Chlorine peroxide radical, and production method, composition, quantification method and use thereof

Abstract

The present disclosure provides a medicinal agent that is useful for disinfection of microorganisms and the like. The present disclosure provides a chlorine peroxide radical. The chlorine peroxide radical is preferably provided as a liquid. Also provided are: a method for producing the chlorine peroxide radical; a method for killing microorganisms using the chlorine peroxide radical; a microorganism-killing agent; a method for measuring bactericidal activity; a device for measuring the chlorine peroxide radical; and a kit for preparing the chlorine peroxide radical. This method for producing the chlorine peroxide radical includes a step for bringing a chlorine oxide into contact with an excess of hydrogen ions.

Description

Chlorine peroxide radicals and methods for making, compositions, quantitation and uses thereof

[0001] FIELD OF THE DISCLOSURE The present disclosure relates to chlorine peroxide radicals, methods for their preparation, compositions, quantification methods and uses.

[0002] There are various types of chlorine oxides, many of which are used as disinfectants, but their details are not fully understood.

[0003] As a result of extensive research, the present inventors have discovered a chlorine peroxide radical and a method for producing the same, and have also found that the chlorine peroxide radical is involved in the sterilization of microorganisms.

[0004] The present disclosure has discovered a method for producing chlorine peroxide radicals. This method involves contacting a solution containing chlorine oxides containing two or more oxygen atoms per chlorine atom with an excess of hydrogen ions relative to the chlorine oxides. It has also been discovered that these chlorine peroxide radicals exist stably. Briefly, the method is as follows.

[0005] The active molecular species in a solution in which chlorine oxide was contacted with excess hydrogen ions were studied using an electron spin spectrometer (ESR) and a spectrophotometer (UV). The ESR spectrum showed the presence of chlorine peroxide radicals (Cl-O-O.). The concentration of these chlorine peroxide radicals depended on the amount of hydrogen ions. The concentration of the chlorine peroxide radicals, which are active molecular species involved in sterilization, was measured using the DPD method, a common colorimetric method for measuring the residual chlorine concentration in swimming pools, etc., as a value (free chlorine concentration), absorbance (A ３５０ ), chlorite ions (ClO) before acidic conditions ２ －) concentration. This shows that the active molecular species in a solution in which chlorine oxide is in contact with an excess amount of hydrogen ions is a chlorine peroxide radical, and that the concentration of this chlorine peroxide radical can be quantified using the value (free chlorine concentration) obtained by a colorimetric method (DPD method, colorimetric methods also include the TMB method and KI method). It has become clear that if these values ​​are controlled together with the content (total chlorine amount) related to safety obtained by potassium iodide reduction titration, it will be possible to safely and reliably enjoy the bactericidal effect against bacteria and the inactivation effect against viruses.

[0006] In this disclosure, chlorite (NaClO ２ By using a spectrophotometer and an electron spin resonance spectrometer to observe a solution of methylparaben (a compound of methylparaben) mixed with an excess amount of a GRAS acid (e.g., hydrochloric acid), the active molecular species were elucidated from both ion chemistry and radical chemistry approaches. Furthermore, methods for qualitatively and quantitatively evaluating these active molecular species and the causal relationship between their antimicrobial effects and bactericidal and disinfectant effects were also clarified.

[0007] For example, the present disclosure provides the following: (Item 1) Chlorine peroxide radical. (Item 2) A method for producing chlorine peroxide radical, comprising the step of contacting chlorine oxide having a moiety containing two or more oxygen atoms per chlorine atom with an excess amount of hydrogen ions. (Item 3) The method of item 2, wherein the excess amount is greater than 0.07 milliequivalents of hydrogen ions per gram of sodium chlorite-equivalent mass of the chlorine oxide. (Item 4) The method of item 2 or 3, wherein the excess amount is greater than 0.15 milliequivalents of hydrogen ions per gram of sodium chlorite-equivalent mass of the chlorine oxide. (Item 5) The method of any one of items 2 to 4, wherein the chlorine oxide is chlorine dioxide, chlorine dioxide gas, aqueous chlorine dioxide, stabilized chlorine dioxide (aqueous chlorine dioxide), chlorous acid, chlorite, chlorite ions, chloric acid, chlorate, chlorate ions, perchloric acid, perchlorate, and / or perchlorate ions. (Item 6) The method according to any one of items 2 to 5, wherein the chlorine oxide is an aqueous solution. (Item 7) The method according to any one of items 2 to 6, wherein the step of contacting hydrogen ions comprises adding an acid. (Item 8) The method according to item 7, wherein the acid is a GRAS acid. (Item 9) The step of contacting hydrogen ions comprises adding H ＋The method of any one of items 2 to 8, comprising using a chlorine peroxide radical-type ion exchange resin. (Item 10) The method of any one of items 2 to 9, wherein the chlorine peroxide radical is present in an aqueous solution. (Item 11) A composition comprising chlorine peroxide radical. (Item 12) The composition of item 11, wherein the composition is an aqueous solution. (Item 13) The composition of items 11 or 12, wherein the chlorine peroxide radical is present as an active ingredient in a disinfectant. (Item 14) A method for killing viruses, bacteria, and / or fungi, comprising contacting chlorine peroxide radical with viruses, bacteria, and / or fungi. (Item 15) Use of chlorine peroxide radical in the manufacture of a disinfectant for killing viruses, bacteria, and / or fungi. (Item 16) A method for confirming the presence and / or quantifying the presence of chlorine peroxide radical in a liquid, comprising detecting chlorine peroxide radical and / or measuring the amount of chlorine peroxide radical by ESR. (Item 17) A method for measuring the bactericidal activity of an agent that kills viruses, bacteria, and / or fungi, comprising the step of measuring the concentration of chlorine peroxide radicals in the agent. (Item 18) Use of chlorine peroxide radicals to kill viruses, bacteria, and / or fungi.

[0008] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0009] A new substance, chlorine peroxide radical, is obtained. A method for sterilizing microorganisms using chlorine peroxide radical is obtained. A method for measuring chlorine peroxide radical is obtained, and necessary and sufficient sterilization can be appropriately performed.

[0010] Figure 1A shows the ESR spectrum of radical species in an aqueous solution obtained by adding sodium chlorite to an acid. This shows the ESR spectrum of radical species observed in the aqueous solution after mixing with an HCl solution for 240 seconds to obtain a final HCl concentration of 1.25 M. The sodium chlorite concentration is indicated in the figure. Figure 1B shows the ESR spectrum of radical species in an aqueous solution obtained by adding sodium chlorite to an acid. The dependence of the ESR spectrum of the aqueous solution on the HCl concentration is shown. The final HCl concentration is indicated in the figure. The ESR spectrum was recorded 240 seconds after mixing 0.1 M aqueous sodium chlorite and HCl solutions. Figure 2A shows the time-dependent change in the ESR spectrum after mixing 0.1 M sodium chlorite solution with 0.08 M hydrochloric acid in a 1:1 ratio. Figure 2B shows the ESR signal intensity and UV-Vis absorption spectrum of the aqueous solution obtained by adding sodium chlorite to an acid. The absorption spectrum of an ASC aqueous solution under the same conditions as in Figure 2A is shown. Figure 2C shows the relationship between the chlorine peroxide radical ESR signal intensity obtained from Figures 2A and 2B and the absorbance at 354 nm. Figure 3A shows the relationship between the active chlorine concentration at 354 nm and the consumption of active chlorine in a solution obtained by adding an acid to sodium chlorite. Figure 3A shows the relationship between the absorbance at 354 nm and the free chlorine concentration obtained by the DPD method under the same reaction conditions as Figure 2A. Figure 3B shows the phenomenon of the gradual decrease in absorbance at 354 nm in an aqueous solution obtained by adding an acid to sodium chlorite when thiosulfate is added. Data are mean ± SD (n = 3). Figure 4 shows the optimized structure, charge distribution, and SOMO of the chlorine peroxide radical. Cl atoms and oxygen atoms are each represented by a sphere. The values ​​indicate the Mulliken charge distribution of each atom, and the mesh surface indicates the spin density of the SOMO. Spin-trapping ESR measurements of an aqueous solution obtained by adding an acid to sodium chlorite using 1.5 M DMPO at various concentrations of HCl. NaClO ２ The change in absorbance after adding HCl solution to the solution is shown.

[0011] The present disclosure will be described in more detail below. Throughout this specification, unless otherwise specified, singular expressions should be understood to include the plural concept. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the relevant field unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present specification (including definitions) will prevail.

[0012] Abbreviations used herein have their conventional meaning within the art unless otherwise specified.

[0013] References herein to "about" a value or parameter include the variation that is directed to the value or parameter itself. For example, "about X" includes "X" itself as well as values ​​that allow for ±10% of error, unless otherwise specified.

[0014] As used herein, "chlorine peroxide radical" refers to a peroxide radical having two oxygen atoms per chlorine atom, where the radical is on the oxygen atom, not the chlorine atom. Therefore, it differs from the chlorine dioxide radical (O=Cl·=O), which has a radical on the chlorine atom. The fact that the chlorine peroxide radical differs from the chlorine dioxide radical can be confirmed by ESR measurement. The chlorine peroxide radical contains a peroxide radical and is considered to have a Cl-O-O· structure, and can be said to be different from the chlorine dioxide radical (O=Cl·=O). The chlorine peroxide radical has a maximum absorbance wavelength of 350 to 360 nm. As a radical, it has low reactivity and can exist stably for a long period of time.

[0015] As used herein, "chlorine oxide" refers to any oxide of chlorine. Examples include hypochlorous acid, chlorous acid, chloric acid, perchloric acid and their salts, esters of these acids (such as alkyl esters), dichlorine heptaoxide, dichlorine hexaoxide, dichlorine trioxide, chlorine dioxide, dichlorine monoxide, sodium hypochlorite, calcium hypochlorite (high-grade bleaching powder), sodium chlorite, sodium chlorate, sodium perchlorate, etc. In the present disclosure, chlorine oxides containing two or more oxygen atoms per chlorine atom are preferred.

[0016] In this specification, "chlorine oxides containing two or more oxygen atoms per chlorine atom" refers to chlorine oxides containing two or more oxygen atoms per chlorine atom. ２ ), chloric acid (HClO ３ ), perchloric acid (HClO ４ Dichlorine heptaoxide is OCl(O) ２ —O—Cl(O) ２ Dichlorine trioxide has the structure OCl-ClO, and contains two or more oxygen atoms per chlorine atom, so it falls under the category of "chlorine oxides containing two or more oxygen atoms per chlorine atom." Dichlorine trioxide is OCl-ClO ２ or Cl—O—ClO ２ It is believed to have the structure "ClO ２ " portion, it falls under the category of "chlorine oxides containing two or more oxygen atoms per chlorine atom."

[0017] "Sodium chlorite equivalent mass" refers to the amount of chlorite ions present in chlorine oxide converted into sodium chlorite (NaClO ２ The amount of chlorite ions can be measured by potassium iodide reduction titration.

[0018] The "equivalent" of hydrogen ions refers to the amount taking into account the number of hydrogen atoms that can replace the hydrogen ions contained in one molecule. When hydrochloric acid is used as the acid that supplies protons, there is one equivalent (eq) of hydrogen ions per mole of hydrochloric acid, and in sulfuric acid, there are two equivalents (eq) of hydrogen ions per mole. For example, H＋ 50 g of type ion exchange resin is 96 meq (for convenience, H ＋ For ion exchange resins, 1 ml is calculated as 1 g, and if the specific gravity is specified on the ion exchange resin to be used, take that into consideration.

[0019] An "excess amount" of hydrogen ions refers to an amount of hydrogen ions greater than the lower limit at which chlorine peroxide radicals are generated in the production of chlorine peroxide radicals. This can be determined by determining the lower limit at which chlorine peroxide radicals are generated. It can be measured by measuring absorbance at 350 nm, ESR, or the like. The excess amount can be expressed as the hydrogen ion equivalent per gram of sodium chlorite mass (e.g., 0.07 meq / g, 0.15 meq / g).

[0020] As used herein, "H ＋ "Ion exchange resin" refers to a type of synthetic resin that has a structure that ionizes as an ion exchange group in part of its molecular structure. Those that release hydrogen ions are preferred. For example, Amberlite IR120B can be mentioned.

[0021] As used herein, "GRAS acid" refers to acids that are generally recognized as safe substances. GRAS acids are, but are not limited to, those that can be used as food ingredients. It is believed that if a product is used as a food ingredient, it can be easily sold as a food product after disinfection using a preparation produced by this method. The classification of GRAS acids used herein is shown below.

[0022]

[0023]

[0024]

[0025] In one embodiment, the GRAS acid is selected from the group consisting of glutamic acid, hydrochloric acid, phosphoric acid, ascorbic acid, erythorbic acid, thiodipropionic acid, acetic acid, aconitic acid, adipic acid, alginic acid, benzoic acid, caprylic acid, citric acid, lactic acid, linolenic acid, malic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid, sulfamic acid, and formic acid. In a preferred embodiment, the GRAS acid is selected from the group consisting of glutamic acid, hydrochloric acid, phosphoric acid, ascorbic acid, erythorbic acid, thiodipropionic acid, acetic acid, aconitic acid, adipic acid, alginic acid, benzoic acid, caprylic acid, citric acid, lactic acid, linolenic acid, malic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, and tartaric acid. Advantageously, these preferred GRAS acids have been approved by the U.S. Food and Drug Administration (FDA) as being suitable for direct use. More preferably, the GRAS acid is selected from the group consisting of sulfuric acid, phosphoric acid, citric acid, lactic acid, acetic acid, and hydrochloric acid, because when these GRAS acids are used, it has been confirmed in the examples that a double-humped peak is maintained, i.e., there are specific absorption regions at wavelengths of 354 nm and 260 nm, and it is understood that the bactericidal activity is maintained stably for a long period of time.

[0026] In this specification, "free chlorine," "free chlorine concentration," or "free residual chlorine concentration" refers to a value measured by Appendix 3 of the "Testing Method for Free Residual Chlorine and Combined Chlorine Established by the Minister of Health, Labour and Welfare Pursuant to the Provisions of Article 17, Paragraph 2 of the Enforcement Regulations of the Water Supply Act" (hereinafter referred to as the colorimetric method (DPD indicator)), and is a value obtained by oxidizing the DPD indicator.

[0027] As used herein, "microorganisms" refers to organisms so small they cannot be seen with the naked eye, such as viruses, bacteria, and fungi. Rickettsiae and spirochetes are bacteria. As used herein, "microorganisms" does not include parasites (protozoa, trematodes, actinozoans, nematodes, etc.). As used herein, "pathogenic microorganisms" refers to microorganisms that infect humans or through the environment. Infected individuals can spread microorganisms through diarrhea, vomiting, coughing, etc., and become infected through secondary infection.

[0028] "Aqueous chlorine dioxide" refers to an aqueous solution of chlorine dioxide.

[0029] The terms "aqueous chlorine dioxide," "stabilized chlorine dioxide," and "stabilized chlorine dioxide solution" are all used interchangeably and refer to chlorine oxides in which chlorine dioxide gas is stabilized in an aqueous solution, such as those commercialized by Bio-Cide International, Inc.

[0030] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0031] The present disclosure relates to chlorine peroxide radicals, methods of making, measuring, and other applications, various aspects of which are described below.

[0032] (Chlorine peroxide radical) In one aspect of the present disclosure, a chlorine peroxide radical is provided. The chlorine peroxide radical discovered in the present disclosure is a peroxide radical having the structure "Cl-O-O.". It has been known that chlorine dioxide can be generated from chlorites such as sodium chlorite, but it was not previously known that the chlorine dioxide radical has the structure "Cl-O-O." rather than the structure "O=Cl.=O," and that this radical exists stably. The chlorine peroxide radical preferably exists in water. The chlorine peroxide radical can exist stably, and can exist for a longer period of time when stored in a container that does not lose radicals, such as a glass bottle, in a cool, dark place. Although not limited thereto, in a preferred embodiment, the chlorine peroxide radical can be stable for a long period of time when produced at a molar yield of 10% or less. However, despite its stability, it maintains its radical properties and can be taken up by microorganisms, such as bacteria, that take up radicals, thereby killing the microorganisms. Chlorine peroxide radicals have a maximum absorbance at wavelengths of 350 to 360 nm, and the change in their amount over time can be easily confirmed by UV measurement.

[0033] (Method for Producing Chlorine Peroxide Radicals) The chlorine peroxide radicals of the present disclosure can be produced by contacting an excess amount of hydrogen ions with a solution containing chlorine oxide having a moiety containing two or more oxygen atoms per chlorine atom. The excess amount can be determined by an increase in the maximum absorption at 354 nm in the UV spectrum. The concentration at which an increase in absorbance at 354 nm is first observed can be determined to be the lower limit for the generation of chlorine peroxide radicals. It is important that the amount of hydrogen ions is in excess relative to chlorine oxide. It has been found that adding an excess amount of hydrogen ions produces chlorine peroxide radicals, which are peroxide radicals, from chlorine oxide. Due to the excess hydrogen ions, the pH can be less than 3, for example, less than 2.5, less than 2.3, 2.0 or less, 1.5 or less, or 10 or less. The reaction can be carried out at room temperature in the atmosphere. In the case of chlorine oxides containing more than two oxygen atoms (e.g., 2.5, 3, 3.5, or 4) per chlorine atom, such as chloric acid or perchloric acid, as a raw material, chlorine peroxide radicals can be generated by adding an excess amount of hydrogen ions to the chlorine oxides. Without wishing to be bound by theory, the presence of the generated chlorine peroxide radicals can be confirmed by ESR.

[0034] The excess amount may be an amount of hydrogen ions greater than 0.07 milliequivalents per gram of the mass of the chlorine oxide converted into sodium chlorite. The excess amount is 0.08 milliequivalent, 0.09 milliequivalent, 0.10 milliequivalent, 0.11 milliequivalent, 0.12 milliequivalent, 0.13 milliequivalent, 0.14 milliequivalent, 0.15 milliequivalent, 0.2 milliequivalent, 0.25 milliequivalent, 0.3 milliequivalent, 0.35 milliequivalent, 0.4 milliequivalent, 0.45 milliequivalent, 0.5 milliequivalent, 0.55 milliequivalent, 0.6 milliequivalent, 0.7 milliequivalent, 0.8 milliequivalent, 0.85 milliequivalent, 0.9 milliequivalent, 1 milliequivalent, 1.5 milliequivalent, 2 milliequivalent, 2.5 milliequivalent, 2.75 milliequivalent, 3 milliequivalent, 3.5 milliequivalent, 4. The amount of hydrogen ions may be 4 milliequivalents, 4 milliequivalents, 4.5 milliequivalents, 4.75 milliequivalents, 5 milliequivalents, 5.5 milliequivalents, 6 milliequivalents, 6.5 milliequivalents, 7 milliequivalents, 8 milliequivalents, 8.5 milliequivalents, 9 milliequivalents, 10 milliequivalents, 11 milliequivalents, 12 milliequivalents, 13 milliequivalents, 14 milliequivalents, 15 milliequivalents, 16 milliequivalents, 18 milliequivalents, 20 milliequivalents, 25 milliequivalents, 30 milliequivalents, 35 milliequivalents, 40 milliequivalents, 50 milliequivalents, 60 milliequivalents, 70 milliequivalents, 80 milliequivalents, 85 milliequivalents, 90 milliequivalents, 95 milliequivalents, 100 milliequivalents or more, or any number between these values.

[0035] In the case of chlorine oxides containing more than two oxygen atoms per chlorine atom, without wishing to be bound by theory, it is believed that the oxygen atoms combine with hydrogen ions and leave the chlorine oxide as "OH". ３ ), it is thought that when it bonds with a hydrogen ion, one oxygen atom is lost, resulting in a chlorine peroxide radical (Cl-O-O.) which contains two oxygen atoms per chlorine atom.

[0036] In one aspect of the present disclosure, a method for producing chlorine peroxide radicals is provided, comprising contacting a chlorine oxide containing two or more oxygen atoms per chlorine atom with an excess of hydrogen ions.

[0037] In one embodiment of the present disclosure, the chlorine oxide is chlorine dioxide, chlorine dioxide gas, aqueous chlorine dioxide, stabilized chlorine dioxide (aqueous chlorine dioxide), chlorous acid, chlorite, chlorite ions, chloric acid, chlorate, chlorate ions, perchloric acid, perchlorate, and / or perchlorate ions.

[0038] In one embodiment of the present disclosure, the chlorine oxide is in the form of an aqueous solution, which may be a desirable embodiment because the aqueous solution is easy to handle and relatively easily promotes the generation of chlorine peroxide radicals.

[0039] In one embodiment of the present disclosure, the step of contacting hydrogen ions includes adding an acid. In one embodiment of the present disclosure, the acid is a GRAS acid. GRAS acids are substances generally regarded as safe and can help to safely produce and use chlorine peroxide radicals.

[0040] In one embodiment of the present disclosure, the step of contacting hydrogen ions comprises contacting H ＋ This involves using a type H ion exchange resin. ＋ The type ion exchange resin may be any type that provides hydrogen ions. ＋ The ion exchange resin may be added directly to the chlorine oxide in the form of a powder, or the chlorine oxide may be passed through a column containing the ion exchange resin.

[0041] In one embodiment of the present disclosure, the chlorine peroxide radical is present in an aqueous solution. The chlorine peroxide radical may be stable in an aqueous solution.

[0042] In one aspect of the present disclosure, a composition containing chlorine peroxide radicals is provided, and preferably, an aqueous solution containing chlorine peroxide radicals is provided. In the aqueous solution containing chlorine peroxide radicals of the present disclosure, the chlorine peroxide radicals can exist stably for a long period of time, for example, for 24 hours, 7 days, or even for one month or more.

[0043] In one embodiment of the present disclosure, the chlorine peroxide radical is present as an active ingredient of a disinfectant. As described above, the chlorine peroxide radical can exist stably in an aqueous solution for a long period of time, for example, for 24 hours, 7 days, or even for one month or more. Therefore, in the present disclosure, the composition of the present disclosure can be provided as a long-lasting disinfectant.

[0044] In one aspect of the present disclosure, there is provided a method for killing microorganisms, comprising contacting a chlorine peroxide radical with the microorganism. The microorganisms include viruses, bacteria, and / or fungi. The method of the present disclosure may be used to treat humans. In another aspect of the present disclosure, there is provided a method for preventing or treating a disease or disorder caused by the microorganism, comprising contacting an effective amount of a chlorine peroxide radical with the microorganism.

[0045] In one aspect of the present disclosure, there is provided the use of a chlorine peroxide radical in the manufacture of a disinfectant for killing microorganisms, including viruses, bacteria, and / or fungi.

[0046] In one aspect of the present disclosure, a method is provided for confirming the presence and / or quantifying chlorine peroxide radicals in a liquid, the method comprising detecting and / or measuring the amount of chlorine peroxide radicals by ESR.

[0047] In one aspect of the present disclosure, there is provided the use of chlorine peroxide radicals to kill viruses, bacteria, and / or fungi.

[0048] Free radicals in liquids can be measured by ESR: chlorine peroxide radicals can be identified by four lines with an intensity ratio of 1:1:1:1 with ESR parameter values ​​of g=2.0114 and α=1.85 mT.

[0049] The ESR signal intensity of chlorine peroxide radicals shows a linear relationship with the absorbance at 354 nm in the UV-Vis absorption spectrum, and chlorine peroxide radicals can be quantified by measuring the UV absorption spectrum. The absorbance at 354 nm shows a linear relationship with the free chlorine concentration measured by the DPD method, and chlorine peroxide radicals can also be quantified by measuring the free chlorine concentration by the DPD method.

[0050] In one aspect of the present disclosure, a method for measuring the bactericidal activity of a drug for killing microorganisms is provided, comprising the step of measuring the concentration of chlorine peroxide radicals (Cl-O-O.) in the drug. Examples of microorganisms include viruses, bacteria, and / or fungi. The concentration of chlorine peroxide radicals (Cl-O-O.) can be measured by electron spin resonance. The measurement of the concentration of chlorine peroxide radicals (Cl-O-O.) can include measuring the absorbance at an absorption maximum wavelength of 350 to 360 nm by ultraviolet-visible absorption spectroscopy. The measurement of the concentration of chlorine peroxide radicals (Cl-O-O.) can be performed by measuring the concentration of free chlorine and calculating the concentration of chlorine peroxide radicals (Cl-O-O.). The measurement of the concentration of chlorine peroxide radicals (Cl-O-O.) can be performed by colorimetry or potassium iodide titration. The measurement of the concentration of chlorine peroxide radicals (Cl-O-O.) can be performed by DPD or TMB.

[0051] In one aspect of the present disclosure, a kit for preparing chlorine peroxide radical (Cl—O—O.) may be provided, the kit including chlorite, an acid, and instructions for contacting the chlorite with the acid.

[0052] (Comparison and evaluation of the antimicrobial effect of chlorine peroxide radicals) A problem in comparing and evaluating the antimicrobial effect of chlorine peroxide radicals is that chlorine oxide concentrations are expressed as either effective chlorine concentration or free chlorine, and the antimicrobial effect depends on free chlorine, which is the source of oxidizing power. While the relationship between free chlorine and effective chlorine concentration for sodium hypochlorite is approximately 1:1, it is known that the effective chlorine concentration and free chlorine do not coincide with each other for chlorous acid, as with sodium hypochlorite. Therefore, when evaluating the bactericidal power of chlorine peroxide radicals, we use the oxidizing power, which indicates the antimicrobial effect, that is, free chlorine, rather than the effective chlorine concentration, to compare them.

[0053] The oxidizing power of chlorine oxide chemicals is typically determined using colorimetric methods such as the DPD method or the TMB method. However, unlike sodium hypochlorite, there are no standards for measuring free chlorine for chlorine peroxide radicals. Therefore, a calibration curve is created by setting 1 mg / L of free chlorine (as Cl) for sodium hypochlorite as oxidizing power 1. Note that oxidizing power can be expressed in free chlorine (as Cl). When comparing the same free chlorine, the free chlorine (as Cl) for sodium hypochlorite is generated from Cl radicals. However, if the free chlorine for chlorine peroxide radicals is evaluated based on the free chlorine for chlorine peroxide radicals, the comparison becomes difficult. Therefore, as with sodium hypochlorite, oxidizing power 1 is calculated as 1 mg / L of free chlorine (as Cl), and by using the same standard, the oxidizing power can be compared and evaluated on the same basis as sodium hypochlorite.

[0054] The method for measuring free chlorine (as Cl) is to add a buffer solution and a DPD indicator to the sample and measure the wavelength at 510 nm with an absorptiometer, while the measurement of free chlorine (as Cl) in the presence of organic matter is performed using a TMB reagent at a wavelength of 655 nm, and the concentration is calculated from the measured value. Furthermore, the method for confirming the bactericidal effect is to prepare the free chlorine (as Cl) of the test agent using the DPD method, contact each agent with an organic matter-containing bacterial solution, and after a certain period of time, neutralize it with sodium thiosulfate, and check the number of surviving bacteria in this neutralized solution.

[0055] It should be understood that all references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0056] The present disclosure has been described above by showing preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only, and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0057] Example 1: Identification of chlorine peroxide radicals in aqueous solutions obtained by adding an acid to sodium chlorite. In this study, we investigated the feasibility of identifying active radical species in aqueous solutions obtained by adding an acid to sodium chlorite and quantifying these species using the diethylphenylenediamine (DPD) method. Electron spin resonance (ESR) spectroscopy was used to identify the active radical species in aqueous solutions obtained by adding an acid to sodium chlorite (NaClO ２ We identified the active species generated in a solution containing chlorine peroxide radical (Cl-O-O-). The ESR signal was directly observed at room temperature in an aqueous solution obtained by adding an acid to sodium chlorite. This ESR signal was very long-lived, indicating that the radical was thermodynamically stable. The ESR parameters of this signal did not match previously reported values ​​for the chlorine radical (Cl·) or chlorine dioxide radical (O=Cl·-O and O=Cl-O·). We attribute this signal to the chlorine peroxide radical (Cl-O-O·). Quantum chemical calculations revealed that the optimal structure of the chlorine peroxide radical is much more thermodynamically stable than that of the chlorine dioxide radical. The UV-visible spectrum of the chlorine peroxide radical showed an absorbance maximum at 354 nm. This absorbance was linearly correlated with the chlorine peroxide radical ESR signal intensity. Quantification of free chlorine concentration using DPD revealed a linear relationship with the absorbance maximum at 354 nm, which also showed a linear relationship with the chlorine peroxide radical ESR signal intensity. These linear relationships suggest that the DPD method can quantify chlorine peroxide radicals, which are believed to be the active species.

[0058] Chlorine dioxide-based chlorine-free bleaching (ECF) technology has long been the dominant process in the pulp and paper industry for producing bleached chemical pulp. Chlorine dioxide is typically produced when sodium chlorate or sodium chlorite under strongly acidic conditions is reduced using hydrochloric acid, sulfuric acid, and hydrogen peroxide. In this process, chlorous acid (HClO ２ ) is a reactive intermediate and has been shown to be disproportionate to the production of chlorine dioxide under a variety of conditions.

[0059] NaClO ２ In an acidic solution of ClO ２ It is well known that NaClO is formed by the following reaction: ２ + H+ → HClO ２ +Na ＋ (1) 4HClO ２ →2ClO ２ +ClO ３ － +Cl － +2H++H ２ O (2)

[0060] Equation (2) is the stoichiometry of the disproportionation of chlorous acid reported by several researchers. This reaction under acidic conditions forms chlorate ions, chlorine dioxide, and chloride ions. It has been reported that the disproportionation of chlorous acid proceeds through stepwise reactions under strongly acidic conditions (Ni Y, Yin G. Disproportionation of chlorous acid at a strong acidity. Ind Eng Chem Res. 275 1998;37: 2367-2372). 2HClO ２ →HOCl+HClO ３ (3) ClO ２ - + HClO ２ →HOCl+ClO ３ － (4) HOCl+ClO ２ － →OH － +Cl-ClO ２ (5) Cl-ClO ２ +HClO ２ →H++Cl － +2ClO２ (6)

[0061] In this process, the reaction is initiated by hypochlorous acid, which is simultaneously produced from the disproportionation of one or two chlorous acid molecules with one chlorite ion. The hypochlorous acid then reacts with the chlorite ion to form Cl-ClO, an intermediate for dichlorine dioxide. ２ The dichlorine dioxide then reacts with chlorous acid to form two molecules of chlorine dioxide.

[0062] ASC solution, an aqueous solution obtained by adding acid to sodium chlorite and adjusting the pH to 2.3-2.9, has recently attracted considerable attention as a disinfectant in the food industry. While several studies have clarified the bactericidal and disinfectant effects of chlorous acid, the primary active ingredient in this ASC solution, there is no evidence to indicate which active molecule produces this bactericidal and disinfectant effect. Therefore, the objective of this study was to use ESR spectroscopy on an aqueous solution obtained by adding acid to sodium chlorite, which has only an absorption maximum at 354 m, to identify the primary active species of chlorous acid and then quantify this species using DPD.

[0063] (Materials and Methods) (Chemicals) Sodium thiosulfate, sodium sulfate anhydrous, sodium chlorite (purity: 80%), sodium hypochlorite, hydrochloric acid, sulfuric acid, potassium iodide, and N,N-diethyl-p-phenylenediamine (DPD) were purchased from FUJIFILM Wako Pure Chemical Industries, Ltd. Starch was purchased from Kanto Chemical Co., Ltd. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) was purchased from Sigma-Aldrich.

[0064] (Preparation of aqueous sodium chlorite solutions) Aqueous sodium chlorite solutions were prepared by mixing 0.1 M sodium chlorite with 2.5 M hydrochloric acid to the desired concentration using deionized water. The sodium chlorite concentration was determined by standard iodometric titration.

[0065] (Measurement of free chlorine concentration) The free chlorine concentration was measured by the DPD method previously reported (Goda H, Yamaoka H, ​​Nakayama-Imaohji H, Kawata H, Horiuchi I, Fujita Y, et al. Microbicidal effects of weakly acidified chlorous acid water against feline calicivirus and Clostridium difficile spores under protein-rich conditions. PLoS One. 2017; 12: e0176718).

[0066] (Back Titration of Active Radical Species in Sodium Chlorite with Acid) The presence of active radical species at 354 nm can be determined by thiosulfate titration. The active radical species at 354 nm can be determined by thiosulfate titration. The active radical species were prepared by mixing a 1:1 solution of 0.1 M sodium chlorite with 0.62 M hydrochloric acid for 20 minutes. The absorbance at 354 nm was measured 10 minutes after the addition of sodium thiosulfate. The decrease in average absorbance at 354 nm was plotted against the final concentration of sodium thiosulfate to reveal the relationship between active radical species and absorbance at 354 nm.

[0067] (Spectrophotometric measurement of aqueous solutions obtained by adding acid to sodium chlorite) UV-vis absorption spectra were recorded on a U-5100 differential beam spectrophotometer (HITACHI). When the absorbance exceeded 1.5, the sample was appropriately diluted with ion-exchanged water. The absorbance of such a sample was calculated by multiplying the dilution factor by the absorbance at 354 nm.

[0068] ESR Analysis of an Aqueous Solution Obtained by Adding Acid to Sodium Chlorite: An aqueous solution obtained by adding an acid to sodium chlorite was placed in a glass capillary tube with a length of 100 mm and an inner diameter of 2 mm. The glass capillary was transferred to a capillary cell attached to an ESR cavity, and ESR measurements were performed at room temperature using an X-band spectrometer (JEOL FA-100ESR) operating at 9.43 GHz. The magnetic field was modulated at 100 kHz. The ESR spectrum was measured under the following conditions: resonance frequency = 9.4599 GHz, microwave power = 4 mW, observed magnetic field = 336.0 ± 10 mT, magnetic field modulation width = 0.1 mT, sweep time = 2 min, and time constant = 0.1 s. The signal intensity was normalized with respect to the MnO marker, and the concentration of the stable radical product was determined based on the signal height using the external standard TEMPOL. The radical species found in the aqueous solution obtained by adding acid to sodium chlorite were quantified using integrated software to determine the spectral region mounted on the ESR device.

[0069] Theoretical Analysis: A model of the chlorine peroxide radical (Cl-O-O·) was created by drawing the molecular structure in GaussView 6.0, and the compound was optimized using density functional theory (DFT). The long-range dispersion-corrected functional ωB97X-D was then considered in Gaussian 16 using the 6-311G(d,p) basis set (Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian 16, Revision A.03. Wallingford, CT: Gaussian, Inc.; 2016). The solvation effects of the model were calculated using a conductor-like polarizable continuum model (CPCM). Water was used as the solvent. The optimized molecular structure was verified by vibrational analysis, which confirmed that the equilibrium structure had no imaginary frequencies and that the total energy was corrected by the zero-point vibrational energy.

[0070] (Results and Discussion) (ESR Analysis of Radicals Found in the Aqueous Solution Obtained by Adding Acid to Sodium Chlorite) Free radicals in the aqueous solution obtained by adding acid to sodium chlorite were measured using ESR measurements. Aqueous solutions of sodium chlorite with concentrations varying from 0.1 M to 0.0063 M were mixed with 2.5 M HCl aqueous solution in a 1:1 ratio. The solution was measured by ESR spectroscopy after 4 minutes. Figure 1A shows the ESR spectrum of this sample at room temperature. Surprisingly, this solution exhibited a stable ESR signal under ambient conditions. This ESR signal was observed for at least two weeks after preparation of the solution obtained by adding acid to sodium chlorite. The lifetime of this ESR signal was very long, and the intensity of the ESR signal increased as the concentration of sodium chlorite increased. Free radicals accounted for up to 30% of the chlorite concentration. At high concentrations of NaClO ２ The broadening of the ESR signal observed for HCl is probably due to spin-spin interactions of the radicals.

[0071] Figure 1A. ESR spectrum of radical species in an aqueous solution obtained by adding an acid to sodium chlorite. (Figure 1A) ESR spectrum of radical species observed in the aqueous solution after mixing with an aqueous HCl solution to obtain a final HCl concentration of 1.25 M after 240 seconds. The sodium chlorite concentration is indicated in the figure. (Figure 1B) Dependence of the ESR spectrum of an aqueous solution obtained by adding an acid to sodium chlorite on the HCl concentration. The final HCl concentration is indicated in the figure. The ESR spectrum was recorded 240 seconds after mixing 0.1 M aqueous sodium chlorite and HCl solutions.

[0072] The ESR spectrum was divided into four lines with an intensity ratio of 1:1:1:1. The ESR parameter values ​​were g = 2.0114 and α = 1.85 mT, which is consistent with the data reported by Ozawa et al. (Ozawa T, Kwan T. Electron spin resonance studies of chlorine dioxide (ClO) ２) in aqueous solutions. Chem Pharm Bull. 1983; 31:2864-2867; Ozawa T, Miura Y, Ueda J-I. Oxidation of spin-traps by chlorine dioxide (ClO ２ ) radical in aqueous solutions: first ESR evidence of formation of new nitroxide radicals. Free Radic Biol Med. 1996;20:837-841) (g=2.0106, α=1.85mT). E=hν=gμBH ０ +αIS, I = 1 / 2 and S = 3 / 2 were calculated. Therefore, S = 3 / 2 indicates the chlorine isotopes 35Cl and 37Cl. Furthermore, g = 2.0114 corresponds to g = hν / μBH ０ from the magnetic field (H ０ ) = 336 mT. ０ ) = 336 mT is the center of the four lines. ０ Two intensity ratios (four ratios in total) were observed, extending on either side of the ≈336 mT peak (Figures 1A and 1B). This signal pattern therefore indicates two consecutive oxygen atoms relative to a chlorine nucleus (Cl→O-O).

[0073] To further investigate the signal, the data were compared with previously reported ESR parameters of radical molecules. Hashimoto et al. (Hashimoto M, Nakai Y, Kohno M, Tajima K, Kanaori K, Endo N, et al. DMPO spin trapping of superoxide anion in strong alkaline DMSO solution. Chem Lett. 1997;26:71-72) reported that superoxide anion (O) was trapped in a strong alkaline DMSO solution containing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in the low-temperature solid state (77 K). ２ ・－ The resulting ESR spectrum was

[0074] It is made up of only wide lines, ２ ・－ The formation of " is shown in the g obtained in isotropic superoxide solution. ０ The value is

[0075] It was. ２ ・－ The g value of the adduct was consistent with that of the radical observed in the aqueous solution obtained by adding an acid to sodium chlorite (Fig. 1A). The ESR parameters reported by Adrian et al. ｚ = 2.0100, g ｘ = 1.9987, and g ｙ = 1.9915 (Adrian FJ, Cochran EL, Bowers VA. Electron spin resonance spectrum of ClCO and ClOO ＊ J Chem Phys. 1972;56:6251-6255). This "Cl-O-O" structure is ２ , 0.1% O ２ The same photolysis of a sample consisting of 98.8% Ar gas and 10% Cl 2 HCl was performed. The observed free radicals were measured at cryogenic temperatures, suggesting that they are nuclear chlorine atoms. Therefore, the g of chlorine radical (Cl ⋅) (or chlorine dioxide radical (O-Cl ⋅ -O)) gas is likely to be a chlorine radical with high electron density on the chlorine nucleus. ０ The value is g ０ = (g ｚ +g ｘ +g ｙ ) / 3 = 2.000. ０ The g values ​​are completely different from those reported in this study. These results suggest that the "Cl-O-O" formation in Figure 1A may have a molecular structure similar to that of superoxide.

[0076] In the case of Eachus et al., the reported ESR parameters are g ｘ = 1.9983 and α ｘ = 0.53 mT, g ｙ = 2.0017 and α ｙ = 0.72 mT, and g ｙ= 2.0130 and α ｚ = 1.49 mT (Eachus RS, Edwards PR, Subramanian S, Symons MCR. A peroxy-radical of chlorine, Cl-O-O.Chem Commun (London) 1967; 20:1036-1037. g ０ The value of is g ０ = (g ｚ +g ｘ +g ｙ ) / 3=2.0043 and α ０ = (α ｚ +α ｘ +α ｙ ) / 3 = 0.913 mT. This "chlorine perchlorate" was obtained by irradiating it with gamma rays at 195 K. ４ However, the reported value g ０ = 2.009 is the amount of superoxide anion (O ２ ・－ ) was similar to that of the oxygen radical (O ). ０ = 2.0043 is not g = 2.0114, but the value of chlorine radical (Cl.) g ０ = 2.000. Furthermore, α ０ = 0.913 mT does not correspond to α = 1.85 mT. The sample structure of Eachus et al. may be a chlorine dioxide radical (O=Cl-O.) with a radical present on the oxygen atom.

[0077] Based on the above, the quadrupole splitting seen in Figure 1A should be assigned to the ESR signal of "Cl-OO·", which has an oxygen radical, rather than the electron-dense chlorine dioxide radical (O=Cl·-O) at the nucleus of the chlorine atom. Therefore, this radical active species should be called "Cl-OO·" or chlorine peroxide radical.

[0078] Figure 1B shows the ESR spectra after mixing 0.1 M aqueous sodium chlorite with aqueous HCl solutions ranging from 2.5 M to 0.008 M in a 1:1 ratio. The ESR signal intensified with increasing HCl concentration. This result indicates that chloroperoxy radicals were formed only under acidic conditions.

[0079] (Relationship between Chlorine Peroxide Radical Intensity and Absorbance) As described above, when an aqueous solution of sodium chlorite is acidified, chlorine peroxide radicals are formed. Figure 2A shows the time-dependent change in the chlorine peroxide radical signal after the addition of HCl. The chlorine peroxide radical ESR signal intensity gradually increased at relatively low concentrations of HCl, and this ESR signal remained stable for more than a week when stored in the dark. Figure 2B shows the UV-vis absorption spectrum of an aqueous solution obtained by adding acid to sodium chlorite. The absorption spectrum was measured under the same conditions as in Figure 2A. As the time after mixing increased, the absorption band at 354 nm also increased. Figure 2C shows the relationship between the ESR signal intensity and the absorbance at 354 nm obtained from Figures 2A and 2B. There is a clear linear relationship between the chlorine peroxide radical signal intensity and the absorbance at 354 nm. This result indicates that the 354 nm absorption band typically reported for aqueous solutions obtained by adding acid to sodium chlorite is due to chlorine peroxide radicals.

[0080] Figures 2A to 2C. ESR signal intensity and UV-Vis absorption spectrum of an aqueous solution obtained by adding an acid to sodium chlorite. (Figure 2A) Time-dependent change in the ESR spectrum after mixing 0.1 M sodium chlorite solution with 0.08 M hydrochloric acid in a 1:1 ratio. (Figure 2B) Absorption spectrum of an aqueous solution obtained by adding an acid to sodium chlorite under the same conditions as in Figure 2A. (Figure 2C) Relationship between the chlorine peroxide radical ESR signal intensity and absorbance at 354 nm obtained from Figures 2A and 2B.

[0081] Figure 3A shows the relationship between absorbance at 354 nm and free chlorine concentration in aqueous solutions after mixing sodium chlorite with HCl. As shown in Figure 2B, the absorbance at 354 nm increased in a time-dependent manner. The free chlorine (free, unbound, and total) concentrations were measured simultaneously with UV absorption spectroscopy using DPD. The free chlorine concentration also increased in a time-dependent manner and showed a good linear relationship with the absorbance at 354 nm, indicating a strong correlation between the two. Figure 3B shows an experiment to quench free chlorine by adding thiosulfate. As the thiosulfate concentration in the aqueous solution obtained by adding acid to sodium chlorite increased, the absorbance at 354 nm decreased linearly, indicating that the absorbance at 354 nm is closely related to the active chlorine species. These data indicate that the absorbance at 354 nm is due to the active chlorine reacting with thiosulfate, which was detected by DPD. In Figure 2B, chlorine peroxide radicals exhibit an absorption maximum at 354 nm, which correlates closely with the active chlorine species that react with thiosulfate. This finding suggests that chlorine peroxide radicals may be the active species in the aqueous solution obtained by adding acid to sodium chlorite. The absorbance at 354 nm was also long-lived. The absorption band at approximately 354 nm was measured three months after the preparation of the aqueous solution obtained by adding acid to sodium chlorite.

[0082] Figure 3A shows the relationship between absorbance at 354 nm, the active chlorine concentration in the aqueous solution obtained by adding an acid to sodium chlorite, and the consumption of active chlorine. Figure 3A shows the relationship between absorbance at 354 nm and the free chlorine concentration obtained by the DPD method under the same reaction conditions as Figure 2A. Figure 3B shows the gradual decrease in absorbance at 354 nm of the aqueous solution obtained by adding an acid to sodium chlorite when thiosulfate is added. Data are average ± SD (n = 3).

[0083] The formation of this absorption band at approximately 354 nm, which is due to chlorine peroxide radicals, does not change in the presence or absence of molecular dioxygen in solution (Fig. 6), suggesting that molecular oxygen is not involved in the formation of chlorine peroxide radicals.

[0084] It is well known that chlorite has no antibacterial activity. Total chlorine concentration includes both inactive species, such as chlorite, and active free radicals, such as chlorine peroxide radicals. Therefore, when the concentration of an aqueous solution of ASC obtained by adjusting the pH of sodium chlorite to 2.3-2.9 with an acid is determined by standard iodometric titration, the concentration of active species in this aqueous solution is overestimated, even though it is not sufficient to cause a sterilizing effect. As described above, the DPD method and absorbance at 354 nm showed a linear relationship with the radicals that are the main active species of chlorous acid, the main active ingredient in aqueous ASC, determined by ESR analysis. This indicates that chlorine peroxide, as an active free radical, in aqueous ASC obtained by adjusting the pH of sodium chlorite to 2.3-2.9 with an acid can be quantified using DPD or absorbance at 354 nm.

[0085] Theoretical Analysis of the Chlorine Peroxide Radical: A molecular model of the aqueous phase chlorine peroxide radical (Cl-O-O·) was created by constructing the molecular structure using GaussView 6.0 and optimized using DFT. The stability of the chlorine peroxide radical was quantified in aqueous solution via a conductor-like polarizable continuum model (CPCM) using water as the solvent.

[0086] The optimized structures of the chlorine peroxide radicals and their Singly Occupied Molecular Orbitals (SOMOs) are shown in Figure 4. The Cl-O bond length was 0.245 nm, while the O-O bond length was 0.119 nm. The O-O bond is the same as the bond of molecular oxygen (O ２ , 0.121 nm), and the bond of the dioxygenyl salt (O ２ ＋ , 0.091-0.121 nm at room temperature) is longer than or similar to (Mazej Z, Goreshnik E. Synthesis of dioxygenyl salts by photochemical reactions in liquid anhydrous hydrogen fluoride: X-ray crystal structures of α- and β-O ２ Sn ２ F ９ , O２ Sn ２ F ９ 0.9HF, O ２ GeF ５ ・HF, and O ２ [Hg(HF)] ４ (SbF ６ ) ９ Inorg Chem. 2020;59:2092-2103). According to the Mulliken charge distribution shown in Figure 4, the negative charge was localized on the Cl atom. These structural characteristics suggest that the Cl atom of the chlorine peroxide radical is anionic, whereas the oxygen atom is cationic, like the dioxygenyl cation.

[0087] Figure 4. Optimized structure, charge distribution, and SOMO of the chlorine peroxide radical. Cl and oxygen atoms are shown as spheres. The values ​​indicate the Mulliken charge distribution of each atom, and the mesh surface indicates the spin density of the SOMO.

[0088] [ClO ２ ] ＊ The total energies of the optimized structures of the candidate chlorine peroxide radical (Cl-O-O.) and chlorine dioxide radical (O=Cl.-O) are shown in Table 1. The total energy of the chlorine peroxide radical (Cl-O-O.) was 274 kJ / mol lower than that of the chlorine dioxide radical (O=Cl.-O). This significant difference indicates that the chlorine peroxide radical (Cl-O-O.) is more stable than the chlorine dioxide radical (O=Cl.-O) in the aqueous phase. The theoretical results are consistent with the experimental observation that the chlorine peroxide radical (Cl-O-O.) exhibited a much longer lifetime than the chlorine dioxide radical (O=Cl.-O). Therefore, these results support the theory that the active radical species observed in the ESR measurements is the chlorine peroxide radical (Cl-O-O.).

[0089]

[0090] Figure 5. Spin-trapping ESR measurements of aqueous solutions of sodium chlorite with 1.5 M DMPO at various concentrations of HCl.

[0091] NaClO ２The concentration was 50 mM. The final HCl concentration is shown in the figure. Figure 5 shows the ESR signals obtained by spin trapping with DMPO in an aqueous solution obtained by adding acid to sodium chlorite. The signal strength of these signals was estimated by comparison with the standard TEMPOL solution and the manganese oxide marker signal observed on either side of the DMPO spin trapping signal.

[0092] NaClO by adding HCl solution ２ Although the ESR signal intensity of DMPO-X increased upon acidification of DMPO, the DMPO-OH signal resulting from spin trapping of hydroxyl radicals by DMPO was not detected. The observed DMPO-X signal was in the range of 5 to 8 μM.

[0093] (Figure 6. NaClO ２ Change in absorbance after adding HCl solution to the solution) Next, ２ and 12 M HCl in Milli-Q grade water to obtain 1 mM NaClO ２ and 400 mM HCl were obtained. The absorbance was measured using a Shimadzu Multispec 1500 UV-visible absorption spectrometer. The aqueous solution used as the test sample was continuously stirred during the measurement. For a nitrogen gas atmosphere, Milli-Q grade water was degassed by bubbling nitrogen gas for 30 minutes before the experiment. NaClO was added to aerated or degassed Milli-Q water in a screw-top cuvette. ２ After adding HCl, the cuvette was closed with a screw cap and applied for measurement.

[0094] The formation of chlorine peroxide radicals, measured by absorbance at 354 nm, was unchanged in the presence or absence of oxygen. ２ This indicates that chlorine peroxide radicals were not formed by the reaction of the derived radicals with dissolved molecular oxygen in the solution. The mechanism of chlorine peroxide radical formation is unknown.

[0095] Conclusions: Free radicals in the aqueous solution obtained by adding sodium chlorite to an acid were measured by ESR spectroscopy, and the radical present was assigned to be the chlorine peroxide radical (Cl-O-O·), rather than the chlorine dioxide radical (O=Cl·-O). Total energy calculations using DFT indicated that the chlorine peroxide radical is more stable than the chlorine dioxide radical. The active chlorine peroxide radical in the aqueous solution obtained by adding sodium chlorite to an acid can be quantified using the DPD method or absorbance measurements at 354 nm. Therefore, the oxidizing power of the chlorous acid obtained by adding sodium chlorite to an acid may depend on the highly stable chlorine peroxide radical.

[0096] In a highly concentrated sodium chlorite solution, chlorine peroxide radicals polymerize, reducing the oxidative activity. To create a solution with highly effective bactericidal activity, it is necessary to adjust (optimize) the sodium chlorite concentration and hydrochloric acid concentration (pH).

[0097] The active molecular species involved in sterilization of microorganisms is the main active ingredient, chlorous acid (HClO ２ ), but rather the radical species, chlorine peroxide radical (Cl-O-O.). The amount of chlorine peroxide radical (Cl-O-O.) produced can be determined by ESR or spectroscopic measurements, so it is possible to control the amount of GRAS, which also makes it possible to control the bactericidal activity. By controlling and stabilizing the amount of the active molecular species, chlorine peroxide radical (Cl-O-O.), it can be commercialized as a new disinfectant.

[0098] Chlorite (NaClO ２ ) and chlorous acid (HClO ２ The active molecular species of chlorine dioxide (ClO) is commonly known as ２ ) molecule, but a peroxide radical (R-O-O.). Moreover, this peroxide radical did not show any signal of a radical attached to a chlorine atom (O=Cl.=O). This indicates that chlorous acid (HClO), which is involved in antimicrobial action, so-called bactericidal power and disinfection effects, is ２The active molecular species of this radical is the chlorine peroxide radical (Cl-O-O.), which has been revealed by ESR observations. Therefore, we believe that this chlorine peroxide radical (Cl-O-O.) should not be called the chlorine dioxide radical, but should be called the chlorine peroxide radical.

[0099] In addition, chlorite (NaClO ２ When GRAS acid (hydrochloric acid) is added to HCl, if an excess of hydrogen ions is supplied (protons are supplied), chlorite ions (ClO), which have a maximum absorption at 260 nm, are generated. ２ － ) is first absorbed by chlorous acid (HClO ２ ) to produce chlorine peroxide radicals (Cl-O-O.), which have an absorption maximum at 350 nm to 360 nm.

[0100] Furthermore, it was confirmed that this chlorine peroxide radical (Cl-O-O.) has a positive correlation with the value (free chlorine concentration) obtained by the colorimetric DPD method, and it was found that it can be easily measured using this method.In addition, it is known that there is a strong correlation between the value (free chlorine concentration) obtained by this colorimetric DPD method and the phenolic acid coefficient, which has a strong causal relationship with antimicrobial activity, also known as bactericidal power and disinfection effect.

[0101] From the above results, it was found that this chlorine peroxide radical (Cl-O-O.) can be easily measured using the value (free chlorine concentration) obtained by the DPD method, which is a colorimetric method, and that if this is managed together with the content (total chlorine amount) obtained using the potassium iodide reduction titration method, it will be possible to safely and reliably enjoy the antimicrobial effect, also known as bactericidal power and disinfection effect, as well as the antiviral inactivation effect.

[0102] Example 2: Microbial Sterilization Bacterial Strains and Growth Media A total of 30 Campylobacter strains, including 15 C. jejuri strains (6 clinical, 5 bovine, and 4 chicken) and 15 C. coli strains (8 clinical and 7 porcine), will be used in this study. Each strain will be isolated from a different host and individual, and the species will be identified by species-specific multiplex PCR based on the cdt gene (Asaura et al., 2008, FEMS Immunology and Medical Microbiology, 52, 260-266). Campylobacter will be grown under anaerobic conditions (10% CO ２ , 10%H2, 80%N ２ ) for at least 48 hours at 37°C on blood agar (Blood Agar Base No. 2 (Oxoid Ltd., Basingstoke, United Kingdom) supplemented with 5% (v / v) defibrinated horse blood (Nippon Bio Supp. Center, Tokyo, Japan).

[0103] Bactericidal activity of chlorine peroxide radicals in Bolton broth C. jejuri strains 81-176, ATCC 33560, and Chi2, and C. coli strains ATCC 33559 and Cc4 were cultured on the blood agar described above. Bacterial colonies were suspended in PBS(-) at pH 7.4, and the bacterial concentration was adjusted to approximately 10 ４ The bacterial count is adjusted to CFU / mL. Then, 100 μL of the bacterial suspension is inoculated into 5 mL of Bolton broth (Oxoid Ltd.) and cultured at 37°C for 48 hours under anaerobic conditions as described above. Two hundred 50 μL of the bacterial culture is transferred to a 1.5 mL tube and mixed with an equal volume of PBS(-) at pH 7.4, or with 400 ppm, 2,000 ppm, or 4,000 ppm of chlorine peroxide radical. The mixture is then maintained at room temperature for 3 minutes. Afterwards, 250 μL of 50 mM sodium thiosulfate (Nacalai Tesque, Inc. Kyoto, Japan) is added to inactivate the compound. The remaining viable bacteria are examined by colony counting using blood agar plates.

[0104] Similarly, all Campylobacter strains are treated with PBS(-), pH 7.4, or 400 ppm chlorine peroxide radical. 100 μL of each of these treated bacterial cultures is then inoculated into 5 mL of Bolton broth, and the cells are grown under anaerobic conditions for 48 hours at 37°C. A loopful of culture is then streaked onto a blood agar plate, and the plate is incubated as before.

[0105] As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present invention should be interpreted solely by the claims. This application claims priority to Japanese Patent Application No. 2021-81185 (filed May 12, 2021), the contents of which are incorporated herein by reference in their entirety. It is understood that the patents, patent applications, and other documents cited herein should be incorporated herein by reference in their entirety as if the contents themselves were specifically set forth herein.

[0106] It is possible to provide agents that are useful as antibacterial agents, bactericides, disinfectants, antiseptics, antiviral agents, and food additives (bactericides).

Claims

1. A method for producing chlorine peroxide radicals comprising contacting an excess of hydrogen ions with a chlorine oxide having moieties containing two or more oxygen atoms per chlorine atom.

2. 2. The method of claim 1, wherein the excess amount is greater than 0.07 milliequivalents of hydrogen ions per gram of sodium chlorite-equivalent mass of the chlorine oxide.

3. 2. The method according to claim 1, wherein the excess amount is an amount of hydrogen ions of 0.15 milliequivalents or more per gram of sodium chlorite-equivalent mass of the chlorine oxide.

4. 4. The method of any one of claims 1 to 3, wherein the chlorine oxide is chlorine dioxide, chlorine dioxide gas, aqueous chlorine dioxide, stabilized chlorine dioxide, chlorite, chlorite ions, chlorate, chlorate, chlorate ions, perchlorate, perchlorate, and / or perchlorate ions.

5. The method according to any one of claims 1 to 3, wherein the chlorine oxide is in an aqueous solution.

6. The method of any one of claims 1 to 3, wherein the step of contacting hydrogen ions comprises adding an acid.

7. The method of claim 6 , wherein the acid is a GRAS acid.

8. The step of contacting hydrogen ions is ＋ The method according to any one of claims 1 to 3, comprising using a type ion exchange resin.

9. The method according to any one of claims 1 to 3, wherein the chlorine peroxide radical is present in an aqueous solution.

10. Chlorine peroxide radical.

11. A composition comprising a chlorine peroxide radical.

12. The composition of claim 11 , wherein the composition is an aqueous solution.

13. 13. The composition of claim 11 or 12, wherein the chlorine peroxide radical is present as an active ingredient of a germicide.

14. 1. A method of killing viruses, bacteria, and / or fungi comprising contacting a chlorine peroxide radical with the viruses, bacteria, and / or fungi.

15. Use of a chlorine peroxide radical in the manufacture of a germicide for killing viruses, bacteria, and / or fungi.

16. A method for confirming the presence and / or quantifying chlorine peroxide radicals in a liquid, comprising the step of detecting and / or measuring the amount of chlorine peroxide radicals by ESR.

17. 1. A method for measuring the germicidal activity of an agent for killing viruses, bacteria, and / or fungi, comprising the step of measuring the concentration of chlorine peroxide radicals in the agent.

18. Use of chlorine peroxide radicals to kill viruses, bacteria, and / or fungi.