RU2789083C2 - Method for obtaining a dextranase preparation and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method for obtaining a dextranase preparation Aspergillus insuetus VKMP F - 342. The method includes: obtaining a working culture on a slant agar; preparation of deep sowing culture in flasks; preparation of a deep seed culture in a fermenter; carrying out the process of biosynthesis in fermenters; separation on the flow separator; concentration of the native solution in an ultrafiltration unit; mixing with mannitol, sterilizing filtration, freezing; freeze drying, while mannitol is added at the rate of 1 g per 8000-12000 units activity. The invention also relates to a dextranase preparation Aspergillus insuetus VKMP F - 342, which has an increased activity and applications of this drug for the treatment and prevention of dental caries and gingivitis. In addition, the invention is directed to oral care products using the preparation.

EFFECT: invention makes it possible to obtain a dextranase preparation with increased activity. 7 cl, 8 tbl

Description

Field of invention

The invention relates to a method for producing a drug based on Aspergillus insuetus VKMP F-342 dextranase, directly to a drug with certain activity and stability characteristics, to the use of said dextranase drug (or dextranase recovered from the drug) for the prevention and treatment of periodontal diseases, including caries teeth, gingivitis and periodontitis; for the destruction of dextran diffusion juice in the production of sugar from sugar beets, as well as compositions and products for certain purposes using this drug.

State of the art

Initial interest in dextranases was expressed with regard to their possible use in the commercial production of clinical dextran, i.e. a sterile solution of dextran with a specific molecular weight, which would be used to restore blood volume in patients suffering from shock due to blood loss (Kim, D., and D. F. Day 1994 A new process for the production of clinical dextran by mixed-culture fermentation of Lipomyces starkeyi and Leuconostoc mesenteroides Enzyme Microb Technol 16:844-848 Lee, J. M. and P. F. Fox 1985 Purification and characterization of Paecilomyces lilacinus dextranase Enzyme Microb Technol 7:573-577). Relatively low molecular weight clinical dextrans have previously been prepared from dextran by controlled acid hydrolysis followed by organic solvent fractionation. However, the yield is low (10-12%) due to losses during hydrolysis and fractionation. The enzymatic method appears to have had the potential to replace acid hydrolysis for the clinical production of dextran, and such processes were patented in the 1950s. The enzymatic method requires less energy and simpler equipment and results in a more homogeneous product with a yield of 25% to 52%.

The highest yield of clinical dextran, 94% of total dextran production, was obtained from the mixed culture fermentation of Lipomyces mesenteroides and the constitutive dextranase mutant Lipomyces starkeyi in the presence of sucrose. A simple industrial fermentation has been developed to produce controlled size dextrans with a low polydispersity index (Kim, D., and D.F. Day. 1994. A new process for the production of clinical dextran by mixed-culture fermentation of Lipomyces starkeyi and Leuconostoc mesenteroides. Enzyme Microb. Technol 16:844-848; Lee, J.M., and P.F. Fox 1985. Purification and characterization of Paecilomyces lilacinus dextranase Enzyme Microb Technol 7:573-577; US 5,229,277).

Dental lamina (plaque), a bacterial film that adheres to tooth surfaces, is composed of densely packed bacteria and non-cellular material. Approximately 20% of the dry weight of plaque is water-insoluble glucans. Degradation and removal of these glucans has been suggested to prevent oral diseases such as caries. Dextranase can inhibit the synthesis of insoluble glucans (Marotta, M., A. Martino, A. Rosa, E. Farina, M. Carteni, and M. Rosa. 2002. Degradation of dental plaque glucans and prevention of glucan formation using commercial enzymes. Process Biochem. 38:101-108), as well as streptococcal adherence (Schilling, K. M., and W. H. Bowen. 1992. Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans. Infect. Immun. 60: 284-295).

The pharmacological properties of dextranase are based on its ability to catalyze the cleavage of dextran bonds, which is part of dental plaque. Dextranase intensively (up to 94%) breaks down plaque dextran, has a pronounced antibacterial effect against cariogenic microflora (US 6,254,856, US 4,438,093). It is known that the higher the oral hygiene, the better the condition of the periodontium. But even regular brushing of teeth does not completely and permanently eliminate plaque, especially since sugar can remain in the microcracks of the gums. Dextranase is the only agent that prevents the formation of plaque and in this sense, the action of the drug can be considered a breakthrough in the field of treatment, prevention of caries and periodontal disease, and this is what distinguishes dextranase from other drugs on the market.

The use of the drug for prophylactic purposes ensures the complete removal of plaque. Prevents the formation of stones and caries, strengthens the gums, whitens teeth, provides 100% prevention of caries in children (US 4,438,093).

For the treatment and prevention of plaque formation, various compositions have been proposed that contain enzymes that hydrolyze or inhibit glucans (WO 01/66570; Schilling, K. M., and W. H. Bowen. 1992. Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans Infect Immun 60:284-295; US 6,254,856; US 4,438,093).

Leuconostoc mesenteroides infections are a common cause of spoilage in sugar beet and sugar cane, especially in humid and warm conditions. L. mesenteroides form dextrans (α-(1→6)-α-d-glucans) and other degradation products, including mannitol and D-lactic acid, which in moderate to severe cases can interfere with the normal manufacturing process. Freezing and subsequent thawing damage the sugar beet and leave it vulnerable to microbial infestation, especially L. mesenteroides, if warm weather follows. The formation of high molecular weight dextran has a detrimental effect on the crystallization (formation) of calcium carbonate during the clarification process. As a result, finer particles of calcium carbonate are formed, which, by increasing the filtration pressure, adversely affect the filtration of the juice of the 2nd carbonation. This, in turn, leads to a reduction in beet processing. The increased viscosity seen at high concentrations of dextran can also lead to sugar crystallization problems, but this is more common in sugar cane production.

Dextrans isolated from various sugarcane products have a very similar structure, 95% α-1,6 bonds and 5% branching, probably through α-1,3 bonds (Khalikova E. Microbial dextran-hydrolyzing enzymes: fundamentals and application / E. Khalikova, P. Susi, T. Korpela // Microbiology and molecular biology reviews, Jue, 2005, pp. 306-325).

Commercial dextranases (1→6)-α-glucanohydrolases, EC 3.2.1.11) have been used in beet and cane plants to cleave dextran by hydrolysis of α-(1→6) bonds at random endogenous sites. P. Lilacinum dextranase from Novo (Denmark) and Hutten DL-2 dextranase Chaetomium gracile (Japan) were successfully used for dextran hydrolysis of contaminated sugar (Rogalski, J., J. Szczodrak, G. Gl'owiak, M. Pleszczynska, Z Zinchenko, O. N., V. I. Shishlo, O. V. Krivosheeva, and A. G. Lobanok 1993. Fungal and yeast dextranases: properties and prospects for application in sugar industry Prikl Biokh Mikrobiol 29:851-855). Hutten DL-2 dextranase Chaetomium gracile in liquid form (56% solution in glycerol) was tested at the Yagotinsky pilot plant of NPO Sakhar (Purification of diffusion juice in sugar production / edited by Lovkis Z.V. 2013. 232 p.) . Chaetomium gracile dextranase from Sugazym DX L (Germany) is successfully used in the production of sugar (Abraham K., Hagen S., Schlumbach K., Rode A., Fluther E. Some aspects of the use of dextranase in sugar solutions. Sugar. - No. 5. - pp. 34-42.). The enzyme concentration is 1-5 g/t, the reaction time is 10-20 minutes, the optimum action is pH 4-8, t is 55-75°C. These conditions basically coincide with the properties of Aspergillus insuetus VKMP F-342 dextranase.

Most commercial dextranases in the US are made from Chaetomium gracile and Chaetomium erraticum, which are available in liquid form and are recognized as safe - GRAS status (Eggleston D., Dilks E., Blowers M., Winters K. 2017. Successful use of dextranase in beet processing plants. Sugar - No. 3. - S. 30-40).

Disclosure of invention

The proposed invention solves the problem of providing a method for obtaining a dextranase preparation of the strain Aspergillus insuetus VKMP F - 342, which has increased activity, as well as the use of a drug obtained by this method for the prevention and treatment of periodontal diseases, including dental caries, gingivitis and periodontitis; as well as for the destruction of dextran diffusion juice in the production of sugar from sugar beets. In addition, the invention is directed to oral care products using the preparation.

Method for obtaining microbial dextranase

Micromycetes are known to be the most common source of extracellular endodextranases (EC3.2.1.11) and exhibit higher enzyme activity than dextranases from bacteria and yeasts.

For the production of dextranase, a strain producing Aspergillus insuetus VKMP F - 342, obtained from a strain of Aspergillus insuetus G-116 without the use of mutagens, was used. Aspergillus insuetus strain VKMP F - 342 was tested for pathogenicity in two types of laboratory animals: rats and mice. The doses administered were 10 ⁷ and 10 ⁸ cells per animal. As a result of the tests, the death and dissemination of parenchymal organs in animals were absent.

Scheme for obtaining dextranase Aspergillus insuetus VKMP F - 342

Obtaining the enzyme preparation of dextranase was carried out according to the following steps:

- obtaining a working culture on slant agar (wort agar, Czapek's medium);

- preparation of deep sowing culture in flasks;

- preparation of deep seed culture in the fermenter V=10 l;

- carrying out the process of biosynthesis in fermenters with a capacity of 250 l;

- separation on the flow separator - Sulfur Z-61;

- concentration of the native solution in the ultrafiltration unit;

- mixing with mannitol, sterilizing filtration, freezing;

- sublimation drying.

The composition of the nutrient medium for the cultivation of the producer (g/l):

Polyglukin 5.0

Yeast autolysate (extract) 3.0

NaNO ₃ 3.0

KN ₂ RO ₄ 1.0

KCl 0.5

MgSO ₄ ⋅ 7H ₂ O 0.5

Sofaxil-1520 0.1 ml

pH=5.0-5.2.

The flasks are placed on a shaker (190-220 rpm), the cultivation is carried out at a temperature of 29±10 C for 48 hours. The volume of the medium in flasks is 200 ml.

Two flasks are seeded with a seed fermenter with a volume of 10 liters, a working volume of 6-7 liters.

The cultivation process is carried out in the following mode:

- temperature 30±10°С

- aeration with compressed air 6 l/min,

- mixing of the medium with a stirrer 400 rpm.

Then 6-7 liters of seed material was mixed into a BIOR-025 type fermenter with a working volume of 160 liters with the above nutrient medium composition.

Values of technological parameters of the micromycete cultivation process: temperature, °С 30+10°С

pH mixing, rpm 350

aeration, rpm 0.5 for the first 10 hours, then

1.0

the duration of the cultivation of the producer is 50-70 hours according to the value of the enzyme activity (50-80 units/ml) at a pH above 8.0.

Biomass separation

Separation of biomass was carried out on a flow separator Sera Z-61 at a rotor speed of 16000 rpm. The separation rate was chosen such that the filtrate sample at the outlet of the separator was visually transparent. The rate of supply of the culture fluid to the separator was maintained in the range of 30-40 l/h.

Receive 150-155 l of the filtrate of the culture fluid with an activity of 50-80 units/ml.

These stages of obtaining dextranase Aspergillus insuetus VKMP F - 342 were carried out in accordance with the author's certificates SU 1756355 and SU 1808873. The scheme for the isolation and purification of dextranase from the culture fluid is not described in the certificates. It is only indicated that the enzyme preparation is obtained in a known manner by precipitation with ethyl alcohol.

Steps for obtaining a purified dextranase preparation

filtrate ultrafiltration

The concentration of the native solution was carried out on the solution using an ultrafiltration unit with an AR-3-15 module (NPK Biotest, Kirishi) with a pore diameter of 15 kDa. Pressure drop - 0.18-0.20 MPa.

The filtration rate is about 10-12 l/hour. The final volume is 7-8 liters.

Evaporation is carried out until the activity of the concentrate is 600-800 units/ml.

It is known that deep purification of enzymes leads to a significant loss of drug activity, and is also very expensive. Moreover, highly purified proteins are less stable than unpurified ones. Therefore, when using soluble enzymes, complete purification is rarely used. Moreover, depending on the scope of application, the requirements for the purity of enzyme preparations are different [Biotechnology / T.G. Volova. - Novosibirsk: Publishing House of the Siberian Branch of the Russian Academy of Sciences, 1999. - 252 p.].

But, in order to maintain the stability of the enzyme at the stage of lyophilization, as a rule, it is necessary to introduce auxiliary ingredients into native solutions that optimize the technical characteristics of drugs. The main condition for obtaining a high-quality drug is the selection of the optimal ratio between the auxiliary and the main substances.

For enzyme and other protein preparations, components are used as fillers that stabilize protein molecules with respect to external influences.

The study of homogeneous endodextranase Penicillium fimiculosum 15 showed that dextranase is a glycoprotein. The enzyme contains residues of mannose, galactose and fructose in a ratio of 6:2:1. Carbohydrates are covalently linked to the protein portion of dextranase and play an important role in the stability of the enzyme.

Samples of homogeneous dextranase Aspergillus insuetus VKMP F - 342 were obtained.

Homogeneous dextranase preparations are characterized by the following indicators:

- molecular weight 71000±2000;

- isoelectric point 4.5±0.05;

- the activity of homogeneous dextranase is 1700-2000 units/mg, the protein content is 48-50% protein;

- specific activity - 900±100 units/mg protein;

- the enzyme contains 6.0 ± 0.5% carbohydrates covalently linked through glucosamine to the protein, which play an important role in the stability of the enzyme. The carbohydrate component contains residues of mannose, galactose and fructose in a ratio of 6:2:1. Obviously, most endodextranases are glycoproteins. These studies show that the most promising stabilizing fillers are low molecular weight sugars or auxiliary substances close to them. Among the known excipients allowed in the preparation of medicines, mannitol (mannitol) is used (Khabriev R.U., Kamaev N.O., Danilova T.I., Kakhoyan E.G. Development of a technology for obtaining microbial hyaluronidase and a drug based on it. - Issues of Biomedical and Pharmaceutical Chemistry - No. 1. - 2016. - P. 4-11). This sugar alcohol is very close in its structure to the main component of the carbohydrate part of the dextranase molecule - mannose. It has been experimentally proven that the stabilizer of Penicillium fimiculosum 15 endodextranase is the products of deep hydrolysis of dextran, containing mainly monosaccharide and disaccharide - glucose and isomaltose, respectively. Mannitol is a dextranase stabilizer, preserving the native structure of the enzyme molecule during drying and further use.

Sterilizing filtration of concentrate

Mannitol is added to the concentrate at the rate of 1 g per 8000-12000 units of activity. Sterilizing filtration of the concentrate is carried out on a microfiltration unit with a microfiltration module Microza of the P series (Pall). The filtration area is 0.2 m2, the pore diameter is 0.1 μm. The plant uses a centrifugal pump at a pressure of 2.8 bar.

The filtrate from the module is taken into a sterile container and transferred to a box for filling into cuvettes. The intermediate product is poured into pallets (400-500 cm ³ ) and frozen at a temperature of minus 40°C during the day. The rest of the concentrate from the module is transferred for disposal.

Drying of the enzyme preparation.

Sublimation drying of the enzyme preparation is carried out in vacuum dryers of the KS-30 brand. Cuvettes with sensors frozen into the control samples are installed in the middle of the shelves. All sensors are connected to a recording device with recorders installed on the control panel.

After loading, close the chamber door, open the valve on the vacuum line. Upon reaching a working vacuum not exceeding 100 microns, the preparation must be cooled to minus (30-35)°C and maintained at the specified temperature for 45-60 minutes.

When drying the drug, regulate the temperature on the shelves in such a way as to reach 0°C in the drug by 16-18 hours of drying, then, gradually increasing the temperature, reach (27-30)°C in the material, at which the drug is kept for 8-10 hours.

The duration of the drying process is 24-28 hours. The standard dextranase preparation has an activity of 50.0±5.0 units/mg.

This amount of dextranase is taken as a unit of dextranase activity. which, acting for 1 min on a 0.15% solution of polyglucin at a temperature of 37°C and pH 4.9-5.1, forms oligosaccharides in an amount equivalent to 1 μmol of glucose.

0.75 ml of a 0.15% solution of polyglucin is added to 3 test tubes (2 experimental and 1 control) and thermostated at 37°C for 1-2 minutes. 0.25 ml of water is added to the control tube, and 0.25 ml of a 0.0004% solution of the drug with a concentration of 0.004 mg / ml is added to 2 experimental tubes. The mixtures are incubated for 10 minutes (with a stopwatch) in a thermostat at 37°C, after which 3 ml of a 0.06% solution of potassium ferricyanide are added to each test tube. The test tubes are placed in a boiling water bath (according to the stopwatch) for 10 min, then cooled in a bath of cold water. The optical density of the test and control solutions is measured on a photoelectric colorimeter at a wavelength of 400 nm in a cuvette with a thickness of 5 mm. Distilled water is used as a reference solution.

Dextranase activity (A) is expressed as the number of units per I mg of the drug (u/mg) and is calculated by the formula:

where Yx is the glucose content in the test sample, determined from the calibration graph built for D-glucose, in micrograms:

Gk - glucose content in the control sample, determined by the calibration graph, built on D-glucose, in micrograms;

- dilution factor of the reaction mixture,

ISO - molecular weight of glucose,

10 - hydrolysis time, in minutes;

m is the concentration of the solution of the dextranase preparation, mg/ml.

Dextranase activity of 1 mg of the drug should be 50 units ± 5 units.

Construction of a calibration graph. Standard solutions of D-glucose are prepared by diluting the stock solution with water according to Table 1. To prepare the stock solution, exactly 0.075 g of anhydrous D-glucose (GOST 6038-79) is dissolved in water in a 500 ml volumetric flask and the volume of the solution is adjusted to the mark.

Preparation of standard glucose solutions

Add 3 ml of a 0.06% solution of potassium ferricyanide to test tubes with standard solutions, place in a boiling water bath for 10 minutes (according to a stopwatch), then cool in a bath of cold water. Measure the optical density of solutions on a photoelectric colorimeter at a wavelength of 400 nm ± 2 nm in a cuvette with a layer thickness of 5 mm.

Distilled water is used as a reference solution.

A calibration graph is built by plotting along the abscissa axis - the content of D-glucose in micrograms in 1 ml of standard solutions, and along the ordinate axis - the optical densities corresponding to them.

The obtained dependence of optical density on concentration is linear (correlation coefficient /y/=0.998). According to the calibration graph, the values of Vx and Vk are found, corresponding to the optical densities of the experimental and control solution, measured at a wavelength of 400 nm ± 2 nm. and substitute these values into the formula for calculating dextranase activity.

Table 1 shows the characteristics of the proposed method for obtaining purified dextranase.

Table 2 shows the scheme for the production of purified dextranase Aspergillus insuetus VKMP F - 342 by stages

Also, the drug was studied in dental practice and in the processing of sugar beet, which is discussed in more detail below.

Dextranase can be used in the processing of sugar beets affected by mucous bacteriosis, as well as for the processing of raw sugar in order to increase the production and quality of white sugar, save raw material costs (sugar beet); increase equipment life and reduce filter wear.

An important problem for sugar beet production is the large volume of raw materials (root crops) affected by mucous bacteriosis.

As a result of the vital activity of microorganisms (bacteria), high-molecular polysaccharides - dextrans are formed, which increase the viscosity of sugar solutions, significantly reducing their technological qualities. It is almost impossible to remove dextran from granulated sugar solutions by filtration or chemical additives. Being a powerful biocatalyst, dextranase breaks dextran molecules into shorter chains, then oligosaccharides appear and the end product is glucose. At the same time, the viscosity of the syrup is reduced to a value corresponding to the viscosity of a pure sugar solution, mucus and turbidity disappear, and filtration is restored.

Dextranase is used in the first stages of the technological process, after obtaining juice from a diffusion plant.

The drug consumption is 2.5-5.0 g per 1 cubic meter of juice at a temperature of 45-50°C, the duration of action is 5-10 minutes. The use of dextranase in the processing of raw material is carried out during the first phase of dissolution at a temperature of 45-50°C. The duration of action is 10-15 minutes.

As a result of using the drug, the juice filtration rate increases by 2.5-3 times. The acceleration of crystallization of sucrose increases by 1.5-3 times. Saving raw materials up to 20% of the total volume due to the possibility of processing mucilaginous beets. Additional production of sugar from beets increases by 9-11% of the total output. Adding the active profit from the sale of additional volume of sugar and the cost savings on raw materials and filter replacement increases profitability.

The presence of even small amounts of dextran in sugar beet complicates the technological process of sugar and then refinery production. Among the technological processes of sugar production, a significant place is occupied by the filtration process. With a high content of polysaccharides in sugar beet and diffusion juice, difficulties are observed in filtering juices of I and II saturation. Difficulties in filtering increase when frozen and thawed beets with a long shelf life are sent for processing. High molecular weight dextran passes from beets of this quality into diffusion juice, which prevents the formation of large CaCO ₃ aggregates, and leaves a slimy residue on the filter surface. Destruction of dextran in juices can be done with the enzyme dextranase.

With lime-carbon dioxide purification of diffusion juice, 20-25% of dextran is removed, the rest goes into purified juice. Dextran, having a positive charge, is not sorbed on calcium carbonate, only a small part of it forms a gelatinous, poorly filtered precipitate with particles of the latter. Moreover, in the presence of dextran, the rate of formation of calcium carbonate nuclei significantly exceeds the rate of their growth; as a result, the latter precipitates in the form of a fine-grained, slightly agglomerated precipitate, which makes filtering difficult. Thus, during the processing of frozen and thawed sugar beets stored for 7 days, the second saturation juice obtained from it contained CaCO ₃ precipitate crystals 1–3 µm in size, while from beets that did not contain dextran - 5.7 µm. The adsorption of dextran on the surface of the filter cloth contributes to the slowing down of the filtration process. Basically, filtration is slowed down by high-molecular fractions of dextran with a molecular weight of 10 ⁵ -10 ⁷ , and fractions with a mass of 10 ⁴ and below practically do not affect its speed.

Further downstream, dextran leads to delayed crystallization of sucrose and centrifugation of massecuites, does not contribute to the depletion of molasses, and degrades the quality of white sugar. During the crystallization of sucrose from sugar solutions containing dextran, the sugar crystals are deformed, elongated, needle-shaped crystals are formed. During the crystallization of sucrose by evaporation, the massecuite crystals of the first stage of crystallization retain from 18 to 25% of dextran to its initial content in solution. Such a massecuite is poorly centrifuged, the conditions for separating edema worsen, and the loss of sugar in molasses increases.

Recrystallization of the sugar removes approximately 80% of the dextran. Crystalline sugar can contain up to 100 mg/kg of dextran, and molasses sometimes contains several thousand mg/kg.

The presence of dextran in white sugar used for the production of non-alcoholic and alcoholic beverages, confectionery adversely affects both the quality of the product and the processes of its production. So, the presence of dextran in sugar causes mucilage of drinks, kvass, kvass wort; the process of filtering sugar syrup is difficult or completely stops, pipelines are clogged. Sometimes a mucous plug can form in the pipeline, which completely blocks the flow. Due to the decomposition of sucrose, the amount of sugar required by the recipe decreases, and the yield of the product decreases. Dextran is resistant to alkalis, it is difficult to hydrolyze. The enzyme dextranase breaks down dextran to isomaltose and higher isomaltosaccharides. When obtaining dextranase, various strains of mold fungi Penicillium and Chaetomium are used, which are grown on a nutrient medium containing dextran, nitrogen, phosphorus and mineral nutrition. The presence of dextran in the nutrient medium induces the formation of the enzyme dextranase in molds. Complete hydrolysis of dextran is ensured under the following conditions: introduction of dextranase in an amount of 0.04%, ensuring pH ₂₀ 5.5 and temperature 50°C for 35 minutes.

Effect of temperature and pH of sugar solutions on the enzymatic degradation of dextran

The influence of temperature on the effectiveness of the action of the dextranase enzyme preparation was studied by determining the rate of decrease in the viscosity of a diffusion juice containing dextran and having a CB=10%; pH=5.5 under isothermal conditions in the temperature range of 30-70°C. 0.5% dextran with a molecular weight of 5×10 ⁵ Da was added to the diffusion juice, heated to the required temperature, dextranase was introduced in an amount of 1 mg/dm ³ , thermostated for 5-40 min at a given temperature directly in the viscometer tube and the viscosity was determined . The results of the determination are presented in the table.

The research results indicate that the studied dextranase preparation is a relatively heat-resistant enzyme preparation, the activity of which is manifested in the temperature range of 30-50°C, and is completely inactivated at temperatures above 60°C.

The effect of pH on dextranase activity was also studied by the rate of decrease in dynamic viscosity during thermostating directly in the viscometer tube for 30 min at a temperature of 40°C in diffusion juice (CB=12%) and in a sugar solution (CB=10%). The pH range was pre-set within 4.3-7.0 for diffusion juice and 3.0-8.0 for sugar solutions using citrate buffer. The concentration of added dextran was 0.4% and the dose of dextranase was 0.4 mg/dm ³ . The results of the determination are presented in tables

Judging by the rate of decrease in viscosity, both in diffusion juice and in sugar solutions, the highest activity of dextranase is manifested in the region of pH=4. At pH more and less than 4, the rate of viscosity decrease slowed down and was the smallest at 3 < pH < 7, which should be taken into account when determining the dose of the drug required to destroy dextran.

It is known that the higher the content of dextran in a sugar solution, the greater the dose required for its destruction to a given concentration. The influence of dextran content in diffusion juice in the range (500-5000 mg/dm ³ ), the duration of enzyme treatment (5 and 15 min) and pH (5.1-6.6) on the residual concentrations of dextran at a dose of dextranase introduced into the diffusion juice was studied. juice, in the range of 0.1-8.0 mg / dm ³ . In the experiments, dextran was added to the diffusion juice; then, after thermostating at 45°C, the residual content of dextran was determined using the turbidimetric method. The results are in the table below.

As can be seen from the data obtained; the consumption of the enzyme preparation increases significantly with an increase in the initial content of dextran in the diffusion juice; reducing the duration of thermostating and increasing pH.

Based on the received data; assuming; that the residual amount of dextran in the diffusion juice should not exceed 100 mg/dm ³ ; we calculated the approximate doses of the studied drug, which must be introduced into the diffusion juice containing dextran (table 6).

When the content of dextran in the diffusion juice is 500 mg/DM ³ and the duration of treatment is 15 minutes, the consumption of the enzyme preparation was 0.2-0.6 mg/DM ³ ; with an increase in the content of dextran to 2000-5000 mg/DM ³ the dose of dextranase increases to 2.0-4.0 mg/DM ³ . Thus, the consumption of the studied drug turned out to be 5-15 times less than the known dextranase preparations from other companies, which indicates its high activity.

The indicated doses are indicative, since they depend on many factors, including the duration of the enzymatic treatment, temperature, pH, the chemical composition of the diffusion juice and other factors that affect the course of the enzymatic decomposition of dextran.

In order to test the effectiveness of dextranase in diffusion juice, as close as possible to production, diffusion juice was obtained in the laboratory from healthy sugar beets and beets infected with Leuconostoc bacteria - dextran producers. The content of dextran in the diffusion juice from beetroot affected by bacteria was 6000 mg/dm ³ . Next, samples of diffusion juice were prepared with a dextran content of 600 to 6000 mg/dm ³ and a pH of 4.5-5.0 by mixing healthy beet juice and diseased beet juice in various proportions. The samples were subjected to enzymatic treatment at a dose of dextranase 0.2-8.0 mg/DM ³ for 15 min at a temperature of 45°C, after which they determined the residual amount of dextran. The research results are presented in table 7.

From the data presented in the table, it can be seen that the observed patterns are similar to those obtained during the destruction of dextran with a molecular weight of 5×10 ⁵ Da when it is added to the diffusion juice (table 5).

The same patterns are observed when determining the approximate doses of the consumption of the enzyme preparation, calculated on the basis of the data obtained for the infected diffusion juice (table 8).

When ^the content of dextran in the diffusion juice was 500-1000 mg/ ^dm 10 ⁵ Yes (Table 6). With a higher content of dextran in the diffusion juice (more than 2000 mg/dm ³ ), the consumption of the enzyme preparation for the infected diffusion juice was higher, which, apparently, is due to a change in the chemical composition of the infected juice and worsening conditions for the enzymatic destruction of dextran.

The effect of the drug on diseases of the oral cavity was also investigated.

The pharmacological properties of dextranase are based on its ability to catalyze the cleavage of dextran bonds, which is part of dental plaque. Dextranase intensively (up to 94%) breaks down plaque dextran, has a pronounced antibacterial effect against cariogenic microflora, as well as a high anti-caries effect. It is known that the higher the oral hygiene, the better the periodontal condition. But even regular brushing of teeth does not completely and permanently eliminate plaque, especially since sugars remain in the microcracks of the gums. It is the absence of plaque that simply prevents caries from forming. Dextranase is the only agent that suppresses the formation of plaque. The use of the drug for prophylactic purposes ensures the complete removal of plaque. Prevents the formation of stones and caries, strengthens the gums, whitens teeth, provides 100% prevention of caries in children.

With periodontal diseases, it allows you to quickly stop bleeding gums, stop the inflammatory process, close "pockets", restore periodontium around the neck of the teeth and stop loosening and falling out of teeth, whiten teeth and get rid of bad breath

As you know, the leading etiological and pathogenetic factor in the development of dental caries and gingivitis is the formation of plaque, which consists mainly of microorganisms, proteins and polysaccharides, in particular dextrans, which ensure adhesion of plaque to the tooth surface with subsequent destruction of enamel.

In an experimental study of the substance and dosage form of dextranase (tablets containing 0.006 g - 30 units) in experiments in vitro in vivo on experimental models, it was found that the drug intensively hydrolyzes plaque dextran (up to 94%), has a pronounced antibacterial effect against caries genetic microflora. Based on the effect of the drug on plaque revealed in the experiment, it was recommended by the developers for its clinical study in dental practice.

A clinical study of the drug "Dextranase" was held at the Central Research Institute of Dentistry in the Department of Therapeutic Dentistry (the active principle of the drug "Dextranase" is dextranase).

Material and research methodology

The study of the action of dextranase in the clinic was carried out in 53 patients aged 25 to 55 years with periodontal diseases, of which 38 patients had gingivitis, 20 patients had periodontitis, 38 patients were diagnosed with mild generalized catarrhal gingivitis, 20 patients had chronic generalized moderate periodontitis.

All patients before receiving the drug were trained in oral hygiene (brushing teeth 2 times a day after meals).

Previously, all groups of patients had dental plaque and tartar removed mechanically.

The scheme for prescribing the drug "Dextranase" was as follows: patients were recommended to suck the tablets of the drug for 18-20 minutes after brushing their teeth with toothpaste twice a day - in the morning and in the evening, 1 tablet; course - 1 month.

Control examinations were performed immediately after the dextranase prophylaxis course (one month after the start of the drug), 1, 3 and 6 months after the end of the dextranase prophylaxis course. The patients received no other treatment.

Criteria for evaluating the action of dextranase: 1. the state of clinical signs; 2. the amount of plaque was determined by the hygienic Green-Vermillion indices; 3. The state of the periodontium was determined by the index PMA and PI.

The control group (20 people) consisted of patients with similar diseases (10 people with gingivitis and 10 people with periodontitis), who, after removing dental deposits, did not receive dextranase, but carried out regular hygienic toothbrushing with paste 2 times a day according to the generally accepted method. Control examinations were carried out immediately after the removal of dental deposits, 1, 3 and 6 months after this procedure.

The bioavailability of dextranase was also studied - exit into saliva.

RESULTS

The results of the treatment are presented in tables 1 and 2. In patients with gingivitis during the initial examination, the Green-Vermillion hygienic index ranged from 2.5 to 3.0 points, in patients with periodontitis, respectively, from 2.8 to 3.0 points.

The condition of the periodontium during the initial examination in patients of the experimental and control groups, according to periodontal indices, was characterized by the following indicators:

in patients with gingivitis - PMA 100%, PI 2.0

in patients with periodontitis - PMA 100%, PI 2.5.

The Schiller-Pisarev test in patients with these diseases was positive.

All patients with periodontal disease noted bleeding gums after brushing their teeth, bad breath, bad taste in the mouth, itching in the gums. Edema, hyperemia of the gingival margin were clinically noted.

Immediately after the removal of dental deposits, the hygienic index in patients of both groups was expressed in the following data:

in patients with gingivitis - Green-Vermillion 0.5-0.6,

in patients with periodontitis - 0.5 and 0.6 points, respectively. Periodontal indices remained at their original numerical values. The results of the action of the drug "Dextranase" in the experimental group of patients with periodontal diseases in dynamics on plaque and periodontal tissues

The state of oral hygiene and the state of periodontal tissues in patients of the control group

As can be seen from tables 1 and 2, the state of periodontal tissues is directly dependent on the hygienic state of the oral cavity - the higher the hygiene, the less pronounced are the signs of inflammation in the periodontium.

Prophylactic administration of the drug "Dextranase" keeps a good condition of the oral cavity up to 3 months after the removal of dental deposits. After a similar therapeutic intervention in the control group of patients, a good hygienic condition of the oral cavity was maintained only for 1 month.

The absence of plaque during this period has a beneficial effect on periodontal tissues. What is revealed and clinically - the gum becomes pale pink, there is no bleeding.

The results of studying the bioavailability of the drug in patients confirmed the data obtained in the experiment - we observed the highest concentration of dextranase in saliva after 18-20 minutes of sucking the tablet.

Clinical trials of the drug "Dextranase" in tablet form were carried out in the prevention department of the polyclinic for children and adolescents of the Central Research Institute of Medical Sciences on 113 adolescents aged 12-15 years.

Method of application. All subjects were divided into 3 groups depending on oral hygiene and the type of drug used: group 1 consisted of 42 adolescents with a high level of oral hygiene who received placebo tablets; group 2 consisted of 43 adolescents with a high level of oral hygiene who received dextranase tablets; Group 3 consisted of 28 adolescents with a low level of hygiene (with dental plaque resistant to conventional brushing), who received dextranase tablets.

Previously, all subjects underwent a special training course on the rules of oral hygiene, as a result of which individuals with increased plaque formation were identified, which were included in group 3.

According to the program, all examined were prescribed 1 tablet of dextranase or placebo for sucking within 10 minutes after brushing their teeth 2 times a day - in the morning and in the evening. Dental examinations were performed before the start of the drug (baseline), after one week and one month of the drug, as well as one week and one month and three months after the end of the course.

Evaluation of the effectiveness of dextranase tablets was carried out according to the OHI-S indices (according to its DI-S component) and PMA. Particular attention was paid to the identification of allergic reactions and discomfort in the oral cavity.

After the completion of the research, the obtained digital data were subjected to statistical processing according to the Student's method.

Results. An analysis of the results of dynamic observation indicates that the state of the periodontium and oral hygiene in adolescents of the first and second groups did not differ significantly from each other and practically did not change throughout the entire observation period (see Table 1). As for the patients of the third group, after one week and one month of using dextranase, they noted an improvement in the hygienic state of the oral cavity and a decrease in inflammation in the gingival margin. A significant decrease in the DI-S and PMA indices was established already after a week of using the drug, and after the completion of the course (1 month), these indicators decreased by almost 2 times, which indicates the high efficiency of dextranase in reducing plaque formation in adolescents with dental plaque resistant to conventional brushing. .

However, a month after the end of the use of the drug, a deterioration in the hygienic state of the oral cavity and an increase in inflammation in the area of the alveolar margin were noted, and after three months the condition of adolescents in this group practically did not differ from the initial level. This indicates the need to repeat the course of dextranase tablets with an interval of no more than 3 months.

As a result of clinical testing of dextranase tablets, a significant inhibitory effect of this drug on the formation of plaque has been established, especially in individuals with poor oral hygiene. In this regard, this drug can be considered as a tool for the prevention of caries and gingivitis, as well as an adjunct in the treatment of inflammatory periodontal diseases, helping to maintain a stable effect due to the reduction of plaque in patients with gingivitis and periodontitis. The intervals between courses of taking dextranase tablets should not exceed 3 months.

Clinical testing of the drug dextranase was carried out in the clinic and hospital of the Department of Hospital Therapeutic Dentistry, MMOSI named after A.I. ON THE. Semashko.

112 patients aged 22 years and older (50 men and 62 women) were examined. Of these, the study of the action of dextranase was carried out in 61 patients with caries and periodontal diseases (chronic catarrhal gingivitis - 36, chronic generalized mild periodontitis - 25, including 18 patients, the drug was used for plaque hydrolysis in the postoperative period). In addition, dextranase was used in 4 patients with lichen planus. The control group consisted of 51 people.

The use of the drug was preceded by teaching patients methods of rational oral hygiene. The administration of dextranase was carried out according to the instructions for the clinical study of the drug. Patients in the control group received a placebo.

Early signs of inflammation in the periodontium were identified according to the analysis and clinical methods of examination, hygiene indices were determined according to Fedorov-Volodkina and Green-Vermillion, as well as PMA and IC.

An analysis of the results of a clinical trial of dextranase, based on clinical examination data and subjective feelings of patients, showed the following:

after application, the impression of cleanliness of the mouth is created,

the surface of the teeth becomes smooth, patients notice whitening of the teeth,

the tolerance of the drug is satisfactory, the tablets are pleasant to the taste,

the tablet form of the drug is resistant to light and humidity, does not change color, smell, taste, consistency.

Under the influence of the drug (table), there was a significant decrease in the indices of oral hygiene and the state of periodontal tissues at the end of treatment.

IG ₁ according to Fedorov-Volodkina - 36.5%

IG ₂ by Green-Vermillion - 47.96%

PMA - 35.2%

IR - 64.7%.

It should be noted that several patients experienced nausea during treatment, which did not require discontinuation of the drug. Sometimes patients noted inconvenience from the need to keep the tablet in the oral cavity for a long time (18-20 minutes) without swallowing saliva.

In periodontal diseases complicated by lichen planus (4 patients), accompanied by a violation of the integrity of the mucosa, after a single application of dextranase, bleeding and a burning sensation were observed, which required discontinuation of the drug. These patients were excluded from the overall evaluation of the effectiveness of dextranase.

CONCLUSIONS

Given all of the above, the drug dextranase can be recommended for widespread medical use in the prevention and treatment of caries and periodontal disease.

With red lichen, accompanied by a violation of the integrity of the mucous membrane, the drug should be used with caution.

It is advisable to create new dosage forms of the drug (toothpaste, chewing gum, etc.).

Employees of the Department of Therapeutic Dentistry of OSMI conducted the first stage of testing dextranase.

Test task:

To reveal the effect of dextranase on dental plaque.

Material and method:

The total number of patients in the experimental group was 34 people. There are 7 people in the control group.

The duration of one course of treatment is 30 days. Within a year it is supposed to repeat a course in 6 months.

Patient selection

The drug was used in OSMI students aged 18 to 27 years to prevent dental caries and gingivitis, as well as as part of complex therapy in the treatment of periodontal diseases.

Criteria for inclusion of patients in trials

The presence of plaque in patients on the smooth surfaces of the crowns of the teeth in the cervical region, which is determined by the hygienic indices of Green-Vermillion and Fedorov-Volodkina.

Predisposition to dental caries (KPU index - at least 3).

The presence of early signs of inflammation in the periodontium (complaints of itching in the gums, bleeding when brushing teeth, etc.).

Criteria for exclusion of patients from trials

The tests did not include patients with advanced stage of periodontitis - with a deep dentogingival pocket (more than 3 mm) and symptoms of suppuration.

Prescribing scheme

The use of the drug is preceded by training patients in methods of rational oral hygiene.

The drug was prescribed 1 tablet 2 times a day (morning and evening) after brushing your teeth for 30 days.

Control examinations were performed immediately after the end of the course of treatment.

The patients were sanitized.

Conventional methods for the treatment of gingivitis have been carried out.

Scheme of examination of the patient

Filling out a medical card of a dental patient (form No. 043 / y).

The amount of plaque was determined by the Green-Vermillion hygienic indices (OH 1-5, taking into account its DI-S component, which characterizes the amount of soft dental deposits) and the Fedorov-Volodkina index.

The condition of the hard tissues of the teeth was determined by the CPH index.

The condition of the periodontium was determined by the indications of the PMA index.

The data obtained allow us to conclude:

Patients taking dextranase note a decrease in bleeding or its complete disappearance, a decrease in the amount of plaque.

Four people complained about the appearance of hyperesthesia of the hard tissues of the teeth after taking the drug.

Statistical processing of the preliminary results of clinical trials made it possible to establish the following:

The PMA index in the control (placebo) and experimental (dextranase) groups did not differ.

M ₁ ±w=13.007±2.026 (control)

M ₂ ±w=13.918±1.480 (experiment) p>0.05

In the course of testing the drug (after 30 days), a slight (statistically unreliable) decrease in the values of the PMA index in the control group to 10.932±2.026 (p>0.05) was established; p<0.001.

Comparing the values of the PMA index in the control and experimental groups after a certain period of time from the start of the drug trials (30 days), it should be noted that although there was a decrease in the PMA values in both groups, however, there were no statistically significant differences during the second examination of the groups.

M ₁ ±w=10.392±2.026 (control)

M ₂ ±b=7.906±1.480 (experiment) p>0.05

When comparing the oral hygiene index (Fedorov-Volodkina index) in the control and experimental (dextranase) groups, the following was noted:

The initial indicators of the oral hygiene index in the control and experimental groups did not differ statistically significantly.

During the trials of the drug (after 30 days), a statistically significant (p<0.001) decrease in the hygiene index in the experimental group (dextranase) was established, while in the control group (placebo) its values did not change significantly.

When comparing the values of the oral hygiene index (Green-Vermillion Hygiene Index).

The initial values of the Green-Vermillion hygiene index in the control and experimental groups did not differ.

In the course of testing the drug (after 30 days), a statistically significant decrease in the values of the studied indicator was established both in the control (p<0.01) and experimental groups (p<0.001).

There were no statistically significant differences in the level of oral hygiene (Green-Vermillion index) between the experimental and control groups after 30 days of clinical trials of the drug (p>0.05).

Conclusion

Clinical trials have revealed the effect of dextranase on plaque. This is confirmed by a decrease in the accumulation of plaque on the teeth (a decrease in the values of the Fedorov-Volodkina, Green-Vermillion hygiene indices) and a decrease in periodontal inflammation (a decrease or complete disappearance of gum bleeding was noted).

The present invention is also directed to an oral care agent.

The oral care agent may be a toothpaste, elixir or chewing gum containing the proposed preparation.

The main components of toothpastes.

Polyhydric hydrocarbon alcohols.

Mannitol or mannitol (Latin mannitum, English mannitol) is a six-atomic hydrocarbon alcohol from the group of sugars. Mannitol is a stereoisomer of sorbitol. The empirical formula of mannitol is C ₆ H ₈ (OH) ₆ . Molar mass 182.172 mol-1.

Mannitol is widely used in the pharmaceutical and food industries. In modern medicine, mannitol is considered mainly as an intravenously administered osmotic diuretic, a means that promotes the excretion of fluid from the body. In pharmaceuticals, mannitol is often used as an auxiliary, inactive substance (Maalox, Panum, Talcid, Gastrozole, Omeprazole-acry, Gastal, Gastracid, Gaviscon, Motilium, Pariet, Omez, Imodium, Rabelok, Zulbeks and others).

Mannitol (mannitol) is a food additive E421 approved for use in Russia, Ukraine and other countries. In the food industry, mannitol is used as a sweetener and anti-caking agent. It is used in the manufacture of toothpastes and chewing gum and other oral care products.

Mannitol and sorbitol are used as stabilizers-fillers of enzyme preparations at the stage of lyophilization [patent No. 2111426 (RF), 1998 "Method of freeze drying of biological products / E.Yu. Shalaev, Franke Felix, N.A. Varaksin, M.Yu. Rukovishnikov]. In our studies, mannitol was chosen as a filler-stabilizer of the dosage form based on microbial hyaluronidase. Preparations with mannitol make it possible to obtain hyaluronidase preparations with a sufficiently high yield of activity at the lyophilization stage [Khabriev R.U., Kamaev N.O., Danilova T.I., Kakhoyan E.G. Development of a technology for obtaining microbial hyaluronidase and a drug based on it. - Issues of biological, medical and pharmaceutical chemistry. - No. 1. - 2016. - S. 4-11].

Mannitol is the main component of the dextranase-based drug formulation developed by us for the prevention and treatment of dental caries. The drug is used in tablet form. Each tablet contains 0.006 g (30 units) of dextranase.

Sorbitol - food additive E421. It is mainly used as a sweetener and complexing agent, texturizer (appointment - improving the structure of the product), as well as a moisture-retaining agent (prevents products from drying out and the appearance of a rough dry crust on the surface of products). In toothpastes, sorbitol is used as a flavoring agent. It is a synthetic sugar substitute that gives toothpaste a pleasant taste without causing cavities. In addition, sorbitol is added to prevent the paste from drying out. Usually toothpastes use sorbitol 70% at a concentration of 20-40 wt. %.

Glycerol (glycerol) - food additive E422 is used in the confectionery industry. Widely used in oral care products. A glycerin-based medication is used as a laxative. Glycerin is not recommended for people with kidney and cardiovascular diseases. In toothpastes, glycerin is used as a thickener. Concentration - 10-50 wt. %

Xylitol (xylitol) food additive E967 is used in the food industry as a sweetener. The empirical formula of xylitol is a - C ₅ H ₇ (OH) ₅ . It is used as a sugar substitute for diabetes. Xylitol inhibits the growth of bacteria in the oral cavity, has a remineralizing effect (important for formulating oral care). This effect on plaque is due to the presence of OH groups, xylitol prevents tooth decay, increases salivation, which helps restore acid-base balance, prevents the development of microorganisms, and does not harm when interacting with enamel. At the same time, to achieve a tangible result from the use of anticaries toothpaste, the content of xylitol in it should be 5-20, for example, about 10 wt. %.

Studies show that all of the above polyhydric alcohols provide high stability of Aspergillus insuetus VKMP F-342 dextranase.

Low molecular weight substances, such as sorbitol, mannitol, xylitol, penetrate into plaque, but due to the low activity of the enzyme that converts them into fructose, lactic acid is formed in small quantities and does not lower the pH.

Abrasives (cleaning component) - chalk, Ca phosphates, for example, dicalcium phosphate dihydrate, silicon dioxide, aluminum hydroxide, aluminum oxide, sodium methyl methacrylate.

The toothpaste abrasiveness index (or RDA) should be in the range of 60-80. Recently, there has been a tendency to reduce the allowable abrasiveness of toothpastes, so below 60 is also good.

Chalk-based pastes have an alkaline pH value (up to 11.0), which is not suitable for dexranase and hyaluronidase.

It is known that an excess of aluminum reduces the retention of calcium in the body, reduces the adsorption of phosphorus and leads to disorders of the nervous and regulatory systems. And therefore, the main danger of aluminum in the composition of toothpaste is that it can accumulate in the body, which is monitored quite difficultly, does not lead to “quick” consequences, but causes significant harm to health.

Silicon dioxide (hydrated silica) is highly compatible with fluorine compounds and other active ingredients, has controlled abrasiveness, which allows creating pastes with a wide range of desired properties. It also provides an optimal pH - 6.8 7.0 (optimal for dextranase), due to which silica-based pastes normalize the acid-base balance. More commonly used hydrated silicon dioxide (SiO ₂ × H ₂ O) in the amount of 15-25% by weight.

The use of silicon dioxide has several advantages:

- these abrasives are practically indifferent to a large number of active components. Modern technologies allow for sufficiently high-quality cleaning from traces of Ca, Mg, Al, Fe and others ions, and therefore, if necessary, compatibility with fluorine-containing substances is achieved to a maximum;

- possibility of regulation of abrasiveness and clearing ability in a wide range;

- destruction of tartar, regulation of acid-base balance;

- the possibility of developing transparent toothpastes, i.e. such pastes in which the refractive index of the abrasive is equal to the refractive index of the liquid phase in which it is suspended. Gel-like pastes have a high foaming ability, have a pleasant taste and attractive appearance. In addition, due to the formation of aqueous internal structures, the gel allows you to include chemically incompatible substances in its composition, since the aqueous shell prevents the chemical reaction between them. However, the cleaning power of such pastes is lower than those based on abrasive systems. With immaturity or increased abrasion of hard tissues of the teeth, the low abrasiveness of gel-like pastes makes them more preferable.

Dicalcium phosphate dihydrate (DCPDG) is the only widely used abrasive that is close in composition to that of tooth enamel. It helps to maintain the constancy of the composition of dental tissues by preventing the dissolution of calcium and phosphorus ions of tooth enamel and by regulating pH. The use of dicalcium phosphate dihydrate is the most attractive in terms of high polishing power and good perfume compatibility. In addition, DCPDG-based pastes improve odor stability. The value of the pH value of toothpaste with DCFDG is in the range of 6.0-8.0. True, in the presence of fluorine ions, there is a high probability of a change in pH in the direction of increasing acidity, up to 5.0. Therefore, this abrasive should be used in a fluoride-free toothpaste. The concentration of this component in the paste ranges from 30 to 40 wt. %.

Dicalcium phosphate dihydrate and silica are polymeric carriers which, due to their properties, provide a reversible bond (immobilization) with proteins, in particular enzymes, contained in the composition according to the invention.

Thus, both silica and dicalcium phosphate dihydrate are most suitable for dextranase.

Detergents (surfactants) or tensides at a concentration of 0.5-2.0% are used to stimulate the processes of foam formation. For these purposes, sodium lauryl sulfate, sodium lauryl sarcosinate are most often used.

Sodium lauryl sulfate provides foaming and destroys bacteria in the oral cavity, prevents the subsequent reproduction of bacteria. High concentrations of this substance are dangerous for the human body: they can cause cataracts, contribute to hair loss, and can accumulate in the human body. In addition, it is the strongest abrasive, the use of which leads to a thinning of the enamel.

Sodium lauryl sulfate is practically not used in the composition of children's toothpastes, since irritation of the mucous membranes occurs.

Sodium lauryl sulfate can be successfully replaced with saponin, xylitol. Castor oil has a good foaming effect.

Saponin is an extract from the bark of a quilaia, an evergreen tree native to Latin America. The average molecular weight is 1800-2000 daltons. Quilaya extract is considered a gentler substitute for surfactants used in oral hygiene products. The quilaia extract is acceptable for many characteristics: it has a pleasant taste in the mouth, and quilaia saponins are generally well compatible with other ingredients used in toothpastes, such as xylitol, glycerin and sorbitol. At the same time, the quality of the foam remains optimal: the bark extract of this tree forms small, neat, uniform bubbles and maintains excellent rheological properties of the product. Quilaia extract is also used as an auxiliary ingredient - as a thickener, stabilizer, as well as an agent that ensures the adhesion of cosmetics to the skin and oral mucosa. At the same time, quilaia bark extract shows the greatest potential in complex mixtures.

Thinners (glycerin, polyethylene glycol) - provide elasticity, viscosity of the paste. In our case, it is most advisable to use glycerin at a concentration of 10-50 wt. %.

Thickeners (aluminum lactate, sorbitol, ethylene glycol).

Aluminum lactate is a salt of aluminum and lactic acid. This substance reduces sensitivity, seals the gums and stops bleeding.

For the above reasons, aluminum compounds are not desirable for use in toothpastes.

In our case, it is most expedient to use sorbitol at a concentration of 20–40 wt. %.

gelling agents. The best known gelling agent is NaKMC, the sodium salt of carboxymethyl cellulose. It is a product of the interaction of cellulose with monochloroacetic acid. White powdery substance, odorless and tasteless. Obtained as a by-product of cotton processing. It has emulsifying, thickening, foaming, stabilizing, softening, gelling, film-forming properties. NaCMC is widely used in toothpastes, creams and cosmetic masks, shampoos, and shaving creams. Non-toxic, highly soluble in water. The viscosity of its solutions is practically independent of pH. It binds water well, solutions are resistant to monovalent salts.

NaKMC and carrageenan are polymeric carriers that, due to their properties, provide a reversible bond (immobilization) with proteins, in particular enzymes, contained in the composition according to the invention.

The concentration of NaKMC in our invention is 0.5-2.0, for example, 1.1 wt. %.

Fluorine compounds. Anti-caries dentifrices are divided into two groups: containing fluoride and not containing fluoride. Of the fluorine compounds in toothpastes, sodium monofluorophosphate, sodium fluoride, tin fluoride, and organic fluorine-containing compounds are used. Fluorine compounds are used in toothpaste because fluoride ions stop glycolysis (fermentation) and strengthen tooth enamel. However, when interacting with water, fluorides, which are part of toothpastes, dissociate, and fluoride ions are related to substances of the first hazard class in terms of acute toxicity, their entry into the respiratory tract or digestive tract can lead to death [Matelo S.K. Clinical and experimental study of new therapeutic and prophylactic toothpastes and gels that do not contain fluorine and have a remineralizing effect: abstract of the thesis of a candidate of medical sciences: - St. Petersburg, 2009. - 23 p.]. Note that the lethal dose of sodium fluoride for a person weighing 70 kg is 5-10 g, and the legal content of this compound in toothpaste is 150 mg/100 g for adults and 50 mg/100 g for children. Consequently, the lethal dose (when ingested by a person weighing 70 kg) of adult toothpaste is about 3 kg, and for children - 10 kg. But, nevertheless, it is recommended to choose a children's toothpaste without fluorine, since children have a strongly developed swallowing reflex, and while brushing their teeth, the child will certainly swallow some of the foam.

In some regions of Russia, there is a high content of fluorine in water (Bashkortostan, Krasnodar Territory, Moscow, Moscow and Ryazan Regions). The concentration of fluorine in drinking water at the level of 1 mg/dm3 is optimal. Pastes with a fluorine content of 1000 ppm i.e. 0.1% is acceptable for daily brushing. This concentration refers to the content of fluorine ions, therefore, the concentration of the substance in which this ion is introduced into the paste should be higher. So, the concentration of sodium monofluorophosphate (Na ₂ PO ₃ F) can reach 0.80%

In the absence of fluorine, calcium compounds are responsible for strengthening enamel in toothpaste: calcium glycerophosphate, calcium lactate and calcium citrate, synthetic hydroxyapatite, and many others. Calcium-containing pastes are excellent for permanent use.

Calcium glycerophosphate - calcium salt of 1,2,3-propanetriol monohydrogen phosphate provides the remineralization process. This substance is available in the form of tablets for oral administration, replenishes calcium deficiency and stimulates anabolic processes. Contraindication - hypercalcemia, and everything connected with it. Unlike fluorine, calcium glycerophosphate is a natural semi-finished product of phosphorus-calcium metabolism, therefore, there are no contraindications in the composition of toothpaste, its concentration ranges from 0.1 to 0.15, on average, about 0.13 wt. %.

Bleaching agents are highly abrasive and contain peroxides [[Matelo S.K. Clinical and experimental study of new therapeutic and prophylactic toothpastes and gels that do not contain fluorine and have a remineralizing effect: abstract of the thesis of a candidate of medical sciences: - St. Petersburg, 2009. - 23 p.]

Highly abrasive components contain sodium bicarbonate, i.e. baking soda, calcium carbonate, calcium lactate, texapone, titanium dioxide, their use leads to the fact that the enamel is erased and the teeth become lighter. In the second case, hydrogen peroxide is used, which, when interacting with enamel, acts as an oxidizing agent. Long-term contact with the oral cavity can cause irritation, and even burns. That is, having achieved the desired result, you are faced with such consequences as increased sensitivity of the enamel, irritation or even loss of taste sensations. It is not recommended to use whitening toothpastes for a long time, because. they break the structure of the enamel. In addition, the original shade of enamel is rarely perfectly white.

Preservatives (antimicrobial components). It is common practice to add preservatives to toothpastes to protect against microorganisms. Usually, methyl, propyl esters of paraoxybenzoic acid, or sodium benzoate are used for these purposes.

Sodium benzoate is used as a preservative used in the preparation of the composition of the present invention, which may be included in an amount of from about 0.1% to about 2% by weight.

Perfumes themselves have some antibacterial activity, as do some currently used active ingredients (for example, triclosan).

Chlorhexidine - 1,6-di-(para-chlorophenylguanido)-hexane. It prevents the formation of plaque on the teeth, is active against gram-positive and gram-negative bacteria (except mycobacterium tuberculosis), protozoa and the herpes virus, and is also used for washing surgical wounds. But it is not meant to be taken orally! And, in addition, the natural microflora is also exposed to the antiseptic action of chlorhexidine, which negatively affects oral health.

As for triclosan, it has similar properties in the composition of toothpaste, but is more chemically active than chlorhexidine. It is an ether, the hydrocarbon radicals in which are dichlorobenzyl and chlorophenyl. Studies have shown the danger of this compound is that it can cause mutations in microorganisms, so it is not recommended to use triclosan containing for oral hygiene.

Chlorophyll is directly involved in the process of photosynthesis in the green part of plants, successfully replacing triclosan. The advantage over triclosan is that chlorophyll is a natural substance, not synthetic, and therefore its action is not aimed at killing bacteria, but at growing natural microflora. Chlorophyll preparations help stop bleeding and activate tissue regeneration processes. The same applies to herbal extracts: organic non-synthetic substances are closer to us, and the safest, but they are less active, and their content in toothpaste is extremely small. Chlorophyll is used at a concentration of 4.2-5.0 wt. %.

Examples of sweeteners useful in making the dentifrice of the present invention may include aspartame, saccharin, sucralose, or a mixture thereof in an amount of from about 0.1% to about 2% by weight.

The closest in technical essence to the proposed one is toothpastes (prototypes) according to the patent RU No. RU 2407512 and patent No. 2535045

The invention encompasses oral care compositions and methods of using the same that are effective for inhibiting or reducing the accumulation of plaque on teeth, reducing acid produced by (cariogenic) bacteria, remineralizing teeth, and inhibiting or attenuating gingivitis. The invention also encompasses compositions and methods for cleaning the oral cavity and relates to improved methods for promoting oral health and/or general health (including cardiovascular health) by reducing the possibility of receiving systemic infection through the tissues of the oral cavity, reduce inflammation in the oral cavity by 80 -85%.

Therefore, the invention includes an oral care composition (composition according to the invention), for example, a dentifrice, containing the enzyme dextranase of microbial origin.

An effective amount of an enzyme, eg, dextranase, eg, present in an amount of at least about 0.03%, eg, from about 0.03 (about 1500 units) to about 0.1% (about 5000 units).

A particulate material, such as silica or dicalcium phosphate, that has a pH in unbuffered solution of less than about 7.5, such as from about 6.5 to about 7.4, such as from about 6.8 to about 7.4 eg approximately neutral pH. This is important for maintaining the activity and stability of the enzyme in the composition.

Polyhydric alcohols, for example, glycerol, sorbitol, xylitol, or a mixture thereof, are used in the present invention as moisturizing agents that maintain the activity and stability of the dextranase enzyme at a concentration of at least about 10 wt. %, for example from about 10 to 50 wt. %.

Detergents (surfactants), for example, sodium lauryl sulfate or saponin at a concentration of at least about 0.5 wt. %, for example from about 0.5 to 2.0 wt. %

The composition according to the invention contains gelling compounds, for example sodium carboxymethyl cellulose.

The sodium salt of carboxymethyl cellulose ensures the immobilization of dextranase and, accordingly, the stability of the enzyme.

The composition of the invention contains sodium carboxymethyl cellulose or carrageenan at a concentration of at least about 0.5 wt. % for example, from about 0.5 to 2.0 wt. %

The composition according to the invention does not necessarily contain a fluorine compound, for example, sodium monofluorophosphate is less than about 0.80, for example, from about 0.40 to about 0.75 wt. %.

The composition according to the invention does not necessarily contain, for example, calcium glycerophosphate, at a concentration of at least about 0.1 wt. % for example, from about 0.1 to 0.15 wt. %

The specified toothpaste according to examples 1,2 is prepared as follows: all components are successively dissolved in demineralized water, except for sodium CMC, dicalcium phosphate dihydrate and silicon dioxide, then sodium CMC and dicalcium phosphate dihydrate or silicon dioxide are added until a gel is formed. The resulting toothpaste is mixed, evacuated and homogenized.

Careful selection of the ingredients of the composition according to the invention and the method of preparation of toothpaste provide high residual activity and stability of dextranase for 2 years.

It is toothpastes containing dextranase that are recommended for oral hygiene in the treatment of periodontal and oral mucosa diseases. Pastes containing dextranase - provide a reduction in inflammation in the oral cavity by 85.8%.

After a 5-minute brushing session, Formula 1-2 toothpastes with positive effects resulted in a reduction in microorganisms from baseline of 45-97% for the gingival sulcus, 78-96% for the vestibular and lingual surfaces of the teeth, and 94-98% for the vestibular and lingual surfaces of the gums. On the buccal mucosa, a decrease in microorganisms by 60-95% was observed.

The positive effect after brushing the teeth lasted for 2 hours.

A change in the pH of the oral fluid to the range of neutral and slightly alkaline values can also be considered as a positive effect of the paste, since this action contributes to the remineralization of tooth enamel. Changing the brushing time from 5 to 3 minutes reduced the effectiveness of the pastes used.

Formula 1-2 toothpastes reduce plaque formation by at least 70%, and are harmless and safe for long-term use.

Clinical trials of the specific action of Aspergillus insuetus VKMP F - 342 dextranase tablets as a means of preventing dental caries. 114 people were examined. Three groups were formed from these people. The first included people who underwent only sanitation of the oral cavity. The second group received fluoride gel on the dentition for ten days. Prevention in the third group was carried out with the help of the drug "Dekstral". Each tablet contained 0.006 g (30 units) of dextranase. The drug "Dextranase" was used once a day in the morning after brushing your teeth for 30 days. The tablet was kept by the patient in the oral cavity for up to 20 minutes, moved with the tongue, and after complete resorption of the tablet, the patient was not recommended to rinse his mouth for up to 1 hour.

The conducted research allowed to establish the following. In the first group, that is, in persons who underwent only oral cavity sanitation, the increase in caries per year according to the KPU index was 1.556 + 0.239, according to the KPU index - 2.204 + 0.301.

In persons of the third group, where the drug "Dextranase" was used as a caries prophylactic, the growth rate of caries for the year was the lowest. The increase in caries according to the KPU index (DKPU) was 0.588+0.125, and according to the KPUP index (DKPUP) -1.353+0.166.

Thus, a comparison of the effectiveness of the studied caries prophylactic agents made it possible to establish the following. The greatest caries prophylactic effect was obtained during the sanitation of the oral cavity and the appointment of the drug "Dekstral", the smallest - in persons who underwent only sanitation of the oral cavity. The use of a fluorine-containing gel as a caries preventive drug had little effect.

As a result, it recommends the use of Dextral tablets containing dextranase at a therapeutic dose of 30 units as a caries prophylactic agent for individuals with medium, low and very low levels of tooth resistance.

Claims (7)

1. The method of obtaining the drug dextranase Aspergillus insuetus VKMP F - 342, including: obtaining a working culture of Aspergillus insuetus VKMP F - 342 on slant agar; preparation of deep sowing culture in flasks; preparation of a deep seed culture in a fermenter; carrying out the process of biosynthesis in fermenters; separation on the flow separator; concentration of the native solution in an ultrafiltration unit; mixing with mannitol, sterilizing filtration, freezing; freeze drying, while mannitol is added at the rate of 1 g per 8000-12000 units. activity. 2. The preparation of dextranase Aspergillus insuetus VKMP F - 342, obtained by the method according to p. activity, for the treatment and prevention of dental caries and gingivitis, as well as for the treatment of periodontal diseases as part of complex therapy. 3. The use of the dextranase preparation according to claim 2 for the treatment and prevention of dental caries and gingivitis, as well as for the treatment of periodontal diseases as part of complex therapy. 4. An oral care product containing the preparation according to claim 2. 5. An oral care product according to claim 4, which is a toothpaste. 6. Oral care agent according to claim 4, which is an elixir. 7. Oral care agent according to claim 4, which is chewing gum.