Innovative Food Science and Emerging Technologies 15 (2012) 3849

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Innovative Food Science and Emerging Technologies

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Electro-activated aqueous solutions: Theory and application in the food

industry and biotechnology
Mohammed Aider a, b,, Elena Gnatko c, Marzouk Benali d, Gennady Plutakhin e, Alexey Kastyuchik a
a
Department of Food Engineering, Universit Laval, Quebec, Qc, Canada G1V 0A6
b
Institute of Nutraceuticals and Functional Foods (INAF), Universit Laval, Quebec, Qc, Canada G1V 0A6
c
Ukrainian State University of Chemical Engineering, Dnepropetrovsk, 49005, Ukraine
d
Natural Resources Canada/CanmetENERGY, 1615 Lionel-Boulet Blvd., P.O. Box 4800, Varennes, Quebec, Canada J3X 1S6
e
Department of Biotechnology, Biochemistry and Biophysics, Kuban State Agrarian University, Kalinin Str. 13, Krasnodar, 350044, Russia

a r t i c l e i n f o a b s t r a c t

Article history: The present review highlights the state-of-the-art electro-activation as a science and the applications of
Received 22 September 2011 electro-activated aqueous solutions in biotechnology and the food industry. The science behind electro-
Accepted 3 February 2012 activation remains unknown. Hence, this review focuses on understanding the mechanisms governing the
process of obtaining electro-activated aqueous solutions. Several applications in biotechnology and the
Editor Proof Receive Date 5 March 2012
food industry are discussed. Among the potential applications of this technology, reagentless chemical catal-
Keywords:
ysis and food safety seem to be the most promising.
Electro-activation Industrial relevance: Electro-activated solution can be successfully used in the food industry and biotechnology for:

Aqueous solution Selective protein and ber extraction from different meal residues.

Acidic anolyte Self-generation of acidic and alkaline conditions for different catalytic applications.

Alkaline catholyte Electro-activated solutions can be used as sanitizing agents for work area cleaning in food processing
Reagentless chemical reaction industries.
Sanitizing
Electro-activated solutions can be used for prevention of bio-lms formation in food processing
equipments.
2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2. Fundamentals of electro-activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.1. Electrolysis and generation of electro-activated water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2. Thermodynamic considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.3. Electro-activation systems and technical requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.4. Design of the electro-activation reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.5. Raman scattering of light by electro-activated water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3. Applications of electro-activated solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.1. Activation of antioxidant enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.2. Baking wheat bread with electro-activated aqueous solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.3. Yeast inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.4. Electrochemical inactivation of bacteria, viruses and bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.5. Biolms prevention/treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.6. Poultry spraying and chilling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.7. Inactivation of endospore-forming bacteria and toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.8. Inactivation of Staphylococcal enterotoxin-A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Corresponding author at: Laval University, 2425 Rue de l'Agriculture, Pavilion P.Comtois # 2403, Quebec, Qc, Canada G1V 0A6. Tel.: + 1 418 656 2131 # 4051; fax: + 1 418 656
3723.
E-mail address: mohammed.aider@fsaa.ulaval.ca (M. Aider).

1466-8564/$ see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2012.02.002
 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849 39

3.9. Other agro-food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

1. Introduction cathode are donated to positively charged ions, such as hydrogen

Water is an important constituent of biological systems that plays a
major role in the physico-chemical properties of molecules in aqueous
solutions. From a chemical point of view, water is composed of two
hydrogen atoms bonded to one oxygen atom. All biological, bioche-
mical and physico-chemical reactions in living organisms only occur
in aqueous media (Stewart, 2009).
During the past decades, there has been an increase in scientic inter-
est worldwide in using water as a potential carrier of non-conventional
chemical reactions (Kirpichnikov, Bakhir, & Hamer, 1986). Several studies
have shown that the existing standards for water properties and aqueous
solutions are not perfect and do not take into account many parameters
that characterize the biological usefulness and activity of these solutions.
The physiological properties of water are affected by its chemical compo-
sition, degree of purication and a number of other complex physical
parameters that characterize water as a complex structured system,
particularly from an energetic point of view (Kloss, 1988). This complex
structured system is more evident when water is in a non-equilibrium
thermodynamic state (Petrushanko & Lobyshev, 2001a, 2001b, 2001c).
The activation of water and its transfer to non-equilibrium thermody-
namic states can be made via physical, chemical or biological methods.
Among these methods, the most effective are resonance and electro-
activation (Bahir, 1996).
Activated water is characterized by high physico-chemical and bi-
ological activity (Kim, Hung, & Brackett, 2000). One of the most impor-
tant parameters of water is its oxido-reduction potential. To be highly
efcient for physiological activities in humans, the oxido-reduction
potential of drinking water should be negative (Petrushanko &
Lobyshev, 2001a, 2001b, 2001c; Petrushanko & Lobyshev, 2004). The
physico-chemical properties of water and aqueous solutions can be
modied by means of electro-activation. Generally, this modication
occurs at the near-electrode interface in an electrolysis-based system.
In the presence of an electric eld, solutions become activated and can
be used in different chemical reactions and catalysis. These solutions
can also be used in biological systems and enzymology (Gnatko,
Kravets, Leschenko, & Omelchenko, 2011). The modied oxido-
reduction potential and critical pH of electro-activated solutions in a
metastable state make these solutions highly reactive and convenient
for non-conventional chemical reactions and different applications in
the food industry and biotechnology, including food safety (Izumi,
1999; Pastukhov & Morozov, 2000; Suzuki et al., 2002).
The present review summarizes the fundamental aspects of the electro-
activation of water and aqueous solutions as well as different applications of
electro-activated aqueous solutions in food and biotechnology.
2. Fundamentals of electro-activation
2.1. Electrolysis and generation of electro-activated water
The electro-activation of aqueous solutions is based on a phenom-
enon called electrolysis (Shaposhnik & Kesore, 1997). When an aque-
ous solution is subjected to an external electric eld, charged species
migrate toward the electrode of opposite charge. In electrolysis, two
phenomena of oxidation and reduction take place. In water or any
aqueous solution, a reduction reaction occurs at the negatively
charged electrode called a cathode and electrons (e ) from the
cations, to form hydrogen gas.
Reduction at the cathode : 2H 2e H2 g
Cathode reduction : 2H2 Ol 2e H2 g 2OH aq
At the positively charged electrode, called an anode, an oxidation
reaction takes place. In this case, free electrons migrate to the anode. A
simple example is the negatively charged oxygen that migrates to-
ward the anode. This migration generates oxygen gas (O2) by transfer-
ring electrons to the anode to complete the following reactions:
Anode oxidation; 2H2 OO2 g 4H 4e ;

Anode oxidation; 4OH aqO2 g 2H2 Ol 4e
When only hydrogen and oxygen molecules are present and when
there are charged species involved in the oxido-reduction reaction
that occurs in an electrolyzer, the number of hydrogen molecules pro-
duced is twice that of oxygen molecules. Under the same temperature
and pressure conditions for both gases, the volume of hydrogen gas pro-
duced is twice that of oxygen gas produced. The number of electrons
pushed through the water doubles, and the number of hydrogen and
oxygen molecules generated quadruples, respectively.
The split or decomposition of pure water into hydrogen and oxygen
that occurs at standard temperature and pressure is thermodynamically
unfavorable.
o
Anode oxidation : 2H2 OlO2 g 4H aq 4e Eox 1:23V
o
Cathode reduction : 2H aq 2e H2 g Ered 0:00V
Based on the Nernst Equation, the standard potential of the water
electrolysis cell is equal to 1.23 V at 25 C and pH 0 (molar concentration
of H+ = 1 M). The same standard potential is also 1.23 V at 25 C and pH
7 (molar concentration of H+ = 1 10 7 M). In such system, the nega-
tive voltage is an indication that the Gibbs free energy for the electrolysis
of water is > 0. This can be found using the following equation:
G nFE
where n and F are the number moles of electrons and Faraday constant,
respectively.
The reaction is impossible without adding sufcient external energy,
which is usually provided by an applied external electrical eld E.
Electro-activation is a relatively novel science, and yet the thermo-
dynamics behind the process of electro-activated water generation is
still unknown (Shirahata, Hamasaki, & Teruya, 2012). From a techni-
cal point of view, the design engineering of any electro-activation
reactor is challenging due to the maximum intensity of the electro-
physical effect on water molecules occurring predominantly at the in-
terface of the electrode/solution (close proximity to the electrode sur-
face) in an electric double layer. In this layer, the local electric eld
intensity reaches hundreds of thousands of volts per centimeter in
comparison to its mean value in the bulk solution. Because the elec-
tric double layer is very thin in weak solutions and thinner in
40 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
concentrated solutions, the electro-activation reactor must be
designed to ensure the maximum treatment of micro-volumes of
water as they are within close proximity to the electrode/solution inter-
face. The generation of an electro-activated solution is also dependent
on physical factors, such as the temperature, the solution ow rate
and the salt concentration (Hsu, 2003). Moreover, storage conditions
can also affect the physico-chemical and antibacterial properties of
electro-activated solutions (Hsu & Kao, 2004).
2.2. Thermodynamic considerations
Water molecules interact with an applied external electric eld.
The dipole moment of each water molecule and high oxygen atom
electronegativity are responsible for these interactions. Moreover,
water molecules excited by an electric eld are known to behave dif-
ferently in different environments inuenced by an electric eld. For
example, water streams and ice growth patterns can signicantly
change in the presence of an electric eld (Libbrecht & Tanusheva,
1998). A novel concept called water wire has interested scientists
due to its importance in biological systems and nanomaterial science.
Several studies have been reported on the effects of electric elds on
the collective phenomena of liquid water and large water clusters
(Vegiri, 2004). Electric elds also affect the structural and energetic
changes of water clusters (Dykstra, 1999). Water activation is the pro-
cess of water transfer into a non-equilibrium thermodynamic state,
which is accompanied by a change in water structure. Furthermore,
water acquires a resonant microcluster structure. The anomalies in
the pH and RedOx potential of electro-activated water have been
reported to result from stable, high-energy resonant water microclus-
ters that are based on co-vibrating dipoles of water molecules and
charged species at near-electrode interfaces (Shironosov &
Shironosov, 1999). In a static state, such dipole systems have been ob-
served to be unstable due to the collapse effect. However, in a dynamic
state, there is a stabilization effect. An alternating eld formed on the
base of two dipoles co-vibrating in an antiphase has a narrow frequen-
cy range called a resonance effect. Anomalous properties of electro-
activated water, such as relaxation periods, activation effects, and
cluster structures, can be partly explained by high-quality microclus-
ter structures (Shironosov, Shironosova, Minakov, & Ivanov, 2003).
The electro-activation of aqueous solutions generally occurs in an
electrolysis device (cell). From a fundamental point of view, one can
consider an electrolysis cell to be composed of a pair of electrodes
submerged into an electrolyte to ensure the conduction of ions
when the system is subjected to an external electric eld (direct or al-
ternative current). The connection between the anode and the cath-
ode is essential for the continuity of the circuit. Continuity is
ensured by the ow in an electrolyte solution of positively and nega-
tively charged ions and molecules. In such a system, the thermody-
namic electrode potential (ET) can be expressed by the Nernst
equation as follows (Fidaleo & Moresi, 2006; Prentice, 1991):
! electro-activated solution with oxidizing properties (anolyte). In
0 RG T K Si
ET E T ln ai
nF i electro-activation apparatus. On the other hand, the versatility of
where
ET0 is the standard potential at 25 C and unit activity;
ai is the activity of the ionic species I;
si the corresponding stoichiometric coefcient (>0 for prod-
ucts or b 0 for reactants);
F is the Faraday's constant (96,486 C mol 1);
RG is the universal gas constant (8.31 J mol 1 K 1);
TK is the absolute temperature (Kelvin);
n is the overall number of electrons participating in the
reaction.
In an electrolyzer cell with two electrodes submerged in NaCl so-
lution, an electrochemical cell with a thermodynamic potential differ-
ence (E ) at 25 C will be established, which can be expressed by the
following
d
equation:
Ed 1:358 0:059pH0:059 logC Cl
where c is the molar concentration of chloride ions.
Cl
In an electrolysis system, the ionic species will continuously mi-
grate to the electrode of the opposite site. To ensure the production
of electro-activated aqueous solutions with metastable properties, it
is imperative to maintain the targeted charged species in one section.
In this case, the anode and cathode sections are separated by a dia-
phragm, which can be either a monopolar or neutral membrane.
2.3. Electro-activation systems and technical requirements
The heart of the electro-activation technology of aqueous solu-
tions is electrolysis; the basic principles which were studied in the
early 19th century (Tanaka et al., 1999). The rst electrolysis cell
was described by Nicholson, Carliebe, & Cruichank, 1800 (Stoner,
Cahen, Sachyani, & Gileadi, 1982). The development of electrochemis-
try and the production/synthesis of new materials have signicantly
improved apparatus design of electrolysis systems and at this time,
the rst mention of electro-activation of aqueous solutions was
used. It was a revolutionary discovery in the eld of applied electro-
chemistry. Later, the discovery of the bactericidal effect of the
electro-activated solutions, along with the increased economic and
environmental requirements of the industry over the past 20 years,
sparked signicant growth in the number of scientic developments
and publications in this research eld. As the scientic publications
on the design of electro-activation systems increased, the need of sys-
tematical classication of the electro-activation processes and devices
is thus required.
Basically, the devices used and technical requirements of the
electro-activation systems can be distinguished by the feeding meth-
od of the treated aqueous solution. There are periodic and continuous
systems. Installations working in a continuous mode are mainly used
in the processing of industrial volumes. They are embedded in the
technological line and work for a long time. To this type of installa-
tions, one can also attribute electro-activation devices manufactured
for personal needs of the population. These units are mounted in
the drinking water supply (Morita et al., 2000). Apparatus and
electro-activation devices working in a batch mode are applied main-
ly in the laboratory conditions because of the large number of sam-
ples to be analyzed in the study of the experimental conditions.
Electro-activation systems are used to produce electro-activated solu-
tions with specic physico-chemical and biological properties. The
electro-activation device can be used to obtain predominantly
this case, the targeted solution is processed in the anodic side of the
the electro-activation technology allows obtaining electro-activated
solution with reducing properties (catholyte). In this case, the
targeted solution is obtained from the cathode side of the electro-
activation device. In such cases, the basic design of an electro-
activation installation comprises two compartments which are divid-
ed into two sections by a diaphragm: the anodic and cathodic
sections. The rst electro-activation systems used diaphragms with
a structure that is characterized by a non-selective permeability to
charged species. It was usually made of ceramics such as porcelain
and earthenware, canvas, nylon, synthetic materials such as polyvinyl
chloride and polytetrauoroethylene fabric. Later developments in
material sciences and particularly ion selective membranes allowed
to signicantly improving the performances of the electro-activation
 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
systems and apparatus. Indeed, ion selective membranes can replace
the inert diaphragm in order to easily modulate the physico-chemical
properties and compositional structure of the electro-activated solu-
tions. For example, the pH and oxidationreduction potential of the
anolyte and catholyte can be modulated by an appropriate selection
of cation or anion exchange membrane. However, for food applica-
tions, some restrictions apply and the membrane material must be
compatible with foods. However, there is electro-activation processes
in which the separation diaphragm is not required or not suitable. In
this case, the electro-activation systems are divided into systems with
and without separated anolyte and catholyte sections. Moreover, a
limited number of electro-activation systems use inert electrodes
(anode and cathode) (Yakimenko, 1977). In such devices, the polarity
of the electrodes can be reversed. Hence, on the basis of this particu-
larity, electro-activation systems can be classied as systems with
and without reversible electrodes. To the electrode material and re-
gardless of its construction, a number of general requirements are
needed (Yahagi et al., 2000): (a) The material used for the manufac-
ture of the electrode must have good electrical conductivity, high cat-
alytic activity and selectivity for the target electrochemical reaction;
(b) The electrode material should have sufcient mechanical strength
to maintain its original properties for long term use; (c) It must have
the lowest possible cost; (d) Location and shape of the electrodes
must ensure uniform distribution of the electric current density; (e)
For food applications, the electrode must be inert, insoluble in the
product when a voltage is applied.
2.4. Design of the electro-activation reactors
The electroactivated aqueous solutions are produced inside the
electrochemical reactors, i.e. electrolyzers, widely varying in design
and production process. For example, only in Japan, electro-
activation was performed, in 1999, using about 30 reactors having
different constructional characteristics (Tanaka, Fujisawa, Daimon,
Fujiwara, Tanaka, et al., 1999). In essence, it means that a certain de-
gree of electro-activation could be achieved by using various electro-
lyzers, if proper technological parameters of the process, such as
voltage, content and ow rate of electrolyte fed into the reactor, dura-
tion of the process, etc., are chosen. Notably, the studies on interrela-
tionship between constructional characteristics of the electrolyzers
and technological parameters of the process, on one side, and func-
tional properties of electroactivated solutions, on another side, are
practically absent in scientic literature. Usually, the desirable param-
eters of the production process are adjusted empirically.
Electrochemical treatment of water and aqueous solution had
been performed using the batch-type and ow-through electrolyzers
(Morita et al., 2000; Stoner et al., 1982). Contrary to the ow-
through reactors, electro-activation of immobile solutions for the
xed duration of time, e.g., 3115 min , is performed inside the cham-
bers of the batch-type reactors having volume from 1 to 15 l . Usually,
the volumetric ow rates inside the ow-through electrolyzers range
0.51.9 l/min. The voltages used for electro-activation range 9120 V
and the values of direct current passing through the electrolyzer are
set from 0.7 to 20 A. Extremely high voltage up to 1100 V was
employed for activation of distilled water; however, the electric cur-
rent during the electrolysis was about few milliamps due to a very
low electrical conductance of the treated liquid (Yahagi et al., 2000).
Quite rarely, the alternating current generated by low frequency
(~1 Hz or less) voltage signal ranged 515 V is used for electro-
activation inside the either ow-through or batch-type reactors. In
general, the reactors for electro-activation differ from each other in
terms of shape and material of their parts, such as casing, electrodes
and diaphragms, if latter are present. The coaxial cylindrical ow-
through electrolyzers represent one of the most common designs
(Rossi-Fedele, Dogramaci, Steier, & de Figueiredo, 2011). Such devices
could be easy assembled in a compact electrochemical module
41
comprising up to eight electrolyzers. Flow-through parallel-plate
electrolyzers use plates as cathode and anode. In some instances,
multiple and bipolar electrodes were employed. A peculiar reactor is
described in the work of (Sergunina, 1968). The electrolyzer com-
prised two columns lled with the grains of magnetite or graphite,
each of which could virtually be considered as a bipolar electrode.
The electrolyzer consisted of 2 columns linked in the lower parts by
a connector. Chemical stability of electrodes is important for the qual-
ity of produced solutions and for longevity of the reactors. Platinum,
platinum with addition of iridium and rhodium oxides, platinum-
plated titanium, titanium coated with active RuO2 layer, titanium
coated with Fe3O4 , and graphite have been used for manufacturing
of anodes. Titanium, platinum or graphite are used as cathodes
(Lovtsevich & Sergunina, 1968). A random-oriented graphite-epoxy
matrix composite material was also employed to fabricate both elec-
trodes. Electrochemical activation of aqueous solution had been per-
formed using both the batch-type and ow-through reactors with
or without porous diaphragm (Shimada, Ito, & Murai, 2000). The dia-
phragms allow for production of electroactivated solutions with spe-
cic functional properties, such as acidic (anolytes), alkaline
(catholytes) or nearly neutral solutions. The latter could be obtained
by the controlled mixing of anolytes and catholytes. The main re-
quirements for the diaphragms are high porosity, hydrophilicity and
low electric resistance. A high-quality porous ceramic diaphragms
were used in (Middleton, Chadwick, Sanderson, & Gaya, 2000).
Electro-conductive cationic diaphragm made of Naon-450 and pro-
prietary ion-exchange membranes were used in (Morita et al.,
2000) along with low-conductive polyester and polyethylene dia-
phragms (Tanaka et al., 1999). The feeding aqueous solutions sub-
jected to electro-activation vary in terms of mineralization from
distilled water and tap water to electrolytes containing from 0.1 to
120 g/l NaCl (Inoue et al., 1997). Produced electroactivated anolytes
are characterized by pH ranging 2.3-6.5, positive redox potential of
10001200 mV, and by about 30300 ppm of dissolved chlorine
(Tanaka et al., 1996). At the same time, the catholyte is characterized
by pH 6.2 and negative redox potential 329 mV and by pH 8.8 and
negative redox potential 390 mV, if produced by electrolysis of dis-
tilled water or 10 4 M NaCl, respectively (Petrushanko & Lobyshev,
2001a, 2001b, 2001c).
2.5. Raman scattering of light by electro-activated water
According to the reports on electro-activated aqueous solutions,
these solutions were observed to be transformed to a metastable
state after excitation by an external electric eld. The reactivity of
electro-activated solutions was observed to signicantly increase
under this state compared to a normal state. Researchers explain
this phenomenon using the vibrational spectrum of water
(Antonchenko, Davydov, & Iliin, 1991). In relation to the vibrational
spectrum of water, Pastukhov and Morozov (2000) studied the
Raman spectra of electro-activated water. To elucidate special fea-
tures of the vibrational spectrum of electro-activated water, the au-
thors investigated the vibrational spectrum of chemically acidied
or alkalinized water (analogues of electro-activated water based on
pH properties). In this study (Pastukhov & Morozov, 2000), water
was activated by electrolysis with the addition of 0.4 10 3 M sodi-
um hydrosulfate to allow the passage of the electric eld without dis-
sociating water molecules. Molecular oxygen and hydrogen were
released from the anode and cathode regions, respectively. For
Raman spectral studies, water samples were taken from the near-
anode (anolyte) and near-cathode (catholyte) zones. The pH values
of these water samples were 4.0 and 10.0 for the anolyte and catho-
lyte, respectively. To compare the spectra of electro-activated water
samples with those of analogues, the electro-activated anolyte was
simulated by the addition of sulphuric acid to pure water. Additional-
ly, the catholyte was simulated by the addition of sodium hydroxide.
42 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
Rectangular single-pass quartz cells were used to study the Raman
spectra of electro-activated and control water samples (acidied
and alkalinized). The Raman spectra were excited by a 200-mW
488-nm line of an argon laser and recorded at an angle of 90 to the
laser beam. The spectra were recorded in the 504000-cm 1 spectral
region at 20 C.
The results demonstrated that Raman spectra between 700 and
2700 cm 1 of the electro-activated water taken in the near-anode
(anolyte) and near-cathode (catholyte) regions were signicantly dif-
ferent from those of chemically acidied and alkalinized water
(Fig. 1) (Pastukhov & Morozov, 2000). The Raman spectra of the ano-
lyte and catholyte were intense in this region, which was absent in
the spectra of chemically acidied and alkalinized water samples.
The authors specied that the addition of sodium hydrosulfate to
water resulted in vibrational bands within the region of
10001500 cm 1 of the Raman spectrum. The scattering intensity
was also demonstrated to be time-dependent. After 24 h, the scatter-
ing intensity of the near-cathode sample (catholyte) decreased signif-
icantly. However, the decrease in the scattering intensity of the near-
anode (anolyte) solution was not drastic after 24 h. The authors
mixed the anolyte and catholyte in equal volumes and observed a de-
creased scattering intensity in the region of 7002700 cm 1 com-
pared to the initial intensities of individual electro-activated water
samples. Usually Raman spectra of water and aqueous solutions are
interpreted based on the time-dependence of the changes in the
strength of the hydrogen bond (Cureton & Goodall, 1983; Moskovits
& Michaelian, 1978). The broad Raman band of the near-anode (ano-
lyte) and near-cathode (catholyte) water samples in the region of
7002700 cm 1 is related to the presence of excess hydrates of H +
or OH ions. These ions result from the electrolysis procedure,
which produces and accumulates H + and OH ions in the anolyte
and catholyte, respectively. These ions are responsible for the acidic
and alkaline properties of electro-activated water solutions. More-
over, the infrared absorption spectra of concentrated acid and alka-
line solutions have been reported to exhibit bands similar to the
vibrational bands observed in Raman spectra of the anolyte and cath-
olyte (Pastukhov & Morozov, 2000). This nding suggests that
electro-activated solutions may possess properties similar to those
of concentrated acids and bases, even if their mineral content is
Fig. 1. Raman spectra of (1) pure water, (2) 0.4 10 3 M sodium hydrosulfate in
water, (3) near-cathode solution (catholyte), (4) near-anode solution (anolyte),
(5) chemically alkalinized water (catholyte analogue), and (6) chemically acidied
water (anolyte analogue). Adapted from (Pastukhov & Morozov, 2000).
very low. The following two phenomena were reported in electro-
activated solutions: high polarizability in AH + A or BH B - hy-
drogen bonds and a continuous distribution of the energy of protons
residing in complex A (or B) interacting at different strengths
(Janoschek, Weidemann, Pfeiffer, & Zundel, 1972). Excess hydroxyl
ions in the near-cathode solution (catholyte) can participate in the
formation of a more symmetric and weak hydrogen bond
(OHO) . Simultaneously, in the near-anode solution (anolyte),
a strong hydrogen bond was observed in close intensities of the broad
Raman and valence bands examined. This phenomenon was used to
explain the higher scattering in the anolyte solution compared to
that of the catholyte. In the near-anode solution, which is considered
a strong acid media, the continuous absorption in the infrared spec-
trum is caused by H5O2 groupings. In the near-cathode solution, the
continuous absorption in the infrared spectrum is caused by H3O 2
groupings (Janoschek et al., 1972). Furthermore, the electrochemical
dissociation of water molecules at the near-electrode interface results
in the formation of unstable complexes, such as (OO), (OO) +, and
(HH) +. These complexes are considered as intermediates and their
vibrational modes can contribute to scattering in the corresponding
spectral regions. The addition of a small amount of sodium hydrosul-
fate did not affect the Raman spectra of electro-activated solutions.
This observation supports the metastable state, in which the near-
electrode solution is located, as the cause of the activity of these solu-
tions (Delimarskii, 1982).
3. Applications of electro-activated solutions
3.1. Activation of antioxidant enzymes
The effects of electro-activated solutions on antioxidant enzymes
have been reported (Podkolzin et al., 2001). Podkolzin et al. studied
the effects of electrochemically activated solutions on catalase, perox-
idase, and superoxide dismutase activities in an animal model system
(Fig. 2). They used peripheral blood erythrocytes from healthy Chin-
chilla rabbits. According to the protocol used, the erythrocytes were
centrifuged and lysed with 5 mM TrisHCl buffer at pH 7.8. For hemo-
globin precipitation, 0.25 ml of 96% ethanol and 0.15 ml of chloroform
were added to 1 ml of lysate, mixed on ice for 15 min, and centrifuged
at 10,000 g for 15 min at 4 C. Catalase activity was then measured by
the reaction of hydrogen peroxide (H2O2) with ammonium molyb-
date. Peroxidase activity was measured by the reaction with indigo
carmine, and superoxide dismutase activity was determined by the
inhibition of hematocrit (HCT) reduction with superoxide anions
generated in the reaction of NADPH with phenazine methosulfate.
The effects of electrochemically activated solutions on the activity of
extra-pure human recombinant superoxide dismutase was obtained
by passing a NaCl solution (12 g/l) through an Izumrud device
equipped with 2 reactor compartments. This procedure allowed the
oxido-reduction potential (ORP) of the electrochemically activated
solutions to vary from +200 mV to 70 mV. The ORP was continu-
ously measured using a pH meter with platinum electrodes under
standard conditions. The authors showed that electrochemically acti-
vated systems normalized the activity of antioxidant enzymes such as
catalase, peroxidase, and superoxide dismutase. Additionally, the
baseline activity of the targeted antioxidant enzymes was observed
to vary considerably in humans and animals. The effect of the electro-
chemically activated systems was characterized by a negative oxida-
tionreduction potential, which was probably related to a training
effect of excess electrons. Podkolzin et al. (2001) showed that prein-
cubation of erythrocyte lysate with various concentrations of the
electro-activated solution for 5, 10, and 20 min caused a bi-phasic re-
sponse of rapid activation of the enzyme followed by normalization
or slight inhibition of its activity. The time- and dose-dependent ef-
fects of the electro-activated solution were also observed at various
oxido-reduction potentials at constant reaction times. These effects
 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
Fig. 2. Effects of electrochemically activated systems (ECAS) on catalase (a), peroxidase
(b), and superoxide dismutase (c) activities in rabbit erythrocytes. Incubation for 5 (1),
10 (2), and 15 min (3). Adapted from Podkolzin et al. (2001).
showed that higher concentrations of electro-activated solutions dur-
ing preincubation with erythrocytes resulted in shorter preincubation
times for the maximum activation of rabbit peroxidase and superox-
ide dismutase. Electro-activated solutions have also been demon-
strated to produce similar effects on human recombinant
superoxide dismutase. The authors interpreted these changes by the
ability of electro-activated solutions to generate superoxide radicals,
producing moderate training effects on antioxidant enzymes. Indeed,
negatively charged electro-activated solutions contain excess elec-
trons formed after electrochemical activation, which contribute to
the generation of superoxide anions. However, components of the
main reaction were also reported to be inactivated at high negative
oxido-reduction potential values (170 mV). After a 5-min incuba-
tion with the electro-activated solution, the reaction of NADPH with
phenazine methosulfate and the HCT reduction were determined to
be inhibited by 2225%. Thus, the effects of ECAS on antioxidant en-
zymes depend on ORP, electro-activated solution concentrations,
43
and the time of preincubation with erythrocytes. After maximum en-
zyme activation occurs, the activating effect of electro-activated solu-
tions becomes inhibitory. These changes are probably associated with
the ability of electro-activated solutions to generate superoxide radi-
cals, which, in low concentrations, activate antioxidant enzymes. Sim-
ilar observations have been reported (Khasanov, 1986; Makats,
Podkolzin, & Dontsov, 1996; Podkolzin et al., 1997).
3.2. Baking wheat bread with electro-activated aqueous solutions
Nabok and Plutahin (2009) explored the possibility of making
wheat bread with electro-activated water in the laboratory of bio-
technology, biochemistry and biophysics at the Kuban State Agrarian
University, Krasnodar, Russia. The authors hypothesized that bread
quality depends upon the quality of the ingredients and water.
According to Nabok and Plutahin (2005), water quality is a key ele-
ment in bread making in which, until now, the existing water quality
estimation standards are not perfect and do not consider a number of
important parameters that affect the biological function and potency
of water. The oxido-reduction potential, which must be negative for
drinking water, was highlighted as the most important water criteria.
An electro-activation device was developed by Nabok and Plutahin
(2005) to produce electro-activated drinking water and aqueous so-
lutions with high quality resonance microcluster structures. The au-
thors considered the effect of such solutions on biological processes
in live cells by investigating their activating or inhibitory inuences
on the reactivation of baking yeast and preparation of fermented
dough. Water was activated using a non-contact method. The activa-
tion procedure was carried out in a tank lled with electrolyte solu-
tion in an unforced circulation mode. Using this technology, anolyte,
catholyte, drinkable catholyte and drinkable anolyte solutions were
produced. The obtained electro-activated solutions were used to acti-
vate yeast used in wheat dough making. The following procedure was
used to activate dry yeast: a 5% (w/v) sugar solution was prepared in
activated aqueous solutions at 30 C. The intensity of yeast activation
was evaluated according to the volume of the solution, which rose
proportionally to the amount of carbon dioxide produced by the
yeast. The fermentation process occurred at 30 2 C and relative hu-
midity of 7580%. Baking was carried out at 230 5 C over 20 min.
Five types of electro-activated water samples were used to make
bread. Four samples were produced by a contact method and one
by a non-contact activation method. All water samples were evaluat-
ed for salinity, pH and RedOx potential (ORP). The control water
exhibited an oxido-reduction potential of 220 mV, mineralization of
470 ppm, and a pH of 7.94. Non-contact activation yielded water
with an ORP of 110 mV without signicantly changing the pH and
mineralization compared to the water control. The drinking catholyte
and anolyte exhibited ORP values of 3.8 and 67 mV, respectively.
However, the cathode and anolyte obtained at the anode sections
were characterized by ORP values of 767 and 905 mV, respectively.
The mineralization of these solutions was 8000 and 7800 ppm, re-
spectively. The yeast activated in the catholyte solution showed the
highest activity followed by the yeast activated in water generated
from a non-contact method. The effect of the drinkable catholyte so-
lution on yeast activity was lower compared to the abovementioned
results. Despite its active chlorine content, the anolyte solution was
not observed to completely inhibit the process of fermentation. The
wheat bread obtained with yeast activated in electro-activated aque-
ous solutions is shown in Table 1. Based on the results of this study,
the activation time used for yeast activation using catholyte solution
was determined to result in the loss of yeast activity. Thus, to obtain
high-quality bread, the activation time needs to be controlled (opti-
mized). The anolyte ORP was 905 mV with the presence of active
chlorine, resulting in yeast activation. The biological value of the pro-
teins in the bread was evaluated, and the results suggested that the
proteins exhibited the highest biological values when used with
44 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
Table 1
Bread physico-chemical characteristics baked with different types of electro-activated
water (adapted from Nabok & Plutahin, 2005).
Type of water Bread Mass Humidity, Acidity Porosity,
volume, ml volume, g/ml % %
Tap water (control) 340 0.405 41.0 1.2
Non-contact 410 0.371 42.0 1.5
activated water
Drinking catholyte 390 0.378 40.0 1.2
Drinking anolyte 410 0.379 39.5 1.4
Catholyte 300 0.459 39.5 1.5
Anolyte 460 0.332 41.5 1.4
electro-activated water obtained by a non-contact method. The au-
thors (Nabok & Plutahin, 2005) reported that both the activated
water obtained from a non-contact method and catholyte exhibited
negative ORP values. They argued that this was a result of the faster
fermentation of yeast taking place in these solutions. The volume of
both bread samples was signicantly higher than that of the control
sample. The bread baked with a drinkable anolyte solution was char-
acterized as having the highest volume. However, a comparison be-
tween the six bread samples demonstrated that the bread made
with water resulting from non-contaction had the best qualitative
indices.
3.3. Yeast inactivation
Over the past decade, critical analyses of nutrition and diet have
indicated a tendency of consumers gravitating toward fresh-like
food products (Guillou, Besnard, El Murr, & Federighi, 2003). From
an engineering perspective, there has been a noticeable interest in
the investigation of the possible uses of non-thermal processes for
food preservation and safety. Among the new technological advances,
the use of electro-activated aqueous solutions seems to be highly
promising. Guillou and El Murr (2002) reported a method for the
use of low intensity electric elds for microbial (yeast) inactivation
(Guillou and El Murr (2002); Guillou et al., 2003) for the stabilization
of semi-sweet white wines. Electro-activation was used as a substi-
tute for the addition of SO2 in wine stabilization (Godet, Poulard,
Guillou, & El Murr, 1999). In the same context, Guillou et al. (2003)
studied the injury and recovery of Saccharomyces cerevisiae after elec-
trolysis. The electro-activation treatment by means of electrolysis was
conducted on microbial suspensions of 2 10 6 cfu/ml) in 0.1 mol/l
phosphate buffer at pH 7.1. The yeast suspension was submitted to
an electro-activation treatment in an electrolysis device (Guillou &
El Murr, 2002). This treatment was conducted directly under a con-
stant electric eld having an amperage of 0.5 A and completed in
3 h. According to the information reported by the authors (Table 2),
the experiment was conducted at 20 C. After 3 h of treatment
under an electric eld with a constant amperage of 0.5 A, the yeast
cell viability was 4.2 0.60% and 2.5 0.98% according to FUN-1
and the plate counts method, respectively. After treatment in the
electrolysis device, the yeast cells were examined to determine
whether they could be resuscitated at 4 and 20 C. However, no resus-
citation was observed. The authors reported that recovery from po-
tential lethal damage is a rare phenomenon at 4 C and also
depends on the severity of the injury (Graumlich & Stevenson,
1978; Guillou et al., 2003; Schenberg-Frascino, 1972). Furthermore,
the authors conrmed the lethal effect of electrolysis (Guillou et al.,
2003). However, the number of viable cells depended on the method
used to evaluate the number of viable cells. The results demonstrated
that the colony count method gave lower viability than other
methods, such as Live/Dead Yeast Viability Kit from Molecular Probes
(Interchim, Montlucon, France). However, they also observed an in-
crease in the viability counts of 0.6 Log10 during storage at 20 C.
They rationalized this observation by the ability to a repair the
Table 2
Inuence of storage at 20 C on yeast viability assessed by plate counts and ATP deter-
mination (Adapted from Guillou et al., 2003).
Yeast suspension Description After electrolysis After storage at
20 C for 5 days
74.0 Log10 ATP Log10 N Log10 ATP Log10 N
76.5 (fg ml 1) (cfu ml 1) (fg ml1) (cfu ml1)
Control Mean 8.18 6.04 8.32 5.06
73.0
S.D. 0.99 0.02 0.09 0.03
73.6
Electrolysis-a treated Mean 6.98 4.61 8.69 5.18
74.3
S.D. 0.35 0.28 0.19 0.20
82.0
a
Electrolysis conditions: 0.5 A (direct current) for 3 h in 0.1 mol l 1 phosphate buff-
er (pH 7.1).
damaged cells. The authors reported that the lethal effect of electrol-
ysis might be overestimated by 0.6Log10. They suggested that more
studies were necessary to understand the effect of electro-activation
(electrolysis) on different microorganisms. Based on this study, one
should not exclude the industrial application of electro-activation or
electrolysis for food safety (Guillou et al., 2003).
3.4. Electrochemical inactivation of bacteria, viruses and bacteriophages
The inactivation of bacteria and yeast cells by electrochemical
means has been reported in several studies (Bari, Sabina, lsobe,
Uemura, & Isshiki, 2003; Gaskova, Sigler, Janderova, & Plasek, 1996;
Grahl & Markl, 1996; Tokuda & Nakanishi, 1995; Velizarov, 1999).
Electric elds have been shown as effective disinfecting agents of
drinking water and vegetables (Beuchat et al., 2001) that can reduce
the numbers of microorganisms in food products. Disinfection of
drinking water is a topic of concern, and research has been carried
out to identify potential substitution methods of conventional
chlorine-containing disinfectants (Matsunaga et al., 1992; Patermarakis
& Fountoukidis, 1990). Drees et al. reported the use of electro-activation
to inactivate bacteria and bacteriophages (Drees, Abbaszadegan, &
Maier, 2003). There has been limited research regarding the effectiveness
of electro-activation on the inactivation of viruses. Drees et al. compared
the ability of bacteria and bacteriophages to survive when exposed to a di-
rect electric eld in an electrochemical cell (Drees et al., 2003). Bacteria
were subjected to irreversible membrane permeabilization and the direct
oxidation of cellular/viral constituents by an electric current. The disinfec-
tion was caused by electrochemically generated oxidants. Suspensions of
Escherichia coli and Pseudomonas aeruginosa as well as the bacteriophages
MS2 and PRD1 at high (1106 CFU or PFU/ml) and low (1103 CFU or
PFU/ml) population densities were used (Drees et al., 2003). Cultures
were exposed to an electric current intensity from 25 to 350 mA. More-
over, the electric eld was applied as 5 s pulses. After treatment, the bac-
teria were counted. The authors observed that (Drees et al., 2003) post-
exposure plaque counts of bacteriophages were proportionally higher
than bacterial culturable counts under corresponding experimental con-
ditions. E. coli and the MS2 bacteriophage were studied at both high and
low population densities. The electro-activation cell was used under the
following conditions: an electric eld intensity of 5 mA was applied
over a treatment period of 20 min. The results showed that the inactiva-
tion rate for E. coli was 2.14.3 times greater than that of the MS2 bacte-
riophage. Moreover, the population density effect was signicant. Both
bacteria and bacteriophages were more resistant to direct electric eld
at higher population densities. The authors suggested that reduced inacti-
vation within electrochemical cells was observed when the glutathione
reducing agent was used. The authors also suggested that the results
may be used in food technologies to reduce the numbers of microbes in
food and water. The bacteriophages were more resistant than the bacteria
tested by Drees et al. (2003).
Electro-activation of aqueous solutions is a physical process that
involves different electrochemical reactions at electrode/solution
 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
interfaces. The antimicrobial or antiviral effect of these solutions re-
sults from different chemical oxidants that are generated when an
electric eld is applied to an aqueous solution. In suspensions con-
taining microbes, the immersed electrodes create an electrolysis phe-
nomenon at the electrodes, generating a variety of oxidants (Liu,
Brown, and Elliot, 1997). As a result, the solution is saturated with ac-
tive oxygen and other oxidants, including hydrogen peroxide and
ozone. In the presence of chloride ions, free chlorine and chlorine di-
oxide are also formed. Therefore, the antimicrobial effects of electro-
activated solutions are a result of the action of such oxidants (Davis,
Shirtliff, Trieff, Hoskins, & Warren, 1994). However, due to a lack of
research on the antibacterial effects of electro-activated solutions,
most of the current research in this eld supports the hypothesis
that antimicrobial agents and electric currents act synergistically to
inactivate bacteria and viruses (Costerton, Ellis, Lam, Johnson, &
Khoury, 1994; Khoury, Lam, Ellis, & Costerton, 1992).
Anolytes, which are generated at the anode/solution interface, are
well-known oxidizing agents that produce a mixture of free radicals
that have antimicrobial effects. From a physico-chemical point of
view, as a result of anode electrochemical activation, the surface ten-
sion of the electro-activated aqueous solution decreases, electric con-
ductivity increases, and water structure changes. The bacterial cell
membrane provides an osmotic barrier for the cell and catalyses the
active transport of substances into the cell. Irreversible modications
of the transmembrane potential caused by the action of electron
donors/acceptors could be associated with the powerful electro-
osmotic processes accompanied by water diffusion against oxida-
tionreduction gradients, resulting in membrane rupture and an out-
ow of bacterial cell contents. Moreover, the bacterial membrane cell
has been established as being electrically charged (Wilson, Wade,
Holman, & Champlin, 2001). Thus, excess anions present in the ano-
lyte solution can react with the cell membrane and modify solution
transport or availability. This phenomenon can disrupt the vital func-
tions of the bacterial cell (Mozes et al., 1987). Moreover, solute trans-
port is largely dependent on the electrostatic interactions and small
charged molecules that are transported across the cell membrane by
means of an electro-chemical gradient (Veld, Driessen, & Konings,
1993). Thus, any signicant change in the oxidationreduction poten-
tial of the immediate medium of the bacterial cell can cause lethal
consequences for the cell. Electro-activation of chlorine-containing
aqueous solutions generates hypochlorous acid, which is more active
than the sodium hypochlorite generated by dissolving this salt in
water.
The effect of electro-activated water on microorganisms is com-
plex. However, some explanations may be made based on generated
active species, such as hydroxyl ions and hypochlorite. Other explana-
tions are based on the fact that electro-activation is an electrochemi-
cal process based on electrolysis, including the excitation of electrons
at low current intensities to their destruction at high current intensi-
ties. The efcacy of electro-activated water against microorganisms is
also based on its oxidative power, which allows electro-activated
water to act as an electron acceptor as a result of the high electron de-
ciency in the water cluster.
3.5. Biolms prevention/treatment
Biolms are a serious concern for the modern food industry, as
they are potential sources of contamination of food products in pro-
cessing plants. One of the most important particularities of the bio-
lms is that they frequently support sanitizer treatments. The
combined effect of alkaline and acidic electro-activated (electrolyzed)
water in the inactivation of Listeria monocytogenes biolms on stain-
less steel surfaces was investigated. Biolms were grown on rectan-
gular stainless steel in a 1/10 dilution of tryptic soy broth that
contained a ve-strain mixture of L. monocytogenes for 48 h at 25 C.
The coupons with biolms were then treated with acidic electro-
45
activated water, alkaline electro-activated water or alkaline electro-
activated water followed by acidic electro-activated water produced
by applying an electric eld intensity of 14 and 20 A for 30, 60, and
120 s. Alkaline electro-activated water alone was not observed to sig-
nicantly reduce L. monocytogenes biolms when compared with the
control. The authors also showed that treatment with acidic electro-
activated water for 30 to 120 s reduced the number of viable bacterial
populations in biolms by 4.3 to 5.2 log CFU per coupon. The com-
bined treatment of alkaline electro-activated water followed by acidic
electro-activated water produced an additional 0.3- to 1.2-log CFU
per coupon reduction. The population of L. monocytogenes reduced
by treatments with acidic electro-activated water increased signi-
cantly with an increase in the time of exposure. No signicant differ-
ences occurred between treatments with electro-activated water
produced with an electric eld intensity of 14 and 20 A, respectively.
The authors concluded that the obtained results suggest that alkaline
and acidic electro-activated water can be used together to achieve
better inactivation of biolms than when applied separately
(Ayebah, Hung, & Frank, 2005; Ayebah, Hung, Kim, & Frank, 2006).
The results of this study corroborate those of the inactivation of Lis-
teria monocytogenes biolms by electrolyzed oxidizing water (Kim,
Hung, Brackett, & Frank, 2001).
3.6. Poultry spraying and chilling
In the poultry industry, the chlorination of water is an established
procedure that has been used to ensure product safety, reduce micro-
bial contamination, and cross contamination of chicken carcasses
(Sanders & Blackshear, 1971). However, several research works dem-
onstrated that chlorine efcacy was a concentration-dependent prop-
erty. Water with a chlorine concentration of 5 to 200 ppm has been
demonstrated to have a weak effect on Salmonella, which is usually
reduced in chicken carcasses by not more than 1 log CFU/carcass.
However, chlorine containing water was observed to enhance the
prevention of cross-contamination (James, Brewer, Prucha,
Williams, & Parham, 1992). In relation to the microbial safety of the
poultry industry, the antibacterial efcacy of electrochemically acti-
vated aqueous solutions was studied, focusing on poultry spraying
and chilling (Yang, Li, & Slavik, 1999). The antibacterial efcacy of
an electrochemically activated solution against Salmonella typhimur-
ium on chicken carcasses at 20 C was examined. The electro-
activated solution was sprayed on chicken carcasses under a pressure
of 413 kPa for 17 s. Then, the treated product was chilled at 48 C for
45 min. Electro-activated aqueous solutions were generated by an
electrochemical reactor consisting of 8 electrochemical cells placed
in a parallel mode (Bakhir et al., 1997). Tap water was mineralized
by means of 15% (w/v) NaCl to obtain a 3 g/l NaCl solution, which o-
wed into the cathode and catalyst chamber, respectively. A direct
electric eld current at a voltage of 30 V was applied to the electro-
chemical device. Under these conditions, an electric eld intensity
of 9 A was obtained. The total concentration of oxidants in the
electro-activated solution was 300 ppm of free chlorine at pH 6.5,
which was controlled by drawing the catholyte through the bypass
of the electrochemical device. Different dilutions were made with
this electro-activated water and used for the spraying and chilling
of chicken carcasses based on conventional chlorine treatments in
poultry processing (Thomson, Cox, & Bailey, 1976). An electro-
activated solution with 50 ppm of oxidants expressed as free chlorine
was observed to reduce Salmonella on carcasses by 1.39 log10 CFU/
carcass. Additionally, tap water with 50 ppm of free chlorine and hy-
pochlorite reduced Salmonella on chicken carcasses by 0.86 and 0.87
log10 CFU/carcass, respectively. The authors also reported that addi-
tional chilling of the carcasses in an iced electro-activated solution
with 50 ppm of chlorine did not reduce Salmonella. The authors sug-
gested that more studies be performed to understand whether the
electro-activation treatment affects the sensory attributes and
46 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
physico-chemical properties of chicken meat obtained from carcasses
treated with electro-activated water (Yang et al., 1999). Fabrizio,
Sharma, Demirici, and Cutter (2002) reported that food borne patho-
gens in cell suspensions or attached to surfaces can be reduced by
treatment with electro-activated (electrolyzed) oxidizing water.
They used this sanitizing approach against pathogens associated
with poultry. Acidic electro-activated water with 20 to 50 ppm chlo-
rine and an oxidationreduction potential of 1150 mV at pH 2.6,
basic electro-activated water with an oxidationreduction potential
of 795 mV, chlorine solution, ozonated water, acetic acid and triso-
dium phosphate at pH 11.6 were each applied to broiler carcasses in-
oculated with Salmonella Typhimurium and submerged at 4 C for
45 min. The remaining bacterial populations were determined and
compared at days 0 and 7 of aerobic, refrigerated storage. At day 0,
submersion in trisodium phosphate and acetic acid reduced Salmonel-
la typhimurium by 1.41 log10, whereas acidic electro-activated water
reduced Salmonella typhimurium by 0.86 log10. After 7 days of stor-
age, acidic electro-activated water, ozone, and acetic acid reduced Sal-
monella typhimurium with detection only after selective enrichment.
The authors reported that spray-washing treatments with any of the
used solutions did not reduce Salmonella typhimurium at day 0.
After 7 days of storage, acetic acid and acidic electro-activated water
reduced Salmonella typhimurium by 2.31 and 1.06 log10, respectively.
The treatment of poultry carcasses with acidic electro-activated solu-
tions was reported as less economically and environmentally effec-
tive. The authors concluded that electro-activated water can reduce
Salmonella typhimurium on poultry surfaces following extended re-
frigerated storage.
3.7. Inactivation of endospore-forming bacteria and toxins
In the food industry, particularly in canned foods, the inactivation
of bacterial spores is important for ensuring product safety. Products
with a long shelf life are sterilized to ensure their stability. Gram-
positive bacteria belonging to the genera Bacillus, Clostridium, and
Sporosarcina grow easily and divide in nutrient-rich media. However,
under conditions where one or more essential nutrient is decient or
limiting, the microorganisms initiate the process of sporulation,
resulting in the formation of dormant spores (Chander, Setlow, &
Setlow, 1998; Setlow, 1994). Under favorable conditions, these spores
can grow, alter the food products, and form toxins. This ability results
from these dormant spores lack ATP, NADH, and energy reserves,
such as 3-phosphoglyceric acid (Chander et al., 1998; Loshon &
Setlow, 1993). Traditionally, aqueous and gaseous decontaminants
and sterilants have been used against endospore-forming microor-
ganisms. However, it is well established that most of these deconta-
minants are costly. Considering this fact, alternative tools and
technologies for the endospore-forming bacteria inactivation have
been explored. Bacillus anthracis spore inactivation using an electro-
chemically activated solution was reported (Rogers, Ducatte, Choi, &
Early, 2006) and suggested as a potential alternative. This solution,
known to have oxidative potential, is obtained by the electrolysis of
an aqueous solution of sodium chloride or any other salt. The oxida-
tive properties of this solution are due to the reaction involved at
the near-anode interface. The solution electro-activation is carried
out by the electrolysis of saline solution, which is circulated through
an electrolyzer containing both an anode and a cathode. These elec-
trodes are generally separated by a diaphragm made of a membrane.
At the anode section, an anolyte solution with oxidative properties
was produced, whereas at the near-cathode interface an alkalinized
solution was generated. Generally, the anolyte is characterized by a
high oxidation potential within the range of +400 to +1200 mV.
The pH of the anolyte is acidic and may vary to values near 1.5-3.
This anolyte is typically used as an antimicrobial agent. The catholyte
is characterized by a reduction potential with values between of 80
to 900 mV and pH 712. It can also be used as a cleaning agent
(Marais, 2000; Marais & Brozel, 1999; Marais & Williams, 2001;
Rogers et al., 2006; Solovyeva & Dummer, 2000). In the study of
Rogers et al. (2006), ve different electro-activated solutions with
different concentrations of free chlorine and oxidative-reduction po-
tentials were generated using the electrolysis device. Bacillus anthra-
cis and spores were suspended in various electro-activated saline
solutions for 30 min. Subsequently, the decontamination efcacy of
this procedure was analyzed. The authors used a 5% high-test hypo-
chlorite calcium solution as a positive control. The electro-activated
solutions were characterized by concentrations of active, free chlo-
rine ranging from 305 to 464 ppm. The mean oxido-reduction poten-
tials of the anolyte (oxidant) solutions ranged from +826
to +1000 mV. Treatments with all electro-activated saline sand 5%
high-test hypochlorite calcium solutions resulted in reductions by
more than 7 log in both Bacillus anthracis and spores. Based on
these results, the authors concluded that the electro-activated solu-
tion, containing the minimum active, free chlorine concentration of
300 ppm and having a RedOx potential of + 800 mV, is able to inacti-
vate the Bacillus anthracis spores in suspension. This effect was similar
to that of the 5% high-test hypochlorite calcium solution. The ndings
of this study may be used in the food industry to ensure product safe-
ty in thermally processed foods.
3.8. Inactivation of Staphylococcal enterotoxin-A
Food safety is a major priority of the food industry and food regu-
latory agencies. Food-borne diseases can be divided into food infec-
tion and food poisoning. Food poisoning is caused by consuming
foods that contain toxins, whereas food borne infections are caused
by infectious pathogens in food. In the case of food poisoning, toxins
can be produced by bacteria or occur in food, such as in some mush-
rooms. Toxins can also be contaminants and directly affect biological
reactions taking place in the host. At sufciently high concentrations,
the effects of toxins are acute and take place over the course of a few
hours after consumption of contaminated food. The symptoms associ-
ated with food poisoning can include nausea and vomiting. In some
cases, the effects are lethal. The toxins can have various origins. In
food poisoning, the two most well known bacterial toxins are pro-
duced by Staphylococcus aureus and Clostridium botulinum. However,
some toxins, such as mycotoxins, have been determined to have
long-term effects, even at small concentrations. The other problem
related to toxins involved in food poisoning is their resistance to
heat; thus, they cannot be easily eliminated by cooking. Staphylococ-
cal enterotoxins are proteins with molecular weights between 27 and
30 kDa. To date, at least eight staphylococcal enterotoxins have been
reported (Su & Wong, 1995; Suzuki et al., 2002), the most dangerous
and resistant being Staphylococcal enterotoxin-A. A total mass below
200 ng of Staphylococcal enterotoxin-A is sufcient to cause food poi-
soning in humans. Toxin concentrations of 0.4 to 0.8 ng/ml can cause
illness in a short period of 35 h after the intake of contaminated
food. The Staphylococcal enterotoxin-A is thermo-, acid- and alkali-
resistant. Thus, it is very important to inactivate Staphylococcal
enterotoxin-A (Huang, Hughes, Bergdoll, & Schantz, 1987; Suzuki et
al., 2002). Suzuki et al. (2002) used an electro-activated aqueous so-
lution to inactivate Staphylococcal enterotoxin-A. The generation of
the electro-activated solution was performed in an electrolyzer. This
device permitted the production of near-anode and near-cathode
NaCl solutions. The anode and cathode sections were separated by a
diaphragm. The electrolysis of the NaCl solution was conducted for
12 min at ambient temperature. A feed solution of 0.1% NaCl was
used and prepared by dissolving salt in deionized water. The electro-
lyzer was submitted to a direct electric current at a voltage between 9
and 11 V. After electrolysis, strong acidic solutions at pH 2.52.8 and
available chlorine of 36.3 ppm were produced. This chlorine concen-
tration was estimated to be equivalent to 0.67 mM HOCl. The anolyte
solution produced at the near-anode interface had an oxidation/
 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
reduction potential of + 1180 mV. Simultaneously, a strong alkaline
solution at pH 11.612.0 with an oxidation/reduction potential
below 880 mV was produced in the cathode compartment. The dis-
infectant properties of the electrolyzed NaCl (anolyte and catholyte)
solutions were tested immediately after they were produced because
electro-activated solutions are generally not stable and because the
bactericide effect is the highest within the rst hours after the solu-
tions are made. Fixed quantities of Staphylococcal enterotoxin-A
were mixed with different ratios of anolyte solution (Suzuki et al.,
2002). Different analytical methods were used to evaluate the inacti-
vation efciency of the electro-activated NaCl solution produced at
the near-anode interface. The following analytical methods the used
after 30 min incubations: reverse-phase passive latex agglutination
tests, immunoassays, native polyacrylamide gel electrophoresis
(PAGE), and amino acid analysis. Exposure to 70 ng (corresponding
to 2.6 pmol) of Staphylococcal enterotoxin-A in 25 L of PBS diluted
10-fold with anolyte solution has been reported to cause a loss of
immuno-reactivity between Staphylococcal enterotoxin-A and a spe-
cic anti-Staphylococcal enterotoxin-A antibody. Native PAGE analy-
sis showed that the anolyte solution fragmented the Staphylococcal
enterotoxin-A. Moreover, amino acid analysis indicated a loss in
amino acid content in the structure of Staphylococcal enterotoxin-A.
Amino acids, such as Met, Tyr, Ile, Asn, and Asp, were the mostly af-
fected. Staphylococcal enterotoxin-A excreted into culture broth
was also observed to be inactivated by adding an excess of the
electro-activated solution with a positive oxidation/reduction poten-
tial. Thus, the anolyte solution can be used to ensure food safety in
food processing industries (Suzuki et al., 2002).
3.9. Other agro-food applications
The effectiveness of nishing young cattle on untreated silage or
silage prepared with an electroactivated solution (EAS) of sodium
chloride was studied (Zinchenko et al., 1990). After an adjustment pe-
riod of 10 days, young bulls were fed for 147 days on the same basal
diet received during the adjustment period or a diet with silage
replaced with silage treated with an EAS of NaCl. The average daily
body weight gain was 1040 and 1120 g, respectively. The electro-
activated NaCl silage resulted in a slight increase in the digestibility
of crude ber and nitrogen-free extracts by the young bulls. However,
the amount of total dry matter and crude protein and fat decreased.
The nitrogen bioavailability in bulls was measured to be 32.3 and
35.47%, respectively. The authors estimated that bulls metabolized
72.9 and 67.6 MJ of energy and gained 712.0 and 783.2 g/kg of
crude protein, respectively.
A recent study on the electro-activation of zebrash (Danio rerio)
eggs (Cardona-Costa, Perez-Camps, & Garcia-Ximenez, 2011) aimed
to establish the electrical parameters combined with other experi-
mental conditions on improving the activation of zebrash eggs at
0 h, 1 h or 2 h after ovulation. This study was based on the hypothesis
that oocyte activation in mammals is induced by electrical sequences
associated with somatic cloning by nuclear transplant (Okahara-
Narita, Tsuchiya, Takada, & Torii, 2007; Onishi et al., 2000), intracyto-
plasmic sperm injection, (Mansour et al., 2009; Zhang et al., 1999) or
obtaining parthenogenetic haploid/diploid embryos (Escriba &
Garcia-Ximenez, 1999). The electro-activation of eggs in sh nuclear
transplants has been tested on medaka sh (Bubenshchikova et al.,
2007; Wakamatsu, 2008). Cardona-Costa et al. (2011) stated that
the nuclear transplant of somatic nucleus in zebrash is able to in-
duce embryo development when activation is only promoted by
water (Huang, Ju, Lee, & Lin, 2003; Perez-Camps, Cardona-Costa,
Francisco-Simao, & Garcia-Ximenez, 2010; Siripattarapravat, Busta,
Steibel, & Cibelli, 2009). However, the authors reported that some
benet could be provided in the case of nuclear transplants in zebra-
sh by supplement activation via electric eld pulses. The electro-
activation procedure was conducted in an Electro Cell Manipulator
47
equipped with an oscilloscope. First, 5 to 20 inactivated eggs con-
tained in Hanks solution were selected. Then, the eggs were intro-
duced in a pulsing chamber containing water as the electro-
activation medium. An established direct electric eld current square
pulse was applied to the medium containing the eggs. Finally, the
electro-pulsed eggs were incubated in Petri dishes at 28.5 C. The con-
trol group was comprised of eggs activated by non electro-activated
water. The egg damage/lysis and functional activation was veried
1 h after the treatment. The electro-activation efciency was evaluat-
ed by the number of eggs that showed at least one abortive cleavage,
using methods based on previously reported work (Lee, Webb, &
Miller, 1999). The following levels of couple [electric eld voltage
(V) pulses] (Cardona-Costa et al., 2011) were used: 2.76 1,
2.76 2, and 2.76 3; and 5.40 1, 5.40 2, and 5.40 3). Eggs
electro-activated by the couple [electric eld voltage (V)*pulses] of
5.40 3 showed the best results with a yield of 32% activated eggs.
Other experiments were conducted in which electrical treatments of
20 min, consisting of a sequence of three equal electrical stimuli for
10 min each of 1 or 3 consecutive, direct current square pulses for
20 s each, were applied at two voltage levels of 2.76 V and 5.4 V.
Cardona-Costa et al. (2011) reported that the number of pulses nega-
tively affected the rates of damaged and lysed eggs. Only the 20-min
treatment with a combination of 3 consecutive pulses at 2.76 V
showed signicant differences compared to the control group such
that 43% versus 18% eggs were activated, respectively. The authors
concluded that the electro-activation stimulus can be an effective
tool for the activation of zebrash eggs.
An electro-activation device was successfully used by Nabok and
Plutahin (2005) to extract protein from sunower seed meal. The
electro-activation device was used to create optimal conditions for
protein extraction at the cathode compartment in which a pH 11 so-
lution was generated. After extraction, the solution was passed
through the anodic compartment in which the pH was controlled to
precipitate the proteins at their isoelectric points ( pH 4.5). This ap-
proach permitted the extraction of 34% of total protein contained in
the meal. The extraction with electro-activation was compared to
that with concentrated NaOH. The results reported by Nabok and
Plutahin (2005) showed that extraction with NaOH yielded 39% pro-
tein. However, extraction with NaOH also yielded 15.4% ber, where-
as the electro-activation technology allowed for the extraction of
protein without ber. According to Nabok and Plutahin (2005), the
electro-activation technology for protein extraction from sunower
seed meal can be improved and optimized by controlling different pa-
rameters such as ow rate, electrode area and meal particle size.
4. Conclusions
Based on the literature on electro-activation of water and aqueous
solutions, we can conclude that the electro-activation of water and
aqueous solutions is feasible in electrolysis-based systems. However,
the most activated solution is obtained at the near-electrode inter-
face. Moreover, electro-activated water and aqueous systems are in
a metastable state, making them highly reactive and useful in
physico-chemical and biological reactions. For practical applications,
electro-activated water and aqueous solutions are powerful tools
used to ensure food safety and reduce the use of conventional and
costly disinfecting methods. However, further research is needed to
understand the thermodynamics behind the electro-activation phe-
nomena of water and aqueous solutions.
Activated solutions have been conclusively shown to exceed
chemically derived equivalents both in low dosage effectiveness and
physico-chemical purity. This increased biocidal capacity permits
the use of electro-activated solutions at lower dose rates relative to
traditional chemical solutions, thereby obviating the risk of intoxica-
tion and adverse environmental impacts. However, it is important
to mention that electro-activated anolytes demonstrate different
48 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 3849
efciencies against a variety of bacteria and viruses. This indicates
that the sensitivity of bacteria to electro-activated anolytes is not an
uncommon phenomenon and that many microorganisms are intrinsi-
cally more or less tolerant of antimicrobial substances. Gram-positive
and Gram-negative bacteria can also react differently to treatment
with electro-activated solutions.
Acknowledgements
The nancial support of the MAPAQ (Ministre de l'agriculture des
pcheries et de l'alimentation du Qubec) is gratefully recognized.
Ces travaux ont t raliss grce une aide nancire du Pro-
gramme de soutien l'innovation en agroalimentaire, un programme
issu de l'accord du cadre Cultivons l'avenir conclu entre le ministre de
l'Agriculture, des Pcheries et de l'Alimentation du Qubec, et Agri-
culture et Agroalimentaire Canada.
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