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Inactivation of Saccharomyces cerevisiae in solutionby low-amperage electric treatment

S. Guillou and N. El MurrLaboratoire d’Analyse Isotopique et Electrochimique de Me ´ tabolismes, Universite ´ de Nantes, Faculte ´ des

Sciences et des Techniques, Nantes, France

2001/79: received 26 April 2001, revised 11 September 2001 and accepted 27 November 2001

S . G U I L L O U A N D N . E L M U R R . 2 002.

Aims: The objectives of this study were to investigate the potential application of a

low-amperage direct electric current as a non-thermal process for inactivation of Saccharomyces

cerevisiae.

Methods and Results: Electric current was generated using a direct current power supply

connected to a traditional electrochemical cell with two platinum electrodes immersed in

conducting solution containing a population of S. cerevisiae. This treatment provokedinactivation of the yeast cells. The microbial destruction illustrated by D-values calculated from

survival curves was shown to be proportional to the current amperage (i) (D varies from

1547 min to 140 min when i varies from 0Æ1 to 1 A, respectively). The efficacy of the treatment

was shown to be better at pH < 7. Statistical analysis showed no significant effect (P > 0Æ05) of

ionic strength on yeast lethality induced by electrolysis.

Conclusions: The lethal effect of the electric treatment on S. cerevisiae in phosphate buffer

was shown to be due to neither ohmic heating nor toxic hydrogen peroxide. A synergistic effect

of temperature and electrolysis was observed when the temperature became lethal for the yeast.

Significance and Impact of the Study: The method described for yeast lethality induced by

electrolysis has potential for soft sterilization, particularly when combined with the synergistic

effect of moderate heat.

INTRODUCTION

Inactivation of micro-organisms is important in the medical

field and in the food-processing industry. Many methods,

such as heat sterilization, u.v. irradiation and the addition of

chemicals (antibiotics, metabolic inhibitors, biocides and

preservatives), are currently used to inactivate micro-

organisms. However, heat treatments cause a loss of

organoleptic properties in food and hence, the development

of efficient alternative food preservation systems is required.In order to preserve freshness qualities of foods, new non-

thermal techniques of sterilization, such as pulsed electric

fields, have been widely studied (Qin et al . 1996). The

application of high electric field serial pulses to liquid foods

has been shown to be an efficient method of sterilization.

However, the industrial plant required for such treatments

represents a major investment for manufacturers. The

method proposed here uses low intensity electric current

and is therefore potentially much simpler and cheaper to

implement.

Recently, the effects of low-amperage electric current on

the inactivation of micro-organisms have been investigated.

It has been postulated that microbial cells are killed by toxic

substances, such as free chlorine (Pareilleux and Sicard1970; Stoner et al . 1982; Davis et al . 1994; Liu et al . 1997),

H2O2 (Shimada and Shimahara 1982; Liu et al . 1997) and

metallic ions (Rosenberg et al . 1965; Berger et al . 1976),

which were generated by the electrode reaction. Use of

electric current to sterilize food products by the Joule effect

is well known. Some investigators (Anderson and Finkel-

stein 1919; Palaniappan et al . 1992) have assimilated the

lethal effect of electricity to a simple thermal effect called

ohmic heating. Palanappian et al . (1992) compared the

antimicrobial effect of ohmic heating with conventional

Correspondence to: Dr Nabil El Murr, Groupe Electrochimie, Laboratoire

d’Analyse Isotopique et Electrochimique de Me tabolismes, UMR CNRS 6006,

Universite de Nantes, Faculte des Sciences et des Techniques, 2, rue de la

Houssiniere, BP 92208-44322 Nantes Cedex 3, France (e-mail: nabil.elmurr@

chimbio.univ-nantes.fr).

ª 2002 The Society for Applied Microbiology

Journal of Applied Microbiology 2002, 92, 860–865

8/12/2019 6547200


heating and concluded that the lethal effect of electric

current was primarily due to thermal effects. There are few

reports dealing with the antimicrobial activity of low

amperage per se. El Murr (1990) described a sterilization

process based on electrolysis current. Liu et al . (1997)

showed that the antibacterial activity of direct currentremained after the removal of chloride ions and oxygen from

the test solution. They postulated that the mechanism of

activity may include the disruption of bacterial membrane

integrity or the electrolysis of molecules on the cell surface.

Matsunaga and Namba (1984) and Matsunaga et al . (1984)

established that the electrochemical destruction of microbial

cells was based on the direct electron transfer between cells

and an electrode, and demonstrated that coenzyme A was

oxidized to dimeric coenzyme A; they also showed that the

resulting microbial cell death was due to the inhibition of

respiratory activity.

In the present study, a simple medium (phosphate buffersolution) inoculated with Saccharomyces cerevisiae was

chosen for the electrochemical treatment in order to reduce

the production of toxic substances by electrolysis. The

objectives of this research were to verify the effectiveness of

the process, to show the influence of processing factors such

as temperature, pH and ionic strength on the inactivation of

the yeast, and to observe the relationship between microbial

death and current intensity under non-lethal thermal

conditions.

MATERIALS AND METHODS

Micro-organisms

Saccharomyces cerevisiae FERMIVIN No. 7013 (Gist-Bro-

cades) yeast cells were cultured in a 250 ml Erlenmeyer flask

containing 100 ml nutritive broth (Yeast Extract (Merck)

10 g l –1; pancreatic peptone (Difco) 10 g l –1; D-glucose

(Fluka) 20 g l –1

; chloramphenicol 0Æ25 g l –1

) at 28C with

continuous agitation at 110 rev min –1

in a shaker. The

microbial cells were grown to late exponential growth phase

as measured by turbidimetry using a spectrocolorimeter

(Milton Roy Spectronic 401, Rochester, NY, USA). Late

exponential phase growth resulted in approximately 1 · 108

S. cerevisiae cfu ml –1

. An equal amount of the yeast

suspension was dispensed into four 30 ml sterile centrifuge

tubes and centrifuged at 1118 g and 10C for 5 min (Sigma

3K15). The supernatant fluids were discarded. Each pellet

was rinsed with 25 ml 0Æ1 mol l –1 pH 7Æ1 phosphate buffer

(KH2PO4 –K2HPO4; Panreac) under the same centrifugation

conditions (speed, temperature and duration). The super-

natant fluids were discarded and each pellet was suspended

in 12Æ5 ml of the treatment medium. The contents of the

four tubes were transferred to a 100 ml sterile Erlenmeyer

flask to constitute a microbial suspension of approximately

1 · 108 cfu ml –1. Approximately 1 ml of this microbial

suspension was added to the electrolytic vessels, containing

50 ml of the treatment medium, to reach a final cell

concentration of 2 · 106 cfu ml –1.

Electric treatment

Suspensions of micro-organisms, 2 · 106

cfu ml –1

, were

made in phosphate buffers. For each experiment, two

thermostatically-controlled electrolytic vessels were used.

One of the electrolytic vessels was fitted with two plain

platinum electrodes (23 · 20 mm) parallel to each other and

20 mm apart. The two electrodes were connected to a d.c.

power supply (Metrix AX322, Annecy Le Vieux, France).

The other vessel, containing the same microbial suspension,

received no electric treatment and was used as a control of

microbial death without electric current. The temperature of

the yeast suspensions was maintained by circulating water ata controlled temperature through the thermostatic jackets of

both vessels. The circulating water was maintained at

20 ± 1C (except when the effect of temperature was

studied) in order to minimize the Joule effect in the first

vessel during electrolysis.

Effect of various parameters on micro-organisminactivation

Influence of amperage. The effect of current intensity on

microbial inactivation was studied by applying a current

ranging from 0Æ1 to 1Æ0 A to suspensions of 2 · 106 cfu ml –1

S. cerevisiae in 0Æ1 mol l –1 phosphate buffer (pH 7

Æ1) at

20C. For the study of the effects of other parameters, the

middle-range value (0Æ5 A) was used. This had the advant-

ages of high inactivation and easy control of the Joule effect.

Influence of temperature. Saccharomyces cerevisiae sus-

pensions (2 · 106

cfu ml –1

) in 0Æ1 mol l –1

pH 7Æ1 phosphate

buffer were treated with 0Æ5 A for 3 h at four temperatures:

10, 20, 30 and 35C.

Influence of pH. The influence of pH on microbial

inactivation was studied by applying 0Æ5 A for 3 h at 20C

to S. cerevisiae suspensions (2 · 106

cfu ml –1

) in 0Æ1 mol l

–1

phosphate solutions at pH 5Æ0, 6Æ0, 7Æ1 and 8Æ5.

Influence of ionic strength. The ionic strength of the

buffer solutions was not adjusted by the addition of KCl

because chlorides can be transformed into toxic chlorine by

electrolysis. To study the effect of ionic strength, pH 7Æ1

buffer solutions were prepared at four different concentra-

tions: 0Æ05, 0Æ1, 0Æ2 and 0Æ4 mol l –1

. Saccharomyces cerevisiae

suspensions (2 · 106 cfu ml –1) in phosphate solutions were

treated with 0Æ5 A for 3 h at 20C.

I N A C T I V A T I O N O F S A C C H A R O M Y C E S 861

ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology , 92, 860–865

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Plate counts

During electrolysis, samples were taken every 30 min to

estimate surviving fractions. Then, 0Æ1 ml of the serially-

diluted samples was surface-plated in duplicate on a YGC

medium (Merck). Several dilutions were used so that the

number of colonies appearing after 48 h of incubation at

28C would be large enough to be statistically meaningful

(between 30 and 300).

Data analysis

All treatments were performed in duplicate and the average

is reported for each experiment. Results are presented in

the form of mean curves representing the log (N/N0) as a

function of the treatment time, where N0 is the concen-

tration of viable yeast before treatment (approximately

2 · 106

cfu ml –1

) and N, the population of viable yeast

after a definite time of electrolysis. For each experiment, amean curve was drawn in which the error was represented

by the mean standard deviation calculated from the two

curves. To represent the effect of inactivation of the micro-

organisms by electric current, the same mathematical

equation as that used for thermal inactivation (survival

curve) was applied, i.e. log (N/N0) ¼ – t/D, where N0 ¼

initial population of micro-organisms, N ¼ population of

micro-organisms after electric treatment, t ¼ duration of

electric treatment, D ¼ death rate constant, which corres-

ponds to one log-cycle reduction of the cell population.

D-values were estimated from the slope of the regres-

sion line obtained from the linear portion of the survivalcurve.

Statistics

Statistical analysis of the effects of temperature, pH and

ionic strength on microbial inactivation was carried out by

analysis of variance, with differences determined by the

method of least significant difference at the 5% (P < 0Æ05)

level.

Determination of hydrogen peroxide

Hydrogen peroxide was measured by spectrophotometri-

cally according to the method described by Bergmeyer

et al . (1983). In the assay solution, hydrogen peroxide

reacts with o-dianisidine chlorhydrate (Sigma) in the

presence of horse-radish peroxidase (EC 1.11.1.7; Boeh-

ringer Mannheim), leading to a coloured complex whose

absorbance measured at 436 nm is proportional to H2O2

concentration.

RESULTS

Effect of current intensity on the viabilityof yeast cells treated by electrolysis

As shown in Fig. 1, inactivation of S. cerevisiae increased

when the current intensity was increased. After 90 min of

treatment, the population of S. cerevisiae treated with direct

electric current from 0Æ1 to 1Æ0 A decreased linearly.

Figure 2 shows that the representation of D-values could

be assimilated to an inverse function of the current intensity,

indicating that the lethality induced by electrolysis was

proportional to the current intensity.

–1·5

–1

–0·5

0

0 30 60 90 120 150 180

Time (min)

l o g

( N / N 0

)

Fig. 1 Effect of amperage on lethality to

Saccharomyces cerevisiae induced by electro-

lysis in 0Æ1 mol l –1 pH 7Æ1 phosphate buffer

solution (20C): (s) 0Æ1 A; (n) 0Æ25 A; (h)

0Æ5 A; (d) 0Æ75 A; (m) 1Æ0 A. Error bars

indicate standard deviations from two

experimental replicates

862 S . G U I L L O U A N D N . E L M U R R


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Effect of temperature on the viability of yeastcells exposed to the electric treatment

No effect of temperature alone was observed under these

experimental conditions until the temperature had reached

35C, at which point weak microbial inactivation of about

0Æ5 log cycle was noticed (Fig. 3a). In contrast, when micro-

organism populations were subjected to an electric current

(i ¼ 0Æ5 A), inactivation occurred, even at low temperatures.

Figure 3b shows the combined effects of temperature and

electrolysis on inactivation of S. cerevisiae in 0Æ1 mol l –1

pH 7Æ1 phosphate buffer. An ANOVA analysis was per-

formed on means of D-values calculated from tests

performed under electrolysis at 10, 20 and 30C. The effect

of temperature on the lethality induced by electrolysis

(i ¼ 0Æ5 A) was found to be significant (P < 0Æ05) at these

temperatures.

At 35C, for a current intensity of 0Æ5 A, a drastic

microbial reduction was observed. A difference of about 1

log cycle was obtained between the experiments performed

at 30C and 35C at the same current intensity. This

could not be attributed to the lethal effect of the increase

in temperature alone. Therefore, a synergistic effect of

temperature and electrolysis was produced when thetemperature became lethal for the micro-organism.

Lethal effect of pH associated with electrolysis

The influence of pH on the inactivation of S. cerevisiae by

electrolysis at 0Æ5 A at 20C was studied. Figure 4 repre-

sents D-values calculated for electrolysis performed in

0Æ1 mol l –1

phosphate solutions at pH ¼ 5, 6, 7Æ1 and 8Æ5.

The effect of pH was shown to be significant (P < 0Æ05).

The least significant difference (P < 0Æ05) showed that the

D-values fell into two distinct groups. The first group

included the D-values obtained at pH 5 and 6 and the

second, for pH 7Æ1 and 8Æ5. These results suggest that the

inactivation is more severe at acidic pH.

0

500

1 000

1 500

2 000

2 500

0·00 0·25 0·50 0·75 1·00

D

v a l u e

s ( m i n )

Amperage (A)

Fig. 2 Microbial destruction rate, D-values vs amperage. D-values

were calculated from the linear portion of the survival curves presented

in Fig. 1. D-values are means based on data from two experiments, and

standard deviations are indicated by error bars. Electrolysis was

performed in 0Æ

1 mol l

–1

pH 7Æ

1 phosphate buffer solution at 20

C

–2·5

–2

–1·5

–1

–0·5

0

0·5

–2·5

–2

–1·5

–1

–0·5

0

0·5

0 30 60 90 120 150 180

Time (min)

l o g ( N / N

0 )

l o g ( N / N 0 )

(b)

0 30 60 90 120 150 180

Time (min)(a)

Fig. 3 Effect of temperature (a) and temperature associated with

electrolysis (b) on viability of Saccharomyces cerevisiae in 0Æ1 mol l –1

pH 7Æ1 phosphate buffer during electrolysis at 0 Æ5 A. (s) 10C; (n)

20C; (h) 30C; (d) 35C

0

10

20

30

40

50

60

70

80

90

5 6

pH

7·1 8·5

D

v a l u e s ( m i n )

Fig. 4 Influence of pH on lethality to Saccharomyces cerevisiae induced

by 0Æ5 A electrolysis in 0Æ1 mol l –1 phosphate solutions. Bars represent

D-values calculated from the linear portion of the survival curves

resulting from 0Æ5 A electrolysis in 0Æ1 mol l –1 phosphate solutions at

different pH. D-values are means based on data from two experiments,

and standard deviations are indicated by error bars



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Production of H+ and OH – by the electrolysis of water

may generate modifications in the pH of cell suspensions.

However, measurements of pH in bulk suspensions before

and after the electric treatment showed that no change in pH

was induced by electrolysis. The influence of pH (from 5 to

8Æ5) on yeast viability was also studied using 0

Æ1 mol l

–1

phosphate solutions. No lethal effect of pH was observed

(log N/N0 » 0), indicating that the inactivation of yeast by

electrolysis could not be attributed to local modifications of

pH, if any were present.

Lethal effect of ionic strength associatedwith electrolysis

Figure 5 shows the influence of the ionic concentration of

phosphate solutions at pH 7Æ1 on yeast inactivation by

electrolysis at 0Æ5 A and 20C. No significant effect of ionic

strength on microbial destruction induced by electrolysiswas observed (P > 0Æ05). Yeast inactivation by electrolysis

was approximately 1Æ5 log cycles, regardless of buffer

concentration.

Production of toxic substances

Although some researchers have attributed the lethality

induced by low-amperage electric current to hydrogen

peroxide (Shimada and Shimahara 1982; Liu et al . 1997), no

hydrogen peroxide was detected during electrolysis under

the present experimental conditions. In addition, the lethal

effect of electrolysis ceased as soon as the direct electric

current was switched off. Moreover, a phosphate buffer

treated by electrolysis just before the addition of yeast cells

was not toxic for yeast. This suggests that the lethal effect of

direct electric current is not due to the production of toxic

substances.

DISCUSSION

The inactivation of S. cerevisiae by electrolysis was shown to

be proportional to the amplitude of the current. Such a

relationship could be used to determine the current intensity

and treatment duration necessary to reach a definitepopulation reduction of a certain micro-organism in a

certain medium. Electric current and temperature seem to

act synergistically for the inactivation of micro-organisms.

This interesting phenomenon could be exploited in the

sterilization of thermo-sensitive food products by electro-

lysis associated with moderate heat treatment.

The role of pH in the survival of micro-organisms is

related to the ability of the organisms to control cytoplas-

mic pH. The intracellular pH of S. cerevisiae cells is

relatively constant at 5Æ2 ± 0Æ4 pH units through the

activities of the cell membrane H+

-ATPase (Cimprich

et al . 1995). The results reported here show that electro-lysis inactivates yeast more efficiently in phosphate solu-

tions at pH < 7, even though S. cerevisiae, like all yeasts,

prefers a slightly acidic medium with an optimum pH

between 4Æ5 and 6Æ5 (Viljoen and Heard 2000). Greater

inactivation at acid pH has also been observed with other

physical treatments, such as pulsed electric fields (Wouters

et al . 1999) and high hydrostatic pressure (ter Steeg et al .

1999). During electrolysis, pH-dependent oxidation and

reduction reactions may occur at the cellular surface and

induce yeast mortality.

No significant effect (P > 0Æ05) of ionic strength was

observed when yeast was submitted to electrolysis in pH 7 Æ1

phosphate buffer. Similar results were obtained with

buffered solutions at concentrations of 0Æ05, 0Æ1, 0Æ2 and

0Æ4 mol l –1

.

Few studies have addressed the lethal effects of low-

amperage electric currents. Shimada and Shimahara (1982)

showed that the application of an alternating current to

microbial suspensions in phosphate solutions caused micro-

bial destruction, mainly as a result of the production of

hydrogen peroxide by the electrode. No hydrogen peroxide

was detected under the experimental conditions used here,

and the absence of an antimicrobial effect after the electric

current had ceased indicates that the lethality induced by

electrolysis is not due to toxic substances. Moreover, itseems that the lethal effect of the electrochemical treatment

could not be attributed to the single effects of temperature

or pH. More research is needed to understand the

mechanism by which yeast is inactivated during electrolysis.

The effects of electrolysis on cell viability and ATP contents

are currently being studied.

When combined with additional parameters such as pH

and temperature, electrolysis could be used effectively in

food preservation, particularly in the stabilization of thermo-

sensitive foods.

–2·5

–2

–1·5

–1

–0·5

0

0·50 30 60 90 120 150 180

Time (min)

l o g ( N / N

0 )

Fig. 5 Effect of ionic strength associated with electrolysis on lethality

to Saccharomyces cerevisiae at 0Æ5 A in pH 7Æ1 phosphate buffer

solution. Buffer concentrations: (s) 0Æ05 mol l –1; (n) 0Æ1 mol l –1;

(h) 0Æ2 mol l –1

; (d) 0Æ4 mol l –1

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ACKNOWLEDGEMENTS

The authors thank Professor M. Federighi (Ecole Nationale

Veterinaire, Nantes, France) for stimulating discussions.

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