Growth inhibitor for fusobacterium bacteria, and therapeutic / prophylactic agent and oral microflora improving agent using same

Abstract

[Problem] The purpose of the present invention is to provide a composition capable of inhibiting diseases other than oral diseases. [Solution] The present invention is a growth inhibitor for Fusobacterium bacteria, and contains cyclodextran and / or a derivative thereof. The inhibitory effect of Fusobacterium bacteria contributes to the treatment and prevention of ulcerative colitis, cirrhosis, obesity, colon cancer, esophageal cancer, oral cancer, breast cancer, ovarian cancer, cervical cancer, oral squamous cell carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma, premature birth, endometriosis, and the like.

Description

Growth inhibitor for Fusobacterium bacteria, as well as therapeutic and preventive agents and oral microbiota improving agents using the same

[0001] The present invention relates to a growth inhibitor for Fusobacterium bacteria, as well as therapeutic and preventive agents and oral microbiota improving agents using the same.

[0002] Dental caries refers to those caused by biological factors among the substantial tooth defects, and it is known that the main cause is the demineralization of teeth by acids produced by oral bacteria from carbohydrates. Streptococcus mutans, a causative bacterium of dental caries, is a kind of gram-positive facultative anaerobic coccus. This Streptococcus mutans synthesizes glucan, a sticky polysaccharide, from sucrose as a raw material by glucosyltransferase (GTF) produced by itself. This glucan forms a mass called plaque together with other oral bacteria on the tooth surface and produces acid inside, which erodes the tooth surface and demineralizes the tooth, thus forming dental caries. If glucan is not formed, it becomes difficult for oral bacteria such as Streptococcus mutans to adhere to the tooth surface, leading to the suppression of dental caries.

[0003] Conventionally, it has been known that some cyclic oligosaccharides have the effect of inhibiting the enzyme activity of GTF of Streptococcus mutans and suppressing dental caries. For example, an anti-caries agent containing cyclic isomaltooligosaccharide as an active ingredient has been known (see Patent Document 1).

[0004] Japanese Patent No. 3400868

[0005] On the other hand, in recent years, it has been pointed out that oral diseases such as dental caries may also affect diseases other than oral diseases, and the medical importance of suppressing dental caries has been increasing.

[0006] Therefore, an object of the present invention is to provide a composition capable of suppressing diseases other than oral diseases.

[0007] As a result of diligent research, the inventors have discovered that rinsing with a solution containing cyclodextran and / or cyclodextran derivatives not only inhibits the enzyme activity of GTF in Streptococcus mutans, but also prevents dysbiosis of the oral microbiota, thus completing the present invention. Furthermore, they have found that cyclodextran and / or cyclodextran derivatives contribute to the suppression of growth of Fusobacterium bacteria, in particular, among oral bacteria involved in biofilm formation, and may contribute to the treatment or prevention of diseases other than oral diseases caused by Fusobacterium bacteria, such as ulcerative colitis, cirrhosis, obesity, colorectal cancer, esophageal cancer, oral cancer, breast cancer, ovarian cancer, cervical cancer, oral squamous cell carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma, premature birth, and endometriosis. Specifically, the present invention provides the following.

[0008] The invention relating to the first feature provides a growth inhibitor for Fusobacterium bacteria, comprising at least one compound selected from cyclodextran and cyclodextran derivatives detected from a culture solution of Bacillus microorganisms using dextran as a substrate and / or a reaction solution of cyclic isomaltose synthase using dextran as a substrate.

[0009] The invention relating to the second feature provides a therapeutic or preventive agent for one or more diseases selected from ulcerative colitis, cirrhosis of the liver, obesity, colorectal cancer, esophageal cancer, oral cancer, breast cancer, ovarian cancer, cervical cancer, oral squamous cell carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma, premature birth, and endometriosis, which contains the growth inhibitor of the invention relating to the first feature.

[0010] The invention relating to the third feature provides an oral microbiota improving agent containing cyclodextran and at least one compound selected from cyclodextran and cyclodextran derivatives detected from a culture solution of Bacillus microorganisms using dextran as a substrate and / or a reaction solution of cyclic isomaltose synthase using dextran as a substrate.

[0011] According to the present invention, it is possible to provide a composition that can suppress not only oral diseases such as dental caries, but also diseases other than oral diseases.

[0012] Figure 1 shows an example of the structural formula of cyclodextran (CI). Figure 2 shows an example of the structural formula of cyclodextrin (CD). Figure 3 shows the results of next-generation sequencing (NGS) and genus-level bacterial flora analysis using the results. Figure 4 shows the results of intergroup comparative analysis (LEfSe, Linear discriminant analysis effect size) in saliva. Figure 5 shows the turbidity (OD) when three types of oral bacteria were cultured in four patterns. ６６０ Figure 6 is a schematic diagram illustrating the crosslinking function of Fusobacterium nucleatum in biofilm formation.

[0013] The following describes specific embodiments of the present invention in detail with reference to the drawings. However, the present invention is not limited in any way to the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention.

[0014] <Growth Inhibitor> The growth inhibitor according to this embodiment contains cyclodextran and at least one compound selected from cyclodextran and cyclodextran derivatives detected from the culture solution of Bacillus microorganisms that use dextran as a substrate and / or the reaction solution of cyclic isomaltose synthase that uses dextran as a substrate.

[0015] Figure 1 shows an example of the structural formula of cyclodextran (CI). Cyclodextran is a cyclic isomaltoligosaccharide in which 4 to 33 glucose molecules are linked cyclically by α-1,6-glycosidic bonds.

[0016] Derivatives of cyclodextran include both naturally occurring compounds and artificially synthesized compounds. In cyclodextran derivatives, at least one of the alcoholic hydroxyl groups of cyclodextran is substituted with another functional group. The other functional group is not particularly limited, but may be one or more selected from chloro, bromo, iodo, alkoxy, glycosyl, thiol, sulfide, selenol, amino, amide, and phosphate groups. Among these, the functional group (substituent) substituted from the alcoholic hydroxyl group is preferably a polar group. These functional groups may substitute a single hydroxyl group of one molecule of cyclodextran, multiple hydroxyl groups may be substituted with the same functional group, or multiple hydroxyl groups may be substituted with different functional groups.

[0017] Preferred compounds as derivatives of cyclodextran include branched cyclodextrans (where the alcoholic hydroxyl group is replaced by a glycosyl group) and amino-substituted cyclodextrans (where the hydroxyl group of some glucose residues is replaced by an amino group). Among the monosaccharide residues included in the latter, those in which the hydroxyl group at position 2 of the glucose residue is replaced by an amino group are generally called glucosamine residues.

[0018] For example, cyclodextran and its derivatives can be obtained from a culture medium of Bacillus microorganisms that use dextran as a substrate, or from a reaction solution of cyclic isomaltoligosaccharide synthase that uses dextran as a substrate (see Japanese Patent Nos. 3075873 and 3117328).

[0019] Examples of Bacillus microorganisms capable of producing cyclodextran include Bacillus circulans T-3040 strain (later renamed Paenibacillus agaridevorans T-3040 strain) and Paenibacillus sp. 598K strain. An example of a cyclic isomaltoligosaccharide synthase is cyclic isomaltoligosaccharide glucanotransferase (CITase), which is obtained by purifying the culture medium of Bacillus microorganisms capable of producing cyclodextran.

[0020] As mentioned above, specific examples of preferred compounds as derivatives of cyclodextran include branched cyclodextran (where alcoholic hydroxyl groups are replaced with glycosyl groups) and amino-substituted cyclodextran (where hydroxyl groups of some glucose residues are replaced with amino groups). However, these are merely examples of known cyclodextran derivatives, and not all cyclodextran derivatives obtained by microbial and / or enzymatic reactions have been identified.

[0021] Methods for measuring the state of cyclodextran derivatives include chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR). However, these methods can only observe specific components, making it impossible to analyze all of the extremely large number of trace components, such as products resulting from microbial and / or enzymatic reactions. In particular, in the case of carbohydrates like cyclodextran, the UV absorption in chromatography is weak, requiring the use of low-sensitivity IR detectors, making the detection of trace components itself difficult. Even if the main component can be detected, chromatography is not a method for identifying its structure, and there are currently no suitable columns available to separate the countless cyclodextran derivatives.

[0022] Furthermore, even when these methods are combined with mass spectrometry (LC-MS) for analysis, it is extremely difficult to clearly identify which parent peak each mass peak corresponds to based on data obtained from peaks with insufficient separation. In addition, cyclodextran derivatives are difficult to ionize, making the detection of trace components itself difficult. Moreover, while methods such as NMR are effective for structural analysis of carbohydrates, it is not practical to analyze all derivatives in a mixture of countless carbohydrates with unknown structures.

[0023] Even if it were possible to identify all cyclodextran derivatives produced by microbial and / or enzymatic reactions by utilizing equipment with extremely low detection limits, this would require a vast number of trials and enormous costs, making it impractical.

[0024] Therefore, there are currently no appropriate means of measurement and analysis to comprehensively cover all types of cyclodextran derivatives contained in the reaction products of microbial and / or enzymatic reactions. Consequently, it is not possible to comprehensively describe the specific forms of cyclodextran derivatives contained in the reaction products of microbial and / or enzymatic reactions, and it is impossible or impractical to directly identify such substances by their structure or properties. It cannot be said that it is unclear to describe cyclodextran derivatives as "at least one compound detected from the culture solution of Bacillus microorganisms that use dextran as a substrate and / or the reaction solution of cyclic isomaltose synthase that uses dextran as a substrate."

[0025] (Note that the contents of the references are incorporated into this specification by reference, but matters specifically stated herein shall be governed by the provisions of this specification.) When cyclodextran is obtained using microorganisms or enzymes, it is obtained as a mixture of cyclodextran and a branched cyclodextran. This mixture of cyclodextran and a branched cyclodextran can be separated, if necessary, into a composition in which the concentration of cyclodextran is 75% or more, preferably 85% or more, more preferably 99% or more, or a composition in which the concentration of the branched cyclodextran is 75% or more, preferably 85% or more, more preferably 99% or more, by using conventionally known affinity chromatography or gel filtration chromatography. The caries-inhibiting composition according to this embodiment may be any of the following, as long as it does not impair the objectives of the present invention: a mixture of cyclodextran and a branched cyclodextran; a composition in which the concentration of cyclodextran is 75% or more, preferably 85% or more, and more preferably 99% or more; or a composition in which the concentration of the branched cyclodextran is 75% or more, preferably 85% or more, and more preferably 99% or more.

[0026] Furthermore, in this specification, when "cyclodextran" is referred to, it refers to cyclodextran, which is a cyclic isomaltoligosaccharide that does not have a branched structure, and those that have a branched structure are distinguished from "cyclodextran" as "branched cyclodextran."

[0027] Cyclodextrin (CD) is known as a compound that is mutually similar to cyclodextran (CI). Figure 2 shows an example of the structural formula of cyclodextrin (CD).

[0028] CI and CD are both cyclic oligosaccharides in which multiple glucose molecules are linked in a ring, but they differ in the following ways: Firstly, CI consists of 4 to 33 glucose molecules, while CD consists of 6 to 8 glucose molecules. Secondly, in CI, the glucose molecules are linked by α-1,6-glycosidic bonds, while in CD, they are linked by α-1,4-glycosidic bonds. Thirdly, the molecular structure of CI is large and shallow, while the molecular structure of CD is small and deep. Fourthly, CI is highly soluble in water, while CD is poorly soluble in water.

[0029] [Uses of the Growth Inhibitor] [Growth Inhibition against Fusobacterium bacteria] The growth inhibitor contributes to the inhibition of growth against Fusobacterium bacteria. Fusobacterium bacteria are causative agents of ulcerative colitis, liver cirrhosis, obesity, colorectal cancer, esophageal cancer, oral cancer, breast cancer, ovarian cancer, cervical cancer, oral squamous cell carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma, premature birth, and endometriosis, and it is known that oral diseases such as dental caries can affect diseases other than oral diseases involving Fusobacterium bacteria. According to the present invention, since the proliferation of Fusobacterium bacteria can be significantly suppressed, it can contribute not only to the suppression of oral diseases such as dental caries, but also to the treatment or prevention of ulcerative colitis, colorectal cancer, esophageal cancer, oral cancer, and endometriosis.

[0030] Examples of bacteria belonging to the genus Fusobacterium include F. necrophorum, F. nucleatum, F. varium, and F. mortiferum.

[0031] The first technical significance of the present invention lies in the discovery that cyclodextran or its derivatives themselves have an inhibitory effect on the growth of Fusobacterium bacteria that do not possess a gene encoding the GTF enzyme.

[0032] Previous knowledge about cyclodextran was limited to inhibiting dental caries by preventing the production of glucosyltransferase (GTF) by Streptococcus mutans. When sugary foods are ingested, Streptococcus mutans produces glucan synthase GTF-I using sucrose as a raw material to create insoluble glucans that are sticky and have α-1,3 linkages as their main chain. Once glucans are formed, they are involved in the attachment and colonization of Streptococcus mutans to the tooth surface, forming clumps with other oral bacteria on the tooth surface. This is called plaque, and it is the biggest cause of the onset and progression of tooth decay and periodontal disease. The technical idea based on previous knowledge was limited to suppressing dental caries caused by Streptococcus mutans by utilizing the enzyme inhibitory effect of cyclodextran on GTF to prevent the production of glucan synthase GTF-I.

[0033] In contrast, bacteria of the genus Fusobacterium do not possess a protein with the Glucosyltransferases domain in the center of the gtf enzyme and therefore do not have GTF activity. The sequence of the gene encoding the GTF enzyme in Streptococcus mutans was registered in GenBank on January 12, 2019, with accession number AP019720.1. According to this, the gene sequences encoding the GTF enzymes (gtfB, gtfC, gtfD) are located at complement(1099083..1103123), complement(1094520..1098887), and complement(1185581..1189969), respectively. In contrast, the gene sequence of Fusobacterium was registered in GenBank on January 31, 2014, with accession number AE009951.2, and according to this, the gene sequence encoding the GTF enzyme does not exist.

[0034] Therefore, the effect of cyclodextran, which is to suppress the growth of Fusobacterium bacteria that do not possess the gene encoding the GTF enzyme, can be said to be an exceptional effect that even those skilled in the art could not have predicted.

[0035] Furthermore, since CI can suppress the growth of Fusobacterium bacteria, which are oral bacteria and can also affect diseases other than oral diseases, rinsing with a CI-containing solution can prevent dysbiosis of the oral microbiota, and as a result, CI can be applied as an oral microbiota improving agent.

[0036] [Dosage Form] The dosage form is not particularly limited, and the growth inhibitor can be used as a caries inhibitor, toothpaste composition, mouthwash composition, etc. These caries inhibitors, toothpaste compositions, and mouthwash compositions may be classified as pharmaceuticals, quasi-drugs, or cosmetics, and may contain medicinal or cosmetic ingredients.

[0037] [Other components that may be included in the caries inhibitor, toothpaste composition, and mouthwash composition] The caries inhibitor, toothpaste composition, and mouthwash composition according to this embodiment may, by using other medicinal components in combination as needed, treat, suppress, or prevent oral diseases such as bad breath, periodontal disease, and hypersensitivity, or they may be able to strengthen tooth enamel. Other medicinal components that may be included in the caries inhibitor, toothpaste composition, and mouthwash composition according to this embodiment, other than cyclodextran and / or cyclodextran derivatives, include conventionally used ones such as fluoride, xylitol, propolis, Swertia japonica extract, and hydroxyapatite. Specific embodiments of the toothpaste composition and mouthwash composition include oral care products such as toothpaste, mouthwash, gargle, and mouth spray, and oral care foods such as edible films, tablets, chewables, candies, gummies, gum, and beverages.

[0038] Furthermore, caries inhibitors, toothpaste compositions, and mouthwash compositions may contain any conventionally used additives, depending on their form. Such additives are not limited to those that do not hinder the objectives of the present invention, but include abrasives, binders, colorants, fragrances, sweeteners, and the like.

[0039] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0040] <Subjects> The subjects of this test were recruited from students of the School of Dentistry and graduate students of the Graduate School of Dentistry, Asahi University. All subjects met the following selection criteria and did not conflict with the exclusion criteria. [Selection Criteria] - Persons aged 20 or older at the time of obtaining consent - Persons with healthy gums (no areas with a periodontal pocket of 4 mm or more, no bleeding during probing) - Persons without untreated dental caries [Exclusion Criteria] - Persons taking medications that may affect the test, such as antibacterial drugs - Persons who had received dental treatment within 3 months from the date of obtaining consent, or persons currently undergoing dental treatment

[0041] Age and gender were asked orally. Also, the depth of the periodontal pocket and the presence or absence of bleeding during probing were evaluated based on the Community periodontal index. Furthermore, the presence or absence of untreated dental caries was confirmed by visual inspection. As a result, 19 students (18 males, 1 female, average age 24.2 years) were selected as subjects.

[0042] <Method> [Mouth rinsing method] The CI-containing solution was prepared at a concentration of 1% by dissolving CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) in distilled water. The subjects rinsed their mouths with distilled water (day 1) and the CI-containing solution (day 2) before going to bed. They rinsed their mouths for 30 seconds to 1 minute with a volume of 20 ml each time.

[0043] [Collection of saliva samples] Saliva samples were collected using a saliva collection kit ITM (reagent for virus inactivation, preservation and transportation) (manufactured by Japan Genetics Co., Ltd.) according to the protocol of the kit. The total volume of the collected liquid was centrifuged and the supernatant was removed. Then, DNA was extracted using the QIAamp PowerFecal Pro DNA Kit (manufactured by QIAGEN, Netherlands) according to the instructions of the manufacturer.

[0044] The saliva samples were collected at the times before going to bed and after waking up. Before going to bed, samples were collected avoiding eating, drinking and oral cleaning for more than 2 hours before rinsing. Also, after waking up, samples were collected immediately after waking up.

[0045] [Method for Analyzing Oral Microbiota] [Next-Generation Sequencing (NGS) of 16S rRNA Genes] The V3-V4 region of the bacterial 16S rRNA gene was analyzed using a primer set consisting of SEQ ID NO: Pro341F (5′-AATGATACGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC TCTTCCATCTCCTACGGGAGGCAGCAGCCTACGGGNBGCASCAG-3′) and SEQ ID NO: Pro805R (5′-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCA GACGTGTGCTCTTCCGATCTGACTACNVGGGTATCTAATCC-3′) (Takahashi S, Tomita J, Nishioka K, Hisada T, Nishijima M. Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing. PLoS One. 2014 Aug The assay was amplified by PCR for 25 cycles (21;9(8):e105592). Sequencing was performed at Bioengineering Lab Co., Ltd. Paired-end sequencing (2 x 300 bp) was performed using the Illumina MiSeq platform (Illumina, Inc., USA) with MiSeq Reagent Kit ver. 3 (Illumina, Inc.).

[0046] [Bioinformatics Analysis] Bioinformatics analysis was performed using MicrobiomeAnalyst 2.0 (https: / / www.microbiomeanalyst.ca / ). Paired-end compressed FASTQ files obtained from 16S sequencing were processed using default parameters. The read truncation length was set to 270 (p-trunc-len 270). Taxonomic assignment was performed using the RDP database (trainset 18) (https: / / academic.oup.com / nar / article / 42 / D1 / D633 / 1063201?login=true).

[0047] [Questionnaire Survey] The subjective effects of mouthwash were evaluated using a questionnaire upon waking. Participants were asked to rate the following four questions on a five-point scale (not at all, almost none, a little, quite a bit).

[0048] [Culture Test] The bacterial strains used were Streptococcus anginosus JCM 12993 (hereinafter SS), Streptococcus mutans JCM 5705 (hereinafter SM), and Fusobacterium nucleatum JCM 8532 (hereinafter FN). For liquid culture, the improved RF medium described in the literature was used (Le Blay G, Lacroix C, Zihler A, Fliss I. In vitro inhibition activity of nisin A, nisin Z, pediocin PA-1 and antibiotics against common intestinal bacteria. Lett Appl Microbiol. 2007 Sep;45(3):252-7.). Simply put, RF medium is made by adding 5g of yeast extract (Becton Dickinson) and 5g of potassium per liter to Brain Heart Infusion Broth (Thermo Fisher Scientific). ２ HPO ４ (Fujifilm-Wako), 8g glucose (Fujifilm-Wako), 0.5g L-cysteine ​​hydrochloride (Sigma Aldrich), 1g Tween 80 (Tokyo Chemical Industries, Ltd.), 0.005g hemin (Fujifilm-Wako), 0.002g vitamin K1 (Fujifilm-Wako), 0.001g rezazurin sodium salt (Tokyo Chemical Industries, Ltd.), 0.025g acetic acid (Fujifilm-Wako), and 0.01g MgSO ２ 7H ２ The solution was adjusted to pH 6.8 by adding oxygen (O) from Fujifilm-Wako.

[0049] Each strain was inoculated into RF medium containing a final concentration of 0.5% sucrose and incubated at 37°C for 23 hours using the AnaeroPack system (Mitsubishi Gas Chemical Company, Inc., Tokyo). OD of SS, SM, and FN strains was measured. ６６０ These values ​​were 2.1, 0.24, and 0.93, respectively.

[0050] RF medium containing sucrose at various concentrations (0 and 0.5%) and high-purity CI at various concentrations (0 and 0.5%) (i.e., 4 patterns) was dispensed into 96-deep-well plates (AxyGen Scientific). Bacteria were then inoculated into each well as follows, and incubated at 37°C for 28 hours using the AnaeroPack system (N=4). High-purity CI refers to CI containing 90% or more of CI-7 (CI with 7 carbon atoms in the ring) to CI-12 (CI with 12 carbon atoms in the ring). (1) SS 15 μL (2) SM 30 μL (3) FN 15 μL (4) SS 10 μL + FN 20 μL (5) SM 10 μL + FN 20 μL

[0051] After culturing is complete, 20 μL of these cultures are suspended in 180 μL of water in a 96-well flat-bottom plate (product name: 4845-96F; manufactured by Watson Bio Lab), and the turbidity (OD) is measured. ６６０ The values ​​were measured using a microplate reader (product name SpectraMax M2; manufactured by Molecular Devices).

[0052] <Results> [Analysis of oral microbiota] NGS and genus-level microbiota analysis were performed using the results. The results are shown in Figure 3.

[0053] Figure 3 shows only the morning results for the Ctrl and CI groups as a representative example, but the pattern was similar at night, with Prevotella being the most dominant genus in both cases. There was no difference in α diversity or β diversity between the Ctrl and CI groups.

[0054] To statistically analyze the changes in genera in detail, we performed a linear discriminant analysis effect size (LEfSe) between groups.

[0055] Figure 4 shows the results of LEfSe analysis in saliva. In saliva, the CI group had significantly fewer Fusobacterium bacteria in the morning compared to the CTrl group, and the CI group had fewer Fusobacterium bacteria in the morning compared to the evening. No decrease in Fusobacterium bacteria was observed when comparing the evening and morning in the CTrl group.

[0056] [Survey Results] Responses were received from 14 people. The results are shown in Table 1.

[0057] In all question categories, fewer people answered "yes" after using CI-containing mouthwash compared to after using distilled water, while more people answered "almost none" or "never." In particular, for the questions "How dirty is your tongue?" and "How dirty is your mouth?", the number of people who answered "never" or "almost none" after using CI-containing mouthwash was double that of those who used distilled water. These results suggest that many people felt less tongue dirt and less discomfort in their mouths after using CI-containing mouthwash.

[0058] [CI utilization of three oral bacteria] Figure 5 shows the turbidity (OD) when three oral bacteria, SS, SM, and FN, were cultured in four patterns. ６６０ The results of the culture tests suggested that these bacteria increase their growth rate by using CI as a carbon source, regardless of the presence or absence of sucrose.

[0059] <Discussion> In saliva, the CI group had significantly fewer Fusobacterium bacteria in the morning compared to the CTrl group, and the CI group had fewer Fusobacterium bacteria in the morning compared to the evening. Furthermore, no decrease in Fusobacterium bacteria was observed when comparing the evening and morning in the CTrl group. These results suggest that CI has the effect of reducing the prevalence of Fusobacterium bacteria in the oral cavity.

[0060] Regarding CI, it is known to inhibit the activity of glutosyltransferase (GTF) produced by Streptococcus mutans, thereby suppressing biofilm formation (Kobayashi M, Funane K, Oguma T. Inhibition of dextran and mutan synthesis by cycloisomaltooligosaccharides. Biosci Biotechnol Biochem. 1995 Oct;59(10):1861-5). In this study, it is hypothesized that CI inhibited the activity of GTF produced by Streptococcus mutans and others, resulting in suppression of biofilm formation in the oral cavity, and consequently, a decrease in the abundance of Fusobacterium bacteria that lost their foothold (Figure 6).

[0061] However, while biofilm formation by GTF is thought to be related to biofilms formed on tooth surfaces, the oral cavity contains various areas with different environments, and the oral bacterial flora in saliva is thought to be greatly influenced not by the tooth surface, but rather by the bacterial flora on the oral mucosa. In this study, the proportion of Streptococcus species in the oral bacterial flora in saliva was very small (Figure 3), and it is thought that the inhibitory effect of CI on GTF activity had little impact on the reduction of Fusobacterium species.

[0062] Furthermore, as shown in Figure 6, Fusobacterium nucleatum, a bacterium of the genus Fusobacterium, is known to interact with Porphyromonas gingivalis and Treponema denticola, which are known as the red complex bacteria of periodontal disease, and is known to contribute to the biofilm establishment of these red complex bacteria. However, from the results of this study, the proportion of Porphyromonas and Treponema species in the oral microbiota in saliva did not decrease, suggesting that CI has a mechanism that specifically reduces Fusobacterium bacteria.

Claims

1. A growth inhibitor for Fusobacterium bacteria, comprising at least one compound selected from cyclodextran and cyclodextran derivatives detected from a culture solution of Bacillus microorganisms using dextran as a substrate and / or a reaction solution of cyclic isomaltose synthase using dextran as a substrate.

2. A therapeutic or prophylactic agent comprising the growth inhibitor described in claim 1 for one or more diseases selected from ulcerative colitis, cirrhosis of the liver, obesity, colorectal cancer, esophageal cancer, oral cancer, breast cancer, ovarian cancer, cervical cancer, oral squamous cell carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma, premature birth, and endometriosis.

3. An oral microbiota improving agent containing cyclodextran and at least one compound selected from cyclodextran derivatives detected from a culture solution of Bacillus microorganisms that use dextran as a substrate and / or a reaction solution of cyclic isomaltose synthase that uses dextran as a substrate.

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