Biofilm formation-inhibiting composition

Abstract

A biofilm formation-inhibiting composition comprising at least one substance (X) selected from the group consisting of a phosphoric acid compound (X1) and salts thereof, wherein the content of the substance (X) based on the weight of the composition is 0.03-5.5 wt%.

Description

Biofilm Formation Inhibiting Composition

[0001] The present invention relates to a biofilm formation inhibiting composition.

[0002] A biofilm is a film-like structure formed by microorganisms attached to the surface of a solid or liquid. For example, in the oral cavity, oral bacteria attach to the receptors of the proteins constituting the pellicle (a saliva-derived protein-based coating that first forms on the tooth surface after tooth brushing) in the pellicle and begin to form colonies. Thereafter, co-aggregation of bacteria that can cause periodontal disease and dental caries (hereinafter also referred to as "causative bacteria") occurs, generating a biofilm in the oral cavity. Furthermore, the biofilm matures through the proliferation of the causative bacteria and co-aggregation with other causative bacteria. The development and maturation of biofilms leads to infections such as periodontal disease, gingivitis, and periodontitis.

[0003] Dental plaque is an aggregate in which bacteria proliferate inside and in the deep part thereof. Within dental plaque, microorganisms such as bacteria synthesize a slime matrix or glycocalyx (biofilm) from polysaccharides, glycoproteins, and dietary sugars (such as sucrose) that are abundantly present in the oral environment. Dental plaque eventually becomes a characteristic dental biofilm that is highly structured and contains diverse microbial populations embedded in the matrix.

[0004] In the biofilm generated in this way, the proliferation of bacteria causes infections such as periodontal disease, gingivitis, and periodontitis. Therefore, various studies have been conducted to suppress the formation of biofilms in the oral cavity (for example, refer to Patent Document 1). Patent Document 1 proposes an antibacterial agent effective against Streptococcus mutans.

[0005] Japanese Unexamined Patent Application Publication No. 2022-027681

[0006] However, in suppression methods using antimicrobial agents that target the causative bacteria, as proposed in Patent Document 1, the already formed oral biofilm hinders the penetration of the antimicrobial agent, making it difficult to achieve the desired suppressive effect. Therefore, controlling co-aggregation, which causes the formation and maturation of biofilms, is considered a safer and more effective countermeasure than suppression methods using antimicrobial agents that target the causative bacteria.

[0007] The object of the present invention is to provide a composition that can suppress co-aggregation, which is the cause of biofilm formation and maturation.

[0008] The inventors of this invention have diligently studied and investigated the above problems, and as a result have arrived at the present invention.

[0009] In other words, the present invention is a biofilm formation inhibitory composition containing at least one substance (X) selected from the group consisting of phosphate compounds (X1) and salts thereof, wherein the content of the substance (X) based on the weight of the composition is 0.03 to 5.5% by weight.

[0010] According to the present invention, it is possible to provide a composition that can suppress co-aggregation, which is the cause of biofilm formation and maturation.

[0011] The biofilm formation inhibitory composition of the present invention (hereinafter sometimes abbreviated as "the composition of the present invention") contains at least one substance (X) selected from the group consisting of phosphate compounds (X1) and salts thereof. In this specification, "biofilm formation inhibition" includes suppressing the generation and / or maturation of biofilms, at least by suppressing bacterial co-aggregation.

[0012] [Substance (X) / Phosphate compound (X1)] Examples of the phosphate compound (X1) include polyphosphate (chain compound) with a degree of polymerization of 2 to 150, and metaphosphate (cyclic compound) with a degree of polymerization of 2 to 150. Examples of polyphosphate with a degree of polymerization of 2 to 150 include pyrophosphate, tripolyphosphate (triphosphate), tetrapolyphosphate, pentapolyphosphate, and polyphosphate with a degree of polymerization of 10 to 150. Examples of metaphosphate with a degree of polymerization of 2 to 150 include hexametaphosphate.

[0013] The salt of the phosphate compound (X1) is not particularly limited and includes, for example, alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; and salts with organic bases such as triethylamine, triethanolamine, and pyridine. Among these, alkali metal salts are preferred, and sodium salts and potassium salts are more preferred.

[0014] The substance (X) is preferably at least one selected from the group consisting of polyphosphates with a degree of polymerization of 2 to 150, metaphosphates with a degree of polymerization of 2 to 150, and alkali metal salts thereof, and more preferably at least one selected from the group consisting of pyrophosphate, tripolyphosphate, hexametaphosphate, pentapolyphosphate, and polyphosphates with a degree of polymerization of 10 to 130, and alkali metal salts thereof. More specifically, examples include sodium pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate (a heated condensate of sodium dihydrogen phosphate, also called Graham salt), sodium pentapolyphosphate, and short-chain, medium-chain, or long-chain sodium polyphosphates with an average chain length of 14 to 130. Two or more phosphoric acid compounds (X1) and their salts contained in substance (X) may be used.

[0015] In the present invention, the content of substance (X) based on the weight of the composition is 0.03 to 5.5% by weight. By having a content of substance (X) within the above range, co-aggregation of bacteria in the oral cavity can be suppressed, thereby inhibiting the formation and maturation of biofilms.

[0016] From the viewpoint of having an excellent effect in suppressing biofilm formation, the content of substance (X) is preferably 0.05 to 5.5% by weight, and more preferably 0.05 to 5.0% by weight, based on the weight of the composition.

[0017] [Other Components] The composition of the present invention may contain other components (additives) other than the above-mentioned substance (X). Other components can be selected according to the method of use and application of the composition.

[0018] For example, when used as an oral composition, additives include lubricants such as waxes, antioxidants, UV absorbers, defoamers, preservatives, fragrances, and solvents [water and organic solvents (ethanol, etc.)].

[0019] When the composition is used as a food, it may contain ingredients that serve as the base material for the food. The base material can be appropriately selected depending on the form of the food. For example, if the food is candy, it may contain corn syrup as the base material. Examples of food forms include candy, gummies, gum, chocolate, tablet candies (such as chewing soft candies), and jelly (solid at 25°C).

[0020] [Composition] The pH of the composition of the present invention can be selected according to the method of use and application site. For example, when the composition of the present invention is used in the oral cavity, the pH is preferably 6 to 8. The pH may be adjusted as appropriate by adding a pH adjusting agent. The pH is the value measured at 25°C using a pH meter (method in accordance with JIS Z 8802:2011).

[0021] The composition of the present invention can be manufactured by blending the components and mixing them uniformly at room temperature or, if necessary, by heating (e.g., 30 to 70°C). The order and method of blending the components are not particularly limited.

[0022] [Method of Use] When using the composition of the present invention for the purpose of inhibiting the formation of biofilm in the oral cavity, it can be used by bringing it into contact with a part of the oral cavity [oral mucosa (tongue, gums, gingiva, etc.)]. Depending on the application, the composition of the present invention can also be used as a pharmaceutical, medical material (medical adhesive and wound healing material, etc.), quasi-drug, cosmetic, and food by using components other than substance (X), and all of the above uses can help prevent periodontal disease, gingivitis, and periodontitis at the application site. Specifically, the above uses include toothpaste, mouthwash, oral gel, oral care food, supplement, pet care product, medical adhesive, wound healing material, etc.

[0023] [Effects] The composition of the present invention exhibits an inhibitory effect on the co-aggregation of bacteria and other organisms that form biofilms. When the composition of the present invention is used as an oral composition, by inhibiting the co-aggregation of bacteria and other organisms, it can suppress the generation and maturation of biofilms that serve as a foothold for bacteria to settle in the oral cavity, thereby preventing infections such as periodontal disease, gingivitis, and periodontitis.

[0024] Bacteria that may occur in the oral cavity and on which the composition of the present invention can act include: Streptococcus mutans, Streptococcus pyogenes, Pseudomonas aeruginosa, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Candida albicans, Candida glabrata, Treponema denticola, Tannerella forsythia, Streptococcus salivarius, Streptococcus mitis, and Streptococcus Examples include *Sanguinis*, *Streptococcus oralis*, and *Streptococcus gordonii*.

[0025] The composition of the present invention can suppress co-aggregation by bacteria in the oral cavity and inhibit the formation and maturation of biofilms. As a result, the present invention can prevent infections such as periodontal disease, gingivitis, and periodontitis caused by bacterial proliferation in the oral cavity.

[0026] This specification discloses the following: Disclosure (1) is a biofilm formation inhibitory composition containing at least one substance (X) selected from the group consisting of phosphate compounds (X1) and salts thereof, wherein the content of the substance (X) based on the weight of the composition is 0.03 to 5.5% by weight. Disclosure (2) is the biofilm formation inhibitory composition according to Disclosure (1), wherein the substance (X) is at least one selected from the group consisting of polyphosphate with a degree of polymerization of 2 to 150, metaphosphate with a degree of polymerization of 2 to 150, and alkali metal salts thereof.

[0027] The present invention will be further described below with reference to examples and comparative examples, but the present invention is not limited thereto.

[0028] <Examples 1-35 and Comparative Examples 2-8> Each of the following substances (X-1) to (X-7) was mixed with deionized water at a temperature of 25°C, and neutralized with disodium hydrogen phosphate or potassium dihydrogen phosphate to a pH of 7 to obtain biofilm formation inhibitory compositions according to Examples 1-35 and Comparative Examples 2-8, containing substances (X-1) to (X-7) at the concentrations shown in Tables 1-4.

[0029] <Comparative Example 1> A PBS(-) aqueous solution (phosphate buffer aqueous solution) was used instead of the composition.

[0030] <Components> The components used in the examples and comparative examples are as follows: [Substance (X)] (X-1): Sodium pyrophosphate (tetrasodium phosphate) (molecular weight: 265.90) ​​(X-2): Sodium tripolyphosphate [pentasodium triphosphate, molecular weight: 367.86, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] (X-3): Sodium hexametaphosphate [heat condensate of sodium dihydrogen phosphate, (NaPO ３ ) ｍ [m: 10-23, mixture of compounds with molecular weights of 1019.6-2345.1, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] (X-4): Sodium pentapolyphosphate [Na ７ P ５ O １６ (X-5): Short-chain sodium polyphosphate [number average molecular weight 1489.4, average chain length 14, manufactured by Taihei Chemical Industry Co., Ltd.] (X-6): Medium-chain sodium polyphosphate [number average molecular weight 6179.7, average chain length 60, manufactured by Regentis Co., Ltd.] (X-7): Long-chain sodium polyphosphate [number average molecular weight 13317.0, average chain length 130, manufactured by Regentis Co., Ltd.]

[0031] <Evaluation Test: Co-aggregation Inhibition Effect Confirmation Test> Using the biofilm formation inhibitory compositions described in Examples 1 to 35 and Comparative Examples 2 to 8, a co-aggregation inhibition effect confirmation test was conducted on Fusobacterium nucleatum using the following method, and the co-aggregation inhibition effect was evaluated. The results are shown in Tables 1 to 4. As a comparative test to serve as a standard for evaluating the co-aggregation inhibition effect, a test was conducted in which the biofilm formation inhibitory composition was changed to a PBS(-) aqueous solution (phosphate buffer aqueous solution), and the results are shown in Table 4 as Comparative Example 1.

[0032] (Bacteria used in the test) Fusobacterium nucleatum, Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythia were used. In the co-aggregation inhibition effect confirmation test, the degree of co-aggregation of each other bacterium (hereinafter referred to as control bacteria) against Fusobacterium nucleatum was evaluated for each target bacterium.

[0033] Of the above bacteria, Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis are bacteria that may be involved in the formation of biofilms, while Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythia are bacteria that may cause biofilm maturation and periodontal disease. In particular, Fusobacterium nucleatum is known to co-aggregate with other bacteria to form strong oral biofilms.

[0034] (Culture medium) Modified GAM broth "Nissui" was used as the culture medium for Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia. Brainheart Infusion medium (Becton Dickinson) was used as the culture medium for Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis. New Oral Spirochete (NOS) medium (12.5 g / L Brain Heart Infusion medium, 10 g / L Triptase soy broth, 2.5 g / L yeast extract, 0.5 g / L sodium thioglycolate, 1 g / L cysteine, 0.25 g / L asparagine, 0.2% sodium bicarbonate, 50 mL fetal bovine serum, 0.06% thiamine pyrophosphate) was used as the culture medium for Treponema denticola. For Tannerella forsythia, the culture medium used was: 37 g / L Brain Heart Infusion Medium, 10 g / L Yeast Extract, 0.5 g / L Cysteine, 0.008 g / L Sodium Chloride, 0.008 g / L Magnesium Chloride, 0.04 g / L Dipotassium Hydrogen Phosphate, 0.04 g / L Potassium Hydrogen Phosphate, 0.04 g / L Sodium Bicarbonate, 0.04 g / L Sodium Chloride, 5 ppm Hemin, 0.5 ppm Menadione, 0.015 g / L N-Acetylmuramic Acid, 5% Horse Serum.

[0035] (Test Method) Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythia were tested under anaerobic conditions, while Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis were tested under aerobic conditions. Nucleatum and each control bacterium were cultured overnight in the above medium at 37°C, and the cultures of each bacterium were centrifuged to remove the supernatant. PBS(-) aqueous solution (phosphate buffer aqueous solution) was added to the cultures of each bacterium after supernatant removal to prepare suspensions in which the absorbance at 600 nm was 1.0 for each culture. Next, 250 μL of the Fusobacterium nucleatum suspension (a suspension obtained from the culture medium of Fusobacterium nucleatum) and 250 μL of the control bacterial suspension (a suspension obtained from the culture medium of the control bacteria) prepared above were placed in a sterile cuvette. Then, 500 μL of either the biofilm formation inhibitory composition according to Examples 1 to 35 and Comparative Examples 2 to 8, or the PBS(-) aqueous solution (Comparative Example 1) was dispensed and mixed in a vortex mixer for 10 seconds to prepare the bacterial suspension mixture for the co-aggregation inhibition effect confirmation test according to Examples 1 to 35 and Comparative Examples 1 to 8. After the sterile cuvette containing the prepared bacterial suspension mixture was left to stand at room temperature for 1 hour, the sterile cuvette was centrifuged (2000 rpm, 2 minutes), the obtained supernatant was dispensed into a new sterile cuvette, and the absorbance at 600 nm was measured. The degree of co-aggregation between Fusobacterium nucleatum and the control bacteria was confirmed by applying the absorbance values ​​measured for each control bacterium in each of Examples 1 to 35 and Comparative Examples 1 to 8 to the following formula. The results are shown in Tables 1 to 4. Degree of co-aggregation = [Absorbance in each example] / [Absorbance in Comparative Example 1] In the formula, "Absorbance in each example" refers to the absorbance measured for each control bacterium in each of Examples 1 to 35 and Comparative Examples 1 to 8.Further, the "absorbance in Comparative Example 1" is the absorbance in Comparative Example 1 for the same bacterium as the control bacterium applied to the "absorbance in each Example". When co-aggregation does not occur, the bacteria remain in a suspended state without sedimentation, so the absorbance does not decrease. On the other hand, Comparative Example 1 uses a PBS(-) aqueous solution that has no effect of inhibiting co-aggregation, and the absorbance decreases due to sedimentation of co-aggregates. Therefore, the higher the co-aggregation degree (the ratio to the absorbance in Comparative Example 1) obtained by the above calculation formula, the less the generation of co-aggregates compared to Comparative Example 1. That is, if the value of the co-aggregation degree is greater than 1.0, it means that the composition has an effect of inhibiting co-aggregation, and a larger value means a more excellent effect.

[0036]

[0037]

[0038]

[0039]

[0040] In each table, "-" means that the corresponding component is not contained. As shown in each table, it was confirmed that the compositions of the Examples have a higher effect of inhibiting co-aggregation of oral bacteria than the products of the Comparative Examples. It can also be seen that the compositions of the Examples effectively inhibit the co-aggregation of bacteria that can be involved in the formation and maturation of biofilms.

[0041] From the above, it is found that according to the present invention, a composition capable of inhibiting co-aggregation that causes the formation and maturation of biofilms can be provided.

Claims

1. A biofilm formation inhibitory composition comprising at least one substance (X) selected from the group consisting of phosphate compounds (X1) and salts thereof, wherein the content of the substance (X) based on the weight of the composition is 0.03 to 5.5% by weight.

2. The biofilm formation inhibitory composition according to claim 1, wherein the substance (X) is at least one selected from the group consisting of polyphosphate having a degree of polymerization of 2 to 150, metaphosphate having a degree of polymerization of 2 to 150, and alkali metal salts thereof.

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