Biofilm treatment agent and biofilm treatment method

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 a food additive, and more specifically, to a biofilm treatment agent and a method for treating a biofilm for preventing harm caused by biofilms in various fields involving microorganisms. [Background technology]

[0002] Biofilms, also called bacterial membranes, are viscous deposits formed by bacteria and are found in various everyday environments. Biofilms are thought to act as a barrier to protect bacteria and as a transport route for water and nutrients, protecting the bacteria inside from environmental changes and chemicals. Biofilms cause many problems in various industrial fields. For example, in the medical field, when biofilms form inside catheters, contact lenses, dentures, etc., bacteria grow within the biofilm, causing infections. When biofilms form in the piping of food factories, they can peel off and not only lead to foreign matter contamination in the product, but also cause food poisoning due to toxins derived from microorganisms. Furthermore, biofilm formation on metal surfaces can cause metal corrosion and accelerate the aging of equipment.

[0003] As biofilm treatment agents for facilities such as medical equipment, food, and cosmetics manufacturing plants, there is a demand for agents that are composed of ingredients that are highly safe for the human body, such as food ingredients, food additives, and enzymes. In terms of biofilm countermeasures, it is desirable for them to have two actions: to inhibit the formation of biofilms and to remove accumulated biofilms.

[0004] Enzyme-based cleaning agents and sodium hypochlorite are commonly known as conventional biofilm countermeasures in the food industry. Enzyme-based cleaning agents are types that remove accumulated biofilms by enzymatic decomposition and cleaning with surfactants, but since they are generally highly effective in alkaline conditions, it is difficult to say that they are safe for the human body. In addition, since they have no effect in suppressing the formation of biofilms, if the biofilm is not completely removed, the bacteria in the remaining biofilm will grow again, causing the biofilm to increase further. Sodium hypochlorite is a type that suppresses the formation of biofilms by suppressing the growth of bacteria with its bactericidal action, but its biofilm removal effect is low. In addition, since it causes corrosion of metal materials, it is difficult to use it at a concentration that exerts a sufficient bactericidal effect.

[0005] Therefore, there is a demand for an agent that is highly safe for the human body, inhibits the formation of biofilms, and removes biofilms while suppressing deterioration of components.

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

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

[0008] [Patent Document 1] JP 2012-246234 A [Patent Document 2] JP 2012-246235 A [Non-patent literature]

[0009] [Non-Patent Document 1] O Fleitas Martinez., et al. Frontiers in Cellular and Infection Microbiology. April 2019 Volume9 Article74 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a biofilm treatment agent composed of a food additive, which has excellent biofilm formation inhibitory effects and biofilm removal effects, and a biofilm treatment method. [Means for solving the problem]

[0011] In order to solve the above problems, the present inventors have conducted extensive research into the possibility of inhibiting biofilm formation and removing biofilms using various types of food additives. As a result, the inventors have found that a biofilm treatment agent containing at least one of the following A1 to A3 as an essential component has excellent biofilm formation inhibiting and biofilm removing effects, and have completed the present invention. A1: γ-lactone having 6 or 7 carbon atoms A2: δ-lactone with 8 carbon atoms A3: 4-pyrones having hydroxyl and alkyl groups in the molecule

[0012] That is, the present invention provides <1> A biofilm treatment agent comprising at least one of the following A1 to A3 as an essential component: A1: γ-lactone having 6 or 7 carbon atoms A2: δ-lactone with 8 carbon atoms A3: 4-pyrones having hydroxyl and alkyl groups in the molecule <2> The alkyl group of A3 is a methyl group or an ethyl group. <1> The biofilm treatment agent according to claim 1, <3> A1 is represented by the following general formula (1), A2 is represented by the following general formula (2), and A3 is represented by the following general formula (3). <1> The biofilm treatment agent according to claim 1, General formula (1) TIFF2025011318000001.tif3545R1: Straight-chain alkyl group with 2 or 3 carbon atoms General formula (2) TIFF2025011318000002.tif3545 R2: A straight-chain alkyl group with 3 carbon atoms General formula (3) TIFF2025011318000003.tif3138 R3 to R6: each represents a hydrogen atom, a hydroxyl group, a methyl group, or an ethyl group, and each has one hydroxyl group, one methyl group, or one ethyl group, and the rest are hydrogen atoms. <4> Contains alcohol and / or a surfactant, <1> The biofilm treatment agent according to claim 1, <5> The alcohol is an aromatic monoalcohol. <4> The biofilm treatment agent according to claim 1, <6> The surfactant is a microbial surfactant or a synthetic surfactant; <4> The biofilm treatment agent according to claim 1, <7> Contains 0.0001 to 0.1 mass% of at least one of A1 to A3, 0.0001 to 0.1 mass% of alcohol, and / or 0.0001 to 0.05 mass% of a surfactant. <4> The biofilm treatment agent according to claim 1, <8> The present invention is characterized in that it is used against at least one species of bacteria belonging to the phylum Proteobacteria, Bacteroidetes, and Bacillota. <7> The biofilm treatment agent according to claim 1, <9> It is characterized in that it 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. <7> The biofilm treatment agent according to claim 1, <10> It is characterized in that it 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. <7> A method for treating a biofilm using the biofilm treatment agent described in the above. Effect of the Invention

[0013] According to the present invention, it is possible to obtain more excellent effects in inhibiting biofilm formation and removing biofilm than conventional treatment agents. In addition, these active ingredients are not reactive like oxidizing agents and can be used at neutral pH, so they are less likely to cause corrosion of the members to which they are applied, and there is an advantage in that the safety of workers can be easily ensured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below.

[0015] (Biofilm treatment agent) The biofilm treatment agent of the present invention contains at least one of the following A1 to A3 as an essential component. A1: γ-lactone having 6 or 7 carbon atoms A2: δ-lactone with 8 carbon atoms A3: 4-pyrones having hydroxyl and alkyl groups in the molecule

[0016] In the present invention, a biofilm treatment agent refers to an agent that has at least a biofilm formation inhibitory effect and a biofilm removal effect.

[0017] Although the biofilm treatment agent of the present invention has the effect of the present invention when each of A1, A2, and A3 is used alone, they may be used in combination, which is preferable because the combined use provides a more excellent biofilm formation inhibitory effect and biofilm removal effect.

[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 in the 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 ring containing the ester functional group in the γ-lactone used in the present invention is a five-membered ring, and the ring containing the ester functional group in the δ-lactone is a six-membered ring. The γ-lactone and δ-lactone used in the present invention are preferably represented by the following general formula (1) or (2), respectively. 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) TIFF2025011318000004.tif3545 R1: A straight-chain alkyl group having 2 or 3 carbon atoms

[0020] General formula (2) TIFF2025011318000005.tif3545 R2: A straight-chain alkyl group with 3 carbon atoms The 4-pyrones used in the present invention are a type of unsaturated heterocyclic pyrone compound, and are preferably represented by the following general formula (3). Specific examples of the 4-pyrones of the general formula (3) include maltol and ethyl maltol.

[0021] General formula (3) TIFF2025011318000006.tif3138 R3 to R6: each represents a hydrogen atom, a hydroxyl group, a methyl group, or an ethyl group, and each has one hydroxyl group, one methyl group, or one ethyl group, and the rest are hydrogen atoms.

[0022] A more preferred embodiment of the biofilm treatment agent of the present invention contains at least one of A1 to A3, an alcohol, and a surfactant, and can provide a more excellent effect of inhibiting biofilm formation and removing biofilms. Examples of the surfactant include a microbial surfactant and a synthetic surfactant, which will be described later.

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

[0024] General formula (4) TIFF2025011318000007.tif3545 R7: A linear alkyl group having 1 to 3 carbon atoms in which any one hydrogen atom is replaced with a hydroxyl group.

[0025] General formula (5) TIFF2025011318000008.tif3545 R8: A linear alkyl group having 1 to 3 carbon atoms in which any one hydrogen atom is replaced with 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 the alcohols, from the viewpoint of biofilm formation inhibition and biofilm removal effects, 1-phenylethanol, 2-phenylethanol, 2-phenoxyethanol, 3-phenoxy-1-propanol, and cinnamyl alcohol are preferred.

[0028] The microbial surfactant is not particularly limited, but from the viewpoint of biofilm removal effect, an amino acid type or a glycolipid type is preferable. Specific examples of amino acid type microbial surfactants include surfactin, and specific examples of glycolipid type microbial surfactants include rhamnolipid, sophorolipid, etc. These can be used alone or in combination of two or more types.

[0029] The synthetic surfactant is not particularly limited, but is preferably anionic or nonionic from the viewpoint of biofilm removal effect. Examples of anionic synthetic surfactants include alkyl sulfate ester salts (e.g., sodium dodecyl sulfate, potassium dodecyl sulfate, etc.), alkylbenzene sulfonate salts (e.g., sodium dodecylbenzene sulfonate, triethanolamine dodecylbenzene sulfonate, etc.), polyoxyethylene alkyl ether sulfate salts (e.g., sodium polyoxyethylene lauryl ether sulfate, etc.), dialkyl sulfosuccinate salts (e.g., sodium dihexyl sulfosuccinate, sodium dioctyl sulfosuccinate, sodium didecyl sulfosuccinate, sodium didecyl sulfosuccinate, etc.). Examples of nonionic synthetic surfactants include alcohol ethoxylates (e.g., polyoxyethylene lauryl ether, etc.), glycerin fatty acid esters (e.g., glycerin monostearate, etc.), and sucrose fatty acid esters (e.g., sucrose laurate, etc.). These can be used alone or in combination of two or more.

[0030] From the viewpoint of inhibiting biofilm formation and removing biofilms, the contents of A1 to A3, alcohol and / or surfactant are preferably 0.0001 to 0.1 mass% of at least one of A1 to A3, 0.0001 to 0.1 mass% of alcohol and / or 0.0001 to 0.05 mass% of surfactant.

[0031] (Minimum Inhibitory Concentration (MIC)) The biofilm formation-inhibiting effect and biofilm removal effect of the biofilm treatment agent of the present invention were evaluated in advance by determining the minimum inhibitory concentration (MIC) of each component of the biofilm treatment agent against biofilm-forming bacteria, and the evaluation was performed at a concentration below the MIC.

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

[0033] The method for calculating the MIC in the present invention is as follows. The chemical substances that are components of the biofilm treatment agent (hereinafter referred to as the evaluation target substances) are serially diluted in bouillon medium for susceptibility testing to prepare a dilution series of 10 mL in total (1.1 times the target concentration). 8 Add 20 μL of the bacterial solution adjusted to cfu / mL and shake culture (2000 rpm) in a 96-well microplate mixer at 37°C for 24 hours. The lowest concentration in the dilution series that does not visually become cloudy is the MIC.

[0034] In the biofilm treatment agent of the present invention, biofilm-forming bacteria grow and the effect of the present invention is exhibited at a biofilm treatment agent concentration of each component less than the MIC. In other words, the biofilm treatment agent of the present invention does not exhibit the effect of the present invention by killing or inhibiting the growth of biofilm-forming bacteria themselves.

[0035] In the present invention, the biofilm formation inhibitory effect refers to an effect of inhibiting the formation of a biofilm by bacteria. For example, a method for confirming the presence or absence of the effect of the evaluation target substance is a method of comparing the amount of biofilm obtained by culturing bacteria for a certain period of time in a medium containing the evaluation target substance with the amount of biofilm obtained by culturing the bacteria in a medium not containing the evaluation target substance (control). In this case, if the amount of biofilm is smaller than that of the control, the evaluation target substance can be determined to have a biofilm formation inhibitory effect.

[0036] In the present invention, the biofilm removal effect refers to the effect of removing the biofilm formed by bacteria. For example, the method of evaluating the biofilm removal effect of the evaluation target substance includes a method of comparing the amount of biofilm remaining after contacting the evaluation target substance with the biofilm obtained by culturing bacteria for a certain period of time with the amount of biofilm remaining after a certain period of time has passed without contacting the evaluation target substance (control). In this case, if the amount of biofilm is smaller than that of the control, it can be determined that the evaluation target 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, was pre-cultured in TSB (Triptic Soy Broth, Bacto: manufactured by Difco Laboratories) medium containing 0.5% glucose at a final concentration at 37°C and 130 rpm (pre-culture solution). (2) Add the target substance to the medium at an appropriate concentration below the MIC of the target substance, and adjust the medium to pH 7.0 with hydrochloric acid or sodium hydroxide as necessary. Use the medium without the target substance as the control (pH 7.0). (3) The OD (turbidity) of the preculture solution is adjusted to 0.6 with medium, and added to the medium prepared in (2) to a final concentration of 10% (v / v), and 2 mL of the solution is dispensed into a 12-well plate. Hereinafter, OD (turbidity) refers to the value measured at a wavelength of 630 nm using a spectrophotometer (iMark microplate reader: Bio-Rad) with distilled water as the blank. (4) Culture the mixture at 37°C and 130 rpm for 4 hours to form a biofilm. (5) Remove the culture medium from each well and rinse twice with distilled water. (6) Add 4 mL of a crystal violet aqueous solution (0.4 w / v%, 20 w / v% ethanol) to the biofilm attached to each well, leave it for 3 minutes, stain it, and 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 elution solution (50 w / v% ethanol, 2 w / v% sodium dodecyl sulfate) to each well and leave for 1 hour to elute the crystal violet from the stained biofilm, and measure the absorbance at 595 nm. Hereinafter, the absorbance refers to the value at 595 nm measured using a spectrophotometer (iMark microplate reader: Bio-Rad) with distilled water as the blank. (8) The absorbance of the control and each substance to be evaluated is the average of the absorbance measured in three or more wells, and the biofilm formation inhibition rate is calculated using the following formula. Biofilm formation inhibition rate (%) = {1-(absorbance of the substance to be evaluated / absorbance of the control)} x 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 inhibition effect Formation inhibition rate: 30% to 70%: Inhibitory effect Formation inhibition rate: 10% to 30%: Weak inhibition effect Formation inhibition rate less than 10%: no inhibition effect A biofilm formation inhibition rate of 30% or more is at a practical level.

[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, was pre-cultured in TSB (Triptic Soy Broth, Bacto: manufactured by Difco Laboratories) medium containing 0.5% glucose at a final concentration at 37°C and 130 rpm (pre-culture solution). (2) The preculture solution is adjusted to an OD (turbidity) of 0.1, diluted with TSB medium to a final concentration of 0.000005% (v / v), and dispensed in 2 mL portions into 12-well polystyrene plates. (3) Culture the mixture at 37°C and 130 rpm for 17 hours to form a biofilm. (4) Remove the culture medium from each well and rinse once with sterile medium. (5) Add the substance to be evaluated to the medium at an appropriate concentration below the MIC of the substance, and adjust the medium pH to 7.0 with hydrochloric acid or sodium hydroxide. Place 3 mL of the medium containing the substance to be evaluated in each well, and as a negative control, place 3 mL of sterile medium (pH = 7.0) in each well. (6) After contacting the biofilm with the medium containing the substance to be evaluated by shaking at 130 rpm at 37°C for 3.5 hours, the medium and detached biofilm in each well are removed and rinsed twice with distilled water. (7) Add 4 mL of a crystal violet aqueous solution to the biofilm attached to each well, leave it for 3 minutes, stain it, and 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 elution solution to each well, leave it for 1 hour, elute the crystal violet from the stained biofilm, and measure the absorbance. (9) The absorbance of the control and each substance to be evaluated is the average of the absorbance measured in three or more wells, and the biofilm removal rate is calculated using the following formula. Biofilm removal rate (%) = {1-(absorbance of the substance to be evaluated / absorbance of the control)} x 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 effect Removal rate: 30% to 70%: Effective Removal rate: 10% to 30%: Weak removal effect Removal rate less than 10%: No removal effect A biofilm removal rate of 30% or more is considered to be at a practical level.

[0039] The product of the present invention may be in the form of the original product or may be in the form of a solution diluted with any medium, a dispersion, a solid such as a gel, granules, or tablets, but when applied to a biofilm, it is usually used in the form of an aqueous solution. The concentration of the diluted biofilm treatment agent is not particularly limited, but it is required to be at a concentration that provides the effect of the present invention when applied to biofilm-forming bacteria.

[0040] The product of the present invention may contain, within the scope of the present invention, gamma-lactones having 6 or 7 carbon atoms, delta-lactones having 8 carbon atoms, 4-pyrones having hydroxyl and alkyl groups in the molecule, alcohols, thickeners other than surfactants, viscosity adjusters, 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, within the scope of the present invention, the product may further contain substances shown in Patent Documents 1 and 2, specifically gamma-octanolactone, gamma-decanolactone, and delta-nonalactone. The content of these substances is preferably 0.1% or less.

[0041] (How to use biofilm treatment agent) The preferred conditions for using the product of the present invention will be described below. The use concentration of the product of the present invention is a concentration at which the use concentration of each component contained in the biofilm treatment agent is less than the MIC of the main causative bacterial species that constitute the biofilm. When used simultaneously against multiple causative bacterial species, the concentration used for the bacteria with the lowest MIC of the target bacterial species is applied. By using at a concentration less than the MIC, the death of biofilm-forming bacteria is suppressed and non-specific adsorption of the dead bacteria to the surface can be suppressed, which leads to the suppression of a hotbed for new biofilms. Although a biofilm treatment agent made into a single agent is preferable in terms of handling, each component may be prepared separately and mixed when contacting with biofilm-forming bacteria. In addition, a mixture of each component at a high concentration may be diluted 200 to 20,000 times or the like and used at a predetermined concentration.

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

[0043] The method of using the product of the present invention includes immersion, circulation, application, or water spraying. Furthermore, physical force such as a sponge, brush, or water flow may be applied. The time of application varies depending on the number of bacteria, the amount of attached biofilm, the concentration of the active ingredient, the application temperature, and the presence or absence of physical force, but is usually in the range of several minutes to several hours. After application, it is desirable to quickly rinse the removed biofilm with running water or the like. In addition, by contacting the product of the present invention with a member on which it is desired to inhibit the formation of a biofilm for several minutes to several hours in advance, the formation of a biofilm can be inhibited by the action of the essential ingredient. In addition, a paint containing the product of the present invention (for example, an acrylic silicone resin or an epoxy resin) can be applied, or the product of the present invention can be kneaded with plastic resins such as polyethylene and polypropylene to form a molded product having a biofilm formation inhibiting effect.

[0044] (biofilm-forming bacteria) The biofilm-forming bacteria to which the present invention is applied include any of the bacteria belonging to the phylum Proteobacteria, Bacteroidetes, and Bacillota that form biofilms.Among these, it is preferable to use the genus Methylobacterium, Ochrobacterium, Aeromonas, Klebsiella, Acinetobacter, Enterobacter, Citrobacter, Stenotrophumonas, Pseudomonas, Rhizobium, and Capriavidus, which belong to the phylum Proteobacteria, the genus Porphyromonas, which belong to the phylum Bacteroidetes, and the genus Staphylococcus, which belong to the phylum Bacilloidetes.In most cases, biofilms are formed by two or more types of bacteria, and the present invention targets biofilms containing one or more types of bacteria belonging to each of the above genera.

[0045] The product 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 cosmetic manufacturing plants or food and beverage manufacturing plants, tanks for aqueous chemicals such as cutting oil, drains and drainage pipes in kitchens, bathrooms, toilets, and kitchens. It can also be applied to cooling water systems such as industrial cooling towers, water treatment membranes, desalination devices, and circulating water systems in paper factories. It can also be applied to medical devices where biofilms are easily formed, such as cleaning agents for endoscopes, catheters, and artificial dialysis machines, and oral hygiene products such as mouthwashes. EXAMPLES

[0046] The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. In the examples, Pseudomonas bacteria (gram-negative bacteria), which are known as representative bacteria that form biofilms, were used as test bacteria to determine the MIC.

[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 a concentration lower than the MIC previously determined above. The test method for the MIC in this example is described below.

[0048] <Test method for MIC (minimum inhibitory concentration)> Using Pseudomonas spp. (Gram-negative bacteria), a representative strain of Proteobacteria known for biofilm formation, as the test bacterium, the MIC of a chemical substance (hereinafter referred to as the substance to be evaluated), which is a component of the biofilm treatment agent, was determined. (1) Test bacterium Pseudomonas aeruginosa (Deposit number: NBRC106052 strain) (2) Substance to be evaluated The chemical substances shown in Table 1 were used as the substances to be evaluated. (3) Test method The substance to be evaluated was 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 adjusted 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 defined 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. Regarding the substances shown in the examples, the following biofilm formation inhibition evaluation and biofilm removal evaluation were performed 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 evaluation target substance at a concentration (ppm) less than that shown in Table 1, according to the evaluation method defined in the present invention. Substances that were not used as evaluation targets in each Example are indicated with "-". The evaluation results of A1 to A3 and combinations thereof are shown in Table 2, the evaluation results of comparative examples of A1 to A3 in Table 3, and the evaluation results of combinations of A1 to A3, alcohol, and surfactant in Tables 4 and 5, respectively. Note that in Tables 2 to 4, the name of the lactone and its carbon number may be shown together.

[0051] [Table 2]

[0052] [Table 3]

[0053] [Table 4]

[0054] [Table 5]

[0055] It can be seen from Examples 1 to 7 in Table 2 that at least one of A1 to A3 of the present invention has a biofilm formation inhibitory effect and a biofilm removal effect. Furthermore, it can be seen from Examples 8 to 10 that a combination of A1 to A3 provides a more excellent effect.

[0056] Comparative examples are shown in Table 3. Comparative Examples 1 to 6 are comparative examples of Examples 1 to 4 in which A1 was used, and Comparative Examples 7 to 9 are comparative examples of Example 5 in which A2 was used, which are substances described in Patent Documents 1 and 2, respectively. It can be seen that Comparative Examples 1 to 9 have no or weak biofilm formation inhibitory effect and biofilm removal effect at the more preferred concentrations described in the literature. Comparative Examples 10 and 11 are comparative examples of Examples 6 to 7 in which A3 was used, and it can be seen that they have no or weak biofilm formation inhibitory effect and biofilm removal effect at concentrations below the MIC.

[0057] From Table 4, it can be seen that the biofilm formation inhibitory effect and biofilm removal effect are superior when A1 to A3 are combined with an alcohol or a surfactant, compared to A1 to A3 alone or any combination (for example, comparison between Examples 11 and 12 and Example 8). It can also be seen that the biofilm formation inhibitory effect and biofilm removal effect are superior when A1 to A3 are combined with an alcohol and a surfactant, compared to A1 to A3 alone or any combination (for example, comparison between Example 16 and Examples 14 and 15). It can also be seen that the same excellent effect is exhibited when at least one of A1 to A3, an alcohol, or a surfactant is combined with any of the substances of Comparative Examples 1, 2, and 7 (Examples 25 to 27).

[0058] From Table 5, it can be seen that any of the combinations A1 to A3 combined with an alcohol and a surfactant exhibits excellent biofilm formation inhibitory effects and biofilm removal effects against biofilms of bacteria belonging to each genus. In addition, for the substances shown in the examples, Methylobacterium aquaticum, Porphyromonas gingivalis, and Staphylococcus epidermidis, the proliferation of each bacterium was confirmed by turbidity at the concentrations added in the examples, and it is clear that the concentrations are below the MIC. [Industrial Applicability]

[0059] According to the present invention, by using as essential components at least one of a γ-lactone having 6 or 7 carbon atoms, a δ-lactone having 8 carbon atoms, and a 4-pyrones having a hydroxyl group and an alkyl group in the molecule, it is possible to provide a biofilm treatment agent that is effective in inhibiting biofilm formation and removing biofilms. In particular, the present invention can provide a treatment agent that is effective against biofilms that form on medical equipment, equipment in cosmetics manufacturing plants or food and beverage manufacturing plants, kitchens, bathrooms, toilet bowls, kitchen drains, drainage pipes, freshwater membranes, cooling towers, and tanks for diluting aqueous chemicals such as cutting oil. Furthermore, since the treatment agent of the present invention has a biofilm formation inhibitory effect and a biofilm removal effect at a neutral pH, it has little effect on corrosion of the human body and equipment used.

Claims

1. A biofilm treatment agent characterized by comprising, as essential components, a combination of two or more groups selected from the following groups A1 to A3. A1: γ-lactone of the following general formula (1) A2: δ-lactone having 8 carbon atoms A3: 4-pyrones having a hydroxyl group and an alkyl group in the molecule General formula (1) R1: a linear alkyl group having 2 or 3 carbon atoms

2. The biofilm treatment agent according to claim 1, characterized in that it contains A3, and the alkyl group of A3 is a methyl group or an ethyl group.

3. A biofilm treatment agent characterized by having as essential components a combination of two or more groups selected from the group A1 represented by the following general formula (1), the group A2 represented by the following general formula (2), and the group A3 represented by the following general formula (3). General formula (1) R1: a linear alkyl group having 2 or 3 carbon atoms General formula (2) R2: a linear alkyl group having 3 carbon atoms General formula (3) R3 to R6 each represent a hydrogen atom, a hydroxyl group, a methyl group, or an ethyl group, and each has one hydroxyl group, one methyl group, or one ethyl group, and the rest are hydrogen atoms.

4. The biofilm treatment agent according to any one of claims 1 to 3, characterized in that it contains an alcohol and / or a surfactant, and 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 has been substituted with a hydroxyl group General formula (5) R8: a linear alkyl group having 1 to 3 carbon atoms in which any one hydrogen atom has been substituted with a hydroxyl group

5. The biofilm treatment agent according to claim 4, wherein the surfactant is a microbial surfactant or a synthetic surfactant.

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

7. The biofilm treatment agent according to claim 6, which is used against at least one species of bacteria belonging to the phylum Proteobacteria, Bacteroidetes, or Bacillota.

8. The biofilm treatment agent according to claim 6, 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.

9. A method for treating a biofilm using the biofilm treatment agent according to claim 6, characterized in that the agent 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.

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