JPH04178320A - Production of anticariogenic agent and anticariogenic substance and anticariogenic food containing the same - Google Patents

Abstract

PURPOSE:To obtain an anticariogenic agent free from harmful action on human body by using an extract of tea leaf having glucosyl transferase-inhibiting action as an active component. CONSTITUTION:Leaves of tea, preferably fermented tea such as oolong tea and black tea are extracted with water or a mixture of water and a polar solvent such as lower alcohol or acetone and the obtained extract is subjected to the adsorption treatment with a synthetic adsorbent (e.g. styrene- divinylbenzene copolymer and methacrylic acid polymer) to obtain a non- adsorbed fraction. Both of the above extract and the non-adsorbed fraction are active anticariogenic components and used by compounding to an anticariogenic agent, a mouth hygienic agent or anticariogenic food. An agent containing the non-adsorbed fraction has strong glucosyl transferase-inhibiting action and is free from peculiar taste and odor and, accordingly, it can be compounded at an arbitrary ratio.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an anti-caries agent and a method for producing an anti-caries substance,
More specifically, [prior art] relating to an anti-caries agent containing an extract from tea leaves having a glucosyltransferase inhibitory effect as an active ingredient, a method for producing an anti-caries substance from tea leaves, and an anti-caries food containing the same. Although various hypotheses have been proposed in the past regarding the cause of this disease, it is now recognized that it is a type of bacterial infection based on Miller's chemical bacteria theory.

The caries development mechanism based on this theory is as follows.

That is, oral streptococci, especially Streptococcus mutans
) produces an enzyme called glucosyltransferase, which first uses sucrose in the mouth as a substrate to produce a sticky, insoluble polysaccharide (glucan). Bacterial cells of Streptococcus mutans (hereinafter abbreviated as rS, mutans) adhere to the tooth surface and form plaque (dental plaque) using the glucan produced. In this plaque, S,
Various microorganisms, including P. mutans, coexist and reproduce, and the metabolism of these microorganisms produces organic acids, which lower the pH of the tooth surface and cause demineralization on the enamel surface. , caries occurs and progresses.

In addition to dental caries, plaque formation is also thought to be a cause of periodontal disease and bad breath.

[Problems to be Solved by the Invention] Based on this knowledge, as a method of caries prevention, antibacterial agents for oral microorganisms, inhibitors of the above-mentioned glucosyltransferases, and glucosyltransferases that degrade polysaccharides formed using sucrose as a substrate have been proposed. Various studies have been conducted on enzymes that do this. However, no effective countermeasure has yet been found.

In particular, since dental plaque formed by oral streptococci, mainly S. mutans, causes dental caries, it is possible to suppress plaque formation by inhibiting the glucosyltransferase activity of S. mutans. Although it is believed that glucosyltransferase activity can be an effective means for preventing the development of dental caries, no substance that actually inhibits glucosyltransferase activity has been found.

[Means for Solving the Problems] The present inventors conducted intensive research to find a substance that effectively inhibits glucosyltransferase and does not have harmful effects on the human body. They discovered that there is a substance in the extract that very effectively inhibits the activity of glucosyltransferase that produces S and mutans, and that this substance is not adsorbed even by synthetic adsorbents, leading to the completion of the present invention. .

That is, the first object of the present invention is to provide an anti-caries agent containing as an active ingredient an extract from tea leaves that has a glucosyltransferase inhibitory effect.

Another object of the present invention is to provide an anti-caries agent containing as an active ingredient a fraction not adsorbed to a synthetic adsorbent obtained by subjecting a solvent extract of tea leaves to adsorption treatment using a synthetic adsorbent. be.

Furthermore, another object of the present invention is to obtain a method of inhibiting glucosyltransferase by extracting tea leaves with a solvent and then adsorbing the extract using a synthetic adsorbent to obtain a non-adsorbed fraction. An object of the present invention is to provide a method for producing an anti-cariogenic substance.

The active ingredient of the anti-caries agent of the present invention is the fraction that does not adsorb to the adsorbent (hereinafter referred to as
(abbreviated as “non-adsorbed fraction”).

The non-absorbed M fraction does not contain catechins or caffeine, which are typical components of tea leaves, or contains only a small amount.

In the production of tea leaf extracts having glucosyltransferase inhibitory activity, which is the active ingredient of the anti-caries agent of the present invention, the tea leaves used as raw materials include unfermented green teas such as sencha, gyokuro, and matcha, as well as oolong tea, black tea, and puerh tea. Although any of the fermented teas can be used, it is more preferable to use fermented teas because extracts of unfermented teas have weak glucosyltransferase inhibitory activity.

The solvent used for extraction is water alone, water and methanol,
Any mixture with one or more polar solvents such as a lower alcohol such as ethanol or acetone may be used, but since the active ingredient of the present invention cannot be efficiently extracted with a polar solvent alone, it must be a mixture with water. It is preferable that the mixing ratio of the solvent is 90% by volume or less. Among these solvents, it is preferable to use water, ethanol, or a mixture thereof from the viewpoint of safety, considering that the extract and the like will ultimately be incorporated into oral preparations and foods.

Although the ratio of tea leaves to solvent during extraction is not particularly limited, it is preferably 2 to 1000 times the weight of the solvent to 1 part of the tea leaves, particularly 20-100 times the weight from the point of view of extraction operation and efficiency.

It is convenient for the extraction temperature to be within the range of room temperature to the boiling point of the solvent under normal pressure, and the extraction time varies depending on the extraction temperature, but
It is preferable to set it as the range of 0 minutes - 24 hours.

In order to obtain a non-adsorbed fraction from the tea leaf extract thus obtained, this extract may be treated with a synthetic adsorbent.

The synthetic adsorbents used for the separation treatment of tea leaf extracts are aromatic synthetic adsorbents manufactured by polymerizing styrene and divinylbenzene and methacrylic synthetic adsorbents manufactured by polymerizing methacrylic acid. Among the aromatic synthetic adsorbents, Diamondion HP20, Diamondion HP21 (Mitsubishi Chemical Industries), Amberlite [F]XAD 2, Diamondion
AD4 (Rohm and Haas, USA), etc. are used as methacrylic synthetic adsorbents such as Sepabead HPIMG and HP2.
Examples include MG (Mitsubishi Chemical Industries), Amberlite XAD7, and Amberlite XAD8 (Rohm and Haas, USA).

The synthetic adsorbent treatment is preferably carried out by filling a column with the adsorbent, passing the tea leaf extract through the column, and washing the resin with water.

When treating tea leaf extracts with these synthetic adsorbents, in order to ensure complete fractionation, pretreatment is performed such as removing the organic solvent in the extract by vacuum concentration, etc., or sufficiently diluting it with water. It is preferable.

The tea leaf extract and non-absorbed M fraction obtained as described above may be extracted or treated with a synthetic adsorbent as is, concentrated, dried after removing the solvent from the eluate, etc.
Although it can be used in any state, it is preferable to use it in a dry state in terms of storage stability and safety of organic solvents.

The anti-caries agent of the present invention is prepared by blending the above-mentioned extract and non-adsorbed fraction with various conventionally used ingredients.

Preferred dosage forms of the anti-caries agent of the present invention include oral preparations such as toothpaste, mouthwash, and troches, as well as sweeteners such as sugar, kneaded bean paste, castella cake, water yokan, and dorayaki skin.
Sponge cake, butter cake, bavarois, custard cream, butter cream, custard pudding, kutsky, sweet bread, steamed bread, jam, lactic acid bacteria drink, carbonated drink, coffee drink, coffee jelly, caramel,
Examples include dosage forms added to foods such as ice cream, chewing gum, juice, candy, and chocolate. In the production of these oral preparations and foods, appropriate ingredients that are normally used can be used depending on the type.For example, for oral preparations, calcium carbonate, dicalcium phosphate, silicic anhydride, magnesium carbonate, glycerin , sorbitol, propylene glycol, polyethylene glycol, carboxymethylcellulose, methylcellulose, sodium alginate, carrageenan, carboxyvinyl polymer, sodium dioctylsulfosuccinate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, methyl paraoxybenzoate, hinokitiol, allantoin, glycyrrhizin, alcohol , gum arabic, starch, cornstarch, sodium saccharin, stevioside, glucose, lactose, magnesium stearate, dipotassium phosphate, dipotassium phosphate,
In addition to menthol, eucalyptus oil, pelapermint, spearmint, pigments, etc., fluorides such as sodium fluoride and sodium monofluorophosphate, anti-inflammatory agents such as lysozyme chloride and azulene, sodium chloride, etc. can be appropriately blended.

In addition, food products include glucose, fructose, sucrose, maltose,
Sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-
Ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic, carrageenan, casein, gelatin, pectin,
It can be manufactured by suitably blending those used as ordinary food raw materials, such as agar, B vitamins, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, and preservatives.

In addition, when adding the caries-resistant ingredients of the present invention to foods to produce caries-resistant foods as shown in Examples, when adding sugar and the caries-resistant ingredients, Examples 9 to 11 described below may be used instead.
It is also possible to use caries-resistant tactile sugars such as those shown in .

Tea has been widely drunk around the world since ancient times, and the extract and its non-adsorbed fraction obtained from it have no safety issues. It is preferable to set the concentration in the range of 0.0001 to 0.5% in terms of dry weight from the viewpoint of anti-caries activity and taste, aroma, color tone, etc. when added. Further, it is preferable that the amount added in foods is also within the above range at the time of use.

[Action and Effect of the Invention] It has been known that catechins have a growth-inhibiting effect on S. mutans. Little anti-cariogenic activity can be expected at the concentrations available.

Furthermore, as shown in the Examples below, even the non-adsorbed fraction, which does not contain catechins or caffeine or contains only a very small amount, has excellent glucosyltransferase inhibitory activity. It is clear that the effect is not related to catechins.

Therefore, the anti-caries engraving of the present invention can be used for the prevention of dental caries, etc. as a new type of anti-caries engraving that utilizes glucosyltransferase inhibitory activity.

In particular, when a non-adsorbed fraction is used as an active ingredient, this fraction has a strong glucosyltransferase inhibitory activity and has no peculiar taste or odor, so it can be incorporated in any amount into anti-caries agents. It can be used as an extremely effective caries resistant material.

[Example] Next, the present invention will be explained in more detail by giving examples related to the method for producing the tea leaf extract and its non-adsorbed fraction, glucosyltransferase inhibitory activity test, and production of anti-caries. The examples are not limited.

Example 1 Extract production example: Put 100g of oolong tea into a 2000ml Erlenmeyer flask,
Hot water of 10,100 O was added and the mixture was boiled at 90° C. for 15 minutes for extraction. This was filtered through Celite, and the obtained filtrate was freeze-dried to obtain 16.5 g of an extract.

For black tea and Bouar tea, 16.4 g and 17.4 g of extracts were obtained, respectively, in the same manner as above.

Example 2 Extract production example: Put 100g of oolong tea into a 2000ml Erlenmeyer flask,
Add 10,100 O of 50 volume % ethanol, and at room temperature,
Extraction was performed for 3 hours with gentle stirring every hour. This was filtered through Celite, and the resulting filtrate was concentrated under reduced pressure to remove ethanol, and then water was added and freeze-dried to obtain 29.2 g of an extract.

For black tea and Bouar tea, 30.4 g and 31.3 g of extracts were obtained, respectively, in the same manner as above.

Example 3 Extract production example: Put 100g of oolong tea into a 2000ml Erlenmeyer flask,
Add 10,100O of 90% ethanol, and at room temperature,
Extraction was carried out for 24 hours with gentle stirring every hour. This was filtered through Celite, and the resulting filtrate was concentrated under reduced pressure to remove ethanol, and then water was added and freeze-dried to obtain 1.2 g of an extract.

Black tea and Pu'er tea were treated in the same manner as above to obtain 1.4 g and 1.2 g of extracts, respectively.

Example 4 Example of manufacturing non-absorbed M fraction: 15 g of oolong tea extract obtained according to the method of Example 1
Dissolve it in 600ml of water and add it to Diaion@H
It was passed through a column (4.4 x 20 cm) packed with P21 and adsorbed.

The fraction not adsorbed to the adsorbent and the washing solution washed with water at step 21' after the adsorption operation were combined to form a non-adsorbed fraction. The non-adsorbed fraction was concentrated under reduced pressure and then lyophilized to obtain 6.4 g of a sample.

For black tea and Pu'er tea, 7.7 g and 6.3 g of non-adsorbed fractions were obtained, respectively, in the same manner as above.

Example 5 Assay of glucosyltransferase inhibitory activity: (N stock solution) Enzyme solutions were prepared using S. mutans MT according to the method of Hamada et al.
8148 shares were purchased by Todd-Hewit.
tt) cultured in medium and extracted from hyphae with 8M urea (S, Hamadaet, al, , J, Ge
n Microbiol, 135, 335-344
(2989)).

(Measurement method) 5% sucrose, 0.5% dextran TlO20.5%
500mM sodium phosphate buffer containing sodium azide (ρ) 16.0) 0.6ml, 2000ppm
A reaction system was created by adding 0.15 ml of the aqueous solution of the sample prepared above, the upper 3-enzyme solution, and ① water for a total volume of 3 ml.
React in a glass test tube. At this time, the amount of enzyme was 37°
C The absorbance at 550 nm is set to be approximately 1.0 after 13 hours of reaction.

The produced insoluble glucan was disrupted by ultrasonication, and the absorbance (A) at 550 nm was measured. Using the absorbance obtained when water was used instead of the sample as a control (B), the inhibition rate (%) was determined using the following formula.

100 The glucosyltransferase inhibitory activity of catechins and catechins was measured.

The results are shown in Table 1 (blank below).
51 Black tea extract of Example 1
87 Black tea extract of Example 2
89 Black tea extract of Example 3
36 Non-adsorbed fraction of the good quality tea extract of Example 4 81 Black tea extract of 1M fruit 4
Non-adsorbed fraction of black tea extract of 94 Example 4 Bouar tea extract of 94 Example 4
74 Epigallocatechin
11 categories
13 shrimp catechin
14 Shrimp catechin gallate
19 Epigallocatechin gallate
17 Example 6 Toothpaste: (Composition) (Parts by weight) Calcium diphosphate 42 Glycerin
18 carrageenan 0.9
Sodium lauryl sulfate 1.2 Sodium saccharin 0.09 Butyl roseoxybenzoate 0.005 Tea leaf extract! 0・
05 fragrance
1 total quantity
100 Extract of the good quality tea leaves obtained in Example 1.

Example 7 Mouth rinse: (Composition) (Parts by weight) Sodium lauryl sulfate 0.8 Glycerin
7 Sorbitol 5 Ethyl alcohol 15 Tea leaf extract-0.05 1-Menthol 0.05 Flavor 0.04 Saccharin sodium 0.1 Water
Full amount remaining
Black tea leaf extract obtained in Example 1.

Example 8 Lozenge: (Composition) (Parts by weight) Gum Arabic 6 Fudo W
73 Tea leaf extract content 0.02
Potassium phosphate 0.2 Potassium phosphate 0.1 Lactose
17 fragrances
0,1 Magnesium Stearate Total amount remaining
100 Bouar tea leaf extract obtained in Example 2.

Example 9 Production of caries-resistant sugar (powder) = (Production method) A solution of the following formulation is heated and dissolved at 80 to 90°C, transferred to a rectangular stainless steel hat, and dried in a dryer at 105°C. During drying, the mixture was stirred every hour. After drying, it was ground in a mortar to prepare powdered sugar.

(Composition) Sugar 200 parts Tea leaf extract 1 1 part Water 30 parts *As a tea leaf extract, implementation f! Any of the gyokuro, matcha, ryono tea, black tea, and bouar tea extracts obtained in A1 to A4 can be used.

Example 10 Production of caries-resistant sugar (granular): (Production method) Using the following formulation, a spray granulator (Flow Coater Multi T
The sugar of the present invention was spray granulated using L○-5M Ookawa Original Manufacturing Co., Ltd.). In other words, put sugar in a raw material container and heat air at a temperature of 90.
Pre-drying was carried out for about 2 hours at "C". Tea leaf extract dissolved in water was sprayed onto the sugar using a spray gun for 10 minutes.
0 m1/min; 30 seconds), then the spraying was stopped and intermediate drying was performed for 20 minutes. After repeating spraying and intermediate drying four times, final drying was performed for 20 minutes, and cooling was further performed for 20 minutes to obtain a tactile sugar with resistance.

(Composition) Sugar 200 parts Tea leaf extract 8
1 part water 10 parts *As the tea leaf extract, any of the gyokuro, matcha, ryono tea, black tea, and bouar tea extracts obtained in Examples 1 to 4 can be used.

Example 11 Production of anti-cariogenic sugar (syrup): (Production method) Add 0.75 parts of tea leaf extract to 50 parts of hot water and dissolve.
1.150 parts of sand is added to this solution to obtain the anticorrosive sugar (syrup) of the present invention.

As the tea leaf extract, any of the gyokuro, matcha, ryono tea, black tea, and bouar tea extracts obtained in Examples 1 and 2 can be used.

In the cariogenic @sugar obtained in Examples 9 to 11 above, the ratio of sugar to cariogenic substance is preferably about 0.1 to 10 parts of the cariogenic agent per 1000 parts of sugar.

Example 12 Comparison of sweetness of caries-resistant sweeteners: The sweetness of the caries-resistant sugar of the present invention was compared with sugar and palatinose by sensory evaluation as follows.

(Test sample) Anti-cariogenic sugar of the present invention; Example 1 using oolong tea extract.

Sugar: Super white sugar Palatinose: Crystalline palatinose (evaluation method) A 5% sugar solution was prepared and used as a control.

Anti-caries sugar makes 3%, 4%, 5%, 6%, 7% solutions, and palatinose makes 6%, 8%, 10%, 12%, 1
A 4% solution was prepared, and 10 panelists conducted a sensory evaluation at room temperature using the two-point discrimination method to examine cariogenic sugar solutions and palatinose solutions that had the same sweetness as a 5% sugar solution.

The results are shown in Table 2.

Table 2 Sample name Sweetness sand @ 1.0 Anti-caries sand 1i 1.0 Palatinose 0.4 From these results, the anti-caries sugar of the present invention shows the same sweetness as sugar, but the anti-caries sugar It can be seen that the substitute, palatinose, has less than half the sweetness of sugar.

Example 13 Solubility of anti-caries sugar: The solubility of sugar, anti-caries sugar and palatinose in water was compared in terms of solubility as follows.

(Test sample) Anti-cavity sugar of the present invention; Oolong tea extract used in Example 1 Sugar; Super sugar Palatinose; Crystalline Palatinose (Evaluation method) Distilled water at 10°C, 130°C, 50°C, and 70°C Sugar, anti-caries sugar and palatinose were each added until completely dissolved.

Table 3 shows the solubility at each temperature at that time.

Table 3 10℃30℃50℃70”C Sand W 63 68 72
74 Anti-cariogenic sweeteners 63 68 72
74 Palatinose 21 32 44
58 As is clear from this result, the caries-resistant sugar of the present invention exhibits the same solubility as sugar.

On the other hand, since palatinose, which is a typical caries-resistant sugar substitute, has poor solubility especially at low temperatures, it is clear that the caries-resistant sugar of the present invention is easier to use than palatinose.

Therefore, the anti-caries sugar of the present invention is suitable for low temperature foods, especially
For example, it can be advantageously used in ice cream, frozen sweets, and other foods that require storage at low temperatures.

Example 14 Anti-caries test: Three samples of sugar (superfine sugar), anti-caries sugar (Example 9), and palatinose (crystalline palatinose) were compared for their glucan production amounts using the following method. That is,
10% sample reservoir? e! 0,3 ml, 1,0% dextran Tl01 containing 1.0% sodium azide
00mM sodium phosphate buffer (pH 6,0) 0.3
A reaction system is prepared by adding water to a total volume of 3 ml to ml and glucosyltransferase enzyme solution, and the reaction is carried out in a glass test tube. At this time, the amount of enzyme is set so that the absorbance at 550 nm is approximately 1.0 when sugar (white sugar) is used as a sample and reacted for 3 hours at 37°C.

The produced insoluble glucan was disrupted by ultrasonication, and the absorbance (A) at 550 nm was measured. Using the absorbance when sugar was used as a test sample as a control (B), the relative amount (%) of produced glucan to sugar was determined using the following formula. The results are shown in Table 4.

100 X A Relative of produced glucan! (%) = Glucosyltransferase enzyme, base solution is S,
mutans MT8148 stock to Todd Hoewit (
Todd-Hewitt) medium and extracted from the bacterial cells with 8 molar urea (S, Hamada).
et,al,J,Gen,Microbiol.

135.335-344. (1989)').

(Results) Table 4 Sample name Relative amount of glucan produced (%) Sand! (control) 10
0 Anti-caries sugar 6 Palatinose 2 As is clear from these results, although the anti-caries sugar of the present invention has sugar as its main component, it is less likely to produce glucan compared to sugar, and is similar to palatinose in suppressing glucan production. It is clear that it has an effect.

Example 15 Anti-cariogenic property test: For sugar (white sugar), anti-cariogenic sugar (using high-grade tea extract in Example 9) and palatinose (crystalline palatinose), dental plaque (plaque) was tested in a test tube. ) A formation test was conducted. For the test, the medium was Dulbecco's modified Eagle's medium to which 5% of each test sample was added.
6 ml of this medium was added to a glass test tube, and S. mutans MT8148 strain was inoculated. 37°C under anaerobic conditions
After culturing for 11 days, the amount of plaque formed on the test tube wall was measured. The results are shown in Table 5.

(Results) Table 5 Sample Amount of plaque formation (mg) Sand fi 25.62 ± 1.7
8” Anti-caries sugar 3.21 ± 0.54” Palatinose 2.01 ± 0.22” 8 Mean value Standard deviation Example 16 Gum: (Composition) (Parts by weight) Gum paste
20 calcium carbonate
2 Stevioside 0.1
Tea leaf extract” 0.01 milk
1i 76.89 total
Amount 100*
Liangno tea extract obtained in Example 3.

Example 17 Youth: (Composition) (Parts by weight) Frozen concentrated unshiu mandarin juice 5-fructose glucose liquid sugar
11 Citric acid 0.2
L-ascorbic acid 0.02 non-adsorbed fraction”
0.01 fragrance
0.2 dye
0.1 water
Amount of remaining umbrellas
100*Non-adsorbed fraction of black tea leaf extract obtained in Example 4.

Example 18 Candy: (Composition) (Parts by weight) Powdered sorbitol 99.745 Flavor
0.2 promotion fraction”
0.005 sorbitol seeds
0.05 total amount
100*Non-adsorbed fraction of Bouar tea leaf extract obtained in Example 4.

Procedural amendment (voluntary) April 15, 1991

Claims (1)

[Scope of Claims] (1) An anti-caries agent containing as an active ingredient an extract from tea leaves that has a glucosyltransferase inhibitory effect. (2) The anti-caries agent according to claim 1, which contains 0.0001 to 0.5% of an extract from tea leaves having a glucosyltransferase inhibitory effect. (3) An oral hygiene agent whose active ingredient is an extract from tea leaves that has a glucosyltransferase inhibitory effect. (4) A food whose active ingredient is an extract from tea leaves that has a glucosyltransferase inhibitory effect. (5) An anti-caries agent containing as an active ingredient a synthetic adsorbent-unadsorbed fraction obtained by subjecting a solvent extract of tea leaves to adsorption treatment using a synthetic adsorbent. (6) Content of non-adsorbed fraction of synthetic adsorbent is 0.0001 to 0
．． The anti-caries agent according to claim 5, wherein the amount is 5%. (7) An oral hygiene agent containing as an active ingredient a synthetic adsorbent-unadsorbed fraction obtained by subjecting a solvent extract of tea leaves to adsorption treatment using a synthetic adsorbent. (8) A food product containing as an active ingredient a synthetic adsorbent-unadsorbed fraction obtained by subjecting a solvent extract of tea leaves to adsorption treatment using a synthetic adsorbent. (9) A method for producing an anti-cariogenic substance having a glucosyltransferase inhibitory effect, which comprises extracting tea leaves with a solvent, adsorbing the extract using a synthetic adsorbent, and obtaining the non-adsorbed fraction. . (10) The method for producing an anti-caries substance having a glucosyltransferase inhibiting effect, wherein the tea leaves are fermented tea.