterilization and radiation sterilization. Heat sterilization can’t sterilization of fresh foods, Radiation sterilization has not been approved in terms of safety. To solve this problem, in the new sterilization methods of the packaging foods, there is pulsed discharge sterilization and high frequency pulsed electric field (PEF) sterilization[1][2]. Sterilization by PEF is the method of applying PEF of the rectangular wave of microsecond order to an object of sterilization, and is being studied as practical method of sterilizing a liquid food, such as juice [3]. This method has an advantage of not losing the flavor or nutrient of the food because of no-heating. In addition, PEF can sterilize inside of the liquid food. On the other hand, The pulsed discharge sterilization is suitable for solid fresh foods as salad and meet. Namely, Use both methods for sterilization of the inside of package of the sterilization target. In this study, focuses on nanosecond electric field sterilization, and to sterilize the highly conductive foods as pickle and milk. The Packed food with bacteria like Figure 1(a) can be written as the equivalent circuit as shown in Figure 1(b) by capacitors and resistors. In the equivalent circuit, the package is written as capacitors. The liquid food is written as the resistor. Since electrical conductivity of the package is small, the impedance of the package is large. Therefore, when the voltage is applied to the packed food, the voltage concentrates to the package, and the electric field is not applied to the bacteria. As the solution of this problem, we have worked out how to lower the impedance of the package by applying the voltage of high frequency, because the package is written as capacitors. Cells of bacteria can be written as the equivalent circuits as shown in Figure 2 by capacitors and resistors [4]. In the equivalent circuit, cell membrane is written as capacitor (a)Pattern diagram (b)Equivalent circuit Figure1. Equivalent circuit of package food because the cell membrane is almost insulator and very thin. Cytoplasm is written as resistor because the cytoplasm is filled with electrolyte. Therefore, when the voltage is applied to the cells, the constituent of cell that the voltage is applied to is different at frequency of the voltage. At low frequency, breakdown occurs in the cell membrane since the voltage is applied to the cell membrane. At high frequency, the electric field affects DNA and contents since the voltage is applied to the cytoplasm. In fact, it has been shown in the references of [5] and [6] that short pulse electric field with high frequency components can sterilize bacteria. In our laboratory, it has been applied to sterilization by nanosecond pulsed electric field having a high frequency component can be reduced impedance of packages. In this paper, we aim to apply for sterilization of a variety of food using nanosecond pulsed electric field. As pre-stage of the packaging sterilized, it was investigated conditions for improving the effect of the electric field sterilization at high frequency pulse. Specifically, It was investigated for effect on the sterilization speed by the salinity and temperature of the brine with S. cerevisiae was changed as parameters. II. EXPERIMENTAL SAMPLES In this study, it has been included in the general fresh food, using a yeast (Saccharomyces Cerevisiae: NBRC0565) having a high degree of safety. Figure 3. shows appearance of yeast. Sterilization subject is a high conductivity food, such as pickles. Accordingly, Simulate by 3% saline solution with high conductivity as same as Pickles solution (Conductivity with 63.3mS/cm). Yeast is a unicellular microorganisms in eukaryotic, the cell structure has a cell nucleus into the cell. The shape of cross section is circular or oval with a diameter of about 5-10 µm. Cell membrane is a lipid bilayer structure having a thickness of about 7 nm with a property as an insulator. Using the YM Broth liquid medium in the culture, It was performed for 48 hours static culture in keeping within the incubator at 28°C. Figure 4. shows the growth curve of yeast. When investigation of the growth curve in a state where the initial number of bacteria at 1000 CFU/ml, Results were obtained that number of bacteria is saturated at 6.5 × 107CFU/ml at 30 hours later. This result, when initial number of bacteria of about 1000 CFU/ml, it can be used as experimental samples in about 30 hours. Although the number of bacteria has become constant at 30 hours later, in consideration of variation of the initial number of bacteria, it was used as the culture 48 ± 3 hours. III. EXPERIMENTAL CIRCUIT In this experiment, using a Blumlein line to the generation of nanosecond pulses. Figure 5 shows the photos, the structure and the equivalent circuit. As shown in Figure 5 (a), Blumlein line is produced sandwiching the dielectric to the conductive Endosome Mitochondria About 5µm Lysosome Smooth Endoplasmic Reticulum Ribosome (a)Electrical equivalent circuit 10μm Cytoplasm Cell membrane Peroxisome (a)Micrograph of yeast (b)Cell schematic diagram of yeast Figure 3. Appearance of yeast (b)Frequency characteristic Figure2. Voltage distribution of cell membrane for frequency Figure4. Growth curve of yeast 14 12 10 Voltage[kV] plate. Using Kapton as a dielectric (DU PONT-TORAY CO., LTD., 500H/V, thickness with 125μm), aluminum plate having a thickness of 1mm as a conductive plate.Table1 shows circuit condition. The size of the Blumlein line in 200mm × 8.1mm(salinity of 0.3%) and 200mm × 41mm(salinity of 3%), the capacitance C is 9.63nF. It is added capacity shifting circuit for Blumlein charge. Using ceramic resistor (TOKAI KONETSU KOGYO CO., LTD., AS20 : 10kΩ) as resistance, ceramic capacitor (Murata Manufacturing Co., Ltd. N4700: breakdown voltage 30kV) as capacitor C0 in the circuit respectively. To 100% of the energy efficiency of the transition, the capacity of the C0 was 1.26nF(salinity of 0.3%) and 10.7nF(salinity of 3%). Using the trigatron spark gap switches and gas gap switch (SF6,0.4MPa) as a switch for operating the device. Operation sequence of the circuit is below. First the capacitor C0 is charged through resistor R of the circuit by a DC high-voltage power supply. After a trigger pulse is outputted from the trigger circuit to the trigger gap switch, switch is turned on by the discharge of the gap occurs. Then, charge of C0 transitions to C of the Blumlein line, to charge the Blumlein line. The voltage charged in the Blumlein line reaches the breakdown voltage of the gas gap switch with SF6 and occurs destruct discharge, square wave is output by a Blumlein line, it is transmitted to the terminating resistor. Figure 6 shows the waveform of the output pulse. Because of the rising velocity of the gas gap switch is insufficient, in Figure 6, Output voltage is the maximum voltage 13kV with pulse width is 7.29ns (FWHM). 8 6 4 2 0 -2 90 95 Aluminum plate (Conductive plate) Load Kapton (Dielectric) Aluminum (a) Blumlein line of photo and structure TABLE I. 115 120 CIRCUIT CONDITION Salinity [%] 0.3 3 Inductance L[nH/m] 196.5 19.4 C[nF/m] 4.53 45.85 C0[nF] 1.26 10.7 SF6 [atm] 4 4 Impedance Z0[Ω] 13.2 1.3 Repetition rate [pps] 1 1 Capacitor EXPERIMENTAL METHOD Cultures of yeast of sterilization sample was diluted 104 times by saline solution, Conductivity was appropriately adjusted. Beforehand has been confirmed that when yeast diluted by saline solution with salt concentration of 3% or less, yeast hardly dies by putting in saline. For applying IPEF, the experimental sample is poured in 200µl to a cuvette with the electrode for electroporation. Cuvette was prepared two pattern that for the application of an electric field and for positive control under the same conditions but without electric field is applied. Application conditions of pulse electric field is summarized in table 2. The temperature of the sample at the time of pulse electric field application is managed by the hot water bath or cooling device and the pump. For positive control stored in an incubator set to the same temperature as the hot water bath, while one sample is applying an electric field. After the pulse electric field application, the supernatant was removed 100μℓ of bacterial solution in the cuvette, diluted 10 times. After extraction of the diluted solution to the agar medium, it was performed for 48 hours culture in keeping within the incubator at 28°C. Using colony counting method for the measurement of the number of bacteria. TABLE II. (b) Equivalent circuit of Blumlein line Figure 5. Equivalent circuit of the ultra-short pulsed high voltage generator and structure of Blumlein line 105 110 Time[ns] Figure 6. Output waveform from the Blumlein line (Input voltage : 20kV, FWHM : 7.29ns) IV. Gas gap switch (SF6) 100 EXPERIMENTAL CONDITIONS Electric field strength E [kV/cm] 70, 100, 130 Pulse applying number [shot] 50, 100, 250, 500, 750, 1000 EXPERIMENTAL RESULTS AND CONSIDERATION B. Changes in temperature and salinity A sample solution obtained by adjusting the electrical conductivity with 63.3ms/cm, 6.27mS/cm by dilution of the culture medium with 3%, 0.3% saline solution respectively. Application conditions of pulse electric field is summarized in table 3. Figure 8(a) shows the sterilization result of the yeast of each temperature in 3% saline (conductivity 63.3ms/cm). This experiment trials is 4 times. When comparing the sample temperature 22.5 ℃ and 30 ℃, because almost no change in the number of bacteria, temperature of the sample in the vicinity ordinary temperature, there is little difference in the sterilization rate. On the other hand, when comparing the sample temperature 22.5 ℃ and 50 ℃, because the number of pulses necessary to sterilization is less at 50 ℃, sterilization speed increases. From these, we said that the sample temperature is a critical parameter in sterilization. Figure 8(b) shows the sterilization results of yeast in each temperature in 0.3% saline solution (conductivity 6.27ms/cm). This experiment trials is 3 times. In the result of a 3% saline solution as well as 0.3% saline, sterilization rate of nanosecond pulsed electric field sterilization by temperature increased is increased. As the cause of the result, we thought that lipid bilayer of the cell membrane properties is changed by temperature. Figure 9 shows The state of the cell membrane for each temperature. The treatment temperature (atmospheric temperature) is increased, the vibration of the lipid molecules TABLE III. EXPERIMENTAL CONDITIONS E [kV/cm] 70, 100, 130 Pulse applying number [shot] 50, 100, 250, 500, 750, 1000 Temperature [℃] 10, 22.5, 30, 40, 50 Saline solution [%] 0.3, 3 Number of yeast [CFU/ml] Electric field strength 10 4 10 3 Negative control Positive control 10 2 10 1 70kV/cm 100kV/cm 130kV/cm 1 1 10 2 3 10 10 Number of shots [Shots] 10 4 Figure 7: Sterilization result for each electric field strength 1.2 10°C 22.5°C 30°C 40°C 50°C 1 Survival ratio[-] A. Effect of the electric field strength Figure 7 shows the sterilization results of yeast per field strength of a sample solution obtained by adjusting the electrical conductivity with 63.3ms/cm by dilution of the culture medium with 3% saline solution. Number of trials is 3 to 4 times. In 70kV/cm, Because of the electric field strength is not sufficient, Could hardly sterilize. In 130kV/cm, as the number of applications increases, it has been almost sterilized. Number of bacteria in subsequent 1000shots is reduced to one of about 100 minutes. In 100kV/cm, that is the middle of the sterilization result, the number of dead yeasts increases as the number of applied pulses increases. Samples sterilized by applying a nanosecond pulses, it is necessary field strength greater than 70kV/cm. In addition, the number of applications and the survival rate is inversely proportional. However, Because of the sample is sufficiently sterilized, it is necessary to apply the 1000shots, we have to investigate the possibility of sterilization speed improvement. As means for improving the sterilization speed can be mentioned that the electric field strength is increased, the pulse width is increased. The former, because of the maximum output voltage that can be stably output of the experimental apparatus was 130 kV/cm, it was not able to experiment. In the future, we are implementing the experiment after improved device. The latter, because of the impedance of the package becomes higher, It does not make sense to increase the more the pulse width. Therefore, we investigated that the salinity and temperature of the brine with S. cerevisiae was changed as parameters. 0.8 0.6 0.4 0.2 0 10 2 10 Number of shots[shots] 3 10 (a) salinity of 3% 1.2 1 Survival ratio[-] V. 0.8 0.6 0.4 0.2 0 10 10°C 30°C 50°C 2 10 Number of shots[shots] 3 10 (b) salinity of 0.3% Figure 8 : The sterilization results for the electric field strength of 130kV / cm with salinity of 0.3% and 3% it is found that there is no difference in the effect of sterilization of pulsed electric field due to salinity differences. We thought that a strong influence of the temperature dependence than the synergistic effect of saline, there was no difference in the effects of sterilization. Lamellar gel phase Ripple gel phase Liquid crystal phase VI. Temperature Pulse-sensitive Low High (Sterilization rate) Figure 9 : The state of the cell membrane for each temperature 1.2 Survival ratio[-] 1 0.3% 10°C 3% 10°C 0.3% 50°C 3% 50°C 0.8 0.6 0.4 0.2 0 10 100 Number of shots[shots] 1000 Figure 10 : Sterilization comparison of the results of yeast by the salinity difference making up the cell membrane becomes vigorous, Accompanying this, the cell membrane is "coarse". We thought that the sensitivity of roughened cell membrane is higher to pulsed electric field. At low temperatures, because the cell membrane becomes more rigid crystalline state from liquid crystalline state, susceptibility to pulsed electric field is greatly reduced. That is not likely to sterilization. Sample temperature is the high temperature state at 40 ℃, 50 ℃, because of cell membrane of yeast has become more sterilized easy liquid crystal state, sterilization rate was improved than the lowtemperature state. Figure 10 shows a comparison of the sterilization result of yeast by the salinity difference. In nanosecond pulsed electric field sterilization of sample temperature at 10 ℃ , while satisfactory sterilization result has not been obtained with 0.3% saline, Sample could be sterilized to survival rate by about 20% in 3% saline solution and application of 500shots. From this result, we confirmed that influence of sterilization by salinity is remarkable at a low temperature state. Saccharomyces cerevisiae hardly dies by putting in saline although have a nonsalt-resistant. However, we thought that if the high salt concentration, when applying a pulse electric field stressed osmotic pressure, improved sterilization speed. We thought that if bacteria non-salt tolerant without limiting to S. cerevisiae, sterilization tends comparable is obtained. It is considered that the difference of osmotic pressure of the salinity and cytoplasm helps that the pulsed electric field gives the cell the sterilization effect. However, considered requires further investigation to compare whether undergoing degree of osmotic pressure. Further, in the high temperature state (50℃), STERILIZATION OF THE SAMPLE SIMULATING THE PACKAGED FOOD A. Experimental setup Package was manufactured using Kapton, Teflon sheets and double-sided tape. A procedure is shown below. Forming the external electrode size similar hole (11mm×11mm) in the cuvette in the heart of the Teflon sheet (30mm×30mm×0.5mm). Subsequently, It was the package that Kapton (20mm×20mm×12.5μm) is adhered to the Teflon sheet using a double-sided tape(thickness of 0.5mm). The thickness of the package is 1mm by the sum of the thickness of the Teflon sheet and the double-sided tape, has a structure that can handle samples of 121μl. Figure 11 shows appearance of sterilization of the packaging sample. Application conditions of pulse electric field is summarized in table 4. This application condition is conditions that is obtained the most sterilization effect in the cuvette. When packages are used as loads, the load matching with the Blumlein line becomes incompatible. Therefore, Packages add the cuvette with saline of 63.3mS/cm in parallel to match the impedance of the Blumlein. B. Experimental result and consideration Figure 12 shows sterilization result of the packaging sample. Figure 13(a) shows output voltage waveform. Number of trials is 3 times. From Figure 12, a significant reduction in yeast due to the application of a pulsed electric field wasn't observed. Therefore, consider the reason for yeast has not decreased. Figure 13(b) shows the frequency characteristic in the output voltage waveform. It showed a peak at 38.1MHz and 82.1MHz from the frequency characteristics. Namely, nanosecond pulses with this frequency are applied to packages. From here, it was calculated voltage being applied to the package. Table 5 shows the calculation results. 93MHz is the frequency of the rising time in output voltage of figure 13(a). Figure 11 : Appearance of sterilization of the packaging sample TABLE IV. EXPERIMENTAL CONDITIONS Electric field strength E [kV/cm] 130 Pulse applying number [shot] 25, 50, 100, 250, 1000 Temperature [℃] 22.5, 50 Saline solution [%] 3 From Figure 7, it is necessary to apply nanosecond pulses with 130 kV / cm for enable yeast sterilization. From Table 5, since the intensity of the electric field has been insufficient for the sterilization in the package, the sample has not been sterilized. 1.2 Survival ratio[%] 1 0.8 0.6 0.4 22.5℃ 50℃ 0.2 0 10 100 1000 10 VII. CONCLUSION In this paper, we aim to sterilization of packaged food, it was carried out sterilization experiments in high-frequency high voltage pulse of the order to reduce the impedance of the package. As a result of applying a high frequency pulse electric field, It has been found that it takes a considerable amount of time in order to obtain a sufficient sterilization result in the electric field strength 130kV / cm. Therefore, we investigated that sterilization effect when allowed to salinity and temperature changes of the sample. At 50 ℃, it was found that it is possible to improve the sterilization rate. It can potentially be sterilized at a low temperature of about 50 ° C. with the use of a pulsed electric field. It is considered that the difference of osmotic pressure of the salinity and cytoplasm helps that the pulsed electric field gives the cell the sterilization effect. However, the higher frequency component is needed in order to sterilize the packaged food with high conductivity. 4 Number of shots[shots] REFERENCES Figure 12 : Sterilization result of the packaging sample [1] 30 30 20 25 10 20 0 15 -10 10 -20 5 -30 0 Input voltage[kV] 35 -40 0 50 100 Time[ns] 150 -5 200 [2] [3] Output voltage[kV] 40 0.35 0.3 Amplitude[kV] 0.25 0.2 38.1MHz 0.1 82.1MHz 0.05 0 -0.05 0 50 100 Frequency[MHz] 150 (b)FFT Figure 13 : Output voltage waveform TABLE V. THE ELECTRIC FIELD INTENSITY APPLIED TO THE INSIDE OF THE PACKAGE Frequency [MHz] 38.1 82.1 93.0 [5] [6] (a)Output voltage 0.15 [4] Inside electric field [kV/cm] 6.8 14.4 31.8 T. Ohshima, K. Okuyama, M. Sato, “Effect of culture temperature on high-voltage pulse sterilization of Escherichia coli,” J. Electrostatics, Vol. 55, pp. 227-235, 2002. D. Garcia, N. Gomez, S. Condon, J. Raso, R. 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