JPH022856B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition for preventing dental caries, which contains a polymeric acidic protein substance having an inhibitory effect on glucosyltransferase activity. The mechanism of dental caries development is that the action of glucosyltransferase produced by Streptococcus mutans, which resides in the oral cavity, causes adhesive exopolysaccharides to be absorbed in the presence of sucrose. Generates certain insoluble glucans. This sticky insoluble glucan adheres to the tooth surface and adsorbs and deposits oral bacteria in the oral cavity, and these oral bacteria are also deposited on the tooth surfaces, grooves, and spaces between teeth. It metabolizes food, especially carbohydrates, to produce organic acids such as lactic acid and pyruvic acid. This organic acid first dissolves the calcium content of the enamel, causing it to soften and disintegrate, and further erodes the enamel and dentin. Furthermore, since this sticky insoluble glucan coats the entire tooth surface with adhesive, the surface area becomes more anaerobic, creating favorable conditions for oral bacteria, and these oral bacteria invade and multiply. It is recognized that this promotes the occurrence of tooth decay. The present inventors have discovered, from cultures of various microorganisms, various high-molecular acidic protein-based substances with molecular weights of about 30,000 to 60,000 that have an inhibitory effect on at least the glucosyltransferase activity produced by Streptococcus miutans. When these substances were applied to Streptococcus miutans, a caries-causing bacterium, in the presence of sucrose, they very effectively inhibited the production of sticky insoluble glucan, resulting in good results. It was discovered that it has a preventive effect on tooth decay. First, the high-molecular acidic protein-based substance having an inhibitory effect on glucosyltransferase activity used in the present invention is, for example, one that has an effect of inhibiting at least the glucosyltransferase activity produced by Streptococcus miutans and has a molecular weight of Any high-molecular acidic protein substance with a molecular weight of about 30,000 to 60,000 may be used, such as the M5071 substance obtained from a culture of the Arthrinium sp. M5071 strain belonging to the genus Arthrinium, Fusarium solani belongs to the genus Fusarium.
M5071-like substance M5084 substance and M5071 substance obtained from the culture of M5084 (Fusarium solani M5084) strain
M5084 substance and Macrophomina hazeolie IFO7318 (Macrophomina) belonging to the genus Macrophomina.
phaseoli IFO7318) obtained from a culture of the bacterial strain
M5071-like substance M4400 substance, M5071-like substance M5093 substance obtained from a culture of Gnomoniella sp. M5093 strain belonging to the genus Gnomoniella, Nodulisporium sp.・P・M5094
(Nodulisporium sp・M5094) M5071-like substance M5094 substance obtained from the culture of the bacterial strain (Japanese Patent Application No. 55-5128, Japanese Patent Application No. 104056-1982, and the patent application dated December 10, 1982) (See each specification) In addition, the above-mentioned M5071 substance, M5071-like substance M5084
Substances, M5071-like substances M5084 substances, M5071-like substances M4400 substances, M5071-like substances M5093 substances, and
The physical and chemical properties and actions of the M5071-like substance M5094 are shown in Table 1. [Table] [Table] Judging the effect. If the reaction mixture exhibits an inhibitory effect on glucosyltransferase, the reaction solution remains clear; if it does not exhibit an inhibitory effect on glucosyltransferase, insoluble glucan is synthesized in the reaction solution, forming a precipitate. .
M5071 substance M5071-like substance M5084 substance of the present invention,
M5071-like substance M5084 substance, M5071-like substance M4400 substance, M
The reaction solutions of the 5071-like substance M5093 and the M5071-like substance M5094 were clear, and from this fact, they were recognized to have the effect of inhibiting the synthesis of insoluble glucan from sucrose by glucosyltransferase. It is something.
From the above physical and chemical properties and actions, each substance is
They are recognized as polymeric acidic protein-based substances that have the effect of inhibiting the synthesis of insoluble glucan by glucosyltransferase, and since they are similar substances, they are collectively referred to simply as M5071-based substances. The potency of the inhibitory effect of M5071-based substances on glucosyltransferase activity can be measured, for example, by observing the degree of inhibition of insoluble glucan synthesis using glucosyltransferase in the presence of sucrose. These are as follows. Streptococcus miutans OMZ176
Streptococcus miutans was cultured for 2 days in BHI medium, and after sterilization, the culture solution was precipitated with ammonium sulfate, and the precipitate was collected, dissolved in water, dialyzed, and concentrated to about 50 times the culture solution. A reaction solution consisting of 0.1 ml of a solution containing glucosyltransferase that catalyzes insoluble glucan synthesis, 0.1 ml of a solution containing each M5071 substance, 0.1 ml of 0.5 M phosphate buffer, and 0.1 ml of 1 M sucrose is incubated at 37°C. On the other hand, as a control, water was used instead of each M5071 substance-containing solution. In addition, each M5071 substance-containing solution is diluted in several stages in advance. After 2 hours, it is confirmed that insoluble glucan is synthesized in the control case. The activity is determined by observing the state of each diluted M5071-like substance-containing solution, and the highest dilution at which the synthesis of insoluble glucan is inhibited is determined as the potency (for example, a 100-fold dilution shows an inhibitory effect, If no inhibitory effect is shown at 120-fold dilution, the titer is set as 100). 1 of the desired M5071-like substance-containing liquids of the present invention
The titer (A) per ml is determined as follows: A=a×10 (Unit/ml) (a indicates the highest dilution). In addition, Arthrinium sp. M5071 strain, which is a producing bacterium of M5071-based substances (microbial accession number notification, microorganism accession number "Feikoken Bibori No. 5352,
FERM-P No.5352”), Fusarium solani
Strain M5084 (Microorganism Accession Number Notification, Microorganism Accession No. 5546, FERM-P No.
5546''), Gnomoniera S.P. strain M5093 (Microorganism accession number notification, Microorganism accession number FERM-P No. 5799), Nodurisporium S.P. strain M5094 (Microorganism accession number FERM-P No. 5799)) Number Notification Consignment number “Feikoken Bibori No. 5800”
The mycological properties of FERM-P No. 5800 are as follows. First, the Arthrinium sp. M5071 strain will be described as follows. (1) Growth status on various media Malt extract agar medium Growth is very good, reaching an inner diameter of 85 mm in 10 days at 26°C.
Reach the entire surface of the Petri dish in mm. Aerial mycelia are fluff-like and white at the beginning of culture, but as growth progresses after a week, they gradually turn light and white in places.
Although the color is Light Tan (hue 3gc), there are many white parts in the village as a whole. Light
Light Amber (hue 3ic) diffusible dye is released into the medium. The color of the back side is dark brown [Dark Brown (hue 2pn) ~
Maple (hue 4le). Potato glucose agar medium Growth is very good, inner diameter 85 after 10 days at 26℃
Reach the entire surface of the Petri dish in mm. Aerial hyphae are fluff-like and white in the initial stage of culture, but as growth progresses over a week, the entire surface gradually becomes camel (hue 3ie). A light amber (hue 3ic) diffusible dye is released into the medium. The back side is dark brown (hue
2pn). Tuapetsk agar medium Growth is very poor, with only very thin, almost colorless mycelia growing on the agar medium after 10 days at 26°C. The back side is colorless. (2) Microscopic characteristics Hyphae are 2.5-3.0μ in diameter, smooth walls, and light brown.
Conidiophores are smooth or have small knob-like ridges, pale brown, with occasional septa. Conidia grow singly at the tip of a short projection on the side of the conidiophore. Spherical or oval, 2.5-10 x 2.6-6μ, brown, walls smooth, sometimes with vertical fissures. (3) Physiological properties Growth temperature: 8-40℃ Optimum growth temperature: 22-32℃ Growth PH: 2.5-9.5 Optimum growth PH: 4-8 The above mycological properties include dark brown colonies and a spherical shape. This strain M5071 is recognized as belonging to the genus Arthrinium due to its characteristics such as having dark brown conidia with vertical slits in the shape of ellipsoids, and is classified as Arthrinium sp.
It was named M5071. Please note that the colors are shown in the Color Harmony Manual.
(Container Corporation of America 1958) marking law. In addition, when setting up this bacteria, GL
Barron, 1968, The genera of Hyphomycetes
from soil, and M Bellis, 1971,
Cited Dematiaceous Hyphomycetes. Furthermore, the Fusarium solani M5084 strain is as follows. (1) Growth status on various media Tuapetsk agar medium Growth is fast, and the diameter decreased after 7 days of culture at 26°C.
Reaching 65-70mm. At the initial stage of culture, it is white and fluffy, and when it matures, it becomes slightly creamy [Cream (1
1/2ca)] and partially Dusty Aqua (18qe). Exudates and diffusible dyes are not released. The colors on the back are Light Ivory (2ca) ~ Light Wheat (2ea).
Partially Dusty Aqua (18ge). Malt extract agar medium Growth is fast, with a diameter of 50 ~ after 7 days of culture at 26℃
Reaches 53mm. At the early stage of culture, it is white and fluffy, and when it matures, the aerial hyphae become Flesh Pink (6ca) and the basal hyphae become Red Wood (6ie). Exudates and diffusible dyes are not released. The back side is written as Red and Wood (6ie). Potato Glucose Agar Medium Growth is fast, with a diameter of 57 cm after 7 days of culture at 26°C.
Reach ~60mm. At the early stage of culture, it is white and fluffy.
Slightly pearly pink when mature.
Pink (3ca)]. No exudate is released. Cultivate the diffusible pigment of Cedar (6 1/2le). The reverse side is Deep Brown Mahogany (6PL). (2) Microscopic characteristics The vegetative hyphae are colorless, smooth, and 3 to 4 μ in width. The conidia formation mode is phialide type, and conidia of two types, large and small, are formed. Small conidiophores develop at right angles to the vegetative strain, are solitary, and are unbranched or branched once or twice. Relatively long, 30~150×2.5
~3μ, colorless, smooth surface, with zero to several septa.
Small conidia are formed in large numbers in a pseudocephalic shape. 5~
23×2~5μ, colorless, smooth, oval or oval,
No septa or 1 septum. Macroconidia have one to several septa, are crescent-shaped, and have a pointed tip.
Colorless, smooth surface, 30-50 x 3-5μ. Well-developed pedicel cells are observed at the junction with the phialide. (3) Physiological properties Temperature for growth: 10-35℃ Optimum growth temperature: 24-28℃ Growth pH: PH2-12 Optimal growth PH: PH3-10 In the above mycological properties, the proportion of this bacterium is The mode of spore formation is phialide, forming two types of large and small conidia, and the macroconidia are colorless, crescent-shaped, have one to several septa, and have setae or other appendages. It belongs to the genus Fusarium (JAvon Arx, 1974.The Genera of Fungi).
Sporulating in Pure Culture.J. Cramer,
Lehre, 315pp.GLBarron.1968. The Genera of Hyphomycetes from Soil. Williams & Wilkins, Baltimore, 364pp). Furthermore, as a result of detailed observation of this bacterium, it was found that it formed cream-colored colonies on Czapetsk agar medium, that the size of large conidia was 30-50 x 3-5μ, and that small conidia were conidial cells. Fusarium.
It matches well with the characteristics of Solani (C. Booth, 1971).
The Genus Fusarium, CMI, kew, 237pp), and this bacterial strain M5084 was identified as Fusarium solani.
It was named M5084. In addition, Macrofomina Hazeoli
The IFO3718 strain is a strain deposited with the Institute for Fermentation (INSTITUTE FOR FERMENTATION).
OSAKA LIST OF CULTURES 1978.SIXTH
EDITION). Furthermore, Gnomoniera S.P. M5093
The following is a description of the strain: (1) Growth status on various media Malt extract agar medium Growth is very fast, reaching the entire surface of a Petri dish with an inner diameter of 885 mm in one week at 26°C. The bacterial flora is flat and slightly fluffy. The color is dark olive.
Dark Olive Gray (near grays)
1ml)]. The area around the village is almost smooth. Does not release diffusible dyes or exudates. The reverse side is Midnight Blue (14pm). Potato glucose agar medium Growth is normal, 30 to 40 cells per week at 26℃
reaching mm. The bacterial flora is somewhat thick and the surface is uneven. The color is dark olive gray (1ml). The periphery is extremely spider-like (Arachnoid). Does not release diffusible dyes or exudates. The back side is dark blue.
Blue (13 1/2pn)]. Tuapetsk agar medium Growth is rather slow, at 26°C, 20~20% in one week
Reaches 28mm. The bacterial flora is thin and flat. The surface is velvety. The color is Dark Olive Green (24 1/2pn). The periphery is extremely spider web-like. Does not release diffusible dyes or exudates. The back is dark olive green (24 1/2pn). (2) Microscopic characteristics: Ascocytes are formed singly or in clusters on the surface or inside the agar medium. Dark brown or black. The diameter of the shell body is approximately 200μ. It has a thick and long neck that is indistinguishable from the body of the shell. Its size is 400~900×70~
130μ, sometimes bifurcated at the base or top. The ascus is formed from the wall of the ascus shell and has a club-shaped short stalk. 40～80×10～
13μ. The ascospores are eight in number and formed in two rows.
The top is Melzer's Reagent.
is not stained. The ascospores are somewhat irregular spindle-shaped and slightly curved. 1 cell, colorless to pale yellowish brown, 13-20 x 5-7μ. The walls are smooth or slightly knobby. (3) Physiological properties PH for growth: 3-8.5 Optimal growth PH: 4-7.5 Temperature for growth: 13-38℃ Optimal growth temperature: 23-28℃ Ascomycetes due to the formation of cysts
It is recognized that it belongs to. Furthermore, an ascospore with a long and stiff neck is formed, and the ascospore develops from the wall of the ascus shell and is club-shaped, and the ascospore is a single cell and has a nearly colorless color. Based on its characteristics, it was identified as belonging to the genus Gnomoniella, and the fungus was named Gnomoniella sp. M5093. For the identification of this bacterium, FEClements.&
CLShear, 1930, The Genera of
Fungi.496pp.Hafner Pub.Co., USA, JAvon
Arx, 1974, The Genera of Fungi
Sporulating in Pure Culture, 315pp.Cramer,
Germany, RWGDennis, 1968, British
Ascomycetes 455pp. Cited Cramer, Germany. Furthermore, Nozurisporium S.P. M5094
The bacterial strains are as follows. (1) Growth status on various media Malt extract agar medium Growth is very fast, and at 26°C, it will grow on the entire surface of a Petri dish with a diameter of 85 mm in one week. The bacterial flora is
The aerial mycelia are flat and fluffy, and the color is covert.
Covert Tan (2ge). The area around the village is spider web-like. It emits diffusible pigment and has a honey color.
Gold [Honey Gold (2ic)]. No exudate is released. The reverse side is Midnight Blue (14pn). Potato Glucose Agar Medium Growth is fast, with a diameter of 65~ in one week at 26℃
Reaches 80mm. The bacterial flora is flat, but somewhat thick. Aerial mycelia are fluffy and covert in color.
Brown [Covert Brown (2li)]. The area around the village is spider web-like. It emits a diffusible pigment and the color is honey gold (2ic). No exudate is released.
The reverse side is Dark Spruce.
(20pn)]. Tuapetsk agar medium Growth is fast, with a diameter of 45 ~ in one week at 26℃
Reaching 65mm. The bacterial flora is thin and flat. The aerial mycelia are slightly downy and the color is covert tan (2ge). The area around the village is spider web-like. It emits a slight amount of diffusible pigment, and the color is Bamboo.
(2gc)]. No exudate is released. The reverse side is Beige Brown (3ig). (2) Microscopic characteristics The vegetative hyphae are colorless to light brown, with smooth walls and septa. Occasionally, granular material accumulates around the hyphae. Conidiophores are 40~200×2.5~
3.5μ, with septa, colorless to light brown. The walls are rough;
Branches relatively regularly with 2 to whorled length. Conidiospore-forming cells are formed singly or regularly in whorls;
Light tan to light brown, 10-20 x 2.5-3.5μ, rough surface.
Conidia are formed in a Sympodulae shape on several small protrusions that appear at the tips of swollen conidiogenic cells. Slightly irregular oval shape with a flattened lower end, colorless ~
Light yellowish brown, 4.5-6 x 2.5-3μ, walls smooth. (3) Physiological properties Able to grow. PH: 3-11 Optimum growth PH: 4-8 Temperature for growth: 12-45°C Optimal growth temperature: 23-37°C With the above mycological properties, this bacterium grows by exogenous conidia. Therefore, it is recognized as belonging to the Hyphomycetes. In addition, the conidiophores are relatively long and branch somewhat regularly, the conidial formation mode is Sympodulae type, and the walls of the conidial cells are rough, with extremely large Based on the characteristics such as not becoming thin and the lower end of the conidia being flat, this bacterial strain M5094 was identified as belonging to the genus Nodurisporium, and was therefore named Nodurisporium S.P. M5094. For the identification of this bacterium, MBERlis, 1971,
Dematiaceous Hyphomycetes, 608pp, CMI,
Englamd, G.L.Barrom, 1969, The Genera of
Hyphomycetes from Sol, 364pp.The
Cited Williams S Wilkins Co., USA. In addition, to give an example when obtaining M5071-based substances,
M5071 substance-producing bacteria belonging to the genus Arthrinium, preferably Arthrinium sp. M5071 strain, M5084 a substance similar to M5071 belonging to the genus Fusarium
substance, M5071-like substance M5084 substance-producing bacteria, preferably the aforementioned Fusarium solani M5084 strain, or M5071-like substance belonging to the genus Macroformina
M4400 substance-producing bacteria, preferably the above-mentioned Macroformina hazeolii IFO7318 strain M5071-like substance M5093 substance-producing bacteria belonging to the genus Gnomoniera, preferably the above-mentioned Gnomoniella sp. M5093
The strain also belongs to the genus Nodurisporium
A microorganism producing the M5071-like substance M5094, preferably the aforementioned Nodurisporium sp. M5094 strain, is cultured by a conventional method for producing antibiotics, enzymes, and the like. As for the form of culture, it is usually advantageous to use liquid culture. As a nutrient source for the culture medium,
A wide variety of materials commonly used for culturing microorganisms can be used. The carbon source may be any assimilable carbon compound, preferably sucrose, glucose, lactose, maltose, fructose, molasses, dextrin, etc. The nitrogen source may be any available nitrogen compound, such as polypeptone, corn stew liquor, meat extract, yeast extract, soybean flour, casein hydrolyzate, and the like. Others include phosphates, carbonates, sulfates, magnesium,
Calcium, potassium, sodium, iron, zinc,
Salts such as manganese are used as necessary. The culture temperature can be changed as appropriate within the range that allows the bacteria to grow and produce each M5071-like substance of the present invention, but it is usually 26 to 30 days.
℃, preferably about 26℃, and the culture time varies depending on the conditions, but it is sufficient to determine the time when each M5071 substance reaches its maximum yield and terminate the culture at an appropriate time. ~4 days. Next, each M5071 substance is collected from the culture obtained in this way, but first the obtained culture is filtered or centrifuged to remove solid matter and the culture solution is recovered. do.
M5071, each M5071-based substance, was extracted from the obtained culture solution.
Substance, M5071-like substance M5084 substance, M5071-like substance
M5084 substance, M5071-like substance M4400 substance, M5071
When isolating the M5093-like substance M5093 substance and the M5071-like substance M5094 substance, it is preferable to isolate the M5071-like substance based on the solubility of each M5071 substance in a solvent and a salting-out method. The resulting precipitate may be collected by means such as centrifugation. Furthermore, this precipitate is dissolved in a buffer having a stable pH range, dialyzed with a dialysis membrane, purified using adsorption chromatography or gel filtration means such as DEAE-cellulose, DEAE-Sephadex, Sephadex, etc. Collect the active fraction by electrofocusing method,
M5071 substance purified to a single component, M5071-like substance M5084 substance, M5071-like substance M5084 substance,
Purified products of the M5071-like substance M4400 substance, the M5071-like substance M5093 substance, and the M5071-like substance M5094 substance may be obtained and freeze-dried. When carrying out the present invention, the polymeric acidic protein substance having an inhibitory effect on glucosyltransferase activity obtained as described above is mixed into a dentifrice composition, for example, in the form of a powder, an aqueous solution, or an alcoholic solution. It can then be added to oral compositions such as toothpaste, powdered toothpaste, foamed toothpaste, gargling tablets, pine surge cream, etc., gum base-containing compositions such as chewing gum, and other food and drink compositions using conventional methods. It may be processed into a product using conventional methods. In addition, as a toothpaste composition, for example, as an abrasive, calcium carbonate, dicalcium phosphate, insoluble sodium metaphosphate, aluminum oxide,
Aluminum hydroxide, amorphous silica, crystalline silica, etc. Thickeners include methylcellulose, sodium alginate, carrageenan, carboxymethylcellulose, silicic anhydride, etc. Viscous humectants include sorbitol, xylitol, glycerin, propylene glucose, polyethylene glucose, etc. , activators such as sodium lauryl sulfate, sodium dodecylbenzenesulfonate, sucrose fatty acid ester, sodium acylsarcosine, etc.
Oral compositions are prepared by appropriately combining other fluorinated compounds, lysozyme chloride, dextranase, hemostatic agents such as ε-aminocaproic acid and tranexamic acid, salt, chlorophyll, sweeteners, other fragrances, preservatives, and bactericides. It can be processed as . Furthermore, in such processing, the polymeric acidic protein-based substance used has an inhibitory effect on glucosyltransferase activity, for example, in a dentifrice composition as an oral composition, which is usually used per use.
The concentration may be adjusted to 10 units or more, and generally, the concentration may be adjusted to 1γ/g or more. The caries preventive composition obtained in this way is
Due to the action and effect of the polymeric acidic protein substance that has the effect of inhibiting glucosyltransferase activity, it inhibits the production of insoluble glucan even in the presence of sucrose, suppresses the adhesion of caries-causing bacteria to the tooth surface, and has a good effect. It was a good product that had a caries-preventing effect. Next, the present invention will be specifically described with reference to Examples and Reference Examples, but the present invention is not limited by these in any way. Example 1 (Inhibition of insoluble glucan synthesis by M5071 substance) (1) Streptococcus miutans OMZ175
was cultured in BHI medium for 2 days, and after removal, the culture solution was precipitated with ammonium sulfate, and this precipitate was collected, dissolved in water, dialyzed, and concentrated to about 50 times the culture solution.
20 ml of glucosyltransferase-containing solution that catalyzes insoluble glucan synthesis by Streptococcus miutans, 200 ml of 0.1M phosphate buffer (PH7.0), 100 ml of 50% sucrose solution,
Mix 20ml of a solution containing 5000 units of M5071 substance and hold at 37°C.
It reacted for 18 hours. As a control, 20 ml of water to which M5071 substance was not added was used. After the reaction, each reaction solution was centrifuged at 1000 rpm for 10 minutes to obtain a precipitate and a supernatant. The precipitate was washed several times with water and dried to obtain an insoluble glucan. The supernatant was also dialyzed against water, and twice the amount of ethanol was added to the dialyzed solution, left at 4°C for 18 hours, centrifuged at 15,000 rpm for 5 minutes, and the precipitate was mixed with ethanol:water (2:1). After washing with the mixed solution several times, it was dried, and this was used as a soluble glucan. As a result, the amount of each glucan in each reaction solution is
The results were as shown in Table 5. [Table] Gibbons et al [Archs.oral.Biol.
13, 1249-1260 (1968)]. Furthermore, from the results shown in Table 5 above, it is clear that substance M5071 significantly inhibits (by 95%) the synthesis of insoluble glucan, which is necessary for Streptococcus miutans to adhere to tooth surfaces, etc. (2) The glucose binding mode of the insoluble glucan and soluble glucan without the addition of the M5071 substance and the soluble glucan with the addition of the M5071 substance obtained as above was investigated. That is, each sample was Mr. Hakomori [J.Biochem. 55 , 205,
(1964)], and after hydrolysis, the methylated sugars were analyzed by gas chromatography. The results were as shown in Table 6. [Table] As a result, 2.4.6-trimethylated sugar, which is often seen in the case without M5071 substance, was significantly reduced in soluble glucan when M5071 substance was added.
It is estimated that it inhibits the synthesis of glucans having α1-3 linkages shown by 2.4.6-trimethylated sugars. Example 2 (Suppressing effect on the adhesion of caries-causing bacteria Streptococcus miutans to tooth surfaces) (1) Regarding the adhesion of caries-causing bacteria to tooth surfaces, sterilized human-derived bacteria were added to BHI medium containing 5% sucrose. Two test tubes in which tooth pieces (5 x 5 mm) were suspended with wires (stainless steel wire) were prepared. Add 1000 units of the M5071 substance to this one bottle, and do not add it to the other one. In each test tube,
A bacterial solution of Streptococcus miutans OMZ176 cultured in BHI medium at 37°C for 18 hours was added to a final concentration of 10%. These were then cultured at 37° C. for 6 hours, and then the tooth pieces were taken out, washed with water, and then observed under a scanning electron microscope at 3000x magnification. the result,
The photograph of the tooth fragment without the addition of the M5071 substance is shown in FIG. 13, and many strains of Streptococcus miutans OMZ176 were adhered to the surface of the tooth. On the other hand,
The photograph of the tooth fragment in the case of M5071 substance addition is shown in
As shown in Figure 4, almost no adhesion of the above bacterial strain was observed, and it is recognized that the M5071 substance suppressed the adhesion of the bacterial cells by inhibiting the synthesis of insoluble glucan, which has extremely good adhesion. (2) Regarding the colony formation of caries-causing bacteria on tooth surfaces, Streptococcus miutans OMZ176
After culturing in BHI medium at 37℃ for 18 hours, the bacterial cells were collected and added to 0.05M phosphate buffer (PH6.8).
After washing with water, suspend in the same buffer solution, add sucrose to this solution to a final concentration of 5%, and culture at 37°C for 1 hour to produce glucan around the bacterial cells. After completion of the reaction, centrifugal washing was performed to remove sucrose and soluble glucan from this reaction, and the thoroughly washed bacterial cells were resuspended.
It was suspended in 0.05M phosphate buffer (PH6.8).
Next, sterilized tooth pieces obtained from humans were placed in this suspension and left at room temperature for 30 minutes. Thereafter, this was thoroughly washed with water to remove unnecessary bacterial cells. Furthermore, this tooth piece was suspended with a wire in a BHI medium supplemented with 10% sucrose. At that time, this
1000 units of M5071 substance was added, and no additive was used as a control. After culturing this at 37° C. for 6 hours, the tooth pieces were washed with water, and each tooth piece was observed using a scanning electron microscope at 3000x magnification. As a result, in the case where M5071 substance was not added, as shown in Figure 15, the attached bacterial cells of Streptococcus miutans OMZ176 proliferated on the tooth surface and formed bacterial plaque together with glucan. It is something. on the other hand,
In the case of addition of the M5071 substance, as shown in FIG. 16, no plaque formation as described above occurred.
Dental plaque is thought to be the result of the production of insoluble glucan and the formation of bacterial colonies as bacteria proliferate, but substance M5071 inhibits the production of the insoluble glucan necessary for this.
It is recognized that plaque formation was effectively suppressed. Example 3 Instead of the M5071 substance in Example 2 (1),
M5071-like substance M5084 substance, M5071-like substance M5084
Substance, M5071-like substance M4400 substance, M5071-like substance
Using M5093 substance, M5071-like substance M5094 substance,
The following procedure was carried out in the same manner as in Example 2 (1), and each
As shown in Figures 18, 19, 20, and 21, almost no adhesion of caries-causing bacteria was observed, indicating that the synthesis of sticky insoluble glucan was significantly inhibited. . Example 4 Thoroughly mix 30 parts of glycerin and 20 parts of water,
Add 0.1 part of an aqueous solution containing 1% M5071 substance and mix.
1 part of methylcellulose and 0.5 part of saccharin sodium were mixed and dissolved in this, and 45 parts of dibasic calcium phosphate was added and mixed thoroughly.
Next, 1 part of fragrance was added and thoroughly kneaded with defoaming in a kneader equipped with a vacuum device to obtain a toothpaste.
This product has a filling amount of 100 to 300g, and is approximately 1 serving per person.
It can be used in g. Example 5 A toothpaste having the following composition was prepared. Dibasic calcium phosphate 50 parts Glycerin 30 parts Sodium lauryl sulfate 1.5 parts Carboxymethyl cellulose 1 part Saccharin sodium 0.4 parts Flavoring 0.6 parts Fructose 4 parts Sodium monofluorophosphate 0.5 parts Sucrose monostearate 2 parts 1% M5071 substance-containing aqueous solution 0.1 part Water Remaining Example 6 A toothpaste powder having the following composition was prepared. Dibasic calcium phosphate 50 parts Calcium carbonate 25 parts Sorbiturate 10 parts Sodium lauryl sulfate 1 part Saccharin sodium 0.3 parts Flavor 0.7 parts Dextranase 1 part Fructose 5 parts 1% M5071 substance-containing aqueous solution 0.1 part Water Balance Example 7 The following composition A gargling tablet having the following properties was prepared. Sodium bicarbonate 54 parts Citric acid 15 parts Sodium phosphate 15 parts Polyethylene glycol 6000 3 parts Flavoring 4 parts Sucrose monolaurylate 1 part Maltose 5 parts Sodium monofluorophosphate 0.8 parts Dextran 2 parts M5071 substance 0.001 parts Example 8 20 parts of gum base was heated to 40°C, and to this was added 59.9 parts of sorbitol and an aqueous solution containing 1% M5071 substance.
0.1 part of the mixture was added thereto, and then 9.5 parts of mannitrate, 10 parts of 70% sorbitol, and an appropriate amount of fragrance were added and thoroughly kneaded in a mixer, formed with a roller, and cut to obtain chewing gum. Example 9 Chewing gum having the following composition was prepared. Gum base 20 parts Calcium carbonate 2 parts Starch syrup 15 parts Powdered sugar 30 parts Maltose 10 parts Fructose 10 parts Sucrose monostearate 3 parts Flavor 1 part M5071 substance 0.001 part Water Remaining Example 10 Satsuma mandarin orange (1/5 concentrated, solid content 53.6%) 560 parts, add 500 parts of dextrin and blow 130 to 140 parts.
The powdered fruit juice was obtained by spray drying at 75-80°C with exhaust air. Next, 0.001 part of the M5071 substance was added to 100 parts of this powdered fruit juice and thoroughly mixed to obtain the desired fruit juice. Example 11 Instead of the M5071 substance of Example 5, M5071-like substance M5084 substance, M5071-like substance M5084 substance,
The same procedure was carried out using 0.1 part of an aqueous solution containing 1% each of M5071-like substance 4400 substance, M5071-like substance M5093 substance, and M5071-like substance M5094 substance to obtain the desired toothpaste. Example 12 Instead of the M5071 substance in Examples 6 and 7, M5071-like substance M5084 substance, M5071-like substance
The same procedure was carried out using the same amounts of each of the M5084 substance, the M5071-like substance 4400 substance, the M5071-like substance M5093 substance, and the M5071-like substance M5094 substance to obtain the desired toothpaste powder and gargling tablets. Example 13 M5071-like substance instead of M5071 substance in Example 8
M5084 substance, M5071-like substance M5084 substance,
The same procedure was carried out using 0.1 part of an aqueous solution containing 1% each of M5071-like substance 4400 substance, M5071-like substance M5093 substance, and M5071-like substance M5094 substance to obtain the desired chewing gum. Reference example 1 Sucrose 1.5%, dextrin 1.5%, polypeptone 2%, corn steep liquor 2%,
KH ₂ PO ₄ 0.2%, MgSO ₄・7H ₂ O 0.1%, FeSO ₄・
100ml medium consisting of 7H ₂ O 0.01% and Celite 1%
(PH6.5) in a 500ml Erlenmeyer flask (120℃,
Sterilized for 20 minutes, 5 bottles each), arthrinium.
S.P. M5071 strain (FERM-P, No.5352)
was inoculated and cultured with shaking at 26°C for 5 days. Next, 5 ml each of this type of culture was added to 500 ml Erlenmeyer flasks (100 flasks) containing 100 ml of a medium having the same composition as above.
and cultured with shaking at 26°C for 4 days. After culturing,
The culture was filtered through Celite and washed with a small amount of water to obtain approximately 10 culture fluids (protein content: 1720 g, specific activity: 0.23). 5.16 kg of ammonium sulfate was added to the obtained culture solution, stirred, and then left in an ice chamber (4° C.) overnight. The resulting precipitate was then collected by centrifugation and added to 0.01M phosphate buffer (PH6.3).
After dissolving in 500 ml, it was dialyzed against 0.01M phosphate buffer using cellophane tube as a dialysis membrane.
Change the external dialysis fluid every 12 hours, and use 120 dialysis fluids.
Dialysis was performed for 72 hours. After dialysis, and in advance
DEAE blunted with 0.01M phosphate buffer (PH6.3)
- Cellulose (Seikagaku Corporation) column (diameter 5
x 40cm) and washed with 0.01M phosphate buffer (PH6.3), then 0.01M phosphate buffer (PH6.3) containing 2 to 0.5M NaCl (0.01M phosphate buffer (PH6.3)). Elution was carried out using a concentration gradient method using PH6.3)2 to obtain an elution section of 460ml of 0.01M phosphate buffer (PH6.3) containing 0.15M NaCl (protein amount 1.27g, specific activity 83), and then membrane PM -10 (manufactured by Amicon). Furthermore, this concentrate
Dialysis was carried out against 0.01M phosphate buffer using cellophantium as a dialysis membrane, and the external dialysis solution was changed every 12 hours, and dialysis was carried out with 80 dialysis solutions for 48 hours. The dialyzed solution was prepared in advance with 0.01M phosphate buffer (PH6.3).
Charge and adsorb onto a column (diameter 3 x 20 cm) of DEAE A-25 (manufactured by Seikagaku Corporation) smoothed with 0.01M phosphate buffer (PH6.3).
After washing with 0.01M phosphate buffer (PH6.3)2
~0.01M phosphate buffer containing 0.3M NaCl (PH6.3)
Elute with concentration gradient method using 0.01M
400ml of active fraction eluted with 0.01M phosphate buffer (PH6.3) containing NaCl (protein content 105mg, specific activity 510)
This was concentrated using Membrane PM-10 to a volume of about 20 ml. Furthermore, this concentrated solution was added to Sephadex G-100, which had been smoothed with 0.01M phosphate buffer (PH6.3).
(Seikagaku Corporation) column (diameter 4.5 x 60 cm), elute with 0.01M phosphate buffer (PH6.3) (1 fraction 4.8ml), and elute the eluted fraction with high specific activity ( Fraction No.88-104)
were collected and combined (protein amount 38.5 mg, specific activity 1300),
Lyophilized. Next, this freeze-dried product was desalted using a Sephadex G-25 course (manufactured by Seikagaku Kogyo Co., Ltd.) that had been equilibrated with water in advance, and this was freeze-dried to obtain 14.7 mg. Furthermore, after dissolving this in a small amount of 0.01M phosphate buffer (PH6.3), carrier ampholite (LKB
Isoelectric focusing (700V, 24
After electrophoresis, the active fraction was fractionated into 2 ml portions to recover the active fraction, and then charged to a column (diameter 2.5 x 50 cm) of Sephadex G-50 Fine (manufactured by Seikagaku Corporation) that had been equilibrated with water. death,
Fractionate each fraction (4.8 ml) with water, collect fractions No. 21 to 25, and freeze-dry them to obtain 4.1 mg of purified white powder of M5071 substance (this product is based on the culture solution). The specific activity was 18,000 times higher, the yield was about 11%, and a single band was observed in polyacrylamide gel electrophoresis (PUA). Furthermore, the amino acid analysis, constituent sugars, and other effects of the M5071 substance are as follows. Amino acid analysis M5071 substance was hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours, and then amino acid analysis was performed using an amino acid autoanalyzer. In addition, tryptophan was determined by ultraviolet absorption measurement. As a result, as shown in Table 2, results for each amino acid and its molar ratio were obtained. [Table] Constituent sugar According to the phenol-sulfuric acid method, the amount was 80γ in terms of glucose and 68γ in terms of mannose per 1 mg of M5071 substance. Other effects Substance M5071 has been confirmed to have an enzyme effect that degrades insoluble glucan produced by Streptococcus miutans OMZ176, although it is extremely weak. (a) Streptococcus miutans OMZ176
Add 200 μg (2000 units) of M5071 substance to 1.0 ml of a homogeneous suspension of purified insoluble glucan produced by
0.1 ml of the containing solution was added and incubated at 37°C. After 20 hours, the turbidity was measured at 550 mμ. As a control, water was used instead of the M5071 substance-containing solution. The results were as shown in Table 3. [Table] As a result, a slight decrease in turbidity was observed in the case of the liquid containing the M5071 substance. (b) Streptococcus miutans OMZ176
Add 50 μg of each M5071 substance to 0.5 ml of a homogeneous suspension of 750 μg of purified insoluble glucan produced by
0.5 ml of a solution containing 200 μg (2000 units) and 200 μg (2000 units) was added and incubated at 37°C. After 2 hours, the reducing sugar was colorimetrically determined as a glucose equivalent value using the Nelson-Somogi method. Also, as a control, water was used instead of the M5071 substance.
The results are shown in Table 4. [Table] As a result, slightly more reducing sugars were detected compared to the control. From the above results, it is determined that the M5071 substance has an extremely weak enzymatic action to degrade insoluble glucan. Reference example 2 Sucrose 1.5%, dextrin 1.5%, polypeptone 2%, corn steep liquor 2%,
KH ₂ PO ₄ 0.2%, MgSO ₄・7H ₂ O 0.1%, FeSO ₄・
100ml medium consisting of 7H ₂ O 0.01% and Celite 1%
(PH6.5) in a 500ml Erlenmeyer flask (120℃,
Sterilize for 20 minutes, 5 bottles) to remove Fusarium solani.
Inoculated with M5084 strain (FERM-P, No. 5546),
The cells were cultured with shaking at 26°C for 4 days. Next, 5 ml each of this seed culture was added to 100 ml of a medium having the same composition as above.
The cells were transplanted into 500 ml Erlenmeyer flasks (100 flasks) and cultured with shaking at 26°C for 4 days. After culturing, the culture was filtered through Celite and washed with a small amount of water to obtain about 10 culture fluids (protein content: 1503 g, specific activity: 0.34). 5.16 kg of ammonium sulfate was added to the obtained culture solution.
After stirring, the mixture was left in an ice chamber (4°C) overnight. Next, the resulting precipitate was collected by centrifugation, dissolved in approximately 500 ml of 0.01M phosphate buffer (PH6.3), and then filtered using cellophane tube as a dialysis membrane.
Dialysis was performed against 0.01M phosphate buffer (PH6.3).
Change the external dialysis fluid every 12 hours, and use 120 dialysis fluids.
Dialysis was performed for 72 hours. After dialysis, and in advance
DEAE blunted with 0.01M phosphate buffer (PH6.3)
- Cellulose (Seikagaku Corporation) column (diameter 5
x 40cm) and washed with 0.01M phosphate buffer (PH6.3), then 0.01M phosphate buffer (PH6.3) containing 2-0.5M NaCl ( PH6.3)2 was used to elute using the concentration gradient method, and 520ml of 0.01M phosphate buffer (PH6.3) containing 0.1M NaCl was used for elution (protein amount: 1.5g, specific activity).
87) was further concentrated using membrane PM-10 (manufactured by Amicon). Further, this concentrated solution was dialyzed against 0.01M phosphate buffer using cellophane tube as a dialysis membrane, and the dialysis solution was changed every 12 hours, and dialysis was carried out with 80 dialysis solutions for 48 hours. The dialyzed solution was charged to a column (diameter 3 x 20 cm) of DEAE-Sephadex A-25 (manufactured by Seikagaku Corporation), which had been smoothed with 0.01M phosphate buffer (PH6.3), and was adsorbed with 0.01M phosphate buffer (PH6.3). M phosphate buffer (PH6.3)
After washing with 0.01M phosphate buffer (PH6.3)2
~0.01M phosphate buffer containing 0.3M NaCl (PH6.3)
Elute with concentration gradient method using 0.05M
Active section containing M5071-like substance M5084 eluted with 0.01M phosphate buffer (PH6.3) containing NaCl
150ml (33mg protein, specific activity 507), and 0.07M
M5071-like substance eluted with NaCI-containing 0.01M phosphate buffer (PH6.3) Activity category 50 containing M5084 substance
ml (protein amount: 23 mg, specific activity: 481) and each was freeze-dried. Furthermore, this M5071-like substance 5084-containing lyophilized product was dissolved in a small amount of water, and the column (diameter 2.5 x 60cm) and elute each fraction (4.8ml) with 0.01M phosphate buffer (PH6.3).
Nos. 55 to 62 were collected and combined (protein content 21 mg, specific activity
1470), lyophilized. Next, this freeze-dried product was dissolved in a small amount of water, desalted using a Sephadex G-25 course (manufactured by Seikagaku Corporation) that had been equilibrated with water, and then freeze-dried to obtain a purified M5071-like substance. 19 mg of the M5084 substance was obtained (the specific activity of this product was about 4500 times higher than that of the culture solution, and it showed a single band in polyacrylamide gel electrophoresis). A lyophilized product containing M5084-like substance was dissolved in a small amount of water and charged to a Sephadex G-100 column (diameter 2.5 x 60 cm) equilibrated with 0.01M phosphate buffer (PH6.3). Elute each fraction (4.8ml) with phosphate buffer (PH6.3), and collect fraction No.56.
~60 was collected and combined (protein content 13 mg, specific activity
1490), freeze-dried. Next, this freeze-dried product is
It was dissolved in a small amount of water, desalted using a Sephadex G-25 course equilibrated with water, and lyophilized to obtain 11 mg of the purified M5071-like substance M5084 (this product is a The specific activity was approximately 4,800 times higher than that of 1%, and showed a single band in polyacrylamide gel electrophoresis.Reference Example 3 Glucose 1%, Protoflower 1%, Peptone 1%, Corn Steep. 1% liquor,
500 ml with 100 ml of medium (PH6.5) consisting of KH ₂ PO ₄ 0.1%, MgSO _{4.7H 2} O 0.1%, Celite 1%
Erlenmeyer flasks (sterilized at 120℃ for 20 minutes, 5 each)
book), Macrophomina hazeolii
IFO7318 strain was inoculated and cultured with shaking at 26°C for 4 days. 5 ml each of this seed culture was then treated with sucrose.
1.5%, dextrin 1.5%, polypeptone 2%,
Corn steep liquor 2%, KH ₂ PO ₄ 0.2%,
Contains 100 ml of medium (PH6.5) consisting of 0.1% MgSO ₄ 7H _{2 O} , 0.01% FeSO ₄ 7H _{2 O} , and 1% Celite.
500ml Erlenmeyer flask (sterilized at 120℃ for 20 minutes,
100 plants each) and cultured with shaking at 26°C for 3 days.
After culturing, strain the culture using Celite,
After washing with a small amount of water, a culture solution of approximately 8.4 (protein content: 1637 g, specific activity: 0.29) was obtained. After adding 4.6 kg of ammonium sulfate to the obtained culture solution and stirring, the mixture was left in an ice chamber (4°C) overnight. The resulting precipitate was then collected by centrifugation and added to 0.01M phosphate buffer (PH6.3).
After dissolving in 500 ml, the solution was dialyzed against 0.01M phosphate buffer (PH6.3) using cellophane tube as a dialysis membrane. Change the external dialysis solution every 12 hours,
The patient was dialyzed for 72 hours with the same dialysate. After dialysis, the cellulose was charged and adsorbed onto a DEAE-cellulose column (diameter 5 x 40 cm) that had been smoothed with 0.01M phosphate buffer (PH6.3), and then the cellulose was absorbed with 0.01M phosphate buffer (PH6.3). After washing, add 0.01M phosphate buffer (PH
6.3) Elute by concentration gradient method using 0.01M phosphate buffer (PH6.3) containing 2-0.5M NaCl,
Obtain 393 ml of elution section (protein amount 1.6 g, specific activity 73) of 0.01 M phosphate buffer (PH6.3) containing 0.15 M NaCl,
It was further concentrated using membrane PM-10. Furthermore, this concentrated solution was dialyzed against 0.01M phosphate buffer using cellophane tube as a dialysis membrane,
Change the external dialysis fluid every 12 hours, and use 48% of dialysate for 80% of dialysate.
Dialyzed for hours. The dialyzed solution was 0.01M in advance.
Charge and adsorb onto a DEAE-Sephadex A-25 column (diameter 3 x 22 cm) smoothed with phosphate buffer (PH6.3), and wash with 0.01M phosphate buffer (PH6.3). , 0.01M phosphate buffer (PH
6.3) Elute by concentration gradient method using 0.01M phosphate buffer (PH6.3) containing 2-0.3M NaCl,
372 ml of active fraction containing M5071-like substance M4400 substance (protein amount 102 mg, specific activity 511) was obtained by elution with 0.01 M phosphate buffer (PH6.3) containing 0.01 M NaCl, and concentrated with membrane PM-10. Approximately 20ml was obtained. Furthermore, this concentrated solution was smoothed with 0.01M phosphate buffer (PH6.3) on a Sephadex G-100 column (diameter:
4.5 x 60 cm), elute each fraction (4.8 ml) with 0.01 M phosphate buffer (PH 6.3), collect fractions No. 79 to 92, and combine them (protein amount 34 mg, ratio activity 1431), lyophilized.
The lyophilized product was then desalted using a Sephadex G-25 course equilibrated with water and lyophilized to obtain 32 mg. Furthermore, add a small amount of this
After dissolving in 0.01M phosphate buffer (PH6.3), isoelectric focusing (700V, 24 hours) using carrier amphorite (manufactured by LKB) with pH 2.4 to 4 in glycerin was performed. After electrophoresis, fractionate 2 ml each, collect the active fraction, and use a Sephadex G-50 fine (manufactured by Seikagaku Corporation) column (diameter 2.4 x 40 cm) equilibrated with water in advance.
Charged to 4.8ml each fraction with water, collected fractions No. 20 to 25, and freeze-dried them to obtain 11mg of white powder of purified M5071 substance M4400 substance (this product is The specific activity was approximately 9,000 times higher than that in the culture solution, and a single band was observed in polyacrylamide gel electrophoresis). Reference example 4 Glucose 1%, Protoflour 1%, Peptone 1%, Corn steep liquor 1%,
500 ml with 100 ml of medium (PH6.5) consisting of KH ₂ PO ₄ 0.1%, MgSO _{4.7H 2} O 0.1%, Celite 1%
Erlenmeyer flasks (sterilized at 120℃ for 20 minutes, 5 each)
Gnomoniera sp. strain M5093 was inoculated into this strain, and cultured with shaking at 26°C for 4 days. 5 ml each of this seed culture was then mixed with 1.5% sucrose, 1.5% dextrin, 2% polypeptone, 2% corn steep liquor, 0.2% KH ₂ PO ₄ , MgSO ₄ .
7H ₂ O 0.1%, FeSO ₄・7H ₂ O 0.01%, Celite 1%
500ml Erlenmeyer flasks containing 100ml of medium (PH6.5) (sterilized at 120°C for 20 minutes, 100 flasks each)
and cultured with shaking at 26°C for 3 days. After culturing,
The culture was filtered through Celite, washed with a small amount of water, and diluted with approximately 10 culture solutions (protein content: 1611 g,
A specific activity of 0.19) was obtained. Add ammonium sulfate to the obtained culture solution.
After adding 5.2 kg and stirring, the mixture was left in an ice chamber (4°C) overnight. Next, the resulting precipitate is collected by centrifugation, and added to approximately 500 ml of 0.01M phosphate buffer (PH6.3).
After dissolving in 0.01 M phosphate buffer using cellophane tube as a dialysis membrane. Dialysis was performed for 72 hours with 120 dialysis fluid, changing the external dialysis fluid every 12 hours. After dialysis, a column (diameter 5 x 40 cm) of DEAE-cellulose (manufactured by Seikagaku Corporation), which had been smoothed in advance with 0.01M phosphate buffer (PH6.3), was added.
After charging and adsorbing with 0.01M phosphate buffer (PH6.3), 0.01M phosphate buffer (PH6.3)
6.3) Elute by concentration gradient method using 0.01M phosphate buffer (PH6.3) containing 2-0.5M NaCl,
415 ml of elution area (protein amount 1.81 g, specific activity 91) near 0.2 M NaCl-containing 0.01 phosphate buffer (PH6.3) was obtained, and membrane PM-10 (manufactured by Amicon) was obtained.
So it was concentrated. Furthermore, this concentrated solution was dialyzed against 0.01M phosphate buffer using cellophane tube as a dialysis membrane, and the dialysis solution was changed every 12 hours, and dialysis was carried out with 80 dialysis solutions for 48 hours. The dialyzed solution was charged and adsorbed onto a column (diameter 3 x 27 cm) of DEAE-Sephadex A-25 (manufactured by Seikagaku Corporation), which had been smoothed with 0.01M phosphate buffer (PH6.3). 0.01M phosphate buffer (PH6.3)
After washing with 0.01M phosphate buffer (PH6.3)2
~0.01M phosphate buffer containing 0.3M NaCl (PH6.3)
Elute with concentration gradient method using 0.1M
M5071-like substance eluted near 0.01M phosphate buffer (PH6.3) containing NaCl.Activity category containing M5093 substance.
208 ml (protein amount: 93 mg, specific activity: 192) was obtained and freeze-dried. Furthermore, this dry product containing the M5071-like substance M5093 substance was dissolved in a small amount of water, and the column (diameter 2.5× 60cm) and elute each fraction (4.8ml) with 0.01M phosphate buffer (PH6.3).
Nos. 69 to 80 were collected and combined (protein content 25 mg, specific activity
1330), freeze-dried. Next, this freeze-dried product was dissolved in a small amount of water, and Sephadex G-25 course (manufactured by Seikagaku Corporation) was equilibrated with water in advance.
The product was desalted and lyophilized to obtain 23 mg. Furthermore, after dissolving this in a small amount of 0.01M phosphate buffer (PH6.3), isoelectric focusing was performed using carrier ampholite (manufactured by LKB) with pH2.4 to 4 in glycerin. Perform electrophoresis (700V, 24 hours). After electrophoresis, fractionate 2 ml each and collect the active fraction.
Furthermore, it was charged to a column (diameter 2.4 x 40 cm) of Sephadex G-50 Fine (manufactured by Seikagaku Kogyo Co., Ltd.) equilibrated with water in advance, and each fraction (4.8 ml) was eluted with water, and the active fraction No. 21
~25 was recovered and purified by lyophilization.
20mg of white powder of M5071-like substance M5093 substance was obtained (this product has a specific activity of about 8200 compared to that of the culture solution).
), and a single band was observed in SDS-polyacrylamide electrophoresis (PH9). Reference example 5 Glucose 1%, Protoflour 1%, Peptone 1%, Corn steep liquor 1%,
500 ml with 100 ml of medium (PH6.5) consisting of KH ₂ PO ₄ 0.1%, MgSO _{4.7H 2} O 0.1%, Celite 1%
Erlenmeyer flasks (sterilized at 120℃ for 20 minutes, 5 each)
book), Nozurisporium S.P. M5091
The strain was inoculated and cultured with shaking at 26°C for 4 days. Then, 5 ml each of this seed culture was added to 1.5% sucrose,
Dextrin 1.5%, Polypeptone 2%, Corn Steep Liquor 2%, KH ₂ PO ₄ 0.2%,
Contains 100 ml of medium (PH6.5) consisting of 0.1% MgSO ₄ 7H _{2 O} , 0.01% FeSO ₄ 7H _{2 O} , and 1% Celite.
500ml Erlenmeyer flask (sterilized at 120℃ for 20 minutes,
100 plants each) and cultured with shaking at 26°C for 3 days.
After culturing, strain the culture using Celite,
Wash with a small amount of water and add approximately 9% of the culture solution (protein content).
1302g, specific activity 0.21) was obtained. After adding 4.6 kg of ammonium sulfate to the obtained culture solution and stirring, the mixture was left in an ice chamber (4°C) overnight. Next, the resulting precipitate was collected by centrifugation, and added to 0.01M phosphate buffer (PH6.3) at 500%
ml, and then dialyzed against 0.01M phosphate buffer (PH6.3) using cellophane tube as a dialysis membrane. Dialysis was performed for 72 hours with 120 dialysis fluid, changing the external dialysis solution every 12 hours. After dialysis, the cells were smoothed with 0.01M phosphate buffer (PH6.3) in advance.
Charge and adsorb onto a DEAE-cellulose column (diameter 5 x 40 cm) and 0.01M phosphate buffer (PH
6.3) After washing with 0.01M phosphate buffer (PH6.3)
0.01M phosphate buffer containing 2-0.5M NaCl (PH
6.3) Elute with concentration gradient method using 2,
Elution section around 0.01M phosphate buffer (PH6.3) containing 0.1M NaCl 433ml (protein amount 2.42g, specific activity 88)
was obtained and further concentrated using membrane PM-10.
Furthermore, this concentrated solution was dialyzed against 0.01M phosphate buffer using cellophane tube as a dialysis membrane, and the external dialysis solution was changed every 12 hours, and dialysis was performed with 80 dialysis solutions for 48 hours. The dialyzed fluid should be prepared in advance.
DEAE equilibrated with 0.01M phosphate buffer (PH6.3)
-Sephadex A-25 column (diameter 3 x 25 cm)
After charging and adsorbing with 0.01M phosphate buffer (PH6.3), wash with 0.01M phosphate buffer (PH6.3) and 0.01M phosphate buffer (PH6.3) containing 2 to 0.3M NaCl. 3) Elute with concentration gradient method using 2,
283 ml of active fraction (protein content: 85 mg, specific activity: 201) containing M5071-like substance M5094 substance eluted near 0.05M NaCl-containing 0.01M phosphate buffer (PH6.3) was obtained and lyophilized. Furthermore, this dried product containing M5071-like substance M5094 substance was dissolved in a small amount of water and charged to a Sephadex G-100 column (diameter 2.5 x 60 cm) equilibrated with 0.01M phosphate buffer (PH6.3).
Elute each fraction (4.8ml) with 0.01M phosphate buffer (PH6.3), and collect fractions No. 85~
97 was collected and combined (protein amount 23 mg, specific activity 1121),
Lyophilized. Next, this lyophilized product was dissolved in a small amount of water, desalted using a Sephadex G-25 course equilibrated with water, and lyophilized to give 22 mg.
I got it. Furthermore, after dissolving this in a small amount of 0.01M phosphate buffer (PH6.3), the pH was dissolved in glycerin.
2. Perform isoelectric focusing (700V, 24 hours) using carrier amphorite of 4 to 4. After electrophoresis, fractionate into 2 ml portions, collect the active fraction, and equilibrate with water in advance. Sephadex G-50 fine column (diameter 2.4 x 40 cm)
The active fractions No. 21 to 25 were collected, and each fraction was eluted with water (4.8 ml).
M5071-like substance purified by freeze-drying this
17 mg of white powder of substance M5094 was obtained (this product has a specific activity approximately 6800 times higher than that of the culture solution, SDS
- It showed a single band in polyacrylamide electrophoresis (PH9)).

[Brief explanation of drawings]

Figure 1 is the PH stability curve of the M5071 substance, Figure 2 is the PH stability curve of the M5071-like substance M5084,
Figure 3 is the PH stability curve of the M5071-like substance M5084 substance, Figure 4 is the PH stability curve of the M5071-like substance M4400 substance, and Figure 5 is the PH stability curve of the M5071-like substance M5093 substance.
Stability curve, Figure 6 is the PH stability curve of the M5071-like substance M5094 substance, Figure 7 is the thermal stability curve of the M5071 substance, Figure 8 is the thermal stability curve of the M5071-like substance M5084 substance, Figure 9 The figure shows the thermal stability curve of the M5071-like substance M5084, and Figure 10 shows the M5071-like substance M4400.
Curve of thermal stability of substances, Figure 11 is M5071-like substance
Thermal stability curve of M5093 material, Figure 12 is M5071
Figure 13 shows the thermal stability curve of M5094-like substance.
Figure 14 is an electron micrograph showing the adhesion of caries-causing bacteria to the tooth surface when M5071 substance is not added. The figure is an electron micrograph showing the colony formation of caries-causing bacteria on the tooth surface when M5071 substance is not added. Figure 16 is an electron microscope photo showing the suppression of the colony formation of caries-causing bacteria on the tooth surface when M5071 substance is added. The photo, Figure 17 is M5071-like substance M5084
Electron micrograph showing suppression of adhesion of caries-causing bacteria to the tooth surface when the substance is added. Figure 18 shows an M5071-like substance.
Figure 19 is an electron micrograph showing the suppression of adhesion of caries-causing bacteria to the tooth surface when M5084 substance is added.
Electron micrograph showing suppression of adhesion of caries-causing bacteria to the tooth surface when M5071-like substance M4400 substance is added, 2nd
Figure 0 is an electron micrograph showing the suppression of adhesion of caries-causing bacteria to the tooth surface when the M5071-like substance M5093 is added, and Figure 21 is an electron micrograph showing the adhesion of caries-causing bacteria to the tooth surface when the M5071-like substance M5094 is added. Electron micrographs showing inhibition are shown.

Claims (1)

[Scope of Claims] 1. A composition for preventing dental caries, comprising a polymeric acidic protein-based substance having an inhibitory effect on glucosyltransferase activity. 2 Polymeric acidic protein-based substances that have an inhibitory effect on glucosyltransferase activity include M5071 substance, M5071-like substance M5084 substance, M5071-like substance
M5084 substance, M5071-like substance M4400 substance, M5071
The composition for preventing dental caries according to claim 1, which is the M5093-like substance and the M5071-like substance M5094. 3. The content of a polymeric acidic protein substance that has an inhibitory effect on glucosyltransferase activity is
The composition for preventing dental caries according to claim 1 or 2, which has a content of 0.0001 W/W% or more. 4. The composition for preventing tooth decay according to claim 1, 2 or 3, wherein the composition for preventing tooth decay is a dentifrice composition. 5. The composition for preventing dental caries according to claim 1, 2 or 3, which is an oral hygiene composition. 6. The composition for preventing tooth decay according to claim 1, 2 or 3, wherein the composition for preventing tooth decay is a composition containing a gum base. 7. The composition for preventing dental caries according to claim 1, 2 or 3, which is a food or drink composition.