Acid Tolerance of <i>Lactobacillus acidophilus</i> LA-K as Influenced by Various Pulsed Electric Field Conditions

doi:10.4236/aim.2012.23044

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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

Email: [email protected]

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-

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

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

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

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

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

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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