A growth inhibitor for bacteria of the genus Fusobacterium, and a therapeutic / preventive agent and oral microbiota improvement agent using the same.

Abstract

The objective is to provide a composition that can suppress diseases other than oral diseases. [Solution] The present invention is a growth inhibitor for Fusobacterium bacteria containing cyclodextran and / or its derivatives. Due to its inhibitory effect on Fusobacterium bacteria, it contributes to the treatment and prevention 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 ulcers.

Description

Technical Field

[0001] The present invention relates to a growth inhibitor for bacteria of the genus Fusobacterium, as well as a therapeutic / preventive agent and an agent for improving the oral bacterial flora using the same.

Background Art

[0002] Tooth decay refers to tooth decay caused by biological factors among dental substance defects, and it is known that demineralization of teeth by acids produced by oral bacteria from carbohydrates is the main cause. Streptococcus mutans, the causative bacterium of tooth decay, 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, thereby forming dental caries. If glucan is not formed, oral bacteria such as Streptococcus mutans are less likely to adhere to the tooth surface, leading to the suppression of tooth decay.

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

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

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

[0006] Therefore, the present invention aims to provide a composition that can suppress diseases other than oral diseases. [Means for solving the problem]

[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, 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 prophylactic 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. [Effects of the Invention]

[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. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows an example of a structural formula for cyclodextran (CI). [Figure 2] Figure 2 shows an example of a cyclodextrin (CD) structural formula. [Figure 3] Figure 3 shows the results of next-generation sequencing (NGS) and genus-level microbial community analysis using the results. [Figure 4] Figure 4 shows the results of the intergroup comparison analysis (LEfSe, Linear discriminant analysis effect size) using saliva. [Figure 5] Figure 5 shows the turbidity (OD660) when three types of oral bacteria were cultured in four different patterns. [Figure 6] Figure 6 is a schematic diagram illustrating the crosslinking function of Fusobacterium nucleatum in biofilm formation. [Modes for carrying out the invention]

[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 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, seleno, 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] Specific examples of compounds preferred as derivatives of cyclodextrin include branched cyclodextrins in which cyclodextrin is branched from some glucose residues (those in which alcoholic hydroxyl groups are substituted with glycosyl groups), and amino-substituted cyclodextrins in which hydroxyl groups of some glucose residues are substituted with amino groups. Among the monosaccharide residues included in the latter, those in which the hydroxyl group at the 2-position of the glucose residue is substituted with an amino group are generally referred to as glucosamine residues.

[0018] As an example, cyclodextrin and its derivatives can be obtained from a culture broth of a microorganism of the genus Bacillus using dextran as a substrate or a reaction solution of cyclic isomaltooligosaccharide synthase using dextran as a substrate (see Patent Nos. 3075873 and 3117328 for reference;

[0019] Examples of microorganisms of the genus Bacillus having cyclodextrin-producing ability include Bacillus circulans T-3040 strain (later renamed Paenibacillus agaridevorans T-3040 strain), Paenibacillus sp. 598K strain, etc. Examples of cyclic isomaltooligosaccharide synthase include cyclic isomaltooligosaccharide glucanotransferase (CITase) obtained by purifying a culture broth of a microorganism of the genus Bacillus having cyclodextrin-producing ability.

[0020] As described above, specific examples of compounds preferred as derivatives of cyclodextrin include branched cyclodextrins in which cyclodextrin is branched from some glucose residues (those in which alcoholic hydroxyl groups are substituted with glycosyl groups), and amino-substituted cyclodextrins in which hydroxyl groups of some glucose residues are substituted with amino groups. However, these are merely examples of known cyclodextrin derivatives, and not all cyclodextrin derivatives obtained by microbial reactions and / or enzymatic reactions can be specified.

[0021] Methods for measuring the state of cyclodextran derivatives include chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. 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, for 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 using equipment with extremely low detection limits, this would require an enormous 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 as part of 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 has a large and shallow opening, while the molecular structure of CD has a small and deep opening. Fourthly, CI is highly soluble in water, while CD is poorly soluble in water.

[0029] [Uses of growth inhibitors] [Growth inhibition against Fusobacterium bacteria] The growth inhibitor contributes to inhibiting the growth of 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 growth 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 on the tooth surface together with other oral bacteria. 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 a 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 ingredients that may be included in caries inhibitors, toothpaste compositions, and mouthwash compositions] The caries inhibitor, toothpaste composition, and mouthwash composition according to this embodiment may, if necessary, be used in combination with other medicinal ingredients to treat, suppress, or prevent oral diseases such as bad breath, periodontal disease, and hypersensitivity, or to strengthen tooth enamel. Other medicinal ingredients besides cyclodextran and / or cyclodextran derivatives that may be contained in the caries inhibitor, toothpaste composition, and mouthwash composition according to this embodiment include conventionally used ingredients 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, as well as 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. [Examples]

[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> Participants in this study were recruited from undergraduate students of the Faculty of Dentistry and graduate students of the Graduate School of Dentistry at Asahi University. All participants met the following selection criteria and did not violate any exclusion conditions. [Selection Criteria] • Persons aged 20 or older at the time of obtaining consent • Individuals with healthy gums (those without periodontal pockets of 4mm or more, and those who do not bleed during probing) • Individuals without untreated dental caries [Exclusion criteria] • Individuals taking medications that may affect the test, such as antibiotics. • Individuals who have received dental treatment within three months of obtaining consent, or who are currently undergoing dental treatment.

[0041] Age and sex were asked verbally. Periodontal pocket depth and the presence or absence of bleeding on probing were evaluated based on the Community Periodontal Index. In addition, the presence or absence of untreated caries was checked visually. As a result, 19 students (18 males, 1 female, average age 24.2 years) were selected as subjects.

[0042] <Method> [How to rinse your mouth] The CI-containing solution was prepared by dissolving CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) in distilled water to a concentration of 1%. Subjects rinsed their mouths with distilled water (day 1) and the CI-containing solution (day 2) before going to bed. Each rinse was 20 ml and lasted for 30 seconds to 1 minute.

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

[0044] Saliva samples were collected before going to bed and after waking up. Before going to bed, a sample was collected before rinsing, after avoiding eating, drinking, and oral hygiene for at least two hours. After waking up, a sample was collected immediately upon waking.

[0045] [Methods for analyzing oral microbiota] [Next-Generation Sequencing (NGS) of 16S rRNA genes] The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR for 25 cycles 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 21;9(8):e105592.). Sequencing was performed at Bioengineering Lab Co., Ltd. Paired-end sequencing (2 × 300 bp) was performed using the Illumina MiSeq platform (Illumina, Inc., USA) with the 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, while 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] [Survey] The subjective effects of mouthwash were evaluated using a questionnaire upon waking. Participants were asked to rate four questions on a five-point scale (not at all, almost none, a little, quite) regarding stickiness in their mouth, coating of their tongue, discomfort in their mouth, and dryness in their mouth.

[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.). In short, the RF medium was prepared by adding 5 g of yeast extract (Becton Dickinson), 5 g of K2HPO4 (Fujifilm-Wako), 8 g of glucose (Fujifilm-Wako), 0.5 g of L-cysteine ​​hydrochloride (Sigma Aldrich), 1 g of Tween 80 (Tokyo Chemical Industries, Ltd.), 0.005 g of hemin (Fujifilm-Wako), 0.002 g of vitamin K1 (Fujifilm-Wako), 0.001 g of rezazrin sodium salt (Tokyo Chemical Industries, Ltd.), 0.025 g of acetic acid (Fujifilm-Wako), and 0.01 g of MgSO2·7H2O (Fujifilm-Wako) per liter to Brain Heart Infusion Broth (Thermo Fisher Scientific), and adjusting the pH to 6.8.

[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 obtained. 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). Note that high-purity CI refers to products 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) SM30μL (3) FN 15 μL (4) SS 10 μL + FN 20 μL (5) SM10μL + FN20μL

[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] <Result> [Analysis results of oral microbiota] NGS and genus-level microbial flora analysis using the results were performed. 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 Ctrl group at night and in the morning.

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

[0057] In all survey questions, fewer respondents answered "yes" after using a CI-containing mouthwash compared to after using a distilled water rinse, while more respondents answered "almost none" or "never." In particular, for the questions "How dirty is your tongue?" and "How uncomfortable is your mouth?", the number of respondents who answered "never" or "almost none" after using a CI-containing mouthwash was double that of those who used a distilled water rinse. These results suggest that many people felt less tongue dirt and less discomfort in their mouths after using a CI-containing mouthwash. [Table 1]

[0058] [CI utilization of three oral bacteria] Figure 5 shows the turbidity (OD) when three types of 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] <Consideration> In saliva samples, 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 Ctrl group's morning and evening. 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 glucosyltransferase (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 more significantly influenced by the bacterial flora on the oral mucosa rather than the tooth surface. 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, since the proportion of Porphyromonas and Treponema species in the oral microbiota in saliva did not decrease, it is thought that CI has a mechanism that specifically reduces Fusobacterium bacteria.

Claims

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

2. A therapeutic or prophylactic 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, comprising the growth inhibitor described in claim 1.

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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