Advances in Microbiology, 2012, 2, 358-363
http://dx.doi.org/10.4236/aim.2012.23044 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Acid Tolerance of Lactobacillus acidophilus LA-K as
Influenced by Various Pulsed Electric Field Conditions
Olga Cueva1, Kayanush J. Aryana1,2
1School of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, USA
2Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, USA
Received June 29, 2012; revised July 29, 2012; accepted August 10, 2012
ABSTRACT
Pulsed electric field (PEF) processing involves the application of pulses of voltage for less than one second to fluid
foods placed between two electrodes. Lactobacillus acidophilus is an important probiotic bacterium used for the pro-
duction of fermented dairy products. Acid tolerance is an important probiotic characteristic. The influence of PEF on
acid tolerance of Lactobacillus acidophilus is not known. Objective of this study was to elucidate the influence of cer-
tain PEF conditions on the acid to lerance of Lactobacillu s acidophilus LA-K. Freshly thawed Lactobacillus acidophilus
LA-K was suspended in sterile peptone 0.1% w/v distilled water and treated in a pilot plant PEF system. The treatments
were pulse width (3, 6 and 9 µs), pulse period (10,000; 20,000 and 30,000 µs) and voltage (5, 15 and 25 kV/cm). Con-
trol was run through PEF system at 60 mL/min without receiving any pulsed electric field condition. Data were ana-
lyzed using the PROC GLM of the Statistical Analysis Systems (SAS). Differences of least square means were used to
determine significant differences at P < 0.05. The control and the three different bipolar pulse widths studied were sig-
nificantly different from each other. The acid tolerance of the control was significantly the highest, followed by the acid
tolerance of the culture subjected to 3 µs and 6 µs. The acid tolerance of culture subjected to 9 µs was the lowest. The
acid tolerance of the contro l was significantly the highest, followed by the acid tolerances subjected to the pulse period
of 30,000 and 20,000 µs. The acid tolerance of culture subjected to pulse period 10,000 µs was significan tly the lowest.
The acid tolerance of the control was significantly the highest followed by the acid tolerances of culture subjected to
electric field strength of 5 and 15 kV/cm. The acid tolerance of culture subjected to 25 kV/cm was significantly the
lowest. Acid tolerance of Lactobacillus acidophilus LA-K lowered by increasing pulse widths and voltages but lower-
ing puls e pe riods.
Keywords: Lactobacillus acidophilus; Pulsed Electric Field; Probiotic
1. Introduction
This High intensity pulsed electric field (PEF) processing
involves the application of pulses of high voltage (typi-
cally 20 - 80 kV/cm) for short time periods (less than 1
second) to fluid foods places between two electrodes [1].
Application of PEF is restricted to foods products that
can withstand high electric fields, have low electrical
conductivity, and do not contain or form bubbles (e.g.,
liquid foods as milk or fruit juices) [2]. PEF technology
is considered better than heat treatment of foods because
it achieves high microbial inactivation, avoids or greatly
reduces detrimental changes in the sensory and physical
properties of food, and inactivates some enzymes [3].
Pulsed electric field is more energy efficient than thermal
pasteurization. This nonthermal technology would add
only $0.03 - $0.07 to final food costs [4]. Several theo-
ries have been proposed to explain microbial inactivation
by PEF. The most commonly accepted theory is electro-
poration, which is the phenomenon in which the lipid
bilayer and proteins of cell membrane exposed to high
intensity electric field pulses are temporarily destabilized
[5].
Lactobacillus acidophilus is a bacterium with several
health benefits, including enhancement of immune sys-
tem, reduction of various types of diarrhea in humans,
alleviation of Crohn’s disease, lower cholesterol, im-
prove symptoms of lactose intolerance, and balancing of
intestinal microflora through the growth modulation of
bacteria present in the gastrointestinal tract [6]. Lactoba-
cillus acidophilus is used extensively for the production
of fermented dairy products and is increasingly applied
in the area of health improvement, as probiotics, in the
form of yogurts and dietary supp lements.
Several PEF process factors namely electric field
strength, pulse waveshape, treatment time and treatment
temperature influence microbial inactivation [7]. Influ-
C
opyright © 2012 SciRes. AiM

O. CUEVA, K. J. ARYANA 359
ence of PEF on acid tolerance of Lactobacillus acido-
philus is not known. The objective was to study the in-
fluence of pulsed width, pulse period and kV on the acid
tolerance Lactobacillus acidophilus LA-K.
2. Materials and Methods
2.1. Experimental Design
Control and Pulsed Electric Field (PEF) treatment sam-
ples were inoculated with Lactobacillus acidophilus LA-K
(F-DVS LA-K, Chr. Hansen’s Laboratory, Milwaukee,
WI, USA). The treatments were pulse width (3, 6, and 9
µs), pulse period (10,000 µs, 20,000 µs, and 30,000 µs),
electric field strength (5 , 10, and 15 kV /cm). Control was
run through the PEF equipment at 60 mL/min without
receiving any pulsed electric field treatment. Acid toler-
ances were determined in the control and PEF treatment
samples. Acid tolerance was evaluated at 0, 5, 10 and 15
minutes of incubation. The experimental design was a
repeated measure design. Three replications were con-
ducted.
2.2. Control and PEF Treatment Samples
Preparation
Control and PEF treatment samples for the acid to lerance
analyses were prepared by inoculating 1% (v/v) of Lac-
tobacillus acidophilus (F-DVS LA-K, Chr. Hansen’s
Laboratory, Milwaukee, WI, USA) in peptone water
(0.1% wt/v) at room temperature (21˚C). Lactobacillus
acidophilus LA-K in control and PEF treatment samples
for protease analysis was inoc ulated at 10% (v/v).
2.3. Acid Tolerance Test
The acid tolerance of Lactobacillus acidophilus LA-K
was conducted as described earlier [8] with slight modi-
fications. Control and PEF treated samples were inocu-
lated (10% [v/v]) into acidified MRS broth (Criterion™,
Hardy Diagnostics, Santa Maria, CA) previously ad-
justed to pH 2.0 with 1N HCl. The acidified MRS broth
mixtures were incubated in a water bath at 37˚C for 15
minutes. One milliliter samples were taken at various
times (0, 5, 10, and 15 min), serially 10-fold diluted in
peptone water, and plated in duplicate onto MRS agar
(Difco, Detroit, MI). The plates were incubated at 37˚C
for 24 h under anaerobic condition before enumeration.
2.4. Statistical Analysis
Data were analyzed using the General Linear Model
(PROC GLM) of the Statistical Analysis Systems (SAS).
Differences of least square means were used to determine
significant differences at P < 0.05 for main effects (pulse
width, pulse period, voltage) and interaction effects
(pulse width * time, pulse period * time, voltage * time).
Data are presented as mean ± standard error of means.
Significant differences were determined at α = 0.05.
3. Results and Discussion
3.1. Pulse Width
The acid tolerance at different bipolar pulse widths over
the four time points of 0, 5, 10 and 15 minutes are shown
in Figure 1. Various pulse widths applied are shown in
Table 1. Bipolar pulse width * minute interaction effect
was significant (P < 0.0001) (Table 2). From minutes 0
to 15 there were significant differences between th e con-
trol and the different bipolar pulse widths. At minute 0,
among the three different bipolar pulse widths, the acid
tolerance of culture subjected to b ipolar pulse widths of 3
µs was significantly higher than 6 µs and 9 µs. At minute
5 the acid tolerances of culture subjected to bipolar pulse
widths of 3 µs and 6 µs were significantly higher com-
pared to 9 µs. The acid tolerance of culture subjected to 3
µs was significantly the highest at minu te 10 followed by
6 µs and 9 µs consecutively. There were no significant
differences among the three different bipolar pulse
widths at 15 minutes. Bipolar pulse width effect had a
significant (P < 0.0001) influence on the acid tolerance
(Table 2). The control and the three different bipolar
pulse widths studied were significantly different from
each other (Table 3). The acid tolerance of the control
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
051015 20
In cuba tion time
control
3µs
6µs
9µs
Lac tobac illi count ( log cf u/mL)
(
min
)
Figure 1. Pulse width influenc e on acid tolerance of LA-K.

O. CUEVA, K. J. ARYANA 360
Table 1. PEF treatment co nditions applied during the study
of the influence of various pulse widths on LA-K.
Parameter Condition
Bipolar pulse width (µs) 3, 6, 9
Electric field strength (kV/cm) 25
Pulse period (µs) 10,000
Delay time (µs) 20
Flow rate (mL/min) 60
Table 2. Mean square (MS) and Pr > F of pulse width, min-
ute and their interaction for acid tolerance.
Acid tolerance
Source MS Pr > F
Pulse width 4.436 <0.0001
Minute 78.519 <0.0001
Pulse width*
minute 0.169 <0.0001
Error 0.009
Table 3. Least square means for acid tolerance as influ-
enced by pulse width.
Acid tolerance
Treatment LS Mean
Control 3.933A
3 µs 2.924B
6 µs 2.754C
9 µs 2.575D
was significantly the highest, followed by the acid toler-
ance of culture subjected to 3 µs and 6 µs. The acid tol-
erance of culture subjected to 9 µs was the lowest (Table
3). The majority of studies have used exponential decay
pulses when studying microbial inactivation by PEF.
However, square wave pulses are more energy and le-
thally efficient as well as more accurate for calculation of
treatment time at a given electric field strength [9]. The
bipolar pulse widths applied in this study were square
wave pulses. According to Qin et al., [10] bipolar pulse
widths are more lethal than monopolar pulses because
PEF causes a movement of charged molecules in the cell
membranes of microorganisms, and reversal in the ori-
entation or polarity of the electric field causes a corre-
sponding change in the direction of charged molecules.
3.2. Pulse Width
The acid tolerance at different pulse periods over the four
time points of 0, 5, 10 and 15 minutes are shown in Fig-
ure 2. Various pulse periods applied are shown in Table
4. Pulse period * minute interaction effect was significant
(P < 0.0001) (Table 5). At minute 0 there was a signifi-
cant difference between the control and 10,000 µs. From
minutes 5 to 15 there were significant differences be-
tween the control and the different pulse periods. From
minutes 0 to 15 there were no significant differences
between the acid to lerance of culture subjected to 20,000
µs and 30,000 µs. From minutes 0 to 10 the acid toler-
ance of culture subjected to pulse periods of 10,000 µs
was significantly lower than the acid tolerance of culture
subjected to pulse periods of 30,000 µs. At minute 15
there were no significant differences among the three
different pulse periods. Pulse period had a significant (P
< 0.0001) influence on the acid tolerance (Table 5). The
control and the three different pulse periods evaluated
were significantly different from each other (Table 6).
The acid tolerance of the control was significantly the
highest, followed by the acid tolerances of culture sub-
jected to 30,000 µs and 20,000 µs consecutively. The
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
05101520
In cu ba tion time (min )
control
10,000 µs
20,000 µs
30,000 µs
Lactob acilli count ( l og cfu/ mL
)
Figure 2. Pulse period effect on acid tolerance of LA-K.

O. CUEVA, K. J. ARYANA 361
able 4. Pulsed electric field (PEF
Parameter Condition
T) treatment conditions ap-
plied during the study of the influence of various pulse pe-
riods on Lactobacillus acidophilus LA-K.
Bipolarµs) pulse width (3
Electri) 2
10,000; 200,000
Flow r)
c field strength (kV/cm5
Pulse period (µs) ,000; 3
Delay time (µs) 20
ate (mL/min60
able 5. Mean square (MS) and Pr > F of pulse period, min-
Acid tolerance
T
ute and their interaction for acid tolerance.
Source MS Pr > F
Pulse period 2.827 <0.0001
Minute 75.104 <0.0001
Pulse width*
minute 0.383 <0.0001
Error 0.007
able 6. Least square means for acid tolerance as influ-
Acid tolerance
T
enced by pulse period.
Treatment LS Mean
Control 3.852
10,000 µs 2.782D
20,000 µs 2.916C
30,000 µs 2.993B
A
acid tolerance of culture subjected to 10,000 µs was sig-
3.3. Electric Field Strength
electric field strengths
nificantly the lowest. In this study Lactobacillus aci-
dophilus LA-K subjected to any of the different pulse
widths and pulse periods studied did not survive after 15
minutes at pH 2.0. In a study carried out by Pereira and
Gibson [8] it was shown that the viability of Lactobacil-
lus pentosus (B) and Streptococcus thermophilus DSM
20617 was lost in less than 15 minutes at pH 2.0. They
also found that Lactobacillus fermentum KC5b, Lactoba-
cillus delbrueckii JCM 1002, and Lactobacillus acido-
philus johnsonii were the most acid tolerant strains by
retaining around 100% viability for up to 2 hours at pH
2.0.
The acid tolerance at different
over the four time points of 0, 5, 10 and 15 minutes are
shown in Figure 3. Various electric field strengths ap-
plied are shown in Table 7. Electric field strengths *
minute interaction effect was significant (P < 0.0001)
(Table 8). From minutes 0 to 10 the acid tolerance of the
control along with the acid tolerance of culture subjected
to 5 kV/cm were significantly higher than the acid toler-
ances of culture subjected to 15 kV/cm and 25 kV/cm.
Moreover, at this same time interval, the acid tolerance
of culture subjected at 25 kV/cm was significantly the
lowest, followed by the acid tolerance of culture sub-
jected to 15 kV/cm. At minute 15 the acid tolerance of
the control was significantly the highest compared to the
other electric field strengths, followed by the acid toler-
ance of culture subjected to 5 kV/cm. At this same min-
ute there were no significant differences between the acid
tolerances of culture subjected to 15 kV/cm and 25
kV/cm. Electric field strength had a significant (P <
0.0001) influence on the acid tolerance (Table 8). The
control and the three different electric field strengths
studied were significantly different from each other (Ta-
ble 9). The acid tolerance of the control was significantly
the highest followed by the acid tolerances of culture
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0510 15 20
Incubation time (min )
control
5 kV /cm
15 kV /cm
25 kV /cm
Lactobacilli count (log cf u/m L )
Figure 3. Electric field strength influence on acid tolerance of LA-K.

O. CUEVA, K. J. ARYANA 362
able 7. Pulsed electric fie
Parameter Condition
Tld (PEF) treatment conditions
applied during the study of the influence of various electric
field strengths on Lactobacillus acidophilus LA-K.
Bipolar pulse width (µs) 3
Electric field strength (kV/cm) 5, 15, 25
in)
Pulse period (µs) 30,000
Delay time (µs) 20
Flow rate (mL /m60
able 8. Mean square (MS) and Pr > F of electric field
Acid tolerance
T
strength, minute and their interaction for acid tolerance.
Source MS Pr > F
Electric field strength 3.981 <0.0001
Minute 70.209 <0.0001
Electric field strength*
minute 0.198 <0.0001
Error 0.012
able 9. Least square means for acid tolerance as in
Acid tolerance
T flu-
enced by voltage.
Treatment LS Mean
Control 3.871
5 kV/cm 3.756B
15 kV/cm 2.952C
25 kV/cm 2.716D
A
bjected to 5 kV/cm. The acid tolerance of culture sub-
ycles, compared with 3.16
h and pulse period significantly low-
. Voltage significantly influenced acid
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