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Trends in Food Science & Technology 13 (2002) 195204

Review

Milk processing by
high intensity
pulsed electric
elds

needed to commercialize PEF treatment for milk and dairy


products processing.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: High intensity pulsed electric elds; Milk; Dairy; Enzymes; Microorganisms; Food components;

Introduction

Slvia Bendichoy,
Gustavo V. Barbosa-Canovas{
and Olga Martny,*
y

CeRTA-UTPV, Departament de Tecnologia


dAliments, Universitat de Lleida, Alcalde Rovira
Roure 191, 25198 Lleida, Spain (tel: +34-973-702593;
fax: +34-973-702596; e-mail: omartin@tecal.udl.es)
{
Department of Biological Systems Engineering,
Center for Nonthermal Processing of Food,
Washington State University, Pullman, WA 991646120, USA
Pulsed electric eld (PEF) processing of food consists of
treatment with very short electric pulses (ms) at high electric
eld intensities at moderate temperatures. This treatment
could be an alternative to traditional thermal processes
since it is capable of destroying micro-organisms and some
enzymes while maintaining the freshness of food products.
A product most commonly used to study the eects of PEF
has been milk, and other dairy products. Research has been
focused on evaluating the eects of PEF on micro-organisms
and enzymes, and the eectiveness of this nonthermal
technique has been demonstrated. The eectiveness of PEF
treatments depends on several factors, such as electric eld
intensity, treatment time, temperature of food, and type of
micro-organism or enzyme. As well as studies on microorganisms and enzymes, some studies exist on the eects of
PEF on milk quality and composition, proving that this
treatment causes fewer changes in the original foods composition than thermal treatments. Further investigation is

* Corresponding author.

Electrical processing of foods began in the early


1900s. In the beginning, electrical pasteurization inactivated micro-organisms by increasing the temperature of
samples by means of an electrical resistance (ohmic
heating), milk being the rst electrically pasteurized
product. Beattie (1915) and Beattie and Lewis (1925)
designed the equipment to process milk electrically,
demonstrating the lethal eects of electrical discharges
on micro-organisms when voltages of 30004000 V were
applied. Fetterman (1928) processed milk using the
ElectroPure Process, which consisted of heating the
milk to 70 C and then passing it through carbon electrodes in an electric heating chamber to inactivate
Mycobacterium tuberculosis and Escherichia coli.
Getchell (1935) pasteurized milk by heating it for 15 s at
71 C with an alternating current of 220 V and Moses
(1938) reported that between 1928 and 1938, more than
200 million liters of electrical pasteurized milk were
consumed. Nevertheless, this process was soon forgotten for no apparent reason (Hall & Trout, 1968;
Paleniappan & Sastry, 1990; Martn, Zhang, Castro,
Barbosa-Canovas, & Swanson, 1994).
In the last decade, several studies have been performed in order to develop nonthermal electrical pasteurization processes. Dierent types of equipment for
the application of high intensity pulsed electric elds
(PEF) have been patented and several studies have
demonstrated the eectiveness of this nonthermal
technique in food processing.
A great part of the PEF research is focused on studying its eect on milk and dairy products due to the
importance of the dairy industry. Most of the studies
carried out with these products have been performed to
evaluate PEFs eect on microbial inactivation. The
level of microbial inactivation achieved with PEF treatment mainly depends on the eld strength and the
number of pulses applied during the process (Martn,

09242244/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 92 4 - 2 24 4 ( 0 2 ) 0 0 13 2 - 2

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S. Bendicho et al. / Trends in Food Science & Technology 13 (2002) 195204

Qin, Chang, Barbosa-Canovas, & Swanson, 1997;


Pothakamury, Barbosa-Canovas, Swanson, & Spence,
1997; Qin et al., 1995). As regards to the eect of PEF
on enzymes, some controversial results have been
obtained. In several cases, high levels of inactivation
have been achieved, whereas in other cases no eect or
increase in initial activity has been detected (Bendicho,
Estela, Fernandez-Molina, Barbosa-Canovas, & Martn, 2002; Bendicho, Mart, Fernandez-Molina, Barbosa-Canovas, & Martn, 2001e; Castro, Swanson,
Barbosa-Canovas, & Zhang, 2001; Van Loey, Verachtert, & Hendrickx, 2002; Vega-Mercado, Powers,
Barbosa-Canovas, & Swansen, 1995; Vega-Mercado,
Powers, Martn-Belloso, Luedecke, Barbosa-Canovas,
& Swanson, 2001).
PEF, as a nonthermal process, is believed to maintain
the original composition of milk. If this technique keeps
the initial content of minority food components, such as
vitamins (Barbosa-Canovas, Gongora-Nieto, Pothakamury, & Swanson, 1999; Barsotti & Cheftel, 1999;
Bendicho, Espachs, Arantegui, & Martn, 2002a; Grahl
& Markl, 1996), it could be introduced to the dairy
industry for obtaining products with excellent
nutritional and sensory qualities.

Eects of PEF on micro-organisms


The eects of the PEF process has been studied in a
model solution similar to milk ultraltrate (SMUF), in
milk with dierent fat contents, and in yogurt. In all of
these cases, the destruction of micro-organisms has been
demonstrated.
Signicant inactivation levels have been achieved in
several micro-organisms inoculated in SMUF. When
treatment was applied to samples inoculated with E. coli
reductions of 6 and 9 log cycles were achieved, respectively, after applying 50 pulses of 60 kV/cm or 80 pulses
of 70 kV/cm (Qin, Barbosa-Canovas, Swanson, Pedrow,
& Olsen, 1998; Zhang, Qin, Barbosa-Canovas, &
Swanson, 1995). In the case of SMUF inoculated with
Staphylococcus aureus, its population could be reduced
up to 5 log cycles after subjecting the samples to 40
pulses at 60 kV/cm (Qin et al., 1998). The eect of PEF
treatments was also evaluated on samples of SMUF
inoculated with Lactobacillus delbrueckii and Bacillus
subtilis, in this case reductions of 4 and 5 log cycles were
achieved, respectively, after applying treatments of 50
and 40 pulses of 16 kV/cm (Pothakamury, MonasalveGonzalez, Barbosa-Canovas, & Swanson, 1995).
Dunn and Pearlman (1987) inoculated pasteurized
milk with E. coli (8.1106 cfu/ml), in which up to
approximately 3 log reductions of inactivation was
reached after a PEF treatment of 23 pulses at 42.8 kV.
A similar study was carried out inoculating Salmonella
dublin (3.8103 cfu/ml), in this case, after a PEF treatment of 40 pulses at 36.7 kV the treated samples showed
no S. dublin and less than 20 milk bacteria of the

background microora. After 8 days stored at 79 C,


the control samples showed substantial bacteria growth
with a count greater than 107 cfu/ml. The treated samples, on the other hand, after 8 days only had a bacteria
count of approximately 400 cfu/ml and no S. dublin
were observed during the entire period of the test. Fernandez-Molina (2001) after inoculating Listeria innocua
(1.2109) or Pseudomonas uorescens (3.8107) in pasteurized skim milk, subjected the samples to PEF treatments up to 200 ms at 50 kV/cm and achieved reductions
of 2.62.7 log cycles for these bacteria.
The eect of PEF treatment on raw milk has also
been studied. Raw milk was subjected to several treatment conditions and, if stored under refrigeration, the
PEF-processed milk was found to have a microbial shelf
life of 2 weeks (Qin et al., 1995). Also in raw milk, Raso,
Gongora, Calderon, Barbosa-Canovas, and Swanson
(1999) observed that Staphylococcus aureus, and coagulase negative Staphylococcus sp. could be inactivated 4
and 2 log cycles, respectively, after PEF treatment and,
to the contrary, no reduction of other micro-organisms
such as Corynebacterium sp. or Xanthomonas maltophilia was observed. Hence, the shelf life of PEF-processed milk depends on the initial concentration of these
PEF-resistant micro-organisms, as well as on their ability to grow at refrigeration temperatures (Raso et al.,
1999).
Dunn and Pearlman (1987) studied the eects of PEF
on several micro-organisms inoculated in yogurt (Lactobacillus brevis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae) and
reported 2 log reductions in each case.

Microbial inactivation mechanism


Studies have been focused on elucidating the
mechanism of inactivation of vegetative bacterial cells
and on obtaining the parameters related to treatment
eectiveness. It has been observed that PEF treatment
causes the electroporation of the cell membrane and
consequently the micro-organism destruction (Pothakamury et al., 1997). There are several theories to
explain how pores are formed but it is still unclear
whether it occurs in the lipid or the protein matrices
(Barbosa-Canovas et al., 1999), but what is sure is that
electric elds induce structural changes in the microbial
cells and membranes of micro-organisms (Pothakumury
et al., 1997). When Pothakamury et al. (1997) treated
cells of S. aureus suspended in SMUF with 64-pulse
treatments of 20, 30 and 40 kV/cm, they observed by
electron microscopy that cells exhibited rough surfaces,
and that the ones treated under more severe conditions
showed small holes in the membrane and leakage of
cellular contents. Therefore, the increase of microbial
inactivation with the eld strength is related to the
increase of cell deterioration. Moreover, it was observed
that the mechanism of inactivation was dierent from

S. Bendicho et al. / Trends in Food Science & Technology 13 (2002) 195204

197

the thermal one, since heat treatment (66 C for 10 min)


resulted in a great damage of the cell organelles whereas
they do not suer cell wall rupture as induced by PEF
treatments (Pothakumary et al., 1997).

where bE is the regression coecient, E is the applied


electric eld, Ec (kV/cm) and tc (ms) are the extrapolated
critical t and E values and k an independent constant
factor (kV/cm).

Relevant factors to PEF treatment eectiveness


Process factors

The main factors aecting inactivation of microorganisms are electric eld intensity and treatment time
(Fernandez-Molina, 2001; Martn et al., 1997; Pothakamury et al., 1997; Qin et al., 1995) (Fig. 1). Several
research groups (Bendicho et al., 2001d; Martn et al.,
1997; Qin et al., 1998; Sensoy, Zhang, & Sastry, 1997;
Sobrino, Rosell, Bendicho, Sanchis, & Martn, 2001;
Zhang, Monsalve-Gonzalaz, Barbosa-Canovas, &
Swanson, 1994) reported that the level of surviving
micro-organisms after PEF treatment in milk or SMUF
tted dierent mathematical models related to the eld
strength (E) and/or the treatment time (t) such as exponential equations [eq (1)] (Sobrino et al., 2001), Hulsheger [eqs (2), (3) and (4)] (Hulshegar, Potel, &
Niemann, 1981, 1983) or Fermis [eq (5)] (Peleg, 1995)
models.
S expkE t

where S is the survival ratio, kE a constant, and t the


treatment time (ms).
ln S bE E  Ec

lnS bt lnt=tc

c
 EE

k
t
S
tC

1


E  Eh
1 exp
a

where Eh (kV/cm) is a critical level of E where S is 50%,


and the a parameter (kV cm1) indicates the steepness
of the curve around Eh.
Treatment time is the product of the number of pulses
applied and the duration of each pulse. Therefore, for
treatments with similar eld strength and number of
pulses, inactivation of E. coli in skim milk increased
with the increase in pulse width (Martn et al., 1999)
(Fig. 2).
Microbial inactivation has also been related to the
treatment temperature (Table 1). Pothakamury, Vega,
Zhang, Barbosa-Canovas, and Swanson (1996) subjected SMUF inoculated with E. coli to PEF in a range
of temperatures from 3 to 40 C and deduced that inactivation rates increased with the temperature. Reina,
Jin, Zhang, and Yousef (1998) also observed that an
increase in treatment temperature (50 C) leads to higher
eectiveness in the inactivation of Listeria monocytogenes. Sensoy et al. (1997) proposed a rst-order
kinetic model to describe microbial inactivation [eq (1)]
where the rst-order constant depended on the treatment
temperature, following the Arrhenius model [eq (4)].
k kexpEz =RT

Fig. 1. Eect of the eld strength and number of pulses on the


inactivation of Escherichia coli in skim milk using high intensity
pulsed electric elds (Martn et al., 1997).

where Ea is the energy of activation, R the constant of


the ideal gases and T the temperature.
In general, continuous processes reached higher inactivation rates than those in batch mode. Martn et al.
(1997) reported a reduction of approximately 2 log
cycles after processing milk inoculated with E. coli, by

Fig. 2. Eect of pulse duration in the inactivation of Escherichia coli


in skim milk, after a 25 kV/cm-HIPEF treatment (Martn et al., 1997).
Pulse duration: ~, 0.7 ms; *, 1.8 ms.

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S. Bendicho et al. / Trends in Food Science & Technology 13 (2002) 195204

Table 1. Eects of treatment temperature on the eectiveness of the microbial inactivation by high intensity pulsed electric elds
Mediaa

Bacteria

Treatment conditions

Microbial reduction
(log reductions)

Reference

SMUF

Escherichia coli

7 C, 36 kV/cm, 16 pulses
20 C, 36 kV/cm, 8 pulses
33 C, 36 kV/cm, 8 pulses

2-3
2.5
2.5

Zhang et al., 1995

SMUF

Escherichia coli

7 C, 36 kV/cm, 64 pulses
20 C, 36 kV/cm, 64 pulses

4
5

Pothakumury et al., 1996

Skim milk

Salmonella dublin

30 C, 25kV/cm, 100 pulses


50 C, 25kV/cm, 100 pulses

1
2

Sensoy et al., 1997

Whole milk

Listeria monocytogenes

25 C, 30 kV/cm, 400 pulses


50 C, 30 kV/cm, 400 pulses

2.5
4

Reina et al., 1998

SMUF: simulated milk ultraltrate.

applying a batch mode 64-pulse treatment at 35 kV/cm.


A similar reduction was achieved by a milder process on
continuous mode, applying 25 pulses at 25 kV/cm. It
has also been found that square-shaped pulses are more
lethal than exponential decay pulses, since after the
application of similar treatments (100 ms at 36 kV/cm)
to samples of SMUF inoculated with E. coli, treatments
of squared pulses lead to 99% inactivation, whereas
that of exponential waveform reduced the population a
93% (Pothakumury et al., 1996).

Medium and inoculum factors


Several authors reported that a high initial concentration of inoculum negatively aects microbial
inactivation (Zhang et al., 1994); however, in the case of
E. coli, it has been observed that an initial concentration
of 1.151037.14108 cfu/ml in SMUF, did not aect
the inactivation rates obtained (Zhang et al., 1995). The
growth stage of micro-organisms also inuences the
inactivation results. Pothakamury et al. (1996), also in
SMUF, found that E. coli cells in the logarithmic phase
were more sensitive to PEF treatment than in the lag or
stationary phase (Fig. 3).
The treatment media can also play a role in the eectiveness of PEF (Table 2). Grahl and Markl (1997)
subjected dierent media (milk 1.5 and 3.5% fat, solutions of sodium-alginate) inoculated with E. coli, L.
brevis, P. uorescens and S. cerevisiae to PEF. When
comparing the eectiveness of treatment as a function
of the media, it was observed that the critical eld
strength (Ec), which is the minimum eld strength (E)
needed to start inactivation (Hulsheger et al., 1981) and
the critical treatment time (tc), that is the minimum
treatment time needed to start inactivation (Hulsheger
et al., 1981), depended on the cell characteristics as well
as on the type of medium in which the cells are suspended. They observed that microbial cells of large diameter (e.g. yeast cells) were killed at lower electric eld

strengths than smaller cells, such as typical bacterial


cells. Moreover, they noticed that the fat particles of
milk seemed to protect the bacteria against electric pulses (Grahl & Markl, 1996). It was also observed that less
inactivation of E. coli by PEF was achieved in skim milk
than in a buer solution when exposed to similar treatment conditions, (Hulsheger et al., 1983; Martn et al.,
1997). This dierence was thought to be due to the
more complex composition of milk, its low resistivity,
and the presence of proteins. Reina et al. (1998) also
compared the eect of PEF in milk with dierent fat
content. They inoculated L. monocytogenes into skim
milk, 2% fat milk, and whole milk, and evaluated the
eects of the fat content on the inactivation rates, but
no dierences were observed among the results.
Another factor of the treatment media that signicantly aects the PEF eect is pH. In SMUF, the
inactivation of E. coli was greater at low pH (5.69) than
at high pH (6.82) (Vega-Mercado, Porthakamury,
Chang, Barbosa-Canovas, & Swanson, 1996) (Table 2).
Ionic strength of the medium also plays an important
role in inactivation levels. Evidence of a relation
between the inactivation rate and the ionic strength was

Fig. 3. Eects of growth stage on the inactivation of Escherichia coli


in SMUF. Samples were subjected to a eld strength of 36 kV/cm
(Pothakamury et al., 1996).

S. Bendicho et al. / Trends in Food Science & Technology 13 (2002) 195204

199

Table 2. Inuence of substrate on the eectiveness of high intensity pulsed electric eld treatments in microbial inactivation
Mediaa

Bacteria

Treatment conditions Eects

Reference

Sodium-alginate solution Escherichia coli


515 kV, 520 pulses The simpler the media composition Grohl & Markl, 1996
Milk 1,5% fat
Lactobacilus brevis
the higher the inactivation.
Milk 3,5% fat
Pseudomonas uorescens
SMUF
Skim milk

Escherichia coli

1525 kV/cm, 530


pulses

Lower inactivation in milk


than in SMUF.

Martn et al., 1997

Milk 0.2% fat


Milk 2% fat
Milk 3.5% fat

Listeria monocytogenes

30 kV/cm, 66400
pulses

No dierences in inactivation.

Reina et al., 1998

SMUFpH 5.69
SMUFpH 6.82

Escherichia coli

2055 kV/cm, up to 8 The lower the pH the higher the


pulses
inactivation.

Vega-Mercado et al., 1996

SMUF 0.028 M
SMUF 0.168 M

Escherichia coli

40 kV/cm, up to 32
pulses

Vega-Mercado et al., 1996

The lower the ionic strength


the higher the inactivation.

SMUF: simulated milk ultraltrate.

veried by Vega-Mercado et al. (1996) who obtained a


dierence of 2.5 log cycles in the inactivation level
between 0.168 and 0.028 M solutions (Table 2).

Combination of PEF with other hurdles


The inactivation rates achieved by PEF can be
improved by combining the electrical treatment with
other processes. Sobrino et al. (2001) observed that
inactivation achieved by PEF could be increased if
moderate heating was applied after the PEF process.
Fernandez-Molina, Barbosa-Canovas, and Swanson
(2000) increased the shelf life of milk treated by PEF up
to 30 days (stored refrigerated) by applying a mild
thermal treatment before the PEF process (Fig. 4). Calderon-Miranda, Barbosa-Canovas, and Swanson (1999)
inoculated L. innocua in skim milk and then subjected it
to dierent PEF treatment conditions; immediately
after, nisin was added to the samples at concentrations
of 10 or 100 IU/ml. It was observed that the combination of both treatments was additive with low eld
strengths and synergistic when higher eld strength
treatments were applied (Fig. 5). By means of electronic
microscopy, it was observed that cells treated with a
combination of nisin and PEF presented more damage
than cells treated with PEF alone (Calderon-Miranda,
Barbosa-Canovas, & Swanson, 1999b). It has been
reported that the combination of PEF with the addition
of acetic or propionic acid in skim milk can signicantly
improve the inactivation of L. innocua, but not the
inactivation of P. uorescens (Fernandez-Molina, 2001).

Swanson (1998) investigated the inactivation of B. subtilis spores in SMUF using PEF. Spores were not inactivated when PEF treatment (75 pulses at 60 kV/cm)
was used alone. The combination of PEF and moderate
temperatures around 60 C, and/or the activation of
spore suspension prior to HIPEF treatment (80 C for
10 min), and/or the use of up to 5000 IU/ml lysozyme,
did not inactivate spores. Only the use of high pressure
(1500 atm during 30 min at 40 C) led to the initiation of
germination and then PEF could inactivate the vegetative forms of these micro-organisms (Pagan et al., 1998).

Eects of PEF on sporulated micro-organisms


Endospore inactivation has been reported as insignificant, as no lethal eects were observed with Clostridium
tyrobutyricum, Bacillus cereus, and Bacillus nivea spores
after a PEF treatment (Grahl & Markl, 1996). Pagan,
Esplugas, Gongora-Nieto, Barbosa-Canovas, and

Fig. 4. Total aerobic bacteria counts in skim milk during refrigerated


storage (4 C). Treatment conditions: Control untreated skim milk;
heat treated skim milk, 73 C for 6 s; heat+PEF treated skim milk at
50 kV/cm with 10, 20 or 30 pulses at a frequency of 4 Hz and total
treatment time of 60 ms (Fernandez-Molina et al., 2000).

200

S. Bendicho et al. / Trends in Food Science & Technology 13 (2002) 195204

Eects of PEF on enzymes


In general, enzymes require more severe PEF treatment than micro-organisms to obtain a signicant
reduction (Ho, Mittal, & Cross, 1997). This fact is
important because some enzymes are useful for the
dairy industry, thus milk could be treated to destroy
micro-organisms while maintaining the activity of
enzymes.
Several enzymes in milk have been studied (Table 3).
It has been observed that variation in enzymatic activity
depends on the electric eld intensity, the number of
pulses applied during the process, and the characteristics
of the enzyme (Bendicho et al., 2001e; Bendicho et al.,
2002b; Castro et al., 2001; Grahl & Markl, 1996; VegaMercado et al., 1995, 2001). The inactivation levels also
depend on the media containing the enzyme (Bendicho et
al., 2001e; Castro et al., 2001; Vega-Mercado et al.,
2001), treatment temperature [12], and enzyme concentration (Castro et al., 2001).

Enzyme inactivation by PEF


Alkaline phosphatase (ALP) is an enzyme present in
milk that is used to indicate the adequacy of thermal
pasteurization. Castro et al. (2001) studied the PEF
inactivation of ALP in SMUF, nonfat milk, 2% fat
milk, and whole milk. PEF treatment was able to reduce
up to 65% of ALP activity after 70 pulses at 22 kV/cm
in SMUF, and at 18.8 kV/cm in skim milk. When 2%
milk and whole milk were treated, ALP activity was
reduced up to 59% after a 70-pulse treatment at 18.8
kV/cm (Table 3); however, Grahl and Markl (1996), Ho

Fig. 5. Eect of the addition of nisin after a HIPEF treatment of 32


pulses at 30 kV/cm (a) and 32 pulses at 50 kV/cm (b) on the inactivation of Listeria innocua (Calderon-Miranda et al., 1999a).

et al. (1997) and Van Loey et al. (2002) did not observe
any signicant enzyme reduction in either milk or aqueous solution. It might be due to the dierence on some
electrical parameters due to the use of dierent PEF
systems. Peroxidase, another milk enzyme, showed no
inactivation when suspended in milk (Grahl & Markl,
1996; Van Loey et al., 2002), whereas a signicant
reduction was observed when suspended in a phosphate
buer (Ho et al., 1997) (Table 3).
The eect of PEF on proteases also was studied.
Inactivation of plasmin by PEF was evaluated in
SMUF by applying a continuous 50-pulse treatment at
eld strengths of 15, 30 and 45 kV/cm (Vega-Mercado
et al., 1995). Plasmin activity decreased by 90% after 50
pulses with eld strengths of 30 or 45 kV/cm at 15 C,
but was only decreased by 60% when treatment temperature was 10 C (Table 3). As for microbial proteases,
Vega-Mercado et al. (2001) achieved an 80% inactivation of an extracellular protease from P. uorescens
when the enzyme was suspended in tryptic soy broth
enriched with yeast extract. Proteolytic activity
increased or decreased signicantly when the medium
was skim milk depending on the PEF treatment applied;
low eld strengths (14 and 15 kV/cm) and frequencies of
1 and 2 Hz resulted in reductions of 40 and 60% after
32 and 98 pulses, respectively (Table 3), whereas samples subjected to higher eld strengths (25 kV/cm) and
lower frequencies (0.6 Hz) showed an increase of the
proteolytic activity. On the other hand, it is interesting
to note that when the enzyme was suspended in casein
Tris buer, a mix of casein with Tris buer (pH 7.5), no
signicant inactivation of protease was obtained. This
indicates that casein has a protective eect on protease
against PEF treatment. Bendicho et al. (2001e) studied
the eect of PEF on a commercial protease from B.
subtilis suspended in SMUF. A maximum inactivation
of 10% was achieved after a batch mode treatment,
whereas inactivation rose to 15% when PEF was
applied in continuous ow; however, when the enzyme
was suspended in skim milk no reduction in enzymatic
activity was reported after batch mode treatments.
When treatments were in continuous ow, the variation
in activity depended on the frequency. In this case, most
of the treatments at high frequencies (4 Hz) caused no
changes on protease activity, on the contrary, a slight
enhancement (1015%) of the activity was observed
after PEF treatments at low frequencies (2 Hz) (Bendicho et al., 2001e) (Table 3).
Lipase is another endogenous milk enzyme that has
been studied. A commercial lipase from P. uorescens
suspended in SMUF could be inactivated up to 62%
after a batch mode treatment (Fig. 6), whereas, only a
13% inactivation was reached with a continuous process (Bendicho et al., 2002b). In this case, oppositely
that for protease, the batch mode treatment showed
more eectiveness than that on continuous, possibly due

S. Bendicho et al. / Trends in Food Science & Technology 13 (2002) 195204

to the superiority of the voltage applied, the slowness of


the treatment or the length of the pulse delay (Bendicho
et al., 2002b). Grahl and Markl [18] also observed a
signicant reduction (about 60%) of lipase content in
milk (Table 3).
Inactivation of lipase followed single exponential models
related to treatment time or eld strength, and the equa-

201

tions of Hulsheger et al. (1983) and Fermi (Peleg, 1995;


Bendicho, Giner, Barbosa-Canovas, & Martin, 2001).

Enzyme inactivation by combining PEF with other


hurdles
Because of the resistance of several enzymes to PEF
treatments, combined processes of PEF with other hurdles

Table 3. Eects of high intensity pulsed electric eld processing on milk enzymes
Enzyme

Mediaa

Alkaline
SMUF
phosphatase
Skim milk (SM)
2% fat milk (SSM)
Whole milk (WM)

Treatment conditions
b

Eects

Reference

18.8 and 22 kV/cm, up to 70 pulses SMUF and SM: 65% inactivation


WM and SSM: 59% inactivation

Castro et al., 2001

Grohl & Markl, 1996

Milk 1.5% fat


Milk 3.5% fat

21.5 kV/cm, up to 20 pulses

Less than 10% inactivation

Buer pH 9.8
Raw milk

b
b

4080 kV/cm, 30 pulses


6.720 kV/cm, up to 200 pulses

Approx. 5% inactivation
Ho et al., 1997
No inactivation (process at room T) Van Loey et al., 2002
74% inactivation (process at 70 C)

Plasmin

SMUF

1545 kV/cm, 1050 pulses

Up to 90% inactivation

Vega-Mercado et al., 1995

Peroxidase

Milk

21.5 kV/cm, up to 20 pulses

Approx. 30% inactivation

Grohl & Markl, 1996

Approx. 30% inactivation

Ho et al., 1997

> 3% inactivation
(process at room T)
213% inactivation
(process at 4452 C)

Van Loey et al., 2002

SM: 60% inactivation


CS: no signicant eects
SM: enzyme activation
CS: no signicant eects

Vega-Mercado et al., 2001

Potassium phosphate b 4073.3 kV/cm, 30 and 100 pulses


100 mM
Raw milk

Protease from Casein solution (CS)


P. uorescens Skim milk (SM)

1319 kV/cm, up to 200 pulses

1415 kV/cm, up to 98 pulses

25 kV/cm, up to 16 pulses
23.3/31.6 kV/cm, up to 32 pulses

Protease from SMUF Skim milk (SM) b16.427.4 kV/cm, up to 100 pulses SMUF: 10% inactivation
SM: no signicant eects
B. subtilis
c
17.625.2 kV/cm, up to 100 pulses. SMUF: 15% inactivation
SM: eects depended on
the frequency
b
SMUF
16.427.4 kV/cm, up to 60 pulses+ About 50% inactivation
Acidication (pH 5)
b
16.427.4 kV/cm, up to 60 pulses+ About 30% inactivation
SMUF
Mild heat treatment (63 CC-5 min)
Lipase

Milk

Lipase from SMUF


P. uorescens

Bendicho et al., 2001e


Bendicho et al., 2001e
Bendicho et al., 2001a
Bendicho et al., 2001a

21.5 kV/cm, up to 20 pulses

About 60% inactivation

Grohl & Markl, 1996

16.427.4 kV/cm, up to 100 pulses

About 62% inactivation

Bendicho et al., 2002b

SMUF
SMUF
a
b
c

26.137.3 kV/cm, up to 100 pulses About 13% inactivation


16.427.4 kV/cm, up to 60 pulses+ About 52% inactivation
Acidication (pH 5)
b
16.427.4 kV/cm, up to 100 pulses+ About 35% inactivation
Mild heat treatment (63 C-15 min)
b

SMUF: simulated milk ultraltrate.


Batch mode HIPEF treatment.
Continuous mode HIPEF treatment.

Bendicho et al., 2001b


Bendicho et al., 2001c

202

S. Bendicho et al. / Trends in Food Science & Technology 13 (2002) 195204

Fig. 6. Inhibition of lipase activity after high intensity pulsed electric eld (HIPEF) treatments. Plotted lines correspond to experimental data tted to a rst-order kinetic model (Bendicho et al.,
2002b).

such as mild heat or acidication of the media were


studied. Van Loey et al. (2002) observed that by
increasing the process temperature, higher rates of
inactivation on alkaline phosphatase and lactoperoxidase could be achieved than when samples were
processed at room temperature (Table 3).
Bendicho, Estela, Barbosa-Canovas, and Martn
(2001a, 2001b) combined the acidication of the media
(with HCl, to pH 5) with the application of a batch PEF
process of 60 pulses at 27.5 kV/cm and achieved
approximately 52% inactivation of a lipase from P.
uorescens and approximately 30% inactivation of a
protease from B. subtilis (Table 3). This demonstrated
that the eectiveness of PEF can be increased by
lowering the medium pH.
The application of mild heat after PEF process was
studied to enhance the inactivation of a protease from
B. subtilis and a lipase from P. uorescens; however, this
combination did not provide great improvements on the
inactivation of these enzymes over PEF alone (Bendicho
et al., 2001a; Bendicho, Estela, Barbosa-Canovas, &
Martn, 2001c) (Table 3).

Eects of PEF on milk quality and milk


micronutrients
There have been only a few studies published that
evaluate the eects of PEF on the quality attributes of
milk. As previously stated, PEF is a nonthermal process, so it does not destroy sensitive minor compounds
such as vitamins (Barbosa-Canovas et al., 1999; Barsotti
& Cheftel, 1999; Bendicho et al., 2002a; Grahl & Markl,
1996) . Thus, if this treatment can achieve microbial
stabilization preserving nutrient composition, a new
variety of high quality nutritive products could be produced.
Milk with 2% fat content was processed by PEF,
aseptically lled into packaging bags, and stored at 4 C.
Its physical and chemical properties (not detailed) were

not inuenced by the PEF treatment and its microbial


shelf life was 2 weeks. A sensory panel found no dierences between heat-pasteurized skim milk and PEFpasteurized (Qin et al., 1995).
As for the eect of PEF on the vitamins in milk,
Bendicho et al. (2002b) evaluated its eect on several
water-soluble vitamins (riboavin, thiamin, and ascorbic acid) and fat-soluble vitamins (cholecalciferol and
tocopherol) applying treatments of up to 400 ms at eld
strengths from 18.3 to 27.1 kV/cm. No changes in the
vitamin content were reported except for ascorbic acid;
observing that milk retained more ascorbic acid after a
400 ms treatment at 22.6 kV/cm (93.4%) than after
either a LTLT (low temperature long time, 30 min at
63 C, 49.7% retained) or a HTST (high temperature
short time, 15 s-75 C, 86.7% retained) heat pasteurization treatments. Grahl and Markl (1996) reported that
the ascorbic acid content of milk was reduced considerably after PEF treatment (90%, data not shown),
whereas the content of vitamin A did not show any
large-scale inactivation, and the avor showed no
signicant change.

Final remarks
The PEF process has good prospects for being used in
the dairy industry, since promising results has been
obtained when processing milk. The treatment could
provide an alternative to thermal pasteurization, since it
provides such advantages as low destruction of avor or
nutrients, which results in a fresh-like product. It maintains the avor and nutritional value of fresh products,
both of which are fundamental objectives in the dairy
industry. Further investigation is required to learn more
about the parameters inuencing this treatment, to
achieve higher rates of inactivation and to control with
more precision all of the critical points involved; it
would be interesting to identify target pathogenic spoilage micro-organisms that would enable the validation
of the process to ensure microbiological eectiveness.
There is also need of evaluating the equivalence of PEF
to heat pasteurization treatment an studying the acceptance of the consumers used to drink thermally treated
milk. Moreover, current studies only include laboratory
scale and pilot plant equipment. Therefore, to introduce
successfully this technique to the dairy industry, systems
with higher rates of production and with identical or
improved eectiveness must be designed taking into
consideration the cost criteria.

Acknowledgements
The authors wish to thank the Comision Interministerial de Ciencia y Tecnologa (CICYT) of Spain
for its support of Project ALI97 0774, and the Ministerio de Educacion, Cultura y Deportes for nancial
support granted to Slvia Bendicho throughout her
Doctoral studies.

S. Bendicho et al. / Trends in Food Science & Technology 13 (2002) 195204

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