GB2069499A - Proteins which inhibit glucosyltransferase for use in prevention of dental caries - Google Patents

Abstract

Acidic proteins having a molecular weight of from 30,000 to 60,000 inhibit the synthesis of insoluble glucans by glucosyltransferase and can be incorporated as the active ingredient in compositions, such as a toothpaste or dentifrice, for preventing dental caries. The proteins may be produced by culturing certain strains of microorganisms of the genera Arthrinium, Fusarium Gnomoniella, Moarophomina or Nodulisporium.

Description

SPECIFICATION Proteins which inhibit glucosyltransferase for use in prevention of dental caries This invention relates to substances which inhibit glucosyltransferase, the production of these substances, compositions for preventing dental caries containing these substances and cultures of the microorganisms used in the production of the substances.

Dental caries formation results from the formation of a sticky exopolysaccharide insoluble glucan from sucrose by the action of glucosyltransferase produced by indigenous oral bacteria Streptococcus mutans. The insoluble glucan adheres to the surface of teeth and absorbs oral bacteria. These oral bacteria metabolize food residue such as sugar on the surface, on striae or in gaps of teeth to form organic acids such as lactic acid and pyruvic acid. The organic acids dissolve the calcium content of teeth enamel, and soften and cause the disintegration of the teeth, further eroding the enamel or dentines. The sticky insoluble glucan on the surface of teeth provides anaerobic conditions for bacterial growth and thereby stimulates dental caries.

It has now been found that acidic proteins (glycoproteins) of molecular weight of 30000 60000 inhibit glycosyltransferase produced by the bacteria Streptococcus mutans which cause dental caries. The acid proteins inhibit the production of the insoluble sticky glucans by Streptococcus mutans in the presence of sugar, and consequently are effective in preventing dental caries.

We have found that the microorganism strain of Fungi imperfecti Arthrinium sp. M507 1 which was isolated from plant leaves (not identified) from Chichi-jima, Ogasawara-mura, Tokyo, produced a glucosyltransferase inhibitor. This substance is referred to herein as substance M5071.

Substance M507 1 has the following physicochemical properties: (1) Molecular weight: about 34000 (gel filtration method using "Sephadex" G-1 00; "Sephadex" is a Registered Trade Mark), and about 45000 (polyacrylamide electrophoresis method).

In determining the molecular weight of substance M 5071 by the polyacrylamide electrophoresis method, 50 y9, 30 g and 20 y9, respectively, of the substance, treated with 0.1% sodium dodecylsulfate at 1000C for 5 minutes, were charged onto an acrylamide gel plate (10% acrylamide) adjusted to pH 9, and electrophoresis was effected for 4 hours with a 25 mA current. Simultaneously bovine albumin (molecular weight about 68000), agg albumin (molecular weight about 45000) and chymotrypsinogen-A were subjected to electrophoresis. After 4 hours separated gel plates were stained with Coomassie blue and destained by 1% acetic acid. As shown in Figure 1 of the accompanying drawings, substance M507 1 shows single band at any concentration.Molecular weight is determined as about 45000 comparing with the migration distances of bovine albumin, egg albumin and chymotrypsinogen-A.

(2) Isoelectric point: pH 3.5 (determined by isoelectric focusing using carrier ampholite).

(3) Ultraviolet absorption spectrum: Figure 2 of the accompanying drawings.

Amax = 280 nm, Amax (shoulder) = 290 nm.

[0.5 mg/ml in 0.1 M phosphate buffer (pH 6.3)] (4) Solubility: soluble in water, insoluble in methanol, acetone, chloroform and ethyl acetate.

(5) Colour reaction: positive Biuret, xanthoproteic and Folin reactions.

(6) Optical rotation: [hZ]2D = 520 (c = 0.1%, pH 6.3, 0.1 M phosphate buffer).

(7) Color: white (lyophilizate).

(8) Action: inhibits the synthesis of insoluble glucan by glucosyltransferase. Streptococcus mutans OMZ 176 is incubated in BHl medium for 2 days. The culture filtrate is taken by salting-out using ammonium sulfate. The precipitate is dissolved in water and is dialyzed up to 50 times concentration to obtain a glucosyltransferase solution. A mixture of 0.1 ml of each of this glucosyltransferase solution, a sample solution, 0.5 M phosphate buffer (pH 6.5) and a 1 M sucrose solution is incubated at 370C. After 2 hours incubation, the sample containing substance M5071 is transparent. However, the sample without substance M507 1 shows the formation of a precipitate of insoluble glucan. These results indicate that substance M507 1 has an inhibitory action on glucosyltransferase activity.

(9) pH stability: pH 5 - 8 as shown in Figure 3 of the accompanying drawings. The pH stability was determined by heating at 400C for 60 min. solutions at each pH (pH 2.9: 0.1 M citrate buffer, pH 4 - 5: 0.1 M acetate buffer, pH 6 - 0.1 M phosphate buffer, pH 8 - 9: 0.1 M borate buffer) and assaying the activity remaining according to the assay method described hereinafter.

(10) Heat stability: stable up to 400 C, almost 100% denatured at 600C as shown in Figure 4 of the accompanying drawings.

Other physico-chemical properties of substance M 5071 are exemplified as follows: Amino Acid Analysis: Substance M5071 was hydrolysed with 6N HCI at 11000 for 24 hours, and analysed by an amino acid autoanalyzer. Tryptophane is measured by the UV absorption method. The results are shown in Table 1.

TABLE 1.

Amino acid: Molar ratio: aspartic acid 33.3 threonine 23.9 serine 35.4 glutamic acid 23.5 glycine 28.6 alanine 27.4 valine 20.9 methionine 1.7 isoleucine 12.4 leucine 13.7 tyrosine 12.5 phenylalanine 12.6 lysine 11.8 histidine 2.8 arginine 2.9 proline 13.6 tryptophane 29.2 Sugar composition: measured by phenol sulfuric acid method.

80 çl/mg as glucose or 68 A/mg as mannose.

Other action: very weak hydrolytic activity on insoluble glucan produced by Streptococcus mutans OMZ 176.

(a) A homogeneous suspension (1.0 ml) of insoluble glucan produced from Streptococcus mutans OMZ 176 and substance M5071 (200 y9, 2000 units) (1 ml) are incubated at 370C. After 20 hours turbidity is measured at 550 nm. Water is used as a control. The results are shown in Table 2.

TABLE 2.

Optical density: at time zero after 20 hours Substance M5071: 0.18 0.15 control (water): 0.21 0.21 As shown in the table, the mixture containing substance M507 1 reveals a slight increase of turbidity on incubation.

(b) To a suspension (0.5 ml) of purified insoluble glucan produced by Streptococcus mutans OMZ 1 76 (750 y9) is added a solution (0.5 ml) of 50 g (500 units) or 200 Ftg (2000 units) of substance M507 1 and incubation is effected at 370C for 2 hours.

Reducing sugar is colorimetrically measured as glucose by the Nelson-Somogyi method. Water is used as a control. The results are shown in Table 3.

TABLE 3.

glucose (llg/ml) 50,ag 20 substance M5071 200 ups 35off5 control (water) 9 A slightly higher value of reducing sugar is observed. Therefore, substance M5071 has a weak hydrolysing enzymatic activity for insoluble glucan.

It has also been found that Fungus strain M5084 isolated from a soil sample of a corn field in Hahajima, Ogasawara-mura, Tokyo, produces analogues of substance M507 1. These analogues are referred to herein as substance M50841 and substance M508411. Both substances are acid glycoproteins and have glucosyltransferase inhibiting activity. It was found that Macrophomina phaseoli IFO 7318 (Institute for Fermentation Osaka, List of Cultures, 1978, 6th Ed.) produces a substance analogous to substance M5071 having glucosyltransferase inhibiting activity. This analogue is designated substance M4400 herein.Additionally, it was found that Fungus strain M5093 belonging to genus Gnomoniella and strain M5094 belonging to genus Nodulisporium produce analogues of substance M5071 which are acid glycoprotein having glucosyltransferase inhibiting activity. These analogues are designated substance M5093 and substance M5094. The physico-chemical properties of each analogue are shown in Table 4.

TABLE 4.

substance substance substance substance substance M5084t M508411 M4400 M5093 M5094 (1) Molecular weight tE;el-fi Itration about about about about about method using 54000 54000 41000 59000 33000 "Sephadex" G-100] (2) Isoelectric point [isoelectric around around around around around focusing method ph 4.8 pH 5.6 ph 3.8 pH 3.1 pH 3.5 using carrier amphol ite3 (3) Ultraviolet absorption -spectrum Amax = 280 nm [0.5 mg/ml, 0.1 M phosphate buffer (pH 6.3) Amax (shoulder) = 290 nm (4) Solubility Soluble in : water Insoluble in : methanol, acetone, chloroform, ethyl acetate. (5) Color reaction Positive Biuret, xanthoproteic and Folin reactions. (6) Color white. (7) Action At least inhibit the synthesis of insoluble glucan by glucosyl transferase.

TABLE 4-. Continued.

substance substance substance substances substancE M50841 M508411 M4400 it5093 M5094 (8) pH stability* as shown as shown as shown as shown as shown in Fig. 5 in Fig. 6 in Fig. 7 in Fig. 8 in Fig. 9 of the of the of the of the of the accompany- accompany- accompany- accompany- accompany ing drawings ing drawings ing drawings ing drawings ing drawings pH 5 - 7 pH 5 - 7 pH 4 - 8 pH 4 - 8 pH 4 - 7 (9) Heat stability** as shown as shown as shown as shown as shown in Fig. 10 in Fig. 11 in Fig. 12 in Fig. 13 in Fig. 14 of the of the of the of the of the accompany- accompany- accompany- accompany- accompany ing drawings ing drawings ing drawings ing drawings ing drawings stable at stable at stable at stable at stable at 5000. 50"C. 50"C. 60"C. 4000.

100% 100% 100% 100% 100% denatured denatured denatured denatured denatured art7000. art7000. art7000. at 80 C. art6000.

*at 40 C for 60 minutes. pH 2 - 3: glycine + NaCI-HOl buffer.

pH 4 - 5: acetic acid -- sodium acetate buffer.

pH 6 - 7: KH2PO4 - NaHPO4 buffer.

pH 8 - 10: borate + KCI - Na,CO, buffer.

**heated in 0.1 M phosphate buffer (pH 6.5) for 60 minutes.

The taxonomical properties of the microorganism strain M5071 used in this invention are as follows: (1) Growth on Various Media: 1) Malt extract agar: Good growth. Fully covered Petri dish (inner diameter 85 mm) after 10 days culture at 260C. Downy aerial mycelia, white at early stage of culture and after one week of growth, gradually Light Tan (hue 3 gc) in some part. Most of colony: white. Soluble pigment: Light Light Amber (hue 3ic). Color of reverse side: Dark Brown (hue 2pn) - Maple (hue 41 e).

2) Potato glucose agar: Good growth. Fully covered Petri dish (inner diameter 85 mm) after 10 days culture at 260 0. Downy aerial mycelia. White at an early stage of culture, after one week of growth gradually turning Camel (hue 3ie) over whole surface. Soluble pigment: Light Amber (hue 3ic). Reverse side: Dark Brown (hue 2pn).

3) Czapek agar: Poor growth. Colorless mycelia slightly covers the surface of an agar medium after 10 days culture at 260C. Reverse side: colorless.

(2) Microscopic Observation: Mycelia: diameter 2.5-3.0 p. Smooth, light brown walled. Conidiophores; with smooth or knotty small protuberances. Light brown. septa in some places. Single conidia are attached to the top of short spinae in the sides of conidiophores. Globose or elliptical. 2.5 - 1 0 x 2.6 - 6 p. Brown. Smooth walled, with sometimes vertical slits.

(3) Physiological Properties: 1) Growth temperature: 8 - 4000.

2) Optimum growth temperature: 22 -- 320C.

3) Growth pH: 2.5 - 9.5.

4) Optimum growth pH: 4 - 8.

On the basis of the dark brownish color of the colonies and dark brownish globose or elliptical conidia with vertical slits, the strain M5071 is referred to genus Arthrinium, and is designated Arthrinium sp. M507 1. The strain has been deposited on 9th January, 1980 in the Fermentation Institute, Japan under permanent culture collection No. FERM-P No. 5352.

Color indications are on the basis of the "Color Harmony Manual" (Container Corp. of America, 1958). Taxonomical properties were assessed in conjunction with G.L. Barron, 1968. "The Genera of Hyphomycetes from soil" (1971) and M.B. Ellis, "Dematiaceous Hyphomycetes" (1971).

Taxonomical properties of strain M5084 are as follows: (1) Growth on Various Media: 1) Czapek agar: Rapid growth. Diameter 65 - 70 mm after 7 days culture at 260C. White downy at early stage of culture and on growing slightly Cream (hue 12 ca) with partly Dusty Agua (hue 18ge). No exudate or soluble pigment. Reverse side color: Light Ivory (hue 2ca) - Light wheat (hue 2ea), partly Dusty Agua (hue 18 ge).

2) Malt Extract Agar: Rapid growth. Diameter 50-53 mm after 7 days culture at 260C. White downy at early stage of culture and on growing, aerial mycelia are Flesh Pink (hue 6ca) and substrate mycelia are Red Wood (hue 6ie). No exudate or soluble pigment. Reverse side color: Red Wood (hue 6ie).

3) Potato Glucose Agar: Rapid growth. Diameter 57 - 60 mm after 7 days culture at 260C. White downy at early stage of culture and on growing Pearl Pink (hue 3ca.). No exudate. Soluble pigment: Cedar (hue 62 le). Reverse side color: Deep Brown Mahogany (hue 6pl).

(2) Microscopic Observation: Vegetative hyphae: colorless, smooth surface, 3 - 4 p in width.

Conidia formation: Phiaride type, two types of macro- and micro-conidia. Micro-conidiophores are verticaliy grown against vegetative hyphae. Single form. No branching or 1 - 2 branching.

Comparatively long, 30- 1 50 x 2.5-3 p, colorless, smooth surface with no or several septa. Many micro-conidia are pseudoglobose. 5 - 23 x 2 - 5 p, colorless, smooth surface, ovoid to elliptical. No septa or a single septum. Macro-conidia: one to several septa, meniscoid shape, sharp cusp. Colorless, smooth surface, 30 -- 50 x 3 - 5 p. Connecting part of phiaride: good grown staff cells.

(3) Physiological Properties: 1) Growth temperature: 10 -- 350C.

Optimum growth temperature: 24 -- 280C.

2)GrowthpH:pH2-12.

Optimum growth pH: pH 3 - 10 On the basis of the Phiaride type conidia formation, two types of macro- and micro-conidia and colorless meniscoid, one to several septa, and no stiff flagella and others, this strain is referred to genus Fusarium. [J.A. von Arx, The Genera of Fungi Sporulating in Pure Culture, (1974), J. Cramer, Lehre, 31 5 pp. G.L. Barron, The Genera of Hypomycetes from Soil, 364 pp. (1968), Williams s Wilkins, Baltimore.].

Further, the properties of having cream color colonies on Czapek agar, 30 -- 50 x 3 - 5 p macroconidia, microconidia on the cusp of conidia forming cells in many pseudoglobose, 5 - 23 x 2 -- 5 u and ovoid or elliptical shape are of Fusarium solani [C. Booth, The Genus Fusarium, CMI, kew, 237 pp, (1 971)]. The strain M5084 is therefore referred to as Fusarium solani M5084 and was deposited on 31st May 1980 in the Fermentation Institute, Japan as deposit No. FERM-P No. 5546.

The taxonomical properties of the strain M5093 are as follows: (1) Growth on various media: 1) Malt extract agar: Rapid growth. Fully covered the surface of a Petri dish (inner diameter 85 mm) after 7 days culture at 260C. Microbial flora are flat and downy. Color: Dark Olive Gray (hue near grays 1 ml). Edges of colony: smooth. No soluble pigment or exudate. Reverse side color: Midnight Blue (hue 14 pn).

2) Potato glucose agar: Moderate growth. 30 -- 40 mm after 7 days culture at 260 C. Microbial flora: slightly thick with rough surface. Color: Dark Olive Gray (hue 1 ml). Edge of colony: heavy Arachnoid. No soluble pigment or exudate. Reverse: Dark Blue (hue 133pun).

3) Czapek agar: Slow growth. 20 -- 28 mm after 7 days culture at 260C. Microbial flora: thin and flat. Surface: velvet. Color: Dark Olive Green (hue 242pun). Edges: heavy Arachnoid. No soluble pigment and exudate.

Reverse: Dark Olive Green (hue 24+pun).

(2) Microscopic Observation: Ascocarps are formed on the surface or inner part of agar with several groups on singles. Dark brown or black. About 200,u in diameter. Long and thick neck which is indistinguishable from ascocarp.

Size in 400 -- 900 x 70 - 130 p. Branched at base or upper part. Asci are formed from wall of ascocarps. Rod and short sTem. 40 - 80 x 10 - 1 3p. Ascospores are eight with biserial. Heads do not stain by Meizer's reagent. Ascospores are irregular spindle spores slightly curved. One-celled. Colorless to pale yellowish brown. 13 - 20 x 5 - 7 p. Smooth wall or with knotty spinae.

(3) Physioiogicai Properties: Growth pH: 3 - 8.5.

Optimum growth pH: 4 - 7.5.

Growth temperature: 13 -- 380C.

Optimum growth temperature: 23 -- 280C.

The strain is confirmed to belong Ascomycetes due to the formation of ascospores. Further properties such as the long solid necked ascocarp, ascospores from the wall of an ascocarp, rod-shaped, one celled ascospores and colorless are characteristics of the genus Gnomoniella. This strain is therefore referred to as Gnomoniella sp. M5093 and was deposited on 9th December, 1980 in Fermentation Institute, Japan as permanent culture collection PERM-P No. 5799. The references F.E.

Clements and C.L. Shear, "The Genera of Fungi," 495 pp., (1930), Hafner Pub. Co., U.S.A., J.A. von Arx, "The Genera of Fungi Sporulating in Pure Culture," 315 pp. (1974), and Cramer, Germany, R.W.G.

Dennis, "British Ascomycetes," 455 pp. (1968) were consulted in identifying strain M5093.

Taxonomical properties of the strain M5094 are as follows: (1) Growth Conditions on Various Media: 1) Malt extract agar: Rapid growth. Fully covered a Petri dish (diameter 85 mm) after 7 days culture at 260 C. Microbial flora: flat, downy aerial mycelia with Covert Tan (hue 2 ge). Edges of colony: Arachnoid. Honey Gold (hue 2id) soluble pigment. No exudate. Reverse side color: Midnight Blue (hue 14 pn).

2) Potato glucose agar: Rapid growth. 65 -- 80 mm in diameter after 7 days culture at 260C. Microbial flora: flat and slightly thick. Downy aerial mycelia with Covert Brown (hue 21i). Edges of colony: Arachnoid. Honey Gold (hue 2ic) soluble pigment. No exudate. Reverse side color: Dark Spruce (hue 20 pn).

3) Czapek agar: Rapid growth. 45 -- 65 mm in diameter after 7 days culture at 260C. Microbial flora: flat. Slightly downy aerial mycelia with Covert Tan (hue 2ge). Edges of colony: Arachoid. Trace soluble pigment with Bamboo (hue 2 gc). No exudate. Reverse: Beige Brown (hue 3ic).

(2) Microscopic Observation: Vegetative hyphae: colorless to pale brown, smooth wall with septa. Sometimes pellet-substances accumulate in the edges of mycelia. Conidiophores; 40 -- 200 x 2.5 - 3.5 p, septa, colorless to pale brown. Rough wall forming circularly and regularly, pale yellowish brown to pale brown. 10 -- 20 x 2.5 -3.5 p. Rough surface. Conidia; Sympodulae type formation on the several small spinose on the top of swolien conidia forming cells. Irregular oval cutted at base edge. Colorless to pale yellowish brown. 4.5 - 6 x 2.5 - 3 p. Smooth wall.

(3) Physiological Properties: Growth pH;3- 11.

Optimum growth pH: 4 - 8.

Growth temperature: 12 - 4500.

Optimum growth temperature: 23 -- 370C.

The strain is confirmed as belonging to Hyphomycetes due to growth by exogrown conidia. The properties such as comparatively long conidiophores with regularly branched Sympodule type of conidia formation, rough wall conidia forming cells with sharpless top and flat edge of conidia show that the strain M5094 belongs to the genus Nodulisporium. The strain is referred to Nodulisporium sp. M5094 and was deposited on 9th December, 1 980 in the Fermentation Institute, Japan as permanent culture collection FERM-P No. 5800. The references M.B. Ellis, "Dematiaceous Hyphomycetes", 608 pp., (1971), C.M.I. Ingland, G. L. Barron, "The Genera of Hyphomycetes from Soil," 364 pp. (1968), The Williams and Wilkins Co., U.S.A. were consulted in identifying strain M5094.

The strains herein mentioned are illustrative and the proteins of the inventions can be produced from artificial or natural mutants of these strains. In the production of proteins of the invention, microorganisms producing these proteins, preferablyArthrinium sp. M5071, Fusarium solani M5084, Macrophomina phaseoli IFO 7318, Gnomoniella sp. M5093 or Nodulisporium sp. M5094, are cultured in a conventional medium for antibiotics or enzyme production. Submerged aeration culture is preferable.

Conventional nutrient sources can be used. Assimilable carbon sources such as glucose, lactose, maltose, fructose, molasses and dextrin, or preferably sucrose, can be used. Assimilable nitrogen sources such as polypeptone, corn steep liquor, meat extract, yeast extract, soy bean powder and casein hydrolysate can be used. Phosphate, carbonate or sulfate and salts of magnesium, calcium, potassium, sodium, iron, zinc or manganese can also be used if necessary. The culture temperature can be selected with the range of growth of microorganisms and the production of the desired protein of the invention, and is generally 26 - 300 C, preferably 260 C. Culturing time depends on the conditions and can be terminated when the production of the desired protein of the invention has reached a maximum.

Generally culturing is effected for 2 - 4 days.

A protein of the invention can be isolated from the cultured mass by filtration and centrifugation of the cultured mass to separate the mycelia. Isolation of the protein from the resulting culture filtrate can preferably be effected by using the differing solubility of the substance in various solvents or salting-out techniques. For example ammonium sulfate can be added to the culture filtrate for salting-out, and the resulting precipitate is collected by centrifugation. The precipitate is dialyzed against a stable pH buffer and purified by adsorption chromatography and gel-filtration using, for example, DEAE- "Sephadex," DEAE-cellulose or "Sephadex." Further purification can be effected by the isoelectric focusing method to obtain a purified substance after lyophilization.

The thus obtained proteins of the invention have an inhibitory action on the synthesis of insoluble glucan by glucosyltransferase. In particular they inhibit the formation of sticky insoluble glucan on the surface of teeth in the presence of sucrose microorganisms Streptococcus mutans which cause dental caries. As a result of their inhibitory action, the adhesion of various dental caries bacteria on the surface of teeth by means of the sticky insoluble glucan is inhibited and dental caries formation is prevented.

The substances of the invention (acid glycoprotein having inhibitory action for glucosyltransferase) can be incorporated as the active ingredient in compositions for use in the prevention of dental caries.

Such compositions also comprise suitable adjuvants. For example, a protein of the invention may be mixed in a dentifrice as a powder or in an aqueous or alcoholic solution to prepare dental preparations such as toothpaste, tooth powder, foaming preparation, gargling tablet or massage cream, or to prepare gum base compositions such as chewing gum, or to prepare other foodstuffs. In the dentifrice, conventional additives are added.Examples of additives are abrasives such as calcium carbonate, calcium phosphate, insoluble sodium metaphosphate, aluminium oxide, aluminium hydroxide, amorphous silica and crystal silica, viscidics such as methylcellulose, sodium arginate, caraginane, carboxymethylcellulose and anhydrous silica, moisturising agent such as sorbitol, xylytol, glycerol, propylene glycol and polyethyleneglycol, activators such as sodium laurylsulfate, sodium dodecylbenzenesulfonate, sucrose fatty acid ester and sodium acylsarcosine, others such as fluoride, lysozyme hydrochloride, dextranase, styptics such as E-aminocaproic acid or tranexamic acid, sodium chloride, chlorophyll, sweetenings and other aromatics, antiseptics or microbicides. The proteins of the present invention can be added, for example in a dentifrice, in an amount of 10 units or more per single use.Generally the concentration of protein in the compositions of the invention is 0.0001 w/w%. The thus prepared compositions prevent the synthesis of insoluble glucan even in the presence of sucrose by the action of the glucosyltransferase inhibitor and inhibit the adhesion of bacteria causing dental caries on the surface of teeth, thereby preventing tooth decay.

Determination of Activity.

The assay method for the activity of the proteins of the invention is based on inhibitory activity on the synthesis of insoluble glucan using glucosyltransferase in the presence of sucrose.

Streptococcus mutans OMZ 1 76 is incubated in BHl medium for 2 days. The culture filtrate is taken by salting-out using ammonium sulfate. The precipitate is dissolved in water and is dialyzed up to 50 times concentration to obtain a glucosyltransferase solution. A mixture of 0.1 ml of each of the glucosyltransferase solution, a solution of a protein of the invention 0.5 M phosphate buffer and 1 M sucrose is incubated at 370C. Water is used as a control. The solution of the protein of the invention is aliquotly diluted. After 2 hours incubation, formation of insoluble glucan is confirmed.

Activity is determined as the maximum dilution of the protein of the invention which completely inhibits the synthesis of insoluble glucan. For example when a 100 times dilution shows inhibitory action and a 1 20 times dilution does not, the potency is defined as 100. A potency per milliliter of a protein of the invention (A) is defined by the following equation.

A=a x 10(unitiml) wherein a means maximum dilution.

The inhibitory action of the proteins of the invention preventing the adhesive adsorption onto the surface of teeth of bacteria Streptococcus mutans which cause dental caries is explained below.

(1) Adhesive adsorption on the surface of teeth of bacteria causing dental caries.

A sterilized piece of a human tooth (5 x 5 mm) is hung by a stainless steel wire in BHl medium containing 5% sucrose in each of two test tubes. Into one test tube substance M507 (1000 units) is added. The other is used as a control and no substance M5071 is added. Cultured broth of Streptococcus mutans cultured at 370C for 18 hours in BHl medium is added up to 10% of final concentration. After incubation at 370C for 6 hours, each piece of tooth is taken out and washed, then observed and photographed by a scanning electron microscope at a magnification of 3000. The result of the control is shown in the photograph of Figure 1 5 of the accompanying drawings.Many microbes of Streptococcus mutans OMZ 176 are adhesively adsorbed on the surface of the piece of tooth. Figure 1 6 of the accompanying drawings is a photograph of the piece of tooth cultured in the presence of substance M5071. The same experiment was also carried out using substances M50841, M508411, M4400, M5093 and M5094. The results of these experiments are shown in Figures 17-21 respectively of the accompanying drawings. Almost no adhesively adsorbed microbes can be observed in Figures 1 6-21. These results may be taken as an effect of inhibitory action of the synthesis of insoluble glucan.

(2) Colony formation on the surface of teeth of bacteria causing dental caries.

Streptococcus mutans OMZ 176 is cultured in BHl medium at 370C for 18 hours. Bacterial cells are collected, washed with 0.05 M phosphate buffer (pH 6.8), suspended in the same buffer and added sucrose up to 5% of final concentration. The suspension is cultured at 370C for one hour to form glucan surround of the cells. The cultured cells are washed by centrifugation in order to remove any remaining sucrose and soluble glucan. The washed cells are again suspended in 0.05 M phosphate buffer (pH 6.8).

A sterilized piece of human tooth is added to the suspension, which is then allowed to stand for 30 minutes at room temperature. The tooth piece is washed with water to remove unnecessary cells and hung by a steel wire in BHl medium containing 10% sucrose. Substance M5071 (1000 units) is added to a test group and no addition of substance M5071 is used as a control. Both groups are incubated at 370C for 6 hours and the pieces of teeth are washed with water and observed and photographed by a scanning electron microscope at a magnification of 3000.

In the control group Streptococcus mutans OMZ 176 cells adhered and grew on the surface of tooth, and bacterial plaques (sordes on the tooth) together with glucan are formed as shown in Figure 22 of the accompanying drawings. As shown in Figure 23 of the accompanying drawings no formation of bacterial plaques (sordes on the tooth) occurred for the group to which substance M5071 was added.

Sordes on the tooth form insoluble glucan according to the growth of bacteria and form bacterial colonies. Substance M5071 inhibits the formation of insoluble glucan and thereby prevents the formation of sordes on the tooth.

The following Examples illustrate the present invention. Percentages and parts are by weight.

EXAMPLE 1 Arthrinium sp. M5071 was inoculated into a medium (100 ml, pH 6.5) containing sucrose 1.5%, dextrin 1.5%, polypeptone 2%, corn steep liquor 2%, KH2PO4 0.2%, MgSO4.7H20 0.1%, FeSO4.7H20 0.01% and Celite (diatomaceous earth) 1% in each of five 500 ml Erlenmeyer flasks (sterilized at 1200C for 20 minutes) and shake cultured at 260C for 5 days. This seed culture was inoculated in 5 ml portions into 100 flasks of the same size and medium as above and cultured at 260C for 4 days. The culture filtrate (10 lit.) was obtained by filtration using Celite (total protein 1 720 g, specific activity 0.23 U/mg).Ammonium sulfate (5.16 kg) was added to the filtrate, stirred and then allowed to stand in a cool room (40C) over-night. The precipitate that had formed was collected by centrifugation, dissolved in 0.01 M phosphate buffer (pH 6.3, 500 ml) and dialyzed using a cellophane tube against the same phosphate buffer. Every 12 hours the dialyzer solution was changed and in total 120 lit. of buffer solution was used for dialysis in 72 hours. After dialysis, the solution was charged onto a column (5 x 40 cm) of DEAE-cellulose which had been previously equilibrated with 0.01 M phosphate buffer (pH 6.3). The column was washed with the same phosphate buffer, and gradiently eluted with 0.01 M phosphate buffer (pH 6.3, 2 lit.) -0.01 M phosphate buffer containing 0.5 M NaCI (pH 6.3, 2 lit.).

Fractions (460 ml, protein 1.27 g, specific activity 83 U/mg) eluted with 0.01 M phosphate buffer containing 0.15 M NaCI (pH 6.3) were collected and concentrated by membrane PM-10 (Amicon Co.).

The concentrate was dialyzed using a cellophane tube against 0.01 M phosphate buffer. The dialyzer solution was changed every 12 hours. The concentrate was dialyzed against 80 lit. of buffer for 48 hours. The dialyzate was charged onto a column (3 x 20 cm) of DEAE-"Sephadex" A-25 which had been previously equilibrated with 0.01 M phosphate buffer (pH 6.3). The column was washed with the same phosphate buffer, and gradiently eluted with 0.01 M phosphate buffer (pH 6.3,2 lit.) -0.01 M phosphate buffer containing 0.3 M NaCI (pH 6.3, 2 lit.). Fractions (400 ml, protein 105 mg, specific activity 510 U/mg) eluted with 0.01 M phosphate buffer containing 0.01 M NaCI were collected and concentrated with membrane PM-10 to obtain a concentrate (20 ml). This concentrate was charged onto a column (4.5 x 60 cm) of "Sephadex" G-l 00 previously equilibrated with 0.01 M phosphate buffer (pH 6.3). This column was eluted with 0.01 M phosphate buffer (pH 6.3) (4.8 ml per fraction).

Fractions No. 88 - 104 were collected (protein 38.5 mg, specific activity 1300 U/mg) and lyophilized.

The lyophilizate was desalted by "Sephadex" G-25 Coarse and again lyophilized to obtain a powder (14.7 mg). The lyophilized powder was dissolved in a small amount of 0.01 M phosphate buffer (pH 6.3) and subjected to isoelectric focussing (700 V, 24 hours) by carrier ampholite (pH 2.4 - 4) in glycerol.

Active fractions (2 ml per fraction) were collected and charged onto a column (2.5 x 50 cm) of "Sephadex" G-50 Fine previously equilibrated with water. Elution was carried out with water for each 4.3 ml fraction. Active fractions (No.21-25 were collected and lyophilized to obtain purified substance M5071 as a white powder (4.1 mg). The specific activity of the powder was 18000 times that of the culture filtrate. Yield: about 11%. A single band was observed by disc electrophoresis using polyacrylamide gel.

Referential Example 1.

Inhibitory action of substance M5071 on the synthesis of insoluble glucan: (1) a glucosyltransferase solution prepared by cultivation as shown in the Assay method hereinbefore (20 ml), 0.1 M phosphate buffer (pH 7.0,200 ml), 50% sucrose solution (100 ml) and a solution (20 ml) of 5000 units of substance M5071 are mixed and incubated at 37"C for 18 hours. Water is added in place of the solution of substance M5071 in a control experiment.

Each incubated mixture was centrifuged at 1000 r.p.m. for 10 minutes to obtain a precipitate and a supernatant solution. The precipitate was washed several times with water to obtain insoluble glucan.

The supernatant solution was dialyzed against water. Twice the volume of ethanol was added to the dialyzate and allowed to stand at 40C for 1 8 hours, then centrifuged at 1 5000 r.p.m. for 5 minutes. The precipitate was washed several times with a mixture of ethanol and water (2:1) and dried to obtain soluble glucan.

The glucan content in each reaction mixture is shown in Table 5.

TABLE 5.

Glucan formed (mg) Glucans M5071 substance control insoluble glucan 45.47 843.98 soluble glucan 1088.52 723.60 Gibbons et al reported that in order for the bacteria causing dental caries to adhere to the surface of teeth, the insoluble glucan produced by Streptococcus mutans plays an important role. [Archs. Oral.

Biol., 13, 1249 - 1260 (1968).] The results of Table 5 show that substance M5071 inhibits the synthesis of the insoluble glucan necessary for the adhesion of Streptococcus mutans to the surface of teeth (95% inhibition). (2) The glucose bonding structures of the insoluble glucan and soluble glucan produced without the addition of substance M5071 and the soluble glucan produced by the addition of substance M5071 were analyzed. Each sample was methylated, hydrolysed and the methylated sugars were analyzed by gas-chromatography according to a method by Hakamori [J. Biochem., 55 205 (1964)]. The results are shown in Table 6.

TABLE 6.

Methylated Sugar Yield (%) No addition of Addition of substance M5071 substance M5071 Soluble Insoluble Soluble glucan glucan glucan 2,3,4,6-tetra- 13.5 14.6 20.8 2,4,6-tri- 2.0 50.5 3.2 2,3,4-tri- 72.1 20.9 56.8 294-di- 12,4 14.0 19.2

The results show that the content of the 2,4,6-trimethylated sugar is decreased in the soluble glucan of the group to which substance M5071 has been added compared with the control group.

Therefore substance M507 1 is presumed to inhibit the synthesis of the glucan having an a1 -3 linkage.

EXAMPLE 2 Fusarium solani M5084 FERM-P No. 5546 was inoculated into five 500 ml Erlenmeyer flasks (sterilized at 1200C for 20 minutes) each containing a medium (100 ml, pH 6.5) containing sucrose 1.5%, dextrin 1.5%, polypeptone 2%, corn steep liquor 2%, KH2PO4 0.2%, MgSO4.7H20 0.1 %, FeSO4.7H20 0.01% and Celite 1% and was shake cultured at 260C for 4 days. 5 ml portions of this seed culture was inoculated into 100 flasks containing the same medium and shake cultured at 260C for 4 days. The culture filtrate was obtained by filtration using Celite (10 lit., protein 1 503 g. specific activity 0.34 U/mg).Ammonium sulfate (5.16 kg) was added to the cultured broth filtrate, stirred and allowed to stand over-night in a cool room (40C). The precipitate that formed was collected by centrifugation, dissolved in 0.01 M phosphate buffer (pH 6.3, about 500 ml) and dialyzed against 0.01 M phosphate buffer (pH 6.3) using a cellophane tube. Every 1 2 hours the dialyzer solution was changed and in total 120 lit. of dialyzer solution were used in 72 hours. The dialyzate was charged onto a column (5 x 40 cm) of DEAE-cellulose previously equilibrated with 0.01 M phosphate buffer. The column was washed with 0.01 M phosphate buffer (pH 6.3) and gradiently eluted with 0.01 M phosphate buffer (pH 6.3, 2 lit) - 0.01 M phosphate buffer containing 0.5 M NaCI (pH 6.3, 2 lit.).The fractions eluted at 0.01 M phosphate buffer containing 0.1 M NaCI were collected (520 ml, protein 1.5 g, specific activity 87 U/mg), and concentrated by membrane PM-10 (Amicon Co.). The concentrate was dialyzed against 0.01 M phosphate buffer using a cellophane tube. The dialyzer solution was changed every 12 hours and in total 80 liters of dialyzer solution were used for 48 hours dialysis. The dialyzate was charged onto a column (3 x 20 cm) of DEAE- "Sephadex" A-25 previously equilibrated with 0.01 M phosphate buffer (pH 6.3). The column was washed with 0.01 M phosphate buffer (pH 6.3), and gradiently eluted with 0.01 M phosphate buffer (pH 6.3, 2 lit.) - 0.01 M phosphate buffer containing 0.3 M NaCI (pH 6.3, 2 lit.).Active fractions (150 ml, protein 33 mg, specific activity 507 U/mg) containing the M5071 analogue, substance M50841, eluted at a 0.05 M NaCI concentration in phosphate buffer and fractions (50 ml, protein 23 mg, specific activity 481 U/mg) containing the M5071 analogue, substance M508411, eluted at a 0.07 M NaCI concentration, were collected and lyophilized.

The lyophilizate of the M507 1 analogue, substance M50841, was dissolved in a small amount of water and was charged onto a column (2.5 x 60 cm) of "Sephadex" G-100 previously equilibrated with 0.01 M phosphate buffer and eluted in 4.8 ml fractions by 0.01 M phosphate buffer (pH 6.3). Fractions No.

55 - 62 were combined (protein 21 mg, specific activity 1570 U/mg) and lyophilized. The lyophilizate was dissolved in a small amount of water, desalted by "Sephadex" G-25 Coarse and lyophilized to obtain the purified M5071 analogue, substance M50841 (19 mg). The product was purified about 4500 times in specific activity as compared with cultured broth, and showed a single band in electrophoresis using polyacrylamide.

The lyophilized M5071 analogue, substance M508411, was dissolved in a small amount of water, charged onto a column (2.5 x 60 cm) of "Sephadex" G-l 00 equilibrated by 0.01 M phosphate buffer (pH 6.3) and eluted in 4.8 ml fractions with 0.01 M phosphate buffer (pH 6.3). Fractions No. 56 - 60 were collected, combined (protein 1 3 mg, specific activity 1490 U/mg) and lyophilized. The lyophilizate was dissolved in a small amount of water, desalted by "Sephadex" G-25 Coarse and lyophilized to obtain purified substance M508411 (11 mg). This substance was purified about 4800 times in specific activity as compared with culture filtrate and showed a single band in electrophoresis using polyacrylamide gel.

EXAMPLE 3.

Macrophominae haseoli IFO 731 8 was inoculated into five 500 ml Erlenmeyer flasks (sterilized at 12000for20 minutes) each containing a medium (100 ml, pH 6.5) containing glucose 1%, protoflower 1%, peptone 1%, corn steep liquor 1%, KH2PO4O.1%, MgSO4.7H20 0.1%, and Celite 1% and was shake cultured at 260C for 4 days. This seed culture was transferred in 5 ml portions into a medium in 100 500 ml Erlenmeyer flasks containing sucrose 1.5%, dextrin 1.5%, polypeptone 2%, corn steep liquor 2%, KH2PO4 0.2%, MgSO4.7H20 0.1% FeSO4.7H20 0.01% and Celite 1 % (pH 6.5, each 100 ml, sterilized at 1200C for 20 minutes) and shake cultured at 260C for 3 days.The culture filtrate was obtained by filtration using Celite to obtain culture filtrate (about 8.4 lit. protein 1 637 g, specific activity 0.29 U/mg).

Ammonium sulfate (4.6 kg) was added in the filtrate, stirred and allowed to stand in a cool room (40C) over-night. The precipitate that formed was collected by centrifugation, dissolved in 0.01 M phosphate buffer (pH 6.3, 500 ml) and dialyzed against 0.01 M phosphate buffer (pH 6.3) using a cellophane tube.

The dialyzer solution was changed every 1 2 hours and in total 120 1 of solution were used for every 72 hours dialysis. The dialyzate was charged onto a column (5 x 40 cm) of DEAE-cellulose equilibrated with 0.01 M phosphate buffer (pH 6.3), washed with 0.01 M phosphate buffer (pH 6.3) and gradiently eluted by 0.01 M phosphate buffer (pH 6.3, 2 lit.) - 0.01 M phosphate buffer containing 0.5 M NaCI (pH 6.3, 2 lit.) to obtain an eluate (393 ml, protein 1.6 g. specific activity 73 U/mg) eluted at a concentration of 0.15 M NaCI. The eluate was concentrated by membrane PM-10. The concentrate was dialyzed against 0.01 M phosphate buffer using a cellophane tube. The dialyzer solution was changed every 12 hours and in total 80 lit. of solution was used for 48 hours dialysis. The dialyzate was charged onto a column (3 x 22 cm) of DEAE-"Sephadex" A-25 equilibrated with 0.01 M phosphate buffer, washed with 0.01 M phosphate buffer, and gradiently eluted with 0.01 M phosphate buffer (2 lit., pH 6.3) - 0.01 M phosphate buffer containing 0.3 M NaCI (pH 6.3, 2 lit.). Active fractions containing the M5071 analogue, substance M4400, (372 ml, protein 102 mg, specific activity 511 U/mg) were eluted at a concentration of 0.01 M NaCI. This eluate was concentrated by membrane PM-10 down to a volume of 20 ml. The concentrate was charged onto a column (4.5 x 60 cm) of "Sephadex" G-l 00 equilibrated by 0.01 M phosphate buffer (pH 6.3) and eluted with 0.01 M phosphate buffer in 4.8 ml fractions.Fractions No. 79 - 92 were collected, combined (protein 34 mg, specific activity 1431 U/mg) and lyophilized. The lyophilizate was desalted by "Sephadex" G-25 Coarse and lyophilized (32 mg). Further the powder was dissolved in a small amount of 0.01 M phosphate buffer (pH 6.3) and subjected to isoelectric focusing (700 V, 24 hours) using carrier ampholite (pH 2.4 - 4 in glycerol.

Active fraction in each 2 ml fractions were collected, charged onto a column (2.4 x 40 cm) of "Sephadex" G-50 Fine equilibrated with water, and fractionated for each 4.8 ml fraction with water.

Fractions No.20-25 were collected and lyophilized to obtain substance M4400 (11 mg) as a white powder. Specific activity was increased about 9000 times compared with that of the culture filtrate.

Single band by polyacrylamide electrophoresis.

EXAMPLE 4 Gnomoniella sp. M5093 was inoculated into five 500 ml Erlenmeyer flasks (sterilized at 1200C for 20 minutes) each containing a medium (100 ml, pH 6.5) containing glucose 1%, protoflower 1%, peptone 1%, corn steep liquor 1%, KH2PO4 0.1 %, MgSO4.7H20 0.1%, and Celite 1% and was shake cultured at 260C for 4 days.This seed culture was transferred in 5 ml portions into 100 500 ml Erlenmeyer flasks containing a medium containing sucrose 1.5%, dextrin 1.5%, polypeptone 2%, corn steep liquor 2%, KH2PO4 0.2%, MgSO4.7H20 0.1%, FeSO4.7H200.01%andCelite 1 % (pH 6.5, each 100 ml, sterilized at 1200C for 20 minutes) and shake cultured at 260C for 3 days. The culture filtrate was obtained by filtration using Celite to obtain culture filtrate (about 10 lit. protein 1 611 g, specific activity 0.19 U/mg). Ammonium sulfate (5.2 kg) was added to the filtrate, stirred and allowed to stand in a cool room (40 C) over-night.The precipitate that formed was collected by centrifugation dissolved in 0.01 M phosphate buffer (pH 6.3, 500 ml) and dialyzed against 0.01 M phosphate buffer (pH 6.3) using a cellophane tube. The dialyzer solution was changed every 12 hours and in total 120 1 of solution were used for 72 hours dialysis. The dialyzate was charged onto a column (5 x 40 cm) of DEAE-cellulose equilibrated with 0.01 M phosphate buffer (pH 6.3), washed with 0.01 M phosphate buffer (pH 6.3) and gradiently eluted by 0.01 M phosphate buffer (pH 6.3, 2 lit.) - 0.01 M phosphate buffer containing 0.5 M NaCI (pH 6.3, 2 lit.) to obtain the eluate (415 ml, protein 1.81 g, specific activity 91 U/mg) eluted at a concentration of 0.2 M NaCI. The eluate was concentrated by membrane PM-10. The concentrate was dialyzed against 0.01 M phosphate buffer using a cellophane tube.The dialyzer solution was changed every 1 2 hours and in total 80 lit. of solution was used for 48 hours dialysis. The dialyzate was charged onto a column (3 x 27 cm) of DEAE-"Sephadex" A-25 equilibrated with 0.01 M phosphate buffer, and gradiently eluted with 0.01 M phosphate buffer (2 lit., pH 6.3) - 0.01 M phosphate buffer containing 0.3 M NaCI (pH 6.3,2 lit.). The active fractions containing the M5071 analogue, substance M5093, (208 ml, protein 93 mg, specific activity 1 92 U/mg) were eluted at a concentration of 0.1 M NaCI and lyophilized. The substance M5093 was dissolved in a small amount of water, charged onto a column (2.5 x 60 cm) of "Sephadex" G-l 00 equilibrated by 0.01 M phosphate buffer (pH 6.3) and eluted with 0.01 M phosphate buffer in 4.8 ml fractions.Fractions No. 69 - 80 were collected, combined (protein 25 mg, specific activity 1330 U/mg) and lyophilized. The lyophilizate was desalted by "Sephadex" G-25 Coarse and lyophilized (33 mg). Further the powder was dissolved in a small amount of 0.01 M phosphate buffer (pH 6.3) and subjected to isoelectric focusing (700 V, 24 hours) using carrier ampholite (pH 2.4 - 4) in glycerol. Active fraction in each 2 ml fractions were collected, charged on a column (2.4 x 40 cm) of "Sephadex" G-50 Fine equilibrated with water, and fractionated for each 4.8 ml fraction with water. Fractions No. 21 - 25 were collected and lyophilized to obtain purified substance M5093 (20 mg) as a white powder. Specific activity was increased about 8200 times compared with that of the culture filtrate.Single band by SDS polyacrylamide electrophoresis (pH 9).

EXAMPLE 5.

Nodulisporium sp. M5094 was inoculated into five 500 ml Erlenmeyer flasks (sterilized at 1 200C for 2 minutes) each containing a medium (100 ml, pH 6.5) containing glucose 1%, protoflower 1%, peptone 1%, corn steep liquor 1%, KH2PO4 0.1 %,MgSO4.7H2O0.1%, and Celite 1% and was shake cultured at 260C for 4 days.This seed culture was transferred in 5 ml portions into 100 500 ml Erlenmeyer flasks containing a medium containing sucrose 1.5%, dextrin 1.5%, polypeptone 2%, corn steep liquor 2%, KH2PO4 0.2%, MgSO4.7H20 0.1 %, FeSO4.7H20 0.01 %, and Celite 1 % (pH 6.5, each 100 ml, sterilized at 1 200C for 20 minutes) and shake cultured at 260C for 3 days. The culture filtrate was obtained by filtration using Celite to obtain the culture filtrate (about 9 lit. protein 1 302 g, specific activity 0.21 U/mg). Ammonium sulfate (4.6 kg) was added in the filtrate, stirred and allowed to stand in a cool room (40 C) over-night. The precipitate that formed was collected by centrifugation, dissolved in 0.01 M phosphate buffer (pH 6.3, 500 ml) and dialyzed against 0.01 M phosphate buffer (pH 6.3) using a cellophane tube. The dialyzer solution was changed every 12 hours and in total 120 1 of solution were used for 72 hours dialysis. The dialyzate was charged onto a column (5 x 40 cm) of DEAE-cellulose equilibrated with 0.01 M phosphate buffer (ph 6.3), washed with 0.01 M phosphate buffer (pH 6.3) and gradiently eluted by 0.01 M phosphate buffer (pH 6.3, 2 lit.) - 0.01 M phosphate buffer containing 0.5 M NaCI (pH 6.3, 2 lit.) to obtain the eluate (433 ml, protein 2.42 g, specific activity 88 U/mg) eluted at concentration of 0.1 M NaCI. The eluate was concentrated by membrane PM-10.The concentrate was dialyzed against 0.01 M phosphate buffer using a cellophane tube. The dialyzer solution was changed every 12 hours and in total 80 lit of solution were used for 48 hours dialysis. The dialyzate was charged onto a column (3 x 25 cm) of DEAE-"Sephadex" A-25 equilibrated with 0.01 M phosphate buffer, washed with 0.01 M phosphate buffer, and gradiently eluted with 0.01 M phosphate buffer (2 lit., pH 6.3) - 0.01 M phosphate buffer containing 0.3 M NaCI (pH 6.3, 2 lit.). Active fractions containing the M5071 analogue, substance M5094, (283 ml, protein 85 mg, specific activity 201) were eluted at a concentration of 0.05 M NaCI and lyophilized.The substance M5094 was dissolved in a small amount of water and was charged onto a column (2.5 x 60 cm) of "Sephadex" G-1 00 equilibrated by 0.01 M phosphate buffer (pH 6.3) and eluted with 0.01 M phosphate buffer in 4.8 ml fractions. Fractions No. 85 - 97 were collected, combined (protein 23 mg, specific activity 1121 U/mg) and lyophilized. The lyophilizate was desalted by "Sephadex" G-25 Coarse and lyophilized (22 mg). Further the powder was dissolved in a small amount of 0.01 M phosphate buffer (pH 6.3) and subjected to isoelectric focusing (700 V, 24 hours) using carrier ampholite (pH 2.4 - 4) in glycerol. Active fraction in each 2 ml fractions were collected, charged onto a column (2.4 x 40 cm) of"Sephadex" G-50 Fine equilibrated with water, and fractionated for each 4.8 ml fraction with water.Fractions No. 21 - 25 were collected and lyophilized to obtain purified substance M5094 (17 mg) as white powder. Specific activity was increased about 6800 times compared with that of the culture filtrate. Single band by SDS polyacrylamide electrophoresis (pH 9).

EXAMPLE 6 Glycerol (30 parts) and water (20 parts) were mixed well and an aqueous solution of substance M5071 (0.1 part) was added to the mixture. Methyl cellulose (1 part) and saccharin sodium (0.5 part) were added to and dissolved in the resulting mixture. Calcium secondary phosphate (45 parts) was added and mixed in well. Further aroma (1 part) was added and the mixture was kneaded under defoaming by an attached vacuum apparatus to obtain a tooth paste.

The product is packed in amounts of 100 -- 300 g and is used in an amount of about 1 g per person at one time.

EXAMPLE 7 A tooth past of the following composition was prepared: Calcium secondary phosphate 50 parts Glycerol 30 Sodium lauryl sulfate 1.5 Carboxy methyl cellulose 1 part Saccharin sodium 0.4 Aroma 0.6 Fructose 4 parts Sodium monofluorophosphate 0.4 part Monostearate sucrose 2 parts 1% aqueous solution of substance M5071 0.1 part Water rest.

EXAMPLE 8 A tooth powder comprising the following composition was prepared: Calcium secondary phosphate 50 parts Calcium carbonate 25 Sorbitol 10 Sodium laurylsulfate 1 part Saccharin sodium 0.3 Aroma 0.7 Dextranase 1 Fructose 5 parts 1% substance M5071 solution 0.1 part Water rest.

EXAMPLE 9.

Tablets for gargling comprising the following composition were prepared: Sodium hydrogen carbonate 54 parts Citric acid 15 Sodium phosphate 15 Polyethylene glycol 6000 3 Aroma 4 Sucrose monolaurylsulfate 1 part Maltose 5 parts Sodium monofluorophosphate 0.8 part Dextran 2 parts M5071 substance 0.001 part EXAMPLE 10 A gum base (20 parts) was heated at 40 C and sorbitol (59.9 parts) and 1% of a solution of substance M5071 (0.1 part) were added. Mannit (9.5 parts) and 70% sorbitol (10 parts) and aroma were further added. The mixture was kneaded well and formed and cut to prepare the chewing gum.

EXAMPLE 11.

A chewing gum of the following composition was prepared: Gum base 20 parts Calcium carbonate 2 Starch syrup 15 Powder sugar 30 Maltose 10 Fructose 10 Sucrose monostearate 3 Aroma 1 part M5071 substance 0.001 Water rest.

EXAMPLE 12.

Dextrin (500 parts) in a mandarin orange concentrate (1/5 volume concentration, solid 53.6%) (560 parts) was spray dried (intake temperature 130 - 1400C, outlet temperature 75 -- 800C) to prepare a powder juice. The powder juice (100 parts) was mixed with substance M5071 (0.001 part) and mixed well to prepare a powder juice.

EXAMPLE 13 In Example 7, substance M5071 was replaced by substance M50841, substance M508411, substance M4400, substance M5093 and substance M5094 respectively (each as 1% aqueous solutions) to prepare tooth pastes.

EXAMPLE 14.

In Examples 8 and 9, substance M5071 was replaced by substance M50841, substance M508411, substance M4400, substance M5093 and substance M5094 respectively to prepare tooth powders and tablets for gargling.

EXAMPLE 15.

In Example 10, substance M507 1 was replaced by substance M50841, substance M508411, substance M4400, substance M5093 and substance M5094 respectively (each as 0.1% aqueous solutions, 0.1 part) to prepare chewing gums.

Claims (31)

1. An acidic protein having a molecular weight of from 30,000 to 60,000 which inhibits the synthesis of insoluble glucans by glucosyltransferase.

2. A protein according to claim 1 which is designated herein substance M507 1 and which has the following physico-chemical properties: Molecular weight: about 34000 (determined by gel-filtration method), and about 45000 (determined by polyacrylamide gel electrophoresis), Isoelectric point: around pH 3.5 (determined by isoelectric focusing using carrier ampholite), Ultraviolet absorption spectrum: Amax = 280 nm, Amax (shoulder) = 290 nm, Solubility: soluble in: water, and insoluble in: methanol, acetone, chloroform and ethyl acetate, Color reaction: positive:Biuret, xanthoproteic and Folin reactions, Optical rotation: [2D = 520 (c = 0.1%, pH 6.3, 0.1 M phosphate buffer), Color: white, pH stability: pH 5 - 8, and Heat stability: stable up to 400C almost 100% denatured at 600C.

3. A protein according to claim 1 which is designated herein substance M50841 and which has the following physico-chemical properties: Molecular weight: about 54000 (determined by gel-filtration method), Isoelectric point: around pH 4.8, Ultraviolet absorption spectrum: Amax = 280 nm, Amax (shoulder) = 290 nm, Solubility: soluble in: water and insoluble in: methanol, acetone, chloroform and ethyl acetate.

Color reaction: positive: Biuret, xanthoproteic and Folin reactions, Color: white, pH stability: pH 5 - 7, and Heat stability: stable up to 500C, almost 100% denatured at 700C.

4. A protein according to claim 1 which is designated herein substance M508411 and which has the following physico-chemical properties: Molecular weight: about 54000 (determined by gel-filtration method).

Isoelectric point: around pH 5.6, Ultraviolet absorption spectrum: Amax = 280 nm, Amax (shoulder) = 290 nm, Solubility: soluble in: water, and insoluble in: methanol, acetone, chloroform and ethyl acetate, Color reaction: positive: Biuret, xanthoproteic and Folin reactions, Color: white, pH stability: pH 5 -- 7. and Heat stability: stable up to 500 C, almost 100% denatured at 70"C.

5. A protein according to claim 1 which is designated herein substance M4400 and which has the following physico-chemical properties: Molecular weight: about 41000 (determined by gel-filtration method), Isoelectric point: around pH 3.8, Ultraviolet absorption spectrum: Amax = 280 nm, Amax (shoulder) = 290 nm, Solubility: soluble in: water, and insoluble in: methanol acetone, chloroform and ethyl acetate, Color reaction: positive: Biuret, xanthoproteic and Folin reactions, Color: white, pH stability: pH 4 - 8, and Heat stability: stable up to 500C, almost 100% denatured at 700C.

6. A protein according to claim 1 which is designated herein substance M5093 and which has the following physico-chemical properties: Molecular weight: about 59000 (determined by gel filtration method), Isoelectric point: around pH 3.1, Ultraviolet absorption spectrum: Amax = 280 nm, Amax (shoulder) = 290 nm, Solubility: soluble in: water, and insoluble in methanol, acetone, chloroform and ethyl acetate, Color reaction: positive: Biuret, xanthoproteic and Folin reactions, Color: white, pH stability: pH 4 - 8, and Heat stability: stable up to 600C, almost 100% denatured at 800C.

7. A protein according to claim 1 which is designated herein substance M5094 and which has the following physico-chemical properties: Molecular weight: about 33000 (determined by gel-filtration method), Isoelectric point: around pH 3.5, Ultraviolet absorption spectrum: Amax = 280 nm, Amax (shoulder) = 290 nm, Solubility: soluble in: water, and insoluble in methanol, acetone, chloroform and ethyl acetate, Color reaction: positive: Biuret, xanthoproteic and Folin reactions, Color: white, pH stability: pH 4 - 7, and Heat stability: stable up to 400 C, almost 100% denatured at 60"C.

8. A composition for use in preventing dental caries which comprises, as active ingredient, an acidic protein as claim in any one of the preceding claims, together with a suitable adjuvant.

9. A composition according to claim 8 which contains at least 0.0001 W/W % of the acidic protein.

10. A composition according to claim 8 or 9 which is a dentifrice.

11. A composition according to claim 8 or 9 which is a dental hygienic composition.

12. A composition according to claim 11 which is a toothpaste, tooth powder, foaming preparation, gargling tablet or massage cream.

13. A composition according to claim 8 or 9 which is a gum base.

14. A composition according to claim 8 or 9 which is a beverage or comestible.

1 5. A composition for use in preventing dental caries substantially as hereinbefore described in any one of Examples 6 to 12 or with reference to any one of the compositions of Example 13 or 14 or 1 5.

1 6. A process for the preparation of susbstance M507 1 as defined in claim 2, which process comprises culturing cells of the microorganism Arthrinium sp. M5071 in a nutrient culture medium therefor and isolating the substance M507 1 that is produced.

1 7. A process for the preparation of substance M507 1 as defined in claim 2 substantially as hereinbefore described in Example 1.

1 8. A process for the preparation of substance M50841 as defined in claim 3, which process comprises culturing cells of the microorganism Fusarium solani M5084 in a nutrient culture medium therefor and isolating the substance M50841 that is produced.

1 9. A process for the preparation of substance M50841 as defined in claim 3 substantially as hereinbefore described in Example 2.

20. A process for the preparation of substance M508411 as defined in claim 4, which process comprises culturing cells of the microorganism Fusarium solani M5084 in a nutrient culture medium therefor and isolating the substance M508411 that is produced.

21. A process for the preparation of substance M508411 as defined in claim 4 substantially as hereinbefore described in Example 2.

22. A process for the preparation of substance M4400 as defined in claim 5, which process comprises culturing cells of the microorganism MacrophominaphaseolllFO 7318 in a nutrient culture medium therefor and isolating the substance M4400 that is produced.

23. A process for the preparation of substance M4400 as defined in claim 5 substantially as hereinbefore described in Example 3.

24. A process for the preparation of substance M5093 as defined in claim 6, which process comprises culturing cells of the microorganism Gnomoniella sp. M5093 in a nutrient culture medium therefor and isolating the substance M5093 that is produced.

25. A process for the preparation of substance M5093 as defined in claim 6 substantially as hereinbefore described in Example 4.

26. A process for the preparation of substance M5094 as defined in claim 7, which process comprises culturing cells of the microorganism Nodulisporium sp. M5094 in a culture medium therefor and isolating the substance M5094 that is produced.

27. A process for the preparation of substance M5094 as defined in claim 7 substantially as hereinbefore described in Example 5.

28. A culture of the microorganism Arthrinium sp. M5071 in a culture medium containing a source of assimilable carbon, a source of assimilable nitrogen and, optionally, mineral elements, said culture being substantially free of other microorganisms.

29. A culture of the microorganism Fusarium solani M5084 in a culture medium containing a source of assimilable carbon, a source of assimilable nitrogen and, optionally, mineral elements, said culture being substantially free of other microorganisms.

30. A culture of the microorganism Gnomoniella sp. M5093 in a culture medium containing a source of assimilable carbon, a source of assimilable nitrogen and, optionally, mineral elements, said culture being substantially free of other microorganisms.

31. A culture of the microorganism Nodulisporium sp. M5094 in a culture medium containing a source of assimilable carbon, a source of assimilable nitrogen and, optionally, mineral elements, said culture being substantially free of other microorganisms.