JPS59231011A - Two enzyme dentifrice - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to toothpaste compositions, and more particularly to toothpaste compositions that, during use in the oral cavity, produce hypothiocyanate therein as a bacteria-inhibiting substance. The present invention relates to an antiseptic toothpaste composition.

(Conventional technology) Toothpastes include powder, paste, cream, and more.

It is used for both cosmetic and therapeutic purposes in liquid and liquid form. Consistent with these objectives, toothpastes are formulated to contain active ingredients such as cleansing and abrasive substances, as well as various antibacterial and prophylactic agents used with the purpose of preventing tooth decay. ing. Also,
It has also been suggested in the prior art that chewing gums, toothpastes such as chewing tablets, and lozenges are comprised of preservative-type compositions to effectively treat teeth.

Some types of tooth decay are caused by bacteria and food particles in the mouth.
It is generally known in dental technology that tooth enamel begins to undergo acid erosion due to acid, which is a metabolite produced as a result of the action of enzymes. Dental plaque (a soft accumulation on the tooth surface consisting of a composition formed by microorganisms, protein-like carbohydrate substances, epithelial cells and food particles) contributes to the development of various pathological conditions of the teeth and soft tissues of the oral cavity. It is generally accepted that this is a contributing factor. It has also been suggested that oral glycolytic organisms cause demineralization beneath the plaque matrix through metabolic activity that causes accumulation and local concentration of organic acids that are associated with plaque. Erosion and demineralization of enamel will continue until it reaches the pulp.

Various substances have been considered as caries preventive agents to be used in toothpaste compositions. Examples of such substances are P-aminobenzoic acid, a combination of urea and urease such as those that produce ammonia during the use of toothpaste in the oral cavity, chlorophyll, perfluorinated long-chain organic compounds, and combreno-sugar. hydrogen peroxide, penicillin, benzohydroxamic acid, and glucose oxidase, which produces hydrogen peroxide during the use of toothpaste in the oral cavity. In addition, substances used for toothpaste include:
Peroxides and perborates such as carbolic acid, menthol, thymol and eucalyptus, calcium peroxide and sodium perborate, and as redox enzymes that produce hydrogen peroxide during oral use of toothpaste. Examples include glucose oxidase.

US Pat. No. L171,392 describes a preservative chewing gum containing chicle, glucose and sugar, along with a mixture of powdered chalk and a preservative such as carbolic acid, menthol, thymol or eucalyptus.

US Pat. No. 2,290,862 describes a preservative chewing gum containing chicle, glucose, flavorings and sugar, along with a mixture of hydrogenated peanut oil and calcium peroxide.

Commercially available glucose oxidase also contains catalase, and the enzymatic reaction facilitates its use in the food and beverage industry as a substance to prevent spoilage in sensitive packaging products in the presence of oxygen and/or glucose. has been done.

This enzymatic reaction consumes oxygen and glucose,
Hydrogen peroxide is produced as an intermediate product, and the final product of the enzymatic reaction is gluconic acid.

U.S. Pat. No. 2,891.868 discloses that chewing gum is composed of oxygen-sensitive flavorings such as glucose,
that the seasoning is protected against oxidative deterioration by introducing into its composition an enzyme system containing glucose oxidase and catalase, and that this protection is effective only in the presence of bound water and does not require free water; is listed.

U.S. Pat. No. 4.150.113 and U.S. Pat.
No. 178.362 describes an enzyme-based toothpaste and an enzyme-based chewing toothpaste containing glucose oxidase, which acts on glucose present in saliva and dental plaque to produce hydrogen peroxide. The patentee states that oral bacteria accomplish glycolysis of sugar-containing food products by an enzyme system having S H groups,
They also point out that lactoperoxidase, present in saliva, provides a means of transporting oxygen from hydrogen peroxide to oral bacteria, resulting in oxidation of SH-containing enzymes to inactive disulfide enzymes.

U.S. Pat. No. 4,269,822 discloses an oxidizable amino acid substrate and an oxidoreductase that has specificity for the substrate and produces hydrogen peroxide and ammonia when toothpaste is used in the oral cavity. An antiseptic toothpaste is described which maintains stability before use by limiting the amount of water in the toothpaste.

Morrison et al., Oral Biology [American Association for the Advancement of Science, No.
Ciaton for the Advance
ment of 5ci-ence), 1
Published in 1968], pages 89-110, states that lactoperoxidase, sodium thiocyanate, and hydrogen peroxide constitute a bacterial inhibitory system, and that the in vivo production of hydrogen peroxide is thought to occur by microorganisms, and that the lactoperoxidase antimicrobial system (also containing hydrogen peroxide and thiocyanate) is described to be reversed by catalase, which competes with lactoperoxidase for available hydrogen peroxide.

Caries Research (Iloogendoorn, etc.)
), pp. 119.77-84, 1977.
Hypothiocyanate ion is lactoperoxidase,
It has been described that it is a bacterial inhibitor formed by a system containing thiocyanate and hydrogen peroxide, and it has also been described that high concentrations of hydrogen peroxide inactivate lactoperoxidase.

Thomas et al., Journal of Dental Research
Re-5earch), 60 (Vol. 41, 785-79
Page 6, April 1981, concerning the salivary antibacterial system consisting of peroxidase enzymes, hydrogen peroxide, and thiocyanate ions, (b) that al peroxidase is synthesized by the salivary glands; (d) The antibacterial activity of the peroxidase system is due to the production of thiocyanate ions (C1 salivary glands collect thiocyanate from the blood; and (d) the antibacterial activity of peroxidase systems. SCN) to hypothiocyanate ion (OSCN), and (e) the yield or accumulation of hypothiocyanate from the antibacterial system is has been described to be increased by the presence of aminohexoses, namely glucosamine and N-acetyl glucosamine.

Glucose oxidase toothpaste as a bacterial inhibitor by formation of hypothiocyanite (U.S. Pat. No. 4)
．． 150,113 and U.S. Pat. No. 4.178.362
The effectiveness of No. 1) depends to a considerable extent on the concentrations of glucose, potassium thiocyanate and lactoperoxidase present in the oral cavity, as well as hydrogen peroxide during oral use in toothpaste. The concentrations of these components supplied by saliva, including potassium thiocyanate and lactoperoxidase, vary in direct proportion to microbial production and saliva flow rate. Thus, when saliva flow is reduced, as occurs naturally or from certain medications, potassium thiocyanate and lactoperoxidase, which are factors that limit oral production of hypothiocyanate bacterial inhibitors, are reduced. The oral concentration of will be correspondingly reduced. Furthermore, when the oral concentration of lactoperoxidase decreases due to decreased salivary flow, U.S. Pat.
As described in US Pat. It will rise to the level.

Therefore, in monoenzyme-based toothpastes such as those described in U.S. Pat. No. 4,150,113 and U.S. Pat. There was a problem in that it was dependent on the flow rate and a stable and excellent bacteria suppression effect could not be obtained.

(Objective of the Invention) The object of the present invention is to provide a bienzyme-based toothpaste that is not affected by the amount of microorganisms produced or the flow rate of saliva and is stable over a long period of time and exhibits an excellent bacteria-inhibiting effect.

(Structure of the Invention) When using toothpaste in the oral cavity, the present invention does not depend on the intraoral concentration of potassium glucose thiocyanate or lactoperoxidase, which only exhibits naturally occurring and effective antibacterial properties, and the present invention does not depend on the concentration of potassium thiocyanate or lactoperoxidase in the oral cavity, which is a naturally occurring amount of potassium thiocyanate or lactoperoxidase. This invention was developed based on the fact that it would be advantageous to provide a dienzyme toothpaste that itself contains these ingredients and produces hypothiocyanate.

That is, the present invention has specificity for oxidizable substrates of 0.015 to 0.6 mmol per gram of toothpaste, and produces hydrogen peroxide when the toothpaste is used in the oral cavity. 0.5 to 500 international units of oxidoreductase, 000001 to 0.01 mmol of thiocyanate, and 0 lactoperoxidase, which interacts with hydrogen peroxide and produces a hypothiocyanate bacterial inhibitor. ．．
01-5° International Units, and the International Units concentration of lactoperoxidase is at least 2% of the International Units concentration of redox enzymes, thereby hydrogen peroxide against lactoperoxidase during oral use of the toothpaste. This is a two-enzyme toothpaste characterized by limiting the proportion of

In the present invention, "toothpaste" refers to chewing gum, chewable tablets, tablets that dissolve in the oral cavity, troches, lozenges, drops, etc., as well as oral compositions in the form of powders, pastes, creams, and liquids, and also includes flossing materials. It includes.

The bienzyme toothpaste of the present invention has an oxidizable substrate and specificity for the substrate, and when the toothpaste is used in the oral cavity, it produces oxygen or oxygen and water as additional reactants. The first enzyme system includes an oxidoreductase that produces hydrogen peroxide in combination with the chemical environment of the oral cavity that causes an enzymatic reaction.

The first enzyme component that can be mixed into the toothpaste composition and generates hydrogen peroxide when the toothpaste is used in the oral cavity includes substrate/enzyme combinations as shown in Table 1. Illustrated.

Table 1 Oxidizable substrates Redox enzyme ialβ-D-glucose Glucose oxidase (b) D-galactose Galactose oxidase (C) Urate Urate oxidase +
dl choline choline oxidase (81
D-amino acid D-amino acid oxidase (f
l D-glutamate D-glutamate oxidase (a glycine glycine oxidase (hl glycolate glycolate oxidase (ILL-sorbose oxidase 0) primary alcohol alcohol oxidase (k) primary amine amine oxidase Table 1 The reactions of representative enzyme systems shown are shown in the second section.
As shown in the table, these are activated under the chemical environment of the oral cavity to produce hydrogen peroxide.

Table 2 (a) Glucose oxidase is β-D-glucose,
Acts as a catalyst for the interaction of water and oxygen, producing hydrogen peroxide and gluconic acid.

(bl Galactose oxidase catalyzes the interaction of D-galactose and oxygen, producing hydrogen peroxide and D-galacto-hexo-sialdose.

(C1 urate oxidase catalyzes the interaction of urate, water and oxygen to produce hydrogen peroxide, allantoin and carbon dioxide.

(dl choline oxidase catalyzes the interaction of choline and oxygen to produce hydrogen peroxide and betaine aldehyde.

(el D-amino acid oxidase catalyzes the interaction of one amino acid, such as the D isomer of proline, methionine, isoleucine, alanine, valine, and phenylalanine, with water and oxygen, hydrogen peroxide, ammonia, and the corresponding It produces an α-keto acid.

(f) D-glutamate oxidase catalyzes the interaction of D-glutamate, water, and oxygen to produce hydrogen peroxide, ammonia, and 2-oxoglucurate.

(glglycine oxidase catalyzes the interaction of glycine, water and oxygen to produce ammonia hydrogen peroxide and glyoxylic acid.

Table 3 shows the characteristics of typical oxidoreductases obtained from specific raw materials shown in Table 1.

Table 3 (i) Molecular weight: 150.000 (Paz
ur ) et al., 1965) i 153,000
(Swoboda, 1969) (
11) Composition: Glycoprotein containing two molecules of flavin-adenine dinucleotide (see Merck Index, 9th edition, 1976, p. 532, item 4007 and page 576, item 4291).

Amino acid components have been measured [Pazur
) et al., 1965].

(iii) Isoelectric point: PH 4,2 (iv) Optimal Pi (: 5.5. Wide PH range from 4 to 7.

(v) Inhibitors: monovalent silver, divalent mercury and copper ions.

-1 (1) Molecular weight: 42,000 (Kelly Falco
Notu (Kelly-Falcoz, 1956)
(ii) Composition: metal-binding enzyme containing 1 gram atom of copper per mole [Amaral et al., 1963]
, amino acid components have been measured [Kefly-Falcoz, 1965].

(iii) Optimal PH near (Cooper
) et al., 1959].

(C) Urate oxidase from pig liver or cow liver (
uricase) (i) Molecular weight: 100.000 (Ma
(Mahler et al., 1955)] (ii) Composition: Metal-binding enzyme containing 1 gram atom of copper per mole (Mahler et al., 1955) (iii) Isoelectric point: PH6,3 (iv) Optimum pH: 9 ((11 D-amino acid oxidase from pig kidney (i
) Molecular weight 790,000 (Ammonium (Ant.)

ntni) et al., 1966) (ii) a glycoprotein containing two molecules of flavin-adenine dinucleotide of the set tc', (iii) optimum pH: 9 (iv) an inhibitor: certain heavy metal oxidizable substrates are , usually 0.0 per gram of toothpaste
It is present in the toothpaste in an amount of 15-0.6 mmol, preferably 0.025-0.1 mmol. On the other hand, oxidoreductases having specificity for this substrate usually contain 0.5 to 500 international units per gram of toothpaste (sometimes abbreviated as IU), preferably 1.0 to 401 U. present in toothpaste in quantity. "Millimol" is the number of grams corresponding to the molecular weight of a component divided by 1000. The "international unit" is 1 minute per minute at pH 7, 0, and 25℃.
．． It is the amount of enzyme that causes the catalytic action of 0 micromoles of substrate. The oxidoreductase is suitably supplied in dry or liquid form with a label specifying the concentration in international units per duram or per milliliter.

In addition to the first enzyme system comprising an oxidizable substrate and an oxidoreductase having specificity for the substrate and producing hydrogen peroxide, the bienzyme toothpaste of the invention comprises a thiocyanate;
Interacts with hydrogen peroxide to form hypothiocyanate (HO3C
N) and lactoperoxidase, which is in acid-alkaline equilibrium and produces a bacterial inhibitory substance in the form of a negative monovalent hypothiocyanate ion (O3CN) present in solution. .

Thiocyanates that can be used in the toothpaste of the present invention include sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, and mixtures thereof. The thiocyanate is usually present in an amount of o,oooi to 0,01 mmol per gram of toothpaste, preferably 0
．． It is present in the toothpaste in an amount of 0.001 to 0.006 mmol. When producing bienzyme toothpastes, care must be taken to avoid the use of metal compounds that suppress or inhibit the activity of oxidoreductase and/or peroxidase enzymes.

Lactoperoxidase is a glycoprotein - a commercially available example is a frozen vacuum-dried powder derived from milk. This commercially available peroxidase has an activity of 801 U/■, and a molecular weight of 93 as determined by the L-tyrosine iodination method.
．． It is 000.

The physical-chemical properties reported for lactoperoxidase are molecular weight 78,000; partial specific volume 0.74.
1 mole of i-heme is 1.0. Lactoperoxidase is usually 0°01 to 50 I per gram of toothpaste.
U, preferably present in the toothpaste in an amount of 0.2 to 4.0 fUO.

In order to maintain the integrity of the action of the two-enzyme system, the ratio of hydrogen peroxide to lactoperoxidase must be limited, as excess hydrogen peroxide will inhibit lactoperoxidase. This limitation is achieved by providing a two-enzyme system in which the international unit concentration of lactoperoxidase is at least 2% of the international unit concentration of oxidoreductase. When the concentration of lactoperoxidase is less than 2% of the concentration of oxidoreductase, lactoperoxidase becomes inhibited quickly and the effective effect of the difluorine system is lost long before the end of its use cycle.

The completeness of action of the two-enzyme system is also achieved by the presence of catalase in commercially available glucose oxidase along with the oral surface tissues. Although catalase is redundant to the two-enzyme system of the invention, it competes with lactoperoxidase for hydrogen peroxide. In order to reduce the loss of hydrogen peroxide due to the presence of catalase, it is advantageous to incorporate enzyme inhibitors with specificity for catalase into the bienzyme toothpaste.

Ascorbate salts such as sodium ascorbate, calcium ascorbate, ascorbate palmitate or mixtures thereof can be used as enzyme inhibitors with specificity for catalase. The effective amount of ascorbate for catalase inhibition is toothpaste 1
Gram per o, ooooooi ~ 0.0001 mmol. Iron salts such as ferrous sulfate can be incorporated into bienzyme toothpastes to enhance the effect of ascorbate in its role as a catalase inhibitor.

The bienzyme toothpaste of the present invention advantageously contains aminohexoses such as aminoglucose, such as glucosamine, N-acetylglucosamine or mixtures thereof, in order to increase the yield or accumulation of hypothiocyanate ions. can be added to. Aminoglucose is usually Q, 0001 to 0.00 per gram of toothpaste.
2 mmol, preferably 0.0003-0. , is present in the toothpaste in an amount of OOi mmol.

Since water facilitates the redox reactions of the present invention and is also a reactive component in some reactions, the use of water is recommended in preparing toothpaste compositions to maximize composition stability and shelf life. Concentration levels should be relatively low. To this end, it has been found that it is necessary to limit the water present in the toothpaste to less than 10% by weight, whether bound or unbound water. However, in the chewing form of tooth brushing, unbound water is used in 1. ．． The amount should be limited to 0% by weight or less. Advantageously, a finely divided aqueous desiccant, such as silica airgel, is incorporated into the toothpaste in an amount of 1 to 5% by weight.

If the activated enzyme-based product contains weak organic acids, it is advantageous to add a buffer to the toothpaste to neutralize the organic acids. As a buffering agent, sodium bicarbonate, which is present in the toothpaste in an amount up to about 6% by weight, for example about 4-6% by weight, is suitable.

BACKGROUND OF THE INVENTION In the field of manufacturing and packaging toothpaste chewing gum, chewing tablets, and chewing lozenges, formulation, equipment, and processing techniques are well developed. The two-enzyme system of the invention is applied in combination during these formulations.

However, the enzymes described herein degrade and become inactive under conditions such as high shear and high temperatures. Therefore, processing conditions must be controlled so that the temperature does not exceed 55° C. throughout the period when the enzyme is being mixed with other ingredients to form the final product. To increase storage stability, the mixture used in bienzyme toothpaste production should be substantially free of unbound water, and the final product should minimize exposure to air and moisture. It should be packaged as follows.

Exemplary substrate formulations for chewing gum, chewable tablets, and chewy lozenges that can be used to make bienzyme-based toothpastes are shown in Table 4 as follows.

Table 4 In Table 4, formulations (al and (b) indicate chewing gum compositions and formulations (0) and +d) indicate tablet and lozenge compositions. Saccharin sodium in these formulations can be replaced with aspartame (a hespartame protein-based sweetener).

Toothbrushing The two-enzyme system of the invention is applied in combination during toothpaste. In view of the water limitations mentioned above, it is advantageous to use a non-aqueous fluid carrier during milling so as to provide a formulation with flow properties that are responsive to pressure. Any suitable fluid can be used for this purpose. The use of organic fluid carriers such as glycerin or propylene glycol provides toothpastes that are stable to the enzyme systems of the present invention. The non-aqueous fluid carrier typically contains about 30-60%, preferably about 45-5% during tooth brushing.
Present in an amount of 5% by weight.

In addition to a fluid carrier, typical toothpaste compositions include flavoring agents, sweeteners, colorants, as well as abrasive substances and surfactants. Toothpaste usually also contains humectants and thickening agents.

Any abrasive material that does not unduly abrade the dentin and is compatible with glucose oxidase can be used in the toothpaste compositions of the present invention.

These abrasive substances include, for example, calcium carbonate,
Calcium birophosphate, dicalcium phosphate, zirconium oxide, and aluminum oxide may be mentioned. The abrasive material is
Usually present in toothpaste in an amount of about 20 = 60%.

Surfactants can be those that produce substantial levels of foam, are otherwise acceptable for use in the oral cavity, and are compatible with glucose oxidase. Nonionic surfactants are desirable because they have been found to coexist best with glucose oxidase. Surfactants can be used at concentration levels ranging from about 0.5 to 5.0% by weight.

Enzymatic toothpastes in the form of toothpastes can be prepared in any suitable manner, such as by mixing the dry ingredients into the liquid ingredients and stirring until a well-mixed mixture is obtained.

Addition of surfactant to the mixture should be done at the final stage to minimize foaming of the batch. Mixing should also be done under moderate conditions to avoid any loss of enzyme.

Floss Materials The bienzyme system of the present invention can be incorporated into dental floss, for example, by being deposited in solution onto the floss fibers or mixed into the floss coating.

EXAMPLES Example 1 The following example illustrates various ingredients and concentration levels that can be used to make a bienzyme toothpaste.

Table 5 Sorbitol, crystal 70 70 70 Gum matrix 23 23 23 Glycerol 5 5 5 Flavoring agent
1 1 1 Coloring agent 0.5
0.5 0.5 Sodium hydrogen carbonate 0.5
0.5 0.5100.0 100.0 100.0 Bienzyme glucose oxidase 40,0OOIU -β-D
Glucose 1.0g - Choline oxidase 8,
0OOIU - Colin
1. Og-D-glutamate oxidase 2,50
0IUD-Glutamate
0.1g Lactoperoxidase 4,0OOIIJ 1,50
0rU 1,0OOIU Potassium thiocyanate JA0.01g O,005g -Sodium thiocyanate
0.01g Table 6 Sorbitol, crystal 43 43 43 Corn sugar 2 (12020 Gum substrate
25 25 25 Thickener
1 1 1 Coloring agent 0
．． 5 0.5 0.510 (1,0100, Oto
o.

Two-enzyme system D-amino acid oxidase 5,000■U-D-alanine
0.1g - Glucose oxidase 20.0OOIU 2
,0OOIUβ-D-glucose
0.5g 0.5g Lactoperoxidase 500IU 2,5001
[11,0OOIU Sodium Thiocyanate 0.0
1g-'--Potassium thiocyanate -0.01g
0.005g Sodium ascorbate 0.01g
-Table 7 Sorbitol, crystal 97 97 97 Glycerol 1.0 1.0 1.0 Thickener 1.0 1.0 1.0
Coloring agent 0.5 0.5 0.5
Sodium hydrogen carbonate 0.5 0.5 0.
5100.0 100.0 100.0 Dienzyme glucose oxidase 10,0OOIU -β-D-
Glucose Ig --- Urate oxidase 10,0OOIU - Urate 0.75 g - Choline oxidase 0.5 g
Lactoperoxidase 200IU 2001U
1,500111 Sodium thiocyanate 0.0
5g 0.08g --Potassium thiocyanate 0.01g Table 8 Sorbitol, crystal 80 80 80 Corn wJ17 17 17 Seasoning
1 1 1 coloring agent
0.5 0.5 0.5 Sodium hydrogen carbonate
0.5 0.5 0.5100.0 100.0
100.0 Two-enzyme system D-glutamate oxidase 10,0OOIU -D-
Glutamate 0.05g - Glucose oxidase 5,0OOIU
-B-D-glucose 0.5g
1g lactoperoxidase 1,500IU 2,0OO
IU 1.0OOIU potassium thiocyanate
0.001g O,005g - Sodium thiocyanate
0.005g Example 2 This example shows the antibacterial effect of the bienzyme toothpaste of the present invention. A bienzyme chewing gum having the following composition was prepared.

Composition Weight, grams Gum substrate 23 Sorbitol, crystals 75 Coloring agent 0.5 Flavoring agent 1.0 β-D-glucose 0.5 Potassium thiocyanate 0.01 Glucose oxidase 0.006 (100,0OOI (t/
g) (600IU) Lactoperoxidase 0.0006 (100,00010/g
) (60 Itl) The above compositions were molded into bars of 3 grams each. This gum stick was given to each of the five people in group (a) with instructions to chew it for 10 minutes.

Saliva samples were collected separately from each person in the following time order: Person (1), immediately after the chewing cycle; Person (2), 60 minutes after the chewing cycle; Person (3), 120 minutes after the chewing cycle; Person (4), 180 minutes after the chewing cycle; Person (5), after the chewing cycle. 240 minutes.

10,000 colonies of Streptococcus mutans
lQmj2 plain containing (strain C67-1)
Five bacterial specimens were prepared by injecting infusion agar into five Patry dishes, respectively.

Immediately after saliva was collected from each person, a 5 ml aliquot was added to the laboratory dish containing the bacterial specimen with stirring, and the resulting mixture was incubated in the open at 35° C. for 10 minutes. At the end of the incubation time, the bacterial specimen mixture was removed from the open tube and the number of visible colonies was measured under a microscope and a microscope to evaluate bacterial inhibition. The same method as described above was repeated for five people in the BL group, except that a chewing gum containing the gum substrate, sorbitol, coloring, and flavoring was used, but without the dienzyme system. The results of this experiment are shown in Table 9. .

Table 9 To the bacterial solution after the chewing cycle * Add saliva Bacterial inhibition (%) Time to chewing (minutes) Kabuto - makeup (control example) No chewing) θ
=la Immediately after lb side 9937 2a 2b 60 99 123
a 3b 120 98 24a
4b 180. 97 05a5b2
40 96 0*Percent bacterial inhibition is the number of bacterial colonies compared to the control colony count? Ii shows a small proportion.

Example 3 The following example shows a first enzyme system consisting of glucose oxidase and β-glucose, and further a combination of lactoperoxidase and thiocyanate to produce a hypothiocyanate inhibitor for oral use. 6° showing an enzymatic toothpaste composition containing a second enzyme system comprising
) 200 J is polyoxyethylene (20) isohexadecyl commercially available as a 100% active paste.
It is a trademark name for ether. In addition, [Silcrone (Sf lcr) was used in this example.

n) G-910J is a trade name for an abrasive containing hydrated silica gel with micron size.

A Composition' Weight, Durham glycerin (99%) 50 Calcium pyrophosphate 40 Sodium bicarbonate 5 Water 1.5 Arlasolve 2002 Glucose oxidase 0.1 (10,000
10) (100,000I [1/g) β-D-glucose 0.5 (0.03 mmol) Lactoperoxidase 0.002 (200
10) (100,000111/g) Sodium thiocyanate 0.04 Colorant
0.5 Seasoning 0.5B Composition Weight, Dharam Glycerin (99%) 46 Titanium dioxide 2 Silcuron G -91040 Water 2 Arlasolp 2002 Glucose oxidase 0.05 (5,0O
OIU ) (100,00010/g) β-D-glucose 1 (0,06 mmol) Lactoperoxidase 0.01 (1,00
01 [1”) (100,00010/g) Potassium thiocyanate 0.005 Coloring agent
0.5 Seasoning 0.5 C 7 Dicalcium phosphate 45 Water 3.5 Arlasolve 2002 Glucose oxidase 0.0008 (80
IU) (100,0OOIU/g) β-D-glucose 0.5 (Q, 03 mmol) Lactoperoxidase 0.005 (500I
U) (100,0001U/g) Sodium thiocyanate 0.01 Colorant
0.5 Seasoning 0.5D Composition Weight, duram glycerin (99%) 50 Calcium pyrophosphate 40 8 Dicalcium phosphate 5 Water 2 Glucose oxidase 0.05 (5,00 anal U) (1
00,00010/g) β-D-glucose 1 (0,06 mmol) Choline oxidase 0,02 (2,0
OOIU) (100,0OOIU/g) ・
Choline 1 Lactoperoxidase 0.008 (8001
0) (100,00010/g) Sodium thiocyanate 0.009 Colorant
0.5 Steel flavoring 0.5 E Composition Weight, duram glycerin (99%) 42 Dicalcium phosphate 6 Titanium dioxide 2 Silcuron G-91038 Water 5 Glucose oxidase 0.4 (40,0OOIIJ) (
100,000tu 7g) β-D-glucose 6 (0.3 mmol) Lactoperoxidase 0.001 (100I
U) (100,0OOIU/g) Sodium thiocyanate 0.01 Colorant
0.5 Seasoning 0.5 3F Composition Weight, Dharam Glycerin (99%) 42 Dicalcium phosphate 6 Titanium dioxide 2 Silcuron G-91038 Water 5 Glucose oxidase 0.02 (2,000 Iυ) (
100,0OOIU/g) β-D-glucose 1 (0,06 mmol) Lactoperoxidase, &;fiO1 (100
IU) (100,00010/g) Sodium thiocyanate 0.01 Colorant
0.5 Seasoning 0.5 G Composition Weight, Lithium glycerin (9
9%) 5〇Titanium dioxide 2 Silcuron G-91040 Water 2 Arlasolve 2002 Glucose oxidase 0゜02 (2,00
01tl ) (100,0OOIU/g) β-D-glucose 2 (0,12 mmol) Rough toperoxidase 0.01 (1,0
0010) (100,0OOIU/g) Sodium thiocyanate 0.01 Colorant
0.5 seasoning agent
0.5H Composition Weight, Duram Propylene Glycol 44 Sodium Bicarbonate 5 Silclon G-91040 Water 6.4 Arlasolve 2002 G) Lt Fucose Oxidase 0.025 (2,
500 IU) (LOO,0QOIU/g) β-D-glucose 1.5 (0,12 mmol) Lactoperoxidase 0.006 (6001
0) (100,000111/g) Potassium thiocyanate 0.005 Coloring agent
0.5 Flavoring agent 0.5N-acetylglucosamine 0.15I Composition Weight, Duram propylene glycol 48 Sodium bicarbonate 5 Silcuron G-91040 Water 2.4 Arlasolve 2002 Glucose oxidase 0.025 (2,50
01tl ) (100,0OOIυ/g) β-D-glucose 1.5 (0,09 mmol) Lactoperoxidase 0.0005 (50
IU) (100,0001 [11' g) Potassium thiocyanate 0.005 Colorant
0.5 Seasoning 0.5 Glucosamine
0.13J Glycerin (99%) 47 Sodium hydrogen carbonate 5 Silcuron G-91040 Water 3.5 Arlasolve 2002 Glucose oxidase 0.04 (4,0O
OIU ) (100,0 OOI [I/g) β-D-glucose 1.5 (0,09 mmol) Lactoperoxidase 0.012 (1,20
011J) (100,0OOIU/g) Sodium thiocyanate 0.05 Colorant
0.5 Flavoring agent 0.5 Glucosamine
0.012N-acetylglucosamine
0.01 Example 4 This example shows that the two-enzyme toothpaste 4D of the present invention was used in combination with enzyme-free toothpaste 4A and U.S. Patent No. 4.15.
This shows that the monoenzyme-based kneading brushes described in No. 0.113 have superior antibacterial effects compared to 4B and 4C.

First, four types of toothpaste with the following compositions were created.

Glycerin 174.0 174.0 174
．． 0 500 Stearyl Al 30.7 30.
7 30.7 -Cholbenzoic acid sodium 0.8 0.8 0.8
- Lamb sodium fluoride 2.2 2.2 2.2
-Karagemate 18.0 18.0
18.0 -(Carraghemate) Aluminum hydroxide 325.0 325.0 325
．． 0 80umsident 15.0 15.0' 15
．． 0 -(Sident) 20 Thiocyanic acid 0.2 0.2 0.2
0.2 potassium aromatic substances 8.0 B, 0 B,
0 - Water 426.0 426
．． 0 426.0 -g/I/:? -su-1,
600IU 20,00010 10,0OOIU Oxidase Amyloglucosi -5,0OOIU 100,0OOI
Udase birophosphate Cal -
300 Sium Sorbitol 4
〇Silicon dioxide
32 ethoxylated iso-stearyl alcohol β-D-gluco-
10-Sodium hydroxide--7 Seasoning 9
Carbomer 3
Methylparaben-1 Lactoperoxy-200
1 U Sidase For each of the above four types of toothpaste, 20~
Evaluation was made by eight 40-year-old people with healthy teeth.

The evaluation method was as follows. First, from the meal 12
Plaque and saliva samples were taken from each person after ~16 hours (-night). Following sample collection, the teeth were quickly brushed with a toothbrush using approximately 3.0 grams of toothpaste. After completing this tooth brushing, further plaque and saliva samples were taken from each person. By this method 3
Evaluations were conducted for consecutive days.

Plaque and saliva samples before and after tooth brushing were
Each specific toothpaste was collected and stored on ice until bacterial counts were determined.

Bacterial evaluation was performed using standard methods to determine total aerobes and total anaerobes. Each sample of dental plaque and saliva collected was divided into substantially equal halves. One half of each sample was transferred to 2 ml of 0.1% peptone solution and cultured at 37° C. for 48 hours under aerobic conditions. The other of each sample was also transferred to the appropriate culture medium.
7 at 37℃ in an atmosphere of 95% hydrogen and 5% carbon dioxide.
The cells were cultured anaerobically for 2 hours.

Pure cultures of each sample were plated on plain heart infusion agar containing 5% defibrinated horse blood.
The number of bacteria was measured.

Regarding each linear tooth brushing, the number of bacteria and bacteria suppression effect before and after tooth brushing for 3 days for dental plaque samples were as shown in Table 10.

Table 10 Bacteria count (colony/mg plaque) 4A 48 4C4D Day 1.

Before tooth brushing 11,000.00012,000,00
012,000.00012,000.000 After brushing teeth 10,000,00010,000,000 9,
000,000 4B, 000 bacteria 1,00
0.000 2,000,000 3.000,000
11,952,000 inhibited bacteria 9.0 16.7 25
99.6 Suppression rate (%) Anus W Before tooth brushing 10,000,00012,000.00
013,000.000 5.000,000 tooth brushing & 9,000.000 9,000,00010
,000,000 60.000 bacteria 1,0
00.000 3,000,000 3,000,00
0 4.940,000 inhibited bacteria 10.0 25 23
9B, 81111 +li'l skewer (%) 1 town before tooth brushing 10,0 (10,00011,000,0
0012,000,0005,000,000 After brushing teeth 9,000,000 9.000,000 9,0
00.000 44.000 bacteria 1,000
,000 2,000,000 3,000,000
4,956,000 inhibited bacteria 10.0 18.2 25
99. r Inhibition rate (%) Similarly, the results measured on saliva samples were as shown in Table 11.

Table 11 Bacteria count (7 ml colony! Saliva) 4A 4
8 4C4D 1st day before tooth brushing 320,000 300.000
300,000 300,000 After brushing teeth 32
C1,000290,0002B0.000 2
60 bacteria 0 10.000 20
,000 299,740 Inhibited bacteria 0 3.3 6.6
．． 99.9 suppression rate (94) 2nd day before tooth brushing 310,000 300,000
320,00a300,000 After brushing teeth 300,
000 300,000 290,000
230 bacteria 10,000 0 3
0,000 299,770 inhibited bacteria 3.2 0 9.3
99.9 suppression jl#I rate (%) 3rd day before tooth brushing 330,000 310,000
30. (100,300,000 after brushing teeth 320,
000 290,000 290,00 (1270
Bacteria 10,000 20,000 1
0,000 299.730 Inhibited bacteria 3.0 6.5 3.3
99. ．． 9 Inhibition rate (%) As is clear from Tables 10 and 11, the two-enzyme toothpaste 4D of the present invention is superior to the enzyme-free toothpaste 4A and US Pat. No. 4,150,113. It has an extremely superior bacteria-inhibiting effect compared to the monoenzyme-based toothpastes 4B and 4C described in the book.

(Effect) According to the bienzyme-based toothpaste of the present invention, the bacteria-inhibiting effect is not affected by the amount of microorganisms produced in the oral cavity or the flow rate of saliva, and can exhibit a stable effect over a long period of time. Compared to other toothpastes, it has a far superior bacteria-inhibiting effect.

Claims (1)

[Claims] (11) 0.0 oxidizable substrate per gram of toothpaste
15-0.6 mmol, oxidoreductase that has specificity for the substrate and produces hydrogen peroxide when toothpaste is used in the oral cavity, 0.5-500 international units, thiocyanate and 0.01 to 5 mmol of lactoperoxidase, which interacts with hydrogen peroxide and produces a hypothiocyanate bacterial inhibitor.
0 International Units, and the International Units concentration of lactoperoxidase is at least 2% of the International Units concentration of redox enzymes, thereby limiting the ratio of hydrogen peroxide to lactoperoxidase during oral use of the toothpaste. A two-enzyme toothpaste that is characterized by: (2) The toothpaste according to claim 1, wherein the oxidizable substrate is β-D-glucose and the oxidoreductase is glucose oxidase. (3) The toothpaste according to claim 1, wherein the oxidizable substrate is D-galactose and the oxidoreductase is galactose oxidase. (4) The toothpaste according to claim 1, wherein the oxidizable substrate is urate and the redox enzyme is uric acid oxidase. (5) The toothpaste according to claim 1, wherein the oxidizable substrate is choline and the oxidoreductase is choline oxidase. (6) Claims in which the oxidizable substrate is a D-amino acid selected from the group consisting of D-isomers of proline, methionine, isoleucine, alanine, valine, and phenylalanine, and the oxidoreductase is D-amino acid oxidase. Tooth brushing as described in paragraph 1. 7) The toothpaste according to claim 1, wherein the oxidizable substrate is D-glutamate and the oxidoreductase is D-glutamate oxidase. (8) The toothpaste according to claim 1, wherein the oxidizable substrate is glycine and the oxidoreductase is glycine oxidase. (9) The thiocyanate is thorium thiocyanate,
The toothpaste according to claim 1, which is a member selected from the group consisting of potassium thiocyanate, ammonium thiocyanate, and mixtures thereof. (10) 7'lucosamine, N-acetylglucosamine,
and a mixture thereof, in an amount of 0.0091 to 0.0 per gram of toothpaste.
The toothpaste according to claim 1, containing the toothpaste in an amount of 0.02 mmol. (11) The amount of oxidizable substrate is 0.00% per gram of toothpaste.
A toothpaste according to claim 1, wherein the toothpaste is present in an amount of 0.025 to 0.1 mmol. (12) 1 to 4 oxidoreductases per gram of toothpaste
0 international single international amount, and lactoperoxidase is present at 0
．． A toothpaste according to claim 9, wherein the toothpaste is present in an amount of 2 to 4.0 I.D. (13) Thiocyanate is 0.0% per gram of toothpaste.
A toothpaste according to claim 1, wherein the toothpaste is present in an amount of 0.001 to 0.006 mmol. (14) Aminoglucose is 0.0% per gram of toothpaste.
A toothpaste according to claim 10, wherein the toothpaste is present in an amount of 0.0003 to 0.004 mmol. (15) The toothpaste according to claim 1, which contains an effective amount of an enzyme inhibitor having specificity for catalase. (16) The toothpaste according to claim 15, wherein the catalase inhibitor is ascorbate in an amount of 0.000001 to 0.0001 mmol per gram of toothpaste. (17) The oxidizable substrate is β-D-glucose, which is present in an amount of 0.025 to 0.1 mmol per gram of toothpaste, and the oxidoreductase is glucose oxidase, which is present in an amount of 0.025 to 0.1 mmol per gram of toothpaste. Present in ~40 international units, thiocyanate per gram of toothpaste.
3. The toothpaste of claim 1, wherein the lactoperoxidase is present in an amount of 0.2 to 4.0 mmoles per gram of toothpaste. (18) The method according to claim 16, which contains aminoglucose selected from the group consisting of glucosamine, N-acetylglucosamine, and mixtures thereof, in an amount of 0.0003 to 0.001 mmol per gram of toothpaste. Brush your teeth.