Methods for producing reactive oxygen species within microorganisms

Abstract

To provide a method for efficiently producing active oxygen in a body of a microorganism by incorporating compounds that have no effect on the human body or a substrate into the body the microorganism, in order to decompose or inactivate compounds that cause stinks and coloring produced by the metabolism of the microorganism, and enzymes and the like involved in the production thereof, to provide a method for controlling microbial metabolism, and to provide a method for controlling microbial metabolites.SOLUTION: Provided is a method for producing active oxygen in a body of a microorganism, which comprises a step (hereinafter referred to as step 1) of contacting a treatment liquid containing the following component (a) with the microorganism to incorporate the component (a) into the body of the microorganism, and performing a reaction to generate hydrogen peroxide by the component (a) and dissolved oxygen in the microorganism and a reaction to generate iron (II) ion by the component (a) and iron (III) ion, in the presence of the iron (III) ion present in a protein structure constituting the microorganism, in the body of the microorganism. Component (a): one or more compounds selected from ascorbic acid and ascorbic acid derivatives.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing reactive oxygen species within microorganisms, a method for controlling the metabolism of microorganisms, and a method for controlling the metabolites of microorganisms. [Background technology]

[0002] In daily life, our interactions with microorganisms such as mold, bacteria, and viruses have been diverse since ancient times, and they are used in food fermentation and pharmaceutical production. On the other hand, microorganisms such as mold, bacteria, and viruses can cause diseases, either by themselves or through their metabolites, or their metabolites can cause foul odors and discoloration, thus reducing the quality of life. To improve these disadvantages to life, killing microorganisms with disinfectants and antibacterial agents is being considered. However, since disinfectants and antibacterial agents destroy the membrane structure and constituent proteins of microorganisms, for example, killing microorganisms on human skin can lead to skin damage, and killing microorganisms on hard materials or base materials such as clothing can lead to deterioration of the base material surface. Furthermore, even if microorganisms are killed, if dead bacterial cells remain, many enzymes remain within the cells, which can lead to the production of compounds that cause foul odors and discoloration. Therefore, it is desirable not only to sterilize the microorganisms themselves, but also to inactivate the enzyme groups within the cells after death, and to neutralize or inactivate any harmful compounds produced by the enzyme system used for sterilization.

[0003] Non-patent document 1 discloses the correlation between the structure of ascorbic acid and its bactericidal activity. Non-patent document 2 discloses the antibacterial activity of ascorbic acid fatty acid esters. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Akira Murata et al., Vitamin Vol. 83, No. 2 (February issue), 2009, pp. 49-52. [Non-Patent Document 2] Shoko Kato et al., Shokuei Magazine, Vol.29, No.5, p.331~335 [Overview of the project] [Problems that the invention aims to solve]

[0005] Approaches to neutralizing microorganisms include treatment from outside the cell with antimicrobial agents such as quaternary ammonium salts, reactive oxygen species, and enzyme inhibitors. However, because microorganisms are protected by membrane structures, the movement of these substances into the cell is limited. Furthermore, when these substances are used at high concentrations, their effects on the human body and substrates must be considered. Therefore, the inventors discovered that if compounds with minimal impact on the human body and substrates can be introduced into microorganisms, and active substances for sterilization and enzyme destruction can be generated within the microorganisms, then microorganisms and compounds derived from microorganisms that have adverse effects on life can be inactivated, leading to the present invention. Reactive oxygen species, which are highly reactive to microorganisms and compounds derived from microorganisms that have adverse effects on life, are expected to cause decomposition and oxidation reactions to the proteins and RNA of the microorganisms themselves, or to compounds produced by microorganisms. Although the generation of reactive oxygen species within some microorganisms is known in metabolic systems such as ATP, there is no known technology to actively generate reactive oxygen species within microorganisms.

[0006] The present invention provides a method for efficiently producing reactive oxygen species within microorganisms, a method for controlling microbial metabolism, and a method for controlling microbial metabolites, in order to decompose or inactivate compounds that cause malodors and discoloration generated by the metabolism of microorganisms, and enzymes involved in their generation, by introducing compounds that have little impact on the human body or substrates into the microorganisms. [Means for solving the problem]

[0007] The present invention relates to a method for producing reactive oxygen species within a microorganism, comprising the steps of: contacting a treatment solution containing the following component (a) with a microorganism to allow the component (a) to be taken into the microorganism; and carrying out a reaction within the microorganism in the presence of iron(III) ions present in the protein structure constituting the microorganism to generate hydrogen peroxide from component (a) and dissolved oxygen in the microorganism, and a reaction in which component (a) and iron(III) ions generate iron(II) ions (hereinafter referred to as step 1). (a) Components: One or more compounds selected from ascorbic acid and ascorbic acid derivatives.

[0008] Furthermore, the present invention relates to a method for producing reactive oxygen species within microorganisms, comprising the step of contacting a treatment solution containing component (a) with microorganisms to react hydrogen peroxide present in the microorganisms with iron(II) ions derived from the microorganisms to generate hydroxyl radicals.

[0009] Furthermore, the present invention relates to a method for controlling microbial metabolism, which involves using reactive oxygen species produced by the method for producing reactive oxygen species within microorganisms according to the present invention to decompose or inactivate enzymes within microorganisms, thereby blocking the metabolic pathways of the microorganisms.

[0010] Furthermore, the present invention relates to a method for controlling microbial metabolites, wherein the reactive oxygen species produced by the method for producing reactive oxygen species within a microbial body according to the present invention decompose or inactivate microbial metabolites released inside and outside the microbial body. [Effects of the Invention]

[0011] The present invention provides a method for efficiently producing reactive oxygen species within microorganisms, a method for controlling microbial metabolism, and a method for controlling microbial metabolites, in order to decompose or inactivate compounds that cause malodorous odors and discoloration produced by the metabolism of microorganisms, as well as enzymes involved in their production, by introducing compounds that have little impact on the human body or substrates into the microorganisms. [Modes for carrying out the invention]

[0012] The mechanism by which the method for producing reactive oxygen species of the present invention generates hydrogen peroxide and hydroxyl radicals, which are the reactive oxygen species of the present invention, within microorganisms is not fully understood, but it is thought to be as follows. However, the present invention is not limited thereto. The method for producing reactive oxygen species according to the present invention begins by bringing a treatment solution containing component (a) of the present invention, which has little impact on the human body, the object to be deodorized, and the environment, into contact with microorganisms, thereby allowing component (a) to be taken up into the microorganisms. It is believed that microorganisms contain relatively hydrophobic parts, such as highly structured protein structures, and hydrophilic parts, such as body fluids, which contain a lot of water. Component (a) taken up into the microorganisms has inherent hydrophilic / hydrophobic properties due to its structure and is distributed in a specific ratio between the hydrophobic and hydrophilic parts of the microorganisms. Some protein structures in microorganisms are complexed with iron(III) ions or stabilized in the form of iron(III) hydroxide, etc. In the hydrophobic parts created by the protein structures, as step 1 of the present invention, in the presence of iron(III) ions, component (a) distributed to the hydrophobic parts and dissolved oxygen in the microorganisms undergo an oxygen reduction reaction, with the iron(III) ions acting as a catalyst, generating hydrogen peroxide. Furthermore, in this hydrophobic region, iron(III) ions present in the protein structure constituting the microorganism undergo a reduction reaction with component (a) to produce iron(II) ions. Some of these reduced iron(II) ions and hydrogen peroxide, having no particular interaction with the protein structure, move to the hydrophilic region. On the other hand, in the hydrophilic sites within the microorganism, the iron(II) ions react with the present hydrogen peroxide, such as the hydrogen peroxide produced in step 1, in a Fenton reaction to generate hydroxyl radicals. In this reaction, the iron(II) ions are oxidized to iron(III) ions. The oxidized iron(III) ions undergo a reduction reaction with component (a) present in the hydrophilic sites to become iron(II) ions, which then react with hydrogen peroxide, allowing for the continuous generation of hydroxyl radicals. In the present invention, since the component (a) can be present in both hydrophobic and hydrophilic sites in a microorganism, hydrogen peroxide can be continuously generated at the hydrophobic site in the microorganism as step 1 of the present invention, and hydrogen peroxide can be continuously supplied to the hydrophilic site in the microorganism. Therefore, as step 2, the production reaction of hydroxyl radicals proceeds efficiently and continuously. As described above, in the method for generating active oxygen of the present invention, it is important that the generation of two types of active oxygen occurs in different environments in the microorganism, that iron (III) ions exist as components of the microorganism itself at the hydrophobic site of the microorganism, and that the component (a) is incorporated and present in both the hydrophobic site and the hydrophilic site. Particularly in a microorganism, the production reactions of these two types of active oxygen can occur efficiently and continuously. Further, when the component (a) has a specific S(Oc) / S(H2O) ratio, after being incorporated into the microorganism, it is distributed in the ratio at the hydrophobic and hydrophilic sites, and since it becomes an environment where step 1 and step 2 occur rapidly, it is considered preferable as a method for generating active oxygen.

[0013] [Method for generating active oxygen in a microorganism] The "active oxygen" of the present invention refers to a molecule having oxygen that easily causes oxidation reactions, radical reactions, etc., and specifically refers to hydrogen peroxide and hydroxyl radicals.

[0014] <Step 1><00000&9>Step 1 is a step of bringing a treatment liquid containing the following component (a) (hereinafter referred to as the treatment liquid of the present invention) into contact with a microorganism to incorporate the component (a) into the microorganism, and in the microorganism, in the presence of iron (III) ions present in the protein structure constituting the microorganism, a reaction of generating hydrogen peroxide from the component (a) and dissolved oxygen in the microorganism and a reaction of generating iron (II) ions from the component (a) and iron (III) ions are carried out. (a) component: one or more compounds selected from ascorbic acid and ascorbic acid derivatives

[0015] <000&093>In the present invention, the microorganism refers to a minute organism whose individual existence cannot be identified by the naked eye of a human, and includes, for example, prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast and mold, lower algae, viruses, and the like. In addition, as the microorganism to be brought into contact with the treatment liquid of the present invention, any microorganism may be used as long as it contains, as a constituent protein, a protein that forms a higher-order structure in which iron (III) ions, for example, form a complex or are stabilized as iron (III) hydroxide or the like. Specifically, one or more selected from Staphylococcus such as Escherichia coli and Staphylococcus aureus, Micrococcus, Moraxella, Acinetobacter, Propionibacterium, Corynebacterium, Legionella, Lactobacillus, Clostridium, Streptococcus, Haemophilus, Shigella, Ralstonia solanacearum, Sphingomonas, Pseudomonas, Xanthomonas, and Aspergillus are mentioned. From the viewpoint of the ability to produce the odor of the bacteria, preferably one or more selected from Escherichia coli, Staphylococcus such as Staphylococcus aureus, Micrococcus, Moraxella, Acinetobacter, Propionibacterium, and Corynebacterium, and more preferably one or more selected from Escherichia coli and Staphylococcus such as Staphylococcus aureus.

[0016] By bringing the treatment liquid of the present invention into contact with the microorganism, the component (a) is taken into the microorganism, and in the presence of iron (III) ions present in the protein structure constituting the microorganism in the microorganism, the component (a) and the dissolved oxygen in the microorganism are reacted to generate hydrogen peroxide. Further, separately and in parallel with the above reaction, the iron (III) ions are reduced by the component (a), and iron (II) ions derived from the microorganism are generated in the microorganism. Specific examples of the protein that forms a structure in which iron (III) ions are present include one or more selected from ferritin, methemoglobin, transferrin, and lactoferrin. From the viewpoint of supplying iron (III) ions to the hydrogen peroxide generation reaction, preferably one or more selected from ferritin and methemoglobin, and more preferably ferritin.

[0017] In the present invention, "bringing the treatment solution of the present invention into contact with microorganisms" is a concept that includes acts such as directly bringing the microorganisms attached to the object to be deodorized into contact with the microorganisms (for example, immersing the object to which microorganisms are attached in the treatment solution, spraying or applying the treatment solution), or after the object has been treated with the treatment solution in advance, such as by washing, spraying or applying, and then dried, etc., to allow component (a) to adhere to the surface of the object, then new microorganisms adhere to the surface of the object, and component (a) on the surface of the object dissolves via moisture such as urine or sweat, and comes into contact with the newly attached microorganisms. In short, it is sufficient that component (a) and microorganisms can come into contact.

[0018] <Process 2> The present invention's method for producing reactive oxygen species within microorganisms preferably further includes a step of reacting hydrogen peroxide obtained in step 1 with iron(II) ions derived from microorganisms within the microorganisms to generate hydroxyl radicals. In step 2, instead of hydrogen peroxide obtained in step 1, hydrogen peroxide present in the microorganism may be reacted with component (a) and iron(II) ions derived from the microorganism to generate hydroxyl radicals. In other words, the present invention provides a method for producing reactive oxygen species within microorganisms, comprising the step (2) of contacting a treatment solution containing component (a) with microorganisms to react hydrogen peroxide present in the microorganisms with iron(II) ions derived from the microorganisms to generate hydroxyl radicals.

[0019] In the present invention, "iron(II) ions derived from microorganisms" refers to iron(II) ions produced when iron(III) ions present in the protein structure within the microorganism in step 1 of the present invention are reduced by component (a), iron(III) ions produced when iron(II) ions are oxidized in the hydroxyl radical generation reaction in step 2 of the present invention are further reduced by component (a) in the vicinity, and iron(II) ions taken into the body by microorganisms from the environment.

[0020] In step 2, the hydrogen peroxide obtained in step 1 or the hydrogen peroxide present in the microorganism and iron(II) ions derived from the microorganism undergo a Fenton reaction within the microorganism, generating hydroxyl radicals, which are reactive oxygen species. These hydroxyl radicals can decompose or inactivate odor compounds produced by the metabolism of microorganisms, as well as enzymes involved in the generation of these odor compounds.

[0021] Odor compounds produced by microorganisms include one or more selected from phenols such as p-cresol, indoles such as skatole, fatty acids such as 3-hydroxy-3-methylhexanoic acid and 3-methyl-2-hexenoic acid, thioalcohols such as 3-mercapto-3-methylhexanol, amines such as ammonia, and steroids such as androstenone. Enzymes involved in the production of odor compounds produced by microorganisms include one or more selected from β-glucuronidase, lipase, aminoacylase, β-lyase, urease, and arylsulfatase.

[0022] Steps 1 and 2 are carried out simultaneously by bringing the treatment solution of the present invention into contact with microorganisms, thereby allowing component (a) to be taken up into the microorganisms. In other words, the method for producing reactive oxygen species within microorganisms according to the present invention may be carried out simultaneously in steps 1 and 2. As a method for bringing the treatment solution of the present invention into contact with microorganisms, when applied to microorganisms present in textile products, for example, a method is to prepare a washing solution by mixing the treatment solution of the present invention with water, and then wash the textile products with the washing solution in a washing machine or the like. After washing, the washed textile products are dried. In this case, steps 1 and 2 are performed during or after washing (during or after drying).

[0023] A method for bringing the treatment solution of the present invention into contact with microorganisms is, when applied to microorganisms present in a textile product, for example, by spraying or applying the treatment solution of the present invention to the textile product. Alternatively, the method for bringing the treatment solution of the present invention into contact with microorganisms may be to spray or apply the treatment solution of the present invention to the textile product and then bring the microorganisms into contact with the textile product when they newly adhere to the textile product. A preferred method for spraying or applying the treatment solution of the present invention to a textile product is to fill the treatment solution of the present invention with a container equipped with a sprayer and spray the treatment solution onto the textile product to bring the treatment solution into contact with the textile product. Alternatively, the treatment solution of the present invention may be applied to the textile product using an applicator such as a cloth or brush to bring the treatment solution into contact with the textile product. Steps 1 and 2 are performed by spraying or applying the treatment solution of the present invention to the textile product.

[0024] <Processing solution> The processing solution of the present invention contains component (a). Component (a) is preferably one or more compounds selected from ascorbic acid and ascorbic acid derivatives, wherein the distribution ratio S(Oc) / S(H2O) of the molar concentration S(Oc) in the n-octanol phase and the molar concentration S(H2O) in the aqueous phase at 20°C, based on the ascorbic acid skeleton, when all components (a) are dissolved in a mixture of equal amounts of n-octanol and water, is 0.0004 or more and 7000 or less.

[0025] (a) The component distribution ratio S(Oc) / S(H2O) is, from the viewpoint of producing hydrogen peroxide and hydroxyl radicals as reactive oxygen species (hereinafter, from the viewpoint of reactive oxygen species production), 0.0004 or more, preferably 0.01 or more, more preferably 0.1 or more, even more preferably 0.3 or more, even more preferably 1 or more, even more preferably 10 or more, and 7000 or less, preferably 1000 or less, more preferably 100 or less, even more preferably 50 or less, and even more preferably 25 or less.

[0026] (a) The distribution ratio S(Oc) / S(H2O) of the molar concentration S(Oc) of component in the n-octanol phase and the molar concentration S(H2O) in the aqueous phase can be determined by either a method that involves dissolving one or more compounds selected from ascorbic acid and ascorbic acid derivatives in a mixed solution of n-octanol and water at 20°C, and then quantifying the ascorbic acid groups present in the n-octanol phase and aqueous phase, or by a method that involves calculating the ratio from the logP value of one or more compounds selected from ascorbic acid and ascorbic acid derivatives.

[0027] [Method for calculating the amount of ascorbic acid groups present in the n-octanol phase and aqueous phase] The distribution ratio S(Oc) / S(H2O) of component (a) can be measured by the method based on JIS Z7260-107. Specifically, all components (a) are dissolved in a mixture of equal volumes of n-octanol and water at 20°C to a concentration of 100 mg / L, and the test container is shaken by a shaker or by hand (5 minutes, approximately 100 times). After immersion, phase separation is performed by centrifugation, and the n-octanol phase and aqueous phase are sampled and their concentrations are measured using high-performance liquid chromatography (HPLC) or the like to calculate the concentration. For example, the concentration of component (a) can be measured by HPLC under the following conditions using UV detection at 255 nm. • Eluent A: 1% v / v phosphate aqueous solution (pH 3.0) Eluent B: Methanol / acetonitrile = 1:1 (v / v) mixture • Gradient conditions: 35% eluent B (0-5 min) → 35%-45% eluent B (5-12 min) → 40-90% eluent B (12-15 min), flow rate: 1.5 mL / min, sample injection volume: 20 μL, column: C18-ODS column

[0028] [Method for calculating from the logP values ​​of ascorbic acid and ascorbic acid derivatives] (a) The component distribution ratio S(Oc) / S(H2O) is calculated for one or more compounds selected from ascorbic acid and ascorbic acid derivatives using the following formula.

[0029]

number

[0030] In the formula, C i is the molar concentration of all (a) components of compound i, and LogP i This is the LogP value of compound i calculated from ChemDraw Professional 17.1 using Crippen's fragmentation method (J. Chem. Inf. Comput. Sci., 27, 21 (1987)).

[0031] (a) Examples of ascorbic acid derivatives of component (a) include one or more selected from ascorbic acid fatty acid esters, ascorbic acid phosphate esters, ascorbic acid sulfate esters, and ascorbic acid glycosides. Specific examples include ethyl ascorbic acid, ascorbyl caprylate, ascorbyl laurate, ascorbyl palmitate, ascorbyl isopalmitate, ascorbyl dipalmitate, ascorbyl diisopalmitate, ascorbyl stearate, ascorbyl isostearate, ascorbyl distearate, ascorbyl diisostearate, ascorbyl myristate, ascorbyl isomyristate, ascorbyl dimyristate, ascorbyl diisomyristate, ascorbyl 2-ethylhexanoate, and di Examples include alkyl fatty acid esters of ascorbate such as ascorbyl 2-ethylhexanoate, ascorbyl oleate, ascorbyl dioleate, ascorbyl tetrahexyldecanoate, glyceryl ascorbate, and caprylyl ascorbate-2-glyceryl ester, ascorbate-2-phosphate ester, ascorbate-3-phosphate ester, DL-α-tocopherol-2-ascorbate phosphate diester, palmityl ascorbate-2-phosphate ester, ascorbate sulfate esters such as ascorbate-2-sulfate and ascorbate-3-sulfate ester, and ascorbic acid glycosides such as ascorbate-2-glucoside, and one or more of these can be used. The ascorbic acid derivative of component (a) can also be salts thereof, and alkali metal salts such as sodium salt and potassium salt, and alkaline earth metal salts such as calcium salt and magnesium salt are preferably used. However, when using these ascorbic acid derivatives, it is preferable that the distribution ratio S(Oc) / S(H2O) of component (a) satisfies the above range. Specifically, the distribution ratio S(Oc) / S(H2O) of component (a) can satisfy the above range by using one or two or more specific ascorbic acid derivatives of ascorbic acid.

[0032] (a) From the viewpoint of reactive oxygen species production, one or more components selected from ascorbic acid fatty acid esters are preferred. From the viewpoint of reactive oxygen species production, the carbon number of the raw material fatty acid of the ascorbic acid fatty acid ester is preferably 6 or more, more preferably 8 or more, even more preferably 10 or more, even more preferably 12 or more, and preferably 26 or less, more preferably 20 or less, even more preferably 18 or less, even more preferably 16 or less, and even more preferably 14 or less. Examples of ascorbic acid fatty acid esters include monoesters, diesters, or triesters, and monoesters are preferred.

[0033] (a) When the component is one type of ascorbic acid fatty acid ester, the number of carbon atoms in the raw material fatty acid is preferably 6 or more, more preferably 8 or more, even more preferably 10 or more, even more preferably 12 or more, and preferably 26 or less, more preferably 20 or less, even more preferably 18 or less, even more preferably 16 or less, and even more preferably 14 or less, from the viewpoint of reactive oxygen species production.

[0034] (a) When component (a) is used in combination from two or more types of ascorbic acid and ascorbic acid fatty acid esters, it is preferable that the distribution ratio S(Oc) / S(H2O) of component (a) as a whole satisfies the above range, and there are no particular restrictions on the combination. When component (a) is used in combination from two types of ascorbic acid and ascorbic acid fatty acid esters, the distribution ratio S(Oc) / S(H2O) of one of the ascorbic acid and ascorbic acid fatty acid esters is preferably 0.0004 or more, preferably 1.0 or less, more preferably 0.5 or less, and even more preferably 0.1 or less, from the viewpoint of reactive oxygen species production, and specifically, ascorbic acid and ascorbic acid fatty acid ester having 2 to 10 carbon atoms in the raw material fatty acid, and from the viewpoint of reactive oxygen species production, ascorbic acid is preferred. On the other hand, the distribution ratio S(Oc) / S(H2O) of the ascorbic acid fatty acid ester is preferably 10 or more, more preferably 100 or more, even more preferably 500 or more, and preferably 8000 or less, and even more preferably 1000 or less, from the viewpoint of reactive oxygen species production. Specifically, the number of carbon atoms in the raw material fatty acid is preferably 12 or more, more preferably 14 or more, and preferably 20 or less, more preferably 18 or less, and even more preferably 16 or less, from the viewpoint of reactive oxygen species production.

[0035] The ratio of the two types of ascorbic acid and ascorbic acid fatty acid esters added is preferably adjusted so that the distribution ratio S(Oc) / S(H2O) of the molar concentration S(Oc) in the n-octanol phase and the molar concentration S(H2O) in the aqueous phase, calculated by averaging their respective distribution ratios S(Oc) / S(H2O), is 0.0004 or higher, preferably 0.01 or higher, more preferably 0.1 or higher, even more preferably 0.3 or higher, even more preferably 1 or higher, even more preferably 10 or higher, and 7000 or lower, preferably 1000 or lower, more preferably 100 or lower, even more preferably 50 or lower, and even more preferably 25 or lower, from the viewpoint of reactive oxygen species production.

[0036] In the case of using component (a) of the present invention for the purpose of treating an object in water, variations in the composition of component (a) adsorbed on the surface of the object can lead to variations in the effect, so it is preferable to constitute the treatment solution of the present invention with one type of ascorbic acid fatty acid ester. Furthermore, in the case of using the treatment solution of the present invention by spraying or applying it to the surface of an object, all of component (a) will be fixed to the surface of the object, so it may be one type of ascorbic acid fatty acid ester or two or more types of ascorbic acid and ascorbic acid fatty acid esters.

[0037] The treatment solution of the present invention may contain a surfactant as component (b). The method for producing reactive oxygen species of the present invention often uses an aqueous solution of component (a) as the treatment solution. However, depending on the structure of component (a), some may have low solubility in water or poor dispersibility, which can reduce the efficiency of uniform treatment and the movement of component (a) into the microorganisms. Therefore, it is preferable to include component (b) in the treatment solution in order to improve the solubility of component (a) in the treatment solution and improve the uptake of component (a) into the microorganisms, and furthermore, to improve the deodorizing effect of the present invention and to provide other values ​​such as cleaning properties. The surfactant is not particularly limited as long as it does not adversely affect the progress of the process of the present invention or the reactive oxygen species production effect of component (a), but examples include one or more selected from (b1) anionic surfactants (hereinafter also referred to as component (b1)), (b2) cationic surfactants (hereinafter also referred to as component (b2)), (b3) nonionic surfactants (hereinafter also referred to as component (b3)), and (b4) amphoteric surfactants (hereinafter also referred to as component (b4)).

[0038] (b1) Examples of anionic surfactants include anionic surfactants having one or more hydrocarbon groups with 8 to 18 carbon atoms and one or more groups selected from the group consisting of sulfonic acid groups, sulfate ester groups and carboxylic acid groups. Examples of anionic surfactants include alkyl or alkenylbenzenesulfonic acid or salts thereof with 8 to 18 carbon atoms, polyoxyalkylene alkyl or alkenyl ether sulfate esters or salts thereof with 8 to 18 carbon atoms, alkyl or alkenyl sulfate esters or salts thereof with 8 to 18 carbon atoms, internal olefin sulfonic acid or salts thereof with 8 to 18 carbon atoms, and fatty acids or salts thereof. The salts of the anionic surfactant are preferably alkali metal salts such as sodium salts and potassium salts.

[0039] (b2) Examples of cationic surfactants include quaternary ammonium salt type cationic surfactants. Examples of quaternary ammonium salt type cationic surfactants include quaternary ammonium salt type cationic surfactants in which one or two of the groups bonded to the nitrogen atom are hydrocarbon groups having 6 or more carbon atoms, preferably 8 or more, and 16 or less, preferably 14 or less, and the remaining group is selected from the group consisting of alkyl groups having 1 to 3 carbon atoms, hydroxyalkyl groups having 1 to 3 carbon atoms, and arylalkyl groups (such as benzyl groups).

[0040] (b3) Examples of nonionic surfactants include polyoxyalkylene alkyl ethers having an alkyl group with 8 to 18 carbon atoms, polyoxyalkylene alkenyl ethers having an alkenyl group with 8 to 18 carbon atoms, polyoxyalkylene sorbitan fatty acid esters having a fatty acid group with 8 to 18 carbon atoms, alkyl glycosides having an alkyl group with 8 to 18 carbon atoms, alkyl polyglycosides having an alkyl group with 8 to 18 carbon atoms, sucrose fatty acid esters having a fatty acid group with 8 to 18 carbon atoms, and alkyl polyglyceryl ethers having an alkyl group with 8 to 18 carbon atoms. One or more of these can be used. (b3) The component is preferably a polyoxyethylene alkyl ether having an alkyl group with 8 to 18 carbon atoms and an average number of ethylene oxide addition moles of 2 to 50.

[0041] The component (b3) is preferably a nonionic surfactant represented by the following general formula (b3). R 31b -O-[(C2H4O) s (C3H6O) t ]-H(b3) [In the formula, R 31b (C2H4O) and (C3H6O) may be a C14 alkyl or alkenyl group having 8 or more carbon atoms, preferably 10 or more, and 18 or less, preferably 16 or less, and may be a residue derived from a primary or secondary alcohol. s and t are the average number of moles added, where s is 2 or more, preferably 4 or more, more preferably 6 or more, even more preferably 10 or more, and 50 or less, preferably 40 or less, more preferably 20 or less, and t is 0 or more, preferably 1 or more, and 5 or less, preferably 3 or less, and t may be 0. (C2H4O) and (C3H6O) may be random polymers or block polymers.

[0042] (b4) Examples of amphoteric surfactants include one or more surfactants selected from betaine-type surfactants and amine oxide-type surfactants. (b4) Specifically, examples of components include one or more surfactants selected from sulfobetaine, carbobetine, and amine oxide.

[0043] Examples of sulfobetaines include N-alkyl-N,N-dimethyl-N-sulfopropylammonium sulfobetaines in which the alkyl group has 10 to 18 carbon atoms, N-alkyl-N,N-dimethyl-N-(2-hydroxy-sulfopropyl)ammonium sulfobetaines in which the alkyl group has 10 to 18 carbon atoms, N-acylaminopropyl-N,N-dimethyl-N-sulfopropylammonium sulfobetaines in which the alkanoyl group has 10 to 18 carbon atoms, and N-acylaminopropyl-N,N-dimethyl-N-(2-hydroxy-sulfopropyl)ammonium sulfobetaines in which the alkanoyl group has 10 to 18 carbon atoms.

[0044] Examples of carboxybetaines include N-alkyl-N,N-dimethyl-N-carboxymethylammonium betaines in which the alkyl group has 10 to 18 carbon atoms and compounds represented by the following general formula (b41).

[0045]

Chemical formula

[0046] [In the formula, R 41b represents an alkyl group or an alkenyl group having 7 to 21 carbon atoms, R 42b represents a propylene group, and R 43b and R 44b each independently represent an alkyl group having 1 to 3 carbon atoms. ]

[0047] As the amine oxide, a compound of the following general formula (b42) is preferable.

[0048]

Chemical formula

[0049] [In the formula, R 45b represents a hydrocarbon group having 7 to 22 carbon atoms, preferably an alkyl group or an alkenyl group, more preferably an alkyl group, and R46b and R 47b represents an alkyl group having 1 to 3 carbon atoms, either identical or different. D represents a -NHC(=O)- group or a -C(=O)NH- group, and E represents an alkylene group having 1 to 5 carbon atoms. m and p represent m=0 and p=0 or m=1 and p=1.

[0050] The processing solution of the present invention contains component (a) in an amount of preferably 0.001% by mass or more, more preferably 0.01% by mass or more, even more preferably 0.05% by mass or more, and preferably 8% by mass or less, more preferably 5% by mass or less, from the viewpoint of reactive oxygen species production.

[0051] The treatment solution of the present invention contains component (b) in an amount of preferably 0.001% by mass or more, more preferably 0.01% by mass or more, even more preferably 0.05% by mass or more, and preferably 20% by mass or less, and more preferably 10% by mass or less, from the viewpoint of reactive oxygen species production.

[0052] In the treatment solution of the present invention, the mass ratio (a) / (b) of the content of component (a) to the content of component (b) is preferably 0.01 or more, more preferably 0.05 or more, even more preferably 0.1 or more, even more preferably 0.3 or more, and preferably 0.8 or less, more preferably 0.7 or less, and even more preferably 0.6 or less, from the viewpoint of reactive oxygen species production.

[0053] The treatment solution of the present invention contains water. The water can be deionized water, distilled water, tap water, or water containing 1 mg / kg to 5 mg / kg of sodium hypochlorite. The treatment solution of the present invention may use water as the remainder of the components constituting the treatment solution, but specifically, it preferably contains 50% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, and preferably 99.9% by mass or less, more preferably 99% by mass or less, and even more preferably 98% by mass or less.

[0054] The treatment liquid of the present invention can be appropriately set according to its purpose. From the perspective of active oxygen generation, the pH at 25°C is preferably 3 or more, more preferably 4 or more, and preferably 12 or less, more preferably 11 or less. The pH is a value measured at 25°C using a glass electrode. Specifically, it is measured by the following method. <Method for Measuring pH> Calibrate the pH electrode (model 6367) in advance with phthalic acid buffer solution (pH 4.01), phosphate standard solution (pH 6.84), and borate standard solution (pH 9.18) for the pH meter D-52 manufactured by Horiba, Ltd., and thoroughly rinse it with ion-exchanged water. Put the pH electrode calibrated and washed as described above into the treatment liquid adjusted to 25°C, and measure using the AUTO HOLD mode of the pH meter until the measured value becomes constant.

[0055] From the perspective of spray suitability in a container equipped with a sprayer, the viscosity of the treatment liquid of the present invention at 25°C is preferably 15 mPa·s or less, more preferably 10 mPa·s or less, still more preferably 5 mPa·s or less, and preferably 1 mPa·s or more, more preferably 1.5 mPa·s or more, still more preferably 2 mPa·s or more. The viscosity of the treatment liquid is measured by attaching a No. 1 rotor to a B-type viscometer (model BM) manufactured by Tokyo Keiki Co., Ltd., filling a 200 mL glass tall beaker with the deodorant composition, adjusting it to 25 ± 0.3°C in a water bath, setting the rotation speed of the rotor to 60 r / min, and taking the indicated value 60 seconds after starting the measurement.

[0056] [Method for Controlling Microbial Metabolism, Method for Controlling Microbial Metabolites] The present invention provides a method for controlling the metabolism of a microorganism, which decomposes or inactivates an enzyme in the microorganism by active oxygen generated by the method for generating active oxygen in the microorganism of the present invention to block the metabolic pathway of the microorganism. The present invention also relates to a method for controlling microbial metabolites, which decomposes or inactivates microbial metabolites discharged inside and outside the microorganism by active oxygen generated by the method for generating active oxygen in the microorganism of the present invention. For example, β-glucuronidase-active bacteria metabolize β-glucuronidase, an enzyme involved in the production of odor compounds within the microbial body, and use this enzyme to break down precursors of odorous substances, thereby generating odorous materials. In this invention, β-glucuronidase-active bacteria are inducing the production of hydroxyl radicals, which are reactive oxygen species, within the microbial body using the reactive oxygen species production method of this invention. These hydroxyl radicals then decompose or inactivate the enzyme β-glucuronidase, which is a metabolite of the β-glucuronidase-active bacteria. This blocks the metabolic pathway of the β-glucuronidase-active bacteria and suppresses the generation of odor-causing substances. [Examples]

[0057] Using the following components, the test solutions shown in Tables 1-3 were prepared, and the resulting compositions were evaluated as described below.

[0058] (a) component • Ascorbyl caprylate: Synthetic product, LogP -0.39 • Ascorbyl laurate: Synthetic product, LogP 1.28 • Ascorbyl palmitate: Manufactured by Tokyo Chemical Industry Co., Ltd., LogP 2.95 • Ascorbyl stearate: Manufactured by Tokyo Chemical Industry Co., Ltd., LogP 3.79 Ascorbic acid: Manufactured by Tokyo Chemical Industry Co., Ltd., LogP -3.36

[0059] Ascorbyl caprylate and ascorbyl laurate were synthesized according to J Am Oil Chem Soc 54: 308-312 (1977).

[0060] <Synthesis of ascorbyl laurate> L-ascorbic acid (8.0 mmol) and lauric acid (10 mmol) were dissolved in concentrated sulfuric acid (25 mL) and stirred at 25°C for 24 hours to carry out the esterification reaction. The reaction mixture was poured into ice water (150 mL), extracted with diethyl ether, and the solvent was removed by evaporation to obtain the crude product. The obtained crude product was purified by washing with hexane. The reaction process and compound identification were performed by measuring TLC and NMR spectra, and a purity of 98% or higher was confirmed.

[0061] <Synthesis of ascorbyl caprylate> Caprylic acid ascorbyl was synthesized in the same manner as ascorbyl laurate, except that lauric acid was replaced with caprylic acid. The reaction process and compound identification were performed by measuring TLC and NMR spectra, and a purity of 98% or higher was confirmed.

[0062] The distribution ratio S(Oc) / S(H2O), which is the molar concentration in the n-octanol phase and the molar concentration in the aqueous phase relative to the ascorbic acid skeleton at 20°C when component (a) of each test solution in Tables 1-3 is dissolved in a mixture of equal volumes of n-octanol and water, was calculated using the following formula.

[0063]

number

[0064] In the formula, C i is the molar concentration of all (a) components of each compound i, and LogP i This is the LogP value of each compound i calculated from ChemDraw Professional 17.1 using Crippen's fragmentation method (J. Chem. Inf. Comput. Sci., 27, 21 (1987)).

[0065] (b) Component • Nonionic surfactant 1: Polyoxyalkylene alkyl ether obtained by sequentially adding ethylene oxide (average 9), propylene oxide (average 2), and ethylene oxide (average 9) to lauryl alcohol. • Nonionic surfactant 2: Polyoxyalkylene alkyl ether (Softanol 70H, manufactured by Nippon Shokubai Co., Ltd.) obtained by adding an average of 7 moles of ethylene oxide to a secondary alcohol with 12 to 14 carbon atoms. • Cationic surfactant 1: N,N-didecyl-N-ethyl-N-methylammonium sulfate ethyl salt (Cotamine D10-ES, manufactured by Kao Corporation) • Cationic surfactant 2: N-ethyl-N,N-dimethyltetradecylammonium sulfate ethyl salt (Cotamin 40ES, manufactured by Kao Corporation)

[0066] Other ingredients • Ferritin: Ferritin (derived from horse spleen) (100 mg / 1 mL) (manufactured by Tokyo Chemical Industry Co., Ltd.), a model protein that possesses iron(III) ions. • BSA: Albumin, derived from bovine serum (BSA), protease-free (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a model protein that does not possess iron(III) ions. • Apoferritin: Apoferritin (derived from horse spleen) (manufactured by Sigma-Aldrich), a model protein that does not contain iron(III) ions. ·DPBS:Dulbecco`s Phosphate-BufferedSaline(DPBS,calcium, magnesium, Thermo FisherScientific) • 0.1M Phosphate Buffer (pH 6.0): 0.1 mol / L phosphate buffer, pH 6.0 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0067] <(a) Confirmation of the location of the component> The test solutions shown in Table 1 were prepared and allowed to stand at 25°C for 2 hours. After standing, ferritin was removed by ultrafiltration using AmiconUltra 100K Devices. The filtrate was diluted 10-fold with methanol to prepare the measurement solution. Next, the components (a) used were diluted with methanol one by one to prepare calibration solutions at concentrations of 0.1 ppm, 0.5 ppm, 1.0 ppm, 5.0 ppm, and 10.0 ppm. The prepared calibration solutions were quantified using a liquid chromatography-mass spectrometer (hereinafter abbreviated as LCMS) under the following conditions to create a calibration curve. Next, the measurement solution was quantified using the LCMS, and the concentration of component (a) in the measurement solution (aqueous phase) was determined from the calibration curve. Then, the concentration of component (a) in ferritin was determined using the following formula. Concentration of component (a) in ferritin = (Total concentration of component (a) in the test solution (25 ppm)) - (Concentration of component (a) in the measurement solution)

[0068] • LCMS system: Shimadzu Corporation LCMS2020 (ESI detection) • Measurement mode: SIM • Measured ions: Ascorbic acid: m / z(-)=175.0, Ascorbyl caprylate: m / z(-)=301.0, Ascorbyl laurate: m / z(-)=357.0, Ascorbyl myristate: m / z(-)=413.2, Ascorbyl stearate: m / z(-)=441.2 • Column: Imtakt Unison UK-C18 HT (50*2 mm) 3 μm • Eluent A: 0.1% formic acid aqueous solution Eluent B:methanol:acetonitrile = 1:1 • Gradient conditions: 50% eluent B (0-2 min) → 50%-90% eluent B (2-3.3 min) → 90% eluent B (3.3-5.3 min) → 90%-50% eluent B (5.3-6.0 min) → 90% eluent B (6.0-8.0 min), flow rate: 0.6 mL / min, sample injection volume: 5 μl, column temperature: 40°C

[0069] [Table 1]

[0070] As shown in Table 1, component (a) is distributed in a specific ratio between the structure formed by ferritin, which acts as a hydrophobic site in the test solution, and the aqueous phase. The distribution ratio does not necessarily coincide with the distribution ratio S(Oc) / S(H2O) value used as an indicator in this invention because ferritin and n-octanol have different degrees of hydrophobicity. However, since the trend matches between the distribution ratio S(Oc) / S(H2O) value and the measured distribution rate for ferritin, the distribution ratio S(Oc) / S(H2O) value serves as a measure of the distribution behavior of component (a) to hydrophobic and hydrophilic sites in cells. Furthermore, ferritin is a model of a protein structure that possesses iron(III) ions and forms hydrophobic sites within microorganisms. The prepared test solution, under a phosphate buffer atmosphere, serves as a model of hydrophilic sites within microorganisms, and as a whole, it serves as a model of the inside of a microbial cell.

[0071] <Analysis of hydrogen peroxide production (Step 1)> Using the above components, the test solutions shown in Table 2 were prepared. To the prepared test solutions, a fluorescent probe BES-H2O2-Ac (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a concentration of 20 μM for the detection of hydrogen peroxide, and the solutions were incubated at 25°C for 30 minutes. The fluorescence intensity (Ex / Em: 485 / 515) of the reaction solution was measured using a microplate reader Infinite(R)200PRO (Tecan). The fluorescence intensity ratio, with Comparative Example 1 set to 1, was calculated as the amount of hydrogen peroxide generated. The results are shown in Table 2. Furthermore, the fluorescent probe reacts immediately with hydrogen peroxide and emits fluorescence. Since no decomposition of hydrogen peroxide or other reactions occur, the hydrogen peroxide production process in step 1 is shown.

[0072] <Analysis of iron(II) ion production (Step 1)> Using the above components, the test solutions shown in Table 2 were prepared. The test solutions were incubated at 37°C for 24 hours. To detect iron(II) ions, 1 μL of a 1 mM DMSO solution of the fluorescent probe FerroOrange (Dojin Chemical) was added to 500 μL of the test solution and reacted at 25°C for 30 minutes, then the solution was analyzed using a microplate reader Infinite. TMThe fluorescence intensity (Ex / Em: 543 / 580) of the reaction solution was measured using a 200PRO (Tecan). The fluorescence intensity ratio, with Comparative Example 1 set to 1, was calculated as the amount of iron(II) ions generated. Note that the aforementioned fluorescent probe reacts specifically with iron(II) ions and emits fluorescence.

[0073] [Table 2]

[0074] Table 2 shows that in Examples 6-10, where component (a) was reacted with ferritin, a model protein containing iron(III) ions, hydrogen peroxide was produced compared to Comparative Examples 1 and 2, where the reaction was carried out without component (a). On the other hand, in Comparative Example 3, where component (a) was reacted with BSA, a model protein that does not contain iron(III) ions, no hydrogen peroxide was produced. Furthermore, in Examples 6-10, it can be seen that a reaction to produce iron(II) ions from component (a) and iron(III) ions occurred simultaneously with the production of hydrogen peroxide. Furthermore, ferritin is a model of a protein structure that possesses iron(III) ions and forms hydrophobic sites within microorganisms. The prepared test solution, under a phosphate buffer atmosphere, serves as a model of hydrophilic sites within microorganisms, and as a whole, it serves as a model of the inside of a microbial cell.

[0075] <Analysis of hydroxyl radical production (steps 1 and 2)> Using the above components, the test solutions shown in Table 3 were prepared. To the prepared test solutions, a fluorescent probe HPF reagent (Hydroxyphenyl Fluorescein (manufactured by Goryo Chemical Co., Ltd.)) for hydroxyl radical detection was added to a concentration of 10 μM, and the mixture was incubated at 37°C for 24 hours. The fluorescence intensity (Ex / Em: 492 / 525) was measured using a microplate reader Infinite(R)200PRO (Tecan). The amount of hydroxyl radical generation was calculated from the fluorescence intensity ratio, with Comparative Example 4 set to 1, using the following formula. The results are shown in Table 3. Furthermore, since the fluorescent probe selectively reacts only with hydroxyl radicals and does not react with hydrogen peroxide, the hydroxyl radical production process in step 2 is shown, utilizing the hydrogen peroxide generated in step 1.

[0076]

number

[0077] [Table 3]

[0078] Table 3 shows that in Examples 11-16, where component (a) was reacted with ferritin, a model protein containing iron(III) ions, hydroxyl radicals, which are reactive oxygen species, were generated compared to Comparative Example 4, where the reaction was carried out without component (a). From the above results and the results in Table 2, it can be said that in Examples 11-16, steps 1 and 2 were performed and hydroxyl radicals were generated. On the other hand, in Comparative Example 6, where component (a) was reacted with apoferritin, a model protein that does not contain iron(III) ions, the generation of hydroxyl radicals was at the same level as in Comparative Example 4. Furthermore, ferritin is a model of a protein structure that possesses iron(III) ions and forms hydrophobic sites within microorganisms. The prepared test solution, under a phosphate buffer atmosphere, serves as a model of hydrophilic sites within microorganisms, and as a whole, it serves as a model of the inside of a microbial cell.

Claims

1. A method for producing reactive oxygen species within the protein structure of ferritin, comprising the steps of: contacting ferritin with a liquid composition containing the following components (a), (b), and water; and carrying out a reaction in the presence of iron(III) ions present in the protein structure constituting ferritin, in which hydrogen peroxide is produced by the reaction of component (a) with dissolved oxygen in the liquid composition, and in which iron(II) ions are produced by the reaction of component (a) with iron(III) ions (hereinafter referred to as step 1). (a) Components: A composition comprising one or more selected ascorbic acid fatty acid esters, or one or more selected ascorbic acid fatty acid esters and ascorbic acid, wherein all (a) components are dissolved in an equal amount of n-octanol and water, and the molar concentration S(Oc) in the n-octanol phase and the molar concentration S(H) in the aqueous phase are based on the ascorbic acid skeleton at 20°C. ２ Distribution ratio of O) S(Oc) / S(H) ２ (O) or (a) the molar concentration S(Oc) in the n-octanol phase and the molar concentration S(H) in the aqueous phase, based on the ascorbic acid skeleton calculated from the logP value of component (a). ２ Distribution ratio of O) S(Oc) / S(H) ２ A compound or composition in which O) is 0.01 or more and 7000 or less. (b) Ingredients: Surfactants

2. Furthermore, the method for producing reactive oxygen species within a ferritin protein structure according to claim 1, comprising the step of reacting hydrogen peroxide obtained in step 1 with iron(II) ions derived from ferritin to generate hydroxyl radicals (hereinafter referred to as step 2).

3. (a) A method for producing reactive oxygen species within a ferritin protein structure according to claim 1 or 2, wherein the component is one or more selected from ascorbic acid fatty acid esters.

4. A method for producing reactive oxygen species within a ferritin protein structure according to any one of claims 2 to 3, wherein step 1 and step 2 are performed simultaneously.

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