Bile tolerance of <i>Lactobacillus acidophilus</i> LA-K as influenced by various pulsed electric field conditions

doi:10.4236/abb.2012.35080

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Advances in Bioscience and Biotechnology, 2012, 3, 620-625 ABB

http://dx.doi.org/10.4236/abb.2012.35080 Published Online September 2012 (http://www.SciRP.org/journal/abb/)

Bile 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 15 June 2012; revised 20 July 2012; accepted 15 August 2012

ABSTRACT

Lactobacillus acidophilus has several health benefits

and is used as an adjunct bacterium in the manufac-

ture of cultured dairy foods. Bile tolerance is an im-

portant probiotic characteristic. Pulsed electric field

(PEF) processing is non-thermal, hurdle technology

which comprises of passing fluid foods between two

electrodes and subjecting them to pulses of voltage

for less than one second. Whether certain mild PEF

conditions can enhance bile tolerance of Lactobacillus

acidophilus is not known. The objective was to study

the influence of certain PEF conditions on the, bile

tolerance of Lactobacillus acidophilus LA-K. Lactoba-

cillus acidophilus LA-K suspended in sterile peptone

0.1% w/v distilled water was treated with pulse

widths of 3, 6 and 9 µs, pulse periods of 10,000;

20,000 and 30,000 µs and electric field strengths of 5,

15 and 25 kV/cm. The control did not receive any

pulsed electric field condition. Bile tolerance was de-

termined hourly for 16 hours. PROC GLM of the

Statistical Analysis Systems (SAS) was used for data

analysis. Significant differences were determined at p

< 0.05. Three replications were conducted. Bipolar

pulse width effect had a significant (p < 0.0001) in-

fluence on the bile tolerance. Bile tolerance of the

control was significantly higher than the bile toler-

ance subjected to any of the bipolar pulse widths

studied. There were no significant differences among

the three different bipolar pulse widths. Pulse period

had a significant (p < 0.0001) influence on the bile

tolerance. The control and the three different pulse

periods studied were significantly different from each

other. The bile tolerance of the control was signifi-

cantly the highest, followed by the bile tolerances

subjected to 30,000 µs and 20,000 µs respect ively. The

bile tolerance subjected to 10,000 µs was significantly

the lowest. Electric field strength had a significant (p

< 0.0001) influence on the bile tolerance. Bile toler-

ance of the control and bile tolerance of Lactobacillus

acidophilus LA-K subjected to 5 kV/cm were signifi-

cantly the highest while the bile tolerance when sub-

jected to 25 kV/cm was significantly the lowest.

Keywords: Bile Tolerance; Lactobacillus acidophilus;

Pulsed Electric Field; Probiotic

1. INTRODUCTION

Application of high electric field strengths (typically 20 -

80 kV/cm) for short time periods (less than 1 second) to

fluid foods places between two electrodes is what high

intensity pulsed electric field (PEF) processing is [1].

There are several PEF factors that influence microbial

inactivation, they are electric field strength, pulse wave

shape, treatment time and treatment temperature.

Electric field strength, is one of the main factors in-

fluencing microbial inactivation, and is determined by

the voltage (kV) across the electrodes and the distance

between the electrodes (cm). Increasing the gap will re-

quire higher voltage to obtain the desired electric field

strength [2]. The microbial inactivation increases with an

increase in the electric field intensity, above the critical

transmembrane potential [3]. Pore formation will occur

when a certain threshold value of the transmembrane

potential formed is exceeded, which was found to be in

the range of 1 V [4]. To achieve this transmembrane po-

tential an electric field strength above 30 kV/cm is re-

quired for most bacteria in liquid systems [5].

Pulse Waves shape can be in the form of exponential

decaying pulses, square wave pulses, oscillatory pulses

or bipolar pulses of electric fields. In terms of pulse

shape, square wave pulses are more energy and lethally

efficient than exponential decaying pulses [6]. Oscilla-

tory pulses are the least efficient for microbial inactiva-

tion because they prevent the cell from being continu-

ously exposed to a high intensity electric field for an

extended period of time [7]. In terms of pulse polariza-

tion, bipolar pulses are more lethal than monopolar

*Corresponding author.

OPEN ACCESS

O. Cueva, K. J. Aryana / Advances in Bioscience and Biotechnology 3 (2012) 620-625 621

pulses. Bipolar pulses produce alternating changes in the

movement of charged molecules, which cause a stress in

the cell membrane and enhance its electric breakdown.

Moreover, bipolar pulse reduces deposition of solids on

the electrode surface, decreases food electrolysis, and is

energy efficient [8].

Treatment time has been earlier defined as the product

of the number of pulses and the pulse width [2]. The

pulse width is defined as the time where the peak field is

maintained for square wave pulses or the time until de-

cay to 37% for exponential decay pulses. Pulse width

influences microbial reduction by affecting the electric

field intensity. Longer pulse widths decrease electric

field intensity, which result in higher inactivation; how-

ever, an increase in pulse width may also result in an

unwanted food temperature increase. Hülsheger et al. [9]

developed a mathematical model that relates microbial

survival fraction with PEF treatment time. The inactiva-

tion of microorganisms increases with an increase in

treatment time [10]. The treatment time for the inactiva-

tion kinetics of tomato juice lipoxygenase by pulsed

electric field was calculated with the following formula:

Treatment time = volume of 1 chamber (mL)/flow rate

(mL/sec)*pulse per second*number of chambers*pulse

width [11].

Treatment Temperature is influenced by constant elec-

tric field strength. Constant electric field strength in-

creases microbial inactivation as well as increases the

temperature in foods. For this reason, proper cooling is

necessary to maintain food temperatures far below those

generated by thermal pasteurization. Vega-Mercado et al.

[12] reported that E. coli reduction was observed to in-

crease from 1 to 6.5 log reduction cycles with a tem-

perature change from 32˚C to 55˚C.

Several health benefits of Lactobacillus acidophilus

have been reported earlier [13]. Any probiotic bacterium

needs to survive the bile conditions in the gastrointestinal

(GI) tract before establishing in the lower GI tract to

confer the health benefits upon the host. It is not known

if mild PEF conditions can enhance the bile tolerance of

this health beneficial bacterium. The objective was to

study the influence of pulsed width, pulse period and kV

on the bile tolerance Lactobacillus acidophilus LA-K.

2. MATERIALS AND METHODS

2.1. Experimental Design

Freshly thawed 1% (v/v) of Lactobacillus acidophilus

(F-DVS LA-K, Chr. Hansen’s Laboratory, Milwaukee,

WI, USA) in peptone water (0.1% wt/v) at room tem-

perature (21˚C) was subjected to various PEF treatment

conditions. The treatments were pulse widths of 3, 6, and

9 µs, pulse periods of 10,000, 20,000, and 30,000 µs,

electric field strengths of 5, 10, and 15 kV/cm. Control

was run through the PEF equipment at 60 mL/min with-

out receiving any pulsed electric field treatment. Bile

tolerances, were determined in the control and PEF

treatment samples hourly over 16 hours of incubation.

All experiments were repeated three times.

2.2. Bile Tolerance Test

Bile tolerance of Lactobacillus acidophilus LA-K was

conducted as described earlier [14] with slight modifica-

tions. Lactobacillus acidophilus LA-K was evaluated for

its ability to grow in MRS-THIO broth [MRS broth

(Criterion™, Hardy Diagnostics, Santa Maria, CA) sup-

plemented with 0.2% (wt/v) of sodium thioglycolate

(Acros Organics, Fair Lawn, NJ)] with bile acids. So-

dium thioglycolate was used in MRS broth as an oxygen

scavenger to achieve microaerophilic conditions. Control

and PEF treated samples were inoculated (10% [v/v])

into MRS-THIO broth with 0.3% (wt/v) oxgall (bovine

bile) (USBiological, Swampscott, MA) at an initial

OD650 of 0.200 ± 0.005, and incubated under anaerobic

conditions at 37˚C for 16 hours. Absorbance in samples

was measured hourly with an UV-Vis Spectrophotometer

(Nicolet Evolution 100, Thermo Scientific; Madison, WI,

USA) at 650 nm. The spectrophotometer was calibrated

by using MRS-THIO broth with 0.3% oxgall as blank.

An average of two readings per treatment was taken, that

is two cuvettes per treatment. An estimate of bacterial

counts (CFU/mL) was calculated from OD650 readings

using a standard curve (Figure 1).

2.3. Statistical Analysis

The General Linear Model (PROC GLM) of the Statisti-

cal Analysis Systems (SAS) was used to analyze the data.

Differences of least square means were used to deter-

mine significant differences at p < 0.05 for main effects

and interaction effects. Significant differences were de-

termined at α = 0.05.

3. RESULTS AND DISCUSSION

3.1. Pulse Width

The OD values at different bipolar pulse widths over the

bile tolerance period of 16 hours are shown in Figure 2.

Various treatments applied are shown in Table 1. condi-

tions are in Bipolar pulse width*hour interaction effect

was significant (p < 0.0001) (Table 2). From hours 4 to

16 there were significant differences between the control

and the three different bipolar pulse widths. Bipolar

pulse width effect had a significant (p < 0.0001) influ-

ence on the bile tolerance (Table 2). The bile tolerance

of the control was significantly higher than the bile tol-

erance subjected at any of the bipolar pulse widths stud-

ied. There were no significant differences among the

three different bipolar pulse widths (Table 3). The bile

O. Cueva, K. J. Aryana / Advances in Bioscience and Biotechnology 3 (2012) 620-625

622

Figure 1. Standard curve for growth of LA-K in MRS-THIO broth with 0.3% oxgall.

Figure 2. Pulse width effect on bile tolerance of Lactobacillus acidophilus LA-K, means ± SE.

Table 1. PEF treatment conditions applied during the study of

the influence of various pulse widths on Lactobacillus aci-

dophilus LA-K.

Table 3. Least square means for bile tolerance as influenced by

pulse width.

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

Bile tolerance

Treatment LS Mean

Control 0.825A

3 µs 0.546B

6 µs 0.542B

9 µs 0.540B

OPEN ACCESS

Table 2. Mean square (MS) and Pr > F of pulse width, hour

and their interaction for bile tolerance.

Bile tolerance

Source MS Pr > F

Pulse width 1.018 <0.0001

Hour 2.445 <0.0001

Pulse width*hour 0.030 <0.0001

Error 0.003

tolerance of different strains of Lactobacillus acidophilus

isolated from human intestinal were studied by Buck and

Gilliand [15]. They found that Lactobacillus acidophilus

ATCC 43121 was significantly more bile tolerant than

isolates C14, G20, G5, H13, H11, J18 and J12. This

strain required only 2 hours for the optical density to

increase by 0.3 units, whereas strains J18 and J12 re-

quired 7 hours to increase.

3.2. Pulse Period

The OD values at different pulse periods over the bile

O. Cueva, K. J. Aryana / Advances in Bioscience and Biotechnology 3 (2012) 620-625 623

tolerance period of 16 hours are shown in Figure 3.

Various treatments applied are shown in Table 4. Pulse

period*hour interaction effect was significant (p <

0.0001) (Table 5). From hours 11 to 16 there were sig-

nificant differences between the control and the three

different pulse periods. However, the 10,000 µs and

20,000 µs pulse periods compared to the control, showed

significant differences from hours 9 and 10 respectively.

From hours 12 to 16, among the different pulse periods,

the 30,000 µs pulse period was significantly higher

compared to 20,000 µs which in turn was significantly

higher compared to 10,000 µs. Bile tolerances at all dif-

ferent pulse periods were significantly different from

each other from hours 14 to 16. Pulse period had a sig-

nificant (p < 0.0001) influence on the bile tolerance (Ta-

ble 5). According to Table 6 the control and the three

different pulse periods studied were significantly differ-

ent from each other. The bile tolerance of the control was

significantly the highest, followed by the bile tolerances

subjected to 30,000 µs and 20,000 µs consecutively. The

bile tolerance subjected to 10,000 µs was significantly

the lowest. Bile resistance of Lactobacillus was arbitrar-

ily classified into four groups according to the delay of

growth [10]. These groups are: resistant strains (delay of

growth d ≤ 15 min), tolerant strains (15 min < d ≤ 40

min), weakly tolerant strains (40 min < d < 60 min) and

sensitive strains (d ≥ 60 min) [16].

3.3. Electric Field Strength

The OD values at different electric field strengths over

the bile tolerance period of 16 hours are shown in Figure

4. Various treatments applied are shown in Table 7.

Electric field strength*hour interaction effect was sig-

nificant (p < 0.0001) (Table 8). The bile tolerance of the

control and the bile tolerance subjected to 5 kV/cm were

significantly different than the bile tolerances subjected

to 15 and 25 kV/cm throughout the entire incubation

time period. There were no significant differences be-

tween the control and 5 kV/cm during all 16 hours period.

Electric field strength had a significant (p < 0.0001) in-

fluence on the bile tolerance (Table 8). The bile toler-

ance of the control and the bile tolerance subjected to 5

kV/cm were significantly the highest compared to 15 and

25 kV/cm (Table 9). The bile tolerance subjected to 25

kV/cm was significantly the lowest.

Table 4. Pulsed electric field (PEF) treatment conditions ap-

plied during the study of the influence of various pulse periods

on Lactobacillus acidophilus LA-K.

Parameter Condition

Bipolar pulse width (µs) 3

Electric field strength (kV/cm) 25

Pulse period (µs) 10,000; 20,000; 30,000

Delay time (µs) 20

Flow rate (mL/min) 60

Figure 3. Pulse period effect on bile tolerance of Lactobacillus acidophilus LA-K, means ± SE.

Table 5. Mean square (MS) and Pr > F of pulse period, hour

and their interaction for bile tolerance.

Bile tolerance

Source MS Pr > F

Pulse period 0.338 <0.0001

Hour 0.473 <0.0001

Pulse width*hour 0.026 <0.0001

Error 0.005

Table 6. Least square means for bile tolerance as influenced by

pulse period.

Bile tolerance

Treatment LS Mean

Control 0.338A

10,000 µs 0.223D

20,000 µs 0.264C

30,000 µs 0.308B

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624

Figure 4. Electric field strength influence on bile tolerance of Lactobacillus acidophilus

LA-K, means ± SE.

Table 7. Pulsed electric field (PEF) treatment conditions ap-

plied during the study of the influence of various electric field

strengths on Lactobacillus acidophilus LA-K.

Parameter Condition

Bipolar pulse width (µs) 3

Electric field strength (kV/cm) 5, 15, 25

Pulse period (µs) 30,000

Delay time (µs) 20

Flow rate (mL/min) 60

Table 8. Mean square (MS) and Pr > F of electric field strength,

hour and their interaction for bile tolerance.

Bile tolerance

Source MS Pr > F

Electric field strength 0.582 <0.0001

Hour 0.746 <0.0001

Electric field strength*hour 0.021 <0.0001

Error 0.005

Table 9. Least square means for bile tolerance as influenced by

electric field strength.

Bile tolerance

Treatment LS Mean

Control 0.494A

5 kV/cm 0.497A

15 kV/cm 0.329B

25 kV/cm 0.296C

4. CONCLUSION

Bipolar pulse width and pulse period significantly low-

ered bile tolerance. Electric field strength significantly

influenced bile tolerance. Bile tolerance of the control

LA-K and LA-K subjected to 5 kV/cm were the highest

while bile tolerance LA-K subjected to 25 kV/cm was

the lowest.

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