Bull Vet Inst Pulawy 48, 371-373, 2004

EFFECT OF PULSED ELECTRIC FIELD (PEF)

ON ESCHERICHIA COLI WITHIN THE LIQUID WHOLE EGG

ADAM MALICKI, MACIEJ OZIEMBOWSKI
1
, JERZY MOLENDA,
TADEUSZ TRZISZKA
1
AND SZYMON BRUEWICZ
2


Department of Food Hygiene and Consumer Health, Veterinary Medicine Faculty,
1
Department of Animal Products Technology, Faculty of Food Science,
Wrocaw Agricultural University, 50-375 Wrocaw, Poland
2
Department of Hygiene, Wrocaw Medical University, 50-345 Wrocaw, Poland
e-mail: malicki@ozi.ar.wroc.pl

Received for publication May 12, 2004.


Abstract

The objective of the present study was to evaluate
the efficiency of pulsed electric field (PEF) against
Escherichia coli contaminating the liquid whole egg (LWE).
The samples of LWE were inoculated with the test bacteria
and subsequently treated for 30 s by the different number of
pulses (20-180) of PEF (32.89 kV x cm
-1
). Application of PEF
resulted in statistically significant reduction of the test
microorganisms, proportional to the number of pulses used.
Depending on the studied strain, treatment with 150-160 PEF
pulses was required to obtain the reduction of initial bacterial
level by 4 log units. Considering the obtained results, PEF
seems to be an effective technique, improving the
microbiological status of LWE, and consequently its industrial
application is highly advisable.

Key words: pulsed electric field, liquid whole
egg, Escherichia coli.


Use of the liquid whole egg (LWE) as a
substitute for shell eggs requires procedures preventing
its spoilage and growth of pathogenic bacteria within the
product. Contamination with Enterobacteriaceae and
particularly with Salmonella sp. and Escherichia coli
constitutes the main microbiological problem related to
LWE (8). Consequently, pasteurisation of the product is
necessary to obtain the microbiological state required by
Polish legislation (11). The thermal treatment, however,
significantly decreases the nutritional value, mainly due
to protein coagulation (4).
Application of pulsed electric field (PEF) seems
to be the profitable alternative for thermal treatment of
food products. Pulsed electric fields were successfully
applied for preservation of numerous liquid products,
and the resulting bacterial reduction was comparable
with that obtained by means of thermal treatment (12).
The effect of PEF is related to the application
of high voltage for very short periods of time (in the
range of nano- or microseconds). Exposure of bacterial
cells to the field changes of the sufficient amplitude
affects the electrical properties of the cell membrane,
reflecting in the decrease in its resistance and the
increase in conductance. Consequently, permeability of
the membrane is altered, which is known as
electroporation (6, 7).
The objective of the present study was to
evaluate PEF efficiency against the test strains of E.coli
contaminating LWE.


Material i Methods

The experiment was performed on the liquid
whole egg (LWE) obtained from the consumer eggs.
The material was inoculated with two strains of E. coli,
PCM 2057 and PCM 224, kindly provided by the
Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, Wrocaw.
The inoculum was prepared from the 18-h
culture in TSB (Oxoid), incubated at 37C. The LWE
samples were homogenised by 150 rotations per min for
4 s, inoculated with 7 x 10
7
CFU x ml
-1
of the test
bacteria and again homogenised for 4 s.
Subsequently, the samples were placed in the
impulse sterilizer and exposed to different number (20,
60, 100, 140 or 180) of PEF pulses (working voltage 25
kV, current 32.89 kV x cm
-1
). The process, lasting 30 s,
was performed at ambient temperature (about 20C).
Microbiological examination of the entire material was
carried out after the treatment. E. coli cells were restored
on chromogenic solid medium (Chromocult Coliform
Agar, Merck). For additional identification, all the
colonies stained from dark blue to violet were treated
with Kovacs reagent for indol. The change of coloration
to cherry-red, appearing in a few seconds (positive result
of indol test), confirmed the isolation of E. coli.
Logarithmic transformation of the bacterial
counts and their statistical analysis were done with the
aid of Microsoft

Excel 2000 and Statistica 5, Version

97 software. The D, 4D and 6D values, i.e. the number
of field pulses required for reduction of the initial

372
bacterial level by 1, 4 and 6 log units, respectively, were
calculated from the regression analysis. The importance
of the mean value differences was established with the
aid of Students test (P<0.05).


Results

The changes in bacterial counts, resulting from
exposition to different number of PEF pulses, are
presented in Fig. 1. Statistically significant reduction of
bacterial number was consonant with the increase in
pulse number (P<0.05). Maximal rate of E. coli
reduction was achieved by 180 pulses of PEF and
reached 4.3 and 4.7 log CFU x ml
-1
for PCM 2057 and
PCM 224 strain, respectively.
The number of survivors differed significantly
for the particular PEF pulse numbers with the exception
of 180 pulses, which gave no statistically important
differences between the counts of both the test strains
studied (P<0.05).
The values of D, 4D and 6D, calculated from
the regression analysis, are given in Table 1. The
differences observed between the test strains were not
statistically significant (P<0.05).


2
.
1
5

F
1
.
7
0

F
6
.
0
2

A
4
.
6
9

B
4
.
0
0

C
3
.
2
5

D
1
.
9
6

F
6
.
6
6

a
5
.
4
3

b
4
.
9
3

c
4
.
3
5

d
3
.
6
0

e
0
1
2
3
4
5
6
7
8
0 20 60 100 140 180
Number of PEF pulses
B
a
c
t
e
r
i
a
l

c
o
u
n
t

[
l
o
g

C
F
U

x

m
l
-
1
]
PCM 2057
PCM 224

Fig. 1. Number of Escherichia coli strains (PCM 2057 and PCM 224) treated for 30 s with different number (20-180) pulses of PEF
(32.89 kV x cm
-1
) within the liquid whole egg (error bars represent standard deviations, A-D, F, a-f = statistically significant
differences, P<0.05).


Table 1
Values of D, 4D and 6D (the number of pulses required for the bacterial reduction by 1, 4 and 6 log units, respectively)
for Escherichia coli strains (PCM 2057 and PCM 224) treated for 30 s with different number (20-180) pulses
of PEF (32.89 kV x cm
-1
) within the liquid whole egg.

PCM 2057 PCM 224
Parameter
Mean Confidence range (95%) Mean Confidence range (95%)
D 26.6 24.7-28.5 35.3 31.5-39.1
4D 150.4 148.6-152.2 160.1 157.2-163.0
6D 252.5 248.6-256.4 251.1 246.0-256.2



Discussion

Implementation of the techniques efficiently
improving the microbiological status of food, without
resulting loss of nutritional value, is particularly
important in case of high protein products, such as liquid
whole egg (LWE). Pulsed electric fields (PEF) are one
of the potential techniques to be applied in that area,
since it is known, that they affect the physico-chemical
properties of the hen egg white at a low extent only (2).
We have evaluated the antibacterial efficiency of PEF
against the two strains of E. coli, since this
microorganism constitutes one of the main
microbiological problems related to poultry products (8).
Antibacterial activity of PEF is dependent on its
electric parameters, microorganism features and
physico-chemical properties of the suspending medium
(6, 7, 13).
The current of PEF and time of exposure were
constant in our experimental model, whereas the number
of field pulses was variable. It was revealed that the
efficiency of PEF against E. coli was increasing
proportionally to the number of pulses applied. Similar
results were obtained in the other studies on Escherichia

373
coli, treated by PEF in the model systems or in the other
food products (1, 5, 9, 10, 12, 13).
Analysis of two different strains of E. coli
revealed some differences in their susceptibility to PEF,
evident particularly by lower number of field pulses.
Some strain or species properties, like shape and size of
the bacterial cell and its growth stage are known to
influence the microbial resistance to PEF treatment (1,
5-7, 13).
The efficiency of PEF significantly depends on
physico-chemical properties of the treated food
products. Recent experiments failed to confirm the
protective effect of proteins and lipids on E. coli
exposed to PEF (9). The effectiveness of the PEF
treatment was also proved in the present study on high
protein environment of LWE.
Low conductivity of the suspending medium is
crucial for the efficiency of PEF treatment (3, 13). Since
the conductivity of LWE is relatively high (6),
sufficiently elevated parameters of PEF are required to
obtain the expected level of bacterial reduction. In the
present study the objective was achieved by increasing
the number of PEF pulses, by the constant, relatively
low electric current. Such approach is quite reasonable,
since the excessive current of PEF results in the
undesirable heating of the treated product, and
consequently the process does not fit the concept of non-
thermal technique (1). On the other hand, the
temperature of the product exposed to PEF should not be
too low. Phospholipids are less ordered and the cellular
membrane has a liquid-crystalline structure at the
temperatures close to 30C. Accordingly, the bacterial
membranes are more susceptible to electroporation and
the antimicrobial efficiency of PEF treatment is maximal
(1).
Synergistic action of PEF and low pH of the
suspending medium against bacteria was proved in
several studies (1, 13). The efficiency of PEF against E.
coli in the model system was the highest at pH values
close to 4.0 (1). Consequently, LWE as a slightly
alkaline product requires elevated parameters of the
treatment, which was proved in the present study.
Concluding, application of PEF in case of the
LWE contaminated with E. coli, seems to be the
effective technique for microbiological status
improvement, providing that the specific features of the
product are considered during the process parameter
adjustment.
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