Composition for oral microbiota

Abstract

To provide novel means for controlling the diversity of oral bacterial species in dental plaque.SOLUTION: Provided is an oral microbiota diversity control composition, comprising cetylpyridinium chloride and DL-pyrrolidonecarboxylic acid salt of N-cocoyl-L-arginine ethyl ester.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a composition for oral flora, etc. More specifically, the present disclosure relates to a composition that can suppress the diversity of oral flora (particularly plaque flora). The contents of all documents (particularly prior art documents) described in this specification are incorporated herein by reference. [Background technology]

[0002] Plaque (also called dental plaque) is a biofilm formed by the aggregation of oral microorganisms, and is thought to be a cause of dental caries and periodontal disease. For this reason, plaque control, especially the inhibition of plaque formation, is important.

[0003] Roughly speaking, plaque is formed as follows. First, a thin membrane of proteins derived from saliva and physiological gingival crevicular fluid called a "pellicle" is formed on the tooth surface, and facultative anaerobic bacteria (initial adhesion bacteria) such as streptococci adhere to the tooth surface through the pellicle. Examples of initially adhesion bacteria include Streptococcus bacteria and Haemophilus parainfluenzae, which are predominant in the oral cavity. Bridge bacteria such as Fusobacterium (particularly Fusobacterium nucleatum), which coaggregates with various oral bacteria, adhere to these early-adherent bacteria, and later-adherent bacteria such as Porphyromonas gingivalis and Filifactor alocis, which are obligate anaerobes, adhere and aggregate via the bridge bacteria, resulting in maturation of the plaque. In particular, it is known that late-adherent bacteria cause periodontal disease, and their proliferation increases the pathogenicity of dental plaque (see, for example, Non-Patent Documents 1 to 4). This highly pathogenic dental plaque is considered to be in a state of dysbiosis, with the bacterial flora disrupted. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6968964 [Non-patent literature]

[0005] [Non-Patent Document 1] Periodontol 2000. 2006;42:47-79. [Non-Patent Document 2] Trends Mol Med. 2015 Mar;21(3):172-83. [Non-Patent Document 3] Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 16: 1552-1576 [Non-Patent Document 4] J Dent Res 90(11):1271-1278, 2011 [Non-Patent Document 5] Front Microbiol. 2022 Jun 30; Vol13:934525 [Non-Patent Document 6] Ammann et al. BMC Microbiology 2012, 12:227 [Non-Patent Document 7] Journal of Applied Microbiology 2005, 98, 624-634 [Non-Patent Document 8] FEMS Immunol Med Microbiol. 2003 Oct 24;39(1):81-6 Summary of the Invention [Problem to be solved by the invention]

[0006] It is believed that the pathogenicity of dental plaque in periodontal disease can be determined by the ratio of early and late-adhering bacteria, which changes from moment to moment as the amount of attached oral bacteria increases. When there are many early-adhering bacteria in dental plaque, it can be judged that the pathogenicity is low, and conversely, when there are many late-adhering bacteria, it can be judged that the pathogenicity is high.

[0007] Furthermore, it is believed that the pathogenicity of dental plaque can be defined by the type of oral bacteria that adheres to it. When the pathogenicity is low, the number of types of oral bacteria that adhere to it is small (a state of low oral bacterial diversity), and conversely, when the pathogenicity is high, the number of types of oral bacteria that adhere to it increases (a state of high oral bacterial diversity).

[0008] In order to prevent periodontal disease, it is important to effectively remove dental plaque, which causes periodontal disease. However, it has been reported that only about 60% of dental plaque is removed by brushing with a toothbrush, and about 40% of dental plaque is left unbrushed. If dental plaque is left unbrushed, oral bacteria will adhere to the unbrushed dental plaque over time, shifting the state to a highly pathogenic state and progressing periodontal disease. Therefore, while it is important to increase the removal of dental plaque, it is also necessary to suppress the pathogenicity of dental plaque, that is, to change it to a low pathogenic state. As mentioned above, in order to achieve a low pathogenic state, it is important to suppress the diversity of oral bacteria (especially the diversity of the bacterial flora of dental plaque).

[0009] To date, bactericides such as cetylpyridinium chloride (hereinafter also referred to as CPC) and chlorhexidine gluconate (hereinafter also referred to as CHX) have been used to prevent periodontal disease. However, it has been reported that repeated treatment of in vitro dental plaque formed using human saliva with the bactericide CPC increased the diversity of oral bacterial species, while the degree of diversity of oral bacterial species was the same as that of saline similarly treated, suggesting that the use of CPC alone may not be sufficient to suppress the pathogenicity of dental plaque. It has also been reported that the use of CHX has an inhibitory effect on the diversity of oral bacterial species in dental plaque, and that its inhibitory effect on said diversity is different from that of CPC and saline (see Non-Patent Document 5).

[0010] Therefore, the present inventors conducted a study to develop a novel means for suppressing the diversity of oral bacterial species in dental plaque. [Means for solving the problem]

[0011] The inventors discovered that the combination of CPC with N-coconut oil fatty acid acyl-L-arginine ethyl-DL-pyrrolidone carboxylate (hereinafter referred to as CAE) can suppress the diversity of oral bacterial species in dental plaque, and have made further improvements.

[0012] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. A composition for inhibiting oral bacterial flora diversity, comprising cetylpyridinium chloride and N-coconut oil fatty acid acyl-L-arginine ethyl-DL-pyrrolidone carboxylate. Section 2. Item 2. The composition according to item 1, wherein the oral bacterial flora is a bacterial flora in plaque. Section 3. Item 3. The composition according to item 1 or 2, which is an oral composition. Section 4. An oral composition comprising cetylpyridinium chloride and N-cocoyl-L-arginine ethyl-DL-pyrrolidone carboxylate, A composition for forming plaque in which the proportion of initially attached bacteria in the plaque bacterial flora is 45% or more. Section 5. An oral composition comprising cetylpyridinium chloride and N-cocoyl-L-arginine ethyl-DL-pyrrolidone carboxylate, A composition for forming plaque in which the total proportion of Haemophilus parainfluenzae and Streptococcus bacteria in the plaque bacterial flora is 45% or more. Section 6. 6. The composition according to any one of items 1 to 5, comprising 0.01 to 0.5% by mass of cetylpyridinium chloride and 0.005 to 0.5% by mass of N-cocoyl-L-arginine ethyl-DL-pyrrolidone carboxylate. Section 7. Item 7. The composition according to any one of items 1 to 6, comprising 0.1 to 50 parts by mass of N-cocoyl-L-arginine ethyl-DL-pyrrolidone carboxylate per part by mass of cetylpyridinium chloride. Effect of the Invention

[0013] The combined use of CPC and CAE can suppress the diversity of oral bacterial species in dental plaque, thereby suppressing the pathogenicity of dental plaque. [Brief description of the drawings]

[0014] [Figure 1A] The Shannon index values ​​when various medicinal solutions were applied to a dental plaque model are shown. [Figure 1B] The total number of bacteria when dental plaque models were treated with various medicinal liquids is shown. [Diagram 2] The percentage of each type of bacteria in the total number of bacteria when a dental plaque model was treated with each type of medicinal liquid is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes compositions for oral flora, particularly compositions that can suppress the diversity of oral flora (especially plaque flora), and compositions for forming plaque with a high proportion of initially attached bacteria, but is not limited thereto, and the present disclosure includes all of the compositions disclosed herein and recognizable by a person skilled in the art.

[0016] The composition for oral flora included in the present disclosure contains cetylpyridinium chloride (CPC) and N-cocoyl-L-arginine ethyl-DL-pyrrolidone carboxylate (CAE). Hereinafter, the composition for oral flora included in the present disclosure may be referred to as the "composition of the present disclosure."

[0017] N-Cocoyl-L-arginine ethyl ester, DL-pyrrolidone carboxylate The content of CPC in the composition of the present disclosure is not particularly limited as long as the effect is exhibited, and may be, for example, about 0.01 to 0.5% by mass. The upper and lower limits of the range are, for example, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, It may be 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, or 0.49% by mass. For example, the range may be about 0.02 to 0.3% by mass, or about 0.03 to 0.1% by mass.

[0018] CAE is a salt of the ethyl ester of coconut oil fatty acid and L-arginine and pyrrolidone carboxylic acid, and is used as an amino acid cationic surfactant. CAE is also called cocoyl arginine ethyl PCA. As CAE, a commercially available product can be purchased and used, for example, "CAE" manufactured by Ajinomoto Healthy Supply Co., Ltd. can be used.

[0019] Examples of fatty acids contained in the coconut oil fatty acid constituting CAE include fatty acids having 8 carbon atoms (more specifically, caprylic acid), fatty acids having 10 carbon atoms (more specifically, capric acid), fatty acids having 12 carbon atoms (more specifically, lauric acid), fatty acids having 14 carbon atoms (more specifically, myristic acid), and fatty acids having 16 carbon atoms (more specifically, palmitic acid). These may be contained alone or in combination of two or more. Among them, it is preferable to contain a fatty acid having 12 carbon atoms (more specifically, lauric acid) and a fatty acid having 14 carbon atoms (more specifically, myristic acid), and it is more preferable to contain a fatty acid having 12 carbon atoms (more specifically, lauric acid).

[0020] The content of fatty acid having 12 carbon atoms (more specifically, lauric acid) in the fatty acids contained in the coconut oil fatty acid constituting the CAE may be, for example, about 40 to 70 mass%. The upper or lower limit of the range may be, for example, 45, 50, 55, 60, or 65 mass%. For example, the range may be about 45 to 65 mass%.

[0021] The content of fatty acid having 14 carbon atoms (more specifically, myristic acid) in the fatty acids contained in the coconut oil fatty acid constituting the CAE may be, for example, about 15 to 35 mass%. The upper or lower limit of the range may be, for example, 20, 25, or 30 mass%. For example, the range may be about 20 to 30 mass%.

[0022] The content of CAE in the composition of the present disclosure is not particularly limited as long as it is within a range in which the effect is exhibited, but may be, for example, about 0.005 to 0.5 mass%. The upper and lower limits of the range are, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0. The range may be 24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, or 0.49% by mass. For example, the range may be about 0.01 to 0.3% by mass, or about 0.02 to 0.1% by mass.

[0023] The content ratio of CPC and CAE in the composition of the present disclosure is, for example, preferably 0.1 parts by mass or more of CAE per 1 part by mass of CPC. Although there is no particular upper limit, it is preferable that the content of CAE is 50 parts by mass or less. The upper or lower limit of the range (0.1 to 50 parts by mass) may be, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49. For example, the range may be 0.2 to 40 parts by mass, or 0.5 to 30 parts by mass.

[0024] The composition of the present disclosure contains CPC and CAE and thus can exhibit an excellent effect of suppressing the diversity of oral bacterial flora.

[0025] In addition, the composition of the present disclosure, by containing CPC and CAE, also exhibits the effect of promoting the formation of plaque with a large proportion of initially attached bacteria. From this viewpoint, it can be said that the composition of the present disclosure can be preferably used to form plaque with a low proportion of initially attached bacteria in the plaque bacterial flora. As a plaque with a low proportion of initially attached bacteria, a plaque in which the number of initially attached bacteria is 45% or more relative to the total number of plaque-forming bacteria (plaque bacterial flora) is preferable, and a plaque in which the number of initially attached bacteria is 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% or more is more preferable. In particular, among the initially attached bacteria, a plaque in which the total number of bacteria of Haemophilus parainfluenzae and Streptococcus genus bacteria is 45% or more relative to the number of plaque-forming bacteria (plaque bacterial flora) is preferable, and a plaque in which the number of plaque-forming bacteria is 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% or more is more preferable. Plaque with a high proportion of early-adhering bacteria is preferable because it has a low proportion of highly pathogenic late-adhering bacteria (and therefore is not a malicious plaque that promotes periodontal disease).

[0026] The composition of the present disclosure can be particularly suitably used as an oral composition. When the composition of the present disclosure is used as an oral composition, the composition may be referred to as the "oral composition of the present disclosure."

[0027] The oral composition of the present disclosure may be, for example, a solid composition or a liquid composition. The oral composition may be used, for example, as a medicine or a quasi-drug. The form of the oral composition of the present disclosure is not particularly limited, and may be, for example, an ointment, a paste, a gel, a liquid, a spray, a mouthwash, a liquid dentifrice, a toothpaste, a gum, or the like (dosage form) according to a conventional method. Among these, mouthwash, liquid dentifrice, toothpaste, ointment, paste, liquid, or gel is preferable.

[0028] The oral composition of the present disclosure may further contain one or more optional components that can be blended in the oral composition, provided that the effect is not impaired.

[0029] For example, the surfactant may be a nonionic surfactant, an anionic surfactant, or an amphoteric surfactant.Specific examples of nonionic surfactants include sugar fatty acid esters such as sucrose fatty acid esters, maltose fatty acid esters, and lactose fatty acid esters; fatty acid alkanolamides; sorbitan fatty acid esters; fatty acid monoglycerides; polyoxyethylene alkyl ethers with a polyoxyethylene addition coefficient of 8 to 10 and an alkyl group with 13 to 15 carbon atoms; polyoxyethylene alkylphenyl ethers with a polyoxyethylene addition coefficient of 10 to 18 and an alkyl group with 9 carbon atoms; diethyl sebacate; polyoxyethylene hydrogenated castor oil; fatty acid polyoxyethylene sorbitan; and the like.Examples of anionic surfactants include sulfate ester salts such as sodium lauryl sulfate and polyoxyethylene lauryl ether sodium sulfate; sulfosuccinate salts such as sodium lauryl sulfosuccinate and polyoxyethylene lauryl ether sodium sulfosuccinate; acylamino acid salts such as sodium cocoyl sarcosine and sodium lauroyl methyl alanine; and sodium cocoyl methyl taurine. Examples of amphoteric surfactants include acetate betaine type surfactants such as lauryl dimethylamino acetate betaine and coconut oil fatty acid amidopropyl dimethylamino acetate betaine; imidazoline type surfactants such as N-cocoyl-N-carboxymethyl-N-hydroxyethyl ethylenediamine sodium; and amino acid type surfactants such as N-lauryl diaminoethyl glycine. These surfactants can be blended alone or in combination of two or more. The blending amount can usually be about 0.1 to 5 mass% based on the total amount of the composition.

[0030] As the flavoring agent, for example, menthol, carboxylic acid, anethole, eugenol, methyl salicylate, limonene, ocimene, n-decyl alcohol, citronellol, α-terpineol, methyl acetate, citronenyl acetate, methyl eugenol, cineole, linalool, ethyl linalool, thymol, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, perilla oil, wintergreen oil, clove oil, eucalyptus oil, pimento oil, d-camphor, d-borneol, fennel oil, cinnamon oil, cinnamaldehyde, peppermint oil, vanillin, and other flavorings can be used. These can be blended alone or in combination of two or more. The blending amount can be, for example, about 0.001 to 1.5% by mass based on the total amount of the composition.

[0031] As sweeteners, for example, saccharin sodium, acesulfame potassium, stevioside, neohesperidyl dihydrochalcone, perillartine, thaumatin, aspartyl phenylalanyl methyl ester, p-methoxycinnamic aldehyde, etc. can be used. These can be blended alone or in combination of two or more. The blending amount can be, for example, about 0.01 to 1% by mass based on the total amount of the composition.

[0032] As the humectant, sorbitol, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, polypropylene glycol, xylitol, maltitol, lactite, polyoxyethylene glycol, etc. can be blended alone or in combination of two or more kinds.

[0033] As preservatives, parabens such as methylparaben, ethylparaben, propylparaben, butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride, etc. can be blended alone or in combination of two or more kinds.

[0034] Colorants that may be blended include legal dyes such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3, mineral dyes such as ultramarine, enhanced ultramarine, and Prussian blue, titanium oxide, etc. These may be blended alone or in combination of two or more kinds.

[0035] As a pH adjuster, citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, or chemically possible salts thereof, sodium hydroxide, etc. may be blended. These may be blended alone or in combination of two or more kinds so that the pH of the composition is in the range of 4 to 8, preferably 5 to 7. The blending amount of the pH adjuster may be, for example, about 0.01 to 2% by weight.

[0036] The oral composition of the present disclosure may further contain, as active ingredients, vitamin E such as dl-α-tocopherol acetate, tocopherol succinate, or tocopherol nicotinate; amphoteric bactericides such as dodecyldiaminoethylglycine; nonionic bactericides such as triclosan, isopropylmethylphenol, and hinokitiol; anionic bactericides such as sodium lauroyl sarcosine; cationic bactericides such as chlorhexidine hydrochloride and benzethonium chloride; enzymes such as dextranase, amylase, protease, mutanase, lysozyme, and bacteriolytic enzymes (retic enzymes); monofluoro Alkali metal monofluorophosphates such as sodium phosphate and potassium monofluorophosphate, fluorides such as sodium fluoride and stannous fluoride, tranexamic acid, epsilon aminocaproic acid, aluminum chlorohydroxyl allantoin, dihydrocholesterol, glycyrrhetinic acid, glycyrrhizinic acid, copper chlorophyllin sodium, glycerophosphate, chlorophyll, sodium chloride, callopeptide, allantoin, carbazochrome, hinokitiol, potassium nitrate, palatinit, etc. can be blended alone or in combination of two or more kinds.

[0037] As bases, it is also possible to add alcohols, silicone, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, plastibase, etc. These can be blended alone or in combination of two or more kinds.

[0038] The oral composition of the present disclosure can be prepared by a known method or a method that can be easily derived from a known method, for example, by appropriately mixing CPC and CAE, and other components as necessary.

[0039] The subject to which the oral cavity composition of the present disclosure is applied is not particularly limited, and examples thereof include humans and non-human mammals. Examples of non-human mammals include livestock and pets, and more specifically, examples thereof include dogs, cats, mice, rats, horses, cows, sheep, and monkeys.

[0040] The above description of the oral composition of the present disclosure can also be applied to compositions of the present disclosure that are not used as oral compositions (for example, compositions used for cleaning dentures).

[0041] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any combination of the constituent elements described in this specification.

[0042] In addition, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0043] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0044] (Study of the diversity of plaque bacterial flora) The index representing the diversity of dental plaque bacterial flora was measured. Specifically, the following procedure was performed.

[0045] Using the Zurich biofilm model (Ammann et al. BMC Microbiology 2012, 12:227), we created a pathogenic biofilm composed of oral bacteria contained in saliva from human saliva. This biofilm was used as a dental plaque model for the study.

[0046] The biofilm was treated with various chemical solutions for a certain period of time, cultured, and the index of diversity was measured after a certain period of time and compared with water (negative control). Measurements were performed by calculating the index of diversity (quantification of diversity) from genomic DNA extracted from the cultured biofilm using a next-generation sequencer and analysis pipeline.

[0047] Each condition is described in more detail below.

[0048] (Collection of human saliva) Resting saliva was collected from a healthy person in his 30s who had no periodontitis or other oral diseases. The collected resting saliva was used as saliva for pellicle formation and as a source of bacteria for biofilm production. For saliva for pellicle formation, the supernatant was collected by centrifugation (11,000 rpm, 4°C, 1 hour) and UV irradiation was performed for 1 hour. The source of bacteria for biofilm production was prepared by diluting the collected resting saliva approximately 100 times with McBain liquid medium (Journal of Applied Microbiology 2005, 98, 624-634).

[0049] (Chemical solution) The following solutions (1) to (4) were prepared and used as each drug solution. As a negative control, (5) water was used. (1):0.05% CPC (CPC0.05 mass% aqueous solution) (2): 0.05% CPC + 0.04% CAE (aqueous solution containing 0.05% by mass of CPC and 0.04% by mass of CAE) (3): 0.05% CPC + 0.08% CAE (aqueous solution containing 0.05% by mass of CPC and 0.08% by mass of CAE) (4): 0.05% CPC + 0.04% CAE alcohol rinse (alcohol rinse containing 0.05% CPC and 0.04% CAE) (5): Water (sterile water)

[0050] In addition, (1) may be referred to as 0.05% CPC (aq), (2) as CPC + 0.04% CAE (aq), (3) as CPC + 0.08% CAE (aq), (4) as CPC + 0.04% CAE (rinse), and (5) as water. In addition, (4) is a chemical solution in which a mixed solution of 10% by mass of ethanol, 10% by mass of glycerin, and the remainder of water (ethanol and glycerin aqueous solution) is used as the solvent.

[0051] (Preparation of biofilms and chemical treatment) Hydroxyapatite disks (HA) (13 mm diameter and 2 mm thickness, with a 1.5 mm hole for tweezers' grip, PEN HOYA Technosurgical, Japan) were used as tooth models. HA was placed in a 24-well plate, and 0.5 ml of saliva for pellicle formation was added. The plate was incubated for 1 hour at 37°C under aerobic conditions to allow a pellicle to adhere to the HA (Pellicle-coated HA). The pellicle-coated HA was then washed twice with phosphate-buffered saline (PBS) and placed in a new 24-well plate, to which 1.4 ml of bacterial source for biofilm production was added and cultured for 24 hours at 37°C under anerobic conditions (BF after 24 hours). After 24 hours, the BF was washed with PBS, immersed in 1 ml of drug solution for 3 minutes, and then transferred to a 24-well plate containing 1.4 ml of new McBain liquid medium and cultured again for 24 hours at 37°C under anerobic conditions (BF after 48 hours - drug-treated once). Thereafter, the same drug solution treatment and culture were repeated twice every 24 hours (BF-drug treatment completed three times after 96 hours).

[0052] (Recovery of pathogenic biofilms) After 96 hours, the HA treated with BF-drug three times was immersed in a 24-well plate containing 1.4 ml of fresh McBain liquid medium, and then P. gingivalis OMZ314 (1 × 10 7 After inoculation, the cells were cultured for 72 hours under aerobic conditions at 37°C. After the culture, HA was harvested and the attached biofilm was collected (harvested BF).

[0053] (DNA extraction from recovered BF) DNA extraction from the recovered BF was performed by standard methods using achromopeptidase and the QIAamp DNA Mini Kit (QIAGEN, Netherlands).

[0054] (Measurement of total bacterial count in recovered BF) The total number of bacteria in the recovered BF was measured using real-time PCR. Specifically, the total number of bacteria was quantified by real-time PCR using universal primers described in Non-Patent Document 8 (FEMS Immunol Med Microbiol. 2003 Oct 24; 39(1): 81-6).

[0055] (Bacterial flora analysis of recovered BF) The bacterial flora analysis of the collected BF was outsourced to Seibutsu Giken Co., Ltd. The analysis was based on amplifying the V3-V4 region of 16S rRNA and sequencing it with Illumina Miseq. The sequence after sequencing was used to perform bacterial flora analysis using the Human Oral Microbiome Database (HOMD). From the results of the bacterial flora analysis, the Shannon index (an index that takes into account the number of bacterial species and the frequency of each species) bacterial flora composition was obtained. The Shannon index is an index that takes into account the number of bacterial species and the frequency of each species, and is used as an index to determine the diversity of the bacterial flora. Therefore, the higher the value of the Shannon index, the higher the bacterial diversity. And, as mentioned above, it is thought that a high diversity of oral bacteria indicates high pathogenicity in pathogenic biofilms.

[0056] The results are shown in Figures 1A, 1B, and 2. Figure 1A shows the Shannon index value, and Figure 1B shows the total bacterial count (Log (viable bacterial count) cells / ml). Figures 1A and 2 show the results of the above-mentioned "bacterial flora analysis of recovered BF", and Figure 1B shows the results of the above-mentioned "measurement of the total bacterial count of recovered BF".

[0057] Figure 2 shows a bar graph of the proportion of various bacteria in the total number of bacteria. From Figure 1B, it can be seen that the total number of bacteria does not differ greatly when each drug is treated, and therefore, although the total number of bacteria present in the biofilm when each drug is treated does not differ greatly, the proportion of various bacteria in each biofilm differs greatly (Figure 2). Considering the results of Figure 1A together, it was found that the bacterial flora diversity does not change significantly when treated with CPC alone (0.05% CPC) compared to when no drug treatment was performed (water), whereas the bacterial flora diversity decreased when CPC and CAE were used in combination.

[0058] Figure 2 also shows the names of each detected species. The first bacterium from the top in each bar graph is Haemophilus parainfluenzae, and the second bacterium is a Streptococcus bacterium, both of which are initially attached bacteria. The ratio (%) of the number of initially attached bacteria of these two types to the total number of bacteria is shown in the following table.

[0059] [Table 1]

[0060] As can be seen from the table, the combined use of CPC and CAE promoted the formation of plaque with a high proportion of early-adhering bacteria. Plaque with a high proportion of early-adhering bacteria also has a low proportion of highly pathogenic late-adhering bacteria, and from this point of view, the reduction in bacterial flora diversity that occurs when CPC and CAE are used in combination is found to be favorable.

Claims

1. A composition for inhibiting the diversity of oral bacterial flora, comprising cetylpyridinium chloride and N-cocoyl-L-arginine ethyl.DL-pyrrolidone carboxylate.

2. The composition according to claim 1 , wherein the oral bacterial flora is the bacterial flora in plaque.

3. The composition according to claim 1 or 2, which is an oral composition.

4. An oral composition comprising cetylpyridinium chloride and N-cocoyl-L-arginine ethyl.DL-pyrrolidone carboxylate, A composition for forming plaque in which the proportion of initially attached bacteria in the plaque bacterial flora is 45% or more.

5. An oral composition comprising cetylpyridinium chloride and N-cocoyl-L-arginine ethyl.DL-pyrrolidone carboxylate, A composition for forming plaque in which the total proportion of Haemophilus parainfluenzae and Streptococcus bacteria in the plaque bacterial flora is 45% or more.

6. The composition according to claim 1, 2, 4, or 5, comprising 0.01 to 0.5% by mass of cetylpyridinium chloride and 0.005 to 0.5% by mass of N-cocoyl-L-arginine ethyl DL-pyrrolidone carboxylate.

7. The composition according to claim 1, 2, 4, or 5, comprising 0.1 to 50 parts by mass of N-cocoyl L-arginine ethyl DL-pyrrolidone carboxylate per 1 part by mass of cetylpyridinium chloride.

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