Innovative Food Science and Emerging Technologies 15 (2012) 38–49
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Innovative Food Science and Emerging Technologies
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Electro-activated aqueous solutions: Theory and application in the foodindustry and biotechnology
Mohammed Aider a,b,⁎, Elena Gnatko c, Marzouk Benali d, Gennady Plutakhin e, Alexey Kastyuchik a
a Department of Food Engineering, Université Laval, Quebec, Qc, Canada G1V 0A6b Institute of Nutraceuticals and Functional Foods (INAF), Université Laval, Quebec, Qc, Canada G1V 0A6c Ukrainian State University of Chemical Engineering, Dnepropetrovsk, 49005, Ukrained Natural Resources Canada/CanmetENERGY, 1615 Lionel-Boulet Blvd., P.O. Box 4800, Varennes, Quebec, Canada J3X 1S6e Department of Biotechnology, Biochemistry and Biophysics, Kuban State Agrarian University, Kalinin Str. 13, Krasnodar, 350044, Russia
⁎ Corresponding author at: Laval University, 2425 Rue3723.
Article history:Received 22 September 2011Accepted 3 February 2012
Editor Proof Receive Date 5 March 2012
Keywords:Electro-activationAqueous solutionAcidic anolyteAlkaline catholyteReagentless chemical reactionSanitizing
The present review highlights the state-of-the-art electro-activation as a science and the applications ofelectro-activated aqueous solutions in biotechnology and the food industry. The science behind electro-activation remains unknown. Hence, this review focuses on understanding the mechanisms governing theprocess of obtaining electro-activated aqueous solutions. Several applications in biotechnology and thefood industry are discussed. Among the potential applications of this technology, reagentless chemical catal-ysis and food safety seem to be the most promising.Industrial relevance: Electro-activated solution can be successfully used in the food industry and biotechnology for:• Selective protein and fiber extraction from different meal residues.• Self-generation of acidic and alkaline conditions for different catalytic applications.• Electro-activated solutions can be used as sanitizing agents for work area cleaning in food processing
industries.• Electro-activated solutions can be used for prevention of bio-films formation in food processing
Water is an important constituent of biological systems that plays amajor role in the physico-chemical properties of molecules in aqueoussolutions. From a chemical point of view, water is composed of twohydrogen atoms bonded to one oxygen atom. All biological, bioche-mical and physico-chemical reactions in living organisms only occurin aqueous media (Stewart, 2009).
During the past decades, there has been an increase in scientific inter-est worldwide in using water as a potential carrier of non-conventionalchemical reactions (Kirpichnikov, Bakhir, &Hamer, 1986). Several studieshave shown that the existing standards forwater properties and aqueoussolutions are not perfect and do not take into account many parametersthat characterize the biological usefulness and activity of these solutions.The physiological properties of water are affected by its chemical compo-sition, degree of purification and a number of other complex physicalparameters that characterize water as a complex structured system,particularly from an energetic point of view (Kloss, 1988). This complexstructured system is more evident when water is in a non-equilibriumthermodynamic 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 themost impor-tant parameters of water is its oxido-reduction potential. To be highlyefficient for physiological activities in humans, the oxido-reductionpotential of drinking water should be negative (Petrushanko &Lobyshev, 2001a, 2001b, 2001c; Petrushanko & Lobyshev, 2004). Thephysico-chemical properties of water and aqueous solutions can bemodified by means of electro-activation. Generally, this modificationoccurs at the near-electrode interface in an electrolysis-based system.In the presence of an electric field, solutions become activated and canbe used in different chemical reactions and catalysis. These solutionscan also be used in biological systems and enzymology (Gnatko,Kravets, Leschenko, & Omelchenko, 2011). The modified oxido-reduction potential and critical pH of electro-activated solutions in ametastable state make these solutions highly reactive and convenientfor non-conventional chemical reactions and different applications inthe 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-activationofwater andaqueous solutions aswell as different applicationsofelectro-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 field, charged speciesmigrate toward the electrode of opposite charge. In electrolysis, twophenomena of oxidation and reduction take place. In water or anyaqueous solution, a reduction reaction occurs at the negativelycharged electrode called a “cathode” and electrons (e−) from the
cathode are donated to positively charged ions, such as hydrogencations, to form hydrogen gas.
Reduction at the cathode : 2Hþ þ 2e
−→H2ðgÞ
Cathode ðreductionÞ : 2H2OðlÞ þ 2e−→H2ðgÞ þ 2OH
−ðaqÞ
At the positively charged electrode, called an “anode”, an oxidationreaction takes place. In this case, free electronsmigrate to the anode. Asimple example is the negatively charged oxygen that migrates to-ward the anode. Thismigration generates oxygen gas (O2) by transfer-ring electrons to the anode to complete the following reactions:
Anode ðoxidationÞ; 2H2O→O2ðgÞ þ 4Hþ þ 4e
−;
Anode ðoxidationÞ; 4OH�ðaqÞ→O2ðgÞ þ 2H2OðlÞ þ 4e−
When only hydrogen and oxygen molecules are present and whenthere are charged species involved in the oxido-reduction reactionthat occurs in an electrolyzer, the number of hydrogen molecules pro-duced is twice that of oxygen molecules. Under the same temperatureand pressure conditions for both gases, the volume of hydrogen gas pro-duced is twice that of oxygen gas produced. The number of electronspushed through the water doubles, and the number of hydrogen andoxygen molecules generated quadruples, respectively.
The split or decomposition of pure water into hydrogen and oxygenthat occurs at standard temperature and pressure is thermodynamicallyunfavorable.
Anode ðoxidationÞ : 2H2OðlÞ→O2ðgÞ þ 4HþðaqÞ þ 4e
−Eoox ¼ 1:23V
Cathode ðreductionÞ : 2HþðaqÞ þ 2e−→H2ðgÞ Eo
red ¼ 0:00V
Based on the Nernst Equation, the standard potential of the waterelectrolysis cell is equal to 1.23 V at 25 °C and pH 0 (molar concentrationof H+=1M). The same standard potential is also 1.23 V at 25 °C and pH7 (molar concentration of H+=1×10−7 M). In such system, the nega-tive voltage is an indication that theGibbs free energy for the electrolysisof water is>0. This can be found using the following equation:
G ¼ −n⋅F⋅E
where n and F are the number moles of electrons and Faraday constant,respectively.
The reaction is impossiblewithout adding sufficient external energy,which is usually provided by an applied external electrical field E.
Electro-activation is a relatively novel science, and yet the thermo-dynamics behind the process of electro-activated water generation isstill unknown (Shirahata, Hamasaki, & Teruya, 2012). From a techni-cal point of view, the design engineering of any electro-activationreactor 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 fieldintensity reaches hundreds of thousands of volts per centimeter incomparison to its mean value in the bulk solution. Because the elec-tric double layer is very thin in weak solutions and thinner in
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concentrated solutions, the electro-activation reactor must bedesigned to ensure the maximum treatment of micro-volumes ofwater as they arewithin close proximity to the electrode/solution inter-face. The generation of an electro-activated solution is also dependenton physical factors, such as the temperature, the solution flow rateand the salt concentration (Hsu, 2003). Moreover, storage conditionscan also affect the physico-chemical and antibacterial properties ofelectro-activated solutions (Hsu & Kao, 2004).
2.2. Thermodynamic considerations
Water molecules interact with an applied external electric field.The dipole moment of each water molecule and high oxygen atomelectronegativity are responsible for these interactions. Moreover,water molecules excited by an electric field are known to behave dif-ferently in different environments influenced by an electric field. Forexample, water streams and ice growth patterns can significantlychange in the presence of an electric field (Libbrecht & Tanusheva,1998). A novel concept called “water wire” has interested scientistsdue to its importance in biological systems and nanomaterial science.Several studies have been reported on the effects of electric fields onthe collective phenomena of liquid water and large water clusters(Vegiri, 2004). Electric fields also affect the structural and energeticchanges 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 inthe pH and RedOx potential of electro-activated water have beenreported to result from stable, high-energy resonant water microclus-ters that are based on co-vibrating dipoles of water molecules andcharged 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 dynamicstate, there is a stabilization effect. An alternating field formed on thebase 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, andcluster 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 anelectrolysis device (cell). From a fundamental point of view, one canconsider an electrolysis cell to be composed of a pair of electrodessubmerged into an electrolyte to ensure the conduction of ionswhen the system is subjected to an external electric field (direct or al-ternative current). The connection between the anode and the cath-ode is essential for the continuity of the circuit. Continuity isensured by the flow 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 Nernstequation as follows (Fidaleo & Moresi, 2006; Prentice, 1991):
ET ¼ E0T−RG⋅TK
n⋅F ⋅ ln ∏aSiii
!
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 coefficient (>0 for prod-
ucts orb0 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
d
) at 25 °C will be established, which can be expressed by thefollowing equation:
Ed ¼ 1:358þ 0:059pH−0:059 logCCl−
where c
Cl−
is the molar concentration of chloride ions.
In an electrolysis system, the ionic species will continuously mi-grate to the electrode of the opposite site. To ensure the productionof electro-activated aqueous solutions with metastable properties, itis 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 theearly 19th century (Tanaka et al., 1999). The first electrolysis cellwas described by Nicholson, Carliebe, & Cruichank, 1800 (Stoner,Cahen, Sachyani, & Gileadi, 1982). The development of electrochemis-try and the production/synthesis of new materials have significantlyimproved apparatus design of electrolysis systems and at this time,the first mention of electro-activation of aqueous solutions wasused. It was a revolutionary discovery in the field of applied electro-chemistry. Later, the discovery of the bactericidal effect of theelectro-activated solutions, along with the increased economic andenvironmental requirements of the industry over the past 20 years,sparked significant growth in the number of scientific developmentsand publications in this research field. As the scientific publicationson the design of electro-activation systems increased, the need of sys-tematical classification of the electro-activation processes and devicesis thus required.
Basically, the devices used and technical requirements of theelectro-activation systems can be distinguished by the feeding meth-od of the treated aqueous solution. There are periodic and continuoussystems. Installations working in a continuous mode are mainly usedin the processing of industrial volumes. They are embedded in thetechnological line and work for a long time. To this type of installa-tions, one can also attribute electro-activation devices manufacturedfor personal needs of the population. These units are mounted inthe drinking water supply (Morita et al., 2000). Apparatus andelectro-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 specific physico-chemical and biological properties. Theelectro-activation device can be used to obtain predominantlyelectro-activated solution with oxidizing properties (anolyte). Inthis case, the targeted solution is processed in the anodic side of theelectro-activation apparatus. On the other hand, the versatility ofthe electro-activation technology allows obtaining electro-activatedsolution with reducing properties (catholyte). In this case, thetargeted 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 cathodicsections. The first electro-activation systems used diaphragms witha structure that is characterized by a non-selective permeability tocharged species. It was usually made of ceramics such as porcelainand earthenware, canvas, nylon, synthetic materials such as polyvinylchloride and polytetrafluoroethylene fabric. Later developments inmaterial sciences and particularly ion selective membranes allowedto significantly improving the performances of the electro-activation
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systems and apparatus. Indeed, ion selective membranes can replacethe inert diaphragm in order to easily modulate the physico-chemicalproperties and compositional structure of the electro-activated solu-tions. For example, the pH and oxidation–reduction potential of theanolyte and catholyte can be modulated by an appropriate selectionof cation or anion exchange membrane. However, for food applica-tions, some restrictions apply and the membrane material must becompatible with foods. However, there is electro-activation processesin which the separation diaphragm is not required or not suitable. Inthis case, the electro-activation systems are divided into systems withand without separated anolyte and catholyte sections. Moreover, alimited number of electro-activation systems use inert electrodes(anode and cathode) (Yakimenko, 1977). In such devices, the polarityof the electrodes can be reversed. Hence, on the basis of this particu-larity, electro-activation systems can be classified as systems withand without reversible electrodes. To the electrode material and re-gardless of its construction, a number of general requirements areneeded (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 sufficient mechanical strengthto maintain its original properties for long term use; (c) It must havethe lowest possible cost; (d) Location and shape of the electrodesmust ensure uniform distribution of the electric current density; (e)For food applications, the electrode must be inert, insoluble in theproduct when a voltage is applied.
2.4. Design of the electro-activation reactors
The electroactivated aqueous solutions are produced inside theelectrochemical reactors, i.e. electrolyzers, widely varying in designand production process. For example, only in Japan, electro-activation was performed, in 1999, using about 30 reactors havingdifferent 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 asvoltage, content and flow 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 electrolyzersand technological parameters of the process, on one side, and func-tional properties of electroactivated solutions, on another side, arepractically absent in scientific literature. Usually, the desirable param-eters of the production process are adjusted empirically.
Electrochemical treatment of water and aqueous solution hadbeen performed using the batch-type and flow-through electrolyzers(Morita et al., 2000; Stoner et al., 1982). Contrary to the flow-through reactors, electro-activation of immobile solutions for thefixed duration of time, e.g., 3–115 min , is performed inside the cham-bers of the batch-type reactors having volume from 1 to 15 l . Usually,the volumetric flow rates inside the flow-through electrolyzers range0.5–1.9 l/min. The voltages used for electro-activation range 9–120 Vand the values of direct current passing through the electrolyzer areset from 0.7 to 20 A. Extremely high voltage up to 1100 V wasemployed for activation of distilled water; however, the electric cur-rent during the electrolysis was about few milliamps due to a verylow 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 5–15 V is used for electro-activation inside the either flow-through or batch-type reactors. Ingeneral, the reactors for electro-activation differ from each other interms of shape and material of their parts, such as casing, electrodesand diaphragms, if latter are present. The coaxial cylindrical flow-through electrolyzers represent one of the most common designs(Rossi-Fedele, Dogramaci, Steier, & de Figueiredo, 2011). Such devicescould be easy assembled in a compact electrochemical module
comprising up to eight electrolyzers. Flow-through parallel-plateelectrolyzers use plates as cathode and anode. In some instances,multiple and bipolar electrodes were employed. A peculiar reactor isdescribed in the work of (Sergunina, 1968). The electrolyzer com-prised two columns filled 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 bya 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, titaniumcoated with Fe3O4 , and graphite have been used for manufacturingof anodes. Titanium, platinum or graphite are used as cathodes(Lovtsevich & Sergunina, 1968). A random-oriented graphite-epoxymatrix 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 flow-through reactors withor without porous diaphragm (Shimada, Ito, & Murai, 2000). The dia-phragms allow for production of electroactivated solutions with spe-cific functional properties, such as acidic (anolytes), alkaline(catholytes) or nearly neutral solutions. The latter could be obtainedby the controlled mixing of anolytes and catholytes. The main re-quirements for the diaphragms are high porosity, hydrophilicity andlow electric resistance. A high-quality porous ceramic diaphragmswere used in (Middleton, Chadwick, Sanderson, & Gaya, 2000).Electro-conductive cationic diaphragm made of Nafion-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 fromdistilled water and tap water to electrolytes containing from 0.1 to120 g/l NaCl (Inoue et al., 1997). Produced electroactivated anolytesare characterized by pH ranging 2.3-6.5, positive redox potential of1000–1200 mV, and by about 30–300 ppm of dissolved chlorine(Tanaka et al., 1996). At the same time, the catholyte is characterizedby pH 6.2 and negative redox potential −329 mV and by pH 8.8 andnegative 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 metastablestate after excitation by an external electric field. The reactivity ofelectro-activated solutions was observed to significantly increaseunder this state compared to a normal state. Researchers explainthis phenomenon using the vibrational spectrum of water(Antonchenko, Davydov, & Iliin, 1991). In relation to the vibrationalspectrum of water, Pastukhov and Morozov (2000) studied theRaman 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 acidifiedor alkalinized water (analogues of electro-activated water based onpH properties). In this study (Pastukhov & Morozov, 2000), waterwas activated by electrolysis with the addition of 0.4×10−3 M sodi-um hydrosulfate to allow the passage of the electric field without dis-sociating water molecules. Molecular oxygen and hydrogen werereleased from the anode and cathode regions, respectively. ForRaman spectral studies, water samples were taken from the near-anode (anolyte) and near-cathode (catholyte) zones. The pH valuesof these water samples were 4.0 and 10.0 for the anolyte and catho-lyte, respectively. To compare the spectra of electro-activated watersamples with those of analogues, the electro-activated anolyte wassimulated 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) 38–49
Rectangular single-pass quartz cells were used to study the Ramanspectra of electro-activated and control water samples (acidifiedand alkalinized). The Raman spectra were excited by a 200-mW488-nm line of an argon laser and recorded at an angle of 90° to thelaser beam. The spectra were recorded in the 50–4000-cm−1 spectralregion at 20 °C.
The results demonstrated that Raman spectra between 700 and2700 cm−1 of the electro-activated water taken in the near-anode(anolyte) and near-cathode (catholyte) regions were significantly dif-ferent from those of chemically acidified 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 inthe spectra of chemically acidified and alkalinized water samples.The authors specified that the addition of sodium hydrosulfate towater resulted in vibrational bands within the region of1000–1500 cm−1 of the Raman spectrum. The scattering intensitywas 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 authorsmixed the anolyte and catholyte in equal volumes and observed a de-creased scattering intensity in the region of 700–2700 cm−1 com-pared to the initial intensities of individual electro-activated watersamples. Usually Raman spectra of water and aqueous solutions areinterpreted based on the time-dependence of the changes in thestrength 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 of700–2700 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 anolyteand catholyte, respectively. These ions are responsible for the acidicand 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 thevibrational bands observed in Raman spectra of the anolyte and cath-olyte (Pastukhov & Morozov, 2000). This finding suggests thatelectro-activated solutions may possess properties similar to thoseof 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 inwater, (3) near-cathode solution (catholyte), (4) near-anode solution (anolyte),(5) chemically alkalinized water (catholyte analogue), and (6) chemically acidifiedwater (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 protonsresiding in complex A (or B) interacting at different strengths(Janoschek, Weidemann, Pfeiffer, & Zundel, 1972). Excess hydroxylions in the near-cathode solution (catholyte) can participate in theformation of a more symmetric and weak hydrogen bond(O···H···O)−. Simultaneously, in the near-anode solution (anolyte),a strong hydrogen bond was observed in close intensities of the broadRaman and valence bands examined. This phenomenon was used toexplain the higher scattering in the anolyte solution compared tothat of the catholyte. In the near-anode solution, which is considereda strong acid media, the continuous absorption in the infrared spec-trum is caused by H5O2 groupings. In the near-cathode solution, thecontinuous absorption in the infrared spectrum is caused by H3O2−
groupings (Janoschek et al., 1972). Furthermore, the electrochemicaldissociation of water molecules at the near-electrode interface resultsin the formation of unstable complexes, such as (OO), (OO)+, and(HH)+. These complexes are considered as intermediates and theirvibrational modes can contribute to scattering in the correspondingspectral 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 enzymeshave been reported (Podkolzin et al., 2001). Podkolzin et al. studiedthe 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 werecentrifuged and lysed with 5 mM Tris–HCl buffer at pH 7.8. For hemo-globin precipitation, 0.25 ml of 96% ethanol and 0.15 ml of chloroformwere added to 1 ml of lysate, mixed on ice for 15 min, and centrifugedat 10,000 g for 15 min at 4 °C. Catalase activity was then measured bythe reaction of hydrogen peroxide (H2O2) with ammonium molyb-date. Peroxidase activity was measured by the reaction with indigocarmine, and superoxide dismutase activity was determined by theinhibition of hematocrit (HCT) reduction with superoxide anionsgenerated in the reaction of NADPH with phenazine methosulfate.The effects of electrochemically activated solutions on the activity ofextra-pure human recombinant superoxide dismutase was obtainedby passing a NaCl solution (1–2 g/l) through an Izumrud deviceequipped with 2 reactor compartments. This procedure allowed theoxido-reduction potential (ORP) of the electrochemically activatedsolutions to vary from +200 mV to −70 mV. The ORP was continu-ously measured using a pH meter with platinum electrodes understandard conditions. The authors showed that electrochemically acti-vated systems normalized the activity of antioxidant enzymes such ascatalase, peroxidase, and superoxide dismutase. Additionally, thebaseline activity of the targeted antioxidant enzymes was observedto vary considerably in humans and animals. The effect of the electro-chemically activated systems was characterized by a negative oxida-tion–reduction potential, which was probably related to a trainingeffect of excess electrons. Podkolzin et al. (2001) showed that prein-cubation of erythrocyte lysate with various concentrations of theelectro-activated solution for 5, 10, and 20 min caused a bi-phasic re-sponse of rapid activation of the enzyme followed by normalizationor slight inhibition of its activity. The time- and dose-dependent ef-fects of the electro-activated solution were also observed at variousoxido-reduction potentials at constant reaction times. These effects
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).
43M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 38–49
showed that higher concentrations of electro-activated solutions dur-ing preincubation with erythrocytes resulted in shorter preincubationtimes 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 recombinantsuperoxide dismutase. The authors interpreted these changes by theability 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 tothe generation of superoxide anions. However, components of themain reaction were also reported to be inactivated at high negativeoxido-reduction potential values (−170 mV). After a 5-min incuba-tion with the electro-activated solution, the reaction of NADPH withphenazine methosulfate and the HCT reduction were determined tobe inhibited by 22–25%. Thus, the effects of ECAS on antioxidant en-zymes depend on ORP, electro-activated solution concentrations,
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 withthe 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 makingwheat bread with electro-activated water in the laboratory of bio-technology, biochemistry and biophysics at the Kuban State AgrarianUniversity, Krasnodar, Russia. The authors hypothesized that breadquality 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 qualityestimation standards are not perfect and do not consider a number ofimportant parameters that affect the biological function and potencyof water. The oxido-reduction potential, which must be negative fordrinking 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 processesin live cells by investigating their activating or inhibitory influenceson the reactivation of baking yeast and preparation of fermenteddough. Water was activated using a non-contact method. The activa-tion procedure was carried out in a tank filled with electrolyte solu-tion in an unforced circulation mode. Using this technology, anolyte,catholyte, drinkable catholyte and drinkable anolyte solutions wereproduced. The obtained electro-activated solutions were used to acti-vate yeast used in wheat dough making. The following procedure wasused to activate dry yeast: a 5% (w/v) sugar solution was prepared inactivated aqueous solutions at 30 °C. The intensity of yeast activationwas evaluated according to the volume of the solution, which roseproportionally to the amount of carbon dioxide produced by theyeast. The fermentation process occurred at 30±2 °C and relative hu-midity of 75–80%. Baking was carried out at 230±5 °C over 20 min.Five types of electro-activated water samples were used to makebread. Four samples were produced by a contact method and oneby a non-contact activation method. All water samples were evaluat-ed for salinity, pH and RedOx potential (ORP). The control waterexhibited an oxido-reduction potential of 220 mV, mineralization of470 ppm, and a pH of 7.94. Non-contact activation yielded waterwith an ORP of −110 mV without significantly changing the pH andmineralization compared to the water control. The drinking catholyteand anolyte exhibited ORP values of 3.8 and 67 mV, respectively.However, the cathode and anolyte obtained at the anode sectionswere 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 thehighest activity followed by the yeast activated in water generatedfrom a non-contact method. The effect of the drinkable catholyte so-lution on yeast activity was lower compared to the abovementionedresults. Despite its active chlorine content, the anolyte solution wasnot observed to completely inhibit the process of fermentation. Thewheat 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 solutionwas determined to result in the loss of yeast activity. Thus, to obtainhigh-quality bread, the activation time needs to be controlled (opti-mized). The anolyte ORP was 905 mV with the presence of activechlorine, resulting in yeast activation. The biological value of the pro-teins in the bread was evaluated, and the results suggested that theproteins exhibited the highest biological values when used with
a Electrolysis conditions: 0.5 A (direct current) for 3 h in 0.1 mol l−1 phosphate buff-er (pH 7.1).
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electro-activated water obtained by a non-contact method. The au-thors (Nabok & Plutahin, 2005) reported that both the activatedwater obtained from a non-contact method and catholyte exhibitednegative ORP values. They argued that this was a result of the fasterfermentation of yeast taking place in these solutions. The volume ofboth bread samples was significantly higher than that of the controlsample. 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 madewith water resulting from non-contaction had the best qualitativeindices.
3.3. Yeast inactivation
Over the past decade, critical analyses of nutrition and diet haveindicated a tendency of consumers gravitating toward fresh-likefood products (Guillou, Besnard, El Murr, & Federighi, 2003). Froman engineering perspective, there has been a noticeable interest inthe investigation of the possible uses of non-thermal processes forfood preservation and safety. Among the new technological advances,the use of electro-activated aqueous solutions seems to be highlypromising. Guillou and El Murr (2002) reported a method for theuse of low intensity electric fields for microbial (yeast) inactivation(Guillou and El Murr (2002); Guillou et al., 2003) for the stabilizationof 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 wasconducted on microbial suspensions of 2×106 cfu/ml) in 0.1 mol/lphosphate buffer at pH 7.1. The yeast suspension was submitted toan electro-activation treatment in an electrolysis device (Guillou &El Murr, 2002). This treatment was conducted directly under a con-stant electric field having an amperage of 0.5 A and completed in3 h. According to the information reported by the authors (Table 2),the experiment was conducted at 20 °C. After 3 h of treatmentunder an electric field with a constant amperage of 0.5 A, the yeastcell viability was 4.2±0.60% and 2.5±0.98% according to FUN-1and the plate counts method, respectively. After treatment in theelectrolysis device, the yeast cells were examined to determinewhether 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 alsodepends on the severity of the injury (Graumlich & Stevenson,1978; Guillou et al., 2003; Schenberg-Frascino, 1972). Furthermore,the authors confirmed the lethal effect of electrolysis (Guillou et al.,2003). However, the number of viable cells depended on the methodused to evaluate the number of viable cells. The results demonstratedthat the colony count method gave lower viability than othermethods, 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
damaged cells. The authors reported that the lethal effect of electrol-ysis might be overestimated by 0.6Log10. They suggested that morestudies were necessary to understand the effect of electro-activation(electrolysis) on different microorganisms. Based on this study, oneshould not exclude the industrial application of electro-activation orelectrolysis for food safety (Guillou et al., 2003).
3.4. Electrochemical inactivation of bacteria, viruses and bacteriophages
The inactivation of bacteria and yeast cells by electrochemicalmeans 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 fields have been shown as effective disinfecting agents ofdrinking water and vegetables (Beuchat et al., 2001) that can reducethe numbers of microorganisms in food products. Disinfection ofdrinking water is a topic of concern, and research has been carriedout to identify potential substitution methods of conventionalchlorine-containing disinfectants (Matsunaga et al., 1992; Patermarakis& Fountoukidis, 1990). Drees et al. reported the use of electro-activationto inactivate bacteria and bacteriophages (Drees, Abbaszadegan, &Maier, 2003). There has been limited research regarding the effectivenessof electro-activation on the inactivation of viruses. Drees et al. comparedthe ability of bacteria andbacteriophages to survivewhenexposed to a di-rect electric field in an electrochemical cell (Drees et al., 2003). Bacteriawere subjected to irreversiblemembrane permeabilization and the directoxidation of cellular/viral constituents by an electric current. The disinfec-tion was caused by electrochemically generated oxidants. Suspensions ofEscherichia coli and Pseudomonas aeruginosa aswell as the bacteriophagesMS2 and PRD1 at high (1×106 CFU or PFU/ml) and low (1×103 CFU orPFU/ml) population densities were used (Drees et al., 2003). Cultureswere exposed to an electric current intensity from 25 to 350 mA. More-over, the electric field 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 higherthan bacterial culturable counts under corresponding experimental con-ditions. E. coli and the MS2 bacteriophage were studied at both high andlow population densities. The electro-activation cell was used under thefollowing conditions: an electric field intensity of 5 mA was appliedover a treatment period of 20 min. The results showed that the inactiva-tion rate for E. coliwas 2.1–4.3 times greater than that of the MS2 bacte-riophage. Moreover, the population density effect was significant. Bothbacteria and bacteriophages were more resistant to direct electric fieldat higher population densities. The authors suggested that reduced inacti-vation within electrochemical cells was observed when the glutathionereducing agent was used. The authors also suggested that the resultsmay be used in food technologies to reduce the numbers of microbes infood andwater. The bacteriophagesweremore resistant than the bacteriatested by Drees et al. (2003).
Electro-activation of aqueous solutions is a physical process thatinvolves different electrochemical reactions at electrode/solution
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interfaces. The antimicrobial or antiviral effect of these solutions re-sults from different chemical oxidants that are generated when anelectric field 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 andozone. 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 ofresearch on the antibacterial effects of electro-activated solutions,most of the current research in this field supports the hypothesisthat antimicrobial agents and electric currents act synergistically toinactivate bacteria and viruses (Costerton, Ellis, Lam, Johnson, &Khoury, 1994; Khoury, Lam, Ellis, & Costerton, 1992).
Anolytes, which are generated at the anode/solution interface, arewell-known oxidizing agents that produce a mixture of free radicalsthat have antimicrobial effects. From a physico-chemical point ofview, 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 cellmembrane provides an osmotic barrier for the cell and catalyses theactive transport of substances into the cell. Irreversible modificationsof the transmembrane potential caused by the action of electrondonors/acceptors could be associated with the powerful electro-osmotic processes accompanied by water diffusion against oxida-tion–reduction gradients, resulting in membrane rupture and an out-flow of bacterial cell contents. Moreover, the bacterial membrane cellhas 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 solutiontransport 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 smallcharged molecules that are transported across the cell membrane bymeans of an electro-chemical gradient (Veld, Driessen, & Konings,1993). Thus, any significant change in the oxidation–reduction poten-tial of the immediate medium of the bacterial cell can cause lethalconsequences for the cell. Electro-activation of chlorine-containingaqueous solutions generates hypochlorous acid, which is more activethan the sodium hypochlorite generated by dissolving this salt inwater.
The effect of electro-activated water on microorganisms is com-plex. However, some explanations may be made based on generatedactive 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 electronsat low current intensities to their destruction at high current intensi-ties. The efficacy of electro-activated water against microorganisms isalso based on its oxidative power, which allows electro-activatedwater to act as an electron acceptor as a result of the high electron de-ficiency in the water cluster.
3.5. Biofilms prevention/treatment
Biofilms are a serious concern for the modern food industry, asthey are potential sources of contamination of food products in pro-cessing plants. One of the most important particularities of the bio-films is that they frequently support sanitizer treatments. Thecombined effect of alkaline and acidic electro-activated (electrolyzed)water in the inactivation of Listeria monocytogenes biofilms on stain-less steel surfaces was investigated. Biofilms were grown on rectan-gular stainless steel in a 1/10 dilution of tryptic soy broth thatcontained a five-strain mixture of L. monocytogenes for 48 h at 25 °C.The coupons with biofilms were then treated with acidic electro-
activated water, alkaline electro-activated water or alkaline electro-activated water followed by acidic electro-activated water producedby applying an electric field intensity of 14 and 20 A for 30, 60, and120 s. Alkaline electro-activated water alone was not observed to sig-nificantly reduce L. monocytogenes biofilms when compared with thecontrol. The authors also showed that treatment with acidic electro-activated water for 30 to 120 s reduced the number of viable bacterialpopulations in biofilms by 4.3 to 5.2 log CFU per coupon. The com-bined treatment of alkaline electro-activated water followed by acidicelectro-activated water produced an additional 0.3- to 1.2-log CFUper coupon reduction. The population of L. monocytogenes reducedby treatments with acidic electro-activated water increased signifi-cantly with an increase in the time of exposure. No significant differ-ences occurred between treatments with electro-activated waterproduced with an electric field intensity of 14 and 20 A, respectively.The authors concluded that the obtained results suggest that alkalineand acidic electro-activated water can be used together to achievebetter inactivation of biofilms 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 biofilms 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 establishedprocedure 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 efficacy was a concentration-dependent prop-erty. Water with a chlorine concentration of 5 to 200 ppm has beendemonstrated to have a weak effect on Salmonella, which is usuallyreduced in chicken carcasses by not more than 1 log CFU/carcass.However, chlorine containing water was observed to enhance theprevention of cross-contamination (James, Brewer, Prucha,Williams, & Parham, 1992). In relation to the microbial safety of thepoultry industry, the antibacterial efficacy of electrochemically acti-vated aqueous solutions was studied, focusing on poultry sprayingand chilling (Yang, Li, & Slavik, 1999). The antibacterial efficacy ofan 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 pressureof 413 kPa for 17 s. Then, the treated product was chilled at 48 °C for45 min. Electro-activated aqueous solutions were generated by anelectrochemical reactor consisting of 8 electrochemical cells placedin a parallel mode (Bakhir et al., 1997). Tap water was mineralizedby means of 15% (w/v) NaCl to obtain a 3 g/l NaCl solution, which flo-wed into the cathode and catalyst chamber, respectively. A directelectric field current at a voltage of 30 V was applied to the electro-chemical device. Under these conditions, an electric field intensityof 9 A was obtained. The total concentration of oxidants in theelectro-activated solution was 300 ppm of free chlorine at pH 6.5,which was controlled by drawing the catholyte through the bypassof the electrochemical device. Different dilutions were made withthis electro-activated water and used for the spraying and chillingof chicken carcasses based on conventional chlorine treatments inpoultry processing (Thomson, Cox, & Bailey, 1976). An electro-activated solution with 50 ppm of oxidants expressed as free chlorinewas 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 0.87log10 CFU/carcass, respectively. The authors also reported that addi-tional chilling of the carcasses in an iced electro-activated solutionwith 50 ppm of chlorine did not reduce Salmonella. The authors sug-gested that more studies be performed to understand whether theelectro-activation treatment affects the sensory attributes and
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physico-chemical properties of chicken meat obtained from carcassestreated 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 bytreatment with electro-activated (electrolyzed) oxidizing water.They used this sanitizing approach against pathogens associatedwith poultry. Acidic electro-activated water with 20 to 50 ppm chlo-rine and an oxidation–reduction potential of 1150 mV at pH 2.6,basic electro-activated water with an oxidation–reduction potentialof −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 for45 min. The remaining bacterial populations were determined andcompared 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 waterreduced 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 theused solutions did not reduce Salmonella typhimurium at day 0.After 7 days of storage, acetic acid and acidic electro-activated waterreduced 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 reduceSalmonella 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 inactivationof bacterial spores is important for ensuring product safety. Productswith a long shelf life are sterilized to ensure their stability. Gram-positive bacteria belonging to the genera Bacillus, Clostridium, andSporosarcina grow easily and divide in nutrient-rich media. However,under conditions where one or more essential nutrient is deficient orlimiting, the microorganisms initiate the process of sporulation,resulting in the formation of dormant spores (Chander, Setlow, &Setlow, 1998; Setlow, 1994). Under favorable conditions, these sporescan grow, alter the food products, and form toxins. This ability resultsfrom 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 decontaminantsand 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 andtechnologies for the endospore-forming bacteria inactivation havebeen 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 ofan aqueous solution of sodium chloride or any other salt. The oxida-tive properties of this solution are due to the reaction involved atthe near-anode interface. The solution electro-activation is carriedout by the electrolysis of saline solution, which is circulated throughan 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 propertieswas produced, whereas at the near-cathode interface an alkalinizedsolution was generated. Generally, the anolyte is characterized by ahigh 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 catholyteis characterized by a reduction potential with values between of−80to −900 mV and pH 7–12. 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 ofRogers et al. (2006), five different electro-activated solutions withdifferent 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 salinesolutions for 30 min. Subsequently, the decontamination efficacy ofthis procedure was analyzed. The authors used a 5% high-test hypo-chlorite calcium solution as a positive control. The electro-activatedsolutions 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 +826to +1000 mV. Treatments with all electro-activated saline sand 5%high-test hypochlorite calcium solutions resulted in reductions bymore than 7 log in both Bacillus anthracis and spores. Based onthese results, the authors concluded that the electro-activated solu-tion, containing the minimum active, free chlorine concentration of300 ppm and having a RedOx potential of +800 mV, is able to inacti-vate the Bacillus anthracis spores in suspension. This effect was similarto that of the 5% high-test hypochlorite calcium solution. The findingsof 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 consumingfoods that contain toxins, whereas food borne infections are causedby infectious pathogens in food. In the case of food poisoning, toxinscan be produced by bacteria or occur in food, such as in some mush-rooms. Toxins can also be contaminants and directly affect biologicalreactions taking place in the host. At sufficiently high concentrations,the effects of toxins are acute and take place over the course of a fewhours after consumption of contaminated food. The symptoms associ-ated with food poisoning can include nausea and vomiting. In somecases, the effects are lethal. The toxins can have various origins. Infood 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 havelong-term effects, even at small concentrations. The other problemrelated to toxins involved in food poisoning is their resistance toheat; thus, they cannot be easily eliminated by cooking. Staphylococ-cal enterotoxins are proteins with molecular weights between 27 and30 kDa. To date, at least eight staphylococcal enterotoxins have beenreported (Su & Wong, 1995; Suzuki et al., 2002), the most dangerousand resistant being Staphylococcal enterotoxin-A. A total mass below200 ng of Staphylococcal enterotoxin-A is sufficient to cause food poi-soning in humans. Toxin concentrations of 0.4 to 0.8 ng/ml can causeillness in a short period of 3–5 h after the intake of contaminatedfood. The Staphylococcal enterotoxin-A is thermo-, acid- and alkali-resistant. Thus, it is very important to inactivate Staphylococcalenterotoxin-A (Huang, Hughes, Bergdoll, & Schantz, 1987; Suzuki etal., 2002). Suzuki et al. (2002) used an electro-activated aqueous so-lution to inactivate Staphylococcal enterotoxin-A. The generation ofthe electro-activated solution was performed in an electrolyzer. Thisdevice permitted the production of near-anode and near-cathodeNaCl solutions. The anode and cathode sections were separated by adiaphragm. The electrolysis of the NaCl solution was conducted for12 min at ambient temperature. A feed solution of 0.1% NaCl wasused and prepared by dissolving salt in deionized water. The electro-lyzer was submitted to a direct electric current at a voltage between 9and 11 V. After electrolysis, strong acidic solutions at pH 2.5–2.8 andavailable chlorine of 36.3 ppm were produced. This chlorine concen-tration was estimated to be equivalent to 0.67 mM HOCl. The anolytesolution produced at the near-anode interface had an oxidation/
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reduction potential of +1180 mV. Simultaneously, a strong alkalinesolution at pH 11.6–12.0 with an oxidation/reduction potentialbelow−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 becauseelectro-activated solutions are generally not stable and because thebactericide effect is the highest within the first hours after the solu-tions are made. Fixed quantities of Staphylococcal enterotoxin-Awere mixed with different ratios of anolyte solution (Suzuki et al.,2002). Different analytical methods were used to evaluate the inacti-vation efficiency of the electro-activated NaCl solution produced atthe near-anode interface. The following analytical methods the usedafter 30 min incubations: reverse-phase passive latex agglutinationtests, immunoassays, native polyacrylamide gel electrophoresis(PAGE), and amino acid analysis. Exposure to 70 ng (correspondingto 2.6 pmol) of Staphylococcal enterotoxin-A in 25 μL of PBS diluted10-fold with anolyte solution has been reported to cause a loss ofimmuno-reactivity between Staphylococcal enterotoxin-A and a spe-cific anti-Staphylococcal enterotoxin-A antibody. Native PAGE analy-sis showed that the anolyte solution fragmented the Staphylococcalenterotoxin-A. Moreover, amino acid analysis indicated a loss inamino 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 brothwas also observed to be inactivated by adding an excess of theelectro-activated solution with a positive oxidation/reduction poten-tial. Thus, the anolyte solution can be used to ensure food safety infood processing industries (Suzuki et al., 2002).
3.9. Other agro-food applications
The effectiveness of finishing young cattle on untreated silage orsilage prepared with an electroactivated solution (EAS) of sodiumchloride was studied (Zinchenko et al., 1990). After an adjustment pe-riod of 10 days, young bulls were fed for 147 days on the same basaldiet received during the adjustment period or a diet with silagereplaced with silage treated with an EAS of NaCl. The average dailybody weight gain was 1040 and 1120 g, respectively. The electro-activated NaCl silage resulted in a slight increase in the digestibilityof crude fiber 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 and35.47%, respectively. The authors estimated that bulls metabolized72.9 and 67.6 MJ of energy and gained 712.0 and 783.2 g/kg ofcrude protein, respectively.
A recent study on the electro-activation of zebrafish (Danio rerio)eggs (Cardona-Costa, Perez-Camps, & Garcia-Ximenez, 2011) aimedto establish the electrical parameters combined with other experi-mental conditions on improving the activation of zebrafish eggs at0 h, 1 h or 2 h after ovulation. This study was based on the hypothesisthat oocyte activation in mammals is induced by electrical sequencesassociated 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) orobtaining parthenogenetic haploid/diploid embryos (Escriba &Garcia-Ximenez, 1999). The electro-activation of eggs in fish nucleartransplants has been tested on medaka fish (Bubenshchikova et al.,2007; Wakamatsu, 2008). Cardona-Costa et al. (2011) stated thatthe nuclear transplant of somatic nucleus in zebrafish is able to in-duce embryo development when activation is only promoted bywater (Huang, Ju, Lee, & Lin, 2003; Perez-Camps, Cardona-Costa,Francisco-Simao, & Garcia-Ximenez, 2010; Siripattarapravat, Busta,Steibel, & Cibelli, 2009). However, the authors reported that somebenefit could be provided in the case of nuclear transplants in zebra-fish by supplement activation via electric field pulses. The electro-activation procedure was conducted in an Electro Cell Manipulator
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 field current squarepulse was applied to the medium containing the eggs. Finally, theelectro-pulsed eggs were incubated in Petri dishes at 28.5 °C. The con-trol group was comprised of eggs activated by non electro-activatedwater. The egg damage/lysis and functional activation was verified1 h after the treatment. The electro-activation efficiency 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 field 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). Eggselectro-activated by the couple [electric field voltage (V)*pulses] of5.40×3 showed the best results with a yield of 32% activated eggs.Other experiments were conducted in which electrical treatments of20 min, consisting of a sequence of three equal electrical stimuli for10 min each of 1 or 3 consecutive, direct current square pulses for20 μs 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-mintreatment with a combination of 3 consecutive pulses at 2.76 Vshowed significant differences compared to the control group suchthat 43% versus 18% eggs were activated, respectively. The authorsconcluded that the electro-activation stimulus can be an effectivetool for the activation of zebrafish eggs.
An electro-activation device was successfully used by Nabok andPlutahin (2005) to extract protein from sunflower seed meal. Theelectro-activation device was used to create optimal conditions forprotein extraction at the cathode compartment in which a pH 11 so-lution was generated. After extraction, the solution was passedthrough the anodic compartment in which the pH was controlled toprecipitate the proteins at their isoelectric points (≅ pH 4.5). This ap-proach permitted the extraction of 34% of total protein contained inthe meal. The extraction with electro-activation was compared tothat with concentrated NaOH. The results reported by Nabok andPlutahin (2005) showed that extraction with NaOH yielded 39% pro-tein. However, extraction with NaOH also yielded 15.4% fiber, where-as the electro-activation technology allowed for the extraction ofprotein without fiber. According to Nabok and Plutahin (2005), theelectro-activation technology for protein extraction from sunflowerseed meal can be improved and optimized by controlling different pa-rameters such as flow rate, electrode area and meal particle size.
4. Conclusions
Based on the literature on electro-activation of water and aqueoussolutions, we can conclude that the electro-activation of water andaqueous 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 ina metastable state, making them highly reactive and useful inphysico-chemical and biological reactions. For practical applications,electro-activated water and aqueous solutions are powerful toolsused to ensure food safety and reduce the use of conventional andcostly disinfecting methods. However, further research is needed tounderstand the thermodynamics behind the electro-activation phe-nomena of water and aqueous solutions.
Activated solutions have been conclusively shown to exceedchemically derived equivalents both in low dosage effectiveness andphysico-chemical purity. This increased biocidal capacity permitsthe use of electro-activated solutions at lower dose rates relative totraditional chemical solutions, thereby obviating the risk of intoxica-tion and adverse environmental impacts. However, it is importantto mention that electro-activated anolytes demonstrate different
48 M. Aider et al. / Innovative Food Science and Emerging Technologies 15 (2012) 38–49
efficiencies against a variety of bacteria and viruses. This indicatesthat the sensitivity of bacteria to electro-activated anolytes is not anuncommon phenomenon and that many microorganisms are intrinsi-cally more or less tolerant of antimicrobial substances. Gram-positiveand Gram-negative bacteria can also react differently to treatmentwith electro-activated solutions.
Acknowledgements
The financial support of the MAPAQ (Ministère de l'agriculture despêcheries et de l'alimentation du Québec) is gratefully recognized.«Ces travaux ont été réalisés grâce à une aide financière du Pro-gramme de soutien à l'innovation en agroalimentaire, un programmeissu de l'accord du cadre Cultivons l'avenir conclu entre le ministère del'Agriculture, des Pêcheries et de l'Alimentation du Québec, et Agri-culture et Agroalimentaire Canada».
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