Innovative Food Science & Emerging Technologies 1 Ž2000.

135᎐149

Effects of pulsed electric fields on cell membranes in real

food systems

Alexander AngersbachU , Volker Heinz, Dietrich Knorr

Department of Food Biotechnology and Food Process Engineering, Berlin Uni¨ ersity of Technology, Konigin-Luise-Str.
¨ 22, D-14195
Berlin, Germany

Received 19 October 1999; accepted 15 March 2000

Abstract

High frequency current and voltage measurements were used to determine passive electrical properties, such as the
polarization effect at intact membrane interfaces and field-induced electropermeability changes in the cellular materials during
direct current pulses. The time sequence of the electropermeabilization at the levelc of the cell membrane showed a similarity to
the breakdown phenomena observed in cell systems Žpotato, apple and fish tissues, as well as plant cell suspension cultures . when
a single pulse with critical or supercritical field amplitude is applied. A slight membrane breakdown phenomenon occurred in the
first few microseconds after the initiation of the pulse at a critical electric field strength of 150᎐200 Vrcm. Significant membrane
breakdown was observed when the field strength of the electric pulses applied directly on the cell systems was in the range of
400᎐800 Vrcm. At various field intensities, the electrical potential across a cell membrane reached a critical value of
approximately 0.7᎐2.2 V. The initiation of conductive channels across the membrane occurred within nanoseconds during the
charging process of the membrane, whereas the formation of a high-conductance membrane due to pore expansion took place
within a few microseconds. The application of a single pulse, even with supercritical field amplitude, does not necessarily cause an
irreversible membrane rupture. The insulating properties of the cell membrane can be completely recovered within several
seconds after the termination of the pulse. The biological and engineering aspects of the membrane permeabilization are
discussed in this paper. These data are utilized as the basis for the design and optimization of high-intensity pulsed electric field
applications in the areas of food science and biotechnology. 䊚 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Pulsed electric fields; Cell membrane; Electropermeabilization

Industrial rele¨ ance: This paper demonstrates the complexity of the membrane permeabilization effects of high intensity electric field pulses and
the potential to convert this basic knowledge into effective processes where controlled membrane permeabilization is required. It is further
encouraging to note how quantifiable the kinetics of reversible or irreversible membrane permeabilization can be identified.

1. Introduction first field effect results in a drastic increase in perme-

The application of an external electric field can
induce a critical electrical potential across the cell
membrane, which leads to rapid electrical breakdown
and local structural changes of the cell membrane. This
U
Corresponding author. Tel.: q49-30-314-71250; fax: q49-30-832-
7663.
E-mail address: foodtech@mailszrz.zrz.tu-berlin.de ŽA. Angersbach..
ability due to the appearance of pores in the mem-
branes. Based on this phenomenon, many practical
applications of high electric fields for the reversible or
irreversible permeabilization of various biological sys-
tems have been studied in the fields of medicine and
biology ŽChang, Chassy, Saunders & Sowers, 1992;
Zimmermann & Neil, 1996; Lynch & Davey, 1996; Ho
& Mittal, 1996.. Most of the studies in food science
have concentrated on the effects of high-intensity elec-
tric field pulse ŽHELP. treatments on the inactivation

1466-8564r00r$ - see front matter 䊚 2000 Elsevier Science Ltd. All rights reserved.
PII: S 1 4 6 6 - 8 5 6 4 Ž 0 0 . 0 0 0 1 0 - 2
136 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149

Nomenclature data are commonly based on studies of secondary,

␶: Polarization time constant post-field effects such as the inactivation rate for mi-
t: Time crobial populations, stress reactions on biological mate-
t pore : Time of beginning pore formation after pulse
rise
rials, or diffusions from cells. The secondary effects can
Am: Membrane area of an average cell in the field also depend on other process parameters, such as pulse
direction duration, energy input and the number of pulses. Very
d: Average cell size little information is available regarding membrane
lm: Membrane thickness permeabilization kinetics, and on reversible᎐irreversi-
␴: Effective electrical conductivity of the sample
␴c : Conductivity of the cell electrolyte
ble structure changes of cells in real food systems
␴a : Conductivity of the extracellular electrolyte during and after the application of HELP. A funda-
␴ma x : Conductivity of the cells system at t ª 0 mental understanding of these phenomena is essential
␴min : Conductivity of intact cells system at t ) 5␶ for optimal process design and for the characterization
␧m : Dielectric constant of cell membrane of critical process parameters of pulsed electric fields in
␧c : Dielectric constant of cell electrolyte
x: Volume fraction of cells
the food industry.
E0 : External field strength, obtained from voltage This paper summarizes part of our ongoing work,
peak amplitude aimed at the identification of crucial parameters of
E: External field strength, obtained from voltage pulsed electric field processing, such as critical external
decay electric field strength, the breakdown voltage of the cell
j: Current density through the sample
jc : Density of current flowing in an average cell
membrane and the voltage range of reversible mem-
ja: Current density through elementary non- brane permeabilization. The time sequence and the
cellular unit dynamics of the membrane electropermeabilization
jma x : Density of the peak current flowing in the process were also studied. We used the fact that the
sample at t ª 0 electric field-induced permeability variation of initially
jmin : Density of current flowing in the extracellular
part of the sample at t ) 5 ␶
insulating biological membranes due to pore formation
jp: Density of polarization current flowing in an is reflected in large conductivity changes in cell mem-
average cell branes and, consequently, in the tissues or cell suspen-
ic: Current flowing in an average cell sions.
i p: Polarization current flowing in an average cell By measuring current and voltage simultaneously
im: Current flowing through the permeabilized
membrane
with a high time resolution, it was possible to monitor
␸: Transmembrane voltage the process of pulse-induced membrane permeabiliza-
Cm : Specific membrane capacitance tion as a function of time. For a better understanding
Aw : Total area of pore surfaces Žporated membrane of these effects, the impact of relatively long, single
area. of an average cell electric field pulses was evaluated. These pulses were
Fw : Fraction of porated membrane surface
Gm : Membrane conductance
generated by storage capacitor discharges with varying
amplitude of the pulse voltage. The data obtained by
pulse repetition gave relevant information about the
recovery of the membrane structure after few pulses,
of microorganisms ŽDoevenspeck, 1961; Sale & Hamil- and of irreversible permeabilization with repeated
ton, 1967; Martin, Zhang, Castro, Barbosa-Canovas & pulses.
Swanson, 1994; Qin, Pothakamury, Vega, Martin, Bar-
bosa-Canovas & Swanson, 1995; Grahl & Markl, ¨ 1996.
The irreversible permeabilization of cell membranes in
plant tissues offers a wide range of process applications 2. Materials and methods
where cell membrane disruption is required, including
the expression, extraction or diffusion of plant 2.1. Cell materials
metabolites, as well as affecting the mass and heat
¨
transfer of the food products ŽDornenburg & Knorr, Tissue samples: Fresh potatoes ŽSpunta. and apples
1993; Knorr, Geulen, Grahl & Sitzmann, 1994; Angers- ŽJonagold. were purchased from a local market. Trout
bach & Knorr, 1997; Knorr & Angersbach, 1998.. The fillets were treated within 1 h after slaughter.
process development must be based upon the Cell culture: Potato cells Ž Solanum tuberosum, ‘De-
knowledge of the cell-specific critical transmembrane siree’. were grown in MS medium ŽMurashige & Skoog,
voltage, ␸c wfor many different types of cell and artifi- 1962. for 14 days at 25⬚C in the dark on a reciprocatory
cial membranes, ␸c is found to be approx. 1 V ŽHo & shaker. Cell suspensions in the exponential growth
Mittal, 1996.x, or of the critical external electric field phase were filtered and resuspended in fresh medium
strength Ec . with a conductivity of 3.5 mSrcm, which was equal to
Published information about Ec is limited and the the conductivity of the cell electrolyte. The volume
 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
ratio of the cells in the suspension was approximately
0.7.
All samples were prepared for treatment as cylinders
Žthe dimensions of the samples perpendicular to the
electrical field: 2 cm2 ; length: 1 cm..
2.2. Electric field pulses protocol
The exponential electric field pulses were applied
with a high intensity pulsed electric field apparatus,
designed and constructed in our department. The cen-
tral components of the pulse generator consisted of a
capacitor charging unit ŽFUG, HCK 800M-20.000,
Rosenheim, D. with pre-selectable charging voltage
and current, a 2-␮F storage capacitor ŽMaxwell,
噛30560, San Diego, CA, USA., a treatment chamber
and a high voltage switch ŽBehlke, HTS 101-80-FI,
FrankfurtrMain, D.. The pulse shape was monitored
on-line with a 100-MHz digital storage oscilloscope
ŽTektronix, TDS 220, Wilsonville, OR, USA.. The volt-
age and current at the treatment chamber were regis-
tered with a 75-MHz high voltage probe ŽTektronix,
P6015A, Wilsonville, OR, USA. and a 100-MHz cur-
rent probe ŽTektronix, A6312, Wilsonville, OR, USA.
connected to an amplifier system probe ŽTektronix, AM
503B, Wilsonville, OR, USA.. The rise time of the
chamber voltage was approximately 200 ns.
The treatment chamber was equipped with two
polished stainless steel cylindrical electrodes which were
placed in the sample containing a PVC tube ŽHeinz,
Phillips, Zenker & Knorr, 1999.. The electrode area
was 2 cm2 . The gap was adjusted to 1 cm. The ohmic
resistance of the treatment chamber containing the cell
material varied in the order of 90᎐2500 ⍀ Žthe resis-
tance of the electrical leads was ; 1 ⍀ . in accordance
with the degree of cell membrane permeabilization.
The peak field strength E0 was gradually increased
from 50 up to 2000 Vrcm, whereas the pulse duration
was adjustable in a range between 0.9 and 25 ms.
The temperature increase in the treated samples due
to Joule heating was low in all experiments. A single
pulse with a maximum field strength of 2 kVrcm
caused a temperature increase of ⌬T s 0.55⬚C in the
sample Ženergy input 2 kJrkg.. Treatment with ten
repeated pulses Žfield strength amplitude E0 s 700
Vrcm. caused a temperature increase of ⌬T s 0.68⬚C
in the sample Žtotal energy input 2.45 kJrkg.. Hence,
temperature related alterations in conductivity were
not considered. The initial sample temperature was
approximately 20⬚C in all experiments.
2.3. Theoretical considerations
2.3.1. Charging time constant and cell model
The intact cell membrane can be regarded as an
137
insulator separating two conducting aqueous phases,
i.e. as a dielectric in a parallel plate capacitor ŽSchwan,
1957, 1977; Pauly, 1964.. The application of an external
electric field induced the charging process at the mem-
brane interfaces. The relaxation time constant, ␶, is a
basic macroscopic characteristic for cell membrane
charge, which depends on cellular size, membrane ca-
pacitance, and the conductivity of cell and extracellular
electrolyte. Therefore, a model for examining systems
containing cells which is sufficiently described by the
relaxation time ␶, should also reflect other electric
properties of a cell.
Theoretical predictions of ␶ for a spherical cell model
and for a planar bilayer membrane cell model Žcubic or
multilayer cell model. are shown in Table 1. The use of
a cubic cell model for the analysis of electrical proper-
ties assumes that the membrane regions involved are
two planar areas on opposite sides of a cell. Consider-
ing only the cell membrane geometry in the field direc-
tion is permissible for large cells in biological tissue
which have a very small membrane-thicknessrcell-size
ratio Ž5 nm:50᎐100 ␮m. ŽHayden, Moyse, Calder,
Crawford & Fensom, 1969; Zhang & Willison, 1991;
Kuang & Nelson, 1998; Angersbach, Heinz & Knorr,
1999.. The application of the cubic cell model accu-
rately describes a simulated behavior during the appli-
cation of electric field in the case of a yeast cell Ž S.
cere¨ isiae. with a linear dimension of 8 ␮m ŽWeaver &
Barnett, 1992..
The comparison between theoretical and experimen-
tally obtained charging time constants in Table 1 shows
that the cell model with planar membrane areas pro-
vides a better description of the passive electrical be-
havior of an individual cell, and, therefore, gives a
more realistic basis for describing the charging process
and the breakdown of cell membranes during applica-
tion of electric field pulses.
In the following, the simplified cubic cell model with
planar geometry is used for examination of cellular
samples.
2.3.2. Discharging pulse and electrical properties of cells
system
A typical time course of the applied discharge pulse
on tissues or suspensions with a high cell volume ratio
is presented in the Fig. 1. The relationship between
current density, j, and field strength, E, gave the in-
stantaneous sample conductivity as ␴ s jrE. For the
calculation of the electrical properties of the cells, it
was assumed that the external electric field E0 in the
initial 8 ␮s after the start of the pulse Žouter rise time
period, approx. 0.2 ␮s. was constant.
A typical current flow across the cells in tissues or
suspensions within a short Ž␮s range. period after the
application of an external electrical field Žassuming the
 138
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
Table 1
Electrophysical properties of intact cell systems a

Sample Average Cell Relaxation time constant of the cell membrane ␶ Ž␮s. calculated according to: Relaxation time
cell size electrolyte Spherical model Multilayer model Cubic cell constant
d c Ž␮m. b conductivity ŽSchwan, 1957, 1977. c Žexperimental. e
ŽKuang & Nelson, 1998. modeld
␴c ŽmSrcm. dc 0.5⭈ ␧ m ⭈ d c q ␧ c ⭈ l m d c ⭈ Cm ␶ Ž␮s.
1 1
␶ s ⭈ Cm ⭈ Ž q . ␶ s ␧0 ␶s
2 ␴a 2 ⭈ ␴c ␴c l m 2 ⭈ ␴c

Potato tissue 100 5.5 1.40 0.40 0.90 0.70

Apple tissue 70 1.2 3.50 0.95 2.30 1.40
Fish tissue 75 6.0 0.94 0.26 0.62 0.63
Suspension cultured 50 3.5 1.10 0.30 0.66 0.45
potato cells
a
Cm is the specific capacitance, for biological membranes Cm is in the order of 1 ␮Frcm2 ŽSchwan, 1977.. Further note: ␧ 0 s 8.85= 10y1 2 Frm; ␧ m s 2.3; ␧ c s 78; l m s 5 nm.
b
Light microscopic examination of wet sections of the tissues investigated in this work showed that examined cells were roughly isometric.
c
Calculated under the assumption that the conductivity of cell electrolytes, ␴c , and extra cellular conductivity, ␴a , are identical.
d
According to a simplistic cell model with one-dimensional planar membrane.
e
Determined directly from experimental relaxation curves during DC pulse with subcritical field strength Žsee Figs. 2 and 4..
 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149 139

Fig. 1. Current density, j, field strength, E, and effective tissue conductivity, ␴, vs. time during a single discharge pulse of a 2-␮F storage
capacitor at various field strength amplitudes, E0 , through potato tissue. For E0 s 66 Vrcm, the peak current density was 0.27 Arcm2 and the
total pulse width was 15 ms.

Fig. 2. Time course of field strength Ža, b. and current density Ža⬘, b⬘. in a potato cylinder in the first 8 ␮s of a pulse with a field strength
amplitude of 66 Vrcm Ža, a⬘. or of 880 Vrcm Žb, b⬘.. The complete pulse course is shown in Fig. 1. The Roman numerals in Ža⬘. are defined
within the text ŽSection 2.3..
140 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
passive electrical properties do not change. is shown in
Fig. 2a. As for a heterogeneous dielectric, the current
time course could be divided into three characteristic
periods. In the first instant, during the rise of the
electric field, electrical polarization and charging of the
‘geometrical’ capacity caused the rapid development of
current peak jmax Žcurrent jump, period I.. During the
second period ŽII., polarization effects arose from the
induced charge accumulation at the interface of intact
membranes ŽMaxwell᎐Wagner effect.. The third period
related to a time range of 3᎐5 ␶. The polarization
process was terminated and the electrical current den-
sity decreased to a minimal value, jmin . When E0 s
constant, jmin remained constant Žperiod III. and was
adjusted through the electrical conductance of the ex-
tracellular liquid.
The same effects for biological matter were postu-
lated in the frequency-domain as ␣- and ␤-dispersion
ŽSchwan, 1957, 1977.. The Laplace transformation may
be applied to translate the Maxwell᎐Wagner effect at
the membrane interface in the DC field Žtime-domain.
to an AC field Žfrequency-domain.. The analysis of
conductance spectra within the ␤-dispersion range of
3᎐50 MHz ŽAngersbach et al., 1999. and of the conduc-
tivity relaxation in the time range from 0.2 to 8 ␮s after
the beginning of the pulse Žsee Fig. 4, curve number 1.
provided identical information for all materials ex-
amined according to the passive electrophysical proper-
ties of the cell system, such as volume ratio of intact
cells in the sample, charging time constant of the cells,
or maximum and minimum values of sample conductiv-
ity Ž ␴max , ␴min ..
The time dependent density of the direct current, j,
flowing through the elementary layer Žorientated per-
pendicular to the electric field. of the system contain-
ing cells after the application of an external electric
field E0 could be described as:
j s x⭈ jc q Ž 1 y x . ⭈ ja
where x is the volume ratio of intact cells; jc is the
density of current flowing in an average cell; and ja is
the current density, flowing through an elementary
non-cellular unit.
Here, it was assumed that the layer thickness was
equal to the average cell size and that the cells were
uniformly distributed within the sample.
The field-induced time-dependent voltage drop across
a cell membrane Žneglecting the natural potential of a
biological membrane, which was in the order of 10y2 V
ŽTsien, Hess, McClesky & Rosenberg, 1987; Tsong,
1996. can be expressed by Eq. Ž2.:
d j
␸s E0 y c
ž /
2 ␴c
The following equation is related to first order differ-
ential equations which describe the interaction of sev-
eral membrane properties, i.e. the parallel combination
of a capacitance Cm and a resistance Rm in the equiva-
lent circuit with cell current ic s jc. Am .
d␸
i c s i p s Cm if t - tpore Ž3.
dt
For t - tpore the polarization current charges the
intact membrane. At t ) tpore , the formation of aqueous
pores on the membrane interface occurred and the
instantaneous current, im , flowing across the membrane
developed. This depended on the fraction of the aque-
ous pores and on decreased transmembrane voltages.
The cell current was given as:
ic s i p q im if t ) t pore Ž4.
For an intact cell, the current ic was equal to the
polarization current ip as a result of the charging of the
insulated membrane through the cell electrolyte Žin
case of high cell concentration . from the pulse genera-
tor. With this assumption, Eq. Ž3. could be transformed
to give the current density, and could be written as
follows:
jc s jp s ␴c ⭈ E0 ⭈ exp Ž ytr␶ . Ž5.
where t is the process time and ␴c is the specific
electrical conductivity of the cell electrolyte. From Eqs.
Ž2. and Ž5. the transmembrane potential for an intact
membrane can be given as:
d
␸s E w 1 y exp Ž ytr␶ .x Ž6.
2 0
Ž1.
Eq. Ž1. shows the relationship between the instanta-
neous current density in the interior of the cells, jc ,
and the total current density in the sample, j, which
could be measured. The values of x and ja in Eq. Ž1.
could be eliminated by analyzing the relationships given
by the exposure to a subcritical electrical field. For cells
with intact membranes at the onset and at the end of
the polarization process waccording to Eq. Ž5.x the fol-
lowing equations applied:
At t ª 0 jmax s x⭈ ␴c ⭈ E0 q Ž 1 y x . ⭈ j a Ž 7a .
At t G 5␶ jmin s Ž 1 y x . ⭈ ja Ž 7b .
where jmax and jmin denote the experimentally ob-
Ž2.
tained current density in the sample at the characteris-
 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
tic time periods t ª 0 and t G 5 ␶, respectively Žsee Fig.
2aX .. Hence, Eqs. Ž1., Ž7a. and Ž7b. give:
jmax y jmin
js ⭈ jc q jmin
␴c ⭈ E0
Eq. Ž8. allowed the calculation of the current jc
flowing in an average cell interior from one experimen-
tal current᎐time curve when the membrane is intact.
During the application of high field strength pulses,
membrane rupture occurs, and the cell current jc can
be determined as follows. Assuming that the contribu-
tion of the current density jmin , flowing through the
extracellular part, to the total current density of the
cell system with intact or permeabilized membranes
remained unchanged, the cell current density jc in the
case of a ruptured membrane could be described as:
␴c ⭈ E0 ⭈ w j y j⬘min x
jc s
jmax y j⬘min
with
jmin ⭈ E0
j⬘min s
Esub
where jmin is obtained from experimental data by the
application of a first pulse on a subcritical field strength
level, Esub Žfor examined cell materials, as a rule lower
than 100 Vrcm.. The values of jmax and j were ob-
tained from recorded current᎐time curves by the appli-
cation of a second pulse with critical or supercritical
field strength Ec in the same sample.
For the ruptured membrane, the relationship
between a membrane current, im , and the transmem-
brane voltage wEq. Ž2.x gave the membrane conduc-
tance, Gm s im r␸, which could be attributed to the sum
of the instantaneous conductances of all individual
transient pores. The determination of the membrane
current, im , deduced from Eq. Ž4., was based on the
assumption that the membrane capacitance, Cm ,
changed only negligibly during the formation and
widening of the pores. This assumption holds because
only a small fraction of the membrane was occupied by
aqueous pores ŽFreeman, Wang & Weaver, 1994; Win-
terhalter, Klotz & Benz, 1996., and the polarization
current ip exponentially decays even further after mem-
brane breakdown.
Assuming that the increase in membrane conductiv-
ity during field-induced breakdown resulted from the
formation of cylindrical pores with ohmic behavior, the
time-dependent total Žaqueous. pore area, Aw , of the
membrane with cell liquid-filled pores was given by:
lm ⭈ Gm
Aw s
␴c
141
where lm is the membrane thickness Ž; 5 nm.. Eq. Ž11.
is valid when the resistance of the lipid area is very
high and the solution conductivity in the individual
pore is unchanged.
Ž8.
3. Results and discussion
In the present study, the pulse width of fully expo-
nential capacitor discharges through the material ex-
amined was strongly dependent on the effective sample
resistancerconductivity, which varied in correspon-
dence to with the applied field strength. In Fig. 1, this
is illustrated using potato tissue.
Obviously, in the experiment with a pulse amplitude
of E0 s 66 Vrcm, the field strength was not sufficiently
high to initiate electropermeabilization of the cell
membranes. The discharge current only passed through
the extracellular matrix of the potato tissue because
Ž9.
the intracellular electrolyte was isolated by intact mem-
branes. The effective conductivity remained at its lowest
value wapprox. 0.33 mSrcm, which is common for the
measurement of passive electrical properties of intact
potato tissue with DC or AC in the 1᎐5 kHz range
Ž 10.
ŽShaw & Galvin, 1949; Hayden et al., 1969; Angersbach
et al., 1999.x. Consequently, the current density and
field strength decayed exponentially, with the highest
time constant of ; 3 ms.
In contrast to the first test for the pulse application
with an amplitude of 880 Vrcm, the field strength E
decayed exponentially with a very low time constant,
approximately 200 ␮s. The total sample conductivity
increased to 5.5 mSrcm as a result of the elec-
tropermeablization of previously insulating cell mem-
branes.
Fig. 2 illustrates the detailed time course of j and E
in potato tissue in the first 8 ␮s after the initiation of
the pulse with amplitudes of 66 and 880 Vrcm Žthe
complete pulse sequence is shown in Fig. 1.. Through
the application of a sub-critical field strength, the exis-
tence of insulated membrane properties was clearly
detected by means of an exponentially decaying polar-
ization current in the cells within the first few mi-
croseconds. With a field strength level of E0 s 880
Vrcm, the current through the samples showed a devi-
ation from exponential decay at 0.4 ␮s, indicating that
electric breakdown in the cell membranes had oc-
curred.
3.1. Membrane polarization and electroporation
The changed conductivity of the total sample in the
pulse experiment yielded integral information about
the extent of the pulse-induced changes in the struc-
tural properties of the complete cell system. The time
Ž 11.
sequence and dynamics of the electropermeabilization
142 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149

at the level of a cell membrane can be characterized by

the present model of cell systems, which is expressed in
Eqs. Ž1. ᎐ Ž11.. The equations were obtained with the
assumption that within this small time period, the ex-
ternal field strength, which is equivalent to the pulse
amplitude, is constant Žsee Fig. 2a,b.. By the applica-
tion of a pulse with a field strength amplitude of 66
Vrcm, the membrane was exponentially polarized to
␸ s 0.3 V without an influence on its structural state.
At higher pulse amplitudes Žsuch as 880 Vrcm., a
current flowed towards the membrane through the
intracellular electrolyte and the transmembrane poten-
tial, ␸, increased exponentially with time Žtime constant
0.7 ␮s.. Close to 0.4 ␮s, ␸ reached a value of 1.7 V, and
later decreased to a residual value of 0.1 V, as a
consequence of pore formation and a drastic increase
in membrane conductance ŽFig. 3.. As soon as the
membrane was ruptured, the membrane current, im ,
was also initiated, which in turn resulted in the sudden
observed increase in cell current, ic .
The decay of the membrane voltage and the simulta-
neous increase of the membrane current, im , indicated
that the membrane conductance, Gm , had drastically
increased. There was a sudden rise in the membrane
conductance from approximately 10y8 S wthe initial
specific conductivity of a cell membrane was of the
Fig. 3. Typical current, voltage and membrane property changes in
order of 10y4 Srcm2 ŽWinterhalter et al., 1996; an individual cell Že.g. a potato cell in tissue. induced from an
Zimmermann, 1996.; the membrane area of the potato external electrical field with supercritical field strength: Ža. currents
cell in the field direction was 10y4 cm2 x to 5 = 10y3 S and Žb. transmembrane potential in an average potato cell; Žc.
in less than 5 ␮s. Such a massive conductivity increase membrane conductance, Gm , and total pore area, Aw , increase in the
membrane surface in the field direction. Field strength amplitude
could be explained by the formation of an aqueous
E0 s 880 Vrcm Žtime course of field strength and current density in
phase at the membrane interface which has been the sample is shown in Fig. 2b,b⬘.. The intracellular current, ic , was
observed for artificial lipids as well as for cell mem- calculated as ic s jc ⭈ Ac by using the Eq. Ž9. in which j⬘min s 0.32
branes ŽZimmermann, 1982; Chernomordik, Sukharev, Arcm2 Žobtained according to Eq. Ž10... im s current through the
Popov, Pastushenko, Sikorko, Abodor & Chizmadzhev, membrane. The polarization current, ip , was calculated as ip s jp ⭈ Ac
with polarization current density, jp , obtained from Eq. Ž5. using
1987; Weaver & Barnett, 1992; Ho & Mittal, 1996.. A
␶ s 0.7 ␮s.
rapid rupture of limited areas of the cell membrane,
but not of the entire membrane, may be possible
ŽChernomordik et al., 1987; Freeman et al., 1994; Ho & aqueous area of the membrane, Fw s Aw rAm , was
Mittal, 1996.. In the case discussed above, the total found to be small and never exceeded 10y2 Ži.e. ap-
area of aqueous pores, Aw , reached a maximum value prox. 1% of the membrane area.. Similarly, 0.01᎐1% of
of up to 46 ␮m2 ŽFig. 3c., suggesting that approxi- lipid bilayer surface was affected by such defects
mately 0.46% of the membrane area in the field direc- ŽChernomordik, Sukharev, Abidor & Chizmadzhev,
tion was filled with an aqueous phase. This small maxi- 1983; Tsong, 1990..
mum value of the fraction of aqueous area of the A better comparison of experimental data through
membrane supports the assumption that the structural the application of various field strengths is given by the
changes within the entire membrane can have a negli- presentation of j᎐E data as effective conductivity ␴ s
gible change in membrane capacitance ŽFreeman et al. jrE0 ŽFig. 4.. For all cell materials examined, the shape
1994; Winterhalter et al., 1996.. of the time behavior of ␴ indicated excellent repro-
The data analysis for all cell materials examined ducibility for the membrane charging processes before
indicated that only a small fraction of the membrane the start of the membrane rupture and also for various
was occupied by pores. This is typical for reversible external field strength E0 . The polarization time up to
electroporation phenomena in biological and artificial the moment of membrane breakdown and the dy-
membrane breakdown ŽHo & Mittal, 1996; Freeman et namics of pore formation were strongly influenced by
al., 1994.. Although dramatic changes occurred in the the field strength. When higher amplitudes than the
membrane conductivity during the pulse, the fractional critical level were used, the conductivity changes of the
 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149 143

Fig. 4. The field induced permeabilization in the first 12 ␮s after pulse start with various field strength amplitude E0 ŽVrcm. for: potato tissue
Ža.: 1᎐100, 2᎐250, 3᎐400, 4᎐680, 5᎐1000, 6᎐1700; apple tissue Žb.: 1᎐100, 2᎐270, 3᎐550, 4᎐750, 5᎐1100, 6᎐1700; culture cell suspension Žc.:
1᎐110, 2᎐250, 3᎐330, 4᎐590, 5᎐1100, 6᎐1800; fish tissue Žd.: 1᎐80, 2᎐300, 3᎐500, 4᎐900. The effective conductivity course was calculated from
monitored current and voltage variation with time. The shortest total pulse width in all experiment was 900 ␮s.

sample show a deviation from exponential decay in the
short time scale after pulse initiation. This indicated
that electric breakdown in the membranes of individual
cells occurred during the charging process, i.e. in the
time period of 3᎐5 ␶. In all experiments, the charging
process was followed by a rapid development of the
membrane current which was caused by a rupture and
the evolution of high conductance membranes, initi-
ated by the critical or supercritical electrical field.
With increasing field strength, the charging time up
to the moment of membrane rupture was reduced, and
the rate of pore widening and the total area of the
pores of the membrane increased. This was reflected by
the rise in conductivity of the samples after apparent
membrane permeabilization ŽFig. 4..
3.2. Biological aspects of the membrane permeabilization
Several hypotheses have been established that rea-
son that in real cell membranes, pore initiation may
occur in lipid domains or at protein channels
ŽChernomordik et al., 1987; Glaser, Leikin, Cher-
nomordik, Pastushenko & Sokirko, 1988; Tsong, 1990;
Zimmermann, 1996; Ho & Mittal, 1996.. In all our
experiments it was observed that the immediate initia-
tion of conductive channels across the membrane and
the rapid formation of a high conductance membrane
due to their expansion took place in less than 1 ␮s. The
speed of pore formation suggests that this action was
located at the lipid domain of the cell membrane
rather than at protein channels. Pore initiation was
most likely triggered by defects in the lipid structure
and occurred in the range of nanoseconds ŽCoster &
Zimmermann, 1975; Benz, Beckers & Zimmermann,
1979; Gruenwald, Frisch & Holzwarth, 1981; Zimmer-
mann, 1996.. The pore widening of the lipid bilayer
happened in the microsecond range ŽSerpersu, Kinosita
& Tsong, 1985; Ho & Mittal, 1996.. Other issues also
needed to be considered. If a protein channel was
punctured by an electrical potential, the local heating
due to excessive membrane current might thermally
denature the protein Žor modify it electrically ., a reac-
tion known to occur in the range of milliseconds᎐sec-
onds ŽKim & Baldwin, 1990; Tsong, 1996.. Within a cell
membrane, the opening and closing of protein channels
could be controlled by the transmembrane electrical
potential. The gating potential of a typical ion channel
is in the range of 50 mV ŽTsien et al., 1987; Tsong,
1996.. The argument that an intense pulse may force
these channels to open before the breakdown of the
lipid bilayer occurs may not be valid, because the
charging curve up to the breakdown point Žand also by
super-critical field intensity . showed an exact exponen-
tial course with a time constant ␶, which was character-
istic of an intact unchanged membrane Žsee Figs. 2 and
4.. The simultaneous opening of protein channels and
144 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
Fig. 5. Critical transmembrane voltage for membranes of cells in
potato tissue Ž䢇., a potato cell culture Ž⽧., apple tissue ŽB. and fish
tissue Ž^. as a function of field strength amplitude. Critical voltage is
defined as the maximal electrical potential across the cell membrane.
initiation of pore formation in the lipid matrix is quite
unlikely. Furthermore, by the application of a single
pulse, we observed only reversible field-induced changes
of the membrane structures. An application of a sec-
ond pulse with a field strength of 700 Vrcm to potato
tissue ŽFig. 7. showed that in the time period between
the pulses, the properties of the sample before perme-
abilization was repeated were almost identical to the
intact state. This suggests an almost complete recovery
of the membrane structure after the first pulse. How-
ever, a recovery of protein channels within 1 s is
unlikely.
Several studies also suggested that the early stages of
electroporation of biomembranes are primarily associ-
ated with the appearance of pores in the lipid matrix,
rather than in the protein-rich domains of membranes
ŽChernomordik et al., 1987; Glaser et al., 1988; Neu-
mann, 1989; Chernomordik, 1992..
3.3. Critical transmembrane potential
Although various cell materials have different mor-
phological structures and electrophysical properties, the
similarity between the voltage dependent membrane
ruptures suggested that the same mechanism was re-
sponsible for the electropermeabilization of the cell
membranes, namely, charging of the insulated mem-
brane up to a critical value. Thus, the direct field
effects on the membrane structure were expressed as
interfacial polarization up to strong transmembrane
fields Žin our experiments in the order of 1400᎐4400
kVrcm, considering a membrane thickness of 5 nm.
which, in turn, induced structural changes ŽNeumann,
1989; Ho & Mittal 1996..
For apple cells, the maximum transmembrane volt-
age achieved Ž1.3 V. was close to the threshold value
Žapprox. 1 V., at which membrane breakdown was first
observed in a series of simulations in which E0 was
varied. When a slightly smaller transmembrane poten-
tial Že.g. ␸ - 0.7 V. was used, no membrane rupture
occurred. Similar results were found with cell mem-
branes in potato and in the corresponding cell suspen-
sions, as well as in fish tissues ŽFig. 5..
The critical transmembrane voltage for most eukary-
otic cells has been reported to be approximately 1 V
Ž0.5᎐1.5 V., independent of the method used ŽSale &
Hamilton, 1967; Coster & Zimmermann, 1975; Ho &
Mittal, 1996; Lynch & Davey, 1996; Zimmermann,
1996.. In our experiments the lowest breakdown volt-
age was approximately 0.7 V Žapple tissue, E0 s 200
Vrcm and potato cultures suspension, E0 s 250 Vrcm..
However, such a value was only observed for the exper-
iments when the charging time of the membrane was
approximately 3᎐5 ␶ Žfor apple tissue, at 5.0 ␮s; for
potato cell suspension cultures, at 1.9 ␮s., i.e. at the
end of the exponential charging process. In this case,
the external field strength could be defined as critical.
For potato tissues, the first indication of membrane
breakdown was observed at E0 s 180 Vrcm Žinduced
breakdown voltages 1.2 V, charging time s 3.0 ␮s..
By the application of super-critical amplitude pulses,
the cell membranes were charged to substantially higher
voltages within a shorter time. For a pulse of 1700
Vrcm amplitude, the membrane in potato cells was
polarized up to 2.2 V within 0.23 ␮s. Although the
classical electroporation theory assumes a breakdown
membrane voltage in the critical or super-critical range,
a tendency towards a rise in the breakdown voltage
with the increasing external field was observed for all
cell materials examined ŽFig. 5.. Similar relationships
were also observed for artificial lipid membranes ŽBenz
& Zimmermann, 1980; Winterhalter et al., 1996.. The
application of Eq. Ž6. for the planar model Žor ␸ s
1.5⭈ d⭈ E⭈ cos␣ for the spherical membrane model. to
calculate the membrane potential induced and to op-
timize the relation between field intensity and pulse
duration should be used cautiously, because these
equations assume a non-variable breakdown voltage.
3.4. Lifetime of the perforated membrane
The data in Fig. 6 show the monitoring of simultane-
ously measured current density and field strength
changes during the first 80 ␮s of the exposure of potato
tissue to an electric field as previously shown in Fig. 1.
The changes in the conductivity of potato tissues indi-
cated that, within this period, the cell conductance did
not increase appreciably during the intensive pulse.
This supported the conclusion that, by the application
of a pulse with super-critical field strength, the forma-
tion of the conductive state of the membrane was
completed within a few microseconds after the start of
 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
the pulse. Under these conditions, pulse parameters
such as magnitude, the shape of the decaying electric
field, and the total pulse duration determined only the
secondary pulse effects, such as lifetime of the pores
already formed.
Once a membrane breakdown occurs, a current, im ,
flows through the aqueous pore. Joule energy is gener-
ated during the field application. An integration of
i2m rGm over the full pulse duration wassuming that at 8
␮s, the value of Gm is approx. 5 = 10y3 S Žsee Fig. 3c.,
and that further conductance of the perforated mem-
brane changes during the pulse proportionally with
decreasing sample conductivityx showed that for the
pulse experiment with E0 s 880 Vrcm presented in
Fig. 1, the transmembrane electric field energy gener-
ated during a pulse of 1 ms duration was approximately
2.5= 10y9 J. The dissipated energy in the small aque-
ous zones of the porated membrane Žat approx. 8 ␮s
after the start of a pulse, considering a total pore area
of 46 = 10y1 2 m2 and a membrane thickness of 5 nm,
yields a volume of approx. 2.3= 10y1 9 m3 . resulted in
an enormous specific energy input, of the order of
MJrkg. In comparison, the energy input into potato
samples was approximately 0.5 kJrkg and the resulting
temperature increase was 0.14⬚C. Obviously, the enor-
mous energy concentration on the pores zone could
produce local thermal fluctuations and some secondary
effects on the elastic modulus of the biological mem-
Fig. 6. Variation of Ža. field strength, E, Žb. current density, j, and
Žc. effective tissue conductivity, ␴, with time for potato tissue during
first 80 ␮s after pulse start with pulse amplitude 440 Vrcm Ž1. and
880 Vrcm Ž2.. Complete pulse sequence is shown in Fig. 1. Effective
tissue conductivity is obtained as ␴ s jrE.
145
branes. Electrical breakdown experiments on cell mem-
branes have also led to the conclusion that local heat-
ing may play an important role after the primary process
of electrical breakdown ŽCoster & Zimmermann, 1975;
Benz et al., 1979..
The high conductance of the membrane during the
voltage relaxation following a pulse with supercritical
field strength amplitude remained unchanged even
when the current and voltage started to decay by the
experiment with E0 s 880 Vrcm ŽFig. 1.. With this
amplitude, the sample conductivity during the total
pulse width did not change significantly. However, it
showed a tendency towards the recovery of the initial
low conductivity state. In experiments with an ampli-
tude of 440 Vrcm, the significant decay of the sample
conductivity began only when the external field was at
half of this level ŽFig. 1.. Furthermore, the decay of the
sample conductivity to its initial state Žthe conductivity
course extrapolated to the end of the pulse was approx.
0.3 mSrcm. indicated that the membrane rapidly re-
sealed during the voltage relaxation. Also, in experi-
ments where the maximum changes of the membrane
conductance were reached Žan experiment of this type
is shown in Fig. 1, by Eo s 880 Vrcm., the measure-
ment of the passive electrical properties Žstarted 2 s
after pulse termination. indicated that the sample con-
ductivity was identical to the measurement before the
pulse was applied Ži.e. the insulation properties of cell
membrane were completely recovered.. A reversible
breakdown of the cell membranes was observed. This
phenomenon is established experimentally for a wide
range of biological cells and is used as a basis for the
electromanipulation of cells in the fields of medicine
and biotechnology ŽChang et al., 1992; Zimmermann &
Neil, 1996; Lynch & Davey, 1996; Ho & Mittal, 1996..
Monitoring of the current during the first 8 ␮s after
starting the repeated supercritical pulse applications
resulted in reversibility after the first pulse and irre-
versible cell membrane rupture after several pulses
ŽFig. 7.. At repeated pulses, with intervals of 1 s between
them, the oscillographic tracking of the charging cur-
rent of the cell membranes for the first and the second
pulse were reproduced with high accuracy. This meant
that the structural properties of the membrane after
the first pulse were identical to those of an intact
membrane. This finding is essential for the appropriate
use of pulsed electric fields. Only if the membrane
structure is restored, does the induction of critical
membrane voltage and membrane breakdown occur
again. Also, for the third pulse, the current course
showed only a slight deviation from the exponential
decaying process. This meant that the membrane struc-
ture was restored to a greater extent within 1 s after
the second pulse. The membrane current was observed
for pulse number 5 in addition to the polarization
current at the start of the pulse. At 0.5 ␮s, a slight
146 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
increase in the current course Žcaused by an increase in
the membrane conductance. was also observed. The
current course of the pulse indicated that an irre-
versible membrane permeabilization had occurred.
Detailed information about irreversible membrane
permeabilization in the cell materials examined, as well
as the dynamics of the process, will be presented in
future publications.
3.5. Engineering aspects
The data presented previously show a general rela-
tionship between field-induced transmembrane voltage
and structural changes in biological membranes. From
an engineering standpoint, it is important to under-
stand how the entire cell system responds to electrical
field pulses of different field strength levels.
The relationship between the pulse amplitude and
the effective conductivity of the examined cell system is
given in Fig. 8. This suggests the existence of typically
three characteristic ranges of external field strength. In
the sub-critical range of field strength Žless than ap-
prox. 150 Vrcm. the electrical behavior is determined
by passive electrical parameters of the cell system,
which results in no detectable change. At E0 f Ec ,
electrical membrane breakdown occurs and no further
fundamental increase of E0 causes an incomplete elec-
Fig. 7. Kinetics of permeabilization of potato cells in tissue during
the application of ten pulses with constant amplitude of field strength
E0 s 700 Vrcm. Ža.: time dependency of field strength, E. Žb.:
current density in the tissue in the first 8 ␮s after pulse start. Pulse
frequency s 1 sy1 ; n s pulse number. Current density trace for ex-
ample without membrane permeabilization Žintact., j⬘, is recon-
structed from relation Ž10., where jsub is registered by application in
the same sample of a single pulse with subcritical field strength level,
Esub s 80 Vrcm.
Fig. 8. Critical field strength range for cell systems examined. Rela-
tive permeability is obtained as w ␴ y ␴min xrw ␴max y ␴min x, where ␴ is
the average effective sample conductivity during the pulse. The
characteristic conductivities for intact sample, ␴ma x and ␴min , are
taken from Fig. 4.
tropermeabilization of the cells. This range of field
strength can be proposed as the transitional range. The
reasons for the different conductivity values of the
various samples are: the dependence of the membrane
rupture on the transmembrane voltage Žwith decreasing
voltage across the membrane, a significant drop in the
membrane conductance occurs. as well as the uneven
cell size distribution in the samples. Compared to the
field strength usually used for irreversible membrane
ruptures of up to several kVrcm, the transitional E0
range is small.
At the super-critical field strength, the formation of
the aqueous phase in the membrane section in the field
direction was so large that the contribution of the cell
membranes to the total electrical conductivity of the
cells system was not significant, and the effective sam-
ple conductivity reached the finite maximum value. The
sample conductivity was determined only by the extra-
and intracellular electrolyte conductivity. These find-
ings led to the conclusion that when a supercritical
field is applied, the maximum conductivity value of the
cell system should be used for the calculation of the
electric field and the energy distribution in the treat-
ment chamber containing cellular material and non-
cellular medium.
This conclusion has been based on tests for plant
tissues Žpotato cubes; size 1 cm. regularly distributed in
media with different conductivities Ž0.5᎐8 mSrcm. in a
large treatment chamber Ždistance between electrodes
s 3 cm; volumes 500 ml; liquid ratio s 0.8. using the
field strength measurement with two isolated needle
electrodes and the detection of temperature increase
 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
directly in the tissue. A field intensity with an ampli-
tude of more than 1 kVrcm was induced directly in the
cell material.
The tests in mixed systems showed that when the
supercritical field intensity is applied, the specific en-
ergy input into particulate tissues per pulse can be
calculated as:
␴max T 2
Qs H0 E Ž t . dt
␳ tially charged to higher voltages as compared to
where ␳ is the tissue density; EŽ t . is the effective field
strength in the particulate as a function of time Žmea-
sured or predicted from electric field distribution in the
treatment chamber with regards to high conductivity
state of the cell material.; T is the total pulse width;
and ␴max is the maximum tissue conductivity. This
consideration has been confirmed with suspended cell
agglomerates and individual cells.
The maximum conductivity value, ␴max , could also be
determined experimentally prior to the supercritical
pulse application via the measurement of passive elec-
trical properties by a DC pulse experiment with subcrit-
ical field strength Žas represented in Fig. 4, ␴max s
jmaxrE0 . or by a high frequency AC field yielding the
so-called ␤-dispersion. For plant or animal tissue, and
for large plant cells in suspension, the electrical
properties of intact or totally ruptured membranes are
identical within the characteristic frequency range from
5 to 50 MHz ŽAngersbach et al., 1999..
4. Conclusion
The time sequence of the electropermeabilization at
the cell membrane level showed similar breakdown
phenomena for different cell systems. When a single
pulse with a critical or super-critical field amplitude
was applied, three different phases were typically in-
volved: Ži. the development of a critical transmembrane
potential due to charge accumulation at the intact
membrane surface; Žii. the termination of the insulat-
ing properties of the membrane Žmembrane break-
down. resulting in development of a conductive mem-
brane; and Žiii. the opening time of the pores and the
tendency towards complete recovery of the initial insu-
lating properties of the membrane.
1. Classical membrane breakdown phenomena oc-
curred for large cells at field strength levels in the
range of 150᎐200 Vrcm. When the electrical po-
tential over the cell membrane reached a critical
value of approximately 0.7᎐2.2 V, it resulted in
membrane breakdown. The polarization time up to
membrane breakdown was influenced by the field
strength. When supercritical electrical fields were
147
applied, the membrane rupture occurred within 1
␮s after pulse initiation. At a very high field
strength, the membrane breakdown should occur
during the voltage rise time. For all of the observed
electroporation behaviors, it was found that the
only attainment of critical transmembrane poten-
tial did not explain all essential aspects of electro-
poration. By the application of a pulse with super-
critical field strength, the membrane was substan-
Ž 12 .
critical field strength. This clearly emphasized the
need to consider the dynamics of the membrane
voltage build up.
2. The formation of a conductive membrane due to
the widening of pores was not an instantaneous
process and was strongly dependent on the field
strength amplitude, if the pulse duration was a few
times more than ␶. The larger the field strength,
the more rapid the dynamics of the pore formation.
The initiation of conductive channels across the
membrane occurred in nanoseconds, while the for-
mation of high conductance membranes due to
their expansion took place in a few microseconds.
A rapid formation of pores in a lipid domain of the
cell membrane might be possible. Although a dras-
tic change in the membrane conductivity during the
pulsation occurred Žin the order of up to 105 ., the
fractional aqueous area of the membrane was found
to be small and never exceeded 1% of the mem-
brane area in the field direction.
3. The originally low conductance state of the cell
material could be reached in all cell materials
examined within a couple of seconds after termina-
tion of the pulse Ži.e. the insulating properties of
cell membrane was completely recovered.. When
critical or slightly lower amplitude pulses were used,
the original low conductance of the membrane
could be reached, even during the pulse. The appli-
cation of a single pulse with super-critical ampli-
tude caused the reversible breakdown of the cell
membranes. The lifetime of the high-conductance
state of the membrane and the reversibility of the
structural changes was found to be the secondary
effect of electric field interactions with the cells
and was also influenced by the energy concentra-
tion on the pores zone of the membrane.
These results are in accordance with the observa-
tions reported for biological and artificial membranes
in classical electroporation phenomena.
From an engineering perspective, the electric field
interactions with real food cell systems can be summa-
rized as follows:
1. The field strength of electric pulses directly applied
to the materials with a cell size of 50᎐120 ␮m must
148 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
be higher than a critical value of 400᎐800 Vrcm
for significant membrane breakdown.
2. The application of a single pulse with super-critical
field amplitude does not necessarily cause an irre-
versible membrane rupture. The determination of
critical values of the external field strength, and the
transmembrane voltage through secondary effects
resulting from irreversible membrane rupture, may
be misleading.
3. The critical breakdown transmembrane potential,
the main parameter for the primary effect of the
high electric field pulse on cell membrane, did not
depend on pulse duration when the pulse width
was several times greater than ␶, which is in the
microsecond range Žfor cells in potato tissue, ␶ s 0.7
␮s; in apple tissue, ␶ s 1.4 ␮s; in fish tissue ␶ s 0.63
␮s; and in plant cell culture suspension ␶ s 0.45
␮s..
4. The charging process and the formation of the
conductive membrane was completed within a few
microseconds after the pulse start, the field strength
being the dominant process parameter. At that
moment, the magnitude and shape of the decaying
electric field, pulse width and the energy intensity
determined only the secondary pulse effects, such
as the opening time of the pores on the membrane
already formed.
5. At super-critical field strength, the effective sample
conductivity reached the finite maximum value
equivalent to the conductivity of the cell system
with totally ruptured membranes. The cell mem-
branes remained in the high-conductance state dur-
ing the voltage relaxation after breakdown oc-
curred.
6. Processing with repeated pulses Žalso in supercriti-
cal range. does not necessarily cause an irre-
versible membrane rupture. For process develop-
ment, the optimum pulse frequency should be based
on the reversibility of the membrane permeabiliz-
ation.
These experimental findings are valuable for process
design and the optimization of the impact of high-
intensity electric field pulses on membrane perme-
abilization or field-induced stress on biological materi-
als.
Acknowledgements
This research project was supported by the FEI
¨
ŽForschungskreis der Ernahrungsindustrie e.V., Bonn.,
the AiF and the German Ministry of Economics ŽPro-
ject No. FV 10611N. as well as by the European
Commission ŽFAIR contract CT97-3044..
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