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Innovative Food Science and Emergin

Bacterial resistance after pulsed electric fields depending

on the treatment medium pH

D. Garcıa, N. Gomez, J. Raso, R. Pagan*

Departamento de Produccion Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, C/ Miguel Servet,

177, 50013, Zaragoza, Spain

Received 16 February 2005; accepted 30 April 2005

Abstract

The objective was to evaluate and compare the pulsed electric field (PEF) resistance of four Gram-positive (Bacillus subtilis, Listeria

monocytogenes, Lactobacillus plantarum, Staphylococcus aureus) and four Gram-negative (Escherichia coli, E. coli O157:H7, Salmonella

serotype Senftenberg 775W, Yersinia enterocolitica) bacterial strains under the same treatment conditions. Microbial characteristics such as

cell size, shape or type of the cell envelopes did not exert the expected influence on microbial PEF resistance. The most PEF resistant bacteria

depended on the treatment medium pH. For instance, L. monocytogenes, which showed the highest PEF resistance at pH 7.0, was one of the

most sensitive at pH 4.0. The most PEF resistant strains at pH 4.0 were the Gram-negatives E. coli O157:H7 and S. Senftenberg. A

subsequent holding of PEF-treated cells in pH 4.0 for 2 h increased the degree of inactivation up to 4 extra Log10 cycles depending on the

bacterial strain investigated. Under these treatment conditions, the most PEF resistant bacterial strains were still the pathogens S. Senftenberg

and E. coli O157:H7.

D 2005 Elsevier Ltd. All rights reserved.

Keywords: Pulsed electric fields; pH; Bacillus subtilis; Escherichia coli O157:H7; Lactobacillus plantarum; Listeria monocytogenes; Salmonella Senftenberg;

Staphylococcus aureus; Yersinia enterocolitica

Industrial relevance: The design of appropriate food preservation processes by PEF requires the selection of an adequate target bacterial strain, which should

correspond to the most PEF resistant microorganism contaminating food. This study indicates that the pH of the treatment medium plays an important role in

determining this target bacterial strain. On the other hand, the combination of PEF and subsequent holding under acidic conditions has been proven to be an

effective method in order to achieve a higher level of microbial inactivation.

1. Introduction

Pulsed electric field (PEF) treatments have been found to

inactivate spoiling and pathogenic microorganisms without

altering nutritional and sensorial properties of foods

(Barbosa-Canovas, Gongora, Pothakamury & Swanson,

1999), so it has been pointed out the possibility of using

PEF treatments to replace or partially substitute traditional

heat pasteurization treatments.

The design of effective PEF treatments that assure the

safety and stability of foods requires identifying the most

1466-8564/$ - see front matter D 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.ifset.2005.04.003

* Corresponding author. Tel.: +34 976 761581; fax: +34 976 761590.

E-mail address: pagan@posta.unizar.es (R. Pagan).

PEF resistant strains among pathogenic and spoiling

contaminating microorganisms. However, limited informa-

tion on variation in microbial resistance to PEF is

available in published literature. Scarce systematic studies

have been carried out under the same experimental

conditions, and an effective comparison of the data

reported in the literature is hampered by the different

environmental conditions and methodologies employed by

its authors, or the contribution to the lethal effect of

temperature increase during continuous operation where

high repetition rates were applied.

In general, microbial inactivation by PEF seems to be

highly dependent on the type of microorganism, species,

strain and even serovar (Alvarez, Manas, Condon &

g Technologies 6 (2005) 388 – 395

D. Garcıa et al. / Innovative Food Science and Emerging Technologies 6 (2005) 388–395 389

Raso, 2003; Aronsson, Lindgren, Johansson & Ronner,

2001; Hulsheger, Potel, & Niemann, 1981, 1983; Lado &

Yousef, 2003; MacGregor, Farish, Fouracre, Rowan &

Anderson, 2000; Pagan, Condon & Raso, 2004; Qin,

Barbosa-Canovas, Swanson, Pedrow & Olsen, 1998; Sale

& Hamilton, 1967; Wouters, Alvarez & Raso, 2002;

Wouters & Smelt, 1997). It is assumed that yeast are

more sensitive than the vegetative bacteria when intense

PEF treatments are applied (Qin et al., 1998; Sale &

Hamilton, 1967), and Gram-positive bacteria more resist-

ant than the Gram-negatives (Aronsson et al., 2001;

Hulsheger et al., 1983; Mazurek, Lubicki & Staroniewicz,

1995; Pothakamury, Monsalve-Gonzalez, Barbosa-Cano-

vas & Swanson, 1995; Sale & Hamilton 1967; Vega-

Mercado, Martın-Belloso, Chang, Barbosa-Canovas &

Swanson, 1996). Since the mechanism of inactivation

by PEF seems to be related to the effects on the

cytoplasmic cell membrane, the thicker and more rigid

cell envelopes of Gram-positive bacteria have been

pointed out to be responsible for their higher PEF

resistance.

The intrinsic resistance of microorganisms also seems to

be related to their cell size and shape (Heinz, Alvarez,

Angersbach & Knorr, 2002; Kehez, Savic & Johnson,

1996; Qin et al., 1998). In fact, the major sensitivity of

yeast to PEF treatments is believed to be due to their

higher cell size. The influence of the cell size on the

inactivating effect of PEF treatments is related to the

transmembrane potential created by an external electric

field. The smaller the cell size, the lower the membrane

potential induced by the action of the electrical field and

therefore a higher microbial resistance to PEF treatments is

achieved (Hulsheger et al., 1983; Zimmermann, Pilwat &

Riemann, 1974). Regarding to the influence of the cell

shape, Heinz et al. (2002) reported that rod-shaped cells

would require a more than 5 times higher electrical field to

be inactivated than spherical cells with the same character-

istic dimension.

Nevertheless, numerous published data are not in

agreement with these general and accepted assumptions.

For example, MacGregor et al. (2000) found that Bacillus

cereus which is a Gram-positive bacterium, was more PEF

sensitive than Pseudomonas aeruginosa which is Gram-

negative. Also, Pagan et al. (2004) reported that Salmo-

nella enterica serovar Senftenberg might be more PEF

resistant than Listeria monocytogenes depending on the

treatment conditions. With regard to the cell size, Heinz et

al. (2002) observed that Yersinia enterocolitica, being one

of the smallest cells tested, was more PEF susceptible than

other larger bacterial strains such as Escherichia coli or

Lactobacillus plantarum. In this sense, Wouters, Bos and

Ueckert (2001) demonstrated that cells of L. plantarum,

sorted by flow cytometry on the basis of different sizes

and shapes, showed a similar degree of permeabilization

when increasing time or energy input of the PEF

treatment.

The influence of unknown or uncontrolled environmental

factors may explain these contradictory results. In fact,

microbial inactivation by PEF has also been found to be

substantially influenced by process parameters such as the

electrical field strength, treatment time, pulse number,

repetition rate of pulses or treatment temperature, and

product parameters such as conductivity, water activity or

pH (Alvarez, Pagan, Condon & Raso 2002; Alvarez, Raso,

Palop & Sala, 2000; Wouters, Alvarez, & Raso, 2002;

Wouters & Smelt, 1997). Besides, pH of the treatment

medium is one of the most investigated parameters (Pagan

et al., 2004; Wouters et al., 2002), its influence on microbial

PEF inactivation is a matter of controversy. In fact, it has

been pointed out that a variation of the pH can increase,

reduce or not modify the microbial PEF sensitivity.

The aim of this work was to evaluate and compare under

the same experimental conditions the PEF resistance of four

Gram-positive (Bacillus subtilis, L. monocytogenes, L.

plantarum, Staphylococcus aureus) and four Gram-neg-

ative (E. coli, E. coli O157:H7; Salmonella serotype

Senftenberg 775W, Y. enterocolitica) bacterial strains

depending on the pH of the treatment medium. The

bacterial strains chosen, which differ in size, shape and

cell wall composition, include food spoiling and pathogens.

The work pretends to determine the role of different

microbial characteristics on microbial PEF resistance

depending on the treatment conditions in order to obtain

a more general picture and help in the design and

optimization of PEF processes.

2. Materials and methods

2.1. Microorganisms and growth conditions

The strains of B. subtilis subsp. niger (NCTC 10073), L.

monocytogenes (ATCC 15313), L. plantarum (ATCC

14917), S. aureus (ATCC 13565), E. coli (NCTC 5934),

S. serotype Senftenberg 775W (ATCC 43485) and Y.

enterocolitica (ATCC 9610) used in this investigation were

supplied by the Spanish Type Culture Collection. The strain

of E. coli O157:H7 used in this investigation is a VTEC–

(Phage type 34) isolated by Dr Chapman (Chapman et al.

1993) and was kindly provided by Dr. B. Mackey

(University of Reading, United Kingdom). During this

investigation the strains were maintained on slants of

Tryptic Soy Agar (Biolife, Milan, Italy) with 0.6% of Yeast

Extract added (Biolife) (TSAYE) (E. coli, E. coli O157:H7

L. monocytogenes, S. aureus, Y. enterocolitica), nutrient

agar (NA) (Biolife) (B. subtilis subsp. niger, S. Senftenberg

775W) or MRS agar broth with Tween 80 (Biolife) (L.

plantarum).

Broth subcultures were prepared by inoculating, with one

single colony from a plate, a test tube containing 5 ml of

sterile Tryptic Soy Broth (Biolife, Milan, Italy) with 0.6% of

Yeast Extract added (Biolife) (TSBYE) (E. coli, E. coli

0 5 10 15 200

5

10

15

20

25

30

Time (µs)

Ele

ctri

c fi

eld

str

eng

th (

kV/c

m)

Fig. 1. Typical pulse used in this investigation.

D. Garcıa et al. / Innovative Food Science and Emerging Technologies 6 (2005) 388–395390

O157:H7, L. monocytogenes, S. aureus, Y. enterocolitica),

Nutrient Broth (NB) (Biolife) (B. subtilis subsp. niger, S.

Senftenberg 775W) or MRS broth with Tween 80 (L.

plantarum). After inoculation the tubes were incubated at 37

-C for 24 h. With these subcultures, 250 ml Erlenmeyer

flasks containing 50 ml of TSBYE (E. coli, E. coli

O157:H7, L. monocytogenes, S. aureus, Y. enterocolitica),

NB (B. subtilis subsp. niger, S. Senftenberg 775W) or MRS

broth with Tween 80 (L. plantarum) were inoculated to a

final concentration of 104 cells ml�1. These flasks were

incubated under agitation (130 rpm; Selecta, mod. Rotabit,

Barcelona, Spain) at 37 -C until the stationary growth phase

was reached.

2.2. PEF equipment

High electric field pulses were produced by discharging a

set of 10 capacitors (6800 pF) (Behlke, C-20C682,

Kronberg, Germany) via a thyristor switch (Behlke, HTS

160-500SCR). The set of capacitors is charged using a high

voltage DC power supply (FUG, HCK 2500M 35000,

Rosenhein, Germany). A function generator (Tektronix,

AGF 320, Wilsonville, OR, USA) delivered the on-time

signal to the switch. A cylindrical plastic tube closed with

two polished stainless steel electrodes was used as the batch

treatment chamber. The distance between electrodes was

0.25 cm and the electrode area was 2.01 cm2. The circuit

configuration generated exponential waveform pulses at

different frequencies (1–60 Hz), pulse widths and electric

field strengths (1–40 kV/cm). The actual electric field

strength and electrical intensity applied were measured in

the treatment chamber with a high voltage probe and a

current probe respectively connected to an oscilloscope

(Tektronix, TDS 3012B, Wilsonville, OR, USA). The PEF

equipment includes provisions for measuring sample tem-

perature. Immediately after the treatment a thermocouple

type K of 0.9 mm diameter, pneumatically activated, enters

into the treatment chamber and the temperature is measured

in the centre of the chamber.

2.3. PEF treatments

Before treatment, each bacterial culture was centrifuged

at 6000�g for 5 min and resuspended separately in citrate–

phosphate buffer of pH 7.0 and 4.0 (Dawson, Elliot, Elliot,

& Jones, 1974) whose concentration was adjusted to an

electric conductivity of 2 mS/cm. Next, 0.5 ml of the

microbial suspensions, at a concentration of approximately

109 CFU/ml, were placed in the batch treatment chamber

with a sterile syringe as it has been previously described

(Raso, Alvarez, Condon & Sala, 2000). Exponential wave-

form pulses (Fig. 1) at an electrical field strength of 25 kV/

cm and a pulse repetition rate of 1 Hz were used in this

study. The specific energy input of each pulse was 3 kJ/kg.

In all experiments the temperature of the samples after

treatment was lower than 35 -C.

2.4. Viable counts

After treatments, samples were adequately diluted and

0.1 ml samples were pour plated onto TSAYE (E. coli, E.

coli O157:H7, L. monocytogenes, S. aureus, Y. enter-

ocolitica), NA (B. subtilis subsp. niger and S. Senftenberg

775W) or MRS agar with Tween 80 (L. plantarum). Plates

were incubated for 24 h (E. coli, B. subtilis subsp. niger,

P. aeruginosa, S. Senftenberg 775W, S. aureus) or 48 h (L.

monocytogenes, Y. enterocolitica, L. plantarum) at 37 -C.Previous experiments showed that longer incubation times

did not influence survival counts. After incubation, colony

forming units (CFU) were counted with an improved

image analyser automatic counter (Protos, Analytical

Measuring Systems, Cambridge, UK) as previously

described (Condon, Palop, Raso & Sala, 1996). Survival

counts were based on mean values obtained from at least

three independent experiments. The error bars on the

figures indicate the mean standard deviations for the data

points.

2.5. Measurement of sensitivity to a subsequent holding in

the treatment medium

After PEF treatments, samples were held in the treatment

medium at room temperature. After 2 h viable counts were

carried out. Previous experiments showed that native cells

were not sensitive under these experimental conditions (data

not shown).

3. Results

3.1. Gram-positive bacterial strains resistance after PEF

depending on the treatment medium pH

The PEF resistance of four Gram-positive bacterial

strains was evaluated depending on the number of pulses

and the treatment medium pH. Table 1 shows the number

of Log10 cycles of inactivation of B. subtilis subsp. niger,

Table 1

Log10 cycles of Gram-positive bacterial strains inactivation after PEF treatments at 25 kV/cm in citrate–phosphate buffer at pH 7.0, 4.0 and after a subsequent

holding for 2 h in citrate–phosphate buffer at pH 4.0

Microorganism shape* size (Am)* Number of pulses pH 7 pH 4 pH 4.0+holding for 2 h in pH 4.0

Mean SD Mean SD Mean SD

B. subtilis; Rods; 0.5–2.5�1.2–10 50 0.81 0.09 3.26 0.36 3.29 0.20

100 1.12 0.05 4.05 0.72 3.96 1.09

150 1.49 0.16 4.08 0.73 3.66 0.62

200 1.82 0.05 3.78 0.31 3.70 0.60

300 2.57 0.07 3.70 0.36 3.69 0.52

L. plantarum; Rods; 0.9–1.2�3–8 50 0.21 0.15 0.80 0.12 2.54 1.39

100 0.51 0.04 1.20 0.14 5.34 0.07

150 0.53 0.15 0.96 0.13 5.46 0.04

200 0.86 0.14 1.42 0.34 5.63 0.15

300 1.14 0.17 1.39 0.73 5.48 0.12

L. monocytogenes; Short rods; 0.4–0.5�0.5–2 50 0.30 0.07 1.72 0.47 3.01 0.10

100 0.42 0.03 2.24 0.69 3.36 0.58

150 0.54 0.04 2.58 0.45 4.12 0.08

200 0.64 0.01 2.78 0.31 4.25 0.01

300 0.86 0.01 3.48 0.49 4.72 0.25

S. aureus; Spheres; 0.8–1 / 50 3.12 0.54 2.31 1.09 3.45 0.34

100 4.16 0.53 3.40 1.24 4.35 0.30

150 4.47 0.53 4.29 1.22 4.83 0.72

200 4.74 0.65 4.20 0.53 4.54 0.05

300 5.49 0.30 4.52 0.72 4.49 0.70

*Data taken from Bergey (1986).

D. Garcıa et al. / Innovative Food Science and Emerging Technologies 6 (2005) 388–395 391

L. plantarum, L. monocytogenes and S. aureus after PEF

treatments at 25 kV/cm for 50, 100, 150, 200 and 300

exponential decay pulses in citrate–phosphate buffer of pH

7.0, pH 4.0 and after a subsequent holding for 2 h in

citrate–phosphate buffer of pH 4.0. The cell size and shape

of each bacterial strain (Bergey, 1986) are also included in

Table 1.

As shown in the table, under the same experimental

conditions, B. subtilis subsp. niger, L. plantarum and L.

monocytogenes showed a higher PEF resistance at pH

7.0 than at pH 4.0, independently of the treatment time

investigated. S. aureus was the most sensitive bacterial

strain at pH 7.0 showing a similar PEF resistance at

both pHs. L. monocytogenes and L. plantarum were the

most PEF resistant at both pH 7.0 and 4.0. Whereas

there were not significant differences between PEF

resistance of L. monocytogenes and L. plantarum shown

at pH 7.0 ( p <0.05), L. plantarum was substantially

more PEF resistant at pH 4.0 at any treatment time

investigated.

With regard to the influence of the treatment time, in

most cases, the longer the treatment time the greater the

number of inactivated cells that were achieved. The

maximum PEF inactivation was reached after 200–300

pulses at 25 kV/cm.

The sensitivity of PEF-treated cells to a subsequent

holding in the same treatment media has also been

evaluated. Whereas the maintenance for 2 h in the treatment

media at pH 7.0 did not significantly ( p<0.05) modify the

number of survivors detected after PEF treatments (data not

shown), the maintenance for 2 h of PEF-treated L.

monocytogenes and L. plantarum cells at pH 4.0 in the

same treatment medium significantly increased the degree

of inactivation ( p <0.05). L. plantarum, which had shown

the highest PEF resistance at pH 4.0, showed the highest

sensitivity to the subsequent holding at pH 4.0 being

inactivated more than 4 extra Log10 cycles of survivors to

the PEF treatment. A PEF treatment of 100 pulses or longer

at 25 kV/cm and a subsequent holding for 2 h in pH 4.0

allowed inactivating more than 5 Log10 cycles of L.

plantarum cells. In contrast, B. subtilis and S. aureus were

not significantly sensitive to the subsequent holding under

acid conditions ( p<0.05).

3.2. Gram-negative bacterial strains resistance after PEF

depending on the treatment medium pH

The PEF resistance of four Gram-negative bacterial

strains under the same experimental conditions was also

evaluated. Table 2 shows the number of Log10 cycles of

inactivation of E. coli, E. coli O157:H7, S. Senften-

berg775W and Y. enterocolitica after PEF treatments at

25 kV/cm for 50, 100, 150, 200 and 300 exponential

decay pulses in citrate–phosphate buffer of pH 7.0, pH

4.0 and after a subsequent holding for 2 h in citrate–

phosphate buffer of pH 4.0. The cell size and shape of

each bacterial strain (Bergey, 1986) are also indicated in

Table 2.

As shown in the table, the four Gram-negative bacteria

were significantly more PEF resistant at pH 4.0 than at

pH 7.0 ( p >0.05), independently of the treatment time

investigated. A PEF treatment of 200 pulses or longer at

Table 2

Log10 cycles of Gram-negative bacterial strains inactivation after PEF treatments at 25 kV/cm in citrate–phosphate buffer at pH 7.0, 4.0 and after a subsequent

holding for 2 h in citrate–phosphate buffer at pH 4.0

Microorganism shape* size (Am)* Number of pulses pH 7.0 pH 4.0 pH 4.0+holding for 2 h in pH 4.0

Mean SD Mean SD Mean SD

E. coli; Straight rods; 1–1.5�2–6 50 2.80 0.17 0.94 0.26 3.76 0.34

100 4.40 0.45 1.35 0.13 3.47 1.23

150 4.83 0.08 1.57 0.12 3.47 0.77

200 5.70 0.39 1.92 0.23 3.67 0.13

300 6.20 0.11 2.69 0.18 3.98 0.36

E. coli; O157:H7; Straight rods; 1–1.5�2–6 50 0.82 0.11 0.33 0.15 2.23 0.02

100 1.38 0.28 0.33 0.15 2.53 0.17

150 2.28 0.62 0.46 0.09 2.80 0.22

200 2.58 0.36 0.64 0.08 2.90 0.07

300 4.47 0.18 1.00 0.12 3.43 0.11

S. Senftenberg; Straight rods; 0.7–1.5�2–5 50 1.93 0.33 0.69 0.19 1.59 0.71

100 2.37 0.45 0.73 0.26 1.54 0.39

150 2.72 0.56 0.70 0.24 1.77 0.54

200 3.18 0.57 0.84 0.26 2.30 0.98

300 4.35 0.55 1.04 0.28 2.79 0.65

Y. enterocolitica; Straight rod to cocobacilli; 0.5–0.8�1–3 50 3.91 1.10 0.80 0.24 3.55 0.89

100 5.52 0.25 1.33 0.22 4.86 1.32

150 4.91 0.30 2.07 0.21 5.11 0.98

200 5.50 0.24 2.69 0.36 5.18 0.50

300 5.97 0.29 4.05 0.73 4.93 0.17

*Data taken from Bergey (1986).

D. Garcıa et al. / Innovative Food Science and Emerging Technologies 6 (2005) 388–395392

25 kV/cm in citrate–phosphate buffer of pH 7.0 allowed

inactivating more than 5 Log10 cycles of both E. coli and

Y. enterocolitica. E. coli O157:H7 and S. Senftenberg

775W showed the highest PEF resistance at both pH 7.0

and 4.0.

Whereas the maintenance for 2 h in the treatment media

at pH 7.0 did not significantly ( p <0.05) increase the degree

of inactivation reached after PEF treatments (data not

shown), when PEF-treated cells were kept for 2 h in the

same treatment medium at pH 4.0 the number of dead cells

increased by 1–3 Log10 cycles. In most cases, the number

of dead cells reached when treated and incubated at pH 4.0

was less than that obtained at pH 7.0.

0 1 2 3 4 5 6 7

Log10 cycles of inactivation

E. coli

Y. enterocolitica

St. aureus

E. coli O157:H7

Salm. Senftenberg

B. subtilis

Lb. plantarum

L. monocytogenes

Fig. 2. Log10 cycles of inactivation of B. subtilis subsp. niger, L.

plantarum, L. monocytogenes, S. aureus, E. coli, E. coli O157:H7, Salm.

Senftenberg 775W and Y. enterocolitica after a PEF treatment at 25 kV/cm

for 300 exponential decay pulses in citrate–phosphate buffer of pH 7.0.

3.3. Comparison of the PEF resistance of Gram-positive

and Gram-negative bacterial strains depending on the

treatment medium pH

Fig. 2 shows the number of Log10 cycles of inactivation

of B. subtilis subsp. niger, L. plantarum, L. monocytogenes,

S. aureus, E. coli, E. coli O157:H7, S. Senftenberg 775W

and Y. enterocolitica after a PEF treatment at 25 kV/cm for

300 exponential decay pulses in citrate–phosphate buffer of

pH 7.0.

As seen in the figure, under the same experimental

conditions, B. subtilis subsp. niger, L. plantarum and L.

monocytogenes were more PEF resistant when treated at pH

0 1 2 3 4 5 6 7

St. aureus

Y. enterocolitica

B. subtilis

L. monocytogenes

E. coli

Lb. plantarum

Salm. Senftenberg

E. coli O157:H7

Log10 cycles of inactivation

Fig. 3. Log10 cycles of inactivation of B. subtilis subsp. niger, L.

plantarum, L. monocytogenes, S. aureus, E. coli, E. coli O157:H7, S.

Senftenberg 775W and Y. enterocolitica after a PEF treatment at 25 kV/cm

for 300 exponential decay pulses in citrate–phosphate buffer of pH 4.0.

0 1 2 3 4 5 6 7

Lb. plantarum

Y. enterocolitica

St. aureus

E. coli

B. subtilis

E. coli O157:H7

Salm. Senftenberg

Log10 cycles of inactivation

L. monocytogenes

Fig. 4. Log10 cycles of inactivation of B. subtilis subsp. niger, L.

plantarum, L. monocytogenes, S. aureus, E. coli, E. coli O157:H7, S.

Senftenberg 775W and Y. enterocolitica after a PEF treatment at 25 kV/cm

for 300 exponential decay pulses in citrate–phosphate buffer of pH 4.0 and

after a subsequent holding for 2 h in citrate–phosphate buffer at pH 4.0.

D. Garcıa et al. / Innovative Food Science and Emerging Technologies 6 (2005) 388–395 393

7.0 than the four Gram-negative bacterial strains inves-

tigated and S. aureus. Whereas this PEF treatment killed

around 1 Log10 cycle of L. monocytogenes or L. plantarum

cells, more than 2 Log10 cycles of B. subtilis and more than

4 Log10 cycles of the other five bacterial strains were

inactivated. The most PEF resistant bacterial strain at pH 7.0

was the pathogen L. monocytogenes.

The comparison of the PEF resistance in citrate–

phosphate buffer of pH 4.0 of the eight bacterial strains

investigated is illustrated in Fig. 3. Under these conditions,

the differences in PEF resistance among the eight bacterial

strains were much smaller being the pathogens E. coli

O157:H7 and S. Senftenberg 775W and the spoiling L.

plantarum the most PEF resistant microorganisms.

Fig. 4 shows the number of Log10 cycles of inactivation

of the eight bacterial strains after a PEF treatment at 25 kV/

cm for 300 exponential decay pulses in citrate–phosphate

buffer of pH 4.0 and a subsequent holding for 2 h in the same

treatment medium. As shown in the figure, the subsequent

incubation caused a drastic increase in the degree of

inactivation of most bacteria tested with the exception of

B. subtilis and S. aureus which were not significantly more

sensitive ( p <0.05).The maintenance for 2 h in pH 4.0

allowed increasing the number of inactivated cells up to 4

extra Log10 cycles depending on the microorganism inves-

tigated. Under these treatment conditions, the most PEF

resistant bacterial strains were still the pathogens S.

Senftenberg 775W and E. coli O157:H7.

4. Discussion

Most published data about the influence of microbial

characteristics such as the structure and thickness of cell

envelopes, cell size and shape on microbial PEF resistance

conclude that Gram-positive bacteria are more PEF resistant

than Gram-negatives (Aronsson et al., 2001; Hulsheger et

al., 1983; Mazurek et al., 1995; Pothakamury et al., 1995;

Sale & Hamilton 1967; Vega-Mercado et al.,1996), smaller

cells more resistant than larger ones and rod-shaped cells

less PEF susceptible than sphere-shaped (Heinz et al., 2002;

Kehez et al., 1996; Qin et al., 1998).

In the present investigation, the PEF resistance of eight

bacterial strains, which differ in the type of cell envelopes,

cell size or shape, has been evaluated and compared under the

same experimental conditions in order to check these general

assumptions and to contribute to determine the ‘‘target’’

bacterial strain depending on the treatment conditions.

The results of this study indicate that the pH of the

treatment medium is a main factor to be considered since it

determines the influence of the microbial characteristics on

the bacterial susceptibility to PEF treatments. For example,

L. monocytogenes, which showed the highest PEF resist-

ance when treated at pH 7.0 (Fig. 2), was one of the most

sensitive strains at pH 4.0 (Fig. 3). However, when PEF-

treated cells at pH 4.0 were kept for 2 h in the same

treatment medium, L. plantarum or Y. enterocolitica were

then more sensitive than L. monocytogenes (Fig. 4).

Whereas at pH 7.0 there were great differences in PEF

resistance among the eight bacterial strains, the differences

were substantially reduced when treated at pH 4.0 showing

a completely different bacterial PEF resistance distribution.

The picture varied again when holding PEF-treated cells at

pH 4.0 for 2 h. For instance, L. plantarum, which had been

proven to be the most PEF resistant Gram-positive bacterial

strain at pH 4.0 (Fig. 3) was the most sensitive strain after

the subsequent holding under acid conditions (Fig. 4).

As illustrated in Fig. 2 and 3, the pH of the treatment

medium set up the behaviour under PEF of the Gram-positive

and Gram-negative bacterial strain groups. With the excep-

tion of S. aureus, the other three Gram-positive bacteria

showed a higher PEF resistance at pH 7.0 than at pH 4.0. The

great sensitivity to PEF shown by S. aureus at pH 7.0 might

not allow observing differences due to the variation of the

treatment medium pH. In contrast, the four Gram-negative

bacteria were more resistant at pH 4.0 than at pH 7.0. This is

not a common bacterial response in comparison to the one

observed when applying other novel or traditional food

preservation methods. Independently of the type of cell wall,

bacteria usually are more resistant at neutral than at acid pHs

when treated by heat (Tomlins & Ordal, 1976) or high

hydrostatic pressure (Alpas, Kalchayanand, Bozoglu & Ray,

2000). When inactivating bacteria by ultrasound (Condon,

Raso & Pagan, 2004) or irradiation (Buchanan, Edelson-

Mammel, Boyd & Marmer, 2004) bacterial resistance does

not depend on the pH of the treatment medium, neither in

Gram-positive nor Gram-negative bacteria. These diverse

results have been obtained depending on the Gram character-

istic and they would allow explaining most published data

about the variable effect of the pH on PEF resistance

depending on the bacterial strain investigated (Pagan et al.,

2004; Wouters et al., 2002). Since damage to the bacterial

cell membrane is believed to be an important event, leading

to the inactivation of cells by PEF (Barbosa-Canovas et al.,

D. Garcıa et al. / Innovative Food Science and Emerging Technologies 6 (2005) 388–395394

1999; Hamilton & Sale, 1967; Pagan et al., 2004; Sale &

Hamilton, 1967; Wouters et al., 2002), the differences in the

structure and the composition between the cell wall of Gram-

positive and Gram-negative bacteria might be responsible for

the different behaviour observed under PEF.

On the other hand, when comparing the eight bacterial

strains at pH 7.0 or 4.0 none of the general assumptions about

the influence of the microbial characteristics on the bacterial

PEF resistance mentioned above exerted the expected

influence. For example, at pH 7.0, as illustrated in Fig. 2,

S. aureus was more PEF sensitive that the Gram-negatives S.

enterica Senftenberg or E. coli O157:H7 despite being a

Gram-positive. Also, Y. enterocolitica, which is one of the

smallest bacterium investigated, was more PEF sensitive than

larger cells like, for instance, L. plantarum. Also the shape of

the bacteria did not exert the expected influence. For

instance, the rod-shaped E. coli cells were as sensitive as S.

aureus or Y. enterocolitica, which are spherical and

cocobacilli cells respectively. At pH 4.0, as seen in Fig. 3,

the situation was not different. For instance, L. plantarum,

which is a large cell, was the most resistant Gram-positive

bacterial strain at pH 4.0. E. coli O157:H7 and S. Senftenberg

were the most PEF resistant strains, and L. monocytogenes,

despite being a Gram-positive and the smallest cell tested,

was more PEF sensitive than L. plantarum or E. coli.

The general assumptions about the influence of the cell

size or shape were proven when studying their influence

within each Gram bacterial strain group at pH 7.0. Our

results indicate that the cell shape would be determinant of

the PEF resistance rather than the cell size. For instance,

besides their smaller size, S. aureus and Y. enterocolitica,

which were the most spherical bacteria, showed the higher

PEF sensitivity within each Gram bacterial group at each

pH. When comparing rod-shaped cells, the smaller the cell

size the greater the PEF resistance that was reached.

Based on the results shown in Table 1 and 2, this study

would indicate that the limiting bacterial strain will depend

on the pH of the treatment medium. At pH 7.0 a Gram-

positive bacterial strain would be expected to be the most

PEF resistant. In this study, L. monocytogenes would be the

target bacterial strain when treated at pH 7.0. However, S.

Senftenberg or E. coli O157:H7 would be the limiting

bacterial strains at pH 4.0 (Fig. 3). S. Senftenberg is also one

of the recommended target microorganisms in the pasteu-

rization of foods such as egg products because of its high

thermal tolerance (Manas, Pagan, Sala & Condon, 2000). In

contrast, E. coli O157:H7 is not a specially heat resistant

microorganism (Stringer, George & Peck, 2000).

Garcıa, Gomez, Condon, Raso, and Pagan (2003)

demonstrated that the higher PEF resistance of E. coli at

pH 4.0 was due to a higher PEF-damage repair capability of

cells treated in acidic media. The authors also observed that

sub-lethally injured PEF-treated E. coli cells in citrate

phosphate buffer at pH 4.0 became sensitive to a subsequent

incubation under acidic conditions. As a result, a higher

level of inactivation was reached.

Following the same procedure the susceptibility of the

eight bacterial strains to a subsequent maintenance for 2 h

at room temperature in citrate–phosphate buffer of pH 4.0

was evaluated. As shown in Fig. 4, most bacteria

investigated were sensitive to the subsequent holding and

consequently the level of inactivation increased up to 4

extra Log10 cycles depending on the microorganism

investigated.

The increased sensitivity of PEF-treated cells to acid

conditions might be due to the damages exerted in the

bacterial cell membrane that affect membrane functions

such as the ability to maintain a transmembrane pH

gradient as described by Simpson, Whittington, Earnshaw

and Russell (1999) in PEF treated L. monocytogenes

cells.

It is remarkable that under these treatment conditions the

differences in PEF resistance among the eight bacterial

strains were substantially reduced. Moreover, the level of

inactivation tended to coincide when Gram-negative bacte-

rial strains were PEF treated at both pH 7.0 and 4.0. That is,

in Gram-negative bacteria the dependence of PEF resistance

on the treatment medium pH would be reduced when

keeping PEF-treated cells under acid conditions for a while.

This drop in the number of survivors when keeping PEF-

treated cells at pH 4.0 should be considered as a possible

cause of the discrepancies observed when comparing the

relative PEF resistance of several bacterial species under the

same treatment conditions, or when studying the influence

of the treatment medium pH on the microbial PEF

resistance. In fact, the sensitivity of the bacterial strains

tested to the subsequent incubation under acid conditions

was not the same. For instance, L. plantarum, which had

been proven to be the most PEF resistant Gram-positive

bacterial strain at pH 4.0 (Fig. 3) showed a great sensitivity

to the subsequent holding under acid conditions (Fig. 4).

Therefore, the holding of the microorganisms in the treat-

ment medium before enumeration may overestimate the

effectiveness of the PEF treatment. In any case, the

increased inactivation obtained after incubating PEF-treated

bacteria at pH 4.0 would help in the design of more effective

PEF treatments.

In conclusion, Gram-positive bacterial cells are not

always more PEF resistant than Gram-negatives, neither

larger nor sphere-shaped cells are more PEF susceptible

than smaller or rod-shaped cells respectively. The influence

of these microbial characteristics on PEF inactivation is

dependant on the pH of the treatment medium. The target

microorganism might also vary depending on the pH of the

treatment medium. The most PEF resistant bacterial strain

at pH 7.0 was the pathogen L. monocytogenes. In contrast,

the pathogens S. Senftenberg 775W and E. coli O157:H7

were the most PEF resistant strains at pH 4.0. The

combination of PEF and subsequent holding under acidic

conditions has been demonstrated to be an effective

method in order to achieve a higher level of microbial

inactivation.

D. Garcıa et al. / Innovative Food Science and Emerging Technologies 6 (2005) 388–395 395

Acknowledgements

This work was supported by CICYT (Project AGL

2000-1222) and Ministerio Espanol de Educacion y

Ciencia, which provided D. Garcıa and N. Gomez with

a grant to carry out this investigation. Our thanks to Mrs.

I. Dumall for her collaboration in the English correction

of this work.

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