NO139590B - DENTAL CARE FOR AA PREVENT THE FORMATION OF DENTAL COATINGS - Google Patents

Abstract

Tooth care agent to prevent the formation of dental plaque.

Description

The present invention relates to dental care products containing enzymes as an active ingredient, to prevent the formation of dental plaque.

It is well known that human dental plaque plays an important role in the development of caries, tartar and dental diseases, which are significant health problems that should be prevented by proper oral hygiene. hygiene.

Dental plaque adheres to the teeth and gum tissue and is held up, at least in part, by polysaccharides and proteins. One of these polysaccharides, a glucan, has previously been recognized as dextran which is subject to enzymatic hydrolysis by dextranases. The incorporation of dextranases into oral hygiene products has been proposed in U.S. Pat. patent no. 3,686,393. In a similar way, the use of plaque-dispersing proteases for oral hygiene has been proposed by Mollé in J. So. Calif. Dent. Assn., 35, 391

(1967) and by Shaver et al. in J. Periodontology, 41, 33 (1970).

It has been found that dental plaque contains another special glucan in amounts approximately equal to dextran, and which resists the hydrolytic action of dextranase, and which has been named "cariogenan", which is a straight-chain glucan with a-(1 —>3) -bonds and a-(1—*2)-bonds in the ratio approx. 1:3.

It has now been shown that previously unknown enzymes are capable of enzymatically breaking down the cariogenan component of dental plaque. These enzymes have been termed "cariogenanases" and their systematic name is α-^^-glucanohydrolase.

Due to the cariogenases' ability to break down cariogenan, which is one of the adhesives in dental plaque, they find use as an ingredient in dental care products such as toothpastes and mouthwashes,

or in chewing gum or in high-velocity jets of water, as a useful adjunct with dextranase.

The present invention relates to a dental care agent to prevent the formation of dental plaque, which agent contains dextranase and possibly protease, and is characterized by the fact that it also contains cariogenanase, which enzymatically cleaves cariogenan present in dental plaque (i.e. straight-chain glucan with a-(l->3) -bonds and a-(l—>2)-bonds in the ratio approx. 3 = 1) > i is» amount of 5 - 20,000 units per 1 g or 1 ml agent, the systematic name of cariogenanase is a~-glucan-3-glucanohydrolase and which is an endo-enzyme that acts on other than the end bonds of the polysaccharide chain, so that the cleavage products are mainly di-, tri- and other oligosaccharides, and which is characterized by a) specificity of such an α-(l—>3)-glucosidic bond, in the vicinity of which there is an α-(1—>2)-glucosidic bond,

b) an isoelectric point of 5.1, and

c) an optimal activity, which covers a wide pH range, with

peak activity at 6.0.

The dental care agent should be composed so that a single treatment will expose the mouth to 10 - 20,000 units of cariogenanase and lOO - 200,000 units of dextranase, and one or more applications per day is desirable.

Human dental plaque was isolated from a number of subjects by physical scraping of the teeth and was found to contain on a dry weight basis from 0.6 to 2.5% of dextranase-resistant polysaccharide.

The same polysaccharide, together with dextran, was shown to be formed, in vitro, by Streptococcus mutans SL-1 (NRRL No. B-5304)

when cultured in a medium containing sucrose as a carbohydrate source, and also when a cell-free Streptococcus mutans broth containing extracellular enzymes was incubated with a sucrose solution.

In each case, the cariogenan was separated from the accompanying dextran by incubation with dextranase. The cariogenan was further purified by deproteinization by standard methods.

The structure of cariogenan was determined by standard methods which include partial acid hydrolysis followed by qualitative and quantitative analysis of the oligosaccharide fractions obtained thereby, and by metaperiodate oxidation and borohydride reduction studies. The structure was thus determined to be an unbranched glucan with α-(l->3) and α-(l->2) bonds in a ratio of approx. 3:1.

Microbial organisms capable of enzymatically hydrolyzing cariogenan are isolated from airborne bacteria by exposing agar plates containing a suitable nutrient medium containing cariogenan as a carbohydrate source and incubating the cultures obtained at a suitable temperature, usually 28-37°C

for up to approx. 10 days. Organisms found to be active are further cultured on similar agar plates to isolate them.

The extracellular enzyme, cariogenanase, is produced in isolable amounts by incubating a culture of a cariogenanase-producing microorganism at a suitable temperature, usually 28 - 37°C, in a nutrient medium in shake flasks until a suitable degree of cariogenanase activity is obtained, usually from 72 to 96 hours. In general, the microorganisms do not require cariogenan as a carbohydrate source in the nutrient medium for building up cariogenanase. One of the organisms, Bacillus sp. MB-2665 (NRRL No. B-5300), used to exemplify the invention, required im-idlex tid cariogenan as a nutrient. By conventional mutation methods, however, a constitutive mutant can be produced which does not require cariogenan in the medium.

The enzyme is isolated from the culture medium by clearing it of cell waste and other insoluble substances by centrifugation or filtration, concentrating the centrifugate 5-15 times, preferably approx. 10 times in vacuum, and precipitate the protein by standard methods such as with an organic solvent, by complexation with metal, or by saturation with a salt such as ammonium sulfate or the like. It is preferable to use several filtrations with intermediate solutions of the precipitate in a buffer solution and clarification of the obtained solutions by filtration or late rif weeks in. The final precipitate in aqueous solution is then dialyzed and stored either as a cold solution or in lyophilized form .

The enzyme thus formed was characterized in that it was specific for an α-(l-»3)-glucosidic bond with a neighboring α-(l->2)-glucosidic bond. It has an isoelectric focusing point of 5.1. It has a wide pH optimum with peak activity at pH 6.0. These properties clearly distinguish cariogenanase from any known type of polysaccharase.

A method for producing the enzyme cariogenanase is described and protected in Norwegian patent no. 138'4H-Sammenligningsforsøk

In vitro coatings on the bottom and walls of 5 small tared culture flasks were formed by cultivation of Streptococcus mutans. These coatings were treated separately with: (1) water, (2) cariogenanase (250 units/ml), (3) dextranase (250 units/ml), (4) cariogenanase + dextranase (250 + 250 units/ml) and (5) cariogenanase + dextranase (250 + 250 units/ml) boiled .

After 3 hours of incubation under gentle oscillating shaking, the supernatant incubation liquid was decanted. The following results were obtained:

It will be seen that (4), the mixture of enzymes, has a greater effect than each of the two enzymes or the sum of their separate effects. The boiled enzyme mixture where the enzymes were denatured is comparable in activity to the water.

Example 1

Example 2 Example 3

Example 4

Claims (1)

Dental care agent to prevent the formation of dental plaque, which agent contains dextranase and possibly protease, characterized in that it also contains cariogenanase, which enzymatically cleaves cariogenan present in dental plaque (i.e. straight-chain glucan with α-(1—*3) bonds and a-(1—£2) bonds in the ratio of approximately 3:1), in an amount of 5 - 20,000 units per 1 g or 1 ml agent, which cariogenase's systematic name is ~9lucan~3~glucanohydrolase and which is an endo-enzyme that acts on other than the terminal bonds of the polysaccharide chain, so that the cleavage products are mainly di-, tri- and other oligosaccharides, and which are characterized by a) specificity of such an α-(l—>3)-glucosidic bond, in the vicinity of which there is an α-(1—42)-glucosidic bond , b) an isoelectric point of 5.1, and "c) an optimum activity, covering a wide pH range, with peak activity at 6.0.