WO2021005897A1 - バイオフィルム処理剤及びバイオフィルム処理方法 - Google Patents
バイオフィルム処理剤及びバイオフィルム処理方法 Download PDFInfo
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- WO2021005897A1 WO2021005897A1 PCT/JP2020/020325 JP2020020325W WO2021005897A1 WO 2021005897 A1 WO2021005897 A1 WO 2021005897A1 JP 2020020325 W JP2020020325 W JP 2020020325W WO 2021005897 A1 WO2021005897 A1 WO 2021005897A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/30—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests characterised by the surfactants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/04—Oxygen or sulfur attached to an aliphatic side-chain of a carbocyclic ring system
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
- A01N31/14—Ethers
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
- B01D65/02—Membrane cleaning or sterilisation ; Membrane regeneration
- B01D65/06—Membrane cleaning or sterilisation ; Membrane regeneration with special washing compositions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
- B01D65/08—Prevention of membrane fouling or of concentration polarisation
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/50—Treatment of water, waste water, or sewage by addition or application of a germicide or by oligodynamic treatment
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/02—Anionic compounds
- C11D1/04—Carboxylic acids or salts thereof
- C11D1/10—Amino carboxylic acids; Imino carboxylic acids; Fatty acid condensates thereof
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/02—Anionic compounds
- C11D1/12—Sulfonic acids or sulfuric acid esters; Salts thereof
- C11D1/14—Sulfonic acids or sulfuric acid esters; Salts thereof derived from aliphatic hydrocarbons or mono-alcohols
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/02—Anionic compounds
- C11D1/12—Sulfonic acids or sulfuric acid esters; Salts thereof
- C11D1/22—Sulfonic acids or sulfuric acid esters; Salts thereof derived from aromatic compounds
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/66—Non-ionic compounds
- C11D1/72—Ethers of polyoxyalkylene glycols
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/20—Organic compounds containing oxygen
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/26—Organic compounds containing nitrogen
- C11D3/30—Amines; Substituted amines ; Quaternized amines
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A20/00—Water conservation; Efficient water supply; Efficient water use
- Y02A20/124—Water desalination
- Y02A20/131—Reverse-osmosis
Definitions
- the present invention relates to a biofilm treatment agent and a biofilm treatment method, which are characterized by containing at least one of an aromatic monoalcohol or anthranilic acid and a microbial surfactant.
- a biofilm is also called a mycelial film and refers to a structure formed by bacteria.
- the formation of the biofilm is carried out as follows. First, bacteria attached to the substrate secrete extracellular polysaccharides and proteins. They act as barriers and transport routes, protecting internal bacteria from environmental changes and chemicals. Bacteria are thought to gradually form a biofilm on the substrate surface while repeating attachment and detachment to the substrate.
- biofilms in the manufacturing process of various products and in heat exchangers, etc. is not desirable because it causes deterioration of product quality and productivity, and in some cases, may cause health hazards.
- bactericides and synthetic surfactants have been used to remove biofilms.
- the disinfectant needs to be used in high concentration in order to come into contact with the bacteria existing inside the biofilm.
- a high-concentration bactericidal agent may have a harmful effect on the human body, and may cause deterioration or corrosion of members to be controlled by forming a biofilm such as a water-forming film.
- a biofilm such as a water-forming film.
- the long-term use of the fungicide will selectively leave biofilm-forming bacteria resistant to the fungicide and concentrate them in the system.
- the bacteria killed by the fungicide non-specifically adsorb to the surface and become a hotbed for new biofilms.
- Synthetic surfactants are mainly used to remove bacteria and biofilms by the physical action of washing, but the effect on biofilm removal when used alone is limited. In addition, some of them are effective at high pH, and there is a concern about a decrease in safety.
- Patent Document 1 describes suppression of the formation of a biofilm containing a combination of two types selected from a plurality of surfactants as an active ingredient.
- Patent Document 2 describes the decomposition of a biofilm by a combination of a vitamin, a metal ion, a synthetic surfactant, and an antimicrobial agent (aromatic alcohol, etc.) (Note that the biofilm in Patent Document 2).
- the decomposing effect is obtained at least by a combination of vitamins, metal ions and synthetic surfactants, and there is no description that explicitly or suggests the biofilm decomposing effect of the antimicrobial agent itself such as aromatic alcohol).
- An object of the present invention is to provide a treatment agent having an excellent biofilm removing ability and a biofilm treatment method using the same.
- the present inventor has been diligently researching the possibility of suppressing and removing the formation of biofilms for various types of chemical substances in order to solve the above-mentioned problems. As a result, they have found that a composition containing at least an aromatic monoalcohol or an anthranilic acid and a microbial surfactant has an excellent biofilm removing effect, and have completed the present invention.
- the present invention ⁇ 1> A biofilm treatment agent containing at least the following A1 and / or A2, A1: Aromatic monoalcohol A2: Anthranylic acid and microbial surfactant ⁇ 2> A biofilm treatment agent containing at least an aromatic monoalcohol, anthranylic acid, a microbial surfactant and a synthetic surfactant.
- a biofilm treatment agent containing at least an aromatic monoalcohol, anthranylic acid, a microbial surfactant and a synthetic surfactant.
- Agent, R 1 A linear alkyl group having 1 to 3 carbon atoms in which any one hydrogen atom is substituted with a hydroxyl group.
- R 2 A linear alkyl group ⁇ 4> anthranilic acid having 1 to 3 carbon atoms in which any one hydrogen atom is substituted with a hydroxyl group is derived from anthranilic acid, methyl anthraniate, ethyl anthranilate, anthranilamide and salts thereof.
- ⁇ 5> The biofilm treatment agent according to ⁇ 1> or ⁇ 2>, wherein the microbial surfactant is an amino acid type or glycolipid type microbial surfactant.
- biofilm treatment agent according to ⁇ 2> above, wherein the synthetic surfactant is at least one selected from sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and polyoxyethylene lauryl ether.
- Aromatic monoalcohol / anthranyl acids / microbial surfactants / synthetic surfactants 1 / 0.25 to 2 / 0.0005 to 2 / 0.005 to 2 in a mass ratio.
- biofilm treatment agent according to any one of ⁇ 1> to ⁇ 7>, wherein the biofilm is formed from a bacterium containing at least a gram-negative bacterium.
- a treatment agent containing an aromatic monoalcohol or a combination of anthranilic acids and a microbial surfactant as an active ingredient provides a higher biofilm removing effect than conventional fungicides and synthetic surfactants.
- active ingredients are not highly reactive substances such as oxidizing agents, which are one of the conventional disinfectants, they have the advantage of being less likely to cause deterioration of the applicable members and the like and being easy to handle. ..
- Biofilm treatment agent contains at least the following A1 and / or A2.
- A1 Aromatic monoalcohol
- A2 Anthranilic acids and microbial surfactants
- the biofilm treatment agent means an agent having at least a biofilm removing effect.
- the biofilm treatment agent may have a biofilm removing effect, and particularly if it has a biofilm formation suppressing effect described later, the biofilm formation is suppressed after the biofilm is removed, which is constant. It is more preferable because it can maintain an environment in which no biofilm is present.
- the biofilm treatment agent of the present invention has the effect of the present invention even if each of A1 and A2 is used alone, but it is preferable to use A1 and A2 in combination.
- the aromatic monoalcohol is not particularly limited, but from the viewpoint of the biofilm removing effect, it is preferably a compound represented by the following formula (1) or formula (2), or a cinnamyl alcohol. These can be used alone or in combination of two or more.
- R 1 A linear alkyl group having 1 to 3 carbon atoms in which any one hydrogen atom is substituted with a hydroxyl group.
- R 2 A linear alkyl group having 1 to 3 carbon atoms in which any one hydrogen atom is substituted with a hydroxyl group.
- aromatic monoalcohol of the formula (1) are 1-phenylmethanol, 1-phenylethanol, 2-phenylethanol, 1-phenyl-1-propanol, 1-phenyl-2-propanol, 3-phenyl-1. -Propanol can be mentioned.
- Specific examples of the formula (2) include 2-phenoxyethanol, 3-phenoxy-1-propanol, 1-phenoxy-2-propanol, 3-phenoxy-2-propanol and the like.
- 1-phenylmethanol, 1-phenylethanol, 2-phenylethanol, 2-phenoxyethanol, and 3-phenoxy-1-propanol are preferable from the viewpoint of the biofilm removing effect.
- Anthranilic acids refer to anthranilic acid (salt) and anthranilic acid derivatives. Specific examples of anthranilic acids include anthranilic acid, methyl anthranilic acid, ethyl anthranilic acid, propyl anthranilic acid, butyl anthranilic acid, 4-chloroanthranilic acid, 6-chloroanthranilic acid, 4-fluoroanthranic acid, and 4-bromoanthranilic acid.
- 6-Bromoanthranilic acid N-acetylanthranilic acid, N-acetoacetylanthranilic acid, anthranilamide, 4-nitroanthranilic acid, 6-nitroanthranilic acid, salts thereof and the like.
- the salt of anthranilic acid is not particularly limited as long as it exhibits the effect of the present invention, and examples thereof include a salt neutralized with an acid or a base.
- the acid addition salt include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, and salts with organic acids such as acetic acid, malic acid, succinic acid, tartaric acid and citric acid.
- the base addition salt examples include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, and salts with amines such as ammonia and triethylamine.
- alkali metals such as sodium and potassium
- alkaline earth metals such as calcium and magnesium
- salts with amines such as ammonia and triethylamine.
- at least one selected from anthranilic acid, methyl anthranilate, ethyl anthranilate, anthranilamide, and salts thereof is preferable from the viewpoint of the biofilm removing effect. These can be used alone or in combination of two or more.
- the microbial surfactant is not particularly limited, but is preferably an amino acid type or glycolipid type microbial surfactant from the viewpoint of the biofilm removing effect.
- amino acid-type microbial surfactants include surfactins and the like.
- glycolipid-type microbial surfactants include ramnolipid and sophorolipid. These can be used alone or in combination of two or more.
- the mass ratio of anthranyl acids and microbial surfactants is preferably 1 / 0.001 to 2 and 1 / 0.01 to 2 from the viewpoint of biofilm removing effect. Is more preferable.
- the biofilm treatment agent of the present invention includes one containing a synthetic surfactant in addition to A1 and A2. By combining these, the biofilm removing effect can be further enhanced.
- the synthetic surfactant is preferably anionic or nonionic, and examples of the anionic synthetic surfactant include alkyl sulfate ester salts (for example, sodium dodecyl sulfate (SDS), potassium dodecyl sulfate, etc.) and alkylbenzene sulfonates.
- alkyl sulfate ester salts for example, sodium dodecyl sulfate (SDS), potassium dodecyl sulfate, etc.
- alkylbenzene sulfonates for example, sodium dodecylbenzene sulfonate (LAS), triethanolamine dodecylbenzene sulfonate, etc.
- polyoxyethylene alkyl ether sulfate for example, sodium polyoxyethylene lauryl ether sulfate (SLS), etc.
- nonionic synthetic surfactant examples include alcohol ethoxylate (for example, polyoxyethylene lauryl ether (POELE)), glycerin fatty acid ester (for example, glycerin monostearate), and sucrose fatty acid ester (for example, sucrose). Lauric acid ester, etc.) and the like.
- alcohol ethoxylate for example, polyoxyethylene lauryl ether (POELE)
- glycerin fatty acid ester for example, glycerin monostearate
- sucrose fatty acid ester for example, sucrose. Lauric acid ester, etc.
- alkyl sulfates, alkylbenzene sulfonates, and alcohol ethoxylates are more preferable
- sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and polyoxyethylene lauryl ether are even more preferable from the viewpoint of biofilm removing effect.
- MIC Minimum inhibitory concentration
- the MIC referred to in the present invention means the minimum concentration (bacteriostatic, antiseptic effect) in which antibiotics and bactericides suppress the growth of microorganisms. Therefore, a concentration less than MIC can be regarded as synonymous with a concentration that does not substantially exhibit a growth inhibitory effect on biofilm-forming bacteria.
- the method for calculating the MIC in the present invention is as follows.
- the compound that is a component of the biofilm treatment agent (hereinafter, may be referred to as the substance to be evaluated) is diluted stepwise with the susceptibility test bouillon medium, and a total of 10 mL dilution sequence (however, 1.1 times the target concentration).
- test strain therein which is a representative strain of biofilm forming bacteria Pseudomonas aeruginosa (accession number: NBRC106052 strain) was 20 ⁇ L added bacterial solution prepared in 10 8 cfu / mL, 37 °C in a 96-well microplate mixer, Shake culture (2000 rpm) for 24 hours.
- the lowest concentration in the dilution column that did not become cloudy visually is defined as MIC.
- the biofilm treatment agent of the present invention allows biofilm-forming bacteria to grow and exerts the effects of the present invention when the concentration of each component contained in the biofilm treatment agent is less than MIC. In other words, the biofilm treatment agent of the present invention does not exert the effect of the present invention by killing or suppressing the growth of the biofilm-forming bacterium itself.
- the biofilm removing effect means an action of removing a biofilm formed by bacteria.
- the amount of biofilm formed after the substance to be evaluated is brought into contact with the biofilm formed by culturing bacteria for a certain period of time, and the contact with the substance to be evaluated.
- the method for evaluating the biofilm removing effect in the present invention is as follows.
- Pseudomonas aeruginosa (deposit number: NBRC106052 strain), which is a representative strain of biofilm-forming bacteria, uses a TSB (Triactive Soy Broth, Bacto: Difco Laboratories) medium with a final concentration of glucose at 120%.
- TSB Triactive Soy Broth, Bacto: Difco Laboratories
- O O.
- the mixture adjusted to D (turbidity) 0.1 is diluted with TSB medium so that the final concentration is 0.000005% (v / v), and 2 mL is dispensed into a 12-well plate.
- TSB Triactive Soy Broth, Bacto: Difco Laboratories
- D refers to a value at a wavelength of 630 nm using distilled water as a blank, measured using a spectrophotometer (iMark microplate reader: manufactured by Bio-Rad).
- Iii Incubate for 17 hours under the conditions of 37 ° C. and 130 rpm to form a biofilm.
- the culture solution in each hole is removed, and each is rinsed twice with distilled water.
- the absorbance refers to a value at a wavelength of 595 nm using distilled water as a blank, measured using a spectrophotometer (iMark microplate reader: manufactured by Bio-Rad).
- the absorbance of the negative control and each substance to be evaluated shall be the average value of the absorbances measured in 4 holes, and the removal rate of the biofilm shall be calculated from the following formula.
- Biofilm removal rate (%) ⁇ 1- (absorbance of substance to be evaluated / absorbance of negative control) ⁇ x 100
- the calculated value is evaluated based on the following criteria.
- the biofilm formation suppressing effect means an action of suppressing the formation of a biofilm by bacteria.
- the method for evaluating the biofilm formation inhibitory effect in the present invention is as follows.
- Pseudomonas aeruginosa (deposit number: NBRC106052 strain), which is a representative strain of biofilm-forming bacteria, uses a TSB (Triactive Soy Broth, Bact: Difco Laboratories) medium with a final concentration of glucose at 120%.
- TSB Triactive Soy Broth, Bact: Difco Laboratories
- the preculture solution adjusted to OD (turbidity) 0.1 was diluted with TSB medium so as to have a final concentration of 0.000005% (v / v), and 2 mL was placed on a 12-well plate. Note.
- V The culture solution in each hole is removed, and each is rinsed twice with distilled water.
- Biofilm formation inhibition rate (%) ⁇ 1- (absorbance of substance to be evaluated / absorbance of negative control) ⁇ x 100 (Ix) The calculated value is evaluated based on the following criteria. ⁇ Criteria> Formation inhibition rate 60% or more: Very high inhibition effect Formation inhibition rate 40% or more and less than 60%: High inhibition effect Formation inhibition rate 20% or more and less than 40%: Inhibition effect Formation inhibition rate Less than 20%: Inhibition effect The biofilm formation inhibitory effect is 40% or more, which is a practical level.
- aromatic monoalcohols and anthranyl acids affect the quorum sensing of biofilm-forming bacteria to remove the biofilm and form the biofilm. It is believed that it has an inhibitory effect.
- the form of the biofilm treatment agent of the present invention may be a solution, a dispersion, a gel or the like diluted with an arbitrary medium as it is, but when it is allowed to act on a biofilm, it is usually used. Used in aqueous solution.
- concentration of the diluted biofilm treatment agent is not particularly limited, but a concentration sufficient to exert the effect of the present invention when acting on biofilm-forming bacteria is required.
- the biofilm treatment agent of the present invention is a thickener, a viscosity regulator, a pH adjuster, a solvent, a fragrance, a colorant, an antioxidant, a preservative, a fluorescent agent, and a shaping agent, as long as the object of the present invention is not impaired.
- Agents, soil release agents, bleaching agents, bleaching activators, powdering agents, granulating agents, coating agents and the like can be blended.
- the concentration of the biofilm treatment agent used it is preferable that the concentration of each component contained in the biofilm treatment agent is less than the MIC of the main causative bacterial species constituting the biofilm.
- the concentration of each component contained in the biofilm treatment agent is less than the MIC of the main causative bacterial species constituting the biofilm.
- the biofilm treatment agent is preferably a single agent in terms of handling, but each component may be prepared individually and mixed when contacted with biofilm-forming bacteria.
- the solution pH when using the biofilm treatment agent can be set as appropriate, but if it is used in the neutral pH range (7.0 to 8.0), the effect on the human body and the water environment used is taken into consideration. There is no need to do it and it is safe.
- the time for the biofilm treatment agent to act varies depending on the amount of attached biofilm, the concentration of the active ingredient, the operating temperature, and the presence or absence of physical force, but is usually in the range of several minutes to several hours. Further, by bringing the biofilm treatment agent into contact with a member whose biofilm formation is desired to be suppressed for several minutes to several hours in advance, the formation of the biofilm can be suppressed by the action of aromatic monoalcohols and anthranilic acids. ..
- biofilm-forming bacteria The biofilm-forming bacteria to which the biofilm treatment agent of the present invention is applied include any of the gram-negative bacteria that form the biofilm. Among these, it should be used for Ochrobactrum, Aeromonas, Klebsiella, Acinetobacter, Enterobacter, Citrobacter, Stenotrovmonas, Pseudomonas, Rhizobium, Capriavidas belonging to the phylum Proteobacteria. Is preferable. In most cases, the formation of a biofilm is carried out by two or more types of bacteria, and a biofilm containing one or more types of biofilm-forming bacteria is the subject of the present invention.
- the biofilm treatment agent of the present invention can be used in a wide range of fields in which a biofilm is formed and becomes a problem.
- it can be applied to drainage ditches and drainage pipes of food manufacturing or beverage manufacturing plants, kitchens, kitchens, bathrooms, toilets, kitchens and the like.
- It can also be applied to cooling water systems such as industrial cooling towers, water treatment membranes, desalination equipment, and circulating water systems such as paper mills.
- cleaning agents for medical devices in which biofilms are easily formed such as endoscopes, catheters, and artificial dialysis machines.
- the biofilm removing effect and the biofilm formation suppressing effect of the biofilm treating agent of the present invention were evaluated in advance by obtaining the MIC of each component of the biofilm treating agent against the biofilm-forming bacteria and at a concentration lower than the MIC.
- the MIC test method is shown below.
- ⁇ MIC (minimum inhibitory concentration) test method Using Pseudomonas (gram-negative bacteria) known as a model bacterium for biofilm formation as a test bacterium, a MIC of a compound (hereinafter, a substance to be evaluated) to be a component of a biofilm treatment agent was obtained.
- Test bacterium Pseudomonas aeruginosa deposit number: NBRC106052 strain
- Substances to be evaluated The compounds shown in Table 1 were used as substances to be evaluated.
- SDS Sodium dodecyl sulfate (synthetic surfactant)
- LAS Sodium dodecylbenzene sulfonate (synthetic surfactant)
- POELE Polyoxyethylene lauryl ether (synthetic surfactant; HLB 12.1)
- DBNPA 2,2-dibromo-3-nitrilopropionamide (organic fungicide)
- the biofilm treatment agent containing at least the A1 component or the A2 component specified in the present invention has a biofilm removing effect as compared with the biofilm treatment agent containing a compound other than the components specified in the present invention. It turns out that it is excellent. Further, it can be seen that the A1 component exhibits superior effects not only in the biofilm removing effect but also in the biofilm formation suppressing effect as compared with the biofilm treating agent containing a compound other than the components specified in the present invention.
- the biofilm treatment agent containing at least both the A1 component and the A2 component specified in the present invention is more than the A1 component or the A2 component alone in comparison with Table 3 or Table 4. , It can be seen that the removal effect is superior. Further, from Tables 5 and 6, the biofilm treatment agent containing at least a synthetic surfactant in addition to both the A1 component and the A2 component specified in the present invention is compared with the case where the synthetic surfactant is not contained. Further, it can be seen that the removal effect is excellent (for example, comparison between Example 19 and Example 26).
- a biofilm treatment agent effective against biofilm-forming bacteria is provided by using at least one of aromatic monoalcohols or anthranyl acids and microbial surfactants as an active ingredient. Can be done.
- an effective treatment agent for a biofilm formed in a path in contact with water such as a water separation membrane, a cooling tower, and a medical device.
- the treatment agent of the present invention since the treatment agent of the present invention has a biofilm formation suppressing effect and a removing effect even at a neutral pH, it is not necessary to consider the influence on the human body and the water environment used, and it is safe.
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Abstract
Description
<1>下記A1及び/又はA2を少なくとも含有するバイオフィルム処理剤、
A1:芳香族モノアルコール
A2:アントラニル酸類及び微生物界面活性剤
<2>芳香族モノアルコール、アントラニル酸類、微生物界面活性剤及び合成界面活性剤を少なくとも含有することを特徴とする、バイオフィルム処理剤、
<3>芳香族モノアルコールが、下記式(1)若しくは式(2)で示される化合物、又はシンナミルアルコールであることを特徴とする、前記<1>又は<2>に記載のバイオフィルム処理剤、
<4>アントラニル酸類が、アントラニル酸、アントラニル酸メチル、アントラニル酸エチル、アントラニルアミド及びこれらの塩から選ばれる少なくとも1種であることを特徴とする、前記<1>又は<2>に記載のバイオフィルム処理剤、
<5>微生物界面活性剤がアミノ酸型又は糖脂質型の微生物界面活性剤であることを特徴とする、<1>又は<2>に記載のバイオフィルム処理剤、
<6>合成界面活性剤が、ドデシル硫酸ナトリウム、ドデシルベンゼンスルホン酸ナトリウム、ポリオキシエチレンラウリルエーテルから選ばれる少なくとも1種であることを特徴とする、前記<2>に記載のバイオフィルム処理剤、
<7>芳香族モノアルコール/アントラニル酸類/微生物界面活性剤/合成界面活性剤=1/0.25~2/0.0005~2/0.005~2の質量比で含むことを特徴とする、前記<2>に記載のバイオフィルム処理剤、
<8>バイオフィルムが、少なくともグラム陰性細菌を含んだ細菌から形成されていることを特徴とする、前記<1>~<7>のいずれか1項に記載のバイオフィルム処理剤、
<9>MIC(最小発育阻止濃度)未満の濃度範囲で使用されることを特徴とする、前記<1>又は<2>に記載のバイオフィルム処理剤を用いたバイオフィルム処理方法、
である。
本発明のバイオフィルム処理剤は、下記A1及び/又はA2を少なくとも含有する。
A1:芳香族モノアルコール
A2:アントラニル酸類及び微生物界面活性剤
本発明のバイオフィルム処理剤が有するバイオフィルム除去効果やバイオフィルム形成抑制効果の評価は、あらかじめ、バイオフィルム処理剤の各成分のバイオフィルム形成細菌に対する最小発育阻止濃度(MIC)を求め、MIC未満の濃度において行った。
バイオフィルム処理剤の成分となる化合物(以下、評価対象物質と称することがある)を感受性試験用ブイヨン培地で段階的に希釈し、合計10mLの希釈列(ただし、目的濃度の1.1倍)を調製する。そこに供試菌株として、バイオフィルム形成菌の代表菌株であるPseudomonas aeruginosa(寄託番号:NBRC106052株)を108cfu/mLに調製した菌液を20μL添加し、96穴マイクロプレートミキサーで37℃、24時間振とう培養(2000rpm)する。目視で白濁しなかった希釈列のうち最も低い濃度をMICとする。
(i)バイオフィルム形成菌の代表菌株であるPseudomonas aeruginosa(寄託番号:NBRC106052株)は、TSB(Triptic Soy Broth, Bacto: Difco Laboratories製)培地にグルコースを終濃度1%としたものを用い、120rpmの条件で前培養液を調製する。
(ii)前培養液のO.D(濁度)=0.1に調整したものを終濃度0.000005%(v/v)となるようにTSB培地で希釈し、12穴プレートに2mL分注する。ここで、以下O.D(濁度)とは分光光度計(iMarkマイクロプレートリーダー:バイオ・ラッド社製)を用いて測定した、蒸留水をブランクとした波長630nmにおける値をいう。
(iii)37℃、130rpmの条件で17時間培養し、バイオフィルムを形成させる。
(iv)各穴の培養液を除去し、それぞれ蒸留水で2回リンスする。
(v)評価対象物質を当該対象物質のMIC未満の適当な濃度で培地に添加し、塩酸若しくは水酸化ナトリウムで培地pH=7.0に調整する。無菌培地(pH=7.0)を各穴に2mL添加したものをネガティブコントロールとする。
(vi)前培養と同じ温度で3.5時間、130rpmで振盪し評価対象物質を含む培地とバイオフィルムを接触させた後、各穴の培地を除去し、蒸留水で2回リンスする。
(vii)各穴内に付着しているバイオフィルムにクリスタルバイオレット水溶液(0.4w/v%,20w/v%メタノール)2mLを加え、2分間静置、染色した後、蒸留水で3回リンスし、バイオフィルムに結合していないクリスタルバイオレット水溶液を除去する。
(viii)各穴に2mLのエタノールを添加、1時間静置し、染色されたバイオフィルムからクリスタルバイオレットを溶出させ、吸光度を測定する。ここで、以下吸光度とは分光光度計(iMarkマイクロプレートリーダー:バイオ・ラッド社製)を用いて測定した、蒸留水をブランクとした波長595nmにおける値をいう。
(ix)ネガティブコントロールと各評価対象物質の吸光度は、4穴測定した吸光度の平均値とし、下記計算式からバイオフィルムの除去率を算出する。バイオフィルム除去率(%)={1-(評価対象物質の吸光度/ネガティブコントロールの吸光度)}×100
(x)算出された値について、下記判定基準に基づいて評価する。
<判定基準>
除去率60%以上:除去効果が非常に高い
除去率40%以上60%未満: 除去効果が高い
除去率20%以上40%未満: 除去効果がある
除去率20%未満: 除去効果がない、又は弱い
バイオフィルム除去効果は、40%以上で実用レベルである。
(i)バイオフィルム形成菌の代表菌株であるPseudomonas aeruginosa(寄託番号:NBRC106052株)は、TSB(Triptic Soy Broth, Bacto: Difco Laboratories製)培地にグルコースを終濃度1%としたものを用い、120rpmの条件で前培養液を調製する。
(ii)前培養液のO.D(濁度)=0.1に調整したものを終濃度0.000005%(v/v)となるようにTSB培地で希釈し、12穴プレートに2mL分注する。
(iii)評価対象物質を当該対象物質のMIC未満の適当な濃度で培地に添加し、塩酸若しくは水酸化ナトリウムで培地pH=7.0に調整する。評価対象物質を含まないものをネガティブコントロールとする(pH=7.0)。
(iv)37℃ 、130rpmの条件で6時間培養し、バイオフィルムの形成を行う。
(v)各穴の培養液を除去し、それぞれ蒸留水で2回リンスする。
(vi)各穴内に付着しているバイオフィルムにクリスタルバイオレット水溶液(0.4w/v%, 20w/v%メタノール)2mLを加え、2分間静置、染色した後、蒸留水で3回リンスし、バイオフィルムに結合していないクリスタルバイオレット水溶液を除去する。
(vii)各穴に2mLのエタノールを添加、1時間静置し、染色されたバイオフィルムからクリスタルバイオレットを溶出させ、波長595nmの吸光度を測定する。
(viii)ネガティブコントロールと各評価対象物質の吸光度は、4穴測定した吸光度の平均値とし、下記計算式からバイオフィルムの形成抑制率を算出した。
バイオフィルム形成抑制率(%)={1-(評価対象物質の吸光度/ネガティブコントロールの吸光度)}×100
(ix)算出された値について、下記判定基準に基づいて評価する。
<判定基準>
形成抑制率60%以上:抑制効果が非常に高い
形成抑制率40%以上60%未満:抑制効果が高い
形成抑制率20%以上40%未満:抑制効果がある
形成抑制率20%未満:抑制効果がない、又は弱い
バイオフィルム形成抑制効果は、40%以上で実用レベルである。
以下、本発明のバイオフィルム処理剤の好ましい使用条件について述べる。
バイオフィルム処理剤の使用濃度は、バイオフィルム処理剤に含まれる各成分の使用濃度がそれぞれ、バイオフィルムを構成する主要な原因菌種のMIC未満であることが好ましい。MIC未満で使用することで、バイオフィルム形成細菌の死滅を抑制し、死滅菌が表面に非特異的に吸着することを抑えることができるため、新たなバイオフィルムの温床の抑制につながる。バイオフィルム処理剤は、1剤化したものがハンドリングの点で好ましいが、各成分を個別に準備し、バイオフィルム形成細菌に接触させるときに混合されてもよい。
本発明のバイオフィルム処理剤が適用されるバイオフィルムの形成細菌は、バイオフィルムを形成するグラム陰性細菌のいずれも含まれる。これらのなかでも、プロテオバクテリア門に属するオクロバクテリウム属、エロモナス属、クレブシエラ属、アシネトバクター属、エンテロバクター属、シトロバクター属、ステノトロフモナス属、シュードモナス属、リゾビウム属、カプリアビダス属に対して用いることが好ましい。なお、バイオフィルムの形成は2種以上の細菌で行われる場合がほとんどであり、バイオフィルム形成細菌が1種以上含まれているバイオフィルムが本発明の対象となる。
バイオフィルム形成のモデル菌として知られているシュードモナス属菌(グラム陰性細菌)を供試菌として、バイオフィルム処理剤の成分となる化合物(以下、評価対象物質)のMICを求めた。
(1)供試菌Pseudomonas aeruginosa(寄託番号:NBRC106052株)
(2)評価対象物質
表1に示す化合物を評価対象物質とした。表1の化合物のうち、1-ペンタノール、2-フェニル-1,3-プロパンジオール、3-フェノキシ-1,2-プロパンジオール、DBNPA(2,2-ジブロモ-3-ニトリロプロピオンアミド)は、以下の試験において比較例として用いた。
(3)試験方法
評価対象物質を感受性試験用ブイヨン培地で段階的に希釈し、合計10mLの希釈列(ただし、目的濃度の1.1倍)を調製した。そこに供試菌株を108cfu/mLに調製した菌液を20μL添加し、96穴マイクロプレートミキサーで37℃、24時間振とう培養(2000rpm)した。目視で白濁しなかった希釈列のうち最も低い濃度をMICとした。
(4)試験結果
結果を表1に示す。表中、「>数値」は、MICが当該数値より大きいことを示す。
A1、A2、合成界面活性剤、A1以外のアルコール、及び有機系殺菌剤の1種であるDBNPA(2,2-ジブロモ-3-ニトリロプロピオンアミド)について、以下のバイオフィルム除去効果の評価及びバイオフィルム形成抑制効果の評価は、MIC未満となる濃度で行った。
SDS:ドデシル硫酸ナトリウム(合成界面活性剤)
LAS:ドデシルベンゼンスルホン酸ナトリウム(合成界面活性剤)
POELE:ポリオキシエチレンラウリルエーテル(合成界面活性剤;HLB12.1)
DBNPA:2,2-ジブロモ-3-ニトリロプロピオンアミド(有機系殺菌剤)
表1に示された各評価対象物質についてのバイオフィルム除去効果及びバイオフィルム形成抑制効果は、評価対象物質をバイオフィルム処理剤成分として含むバイオフィルム処理剤を本発明で定めた評価方法に従って確認した。培地への各評価対象物質の添加濃度を表2に示す。また、芳香族モノアルコール単独のバイオフィルム除去効果及びバイオフィルム形成抑制効果の評価結果を表3に、評価対象物質の組み合わせによるバイオフィルム除去効果の評価結果を表4~表6にそれぞれ示す。
特に、水分離膜、冷却塔、医療機器などの水に接する経路で形成されるバイオフィルムに対して有効な処理剤を提供することができる。
また、本発明の処理剤は、中性pHでもバイオフィルム形成抑制効果及び除去効果を有することから、人体及び使用する水環境への影響を考慮する必要もなく安心である。
Claims (9)
- 下記A1及び/又はA2を少なくとも含有することを特徴とするバイオフィルム処理剤。
A1:芳香族モノアルコール
A2:アントラニル酸類及び微生物界面活性剤 - 芳香族モノアルコール、アントラニル酸類、微生物界面活性剤及び合成界面活性剤を少なくとも含有することを特徴とする、バイオフィルム処理剤。
- アントラニル酸類が、アントラニル酸、アントラニル酸メチル、アントラニル酸エチル、アントラニルアミド及びこれらの塩から選ばれる少なくとも1種であることを特徴とする、請求項1又は2に記載のバイオフィルム処理剤。
- 微生物界面活性剤がアミノ酸型又は糖脂質型の微生物界面活性剤であることを特徴とする、請求項1又は2に記載のバイオフィルム処理剤。
- 合成界面活性剤が、ドデシル硫酸ナトリウム、ドデシルベンゼンスルホン酸ナトリウム、ポリオキシエチレンラウリルエーテルから選ばれる少なくとも1種であることを特徴とする、請求項2に記載のバイオフィルム処理剤。
- 芳香族モノアルコール/アントラニル酸類/微生物界面活性剤/合成界面活性剤=1/0.25~2/0.0005~2/0.005~2の質量比で含むことを特徴とする、請求項2に記載のバイオフィルム処理剤。
- バイオフィルムが、少なくともグラム陰性細菌を含んだ細菌から形成されていることを特徴とする、請求項1~7のいずれか1項に記載のバイオフィルム処理剤。
- MIC(最小発育阻止濃度)未満の濃度範囲で使用されることを特徴とする、請求項1又は2に記載のバイオフィルム処理剤を用いたバイオフィルム処理方法。
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JP7188558B2 (ja) | 2022-12-13 |
KR20220016504A (ko) | 2022-02-09 |
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