Anti-biofilm composition

Abstract

To provide an anti-biofilm composition which exhibits superior performance in inhibiting biofilm formation even with a low concentration bactericidal component.SOLUTION: An anti-biofilm composition comprises: (A) isopropylmethylphenol; (B) polyoxyethylene hydrogenated castor oil having an average added molar number of ethylene oxide of 30 to 70 mol; (C) diol having 3 to 6 carbon atoms; and (D) water. A content of components (A) to (C) is set within the following ranges relative to the mass of the anti-biofilm composition. Component (A): 10 ppm or more and less than 50 ppm. Component (B): 15 ppm or more and less than 100 ppm. Component (C): 15 ppm or more and less than 100 ppm.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an anti-biofilm composition for suppressing the formation of biofilms.

Background Art

[0002] Biofilms are formed by microorganisms using substances they produce themselves (e.g., sticky exopolysaccharides (outer membranes), proteins, etc.). It is thought that microorganisms are protected from external factors by this strong biofilm formation and repeat their growth within it. Such biofilms are formed in various places such as in water, soil, food, medical devices, the surface of teeth, etc., and cause problems such as clogging of water pipes and piping, food spoilage, periodontal disease, infectious diseases, etc. Therefore, various studies have been made to suppress the formation of biofilms as well as to sterilize microorganisms.

[0003] For example, Patent Document 1 discloses an oral composition using 0.05 to 0.5% by mass of isopropylmethylphenol as a sterilizing component and 0.1 to 1% by mass of polyoxyethylene hydrogenated castor oil.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, since the one in Patent Document 1 aims to suppress the formation of biofilms and sterilize the microorganisms that penetrate into them and cause problems, it uses 0.05% by mass or more of isopropylmethylphenol as a sterilizing component. On the one hand, although isopropylmethylphenol itself is evaluated to be relatively safe, due to the increasing health consciousness in recent years, consumers tend to choose products with fewer bactericidal components.

[0006] In view of such circumstances, the present invention aims to provide an anti-biofilm composition that can exhibit excellent performance in suppressing the formation of biofilms even at a low concentration of bactericidal components.

Means for Solving the Problems

[0007] The inventors of the present invention recalled that if the adhesion of microorganisms floating in the surroundings to the substrate is suppressed, even if the concentration is below the minimum inhibitory concentration (MIC) of the bactericidal component, the formation of biofilms can be suppressed, and various problems caused by microorganisms can be solved. Thus, various studies were conducted. As a result, it was found that when (A) isopropylmethylphenol (hereinafter sometimes referred to as "IPMP"), (B) polyoxyethylene hydrogenated castor oil with an average addition mole number of ethylene oxide of 30 to 70 moles, and (C) a diol having 3 to 6 carbon atoms are each contained in a specific amount, the formation of biofilms can be suppressed by the synergistic effect of each component with a smaller amount of bactericidal components than before, and the present invention was thus achieved. Although the reason why the intended purpose can be achieved by the above configuration is not clear, it is considered as follows. That is, (B) polyoxyethylene hydrogenated castor oil having a surface-active effect suppresses the adhesion of bacteria by preferentially arranging on the substrate surface, and also suppresses the production of extracellular polysaccharides, which are important for biofilm formation, with a certain bactericidal effect even at an IPMP concentration considerably lower than the MIC. It is considered that the biofilm formation inhibitory effect is exhibited by these two synergistic effects.

[0008] In order to achieve the above object, the present invention has the following [1] to [2] as its gist. [1] An anti-biofilm composition containing (A) isopropyl methylphenol, (B) polyoxyethylene hydrogenated castor oil with an average addition mole number of ethylene oxide being 30 to 70 moles, (C) a diol having 3 to 6 carbon atoms, and (D) water, wherein the contents of components (A) to (C) are set within the following ranges with respect to the mass of the anti-biofilm composition. Component (A): 10 ppm or more and less than 50 ppm. Component (B): 15 ppm or more and less than 100 ppm. Component (C): 15 ppm or more and less than 100 ppm. [2] The anti-biofilm composition according to [1], further containing at least one selected from the group consisting of a low-molecular surfactant, a polymer surfactant, and an enzyme. [Advantages of the Invention]

[0009] From the above, the anti-biofilm composition of the present invention has a smaller amount of bactericidal components than conventional ones, yet can surely suppress the formation of biofilms. [Embodiments for Carrying out the Invention]

[0010] Next, embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described below.

[0011] In the present invention, when expressed as "X to Y" (X and Y are arbitrary numbers), unless otherwise specified, it means "X or more and Y or less", and also includes the meaning of "preferably greater than X" or "preferably less than Y". Also, when expressed as "X or more" (X is an arbitrary number) or "Y or less" (Y is an arbitrary number), it also includes the meaning of "preferably greater than X" or "preferably less than Y". Furthermore, when expressed as "x and / or y" (x and y are arbitrary configurations), it means at least one of x and y, and includes three cases: only x, only y, and x and y.

[0012] The anti-biofilm composition of the present invention (hereinafter sometimes referred to as "this composition") contains (A) isopropylmethylphenol, (B) polyoxyethylene hydrogenated castor oil with an average addition mole number of ethylene oxide of 30 to 70 moles, (C) a diol having 3 to 6 carbon atoms, and (D) water, and the contents of the components (A) to (C) are set to be within specific ranges with respect to the mass of this composition.

[0013] In the present invention, "biofilm" means the entire aggregate formed by extracellular viscous polysaccharides produced by microorganisms on a solid phase surface, and includes not only dental plaque (dental calculus) formed on the tooth surface in the oral cavity, but also aggregates of extracellular viscous polysaccharides formed on the surfaces of medical devices, water pipes, foods, etc. Therefore, "anti-biofilm" means inhibiting the formation of the above biofilm. Specifically, it means that after 24 hours, the biofilm formation rate is 50% or less compared to the non-added product. Here, since it is known that the formation rate of the above biofilm has a primary correlation with the adsorption amount of crystal violet dye and the dry weight of the biofilm, for example, the biofilm is stained with crystal violet or the like, the stained biofilm is dispersed in a liquid such as ethanol, and the absorbance of the liquid is measured and compared with the non-added product to calculate it.

[0014] Details of each component in this composition will be described below.

[0015] 《Isopropylmethylphenol: Component (A)》 Isopropylmethylphenol (IPMP) used as the component (A) of this composition is a bactericidal component effective against Gram-positive bacteria, Gram-negative bacteria, Mycobacterium tuberculosis, etc. For example, commercially available products manufactured by Osaka Chemical Co., Ltd. or the like can be used.

[0016] The content of the above component (A) is set to be 10 ppm or more and less than 50 ppm with respect to the whole composition, preferably set in the range of 15 to 45 ppm, and more preferably set in the range of 20 to 40 ppm. If it is less than the above range, the adhesion inhibition of bacteria to the substrate and the synergistic effect of bactericidal property tend not to be exhibited. If it is more than the above range, the stability of the formulation such as crystal precipitation of isopropylmethylphenol is impaired, and the safety aspect and cost aspect tend to be disadvantageous, and the synergism of each component tends to be impaired.

[0017] 《Polyoxyethylene hydrogenated castor oil with an average added mole number of ethylene oxide of 30 to 70 moles: Component (B)》 The polyoxyethylene hydrogenated castor oil used as component (B) of this composition has an average added mole number of ethylene oxide of 30 to 70 moles. That is, when the average added mole number of ethylene oxide is less than 30 moles, the effect of suppressing the formation of biofilm may decrease. Therefore, this average added mole number is 30 moles or more, more preferably 40 moles or more, and even more preferably 50 moles or more. Also, when the added mole number of ethylene oxide exceeds 70 moles, this composition tends to thicken or solidify, and the handling tends to deteriorate.

[0018] As the above component (B), from the viewpoint of improving the stability over time at low temperatures, it is preferable to use a purified polyoxyethylene hydrogenated castor oil. Here, the purification treatment means reducing the amount of low molecular weight components such as unreacted substances by methods such as distillation, adsorbent treatment, and solvent washing. The purified polyoxyethylene hydrogenated castor oil is obtained by preparing polyoxyethylene hydrogenated castor oil to 2% by mass using a 50% by mass ethanol aqueous solution and storing it at 5°C for 12 hours or more, and then the appearance becomes transparent. Therefore, by using the purified polyoxyethylene hydrogenated castor oil, the stability over time of this composition can be improved, and the anti-biofilm property can be further enhanced. These can be used alone or in combination of two or more. In addition, the HLB value (Hydrophile-lipophile balance value), which is an index of the hydrophilic-lipophilic balance, is preferably set in the range of 6 to 20, more preferably in the range of 8 to 18. If it is less than the above range, the solubility in water tends to be low and formulation becomes difficult. If it is more than the above range, there is a tendency such as crystallization of isopropylmethylphenol.

[0019] The content of the above component (B) is set to be 15 ppm or more and less than 100 ppm with respect to the whole composition, preferably set in the range of 20 to 90, more preferably set in the range of 25 to 80. If it is less than the above range, the suppression of adhesion of bacteria to the substrate tends to be insufficient. If it is more than the above range, the solubility tends to deteriorate.

[0020] 《Diol having 3 to 6 carbon atoms: Component (C)》 The diol used as the component (C) of the present composition has 3 to 6 carbon atoms and is a compound having a structure in which hydroxyl groups are substituted one by one on two carbon atoms of a chain aliphatic hydrocarbon. When the number of carbon atoms is less than 3 or exceeds 6, it is not preferable in terms of solubilization. Specific examples of the above component (C) include 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, 1,2-hexanediol, 1,6-hexanediol, isoprene glycol, and the like. Among them, 1,2-propanediol, 1,3-butanediol, and isoprene glycol are preferable, and 1,3-butanediol and isoprene glycol are particularly preferable from the viewpoint of the transparency of the diluted aqueous solution. These can be used alone or in combination of two or more.

[0021] The content of the above component (C) is set to be 15 ppm or more and less than 100 ppm with respect to the whole composition, preferably set in the range of 20 to 90, and more preferably set in the range of 25 to 80. If it is less than the above range, each component tends to be difficult to dissolve, and if it is more than the above range, the synergistic effect tends to decrease due to the lower concentration.

[0022] The content ratio (B / A) of the component (B) to the component (A) is preferably set in the range of 8 / 1 to 0.5 / 1, and more preferably set in the range of 6 / 1 to 1 / 1, from the viewpoint of effectively expressing the synergistic effect of suppressing the adhesion of bacteria to the base material and bactericidal property.

[0023] The content ratio (C / A) of the component (C) to the component (A) is preferably set in the range of 8 / 1 to 0.5 / 1, and more preferably set in the range of 6 / 1 to 1 / 1, from the viewpoint of dissolving the component (A) and stabilizing the preparation.

[0024] The content ratio (C / B) of the component (C) to the component (B) is preferably set in the range of 3 / 1 to 0.2 / 1, and more preferably set in the range of 2 / 1 to 0.5 / 1, from the viewpoint of dissolving the component (B) and stabilizing the preparation.

[0025] 《Water: Component (D)》 Examples of the water as the component (D) include pure water, ion-exchanged water, soft water, hard water, distilled water, tap water, physiological saline, etc. These can be used alone or in combination of two or more. Among them, pure water, ion-exchanged water, distilled water, and physiological saline are preferably used in terms of being able to remove contamination of miscellaneous bacteria and impurities.

[0026] 《Low-molecular surfactant: Optional component (E)》 This composition is prepared using the above components (A) to (D) as essential components. Furthermore, as an optional component (E), low-molecular-weight surfactants such as sodium lauryl sulfate, sodium α-olefin sulfonate, lauryldimethylaminoacetic acid, sodium coconut oil fatty acid methyl taurine solution, lauryldimethylaminoacetic acid solution, coconut oil fatty acid amidopropyl betaine, cetylpyridinium chloride, etc. can also be used. These can be used alone or in combination of two or more kinds. When using the above component (E), the content is preferably set in the range of 15 to 45 ppm, more preferably in the range of 20 to 40 ppm, based on the whole composition. If it is less than the above range, the contribution to bactericidal property and / or suppression of bacterial adhesion tends to be low. If it is more than the above range, the synergistic effect of components (A) and (B) tends to be suppressed.

[0027] 《Polymer Surfactant: Optional Component (F)》 This composition can further use, as an optional component (F), polymer surfactants such as polyethyleneimine, polyphenol gallate, polyvinyl alcohol, polyoxyethylene sorbitan stearate, polyoxyethylene lauryl ether, polyethylene glycol, sodium polyacrylate, polyvinylpyrrolidone, polylysine, polyglutamate, etc. These can be used alone or in combination of two or more kinds. When using the above component (F), the content is preferably set in the range of 15 to 45 ppm, more preferably in the range of 20 to 40 ppm, based on the whole composition. If it is less than the above range, the contribution to bactericidal property and / or suppression of bacterial adhesion tends to be low. If it is more than the above range, the synergistic effect of components (A) and (B) tends to be suppressed.

[0028] 《Enzyme: Optional Component (G)》 The composition may further use, as an optional (G) component, enzymes such as dextranase, amylase, protease, tranexamic acid, lauroyl sarcosinate, etc. These can be used alone or in combination of two or more. When using the above (G) component, the content is preferably set in the range of 15 to 45 ppm, more preferably in the range of 20 to 40 ppm, based on the whole composition. If it is less than the above range, the contribution to bactericidal property and / or suppression of bacterial adhesion tends to be low. If it is more than the above range, the synergistic effect of components (A) and (B) tends to be suppressed.

[0029] 《Acid: Optional (H) Component》 The composition may further use, as an optional (H) component, acids such as organic acids, and among them, ascorbic acid, ethylenediaminetetraacetic acid, citric acid, malic acid, succinic acid, etc. can be preferably used. These can be used alone or in combination of two or more. When using the above (H) component, the content is preferably set in the range of 15 to 45 ppm, more preferably in the range of 20 to 40 ppm, based on the whole composition. If it is less than the above range, the contribution to bactericidal property and / or suppression of bacterial adhesion tends to be low. If it is more than the above range, the synergistic effect of components (A) and (B) tends to be suppressed.

[0030] 《Other Components》 In addition to the essential components (A) to (D) and the optional components (E) to (H) described above, the composition may further appropriately contain various optional components. Examples of such optional components include oily components, pH adjusters, water-soluble pigments, etc. These can be used alone or in combination of two or more. The content of such optional components can be contained within a range that does not inhibit the effects of the present invention. For example, it is preferably 1000 ppm or less, more preferably 800 ppm or less, and still more preferably 400 ppm or less of the composition, and it may not be contained.

[0031] However, from the gist of the present invention, the content of additive components other than the above components (A) to (H), such as sugar alcohols, vitamins, fatty acid esters, fragrances, wetting agents, etc., is preferably 1000 ppm or less of the present composition, more preferably 800 ppm or less, still more preferably 400 ppm, even more preferably 200 ppm or less, and particularly preferably not contained at all.

[0032] 《Manufacture of the present composition》 The present composition can be manufactured, for example, as follows. That is, using the essential components (A) to (D) and these optional components contained as necessary, it can be prepared according to the method for preparing a normal antibacterial composition, anti-biofilm composition, etc. Alternatively, a precursor of the present composition can be prepared using components (A) to (C) and optional components if necessary, and the present composition can be prepared by adding water of component (D) thereto.

[0033] The present composition thus obtained can exhibit excellent performance of suppressing the formation of biofilms while having a low concentration of the bactericidal component because components A, B, and C are blended at specific ratios, respectively. Therefore, it can effectively suppress the growth of microorganisms and prevent clogging of water pipes and pipes, food spoilage, periodontal disease, infectious diseases, etc.

Examples

[0034] Next, the examples will be described together with the comparative examples. However, the present invention is not limited to these examples. In the examples, “%” means mass basis.

[0035] Component (A): IPMP Component (B): Polyoxyethylene hydrogenated castor oil with an average addition mole number of ethylene oxide of 60 Component (C): Isoprene glycol (D) Component: Ion-exchanged water (E) Component: Sodium lauryl sulfate (F) Components: Polyvinyl alcohol, polyethyleneimine (G) Component: Dextranase (H) Components: Ascorbic acid, ethylenediaminetetraacetic acid

[0036] [Examples 1 - 8, Comparative Examples 1 - 14] A TSB liquid medium with 0.25% sucrose added to tryptone soy broth (TSB) was prepared. The components (A) - (H) were formulated and mixed in the ratios shown in Tables 1 - 3 below in the above TSB liquid medium to obtain test solutions of the anti-biofilm compositions of the examples and comparative examples.

[0037] Using these test solutions, based on the following criteria, the "biofilm formation inhibitory effect" and the "bactericidal effect on planktonic bacteria" were evaluated. The results are shown together in Tables 1 - 5 below.

[0038] <(Biofilm formation inhibitory effect (present))> · Test method First, a bacterial solution was prepared. The cariogenic bacterium Streptococcus mutans JCM5705 was pre-cultured in a tryptone soy broth (TSB) medium at 37°C under anaerobic conditions for 24 hours. The concentration of the bacterial solution after pre-culture was 1×10 8 cfu / mL. Next, 2 μL of the test solutions of each example and comparative example were dispensed into each well of a 96-well microplate. 198 μL of the pre-cultured bacterial solution was inoculated into each well into which the above test solution had been dispensed. Then, for each test solution, test solutions with 8 different concentrations of the (A) component, isopropylmethylphenol, diluted 2-fold from 400 ppm to 3.1 ppm were prepared using these as the reference concentrations, and the volume of the liquid in each well was adjusted to be the same, and they were cultured at 37°C under anaerobic conditions for 24 hours. After culturing, the test solutions in each well were discarded, and the inside of the well was washed 3 times with 0.9% physiological saline and then dried. To each well after drying, 250 μL of crystal violet solution was added and stained for 20 minutes. Then, the crystal violet solution was discarded, and the inside of this well was washed 3 times with 0.9% saline. After that, 200 μL of ethanol was added to each well to dissolve and extract the crystal violet stained on the biofilm, and its absorbance (OD595) was measured. · Evaluation criteria The biofilm formation rate (%) was calculated using the following formula from the measured absorbance, and the obtained biofilm formation rate (%) was evaluated based on the following evaluation criteria. That is, when a biofilm is formed in each test solution, crystal violet is extracted into the above ethanol, so the absorbance in the above measurement increases. Biofilm formation rate (%) calculated from absorbance =(Absorbance of the well containing the test solution) / (Absorbance of the well without the test solution) × 100 (Evaluation criteria) ◎: The biofilm formation rate is 10% or less. 〇: The biofilm formation rate exceeds 10% and is 50% or less. ×: The biofilm formation rate exceeds 50%.

[0039] <No bactericidal effect on planktonic bacteria> · Test method As described in the test method for evaluating the biofilm formation inhibitory effect above, each test solution was prepared, and the test solution in each well cultured at 37 °C under anaerobic conditions for 24 hours was visually observed. That is, when no bactericidal effect on planktonic bacteria is observed in each test solution, turbidity is observed in the above observation. · Evaluation criteria The presence or absence of turbidity of each test solution was confirmed and evaluated based on the following evaluation criteria. (Evaluation criteria) ◎: Turbidity present ×: No turbidity

[0040]

Table 1

[0041]

Table 2

[0042]

Table 3

[0043]

Table 4

[0044]

Table 5

[0045] As shown in Tables 1 to 5, although the anti-biofilm compositions of the examples contain only low concentrations of bactericidal components, an anti-biofilm effect was observed. This is because polyoxyethylene hydrogenated castor oil, which is a surfactant, covers the surface of the substrate, thereby suppressing the adhesion of bacteria to the substrate at the initial stage of biofilm formation, and suppressing the gene expression necessary for the biofilm formation (production of sticky exopolysaccharides (outer membrane)) of IPMP and / or the original bactericidal property of IPMP (inhibition of bacterial metabolic activity enzymes, physical membrane disruption, etc.). Due to these synergistic effects, it is considered that biofilm formation inhibition could be efficiently achieved even at concentrations considerably lower than the minimum inhibitory concentration (MIC).

Industrial Applicability

[0046] Since the anti-biofilm composition of the present invention can suppress the formation of biofilms while having a low concentration of bactericidal components, it can be preferably used particularly as materials for food, medical devices, and oral use.

Claims

1. An anti-biofilm composition containing (A) isopropylmethylphenol, (B) polyoxyethylene hydrogenated castor oil with an average addition mole number of ethylene oxide of 30 to 70 moles, (C) a diol having 3 to 6 carbon atoms, and (D) water, wherein the contents of components (A) to (C) are set within the following ranges with respect to the mass of the anti-biofilm composition. An anti-biofilm composition. Component (A): 10 ppm or more and less than 50 ppm. Component (B): 15 ppm or more and less than 100 ppm. Component (C): 15 ppm or more and less than 100 ppm.

2. The anti-biofilm composition according to claim 1, further containing at least one selected from the group consisting of a low-molecular surfactant, a polymer surfactant, and an enzyme.

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