JP7640771B1 - Caries-inhibiting composition, and sweetening composition, food, supplement, dentifrice composition, and mouthwash composition using the same - Google Patents

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), the component (A) being cyclodextran and/or a cyclodextran derivative, and the cyclodextran derivative is a composition in which at least one of the alcoholic hydroxyl groups of the cyclodextran is substituted with another polar group. According to the present invention, the composition contains (A) an oligosaccharide having a predetermined cyclic structure whose constituent units are glucose and/or a glucose derivative, and (B) a sugar other than the component (A), and these act in cooperation with each other to inhibit caries, so that caries can be inhibited at a higher level than conventional anti-caries agents.
[Selected Figure] Figure 1

Description

The present invention relates to a caries-inhibiting composition, as well as a sweetening composition, a food product, a supplement, a dentifrice composition, and a mouthwash composition that use the same.

Dental caries refers to substantial tooth loss caused by biological factors, and it is known that the main cause is decalcification of the teeth due to acids produced from carbohydrates by bacteria in the oral cavity.
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 dental caries. 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.

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-dental caries agent containing cyclic isomaltooligosaccharide as an active ingredient is known (see Patent Document 1).

Patent No. 3400868

On the other hand, in recent years, it has been pointed out that oral diseases such as dental caries may affect diseases other than those in the oral cavity, and the medical importance of inhibiting dental caries is increasing. Therefore, the present invention aims to provide a caries-inhibiting composition etc. that can inhibit dental caries at a higher level than conventional anti-caries agents.

As a result of extensive research, the inventors have found that the above 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.

The invention according to the first aspect provides a caries-inhibiting composition that contains (A) an oligosaccharide having a cyclic structure with glucose and/or a glucose derivative as a constituent unit, 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.

The caries-inhibiting composition of the present invention contains (A) a specific cyclic oligosaccharide whose constituent unit is glucose, 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.

FIG. 1 is an example of the structural formula of cyclodextran (CI). FIG. 2 is an example of the structural formula of cyclodextrin (CD). FIG. 3 shows the results of [Test 1] an investigation into the growth rate of Streptococcus mutans in the presence/absence of cyclodextran. FIG. 4 shows the results of [Test 2] measurement of the amount of biofilm formed by Streptococcus mutans in the presence/absence of cyclodextran. FIG. 5 shows the results of [Test 3-1] confirmation of biofilm structure by staining of cells and microscopic observation in [Test 3] observation of biofilm structure caused by Streptococcus mutans. FIG. 6 shows the results of [Test 3-1] confirmation of biofilm structure by staining of cells and microscopic observation in [Test 3] observation of biofilm structure caused by Streptococcus mutans. FIG. 7 shows the results of [Test 3-2] examining the amount of glucan and bacteria in a biofilm in the observation of a biofilm structure caused by Streptococcus mutans in [Test 3]. FIG. 8 shows the results of [Test 4-1] verification of the inhibitory effect on the activity of glucan synthase of Streptococcus mutans, using a commercially available dextran mixture containing 13% or more of cyclodextran. FIG. 9 shows the results of [Test 4-2] verification of the inhibitory effect on the activity of glucan synthase of Streptococcus mutans using a high purity sample of cyclodextran. FIG. 10 shows the results of [Test 5-1] examining the expression level of protein by Western blotting in [Test 5] Verification of the GTF activity inhibitory mode of cyclodextran. 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. 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. 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. 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. 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. 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. 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. FIG. 18 shows the test results of [Test 7] production of catalytic domain (CAT) recombinant protein. FIG. 19 shows the test results of [Test 7] production of catalytic domain (CAT) recombinant protein. FIG. 20 shows the results of investigating the intercellular adhesive strength in a biofilm disruption test (Test 8). FIG. 21 is a flow chart showing the research outline for [Test 9] investigating the effect of inhibiting oral plaque deposition in human tests. FIG. 22 shows the results of [Test 9] a human test to examine the effect of inhibiting oral plaque deposition.

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 present invention.

<Caries-inhibiting 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).

As described above, Streptococcus mutans synthesizes glucan, a sticky polysaccharide, from sucrose as a raw material using glucosyltransferase (GTF) that it produces itself, which results in the formation of plaque on the tooth surface. The caries-inhibiting composition according to this embodiment contains (A) cyclic oligosaccharides whose constituent units are glucose and/or glucose derivatives, and (B) sugars other than the component (A), and thus can exhibit a higher caries-inhibiting effect than a composition containing only (A) cyclic oligosaccharides whose constituent units are glucose and/or glucose derivatives.

[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 substituted with another polar group (hereinafter, also referred to simply as "cyclodextran derivatives").

Among these, cyclodextran derivatives include both compounds found in nature and compounds that are artificially synthesized. Examples of polar groups that may replace the alcoholic hydroxyl groups of cyclodextran include, but are not limited to, chloro, bromo, iodine, alkoxyl, glycosyl, thiol, sulfide, selenol, amino, amide, and phosphate groups. These polar groups may replace a single hydroxyl group in 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.

Furthermore, the term "glucose derivative" refers to the structural units that make up the above-mentioned cyclodextran derivatives other than glucose. Even if the substituent introduced into the cyclodextran derivative is introduced after cyclodextran is synthesized from glucose, the structural units that can be obtained by hydrolyzing the glycosidic bond of cyclodextran other than glucose 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.

[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.

Specific examples of compounds that are 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 have their hydroxyl groups 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.

As an example, cyclodextran can be obtained from the culture medium of a microorganism of the genus Bacillus or the reaction solution of cyclic isomaltooligosaccharide synthase (see Patent No. 3075873 and 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 be separated into a composition having a cyclodextran concentration of 75% or more, preferably 85% or more, more preferably 99% or more, or 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, etc., as necessary. The caries-inhibiting composition according to 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.

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".

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

CI and CD have in common 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 that is large and shallow, while CD has a molecular structure that is small and deep. Fourth, CI has high solubility in water, while CD has low solubility in water.

[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.

[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.

Among these, aldose refers to a monosaccharide represented by _CnH2nOn (where _n is _an integer of 3 or more) and 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.

Ketoses refer to monosaccharides that are expressed as _CnH2nOn (where _n is _an integer of 3 or more) like aldoses, but have 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.

[Disaccharide]
The 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. Among these, it is preferable to adopt sucrose, lactose, and maltose as the 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).

In particular, it is preferable that component (B) contains a disaccharide, and among the disaccharides, it is preferable that component (B) 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 component (A), component (A) and the disaccharide having glucose and/or fructose as a constituent unit act in cooperation with each other, and the caries-inhibiting effect is enhanced compared to the case of component (A) alone. Note that sucrose, one of the disaccharides having glucose and/or fructose as constituent units, is generally known to be a cause of caries, for example, being used as a nutrient source for oral bacteria such as Streptococcus mutans. For this reason, the fact that sucrose itself acts in cooperation with component (A) to enhance the caries-inhibiting effect is far beyond the scope of predictions of those skilled in the art.

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 in the caries-inhibiting composition according to this embodiment.

[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.

[Cyclic oligosaccharides]
A cyclic oligosaccharide capable of producing a mixture containing aldoses and/or ketoses upon hydrolysis refers to a cyclic oligosaccharide having monosaccharides that are aldoses and/or ketoses as constituent sugars. The above-mentioned cyclodextrin is a representative example of a cyclic oligosaccharide, and among these, cyclodextrins having six, seven, or eight glucose units bound thereto are known. These cyclodextrins are sometimes called α-cyclodextrin (cyclohexaamylose, α-CD; six glucose units bound thereto), β-cyclodextrin (cycloheptaamylose, β-CD; seven glucose units bound thereto), or γ-cyclodextrin (cyclooctaamylose, γ-CD; eight glucose units bound thereto). However, the cyclic oligosaccharide is not limited to cyclodextrin, and may be a cyclic oligosaccharide having monosaccharides other than glucose as constituent sugars.

[Polysaccharides]
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.

[Other sugars]
As the sugar other than the component (A) contained in the caries-inhibiting composition according to this embodiment, 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 may be used. 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 the 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.

[Mixing ratio of component (A) and component (B)]
The blending ratio of the (A) component and the (B) component contained in the caries-inhibiting composition according to this embodiment is preferably such that the lower limit of the blending amount of the (A) component is 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. When the lower limit of the ratio of the (A) component satisfies the above condition, acidification in the oral cavity caused by the presence of an excess of the (B) component is easily suppressed, and the caries-inhibiting effect of the caries-inhibiting composition according to this embodiment is enhanced.

Furthermore, with regard to the blending ratios of the (A) component and the (B) component contained in the caries-inhibiting composition according to this embodiment, the upper limit of the blending amount of the (A) component relative to 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, still more preferably 10 parts by mass or less, and particularly preferably 5 parts by mass or less. When the upper limit of the ratio of the (A) component satisfies the above condition, the synergistic action of the (A) component and the (B) component in the caries-inhibiting effect is further enhanced, and the caries-inhibiting effect of the caries-inhibiting composition according to this embodiment is enhanced.

<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.

Among these, foods containing caries-inhibiting compositions are not particularly limited and include not only finished products but also semi-finished products. For example, 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, chocolate confectionery, semi-chocolate confectionery, edible films, candies (gummy candy, 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, pet food, etc.

In addition, examples of supplements containing caries-inhibiting compositions include base supplements, health supplements, optional supplements, etc., and examples of functional nutritional 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.

[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 purpose of the present invention, and may 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 purpose of the present invention.

<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.

[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.

The caries inhibitor, dentifrice composition, and mouthwash composition of this embodiment may contain any additive that is conventionally used depending on the embodiment. Such additives are not limited, so 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 of this embodiment may contain linear, branched, or cyclic polysaccharides similar in structure to component (A) to the extent that they do not impede the object of the present invention.

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

In addition, when 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 as part of this specification by reference, but the descriptions in this specification take precedence over any matters specifically described in this specification.

[Test 1] Examination of the growth rate of Streptococcus mutans in the presence/absence of cyclodextran
100 μL of test bacterial solution of Streptococcus mutans MT8148 strain, which had been cultured in Todd-Hewitt liquid medium (hereinafter also referred to as "TH liquid medium") at 37°C for 18 hours, was subcultured in 10 mL 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 this bacterial solution was measured over time every hour using a visible spectrophotometer (Novaspec plus, manufactured by Amersham Biosciences) at a wavelength of 570 nm. The results are shown in FIG. 3.

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, a clear concentration-dependent delay in the growth rate was observed when cyclodextran was added.

[Test 2] Measurement of the amount of biofilm formed by Streptococcus mutans in the presence/absence of cyclodextran After culturing the test bacteria of Streptococcus mutans MT8148 strain in 10 mL of TH liquid medium at 37 ° C for 18 hours, the cultured test bacteria liquid was diluted in 0.25% sucrose-added TH liquid medium, and 100 μL 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 ° C 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, 25 μL of 1% Crystal violet solution (manufactured by Nacalai Tesque Co., Ltd.) was added and left to stand at room temperature for 15 minutes to stain the attached bacteria, and 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.

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

[Test 3] Observation of biofilm structure by Streptococcus mutans [Test 3-1] Confirmation of biofilm structure by staining of bacteria and microscopic observation Streptococcus mutans MT8148 strain was cultured in TH liquid medium at 37 ° C. for 18 hours, and then the bacteria were collected by centrifugation. The bacteria were stained with 10 mM hexidium iodide (Invitrogen (trademark), Thermo Fisher Scientific Co., Ltd.) and adjusted to a turbidity of 0.1 at a wavelength of 600 nm in a chemically synthesized medium containing 0.5% sucrose to prepare a live bacterial sample. These bacterial solutions and each sample were seeded in 200 μL each in a polystyrene 8-hole Lab-Tek chamber slide system (Nunc (trademark), Thermo Fisher Scientific Co., Ltd.) and cultured at 37 ° C. for 24 hours. CI-Dextran mix (Nissin Sugar Co., Ltd.) was added to the medium so that the final concentration was 0% to 10%. The formed biofilm was observed with a confocal scanning laser microscope LSM780 (Version 4.2, Carl Zeiss Microimaging). Furthermore, the density was calculated using ImageJ (registered trademark). The microscopic image observed with the confocal scanning laser microscope is shown in FIG. 5, and the density of the biofilm calculated using ImageJ is shown in FIG. 6.

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 samples without CI-Dextran mix and samples with CI-Dextran mix added at a concentration of 0.625% or more was statistically significant (Fisher's PLSD).

[Test 3-2] Examination of glucan and bacterial amount in biofilm Streptococcus mutans MT8148 strain was stained with fluorescent nucleic acid staining dye SYTO (registered trademark) 9 green, and then adjusted in 1% sucrose-containing CDM medium. Alexa Fluor (registered trademark) 647-labeled dextran was added and then seeded on a chamber slide. CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) was added to the medium so that the final concentration was 0% to 10% at the timing of starting the culture, 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 FIG. 7.

As is clear from Figure 7, in the presence of cyclodextran, the amount of glucan and the amount of bacteria in the biofilm decreased significantly, and the amount of glucan and the amount of bacteria tended to decrease more as the amount of cyclodextran added increased.

[Test 4] Verification of the activity inhibitory effect 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 a phosphate buffer containing 1% sucrose so that the concentrations ranged from 0% to 1%. 10 μL of rGTFB (recombinant GTFB) was added and cultured at 37°C for 16 hours. After culture, the synthesized water-insoluble glucan was measured at an absorbance of 550 nm to verify the activity inhibitory effect of rGTFB. The results are shown in Figure 8.

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 the specified concentrations was statistically significant (p<0.001, Fisher's PLSD).

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

As is clear from Figure 9, like the CI-Dextran mix above, the high-purity sample of cyclodextran also effectively inhibited the synthesis of water-insoluble glucan, but the high-purity sample of cyclodextran that mainly contains CI7, in which seven glucose units are linked, was particularly effective.

[Test 5] Verification of the GTF activity inhibition mode of cyclodextran [Test 5-1] Examination of protein expression level by Western blotting Streptococcus mutans MT8148 strain was cultured to stationary phase in TH liquid medium to which CI-Dextran mix (manufactured by Nisshin Sugar Co., Ltd.) and sucrose were added at concentrations of 0% to 10%, and then 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 acrylamide concentrations of 10% for separation gel and 3% for 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 a color-developing substrate was added to develop color, and the bands were visualized. The results are shown in Figure 10.

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.

[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 a paranose aniline solution. The results are shown in Figure 11.

As is clear from Figure 11, first, the activity of GTF was examined in sucrose and CI-Dextran mix alone. Strong activity was observed up to 0.125% with sucrose alone, and although activity decreased at 0.25% or higher, 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 higher compared to when the sucrose concentration was 0.25% or lower.

[Test 5-3] Verification of the effect on enzyme activity from the viewpoint 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 absorbance of the synthesized water-insoluble glucan was measured at 550 nm. The results are shown in FIG. 12.

12, in the CI-added group (with CI), the maximum velocity _Vmax decreased by about 50%, and the final amount of glucan synthesis decreased.

Based on this result, the enzyme reaction kinetics analysis of the inhibitory effect of rGTFB on glucan synthase activity was performed using a Lineweaver-Burk plot. The results are shown in FIG. 13. In the Lineweaver-Burk plot, the point where the straight line intersects with the Y axis represents 1/Vmax, and the slope represents _Km / _Vmax , from which _Vmax and _Km values can be obtained. In the CI-free group (without CI), the maximum reaction rate ( _Vmax ) was 0.44 nmol/min, and the Michaelis constant ( _Km value; substrate concentration when the reaction rate is 1/2 of _Vmax ) was 1.54 mM. On the other hand, in the CI-added group (with CI), _Vmax was 0.57 nmol/min, and the _Km value was 4.43 mM.

From these results, the inhibition mode of enzyme activity by cyclodextran is inferred as follows. There are three types of inhibition modes 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 a substrate. Since the substrate and the inhibitor compete to bind to the active site, the _Km value increases, but _Vmax does not change. Uncompetitive inhibition is an inhibition mode in which an inhibitor binds to a site other than the active site of an enzyme and changes the three-dimensional structure of the enzyme to prevent the binding of a substrate. Since the inhibitor binds to a site other than the active site, there is no change in the affinity of the substrate and the _Km value does not change, but the three-dimensional structure of the enzyme changes, so _Vmax decreases. Uncompetitive inhibition is an inhibition mode in which an inhibitor binds to an enzyme-substrate complex and prevents a reaction. Since the inhibitor binds after the enzyme and substrate bind, both the _Km value and _Vmax change. The test results showed that the _Km value increased but 1/ _Vmax did not change, suggesting that the inhibitory effect of cyclodextran on GTFB is likely to be competitive inhibition.

[Test 6] Study on the inhibitory effect on the development of caries caused by Streptococcus mutans and the inhibitory effect on plaque deposition when cyclodextran is added to the feed and drinking water in a rat animal caries model. Male Sprague Dawley rats free of specific microorganisms and parasites (SPF), which were forcibly weaned on the 15th day after birth, were used as experimental animals, and 10 rats were used in each group for the experiment. For two days, on the 15th and 16th days after birth, the oral cavity was treated with antibiotics to facilitate the establishment of the test bacteria. After that, for five days from the 18th to 22nd days after birth, 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, and 100 μL of the inoculum was directly inoculated into the rat cavity using a micropipette once a day to infect the rat. 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%, and 5%, and were allowed to consume the diet freely until the end of the experiment. Distilled water was allowed to be consumed freely. On the 72nd day after birth, the rats were killed under chloroform anesthesia, the jawbone was aseptically extracted, the maxillary teeth were stained with erythrosine, and plaque was determined by the Regorati and Hotz method. Furthermore, the caries scores of the upper and lower molar teeth were calculated by the Keyes method, which was 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%, and 5% added to distilled water, and were allowed to consume the cariogenic diet. The results are shown in Figures 14 to 17.

As is clear from Figures 14 and 17, when CI-Dextran mix was added to feed, the addition of 1.25% CI-Dextran mix had the greatest caries-inhibiting effect. It was also revealed that the inhibition of plaque deposition was significantly reduced even 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 suppress the occurrence of caries by inhibiting plaque deposition.

[Test 7] Preparation of catalytic domain (CAT) recombinant protein GTFB has two functional domains: CAT, which is the sucrose binding site, and GBD, which synthesizes glucan by translocating glucose. Since it was shown that the enzyme inhibitory activity mode may be competitive inhibition, it was thought 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.

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 E. coli BL21 strain, and after mass cultivation, the cells obtained by centrifugation were suspended in PBS buffer and then ultrasonicated to destroy the cell wall of E. coli. The supernatant was obtained by centrifugation, and then purified using a Glutathione Sepharose 4B (registered trademark) column (GE Healthcare), and GST-rGTFB (CAT) was used in the following experiments.

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 Fuji Film Wako Pure Chemical Industries, Ltd.) was added and boiled for 20 minutes, after which Nelson solution (manufactured by Fuji Film 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.

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 eliminated at 0.25% CI-Dextran mix, and the effect of reducing activity was greater than in the absence of sucrose. This shows that cyclodextran binds to the CAT of GTFB in competition with sucrose, inhibiting the decomposition of sucrose into glucose and fructose, and as a result, suppressing the synthesis of glucan.

Similar tests were also carried out using fructose and maltose instead of sucrose. As a result, with fructose, no loss of activity was observed when CI-Dextran mix was used in combination with the monosaccharides, as seen in Figure 19. On the other hand, with maltose, a decrease in activity was observed when CI-Dextran mix was used in combination with the disaccharides, although to a lesser extent than with sucrose.

[Test 8] Study of intercellular binding 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 anaerobic 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 detached biofilm was diluted stepwise with sterile saline, inoculated on Trypticase Soy agar medium (Becton Dickinson & Co.), and cultured for 2 days at 37°C under anaerobic conditions. For the control group, the biofilm was not sonicated, and the cells were similarly collected without sonication and inoculated on Trypticase Soy agar medium. After culturing, the number of colonies was counted, and the ratio of the number of remaining bacteria after sonication to the number of bacteria was calculated as an index representing the intercellular binding strength of the bacteria. The results are shown in Figure 20.

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 no CI-Dextran mix was added with the case where 1% CI-Dextran mix was added, the number of bacteria in the biofilm was reduced by approximately 65% (p < 0.01, Tukey).

[Test 9] Study of oral plaque deposition inhibition effect in human test The flow chart in Figure 21 shows the research outline of 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.

There were 17 subjects, but 6 were excluded because the number of mutans streptococci in saliva was less than 103 CFU/mL (total), which was the exclusion criterion. Eleven volunteers (ages 31 to 59: average age 49.1) participated in the final study. The number of mutans streptococci in the subjects ranged from 1.1×10 ⁴ to 6.4×10 ⁵ CFU/mL, with an average of 2.9×10 ⁴ CFU/mL. After the study, the plaque score per tooth surface was 1.55 for water and 0.97 for the CI-Dextran mix solution, showing a significant difference (p<0.01, Fisher's PLSD). From the above results, plaque deposition was significantly suppressed when drinking the CI-Dextran mix solution compared to when drinking water.

Claims (6)

(A) cyclodextran ; and (B) sucrose ;
A caries-inhibiting 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. (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. (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. (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. (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. (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.