Dental caries suppression composition, and sweet composition, food, supplement, dentifrice composition and mouthwash composition that employ the same

Abstract

To provide, e.g., a dental caries suppression composition that is, in comparison with conventional anti-dental caries agents, capable of providing an even higher level of suppression of caries.SOLUTION: The dental caries suppression composition contains: (A) an oligosaccharide that has a cyclic structure comprising glucose and / or a glucose derivative as a structural unit; and (B) a sugar other than the component (A). The component (A) is a cyclodextran and / or a cyclodextran derivative. The cyclodextran derivative has at least one of the alcoholic hydroxyl groups of cyclodextran substituted with another polar group. According to the present invention, the composition contains: (A) an oligosaccharide that has a prescribed cyclic structure comprising glucose and / or a glucose derivative as a structural unit; and (B) a sugar other than the component (A). The components (A) and (B) interact with one another to suppress dental caries. Consequently, suppression of dental caries at a higher level than that achieved by conventional anti-dental caries agents is realized.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a caries-inhibiting composition, and to a sweet composition, food, supplement, dentifrice composition, and mouthwash composition using the same. [Background technology]

[0002] Dental caries refers to the loss of tooth substance caused by biological factors, and it is known that the main cause is decalcification of teeth due to acids produced from carbohydrates by bacteria in the oral cavity. Streptococcus mutans, a bacterium that causes dental caries, is a gram-positive, facultatively anaerobic streptococcus. Streptococcus mutans synthesizes glucan, a sticky polysaccharide, from sucrose using glucosyltransferase (GTF) that it produces itself. This glucan, along with other oral bacteria, forms a mass called plaque on the tooth surface, producing acid within it, which erodes the tooth surface and demineralizes the tooth, resulting in caries. Without glucan formation, oral bacteria such as Streptococcus mutans find it difficult to adhere to the tooth surface, which helps inhibit caries.

[0003] It has been known that some cyclic oligosaccharides inhibit the enzyme activity of GTF of Streptococcus mutans and suppress dental caries. 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] Patent No. 3400868 Summary of the Invention [Problem 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 thus the medical importance of inhibiting dental caries is increasing. Therefore, an object of the present invention is to provide a caries-inhibiting composition and the like that can inhibit dental caries at a higher level than conventional anti-carious agents. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have found that the above-mentioned problems can be solved by a caries-inhibiting composition containing (A) an oligosaccharide having a specific cyclic structure whose constituent units are glucose and / or a glucose derivative, and (B) a sugar other than component (A), and have thus completed the present invention. Specifically, the present invention provides the following.

[0007] The first aspect of the invention provides a caries-inhibiting composition comprising (A) an oligosaccharide having a cyclic structure whose constituent units are glucose and / or a glucose derivative, and (B) a sugar other than component (A), wherein component (A) is cyclodextran and / or a cyclodextran derivative, and the cyclodextran derivative has at least one alcoholic hydroxyl group of the cyclodextran substituted with another polar group. [Effects of the Invention]

[0008] The caries-inhibiting composition of the present invention contains (A) a specified cyclic oligosaccharide having glucose as a constituent unit and (B) a sugar other than component (A), and these act in cooperation to inhibit caries, so that it can inhibit caries at a higher level than conventional anti-caries agents. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows an example of the structural formula of cyclodextran (CI). [Figure 2] FIG. 2 shows an example of the structural formula of cyclodextrin (CD). [Figure 3]Figure 3 shows the results of [Test 1], an investigation into the growth rate of Streptococcus mutans in the presence / absence of cyclodextran. [Figure 4] Figure 4 shows the results of [Test 2] measurement of the amount of biofilm formed by Streptococcus mutans in the presence / absence of cyclodextran. [Figure 5] Figure 5 shows the results of [Test 3-1] confirmation of biofilm structure by staining the bacterial cells and microscopic observation in [Test 3] observation of biofilm structure caused by Streptococcus mutans. [Figure 6] Figure 6 shows the results of [Test 3-1] confirmation of biofilm structure by staining the bacterial cells and microscopic observation in [Test 3] observation of biofilm structure caused by Streptococcus mutans. [Figure 7] Figure 7 shows the results of [Test 3-2] examining the amount of glucan and bacteria in the biofilm in [Test 3] observation of the biofilm structure caused by Streptococcus mutans. [Figure 8] Figure 8 shows the results of [Test 4-1] verification of the inhibitory effect of glucan synthase activity in Streptococcus mutans using a commercially available dextran mixture containing 13% or more cyclodextran. [Figure 9] Figure 9 shows the results of [Test 4-2] verification of the inhibitory effect of glucan synthase activity in Streptococcus mutans using a highly purified sample of cyclodextran. [Figure 10] FIG. 10 shows the results of [Test 5-1] examining the expression level of proteins by Western blotting in [Test 5] verifying the mode of inhibition of GTF activity by cyclodextran. [Figure 11] FIG. 11 shows the results of [Test 5-2] verification of the effect on enzyme activity using activity staining in [Test 5] verification of the GTF activity inhibitory mode of cyclodextran. [Figure 12]FIG. 12 shows the results of [Test 5-3] verification of the effect on enzyme activity from the viewpoint of enzyme-substrate reaction in [Test 5] verification of the mode of inhibition of GTF activity by cyclodextran. [Figure 13] FIG. 13 shows the results of [Test 5-3] verification of the effect on enzyme activity from the viewpoint of enzyme-substrate reaction in [Test 5] verification of the mode of inhibition of GTF activity by cyclodextran. [Figure 14] Figure 14 shows the results of [Test 6] examining the inhibitory effect of adding cyclodextran to feed in a rat animal caries model on the development of caries caused by Streptococcus mutans. [Figure 15] FIG. 15 shows the results of [Test 6] examining the plaque deposition inhibitory effect when cyclodextran was added to feed in a rat caries experimental model. [Figure 16] FIG. 16 shows the results of [Test 6] examining the inhibitory effect of adding cyclodextran to drinking water on the development of dental caries caused by Streptococcus mutans in a rat animal dental caries model. [Figure 17] FIG. 17 shows the results of [Test 6] examining the plaque deposition inhibitory effect when cyclodextran was added to drinking water in a rat caries experimental model. [Figure 18] FIG. 18 shows the test results of [Test 7] production of catalytic domain (CAT) recombinant protein. [Figure 19] FIG. 19 shows the test results of [Test 7] production of catalytic domain (CAT) recombinant protein. [Figure 20] FIG. 20 shows the results of examining the intercellular binding strength in the biofilm disruption test (Test 8). [Figure 21] FIG. 21 is a flow chart showing the research outline for [Test 9] human test to examine the effect of inhibiting oral plaque deposition. [Figure 22] FIG. 22 shows the results of [Test 9] human test to examine the effect of inhibiting oral plaque deposition. DETAILED DESCRIPTION OF THE INVENTION

[0010] Specific embodiments of the present invention will be described in detail below with reference to the drawings. However, the present invention is not limited to the following embodiments and can be implemented with appropriate modifications within the scope of the object of the present invention.

[0011] <Caries inhibition composition> The caries-preventing composition according to this embodiment contains (A) an oligosaccharide having a cyclic structure whose constituent unit is glucose and / or a glucose derivative, and (B) a sugar other than the component (A).

[0012] As described above, Streptococcus mutans synthesizes glucan, a sticky polysaccharide, from sucrose using glucosyltransferase (GTF) produced by itself, which results in the formation of plaque on the tooth surface. The caries-inhibiting composition of this embodiment contains (A) a cyclic oligosaccharide having glucose and / or a glucose derivative as a constituent unit, and (B) a sugar other than component (A), and therefore can exhibit a higher caries-inhibiting effect than a composition containing only (A) a cyclic oligosaccharide having glucose and / or a glucose derivative as a constituent unit.

[0013] [Component (A): Cyclic oligosaccharides having glucose and / or glucose derivatives as structural units] Examples of the cyclic oligosaccharides (A) having glucose and / or glucose derivatives as constituent units contained in the caries-preventing composition of this embodiment include cyclodextran and / or derivatives in which at least one of the alcoholic hydroxyl groups of these has been substituted with another polar group (hereinafter also referred to simply as "cyclodextran derivatives").

[0014] Among these, cyclodextran derivatives include both compounds found in nature and artificially synthesized compounds. The polar groups that may substitute the alcoholic hydroxyl groups of cyclodextran include, but are not limited to, chloro, bromo, iodo, alkoxy, glycosyl, thiol, sulfide, selenol, amino, amide, and phosphate groups. These polar groups may substitute a single hydroxyl group in a single cyclodextran molecule, or multiple polar groups may be substituted with the same polar group, or multiple polar groups may be substituted with different polar groups.

[0015] Furthermore, the term "glucose derivative" refers to any structural unit other than glucose that constitutes the above-mentioned cyclodextran derivative. Even if the substituent introduced into the cyclodextran derivative is introduced after cyclodextran has been synthesized from glucose, the term "glucose derivative" includes any structural unit other than glucose that can be obtained by hydrolyzing the glycosidic bond of cyclodextran. Therefore, the term "glucose derivative" does not imply that a glucose derivative is used as a synthetic raw material in the synthesis of a cyclodextran derivative.

[0016] [Cyclodextran and cyclodextran derivatives] Fig. 1 shows an example of the structural formula of cyclodextran (CI). Cyclodextran is a cyclic isomaltooligosaccharide in which 4 to 33 glucose units are cyclically linked via α-1,6-glycosidic bonds. In the caries-inhibiting composition according to this embodiment, it is preferable to use cyclodextran as component (A) from the viewpoint of effective caries inhibition.

[0017] Specific examples of preferred compounds as cyclodextran derivatives include branched cyclodextran in which cyclodextran is branched from some glucose residues (in which alcoholic hydroxyl groups are substituted with glycosyl groups), and amino-substituted cyclodextran in which hydroxyl groups of some glucose residues are substituted with amino groups. Among the monosaccharide residues contained 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] For example, cyclodextran can be obtained from the culture medium of a Bacillus microorganism or from the reaction mixture of a cyclic isomaltooligosaccharide synthase (see Japanese Patent Nos. 3075873 and 3117328; the contents of these references are incorporated herein by reference, but the contents of this specification take precedence over any special mention of them). When cyclodextran is obtained using a microorganism or enzyme, it is obtained as a mixture of cyclodextran and branched cyclodextran. This mixture of cyclodextran and branched cyclodextran can be separated, if necessary, into a composition having a cyclodextran concentration of 75% or more, preferably 85% or more, and more preferably 99% or more, or into a composition having a branched cyclodextran concentration of 75% or more, preferably 85% or more, and more preferably 99% or more, using conventional affinity chromatography or gel filtration chromatography. The caries-inhibiting composition of this embodiment may be a mixture of cyclodextran and a branched form of cyclodextran, a composition having a cyclodextran concentration of 75% or more, preferably 85% or more, and more preferably 99% or more, or a composition having a branched form of cyclodextran concentration of 75% or more, preferably 85% or more, and more preferably 99% or more, as long as the object of the present invention is not impaired.

[0019] In this specification, when "cyclodextran" is mentioned, it refers to cyclodextran, which is a cyclic isomaltooligosaccharide that does not have a branched structure, and those that have a branched structure are distinguished from "cyclodextran" as "branched cyclodextran."

[0020] Cyclodextrin (CD) is known as a compound similar to cyclodextran (CI). Figure 2 shows an example of the structural formula of cyclodextrin (CD).

[0021] CI and CD are similar in that they are cyclic oligosaccharides in which multiple glucose units are linked in a ring, but they differ in the following ways. First, CI has 4 to 33 glucose units linked together, while CD has 6 to 8 glucose units linked together. Second, CI has glucose units linked together via α-1,6-glycosidic bonds, while CD has glucose units linked together via α-1,4-glycosidic bonds. Third, the molecular structure of CI is large and shallow, while that of CD is small and deep. Fourth, CI has high solubility in water, while CD has low solubility in water.

[0022] [Component (B); sugars other than component (A)] The sugar other than component (A) contained in the caries-inhibiting composition of this embodiment is not particularly limited as long as it does not impair the effects of the present invention, and any sugar can be used. However, among these sugars, component (B) is preferably a monosaccharide that is an aldose or ketose, or a disaccharide, oligosaccharide, cyclic oligosaccharide, or polysaccharide that can produce a mixture containing aldose and / or ketose upon hydrolysis. Representative examples of such monosaccharides, disaccharides, oligosaccharides, cyclic oligosaccharides, and polysaccharides are listed below.

[0023] [monosaccharide] Examples of the aldose or ketose monosaccharide that may be contained in the caries-inhibiting composition according to this embodiment include conventionally known aldose or ketose monosaccharides.

[0024] Of these, aldose is C n H 2n O n (where n is an integer of 3 or greater) and refers to a monosaccharide having one aldehyde group at its terminal. More specific examples of aldose monosaccharides include glyceraldehyde (C3), erythrose (C4), threose (C4), ribose (C5), arabinose (C5), xylose (C5), lyxose (C5), allose (C5), altrose (C6), glucose (C6), mannose (C6), gulose (C6), idose (C6), galactose (C6), and talose (C6). Among these monosaccharides, glucose is particularly preferred.

[0025] For ketoses, as with aldoses, C n H 2n O n (where n is an integer of 3 or more), but refers to a monosaccharide having one keto group (ketonic carbonyl group) within the chain structure. More specific examples of ketose monosaccharides include dihydroxyacetone (C3), erythrulose (C4), xylulose (C5), ribulose (C5), psicose (C6), fructose (C6), sorbose (C6), tagatose (C6), etc. Among these monosaccharides, fructose is particularly preferred.

[0026] [Disaccharide] Disaccharides capable of producing a mixture containing aldoses and / or ketoses upon hydrolysis refer to disaccharides having aldose and / or ketose monosaccharides as constituent sugars, and more specifically include sucrose, lactose, maltose, trehalose, cellobiose, isomaltose, partinose, xylobiose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, etc. Among these, sucrose, lactose, and maltose are preferably used as the disaccharide, and sucrose is more preferably used. Sucrose is a disaccharide in which glucose and fructose are bonded together via an α(1→2) bond, lactose is a disaccharide in which glucose and galactose are bonded together via a β(1→4) bond, and maltose is a disaccharide in which glucose and glucose are bonded together via an α(1→4) bond.

[0027] In particular, component (B) preferably contains a disaccharide, and among disaccharides, it is preferable to contain a disaccharide having glucose and / or fructose as a constituent unit. When the caries-inhibitory composition contains a disaccharide having glucose and / or fructose as a constituent unit in addition to component (A), component (A) and the disaccharide having glucose and / or fructose as a constituent unit act synergistically, resulting in a stronger caries-inhibitory effect than when component (A) is used alone. It is known that sucrose, one of the disaccharides having glucose and / or fructose as a constituent unit, is used as a nutrient source for oral bacteria such as Streptococcus mutans and is therefore a cause of caries. Therefore, the fact that sucrose itself acts synergistically with component (A) to enhance the caries-inhibitory effect is far beyond the scope of prediction by those skilled in the art.

[0028] Examples of disaccharides having glucose and / or fructose as constituent units include sucrose and maltose. Of these, sucrose is a disaccharide in which glucose and fructose are linked by an α-1,2-glycosidic bond. Maltose is a disaccharide in which two glucose units are linked by an α-1,4-glycosidic bond. Thus, sucrose and maltose have structural similarity with each other in that at least one constituent unit is glucose and is linked by an α-glycosidic bond. It is most preferable to use sucrose as the disaccharide having glucose and / or fructose as constituent units to be incorporated into the caries-preventing composition according to this embodiment.

[0029] [Oligosaccharides] Oligosaccharides that can produce a mixture containing aldoses and / or ketoses upon hydrolysis refer to oligosaccharides that have aldose and / or ketose monosaccharides as constituent sugars, and more specific examples include cellobiose, trehalose, isomaltose, partinose, xylobiose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, galactooligosaccharides, fructooligosaccharides, lactosucrose, isomaltooligosaccharides, and kestose.

[0030] [Cyclic oligosaccharides] A cyclic oligosaccharide capable of producing a mixture containing aldoses and / or ketoses upon hydrolysis refers to a cyclic oligosaccharide having aldose and / or ketose monosaccharides as constituent sugars. Cyclodextrins, as mentioned above, are representative of cyclic oligosaccharides, and known cyclodextrins have six, seven, or eight glucose bonds. These cyclodextrins are sometimes referred to as α-cyclodextrin (cyclohexaamylose, α-CD; six glucose bonds), β-cyclodextrin (cycloheptaamylose, β-CD; seven glucose bonds), or γ-cyclodextrin (cyclooctaamylose, γ-CD; eight glucose bonds). However, cyclic oligosaccharides are not limited to cyclodextrins and may also include cyclic oligosaccharides having monosaccharides other than glucose as constituent sugars.

[0031] [Polysaccharide] Polysaccharides that can produce a mixture containing aldoses and / or ketoses upon hydrolysis refer to polysaccharides having aldoses and / or ketoses as their constituent sugars, and more specifically include amylose, amylopectin, glycogen, galactogen, etc. Among these, amylose, amylopectin, and glycogen are all polysaccharides whose constituent sugar is glucose, and tend to differ from one another in terms of branching structure, etc. Galactogen is a polysaccharide whose constituent sugar is galactose.

[0032] [Other sugars] The sugars other than component (A) contained in the caries-preventing composition of this embodiment may include, in addition to the monosaccharides, disaccharides, oligosaccharides, cyclic oligosaccharides, and polysaccharides described above, amino sugars, deoxy sugars, sugar alcohols, lactones, and disaccharides, oligosaccharides, cyclic oligosaccharides, and polysaccharides containing any of these as constituent sugars. Examples of the amino sugars, deoxy sugars, sugar alcohols, and lactones as constituent sugars include N-acetylglucosamine, N-acetylneuraminic acid, N-glycolylneuraminic acid, deaminoneuraminic acid, D-deoxyribose, L-fucose, L-rhamnose, D-quinovose, sorbitol, xylitol, gluconolactone, maltitol, erythritol, and sucralose. When the sugars other than component (A) contained in the caries-inhibiting composition of this embodiment are disaccharides, oligosaccharides, cyclic oligosaccharides, and polysaccharides, these amino sugars, deoxy sugars, sugar alcohols, and lactones may constitute the disaccharides, oligosaccharides, cyclic oligosaccharides, and polysaccharides together with the above-mentioned aldoses and / or ketoses, or the disaccharides, oligosaccharides, cyclic oligosaccharides, and polysaccharides may be constituted only by the amino sugars, deoxy sugars, sugar alcohols, and lactones.

[0033] [Mixing ratio of component (A) and component (B)] Regarding the blending ratios of component (A) and component (B) contained in the caries-inhibitory composition according to this embodiment, the lower limit of the blending amount of component (A) is preferably 0.1 parts by mass or more, and more preferably 0.2 parts by mass or more, per 100 parts by mass of component (B). When the lower limit of the proportion of component (A) satisfies the above condition, acidification in the oral cavity caused by the presence of excess component (B) is more easily suppressed, and the caries-inhibitory effect of the caries-inhibitory composition according to this embodiment is enhanced.

[0034] Furthermore, with regard to the blending ratios of component (A) and component (B) contained in the caries-inhibitory composition according to this embodiment, the upper limit of the blending amount of component (A) per 100 parts by mass of component (B) is preferably 500 parts by mass or less, more preferably 100 parts by mass or less, even more preferably 50 parts by mass or less, still more preferably 10 parts by mass or less, and particularly preferably 5 parts by mass or less. When the upper limit of the proportion of component (A) satisfies the above condition, the synergistic action of component (A) and component (B) in the caries-inhibitory effect is further enhanced, and the caries-inhibitory effect of the caries-inhibitory composition according to this embodiment is enhanced.

[0035] <Sweetening compositions, foods, and supplements containing caries-inhibiting compositions> The caries-inhibitory composition according to this embodiment may be formulated as a sweet composition, food, or supplement. By formulating the caries-inhibitory composition according to this embodiment as a sweet composition, food, or supplement, it is possible to provide a sweet composition, food, or supplement that is less likely to cause caries when eaten.

[0036] Among these, the food containing the caries-inhibiting composition is not particularly limited, and includes not only finished products but also semi-finished products. Examples of such products include frozen foods, frozen dough, canned foods, bottled foods, chilled foods, pressure-heat sterilized foods, dried foods, processed meat products, processed seafood products, processed vegetable products, processed fruit products, processed grain products, seasonings, beverages, baked goods, Japanese sweets, Western sweets, chocolate, semi-chocolate products, chocolate confectionery, semi-chocolate confectionery, edible films, candies (gummy candies, candy, etc.), gum, tablets, jelly, yogurt, ice cream, ice milk, lacto ice cream, frozen desserts, cream, butter, butter oil, cheese, concentrated whey, concentrated milk, skim concentrated milk, unsweetened condensed milk, unsweetened condensed skim milk, sweetened condensed milk, sweetened condensed skim milk, whole milk powder, skim milk powder, premix products, cream powder, whey powder, protein-concentrated whey powder, buttermilk powder, sweetened powdered milk, modified milk powder, modified liquid milk, fermented milk, lactic acid bacteria drinks, and pet food.

[0037] Furthermore, supplements containing caries-inhibiting compositions include base supplements, health supplements, optional supplements, etc., and the nutritional functional food ingredients contained in each of these include vitamins, minerals, amino acids, enzymes, dietary fiber, coenzyme Q10, placenta, hyaluronic acid, collagen, collagen peptides, astaxanthin, omega-3 fatty acids, glucosamine, chondroitin, etc., so long as they do not impair the effects of the present invention.

[0038] [Other ingredients that may be contained in sweetening compositions, foods, and supplements] The sweetening compositions, foods, and supplements containing the caries-inhibiting composition of this embodiment can be classified as food additives, foods, and dietary supplements, respectively, and may contain additives other than component (A) and component (B) that are commonly used in the fields of food additives, foods, and dietary supplements. Such additives are not particularly limited as long as they do not impair the objectives of this invention, and examples of such additives include acidulants, enzymes, spices, colorants, preservatives, flavorings, sweeteners, seasonings, emulsifiers, thickeners, stabilizers, foaming agents, leavening agents, coloring agents, oxidizing agents, preservatives, confectionery gelatin, confectionery albumin, confectionery starch, bactericides, antioxidants, and fungicides. Furthermore, the sweetening compositions, foods, and supplements containing the caries-inhibiting composition of this embodiment may contain linear, branched, or cyclic polysaccharides structurally similar to component (A), provided that the additives do not impair the objectives of this invention.

[0039] <Caries-inhibiting agent, dentifrice composition, and mouthwash composition containing the caries-inhibiting composition> The caries-inhibitory composition according to this embodiment may be configured as a caries-inhibitory agent, a dentifrice composition, or a mouthwash composition. By configuring the caries-inhibitory composition according to this embodiment as a sweet composition, food, or supplement, users can effectively inhibit caries. These caries-inhibitory agents, dentifrice compositions, and mouthwash compositions may be classified as pharmaceuticals, quasi-drugs, or cosmetics, and may contain medicinal or cosmetic ingredients.

[0040] [Other ingredients that may be contained in the caries inhibitor, dentifrice composition, and mouthwash composition] The caries inhibitor, dentifrice composition, and mouthwash composition according to the present embodiment may be used in combination with other medicinal ingredients as needed to treat, inhibit, or prevent oral diseases such as bad breath, periodontal disease, and hypersensitivity, or may strengthen tooth structure. Examples of medicinal ingredients other than component (A) and component (B) that may be contained in the caries inhibitor, dentifrice composition, and mouthwash composition according to the present embodiment include conventionally used ingredients such as fluoride, xylitol, propolis, Swertia japonica extract, and hydroxyapatite. Specific embodiments of the dentifrice composition and mouthwash composition include oral care products such as toothpaste, mouthwash, mouthwash, and mouth spray.

[0041] The caries inhibitor, dentifrice composition, and mouthwash composition according to the present embodiment may contain any conventionally used additive depending on the embodiment. Such additives are not limited to abrasives, binders, colorants, flavorings, sweeteners, etc., as long as they do not impede the object of the present invention. Furthermore, the caries inhibitor, dentifrice composition, and mouthwash composition according to the present embodiment may contain linear, branched, or cyclic polysaccharides similar in structure to component (A), as long as they do not impede the object of the present invention. [Example]

[0042] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0043] In conducting various tests, the preparation of various media, buffer solutions, and reagents, and the determination of test protocols were carried out with reference to the descriptions in "Molecular Cloning" by Michael R. Green et al., Cold Spring Harbor Laboratory Press (2012). The contents of the references are incorporated herein by reference, but the details specifically stated herein take precedence.

[0044] [Test 1] Examination of the growth rate of Streptococcus mutans in the presence / absence of cyclodextran Streptococcus mutans MT8148 strain, cultured in Todd-Hewitt liquid medium (hereinafter referred to as "TH liquid medium") at 37°C for 18 hours, was subcultured in 100 μL of fresh Todd-Hewitt (TH) liquid medium. CI-Dextran mix (a dextran mixture containing 13% or more cyclodextran, manufactured by Nisshin Sugar Co., Ltd.; the same applies hereinafter) was added to the medium to a final concentration of 0% to 1%. The turbidity of the culture was measured every hour at a wavelength of 570 nm using a visible spectrophotometer (Novaspec plus, manufactured by Amersham Biosciences). The results are shown in Figure 3.

[0045] As is clear from Figure 3, bacterial growth ultimately converged to the same cell number whether cyclodextran was added or not, and no change was observed. However, during the logarithmic growth phase, the addition of cyclodextran clearly slowed the growth rate in a concentration-dependent manner.

[0046] [Test 2] Measurement of biofilm formation by Streptococcus mutans in the presence / absence of cyclodextran Streptococcus mutans MT8148 test bacteria were cultured in 10 mL of TH liquid medium at 37°C for 18 hours. The cultured bacteria were then diluted with 0.25% sucrose-supplemented TH liquid medium and 100 μL of the diluted solution was dispensed into each well of a FALCON® 96-well flat-bottom cell culture microtiter plate (Corning Incorporated) and cultured anaerobically at 37°C for 2 days. CI-Dextran mix (Nissin Sugar Co., Ltd.) was added to the medium to a final concentration of 0.16% to 10%. After removing the suspended bacteria from each well, 25 μL of 1% Crystal Violet solution (Nacalai Tesque, Inc.) was added and the plate was left to stand at room temperature for 15 minutes to stain the attached bacteria. The plate was then washed six times with distilled water. Thereafter, the plate was washed with 95% ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), dried, and then distilled water was added thereto, followed by measurement of absorbance at a wavelength of 570 nm. The results are shown in FIG.

[0047] As is clear from Figure 4, the amount of biofilm formed decreased in a concentration-dependent manner. When a significance test was performed between the sample without CI-Dextran mix and the samples with CI-Dextran mix added at the specified concentrations, the difference between the sample without CI-Dextran mix and all the samples with CI-Dextran mix added at the specified concentrations was statistically significant (p<0.001, Fisher's PLSD).

[0048] [Test 3] Observation of biofilm structure caused by Streptococcus mutans [Test 3-1] Confirmation of biofilm structure by staining bacterial cells and microscopic observation Streptococcus mutans MT8148 strain was cultured in TH liquid medium at 37°C for 18 hours and then harvested by centrifugation. The cells were stained with 10 mM hexidium iodide (Invitrogen™, Thermo Fisher Scientific) and adjusted to a turbidity of 0.1 at 600 nm in a chemically defined medium containing 0.5% sucrose to prepare live bacterial samples. 200 μL of each of these bacterial suspensions and samples was inoculated into an 8-well polystyrene Lab-Tek chamber slide system (Nunc™, Thermo Fisher Scientific) and cultured at 37°C for 24 hours. CI-Dextran mix (Nissin Sugar Co., Ltd.) was added to the medium to achieve a final concentration of 0% to 10%. The formed biofilms were observed using a confocal laser scanning microscope (LSM780, Version 4.2, Carl Zeiss Microimaging). Furthermore, the density was calculated using ImageJ (registered trademark). Microscopic images observed with a confocal scanning laser microscope are shown in Figure 5, and the biofilm density calculated using ImageJ is shown in Figure 6.

[0049] As is clear from Figures 5 and 6, the density of the biofilm structure became sparser and the thickness of the biofilm decreased as the concentration of CI-Dextran mix increased. The difference in density between the sample without CI-Dextran mix and the sample with CI-Dextran mix added at a concentration of 0.625% or higher was statistically significant (Fisher's PLSD).

[0050] [Test 3-2] Examination of glucan and bacterial amount in biofilm Streptococcus mutans MT8148 strain was stained with the fluorescent nucleic acid dye SYTO® 9 green and then adjusted to CDM medium containing 1% sucrose. Alexa Fluor® 647-labeled dextran was added and then seeded onto a chamber slide. At the start of incubation, CI-Dextran mix (Nissin Sugar Co., Ltd.) was added to the medium to a final concentration of 0% to 10%. After 24 hours of anaerobic incubation at 37°C, the formed biofilm was observed using a confocal laser scanning microscope LSM780 (Version 4.2, Carl Zeiss Microimaging). Micrographs are shown in Figure 7.

[0051] As is clear from Figure 7, in the presence of cyclodextran, both the amount of glucan and the amount of bacteria in the biofilm decreased significantly, and there was a tendency for the amount of glucan and the amount of bacteria to decrease more as the amount of cyclodextran added increased.

[0052] [Test 4] Verification of the inhibitory effect on the activity of glucan synthase of Streptococcus mutans [Test 4-1] Verification using a commercially available dextran mixture containing 13% or more cyclodextran CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) was added to 1% sucrose-containing phosphate buffer at concentrations ranging from 0% to 1%. 10 μL of rGTFB (recombinant GTFB) was added, and the cells were cultured at 37°C for 16 hours. After culture, the absorbance of the synthesized water-insoluble glucan was measured at 550 nm to verify the inhibitory effect of rGTFB on the activity. The results are shown in Figure 8.

[0053] As shown in Figure 8, the amount of water-insoluble glucan synthesis decreased as the concentration of CI-Dextran mix increased, indicating that rGTFB activity was inhibited in a concentration-dependent manner. A significance test was performed between samples without CI-Dextran mix and samples with CI-Dextran mix, and the differences between samples without CI-Dextran mix and all samples with CI-Dextran mix added at the specified concentrations were statistically significant (p<0.001, Fisher's PLSD).

[0054] [Test 4-2] Verification using a highly purified cyclodextran sample The inhibitory effect on glucan synthase activity was examined in the same manner as in Test 4, except that a high-purity cyclodextran sample was used instead of the CI-Dextran mix. The results are shown in Figure 9. The cyclodextran was obtained by chromatographic fractionation of the CI-Dextran mix. Among the samples shown in Figure 9, those designated CI7, CI8, CI9, C10, and C11 are samples containing oligosaccharides with a cyclic structure consisting of 7, 8, 9, 10, and 11 linked glucose units, respectively.

[0055] As is clear from Figure 9, like the above CI-Dextran mix, the high-purity cyclodextran sample also effectively inhibited the synthesis of water-insoluble glucan, but the high-purity cyclodextran sample mainly containing CI7, in which seven glucose units are linked, showed the most excellent effect.

[0056] [Test 5] Verification of the inhibitory mode of GTF activity by cyclodextran [Test 5-1] Examination of protein expression levels by Western blotting Streptococcus mutans MT8148 strain was cultured to stationary phase in TH liquid medium supplemented with CI-Dextran mix (Nissin Sugar Co., Ltd.) and sucrose at concentrations ranging from 0% to 10%. The cells were then harvested by centrifugation. Equal volumes of 2x SDS-PAGE buffer were mixed and heated to prepare the SDS-PAGE samples. Electrophoresis was performed using 10% acrylamide for the separating gel and 3% for the stacking gel. After electrophoresis, the acrylamide gel layer was transferred to a transfer membrane by applying an electric current and reacted with rabbit anti-CA-GTF antibody at room temperature for 1 hour. After washing, the membrane was further reacted with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin at room temperature for 1 hour. The bands were visualized by adding a colorimetric substrate. The results are shown in Figure 10.

[0057] As is clear from Figure 10, when the sucrose concentration was 1% and the cyclodextran concentration was varied, strong expression of a band near 145 kDa to 155 kDa, the molecular weight of the GTF protein, was observed at concentrations of 0.25% or higher. This suggests that the presence of glucan generated from sucrose and glucan originally contained in the CI-Dextran mix may have increased GTF expression. On the other hand, when the CI-Dextran mix concentration was 0.5% and the sucrose concentration was varied, a decrease in GTF protein expression was observed at concentrations of 0.5% or higher.

[0058] [Test 5-2] Verification of the effect on enzyme activity using activity staining Samples were prepared in the same manner as in [Test 5-1] and subjected to electrophoresis using SDS-PAGE. The gel was then incubated in a solution containing sucrose and Triton-X at 37°C for 18 hours. The glucans produced by GTFB and GTFC present on the gel were stained with paranose-aniline solution. The results are shown in Figure 11.

[0059] As shown in Figure 11, we first examined the GTF activity in sucrose and CI-Dextran mix alone. Strong activity was observed up to 0.125% sucrose alone. Although activity decreased above 0.25%, comparable activity was observed between 0.25% and 1.5% sucrose. On the other hand, a concentration-dependent increase in GTF activity was observed in CI-Dextran mix alone. When the CI-Dextran mix was fixed at 0.5% and the sucrose concentration was varied from 0% to 1%, a significant decrease in activity was observed at sucrose concentrations of 0.5% and above compared to sucrose concentrations of 0.25% and below.

[0060] [Test 5-3] Verification of the effect on enzyme activity from the perspective of enzyme-substrate reaction 10 μL of rGTFB was added to a phosphate buffer containing 0% (without CI) or 0.25% (with CI) CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) and serially diluted sucrose from 0 mM to 25 mM. After incubation at 37°C for 16 hours, the synthesized water-insoluble glucan was measured for absorbance at 550 nm. The results are shown in Figure 12.

[0061] As is clear from Figure 12, in the CI-added group (with CI), the maximum velocity V max The final amount of glucan synthesis was reduced by approximately 50%.

[0062] Based on these results, a Lineweaver-Burk plot was used to perform an enzyme kinetic analysis of the inhibitory effect of rGTFB on glucan synthase activity. The results are shown in Figure 13. In the Lineweaver-Burk plot, the point where the line intersects with the Y axis represents 1 / Vmax, and the slope is K m / V max To represent this, let V max and K. m In the CI-free group (without CI), the maximum reaction rate (V max ) is 0.44 nmol / min, and the Michaelis constant (K m value; reaction rate V maxThe substrate concentration at half of the original concentration was 1.54 mM. max is 0.57 nmol / min, and K m The value was 4.43 mM.

[0063] From these results, we speculate that the mode of inhibition of enzyme activity by cyclodextran is as follows. There are three types of inhibition of enzyme activity: competitive inhibition, non-competitive inhibition, and uncompetitive inhibition. Competitive inhibition is an inhibition mode in which an inhibitor binds to the active site of the enzyme and prevents the substrate from binding. Since the substrate and inhibitor compete to bind to the active site, K m The value is large, but V max Non-competitive inhibition is a type of inhibition in which an inhibitor binds to a site other than the active site of an enzyme, changing the enzyme's conformation and preventing substrate binding. Since the inhibitor binds to a site other than the active site, there is no change in the substrate affinity, and the K m The value does not change, but the enzyme's conformation changes, so V max Uncompetitive inhibition is a mode of inhibition in which an inhibitor binds to the enzyme-substrate complex and prevents the reaction. Since the inhibitor binds after the enzyme and substrate bind, K m Value and V max The test results show that K m The value becomes larger, 1 / V max Since there was no change in the GTFB activity, it was suggested that the inhibitory effect of cyclodextran on GTFB may be competitive.

[0064] [Test 6] Examination of the inhibitory effect of cyclodextran added to the feed and drinking water on the development of dental caries caused by Streptococcus mutans and on the suppression of plaque deposition in a rat animal caries model The experimental animals were male Sprague-Dawley rats, free of specific microorganisms and parasites (SPF), forcibly weaned on postnatal day 15. Groups of 10 rats were used for the experiment. For two days, on postnatal days 15 and 16, the rats were treated with an antibiotic intraoral treatment to facilitate the establishment of the test bacteria. Subsequently, for five days, from postnatal day 18 to 22, the rats were cultured with Streptococcus mutans MT8148 R strain in Brain Heart Infusion (BHI) liquid medium at 37°C for 18 hours, centrifuged, and suspended in 1 / 100 volume of sterile saline. 100 μL of the inoculum was inoculated directly into the rat's oral cavity using a micropipette once daily for infection. From the day of infection with the test bacteria, rats were fed a cariogenic diet containing 56% sucrose, Diet 2000, supplemented with CI-Dextran mix (Nissin Sugar Co., Ltd.) at 0.625%, 1.25%, 2.5%, or 5%. This diet was allowed to be consumed ad libitum until the end of the experiment. Distilled water was available ad libitum for drinking. On postnatal day 72, rats were sacrificed under chloroform anesthesia, and the jawbones were aseptically removed. The maxillary teeth were stained with erythrosine, and plaque was assessed by the Regorati and Hotz method. Additionally, caries scores for the upper and lower molars were calculated using Keyes' method, modified for rats by Ooshima et al. Similarly, rats were fed a cariogenic diet containing 56% sucrose, Diet 2000, supplemented with CI-Dextran mix (Nissin Sugar Co., Ltd.) at 0.625%, 1.25%, 2.5%, or 5% in distilled water. The results are shown in Figures 14 to 17.

[0065] As is clear from Figures 14 and 17, when CI-Dextran mix was added to feed, the addition of 1.25% CI-Dextran mix was the most effective in inhibiting dental caries. Plaque deposition inhibition was also significantly reduced when CI-Dextran mix was added at a low concentration of 0.625% (p<0.01, Tukey). These results suggest that CI-Dextran mix inhibits plaque deposition, thereby suppressing the occurrence of dental caries.

[0066] [Test 7] Preparation of catalytic domain (CAT) recombinant protein GTFB contains two functional domains: CAT, which is the sucrose-binding site, and GBD, which translocates glucose to synthesize glucan. Since the mode of enzyme inhibition was shown to be competitive inhibition, it was suggested that CI binds to CAT and inhibits the binding of sucrose to CAT. Therefore, we prepared a partial protein of CAT, rGTFB(CAT), and reacted it with sucrose. The glucose produced was measured by the Somogyi-Nelson method, a method for quantifying reducing sugars using a copper reagent.

[0067] Based on the complete nucleotide sequence of the gtfB gene of Streptococcus mutans UA159, the CAT domain was amplified by PCR. Subsequently, protein expression primers pGEX6P-1(CAT)-F and pGEX6P-1(CAT)-R were designed to insert this DNA fragment into the pGEX6P-1 plasmid. The DNA fragment obtained by PCR using these protein expression primers was inserted into the pGEX6P-1 plasmid using PrimeSTAR® Max DNA Polymerase (Takara Bio Inc.), producing the pGEX6P-1(CAT) plasmid. pGEX6P-1(CAT) was transformed into E. coli BL21 for protein expression. After large-scale cultivation, the cells were centrifuged, suspended in PBS buffer, and then sonicated to disrupt the E. coli cell wall. The supernatant was obtained by centrifugation and then purified on a Glutathione Sepharose 4B (registered trademark) column (GE Healthcare), and the resulting GST-rGTFB(CAT) was used in the following experiments.

[0068] CI-Dextran mix (Nissin Sugar Co., Ltd.) was serially diluted from 0% to 1.25% and added to sterile purified water containing GST-rGTFB(CAT) and 1% sucrose, and the mixture was allowed to react at 37°C for 1 hour. Somogyi copper solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and the mixture was boiled for 20 minutes. Nelson solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and the mixture was allowed to stand for 15 minutes. The absorbance at 500 nm was then measured. The results are shown in Figures 18 and 19.

[0069] As shown in Figure 18, the amount of glucose produced by the degradation of sucrose was statistically significantly reduced with the addition of CI-Dextran mix (p<0.05, P<0.0001). Furthermore, as shown in Figure 19, in the presence of sucrose, activity was almost completely eliminated at 0.25% CI-Dextran mix, and the activity reduction effect was greater than in the absence of sucrose. This indicates that cyclodextran binds to the CAT of GTFB in competition with sucrose, inhibiting the degradation of sucrose to glucose and fructose, and consequently suppressing glucan synthesis.

[0070] Similar tests were also performed using fructose and maltose instead of sucrose. As a result, with fructose, no loss of activity was observed when the CI-Dextran mix was used in combination with the monosaccharide, as shown in Figure 19. On the other hand, with maltose, a decrease in activity was observed when the CI-Dextran mix was used in combination with the disaccharide, although to a lesser extent than with sucrose.

[0071] [Test 8] Examination of intercellular binding strength by biofilm disruption test Streptococcus mutans MT8148 strain was cultured and dispensed into a FALCON® 6-well cell culture multiwell plate (Corning). CI-Dextran mix (Nissin Sugar Co., Ltd.) was added at concentrations ranging from 0% to 1.25% and cultured anaerobically at 37°C for 24 hours. After washing with PBS buffer, the plate was resuspended in PBS buffer and sonicated at level 7 for 2 minutes using a Handy Sonic model UR-21P. After washing with PBS buffer, the plate was resuspended in PBS buffer and the biofilm was detached using a cell scraper "Sumilon" (Sumitomo Bakelite Co., Ltd.). The detached biofilm was serially diluted with sterile saline and plated on Trypticase Soy agar (Becton Dickinson & Co.) and cultured anaerobically at 37°C for 2 days. For the control group, unsonicated biofilms were used. Similarly, bacterial cells were collected without sonication and inoculated onto Trypticase Soy agar medium. After incubation, the number of colonies was counted, and the ratio of the number of remaining bacteria after sonication to the total number of bacteria was calculated as an index of the intercellular binding strength of the bacteria. The results are shown in Figure 20.

[0072] As is clear from Figure 20, the number of bacteria in the biofilm decreased as the concentration of CI-Dextran mix increased. When comparing the cases where no CI-Dextran mix was added with the cases where 1% CI-Dextran mix was added, the number of bacteria in the biofilm was reduced by approximately 65% ​​(p < 0.01, Tukey).

[0073] [Study 9] Human trials to examine the effect of inhibiting oral plaque deposition The flowchart in Figure 21 shows the research outline for Study 9. In their daily lives, participants consumed water or cyclodextran solution (approximately 200 mL) three times a day with meals, plus 100 mL between meals and at bedtime. The study period was 7 days. The test beverage was a 1.2% CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) solution or water. Plaque scores and mutans streptococcus counts were evaluated before and after the study. The results are shown in Figure 22.

[0074] There were 17 subjects, but six were excluded because the number of mutans streptococci in saliva (total) was less than 103 CFU / mL, which was the exclusion criterion. The final number of volunteers who participated in the study was 11 (ages 31 to 59: average age 49.1). The number of mutans streptococci in the subjects was 1.1 x 10 4 ~6.4×10 5 CFU / mL, with an average of 2.9 × 10 4 CFU / mL. After the test, the plaque score per tooth surface was 1.55 for water and 0.97 for the CI-Dextran mixed aqueous solution, a significant difference (p<0.01, Fisher's PLSD). These results show that drinking the CI-Dextran mixed aqueous solution significantly inhibited plaque deposition compared to drinking water.

Claims

1. (A) an oligosaccharide having a cyclic structure whose constituent unit is glucose and / or a glucose derivative; and (B) a sugar other than the component (A), the component (A) is cyclodextran and / or a cyclodextran derivative, The caries-inhibiting composition is a cyclodextran derivative in which at least one of the alcoholic hydroxyl groups of cyclodextran is substituted with another polar group.

2. (A) an oligosaccharide having a cyclic structure whose constituent unit is glucose and / or a glucose derivative; and (B) a sugar other than the component (A), the component (A) is cyclodextran and / or a cyclodextran derivative, The cyclodextran derivative is a sweet composition in which at least one of the alcoholic hydroxyl groups of cyclodextran is substituted with another polar group.

3. (A) an oligosaccharide having a cyclic structure whose constituent unit is glucose and / or a glucose derivative; and (B) a sugar other than the component (A), the component (A) is cyclodextran and / or a cyclodextran derivative, The cyclodextran derivative is a food product in which at least one of the alcoholic hydroxyl groups of cyclodextran is substituted with another polar group.

4. (A) an oligosaccharide having a cyclic structure whose constituent unit is glucose and / or a glucose derivative; and (B) a sugar other than the component (A), the component (A) is cyclodextran and / or a cyclodextran derivative, The cyclodextran derivative is a supplement in which at least one of the alcoholic hydroxyl groups of cyclodextran is substituted with another polar group.

5. (A) an oligosaccharide having a cyclic structure whose constituent unit is glucose and / or a glucose derivative; and (B) a sugar other than the component (A), the component (A) is cyclodextran and / or a cyclodextran derivative, The dentifrice composition is characterized in that the cyclodextran derivative has at least one alcoholic hydroxyl group of the cyclodextran substituted with another polar group.

6. (A) an oligosaccharide having a cyclic structure whose constituent unit is glucose and / or a glucose derivative; and (B) a sugar other than the component (A), the component (A) is cyclodextran and / or a cyclodextran derivative, A mouthwash composition in which the cyclodextran derivative has at least one alcoholic hydroxyl group of cyclodextran substituted with another polar group.

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