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European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
12
Copyright © 2015 by Academic Publishing House Researcher
Published in the Russian Federation
European Journal of Molecular Biotechnology
Has been issued since 2013.
ISSN: 2310-6255
E-ISSN 2409-1332
Vol. 7, Is. 1, pp. 12-26, 2015
DOI: 10.13187/ejmb.2015.7.12
www.ejournal8.com
UDC 631.531.027: 635.63
Electrochemically Activited Water:
Biophysical and Biological Effects of Anolyte and Catholyte Types of Water
1* Georgi Gluhchev
2 Ignat Ignatov
3 Stoil Karadzhov
4 Georgi Miloshev
5 Nikolay Ivanov
6 Oleg Mosin
1 Institute of Information and Communication Technologies
Bulgarian Academy of Sciences, Bulgaria
Assoc. Professor, Ph. D.
1113, Sofia, Acad. G. Bonchev Street, 2
*E-mail: gluhchev@iinf.bas.bg
2 The Scientific Research Center of Medical Biophysics (SRC MB), Bulgaria
Professor, D. Sc., director of SRC MB
1111, Sofia, N. Kopernik street, 32
E-mail: mbioph@dir.bg
3 Bulgarian Association of Activated Water, Bulgaria
Professor, D. Sc.
1619, Sofia, Kutuzov blvd., 39
4 Institute of Molecular Biology, Bulgarian Academy of Science, Bulgaria
Assoc. Professor, Ph. D.
1113, Sofia, Acad. G. Bonchev Street, 21
5 Institute of Information and Communication Technologies
Bulgarian Academy of Sciences, Bulgaria
Assis. Professor, Dipl. Eng.
1113, Sofia, Acad. G. Bonchev Street, 2
6 Moscow State University of Applied Biotechnology, Russian Federation
Senior research Fellow of Biotechnology Department, Ph. D.
103316, Moscow, Talalihina ulitza, 33
E-mail: mosin-oleg@yandex.ru
Abstract
This article outlines the results on the antimicrobial action of electrochemically activated
water solutions (anolyte/catholyte), produced in the anode and cathode chamber of the electrolitic
cell. Under laboratory conditions the cell culture and suspensions of classical swine fever (CSF)
virus were treated with the anolyte. After inoculating them with cell cultures, the viral presence
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
13
(the presence of viral antigen) was measured using the immunoperoxidase technique. It was found
that anolyte did not affect the growth of the cell culture PK-15; viral growth during the infection of
a cell monolayer with a cell culture virus was affected in the greatest degree by the anolyte in 1:1
dilution and less in other dilutions; whereas the viral growth at the infection of a cell suspension
with the CSF virus was affected by the anolyte in dilution 1:1 in the greatest degree, and less by
other dilutions; viral growth at the infection with a virus in suspension of the cell monolayer was
affected by the anolyte in all dilutions. Unexpectedly, the stronger biocidal effect of the catholyte
was observed when a strain of E. coli DH5 was treated by the anolyte and catholyte, respectively. In
order to provide additional data about the antiviral activity of the electrochemically activated water
and the distribution of H2O molecules according to the energies of hydrogen bonds, the non-
equilibrium energy spectrum (NES) and differential non-equilibrium energy spectrum (DNES) of
the anolyte and catholyte were measured.
Keywords: anolyte; catholyte; E. coli DH5; CSF virus; NES; DNES.
Introduction
The phenomenon of electrochemical activation of water (EAW) is a set of electrochemical and
electrical processes occur in water in the electric double layer (EDL) type of electrodes (anode and
cathode) with non-equilibrium electric charge transfer through EDL by electrons under the
intensive dispersion in water the gaseous products of electrochemical reactions [1]. In 1985 EAW
was officially recognized as a new class of physical and chemical phenomena.
As a result of the treatment of water by a constant electric current at electric potentials equal
to or greater than the decomposition potential of water (1,25 V), water goes into a metastable state,
accompanied by electrochemical processes and characterized by the abnormal activity levels of
electrons, the redox potential, and other physical-chemical parameters (pH, Eh, ORP) [2].
The main stage of electrochemical treatment of water is the electrolysis of water or aqueous
solutions with low mineralization as aqueous solutions of 0,5–1,0 % sodium chloride (NaCl) [3],
which occurs in the electrolysis cell, consisting of the cathode and the anode separated by a special
semipermeable membrane (diaphragm) which separates water to alkaline fraction – the catholyte
and acidic fraction – the anolyte (Figure 1)
.
When the passing of electric current through water, the
flow of electrons from cathode as well as the removal of electrons from water at the anode, is
accompanied by series of redox reactions on the surface of the cathode and anode [4]. As the result,
new elements are being formed, the system of intermolecular interactions, as well as the
composition of water and the water structure are changed [5, 6].
Figure 1. The diaphragm electrolysis method for the preparation of acid (anolyte) and alkali
(catholyte) solutions via the electrochemical activation of sodium chloride
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
14
The products of electrode reactions are the neutralized aqueous admixtures, gaseous
hydrogen and oxygen generated during the electrolytic destruction of H2O molecules, metal cations
(Al3+, Fe2+, Fe3+) in the case of metal anodes made of aluminum and steel, the molecular chlorine.
Wherein at the cathode is generated the gaseous hydrogen, and at the anode
–
oxygen. Water also
containes a certain amount of hydronium ions (Н3О+) depolarizing at the cathode with formation
of the atomic hydrogen:
Н3О+ + е- → Н + Н2О, (1)
In an alkaline environment there occurs the disruption of Н2О molecules, accompanied by
formation of the atomic hydrogen and hydroxide ion (OH-):
Н2О + е- → Н + ОН-, (2)
The reactive hydrogen atoms are adsorbed on the surfaces of the cathode, and after
recombination are formed the molecular hydrogen H2, released in the gaseous form:
Н + Н → Н2, (3)
At the same time at the anode is released the atomic oxygen. In an acidic environment, this
process is accompanied by the destruction of H2O molecules:
2Н2О – 4е- → О2 + 4Н+, (4)
In an alkaline environment, the source of oxygen source is OH- ions, moving under the
electrophoresis from the cathode to the anode:
4ОН- → О2 + 2Н2О + 4е-, (5)
The normal redox potentials of these reactions compiles +1,23 V and +0,403 V, respectively,
but the process takes place in certain conditions of electric overload.
The cathodes are made of metals that require high electrical voltage (lead, cadmium), allow
to generate the reactive free radicals as Cl*, O*, OH*, HO2*, which react chemically with other
radicals and ions.
In bulk oxidative processes a special role plays products of electrolysis of water – oxygen
(O2), hydrogen peroxide (Н2О) and hydrochlorine acid (HClO). During the electrolysis, an
extremely reactive compound formed
–
Н2О2, the formation of which occurs due to the hydroxyl
radicals (OH*), which are the products of the discharge of hydroxyl ions (OH-) at the anode:
2ОН- → 2OH* → Н2О2 + 2е-, (6)
where ОН* – the hydroxyl radical.
The chlorine-anion is transformed to Cl2:
2Cl- → Cl2 + 2e-, (7)
Gaseous Cl2 forms highly active oxidants: Cl2O; ClO2; ClO-; HClO; Cl*; HO2*. The parameters
of pH, the redox potential, ORP and the electrical conductivity of the anolyte/catholyte depend on
different factors including the ratio of water volumes in the two electric chambers, the material of
electrodes, NaCl concentration, the temperature, electric voltage and processing time [7,8].
The electrolysis cell can be regarded as a generator of the above mentioned products, some of
them, entering into the chemical interaction with each other and water impurities in the
interelectrode space, providing additional chemical treatment of water (electrophoresis,
electroflotation, electrocoagulation) [9]. These secondary processes do not occur on the electrode
surface, but in the bulk water. Therefore, in contrast to the electrode processes they are indicated
as the volume processes. They generally are initiated with increasing the temperature of water
during the electrolysis process and with increasing the pH value.
As a result of the cathode (catholyte) treatment water becomes alkaline: its ORP decreases,
the surface tension is reduced, decreasing the amount of dissolved oxygen in water, increases the
concentration of hydrogen, hydroxyl ions (OH-), decreases the conductivity of water, changes the
structure of hydration shells of ions [10]. By external characteristics the catholyte – is a soft, light,
with an alkaline taste liquid, sometimes with white sediment; its pH = 10
–
11, ORP = -200…-800
mV. On physical and chemical parameters the catholyte has the significantly enhanced electron-
donating properties, and getting into the physiological fluids of an organism can enhance the
electron-background for a few tens of millivolts [11]. The catholyte reportedly has antioxidant,
immunostimulating, detoxifying properties, normalizing ORP, metabolic processes (increases the
ATP synthesis, modification of enzyme activity), stimulates the regeneration of tissues, increases
the DNA synthesis and stimulates the growth and division of cells by increasing the mass transfer
of ions and molecules across the cell membrane, improves trophic processes in tissues and blood
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
15
circulation [12]. It was also reported that catholyte with the redox potential at -700…-100 mV
favorizes the development of anaerobs, whereas the anolyte with the redox potential at
+200…+750 mV supports the growth of aerobs [13]. The antibacterial effect of the catholite is
differentiated: the bactericidal effect is appeared relative to Enterobacteriaceae, resistant to it are
enterococci and the group of streptococci B, and against Gram-negative microorganisms
–
only the
bacteriostatic effect [14].
The electrochemically activated solutions of the catholite, depending on the strength of the
transmitted electric current may be of several types:
C – alkaline catholyte (pH > 9,0; ORP = -700…-820 mV), the active components
–
NaOH,
О2, НО2-, НО2*, ОН-, ОН*, НО2-, О2;
CN
–
neutral catholyte (pH = 9,o; ORP = -300…-500 mV), the active components
–
О2, НО2-,
НО2*, Н2О2, Н+, ОН-.
As a result of the anode (anolyte) treatment water becomes acid reaction, the ORP increases
slightly, the surface tension is slightly reduced, the conductivity increases, the amount of the
dissolved oxygen and chlorine in water also increases, whereas the amount of hydrogen decreases
[15]. The anolyte is a brownish, acid, with a characteristic odor and taste the liquid with a pH = 4
–
5
and ORP = +500…+1100 mV. The specific anolyte toxicity when being administered in the stomach
and applying to the skin refers to the class 4 of harmful substances according to the Russian
Standard GOST 12.1.007-76, with the minimal toxicity within this class. When being inhaled the
anolyte with oxidants content of 0,02 % and total mineralization 0,25
–
0,35 % does not irritate the
respiratory system and mucous membranes of the eyes. When introduced into the organism, the
anolyte has no immunotoxic action and increased chromosomal aberrations in the bone marrow
cells and other tissues, and it has no cytogenetic activity. When being heated to 50 0C the
bactericidal activity of the anolyte is increased by 30
–
100 % [16].
The electrochemically activated solutions of the anolyte are divided into four main types:
A
–
acidic anolyte (pH < 5,0; ORP = +800…+1200 mV), the active components
–
НСlО, Сl2,
НСl, НО2*;
AN
–
neutral anolyte (pH = 6,0; ORP = +600…+900 mV), the active components
–
НСlО,
О3, НО-, НО2*;
ANK
–
neutral anolyte (pH = 7,7; ORP = +250…+800 mV), the active components
–
НСlО,
СlО-, НО2-, Н2О2, О2, Сl-, НО*;
ANKD
–
neutral anolyte (pH = 7,3; ORP = +700…+1100 mV), the active components
–
НСlО, НСlО2, СlО-, СlО2*, НО2*, Н2О2, О2, О3, Сl-, НО-, О*.
The anolyte has antibacterial, antiviral, antifungal, anti-allergic, anti-inflammatory,
antiedematous and antipruritic effect, may be cytotoxic and antimetabolite action without harming
the human tissue cells [17]. The biocide elements in the anolyte are not toxic to somatic cells, as
represented by oxidants, such as those ones produced by the cells of higher organisms.
Studies on the virucidal effect of the anolyte are rare and insufficient, basically on the
possibilities of applying the anolyte in the implementation of effective control of viral diseases in
humans and animals and especially on particularly dangerous viral infections, as staphylococcal
Enterotoxin-A [18]. One of them is the classical swine fever (CSF), prevalent in different regions of
the world and inflicting heavy economic losses. It is caused by enveloped viruses belonging to the
genus Pestivirus of the family Flaviviridae [19, 20]. The resistance and inactivation of the virus of
CSF virus is a subject of extensive research. Although it is less resistant to external stresses other
than non-enveloped viruses, it retains its virulence for a long period of time: in frozen meat and
organs – from a few months up to one year; in salted meat – up to three years; in dried body fluids
and excreta – from 7 to 20 days. In rotting organs it dies for a few days and in urine and faeces –
for approx. 1
–
2 days. In liquid fertilizer it can withstand 2 weeks at 20 0C, and over 6 weeks at 4 0C.
Its thermal resistance may vary depending on the strain type, but the inactivation is dependent
mostly on the medium containing the virus. Although the CSF virus loses its infectivity in cell
cultures at 60 0C for 10 min, it is able to withstand at least 30 min at t = 68 0C in defibrinated
blood. It is relatively stable at pH = 5
–
10, and the dynamic of the inactivating process below pH = 5
depends on the temperature.
According to J.A. Sands [21] and U.S. Springthorpe [22], the effective disinfection of viruses
whose infectivity is associated with the elements of the casing is achieved by disinfectants
dissolving fats, surfactants, disinfectants or fatty acids, organic solvents (ether and chloroform),
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
16
detergents, proteases, and common disinfectants. It is believed that 2 % solution of sodium
hydroxide is most suitable for the disinfection of spaces contaminated with them. It is thought that
to achieve the effective electrochemical disinfection it is necessary to irreversibly damage the
RNA [23].
Investigations conducted by other authors [24] were carried out with E. coli, using as a
desinfectant the anolyte with ORP equal or greater than +1100 mV and pH = 5,5, obtained via
electrolysis of diluted NaCl solution on planktonic cells of a strain of E. coli JM109. It was
demonstrated that within 5 min of influence all cells were inflated and burst. Also, it was occurred
a full destruction of proteins, DNA and RNA. Supposedly the anolyte enters the cells provoking
structural and functional damages on the cell„s membrane and cell„s wall.
Similar research was performed by S.V. Kumar et al. [25]. They evaluated the inactivation
efficacy of anolyte of pH = 2,7 and ORP = + 1100 mV on Escherihia coli O157:H7, Salmonela
enteritidis and Lusteria monocytogenes. As it was demonstrated on five strains of E. coli E06
(milk), E08 (meat), E10 (meat), E16 (meat) and E22 (calf feces), all patogens were significantly
reduced (7,0 logCFU/ml) or fully destroied (8,0 logCFU/ml) after 2 to 10 min inactivation by the
anolyte in the temperature range from 4 0C to 23 0C. Supposedly, the low pH value of the anolyte
makes sensitive the outer cell„s membrane, thus facilitating HClO to enter the cell and further
destroy it.
However, it should be noted that the pharmacological studies of electrochemically activated
solutions of water and their virucidal effects and toxicity have not yet been completely evaluated.
Therefore, the purpose of this research was to study the antiviral virucidal effect: 1) of the anolyte
in different dilutions on classical swine fever virus in cell culture and organ suspensions; 2) of the
anolyte/catholyte on a strain of E. coli DH5a, and 3) to determine how the virocidal effect relates to
local maximums in NES-spectra of the anolyte and catholyte*.
Material and Methods
The studies of the antiviral activity of the anolyte were performed at the National Reference
Laboratory of Classical and African Swine Fever, section “Exotic and Especially Dangerous
Infections” of the National Diagnostic and Research Veterinary Medical Institute (Sofia, Bulgaria).
Experiments were conducted with the anolyte obtained by the electrolysis apparatus
“Wasserionisierer Hybrid PWI 2100” equipped with four titanium electrodes coated with platinum.
0,3 % solution of chemically pure sodium chloride (NaCl) in distilled water was used for the
electrolysis. The obtained anolyte had pH = 3,2 and ORP = +1070 mV. The interaction of the
anolyte with the virus suspension was carried out at a temperature of 22 0C.
A cell culture of porcine origin sensitive to the CSF virus was used: a continuous cell line was
PK-15. Contamination of cell cultures was carried out with the standard cell culture test virus 2,3
(Bulgaria) with a cell titre 107,25 TCID50/ml and organ suspension of internal organs (spleen,
kidney, lymph node) of wild boar originating from the last outbreak of CSF in Bulgaria in 2009.
The titer of the established virus in the suspension was 104,75 TCID50 ml.
To establish the virucidal activity of the anolyte, the inocula prepared for contamination of
cell culture (cell culture virus) were treated with the following dilutions of the anolyte in sterile
distilled water: 1:1 (50 %), 1:2 (33,33 %), 1:3 (25 %), 1:4 (20 %). These dilutions were mixed with
inocula in proportion 1:1 (100 μl of the CSF virus suspension and 100 μl of the appropriate anolyte
concentration). The time of action was conformed to the period, at which it was methodologically
necessary to “capture” any viral presence in the cell culture. Upon the infection of a cell monolayer,
the mixture was removed after the end of the exposure period of 1 h. Upon the infection of a cell
suspension, the mixture, otherwise, was not removed.
To establish the virucidal activity of the anolyte on the CSF virus in the suspension, a
different dilution was used: the inoculum was mixed directly with the concentrated anolyte in
anolyte-inoculum ratios 1:1; 3:1; 7:1 and 15:1 respectively. Since it is known that the growth of the
virus does not cause a cytopathic effect, therefore, for demonstration of its presence,
immunoperoxidase plates dyeing were used. The cells were fixed and the viral antigen was detected
after binding to a specific antibody labeled with peroxidase. The organs exude 1 cm3 of tissue,
* Such a dependence was established between the local maximum (-0,1387 eV; 8,95 µm) in the NES-
spectrum of the catholyte that suppresses the development of tumor cells (Ignatov & Mosin, 2014).
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
17
which was homogenized in a mortar with 9 ml of the cell culture medium containing antibiotics, in
order to obtain 10 % of organ suspension. Sterile sand was added to improve the homogenization.
The samples were kept at room temperature for 1 h, after that they were centrifuged for 15 min at
2500 g. The supernatant was used to infect the cells. In case of cytotoxic effect, the parallel
dilutions of the homogenates were prepared in proportions 1:10 and 1:100. From the suspensions
into multi well (24-well) plates were added 200 μl of the inoculums with coverage of 50
–
80 %. Cell
cultures were incubated at t = 37 0C for 1 h in order to “capture” an eventual virus if presented, then
they were rinsed once with PBS and fresh media were added. Alternatively, the plate was filled
directly (cell suspension), since the preliminary studies had found that the anolyte did not induce a
cytotoxic effect.
The cell cultures were incubated for 72
–
96 h at t = 37 0C in a CO2 incubator. The procedure
with preparation of the positive and negative control samples was similar. The positive control
sample was a reference strain of the CSF virus. The immunoperoxidase technique with using a
horseradish peroxidase was used for the enzymatic detection of antigen-antibody complexes in cell
cultures. The fixation of the plates was carried out thermally for ~3 h at t = 80 0C in a desiccator.
In the processing was used a primary monoclonal antibody C 16, diluted in proportion 1:50, and
secondary antibody RAMPO, diluted in proportion 1:50. For the immunoperoxidase staining was
used 3 % H2O2 and AEC (dimethylformamide and 3-amino-9-ethylcarbazole) in acetate buffer. The
antibody-antigen complex was visualized by the peroxidase reaction with the substrate.
A polymerase chain reaction (PCR) to amplify the segments of the RNA was carried out in
real time scale. The cell culture and organ suspensions were examined for the presence of the CSF
viral genome by the PCR in real time (real-time RT-PCR, one step, TagMan), one-step according to
Protocol of the Reference Laboratory for CSF of EU. For RNA extraction was used the test QIAamp
Vital RNA Mini Kit, Qiagen Hilden (Germany). The initial volume of the biological material was
140 μl, and the elution volume – 60 μl.
For amplification of PCR was used the test Qiagen OneStep RT-PCR Kit in a total volume of
25 μl, and template volume of 5 μl. In the PCR were used primers A 11 and A14, and probe TaqMan
Probe
–
FAM
–
Tamra.
PCR studies were carried out with a thermo cycler machine “Applied Biosystems 7300 Real
Time PCR System” with the temperature control for reverse transcription at t = 50 0C – 30:00 min,
inactivation of reverse transcriptase and activation of Taq at t = 95 0C
–
15:00 min, denaturation at
t = 95 0C
–
00:10 min, extension at t = 60 0C
–
00:30 min for 40 cycles.
The second study on the antimicrobial activity of the anolyte/catholyte was performed at the
Institute of Molecular Biology of the Bulgarian Academy of Sciences (BAS). The two
electrochemical solutions were prepared with using the Activator-I, developed at the Institute of
Information and Communication Technologies at BAS. For this, drinking water without aditional
quantity of NaCl was used. This led to pH = 3,0 and ORP = +480 mV for the anolyte, and pH = 9,8
and ORP = -180 mV for the catholyte.
Bacterial strain used in these experiments was E. coli DH5α with genotype: fhuA2
lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17.
The Colony Forming Units (CFU) technique was used in this study to assess cellular viability.
The conditions for the bacterial cultures growth were as described in our previous paper [26]. The
bacterial cells were cultivated on the LB-medium (pH = 7,5) with 1 % bactotryptone; 0,5 % yeast
extract; 1,0 % NaCl at t = 37 0C. After overnight cultivation of bacteria 100 µl samples of culture
liquids were taken, centrifuged for 1 min at 10000 g and the pellet of bacterial cells was
resuspended in 100 µl of the anolyte or the catholyte. As control samples were used the bacterial
samples, re-suspended in non-electroactivated water. Different dilutions of cells were spread on
LB-agar Petri plates. After the overnight incubation at t = 7 0C the appeared bacterial colonies were
counted. The viable cells were calculated as a percentage from the CFU. The CFU obtained from
culture liguids treated with non-electrochemically activated water were accepted as 100 %.
The NES method was used for the estimation of energy of hydrogen bonds of the anolyte,
catholyte and deionized water in order to make a supposition about the spectrum characteristics.
The device measures the angle of evaporation of water drops from 72 0 to 0 0. As the main
estimation criterion was used the average energy (∆EH...O) of hydrogen O...H-bonds between
individual H2O molecules in water‟s samples. The NES-spectrum of water was measured in the
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
18
range of energy of hydrogen bonds 0,08
–
0,387 eV or = 8,9
–
13,8 µm with using a specially
designed computer program.
Results and Discussion
Research into the effects of electro-activated aqueous NaCl (anolyte) on the
CSF virus
As shown in Figure 2 the cytoplasm of cells infected by the CSF virus was stained in the dark
reddish brown color (positive reaction), whereas in the uninfected cells it was colorless. That
indicates on the presence of viral antigen in the samples.
Figure. 2. The established presence of viral antigen in cell cultures (left)
and a negative control (right)
Table 1 summarizes the results of different experiments of the virucidal action of the anolyte
on the cell culture suspension of the CSF virus upon infecting cell monolayer PK-15. As is shown in
Table 1, upon treatment of the viral inoculum with the anolyte in a 1:1 dilution, there was no viral
growth in the four infected wells of the plate, upon 1:2 dilution there was no growth in two of the
wells, the other two were reported as positive. Upon treatment with the anolyte at dilutions 1:3 and
1:4, the result was identical: no growth in one of the contaminated wells of the plate, and poor
growth – in the other three. The results obtained by infection of CSF virus a cell monolayer and cell
suspension were identical.
Table 2 summarizes the results of studies aimed at the evaluation of the virucidal effect of the
anolyte on organ suspension containing CSF virus upon infecting a cell monolayer PK-15 with the
virus. According to the data, upon treatment of the CSF viral inoculum (organ suspension) with the
anolyte in all dilutions, there was no viral growth in the four infected wells of the plate.
Table 1: The virucidal action of the anolyte on cell culture suspensions of the CSF virus upon
infecting cell monolayer PK-15
Contamination
of CC with:
Dilutions of
anolyte (100 µl)
Total volume
of the
inoculum
(µl)
Concentration
of anolyte in %
Number
of wells:
Result:
positive/
negative:
Virus 200 µl
–
200
–
4
4/0
Virus 100 µl
1:1
200
25
4
0/4
Virus 100 µl
1:2
200
16,51
4
2/2
Virus 100 µl
1:3
200
12.5
4
3/1
Virus 100 µl
1:4
200
10
4
3/1
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
19
Table 2: The virucidal action of the anolyte on organ suspensions containing CSF virus upon
infecting cell monolayer PK-15
Contamination
of CC with:
Dilutions
of anolyte
(100 µl)
Total volume
of the
inoculum (µl)
Concentration
of anolyte in %
Number
of wells:
Result:
positive/ negative:
Virus 200 µl
–
200
–
4
4/0
Virus 100 µl
1:1
200
50
4
0/4
Virus 50 µl
3:1
200
75
4
0/4
Virus 25 µl
7:1
200
87
4
0/4
Virus 12,5 µl
15:1
200
94
4
0/4
Evidently, the anolyte has a destructive influence on the envelope of the CSF virus, wherein
the main antigens (proteins) are localized. Studies of the viral inocula used in the tests by means of
polymerase chain reaction (PCR) in real time demonstrated the presence of a genome (RNA) in
them, also after the treatment with the anolyte. Some shortening of the time was proved (the
decreased number of amplification cycles), required for the formation of a fluorescent signal,
respectively, a positive reaction for a genome, closely correlated with the exposure under the
treatment of the viral inocula. The longer the exposure of processing with the anolyte, the sooner
the presence of the viral RNA in the PCR was detected. According to one of our co-authors (Stoil
Karadzhov), this may serve as an indirect indication that anolyte destroys the CSF virus envelope,
which, in its turn, facilitates the extraction of viral RNA and its more rapid reading by the
fluorescent signal. However, there is still no sufficient convincing evidence on the impact of
different concentrations of the anolyte on CSF viral particles. The analogous experiments carried
out by Russian and German researchers were carried out mainly with the concentrated anolyte.
The maximum virucidal effect detected in those experiments confirmed a strong virucidal action of
the electrochemically activated aqueous solution of NaCl on the CSF virus. The difference in the
results evidently is due to the use of lower concentrations of NaCl in our experiments.
We attributed essential significance to the fact that we determined the concentration limit (25 %) of
the well demonstrated by the virucidal activity. In this aspect the further studies on reducing the
time of the virucidal action, and the conducting of experiments in the presence of biofilms which
protect viruses would be promising.
Research into the antibacterial effects of the anolyte and catholyte on a strain
of E. coli
In order to assess the effect, if any, of the electrochemically activated water solutions
(catholyte/anolyte) on bacterial cells we treated the cultures of a strain of E. coli DH5a by the
catholyte. After the treatment of bacterial cells the colonies appearing on the plates with 2 % agar
were obtained, produced by survived cells, which were further counted by the CFU method.
Therefore, the number of colonies was presented on Figure 3 as a percentage of viable cells. It can
be seen from Figure 3 that bacterial cells of E. coli DH5a treated with the catholyte hardly survived
the treatment with only approximately 15 % of the cells being survived. This clearly shows that the
electrochemically activated water produced from the cathode possesses a strong bacteriocidal
activity on the strain of E. coli DH5a.
Notably, the anolyte also showed slight antibacterial effect. Thus, approximately, 73 % of the
bacterial cells of E. coli DH5a survived the electrochemical treatment with the anolyte. In
summary, it is assumed that both types of the electrochemically activated water solutions
(catholite/anolyte) possess antibacterial effect on the strain of E. coli DH5a, however it is obvious
that the catholyte has a stronger bacteriocide effect than the anolyte.
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
20
Control Catholyte Anolyte
Figure 3. Percentage of viable cells of E. coli DH5a after the electrochemical treatment with the
catholyte and anolyte relative to the non-electrochemically activated water.
Figure 4. The dependence between the acidity and basicity (pH)
of electrochemically activated solution of NaCl and the oxidation-reduction potential (ORP)
on the biosphere of microorganisms.
Figure 4 shows the dependence between the acidity and basicity (pH) of electrochemically
activated solution of NaCl and the oxidation-reduction potential (ORP). The pH value within the
interval from 3 to 10 units and the ORP within the interval from -400 mV to +900 mV characterize
the area of the biosphere of microorganisms. Outside these ranges of pH and ORP the
microorganisms will hardly survive. The disinfecting effect in this case is strengthened by the
residual chlorine in electrochemically activated solution of NaCl, destructing unsaturated fatty
acids, phospholipids and protein in the cell membrane.
NES and DNES methods in spectral analysis of the anolyte and catholyte
Other method for obtaining useful information about the structural changes in water and the
average energy of hydrogen bonds is the measuring of the energy spectrum of the water state.
It was established experimentally that at evaporation of water droplet the contact angle θ decreases
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
21
discretely to zero, whereas the diameter of the droplet changes insignificantly [27]. By measuring
this angle within a regular time intervals a functional dependence f(θ) can be determined, which is
designated as “the spectrum of the water state” (SWS) [28]. For practical purposes by registering
the SWS it is possible to obtain information about the averaged energy of hydrogen bonds in an
aqueous sample. For this purpose the model of W. Luck was used, which consider water as an
associated liquid, consisted of О–Н…О–Н groups [29]. The major part of these groups is
designated by the energy of hydrogen bonds (-E), while the others are free (E = 0). The energy
distribution function f(E) is measured in electron-volts (eV-1) and may be varied under the
influence of various external factors on water as temperature and pressure.
For calculation of the function f(E) experimental dependence between the water surface
tension measured by the wetting angle (θ) and the energy of hydrogen bonds (E) is established:
f(E) = bf(θ) / [1 – (1 + bE)2]1/2, (8)
where b = 14,33 eV-1; θ = arcos(1 – bE)
The energy of hydrogen bonds (Е) measured in electron-volts (eV) is designated by the
spectrum of energy distribution. This spectrum is characterized by non-equilibrium process of
water droplets evaporation, thus the term “non-equilibrium energy spectrum of water” (NES) is
applied.
The difference ∆f(E) = f (samples of water) – f (control sample of water) – is designated as
the “differential non-equilibrium energy spectrum of water” (DNES) [30].
The DNES-spectrum measured in milielectron volts (0,001 eV) is a measure of changes in the
structure of water as a result of external factors. Figure 5 shows the characteristic NES-spectrum of
deionized water made from 25 independence measurements performed in a period of one year.
Figure 5. NES-spectrum of deionized water (chemical purity – 99,99 %; pH – 6,5–7,5; total
mineralization – 200 mg/l; electric conductivity – 10 S/cm). The horizontal axis shows the energy
of the H...O hydrogen bonds in the associates – E (eV). The vertical axis – energy distribution
function – f (eV-1). k – the vibration frequency of the H–O–H atoms (cm-1); λ – wavelength (m)
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
22
The average energy (∆EH... O) of hydrogen Н…O-bonds among individual molecules H2O was
calculated for the catholyte and analyte by NES- and DNES-methods. We studied the distribution
of local maximums in catholyte and anolyte solutions. The local maximum for catholyte in the
NES-spectrum was detected at -0,1285 eV, for anolyte
– at
-0,1227 eV, and for the control sample of
deionized water
– at
-0,1245 eV. The calculations of ∆EH...O for the catholyte with using the DNES
method compiles (-0,004±0,0011 eV) and for anolyte (+1,8±0,0011 eV). These results suggest the
restructuring of ∆EH... O values among individual H2O molecules with a statistically reliable increase
of local maximums in DNES-spectra of the catholyte and anolyte (Table 3).
For the catholyte the biggest local maximum was detected at -0,1387 eV, or at 8,95 μm.
In 1992 A. Antonov performed experiments with the impact of different types of water on tumor
mice cells. It was detected a decrease in the NES-spectrum compared with the control sample of
cells from healthy mice. There was also a decrease of the local maximum at -0,1387 eV, or 8,95 μm
in DNES-spectra. Notably, the local maximum at 8,95 μm was detected with the negative value.
It should be noted that for the catholyte the local maximum in the DNES-spectrum was detected
with the positive value at +133,3 еV-1.
For the catholyte the biggest local maximum in the DNES-spectrum was detected at -0,1312
eV, or 9,45 μm. It should be noted that for the treatment of influenza in medical drugs is included
Al(OH)3 [31]. The local maximum in this case was measured at -0,1326 eV, or at 9,35 μm.
The evaluation of the possible number of hydrogen bonds as percent of H2O molecules with
different values of distribution of energies is presented in Table 4. These distributions are basically
connected with the restructuring of H2O molecules with the same energies. This serves as the base
for evaluating the mathematical model explaining the behavior of the anolyte and catholyte
regarding the distribution of H2O molecules to the energies of hydrogen bonds [32].
Table 3: Local maximums of catholite and anolyte solutions in NES- and DNES-spectra
-Е(eV)
x-axis
Catholyte
Anolyte y-axis
(еV-1)
Control sample
y-axis (еV-1)
DNES
Catholyte
DNES
Anolyte
-Е(eV) x-axis
Catholyte y-
axis (еV-1)
Anolyte y-axis
(еV-1)
Control
Sample y-axis
(еV-1)
DNES
Catholyte
y-axis (еV-1)
0,0937
0
0
0
0
0
0,1187
0
66,7
66,7
-66,7
0,0962
0
0
0
0
0
0,1212
66,7
0
0
66,7
0,0987
0
0
0
0
0
0,1237
0
0
0
0
0,1012
66,7
66,7
33,3
33,4
33,4
0,1262
0
0
66,7
-66,7
0,1037
0
0
33,3
-33,3
-33,3
0,1287
0
0
66,7
-66,7
0,1062
0
0
0
0
0
0,1312
33,3
100
33,3
0
0,1087
0
0
0
0
0
0,1337
33,3
33,3
33,3
0
0,1112
0
0
0
0
0
0,1362
0
0
0
0
0,1137
0
66,7
66,7
-66,7
0
0,1387
200
66,7
66,7
133,3
0,1162
0
0
0
0
0
–
–
–
–
–
Table 4: Energy distribution of catholyte and anolyte solutions
in electrochemical activation of sodium chloride
-Е(eV)
x-axis
Catholyte
y-axis, %
(-Evalue)/
(-Etotal value)
Anolyte
y-axis, %
(-Evalue)/
(-Etotal value)
-Е(eV)
x-axis, %
(-Evalue)/
(-Etotal value)
Catholyte
y-axis, %
(-Evalue)/
(-Etotal value)
Anolyte
y-axis, %
(-Evalue)/
(-Etotal value)
0,0937
0
0
0,1187
0
16,7
0,0962
0
0
0,1212
16,7
0
0,0987
0
0
0,1237
0
0
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
23
0,1012
16,7
16,7
0,1262
0
0
0,1037
0
0
0,1287
0
0
0,1062
0
0
0,1312
8,4
24,8
0,1087
0
0
0,1337
8,4
8,4
0,1112
0
0
0,1362
0
0
0,1137
0
16,7
0,1387
49,8
16,7
0,1162
0
0
–
–
–
Conclusions
The experimental results prove the strong influence of different types of electrochemically
activated water solutions (catholyte/anolyte) on various microbes and viruses. They are in
accordance with the results obtained by other researchers, and demonstrate the strong biocidal
effect of the anolyte toward the CSF virus. Also, the interesting results on the antibacterial effect
were obtained when a strain of E. coli DH5a was treated with the catholyte and anolyte,
respectively. Unexpectidely, the catholyte with ORP ≈ -180 mV and pH = 9,8 demonstrated the
better biocidal effect than the anolyte with ORP ≈ +500 and pH = 3,9. We tried to relate the
antimicrobial and antiviral action of electrochemically activated water with the characteristics of
the NES-spectrum. There is an indication about such a connection but more thorough research is
needed to prove it. For example, the inverse biocidal effect between the catholyte and anolyte in
case of a strain of E. coli DH5a requires a clear explanation.
The results of the research are formulated as follows.
1. The anolyte did not affect the growth of the cell culture PK-15;
2. The anolyte administered at a concentration of 25 %, exerts a strong virucidal effect on a
cell culture virus, and a weaker antiviral activity at concentrations of 16,51 %, 12,5 % and 10 %;
3. The anolyte exerted a strong virucidal effect at concentrations of 50 %, 75 %, 87 % and
94 % over the CSF virus in cell culture suspensions;
4. The catholyte supresses the growth of E. coli up to 85 % while anolyte is at least three
times less effective;
5. The local maximum in the DNES-spectrum of the catholyte was detected at 9,85 μm; there
was a decrease of this local maximum in water with mice tumor cells;
6. The local maximum in the DNES-spectrum of the anolyte was detected at 9,45 μm; at
9,35 μm occurred the effect of inflammation from virus of influenza;
7. The mathematical model of the catholyte and anolyte regarding the distribution of H2O
molecules to the energies of hydrogen bonds was evaluated.
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УДК 631.531.027: 635.63
Электрохимически активированные растворы воды:
Биофизические и биологические эффекты католита и анолита
1* Георгий Глухчев
2 Игнат Игнатов
3 Стоил Караджов
4 Георгий Милошев
5 Николай Иванов
6 Олег Викторович Мосин
1 Институт информации и коммуникационных технологий
Болгарская Академия Наук, Болгария
Доцент, Ph. D.
1113, София, Акад. Г. Бончева ул., 2
*E-mail: gluhchev@iinf.bas.bg
2 Научно-исследовательский центр медицинской биофизики (РИЦ МБ), Болгария
Профессор, доктор наук Европейской академии естественных наук (ФРГ), директор НИЦ
МБ.
1111, София, ул. Н. Коперника, 32/6
E-mail: mbioph@dir.bg
3 Болгарская ассоциация активированной воды, Болгария
Профессор, D. Sc.
1619, София, Кутузов бульв., 39
4 Институт молекулярной биологии
Болгарская Академия наук, Болгария
Доцент, Ph. D.
1113, София, Акад. Г. Бончева ул., 2
5 Институт информации и коммуникационных технологий
Болгарская Академия Наук, Болгария
Доцент, диплом. инженер
1113, София, Акад. Г. Бончева ул., 2
6 Московский государственный университет прикладной биотехнологии, Российская
Федерация
Старший научный сотрудник кафедры биотехнологии, канд. хим. наук
103316, Москва, ул. Талалихина, 33
E-mail: mosin-oleg@yandex.ru
Аннотация. В статье описываются результаты антимикробного действия
электрохимически активированных водных растворов (анолит/католит), полученных в
анодной и катодной камере электролитической ячейки. В лабораторных условиях культура
клеток суспензии вируса свиного гриппа была обработана анолитом. После прививки их с
культурами клеток присутствие вируса (наличие вирусного антигена) было измерено с
использованием иммунопероксидазного метода. Было обнаружено, что анолит не влияет на
рост культуры клеток РК-15; вирусной рост при заражении клеточного монослоя замедлялся
вирусом в наибольшей степени при разведении анолита в пропорции 1:1 и менее в других
European Journal of Molecular Biotechnology, 2015, Vol.(7), Is. 1
26
разведениях; в то время как вирусный рост при инфекции клеточной суспензии с вирусом
замедлялся анолита в наибольшей степени в разведении 1:1, и менее в других разведениях;
вирусный рост при инфекции вирусом суспензии клеток монослоя зависел от присутствия
анолита во всех разведениях. Неожиданно сильный биоцидный эффект католита
наблюдался при обработке штамма E. coli DH5 анолитом и католитом соответственно.
Для получения дополнительных данных о противовирусной активности
электроактивированных растворов воды, а также о структурных изменениях, были
измерены неравновесный энергетический спектр (НЭС) и дифференциальный
неравновесный энергетический спектр (ДНЭС) анолита и католита.
Ключевые слова: анолит; католит; E. coli DH5; вирус свиного гриппа; НЭС; ДНЭС.
