An ideal disinfectant should posses high bactericidal activity, long shelf life, be ready for use without any preliminary activation and utilized after use without negative effect to the environment. Long storing of stable chemicals is available, but their utilization requires equivalent activation by another agent or energy. Therefore, combination of stability and easy utilization is impossible.
In order to exclude the preliminary activation stage before usage of a liquid chemical germicide, it should be noted that all variety of biocidal agents are belong to few classes of chemicals well-known for tens of years.
Appearance of new class of chemicals, which will meet the requirement, is unlikely. Modern tendency in developing new disinfectants is in search for activation means of known disinfectants, and not the creation of new ones. Addition of activators using an extra physical influence, i.e. creating conditions converting active ingredients into metastable state at the moment of disinfection, is one of main directions for the disinfection efficacy improvement.
Interaction disinfectants and microorganisms’ cell membrane.
Due to complexity and multi-functionality of microorganisms’ membrane specific interaction between membrane’s biopolymers and abovementioned chemicals is hardly studied at all.
Cytoplasmic membrane is extremely vitally important structure of any cells including microbes. Organic compounds are part of it and have many reactive groups that cause a high sensitiveness of membrane to damaging factors of different nature. It is known that high concentration of membrane-attacking agents destroy biopolymers of membrane, resulting in damaging lysis of microbe’s cell. The same chemicals in small doses affect membrane functions – change osmotic pressure, permeability, transport processes of molecules and ions through membrane, inhibit metabolic processes, bio-oxidation and cell divisions.
Cationic surfactants (quarternary ammonium compounds) are concentrated at membrane and bind with phosphatidic groups of its lipids; anionic surfactants such as alkaline detergents, alkyl- and arylsulfones, iodophors react with membrane lipids. Phenols and alcohols dissolve lipid’s fragments of membrane.
After disinfection treatment is completed, the moist surfaces get dry, so organic compounds are concentrated in a volume of porous material and turn into superfine and invisible to the eye film. Then it evaporates by sublimation with less intensity than under evaporation during wet treatment. Formed aerosol frequently has no smell that creates illusions of its harmlessness. One should take into account that in accordance with known physical laws each liter of the air in the room contains about some milliards of molecules of matter vaporized with natural course or due to sublimation even if its concentration could be hardly measured and does not exceed hundreds or thousands parts from maximum permissible concentration (MPC). During breathing as well as through the skin and mucous membrane such molecules penetrate to human organism (patients or medical staff) and each one of it keeps realizing its main function – suppression of vital function of cells, but this time in a human body. Stability of liquid chemical germicides creates their accumulation in organism followed by migration through digestive cycle.
Colonies of microorganisms form resistance to dry inefficient disinfectant and start using it as a nutrient medium. Processes as described above have recently become an object of attention; so it is in a stage of study now.
It is quite evident that the development of new liquid chemical germicides which allow bacteria to develop resistance in a short period of time, creates conditions for improvement of mutability mechanism of pathogens and initiates appearance of new isolates of microorganisms.
Today by efficacy of disinfectants is implied its spectrum of biocidal activity. Efficacy also relates to exposure time required for disinfection. However, taking a broad view on the subject we should say that disinfectant is effective only in the case it has a broad spectrum of biocidal activity and does not stimulate microorganism’s adaptation during a long-term use. In other words, effective disinfectant must be used for years with certainty that microorganisms could not form adaptation to it for principal reasons.
Mechanism of Antibacterial defence
Let us consider a mechanism of antibacterial defense created by nature and functions in internal environment of life organisms – from unicellular organisms up to human – over million of years and without any fail.
It is proved that leading role in bactericidal effect of neutrophils belongs to hypochlorous acid (HOCL) made by phagocytes. Under respiratory burst about 28% of oxygen used by neutrophils is spent for formation of HOCL. HOCL is generated from hydrogen peroxide and chloride-ions in neutrophils. Catalyst of this reaction is myeloperoxidase (MPO):
H2O2 + Cl [Cat (МPО)] HOCL + OH [9, 10].
Hypochlorous acid dissociates in aqueous media with formation of hypochlorite-anion and hydrogen-ion:
HOCL ClO + Н+.
Concentrations of HOCL and hypochlorite-anions ClO are almost equal at neutral pH. A decrease in pH shifts reaction balance towards to HOCL, and an increase of pH raises concentration of hypochlorite-anions.
A formation of H2O2 and HOCL in a short time (fractions of a second) in a little volume of aqueous media (parts of microliter, in a volume of active zone of phagocytosis) – inevitably must be followed by reactions of spontaneous decomposition and interaction of reaction products with formation of active particles similar to once formed by radiolysis or electrolysis of water.
Spontaneous decomposition of hydrogen peroxide in aqueous media is followed by formation of highly active biocides (in parenthesis appropriate reactions are presented):
HO2 – hydroperoxide-anion (H2O2 + OH HO2 + H2O);
О22 – peroxide-anion (OH + HO2 O22 + H2O);
О2 – superoxide-anion (O22 + H2O2 O2 + OH + OH );
НО2 – hydrogen peroxide radical (НO + H2O2 H2O + HO2);
HO2 – hydrogen super-oxide (O2 + H2O HO2 + OH).
At the same time it is possible the formation of extremely reactive singlet oxygen 1О2 : (ClO + H2O2 1О2 + H2O + Cl ). Participation of molecular oxygen ion-radical О2 in reactions of phagocytosis is determined experimentally. One of the described above could be the way of its formation.
Formation of free radicals СlO, Сl, НО is possible in aqueous media in presence of НСlО and СlO
HOCL + ClO ClO + Cl + НO.
By modern theory of catalytic processes, a formation of interim activated complex with myeloperoxidase as a catalyst seems also to be most possivle. A dissociation of this complex is followed by formation of О , and medium acidification:
HOCL + ClO [HOCL Cat (МПО) ClO ] 2Сl + 2O + Н+
Active hypochlorite radical СlO can participate in reactions of atomic oxygen (O ) and hydroxyl radical (НO ) formation:
СlO + СlO + ОН Сl + 2O + ОН.
Followed by formation of chlorine radicals:
OH + Cl Cl + OH.
Formed radicals and atomic oxygen take part in microbe’s destruction, oxidizing biopolymers, for example, by the following:
RH2 + OH RH + H2O;
RH2 + Cl RH + HCl;
RH2 + O RH + OH .
A metastable mixture of compounds formed during phagocytosis is a very effective mean for microbe’s destruction due to many spontaneous realized possibilities of changing (irreversible damage) of essential functions of microorganism’s biopolymers at a level of electron transmission. Metastable particles with different values of electrochemical potential possess universal spectrum of action, i.e. they are able to damage all large systematic groups of microorganisms (bacteria, mycobacteria, viruses, funguses, spores) and without damaging of human tissues and other multicellular system organisms.
That can be explained by texture and living activities of cells of that living organisms. Cells of multicellular organisms during their life process, for example, in oxygenase’s reactions of cytochrome P-450, during phagocytosis under microbe’s adhesion and cidal action produce a range of highly efficient oxidants. These cells have a strong chemical system of antioxidant protection with preventing a toxic effect of such compounds on vitally important cellular structures. Antioxidant properties of somatic cells are related to a presence of a strong three-layered lipoprotein’s shell that contains diene conjugates (–С=С–) possessing electron-donor properties and sulfhydric groups (SH). Microorganisms do not have strong mechanisms of antioxidant protection due to absence of mentioned chemical groups.
All somatic cells of living organisms are heterotrophs: their trophism depends on availability of nutritive materials in extracellular medium – glucose, amino acids, fatty acids. Though biological well-being of any somatic cell is up to place it keeps in a process of dispensing of trophic functions of all elements of multicellular system (cell is supported by cell).
Trophic functions of multicellular organisms cells are obeyed to interchangeability law. If a trophism of single cell is disturbed, then this disturbance can be corrected by neurotrophic regulation, functions of adjoining cells, reparative processes, nutritive function of blood and so on.
All microbe’s cells are autotrophs, so their nutrition depends on their own activity, in other words if enzymatic processes in microbe’s cell are depressed, it dies since there is no compensatory mechanism. Microbial cell gets all its trophic functions by enzymatic reactions only. An interaction between microbial cells in their habitat is not a compensatory one, that is to say susceptibility of microbe is in its autonomy.
Natural production of HOCL
Investigations carried out in recent decades indicate that all higher multi-cellular organisms including humans synthesize hypochlorous acid and highly-active meta-stable chlorine-oxygen and hydroperoxide compounds (a meta-stable oxidants’ mixture) in special cellular structures to combat microorganisms and foreign substances. Hypochlorous acid dissociates in aqueous medium forming hypochlorite-anion and hydrogen ion: НOСl OCl– + Н+. When рН values are close to neutral, concentrations of НOСl and hypochlorite-anions OCl– are approximately equal. Lower рН leads to shift of this reaction equilibrium towards higher concentration of НOСl; higher — towards higher concentration of hypochlorite-anions. Sodium hypochlorite demonstrates a considerably lower bactericidal ability than hypochlorous acid.
The highest bactericidal effect of oxygen chlorine compounds is observed with рН varying from 7.0 to 7.6, where concentrations of hypochlorite-ions and hypochlorous acid are comparable. This is due to the fact that the above compounds being conjugated acid and base (НOCl + Н2О + Н3О+ + OCl–; OCl– + Н2О + НOСl + ОН–) form in the given range a meta-stable system capable of generating a number of compounds and particles possessing a much higher antimicrobial ability than hypochlorous acid: 1O2 — singlet molecular oxygen; СlO• — hypochlorite-radical; Сl• — chlorine-radical (atomic chlorine); О• — atomic oxygen; ОН• — hydroxyl radical. Catalysts of reactions with chlorine-oxygen compounds are Н+ and ОН– ions present in water also in approximately equal quantity at рН value close to neutral one.
Chemical production of HOCL
A unique ability of hypochlorous acid to form meta-stable, universal in its scope of antimicrobial action oxidant mixture is widely employed in many disinfectant agents based on cyanuric acid salts (Aquatabs, Deochlor, Chlorsept, Presept, Javelion, Chlor-Clean, Sanival and others) making it possible to decrease active chlorine content in disinfectant working solutions at least 10-fold as compared to sodium hypochlorite solutions, antimicrobial activity of the former being higher. Let us take the mechanism of action of Johnson & Johnson’s Presept tablets as an example. The active ingredient is hypochlorous acid formed in the process of sodium dichloroisocyanurate interaction with water at a рН value of 6.2, maintained by adipic acid contained in the tablets.
However, the use of mentioned disinfectant agents based on cyanuric acid salts is unsafe for human and other warm-blooded organisms since it contain a chlorine organic compound, in particular, sodium dichloroisocyanurate, which, unlike inorganic chlorine-oxygen compounds, does not disappear leaving no traces during desiccation, but accumulates in the environment and human body.
The most efficient antimicrobial agents among all generally known liquid sterilizing and disinfectant means, which demonstrate very low toxicity or no toxicity at all for warm-blooded animals, are electrochemically activated solutions, in particular Neutral electrolyzed Water.
Electrochemical activated HOCL
Maximum use of fundamental difference between living organisms of micro- and macro-biological life is an ideological basis of electrochemical activated biocidal liquids.
As physicochemical process electrochemical activation is an electrophysical and electrochemical influence on water that contains ions and molecules of dissolved substances in it. It takes place under conditions of minimal heat release in the area of dimensional charge at the electrode surface (anode or cathode) of electrochemical system at non-equilibrium charge transfer through the interface “electrode – electrolyte” by electrons.
As a result of electrochemical activation water converts into a metastable (activated) condition showing increased reactivity in different physical-chemical processes during some tens hours. Electrochemical activation allows directly change a composition of dissolved gases, acid-base and redox characteristics of water within the bigger scale then under the equivalent chemical regulation. Chemical reagents (oxidants or reducing agents) in metastable condition can be generated from water and dissolved substances. It is used in processes of water purification and disinfection as well as for water or diluted electrolyte solution transformation into ecologically friendly biocidal (disinfecting/sterilizing solution), cleaning, extractive and other functionally useful liquids.
An Electrolytic Flow Cell is used for electrochemical transformation of water and dissolved substances. A distinctive feature of a Flow Cell is in combination of properties of ideal displacement reactor and ideal mixing reactor in one element as well as high technical and economic characteristics at processing of fresh water and low-mineralized liquids.
Very seldom electrochemically activated solutions (Electrolyzed Water or Super-Oxidized Water) is identified with hypochlorous acid. This is due to inadequate awareness and natural tendency to simplify comprehension by classifying electrochemically activated solutions to well-known hypochlorite ones on the basis of their formal resemblance.
Neutral Electrolyzed Water, unlike 0.5-5.0% hypochlorite solutions possessing only disinfectant ability, is a sterilizing solution at oxidant concentration 0.005 to 0.05%.(5-500ppm)
Benefits of Electrochemical activated HOCL
Active ingredients of Electrochemical activated HOCL (Neutral Electrolyzed Water) are chlorine-oxygen compounds НOСl (hypochlorous acid) and OCl– (hypochlorite-ion).
The combination of active these active chlorine-oxygen substances avoid that microorganisms adapt or become resistant to Neutral Electrolyzed Water, while low total concentration of chlorine-oxygen compounds guarantee absolute safety for man and the environment in the process of its long-term application.
In other words, a mixture of metastable chlorine-oxygen compounds eliminates microbes’ ability for adaptation to bactericidal effect of Neutral Electrolyzed Water. Thus, only a small concentration of chlorine-oxygen compounds guarantees for absolute safety for man and environment under long-term use of Electrolyzed Water.
Neutral Electrolyzed Water is considered non-toxic due to low content of active substances HOCL and OCL–, therefore there is no need to remove it from treated surfaces after treatment.
Total content of active chlorine-oxygen compounds in Neutral Electrolyzed Water oxidant content varies from 50 to 500ppm which is many times lower than in most working solutions of disinfectants routinely used today. Neutral Electrolyzed Water causes no coagulation of protein protecting microorganisms and thanks to its loose structure easily penetrates into micro-channels of living and nonliving matter.
Environmentally friendly electrochemically-activated Neutral Electrolyzed Water has “life time” that is necessary for procedure of disinfection. After its use it spontaneously degrades without formation of toxic xenobiotics and does not require any neutralization before discharging to sewerage.
A chemical potential of molecules and ions in Neutral Electrolyzed Water is much higher than in hypochlorite solutions. A low mineralization of Neutral Electrolyzed Water and its hydration ability helps penetration through cell membrane, creates conditions for intensive osmotic and electro-osmotic oxidant’s transfer into intracellular media. The osmotic transfer of oxidants through shells and membranes of microbe’s cells is more intensive than through membranes of somatic cells due to inherent difference in osmotic gradient of these types of cells. Electrically charged cluster structures formed by dissolved gas molecules in water and electron-active components of medium promote high-speed electro-osmotic carry of oxidants into bacterial cell, because this clusters produce strong local electric fields with high heterogeneity in zones of contact with biopolymers.
Neutral Electrolyzed Water kills microorganisms of bacterial, viral and fungous etiology (Staphylococcus aureus, Pseudumonas aeruginosa, Escherichia coli, hepatitis B virus, poliomyelitis virus, HIV, adenovirus, pathogens of tuberculosis, salmonellosis, dermatomycosis and others). By its efficacy Electrolyzed Water greatly exceeds chloramines, sodium hypochlorite and overwhelming majority of other disinfectants and sterilizing agents.
A sum of active chlorine-oxygen compounds in Neutral Electrolyzed Water (total oxidant content) is within 50 to 500 mg/l, that is many times less than in most solutions of currently used disinfectants. Neutral Electrolyzed Water does not cause coagulation of protein that protects microorganisms and, due to its loosened structure, easily penetrate into pinholes of living and lifeless matter.
Neutral Electrolyzed Water is produced from dilute solution of sodium chloride in drinking water. Total mineralization of initial solution for Neutral Electrolyzed Water is within 0,5 to 5,0 g/l.
To sum up, it can be concluded that the most effective disinfecting liquid in terms of their functional properties and simultaneously very low-toxicity is Neutral Electrolyzed Water (meta-stable low-mineralized chlorine-oxygen antimicrobial solutions), which have no alternative as long as life on Earth is represented by various forms of protein bodies existing in electrolyte of aqueous solutions of mainly sodium and chlorine ions.