Oral bacterial community composition

Abstract

To provide a novel method for suppressing the proportion of highly periodontal disease-causing bacteria among oral bacterial species in dental plaque. [Solution] A composition comprising cetylpyridinium chloride and dodecylpyridinium chloride for suppressing or reducing the proportion of highly periodontal disease-causing bacteria in the oral microbiota.

Description

Technical Field

[0001] The present disclosure relates to compositions for oral microbiota, etc. The content of all documents (especially prior art documents) described in this specification is incorporated herein by reference.

Background Art

[0002] Plaque (also called dental plaque) is a biofilm formed by the aggregation of oral microorganisms and is considered to be a cause of dental caries and periodontal diseases.

[0003] Generally speaking, plaque is formed as follows. First, a thin film of proteins derived from saliva and physiological gingival sulcus exudate called "pellicle" is formed on the surface of teeth, and facultative anaerobic bacteria (initial adherent bacteria) such as Streptococcus attach to the tooth surface through the pellicle. Examples of initial adherent bacteria include bacteria belonging to the genus Streptococcus that are predominantly present in the oral cavity and Haemophilus parainfluenzae. Bridging bacteria such as Fusobacterium (especially Fusobacterium nucleatum), which co-aggregate with various oral bacteria to these initial adherent bacteria, attach, and furthermore, late adherent bacteria such as Porphyromonas gingivalis and Filifactor alocis, which are obligate anaerobic bacteria, attach and aggregate through the bridging bacteria, and the plaque matures. In particular, late adherent bacteria are known to cause periodontal diseases, and the pathogenicity of dental plaque becomes stronger as these bacteria grow (see, for example, Non-Patent Documents 1 to 4). This highly pathogenic dental plaque is in a state of dysbiosis with disrupted microbiota.

Prior Art Documents

Patent Documents

[0004] [License 1] Patent No. 6968964 [Non-licensed literature]

[0005] [Non-licensed Document 1] Periodontol 2000. 2006;42:47-79. [Non-licensed Document 2] Trends Mol Med. 2015 Mar;21(3):172-83. [Non-licensed Document 3] Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 16: 1552-1576 [Non-licensed Document 4] J Dent Res 90(11):1271-1278, 2011 [Non-licensed Document 5] Front Microbiol. 2022 Jun 30; Vol13:934525 [Non-licensed Document 6] Ammann et al. BMC Microbiology 2012, 12:227 [Non-licensed Document 7] Journal of Applied Microbiology 2005, 98, 624-634 [Non-licensed Document 8] FEMS Immunol Med Microbiol. 2003 Oct 24;39(1):81-6 [Non-licensed Document 9] ISME J. 2022 Apr; 16(4): 948-957 [Non-licensed Document 10] Front Microbiol. 2024 Aug 20:15:1431785 [Non-licensed Document 11] J Periodontal Implant Sci. 2020 Jun; 50(3): 171-182 [Non-licensed Document 12] Journal of Oral Science, Vol. 59, No. 2, 201-206, 2017 [Non-Patent Document 13] mBio. 2022 Feb 22;13(1):e0013122. doi: 10.1128 / mbio.00131-22. Epub 2022 Feb 22. [Non-Patent Document 14] Journal of the Japan Society for Biotechnology, Vol. 99, No. 11, pp. 577-579, 2021. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The pathogenicity of dental plaque in periodontal disease can be determined by the constantly changing ratio of early-attached and late-attached bacteria as the amount of attached oral bacteria increases. If the proportion of early-attached bacteria in dental plaque is high, the pathogenicity is considered low, while if the proportion of late-attached bacteria and other highly periodontal pathogenic bacteria is high, the pathogenicity can be considered high.

[0007] Furthermore, regarding the pathogenicity of dental plaque, it is known that when pathogenicity is low, the number of types of oral bacteria that adhere to it is small (a state of low oral bacterial diversity), while conversely, when pathogenicity is high, the number of types of oral bacteria that adhere to it increases (a state of high oral bacterial diversity).

[0008] Therefore, even if dental plaque is present, if the proportion of highly periodontal pathogenic bacteria in the bacterial flora is low (for example, if the proportion of initially attached bacteria is high, more preferably if the proportion of initially attached bacteria is high and the diversity of the bacterial flora is low), the pathogenicity of dental plaque is considered to be low. For this reason, it is considered important to suppress the proportion of highly periodontal pathogenic bacteria in the oral bacterial flora (Non-patent documents 9-14).

[0009] In preventing periodontal disease, it is considered important to effectively remove dental plaque, which is the cause of periodontal disease. However, reports indicate that brushing with a toothbrush only removes about 60% of plaque, leaving about 40% behind. If plaque remains behind, over time, oral bacteria will accumulate on the remaining plaque, shifting it to a highly pathogenic state and causing periodontal disease to progress. Therefore, while improving the removal of dental plaque is important, it is also necessary to suppress the pathogenicity of dental plaque, that is, to change it to a less pathogenic state. As mentioned above, in order to reduce pathogenicity, it is important to suppress the proportion of highly periodontal disease-causing bacteria among oral bacteria.

[0010] To date, bactericides such as cetylpyridinium chloride (hereinafter also known as CPC) and chlorhexidine gluconate (hereinafter also known as CHX) have been used to prevent periodontal disease. However, in in vitro studies using human saliva, repeated treatment with the bactericide CPC increased the diversity of oral bacterial species, and the degree of diversity remained unchanged compared to similarly treated saline solution. Therefore, it has been reported that using CPC alone may not be sufficient to suppress the pathogenicity of dental plaque. Furthermore, it has been confirmed that CHX has an inhibitory effect on the diversity of oral bacterial species in dental plaque, and that this inhibitory effect differs from that of CPC and saline solution (see Non-Patent Document 5).

[0011] Therefore, the inventors conducted research to develop a novel method for suppressing the proportion of highly periodontal disease-causing bacteria among oral bacterial species in dental plaque. [Means for solving the problem]

[0012] The inventors have discovered that by using CPC in combination with dodecylpyridinium chloride (hereinafter also referred to as DPC), it is possible to suppress or reduce the proportion of highly periodontal disease-causing bacteria in the oral microbiota, and have further improved the method.

[0013] The present disclosure encompasses, for example, the subject matter described in the following clauses. Clause 1. A composition for suppressing or reducing the proportion of highly periodontal pathogenic bacteria in the oral microbiota, comprising cetylpyridinium chloride and dodecylpyridinium chloride. Clause 2. The composition according to clause 1, which is an oral composition. Clause 3. The composition according to clause 2, wherein the oral microbiota is the microbiota in plaque or saliva. Clause 4. An oral composition comprising cetylpyridinium chloride and dodecylpyridinium chloride, for forming plaque in which the proportion of Fusobacterium bacteria and Porphyromonas bacteria in the oral microbiota is 10% or less of the total oral microbiota. Clause 5. An oral composition comprising cetylpyridinium chloride and dodecylpyridinium chloride, for forming plaque in which the proportion of Haemophilus bacteria and Streptococcus bacteria in the oral microbiota is 60% or more relative to the total oral microbiota. Clause 6. An oral composition comprising cetylpyridinium chloride and dodecylpyridinium chloride, wherein the proportion of Fusobacterium bacteria and Porphyromonas bacteria in the oral microbiota is 10% or less of the total oral microbiota, and the proportion of Haemophilus bacteria and Streptococcus bacteria is 60% or more relative to the total oral microbiota for forming plaque. Clause 7. The composition according to any one of clauses 1 to 6, comprising 0.01 to 0.5% by mass of cetylpyridinium chloride and 0.004 to 0.5% by mass of dodecylpyridinium chloride. Clause 8. The composition according to any one of clauses 1 to 6, comprising 0.05 to 50 parts by mass of dodecylpyridinium chloride with respect to 1 part by mass of cetylpyridinium chloride.

Advantages of the Invention

[0014] By using CPC and DPC in combination, it is possible to suppress or reduce the proportion of highly pathogenic periodontal disease-causing bacteria (high periodontal disease pathogens) in the oral microbiota (particularly the dental plaque microbiota and salivary microbiota). By using CPC and DPC in combination, it is preferably possible to suppress the proportion of high periodontal disease pathogens in the oral microbiota and increase the proportion of initially attached bacteria, and more preferably to reduce the diversity of the microbiota. [Brief explanation of the drawing]

[0015] [Figure 1] This table shows the bacterial diversity (Inverse Simpson index) values ​​when dental plaque models were treated with various chemical solutions. ** indicates p<0.01, **** indicates p<0.0001, and ns indicates no statistical significance. [Figure 2] This shows the proportion of various bacteria present when a dental plaque model is treated with various chemical solutions. The dotted rectangle indicates the proportion of Haemophilus and Streptococcus bacteria, which are the initial attached bacteria. [Figure 3] The results of examining bacterial diversity (Shannon index) in saliva, supragingival plaque, or subgingival plaque after using a test mouthwash containing both CPC and DPC are shown. [Modes for carrying out the invention]

[0016] The embodiments included in this disclosure will be described in more detail below. This disclosure preferably includes, but is not limited to, compositions for oral microbiota, particularly compositions that can suppress the diversity of oral microbiota (especially plaque microbiota), and compositions for forming oral microbiota with a low proportion of highly periodontal pathogenic bacteria and / or a high proportion of initially attached bacteria. This disclosure includes everything disclosed herein and that can be recognized by those skilled in the art.

[0017] The oral microbiota compositions included in this disclosure include cetylpyridinium chloride (CPC) and dodecylpyridinium chloride (DPC). Hereinafter, the oral microbiota compositions included in this disclosure may be referred to as "the compositions of this disclosure."

[0018] The CPC content in the compositions of this disclosure is not particularly limited as long as the effect is achieved, but for example, it is about 0.01 to 0.5% by mass. The upper or lower limits of this 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, The mass percentage 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%. For example, the range may be approximately 0.02 to 0.3% by mass, or approximately 0.03 to 0.1% by mass.

[0019] The DPC content in the compositions of this disclosure is not particularly limited as long as the effect is achieved, but for example, it is about 0.004 to 0.5% by mass. The upper or lower limits of this range are, for example, 0.005, 0.006, 0.007, 0.008, 0.009, 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. The mass percentages may be 21, 0.22, 0.23, 0.24, 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%. For example, the range may be approximately 0.01 to 0.3% by mass, or approximately 0.02 to 0.1% by mass.

[0020] In the compositions of this disclosure, the content ratio of CPC to DPC is preferably, for example, 0.05 parts by mass or more of DPC per 1 part by mass of CPC. Furthermore, while there is no particular upper limit, it is preferable that the amount of DPC be 50 parts by mass or less. The upper or lower limits within this range (0.05 to 50 parts by mass) are, for example, 0.06, 0.07, 0.08, 0.09, 0.1, 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.

[0021] The composition of this disclosure, by containing CPC and DPC, can exhibit an excellent effect in suppressing oral microbiota diversity.

[0022] Furthermore, the compositions of this disclosure, by containing CPC and DPC, also exhibit the effect of promoting the formation of an oral microbiota (particularly plaque microbiota) with a low proportion of highly periodontal disease-causing bacteria. From this viewpoint, the compositions of this disclosure can also be preferably used to form plaque with a low proportion of highly periodontal disease-causing bacteria in the oral microbiota. As plaque with a low proportion of highly periodontal disease-causing bacteria, plaque in which the proportion of highly periodontal disease-causing bacteria is 45% or less of the total oral microbiota is preferred, and plaque in which it is 40% or less is more preferred.

[0023] Furthermore, the compositions of this disclosure preferably suppress the proportion of Fusobacterium and Porphyromonas species among highly periodontal pathogenic bacteria. The compositions of this disclosure can also be preferably used to form plaque with a low proportion of Fusobacterium and Porphyromonas bacteria in the oral microbiota. Plaque with a low proportion of Fusobacterium and Porphyromonas bacteria preferably has a proportion of 10% or less of Fusobacterium and Porphyromonas bacteria relative to the entire oral microbiota, more preferably 9, 8, 7, 6, 5, or 4% or less, even more preferably 3% or less, and even more preferably 2% or less.

[0024] Furthermore, an oral microbiota with a low proportion of highly periodontal pathogenic bacteria can be said to have a correspondingly higher proportion of less pathogenic initial-attached bacteria. The compositions of this disclosure also exhibit the effect of promoting the formation of an oral microbiota with a high proportion of initial-attached bacteria. From this viewpoint, the compositions of this disclosure can be preferably used to form plaque in which the proportion of initial-attached bacteria in the oral microbiota is high relative to the entire oral microbiota. As for plaque with a high proportion of initial-attached bacteria, plaque in which the number of initial-attached bacteria is 60% or more of the entire oral microbiota is preferable, and plaque in which it is 65% or more is more preferable.

[0025] Furthermore, the compositions of this disclosure can be preferably used to form plaque in which the proportion of Haemophilus and Streptococcus bacteria is particularly high relative to the overall oral microbiota among the initially attached bacteria. As for plaque with a high proportion of Haemophilus and Streptococcus bacteria, plaque in which the proportion of Haemophilus and Streptococcus bacteria is 60% or more of the overall oral microbiota is preferred, and plaque in which it is 65% or more is more preferred. Furthermore, although not particularly limited, the proportion of Streptococcus bacteria relative to the total amount of Haemophilus and Streptococcus bacteria is more preferably 50% or more, even more preferably 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more, and even more preferably 90% or more.

[0026] While not particularly limited, the compositions of the present disclosure are preferably used to achieve the above proportions. They are more preferably used to (i) ensure that the proportion of Fusobacterium and Porphyromonas is 10% or less of the total oral microbiota, and (ii) ensure that the proportion of Haemophilus and Streptococcus bacteria is 60% or more of the total oral microbiota. Furthermore, they are even more preferably used to (iii) ensure that the proportion of Streptococcus bacteria is 50% or more of the total amount of Haemophilus and Streptococcus bacteria. While not particularly limited, the compositions of the present disclosure are preferably used to achieve one or two of (i) to (iii), and most preferably to achieve all of (i) to (iii).

[0027] This proportion is measured by collecting oral microbiota (specifically, plaque, for example), amplifying the V3-V4 region of the 16S rRNA of the bacteria contained therein, and sequencing it (the proportion of bacteria is determined by the proportion of the V3-V4 region of the 16S rRNA derived from the test bacteria out of the total sequence measured).

[0028] The compositions of this disclosure can be used particularly suitably as oral compositions. When the compositions of this disclosure are used as oral compositions, such compositions may be referred to as "oral compositions of this disclosure."

[0029] The oral compositions of this disclosure may be, for example, solid compositions or liquid compositions. These oral compositions can be used, for example, as pharmaceuticals, quasi-drugs, etc. The form of the oral compositions of this disclosure is not particularly limited, but can be made into forms (dosage forms) such as ointments, pastes, pasta preparations, gels, liquids, sprays, mouthwashes, liquid toothpastes, toothpastes, gums, etc., according to conventional methods. Among these, mouthwashes, liquid toothpastes, toothpastes, ointments, pastes, liquids, and gels are preferred.

[0030] The oral compositions of this disclosure may contain, alone or in addition to two or more optional components that can be incorporated into oral compositions, as long as they do not impair the effects.

[0031] For example, nonionic surfactants, anionic surfactants, or amphoteric surfactants can be incorporated as surfactants. Specifically, 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 13 to 15 carbon atoms in the alkyl group; polyoxyethylene alkylphenyl ethers with a polyoxyethylene addition coefficient of 10 to 18 and 9 carbon atoms in the alkyl group; diethyl sebacate; polyoxyethylene hydrogenated castor oil; and fatty acid polyoxyethylene sorbitan. Examples of anionic surfactants include sulfate ester salts such as sodium lauryl sulfate and sodium polyoxyethylene lauryl ether sulfate; sulfosuccinates such as sodium lauryl sulfosuccinate and sodium polyoxyethylene lauryl ether sulfosuccinate; acyl amino acid salts such as sodium cocoyl sarcosinate and sodium lauroyl methylalanine; and sodium cocoyl methyl taurate. Examples of amphoteric surfactants include betaine-type surfactants such as lauryldimethylaminoacetic acid betaine and coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine; imidazoline-type surfactants such as N-cocoyl-N-carboxymethyl-N-hydroxyethylethylenediamine sodium; and amino acid-type surfactants such as N-lauryldiaminoethylglycine. These surfactants can be used individually or in combination of two or more. The amount used is usually about 0.1 to 5% by mass of the total composition.

[0032] As flavoring agents, 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 fragrances can be used. These can be used individually or in combination of two or more. The amount used can be, for example, about 0.001 to 1.5% by mass of the total amount of the composition.

[0033] As sweeteners, for example, sodium saccharin, potassium acesulfamethamate, stevioside, neohesperidyl dihydrochalcone, perillartin, thaumatin, aspartylphenylalanyl methyl ester, p-methoxycinnamic aldehyde, etc., can be used. These can be used individually or in combination of two or more. The amount used can be, for example, about 0.01 to 1% by mass of the total composition.

[0034] As a humectant, sorbitol, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, polypropylene glycol, xylitol, maltitol, lactitol, polyoxyethylene glycol, etc., can be used individually or in combination of two or more.

[0035] As preservatives, parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, sodium benzoate, phenoxyethanol, and alkyldiaminoethylglycine hydrochloride may be included. These can be included individually or in combination of two or more.

[0036] As colorants, legally approved pigments such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3, mineral pigments such as ultramarine, enhanced ultramarine, and navy blue, and titanium dioxide may be included. These can be used individually or in combination of two or more.

[0037] As a pH adjuster, citric acid, phosphoric acid, malic acid, pyrophosphate, lactic acid, tartaric acid, glycerophosphate, acetic acid, nitric acid, or chemically possible salts thereof, or sodium hydroxide may be included. These can be included individually or in combination of two or more so that the pH of the composition is in the range of 4 to 8, preferably 5 to 7. The amount of pH adjuster may be, for example, about 0.01 to 2% by weight.

[0038] The oral compositions disclosed herein further include, as pharmaceutically active ingredients, vitamin E derivatives 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 sarcosinate; cationic bactericides such as chlorhexidine hydrochloride and benzethonium chloride; enzymes such as dextranase, amylase, protease, mutanase, lysozyme, and lytic enzymes (Litec enzyme); and 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, glycyrrhizic acid, sodium copper chlorophyllin, glycerophosphate, chlorophyll, sodium chloride, caropeptide, allantoin, carbazochrome, hinokitiol, potassium nitrate, and palatinite can be formulated individually or in combination of two or more.

[0039] Alcohols, silicones, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, Plastibase, etc., can also be added as bases. These can be used individually or in combination of two or more.

[0040] Furthermore, the oral compositions of this disclosure can be prepared by known methods or by methods readily conceivable from known methods. For example, they can be prepared by appropriately mixing CPC and DPC, and other components as needed.

[0041] The oral compositions of this disclosure are not particularly limited in their application to humans and non-human mammals. Preferred non-human mammals include livestock and pets, and more specifically, dogs, cats, mice, rats, horses, cattle, sheep, monkeys, and the like.

[0042] Furthermore, the above-mentioned descriptions of oral compositions in this disclosure may also apply to compositions of this disclosure that are not used as oral compositions (for example, when used for denture cleaning).

[0043] In this specification, the term "comprising" includes both "consisting essentially of" and "consisting of." Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0044] Furthermore, the various characteristics (properties, structure, function, etc.) described in each embodiment of this disclosure above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein. [Examples]

[0045] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0046] (Examination of plaque bacterial flora diversity) We measured indicators representing the diversity of the dental plaque bacterial flora. Specifically, we did so as follows: Based on the Zurich biofilm model manufacturing method (Ammann et al. BMC Microbiology 2012, 12:227), a pathogenic biofilm composed of oral bacteria contained in saliva was prepared using saliva collected from humans. This biofilm was used as a dental plaque model for the study. The biofilm was treated with various drug solutions for a set period of time and cultured. After the set period, an index representing diversity was measured and evaluated by comparing it with water (negative control). The measurement was performed by calculating an index representing diversity (quantification of diversity) from genomic DNA extracted from the cultured biofilm using a next-generation sequencer and analysis pipeline. The following are more detailed conditions.

[0047] (Collection of human saliva) Resting saliva was collected from one healthy individual in their 30s who had no periodontitis or other oral diseases. The collected resting saliva was used as pellicle-forming saliva and as a bacterial source for biofilm formation. For pellicle-forming saliva, the supernatant was collected by centrifugation (11000 rpm, 4°C, 10 minutes) and irradiated with UV light for 1 hour. For the bacterial source for biofilm formation, the collected resting saliva was diluted approximately 50 times in McBain liquid medium (Journal of Applied Microbiology 2005, 98, 624-634).

[0048] (Medicinal solution) The following solutions (1) to (5) were prepared and used as the respective drug solutions. (1) Water was used as a negative control. (1): Water (sterile water) (2): 0.05% CPC (0.05% by mass aqueous solution of CPC; also written as 0.05% CPC aq) (3): 0.1% CPC (0.1% by mass aqueous solution of CPC; also written as 0.1% CPC aq) (4): CPC + DPC (an aqueous solution containing 0.05% by mass of CPC and 0.03% by mass of DPC; also written as CPC + DPC aq) (5): CPC+CAE (an aqueous solution containing 0.05% by mass of CPC and 0.04% by mass of CAE; also written as CPC+CAE aq)

[0049] CAE stands for N-coconut oil fatty acid acyl-L-arginine ethyl DL-pyrrolidone carboxylate. CAE is a known surfactant and is also known to have bactericidal effects.

[0050] (Biofilm preparation and chemical treatment) Hydroxyapatite discs (HA) (13 mm diameter and 2 mm thickness, with a 1.5 mm hole for tweezers' grip, PEN HOYA Technosurgical, Japan) were used as a tooth model. The HA was placed in a 24-well plate, and 0.5 ml of saliva for pellicle formation was added. The plate was incubated at 37°C aerobic conditions for 1 hour to allow the pellicle to adhere to the HA (pellicle-coated HA). Subsequently, the pellicle-coated HA was washed twice with phosphate-buffered saline (PBS), placed in a new 24-well plate, 1.4 ml of a biofilm-forming bacterial source was added, and the plate was cultured at 37°C anaerobic conditions for 24 hours (24-hour BF). 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 fresh McBain liquid medium. The plate was cultured again at 37°C anaerobic conditions for 24 hours (48-hour BF - drug treatment once). Subsequently, the same drug solution treatment and culture were repeated twice at 24-hour intervals (after 96 hours, BF-drug treatment was performed three times).

[0051] (Recovery of pathogenic biofilms) After 24 hours, BF was collected as the baseline (BL) biofilm (BL biofilm), and after 96 hours, BF was collected as a drug-treated biofilm.

[0052] (DNA extraction from recovered BF) DNA extraction from recovered biofilms (BL biofilms and drug-treated biofilms) was performed using achromopeptidase and the QIAamp DNA Mini Kit (QIAGEN, Netherlands) according to conventional methods.

[0053] (Bacterial flora analysis of recovered BF) The bacterial flora analysis in BL biofilms and drug-treated biofilms was outsourced to Biotechnology Research Institute Co., Ltd. This analysis involved amplifying the V3-V4 region of 16S rRNA and sequencing it using Illumina Miseq. The resulting sequences were then analyzed using the Human Oral Microbiome Database (HOMD: https: / / www.homd.org / ). The Inverse Simpson index was calculated from the results of the bacterial flora analysis. The Inverse Simpson index is used as an indicator to judge alpha diversity (alpha diversity refers to the degree of diversity in species and individuals within a given area). Therefore, a higher Inverse Simpson index indicates higher bacterial diversity. As mentioned above, high oral bacterial diversity is considered to indicate high pathogenicity in pathogenic biofilms. The analysis results are shown in Figure 1, Figure 2, Table 1, and Table 2. Figure 1 shows the Inverse Simpson index, and Table 1 shows the median of the data in Figure 1. In Figure 1, the final "aq" is omitted in the sample notation. Figure 2 shows the composition ratio of the bacterial community at the genus level, and Table 2 shows the percentage of major genera in the healthy bacterial community and major genera in the pathogenic bacterial community in Figure 2. In these figures and tables, BL indicates the results of the analysis of BL biofilms.

[0054] [Table 1]

[0055] [Table 2]

[0056] In Figure 2, genera that make up 1% or more of the bacterial community are shown. (Genealogies that make up less than 1% are grouped under "Others.") The dotted rectangles indicate initially attached bacteria. Figure 1 and Table 1 show that using CPC and DPC in combination can particularly reduce the diversity of the oral microbiota. Furthermore, Figure 2 and Table 2 show that using CPC and DPC in combination increases the proportion of major genera in healthy microbiota and decreases the proportion of major genera in pathogenic microbiota. It should be noted that the genera Streptococcus and Haemophilus, which were listed as major genera in healthy microbiota, are both initial attaching bacteria in dental plaque formation. Therefore, it can also be said that using CPC and DPC in combination can lead to the formation of dental plaque with a higher proportion of initial attaching bacteria.

[0057] (Investigation of the effects of mouthwash containing CPC and DPC on the oral microbiota) Seventeen healthy human subjects (men and women aged 40 years or older) were invited to participate in a randomized, double-blind, parallel-group controlled trial to verify the effectiveness of a mouthwash. For the trial, a placebo mouthwash containing neither CPC nor DPC, and a test mouthwash containing both CPC and DPC were prepared. The 17 subjects were randomly assigned to two groups: a placebo group (8 subjects) and a test group (9 subjects). Participants rinsed their mouths twice daily (morning and evening) with 10 ml of mouthwash (placebo group: placebo mouthwash, test group: test mouthwash) for 20 seconds each time, for two weeks. Immediately before using the mouthwash each day, they used fluoride toothpaste without antibacterial agents. Resting saliva was collected from each subject at three points: before mouthwash use (baseline, BL), after 1 week of mouthwash use (1w), and after 2 weeks of mouthwash use (2w). In addition, a probing pocket depth (PPD) of 4-6 mm was selected around the molar area of ​​each subject, and supragingival and subgingival plaque were collected from at least one site per subject at BL and 2w, for a total of two points.

[0058] The oral microbiota was analyzed using meta-16S analysis. Specifically, genomic DNA derived from oral bacteria was first extracted and purified from saliva, supragingival plaque, or subgingival plaque. The extraction and purification of this genomic DNA was performed using achromopeptidase and the QIAamp DNA Mini Kit (QIAGEN, Netherlands) according to standard procedures, as described above.

[0059] Next, DNA samples were analyzed using a next-generation sequencer (Illumina, MiSeq) to obtain meta-16S nucleotide sequence data. The obtained nucleotide sequence data was then compared with the HOMD database (https: / / www.homd.org / ) using Qiime2, a data platform for next-generation sequencers, to identify the bacterial species, obtain sequence count data for each species, and determine the Shannon index (an index that considers the number of bacterial species plus the frequency of each species) and the bacterial community composition. The Shannon index is an index that considers the number of bacterial species plus the frequency of each species and is used as an indicator to judge bacterial community diversity. Therefore, a higher Shannon index value means higher bacterial diversity.

[0060] Analysis of the bacterial flora in saliva showed no significant difference in the Shannon index at BL (Blackout) between groups, but the test group had significantly lower levels compared to the placebo group at 1 week and 2 weeks (p<0.05). Within the group, the placebo group showed no significant changes at BL, 1 week, or 2 weeks, but the test group showed a significant decrease from BL to 1 week (p<0.05) and a decreasing trend from BL to 2 weeks (p<0.1).

[0061] Analysis of the bacterial flora in supragingival plaque revealed no significant difference in the Shannon index at BL (bleed-through) between groups, but the test group showed a tendency towards lower levels at 2 weeks compared to the placebo group (p<0.10). Furthermore, within the group, no significant changes were observed in the placebo group at BL or 2 weeks, but a significant decrease was observed in the test group from BL to 2 weeks (p<0.05).

[0062] Analysis of the bacterial flora in subgingival plaque revealed no significant differences between the test group and the placebo group at BL (bleed-through) and 2 weeks. Within the group, the placebo group showed no significant changes at BL and 2 weeks, while the test group showed a decreasing trend from BL to 2 weeks (p<0.10).

[0063] These results confirmed that bacterial diversity in saliva, supragingival plaque, and subgingival plaque samples was reduced by the use of mouthwashes containing CPC and DPC.

Claims

1. A composition comprising cetylpyridinium chloride and dodecylpyridinium chloride for suppressing or reducing the proportion of highly periodontal disease-causing bacteria in the oral microbiota.

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

3. The composition according to claim 2, wherein the oral microbiota is the microbiota in plaque or saliva.

4. An oral composition comprising cetylpyridinium chloride and dodecylpyridinium chloride, A composition for forming plaque in which the proportion of Fusobacterium and Porphyromonas bacteria in the oral microbiota is 10% or less of the total oral microbiota.

5. An oral composition comprising cetylpyridinium chloride and dodecylpyridinium chloride, A composition for forming plaque in which Haemophilus and Streptococcus bacteria make up 60% or more of the total oral microbiota.

6. An oral composition comprising cetylpyridinium chloride and dodecylpyridinium chloride, The proportion of Fusobacterium and Porphyromonas bacteria in the oral microbiota is 10% or less of the total oral microbiota. Haemophilus and Streptococcus bacteria make up 60% or more of the total oral microbiota. A composition for causing plaque formation.

7. A composition according to any one of claims 1 to 6, comprising 0.01 to 0.5% by mass of cetylpyridinium chloride and 0.004 to 0.5% by mass of dodecylpyridinium chloride.

8. The composition according to any one of claims 1 to 6, comprising 0.05 to 50 parts by mass of dodecylpyridinium chloride per 1 part by mass of cetylpyridinium chloride.

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