Aqueous germicidal composition

Abstract

The invention provides a freshly prepared Cu+ aqueous solution which is germicidal at aerobic conditions, comprising a water- soluble cuprous complex and an oxidi zed form of a reductant dissolved in water from which oxygen was removed, wherein the solution is exposed to air. Methods of preparing and using the Cu+ solution, and corresponding kits, are also provided by the invention.

Description

[0001] Aqueous germicidal composition

[0002] Field of the invention

[0003] The invention relates to the preparation and use of an aqueous germicidal solution based on the antibacterial, antifungal, and antiviral action of copper.

[0004] Background of the invention

[0005] Copper exists in two oxidation states: the cuprous ion (Cu+) and the cupric ion (Cu2+) . The cuprous ion is very unstable in aqueous solutions; it undergoes auto-oxidation-reduction into copper metal and cupric ion, as follows:

[0006] 2Cu+Cu° + Cu2+(1) or is quickly oxidized in aqueous solutions upon exposure to dissolved oxygen, transforming into the cupric form.

[0007] To maintain reasonably stable concentrations of Cu+in aqueous solutions, anaerobic environment (02-free water) is needed, and the aid of suitable reagents, which form stable complexes with the Cu+cation. Reagents such as acetonitrile (CH3CN) (Parker A. J, Search, 4, 426, 1973; Parker A. J et al., Journal of Solution Chemistry, November 1981, Volume 10, Issue 11, pp 757-774) or benzoic acid (CsHsCOOH) (Saphier M. et al., J. Chem. Soc., Dalton Trans, 1845-1849, 1999) provide (in anaerobic environment) stable concentrations of cuprous (Cu+) ions.

[0008] It has been reported that the cuprous ion shows strong antibacterial activity. But it seems that hitherto, the instability of the cuprous ion in aqueous solutions restricted its range of applications. For example, the cuprous ion was formulated in an ointment as a wound care product; an ointment which maintains a dynamic effective concentration of cuprous ion, based on a reduction environment and confers good protection to the active ingredient against oxidation [see: 1) Magal Saphier, Eldad Silberstein, Yoram Shotland, Stanislav Popov and Oshra Saphier, "Prevalence of Monovalent Copper Over Divalenf in Killing Escherichia coli and Staphylococcus aureus" , Current Microbiology 75, pp . 426-430 (2018) and 2) WO 2018 / 104937] .

[0009] When the cuprous ion was formulated in an aqueous carrier to test its antibacterial action, well-controlled anaerobic environment was maintained during use [See Stanislav Popov, Oshra Saphier, Mary Popov, Marina Shenker, Semion Entus, Yoram Shotland and Magal Saphier, "Factors Enhancing the Antibacterial Effect of Monovalent Copper Ions", Current Microbiology, 77, 361-368 (2020) ] . Cu+was produced in deaerated water by the dissolution of cupric sulfate (CuSO SJUO) and the reduction of the Cu2+ions with metallic copper or ascorbic acid. In the presence of acetonitrile as a stabilizing ligand and Cu° as a reductant, the following reaction took place, creating a stabile form of the Cu+ion in the aqueous solution:

[0010] Then the Cu+solution was tested by injection through a three- way syringe valve to syringes filled with LB broth or M9 medium containing bacteria, to maintain anaerobic conditions. A solution prepared in this manner ( Cu2+ / reductant / ligand in O2- free water to give [Cu+(L)n]+) , stored and used anaerobically, is named herein "Cu+anaerobic aqueous solution" . The solution retains its activity for months, but its storage and use require strict conditions.

[0011] Other antimicrobial compositions of copper are reported in US 2011 / 0236504 (copper sulfate formulated in water in the presence of L-ascorbic acid and L-cysteine) and US 2016 / 0220728 (insoluble forms of cuprous salts) . Interestingly, a series of patents in the sixties, dealing with aqueous solutions of ascorbic acid and cupric salts, reported the limited duration of such solutions, indicating that their antimicrobial activity drops very rapidly, within five minutes after preparation, while ascribing the strong antimicrobial action of the solution to the oxidation of ascorbic acid by the cupric ion (Cu2+) . In US 3, 065, 139 it was explained that because of this reason, "the reacting components must not be brought together in water solution until the time of application, or immediately before this application". In US 3, 681,492, it was proposed to stabilize the solution by addition of nitrogenbearing compounds, which can form complex with cupric ion (Cu2+) , thereby preserving the antimicrobial activity of the solution over longer times.

[0012] The invention

[0013] Experimental work conducted in support of this invention shows that a solution formed by combining a cupric salt, a reductant and a suitable ligand in Os-free water, whereby a stable complex of the cuprous ion is produced, can be held and used aerobically (i.e., left exposed to air) as a disinfectant over a reasonable utilization period (the length of time during which such solution is in contact with air after preparation, without evidence of oxidation and loss of potency) .

[0014] A solution that is prepared in this manner (Cu2+ / reductant / ligand in 02-free water to give [Cu(L)n]+) , and allowed to act under aerobic conditions during its utilization period, is named herein "freshly prepared Cu+aerobic aqueous solution". Experimental results reported below show that a freshly prepared Cu+aerobic aqueous solution exhibits strong sanitization action and retains efficacy comparable to that of Cu+anaerobic aqueous solution, over a utilization period of at least one hour, e.g., at least two hours after preparation. The length of the utili zation period may be extended i f the Cu+aerobic aqueous solution is prepared and put to use at a temperature above room temperature , say, up to 45-50 ° C ; the utili zation period of the freshly prepared solution is usually from 1 to 7 hours .

[0015] Thus , the invention is primarily directed to a freshly prepared Cu+aerobic aqueous germicidal solution, comprising a water- soluble cuprous complex and an oxidi zed form of a reductant . That is , the invention provides a freshly prepared Cu+aqueous solution which i s germicidal at aerobic conditions , comprising a water-soluble cuprous complex and an oxidi zed form of a reductant dissolved in water from which oxygen was removed, wherein the solution is exposed to air .

[0016] Another aspect of the invention is a method of disinf ecting / saniti zing using the Cu+solution, e . g . , comprising treating a contaminated surface , surface prone to microbial contamination, or agricultural produce , with a freshly prepared Cu+aerobic aqueous solution that contains a water-soluble cuprous complex and an oxidi zed form of a reductant , prepared by bringing together in 02- free water a cupric salt , a reductant , and a stabili zing ligand capable of complexing with cuprous ions , and applying the solution when left exposed to air .

[0017] Another aspect of the invention is a method of microbial control in water and / or treated wastewater, comprising providing a freshly prepared Cu+aerobic aqueous solution that contains a water-soluble cuprous complex and an oxidi zed form of a reductant prepared by bringing together in 02- free water a cupric salt , a reductant , and a stabili zing ligand capable of complexing with cuprous ions , adding the freshly prepared solution that was left exposed to air to the target water and / or treated wastewater, wherein the microbial control comprises combatting planktonic bacteria and / or biofilm bacteria on a surface in contact with the water and / or inhibiting biofilm formation on a surface prone to biofilm growth.

[0018] To prepare the Cu+aerobic aqueous solution of the invention, cupric ions are reduced to cuprous ions in 02-free water that is prepared beforehand. Expelling dissolved oxygen from water can be achieved by any acceptable technique, namely, boiling water at atmospheric pressure, boiling water under reduced pressure, purging with nitrogen and sonication under reduced pressure, as described, for example, by Butler IB, Schoonen MA, Rickard DT . Removal of dissolved oxygen from water: A comparison of four common techniques. Taianta, 1994 Feb; 1 (2) : 211-5. For example, nitrogen bubbling for 20-40 min at a flow rate of 25 mL / s can reduce the concentration of dissolved oxygen down to 0.2-0.4 ppm. But the low level of dissolved oxygen achieved by boiling water at atmospheric pressure is also satisfactory for the purposes of the invention, and boiling water was the method of choice in the experimental work reported below, leading to good results. By "02-free water" is meant water with a concentration of dissolved oxygen of up to 3 ppm, e.g., up to 1 ppm (dissolved oxygen in water can be measured by the Winkler method) .

[0019] Thus, the freshly prepared Cu+aqueous solution is provided by reducing cupric ions in 02-free water in the presence of a complex-forming ligand, allowing the resultant solution to be in contact with air prior to application and applying the solution at least 30 minutes after the solution was prepared.

[0020] Cupric ions are supplied to the 02-free water at a concentration of at least 0.001M, e.g., >0.01M, >0.02M, e.g., in the range from 0.001M to 0.05M, e.g., from 0.01 to 0.05M, by addition of a suitable amount a Cu2+source (a salt, a complex) that dissociates in water to release Cu2+ions. When the 02-free water is prepared by boiling water, then the cupric salt is preferably added after the water was allowed to cool to a temperature in the range from 50 to 70°C (in an open or closed vessel) .

[0021] Water soluble cupric salts (or sparingly / slightly soluble) ; such as cupric sulfate [CuSCh] and its hydrated forms, e.g., cupric pentahydrate [CuSCg • 5H2O] , cupric chloride [CUCI2] , cupric carbonate [CuCOs] , cupric acetate [Cu (CH3COO) 2] , cupric nitrate [CU (NC>3) 2] , cupric benzoate [Cu (C6H5CO2) 2] , cupric gluconate [C12H22CUO14] and the hydrated forms of such salts, can be used. Cupric sulfate pentahydrate and cupric gluconate, which are easily soluble in water, are preferred.

[0022] A stabilizing ligand is introduced to the reaction vessel, nearly at the same temperature range mentioned above. That is, a coordination agent that can stabilize indigenously-generated Cu+in the solution, by forming a complex of appropriate stability. The ligand should have higher affinity to Cu+compared to Cu2+. For most contemplated applications, the ligand is a non-toxic, physiologically acceptable substance. Cuprous complexes that are quite stable and are readily soluble in water are formed with organic compounds comprising functional groups such as carboncarbon double (C=C) and triple (C=C) bonds and carbon-nitrogen triple (C=N) bonds, aromatic rings, tertiary amines, or multidentate ligands. On the other hand, primary amines associate strongly with Cu2+, and normally do not stabilize Cu+effectively in mild concentrations and natural pH (at high concentration and high pH they have bacteriocidic action) . Hence, Cu+-stabilizing ligands of the invention usually do not contain amino functionality. Specific examples of suitable ligands include acetonitrile [CH3CN] , succinonitrile [C2H4(CN)2] , thiourea and ethylenethiourea, (optionally in combination with halide ligands) , fumaric acid, maleic acid, beta-carotene, salicylic acid, benzoic acid, acetylsalicylic acid and 2,5,9,12- tetramethyl-2 , 5,9, 12- tetraazatri de cane . The preferred ligand is acetonitrile. It is added to the 02-free water that was previously charged with the cupric salt, at high molar excess relative to the cupric ions. Usually, the number (n) of ligand molecules (e.g., CH3CN) needed to form a stable cuprous complex [Cu (CH3CN)n]+is at least 1, e.g., from 1 to 4, such that concentration of CH3CN in the aqueous reaction mixture is from 0.001M to 0.05M, e.g., 0.06 to 0.4 M, amounting to 0.2- 1.5 wt . % acetonitrile in water mixture.

[0023] Next, a chemical reductant (for most contemplated applications, a non-toxic, physiologically acceptable reducing agent; metals, including Cu°) is added to the reaction mixture. Because E°(cu+ / cu2+)=-0.16V, a suitable reductant (RED) has to satisfy the following requirement: E° (RED / OXI) > -0.16V, e.g., -0.16V <E° (RED / OXI ) < -0.52V, wherein OXI is the corresponding oxidized form [pH 4-7] .

[0024] A suitable reductant is ascorbic acid; namely, the redox pair consists of ascorbic acid (vitamin C; abbreviated "ASC") and its oxidized form dehydroascorbic acid (abbreviated "DHA") . It is a two electron reaction from ASC to DHA, with E° (ASC / DHA) =- 0.06V. Metals worthy of mention are Cu° and Ag° .

[0025] Water soluble derivatives of ascorbic acid which can be used instead of, or in combination with, ascorbic acid, have the following formula: wherein R is an organic moiety, e.g., an alkyl group (e.g., ethyl ascorbic acid) or an acid residue forming an ester bond (-C (0) -alkyl) , such as Ascorbyl Isostearate, Ascorbyl Linoleate, Ascorbyl Tetraisopalmitate, Ascorbyl Palmitate, Ascorbyl Dipalmitate and Ascorbyl Stearate and salts thereof. Salts of ascorbic acid, e.g., sodium ascorbate, is also effective .

[0026] Other ene-diol compounds (with carbon-carbon double bond and two hydroxyl groups attached each to the carbon atoms of the C=C bond, such as reductic, squaric and dihydroxy fumaric acids) , can also be used.

[0027] The reductant (e.g., ascorbic acid) is added to the aqueous solution of the ligand (e.g., water / acetonitrile mixture) in equimolar amount or in slight excess, relative to Cu2+. That is, the concentration of the reductant is from 0.001 to 0.05M. For example, the oxidation-reduction reaction, leading to the generation of cuprous ions, and the complex formation reaction, whereby a stable complex of the cuprous ion is created in the solution, are as follows:

[0028] 2Cu2++ ASC 2Cu++ DHA + 2H4(3)

[0029] The addition of the reductant to the reaction vessel may be carried out at room temperature or above, e.g., from 20 to 45- 50°C, e.g., from 35 to 45°C, at pH in the range of 4 to 7. The oxidation-reduction reaction advances rapidly over this temperature range and reaches completion within 1-5 minutes (reaction rate increases with reaction temperature) . The progress of the reaction can be monitored by the naked eye or spectrophotometrically ; the characteristic blue color acquired by aqueous Cu2+solutions is gradually vanished, as the divalent cation turns into the monovalent cation, with the product solution becoming colorless. The freshly prepared Cu+aerobic aqueous solution is preferably provided by boiling water (e.g., at atmospheric pressure) to remove dissolved oxygen and obtain 02-free water, adding a water- soluble cupric salt and a complex-forming ligand having one or more carbon-carbon double (C=C) bonds, one or more carbon-carbon triple (C=C) bonds or one or more carbon-nitrogen triple (C=N) bonds, cooling the solution at below 45-50°C, e.g., in a closed vessel, and adding a reductant to reduce the cupric ions to cuprous ions. The solution can be left exposed to air until use.

[0030] Thus, a freshly prepared Cu+aerobic aqueous disinfectant solution of the invention comprises from 0.001 to 0.05 M [Cu (CH3CN)n]+(e.g., >0.01M; from 0.01 to 0.05M) and from 0.001 to 0.05 M dehydroascorbic acid.

[0031] The utilization period of the freshly prepared Cu+aerobic aqueous solution is normally from 1 to 7 hours (temperature has a strong effect on the length of utilization period) , during which time the concentration of dissolved oxygen in the solution is usually up to 6 ppm, e.g., in the range from 3 to 6 ppm.

[0032] Experimental results reported below show that the freshly prepared Cu+aerobic aqueous solution is a powerful sanitizer. The addition of the Cu+solution to agar seeded with microorganisms in Petri dishes effectively inhibited the development of bacterial and fungal colonies on the agar surface. Another experimental model used to test the effect of the Cu+solution consisted of processing spoiled food into a pulp and contacting pulp samples with the Cu+solution over different time periods; a strong sterilizing action was achieved even with short contact times (<5 minutes) . Application of the Cu+solution on agricultural produce, i.e., vegetables and fruits, was shown to inhibit decay processes, generating a fairly long-term effect (lasting over 20 days from application) . Thus, the invention can be commercialized and supplied to an end user as a collection of the related materials, i.e., a kit comprising : a) a solid cupric salt, in powder, granular or tablet form; b) a ligand substance, e.g., acetonitrile in a container; and c) a reductant, e.g., ascorbic acid in a powder form; and a label indicating copper as the active ingredient and one or more of the following applications: fruits and vegetable wash, by adding to 02-free water (e.g., boiled water) appropriate dosages of the three components a) , b) and c) at room temperature and up to 45°C, contacting the produce with the freshly prepared solution (e.g., by dipping) , allowing contact time of not less than 1-2 minutes, followed by optional rinsing the produce with water, wherein the utilization period of the freshly prepared solution is also indicated, e.g., it does not exceed two hours at 20°C and five hours at 40°C; surfaces (e.g., made of plastic, wood, glass, metals, ceramics, leather) , by adding to 02-free water (e.g., boiled water) appropriate dosages of the three components a) , b) and c) at room temperature and up to 45°C, contacting surfaces to be treated with the freshly prepared solution (e.g., by dipping, or adding the solution to a sprayer and applying onto a surface) , allowing contact time of not less than 1-2 minutes, followed by optional rinsing the surface with water, wherein the utilization period of the freshly prepared solution is also indicated, e.g., it does not exceed two hours at 20°C and five hours at 40°C.

[0033] Sanitization of agricultural produce by the freshly prepared Cu+aerobic aqueous solution can be integrated with the handling and preparation taking place in a packing house, before shipping and distribution to the market, to confer to the produce resistance to postharvest decay and extend the shelf-life of the produce. Owing to the finding that a freshly prepared Cu+aerobic aqueous solution retains strong sanitizing action over a few hours after preparation, the solution can be produced on-site, on-demand, and put to use in a straightforward manner, with no need to maintain anaerobic conditions during usage. The Cu+aerobic aqueous solution of the invention is compatible with agricultural produce as acetonitrile and copper ions are easily washed from the produce by rinsing the produce with water. After rinsing with water, no acetonitrile was detected by High Performance Liquid Chromatography (HPLC) analysis of the produce, and spectrophotometric determination of copper in the wash water showed that the concentration of copper ions was below 0.3 ppm, using the reagent oxalic acid bis (cyclohexylidene hydrazide) ; cupric ions form a blue complex with the reagent, marked by a strong absorbance peak at 600 nm.

[0034] For example, a freshly prepared Cu+aerobic aqueous solution can be produced by dissolving the ingredients in a tank charged with 02-free water, followed by dipping the produce in the tank. The solution can be used instead of water in conventional washing machines operating in packing houses, such as drum-type washing machines, conveyor belt washing machines, and others. The application rate of the sanitizer of the invention is usually from 0.5 to 2 L of 0.001-0.05M Cu+solution per one kg of produce, with a contact time of at least one minute. Produce that can be sanitized by a freshly prepared Cu+aerobic aqueous solution includes vegetables (e.g., cucumber, tomato, capsicum, carrot) fruits (e.g., strawberry) and mushrooms.

[0035] The cupric salt, the stabilizing ligand and the reductant can be combined in a tank mixer with water (that was boiled beforehand to expel oxygen and cooled to <45°C) . The so-formed solution can be sprayed to protect crops in the field. Another use is as a topical solution. For example, the solution can be administered to an animal to treat or prevent mastitis, by application to the teats and udder of a cow. Thus, the invention provides the use of the freshly prepared Cu+aqueous solution described above, as a sanitizer / disinfectant , for sanitizing / disinf ecting surfaces; for sanitizing / disinfecting agricultural produce; for incorporation into wastewater treatment / tertiary wastewater treatment; in crop protection; and treatment / prevention of mastitis.

[0036] Another aspect of the invention relates to the finding that addition of excess enediol compound, e.g., ascorbic acid (abbreviated ASC) , to a solution of cupric salt in 02-free water serves a twofold purpose, namely, not only as a reductant converting cupric ions to cuprous ions, but also as a stabilizing ligand for the cuprous ions. That is, when added in large molar excess (e.g., >100% molar excess, >200%, >300%, namely, at molar ratio of at least 2:1, at least 3:1, at least 4:1) , part of the ascorbic acid molecules will donate electrons to the Cu2+, transforming into dehydroascorbic acid, with simultaneous association of ascorbic acid molecules as ligands forming [Cu(ASC)n]+complex.

[0037] Thus, the invention provides a process for preparing a germicidal aqueous solution of enediol compound (e.g., ascorbic acid) and copper for nonimmediate use, comprising removing dissolved oxygen from water and reducing cupric ions with excess enediol in 02- free water. The molar ratio enediol : Cu2+is as set out above. For example, the process comprises boiling water, e.g., at atmospheric pressure, to remove dissolved oxygen and obtain 02- free water, adding a water-soluble cupric salt, cooling the solution at below 45-50°C in a closed vessel, and adding the enediol compound in excess thereby reducing cupric ions to cuprous ions .

[0038] Another aspect of the invention is a germicidal aqueous solution of enediol compound and copper for nonimmediate use, comprising cuprous ions, enediol (e.g., ascorbic acid; ASC) and a reduced form of the enediol (e.g., dehydroascorbic acid; DHA) , dissolved in 02-free water, i.e., in water from which oxygen was removed beforehand by the methods described above. Experimental work reported below shows that the Cu+ / ASC / DHA solution retains potent antimicrobial activity even after it has been left exposed to air for, e.g., an hour. Thus, after preparation in 02-free water, i.e., with initial O2 levels <3 ppm (<lppm) , the aqueous solution can be left open, and put to use within a utilization period of at least 10 minutes; >15 minutes; >30 minutes; and is still presumably active when oxygen uptake occurs up to, e.g., 6 ppm. Alternatively, the Cu+ / ASC / DHA aqueous solution can be stored immediately after preparation in airtight packaging and opened just or shortly before application. With the reduction and complex formation reactions:

[0039] 2Cu2++ ASC 2Cu++ DHA + 2H+(5) occurring in oxygen-free water, the very short duration of the antimicrobial action of copper / ascorbic acid solution is extended appreciably. Specifically, there is provided a germicidal aqueous solution of enediol compound and copper for nonimmediate use, comprising from 0.001 to 0.05 M [Cu(ASC)n]+(e.g., >0.01M; 0.01 to 0.05M) and from 0.001 to 0.05 M dehydroascorbic acid.

[0040] Also provided is a kit comprising: a) a solid cupric salt (e.g., CuSCy '51320; CUSO4) in powder, granular or tablet form; b) enediol (e.g., ASC) in a solid form; and a label indicating copper as the active ingredient and instructions to mix the ingredients in 02-free water, e.g., for one or more of the following applications: topical solution, to be prepared by adding to 02-free water appropriate dosages of components a) and b) at room temperature and up to 45°C, charging the solution into a spray or a dropper, and applying onto the skin, wherein the utilization period of the freshly prepared solution is indicated in the label, e.g., not more than two hours at 20°C and five hours at 40°C hours, fruits and vegetable wash, to be prepared by adding to 02-free water appropriate dosages of the components a) and b) at room temperature and up to 45°C, contacting the produce with the freshly prepared solution, allowing contact time of not less than e.g., one minute, followed by optional rinsing the produce with water, wherein the utilization period of the freshly prepared solution is indicated in the label, e.g., not more than two hours at 20°C and five hours at 40°C hours, surface sanitizer, to be prepared by adding to 02-free water appropriate dosages of the components a) and b) at room temperature and up to 45°C, contacting surfaces with the freshly prepared solution, allowing contact time of not less than, e.g., one minute, followed by optional rinsing the surface with water, wherein the utilization period of the freshly prepared solution is indicated in the label, e.g., not more than two hours at 20°C and five hours at 40°C hours.

[0041] When the cupric salt is cupric sulfate, then the label attached to the kit may further indicate to dissolve cupric sulfate in water, add ascorbic acid and mix until the blue color is vanished and the solution becomes colorless.

[0042] Brief description of the drawings

[0043] Figure 1 is a bar graph showing the inhibition of growth on the agar surface of different types of bacteria and fungi by the action of a freshly prepared aerobic Cu+solution. Figures 2A and 2B show Petri dishes seeded with samples of food pulp which was obtained from spoiled food; a sample that was not treated by the Cu+solution (2A) and a sample of spoiled food immersed in the Cu+solution for ten minutes (2B) .

[0044] Figure 3 shows disinfection percentage versus time curves, indicating the utilization period of the freshly prepared solution at different temperatures (the blue and red curves correspond to experiments run at T=25°C and T=40°C, respectively) .

[0045] Figure 4 is a photograph of Petri dishes with carrot slices after 24 hours of incubation: A) control (without treatment) ; B) after washing with water; and C) after immersion for 3, 4 and 10 minutes in the disinfectant solution.

[0046] Figure 5 is a diagram showing the percentage of infected areas in Petri dishes with cucumber, tomato, carrot, strawberry, and champagne mushroom slices after 24 hours of incubation for control (slices without treatment) , after washing with water, and after immersion for three minutes in the disinfection solution .

[0047] Figure 6 is a diagram of the percentage of infected areas of Petri dishes with carrot slices after 24 hours of incubation for control (slices without treatment) , after washing with water, and after immersion for 3 minutes in the disinfection solution at 1, 3, 5, 7 and 12 days after the treatment. Examples

[0048] Materials and methods

[0049] Detection of acetonitrile by HPLC (JASCO model 2012) :

[0050] Column: RP-C18;

[0051] Injection volume: 40 pl

[0052] Mobile phase: [A] methanol 75% [B] water (TDW) 25% Flow: 1.0 ml / min

[0053] Wavelength: X=210 nm (range: 195-201 nm)

[0054] Detection of copper by spectrophotometry (Thermo Fisher Scientific model 4001 / 4) : absorbance measurement at wavelength of 600 nm using the reagent [oxalic acid bis (cyclohexylidene hydrazide) ] , which forms a blue complex with Cu2+ions.

[0055] Examples 1 to 4

[0056] Preparation of Cu+aerobic aqueous solutions

[0057] Water was boiled at atmospheric pressure. The water was allowed to cool to 60 °C. CUSO4 ’5H2O was then added, followed by acetonitrile. The mixture was stirred to obtain a solution. Next, ascorbic acid was added at 40 °C. The resulting mixture was stirred for several minutes to obtain a colorless, clear solution, which was left exposed to air. The concentrations of the components are tabulated in Table 1.

[0058] Table 1 Cuprous-containing aerobic aqueous solutions prepared as described above were put to use at ambient temperature shortly after preparation (within two hours) .

[0059] Example 5

[0060] Germicidal action of Cu+aerobic aqueous solutions: inhibition growth of bacteria and fungi on agar

[0061] The goal of the study was to evaluate the germicidal action of the Cu+aerobic aqueous solutions of Examples 1 to 4 against bacteria (Escherichia coli, Staphylococcus aureus and Bacillus thuringiensis) and fungi (Brewer's yeast) .

[0062] 0.1 ml of a bacterial solution with a population of over 100,000 bacteria per milliliter, or 0.1 ml of yeast solution with a population of over 100,000 yeast cells per milliliter, was seeded on a Petri dish containing LB-Agar. Within twenty minutes, 2 ml of the tested disinfectant solution was added. The Petri plates, including control plates, were placed in an incubator at 37 °C for twenty-four hours [in the control samples, each component (CuSCh ’SJhO, acetonitrile and ascorbic acid) was applied separately at a concentration equal to the treatment plates] .

[0063] Then the Petri plates were visually inspected to assess the proportion of agar surface that was not occupied by bacterial colonies (the test is qualitative because different microorganisms exhibit different patterns of growth, creating colonies with varying shapes) . The results are shown in the form of a bar diagram in Figure 1 for the treatment plates. For each of the four pathogens, a group of four bars is shown (corresponding to Cu+aerobic aqueous solutions of Examples 1 to 4) . The ordinate is the percentage of disinfection achieved by the tested Cu+solution; 100% disinfection indicates that no colonies were developed on the surface of the agar.

[0064] The relationship between the applied concentration of the cuprous ion and the effect observed is clearly seen in Figure 1. All four tested solutions showed strong germicidal action; pathogens that resisted the low-dose treatments (e.g., Bacillus thuringiensis) were effectively controlled by the application of high dose Cu+solutions.

[0065] No effects were observed with the control samples (when CUSO4 ’5H2O, acetonitrile and ascorbic acid were applied separately to the Petri plates) .

[0066] Example 6 (reference, based on Popov et al. , supra) Germicidal action of Cu+anaerobic aqueous solutions

[0067] A reference solution was prepared under anaerobic conditions by the addition of metallic copper (0.5 g, in the form of 0.1mm foil) to an aqueous solution containing 0.025M of CuSCh and 0.26M of CH3CN (Cu° acts as a reductant) . The "reversed disproportionation reaction", i.e., the redox reaction between Cu2+and Cu°, and stabilization of the Cu+formed with the aid of acetonitrile molecules, is shown by the following equation:

[0068] H 0 nCH3CN > 2[Cu(CH3CN)n]+ 102

[0069] The reference solution was tested according to the experimental protocol of Example 5, to control the same pathogens as in Example 5. The results indicated that the Cu+aerobic aqueous solutions of Examples 1 to 4 and the reference Cu+anaerobic aqueous solution show comparable germicidal action. Example 7

[0070] Germicidal action of Cu+aerobic aqueous solutions

[0071] The Cu+solution of Example 4 (0.05M of CuSCh ’SJhO, 0.05M of Ascorbic acid and 0.26M of Acetonitrile in boiled water) was tested for its ability to disinf ect / sterilize spoiled food.

[0072] Food scraps (consisting of potatoes, carrots, tomatoes, wheat pastry, rice and more) , were kept over four days under inadequate storage conditions, at room temperature, to allow the built-up of microbial load. Then the food was ground into a paste (food pulp) and suspended in the Cu+solution (w / v concentration of 1:1) . The mixture was sampled at intervals of one minute over fifteen minutes, to determine the time needed for the Cu+solution to completely sterilize the spoiled food. The presence of bacteria and fungi in the food pulp was determined by adding the sample (0.5g) to a test tube containing 15 ml of sterile water. The added pulp sample was suspended in the water using a vortex. From the suspension, 0.1 ml was taken for seeding in a Petri dish containing a substrate of LB Agar. The Petri dish was placed in an incubator at 37 °C for 24 hours.

[0073] Figures 2A and 2B show photographs of Petri dishes corresponding to t=0 (before the treatment started) and t=10 min (i.e., the pulp was immersed in the Cu+solution for the duration of 10 minutes) , respectively. At t=0, the development of bacterial / fungal colonies on the agar are visible. It should be noted that a disinfection effect was observed as early as t=l min, indicated by colonies-f ree area on the surface of the agar in the Petri plate (results not shown) . At t=10 min, no colonies were observed on the agar (Figure 2B) , i.e., a complete disinfection effect was achieved. Example 8

[0074] Change in potency of Cu+aerobic aqueous solutions over time

[0075] The goal of the study was to measure the loss of potency of the disinfectant over time due to contact with air.

[0076] A set of Cu+solutions with the composition of Example 4, each consisting of 0.05M of CuSCh ’SJhO, 0.05M of Ascorbic acid and 0.26M of Acetonitrile dissolved in boiled water, were prepared in open flasks. Some solutions were kept at room temperature (25°C) and others at elevated temperature (40°C) . All solutions were exposed to air to allow the dissolution of oxygen. Every hour, spoiled food samples processed into food pulp as described in Example 7, were added to a room temperature solution and a hot solution (1:1 w / v ratio) . That is, the experiments were carried out at 25 °C and 40 °C. The disinfection rate was assessed by the method of Example 7 (after ten minutes of contact with the disinfectant solution, a sample (0.5g) was added to a test tube containing 15 ml of sterile water; the added pulp sample was suspended in the water using a vortex; from the suspension, 0.1 ml was taken for seeding in a Petri dish containing a substrate of LB Agar; The Petri dish was placed in an incubator at 37 °C for 24 hours; disinfection percentage achieved by each Cu+solution (at 25 °C and 40 °C) was estimated according to the portion of the surface of the agar that was occupied by bacterial colonies as previously explained) .

[0077] The results are shown in Figure 3 as disinfection percentage versus time plots (the blue and red curves correspond to the experiments at T=25°C and T=40°C, respectively) . Note that discrete values are shown in the abscissa, i.e., the time (in hours) that elapsed between the preparation of a Cu+solution and its application as a disinfectant. As can be seen from Figure 3, a Cu+aerobic aqueous solution can be used as a disinfectant at room temperature within two hours after preparation and within five hours, if the solution is kept / used at a temperature of 40 °C.

[0078] The Cu+aerobic aqueous solution of the invention is compatible with food products as acetonitrile and copper ions are easily washed from the food. After washing with water, HPLC analysis shows that no acetonitrile remains in the food, and spectroscopic measurements indicated that the concentration of copper ions in the wash water was below 0.3 ppm (the permissible concentration of copper ions in drinking water is 1.4 ppm, according to the FDA) .

[0079] Example 9

[0080] The sanitizing effect of Cu+aerobic aqueous solutions on fresh produce

[0081] The goal of the study was to evaluate the ability of a Cu aerobic aqueous solution to sanitize fresh produce.

[0082] The Cu+aerobic aqueous solution of Example 4, consisting of 0.05M of CUSO4 ’5H2O, 0.05M of ascorbic acid and 0.26M of acetonitrile dissolved in boiled water, was used to wash different types of produce: vegetables (carrot, capsicum, tomato, cucumber) , fruits (strawberry) and mushrooms (champagne mushroom) . Each type of produce was divided into three groups. The produce of the first group received no treatment (control) . The sliced produce of the second group was washed with distilled water. The sliced produce of the third group was immersed in 100 ml of the Cu+solution; three different contact times were examined: 3, 4 and 10 minutes. The slices had surface area of about 4 cm2. Immediately after the treatments, the produce was tested for the presence of microorganisms by placing the slice on an LB Agar substrate, followed by incubation at 37 °C for 24 hours. The results are shown in Figures 4 and 5.

[0083] Figure 4 shows photographs of the control plates (without treatment) (Figure 4A) , after washing with distilled water (Figure 4B) and after immersion for 3, 4 and 10 minutes in the disinfection solution at room temperature (Figure 4C) . It is seen that even dipping in the disinfectant solution for just three minutes ensures complete disinfection of the carrot slices .

[0084] Figure 5 shows the degree of inhibition of the growth of bacterial colonies on the agar plates, expressed as a percentage of the agar surface covered with colonies of the microorganisms. Each of the six types of produce (cucumber, tomato, capsicum, carrot, strawberry and champagne mushroom) is represented by three bars, which correspond to the control group (left bar) ; the group washed with distilled water (middle bar) and dipped in the Cu+solution for three minutes (right bar) . A clear effect is observed for the treatment of the invention, i.e., a 3-minute immersion of the produce in the Cu+solution ensures complete disinfection of vegetables, fruits and mushrooms.

[0085] An additional set of tests was performed on the produce to determine the long-term sanitizing effect of the treatment, i.e., one, three, five, seven and twelve days after immersion for three minutes in the Cu+solution. During these periods, the produce was stored at room conditions. The experimental protocol was the same as above; slices of produce from the different groups were placed on an LB Agar substrate, followed by incubation at 37 °C for 24 hours. Then the agar surface was visually inspected to determine the percentage of the surface area on which colonies had developed.

[0086] The results obtained for the carrot are shown in Figure 6; the abscissa indicates the number of days that elapsed between the treatments and the placement of the treated carrot slice in the Petri plate and the ordinate is the degree of inhibition of growth on the agar plate, i.e., percentage of surface area displaying colonies.

[0087] The results in Figure 6 show that the carrot's skin remains free of microorganisms even seven days after the contact with the Cu+solution, indicating the strong sanitizing effect of the Cu+solution. Similar results were obtained with the other types of vegetables, fruits and mushrooms tested.

[0088] Example 10

[0089] Sanitization effect of Cu+ / ascorbic acid aqueous solutions

[0090] A series of copper solutions with varying concentrations of ascorbic acid was prepared. Water was boiled and used to dissolve separately a cupric salt and ascorbic acid to prepare stock solutions respectively:

[0091] 0. IM Cu2+stock solution was prepared by dissolving 2.5 g of CUSO4 ’5H2O (MW=249.7 g / mol) is 100 ml of boiled water, after the water was allowed to cool to 40°C in a closed vessel.

[0092] 0. IM ascorbic acid stock solution was prepared by dissolving 0.17 g of ascorbic acid (MW =176 g / mol) in 100 ml of boiled water, after the water was allowed to cool to 30°C in a closed vessel .

[0093] 10 ml samples were prepared by combining appropriate volumes of the two stock solutions and diluting with boiled water (cooled to 40°C) as shown in Table 2. The solutions were stored in closed vessels at temperature of about 30°C-40°C until the testing.

[0094] Table 2

[0095] The efficacy of the solutions was evaluated immediately after preparation and following exposure to air for different durations. The test included the mixing of 1 ml E.coli solution (103CFU / ml) and 0.5 of the test Cu+ / ASC solution and counting the colonies observed by the CFU method after twenty minutes of contact time. The results are tabulated in Table 3.

[0096] Table 3

[0097] - less than 10 colonies observed + More than 100 and less than 200 colonies observed.

[0098] ++ More than 600 colonies observed.

Claims

Claims1) A freshly prepared Cu+aqueous solution which is germicidal at aerobic conditions, comprising a water-soluble cuprous complex and an oxidized form of a reductant dissolved in water from which oxygen was removed, wherein the solution is exposed to air.2) The aqueous germicidal solution according to claim 1, wherein the water-soluble cuprous complex contains a ligand having a functional group selected from carbon-carbon double (C=C) bond, carbon-carbon triple (C=C) bond and carbon-nitrogen triple (C=N) bond .3) The aqueous germicidal solution according to claim 1, wherein the oxidized form of the reductant is the oxidized form of ascorbic acid.4) The aqueous germicidal solution according to any one of the preceding claims, comprising a cuprous-acetonitrile complex and an oxidized form of ascorbic acid.5) The aqueous germicidal solution of claim 4, comprising from 0.001 to 0.05 M [Cu (CH3CN)n]+, wherein n is an integer from 1 to 4, and from 0.001 to 0.05 M dehydroascorbic acid.6) The aqueous germicidal solution of any one of claims 1 to 5, wherein the concentration of the dissolved oxygen in the solution is up to 6 ppm.7) A fruit and vegetable sanitizer / disinf ectant , comprising the aqueous germicidal solution according to any one of claims 1 to 6.8) A surface sanitizer / disinf ectant , comprising the aqueous germicidal solution according to any one of claims 1 to 6.9 ) A method of disinf ecting / saniti zing, comprising treating a contaminated surface , surface prone to microbial contamination, or agricultural produce , with a freshly prepared Cu+aerobic aqueous solution that contains a water-soluble cuprous complex and an oxidi zed form of a reductant , prepared by bringing together in 02- free water a cupric salt , a reductant , and a stabili zing ligand capable of complexing with cuprous ions , and applying the solution when left exposed to air .10 ) A method of microbial control in water and / or treated wastewater, comprising providing a freshly prepared Cu+aerobic aqueous solution that contains a water-soluble cuprous complex and an oxidi zed form of a reductant prepared by bringing together in 02- free water a cupric salt , a reductant , and a stabili zing ligand capable of complexing with cuprous ions , adding the freshly prepared solution that was left exposed to air to the target water and / or treated wastewater, wherein the microbial control comprises combatting planktonic bacteria and / or biofilm bacteria on a surface in contact with the water and / or inhibiting biofilm formation on a surface prone to biofilm growth .11 ) The method according to claim 9 or 10 , wherein the freshly prepared Cu+aqueous solution is provided by reducing cupric ions in 02- free water in the presence of a complex- forming ligand and allowing the resultant solution to be in contact with air prior to application and applying the solution at least 30 minutes after the solution was prepared .12 ) The method according to claim 11 , wherein the freshly prepared Cu+aerobic aqueous solution is provided by boiling water to remove dissolved oxygen and obtain 02- free water, adding a water-soluble cupric salt and a complex- forming ligand having one or more carbon-carbon double ( C=C ) bonds , one or more carboncarbon triple ( C=C ) bonds or one or more carbon-nitrogen triple(C=N) bonds, cooling the solution at below 50°C in a closed vessel, and adding a reductant to reduce cupric ions to cuprous ions .13) The method according to any one of claims 9 to 12, wherein the complex-forming ligand is acetonitrile and the reductant is ascorbic acid.14) The method according to any one of claims 9 to 13, wherein the freshly prepared Cu+aerobic aqueous solution is provided and applied at temperature in the range from room temperature to 45°C.15) A kit comprising: a) a solid cupric salt, in powder, granular or tablet form; b) a ligand substance; c) a reductant in a solid form; and a label indicating copper as the active ingredient and one or more of the following applications: fruits and vegetable wash, by adding to 02-free water appropriate dosages of the three components a) b) and c) at room temperature and up to 45°C, contacting the produce with the freshly prepared solution, allowing contact time of not less than one minute, followed by optional rinsing the produce with water, wherein the utilization period of the freshly prepared solution is also indicated in the label, e.g., not more than two hours at 20°C and five hours at 40°C hours, surfaces, by adding to 02-free water appropriate dosages of the three components a) b) and c) at room temperature and up to 45°C, contacting surfaces with the freshly prepared solution, allowing contact time of not less than one minutes, followed by optional rinsing the surface with water, wherein the utilization period of the freshly prepared solution is also indicated in the label, e.g., not more than two hours at 20°C and five hours at 40°C hours.16) A kit according to claim 15, comprising acetonitrile as the ligand and ascorbic acid as the reductant.17) Use of a freshly prepared Cu+aqueous solution which comprises a water-soluble cuprous complex and an oxidized form of a reductant dissolved in water from which oxygen was removed, wherein said solution is open to air, as a sanitizer / disinfectant .18) The use according to claim 17 for sanitizing / disinf ecting vegetables, fruits, and mushrooms.19) The use according to claim 17 for sanitizing / disinf ecting surfaces .20) The use according to any one of claims 17 to 19, wherein the aqueous solution comprises a water-soluble cuprous complex with acetonitrile ligands and an oxidized form of ascorbic acid.21) A process for preparing a germicidal aqueous solution of enediol compound and copper for nonimmediate use, comprising removing dissolved oxygen from water and reducing cupric ions with excess enediol in 02-free water.22) A process according to claim 21, wherein the molar ratio enediol : Cu2+is at least 2:1.23) A process according to claim 21 or 22, wherein the enediol is ascorbic acid.24) A process according to any one of claims 21 to 23, comprising boiling water to remove dissolved oxygen and obtain 02-free water, adding a water-soluble cupric salt, cooling the solution at below 50°C in a closed vessel, and adding the enediol compound in excess thereby reducing cupric ions to cuprous ions.25) A germicidal aqueous solution of enediol compound and copper for nonimmediate use, comprising cuprous ions, enediol and a reduced form of the enediol dissolved in water from which oxygen was removed.26) A germicidal aqueous solution of enediol compound and copper for nonimmediate use according to claim 25, stored in airtight packaging .27) A germicidal aqueous solution of enediol compound and copper for nonimmediate use according to claim 25 or 26, wherein the enediol is ascorbic acid, the solution comprising a cuprous- ascorbic acid complex and an oxidized form of ascorbic acid.28) A germicidal aqueous solution of enediol compound and copper for nonimmediate use according to claim 27, comprising from 0.001 to 0.05 M [Cu(ASC)n]+and from 0.001 to 0.05 M dehydroascorbic acid.29) A kit comprising: a) a solid cupric salt, in powder, granular or tablet form; b) ascorbic acid in a solid form; and a label indicating copper as the active ingredient and instructions to mix the ingredients in 02-free water.30) A kit according to claim 29, wherein the label indicates application as a topical solution, to be prepared by adding to 02-free water appropriate dosages of components a) and b) at room temperature and up to 45°C, charging the solution into a spray or a dropper, and applying onto the skin, wherein the utilization period of the freshly prepared solution is also indicated in the label .31) A kit according to claim 29, wherein the label indicates application as fruits and vegetable wash solution, to be preparedby adding to 02-free water appropriate dosages of the components a) and b) at room temperature and up to 45°C, contacting the produce with the freshly prepared solution, allowing contact time of not less than one minute, followed by optional rinsing the produce with water, wherein the utilization period of the freshly prepared solution is also indicated in the label.32) A kit according to claim 29, wherein the label indicates application as surface sanitizer, to be prepared by adding to 02-free water appropriate dosages of the components a) and b) at room temperature and up to 45°C, contacting surfaces with the freshly prepared solution, allowing contact time of not less than one minutes, followed by optional rinsing the surface with water, wherein the utilization period of the freshly prepared solution that is also indicated in the label.33) A kit according to any one of claims 29 to 32, wherein the utilization period indicated in the label is not more than two hours at 20°C and five hours at 40°C hours.34) A kit according to any one of claims 29 to 33, wherein the cupric salt is cupric sulfate, and the instructions indicate to dissolve cupric sulfate in water, add ascorbic acid and mix until the blue color is vanished and the solution becomes colorless.

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