Biofilm treatment agent and biofilm treatment method

Abstract

[Problem] The present invention addresses the problem of providing a treatment agent having an exceptional synergistic effect in terms of biofilm removal ability, and a biofilm treatment method in which said treatment agent is used. [Solution] A biofilm treatment agent characterized by comprising: (A) at least one selected from the group consisting of alkyldiaminoethylglycine having 12 to 14 carbon atoms in an alkyl chain and salts thereof; and (B) at least one selected from the group consisting of 1-phenylethanol, 2-phenylethanol, 2-phenoxyethanol, cinnamyl alcohol, anthranilamide, anthranilic acid, anthranilate, and methyl anthranilate, the blending ratio of (A) and (B) being (B) / (A) = 0.5 to 5 by mass.

Description

Biofilm treatment agent and biofilm treatment method

[0001] The present invention relates to a biofilm treatment agent and a biofilm treatment method.

[0002] Biofilms, also known as pectin membranes, are structures formed by microorganisms such as bacteria. Biofilm formation occurs as follows: First, bacteria attached to a substrate secrete extracellular polysaccharides and proteins. These act as a barrier and transport pathway, protecting the bacteria inside from environmental changes and chemicals. It is believed that bacteria gradually form a biofilm on the surface of the substrate by repeatedly attaching to and detaching from the substrate.

[0003] Biofilms are known to grow in various environments involving water and cause problems. For example, biofilms that grow in product manufacturing processes or heat exchange equipment can cause product contamination, reduced productivity, and energy loss. They can also cause infectious diseases in medical equipment and facilities. In living spaces, they grow on sinks, bathrooms, exterior walls, and other surfaces, which are undesirable because they impair aesthetics and cause unpleasant odors. Conventionally, disinfectants and surfactants have been used to control these biofilms.

[0004] Bactericides are broadly classified into organic and inorganic types, but both must be used at high concentrations to be effective against the bacteria present inside biofilms. High concentrations of bactericides may have harmful effects on the human body and the environment, may cause deterioration or corrosion of the materials targeted for biofilm control, and are not satisfactory in terms of treatment costs.

[0005] Surfactants are generally classified into anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, and microbial surfactants. However, the biofilm control effect of most of these surfactants is limited to a non-bactericidal cleaning action at normal use concentrations. Some cationic surfactants (e.g., benzalkonium chloride) and some amphoteric surfactants (e.g., alkyldiaminoacylglycine) with strong bactericidal activity have traditionally been used as disinfectants and biofilm control agents. However, their bactericidal activity against biofilm-forming bacteria is known to be significantly attenuated, and it is thought that biofilms are re-formed within a short period of time by bacteria that escape cleaning and sterilization (see Non-Patent Document 1). Therefore, cleaning measures using any surfactant require frequent cleaning work within a short period of time, which cannot be said to be satisfactory in terms of labor and cleaning costs. Furthermore, using these surfactants at high concentrations to achieve high effectiveness is not desirable due to concerns about safety to the human body and the environment, foaming, etc.

[0006] Therefore, it cannot be said that removing biofilms solely by using disinfectants or surfactants is necessarily effective.

[0007] The following biofilm control techniques are known to improve these conditions:

[0008] Patent Document 1 describes a non-bactericidal biofilm control technology that uses a combination of specific aromatic alcohols and / or anthranilic acids used at a concentration below the MIC (minimum inhibitory concentration) of the microorganisms with a microbial surfactant. Patent Document 2 also discloses a cleaning composition containing a specific glycine-type amphoteric surfactant and an anionic surfactant. Patent Document 3 discloses an aquarium treatment method and composition that uses a combination of a quaternary ammonium surfactant and 2-phenoxyethanol. However, none of these prior art techniques achieves the desired level of biofilm removal effect.

[0009] International Publication No. 2021 / 005897 Japanese Patent Application Laid-Open No. 2015-017151 International Publication No. 2002 / 003830

[0010] Environmental Infection, 2000, Vol. 15, No. 2, pp. 156-162

[0011] An object of the present invention is to provide a treatment agent that has excellent biofilm removal ability and can improve the efficiency of action, and a biofilm treatment method using the same.

[0012] To solve the above-mentioned problems, the present inventors have conducted extensive research into improving biofilm removal ability and efficiency by using various chemical substances and their combinations, particularly substances that have non-bactericidal activity at low concentrations below the MIC. Efficiency refers to the ability to achieve high biofilm removal ability at lower drug concentrations, which contributes to reducing adverse effects on the human body and the environment and reducing usage costs. Surprisingly, the present inventors discovered that a composition containing a specific combination of substances in a specific mass ratio can achieve excellent biofilm removal effects with efficiency that would be unexpected if the substances were simply combined, leading to the completion of the present invention.

[0013] That is, the present invention provides: <1> a biofilm treatment agent comprising (A) at least one selected from the group consisting of alkyldiaminoethylglycines having an alkyl chain carbon number of 12 to 14 and salts thereof, and (B) at least one selected from the group consisting of 1-phenylethanol, 2-phenylethanol, 2-phenoxyethanol, cinnamyl alcohol, anthranilamide, anthranilic acid, anthranilate salts, and methyl anthranilate, wherein the blending ratio of (A) to (B) is (B) / (A) in mass ratio form of 0.5 to 5; <2> the biofilm treatment agent according to <1>, wherein (A) is dodecyldiaminoethylglycine; <3> a biofilm treatment method comprising the step of contacting the biofilm treatment agent according to <1> or <2> with a biofilm, wherein the biofilm treatment agent is used in the step under conditions such that the total concentration of (A) and (B) is 100 ppm or more and the concentration of (B) is less than the MIC (minimum inhibitory concentration) calculated by the method described below; MIC (minimum inhibitory concentration): A compound that is a component of a biofilm treatment agent (hereinafter referred to as the substance to be evaluated) is serially diluted with a broth medium for susceptibility testing to prepare a dilution series totaling 10 mL (1.1 times the target concentration). 10 mL of Pseudomonas aeruginosa (deposit number: NBRC106052), a representative strain of biofilm-forming bacteria, is added as a test strain. ８ 20 μL of the bacterial solution adjusted to cfu / mL is added, and the mixture is shaken (2000 rpm) at 37°C for 24 hours in a 96-well microplate mixer. The lowest concentration in the dilution series that does not become cloudy when visually observed is selected.

[0014] The biofilm treatment agent of the present invention exhibits an unexpected synergistic effect that exceeds the effect achieved by simply using (A) and (B) individually, thereby achieving superior biofilm removal effects with higher efficiency than conventional bactericides and surfactants. Furthermore, these active ingredients are safer for humans and the environment than conventional bactericides, and are not highly reactive, so they are less likely to cause deterioration of the target components. Furthermore, since the agent can be used in lower amounts than conventional bactericides, it is safer for humans and the environment, has low foaming properties, and can reduce the amount of chemicals used, making it cost-effective.

[0015] The present invention will be described in detail below.

[0016] (Biofilm Treatment Agent) The biofilm treatment agent of the present invention contains at least the following (A) and (B): (A) at least one alkyldiaminoethylglycine having an alkyl chain with 12 to 14 carbon atoms and a salt thereof, and (B) at least one selected from the group consisting of 1-phenylethanol, 2-phenylethanol, 2-phenoxyethanol, cinnamyl alcohol, anthranilamide, anthranilic acid, anthranilate salts, and methyl anthranilate.

[0017] In the present invention, the biofilm treatment agent refers to an agent that has at least the effect of removing a biofilm that has already been formed by microorganisms, but may also have the effect of suppressing the formation of a biofilm.

[0018] The biofilm treatment agent of the present invention is characterized in that by combining (A) and (B) at a predetermined concentration and concentration ratio, a synergistic effect is obtained that exceeds the total biofilm removal effect when (A) and (B) are used alone.

[0019] The alkyldiaminoethylglycine having 12 to 14 carbon atoms in the alkyl chain, which is component (A), is not particularly limited, but dodecyldiaminoethylglycine and its salts are particularly preferred. These components may be commercially available products, such as those available from Sanyo Chemical Industries, Ltd. under the trade names "Levon (registered trademark) S" and "Levon (registered trademark) T-2."

[0020] The component (B) is at least one selected from 1-phenylethanol, 2-phenylethanol, 2-phenoxyethanol, cinnamyl alcohol, anthranilamide, anthranilic acid, anthranilate salts, and methyl anthranilate, and these can be used alone or in combination of two or more.

[0021] The blending ratio of (A) and (B) in the biofilm treatment agent of the present invention is (B) / (A) = 0.5 to 5, which is a mass ratio that provides a more excellent combined effect in terms of biofilm removal effect during use.

[0022] The concentrations of (A) and (B) in the biofilm treatment agent of the present invention are not particularly limited as long as the aforementioned mass ratio is satisfied. However, when treating a biofilm by contacting the agent with the biofilm, the biofilm control effect is most effectively achieved by using the agent under conditions in which the total concentration of (A) and (B) is 100 ppm or more and the concentration of (B) is less than the MIC (minimum inhibitory concentration) calculated by the method described below. Considering the treatment concentration used in an actual use environment, it is more preferable that the concentration of (B) is less than half the MIC of each substance to be evaluated, in terms of safety to the human body and the environment and treatment costs. While a single-component biofilm treatment agent is preferred for ease of handling, each component may be prepared separately and mixed before contacting biofilm-forming bacteria.

[0023] When evaluating the biofilm removal effect of the biofilm treatment agent of the present invention when used at a concentration of (B) below the MIC, the MIC (minimum inhibitory concentration) of each of the (B) components of the biofilm treatment agent against biofilm-forming bacteria is determined in advance, and the biofilm treatment agent of the present invention is used at a concentration such that the concentration of the (B) component is below the MIC.

[0024] In the present invention, the MIC refers to the minimum concentration at which an antibiotic or disinfectant inhibits the growth of microorganisms (bacteriostatic and antiseptic effects). Therefore, a concentration below the MIC can be considered synonymous with a concentration at which the growth inhibitory effect on biofilm-forming bacteria is not substantially exhibited.

[0025] The MIC in the present invention is calculated as follows: A compound that is a component of the biofilm treatment agent (hereinafter, sometimes referred to as the substance to be evaluated) is serially diluted with a broth medium for susceptibility testing to prepare a dilution series totaling 10 mL (1.1 times the target concentration). 10 mL of Pseudomonas aeruginosa (deposit number: NBRC106052), a representative strain of biofilm-forming bacteria, is added as the test strain. ８ Add 20 μL of the bacterial solution adjusted to cfu / mL and culture in a 96-well microplate mixer at 37°C for 24 hours with shaking (2000 rpm). The lowest concentration in the dilution series that does not become cloudy when visually observed is used.

[0026] In the present invention, the biofilm removal effect refers to the action of contacting a biofilm formed by a microorganism with a total concentration of (A) and (B) of 100 ppm or more and a concentration of (B) less than the MIC and removing the biofilm. A method for evaluating the removal effect of a target substance includes, for example, comparing the amount of biofilm formed after contacting the target substance with a biofilm formed by culturing bacteria for a certain period of time with the amount of biofilm formed after a certain period of time has passed without contact with the target substance (negative control). In this case, if the amount of biofilm formed is less than that of the negative control, it can be determined that the target substance has a biofilm removal effect.

[0027] The method for evaluating the biofilm removal effect in the present invention is as follows. (i) For Pseudomonas aeruginosa (deposit number: NBRC106052 strain), a representative strain of biofilm-forming bacteria, a preculture solution is prepared using TSB (Triptic Soy Broth, Bacto: manufactured by Difco Laboratories) medium with glucose at a final concentration of 1% at 130 rpm. (ii) The preculture solution, adjusted to an O.D. (turbidity) of 0.1, is diluted with TSB medium to a final concentration of 0.000005% (v / v), and 1.2 mL is dispensed into a 24-well polystyrene plate. Hereinafter, O.D. is used. D (turbidity) refers to the value measured at a wavelength of 630 nm using a spectrophotometer (iMark microplate reader, manufactured by Bio-Rad) with distilled water as the blank. (iii) The plate was cultured for 17 hours at 37°C and 130 rpm to form a biofilm. (iv) The culture medium was removed from each well and rinsed twice with distilled water. (v) The substance to be evaluated was added to the medium at an appropriate concentration, and the medium pH was adjusted to 7.0 with hydrochloric acid or sodium hydroxide. 1.2 mL of sterile medium (pH 7.0) without the substance to be evaluated was added to each well to serve as a negative control. (vi) The plate was shaken at 130 rpm for 1 hour at the same temperature as the pre-culture to allow contact between the medium containing the substance to be evaluated and the biofilm, after which the medium from each well was removed and rinsed twice with distilled water. (vii) 3 mL of a crystal violet aqueous solution (0.4 w / v%, 20 w / v% methanol) was added to the biofilm adhering to each well, allowed to stand for 2 minutes, and then rinsed three times with distilled water to remove any crystal violet aqueous solution that had not bound to the biofilm. (viii) 3 mL of ethanol was added to each well, allowed to stand for 1 hour, and the crystal violet was eluted from the stained biofilm, and the absorbance was measured. Hereinafter, absorbance refers to the value measured at a wavelength of 595 nm using a spectrophotometer (iMark microplate reader: Bio-Rad) with distilled water as the blank. (ix) The absorbance of the negative control and each substance to be evaluated was the average absorbance measured in three or more wells, and the biofilm removal rate was calculated using the following formula:Biofilm removal rate (%)={1−(absorbance of substance to be evaluated / absorbance of negative control)}×100.

[0028] In the present invention, the method for evaluating the synergistic effect of biofilm removal when (A) and (B) are used in combination at various concentrations is as follows: <Evaluation criteria> Removal rate (c) - removal rate [(a) + (b)] ≦ 0: No synergistic effect Removal rate (c) - removal rate [(a) + (b)] > 0: Synergistic effect The biofilm removal rates (a) to (c) are defined as follows: Removal rate (a): Removal rate (%) when treated with (A) alone Removal rate (b): Removal rate (%) when treated with (B) alone Removal rate (c): Removal rate (%) when (A) and (B) are used in combination However, when multiple components defined in the present invention as (A) and (B) are used in combination, the removal rate (a) and the removal rate (b) are taken as the total concentration of the components corresponding to (A) and the total concentration of the components corresponding to (B), respectively. That is, when the removal rate of two types of components (A) and (B) used in combination is higher than the sum of the removal rates of each component when treated alone at the same concentration as when they are used in combination, the combination is judged to be an effective combination, and is evaluated as having a synergistic effect in the present invention.

[0029] Component (B) contained in the biofilm treatment agent of the present invention does not achieve the effects of the present invention by killing or inhibiting the growth of biofilm-forming bacteria at treatment concentrations below the MIC. Based on previous verification results, the inventors believe that component (B) in the biofilm treatment agent of the present invention exerts a biofilm removal or formation inhibition effect by affecting the quorum sensing of biofilm-forming bacteria. That is, by inhibiting biofilm formation against planktonic bacteria that do not form biofilms, and by inhibiting biofilm growth and reducing the structural robustness of bacteria that have already formed biofilms, it is thought to produce a synergistic effect that maximizes the bactericidal and cleaning effects of component (A). Therefore, in a treatment method that produces a synergistic effect, not only is a high biofilm removal effect achieved, but the amount of active ingredient used can be kept to a minimum, providing excellent features that are easy to use in terms of safety to the human body and the environment, foaming properties, cost, and other concerns typically associated with biofilm countermeasures.

[0030] The biofilm treatment agent of the present invention may be in the form of an original product or may be diluted with any medium and be in the form of a solution, dispersion, gel, etc. When acting on a biofilm, it may be in the form of an aqueous solution, gel, coating, etc., but is usually used in the form of an aqueous solution.

[0031] The biofilm treatment agent of the present invention can be formulated with thickeners, viscosity adjusters, pH adjusters, solvents, fragrances, colorants, antioxidants, preservatives, fluorescent agents, excipients, soil release agents, bleaching agents, bleach activators, powdering agents, granulating agents, coating agents, chelating agents, antifoaming agents, rust inhibitors, and the like, as long as the purpose of the present invention is not impaired.

[0032] The solution pH when using a biofilm treatment agent can be set as appropriate, but if it is used near the neutral pH range (5.8 to 8.6), there is no need to consider the effects on the human body or the water environment in which it is used, so it is safe.

[0033] The time for which the biofilm treatment agent is allowed to act varies depending on the amount of attached biofilm, the concentration of the active ingredient, the acting temperature, and whether or not physical force is applied, but is usually in the range of several minutes to several weeks.

[0034] The biofilm treatment agent of the present invention can be used in a wide range of fields where biofilm formation is a problem. For example, it can be applied to equipment in food manufacturing or beverage manufacturing plants, and to the inside of drains, drain pipes, air conditioners, and humidifiers in kitchens, kitchens, bathrooms, toilets, and other kitchens. It can also be applied to cooling water systems such as industrial cooling towers, water treatment membranes, desalination equipment, and circulating water systems, service water, and chemical tanks in paper mills. Biological fouling, including algae, that occurs on the exterior walls of buildings is a type of complex biofilm, and the agent can also be used to control these. Furthermore, the agent can be used as a cleaning agent for medical devices that are prone to biofilm formation, such as endoscopes, catheters, and artificial dialysis machines.

[0035] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0036] The biofilm removal effect of the biofilm treatment agent of the present invention was evaluated by first determining the MIC of (B) of the biofilm treatment agent against biofilm-forming bacteria, and then determining a concentration at which (B) was less than the MIC. The MIC testing method is described below.

[0037] <MIC (Minimum Inhibitory Concentration) Test Method> Pseudomonas (Gram-negative bacteria), known as a model bacterium for biofilm formation, was used as the test bacterium to determine the MIC of a compound (hereinafter referred to as the evaluation target substance) that is a component of a biofilm treatment agent. (1) Test bacterium Pseudomonas aeruginosa (Deposit number: NBRC106052 strain) (2) Evaluation target substance The compounds shown in Table 1 were used as the evaluation target substances. (3) Test method The evaluation target substance was serially diluted with a broth medium for susceptibility testing to prepare a dilution series totaling 10 mL (however, 1.1 times the target concentration). 10 mL of the test strain was added to the dilution series. ８20 μL of the bacterial solution adjusted to cfu / mL was added and cultured at 37°C for 24 hours with shaking (2000 rpm) in a 96-well microplate mixer. The lowest concentration in the dilution series that did not visually become cloudy was taken as the MIC. (4) Test Results The results are shown in Table 1.

[0038] <Evaluation of Biofilm Removal Effect> The biofilm removal effect of the evaluation target substances (A) and (B), and their components and combinations at different concentrations, was confirmed by preparing biofilm treatment agents containing each of them as an active ingredient and using them according to the evaluation method defined in the present invention. Furthermore, the presence or absence of a synergistic effect in biofilm removal when (A) and (B) were used in combination at various concentrations was confirmed according to the evaluation method defined in the present invention. The concentrations and concentration ratios of each evaluation target substance added to the medium, as well as the respective removal effects and the evaluation results of the combined effects are shown in Tables 2 to 8. From Tables 1 and 2 to 8, it can be said that the evaluation of the biofilm removal effect was such that the concentration of (B) was sufficiently lower than the MIC.

[0039] As comparative examples, biofilm removal effects and the presence or absence of synergistic effects of combinations in which the combination of (A) and (B) was replaced with (A) and (D), or combinations in which the combination was replaced with (C) and (B), were evaluated by preparing biofilm treatment agents containing each active ingredient. The evaluation results are shown in Tables 9 to 11. (C) is benzalkonium chloride, a cationic surfactant, and (D) is 3-phenoxy-1-propanol, a type of aromatic alcohol, or benzyl alcohol. <Evaluation Criteria> Removal rate (c) - removal rate [(a) + (b)] ≦ 0: No synergistic effect Removal rate (c) - removal rate [(a) + (b)] > 0: Synergistic effect The biofilm removal rates (a) to (c) are defined as follows: Removal rate (a): Removal rate (%) when (A) is treated alone, or removal rate (%) when (C) is treated alone. Removal rate (b): Removal rate (%) when (B) is treated alone, or removal rate (%) when (D) is treated alone. Removal rate (c): Removal rate (%) when (A) and (D) are used together, or removal rate (%) when (C) and (B) are used together. In other words, for two types of components used together, if the removal rate when the components are treated together is higher than the sum of the removal rates when each component is treated alone at the same concentration as when they are treated together, it is evaluated as having a synergistic effect, and if it is lower, it is evaluated as not having a synergistic effect.

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] Explanation of symbols in the table: Evaluation: 〇: Synergistic effect exists Evaluation: ×: No synergistic effect exists

[0052] The results in Tables 2 to 11 show that biofilm treatment agents containing (A) and (B) at the concentrations and concentration ratios specified in the present invention exhibit excellent synergistic effects that cannot be obtained from biofilm treatment agents whose concentrations deviate from the concentrations and concentration ratios specified in the present application or from biofilm treatment agents that contain compounds other than (A) and (B).

[0053] According to the present invention, by using a specific alkylaminoethylglycine in combination with a specific aromatic monoalcohol or anthranilic acid, it is possible to provide a treatment agent and treatment method that are effective against biofilms. In particular, it is possible to provide a treatment agent and treatment method that are effective against biofilms that form on medical devices, food manufacturing or beverage manufacturing plant equipment, bathrooms, toilets, air conditioners, the interior of humidifiers, building exterior walls, etc. Furthermore, because the treatment agent of the present invention has a biofilm control effect even at low concentrations or a neutral pH, it is safe and does not need to consider the effects on the human body, the environment, or corrosion of the equipment used.

Claims

1. A biofilm treatment agent comprising: (A) at least one selected from the group consisting of alkyldiaminoethylglycines having an alkyl chain with 12 to 14 carbon atoms and salts thereof; and (B) at least one selected from the group consisting of 1-phenylethanol, 2-phenylethanol, 2-phenoxyethanol, cinnamyl alcohol, anthranilamide, anthranilic acid, anthranilate salts, and methyl anthranilate, wherein the blending ratio of (A) to (B) is (B) / (A) = 0.5 to 5 by mass.

2. The biofilm treatment agent according to claim 1, wherein (A) is dodecyldiaminoethylglycine.

3. A biofilm treatment method comprising a step of contacting a biofilm with the biofilm treatment agent according to claim 1 or 2, wherein the biofilm treatment agent is used in the step under conditions where the total concentration of (A) and (B) is 100 ppm or more and the concentration of component (B) is less than the MIC (minimum inhibitory concentration) calculated by the following method: MIC (minimum inhibitory concentration): A compound that is a component of the biofilm treatment agent (hereinafter sometimes referred to as the substance to be evaluated) is serially diluted with a susceptibility test broth medium to prepare a dilution series totaling 10 mL (1.1 times the target concentration). 10 mL of Pseudomonas eruginosa (deposit number: NBRC106052), a representative strain of biofilm-forming bacteria, is added as a test strain. ８ 20 μL of the bacterial solution adjusted to cfu / mL is added, and the mixture is cultured with shaking (2000 rpm) at 37° C. for 24 hours in a 96-well microplate mixer. The lowest concentration in the dilution series that does not become cloudy visually is selected.

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