Biofilm treatment agent and method for treating biofilms

Abstract

To provide a biofilm treatment agent composed of a food additive and having excellent effects of inhibiting biofilm formation and removing biofilms, and a biofilm treatment method.SOLUTION: Provided are a biofilm treatment agent that is characterized by containing, as an essential component, at least one of the following A1 to A3, and a biofilm treatment method. A1: a γ-lactone having 6 or 7 carbon atoms. A2: a δ-lactone having 8 carbon atoms. A3: a 4-pyrone having hydroxy and alkyl groups in a molecule.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a biofilm treatment agent composed of food additives, and more particularly to a biofilm treatment agent and a method for treating biofilms for preventing hazards caused by biofilms in various fields involving microorganisms.

Background Art

[0002] A biofilm, also called a bacterial film, is a viscous deposit formed by bacteria and exists in various daily environments. The biofilm serves as a barrier to protect bacteria and a transport route for water, nutrients, etc., and is considered to protect the internal bacteria from environmental changes and chemical substances. In various industrial fields, biofilms cause many problems. For example, in the medical field, when biofilms are formed inside catheters, contact lenses, dentures, etc., bacteria grow in the biofilms and become a factor causing infectious diseases. When biofilms are formed in the pipes of a food factory, the biofilms peel off, not only leading to foreign matter contamination in the products, but also possibly causing food poisoning due to toxins derived from microorganisms. Furthermore, the formation of biofilms on metal surfaces causes metal corrosion and accelerates the aging of equipment.

[0003] As a biofilm treatment agent for equipment such as medical devices, food, or cosmetics manufacturing factories, a drug composed of components with high safety for the human body, such as food materials, food additives, enzymes, etc., is required. Also, from the perspective of countermeasures against biofilms, it is desirable to have two functions: suppressing the formation of biofilms and removing the deposited biofilms.

[0004] Traditional methods for combating biofilms in the food industry generally involve enzyme-based detergents and sodium hypochlorite. Enzyme-based detergents remove accumulated biofilms through enzymatic decomposition and surfactant cleaning, but because they are generally alkaline and highly effective, their safety for human use is questionable. Furthermore, they do not inhibit biofilm formation, so if the biofilm is not completely removed, the bacteria in the remaining biofilm will proliferate again, leading to further biofilm growth. Sodium hypochlorite inhibits biofilm formation by suppressing bacterial growth through its bactericidal action, but its biofilm removal effect is low. In addition, it causes corrosion of metal materials, making it difficult to use at a concentration that provides sufficient bactericidal action.

[0005] Therefore, there is a need for a drug that is highly safe for the human body, suppresses material degradation, and inhibits and removes biofilm formation.

[0006] The following are known prior art related to the present invention.

[0007] Patent Document 1 describes an autoinducer-2 inhibitor containing at least one selected from the group consisting of γ-lactones having 8 to 12 carbon atoms as an active ingredient, and Patent Document 2 describes an autoinducer-2 inhibitor containing at least one selected from the group consisting of δ-lactones having 9 to 12 carbon atoms and ε-lactones having 9 to 12 carbon atoms as an active ingredient. These are intended to prevent or treat infectious diseases by inhibiting the synthesis of autoinducer-2 and regulating the production of virulence factors, and there is no description or suggestion of biofilm formation inhibitory effects or biofilm removal effects. Non-patent document 1 states that pyrone analogs have anti-biofilm activity, but it does not mention or provide examples of specific substances or concentrations. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2012-246234 [Patent Document 2] Japanese Patent Publication No. 2012-246235 [Non-patent literature]

[0009] [Non-Patent Document 1] O Fleitas Martinez., et al. Frontiers in Cellular and Infection Microbiology. April 2019 Volume9 Article74 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The present invention aims to provide a biofilm treatment agent and a biofilm treatment method composed of food additives that exhibit excellent biofilm formation inhibitory and biofilm removal effects. [Means for solving the problem]

[0011] To solve the aforementioned problems, the inventors have diligently researched the potential of various types of food additives to inhibit biofilm formation and remove biofilms. As a result, they have discovered that a biofilm treatment agent characterized by containing at least one of the following A1 to A3 as an essential component has excellent biofilm formation inhibiting and biofilm removal effects, leading to the completion of the present invention. A1: γ-lactone with 6 or 7 carbon atoms A2: δ-lactone with 8 carbon atoms A3: 4-pyrone compounds having hydroxyl groups and alkyl groups in the molecule

[0012] In other words, the present invention is <1> A biofilm treatment agent characterized by containing at least one of the following A1 to A3 as an essential component. A1: γ-lactone with 6 or 7 carbon atoms A2: δ-lactone with 8 carbon atoms A3: 4-pyrone compounds having hydroxyl groups and alkyl groups in the molecule <2> The alkyl group of A3 is characterized by being a methyl group or an ethyl group. <1> The biofilm treatment agents described above, <3> A1 is expressed by the following general formula (1), A2 by the following general formula (2), and A3 by the following general formula (3). <1> The biofilm treatment agents described above, General formula (1) TIFF0007894656000001.tif3545R1: A linear alkyl group with 2 or 3 carbon atoms. General formula (2) TIFF0007894656000002.tif3545 R2: A linear alkyl group with 3 carbon atoms. General formula (3) TIFF0007894656000003.tif3138 R3~R6: Represents either a hydrogen atom, a hydroxyl group, a methyl group, or an ethyl group. Each group has one hydroxyl group, one methyl group, or one ethyl group, with the remaining group being a hydrogen atom. <4> Characterized by containing alcohol and / or surfactant, <1> The biofilm treatment agents described above, <5> The alcohol is characterized by being an aromatic monoalcohol. <4> The biofilm treatment agents described above, <6> The surfactant is either a microbial surfactant or a synthetic surfactant. <4> The biofilm treatment agents described above, <7> It contains at least one of A1 to A3 in 0.0001 to 0.1% by mass, an alcohol in 0.0001 to 0.1% by mass, and / or a surfactant in 0.0001 to 0.05% by mass. <4> The biofilm treatment agents described above, <8> This method is characterized by its use against bacteria belonging to at least one species of the phylum Proteobacteria, phylum Bacteroidetes, or phylum Basilota. <7> The biofilm treatment agents described above, <9>The biofilm treatment agent according to <7>, which is used in a concentration range less than the MIC (minimum inhibitory concentration) of bacteria belonging to at least one of the phyla Proteobacteria, Bacteroidetes, and Bacillota. <10>A biofilm treatment method using the biofilm treatment agent according to <7>, which is used in a concentration range less than the MIC (minimum inhibitory concentration) of bacteria belonging to at least one of the phyla Proteobacteria, Bacteroidetes, and Bacillota.

Effect of the Invention

[0013] According to the present invention, in biofilm formation inhibition and biofilm removal, an effect superior to that of conventional treatment agents can be obtained. Further, these active ingredients have no reactivity such as an oxidizing agent and can be used at neutral pH, so they have the merit of being less likely to cause corrosion of members to which they are applied and being easy to ensure the safety of workers.

Modes for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in detail.

[0015] (Biofilm treatment agent)<00001​​​​​​​​​​​​​​

[0018] The γ-lactone and δ-lactone used in the present invention are each a type of lactone, which is a cyclic ester compound containing an ester functional group within its ring. Lactones are classified into α-lactone, β-lactone, γ-lactone, δ-lactone, ε-lactone, etc., depending on the number of atoms constituting the ring containing the ester functional group. The γ-lactone used in the present invention has a 5-membered ring containing the ester functional group, and the δ-lactone has a 6-membered ring. The γ-lactone and δ-lactone used in the present invention are preferably represented by the following general formulas (1) or (2). Specific examples of the γ-lactone of general formula (1) include γ-hexanolactone and γ-heptanolactone. Specific examples of the δ-lactone of general formula (2) include δ-octanolactone.

[0019] General formula (1) TIFF0007894656000004.tif3545 R1: A linear alkyl group with 2 or 3 carbon atoms.

[0020] General formula (2) TIFF0007894656000005.tif3545 R2: A linear alkyl group with 3 carbon atoms. The 4-pyrone compounds used in the present invention are a type of unsaturated heterocyclic pyrone compound, preferably represented by the following general formula (3). Specific examples of 4-pyrone compounds of general formula (3) include maltol and ethylmaltol.

[0021] General formula (3) TIFF0007894656000006.tif3138 R3~R6: Represents either a hydrogen atom, a hydroxyl group, a methyl group, or an ethyl group. Each group has one hydroxyl group, one methyl group, or one ethyl group, with the remaining group being a hydrogen atom.

[0022] A more preferred embodiment of the biofilm treatment agent of the present invention comprises at least one of A1 to A3, an alcohol, and a surfactant, which can provide even better biofilm formation inhibition and biofilm removal effects. Examples of surfactants include microbial surfactants and synthetic surfactants, which will be described later.

[0023] The alcohol is not particularly limited, but from the viewpoint of inhibiting biofilm formation and removing biofilm, it is preferably an aromatic monoalcohol represented by the following general formula (4) or general formula (5), or cinnamyl alcohol. These can be used alone or in combination of two or more.

[0024] General formula (4) TIFF0007894656000007.tif3545 R7: A linear alkyl group having 1 to 3 carbon atoms, in which any one hydrogen atom is replaced by a hydroxyl group.

[0025] General formula (5) TIFF0007894656000008.tif3545 R8: A linear alkyl group with 1 to 3 carbon atoms in which any one hydrogen atom is replaced by a hydroxyl group.

[0026] Specific examples of aromatic monoalcohols of general formula (4) include 1-phenylmethanol, 1-phenylethanol, 2-phenylethanol, 1-phenyl-1-propanol, 1-phenyl-2-propanol, and 3-phenyl-1-propanol. Specific examples of aromatic monoalcohols of general formula (5) include 2-phenoxyethanol, 3-phenoxy-1-propanol, and 1-phenoxy-2-propanol.

[0027] Among alcohols, 1-phenylethanol, 2-phenylethanol, 2-phenoxyethanol, 3-phenoxy-1-propanol, and cinnamyl alcohol are preferred from the viewpoint of inhibiting biofilm formation and removing biofilms.

[0028] While the microbial surfactant is not particularly limited, amino acid-type or glycolipid-type surfactants are preferred from the viewpoint of biofilm removal effect. Specific examples of amino acid-type microbial surfactants include surfactin, and specific examples of glycolipid-type microbial surfactants include rhamnolipid and sophorolipid. These can be used individually or in combination of two or more.

[0029] The synthetic surfactant is not particularly limited, but from the viewpoint of biofilm removal effect, it is preferable that it be anionic or nonionic. Examples of anionic synthetic surfactants include alkyl sulfate salts (e.g., sodium dodecyl sulfate, potassium dodecyl sulfate, etc.), alkylbenzene sulfonates (e.g., sodium dodecylbenzenesulfonate, triethanolamine dodecylbenzenesulfonate, etc.), polyoxyethylene alkyl ether sulfates (e.g., sodium polyoxyethylene lauryl ether sulfate, etc.), and dialkyl sulfosuccinates (e.g., sodium dihexyl sulfosuccinate, sodium dioctyl sulfosuccinate, sodium didecyl sulfosuccinate, sodium didodecyl sulfosuccinate, etc.). Examples of nonionic synthetic surfactants include alcohol ethoxylates (e.g., polyoxyethylene lauryl ether, etc.), glycerin fatty acid esters (e.g., glyceryl monothrea phosphate, etc.), and sucrose fatty acid esters (e.g., sucrose laurate, etc.). These can be used individually or in combination of two or more.

[0030] From the viewpoint of inhibiting biofilm formation and removing biofilm, it is preferable that the content of A1 to A3, alcohol, and / or surfactant be 0.0001 to 0.1% by mass of at least one of A1 to A3, 0.0001 to 0.1% by mass of alcohol, and / or 0.0001 to 0.05% by mass of surfactant.

[0031] (Minimum Inhibitory Concentration (MIC)) The biofilm formation inhibitory effect and biofilm removal effect of the biofilm treatment agent of the present invention were evaluated by first determining the minimum inhibitory concentration (MIC) of each component of the biofilm treatment agent against biofilm-forming bacteria, and then performing the evaluation at concentrations below the MIC.

[0032] In this invention, MIC refers to the minimum concentration at which a chemical substance inhibits the growth of microorganisms (bacteriostatic and antiseptic effect). Therefore, a concentration below the MIC can be considered synonymous with a concentration that does not substantially inhibit the growth of biofilm-forming bacteria.

[0033] The method for calculating MIC in this invention is as follows. The chemical substances that make up the biofilm treatment agent (hereinafter sometimes referred to as the substance to be evaluated) are diluted stepwise in a broth medium for susceptibility testing to prepare a total of 10 mL of dilution series (however, 1.1 times the target concentration). Then, 10 units of the test bacteria are added to each. 8 Add 20 μL of bacterial suspension prepared to cfu / mL and incubate in a 96-well microplate mixer at 37°C for 24 hours with shaking (2000 rpm). The lowest concentration among the dilution series that did not show turbidity visually is taken as the MIC.

[0034] The biofilm treatment agent of the present invention achieves the effects of the present invention when biofilm-forming bacteria grow at biofilm treatment agent concentrations below the MIC of each component. In other words, the biofilm treatment agent of the present invention does not achieve the effects of the present invention by killing or inhibiting the growth of the biofilm-forming bacteria themselves.

[0035] In this invention, the biofilm formation inhibitory effect refers to the effect of inhibiting the formation of biofilms by bacteria. One method for confirming whether or not a substance under evaluation has this effect is to compare the amount of biofilm obtained by culturing bacteria in a culture medium containing the substance under evaluation for a certain period of time with the amount of biofilm obtained by culturing the same bacteria in a culture medium without the substance under evaluation (control). In this case, if the amount of biofilm is less than that of the control, it can be determined that the substance under evaluation has a biofilm formation inhibitory effect.

[0036] In this invention, the biofilm removal effect refers to the effect of removing biofilm formed by bacteria. Methods for evaluating the biofilm removal effect of a substance include, for example, comparing the amount of biofilm remaining after contacting the substance with a biofilm obtained by culturing bacteria for a certain period of time, with the amount of biofilm remaining after a certain period of time without contact with the substance (control). In this case, if the amount of biofilm is less than that of the control, it can be determined that the substance has a biofilm removal effect.

[0037] The method for evaluating the biofilm formation inhibitory effect in the present invention is as follows. (1) Pseudomonas aeruginosa (deposit number: NBRC106052), a representative strain of biofilm-forming bacteria, is used as the pre-culture medium, which is prepared by pre-culturing in TSB (Triptic Soy Broth, Bacto: Difco Laboratories) medium with a final glucose concentration of 0.5% at 37°C and 130 rpm. (2) Add the substance to be evaluated to the culture medium at an appropriate concentration below the MIC of the substance, and adjust the pH of the culture medium to 7.0 with hydrochloric acid or sodium hydroxide as necessary. The sample that does not contain the substance to be evaluated will be used as the control (pH 7.0). (3) The pre-culture medium, adjusted with culture medium to an OD (turbidity) of 0.6, is added to the culture medium prepared in (2) to a final concentration of 10% (v / v), and 2 mL is dispensed into each 12-well plate. Hereinafter, OD (turbidity) refers to the value at a wavelength of 630 nm with distilled water as the blank, measured using a spectrophotometer (iMark Microplate Reader: Bio-Rad). (4) Incubate at 37°C and 130 rpm for 4 hours to allow biofilm formation. (5) Remove the culture medium from each well and rinse each well twice with distilled water. (6) Add 4 mL of crystal violet aqueous solution (0.4 w / v%, 20 w / v% ethanol) to the biofilm attached to each pore, let stand for 3 minutes to stain, then rinse three times with distilled water to remove any crystal violet aqueous solution that is not bound to the biofilm. (7) Add 4 mL of eluent (50 w / v% ethanol, 2 w / v% sodium dodecyl sulfate) to each well, let stand for 1 hour to elute crystal violet from the stained biofilm, and measure the absorbance at a wavelength of 595 nm. Hereinafter, absorbance refers to the value at a wavelength of 595 nm with distilled water as the blank, measured using a spectrophotometer (iMark microplate reader: Bio-Rad). (8) The absorbance of the control and each substance to be evaluated shall be the average value of the absorbance measured in 3 or more wells, and the biofilm formation inhibition rate shall be calculated using the following formula. Biofilm formation inhibition rate (%) = {1 - (absorbance of the substance being evaluated / absorbance of the control)} × 100 (9) The calculated values ​​are evaluated for their biofilm formation inhibitory effect based on the following criteria. <Judgment criteria> Formation inhibition rate of 70% or more: High inhibitory effect Formation inhibition rate of 30% to less than 70%: Inhibitory effect is present. Formation inhibition rate of 10% to less than 30%: Weak inhibitory effect Formation inhibition rate less than 10%: No inhibitory effect. A biofilm formation inhibition rate of 30% or more is considered practical.

[0038] The method for evaluating the biofilm removal effect in the present invention is as follows. (1) Pseudomonas aeruginosa (deposit number: NBRC106052), a representative strain of biofilm-forming bacteria, is used as the pre-culture medium, which is prepared by pre-culturing in TSB (Triptic Soy Broth, Bacto: Difco Laboratories) medium with a final glucose concentration of 0.5% at 37°C and 130 rpm. (2) Dilute the pre-culture medium, adjusted to an OD (turbidity) of 0.1, with TSB medium to a final concentration of 0.000005% (v / v), and dispense 2 mL into a 12-well polystyrene plate. (3) Incubate at 37°C and 130 rpm for 17 hours to allow biofilm formation. (4) Remove the culture medium from each well and rinse once with sterile medium. (5) Add the substance to be evaluated to the culture medium to an appropriate concentration below the MIC of the substance, and adjust the pH of the culture medium to 7.0 with hydrochloric acid or sodium hydroxide. Add 3 mL of the culture medium containing the substance to be evaluated to each well, and as a negative control, add 3 mL of sterile medium (pH=7.0) to each well. (6) Shake at 37°C for 3.5 hours at 130 rpm to allow the culture medium containing the substance to be evaluated to come into contact with the biofilm. After that, remove the culture medium and detached biofilm from each well and rinse twice with distilled water. (7) Add 4 mL of crystal violet aqueous solution to the biofilm attached to each well, let it stand for 3 minutes to stain, then rinse three times with distilled water to remove any crystal violet aqueous solution that is not bound to the biofilm. (8) Add 4 mL of eluent to each well, let stand for 1 hour to elute crystal violet from the stained biofilm, and measure the absorbance. (9) The absorbance of the control and each substance to be evaluated shall be the average value of the absorbance measured with 3 or more wells, and the biofilm removal rate shall be calculated using the following formula. Biofilm removal rate (%) = {1 - (absorbance of the substance being evaluated / absorbance of the control)} × 100 (10) The biofilm removal effect of the calculated values ​​is evaluated based on the following criteria. <Judgment criteria> Removal rate of 70% or more: High removal effectiveness Removal rate between 30% and 70%: Effective in removing the substance. Removal rate between 10% and 30%: Weak removal effect Removal rate less than 10%: No removal effect. A biofilm removal rate of 30% or more is considered practical.

[0039] The present invention may take the form of the raw material, a diluted solution or dispersion in any medium, or a solid substance such as a gel, granules, or tablet. However, when acting on a biofilm, it is usually used in aqueous solution form. The concentration of the diluted biofilm treatment agent is not particularly limited, but it must be at a concentration sufficient to produce the effects of the present invention when acting on biofilm-forming bacteria.

[0040] The present invention may contain, to the extent that it does not impair the objectives of the present invention, γ-lactones having 6 or 7 carbon atoms, δ-lactones having 8 carbon atoms, 4-pyrones having a hydroxyl group and an alkyl group in the molecule, alcohols, thickeners other than surfactants, viscosity modifiers, pH adjusters, solvents, fragrances, colorants, antioxidants, preservatives, fluorescent agents, excipients, soil release agents, bleaching agents, bleaching activators, powdering agents, granulating agents, and the like. Furthermore, to the extent that it does not impair the effects of the present invention, it may also contain substances shown in Patent Documents 1 and 2, specifically γ-octanolactone, γ-decanolactone, and δ-nonalactone. The content of these substances is preferably 0.1% or less.

[0041] (How to use biofilm treatment agents) The preferred usage conditions for the present invention are described below. The concentration of the present invention is such that the concentration of each component contained in the biofilm treatment agent is below the MIC of the major causative bacterial species that make up the biofilm. When used simultaneously against multiple causative bacterial species, the concentration applied is that of the bacterial species with the lowest MIC among the target species. By using it below the MIC, the death of biofilm-forming bacteria is suppressed, and the non-specific adsorption of dead bacteria to the surface is suppressed, thus leading to the suppression of new biofilm formation. A single-component biofilm treatment agent is preferable for handling purposes, but each component may be prepared individually and mixed when in contact with the biofilm-forming bacteria. Alternatively, a mixture of each component at a high concentration may be diluted 200 to 20,000 times or so to use at the desired concentration.

[0042] The pH of the solution when using the present invention can be set as appropriate, but if used in the neutral pH range (6.0 to 8.0), there is no need to consider the impact on the human body or the water environment used, making it safe.

[0043] The present invention can be used by immersion, circulation, coating, or watering. Furthermore, physical force such as a sponge, brush, or water flow may be applied. The duration of application varies depending on the number of bacteria, the amount of attached biofilm, the concentration of the active ingredient, the temperature of application, and whether or not physical force is applied, but is usually in the range of several minutes to several hours. After application, it is desirable to quickly rinse away the removed biofilm with running water. In addition, by contacting the present invention with a material on which biofilm formation is to be suppressed for several minutes to several hours in advance, biofilm formation can be suppressed by the action of the essential components. Furthermore, the present invention can be applied as a coating (for example, acrylic silicone resin or epoxy resin), or kneaded with plastic resins such as polyethylene or polypropylene to create molded products that have a biofilm formation suppression effect.

[0044] (Biofilm-forming bacteria) The biofilm-forming bacteria to which the present invention applies include any bacteria belonging to the Proteobacteria, Bacteroidetes, and Basilota phyla that form biofilms. Among these, it is preferable to use the present invention on the genera Methylobacterium, Ocrobacterium, Aeromonas, Klebsiella, Acinetobacter, Enterobacter, Citrobacter, Stenotrophumonas, Pseudomonas, Rhizobium, and Capriavidus belonging to the Proteobacteria phylum, the genera Porphyromonas belonging to the Bacteroidetes phylum, and the genera Staphylococcus belonging to the Basilota phylum. It should be noted that biofilm formation is almost always carried out by two or more types of bacteria, and biofilms containing one or more bacteria belonging to the above genera are the target of the present invention.

[0045] 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 cosmetic or food and beverage manufacturing plants, aqueous chemical tanks such as cutting oils, and drains and drainpipes in kitchens, bathrooms, toilets, and other kitchen facilities. It can also be applied to cooling water systems such as industrial cooling towers, water treatment membranes, desalination equipment, and circulating water systems in paper mills. Furthermore, it can be applied to cleaning agents for medical devices prone to biofilm formation, such as endoscopes, catheters, and hemodialysis machines, as well as oral hygiene products such as mouthwash. [Examples]

[0046] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. In the examples, Pseudomonas species (Gram-negative bacteria), which are known as representative biofilm-forming bacteria, were used as test organisms, and the MIC was determined.

[0047] In the examples, the biofilm formation inhibitory effect and biofilm removal effect of the biofilm treatment agent of the present invention were evaluated at concentrations below the MIC determined in advance. The method for testing the MIC in these examples is shown below.

[0048] <Test method for MIC (Minimum Inhibitory Concentration)> Using Pseudomonas bacteria (Gram-negative bacteria), a genus of Proteobacteria known as a representative strain for biofilm formation, as the test bacteria, the MIC of the chemical substances (hereinafter referred to as the substances to be evaluated), which are the components of the biofilm treatment agent, was determined. (1) Test bacteria Pseudomonas aeruginosa (Deposit number: NBRC106052 strain) (2) Substances to be evaluated The chemical substances shown in Table 1 were used as the substances to be evaluated. (3) Test method The substances to be evaluated were serially diluted in broth medium for susceptibility testing to prepare a total of 10 mL of dilution series (however, 1.1 times the target concentration). 20 μL of the bacterial suspension prepared to 10 8 cfu / mL was added thereto, and the mixture was cultured with shaking at 37 °C for 24 hours (2000 rpm) using a 96-well microplate mixer. The lowest concentration among the dilution series that did not show turbidity visually was taken as the MIC. (4) Test results The MIC results of each substance to be evaluated are shown in Table 1. In the table, "> numerical value" indicates that the MIC is greater than the numerical value. For the substances shown in the examples, the following biofilm formation inhibition evaluation and biofilm removal evaluation were carried out at concentrations lower than the MIC described in Table 1.

[0049]

Table 1

[0050] <Evaluation of biofilm formation inhibition effect and biofilm removal effect> The biofilm formation inhibitory effect and biofilm removal effect were evaluated for each target substance at concentrations below the values ​​listed in Table 1 (ppm), according to the evaluation method defined in this invention. Substances not used as target substances in each example are indicated with "-". The evaluation results for A1 to A3 and their combinations are shown in Table 2, the evaluation results for comparative examples of A1 to A3 are shown in Table 3, and the evaluation results for combinations of A1 to A3, alcohol, and surfactant are shown in Tables 4 and 5, respectively. Note that in Tables 2 to 4, the name of the lactone and its carbon number may be listed together.

[0051] [Table 2]

[0052] [Table 3]

[0053] [Table 4]

[0054] [Table 5]

[0055] Table 2, Examples 1-7, shows that at least one of the A1-A3 of the present invention has both a biofilm formation inhibitory effect and a biofilm removal effect. Furthermore, Examples 8-10 show that combining A1-A3 yields even better results.

[0056] Table 3 shows comparative examples. Comparative Examples 1-6 are comparative examples of Examples 1-4 using A1, and Comparative Examples 7-9 are comparative examples of Example 5 using A2, and the substances are described in Patent Documents 1 and 2, respectively. Comparative Examples 1-9 show that at concentrations more preferable than those described in the literature, there is no or weak biofilm formation inhibitory effect and biofilm removal effect. Comparative Examples 10 and 11 are comparative examples of Examples 6-7 using A3, and show that at concentrations below the MIC, there is no or weak biofilm formation inhibitory effect and biofilm removal effect.

[0057] Table 4 shows that combining A1-A3 alone or any combination with alcohol or a surfactant results in superior biofilm formation inhibition and biofilm removal effects (for example, comparing Examples 11 and 12 with Example 8). Furthermore, combining A1-A3 alone or any combination with alcohol and a surfactant results in even superior biofilm formation inhibition and biofilm removal effects (for example, comparing Example 16 with Examples 14 and 15). Additionally, combining at least one of A1-A3, alcohol, or a surfactant with any of the substances in Comparative Examples 1, 2, and 7 also shows similarly excellent effects (Examples 25-27).

[0058] Table 5 shows that any combination of A1 to A3 combined with alcohol and a surfactant exhibits excellent biofilm formation inhibitory and biofilm removal effects against the biofilms of bacteria belonging to each genus. Furthermore, for Methylobacterium aquaticum, Porphyromonas gingivalis, and Staphylococcus epidermidis, as shown in the examples, the growth of each bacterium at the concentrations added in the examples was confirmed by turbidity, clearly indicating that the MIC is below the limit. [Industrial applicability]

[0059] According to the present invention, by using at least one of a γ-lactone having 6 or 7 carbon atoms, a δ-lactone having 8 carbon atoms, and a 4-pyrone having a hydroxyl group and an alkyl group in the molecule as essential components, it is possible to provide a biofilm treatment agent that is effective for inhibiting biofilm formation and removing biofilms. This invention provides an effective treatment agent for biofilms that form particularly in medical equipment, cosmetic manufacturing plants, or food and beverage manufacturing plants, kitchens, bathrooms, toilets, drains and pipes in kitchens or other kitchen facilities, water-decomposing membranes, cooling towers, and tanks for diluting aqueous chemicals such as cutting oils. Furthermore, since the treatment agent of the present invention has both a biofilm formation inhibitory effect and a biofilm removal effect at a neutral pH, it has little impact on the human body and corrosion of the equipment used.

Claims

1. A biofilm treatment agent characterized in that it contains as an essential component a combination of two or more groups, each selected from the groups A1 represented by the following general formula (1), A2 represented by the following general formula (2), and A3 represented by the following general formula (3), including A2. General formula (1) R1: A linear alkyl group having 2 or 3 carbon atoms. General formula (2) R2: A linear alkyl group with 3 carbon atoms. General formula (3) R3 to R6 represent one of the following: a hydrogen atom, a hydroxyl group, a methyl group, or an ethyl group. Each group has one hydroxyl group, one methyl group, or one ethyl group, and the remaining group is a hydrogen atom. However, R4 or R6 is a hydroxyl group.

2. The biofilm treatment agent according to claim 1, characterized in that it contains an alcohol and / or a surfactant, wherein the alcohol is an aromatic monoalcohol represented by the following general formula (4) or general formula (5), or cinnamyl alcohol. General formula (4) R7: A linear alkyl group having 1 to 3 carbon atoms, in which any one hydrogen atom is replaced by a hydroxyl group. General formula (5) R8: A linear alkyl group having 1 to 3 carbon atoms, in which any one hydrogen atom is replaced by a hydroxyl group.

3. The biofilm treatment agent according to claim 2, wherein the surfactant is a microbial surfactant or a synthetic surfactant.

4. The biofilm treatment agent according to claim 2, comprising 0.0001 to 0.1% by mass of at least one of A1 to A3, 0.0001 to 0.1% by mass of alcohol and / or 0.0001 to 0.05% by mass of a surfactant.

5. The biofilm treatment agent according to claim 4, characterized in that it is used against bacteria belonging to at least one of the phyla Proteobacteria, Bacteroidetes, and Basilota.

6. The biofilm treatment agent according to claim 4, characterized in that it is used in a concentration range below the MIC (minimum inhibitory concentration) of at least one bacterium belonging to the phylum Proteobacteria, phylum Bacteroidetes, or phylum Basilota.

7. A biofilm treatment method using the biofilm treatment agent according to claim 4, characterized in that it is used for purposes other than medical procedures on humans, within a concentration range below the MIC (minimum inhibitory concentration) of bacteria belonging to at least one species of the phylum Proteobacteria, phylum Bacteroidetes, and phylum Basilota.

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