A Review of Kinetic Models For Inactivating Microorganisms and Enzymes by Pulsed Electric Field Processing

Review
A review of kinetic models for inactivating microorganisms and enzymes

by pulsed electric eld processing
Kang Huang, Hongping Tian, Ling Gai, Jianping Wang
College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China
a r t i c l e i n f o
Article history:
Received 25 May 2011
Received in revised form 21 December 2011
Accepted 5 February 2012
Available online 16 February 2012
Keywords:
Pulsed electric eld (PEF)
Kinetic model
Microbial inactivation
Microorganism
Enzyme
Health-related compound
a b s t r a c t
As a non-thermal technology, pulsed electric eld (PEF) treatment can be utilized in food processing and
bioengineering for the inactivation of microorganisms and quality-degrading enzymes, as well as the
retention of health-related compounds and the extension of shelf-life. Development of kinetic models
that t the degree of microbial inactivation and the loss of food quality is important to improve the ef-
ciency of PEF treatment. The current review aims to provide an overview of the kinetic models used by
PEF for microbial inactivation in liquid foods. Kinetics modeling for the destruction of microorganisms,
inactivation of enzymes, retention of health-related compounds, and extension of shelf-life are discussed.
Additionally, the tting accuracy of several models, as well as issues that need further investigation, are
discussed to promote further understanding and the deployment of PEF technology.
2012 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
2. Definitions for kinetic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
3. Establishing a reliable model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
4. Kinetic models for PEF processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
4.1. First-order kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
4.1.1. Destruction of microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
4.1.2. Inactivation of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
4.1.3. Retention of health-related compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
4.2. Hulsheger. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
4.3. Fermi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
4.4. Weibull distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
4.4.1. Inactivation of microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
4.4.2. Inactivation of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
4.4.3. Retention of health-related compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
4.5. Loglogistic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
4.6. Giner-Segui . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
5. Combined effects of the processing factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
5.1. Destruction of microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
5.2. Inactivation of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
5.3. Retention of health-related compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
0260-8774/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2012.02.007
Corresponding author. Tel./fax: +86 571 88982350.

E-mail address: jpwang@zju.edu.cn (J. Wang).
Journal of Food Engineering 111 (2012) 191207
Contents lists available at SciVerse ScienceDirect
Journal of Food Engineering
j our nal homepage: www. el sevi er. com/ l ocat e/ j f oodeng
1. Introduction
As a non-thermal technology for food processing, pulsed elec-
tric eld (PEF) technology has been demonstrated to be capable
of inactivating microorganisms and enzymes at lower tempera-
tures compared with conventional heat treatment technologies
(Amiali et al., 2007; Gerlach et al., 2008; Riener et al., 2009;
Saldana et al., 2011). In the PEF process, a series of short, high volt-
age pulses are applied, resulting in local instabilities and tension in
the cell membrane attributable to electromechanical compression
and facilitates the formation of pores in the membrane (electropor-
ation) (Barbosa-Canovas et al., 1999; Weaver and Chizmadzhev,
1996; Wouters et al., 2001). The potential of commercializing this
technology to satisfy consumer demands for food products with
physical, chemical, and nutritional characteristics similar to those
of fresh food has recently attracted more attention and interest
from the food-processing industry. The objectives of PEF treatment
for liquid food include the inactivation of microorganisms and
quality-degrading enzymes, retention of health-related com-
pounds, and extension of shelf-life. Various studies have demon-
strated that maximizing microbial inactivation while minimizing
negative effects on valuable food compounds and bioactive compo-
nents is possible under homogenous treatment conditions (Alvarez
et al., 2000; Barbosa-Canovas et al., 1999; Calderon-Miranda et al.,
1999; Castro et al., 1993; Hoover, 1997; Yeom et al., 2000).
Proper design of the treatment chamber is one of the most piv-
otal factors in the development of PEF treatment (Alkhafaji and
Farid, 2007). Therefore, a large number of attempts have been
made to optimize the geometry of the treatment chamber to im-
prove the effectiveness of PEF treatment (Huang and Wang,
2009). To determine the optimum design of the treatment cham-
ber, numerical simulation of PEF treatment for liquid foods has
been developed as a useful tool to evaluate parameters that cannot
be experimentally measured without producing signal perturba-
tion and interferences caused by high intensity electric eld
(Buckow et al., 2010, 2011; Fiala et al., 2001; Gerlach et al.,
2008; Heinz et al., 2003; Jaeger et al., 2009; Lindgren et al., 2002;
Toep et al., 2007). However, such simulations provided detailed
information only on the distribution of the electric eld, ow
velocity, and temperature inside the treatment chamber. The com-
bined application of a numerical simulation and inactivation ki-
netic models is urgently needed because such an approach can
signicantly contribute to the development of efcient PEF equip-
ment (Gerlach et al., 2008).
Models are also necessary to dene the processing conditions
necessary to achieve a certain level of microbial safety. The rst
objective of a preservation technology is to offer a safety product,
then extend shelf-life, and nally, preserve the quality properties
of the fresh product. To fulll this goal, numerical simulation
should involve various disciplines, such as mathematics, microbi-
ology, engineering, and chemistry, and apply mathematical models
to t microorganism and enzyme responses to processing factors
and environmental variables. Wouters et al. (2001) and Van Loey
et al. (2002) reviewed the factors that are important to the out-
come of the inactivation of microorganisms and enzymes by PEF,
including process parameters, microbial characteristics, and prod-
uct parameters. Considering the effect of such factors, the kinetic
models that capture the mechanism of the inactivation of microor-
ganisms and enzymes by PEF have been proposed to analyze
available data, as well as to provide a basis for optimizing treat-
ment chamber geometry and determining optimum process
conditions.
The current review aims to provide an overview of the kinetic
models used by PEF for the destruction of microorganisms,
inactivation of enzymes, retention of health-related compounds,
and extension of shelf-life in liquid foods. Additionally, the tting
accuracy of several models and the issues that require further
investigation are discussed.
2. Denitions for kinetic models
Mathematical models are becoming important tools for describ-
ing and predicting the growth, survival, and inactivation responses
of foodborne microorganisms and enzymes under specic environ-
mental conditions (Mosqueda-Melgar et al., 2008a). Typically, a
predictive kinetic model comprises two parts (McMeekin and Ross,
2002), namely, a primary model that describes the microorganism
(or enzyme) evolution as a function of time and a secondary model
that characterizes factors appearing in primary models as a func-
tion of processing factors and environmental conditions. The com-
bination of primary and secondary models, that is, the tertiary
model, is then capable of predicting the microorganism (or en-
zyme) evolution as a function of time, processing factors, and envi-
ronmental conditions.
The primary processing factors affecting the treatment ef-
ciency of PEF processing include electric eld strength, treatment
time, specic energy input, pulse width, pulse frequency, pulse
polarity, and initial temperature (Barbosa-Canovas et al., 1999;
Wouters et al., 2001). Among these factors, electric eld strength
and treatment time are the most important (Castro et al., 1993;
Huang and Wang, 2009). Correspondingly, the equations for the
inactivation kinetics of microorganisms and enzymes are always
functions of either electric eld strength or treatment time or the
combination of both. In addition, for continuous PEF processing,
specic energy input is an essential parameter to know the tem-
perature increment during treatment (Buckow et al., 2011).
Generally, the survival ratio of microorganisms S is dened as
the microbial load after PEF treatment over the initial cell count
before PEF processing (N
t
/N
0
):
S
N
t
N
0
1
where N
t
represents the number of surviving microorganisms after
a session of PEF treatment, and N
0
is the initial number of
microorganisms.
The residual enzyme activity (RA) is dened by Eq. (2):
RA
A
t
A
0
2
where A
t
is the enzyme activity in the sample after treatment, and
A
0
is the enzyme activity of the untreated sample, which is deter-
mined immediately after processing to avoid the effects of storage
time.
Similar to modeling for the enzyme activity, the relative content
of health-related compounds or relative antioxidant capacity (RC)
is dened by Eq. (3):
RC
C
t
C
0
3
where C
t
and C
0
are the residual and initial contents of the health-
related compound (mg/100 g) or antioxidant capacity (%). RA and RC
values are typically multiplied by 100 and expressed as a percent-
age in the results. According to Eq. (2), RA and RC values range from
zero, if no residual activity was measured after treatment, up to one,
if the measured residual activity after treatment corresponded to
the original activity. The kinetic models used for interpreting the
curves of the destruction of microorganisms and inactivation of en-
zymes, as well as those for the loss of health-related compounds,
show signicant similarity, except for a number of specic rate
192 K. Huang et al. / Journal of Food Engineering 111 (2012) 191207
constants. To describe the following models conveniently, Y is de-
ned as S, RA, or RC.
The goodness of t of the experimental data to the linear and
nonlinear models is measured using the regression coefcient R
2
,
root mean square error (RMSE), and accuracy factor parameter A
f
.
R
2
measures how well further outcomes are likely to be predicted
by a linear or nonlinear model, and an R
2
value closer to one indi-
cates a better tting accuracy of the model (Nagelkerke, 1991).
RMSE is a frequently used measure of the average deviation be-
tween the observed and predicted data sets. As a good measure
of precision, a lower RMSE value indicates less residual variance
and better t of the data for the model:
RMSE
predictedobserved
2
NONP
4
where NO is the number of observations, and NP is the number of
parameters to be estimated.
To measure the accuracy of the estimates obtained by the dif-
ferent assayed models, Ross (1996) suggested the accuracy factor
A
f
, which is dened by Eq. (5):
A
f
exp
1
NO
NO
i1
ln
predicted
observed
_ _
_ _
5
where the subscript i denotes a given pair of observation-prediction
values, and NO is the total number of observations used for the cal-
culations. It should be indicated that A
f
enables to estimate the per-
centage discrepancy or error (%D) between observed and estimated
values as % D = (A
f
1)100 (Baranyi and Pin, 2000). An A
f
value of
one indicates a perfect correspondence between observed and pre-
dicted values. On the contrary, a higher difference between the pre-
dictions of a model and experimental observations makes the A
f
value farther from unity.
3. Establishing a reliable model
Understanding inactivation kinetics will help in the develop-
ment and validation of predictive mathematical models. To estab-
lish appropriate PEF process conditions and to learn the levels of
PEF treatment efciency, mathematical models describing the
kinetics of the inactivation of pathogenic microorganisms and
quality-degrading enzymes, as well as the loss of health-related
compounds, are required. Mathematical models should be based
on reliable experiments without any artifact of the experimental
procedures and on the understanding of the physiological
mechanism underlying the microbial inactivation by PEF (Mosqu-
eda-Melgar et al., 2008a). However, the comparability between
simulation and experimental results is affected by numerous
critical factors, the broad range of experimental conditions used,
and the diversity of equipment employed. Likewise, establishing
a consistent model that accurately expresses the behavior of
bacteria when subjected to different environmental conditions is
of utmost importance (Raso et al., 2000). Static chambers may be
preferable at the early stage of modeling because they avoid the
complexity of continuous treatment and simplify the control of
critical factors. The mathematical models should then be validated
in a continuous process and in real food systems (Wouters et al.,
2001).
Microbial inactivation by different lethal agents is commonly
assumed to follow a linear or nonlinear relationship between the
log of the number of survivors and the treatment factors. Most cur-
rent PEF studies are not only relevant to PEF technology, but are
also relevant to existing heat or mild preservation processes other
than PEF. Therefore, the relevant parameters involved in determin-
ing the inactivation kinetics should be clearly dened before the
experiment is designed. In particular, the temperature in the treat-
ment chamber should be carefully controlled below the critical
temperature for microbial lethality, ensuring that the inactivation
is only achieved by PEF, without the thermal effects. However,
the temperature may have effects at sublethal temperatures, which
inevitably affects the effectiveness of PEF. A multilevel factorial de-
sign is then used to evaluate the effects of various factors on the
microbial inactivation in PEF-treated food products. Then, the
experiment is repeatedly performed, and microbial counts, enzyme
activities, and the residual content of health-related compound are
measured. Means and standard deviations (SD) are calculated for
each treatment. A regressive analysis is performed to obtain the
coefcients of the nal equations for better goodness of t. Finally,
after obtaining the complete kinetics equations, the PEF treatment
conditions are experimentally veried to validate parameter esti-
mation. A wide variety of commercial software packages can be
employed for statistical analyses of microbial inactivation kinetics
by PEF, including Statgraphics
, Statistical Product and Service

Solutions
, Statistical Analysis System
, Minitab
, and so on.
4. Kinetic models for PEF processing
Other than model names, the differences among existing kinetic
models lie in their scope of application, namely, the eradication of
vegetative microorganisms (San-Martin et al., 2007; Yeom et al.,
2000), inactivation of quality-degrading enzymes (Bendicho et al.,
2002b; Giner-Segui et al., 2006), and retention of health-related
compounds (Aguilo-Aguayo et al., 2010a; Cserhalmi, 2006;
Odriozola-Serrano et al., 2008d).
The widespread demand for fresh-like food products has been
evident during the last 20 years. However, such fresh-like prod-
ucts, whichomit anyeffective microbial eliminationstep, usuallyre-
sult in foods that may carry some microorganisms, such as
Escherichia coli, Enterobacter sakazakii, Listeria innocua, Listeria
monocytogenes, Lactobacillus plantarum, Staphylococcus aureus,
Salmonella Enteritidis, Salmonella Dublin, and Yersinia enterocolitica
that may bring about public health problems (Mosqueda-Melgar
et al., 2008a). PEF treatment is capable of killing the vegetative cells
of microorganisms in food products without signicant loss of
avor, color, and nutrients. Mathematical models that describe the
kinetics of inactivation of spoilage and pathogenic microorganisms
are required to yield stable and safe products without over-process-
ing (Wouters et al., 2001). To date, various kinetic models, such as
the traditional rst-order kinetic model, Hulsgegers, Fermis,
Weibull distribution, and the Loglogistic models, have been
proposed to describe the microorganism inactivation by PEF in
liquid foods (Table 1).
Compared with microbial inactivation, the effect of PEF treat-
ment on quality-degrading enzymes is a topic of particular interest
in the eld of non-thermal technology for food processing. On one
hand, some enzymes are positively utilized during food processing
for the recovery of byproducts, development of new food products,
and improvement food quality, including avor. However, some
enzymes may have detrimental effects on food quality (Van Loey
et al., 2002). Food quality defects may be attributed to enzymes
naturally present in food or to enzymes produced by certain micro-
organisms. Therefore, aside from microbial destruction, PEF
technology aims at inactivating enzymes that have negative effects
on treated products.
Kinetic models for studying the effects of PEF treatment on the
activity of enzymes, including peroxidase (POD), lipoxygenase
(LOX), b-glucosidase (b-GLUC), pectinesterase (PE), polyphenol
oxidase (PPO), polygaracturonase (PG), hydroperoxide lyase
(HPL), alkaline phosphatase (ALP), and lactoperoxidase (LPO), have
been extensively used in different process conditions (Table 2).
K. Huang et al. / Journal of Food Engineering 111 (2012) 191207 193
Table 1
Kinetic models for PEF microbial inactivation.
Kinetic model Microorganism Medium Treatment conditions Maximum
reduction(Log
CFU/mL)
R
2
/RMSE/A
f
References
First-order kinetics
models
E. sakazakii CECT 858 Buffered peptone
water
1040 kV/cm, pulse width 2.5 ls, treatment time 360 ls, ow rate 1.8 L/h, 25 C 2.7 R
2
= 0.880.94 Perez et al. (2007)
E. sakazakii CECT 858 Rehydrated infant
formula milk
2
= 0.840.93 Perez et al. (2007)
E. coli Orange juice 1540 kV/cm, pulse width 2.5 ls, treatment time 700 ls, ow rate 60 mL/min,
<55 C
3.83 RMSE = 0.451.16 Rivas et al. (2006)
E. coli O157:H7 Liquid egg yolk 30 kV/cm, pulse width 2 ls, treatment time 210 ls, ow rate 12 mL/min, 40 C 4.9 R
2
= 0.9470.999 Amiali et al. (2007)
L. plantarum 0.6% peptone water 2028 kV/cm, pulse width 2.5 ls, treatment time 30240 ls, ow rate 60 mL/
min, 1035 C
4.9 R
2
= 0.9260.985
A
f
= 1.101.18
Rodrigo et al. (2003b)
Salmonella Enteriditis Liquid egg yolk 30 kV/cm, pulse width 2 ls, treatment time 210 ls, ow rate 12 mL/min, 40 C 4.8 R
2
= 0.9510.999 Amiali et al. (2007)
Hulshegers models E. coli Orange juice 1540 kV/cm, pulse width 2.5 ls, treatment time 700 ls, ow rate 60 mL/min,
<55 C
3.83 RMSE = 0.040.36 Rivas et al. (2006)
E. coli CGMCC 1.90 Carrot juice 525 kV/cm, pulse width 1.5 ls, treatment time 2071449 ls, coaxial treatment
chamber, ow rate 52.5 mL/min, <40 C
3.6 R
2
= 0.9530.992 Zhong et al. (2005)
E. coli K12 Aqueous suspension 12 kV/cm, bipolar pulse width 36 ls, pulse frequency 0.2 Hz, inlet temperature
20 C
3.0 R
2
= 0.991 Hulsheger et al. (1981)
min, 1035 C
4.9 R
2
= 0.9230.959
A
f
= 1.111.28
Salmonella Dublin
(ATCC 15480)
Skim milk 1540 kV/cm, treatment time 12127 ls, 1050 C 4.0 R
2
= 0.870.97 Sensoy et al. (1997)
Fermis model E. coli CGMCC 1.90 Carrot juice 525 kV/cm, pulse width 1.5 ls, treatment time 2071449 ls, coaxial treatment
chamber, ow rate 52.5 mL/min, <40 C
3.6 R
2
= 0.9020.985 Zhong et al. (2005)
L. innocua ACTT 51742 McIlvaine buffer 3545 kV/cm, pulse width 5.3911.55 ls, treatment time 37 ls, ow rate
1200 mL/min, 1565 C
6.1 R
2
= 0.9940.999 San-Martin et al.
(2007)
Salmonella Dublin
(ATCC 15480)
Skim milk 1540 kV/cm, treatment time 12127 ls, 1050 C 4.0 R
2
= 0.970.99 Sensoy et al. (1997)
Weibull
distribution
model
E. sakazakii CECT 858 Rehydrated infant
formula milk
2
= 0.860.97 Perez et al. (2007)
E. sakazakii CECT 858 Buffered peptone
water
2
= 0.910.95 Perez et al. (2007)
E. coli Orange juice 1540 kV/cm, pulse width 2.5 ls, treatment time 700 ls, ow rate 60 mL/min,
<55 C
3.83 RMSE = 0.080.25 Rivas et al. (2006)
min, 1035 C
4.9 R
2
= 0.9730.987
A
f
= 1.051.10
L. innocua ACTT 51742 McIlvaine buffer 3545 kV/cm, pulse width 5.3911.55 ls, treatment time 37 ls, ow rate
1200 mL/min, 1565 C
6.1 R
2
= 0.9550.996 San-Martin et al.
(2007)
L. monocytogenes Citratephosphate
McIlvaine
1528 kV/cm, pulse width 2 ls, pulse frequency 1 Hz, treatment time 0
2000 ls, <32 C
4.77 R
2
= 0.9750.995 Alvarez et al. (2003a)
Salmonella Senftenberg
775 W
Liquid whole egg 2045 kV/cm, square pulse width 3 ls, treatment time 0150 ls, 55 C 3.3 R
2
= 0.9310.957 Monfort et al. (2010)
Loglogistic model Salmonella Senftenberg McIlvaine buffer 1228 kV/cm, pulse width 115 ls, pulse frequency 15 Hz, treatment time 0
600 ls, initial temperature 20 C
6.5 R
2
= 0.992 Raso et al. (2000)
Quadratic response
model
E. coli Melon juice 35 kV/cm, pulse width 4 ls, pulse frequency 217 Hz, treatment time 1440 ls,
40 C
3.7 R
2
= 0.963 Mosqueda-Melgar
et al. (2007)
E. coli Watermelon juice 35 kV/cm, pulse width 4 ls, pulse frequency 188 Hz, treatment time 1727 ls,
40 C
3.6 R
2
et al. (2007)
E. coli O157:H7 Apple juice 2030 kV/cm, pulse width 5125 ls, ow rate 3 L/h, <55 C 3.6 R
2
= 0.978
RMSE = 0.20
Saldana et al. (2011)
E. coli O157:H7 Apple juice + LAE 2030 kV/cm, pulse width 5125 ls, ow rate 3 L/h, <55 C 6.7 R
2
= 0.937
RMSE = 0.55
Saldana et al. (2011)
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E. coli O157:H7 Apple juice 35 kV/cm, bipolar pulse width 4 ls, ow rate 80110 mL/min, <40 C 4.29 R
2
et al. (2008b)
E. coli O157:H7 Pear juice 35 kV/cm, bipolar pulse width 4 ls, ow rate 80110 mL/min, <40 C 4.53 R
2
et al. (2008b)
E. coli O157:H7 Orange juice 35 kV/cm, bipolar pulse width 4 ls, ow rate 80110 mL/min, <40 C 5.16 R
2
et al. (2008b)
E. coli O157:H7 Strawberry juice 35 kV/cm, bipolar pulse width 4 ls, ow rate 80110 mL/min, <40 C 5.56 R
2
et al. (2008b)
G. oxydans Grape juice 35 kV/cm, bipolar pulse width 5 ls, pulse frequency 303 Hz, ow rate 3.33 mL/s,
inlet temperature 15 C, maximum temperature <30.4 C
2.24 R
2
= 0.818 Marselles-Fontanet
et al. (2009)
K. apiculata Grape juice 35 kV/cm, bipolar pulse width 5 ls, pulse frequency 303 Hz, ow rate 3.33 mL/s,
3.88 R
2
et al. (2009)
L. bacteria Grape juice 35 kV/cm, bipolar pulse width 5 ls, pulse frequency 303 Hz, ow rate 3.33 mL/s,
3.54 R
2
et al. (2009)
L. monocytogenes Melon juice 35 kV/cm, pulse width 4 ls, pulse frequency 217 Hz, treatment time 1440 ls,
40 C
3.56 R
2
et al. (2007)
L. monocytogenes Watermelon juice 35 kV/cm, pulse width 4 ls, pulse frequency 188 Hz, treatment time 1727 ls,
40 C
3.41 R
2
et al. (2007)
S. cerevisiae Grape juice 35 kV/cm, bipolar pulse width 5 ls, pulse frequency 303 Hz, ow rate 3.33 mL/s,
3.90 R
2
et al. (2009)
Salmonella Enteriditis Melon juice 35 kV/cm, pulse width 4 ls, pulse frequency 217 Hz, treatment time 1440 ls,
40 C
3.71 R
2
et al. (2007)
Salmonella Enteriditis Watermelon juice 35 kV/cm, pulse width 4 ls, pulse frequency 188 Hz, treatment time 1727 ls,
40 C
3.56 R
2
et al. (2007)
Salmonella Enteriditis Apple juice 35 kV/cm, bipolar pulse width 4 ls, ow rate 80110 mL/min, <40 C 4.34 R
2
et al. (2008b)
Salmonella Enteriditis Pear juice 35 kV/cm, bipolar pulse width 4 ls, ow rate 80110 mL/min, <40 C 4.87 R
2
et al. (2008b)
Salmonella Enteriditis Orange juice 35 kV/cm, bipolar pulse width 4 ls, ow rate 80110 mL/min, <40 C 5.22 R
2
et al. (2008b)
Salmonella Enteriditis Strawberry juice 35 kV/cm, bipolar pulse width 4 ls, ow rate 80110 mL/min, <40 C 4.43 R
2
et al. (2008b)
Staph. aureus Milk 2535 kV/cm, bipolar pulse width 8 ls, pulse frequency 100 Hz, <25 C 4.5 R
2
= 0.84 Sobrino-Lopez et al.
(2006)
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Table 2
Kinetic models for PEF enzyme inactivation.
Kinetic model Enzyme Medium Process conditions Minimum RA
a
(%)
R
2
/A
f
References
First-order kinetics
models
LOX Tomato juice 35 kV/cm, pulse width 3 ls, 50 ls treatment time, ow rate 1 mL/
s, 30 C
20 R
2
= 0.9010.976 Min et al. (2003)
LOX Soymilk 2040 kV/cm, 400 Hz pulse width 2 ls, treatment time 1036 ls,
25 C
12 R
2
= 0.9930.999 Li et al. (2008)
PE Distilled water 24 kV/cm, 400 Hz pulse width 0.02 ms 6.2 R
2
= 0.975 Giner et al. (2000)
PE Orange juice 25 kV/cm, 700 Hz pulse width 2.0 ms, ow rate 0.31 mL/s, 50 C 10 R
2
= 0.91 Yeom et al. (2002)
PE Fresh mixed orange and carrot
juice
2540 kV/cm, bipolar pulse length 2.5 ls, treatment time 340 ls,
60 mL/min
18.6 A
f
= 1.0841.249 Rodrigo et al. (2003a)
PE Orange juice 535 kV/cm, 200 Hz bipolar and monopolar pulse width 4 ls,
treatment time 1500 ls, 60 mL/min, 37.5 C
20 R
2
= 0.9550.996
A
f
= 1.0061.109
Elez-Martinez et al. (2007)
PE Red grape juice 40 kV/cm, 15 Hz pulse width 1 ls, treatment time 100 ls 3.2 R
2
= 0.98 Riener et al. (2009)
PE Distilled water 20 kV/cm, pulse width 40 ls, number of pulses 6, treatment time
8 ms, 25 C
2.1 A
f
= 3.6 Giner et al. (2005a)
PE Distilled water 38 kV/cm, treatment time 340 ls 13.2 R
2
= 0.969
A
f
= 1.073
Giner et al. (2005b)
PG Distilled water 10.28 kV/cm, pulse width 40 ls, treatment time 32.4 ms, 25 C 2 R
2
= 0.9360.982 Giner et al. (2003)
POD Grape juice 2535 kV/cm, 600 Hz bipolar pulse width 4 ls, treatment time
5 ms, ow rate 7.8 mL/s, 40 C
49.4 R
2
= 0.8690.999 Marselles-Fontanet and Martin-Belloso
(2007)
POD Apple juice 2350 kV/cm, 15 Hz pulse width 1 ls, treatment time 100 ls,
50 C
32 R
2
= 0.8640.978 Riener et al. (2008)
PPO NaCl + PVP + McIlvain buffer 24.6 kV/cm, bipolar pulse length 0.02 ms, 6 ms treatment time,
15 C
3.15 R
2
= 0.940.97 Giner et al. (2001)
PPO Peach juice 24.3 kV/cm, bipolar pulse width 0.02 ms, 5 ms treatment time, 30 R
2
= 0.810.99 Giner et al. (2002)
PPO Grape juice 2535 kV/cm, 600 Hz bipolar pulse width 4 ls, treatment time
0 R
2
(2007)
PPO Apple juice 2350 kV/cm, 15 Hz pulse width 1 ls, treatment time 100 ls,
50 C
29 R
2
= 0.7940.923 Riener et al. (2008)
Hulshegers models LOX Tomato juice 35 kV/cm, pulse width 3 ls, 50 ls treatment time, ow rate 1 mL/
s, 30 C
20 R
2
= 0.91 Min et al. (2003)
2
= 0.946 Giner et al. (2000)
juice
2540 kV/cm, bipolar pulse length 2.5 ls, treatment time 340 ls,
60 ml/min
18.6 A
f
= 1.0941.317 Rodrigo et al. (2003a)
20 R
2
= 0.8800.915
A
f
= 1.1041.124
8 ms, 25 C
2.1 A
f
= 2.20 Giner et al. (2005a)
2
= 0.924
A
f
= 1.149
Fermis model LOX Tomato juice 35 kV/cm, pulse width 3 ls, 50 ls treatment time, ow rate 1 mL/
s, 30 C
20 R
2
= 0.8510.987 Min et al. (2003)
25 C
12 R
2
= 0.9730.991 Li et al. (2008)
2
= 0.970 Giner et al. (2000)
20 R
2
= 0.9230.989
A
f
= 1.0251.104
2
= 0.932
A
f
= 1.077
Weibull distribution
model
25 C
12 R
2
= 0.9960.999 Li et al. (2008)
juice
2535 kV/cm, bipolar pulse length 2.5 ls, treatment time up to
340 ls, 60 ml/min
18.6 A
f
= 1.0071.025 Rodrigo et al. (2003a)
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20 R
2
= 0.9590.994
A
f
= 1.0031.117
8 ms, 25 C
2.1 A
f
= 1.902.45 Giner et al. (2005a)
2
= 0.974
A
f
= 1.064
Giner-Seguis model PE Gazpacho 35 kV/cm, 200 Hz monopolar pulse width 4 ls, treatment time
1500 ls, ow rate 60 mL/min, 40 C
3.8 A
f
= 1.055 Giner-Segui et al. (2009)
PG Distilled water 38 kV/cm, 200 Hz monopolar pulse width 4 ls, treatment time
1100 ls, ow rate 60 mL/min, 40 C
23.5 A
f
= 1.059 Giner-Segui et al. (2006)
Quadratic response
model
HPL Tomato juice 35 kV/cm, 250 Hz bipolar pulse width 7 ls, treatment time
1000 ls
10 R
2
= 0.923 Aguilo-Aguayo et al. (2009b)
LOX Tomato juice 35 kV/cm, pulse width 3 ls, 50 ls treatment time, ow rate 1 mL/
s, 30 C
20 R
2
= 0.92 Min et al. (2003)
LOX Tomato juice 35 kV/cm, 250 Hz bipolar pulse width 7 ls, treatment time
1000 ls
81 R
2
LOX Watermelon juice 35 kV/cm, 50 Hz monopolar pulse width 1 ls, treatment time
1000 ls
112.25 R
2
PE Tomato juice 35 kV/cm, 250 Hz bipolar pulse length 7 ls, ow rate 60 mL/min 10 R
2
= 0.91 Aguilo-Aguayo et al. (2009a)
PE Strawberry juice 35 kV/cm, 100 Hz monopolar pulse length 1 ls, ow rate 60 mL/
min
10 R
2
PG Tomato juice 35 kV/cm, 250 Hz monopolar pulse length 7 ls, ow rate 60 mL/
min
45 R
2
PG Strawberry juice 35 kV/cm, 100 Hz monopolar pulse length 1 ls, ow rate 60 mL/
min
75 R
2
POD Grape juice 2535 kV/cm, 600 Hz bipolar pulse width 4 ls, treatment time
49.4 R
2
(2007)
POD Tomato juice 35 kV/cm, 250 Hz bipolar pulse width 7 ls, ow rate 60 mL/min 0 R
2
= 0.85 Aguilo-Aguayo et al. (2008)
POD Watermelon juice 35 kV/cm, 50 Hz monopolar pulse width 1 ls, treatment time
1000 ls
15.25 R
2
PPO Grape juice 2535 kV/cm, 600 Hz bipolar pulse width 4 ls, treatment time
0 R
2
(2007)
a
RA means the residual enzyme activity.
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Considering the complexity of the mediums, most early studies on
enzyme inactivation by PEF were performed on model food sys-
tems, such as distilled water, buffered solution, and simulated milk
ultraltrate (SMUF), but not on real food products (Giner et al.,
2000, 2001, 2003, 2005a,b; Van Loey et al., 2002). Fresh fruit juices
have recently been used to validate the calculated results (Aguilo-
Aguayo et al., 2009a,b; Elez-Martinez et al., 2007; Giner-Segui
et al., 2009; Li et al., 2008; Marselles-Fontanet and Martin-Belloso,
2007; Min et al., 2003; Riener et al., 2008, 2009; Rodrigo et al.,
2003a). A majority of kinetic models used for enzyme inactivation
by PEF treatment are analogous to those used for the inactivation
of microorganisms, such as simple and traditional zero- and rst-
order kinetics. More importantly, the other kinetic models exclu-
sively used for enzyme inactivation will also be discussed in detail
in this section.
Compared with the extensive studies devoted to modeling for
the inactivation kinetics of microorganisms and enzymes by PEF,
limited information are available for modeling the loss of
health-related compounds after PEF treatment and during com-
mercial shelf-life (Bendicho et al., 2002a; Odriozola-Serrano et al.
2008a,b; Torregrosa et al., 2006), which may be attributed to the
application of treatments of equivalent microbial lethality to heat
treatment that are used when evaluating the effect of PEF treat-
ments on food quality properties. However, few currently available
publications indicate the process criteria to pasteurize liquid prod-
ucts by PEF. A few studies have developed models for the retention
of health-related compounds, including lycopene, anthocyanin,
avonoids, Vitamin C, phenolic acids, and antioxidant capacity
(Table 3). The models proposed for microorganism destruction
and enzyme inactivation have also been used for modeling the
Table 3
Kinetic models for bioactive compounds and antioxidant capacity degradation by PEF treatment.
Kinetic model Health-related
compound
Medium Process conditions Maximum
RC
a
(%)
R
2
/A
f
References
First-order
kinetics
models
Anthocyanins Strawberry
juice
35 kV/cm, 232 Hz bipolar pulse width 1 ls, 100 ls treatment
time, ow rate 60 mL/min, 40 C
100.5 R
2
= 0.731 Odriozola-Serrano
et al. (2008d)
Antioxidant
capacity
Tomato juice 35 kV/cm, 100 Hz bipolar pulse width 4 ls, 1500 ls treatment
time, ow rate 60 mL/min, 40 C, storage at 4 C
NR
b
R
2
et al. (2008b)
Antioxidant
capacity
100 R
2
= 0.836
A
f
= 1.039
Odriozola-Serrano
et al. (2008c)
Antioxidant
capacity
Strawberry
juice
25 kV/cm, ow rate 60 mL/min, 40 C NR
b
R
2
et al. (2008d)
Ascorbic acid Milk A static parallel plate treatment chamber, 27.1 kV/cm, 2025 C 93.4 R
2
= 0.936
0.985
Bendicho et al.
(2002a,b)
Lycopene Tomato juice 35 kV/cm, 100 Hz bipolar pulse width 4 ls, 1500 ls treatment
28.3 R
2
et al. (2008b)
Vitamin C Tomato juice 35 kV/cm, 100 Hz bipolar pulse width 4 ls, 1500 ls treatment
86.5 R
2
et al. (2008b)
Vitamin C Tomato juice 35 kV/cm, 250 Hz, ow rate 60 mL/min, 40 C 97 R
2
= 0.987
A
f
= 1.021
Odriozola-Serrano
et al. (2008c)
Vitamin C Strawberry
juice
93 R
2
et al. (2008d)
Fermis model Lycopene Tomato juice 35 kV/cm, 250 Hz bipolar pulse width 1 ls, 2000 ls treatment
137.7 R
2
= 0.992
A
f
= 1.001
Odriozola-Serrano
et al. (2008c)
Weibull
distribution
model
juice
100.5 R
2
et al. (2008d)
Antioxidant
capacity
100 R
2
= 0.984
A
f
= 1.008
Odriozola-Serrano
et al. (2008c)
Antioxidant
capacity
Strawberry
juice
25 kV/cm, ow rate 60 mL/min, 40 C NR
b
R
2
et al. (2008d)
137.7 R
2
= 0.986
A
f
= 1.012
Odriozola-Serrano
et al. (2008c)
Vitamin C Tomato juice 35 kV/cm, 250 Hz, ow rate 60 mL/min, 40 C 97 R
2
= 0.987
A
f
= 1.007
Odriozola-Serrano
et al. (2008c)
juice
93 R
2
et al. (2008d)
Quadratic
response
model
juice
35 kV/cm 250 Hz, bipolar pulse width 1 ls, treatment time
1000 ls
101.9 R
2
et al. (2009)
Antioxidant
capacity
92.3 R
2
et al. (2007)
Antioxidant
capacity
Watermelon
juice
35 kV/cm 200 Hz, bipolar pulse width 7 ls, treatment time 50 ls 100 R
2
= 0.838 Oms-Oliu et al.
(2009)
Antioxidant
capacity
Strawberry
juice
1000 ls
99.8 R
2
et al. (2009)
146.2 R
2
et al. (2007)
Lycopene Watermelon
juice
2
(2009)
Vitamin C Tomato juice 35 kV/cm, 50 Hz monopolar pulse width 1 ls, 1000 ls treatment
99 R
2
et al. (2007)
Vitamin C Watermelon
juice
2
(2009)
juice
1000 ls
100.3 R
2
et al. (2009)
a
RC means the relative content of bioactive compounds or relative antioxidant capacity.
b
NR means not reported.
effects on health-related compounds, such as the rst-order kinet-
ics, Hulshegers, Fermis, Weibull distribution, and the quadratic re-
sponse models. Most of these studies focused on tomato and
strawberry juices, which are rich in Vitamin C and antioxidants.
The following are in-depth discussions for each kinetic model.
4.1. First-order kinetics
4.1.1. Destruction of microorganisms
In 1921, Bigelow (1921) proposed a traditional rst-order mod-
el for heat treatments that expressed the reduction of the survival
fraction as a function of treatment time (Eq. (6)):
logY
t
D
t
6
where t is the treatment time, and D
t
is the decimal reduction time,
mathematically expressed as the negative inverse of the inactiva-
tion curve slope.
This approach was rst used to dene D
t
(decimal reduction
time) and z (dependence of D
t
on temperature) values for thermal
treatment and was subsequently used to describe the linear sec-
tions of inactivation curves obtained from non-thermal treatments,
such as high pressure (Musa and Ramaswamy, 1997) and PEF
treatments (Castro et al., 1993; Sensoy et al., 1997; Rodrigo et al.,
2003b; Rivas et al., 2006; Perez et al., 2007). The classical D
t
value
presents a simple biological signicance, that is, time resulting in a
simple exponential reduction of the surviving population. In many
cases, the survival curves of microorganisms by PEF are linear, and
only 23 log reductions are obtained. In these cases, Bigelows
model was effective in describing the linear relationship between
the logarithm of the survival ratio and the treatment time.
However, some obvious objections have recently been made
against the traditional rst-order kinetics model. The fact that
the single-target theory cannot account for the death rate observed
at the initial stage of survival curves and for the sublethal injury in
microorganisms is disputable (San-Martin et al., 2007). On the
other hand, the different behavior between various microorgan-
isms and enzymes treated by PEF might due not only to treatment
time but also to other important factors, such as medium, PEF sys-
tem, treatment chamber, characteristics of electric pulses and
treatment temperature (Giner et al., 2005b).
Treatment temperature has been proven to be an important fac-
tor in microbial inactivation by PEF, and its effects are rather com-
plex (Floury et al., 2006). The efciency of PEF treatments normally
increases with an appropriate increase in temperature. Based on
the synergy between temperature and electric eld strength, the
application of PEF at mild temperatures has been suggested as a
method for enhancing PEF effectiveness for food preservation
(Sepulveda et al., 2005). However, few models for the effect of tem-
perature on PEF treatment are available. Etsy and Meyer (1922)
developed another rst-order kinetics model, wherein the primary
model uses Bigelows model in exponential form as dened by Eq.
(7):
Y expkt 7
According to Arrhenius equation, the rate constant k increases
with increasing medium temperature T:
k k
T
exp
E
A
RT
_ _
8
where k
T
is a rate constant at the reference temperature, E
A
is the
activation energy (J/kg mol), R is the universal gas constant
(8.314 J/mol K), and T is the medium temperature (K).
For PEF treatments, the rate constants may take different val-
ues, attributing to differences in processing factors (e.g., medium,
treatment chamber, energy delivered to samples, and so on).
However, k cannot be adjusted as a function of electric eld inten-
sity in any model (Bendicho et al., 2002a). This adjustment could
not be conducted because in more severe working conditions,
dielectric sample breakdown occurred. The survival fractions of
E. coli O157:H7 and Salmonella Enteritidis in liquid egg yolk after
PEF were described based on the kinetics model (Eqs. (7) and (8))
(Amiali et al., 2007). As expected, the tting results showed that
the rate constants increased with increasing temperature. The
inactivation rate constants increased from 0.004 ls
1
to
0.098 ls
1
for Salmonella Enteritidis and from 0.009 ls
1
to
0.039 ls
1
for E. coli O157:H7 as processing temperature increased
from 20 C to 40 C. Salmonella Enteritidis was found to be more
resistant to PEF inactivation than E. coli O157:H7 at lower process-
ing temperatures. Different bacterial strains exhibit different resis-
tances to PEF-mediated inactivation even at the same temperature
possible because of differences in morphology (size or shape) or in
cell membranes (composition or structure). However, experimen-
tal or modeling data on comparative inactivation resistances of
microorganisms are scarce in published studies. Moreover, when
the effect of temperature on microbial inactivation by PEF is con-
sidered, treatments should have been applied under isothermal
conditions. If treatments are applied in continuous conditions,
the temperature of the treatment medium varies during PEF treat-
ment, making the evaluation of the effect of temperature on micro-
bial inactivation by PEF difcult (Saldana et al., 2010).
4.1.2. Inactivation of enzymes
The importance of adequate temperature control during PEF
treatment in small batch treatment chambers is often neglected.
Thus, no distinction could be made between thermal and PEF ef-
fects on enzymes (Van Loey et al., 2002). However, in continuous
treatment chambers, treatment temperature plays an important
role in the inactivation of enzymes because of the difference in
heat sensitivity among various enzymes. Accordingly, different en-
zymes require different intensities of PEF treatment, and the tem-
perature variation affects the accuracy of the model. Therefore, the
effect of temperature during the different processing steps has to
be considered when evaluating the process effect (Jaeger et al.,
2010). These steps include the pre-heating, ohmic heating during
PEF treatment, cooling, as well as the occurring holding times in
the pipe-sections. The quantication of thermal and electric eld
effects and their contribution to the overall inactivation revealed
that thermal effects had a signicant effect on enzyme inactivation
during thermal-assisted PEF processing or PEF treatment with the
occurrence of temperature hot spots (Jaeger et al., 2009, 2010).
Additionally, numerous effects need to be considered when study-
ing the PEF effect on enzymes because temperature could be af-
fected by other parameters, such as pulse frequency, treatment
time, and medium conductivity. For instance, the increase in treat-
ment time or in specic energy input would result in enzyme inac-
tivation attributed to thermal effects resulting from the excess
thermal inactivation temperature of the enzyme (El Zakhem
et al., 2007; Grahl and Markl, 1996). To avoid the interference of
thermal effects, the kinetic model should be dened as a PEF-only
inactivation based on the estimation of a homogeneous tempera-
ture increase in the treatment chamber. However, this assumption
has certain limitations, as reported by Jaeger et al. (2009), who
indicated the occurrence of high local temperature in the co-linear
treatment chamber.
Similar to the destruction of microorganisms, the rst-order
kinetics model (Eqs. (7) and (8)) is used to study the kinetics
involving the effect of temperature on enzyme inactivation. Jaeger
et al. (2010) used this model to differentiate temperature and
electric eld effects on the inactivation of ALP and LPO in apple
juice based on the heat inactivation kinetics and PEF preservation
process results. The experimental and calculated PEF-only
inactivation results were in good agreement. Based on this kinetic
model, Riener et al. (2009) studied the combined effects of thermal
and PEF treatments on PE inactivation in red grape juice. A corre-
lation (R
2
= 0.960.98) between relative activity and PEF treatment
time was obtained at low preheating temperatures. However, R
2
(0.740.81) signicantly decreased as the pre-treatment tempera-
ture increased, which possibly resulted from the assisted thermal
effects and the heat labile fraction of PE.
Considering that enzyme activity is proportional to the amount
of remaining enzymes, enzyme activity can be determined by cal-
culating the change in enzyme concentration during the PEF pro-
cess. According to the rst-order kinetics model, the expression
can be easily obtained using Eq. (7), where the rst-order kinetics
constant, k, unlike models for microorganism inactivation, is de-
ned as a function of electric eld strength E. As expressed in Eq.
(9), an exponential relationship is shown between the rate con-
stant and electric eld intensity
k k
E
expx E 9
where k
E
and x are constants that require calculation and are ex-
pressed in the same units as k and in cm/kV, respectively.
The rst-order kinetics model (Eqs. (7) and (9)) has been used to
describe the effect of electric eld intensity, number of pulses, and
pulse width on the enzyme activity (Giner et al., 2000, 2001, 2002,
2003). Enzyme activities signicantly decreased with any increase
in applied pulse number, electric eld intensity, and pulse width.
The range of k values varied when enzyme extracts were exposed
to pulses within different working electric eld strengths. Such a
phenomenon could be explained from two perspectives. First,
according to Eq. (9), electric eld intensity was observed to have
a positive and strong effect on k, depending on the parameters k
E
and x. The values of the constants in Eq. (9) show the important
effect of electric eld intensity on the kinetic rate constant. Second,
on the premise that none of the thermal effects are achieved, the
enzymes sourced from different liquid foods may have varied sen-
sitivity during PEF treatment, which may be attributable to the
huge differences in molecular size and structures displayed among
enzymes in these studies. Both of the rate constants may be af-
fected by a large number of factors and variables, some of which
can be related with their own nature, enzyme structure, and en-
zyme medium properties (Giner et al., 2001).
In continuous PEF processing, the energy input on a sample can
be used to estimate a theoretical temperature increase of this sam-
ple assuming that all the electrical energy delivered in the sample
is dissipated as heat (Van Loey et al., 2002). As the input of energy
density increases, the enzyme activity decreases after PEF treat-
ment. The rst-order kinetics model (Eq. (10)) has been applied
to correlate RA as a function of the total electric energy density Q
(J/m
3
), which is supplied by the pulse generator during PEF
treatment:
RA expk
q
Q 10
where k
q
(m
3
/J) is a constant.
This model was widely used to describe the dependency of RA
on Q for various enzymes (Bendicho et al., 2002b; Giner et al.,
2001, 2002, 2003). For both polarities, the experimental data
clearly showed a decrease in RA as Q increased, and the general
shape of the decrease at any Q seemed to be exponential (Bendicho
et al., 2002b; Giner et al., 2002). Giner et al. (2001) reported that a
higher input electric energy supplied to samples results in a
greater reduction in obtained PPO activity. In addition, to achieve
a required inhibition level on PPO enzyme activity, pear PPO re-
quired signicantly higher energy densities compared with apple,
indicating that the sensitivities of enzymes towards PEF treatment
were certainly somewhat different from one another. Bendicho
et al. (2002b) performed experiments with different PEF devices
working in either batch or continuous mode and reported the same
kind of relationship for microbial lipase in SMUF. Giner et al.
(2003) compared the value of the exponential factor (k
q
) in Eq.
(10) with that obtained by Bendicho et al. (2002b), and results indi-
cated that although different PEF systems are used, a similar
degree of enzyme inactivation is achieved if the same quantity of
energy per volume unit to its respective medium is supplied. The
result conrmed that this model is capable of predicting the deple-
tion of enzymes at different input energy densities with reasonable
accuracy.
Lebenspiel (1972) proposed a rst-order fractional conversion
model (Eq. (11)) that is one of the most useful for describing the
inhibition of enzymes by PEF. This model was rst used for PE
and PG inactivation modeling using thermal and high-pressure
treatment (Fachin et al., 2003; Van den Broeck et al., 2000)
RA RA
1
RA
0
RA
1
expk
p
P 11
where RA
1
(%) is the residual enzyme activity after prolonged treat-
ment (stabilization value); P stands for the treatment time (t, s), the
pulse frequency (f, Hz), the pulse width (s, s), or the electrical
energy density (Q, J/m
3
); and k
p
is a modied rate constant.
In this model, the reduction in enzyme activity is found to be
related to the pulse frequency, pulse width, and electrical energy
density supplied by the pulse generator during PEF treatments.
The residual enzyme activity stabilization value (RA
1
) decreases
with increasing electric eld strength, which plays an important
role in the mechanism of enzyme inactivation by PEF because an
increase in electric eld strength may result in conformational
changes in the enzyme structure. Similarly, the pulse width and
pulse frequency have a signicant effect on the enzyme inactiva-
tion by PEF. However, kinetic rate k
p
is not statistically affected
by the pulse polarity nor the electric eld strength. This model t-
ted with high accuracy to the inactivation of PE in orange juice by
PEF as a function of either pulse frequency f (R
2
= 0.990; A
f
= 1.024)
or pulse width s (R
2
= 0.947; A
f
= 1.094) (Elez-Martinez et al.,
2007). These results were consistent with those reported by Elez-
Martinez et al. (2006), who treated orange juice POD at different
pulse frequencies, and by Bendicho et al. (2003), who studied the
changes induced by variations of pulse width on protease in skim
milk.
Only Elez-Martinez et al. (2007) dened the relationship
between Q and RA using the rst-order fractional conversion mod-
el. The values of R
2
and A
f
were 0.914 and 1.069 for the monopolar
mode and 0.933 and 1.099 for the bipolar mode. The model was
very useful in relating orange juice PE inactivation to the energy
supplied by the pulse generator. The exponential factor (k
P
) in
Eq. (11) took values of 2.92 10
4
and 4.82 10
4
m
3
/MJ when
pulses were applied in monopolar and bipolar modes, respectively.
Monopolar pulses exhibited less effective inactivation of orange
juice PE than bipolar pulses when the same energy density is ap-
plied. These observations were consistent with the results reported
by Giner et al. (2002), who described the inactivation of peach PPO
as a function of Q using an exponential decay model.
4.1.3. Retention of health-related compounds
The rst-order kinetics model (Eqs. (7) and (8)) involving
temperature variation was also applied to study the effects of the
PEF process on the destruction of water- and fat-soluble vitamins
in SMUF and milk at different treatment temperatures (Bendicho
et al., 2002a). The retention of ascorbic acid tted a rst-order
kinetics model for both PEF and thermal processes. The rst-order
constant k values varied from 1.8 10
4
ls
1
to 1.27 10
3
ls
1
for the PEF treatments (18.3 kV/cm to 27.1 kV/cm). The values of
the constant in SMUF were higher than those in skim milk, and
the maximum k values were obtained when low electric eld
intensity was applied. No signicant differences were found be-
tween the results obtained after applying PEF treatments at room
or moderate temperatures. The resultant k values were similar at
different treatment temperatures.
4.2. Hulsheger
Hulsheger and Niemann (1980) proposed a model involving
critical treatment time t
c
:
lnY B
t
lnt lnt
c
12
where B
t
is the curve slope dependent on the treatment time t, and
t
c
is the critical treatment time value (s). Treatment time, t, is de-
ned as the product of the pulse width s and the pulse number n:
t ns nRC 13
where R is the resistance of the eld treatment cell (X), and C is the
value of capacitor (F).
Considering the effect of the electric eld, Hulsheger et al.
(1981) also proposed a function of electric eld strength:
lnY B
E
E E
c
14
where B
E
is the curve slope dependent on the electric eld strength
E. For the assumption of a practically nonconducting sphere in a
conducting medium, E
c
, the critical strength of electric eld (kV/
cm) is found to be a function of cell size, which can be calculated
by Eq. (15) (Sale and Hamilton, 1968; Schwan, 1977; Zimmermann
et al., 1974):
E
c

V
c
1:5a
15
where V
c
is the critical membrane potential, and a is the mean cell
diameter. However, Eq. (15) is invalid for nonspherical cells without
modications, such as E. coli.
Assuming a linear relationship between the logarithm of the
survival ratio and the electric eld strength, as well as a linear rela-
tionship between the logarithm of the survival ratio and the loga-
rithm of treatment time, a mathematical model Eq. (16) is obtained
(Hulsheger et al., 1981):
Y
t
t
c
_ _
EEc
kc
16
where k
c
represents an independent constant factor. The parame-
ters E
c
, t
c
, and k
c
are proposed to be independently determined by
the target microorganism or enzyme.
According to these expressions, the models are based on the
electric eld intensity, E, treatment time, t, or the combination of
both, E and t. For the rst two models, the regression coefcients
B
t
and B
E
, which vary with different mediums, show that the gradi-
ents of the straight survival curves are dependent on treatment
time and electric eld strength. Larger B
t
or B
E
values result in a
higher microbial inactivation rate. However, the B
t
and B
E
values
in Hulshegers models cannot be considered a rate index for micro-
organisms subjected to PEF treatment because they do not always
decrease with increasing treatment time or electric eld strength
(Rivas et al., 2006).
The critical treatment time t
c
and the critical eld strength E
c
are related to microbial characteristics. According to Eqs. (12)
and (14), lower t
c
and E
c
values appear to indicate less resistance
to the PEF treatment. Critical treatment time t
c
is also known as
the time wherein the fastest inactivation rate is achieved. In partic-
ular, E
c
was found to be a function of cell size because it decreased
with increasing cell size, which is attributable to the transmem-
brane potential over the cell that was proportional to the cell size
(Grahl and Markl, 1996; Qin et al., 1998; Barbosa-Canovas et al.,
1999; Mosqueda-Melgar et al., 2008a). On further modeling, Eq.
(16) describes the survival ratio as a combined function of the elec-
tric eld strength and the treatment time in a double-logarithmic
relationship. Similar to B
t
and B
E
, the k
c
value is determined by
the sensitivity of a specic microorganism (or enzyme) to PEF.
For instance, a large value of kinetic constant k
c
indicates a deep
decline in the inactivation rate curve and higher susceptibility to
PEF, whereas a small value implies an insensible gradation of inac-
tivation rate and lower sensitivity to PEF.
Rivas et al. (2006) tted the inactivation of E. coli (ATCC 8739)
suspended in orange juice and milk beverage to the Hulshegers
model (Eq. (12)). The t
c
value decreases from 6.753 ls to
1.091 ls as the electric eld strength increases from 15 kV/cm to
40 kV/cm, in agreement with the conclusions mentioned above.
The killing effect of PEF on E. coli CGMCC 1.90 in carrot juice was
studied by Zhong et al. (2005), and the tting of its inactivation
kinetics was performed by Hulshegers model (Eq. (14)). As the
number of pulses increased, the kinetic constants B
E
and E
c
calcu-
lated based on Hulshegers model varied from 0.3116 cm/kV to
0.3790 cm/kV and from 4.0565 kV/cm to 1.6121 kV/cm, respec-
tively. These data were inconsistent with the value of E
c
(from
4.7 kV/cm to 8.2 kV/cm) obtained by Hulsheger et al. (1981) based
on different electrolytes and that reported by Grahl and Markl
(1996). San-Martin et al. (2007) attempted to describe the inactiva-
tion of L. innocua ATCC 51742 by PEF using Hulshegers model (Eq.
(16)) but failed. Since the calculated critical electric eld for
37.4 ls is smaller than the E
c
value for 25.12 ls, the model is not
adequate for describing the experimental data, probably due to dif-
ferences in experimental conditions.
Accordingly, the Hulshegers models were utilized to describe
the enzymatic inhibition, such as LOX and PE in different mediums
(Min et al., 2003; Rodrigo et al., 2003a). Giner et al. (2000) found
lower values in E
c
(0.7 kV/cm) and higher values in t
c
(480 ls) for
tomato PE than those (E
c
= 4 kV/cm and t
c
= 60 ls) obtained by
Elez-Martinez et al. (2007) who exposed orange juice PE to PEF.
Nevertheless, Giner et al. (2005a,b) treated PE in a commercial en-
zyme preparation (CEP) with PEF and they reported an E
c
of
14.8 kV/cm and a t
c
of 43 ls. It was indicated that the different val-
ues of Hulshegers model constants for enzyme inactivation were
affected by the enzyme source and the PEF-treatment parameters.
4.3. Fermi
Deviations from linearity, particularly in the case of the non-
thermal inactivation of microorganisms and enzymes, are becom-
ing more evident as research progresses. Similarly, Peleg (1995)
proposed another equation called Fermis model for the combined
effects of treatment time and electric eld (Eq. (17)), which also
describes the sigmoid shape of the survival curves:
Y
1
1 exp
EEc n
kc n
_ _ 17
where k
c
is the parameter that indicates the steepness of the sur-
vival curve around E
c
. The modication is that both parameters in
this function, E
c
and k
c
, are exponentially related to the number of
applied pulses, n.
This model indicates the effect of critical electric eld strength
on the survival fraction as a function of treatment time, which is
proportional to the number of pulses with the same pulse width.
The kinetic parameter k
c
refers to the level of microbial inactiva-
tion around E
c
. A lower k
c
value results in faster inactivation of
microorganisms (Zhong et al., 2005). As the number of pulses
increases within a certain range, the value of E
c
continuously
decreases. However, the variation in the number of pulses beyond
this range has an insignicant effect on the E
c
value. For example,
when San-Martin et al. (2007) tted the Fermis function to L.
innocua inactivation by PEF, the value of critical electric eld (E
c
)
decreased with increasing number of pulses from5 to 15; however,
at 20 pulses the E
c
increased and was similar to the E
c
value ob-
tained for 10 pulses. The range of E
c
values vary with the changes
in the conguration of PEF treatment chambers and the genus of
bacteria. L. innocua treated in co-linear treatment chamber (from
19.3 kV/cm to 22.2 kV/cm) showed greater E
c
values than E. coli
treated in coaxial treatment chamber (from 2.5 kV/cm to 8.1 kV/
cm), indicating higher resistance to PEF treatment (San-Martin et
al., 2007; Zhong et al., 2005). Fermis equation successfully mod-
eled the inactivation of different microorganisms by PEF, including
E. coli, L. innocua, Salmonella Dublin, and so on (Peleg, 1995;
San-Martin et al., 2007; Sensoy et al., 1997; Zhong et al., 2005).
Moreover, the feasibility of using the Fermis model to describe
the inactivation of the LOX and PE enzyme was demonstrated
(Elez-Martinez et al., 2007; Li et al., 2008; Min et al., 2003). Differ-
ent from the parameter E
c
, the steepness parameter (k
c
) were
signicantly non-depending on treatment time and pulse polarity
(Elez-Martinez et al., 2007), in conformity with the observations
obtained by Zhong et al. (2005).
4.4. Weibull distribution
In probability theory and statistics, the Weibull distribution is a
continuous probability distribution named after Waloddi Weibull,
who described this model in detail in 1951 (Weibull, 1951). As a
function of treatment time or specic energy density, the Weibull
distribution function is given by the following decimal logarithmic
form (Peleg and Cole, 1998):
log Y bt
p
18
Considering that parameter b of Eq. (18) has no immediate
physical signicance and has the dimensions of time power p,
the model was modied into the following form (Mafart et al.,
2002):
Y exp
v
d
_ _
p
_ _
19
where v is the treatment time t or the specic energy density Q, and
d and p are the scale and shape parameters, respectively. With rare
exceptions, an exponential relationship exists between the values of
d and the electric eld strength E.
4.4.1. Inactivation of microorganisms
In the last 10 years, numerous studies have applied the Weibull
distribution model (Eq. (19)) to describe the survival curves of
thermal and PEF-treated foods. The Weibull distribution function
has two parameters, namely, the scale parameter d and the shape
parameter p. The value of the scale parameter d, which can be con-
sidered a measure of microbial resistance to the treatment (kinetic
parameter), decreases with increasing electric eld strength
(Alvarez et al., 2003a,b). The parameter p describes the shapes of
the survival curve, so that when p < 1, the survival curve is up-
wardly concave, when p > 1, it is downwardly concave, and when
p = 1, it is a straight line on a logarithmic scale (equal to Bigelows
model) (Gomez et al., 2005). This parameter should be useful when
dening PEF processing conditions (San-Martin et al., 2007)
because p > 1 implies that at a certain voltage, a treatment time be-
yond exists, wherein inactivation will drastically increase.
Whereas, p < 1 indicates that a treatment time beyond exists,
wherein no further reduction attributable to PEF treatment is
expected, thus, indicating the opposite.
The specic energy density Q is considered a control factor of
PEF treatments because of its integrated characteristic (Heinz
et al., 1999). However, specic energy density cannot be used as
the only factor that involves the effect of electric eld strength
and treatment time because at a constant specic energy, micro-
bial elimination depends on the applied electric eld strength
(Monfort et al., 2010). When low-frequency pulses were applied
in the treatment chamber, the Weibull distribution model as a
function of Q was well tted to describe the kinetics of microbial
inactivation by PEF. The rst-order model is less complex than
the Weibull distribution model. However, the Weibulls model
always presents enhanced exibility in describing potential differ-
ent patterns of inactivation curves because this model includes an
additional shape parameter. When p = 1, the Weibulls model as a
function of Q may be treated as the rst-order model (Eq. (10)).
Alvarez et al. (2003a) and Monfort et al. (2010), respectively
proposed tertiary models for the inactivation of L. monocytogenes,
Salmonella Enteriditis, and Salmonella senftenberg treated by PEF
in terms of processing factors (eld strength, treatment time, and
specic energy density). Their tertiary models were adequate for
accurately predicting the inactivation of different bacterial strains
and were capable of achieving a high maximum correlation coef-
cient between the predicted and observed data in terms of specic
energy density.
The tting accuracy of the traditional rst-order kinetics,
Hulshegers, Fermis, and Weibull distribution models for microbial
inactivation by PEF treatments were compared in several studies
(Alkhafaji and Farid, 2008; Alvarez et al., 2003b; Grahl and Markl,
1996; Martin et al., 1997; Rivas et al., 2006; Rodrigo et al., 2003b;
San-Martin et al., 2007). The model that best tted the survival
curves was the Weibull distribution function, followed by the tra-
ditional rst-order kinetics model (Rivas et al., 2006; Rodrigo et al.,
2003b; San-Martin et al., 2007). Although the traditional rst-order
kinetics model had slightly lower accuracy than the Weibull distri-
bution in these studies, it was simpler and was considered suf-
cient to describe the linear region of inactivation kinetics, which
occurred at low voltages and short treatment times. On the con-
trary, the Weibull distribution model performed better than the
traditional rst-order kinetics model when applied to estimate
microbial inactivation at high electric elds and long treatment
times (San-Martin et al., 2007).
However, the Weibull distribution function presents a major
drawback, that is, parameter p is structurally strongly correlated
with d values, indicating that the two parameters are not
independent. Thus, an error in d will be balanced by an error in p
(Mafart et al., 2002). Such an autocorrelation results in certain
instabilities of parameter estimates (Peleg and Cole, 1998).
Further investigations were performed by Couvert et al. (2005)
to assess the effect of environmental conditions on the Weibull
model parameters. The environmental factors presented no clear
and regular effect on the shape parameter p, but the scale param-
eter d was dependent on temperature and medium pH. Thus, the
simplied Weibulls model proposed by Couvert et al. (2005) with
a constant p-value for given microbial populations can be applied
for calculations, without considering the minimal effect of param-
eter p variation on the goodness of t.
4.4.2. Inactivation of enzymes
Based on Weibulls distribution function, the kinetic models
were also developed for the LOX in soymilk and the PE in fruit juice
and distilled water (Elez-Martinez et al., 2007; Giner et al.,
2005a,b; Li et al., 2008; Rodrigo et al., 2003a). Rodrigo et al.
(2003a) processed orangecarrot juice by PEF from 25 kV/cm to
35 kV/cm up to 340 ls. The Weibull distribution model was used
to describe the inactivation of PE by PEF, where the value of the
scale parameter d ranged from 120.57 ls to 288.48 ls. Li et al.
(2008) showed the inactivation of soybean LOX as a function of
the Weibulls model with high regression parameters (R
2
= 0.996
to 0.999). The scale parameter d and the shape parameter p
decreased from 904.94 ls to 322.16 ls and from 0.794 ls to
0.492 ls with increasing electric eld strength. Elez-Martinez
et al. (2007) modeled the PEF inactivation of orange juice PE using
this model, which properly tted the experimental data
(R
2
> 0.955) and exhibited good accuracy (A
f
= 1.0021.117). Both
scale and shape parameters (d and p) were found to be signicantly
nondependent on electric eld and pulse polarity. However, the d
values in the present study (from 18,000 ls to 20,000 ls) were
relatively high compared with those reported by Rodrigo et al.
(2003a). Therefore, orange juice PE was more resistant to PEF than
the PE of the mixture of orange and carrot juice according to the d
parameter of the Weibulls model. Moreover, a concave inactiva-
tion curve was indicated in the present study with p-values (from
0.22 to 0.70) lower than one, whereas Rodrigo et al. (2003a)
reported p parameters (from 2.82 to 2.91) higher than one,
implying a convex inhibition curve. This nding indicates that
the shape of the curve of enzyme inactivation related to the en-
zyme source.
The tting accuracy of the rst-order kinetics, Hulshegers,
Fermis, and Weibull distribution models to describe the enzyme
inactivation by PEF were also compared (Elez-Martinez et al.,
2007; Li et al., 2008; Rodrigo et al., 2003a). Elez-Martinez et al.
(2007) indicated that the rst-order fractional conversion model
was most useful for describing the inhibition of orange juice PE
by PEF as a function of treatment time. The Weibull distribution
function described enzyme inactivation at higher levels of electric
eld strength with better accuracy compared with lower levels.
However, both Rodrigo et al. (2003a) and Li et al. (2008) showed
that the Weibull distribution function was the most suitable model
for describing the inactivation of orangecarrot juice PE and
soybean LOX as a function of treatment time and strength.
The results of the inactivation of PE in CEP under PEF were stud-
ied by Giner et al. (2005a,b). Two models on the effect of specic
energy density Q on the RA were compared in the current study,
namely, the rst-order kinetics (Eq. (10)) and the Weibull distribu-
tion models (Eq. (19)) as a function of Q. The statistics for the dis-
tributions obtained for the respective accuracy factors of these
models indicated that the Weibulls model better tted the exper-
imental data compared with the rst-order function, with values of
A
f
at 3.6 and 2.45 for the rst-order kinetics and the Weibulls
models, respectively. In general, these two tested models involving
Q showed weak capability to describe the inactivation of PE under
PEF treatments.
4.4.3. Retention of health-related compounds
The Weibull distribution model is also likely to be a useful tool
for describing the retention of health-related compounds in PEF-
treated fruit juices (Odriozola-Serrano et al., 2008c). Changes in
lycopene, Vitamin C, and antioxidant capacity of PEF-treated toma-
to juice were modeled as a function of electric eld strength and
treatment time (Odriozola-Serrano et al., 2008c). The Weibull
kinetic model was used to predict Vitamin C and antioxidant
retention of PEF-treated tomato juice with good accuracy (R
2
P
0.836; A
f
= 1.0011.010), whereas the Fermis model gave compara-
tively less accurate predictions (R
2
P0.747, A
f
= 1.0181.021).
When the combined effects of both treatment time and electric eld
strength were considered, the Weibull distribution model gave the
most accurate predictions on the retention of health-related com-
pounds (R
2
P0.948; A
f
= 1.0161.017). Odriozola-Serrano et al.
(2008d) modeled the retention of anthocyanins and Vitamin C, as
well as antioxidant capacity, in fresh PEF-treated strawberries using
the rst-order and Weibull distribution functions. Both models
described the relationship between health-related compound
retention and PEF treatment time quite well, irrespective of electric
eld strength. However, R
2
= 0.7310.846 and R
2
= 0.7270.959
for the rst-order kinetics and Weibull distribution models,
respectively.
4.5. Loglogistic
Cole et al. (1993) described the Loglogistic model to t the sur-
vivor curves:
logY a
1
a
2
a
1
1 exp
4rklog t
a
2
a
1
_ _ 20
where a
1
is the upper asymptote (log CFU/ml), a
2
is the lower
asymptote (log CFU/ml), r is the maximum slope of the inactivation
curve, and k is the log time at which the maximum slope is reached
(position of the curve). For PEF treatments, the effect of electric eld
strength on the k value is used to describe microbial inactivation
across the tested electric eld strength range.
The Loglogistic model (Eq. (20)), describing sigmoid curves,
was also proposed to t the inactivation of microorganisms at
different electric eld strengths, justied by a distribution of resis-
tance within the bacterial population. Survival curves of Salmonella
Senftenberg treated by PEF that covered 67 log cycles were mod-
eled by using this model (Raso et al., 2000). When the sigmoid
curve was tted to the experimental data, no signicant change
was observed in the values of a
1
, a
2
, r, and there a relationship
was observed between electric eld strength and the position of
the curve on the log time axis (k value). The effect of electric eld
strength on the microbial inactivation can be estimated from the
position of the curve (k value). The correlation R
2
= 0.992 of the lin-
ear regression analyses between measured and estimated values
showed that the model results were a good t with the measured
data and were in agreement with the results of microbial inactiva-
tion by heat treatment (Anderson et al., 1996; Ellison et al., 1994;
Stephens et al., 1994).
4.6. Giner-Segui
Based on the hypothesis that under PEF, enzymes undergo two
consecutive irreversible steps with the presence of intermediate
active forms of the enzyme between the native and the completely
inactivated forms, Giner-Segui et al. (2006) proposed a kinetic
model as a function of treatment time at xed conditions,
expressed as Eq. (21). The effect of E on K is shown as Eq. (22),
in which a
K
, b
K
, and c
K
are parameters that are to be determined
RA RA
0
e
k
1
t
k
1
K
k
1
k
2
e
k
1
t
e
k
2
t
_ _
_ _
21
K a
K
E
2
b
K
E c
K
22
where k
1
and k
2
are the rate constants for the rst and second steps
of the enzyme inactivation process, respectively; and K is the ratio
between the activities of intermediate and native forms of the
enzyme K k
2
=k
1
, where k
1
and k
2
are the proportional constants
of native and intermediate forms of the enzyme. In this model,
either the electric eld strength or the polarity of pulses exerts no
signicant effect on the value of parameter k
1
. On the contrary,
the rate constant of the second stage k
2
is equally affected by the
electric eld intensity and polarity of pulses. Hence, the value of
k
2
increases with the augmentation of the electric eld intensity
of both polarities of pulses (Giner-Segui et al., 2009). As expressed
in Eq. (22), the model parameter K is related to electric eld inten-
sity. The value of parameter K implies noteworthy differences
between the activities of the same enzyme from different sources,
which may be attributed to the different sensitivities of the en-
zymes to PEF. Although this model is more complex than others
with less parameters, it adds more exibility in addition to accu-
racy. Giner-Segui et al. (2009) demonstrated the excellent accuracy
of the model in predicting the inactivation of PE in gazpacho apply-
ing monopolar (A
f
= 1.0281.055) or bipolar pulses (A
f
= 1.030
1.089). An earlier report by Giner-Segui et al. (2006) using the sim-
ilar kinetic model demonstrated high accuracy (A
f
= 1.0161.059)
when compared with experimental data.
5. Combined effects of the processing factors
The dened functions of E and t notably include other process-
ing factors, such as pulse polarity, electrolyte concentration, and
capacitor value. When these factors are not assumed as constants,
the above mentioned models will reach their limits. Models
involving variations in physiological conditions have been pro-
posed to optimize process conditions so that the highest microbial
inactivation and the lowest energy consumption are obtained.
Generally, a single response analysis of the data is performed as
a rst step to obtain optimum values of each factor with maximal
reduction for every microorganism in each medium. However, to
avoid discrepancies or differences among the obtained values of
the factors, a multiple response analysis is applied to determine
the minimum value of each factor needed to obtain the highest
process efciency in each food product undergoing PEF treatment.
The response surface methodology (RSM), which reduces the
number of experiments and improves statistical interpretations,
consists of mathematical and statistical procedures that study
the relationships between one or more responses (Diniz and
Martin, 1996). RSM is utilized to analyze the effect of multiple
independent variables during PEF treatment. The quadratic re-
sponse function is expressed by the following equation (Myers
and Montgomery, 2002):
Y b
0
3
i1
b
i
X
i
3
i1
b
ii
X
2
i

2
i1
3
ji1
b
ij
X
i
X
j
23
where Y is the response; factor X represents the encoded values of
the variables; and b
0
, b
i
, b
ii
, and b
ij
are the constant, linear, qua-
dratic, and interaction regression coefcients, respectively.
5.1. Destruction of microorganisms
Based on the polynomial response function (Eq. (23)), Sobrino-
Lopez et al. (2006) studied the effects of ve independence vari-
ables on the microbial inactivation of Staphylococcus aureus in milk
by PEF treatment. Among the studied variables, polarity, pulse
number, pulse width, electric eld intensity, and the combined ac-
tion of pulse number with pulse width or electric eld strength sig-
nicantly affected the microbial death of S. aureus. Moreover, the
combined action of variables resulted in higher microbial inactiva-
tion, showing that microbial inactivation did not only result from
single variables, but also from their combinations. Mosqueda-Mel-
gar et al. (2007, 2008b) reported a multilevel factorial design used
to evaluate the effects of treatment time and pulse frequency on
the bacterial reductions of Salmonella Enteritidis, E. coli O157:H7,
and L. monocytogenes populations inoculated in different PEF-trea-
ted fruit juices. Salmonella Enteritidis and E. coli O157:H7 were
found to be more sensitive to PEF treatment than L. monocytogenes,
indicating that inactivation efcacy varies for all microorganisms.
On the other hand, the electric conductivity of melon juice
(5.23 mS/cm 0.03 mS/cm) was higher than that of watermelon
juice (3.66 mS/cm 0.05 mS/cm). When a certain electric eld
strength was applied, the energy density received by the melon
juice was higher than that by the watermelon juice, therefore
resulting in higher temperature and greater sensitivity to PEF
treatment of the former than the latter. Marselles-Fontanet et al.
(2009) evaluated the effect of three processing factors (electric
eld strength, pulse frequency, and treatment time) on a mixture
of microorganisms (Kloeckera apiculata, Saccharomyces cerevisiae,
Lactobacillus plantarum, Lactobacillus hilgardii, and Gluconobacter
oxydans) in grape juice using polynomial response functions.
Single- and multiple-response analyses were performed to opti-
mize the inactivation of spoilage microorganisms by PEF. The opti-
mal inactivation of the mixture of spoilage microorganisms was
predicted by the polynomial response models at electric eld
strength of 35.0 kV/cm for total treatment time of 1000 ls, with
pulse frequency of 303 Hz and pulse width of 5 ls. As predicted
by the model, inactivation was greater for yeasts than for bacteria.
5.2. Inactivation of enzymes
The effect of independent variables on residual enzyme activi-
ties was also modeled using the polynomial response function. Sta-
tistical analyses indicate that the quadratic model is adequate for
accurately describing the changes in enzyme activity. Based on
RSM, various quadratic models of enzyme inactivation were
proposed, considering the effects of a greater number of factors,
including pulse width, pulse frequency, and pulse polarity
(Aguilo-Aguayo et al., 2008, 2009a,b, 2010b; Marselles-Fontanet
and Martin-Belloso, 2007; Min et al., 2003).
The interaction of different treatment factors can result in dif-
ferences in the effect of PEF on the residual enzyme activity RA.
The combined effect of treatment time and temperature on PEF-in-
duced inactivation of tomato juice LOX was evaluated by Min et al.
(2003). The quadratic model tted the observed residual LOX activ-
ity, with a correlation coefcient of 0.92. Marselles-Fontanet and
Martin-Belloso (2007) studied the impact of PEF processing factors,
including electric eld strength, pulse frequency, pulse width, and
total treatment time, on PPO and POD activity using the quadratic
model. The analysis of variance showed that the polynomial
models containing up to second-order terms, including interac-
tions and quadratic factors, explained the variability of raw data
(R
2
PPO
= 0.9147; R
2
POD
= 0.9010) better than models with a single
factor. The predictive models are always developed based on infor-
mation from the analysis of variance considering only the terms
that signicantly affect the response variables and maintaining
the hierarchy of these variables in the models. The effect of pulse
polarity on POD inactivation was studied through this model, ade-
quately describing PEF behavior (Aguilo-Aguayo et al., 2008). PEF
treatment was found to be more effective in the bipolar than in
the monopolar mode in reducing POD activity, obtaining 7.9%
and 37.1% of residual activity, respectively, when tomato juice
was subjected to PEF treatment at 35 kV/cm for 1000 ls using
250 Hz and 4 ls pulse width. The signicant interaction term pulse
frequency and treatment time was included in the model, showing
that different combinations of both variables can result in the same
level of residual POD activity. In addition, the effects of pulse fre-
quency, pulse width, and polarity on the PEF treatments on fruit
juice LOX, HPL, PE, POD, and PG activities were also evaluated
using RSM, and the statistical analysis indicated that the quadratic
models were sufciently accurate to t the experimental data
(Aguilo-Aguayo et al., 2009a,b, 2010b).
5.3. Retention of health-related compounds
Given the complex effects of PEF treatment on health-related
compounds and antioxidants, using the RSM consisting of mathe-
matical and statistical procedures is necessary for studying the
relationships among various factors. The effects of PEF treatment
variables (frequency, pulse width, and pulse polarity) on lycopene,
Vitamin C, and antioxidant capacities of tomato juice were mod-
eled by Odriozola-Serrano et al. (2007), with correlation coef-
cients of 0.9187, 0.7666, and 0.7954, respectively. The quadratic
term of frequency and the combined effect of frequency and pulse
width were found to be signicant. Maximal relative lycopene con-
tent (131.8%), Vitamin C content (90.2%), and antioxidant capacity
retention (89.4%) were attained with PEF treatments of a 1 ls pulse
duration applied at 250 Hz in the bipolar mode for 1000 ls.
Furthermore, Oms-Oliu et al. (2009) determined the effects of elec-
tric eld strength, frequency, pulse width, treatment time, and
polarity mode on health-related compounds on PEF-treated water-
melon juice. As a measure of the prediction accuracy, the correla-
tion coefcients between the polynomial model predictions and
the observed data were 0.882, 0.751, and 0.838 for Vitamin C, lyco-
pene, and antioxidant capacity retention, respectively. Maximal
relative lycopene content (113%), Vitamin C (72%), and antioxidant
capacity retention (100%) were obtained when PEF treatments
were set at 35 kV/cm for 50 ls using 7 ls bipolar pulses at 200 Hz.
Information on quadratic response models for the retention of
health-related compounds is very limited. Such models proved to
be useful in achieving the optimal processing conditions to obtain
fruit juices with high nutritional quality.
Based on published studies, quadratic response models involv-
ing more factors generally give slightly lower correlation coef-
cients compared with simple models. Compared with the
quadratic response model, the simple models require large num-
bers of experiments to obtain a suitable t and have a more limited
range of application. The processing factors without signicant ef-
fects could then be rejected from the quadratic response models.
Removing insignicant factors, improving statistical interpreta-
tions, and indicating whether or not parameters interact are help-
ful for improving such models.
6. Conclusions
An overview of the kinetic models was presented to study the
effects of various PEF processing factors on the destruction of
microorganisms, inactivation of enzymes, retention of health-re-
lated compounds, and extension of shelf-life of food products. Pre-
dictive microbiology models have immediate practical application
for improving microbial food safety and quality, providing a quan-
titative understanding of the microbial ecology of foods and offer-
ing various advantages to the eld of food microbiology, such as
providing tools for difcult analysis in some critical points or expo-
sure assessment for microbial risk assessment. An increased focus
on developing quadratic response models for the retention of bio-
active components is required to fully understand the effect of
multiple processing parameters. In addition, developing a univer-
sal model that has the capability to accurately describe the behav-
ior of microorganisms and enzymes when they are subjected to
different treatment parameters is important.
On the other hand, a increasing number of studies have recently
been conducted on numerical models toward coupled simulations
of the uid ow, electric eld, and heat transfer in the treatment
region. The implementation of inactivation kinetics into the
numerical models is an inevitable trend that is essential for the
development of PEF technology.
Several aspects of the PEF treatment process need to be further
investigated to make this technology a widely used method equiv-
alent to heat pasteurization. These aspects include not only the
conditions for the PEF process, but also the characteristics of
pathogenic microorganisms, quality-related enzymes, and health-
related compounds. Finally, although kinetic models have been
developed to describe and predict microbial inactivation by PEF,
few have been used to establish process criteria for a food-pasteur-
ization equivalent or even higher lethality than the current heat
pasteurization treatments. Thus, publications indicating the
process criteria for the pasteurization of liquid products using PEF
are needed.
Acknowledgements
The authors gratefully acknowledge the nancial support pro-
vided by National High Technology Research and Development
Program (2007AA100405).
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A Review of Kinetic Models For Inactivating Microorganisms and Enzymes by Pulsed Electric Field Processing

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