Caries-inhibiting composition, and sweetening composition, food, supplement, dentifrice composition, and mouthwash composition using the same

Abstract

The present invention provides a caries-inhibiting composition and the like that can inhibit caries at a higher level than conventional anti-caries agents. [Solution] The composition of the present invention is a caries-inhibiting composition that contains (A) an oligosaccharide having a cyclic structure whose constituent units are glucose and / or a glucose derivative, and (B) a sugar other than the component (A), where the component (A) is cyclodextran and / or a cyclodextran derivative, and the cyclodextran derivative has at least one of the alcoholic hydroxyl groups of the cyclodextran substituted with another polar group. According to the present invention, the composition contains (A) an oligosaccharide having a specific cyclic structure whose constituent units are glucose and / or a glucose derivative, and (B) a sugar other than the component (A), which act in cooperation with each other to inhibit caries, and therefore can inhibit caries at a higher level than conventional anti-caries agents.

Description

[Technical field]

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

[0002] Dental caries refers to substantial loss of teeth 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 mouth. Streptococcus mutans, the causative bacterium of dental caries, is a type of gram-positive, facultative anaerobic streptococcus. Streptococcus mutans synthesizes glucan, a sticky polysaccharide, from sucrose using glucosyltransferase (GTF) that it produces. This glucan, together with other oral bacteria, forms a mass called plaque on the tooth surface, producing acid inside the mass, which erodes the tooth surface and demineralizes the tooth, resulting in the formation of cavities. If glucan is not formed, oral bacteria such as Streptococcus mutans are less likely to adhere to the tooth surface, which leads to the suppression of dental caries.

[0003] 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-dental 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, the present invention aims to provide a caries-inhibiting composition and the like that can inhibit dental caries at a higher level than conventional anti-caries agents. [Means for solving the problem]

[0006] As a result of intensive 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 the 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 at least one of the alcoholic hydroxyl groups of the cyclodextran is substituted with another polar group. Effect 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 to a higher level than conventional anti-caries agents. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is an example of the structural formula of cyclodextran (CI). [Diagram 2] FIG. 2 is an example of the structural formula of cyclodextrin (CD). [Diagram 3]Figure 3 shows the results of an investigation into the growth rate of Streptococcus mutans in the presence / absence of cyclodextran (Test 1). [Figure 4] Figure 4 shows the results of measurement of the amount of biofilm formed by Streptococcus mutans in the presence / absence of cyclodextran (Test 2). [Diagram 5] Figure 5 shows the results of [Test 3-1] confirmation of the biofilm structure by staining the bacterial cells and observing it under a microscope in [Test 3] observation of the biofilm structure caused by Streptococcus mutans. [Figure 6] Figure 6 shows the results of [Test 3-1] confirmation of the biofilm structure by staining the bacterial cells and observing it under a microscope in [Test 3] observation of the biofilm structure caused by Streptococcus mutans. [Figure 7] Figure 7 shows the results of [Test 3-2] the examination of glucan and bacterial amounts in biofilms in the observation of biofilm structure caused by Streptococcus mutans in [Test 3]. [Figure 8] Figure 8 shows the results of [Test 4-1] the 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 high-purity sample of cyclodextran. [Figure 10] FIG. 10 shows the results of [Test 5-1] examining the expression level of protein by Western blotting in [Test 5] Verification of the inhibitory mode 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 GTF activity inhibitory mode of 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 GTF activity inhibitory mode of cyclodextran. [Figure 14] FIG. 14 shows the results of [Test 6] investigating 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] investigating 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] investigating the inhibitory effect of adding cyclodextran to drinking water in a rat animal caries model on the development of caries caused by Streptococcus mutans. [Figure 17] FIG. 17 shows the results of [Test 6] investigating the plaque deposition inhibitory effect when cyclodextran was added to drinking water in a rat caries experimental model. [Figure 18] Figure 18 shows the test results of [Test 7] production of catalytic domain (CAT) recombinant protein. [Figure 19] Figure 19 shows the test results of [Test 7] production of catalytic domain (CAT) recombinant protein. [Figure 20] FIG. 20 shows the results of an investigation into the adhesive strength between bacteria in a biofilm disruption test (Test 8). [Figure 21] FIG. 21 is a flow chart showing the research outline for [Test 9] investigating the effect of inhibiting oral plaque deposition in human tests. [Figure 22] FIG. 22 shows the results of [Test 9] a human test to examine the effect of inhibiting oral plaque deposition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 modified as appropriate within the scope of the object of the present invention.

[0011] <Caries inhibition composition> The caries-inhibiting composition according to this embodiment contains (A) an oligosaccharide having a cyclic structure whose constituent units are 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 as a raw material by glucosyltransferase (GTF) produced by itself, which forms plaque on the tooth surface. The caries-inhibiting composition according to this embodiment contains (A) a cyclic oligosaccharide having glucose and / or a glucose derivative as a constituent unit, and (B) a sugar other than the component (A), and thus 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 having glucose and / or glucose derivatives as constituent units contained in the caries-inhibiting composition of this embodiment include cyclodextran and / or derivatives in which at least one of the alcoholic hydroxyl groups thereof has been replaced with another polar group (hereinafter, also referred to simply as "cyclodextran derivatives").

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

[0015] Furthermore, the term "glucose derivative" refers to the structural units constituting the above-mentioned cyclodextran derivatives other than glucose. Even if the substituent introduced into the cyclodextran derivative is introduced after the synthesis of cyclodextran from glucose, the structural units other than glucose that can be obtained by hydrolyzing the glycosidic bond of cyclodextran are also considered to be included in the term "glucose derivative". Therefore, the term "glucose derivative" does not mean 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 is 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 compounds preferred as derivatives of cyclodextran include branched cyclodextran in which some glucose residues are branched (alcoholic hydroxyl groups are replaced with glycosyl groups) and amino-substituted cyclodextran in which some glucose residues are replaced 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 replaced with an amino group are generally referred to as glucosamine residues.

[0018] For example, cyclodextran can be obtained from the culture medium of a microorganism of the genus Bacillus or from the reaction solution of cyclic isomaltooligosaccharide synthase (see Japanese Patent No. 3075873 and Japanese Patent No. 3117328; the contents of the reference documents are incorporated by reference as part of this specification, but the matters specifically described in this specification take precedence). When cyclodextran is obtained using a microorganism or an enzyme, it is obtained as a mixture of cyclodextran and branches of cyclodextran. This mixture of cyclodextran and branches of cyclodextran can also be separated into a composition having a cyclodextran concentration of 75% or more, preferably 85% or more, more preferably 99% or more, or into a composition having a cyclodextran branch concentration of 75% or more, preferably 85% or more, more preferably 99% or more, by using a conventionally known affinity chromatography or gel filtration chromatography, if necessary. 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 the term "cyclodextran" is mentioned, it refers to cyclodextran that is a cyclic isomaltooligosaccharide having no branched structure, and those having 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, while CD has 6 to 8 glucose units linked. Second, CI has glucose units linked by α-1,6-glycosidic bonds, while CD has glucose units linked by α-1,4-glycosidic bonds. Third, CI has a molecular structure with a large and shallow opening, while CD has a molecular structure with a small and deep opening. Fourth, CI has a high solubility in water, while CD has a low solubility in water.

[0022] [Component (B); sugar other than component (A)] The sugar other than component (A) contained in the caries-inhibiting composition according to 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 more) and refers to a monosaccharide having one aldehyde group at the end. More specifically, 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), talose (C6), and the like. Among these monosaccharides, glucose is particularly preferred.

[0025] For ketoses, the same as for 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) inside the chain structure. More specifically, 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] A disaccharide capable of producing a mixture containing aldose and / or ketose by hydrolysis refers to a disaccharide having monosaccharides that are aldose and / or ketose as constituent sugars, and more specifically, sucrose, lactose, maltose, trehalose, cellobiose, isomaltose, partinose, xylobiose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, etc. can be mentioned. Among these, it is preferable to adopt sucrose, lactose, and maltose as the above disaccharide, and it is preferable to adopt sucrose. Note that sucrose is a disaccharide in which glucose and fructose are bonded by α(1→2), lactose is a disaccharide in which glucose and galactose are bonded by β(1→4), and maltose is a disaccharide in which glucose and glucose are bonded by α(1→4).

[0027] In particular, it is preferable that the (B) component contains a disaccharide, and among the disaccharides, it is preferable that the (B) component contains a disaccharide having glucose and / or fructose as a constituent unit. When the caries-inhibiting composition contains a disaccharide having glucose and / or fructose as a constituent unit in addition to the (A) component, the (A) component and the disaccharide having glucose and / or fructose as a constituent unit act synergistically, and the caries-inhibiting effect is enhanced compared to the case of the (A) component alone. In addition, sucrose, which is one of the disaccharides having glucose and / or fructose as a constituent unit, is generally known to be a cause of caries, for example, being utilized as a nutrient source for oral bacteria such as Streptococcus mutans. Therefore, the fact that sucrose itself acts synergistically with the (A) component to enhance the caries-inhibiting 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 bonded by α-1,2-glycosidic bonds. Maltose is a disaccharide in which two glucoses are bonded by α-1,4-glycosidic bonds. Thus, sucrose and maltose have structural similarity with each other in that at least one constituent unit is glucose and is bonded by α-glycosidic bonds. It is most preferable to use sucrose as a disaccharide having glucose and / or fructose as constituent units to be incorporated in the caries-inhibiting composition according to this embodiment.

[0029] [Oligosaccharides] An oligosaccharide that can produce a mixture containing aldoses and / or ketoses upon hydrolysis refers to an oligosaccharide having monosaccharides that are aldoses and / or ketoses as constituent sugars, and more specifically includes cellobiose, trehalose, isomaltose, partinose, xylobiose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, galactooligosaccharides, fructooligosaccharides, lactosucrose, isomaltooligosaccharides, and kestose.

[0030] [Cyclic oligosaccharides] Cyclic oligosaccharides capable of producing a mixture containing aldoses and / or ketoses by hydrolysis refer to cyclic oligosaccharides having monosaccharides that are aldoses and / or ketoses as constituent sugars. Cyclosaccharides are typically cyclodextrins, and among them, cyclodextrins with 6, 7, or 8 glucose units are known. These cyclodextrins are sometimes called α-cyclodextrins (cyclohexaamylose, α-CD; 6 glucose units are bound), β-cyclodextrins (cycloheptaamylose, β-CD; 7 glucose units are bound), or γ-cyclodextrins (cyclooctaamylose, γ-CD; 8 glucose units are bound). However, cyclic oligosaccharides are not limited to cyclodextrins, and may be cyclic oligosaccharides having monosaccharides other than glucose as constituent sugars.

[0031] [Polysaccharide] A polysaccharide capable of producing a mixture containing aldoses and / or ketoses upon hydrolysis refers to a polysaccharide having monosaccharides that are aldoses and / or ketoses as constituent sugars, and more specifically, can 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. Also, galactogen is a polysaccharide whose constituent sugar is galactose.

[0032] [Other sugars] The sugar other than the component (A) contained in the caries-inhibiting composition according to this embodiment may be, in addition to the above-mentioned monosaccharides, disaccharides, oligosaccharides, cyclic oligosaccharides, and polysaccharides, amino sugars, deoxy sugars, sugar alcohols, lactones, and disaccharides, oligosaccharides, cyclic oligosaccharides, and polysaccharides having 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 components (A) and (B)] The blending ratio of the (A) component and the (B) component contained in the caries-inhibiting composition of this embodiment is such that the lower limit of the blending amount of the (A) component 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 the (B) component. (A) By satisfying the above conditions for the lower limit of the component ratio, Acidification in the oral cavity caused by the presence of an excess of component (B) is more easily suppressed, and the caries-inhibiting effect of the caries-inhibiting composition according to this embodiment is enhanced.

[0034] Furthermore, with regard to the blending ratio of the (A) component and the (B) component contained in the caries-inhibiting composition of this embodiment, the upper limit of the blending amount of the (A) component per 100 parts by mass of the (B) component 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, even more preferably 10 parts by mass or less, and particularly preferably 5 parts by mass or less. (A) By satisfying the above conditions, The synergistic effect of component (A) and component (B) in inhibiting dental caries is further enhanced, and the caries-inhibiting effect of the caries-inhibiting composition according to this embodiment is enhanced.

[0035] <Sweetening composition, food, and supplement containing caries-inhibiting composition> The caries-inhibiting composition according to the present embodiment may be formulated as a sweet composition, food, or supplement. By formulating the caries-inhibiting composition according to the present embodiment as a sweet composition, food, or supplement, it is possible to provide a sweet composition, food, or supplement that is unlikely 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, pressurized and heated 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, 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 milk powder, prepared milk powder, prepared liquid milk, fermented milk, lactic acid bacteria beverages, and pet foods.

[0037] Furthermore, examples of 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 included in sweetening compositions, foods, and supplements] The sweetening composition, food, and supplement containing the caries-inhibiting composition according to the present embodiment may be classified as a food additive, food, or nutritional supplement, and may contain additives other than the (A) component and the (B) component that may be generally used in the fields of food additives, food, and nutritional supplements. Such additives are not particularly limited as long as they do not impair the object of the present invention, and examples of such additives include acidulants, enzymes, spices, colorants, preservatives, flavorings, sweeteners, seasonings, emulsifiers, thickeners, stabilizers, foaming agents, leavening agents, colorants, oxidizing agents, preservatives, confectionery gelatin, confectionery albumin, confectionery starch, bactericides, antioxidants, and fungicides. In addition, the sweetening composition, food, and supplement containing the caries-inhibiting composition according to the present embodiment may contain linear, branched, or cyclic polysaccharides having a structure similar to that of the (A) component, as long as they do not impair the object of the present invention.

[0039] <Caries-inhibiting agent, dentifrice composition, and mouthwash composition containing the caries-inhibiting composition> The caries-inhibiting composition according to the present embodiment may be configured as a caries-inhibiting agent, a dentifrice composition, or a mouthwash composition. By configuring the caries-inhibiting composition according to the present embodiment as a sweetening composition, food, or supplement, a user who uses it can effectively inhibit caries. These caries-inhibiting agents, dentifrice compositions, and mouthwash compositions may be classified as medicines, quasi-drugs, or cosmetics, and may contain medicinal ingredients or beauty ingredients.

[0040] [Other components 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 necessary to treat, inhibit, or prevent oral diseases such as halitosis, periodontal disease, and hypersensitivity, or may strengthen tooth structure. Examples of medicinal ingredients other than the (A) and (B) ingredients that may be contained in the caries inhibitor, dentifrice composition, and mouthwash composition according to the present embodiment include conventionally used ingredients such as fluorine, xylitol, propolis, Swertia japonica extract, and hydroxyapatite. Specific examples 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 additive commonly used in the past depending on the embodiment. Such additives are not limited as long as they do not impede the object of the present invention, and may include abrasives, binders, colorants, flavorings, sweeteners, etc. 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) within the scope of not impeding the object of the present invention. EXAMPLES

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

[0043] In addition, the preparation of various media, buffer solutions, and reagents, and the determination of test protocols were carried out with reference to the descriptions in Michael R. Green et al., "Molecular Cloning," Cold Spring Harbor Laboratory Press (2012). The contents of the references are incorporated as part of this specification by reference, but the descriptions in this specification take precedence over any matters specifically described in this specification.

[0044] [Test 1] Examination of the growth rate of Streptococcus mutans in the presence / absence of cyclodextran 100μL of the test solution of Streptococcus mutans MT8148 strain, which had been cultured in Todd-Hewitt liquid medium (hereinafter referred to as "TH liquid medium") at 37℃ for 18 hours, was subcultured in 10mL of fresh Todd-Hewitt (TH) liquid medium. CI-Dextran mix (a dextran mixture containing 13% or more of cyclodextran, manufactured by Nisshin Sugar Co., Ltd.; the same applies below) was added to the medium so that the final concentration was 0% to 1%. The turbidity of the solution was measured over time every hour at a wavelength of 570nm using a visible spectrophotometer (Novaspec plus, manufactured by Amersham Biosciences). The results are shown in Figure 3.

[0045] As is clear from Figure 3, the bacterial growth eventually 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 the amount of biofilm formed by Streptococcus mutans in the presence / absence of cyclodextran Test bacteria of Streptococcus mutans MT8148 strain were cultured in 10mL of TH liquid medium at 37℃ for 18 hours, and the cultured test bacteria liquid was diluted in TH liquid medium supplemented with 0.25% sucrose, and 100μL was dispensed into each well of a FALCON (registered trademark) 96-well flat-bottom cell culture microtiter plate (manufactured by Corning) and cultured anaerobically at 37℃ for 2 days. At that time, CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) was added to the medium so that the final concentration was 0.16% to 10%. After removing the floating bacteria liquid from each well, 25μL of 1% Crystal violet solution (manufactured by Nacalai Tesque, Inc.) was added and left to stand at room temperature for 15 minutes to stain the attached bacteria, and the plate was washed six times with distilled water. Thereafter, 95% ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to wash the sample, and after drying, distilled water was added and the absorbance at a wavelength of 570 nm was measured. 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 to which CI-Dextran mix was not added and the sample to which CI-Dextran mix was added at a given concentration, the difference between the sample to which CI-Dextran mix was not added and all the samples to which CI-Dextran mix was added at a given concentration 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 bacteria and observing under a microscope Streptococcus mutans MT8148 strain was cultured in TH liquid medium at 37°C for 18 hours, and then the cells were collected 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 cell samples. These cell solutions and each sample were inoculated in 200 μL portions into a polystyrene 8-well 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 a final concentration of 0% to 10%. The formed biofilm was 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 at a concentration of 0.625% or more 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 fluorescent nucleic acid staining dye SYTO® 9 green and then adjusted in CDM medium containing 1% sucrose. Alexa Fluor® 647-labeled dextran was added and then seeded on a chamber slide. At the start of the culture, CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) was added to the medium so that the final concentration was 0% to 10%, and after anaerobic culture at 37°C for 24 hours, the formed biofilm was observed with a confocal scanning laser microscope LSM780 (Version 4.2, manufactured by Carl Zeiss Microimaging). Microscopic photographs are shown in Figure 7.

[0051] As is clear from FIG. 7, in the presence of cyclodextran, both the amount of glucan and the amount of bacteria in the biofilm were significantly reduced, 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 synthesis enzyme 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 a phosphate buffer solution containing 1% sucrose at concentrations ranging from 0% to 1%. 10 μL of rGTFB (recombinant GTFB) was added and 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 activity inhibitory effect of rGTFB. The results are shown in Figure 8.

[0053] As is clear from 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 CI-Dextran mix concentration-dependent manner. When a significance test was performed between samples without CI-Dextran mix and samples with CI-Dextran mix, the difference between samples without CI-Dextran mix and all samples with CI-Dextran mix added at a given concentration was statistically significant (p<0.001, Fisher's PLSD).

[0054] [Test 4-2] Verification using a high-purity sample of cyclodextran The inhibitory effect on glucan synthase activity was examined in the same manner as in [Test 4], except that a high-purity sample of cyclodextran was used instead of the CI-Dextran mix. The results are shown in Figure 9. The cyclodextran was obtained by fractionating the above-mentioned CI-Dextran mix by chromatography. Among the samples shown in Figure 9, those represented as CI7, CI8, CI9, C10, and C11 are samples that mainly contain oligosaccharides having a cyclic structure in which 7, 8, 9, 10, and 11 glucose units are linked, respectively.

[0055] As is clear from Figure 9, like the above CI-Dextran mix, the high-purity sample of cyclodextran also effectively inhibited the synthesis of water-insoluble glucan, but the high-purity sample of cyclodextran 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 (manufactured by Nisshin Sugar Co., Ltd.) and sucrose at concentrations from 0% to 10%, and the cells were collected by centrifugation. Equal amounts of 2x SDS-PAGE buffer were mixed and heated to prepare SDS-PAGE samples. Electrophoresis was performed using 10% acrylamide for the separation gel and 3% for the concentration gel. After electrophoresis, the acrylamide gel layer was transferred to a transfer membrane by applying electricity and reacted with rabbit anti-CA-GTF antibody at room temperature for 1 hour. After the reaction, the membrane was washed and reacted with alkaline phosphatase-labeled goat anti-rabbit immunoglobulin at room temperature for another 1 hour, and the bands were visualized by adding a color-developing 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, which is the molecular weight of the GTF protein, was observed at 0.25% or more. 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 more.

[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 electrophoresed using SDS-PAGE. The gel was then shaken for 18 hours at 37°C in a solution containing sucrose and Triton-X. 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 is clear from Figure 11, first, the activity of GTF was examined using sucrose and CI-Dextran mix alone. Strong activity was observed up to 0.125% with sucrose alone, and activity decreased at 0.25% or more, but similar activity was observed between 0.25% and 1.5%. On the other hand, a concentration-dependent increase in GTF activity was observed with 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 when the sucrose concentration was 0.5% or more compared to when the sucrose concentration was 0.25% or less.

[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 culturing at 37° C. for 16 hours, the absorbance of the synthesized water-insoluble glucan was measured at 550 nm. The results are shown in FIG. 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 decreased by about 50%.

[0062] Based on these results, we performed an enzyme kinetic analysis of the inhibitory effect of rGTFB on glucan synthase activity using a Lineweaver-Burk plot. The results are shown in Figure 13. In the Lineweaver-Burk plot, the point where the straight line intersects with the Y axis represents 1 / Vmax, and the slope is K m / V max To represent this, let us use 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; the reaction rate is V maxThe substrate concentration at half the concentration of V was 1.54 mM. max is 0.57 nmol / min, and K m The value was 4.43 mM.

[0063] From these results, the inhibition mode of enzyme activity by cyclodextran is speculated 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 an enzyme and prevents the binding of the substrate. Since the substrate and inhibitor compete to bind to the active site, K m The value is larger, but V max does not change. Noncompetitive inhibition is a type of inhibition in which an inhibitor binds to a site other than the active site of an enzyme and changes the enzyme's three-dimensional structure, preventing substrate binding. Since the inhibitor binds to a site other than the active site, there is no change in the affinity for the substrate, and the K m The value does not change, but the enzyme's conformation changes, so V max In competitive inhibition, the inhibitor binds to the enzyme-substrate complex and inhibits the reaction. Since the inhibitor binds after the enzyme and substrate bind, the K m Value and V max The test results show that K m The value becomes larger and becomes 1 / V max Since there was no change in the GTFB activity, it was suggested that the inhibitory effect of cyclodextran on GTFB was competitive inhibition.

[0064] [Test 6] Examination of the inhibitory effect of cyclodextran on the development of caries caused by Streptococcus mutans and on the suppression of plaque deposition in rat animal caries models The experimental animals were male Sprague Dawley rats that were free of specific microorganisms and parasites (SPF) and were forcibly weaned on the 15th day of life. Ten rats were used in each group for the experiment. For two days, on the 15th and 16th days of life, the oral cavity of the rats was treated with antibiotics to facilitate the establishment of the test bacteria. After that, for five days, from the 18th to 22nd days of life, Streptococcus mutans MT8148 R strain was cultured in Brain Heart Infusion (BHI) liquid medium at 37°C for 18 hours, centrifuged, and suspended in 1 / 100 volume of sterile saline. 100μL of the inoculum was directly inoculated into the rat cavity with a micropipette once a day to infect the rats. From the day of infection with the test bacteria, the rats were fed a cariogenic diet containing 56% sucrose, Diet 2000, with CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) at 0.625%, 1.25%, 2.5%, or 5%, and were allowed to consume the diet ad libitum until the end of the experiment. Distilled water was allowed to be consumed ad libitum. On the 72nd day after birth, the rats were killed under chloroform anesthesia, the jawbone was aseptically removed, the maxillary teeth were stained with erythrosine, and plaque was determined by the Regorati and Hotz method. In addition, the caries scores of the upper and lower molars were calculated using the Keyes method, modified for rats by Ooshima et al. Similarly, a test was conducted in which the rats were fed a cariogenic diet containing 56% sucrose, Diet 2000, with CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) at 0.625%, 1.25%, 2.5%, or 5%, and were allowed to consume the cariogenic diet. 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. The inhibition of plaque deposition was also significantly reduced when CI-Dextran mix was added at a low concentration of 0.625% (p<0.01, Tukey). From these results, it is believed that CI-Dextran mix is ​​able to inhibit the occurrence of dental caries by inhibiting plaque deposition.

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

[0067] Based on the entire base sequence of the gtfB gene of Streptococcus mutans UA159 strain, the CAT domain portion was amplified by PCR, and then the protein expression primers pGEX6P-1(CAT)-F and pGEX6P-1(CAT)-R were designed to insert this DNA fragment into the plasmid pGEX6P-1. The DNA fragment obtained by PCR using these protein expression primers was inserted into the plasmid pGEX6P-1 using PrimeSTAR (registered trademark) Max DNA Polymerase (manufactured by Takara Bio Inc.) to produce the plasmid pGEX6P-1(CAT). pGEX6P-1(CAT) was transformed into the protein expression strain E. coli BL21, and after mass cultivation, the cells obtained by centrifugation were suspended in PBS buffer and then sonicated to destroy the cell wall of E. coli. The supernatant was obtained by centrifugation and then purified on a Glutathione Sepharose 4B (registered trademark) column (GE Healthcare), and the GST-rGTFB(CAT) was used in the following experiment.

[0068] CI-Dextran mix (manufactured by Nisshin 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 enzyme reaction was carried out at 37°C for 1 hour. Somogyi copper solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added and boiled for 20 minutes, after which Nelson solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added and the mixture was allowed to stand for 15 minutes. The absorbance at a wavelength of 500 nm was then measured. The results are shown in Figures 18 and 19.

[0069] As is clear from Figure 18, the amount of glucose produced by glucose production accompanying the decomposition of sucrose was statistically significantly decreased with the addition of CI-Dextran mix (p<0.05, P<0.0001). Also, as is clear from Figure 19, in the presence of sucrose, activity was almost completely lost at 0.25% CI-Dextran mix, and the activity reduction effect was higher than in the absence of sucrose. From this, it was revealed that cyclodextran inhibits the decomposition of sucrose into glucose and fructose by binding to CAT of GTFB in competition with sucrose, and as a result, it suppresses the synthesis of glucan.

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

[0071] [Test 8] Examination of intercellular adhesion strength by biofilm disruption test After culturing Streptococcus mutans MT8148 strain, it was dispensed into FALCON (registered trademark) 6-well cell culture multi-well plate (manufactured by Corning), CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) was added at an amount of 0% to 1.25%, and cultured at 37 ° C under anerobic conditions for 24 hours. After washing with PBS buffer, it was resuspended in PBS buffer and sonicated at level 7 for 2 minutes with Handy Sonic model UR-21P. After washing with PBS buffer, it was resuspended in PBS buffer and the formed biofilm was peeled off using a cell scraper "Sumiron" (manufactured by Sumitomo Bakelite Co., Ltd.). The peeled biofilm was serially diluted with sterile saline, inoculated on Trypticase Soy agar medium (manufactured by Becton Dickinson & Co., Ltd.), and cultured at 37 ° C under anerobic conditions for 2 days. For the control group, biofilms not treated with ultrasound were used, and the cells were similarly collected without ultrasound treatment and inoculated on Trypticase Soy agar medium. After cultivation, the number of colonies was counted, and the ratio of the number of remaining cells after ultrasound treatment to the number of cells was calculated as an index of the binding strength between the cells of the bacteria. The results are shown in Figure 20.

[0072] As is clear from Figure 20, as the concentration of CI-Dextran mix increased, the number of bacteria in the biofilm decreased. When comparing the case where CI-Dextran mix was not added with the case where it was added at 1%, the number of bacteria in the biofilm was reduced by about 65% when CI-Dextran mix was added at 1% (p < 0.01, Tukey).

[0073] [Study 9] Human trials to evaluate the effect of inhibiting oral plaque deposition The flow chart in Figure 21 shows the research outline for Test 9. In daily life, subjects drank water or cyclodextran solution (approximately 200 mL) three times a day with meals, and also drank 100 mL between meals and at bedtime. The test period was 7 days. The test beverage was a 1.2% CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) aqueous solution or water, and the plaque score and mutans streptococcus count were evaluated before and after the test. The results are shown in Figure 22.

[0074] There were 17 subjects, but 6 were excluded because the total number of mutans streptococci in saliva 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, the average was 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 mix aqueous solution, a significant difference (p<0.01, Fisher's PLSD). These results show that drinking the CI-Dextran mix aqueous solution significantly inhibited plaque deposition compared to drinking water.

Claims

1. (A) cyclodextran; and (B) sucrose; A caries-inhibiting composition (excluding those containing a hop extract) in which the ratio of the (A) component to 100 parts by mass of the (B) component is 100 parts by mass or less.

2. (A) cyclodextran; and (B) sucrose; A sweetening composition (excluding those containing a hop extract) in which the ratio of the (A) component to 100 parts by mass of the (B) component is 10 parts by mass or less.

3. (A) cyclodextran; and (B) sucrose; A food (excluding those containing a hop extract), in which the ratio of the (A) component to 100 parts by mass of the (B) component is 10 parts by mass or less.

4. (A) cyclodextran; and (B) sucrose; A supplement (excluding those containing hop extract) in which the ratio of the (A) component to 100 parts by mass of the (B) component is 10 parts by mass or less.

5. (A) cyclodextran; and (B) sucrose; A dentifrice composition (excluding those containing hop extract), in which the ratio of the (A) component to 100 parts by mass of the (B) component is 100 parts by mass or less.

6. (A) cyclodextran; and (B) sucrose; A mouthwash composition (excluding those containing hop extract), in which the ratio of the (A) component to 100 parts by mass of the (B) component is 100 parts by mass or less.

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