ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 21 (2004) 519–525
www.elsevier.nl/locate/jnlabr/yfmic

Control of Lactobacillus plantarum and Escherichia coli by pulsed

electric fields in MRS Broth, Nutrient Broth and orange–carrot juice
! b, M. Valerob, P.S. Ferna! ndeza,*
M.V. Selmaa, M.C. Salmeron
a
Departamento Ingenier!ıa de Alimentos y del Equipamiento Agr!ıcola, Universidad Polit!ecnica de Cartagena, ETSIA, Paseo Alfonso XIII 48, Cartagena
(Murcia) 30203, Spain
b
!
Microbiolog!ıa, Escuela Polit!ecnica Superior de Orihuela, Universidad Miguel Hernandez, Ctra. Beniel Km 3.2, Orihuela Alicante 03312, Spain
Received 25 July 2003; accepted 10 December 2003

Abstract

Pulsed electric fields (PEF) are an emerging non-thermal treatment valid for liquid foods. This novel technology offers a relevant
alternative to traditional thermal methods avoiding thermal damage to the product (loss of flavour and nutritional value). The aim
of this work was to evaluate the influence of PEF treatment conditions, inoculum size (initial cell concentration) and substrate
conditions after PEF treatment on the inactivation and potential growth of Lactobacillus plantarum and Escherichia coli in orange–
carrot juice. Although a maximum inactivation of 1.3 and 2.6 log reductions were achieved for L. plantarum and E. coli, respectively,
after PEF treatment, it was effective inducing sub-lethal injury. An increase in the lag-phase duration was evidenced under
refrigeration conditions. When sub-lethal damage could be repaired, the subsequent growth rate was not affected. An increased
inhibitory effect of PEF, low temperature and low inoculum size on the delay in lag phase was observed.
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Pulsed electric field processing; Lactobacillus plantarum; Escherichia coli; Juice

1. Introduction Lactobacillus plantarum often is a spoilage organism

High voltage pulsed electric fields (PEF) treatment is
an emerging non-thermal process used in liquid foods. It
offers a relevant potential for increasing the shelf-life of
fresh juices. Consumer demand for non-pasteurised
juices and fruit juices mixed with vegetable juices or
milk, has gained importance due to interest in flavour
and nutritional value of food, as well as, heat treatment
is perceived as damaging to these qualities (Martin et al.,
1997; Barsotti et al., 2002).
However, recently Escherichia coli O157:H7 has been
implicated in outbreaks after consumption of fresh
apple and orange juices despite of having a low pH
(Zhao et al., 1993; McDonald et al., 2000). This
microorganism is the most virulent of verotoxigenic E.
coli, causing a high risk of potential lethal complications
(Law, 2000).
*Corresponding author. Tel.: +34-968-325-905; fax: +34-968-325-
433.
E-mail address: pablo.fernandez@upct.es (P.S. Fern!andez).
of orange juice (Davis et al., 1986) that needs to be
controlled to avoid product loss. Thermal damage to
freshly squeezed orange juices is minimal at tempera-
tures below 50 C (Innings, 1998). Using PEF treatment
below this temperature would allow to inactivate
pathogenic and spoilage microorganisms or slow down
their development, under controlled conditions (McDo-
nald et al., 2000), without affecting its sensory quality.
Previous studies in orange–carrot juices have demon-
strated that PEF has non-thermal, lethal effects on
microorganisms including L. plantarum and E. coli
(Rodrigo et al., 2001, 2003). However, there was a
resistant fraction of population since, in many cases,
PEF inactivation kinetics do not follow a first-order
kinetics (Rodrigo et al., 2003). Therefore, the effective-
ness of this technique could be improved combining it
with other treatments as far as they may capitalised on
PEF cellular damages, such as, reversible pore forma-
tion (Simpson et al., 1999; Wouters et al., 2001; Selma
et al., 2003). This implies the need to establish the
parameters that can guarantee their microbiological
safety during the shelf-life of PEF-treated juices.

0740-0020/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2003.12.004
 ARTICLE IN PRESS
520 M.V. Selma et al. / Food Microbiology 21 (2004) 519–525
Very little is known about the parameters that
could influence the results of sub-lethal injury of
PEF. However, previous studies have shown that
product parameters (nutrients, pH and temperature)
have a relevant role on the ability of bacteria to recover
under exposure to a technological treatment (Augustin
et al., 2000; Ulmer et al., 2002; Wuytack et al.,
2003). The aim of this work was to study the effect of
field strength, inoculum size and substrate conditions
after PEF treatment, on the inactivation and potential
growth of L. plantarum and E. coli in orange–carrot
juice.
2. Materials and methods
2.1. Culture preparations
Cultures of L. plantarum CECT 220 and E. coli CECT
516 were obtained from the Spanish Type Culture
Collection (Valencia, Spain). E. coli CECT 516 corre-
sponds to ATCC 8739 used in previous PEF studies, in
apple and orange–carrot juices (Evrendilek et al., 1999;
Rodrigo et al., 2003). This E. coli strain was chosen due
to its ability to growth under acidic conditions
(Evrendilek et al., 1999), as well as, it is recommended
non-pathogenic test organisms in PEF studies (Wouters,
1997). Cultures of L. plantarum and E. coli were
rehydrated in Man Rogosa Sharpe (MRS) Broth
(Scharlab, Spain) and Nutrient Broth (Oxoid, UK),
respectively. Then, they were inoculated onto separate
MRS agar (Scharlab, Spain) and nutrient agar (Oxoid,
UK) plates and incubated for 24 h at 30 C. After
incubation, the stock bacterial culture was kept in agar
slants at 4 C. Single colonies of L. plantarum and E. coli
cultures were inoculated from stock in a flask containing
100 ml of MRS Broth and Nutrient Broth, respectively.
L. plantarum and E. coli culture inoculum were then
incubated at 30 C in a well-shaken waterbath for 24 and
8 h, respectively. Before PEF treatment, cells were
diluted to a level of 105–106 cfu ml 1, in 500 ml buffered
peptone water solution, with the same conductivity as
orange–carrot juiced (0.435 S/m at 20 C), since PEF
effect is influenced by the ionic strength of the
conductive media (Dutreux et al., 2000; Wouters et al.,
2001).
2.2. PEF treatment
Inoculated buffered peptone water solutions were
shaken at low speed and subjected to PEF treatment. An
OSU-4D bench scale continuous PEF system designed
at Ohio State University (USA) (Yin et al., 1997), and
based at the Institute of Agrochemical and Food
Technology (CSIC, Valencia), was used to treat the
inoculated buffered peptone water. A continuous, co-
field system with four and six chambers connected in
series was used in L. plantarum and E. coli experiments,
respectively. A 2.5 ms2-wave pulse was selected for the
treatment. This pulse waveform has been found to be
more lethal than exponential decay pulses (Zhang et al.,
1994). Two cooling coils were connected before and
after each pair of chambers and submerged in a
circulating refrigerated bath to maintain the treatment
temperature below 30 C. It has been previously shown
that this factor has an influences on the membrane
breakdown during PEF treatment (Reina et al., 1998;
Evrendilek et al., 1999). The temperature was recorded
with a data logger. Pulse waveform, voltage and
intensity in the treatment chambers were recorded with
a digital oscilloscope (Tektronix TDS 210, Tektronix,
OR). Flow rate was adjusted to 1.0  10 3 l s 1. For L.
plantarum treatment, peak electric field strength (E) of
28  105, 25  105 and 22  105 V m 1 were applied. The
total treatment time was 11.5  10 5, 7  10 5 and
9  10 5 s for 28  105, 25  105 and 22  105 V m 1,
respectively (Table 1). For E. coli inactivation, while
peak electric field strengths (E) of 40  105 V m 1 were
applied, the total treatment time was 10  10 5, 8  10 5
and 4  10 5 s (Table 2).
2.3. Uninoculated control
A non-inoculated buffered peptone water was run
through the system before inoculated samples were
treated. The total plate count method using MRS and
Nutrient Agar was performed to assess the absence of
microbial contamination in the PEF equipment prior to
treating L. plantarum and E. coli, respectively.
2.4. Orange–carrot juice preparation
Pasteurised orange–carrot juice was obtained by
mixing 20% carrot juice with 80% orange juice.
Table 1
Data relative to conditions of the different PEF treatment applied on
L. plantarum
Electric field (V m 1  105)/ 30/190 30/50 25/300
total treatment time (s  106)
Effective electric field strength 28.70 25.61 22.92
(V m 1  105)
Total number of pulses 46 28 36
T1 ( C) 15.0 14.0 14.0
TF ( C) 25.0 15.0 17.1
DT ( C) 10.0 1.0 3.1
T1 : Average temperature at the entrance of the first treatment
chamber.
TF : Average temperature at the exit of the fourth treatment chamber.
DT: Total temperature increase.
 ARTICLE IN PRESS
M.V. Selma et al. / Food Microbiology 21 (2004) 519–525
Table 2
Data relative to conditions of the different PEF treatment applied on
E. coli
Electric field (V m 1  105)/ 40/100 40/80
total treatment time (s  106)
Effective electric field strength 40.90 40.61
(V m 1  105)
Total number of pulses 40 32
T1 ( C) 7.5 8.0
TF ( C) 22.5 21.0
DT ( C) 15.0 13.0
T1 : Average temperature at the entrance of the first treatment
chamber.
TF : Average temperature at the exit of the fourth treatment chamber.
DT: Total temperature increase.
Orange–carrot juice had a conductivity of 0.45 S m
and a pH 3.8–4.2.
2.5. Recovery conditions
One hundred milliliters of MRS Broth and orange–
carrot juice in 250 ml flasks were inoculated with 1 ml of
diluted L. plantarum inoculum, before and after each
PEF treatment, to give an initial concentration of about
103 cfu ml 1. In the same way, 1 ml of diluted E. coli
inoculum was inoculated in 100 ml of Nutrient Broth
and orange–carrot juice, respectively. Alternatively,
100 ml of each medium was inoculated with 1 ml of
diluted inoculum to give and initial concentration of
about 10 cfu ml 1. Inoculated flasks with L. plantarum
were stored at 5 C, 8 C, 10 C, 12 C and 16 C, while as
E. coli flasks were stored at 5 C, 8 C, 12 C, 16 C and
25 C. Samples were taken at appropriate time intervals,
dilutions were made if necessary in peptone water and
plate counts in TSA were performed. Triplicate growth
curves were obtained and kept for at least 60 days if
growth was not detected.
2.6. Data modelling
Growth curves were fitted using the function of
Baranyi et al. (1993) to estimate the main
growth parameters, i.e. specific growth rate and
lag time. Only growth curves with at least 10 data
point were used for modelling, as suggested by the
authors. The correlation coefficient (R2 ) for each growth
curve (indicating the goodness of fit of experimental
data to the equation fitted by the model) was also
calculated. Finally, an ANOVA analysis was performed
to establish significant differences among the treatments
and recovery conditions tested using MATLABs
software.
521
3. Results and discussion
3.1. Effect of field strength
40/40
3.1.1. L. plantarum
40.01 The effect of three PEF treatments on the ability of L.
plantarum to grow at different temperatures was
16 evaluated. The uninoculated control with buffered
8.0
peptone–water proved the absence of microbial con-
14.0
6.0 tamination in the PEF equipment, since no bacterial
counts were obtained. Both treated and untreated cells
were not able to grow at 5 C or 8 C (Tables 3 and 4).
The level of inactivation achieved by PEF treatments
ranged from 1.370.1 (28  105 V m 1, 11.5  10 5 s) to
0.670.1-log reductions (25  105 V m 1, 7  10 5 s and
22  105 V m 1, 9  10 5 s) and was similar to data
described in the scientific literature (Rodrigo et al.,
1
2003). Although no more than 1.3-log reductions were
obtained by selected treatments, L. plantarum growth in
both substrates (orange–carrot juice and MRS Broth) at
10 C, 12 C and 16 C was affected by PEF treatments.
Furthermore, the ability to grow after PEF treatment
was more affected in orange–carrot juice than in MRS
Broth.
The specific growth rate was not modified, but the lag
period was prolonged (Tables 3 and 4). It indicated that
PEF treatments produced sub-lethal injury since lag-
phase elongation was related to the repair and adapta-
tion period (Shin and Pyun, 1997). The subsequent
growth rate did not vary because, once cells were
recovered, they could grow normally. Similar results
have been shown both for E. aerogenes (Selma et al.,
2003) and L. monocytogenes (Shin and Pyun, 1997)
where lag-phases were longer for injured cells recovered
under stress conditions.
No significant differences were found between the
three PEF treatments tested in MRS Broth, at incuba-
tion temperatures of 10 C, 12 C or 16 C (Table 3,
Fig. 1). When L. plantarum was incubated at 10 C in
MRS Broth, the extension of lag phase in PEF-treated
cells was more evident than at 12 C and 16 C, since it
was prolonged from 20.6 h to 12 days. Inactivation level
achieved by PEF treatment, as well as, subsequent
influence of low temperature and other stresses (i.e. pH)
(Wouters et al., 1999; Valero et al., 2000) can be
an effective way to produce safe foods with a longer
shelf-life.
When L. plantarum was recovered in orange–carrot
juice, both treated and untreated cells were not able to
grow at 10 C (Table 4). It indicates that orange–carrot
juice is not an optimal substrate for supporting L.
plantarum growth, since its proliferation was inhibited at
low temperature. Although growth was observed at
12 C and 16 C, the extension of the lag time was more
evident in juice than in MRS Broth. This effect was also
more significant at 12 C (where it was prolonged from
 ARTICLE IN PRESS
522 M.V. Selma et al. / Food Microbiology 21 (2004) 519–525

Table 3
Growth characteristics of L. plantarum on MRS Broth after different PEF treatments
1
E (V m  105) Treatment time Incubation Lag time Maximum Correlation
(s  106) temperature ( C) (time7s.d.) (h) specific growth coefficient (R2 )
rate (7s.d.) (h 1)

Untreated 5 No growth — —
Untreated 8 No growth — —
Untreated 10 20.6371.92 A 0.01370.001 G 0.997
Untreated 12 12.1771.05 C 0.02870.005 H 0.990
Untreated 16 5.5070.49 E 0.06470.009 I 0.997
28 115 5 No growth — —
28 115 8 No growth — —
28 115 10 315.80710.66 B 0.01670.003 G 0.991
28 115 12 27.1771.48 D 0.02070.003 H 0.992
28 115 16 12.9472.21 F 0.05070.006 I 0.995
25 70 5 No growth — —
25 70 8 No growth — —
25 70 10 292.89710.62 B 0.01370.004 G 0.988
25 70 12 31.0278.96 D 0.02270.004 H 0.999
25 70 16 15.4171.06 F 0.05270.003 I 0.990
22 90 5 No growth — —
22 90 8 No growth — —
22 90 10 311.17716.53 B 0.01670.002 G 0.992
22 90 12 23.7072.80 D 0.02370.005 H 0.997
22 90 16 12.9971.54 F 0.05370.003 I 0.992

s.d.: Standard deviation. Numbers followed by the same letter are not significantly different (Pp0:05) for the same incubation temperature within
columns.

Table 4
Growth characteristics of L. plantarum on orange–carrot juice after different PEF treatments
1
E (V m  105) Treatment time Incubation Lag time Maximum Correlation
(s  106) temperature ( C) (time7s.d.) (h) specific growth coefficient (R2 )
rate (7s.d.) (h 1)

Untreated 5 No growth — —
Untreated 8 No growth — —
Untreated 10 No growth — —
Untreated 12 32.5676.50 A 0.03670.001 E 0.998
Untreated 16 18.5072.01 C 0.05470.002 F 0.999
28 115 5 No growth — —
28 115 8 No growth — —
28 115 10 No growth — —
28 115 12 145.68718.83 B 0.03470.016 E 0.995
28 115 16 24.9471.59 D 0.05370.002 F 0.997
25 70 5 No growth — —
25 70 8 No growth — —
25 70 10 No growth — —
25 70 12 121.00711.36 B 0.03570.002 E 0.994
25 70 16 21.2772.71 D 0.04970.003 F 0.997
22 90 5 No growth — —
22 90 8 No growth — —
22 90 10 No growth — —
22 90 12 118.30714.21 B 0.03370.002 E 0.996
22 90 16 24.3172.83 D 0.05270.002 F 0.998

s.d.: Standard deviation. Numbers followed by the same letter are not significantly different (Pp0:05) for the same incubation temperature within
columns.

32.6 h to 120 h) than at 16 C confirming the ‘‘hurdle
technology’’ effect. No differences were found between
the three PEF treatments tested neither at 12 C nor at
16 C (Table 4). It suggested that cellular injury
mechanisms produced by the three treatments are
similar, possibly associated to reversible pore formation
that makes the cells susceptible to other stresses for a
limited period of time (Simpson et al., 1999).
At 16 C, the specific growth rate was not significantly
different in MRS Broth or juice. However at 12 C, it
 ARTICLE IN PRESS
M.V. Selma et al. / Food Microbiology 21 (2004) 519–525 523

was slower in juice. It was particularly evident when
combined with other stressful conditions, such as
temperatures, nutrients and pH. Therefore, the com-
bined effect of PEF, low temperature and substrate
(orange–carrot juice, in this case) would allow to inhibit
L. plantarum development, even during a relatively long
time (over 60 days).
3.1.2. E. coli
The effect of three PEF treatments on the ability of E.
coli to grow at different temperatures was evaluated.
The uninoculated control with buffered peptone–water
10
Log 10 plate count (CFU mL-1)
proved again the absence of microbial contamination in
the PEF equipment, since no bacterial counts were
obtained. The level of inactivation achieved by PEF
treatments ranged from 1.7370.04 (40  105 V m 1,
4  10 5 s) to 2.670.5-log reductions (40  105 V m 1,
8  10 5 s and 40  105 V m 1, 10  10 5 s), similar to
published work (Rodrigo et al., 2003). Both treated and
untreated cells were not able to grow either at 5 C or
8 C in Nutrient Broth or 5 C, 8 C, 12 C, 16 C and
25 C in orange–carrot juice (Table 5). However, whilst
untreated cells recovered in orange–carrot juice at 12 C,
16 C and 25 C were viable for 5 days, treated cells were
inactivated in 24 h. Therefore, these results indicate that
PEF application in low-pH juices would contribute to

9 the inactivation of E. coli, which could imply, if

8 corroborated for the pathogenic strains, a reduction of
7 the potential risk for the consumers.
6 When E. coli was incubated at 12 C and 16 C in
5 Nutrient Broth, lag phase in PEF-treated cells was
4 found to be significantly longer than in untreated cells
3 (Table 5, Fig. 2). However, no significant differences in
2 lag phase extension at 25 C between control and treated
1 cells were observed. It confirms the need to control
0 product parameters (temperature, pH, nutrients, etc.) in
0 200 400 600 800 order to slow down or inhibit potential growth of
Incubation time (h) surviving cell to PEF treatment. The specific growth rate
of the bacteria was not modified by the treatments, at
Fig. 1. Growth of L. plantarum in MRS Broth before and after
exposure to PEF (2.2  106 V m 1, 9  10 5 s). PEF-untreated cells the different temperatures tested. Again, no significant
incubated at 10 C (~) and 12 C (K); PEF-treated cells incubated at differences were found between the three PEF treat-
10 C (m) and 12 C (X). ments (Table 5). This supports conclusions obtained

Table 5
Growth characteristics of E. coli on MRS Broth after different PEF treatments
1
E (V m  105) Treatment time Incubation Lag time Maximum Correlation
(s  106) temperature ( C) (time7s.d.) (h) specific growth coefficient (R2 )
rate (7s.d.) (h 1)

Untreated 5 No growth — —
Untreated 8 No growth — —
Untreated 12 16.7974.87 A 0.07570.010 G 0.983
Untreated 16 6.8871.00 C 0.15570.012 H 0.997
Untreated 25 2.2470.63 E 0.50470.054 I 0.993
40 100 5 No growth — —
40 100 8 No growth — —
40 100 12 — — —
40 100 16 18.7574.40 D 0.13270.020 H 0.996
40 100 25 4.1070.80 F 0.46870.060 I 0.998
40 80 5 No growth — —
40 80 8 No growth — —
40 80 12 44.71710.62 B 0.06970.012 G 0.995
40 80 16 20.4276.67 D 0.14370.013 H 0.998
40 80 25 4.5471.0 F 0.51170.030 I 0.999
40 40 5 No growth — —
40 40 8 No growth — —
40 40 12 — — —
40 40 16 15.6072.80 D 0.13170.012 H 0.996
40 40 25 3.1370.54 F 0.46770.030 I 0.999

s.d.: Standard deviation. Numbers followed by the same letter are not significantly different (Pp0:05) for the same incubation temperature within
columns.
 ARTICLE IN PRESS
524 M.V. Selma et al. / Food Microbiology 21 (2004) 519–525
Log 10 plate count (CFU mL-1)
10 10

Log 10 plate count (CFU mL-1)

9 9
8 8
7 7
6 6
5 5
4
4
3
3
2
2
1
1
0
0
0 50 100 150 200
0 100 200 300 400
Incubation time (h) Incubation time (h)
Fig. 2. Growth of E. coli in Nutrient Broth before and after exposure
Fig. 4. Growth of L. plantarum in orange–carrot juice after exposure
to PEF (4  106 V m 1, 8  10 5 s). PEF-untreated cells incubated at
to PEF (2.8  106 V m 1, 11.5  10 5 s) and high and low inoculum
12 C (~) and 16 C (); PEF-treated cells incubated at 12 C (n) and concentration. High inoculum level (B103 cfu ml 1) incubated at 12 C
16 C (J). (~) and 16 C (J); Low inoculum level (B10 cfu ml 1) incubated at
16 C (X).

10 damaged population to repair the injury and start

Log 10 plate count (CFUmL-1)

9 growth.
8 This delay was also observed at 12 C, with lag times
7 for high and low inoculum levels of 27 and 51 h,
6 respectively. However, the growth rate was not affected,
5
once the lag time was finished for any of the tested
4
temperatures (Fig. 3). Therefore, the metabolic cap-
3
ability of the cells was fully restored, independently of
2
1
the inoculum size, as it was shown previously (Selma
0 et al., 2003). Furthermore, it was confirmed that this
0 200 400 600 800 delay is due to the need to repair damage and not to
Incubation time (h) population variability, since the lag-phase of untreated
cells was identical at high and low inoculum levels.
Fig. 3. Growth of L. plantarum in MRS Broth after exposure to PEF
When PEF-treated L. plantarum was recovered at
(2.8  106 V m 1, 11.5  10 5 s) and high and low inoculum concentra-
tion. High inoculum level (B103 cfu ml 1) incubated at 12 C (~) and 16 C in orange–carrot juice, the resulting growth curves
16 C (K); Low inoculum level (B10 cfu ml 1) incubated at 12 C (m) showed a similar effect to MRS Broth with the low
and 16 C (X). inoculum levels. The average lag phases presented
significant differences and were of 24.971.6 and
from L. plantarum in this work where lag-phase 35.671.4 h for the high and low inoculum levels,
elongation is proposed as a repair and adaptation respectively. Furthermore at 12 C, it was not able to
period after which the subsequent growth rate is not grow in juice with a low inoculum level, whereas for the
affected. high inoculum size, lag-time was longer than in MRS
Broth (Fig. 4). Again, the maximum specific growth rate
3.2. Effect of initial bacterial inoculum size showed no significant differences between high and low
cell concentrations. These results indicated the inhibi-
Growth curves obtained from treated L. plantarum at tory effect of orange–carrot juice compared to MRS
28  105 V m 1, 11.5  10 5 s inoculated at either high or Broth, to support growth of L. plantarum and its
low initial bacterial concentration, in MRS Broth inhibitory effect with low temperatures during repair of
(Fig. 3) or in orange–carrot juice (Fig. 4) were obtained. injury. Only the most stressful conditions tested (sub-
No significant differences in growth parameters were strate, incubation temperature, PEF treatment and low
observed for untreated L. plantarum at either low or inoculum size) were sufficient to inhibit growth for a 60-
high inoculum levels. When PEF-treated L. plantarum day period. These observations are in agreement with
was incubated in MRS Broth at 16 C, significant Augustin et al. (2000), who found that the effect of small
differences in lag phase were observed for inoculum inoculum size could only be evidenced in severe stress
levels around 103 cfu ml 1 (12.972.2 h) or levels of conditions.
10 cfu ml 1 (26.571.0 h) (Fig. 3). This indicated that the Therefore, to prevent spoilage of orange–carrot juice,
inoculum size determined the time needed for the it would be necessary to combine PEF treatment with
 ARTICLE IN PRESS
M.V. Selma et al. / Food Microbiology 21 (2004) 519–525
refrigeration temperatures during the distribution and
storage, as well as, to guarantee low initial concentra-
tions of these bacteria in fresh-squeezed juice, ensuring a
safe beverage extraction processing. If any of these
factors fail during processing, storage, distribution or
retail, the microbial stability and therefore, the shelf life
of this product could be compromised. It also needs to
be established whether PEF treatment will control
sufficiently the occurrence of foodborne pathogens when
they occur as contaminants. Our results seem to indicate
that they also have a significant effect controlling E. coli
strains, but this point would need to be verified in real
process conditions with the target organisms.
Acknowledgements
This work has been carried out with financial support
from EU and CICYT project 1FD97-0575-C03-03. The
authors are grateful to the Institute of Agrochemical
and Food Technology (CSIC, Valencia) for the support
given to perform part of the experimental work.
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