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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
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