WO2016068230A1

WO2016068230A1 - Biofilm formation inhibiting agent and/or removing agent, and biofilm formation inhibiting method and/or removing method

Info

Publication number: WO2016068230A1
Application number: PCT/JP2015/080536
Authority: WO
Inventors: 五十嵐　亮二; 久保　武
Original assignee: 星光Pmc株式会社
Priority date: 2014-10-31
Filing date: 2015-10-29
Publication date: 2016-05-06
Also published as: CN107072201A; JPWO2016068230A1; JP6066157B2; CN107072201B

Abstract

The objective of the present invention is to provide a highly safe agent that has excellent biofilm formation inhibiting capability and/or formed biofilm removing capability, and a biofilm formation inhibiting method and/or a biofilm removing method using the agent. Provided are a biofilm formation inhibiting agent and/or removing agent containing, as an active ingredient, at least one selected from anthranilic acid or an anthranilic acid derivative represented by general formula (1), or a salt of those, and a biofilm formation inhibiting method and/or removing method using the biofilm formation inhibiting agent and/or removing agent. (In the formula, each of R₁-R₄ independently represents the following. R₁: C(=O)OH (carboxyl group), C(=O)NH₂, C(=O)OC_nH_2n+1 (alkyl ester: n=1-4). R₂, R₃: halogen atom, nitro group, hydrogen atom. R₄: hydrogen atom, CH₃C(=O) (acetyl group), CH₃C(=O)CH₂C(=O) (acetoacetyl group).)

Description

Biofilm formation inhibitor and / or removal agent, and biofilm formation inhibition method and / or removal method

The present invention relates to a biofilm formation inhibitor and / or removal agent, and a biofilm formation inhibition method and / or removal method. More specifically, the present invention relates to a biofilm formation inhibitor and / or removal agent containing anthranilic acid or a specific anthranilic acid derivative or a salt thereof as an active ingredient, and a biofilm formation inhibition method and / or removal method.

Biofilm is also called a fungal membrane and refers to a structure formed by bacteria. The biofilm is formed as follows. First, bacteria attached to the substrate secrete extracellular polysaccharides and proteins. These act as barriers and transport routes to protect internal bacteria from environmental changes and chemicals. Bacteria are thought to gradually form a biofilm on the surface of the substrate while repeatedly attaching to and detaching from the substrate.
By the way, the formation of biofilms in the manufacturing process of various products, heat exchangers, etc. is not desirable because it causes deterioration of product quality and productivity, and in some cases may cause health damage.
In order to suppress the formation of such a biofilm or to remove the biofilm once formed, a bactericidal agent has been conventionally used.
In order to suppress the formation of a biofilm with a bactericidal agent, it is desirable to kill or suppress the growth of floating bacteria before the bacteria form a biofilm. This is because bactericides are effective for sterilization of floating bacteria, but the bacteria that once formed a biofilm are due to the structure of the biofilm covered with extracellular polysaccharides. This is because there is a case where high resistance is exhibited.
Therefore, in order to suppress biofilm formation using a bactericidal agent, a high concentration (for example, a minimum bactericidal concentration (MBC) or a minimum growth inhibitory concentration (MIC) or higher) is required for bacteria to be controlled in a floating state. It is necessary to use in. Moreover, it is necessary to use a disinfectant at a higher concentration for the biofilm once formed.
A high-concentration disinfectant may cause a harmful effect on the human body, or may cause deterioration or corrosion of a member that is a target for formation and control of a biofilm such as a water-forming film. Moreover, there is also a risk that by using the bactericidal agent for a long time, biofilm-forming bacteria resistant to the bactericidal agent are selectively left and concentrated in the system.
In addition, as a feature of the bactericidal agent, when it is less than the MIC, since it does not have a killing action and a growth inhibiting action of floating bacteria, it does not show a biofilm formation inhibiting action or a biofilm removing action.
Therefore, biofilm formation suppression and biofilm removal by a bactericidal agent are not necessarily effective.
From the above, it is possible to suppress the formation of biofilm by sterilizing floating bacteria with a bactericidal agent, or to remove the biofilm by sterilizing the bacteria in the biofilm once formed. There is a demand for a drug that can suppress the formation of a biofilm and remove the biofilm that has been formed.
As techniques for controlling conventional biofilms, techniques shown in Patent Documents 1 to 3 are known.
Patent Document 1 describes a growth inhibitor against bacteria that form a biofilm containing a benzoic acid derivative as an active ingredient. This document only describes the growth-inhibiting activity of the floating bacteria themselves for the benzoic acid derivative, and does not describe the biofilm formation-inhibiting action or the formed biofilm-removing action.
Patent Document 2 describes an antibacterial agent against Legionella which contains anthranilic acid or its derivative as an active ingredient, and Patent Document 3 describes an antibacterial agent containing an aromatic carboxylic acid such as benzoic acid as an active ingredient. Yes.
These references only describe the bactericidal action, bacteriostatic action, and growth-inhibiting action against floating bacteria themselves, but also the biofilm formation-inhibiting action and the formed biofilm removing action. There is no description.

Japanese Patent Laid-Open No. 9-67211 JP 2012-246228 A Special table 2007-512356 gazette

The present invention provides a drug having high safety and excellent biofilm formation inhibiting ability and / or excellent ability to remove formed biofilm, and biofilm formation inhibiting method and / or biofilm removing method using the same. The issue is to provide

In order to solve the above-mentioned problems, the present inventor has conducted intensive research on the possibility of suppressing and / or removing each biofilm formed by many different types of bacteria for various types of chemical substances.
As a result, surprisingly, it has been found that anthranilic acid or a specific anthranilic acid derivative, or a salt thereof has a biofilm formation-inhibiting action and / or removal action, and the present invention has been completed.
That is, the present invention has the following configuration.
[1] A biofilm formation inhibitor and / or removal agent containing, as an active ingredient, one or more selected from anthranilic acid or anthranilic acid derivatives represented by the following general formula (1) or salts thereof.

(Wherein R ₁ to R ₄ each independently represents the following).
R ₁ : C (═O) OH (carboxyl group), C (═O) NH ₂ , C (═O) OC _n H _{2n + 1} (alkyl ester: n = 1 to 4)
R ₂ , R ₃ : halogen atom, nitro group, hydrogen atom R ₄ : hydrogen atom, CH ₃ C (═O) (acetyl group), CH ₃ C (═O) CH ₂ C (═O) (acetoacetyl group ))
[2] The biofilm formation inhibitor according to [1], wherein the active ingredient is used at a concentration that does not substantially exhibit a growth inhibitory action against biofilm-forming bacteria (less than the minimum growth inhibitory concentration (MIC)) / Or remover.
[3] The anthranilic acid derivative is methyl anthranilate, ethyl anthranilate, propyl anthranilate, butyl anthranilate, 4-chloroanthranilic acid, 6-chloroanthranilic acid, 4-fluoroanthranilic acid, 6-fluoroanthranilic acid, 4 Selected from the group consisting of -bromoanthranilic acid, 6-bromoanthranilic acid, N-acetylanthranilic acid, N-acetoacetylanthranilic acid, anthranilamido, 4-nitroanthranilic acid, 6-nitroanthranilic acid and combinations thereof [ The biofilm formation inhibitor and / or removal agent according to [1] or [2].
[4] The biofilm formation inhibitor and / or removal agent according to any one of [1] to [3], wherein the biofilm-forming bacteria are bacteria that appear in a papermaking process, a separation membrane, or a cooling water process. .
[5] The biofilm-forming bacterium is a genus Microbacterium belonging to Actinobacteria, Deinococcus belonging to Deinococcus, Ocrobacterium belonging to Proteobacteria, Acidvolax, Eromonas, Klebsiella, Acinetobacter, The biofilm according to any one of [1] to [4], wherein the biofilm is one or more selected from bacteria belonging to the genus Enterobacter, Citrobacter, Stenotropmonus, Pseudomonas, Rhizobium, Capriavidas Formation inhibitor and / or removal agent.
[6] A method for inhibiting formation and / or removal of a biofilm, the method comprising the step of contacting the following biofilm formation inhibitor and / or remover with a biofilm-forming bacterium.
A biofilm formation inhibitor and / or removal agent;
The biofilm formation inhibitor and / or removal agent which contain as an active ingredient 1 or more types chosen from the anthranilic acid or anthranilic acid derivative shown by following General formula (1), or these salts.

(Wherein R ₁ to R ₄ each independently represents the following).
R ₁ : C (═O) OH (carboxyl group), C (═O) NH ₂ , C (═O) OC _n H _{2n + 1} (alkyl ester: n = 1 to 4)
R ₂ , R ₃ : halogen atom, nitro group, hydrogen atom R ₄ : hydrogen atom, CH ₃ C (═O) (acetyl group), CH ₃ C (═O) CH ₂ C (═O) (acetoacetyl group ))
[7] The step is a step of bringing the active ingredient into contact with the biofilm-forming bacterium at a concentration (less than the minimum growth inhibitory concentration (MIC)) that does not substantially inhibit the growth of the biofilm-forming bacterium. The biofilm formation suppression method and / or removal method according to [6].
[8] The anthranilic acid derivative is methyl anthranilate, ethyl anthranilate, propyl anthranilate, butyl anthranilate, 4-chloroanthranilic acid, 6-chloroanthranilic acid, 4-fluoroanthranilic acid, 6-fluoroanthranilic acid, 4 Selected from the group consisting of -bromoanthranilic acid, 6-bromoanthranilic acid, N-acetylanthranilic acid, N-acetoacetylanthranilic acid, anthranilamido, 4-nitroanthranilic acid, 6-nitroanthranilic acid and combinations thereof [ The biofilm formation inhibitor method and / or removal method according to [6] or [7].
[9] The biofilm formation inhibiting method and / or the removing method according to any one of [6] to [8], wherein the biofilm-forming bacteria are bacteria that appear in a papermaking process, a separation membrane, or a cooling water process. .
[10] A microfilm genus belonging to the Actinobacterium genus, a Deinococcus genus belonging to the Deinococcus genus, an Ocrobacterium belonging to the Proteobacteria genus, an Acidvolax genus, an Aeromonas genus, a Klebsiella genus, an Acinetobacter genus, The biofilm according to any one of [6] to [9], wherein the biofilm is one or more selected from bacteria belonging to the genus Enterobacter, Citrobacter, Stenotropmonus, Pseudomonas, Rhizobium, Capriavidas Formation inhibition method and / or removal method.

According to the present invention, the formation of a biofilm can be efficiently suppressed and the formed biofilm can be removed by a drug containing anthranilic acid or an anthranilic acid derivative or a salt thereof as an active ingredient. Since these active ingredients have a very low sterilizing ability compared to conventional sterilizing agents, they are highly safe for human bodies and the environment. Moreover, since these active ingredients are not highly reactive substances such as oxidants, there is an advantage that they are less likely to cause deterioration of members to be applied and are easy to handle.

(Biofilm formation inhibitor and / or removal agent)
The biofil formation inhibitor and / or removal agent of the present invention is a drug containing, as an active ingredient, one or more selected from anthranilic acid or anthranilic acid derivatives represented by the following general formula (1) or salts thereof.

(In the structural formula, R ₁ to R ₄ each independently represent the following.
R ₁ : C (═O) OH (carboxyl group), C (═O) NH ₂ , C (═O) OC _n H _{2n + 1} (alkyl ester: n = 1 to 4)
R ₂ , R ₃ : halogen atom, nitro group, hydrogen atom R ₄ : hydrogen atom, CH ₃ C (═O) (acetyl group), CH ₃ C (═O) CH ₂ C (═O) (acetoacetyl group ))

R ₂ (hydrogen at the 4-position of the benzene ring) is a halogen atom, a nitro group or a hydrogen atom, and the halogen atom is preferably chlorine, fluorine or bromine, more preferably chlorine.
R ₃ (hydrogen at the 6-position of the benzene ring) is a halogen atom, a nitro group or a hydrogen atom, and the halogen atom is preferably chlorine, fluorine or bromine, more preferably chlorine.
Specific examples of the anthranilic acid derivatives of the present invention include methyl anthranilate, ethyl anthranilate, propyl anthranilate, butyl anthranilate, 4-chloroanthranilic acid, 6-chloroanthranilic acid, 4-fluoroanthranilic acid, 6-fluoro Anthranilic acid, 4-bromoanthranilic acid, 6-bromoanthranilic acid, N-acetylanthranilic acid, N-acetoacetylanthranilic acid, anthranilamido, 4-nitroanthranilic acid, 6-nitroanthranilic acid and the like can be mentioned.

In the present invention, the biofilm formation inhibitor refers to an agent having at least a biofilm formation inhibitory action, and the biofilm remover refers to an agent having at least a biofilm removal action. Therefore, these may have different actions, and cases where both of these actions are included are also included.
In particular, an agent having both actions may be expressed as a biofil formation inhibitor and a removal agent, but when referring to one functional agent (for example, a biofilm formation inhibitor), the other functional agent (biofilm removal agent). ) Is not intended to be excluded.
Moreover, in this specification, the biofilm formation inhibitor and removal agent, the agent having the biofilm formation inhibitory action and the removal action, the biofil formation inhibitor and the remover shall be used synonymously.
The biofilm formation inhibitory action of the present invention refers to an action that inhibits the formation of biofilm by bacteria. As a method for confirming the presence or absence of the action, for example, the amount of biofilm formed by culturing bacteria for a certain period of time in a medium containing a test target substance and the culture of the bacteria in a medium not containing the test target substance There is a method of comparing the amount of biofilm formed (control). In this case, when the amount of biofilm formation is smaller than the control, it can be determined that the test target substance has a biofilm formation inhibitory action.
The biofilm removing action of the present invention means an action of removing a biofilm formed by bacteria. As a method for confirming the presence or absence of the action, for example, a biofilm formed by culturing bacteria is brought into contact with the test target substance after the test target substance is brought into contact with the test target substance for a certain period of time. For example, there is a method of comparing the amount of biofilm formation (control) after a certain period of time. In this case, when the amount of biofilm formed is smaller than the control, it can be determined that the target substance has a biofilm removing action.
The larger biofilm formation inhibitory action is desirable, and the biofilm inhibition rate calculated in the test examples described later is preferably 20% or more. Other preferable suppression rates include 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more.
The larger the biofilm removal action is desirable, and the biofilm removal rate calculated in the test examples described later is preferably 20% or more. Other preferable removal rates include 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more.
As described above, the biofilm formation inhibitor of the present invention is required to have a biofilm formation inhibitory action, but it is desirable that the biofilm formation inhibitor further has other actions depending on the purpose of use. It is more desirable to have
In addition to the biofilm formation inhibitory action, if an agent having a biofilm removal action, that is, a biofilm formation inhibitor and a removal agent is administered in an environment where a biofilm has already been formed, the biofilm is removed, And since formation of a biofilm is suppressed after that and the environment where a biofilm does not exist constantly can be maintained, it is very effective.

(Salt of anthranilic acid or anthranilic acid derivative)
The anthranilic acid salt or anthranilic acid derivative salt of the present invention is a pharmaceutically acceptable salt and is not particularly limited as long as it has a biofilm formation-inhibiting action and / or a biofilm removing action. Examples include addition salts and base addition salts. Examples of acid addition salts include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, organic acids such as acetic acid, malic acid, succinic acid, tartaric acid and citric acid, and salts with chlorine. Base addition salts include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, and salts with amines such as ammonium and triethylamine.

(Usable pH range)
The biofilm formation inhibitor and / or removal agent of the present invention can be appropriately set according to the effect by those skilled in the art depending on the type of each agent, but may be used in the range of pH 5.0 to pH 9.0. preferable. In the present specification, “˜” is used to mean including left and right numerical values. Other preferable ranges include a region greater than pH 5.0 and less than pH 9.0, a region greater than pH 5.0 and less than pH 9.0, pH 5.5 to pH 9.0, pH 5.5 to pH 8.5, pH 5.5. To 8.0.

(Biofilm-forming bacteria)
The biofilm-forming bacteria to which the biofilm formation inhibitor and / or removal agent of the present invention is applied include any bacteria that form biofilms. Among these, the genus Microbacteria belonging to Actinobacteria, Deinococcus belonging to Deinococcus, Ocrobacterium belonging to Proteobacteria, Acidvolax, Eromonas, Klebsiella, Acinetobacter, Enterobacter, Citro Bacteria belonging to the genus Bacter, Stenotrophumonas, Pseudomonas, Rhizobium, Capriavidas are preferred. The formation of biofilm is almost always performed with two or more kinds of bacteria, and a biofilm containing one or more kinds of bacteria belonging to each of the above genera is desirable as an object of the present invention.

(Minimum growth inhibitory concentration (MIC))
The minimum growth inhibitory concentration (MIC) referred to in the present invention refers to the minimum concentration (bacteriostatic and antiseptic effect) at which antibiotics and bactericides suppress the growth of microorganisms. Therefore, the concentration lower than the MIC can be regarded as synonymous with a concentration that does not substantially exhibit a growth inhibitory action against biofilm-forming bacteria.
The MIC can be obtained by methods well known to those skilled in the art. For example, Saitama Industrial Technology Center Research Report Vol. 8 (2010), “Development of Simple Evaluation Method for Antibacterial Agents”, CHEMOTHERAPY, JAN. 1990, p102-105, antibacterial product technology council, test method “(3) Minimum bactericidal concentration measurement method I (2012 version)” and the like.
In this application, the MIC of each biofilm-forming bacterium is determined in advance for each drug to be tested for biofilm formation inhibitory effect and / or biofilm removal effect, and each biofilm-forming bacterium grows at a concentration below MIC. A test was conducted to confirm the above.
Therefore, when the growth of each biofilm-forming bacterium can be confirmed and the biofilm formation inhibitory effect and / or removal effect can be confirmed at the concentration of each drug below MIC, the drug is the drug of the present invention. It can be confirmed that That is, the drug of the present invention does not sterilize the biofilm-forming bacteria themselves, or suppress or eliminate the formation of biofilm by suppressing the growth, but with a different mechanism of action. It is possible to confirm that the drug is capable of suppressing the formation of or forming a biofilm.

(Usage form)
The use amount of the biofilm formation inhibitor and / or removal agent of the present invention can be appropriately determined depending on the application and dosage form, but is usually used in the form of an aqueous solution in the situation where it acts on the biofilm. May be any concentration that exhibits a biofilm formation inhibitory action and / or a biofilm removal action as the active ingredient concentration at the time of contact with the biofilm-forming bacteria.
Specifically, these concentrations are preferably such that the biofilm removal action or biofilm formation inhibitory action is most effectively exerted, and the main causative species constituting the biofilm to be acted on are preferred. It is preferably less than the MIC. In practice, it is preferred to use less than the MIC of the preferred species by specifying the preferred species of the biofilm in advance. Among these, preferable ranges include 0.001 to 10% by weight, 0.01 to 5% by weight, 0.05 to 1% by weight, and the like.
The concentration at the time of contact with the biofilm-forming bacterium refers to the concentration of the active ingredient in the aqueous solution when added to the aqueous solution in which the biofilm is present.
Therefore, the active ingredient is adjusted at a high concentration as a biofilm formation inhibitor and / or removal agent, and is diluted by adding it to an aqueous solution containing a biofilm at the time of use, and enters the above concentration range. It may be a thing. Further, it may be once diluted to an appropriate concentration at the time of use, and finally added to an aqueous solution containing a biofilm so as to finally enter the above concentration range. Also, when removing the biofilm by impregnating it with a sponge or the like, or putting the film with the biofilm attached into a container and immersing it in the solution containing the biofilm formation inhibitor of the present invention and the removal agent to remove it. In such a case, the concentration of the active ingredient impregnated in the sponge or the concentration of the active ingredient in the container (after the addition of the active ingredient) may be in the above-mentioned concentration range.
Furthermore, since the active ingredient of the biofilm formation inhibitor and / or removal agent of the present invention has a property of sufficiently exhibiting biofilm formation inhibition and removal effects even if it is less than MIC, consideration for the environment, etc. It is desirable to use a concentration as low as possible, and a concentration below MIC is more desirable as the concentration at the time of contact with the biofilm-forming bacteria.
Partially inhibiting and killing fungal growth can lead to the emergence of more powerful biofilm-forming bacteria in the long term. Furthermore, the organic matter leaked from the killed fungus carcass promotes the growth of biofilm-forming bacteria, or the adherent and deposited fungus carcasses themselves are used as scaffolds for further biofilm formation on the target vessel wall surface. It may become. In addition, killing bacteria that form biofilms does not always destroy the biofilm structure that has already been formed. Not shown, may even promote biofilm formation as a result.
From these viewpoints, it is desirable that the concentration of the active ingredient used in the biofilm formation inhibitor and / or removal agent of the present invention is less than the MIC that does not affect the growth and viability of the biofilm-forming bacteria. It can be said.

The time for which the biofilm formation inhibitor and / or removal agent of the present invention is allowed to act varies depending on the amount of biofilm attached, the concentration of the active ingredient, the working temperature, and the presence or absence of physical force, but usually from several seconds. It is in the range of several hours.

As the dosage form of the biofilm formation inhibitor and / or removal agent of the present invention, a solution dissolved in a solvent such as water, ethanol, isopropanol, or a solid, gel, emulsion / dispersion, Powders, aerosols, etc. are listed, and can be selected as appropriate. Of course, the product form is adjusted to the working concentration, and it is possible to dilute and use it in a high-concentration product form. It is.

The biofilm formation inhibitor and / or removal agent of the present invention is a thickener, viscosity modifier, pH adjuster, solvent, fragrance, colorant, antioxidant, preservative within the range not impairing the object of the present invention. , Fluorescent agents, excipients, soil release agents, bleaching agents, bleach activators, powdering agents, granulating agents, coating agents and the like can be blended.

The biofilm formation inhibitor and / or removal agent of the present invention can be used in a wide range of fields where biofilms are formed and become problematic. For example, the present invention can be applied to drainage grooves and drain pipes of food production or beverage production plants, kitchens, kitchens, bathrooms, toilets, kitchens, and the like.
It can also be applied to cooling water systems such as industrial cooling towers, water treatment membranes, desalination equipment, and circulating water systems such as paper mills. In addition, the present invention can be applied to a cleaning agent such as an endoscope, a catheter, and an artificial dialysis machine that can easily form a biofilm. Furthermore, since it has high safety, it can be used for cleaning agents for the body, dentifrices, oral care agents, denture care agents, contact lens cleaners, and the like.
Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited thereto.

[Test Example 1] Examination of MIC (Minimum Growth Inhibitory Concentration) For various biofilm-forming bacteria, the MIC of the compound to be tested was determined.
(1) Test bacteria The biofilm-forming bacteria shown in Table 1 were used. These bacteria are bacteria collected and separated from various processes of actual paper mills, separation membranes, plaque, ocean, etc., and the strains in parentheses are the deposit numbers of the strains used to identify each strain. Indicates. Among these, Pseudomonas aeruginosa PAO1 NBRC106052 strain is a representative strain of biofilm-forming bacteria.
(2) Substances to be evaluated The compounds shown in Table 1 were used as substances to be evaluated. Among the compounds in Table 1, DBNPA (2,3-dibromopropionamide) is one type of organic fungicide and was used as a comparative example in the following tests.
The source of each reagent is as follows.
Anthranilic acid, methyl anthranilate: Wako Pure Chemical Industries, Ltd.
Anthranilamide, 4-chloroanthranilic acid, 4-fluoroanthranilic acid, 4-bromoanthranilic acid, 6-chloroanthranilic acid, N-acetylanthranilic acid, N-acetoacetylanthranilic acid, ethyl anthranilic acid: Tokyo Chemical Industry Co., Ltd.
(3) Test method The substance to be evaluated was diluted stepwise with a bouillon medium for sensitivity test to prepare a total of 10 mL dilution rows (1.1 times the target concentration). 20 μL of a bacterial solution prepared with 10 ⁸ cfu / mL of various test strains was added thereto, and cultured with shaking (2,000 rpm) at 37 ° C. for 24 hours in a 96-well microplate mixer. The lowest concentration in the dilution series that did not become cloudy visually was defined as MIC.
(4) Test results The results are shown in Table 1. In the table, “<numerical value” indicates that the MIC is smaller than the numerical value, and “> numerical value” indicates that the MIC is larger than the numerical value.
The following biofilm formation inhibition evaluation test and biofilm removal evaluation test were performed at a concentration at which the final concentration of the evaluation target substance was less than MIC.

[Test Example 2] Biofilm formation inhibition evaluation test
It was examined whether various compounds to be tested exhibited biofilm formation inhibitory action.
(1) Test bacteria Various strains shown in Table 2 were used.
(2) Substances to be evaluated Compounds having concentrations shown in Table 2 were used as substances to be evaluated. The said density | concentration shows the final density | concentration after the addition to a culture medium, and corresponds to the density | concentration less than MIC calculated | required in the said test example 1. FIG.
(3) Test method (i) Each test bacterium was prepared by using TSB (Triptic Soy Broth, Bacto: Difco Laboratories) medium with glucose at a final concentration of 1%, and adjusting the preculture solution under the condition of 120 rpm.
(Ii) Each preculture solution was diluted with each medium so that the concentration was 0.1% (v / v), and 150 μL was dispensed into a 96-well plate.
(Iii) The substance to be evaluated was added to the medium at each concentration, and the medium was adjusted to pH = 7.0 with hydrochloric acid or sodium hydroxide. The thing which does not contain an evaluation object substance was made into negative control (pH = 7.0).
(Iv) Various bacteria were cultured under the following conditions to form a biofilm.
(A) Ochrobacterium pseudointermedium, Klebsiella sp., Acinetobacter baumanii, Citrobacter freundii, and Pseudomonas aeruginosa were cultured at 37 ° C. and 600 rpm for 6 hours to form a biofilm.
(B) Cupriavidus sp., Pseudomonas plecoglossicida, and Microbacterium hominis were cultured at 37 ° C. and 600 rpm for 17 hours to form a biofilm.
(V) The culture solution in each hole was removed, and each well was rinsed twice with distilled water.
(Vi) Add 250 μL of crystal violet aqueous solution (0.4 w / v%, 20 w / v% methanol) to the biofilm adhering in each hole, let stand for 2 minutes, stain, then rinse three times with distilled water, The aqueous crystal violet solution not bound to the biofilm was removed.
(Vii) 250 μL of ethanol was added to each hole, allowed to stand for 1 hour, crystal violet was eluted from the stained biofilm, and the absorbance was measured at a wavelength of 595 nm.
(Viii) The negative control measurement value and each evaluation series were calculated using the average value of 5 holes as the measurement value, and the biofilm inhibition rate was calculated from the following formula. The calculated value was evaluated based on the following criteria.
Biofilm suppression rate (%) = {1− (measurement value for evaluation / measurement value for negative control)} × 100
<Criteria>
◎ High suppression effect (suppression rate of 40% or more)
○ Suppressive effect (suppression rate 20% or more and less than 40%)
× No inhibitory effect (suppression rate less than 20%)
(4) Test results The results are shown in Table 2.

[Test Example 3] Biofilm removal evaluation test
It was examined whether or not the biofilm formed on various compounds to be tested exhibited a removing action.
(1) Test bacteria Various strains shown in Table 3 were used.
(2) Substances to be evaluated Compounds having concentrations shown in Table 3 were used as substances to be evaluated. The said density | concentration shows the final density | concentration after the addition to a culture medium, and corresponds to the density | concentration less than MIC calculated | required in the said test example 1. FIG.
(3) Test method (i) Each test bacterium was prepared by using TSB (Triptic Soy Broth, Bacto: Difco Laboratories) medium with glucose at a final concentration of 1%, and adjusting the preculture solution under the condition of 120 rpm.
(Ii) Each preculture solution was diluted with each medium so that the concentration was 0.1% (v / v), and 150 μL was dispensed into a 96-well plate.
(Iii) Various bacteria were cultured under the following conditions to form a biofilm.
Ochrobacterium pseudointermedium, Cupriavidus sp., Klebsiella sp., Acinetobacter baumanii, Citrobacter freundii, Pseudomonas aeruginosa, Pseudomonas plecoglossicida, and Microbacterium hominis were cultured at 37 ° C. and 600 rpm for 17 hours to form a biofilm.
(Iv) The culture solution in each hole was removed, and each well was rinsed twice with distilled water.
(V) The substance to be evaluated was added to the medium at each concentration, and the medium pH was adjusted to 7.0 with hydrochloric acid or sodium hydroxide. A negative control was prepared by adding 200 μL of sterile medium (pH = 7.0) to each well.
(Vi) Each bacterium was shaken at 600 rpm for 3.5 hours at the same temperature as the pre-culture, and after contacting the medium containing the target substance with the biofilm, the medium in each well was removed and rinsed twice with distilled water. .
(Vii) Add 250 μL of crystal violet aqueous solution (0.4 w / v%, 20 w / v% methanol) to the biofilm adhering in each hole, let stand for 2 minutes, stain, then rinse three times with distilled water, The aqueous crystal violet solution not bound to the biofilm was removed.
(Viii) 250 μL of ethanol was added to each hole, allowed to stand for 1 hour, crystal violet was eluted from the stained biofilm, and the absorbance was measured at a wavelength of 595 nm.
(Vix) The negative control measurement value and each evaluation series were calculated using the average value of 5 holes as the measurement value, and the biofilm removal rate was calculated from the following formula. The calculated value was evaluated based on the following criteria.
Biofilm removal rate (%) = {1− (measurement value to be evaluated / negative control measurement value)} × 100
<Criteria>
◎ High removal effect (removal rate 40% or more)
○ Effective removal (removal rate 20% or more and less than 40%)
× No removal effect (removal rate less than 20%)
(4) Test results The results are shown in Table 3.

[Test Example 4] Difference due to pH (1) Test strain Pseudomonas aeruginosa PAO1 NBRC106052 strain was used.
(2) Substances to be evaluated Compounds having concentrations shown in Table 4 were used as substances to be evaluated. The said density | concentration shows the final density | concentration after the addition to a culture medium, and corresponds to the density | concentration less than MIC calculated | required in the said test example 1. FIG.
(3) Test Method The test method was the same as the test method of Test Example 2, except that the pH of the medium was adjusted to each pH shown in Table 4 in the procedure (iii) of Test Example 2 (3).
(4) Test results The results are shown in Table 4.

[Discussion]
The concentrations of the substances to be evaluated listed in the examples were all less than MIC, and substantially did not have the growth inhibitory action of the evaluated strain (Test Example 1). Therefore, it is considered that the biofilm formation inhibitory action and the removal action by the evaluation substances shown in the Examples are not due to the difference in the number of viable bacteria, but are caused by changes in the existence form of the bacteria.
Since DBNPA (2,3-dibromopropionamide), which is a kind of organic fungicide listed as a comparative example, did not show any biofilm formation inhibition or removal action at a concentration below MIC, It was shown that the mechanism of action of the substances listed in (1) is different from the bactericidal action.
For the substances listed in the examples, a wide range of taxonomic biofilm-forming species, namely α-proteobacteria, β-proteobacteria, γ-proteobacteria, actinobacteria, All showed the biofilm removal effect in addition to the biofilm formation inhibitory effect.
From Test Examples 2 and 3, anthranilic acid, derivatives partially substituted therefor, and salts thereof were effective. For example, it is effective that hydrogen at the 4-position of the benzene ring is substituted with chlorine, fluorine, or bromine, and the chlorine-substituted product at the 6-position also showed an effect. It is considered effective. In addition, since anthranilamides, methyl anthranilate, and ethyl anthranilate, which are substituents of the carboxyl group, were also effective, alkyl esters such as propyl anthranilate and butyl anthranilate are also considered effective.
In addition, N-acetylanthranilic acid and N-acetylanthranilic acid, which are substituted amino groups, were also effective.
Furthermore, from Test Example 4, the biofilm formation inhibitor and removal agent of the present invention were effective in a wide pH range including pH 5.5 to pH 8.0.

According to the present invention, an anthranilic acid or anthranilic acid derivative or a salt thereof is used as an active ingredient, thereby providing a biofilm formation inhibitor and / or removal agent effective against a wide range of taxonomic bacteria. be able to.
In particular, it is possible to provide an effective drug for a biofilm formed in a papermaking process of a paper mill, a water separation membrane, a water-contacting route such as a cooling tower.
In addition, since the drug of the present invention has a biofilm formation inhibitory effect and / or a removal effect even at a low concentration that does not inhibit the growth of the target biofilm-forming bacteria, it has an effect on the microbiota of the aqueous environment used. There is no need to consider it.

Claims

The biofilm formation inhibitor and / or removal agent which contain as an active ingredient 1 or more types chosen from the anthranilic acid or anthranilic acid derivative shown by following General formula (1), or these salts.

(Wherein R 1 to R 4 each independently represents the following).
R 1 : C (═O) OH (carboxyl group), C (═O) NH 2 , C (═O) OC n H 2n + 1 (alkyl ester: n = 1 to 4)
R 2 , R 3 : halogen atom, nitro group, hydrogen atom R 4 : hydrogen atom, CH 3 C (═O) (acetyl group), CH 3 C (═O) CH 2 C (═O) (acetoacetyl group ))
The biofilm formation inhibitor and / or removal according to claim 1, wherein the active ingredient is used at a concentration that does not substantially exhibit a growth inhibitory action against biofilm-forming bacteria (less than the minimum growth inhibitory concentration (MIC)). Agent.
The anthranilic acid derivative is methyl anthranilate, ethyl anthranilate, propyl anthranilate, butyl anthranilate, 4-chloroanthranilic acid, 6-chloroanthranilic acid, 4-fluoroanthranilic acid, 6-fluoroanthranilic acid, 4-bromoanthranilic acid. The selected from the group consisting of acids, 6-bromoanthranilic acid, N-acetylanthranilic acid, N-acetoacetylanthranilic acid, anthranilamido, 4-nitroanthranilic acid, 6-nitroanthranilic acid and combinations thereof 2. The biofilm formation inhibitor and / or removal agent according to 2.
The biofilm formation inhibitor and / or removal agent according to any one of claims 1 to 3, wherein the biofilm-forming bacteria are bacteria that appear in a papermaking process, a separation membrane, or a cooling water process.
Microfilm genus belonging to Actinobacterium gate, Deinococcus genus belonging to Deinococcus genus, Ocrobacterium belonging to Proteobacteria genus, Acidvolax genus, Aeromonas genus, Klebsiella genus, Acinetobacter genus, Enterobacter genus The biofilm formation inhibitor according to any one of claims 1 to 4, which is one or more selected from bacteria belonging to the genus Citrobacter, Stenotrophumonas, Pseudomonas, Rhizobium, and Capriavidas. Or remover.
A biofilm formation suppression method and / or removal method,
The said method including the process of making the following biofilm formation inhibitor and / or removal agent contact a biofilm-forming bacterium.
A biofilm formation inhibitor and / or removal agent;
The biofilm formation inhibitor and / or removal agent which contain as an active ingredient 1 or more types chosen from the anthranilic acid or anthranilic acid derivative shown by following General formula (1), or these salts.

(Wherein R 1 to R 4 each independently represents the following).
R 1 : C (═O) OH (carboxyl group), C (═O) NH 2 , C (═O) OC n H 2n + 1 (alkyl ester: n = 1 to 4)
R 2 , R 3 : halogen atom, nitro group, hydrogen atom R 4 : hydrogen atom, CH 3 C (═O) (acetyl group), CH 3 C (═O) CH 2 C (═O) (acetoacetyl group ))
The step is a step in which the active ingredient is brought into contact with the biofilm-forming bacterium at a concentration (less than the minimum growth inhibitory concentration (MIC)) that does not substantially inhibit the growth of the biofilm-forming bacterium. The biofilm formation suppression method and / or removal method described in 1.
The anthranilic acid derivative is methyl anthranilate, ethyl anthranilate, propyl anthranilate, butyl anthranilate, 4-chloroanthranilic acid, 6-chloroanthranilic acid, 4-fluoroanthranilic acid, 6-fluoroanthranilic acid, 4-bromoanthranilic acid. The selected from the group consisting of acids, 6-bromoanthranilic acid, N-acetylanthranilic acid, N-acetoacetylanthranilic acid, anthranilamido, 4-nitroanthranilic acid, 6-nitroanthranilic acid and combinations thereof 8. The biofilm formation inhibiting method and / or removing method according to 7.
The method for inhibiting and / or removing biofilm formation according to any one of claims 6 to 8, wherein the biofilm-forming bacteria are bacteria that appear in a papermaking process, a separation membrane, or a cooling water process.
Biofilm-forming bacteria are the genus Microbacteria belonging to Actinobacterium, Deinococcus belonging to Deinococcus, Ocrobacterium belonging to Proteobacteria, Acidvolax, Eromonas, Klebsiella, Acinetobacter, Enterobacter The method for inhibiting the formation of biofilm according to any one of claims 6 to 9, wherein the method is one or more selected from bacteria belonging to the genus Citrobacter, Stenotropmonus, Pseudomonas, Rhizobium, and Capriavidas. Or removal method.