Method for producing a disinfectant containing silver ions and an organic acid

Abstract

To provide a bactericide including a silver ion and an organic acid for the safe and practical supply of a silver ion water-based antiseptic solution, and a method for producing the same.SOLUTION: A solution comprises silver ion water, produced through electrolysis using pure water and pure silver (4 N grade), and malic acid (D type) as a chelate material dissolved in concentrations ranging from 0.01 to 10 wt.%. The chelate material is not limited to malic acid; it also includes basic amino acids such as cysteine, tyrosine, glutamic acid, aspartic acid, arginine, lysine, histidine.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a disinfectant containing silver ions and an organic acid, and a method for producing the same. [Background technology]

[0002] Silver was discovered around 4000 BC, and first appeared in human life around 3000 BC. Since then, it has been used for various purposes, starting with currency and decoration, as well as tableware, sterilization, deodorization, etc. In recent years, it has been discovered that cationic silver ions (hereinafter, Ag+) or solutions containing them have strong bactericidal properties against bacteria (prokaryotes with cell membranes composed of fatty acid esters), and so it is now widely used as an antibacterial agent. For example, some products labeled as antibacterial are treated with silver compounds, and the list of applied products is endless. In recent years, Ag+ complexes have been attracting attention in the medical field, especially due to their antibacterial and anticancer effects. It has been strongly suggested that Ag+ ions interact with various proteins.

[0003] The inventors have created a silver ion water production device using pure water and 4N grade (99.99% or more) pure silver through electrolysis, and have put it into practical use. Its effectiveness has been verified through various tests by a third-party testing organization. In particular, the inactivation effect against the new coronavirus (SARS-CoV-2), which is an urgent and important social issue, is expected to be a safe and secure (human-friendly) application of Ag+. We have focused on the fact that Ag+ has particularly strong interactions with some amino acids and peptides and proteins with certain side chains, such as the simulation results showing that Ag+ interacts with the proteins that make up the Ebola virus and inhibits replication by causing structural changes. Furthermore, we have conducted comprehensive studies, such as examining chelating materials to enhance the delivery effect of Ag+. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 6,197,814 [Patent Document 2] Patent No. 6481867 [Non-patent literature]

[0005] [Non-Patent Document 1] Hisanori Tanimoto, Kazuhiro Hashiguchi, Masashi Kamiya, and Daisuke Ichikawa, "Reduction of silver ions and formation of hexagonal plate nanoparticles by visible light irradiation of silver citrate solution", Abstracts of Lectures of the Physical Society of Japan, The Physical Society of Japan, 2018, Vol. 73.2. [Non-Patent Document 2] Yamato Hayashi and Hirotane Takizawa, "Study on solid-liquid metal nanomaterial synthesis technology using ultrasound and microwaves", p. 933, "Powder and Powder Metallurgy", Vol. 63, No. 11, Japan Society of Powder and Powder Metallurgy DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0006] Silver ion water using citric acid (C6H8O7·H2O) has already been put to practical use and is a disinfectant product certified by the US EPA. Citric acid can be used as a surface protective group and as a reducing material, and is used to stabilize the dispersion of precious metal nanoparticles in aqueous solutions, but its reducing power is not high, and heating to 100°C or irradiation with ultraviolet light or high-energy particle beams is usually required. Here, one of the problems with the practical application we are aiming for is that citric acid is poorly soluble in water. In previous research (Non-Patent Document 1), experiments were conducted with an ammonia-added silver citrate solution, and it was shown that silver nanoparticles can be formed even with visible light irradiation. However, in addition to the concentration of citric acid, using a large amount of ammonia (NH3) to completely dissolve it in this way leads to the suppression of the Ag+ effect, which does not meet our purpose. The high concentration of citric acid makes it sticky on the hands, and when it becomes viscous, it is not suitable for purposes such as spraying due to its wettability. Furthermore, our experiments indicated that further improvements were needed, such as shortening the time it takes for silver ion water containing citric acid to inactivate the new coronavirus.

[0007] On the other hand, conventional silver ion water for sterilization generally used silver-loaded zeolite as a raw material (Patent Document 2, etc.), but when using zeolite-based materials, aluminum is eluted, which has a negative effect on the chelate design, which is a problem. Therefore, a method was explored to generate silver ions by electrolysis using pure water and highly pure (4N grade, 99.99% or more) silver. However, the electrolysis of pure silver had the problem that in addition to silver ions (Ag+), silver oxide (AgO) was also generated. Although there is a method to remove silver oxide (AgO) with a filter, it is not possible to remove it sufficiently. Furthermore, silver ions (Ag+) tend to form large clusters and become stable, but in this state, there was also the problem that they were difficult to bind to chelating agents.

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide a disinfectant containing silver ions and an organic acid, and a method for producing the same, for the purpose of supplying a safe and practical disinfectant based on silver ion water. [Means for solving the problem]

[0009] As a result of intensive research in view of the above-mentioned problems to be solved, the present inventors came up with the following idea. After many experiments using various chelating agents such as citric acid, glycolic acid, and gluconic acid, we found that malic acid (C4H6O5) is a chelating material suitable for this purpose. Stable and appropriate chelating properties (overall, properties that turn the silver ions into a compound with "soft" ion-sequestering properties that can react immediately with the new coronavirus) are important. Malic acid has a smaller molecular weight than citric acid and is also more soluble in water, which is also suitable for the purpose. Traditionally, malic acid has been said to improve gastrointestinal function, have antibacterial effects, reduce lactic acid, relieve fatigue, and prevent stiff shoulders and back pain. Malic acid is known to have the effect of healing inflammation within the body and purifying the body, and has an antipyretic effect for feverish illnesses, and an expectorant and anti-inflammatory effect for bronchitis and colds. Thus, the fact that malic acid is a safe organic acid is an important point when selecting a chelate. On the other hand, with regard to the problems of the generation of silver oxide (AgO) and the clustering of silver ions (Ag+), it has been found that, by ultrasonic irradiation, silver oxide (AgO) is reduced to Ag+ and the silver ion (Ag+) clusters are crushed into silver nanoparticles (particle size of about 1 to 100 nm) (Non-Patent Document 2, etc.). The silver ions (Ag+) that have become silver nanoparticles can easily be chelated with organic acids such as malic acid.

[0010] Based on the above considerations, the present invention provides a method for producing a fungicide as follows. First Method A step of generating 6 to 60 ppm silver ion water by electrolysis using pure water and pure silver kept at 10°C or less in a dark room; A step of irradiating the silver ion water with ultrasonic waves at an output of 10 to 1000 W for 1 minute or more; A step of dissolving 0.01 to 10 wt % of an organic acid in the silver ion water; A method for producing a fungicide comprising the steps of: Second Method A step of generating 6 to 60 ppm silver ion water by electrolysis using pure water and pure silver kept at 10°C or less in a dark room; A step of dissolving 0.01 to 10 wt % of an organic acid in the silver ion water; A step of irradiating the silver ion water with ultrasonic waves at an output of 10 to 1000 W for 1 minute or more; A method for producing a fungicide comprising the steps of: Third Method A step of dissolving 0.01 to 10 wt % of an organic acid in pure water to generate an organic acid water; A step of generating 6 to 60 ppm silver ion water by electrolysis using pure silver in a dark room for the organic acid water kept at 10°C or less; A step of irradiating the silver ion water with ultrasonic waves at an output of 10 to 1000 W for 1 minute or more; A method for producing a fungicide comprising the steps of: In the first to third manufacturing methods, The pure silver has a purity of 99.99% or more, The organic acid is preferably malic acid. Alternatively, the organic acid may be one or more basic amino acids selected from the group consisting of cysteine, tyrosine, glutamic acid, aspartic acid, arginine, lysine, and histidine. In any of the above first to third manufacturing methods, it is possible to produce a stable disinfectant solution in a short time by applying ultrasonic technology. In addition, in an actual demonstration experiment, a novel coronavirus (SARS-CoV-2) inactivation experiment was conducted more than one month after the disinfectant solution produced by the method of the present invention was produced. As will be clear from the details of the experiment described below, as a document of the official experimental results by a third-party research institute, it was found that a silver ion + malic acid (silver ion 30 ppm + malic acid 5%) solution has an inactivation effect against the novel coronavirus (SARS-CoV-2). Moreover, it was shown that the infectivity was reduced to the detection limit with a reduction rate of 99.580% in 1 minute and a reduction rate of >99.998% in 10 minutes. This was a result that was significantly superior to silver ion water synthesized with citric acid or hypochlorous acid. Effect of the Invention

[0011] As described above, the method for producing a germicide containing silver ions and an organic acid according to the present invention makes it possible to realize the supply of a safe and practical disinfectant solution based on silver ion water. [Brief description of the drawings]

[0012] [Figure 1] Diagram of the geometry of deprotonated anionic amino acids bound to silver cations. [Diagram 2] Diagram of the interaction of silver ions with cells (in the case of bacteria). [Diagram 3] Diagram of the chemical structures of citric acid and malic acid. [Figure 4] FIG. 1 is a graph showing test results of the antiviral effect of a disinfectant produced by the production method of the present invention. [Diagram 5] FIG. 1 is a graph showing test results of the antiviral effect of a disinfectant produced by the production method of the present invention. [Figure 6] FIG. 1 is a graph showing test results of the antiviral effect of a disinfectant produced by the production method of the present invention. [Figure 7] FIG. 1 is a graph showing test results of the antiviral effect of a disinfectant produced by the production method of the present invention. BEST MODE FOR CARRYING OUT THEINVENTION

[0013] BEST MODE FOR CARRYING OUT THE DISCLOSURE The best mode for carrying out the method for producing a germicide containing silver ions and an organic acid of the present invention will be described in detail below with reference to the accompanying drawings. 1. Discussion Interaction of silver ions with amino acids and proteins In 1893, Swiss botanist Karl Wilhelm von Nageli, discovered that silver and copper ions inactivate the algae Spirogyra. Copper and silver are also well known as protein precipitants, but the overall explanation from the perspective of chemical reactions is as follows. That is, H in the -SH enzyme is replaced by Ag+, R-SH + Ag+ = R-SAg + H+ The above reaction occurs, and the irreversible generation of -SH changes the structure of proteins, destroys cell membranes, and deactivates them by depriving them of their ability to proliferate. This is the basic explanation for the sterilization effect of silver ions. It has also been explained that silver cations are adsorbed to negatively charged bacteria and algae in the water, causing the above reaction. Recent studies have already reported the interaction of silver ions with selected amino acids and proteins (Leee et al. 1998; Shoeibet al. 2002; Novikovet al. 2010; Eckhardt et al. 2013). Theoretical studies also reported that the three basic amino acids arginine, lysine, and histidine are the strongest silver ion binders, whereas the non-polar (aliphatic) amino acids, except for methionine, have the weakest silver ion affinity (Shoeib et al. 2002; Eckhardt et al. 2013). On the other hand, in recent years, research into theoretical elucidation of chemical phenomena of molecules has also progressed, and simulation techniques are rapidly spreading in the field of organic chemistry. In particular, research into the use of density functional theory and other methods to improve molecular design and consideration, such as the proposal of methods with fewer errors from experimental values, is becoming more active. A simulation study (V. Saranya et al.) has reported results suggesting that the compound interacts with a protein that constitutes the Ebola virus (a protein called viral protein 40 (a protein essential for the replication of the Ebola virus)) and causes a structural change, thereby inhibiting replication. There have also been reports that the compound interacts not only with proteins but also with nucleic acids. On the other hand, Andrey et al. (2013) introduced the PBE-D3 and MP2 methods to study the binding between amino acids and silver ions using quantum chemical techniques. In the paper, they considered the binding between deprotonated anionic amino acids and silver cations and found that cysteine, tyrosine, glutamic acid, aspartic acid, etc. showed particularly high interactions (Figure 1). A closer look reveals that all four deprotonated anionic amino acids have different variants of Ag + attachment. For example, it is found that the proton Cys(-H+) of cysteine ​​forms a bidentate complex with Ag+ through bonds with sulfur and carbonyl. Similarly, it is found that the Asp(-H+) of aspartic acid forms a tridentate complex with silver ions through the nitrogen of the amino group and two carbonyls. As such, it is clear that very useful information can be obtained when considering reactions with silver ions. In our invention, we have also utilized such considerations of molecular structure to select appropriate chelates from naturally occurring amino acids. On the other hand, such silver ion complexes are attracting attention in the medical field, especially due to their antibacterial and anticancer properties (Hecel et al.). Another important example for future applications is SiLE. Bacteria that are resistant to silver ions produce a silver resistance protein called SilE, and it has been described that silver ions interact with histidine and methionine residues in proteins (Chabert et al.). In addition, the creation of complexes such as Ag+ and peptides and Ag+ and proteins (Li et al.) is also useful information. According to Pearson's classification (HSAB rule), Ag(I) silver ions are soft cations (positively charged ions), and this has been shown to have a high affinity for soft bases (Pearson). Among the amino acids that make up proteins, it has been pointed out that the side chains of cysteine ​​and methionine are particularly "soft" (due to the presence of sulfur atoms) (Glusker). Furthermore, in the case of bacteria, an outline of how silver ions interact with cells has been summarized (Hecel et al.) (Figure 2).

[0014] Chelate In our invention, we considered the molecular structure as described above, and selected an appropriate chelate from naturally occurring amino acids, and as a result, malic acid was selected. As mentioned above, citric acid can be used as a surface protective group and as a reducing material, and is used to stabilize the dispersion of precious metal nanoparticles in aqueous solutions, but its reducing power is not high, and heating at 100°C or irradiation with ultraviolet light or high-energy particle beams is usually required. Here, one of the problems with the practical application we are aiming for is that citric acid is poorly soluble in water. In Non-Patent Document 1, experiments were conducted with an ammonia-added silver citrate solution, and it was shown that silver nanoparticles can be formed even with visible light irradiation. However, in addition to the concentration of citric acid, using a large amount of ammonia (NH3) to completely dissolve it in this way leads to the suppression of the Ag+ effect, which does not meet our purpose. The high concentration of citric acid makes it sticky on the hands, and when it becomes viscous, it is not suitable for purposes such as spraying due to its wettability. Furthermore, our experiments indicated that further improvements were needed, such as shortening the time it takes for silver ion water containing citric acid to inactivate the new coronavirus. Figure 3 shows the molecular structures of citric acid and malic acid. Malic acid has a smaller molecular weight than citric acid and is highly soluble in water. Malic acid is also an important molecule in stereochemistry, as it has an asymmetric carbon. In short, an asymmetric carbon is a general term for a carbon with all four bonds connected to different groups, and there are molecules with different structures even if they have the same molecular formula. Walden created D-malic acid by reacting L-malic acid with phosphorus pentachloride and reacting it with water via silver oxide. D-malic acid and L-malic acid have symmetrical structures like mirror images, but they are known to have different properties such as optical rotation. Example 1

[0015] In our inactivation experiments against the novel coronavirus, malic acid was used, which is commonly available DL-malic acid (primary, DL-Malic acid HOOCCHOHCH2COOH, Showa Chemical Co., Ltd.). Experiment to evaluate inactivation effect against the new coronavirus A series of evaluation experiments were carried out at the Department of Microbiology and Infectious Diseases, Nara Medical University. The experimental conditions and results are described below. Setting conditions for evaluation experiments Research purpose To clarify whether it has an inactivating effect on the new coronavirus. Test item (sample) Test product: Silver ions + malic acid (silver ions 30 ppm + malic acid 5%) Test microorganism: Novel coronavirus (SARS-CoV-2) VeroE6 / TMPRSS2 cells were infected with the novel coronavirus, and those that showed cytopathic effects were collected and frozen in a -80°C freezer. After two freeze-thaw cycles, the cells were centrifuged and the supernatant was concentrated and purified using an ultrafiltration membrane. This was used as the test virus liquid, and was frozen and stored in a -80°C freezer until testing. SARS-CoV-2 was obtained from the National Institute of Infectious Diseases, and VeroE6 / TMPRSS2 cells were obtained from the JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health and Nutrition.

[0016] Exam Content 20μl of virus solution was mixed with 180μl of test sample and left to stand for 1 minute and 10 minutes, respectively. After the reaction, 1,800 μl of EDTA-containing SCDLP medium was added to stop the reaction. The virus infectivity (PFU / mL) was measured using the recovered solution by the plaque assay. The reaction temperature (room temperature) was 20-25°C. Each test was conducted twice. The inactivation effect was calculated as follows. Inactivation effect (Mv) = log(Ct / C0) - log(Nt / N0) = logCt / Nt Ct: Control infectious titer after t hours C0: Control infectious titer after 0 hours Nt: Infectivity of test item after t hours N0: Infectivity of test item after 0 hours The reduction rate was calculated from the log reduction value as follows. Reduction rate = (1-1 / 10 log reduction value) x 100% All studies were conducted in a biosafety level 3 (BSL3) laboratory at our university under appropriate pathogen containment measures.

[0017] Experimental Results The results are shown in Figures 4 and 5. Figure 4 Table 1. Changes in viral infectivity by test sample Table 2. Virus inactivation effect of test products Figure 5 Changes in viral infectivity by test sample The values ​​are the average of two tests. The test product had a 8.50 x 10 6 From PFU / ml to 3.25 × 10 in 1 minute 4 PFU / ml (reduction rate 99.580%), <1.00 × 10 at 10 min 2 The infectivity was reduced to the detection limit of PFU / ml (reduction rate >99.998%).

[0018] Comparison This paper describes an experiment to evaluate the inactivation effect of this invention against the novel coronavirus, which was conducted to compare it with other combinations of disinfectants and silver ions (different chelators or additives). The experiment was conducted under exactly the same conditions as the "evaluation experiment conditions" for the invention, but the evaluation time settings may differ due to differences in the time required for inactivation, etc. The results are summarized in Figure 6 (Table 3: Virus inactivation effect (comparison experiment results with different chelating agents and additives)). In the experiments, comparative experiments were conducted using different chelating agents or additives, such as alum, citric acid (5% by weight), and hypochlorous acid. At a glance, it can be seen that only the invention using malic acid chelate is able to completely inactivate the new coronavirus in a short time of 1 minute or 10 minutes, compared to other additives. Furthermore, the inactivation rate was <1.00 × 10 2 Since the infectivity was reduced to the detection limit of PFU / ml (reduction rate >99.998%), the disinfection effect itself was superior to other cases.

[0019] Confirmation of inactivation effect against other bacteria Silver ion water has been proven to be highly effective through various bactericidal effect confirmations (acetic acid deodorization test, ammonia deodorization test) and bactericidal effect tests (black mold, feline calicivirus, Escherichia coli, etc.). Here, an experiment was conducted to confirm the inactivation effect against carbapenem-resistant Enterobacteriaceae (CRE). The purpose of this is to reaffirm that the disinfectant of the present invention can be used over a wide spectrum. The results are shown in Figure 7 (Table 4: Inactivation experiment against bacteria). The results reaffirm that silver ions have a high inactivation effect against CRE, just as they do against other bacteria.

[0020] The present invention has made it possible to develop a disinfectant that is safe for people, taking advantage of the performance of silver ions, while also achieving a high level of inactivation effect against the new coronavirus. It is expected that this disinfectant will be put to practical use in the future and contribute to society through a wide range of applications, such as spraying in large areas, disinfecting anything that comes into contact with things such as doorknobs, and spraying on masks.

Claims

1. A process of generating 6 to 60 ppm silver ion water by electrolysis using pure water and pure silver kept at 10°C or less in a dark room; a step of irradiating the silver ion water with ultrasonic waves at an output of 10 to 1000 W for one minute or more; Dissolving 0.01 to 10 wt % of an organic acid in the silver ion water; A method for producing a fungicide comprising the steps of: The organic acid is cysteine, tyrosine, glutamic acid, aspartic acid, arginine, One or more basic amino acids selected from the group consisting of lysine, histidine, or malic acid. A method for producing a disinfectant, comprising:

2. A process of generating 6 to 60 ppm silver ion water by electrolysis using pure water and pure silver kept at 10°C or less in a dark room; Dissolving 0.01 to 10 wt % of an organic acid in the silver ion water; a step of irradiating the silver ion water with ultrasonic waves at an output of 10 to 1000 W for one minute or more; A method for producing a fungicide comprising the steps of: The organic acid is cysteine, tyrosine, glutamic acid, aspartic acid, arginine, One or more basic amino acids selected from the group consisting of lysine, histidine, or malic acid. A method for producing a disinfectant, comprising:

3. A step of dissolving 0.01 to 10 wt % of an organic acid in pure water to produce an organic acid water; A step of generating 6 to 60 ppm silver ion water by electrolysis using pure silver in a dark room for the organic acid water kept at 10°C or less; a step of irradiating the silver ion water with ultrasonic waves at an output of 10 to 1000 W for one minute or more; A method for producing a fungicide comprising the steps of: The organic acid is cysteine, tyrosine, glutamic acid, aspartic acid, arginine, One or more basic amino acids selected from the group consisting of lysine, histidine, or malic acid. A method for producing a disinfectant, comprising:

4. 4. The method according to claim 1, wherein the pure silver has a purity of 99.99% or more. A method for producing the fungicide described above.

5. A method for sterilizing the SARS-CoV-2 virus using a disinfectant produced by the method for producing a disinfectant according to any one of claims 1 to 3.

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