RU2748594C2 - Composition based on bromelain and bacterial lysate for prevention of oral diseases - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to a composition for oral cavity, which has anti-inflammatory action, reduces manifestation of gingivitis, periodontitis, normalizes oral microbiota composition, reduces the risk of caries and symptoms associated with xerostomia, contains a combination of a proteolytic enzyme bromelain and lysate Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis; or lysate Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans; or lysate Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus bulgaricus and Streptococcus Thermophilus, taken in certain ratio.

EFFECT: said solution consists in providing synergetic interaction and anti-inflammatory action of bromelain and lysate of said bacteria, as well as in establishing a balance of oral microbiota, characteristic for a healthy state.

9 cl, 2 dwg, 5 tbl, 8 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to an innovative immunomodulatory complex for use in the oral cavity, which is a composition for use in the oral cavity containing a combination of the proteolytic enzyme bromelain and a bacterial lysate. The invention can be used in the care of the oral cavity, as well as for the prevention of diseases of the teeth, gums and / or oral cavity.

LEVEL OF TECHNOLOGY

. April 2012. Статья он-лайн. URL: <http://www.who.int/mediacentre/factsheets/fs318/en/>). Заболевания ротовой полости отражаются не только в косметическом эффекте; они в значительной мере влияют на качество жизни, социальные аспекты жизни человека, а также обусловливают развитие системных патологий - атеросклероза, болезни Альцгеймера и др. Наиболее сильная ассоциация в этой группе состояний выявлена между гингивитом и атеросклерозом. Обнаружено, что при гингивите часть дисбиотической флоры попадает в кровь и вызывает бактериемию, а кроме того вследствие незавершенного фагоцитоза бактерии могут мигрировать в атеросклеротический очаг внутри фагоцитов. Выделение провоспалительных цитокинов в зоне пародонтита может спровоцировать системный воспалительный ответ, тем самым создав предпосылки к развитию воспаления сосудов. Также отмечено, что пародонтит сильно меняет состав кишечной микрофлоры, снижая ее противовоспалительные способности и вызывая системное воспаление.Oral health is supported by a number of physiological systems that provide homeostasis; important components of these systems are the immune system of the mucous membrane and the microbiota interacting with it in various compartments of the oral cavity. Under unfavorable conditions (especially diet, somatic diseases, smoking, etc.), the normal physiological state of these systems is disrupted, which provokes the development of various diseases of the oral cavity: gingivitis, periodontitis, stomatitis, xerostomia, caries (Kinane DF, 2017; Acharya A, 2017). These diseases place an enormous burden on health systems around the world and are largely controllable (Petersen PE, 2005). According to the WHO, in the world 60-90% of school-age children and up to 100% of adults have karyotic lesions that are preventable. Severe periodontal lesions occur in 15-20% of people aged 35-44 years (Oral health. Fact sheet

... April 2012. Article online. URL: <http://www.who.int/mediacentre/factsheets/fs318/en/>). Diseases of the oral cavity are reflected not only in the cosmetic effect; they significantly affect the quality of life, social aspects of a person's life, and also determine the development of systemic pathologies - atherosclerosis, Alzheimer's disease, etc. The strongest association in this group of states was found between gingivitis and atherosclerosis. It was found that with gingivitis, part of the dysbiotic flora enters the bloodstream and causes bacteremia, and in addition, due to incomplete phagocytosis, bacteria can migrate to the atherosclerotic focus inside phagocytes. The release of pro-inflammatory cytokines in the periodontitis zone can provoke a systemic inflammatory response, thereby creating the prerequisites for the development of vascular inflammation. It was also noted that periodontitis strongly changes the composition of the intestinal microflora, reducing its anti-inflammatory properties and causing systemic inflammation.

An increase in the risk of autoimmune diseases occurs due to the enzymes of P. gingivalis, which modify arginine in proteins and convert it into citrulline without changing the protein itself. Citrullinated proteins of one's own body are able to induce an immune response: in the case of rheumatoid arthritis, antibodies to citrullinated proteins are found in patients.

The presence of key pathogens - P. gingivalis and Fusobacterium nucleatum - is also associated with the risk of oropharyngeal squamous cell carcinoma, pancreatic cancer, and colon cancer.

Diseases of the oral cavity also adversely affect the fetus during pregnancy. There was a decrease in the weight of the newborn, an increase in the risk of premature birth, miscarriage and stillbirth due to the penetration of microorganisms through the placenta and the occurrence of a pro-inflammatory background in the mother's body.

The most common diseases are tooth decay and periodontitis. Caries is characterized by demineralization of the inorganic part of the enamel and the destruction of its organic matrix, which leads to the destruction of the hard tissues of the tooth and to the occurrence of inflammatory complications from the pulp and periodontium. The intake of simple carbohydrates with the diet leads to a decrease in pH, as a result of which acid-fast species survive - S. mutans, lactobacilli, bifidobacteria. In addition to S. mutans, bacteria of other types also play a role in the development of children's caries - Granulicatella, Actinomyces, Actinobaculum, Scardovia, Atopobium, Aggregatibacter, Slackia, Bifidobacteria, Prevotella (Tanner A.C., 2016; Marsh PD, 2015; Arweiler NB, 2016). The role of lactobacilli remains twofold - on the one hand, in the early stages they provide competition with S. mutans, and on the other hand, they are a predictor of the development of caries and are the dominant species in deep lesions in severe caries in children. In this regard, the use of some types of live lactobacilli in the composition of oral hygiene products is undesirable. With the advent of new research methods, it becomes clear that not one microorganism is important in the development of the disease, but the whole community. In particular, the saliva microbiota in children 6-8 years old with caries is very diverse compared to healthy children (Gomez A, 2017; Huang R, 2011; Mark Welch J.L. 2016).

Gingivitis is an inflammation of the gums without compromising the integrity of the periodontal junction. Most often it occurs due to the accumulation of microbial plaque on the teeth, as a result of non-observance of oral hygiene. Bacteria (less often viruses, fungi) are the direct cause of gum inflammation (Fusobacterium nucleatum, Lachnospiraceae, Lautropia, Prevotella oulorum). Gingivitis is a reversible condition that can be eliminated with an adequate hygiene regimen. If untreated, gingivitis can progress to a destructive form of periodontal disease - periodontitis. Periodontitis is an inflammatory disease of the periodontal tissues, characterized by progressive destruction of the normal structure of the alveolar process of the jaw. In severe periodontitis, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Treponema denticola are most often found. Another reason is traumatic, which, in addition to the anatomical features and the presence of orthodontic structures, includes improper use of a toothbrush. For the prevention of periodontitis, it is necessary to adhere to an adequate hygienic regime. As in the case of caries, periodontitis should be considered not as an infectious process, but as a dysbiotic one. The state of the periodontium is normal and periodontitis largely depends on the functioning of neutrophils. The destruction of bacteria by neutrophils normally occurs without the development of an active phase of inflammation. Smoking, poor diet, physical damage leads to an increase in the colonization of the microbial community by the bacterium P. gingivalis, as a result, neutrophils enter the connective tissue, but do not migrate to the site of infection, which leads to increased inflammation. As a result, additional recruitment of neutrophils into tissues and tissue destruction occurs (Uriarte SM, 2016; Olsen I, 2017)

Thus, modern concepts of the pathogenesis of periodontitis are described by the model of "polymicrobial synergy and dysbiosis": key microorganisms change the immune response in the periodontal area, additional pathogens colonize it, leading to increased inflammation. The healthy microbiota of the peri-gingival region is less diverse than the one affected by periodontitis. The complex ecological relationships of the oral microbiota do not imply simple solutions to the incidence of periodontitis and require an integrated approach (Diaz P.I., 2016; Arweiler NB, 2016; Abiko Y, 2003; Campbell EL, 2011).

Less common diseases are xerostomia and stomatitis. Xerostomia is dry mouth caused by decreased or completely stopped salivation. With xerostomia, the risk of developing infectious diseases of the oral cavity, including fungal diseases, increases. Stomatitis is an ulcerative lesion of the oral mucosa caused by the reaction of the immune system to stimuli. Local factors are also considered the cause of its occurrence: non-observance of oral hygiene. Diseases of the gastrointestinal tract, such as gastritis, duodenitis, colitis, as well as helminthic invasion, can cause catarrhal stomatitis. With this disease, the oral mucosa becomes swollen, painful, hyperemic, it can be covered with a white or yellow coating.

For the prevention of the described pathologies, it is necessary to exclude harmful external factors: normalization of the diet, cessation of smoking, adherence to the principles of a healthy lifestyle in general and the use of hygiene products with a complex effect - toothpastes, rinses, etc. The following requirements are imposed on these products: surface cleaning teeth from plaque, prevention of caries, prevention of gingivitis and periodontitis (anti-inflammatory effect).

Thus, the population has a need for a complex agent that would provide a cleansing, brightening, anti-caries, anti-inflammatory, desensitive, immunomodulatory effect and would help to normalize the balance of the oral microbiota.

The authors of the present invention see the need and the possibility of solving this problem through the creation of an innovative complex "SP.WHITE SYSTEM" (alternative name "BIO SP.WHITE SYSTEM") for the oral cavity, consisting of the following components:

Hydroxyapatite is a mineral that helps to remineralize tooth tissues. In addition, HAP is able to close the dentinal tubules in exposed dentin.

The cleansing enhancer consists of particles that gently cleanse and polish the tooth enamel. In the present invention, the cleaning power enhancer can be in the form of silica (silicon dioxide), calcium carbonate, dicalcium phosphate dihydrate, calcium pyrophosphate, alumina, perlite, sodium bicarbonate, charcoal, hydrogen peroxide, calcium peroxide, sodium citrate, sodium pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate, calcium peroxide.

A film former is a substance that prevents the formation of plaque and color of tooth enamel, as well as removes plaque.

Bromelain is a mixture of proteolytic enzymes, which are sulfhydryl proteases. Bromelain breaks down proteins that make up plaque and lysates.

Lysates of microorganisms associated with a healthy oral health, selected from Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis and Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Lugarillonella parvula and Lactobacterus albactus albactus albactus albactus Thermophilus are fragments of cell walls and organelles of bacteria and fungi obtained by chemical, physical or biological action on these microorganisms. According to the studies carried out by the authors of the present invention, these lysates act on cells of the immune system through TLR receptors, and also serve as a prebiotic substrate for beneficial microorganisms.

The synergy of the components of this complex leads to a powerful enhancement of the cleansing action, and by acting on the oral microbiota and restoring the homeostasis of the immune system, prevention of oral diseases and maintenance of sub-inflammatory, that is, normal neutrophil activation status is ensured. All of the above can significantly improve the properties of oral hygiene products.

Oral compositions such as toothpastes, gels, elixirs, balms, ointments, rinses, foams, chewing gums, sprays are designed to remove plaque and / or prevent plaque formation.

The prior art discloses that dental plaque is a multicomponent, constantly evolving biotope of the oral cavity, which is based on almost all representatives of the microflora of the oral cavity in a conglomerate with proteins and polysaccharides. The antigens of these microorganisms stimulate the activity of the factors of immunobiological resistance of the organism. In the process of plaque formation, a pronounced change in the composition of the microflora occurs. In the first phase, lasting 2-4 hours, early dental plaque is formed with a predominance of aerobic and facultative anaerobic bacteria: streptococci, staphylococci, neisseria and lactobacilli. The total bacterial content is no more than 100-1000 per 1 g. In the second phase (4-5 days), aerobic and facultative anaerobic microflora shift towards anaerobic leptotrichia and fusobacteria. The total content of bacteria increases to 1-10 million / g. In the third phase (6-7 days and beyond), the formation of the qualitative composition of dental plaque ends, the content of aerobes and facultative anaerobes sharply decreases, and obligate anaerobes (bacteroids, fusobacteria, veilonella, actinomycetes, peptostreptococci), emitting a complex of toxic substances that cause the destruction of IgA and IgG, a sharp shift in pH to the acidic side, which leads to the progression of microbial invasion, the development of caries and periodontal disease.

Therapeutic and prophylactic toothpaste used for the prevention of inflammatory periodontal diseases is known from RU 2494724 C2 (publ. 10.10.2013). The proposed toothpaste contains as active ingredients 0.001-5.0 wt. % of lyophilized mass of live bifidobacteria, 0.01-5.0 wt. % lysozyme; 0.01-5.0 wt. % papain; 2.00-20.00 wt. % of the abrasive component; 5.00-20.88 wt. % humidifier; 0.05-5.0 wt. % hydrocolloid; 0.10-2.00 wt. % Surfactant; 0.10-0.32 wt. % sodium fluoride; 0.50-5.00 wt. % tetrapotassium pyrophosphate; 0.15-0.40 wt. % preservative; 0.05-1.50 wt. % flavor; 30.00-87.029 wt. % water.

The disadvantage of this toothpaste is that live bifidobacteria in the toothpaste are not viable, including due to the presence of a preservative in the composition. Therefore, the claimed effect cannot be realized in commercial hygiene products intended for general consumption. Even if they are viable, bifidobacteria are acid-producing bacteria, which entails the risk of demineralization and the appearance of karyotic changes in the teeth. In addition, the papain contained in RU 2494724 C2 destroys immunoglobulins (one of the key factors of anti-infectious defense in the oral cavity) to smaller molecules that do not have useful functionality, which violates the immunity of the oral cavity.

The complex declared by the authors of the present invention differs from RU 2494724 C2 in that it contains not a lyophilized mass of live bifidobacteria, the viability of which in the finished composition is extremely low, but a bacterial lysate, which contains antigens already prepared for recognition by the immune system. Unlike papain, bromelain contained in RU 2494724 C2 does not destroy immunoglobulin molecules to small non-functional fragments (Matharu Z, 2012). Also, the claimed complex contains an enhancer of the cleansing ability, CaMgZn-HAP and PVP, which significantly increases the beneficial effects.

From DE 202009011379 U1 (publ. 12/30/2010) it is known containing a probiotic oral care product, characterized by the inclusion of probiotic cultures from the group: Bacilli, Actinobacteria, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus Lactinus crispatus, Lactobacillus gallactus gallactus Lactobacillus paracasej, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salvarius, Bifidobacterium anolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Leuc. Mesenteroides, Ped. Acidilactici, Sporolactobacillus inulinus, Streptococcus thermophilus, Bacilus cereus, Escherichia coli, Propionibacterium freudenreichii and Saccharomyces cerevisiae.

It is disclosed that odor from the mouth, which is not typical for a healthy state of the oral cavity, is caused, among other things, by volatile sulfur compounds that are mixed with exhaled air. These include hydrogen sulfide, 1,5-diaminopentane, and methanethiol. These substances are formed, for example, by bacterial decomposition of food debris or in dead tissue. Local or systemic factors can be considered as a trigger for bad breath. In 80-90% of cases, the smell is caused by local factors acting either in the oral cavity or in the nasopharynx.

Streptococcus mutans is of particular importance among pathogenic bacteria in the oral cavity.

There are various ways to combat bad breath. For this purpose, various agents are used in dental care preparations. The most common are antibacterial agents (eg, triclosan, chlorhexidine). Dental cleaners, available on the market in various forms, primarily serve to clean the surface of teeth and prevent tooth decay. They usually contain a combination of polishes, surfactants, binders, fragrances and active antimicrobial ingredients. However, the use of such agents may have disadvantages in some cases due to the taste characteristics, aesthetic nature, problems with the stability of therapy, and a violation of the natural flora of the oral cavity. The disclosed oral care product effectively fights against bad breath and does not have such disadvantages.

However, probiotic cultures in the composition of the above agent have a very short pot life, therefore, the claimed effect cannot be realized in commercial samples of hygiene products intended for general consumption, in particular, since special storage conditions of the commercial product are required. Also, the indicated complex does not have a cleansing effect.

The complex claimed by the authors differs from DE202009011379U1 in that it contains not probiotic cultures, the viability of which in the finished composition is extremely low, but a lysate of bifidobacteria, which contains antigens already prepared for recognition by the immune system. In addition, the claimed complex contains bromelain, an enhancer of cleansing ability, CaMgZn-HAP and PVP, which complement the properties of lysates in order to effectively cleanse and remineralize tooth enamel and enhance the activity of lysates.

From RU 2496468 C2 (publ. 10/27/2013) known toothpaste containing bacterial lysate, made from the following components: 0.001-5.0% wt. bacterial lysate Streptococcus pyogenes groupe A, Enterococcus faecium, Enterococcus faecalis, Streptococcus sanguis, Staphylococcus aureus subsp. aureus, Klebsiella pneumoniae subsp. pneumoniae, Corynebacterium pseudodiphtheriticum, Fusobacterium nucleatum subsp.nucleatum, Candida albicans, Lactobacillus acidophilius, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. Lactus, 0.01-5.0% wt. lysozyme, 0.01-5.0% wt. papain, 2.00-20.00% wt. abrasive component, 5.00-20.88% wt. humidifier, 0.05-5.0% wt. hydrocolloid, 0.10-2.00% wt. surfactant, 0.05-0.32% wt. sodium fluoride, 0.50-5.00% wt. tetrapotassium pyrophosphate, 0.15-0.40 wt. preservative, 0.05-1.50% wt. flavor, 30.00-87.029% wt. water. The use of the proposed toothpaste allows you to activate phagocytosis, increase the content of salivary lysozyme, increase the number of immunocompetent cells, has a pronounced cleansing, anti-inflammatory and hemostatic effect.

The negative properties of this toothpaste include the fact that bacterial lysates contained in it are not associated with a healthy state of the oral cavity, and, therefore, can be recognized by the immune system as foreign, which can aggravate the existing inflammatory process or lead to the development of inflammation. This is confirmed by the effect of this composition - it increases the number of immunocompetent cells, which directly indicates an increase in their migration and the implementation of an active inflammatory process: an increase in "cellularity", that is, the number of immunocompetent cells is a reliable sign of the development of inflammation.

The complex claimed by the authors differs from RU 2496468 C2 in that it contains bacterial lysates that purposefully modulate the immune response in order to prevent the development of the inflammatory status characteristic of gingivitis and periodontitis, and not to activate phagocytosis. Also, the claimed complex does not increase the number of immunocompetent cells (which would indicate an increase in inflammation), but contributes to the formation of an adequate balance of subpopulations of these cells. In addition, the claimed complex contains bromelain, a cleansing enhancer, CaMgZn-HAP and PVP, which have a synergistic effect.

A composition is known from JP 2004250374 A (publ. 10/27/2013) which is effective in removing plaque and safe for the human body. Such a composition contains lactic acid bacteria or a fermentation solution of lactic acid and gluconate and / or oligosaccharide. Can be used in any form of lactic acid bacteria, for example, powdered lyophilized products. Examples of lactic acid bacteria that can be used are Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus brandarum, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus brevisco, Bactobacillus Streptococillus, Streptocillus coelus, Streptococillus longcocillus, lactobacillus longcocillus, etc., Lactobacillus. The lactic acid fermentation solution is preferably a fermentation solution obtained by fermentation of lactic acid using lactic acid bacteria in a medium containing lactose or glucose as the main component. The lactic acid bacteria used for the fermentation of lactic acid can be any of the lactic acid bacteria commonly used in the production of conventional lactic acid drinks, fermented milk, and the like, and examples thereof include Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus Brantobacaram, salactobacillus Brantobaram, salactobacillus , Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus buffner, Lactobacillus cellobios, Streptococcus thermophilus and the like.

The disadvantage of this composition is that live bacteria in the toothpaste composition are not viable and, therefore, the claimed effect cannot be realized in commercial samples of hygiene products intended for general consumption. In addition, even when vitality is achieved, these lactic acid bacteria will create an acidic environment near the surface of the tooth, which will lead to demineralization and provoke the development of karyotic changes.

The claimed complex differs from JP 2004250374 A in that it contains non-probiotic cultures of acid-producing bacteria, the viability of which, moreover, in the finished composition is extremely low, but the lysate of bifidobacteria, which contains antigens already prepared for recognition by the immune system. It should be noted that bromelain, which enhances the activity of lysates, as well as remineralizing enamel CaMgZn-HAP and PVP with an enhancer of cleansing ability, improve the beneficial properties of this complex.

From EA 001741 B1 (publ. 27.08.2001) known toothpaste or mouthwash containing sphingomyelinase to increase the levels of ceramides in the skin and mucous membranes, using sphingomyelinase of gram-positive bacteria, gram-negative bacteria, lactobacilli or mixtures thereof. Lactobacilli are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus catenaforme, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus jensenii, Lactobacillus leichmanii, Lactobacillus minutus, Lactobacillus plantarum, Lactobacillus rogosae , Lactobacillus salivarius, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium eriksonii, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium plantarum, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, Streptococcus lactis, Streptococcus raffinolactus and Streptococcus thermophilus.

However, this toothpaste or liquid is not without its drawbacks. So, sphingomyelinase does not have positive effects on the mucous membrane, which are reflected in improving the condition of the oral cavity, reducing the risk of caries, periodontitis and other diseases. Increasing the amount of ceramides can be beneficial in the skin, where they are involved in maintaining an adequate level of hydration of the epidermis, but not in the mucous membrane.

The claimed complex differs from JP 2004250374 A in that it contains not sphingomyelinase, which does not have positive effects on the immune system of the oral cavity, but a lysate of bifidobacteria containing antigens already prepared for recognition by the immune system. In addition, the claimed complex contains bromelain, which enhances the activity of lysates, as well as synergistic PVP, an enhancer of cleansing ability, and CaMgZn-HAP.

In addition to the toothpaste compositions disclosed in the aforementioned patent documents, some lactobacilli-containing pastes are on the market.

So, the known toothpaste N-Zim Prebio manufactured by ArtLife ("Toothpaste N-Zim Prebio". Online article. URL: <http://artlife-msk.ru/zubnaja-pasta-n-zim-prebio-100 -gr>) containing sorbitol 70%, silicon dioxide Tixosil 73, silicon dioxide Tixosil 43, dicalcium phosphate dihydrate, polyethylene glycol PEG-400, sodium lauryl sarcosinate Amin LS 30, sodium carboxymethyl cellulose, CEKOL 2000, puffer Tusmint VVM 230, D- sodium lauryl sulfate, lysine, titanium dioxide, papain, sodium fluoride, Rona Care olaflur aminofluoride, allantoin, sodium saccharinate, lactobacillus lysate, sodium methylparaben, sodium propylparaben, stevioside, aloe gorophyte, demineralized water.

However, this toothpaste contains papain, which breaks down immunoglobulins (one of the key factors of anti-infectious defense in the oral cavity) to smaller molecules that do not have useful functionality, which disrupts the immunity of the oral cavity. Bacterial lysates are not associated with oral health.

The claimed complex differs from this toothpaste in that it contains bacterial lysates that purposefully modulate the immune response in order to prevent the development of the inflammatory status characteristic of gingivitis and periodontitis. In addition, the enhancement of the effects of the claimed complex is achieved by the presence in its composition of a cleansing ability enhancer, CaMgZn-HAP and PVP, as well as bromelain, which does not destroy immunoglobulins to small fragments.

Known toothpaste PerioBiotic Toothpaste manufactured by Designs For Health Inc. (“Designs For Health PerioBiotic Toothpaste - Spearmint Flavor.” Online article <URL: http: // https: //www.needs.com/product/Designs_For_Health_PerioBiotic_Toothpaste_Spearmint_Flavor_118/pc> calcium carbonate) , Glycerin, Xylitol, Sodium Bicarbonate, Peppermint Oil (Foeniculum Vulgare), Carrageenan, Maltodextrin, Dental-Lac ™ (Lactobacillus paracasei), Potassium Sorbate, Yucca Powder (Y. filamentosa), Stevia Leaf Extract (Eupatorium rebaudianum bertoni) ... Dental-Lac has been disclosed to compete with unwanted bacterial strains in the mouth, including S. mutans, thereby helping to maintain healthy teeth and gums.

The disadvantage of this toothpaste is that the living bacteria Dental-Lac ™ (Lactobacillus paracasei) in the toothpaste cannot be viable and, therefore, the claimed effect cannot be realized in commercial hygiene products intended for general consumption.

The claimed complex differs in that it contains not probiotic cultures, but a lysate of bifidobacteria, which contains antigens already prepared for recognition by the immune system. The activity of lysates is intensified by bromelain, a cleansing enhancer, CaMgZn-hydroxyapatite and PVP.

Thus, in the prior art, there are no solutions for information on a combined agent based on the proteolytic enzyme bromelain, bacterial lysate, an enhancer of the cleansing ability, PVP and CaMgZn-HAP, allowing to achieve a significant synergistic effect, consisting in enhancing cleansing, anti-inflammatory, anti-caries, immunomodulating effect and correction oral microbiota.

The components that make up the claimed combined agent exhibit a synergistic effect, which is confirmed further.

SUMMARY OF THE INVENTION

The present invention generally relates to an oral composition comprising a combination of the proteolytic enzyme bromelain and a bacterial lysate.

In one embodiment, the invention is an oral composition comprising a combination of the proteolytic enzyme bromelain and a bacterial lysate.

In another embodiment, the bacterial lysate may be a bifidobacterial lysate.

In a further embodiment, the bifidobacterium lysate may be a Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium adolescentis lysate.

In another embodiment, the bacterial lysate may be a Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, and Candida albicans lysate.

In another embodiment, the bacterial lysate may be a Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus bulgaricus and Str. Thermophilus.

In another embodiment, the composition according to the invention may contain 0.005-0.2% wt. the proteolytic enzyme bromelain.

In another embodiment, the composition according to the invention may contain 0.05-1.5% wt. lysate of bacteria.

In another embodiment, the composition of the invention may further comprise a cleaning enhancer which may be selected from the group of silica (silicon dioxide), calcium carbonate, dicalcium phosphate dihydrate, calcium pyrophosphate, alumina, perlite, sodium bicarbonate, charcoal, hydrogen peroxide, calcium peroxide, sodium citrate, sodium pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate, calcium peroxide and mixtures thereof.

In another embodiment, the composition according to the invention may further comprise hydroxyapatite, which may be selected from the group: hydroxyapatite enriched in calcium, magnesium and zinc.

In another embodiment, the composition according to the invention may further comprise a film-forming component (film-former), which can be selected from the group: polyvinylpyrrolidone, polyvinyl alcohol, wax-like substances, and mixtures thereof.

In another embodiment, the composition according to the invention may additionally contain a film former, a cleaning enhancer, hydroxyapatite and auxiliary substances, wt%:

proteolytic enzyme bromelain 0.005-0.2, bacteria lysate 0.05-1.5, film former 0.3-1.0, cleaning power enhancer 2.5-5.0, hydroxyapatite 0.1-10.0, Excipients up to 100.

In another embodiment, the composition is used in the oral cavity for the treatment of diseases of the teeth, gums and / or oral cavity, which are selected from gingivitis, glossitis, caries and xerostomia, post-traumatic inflammation caused by orthodontic appliances

In another embodiment, the composition may be in the form of a toothpaste, gel, elixir, balm, spray, foam, chewing gum, or mouthwash.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1.The data on the diversity of the gingival fluid microbiota are presented.

FIG. 2. presents data on various types of microorganisms at the end of the study in the experimental groups.

DETAILED DESCRIPTION OF THE INVENTION

There is a need in the art to provide alternatives to commercially available oral care products for use in the oral cavity, such as toothpastes and other forms. Expanding the arsenal of such means at the expense of the claimed means will allow to overcome the existing difficulties and disadvantages known to specialists in this field of technology and to reach a new level of cleaning teeth, prevention and treatment of inflammatory conditions.

The authors unexpectedly found that the substances that make up the lysates of bacteria Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis and Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Lactobacterium. Thermophilus has anti-inflammatory properties. This effect is achieved through the induction of an anti-inflammatory immune response (Bifidobacterium longum, Veillonella parvula, Lactobacillus bulgaricus, Str. Thermophilus), the formation of an immune response to cariogenic and other pathogenic types of microorganisms (Bifidobacterium bifidum, Lactobacillus acidobacillus casei, and the action of bacteriocins contained in lysates of probiotic bacteria (Bifidobacterium adolescentis, Streptococcus sanguinis, Lactobacillus fermentum).

The effect of bifidobacteria on mucosal immunity was found. However, some species of bifidobacteria are the so-called allochthonous, or "transit" microflora, i.e. do not colonize mucous membranes, while at the same time managing to exert a regulatory effect on the immune system during the passage. In the oral cavity, the presence of live bifidobacteria is also undesirable, since this genus belongs to the acid-resistant species associated with caries. Since the positive role of bifidobacteria in any case is mediated by the components of their cell wall (membrane) and intracellular structures, it is rational to use not living bacteria, but their lysates. All of the above also necessitates the constant or long-term use of products containing bacterial lysates.

The mechanism of the anti-inflammatory action of lysates on mucous membranes is associated with the expansion of the population of regulatory T-lymphocytes (Treg), in particular, this was demonstrated for B. longum on intestinal cells. The change in the composition of lymphocyte subpopulations was accompanied by suppression of the synthesis of proinflammatory cytokines TNFα and MCP-1, IL-12 and IFNγ, and an increase in the “anti-inflammatory” cytokine IL-10. In cell cultures of B. longum subsp. infantis JCM 1222T also suppressed IL-17A production.

The anti-inflammatory effect of lysates is initially realized upon recognition of their membrane structures by TLR2 and TLR9 receptors of immune cells and Nod receptors. One of the consequences of this is the maintenance of tight contacts between cells (probably due to inhibition of TNFα synthesis) and counteraction to apoptosis of mucosal cells due to an increase in the synthesis of cyclooxygenase COX-2 and prostaglandin PGE (2).

In addition to the components of the cell wall, anti-inflammatory action has also been demonstrated for the secreted components of Bifidobacterium longum, Veillonella parvula, Lactobacillus bulgaricus, Str. Thermophilus, in part by regulating the composition of the microbiota and reducing lipid peroxidation.

There are very few studies in the art on the effect of lysates on the oral mucosa. It has been established that B. bifidum can suppress inflammation caused by mechanical trauma to the oral mucosa, as well as reduce the risk of C. albicans colonization when using dentures.

Thus, the use of bacterial lysates for the prevention and correction of conditions associated with inflammatory processes in the oral mucosa seems reasonable.

The authors unexpectedly found that the substances included in the lysates (mixture) of bacteria Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans, Lactobacillus bulgaricus, Str. Thermophilus also has anti-inflammatory properties. This effect is also achieved by inducing an anti-inflammatory immune response.

Thus, the use of lysates from bacteria Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans, Lactobacillus bulgaricus, Str. Thermophilus for the prevention and correction of conditions associated with inflammatory processes in the oral mucosa also seems appropriate.

The authors also unexpectedly found that combinations of lysates from bacteria Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis and Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicus. Thermophilus and the proteolytic enzyme bromelain have a synergistic effect, which is further confirmed in non-limiting examples.

The authors also unexpectedly found that combinations of lysates of bacteria Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis and Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans, the enzyme bromelain, and the film former have a synergistic effect, which is further confirmed in non-limiting examples.

We also unexpectedly found that the combination of bromelain and lysate of these bacteria potentiates their combined action, leading to synergistic interactions and anti-inflammatory effects, and also helps to establish a balance of the oral microbiota characteristic of a healthy state, which is further confirmed in non-limiting examples.

Some embodiments of the claimed invention are disclosed below.

In one embodiment, the claimed invention is an oral composition comprising a combination of the proteolytic enzyme bromelain and a bacterial lysate.

In another embodiment, the bacterial lysate may be a bifidobacterial lysate.

In a further embodiment, the bifidobacterium lysate may be a Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium adolescentis lysate.

In another embodiment, the bacterial lysate may be a Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, and Candida albicans lysate.

In another embodiment, the bacterial lysate may be a Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus bulgaricus, Str. Thermophilus.

In another embodiment, the composition according to the invention may contain 0.005-0.2% wt. proteolytic enzyme bromelain, preferably 0.05-0.20 wt.%, more preferably 0.1 wt.

In another embodiment, the composition according to the invention may contain 0.05-1.5% wt. lysate of bacteria, preferably 0.5-1.0% wt., more preferably 0.7% wt.

In another embodiment, the composition of the invention may further comprise a cleaning enhancer comprising silica (silicon dioxide), calcium carbonate, dicalcium phosphate dihydrate, calcium pyrophosphate, alumina, perlite, sodium bicarbonate, charcoal, hydrogen peroxide, calcium peroxide, sodium citrate , sodium pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate, calcium peroxide.

These ingredients are traditionally used in toothpastes. The composition according to the invention may contain a cleaning enhancer in an amount of 2.5-5.0% by weight, preferably 3.5-4.0% by weight, more preferably 3.7% by weight.

In another embodiment, the composition of the invention may further comprise hydroxyapatite. This ingredient is traditionally used in toothpastes. The composition according to the invention may contain hydroxyapatite enriched in calcium, magnesium and zinc CaMgZn in an amount of 0.1-10.0 wt.%, Preferably 1.0-8.0 wt.%, More preferably 3.0-6.0 wt.% ., most preferably 5.0% May

In another embodiment, the composition of the invention may further comprise a film former, preferably polyvinylpyrrolidone. This ingredient is traditionally used in toothpastes. The composition according to the invention may contain a film former in an amount of 0.3-1.0% by weight, preferably 0.5-0.8% by weight, more preferably 0.7% by weight.

In another embodiment, a composition of the invention may comprise:

proteolytic enzyme bromelain 0.005-0.2, bacteria lysate 0.05-1.5, film former 0.3-1.0, cleaning power enhancer 2.5-5.0, hydroxyapatite 0.1-10.0, Excipients up to 100.

These auxiliary substances are conventionally used in this field, for example, neutral carriers, target additives, pharmaceutically acceptable auxiliary substances, substances for creating a base or base of the composition or formulation.

In another embodiment, the composition is used in the oral cavity to treat diseases of the teeth, gums and / or oral cavity, which can be selected from gingivitis, caries and xerostomia.

In another embodiment, the composition of the invention can be incorporated into a toothpaste. Such a toothpaste can be used for the prevention and treatment of inflammatory conditions in the oral cavity, such as, but not limited to, gingivitis, glossitis, caries, xerostomia, post-traumatic inflammation caused by orthodontic appliances.

In another embodiment, the invention may relate to the use of the composition in a method for preventing and / or treating diseases of the teeth, gums and / or oral cavity.

In another embodiment, the invention can be a method for preventing and / or treating diseases of the teeth, gums and / or oral cavity, comprising the use of such a toothpaste.

The use of lysate of Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis or Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans allows you to normalize the inflammatory status of the oral cavity tissues and restore the characteristic ratio of microorganisms to the oral cavity. Purposeful introduction of bacterial lysate of Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis or Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans into the complex allows for effective prevention of mucosal gingivitis, bacteriosis, and infections of gingivitis, bacteriosis and infections mouth, tongue, upper respiratory tract, halitosis, glossitis, post-traumatic inflammation caused by orthodontic appliances and other diseases involving the immune system and the microbiota of the oral cavity and upper respiratory tract.

EXAMPLES

The examples included in the present description are not limiting of the claimed invention and are provided only for the purpose of illustration and confirmation of the achievement of the expected technical results. These examples are one of many experimental data obtained by the inventors, which confirm the effectiveness of tools within the scope of the invention.

Clinical studies have been conducted to assess the effectiveness.

Example 1.

In the experimental groups, the patients used the following compositions, which included the claimed complex containing bromelain and bacterial lysates in a percentage of 0.005 and 0.05 wt% as active components (Table No. 1), as well as a film former (polyvinylpyrrolidone) - 0.3 wt% ., an enhancer of the cleaning ability (silicon dioxide) - 2.5% wt., hydroxyapatite (hydroxyapatite, enriched with calcium, magnesium and zinc) in the form of a 10% suspension - 0.1% wt.

Toothpaste with the composition shown in Table 2 was used as a base for the inclusion of components.

A prospective, randomized, double-blind study on 200 subjects (107 men and 93 women), randomized into four equal age and gender groups. The patients applied the toothpaste compositions 2 times a day for 6 weeks.

The parameter for assessing the effectiveness of the drugs was the average gum bleeding index (GBI), determined by the dentist. Changes in local immunity were also determined by assessing the amount of hBD-1 and hBD-2 defensins in the gingival fluid collected for 30 seconds.

For all quantitative data, the group arithmetic mean (M), median (Me), standard deviation (SD) and standard error of the mean (SEM), interquartile ranges, 90% CI were calculated. The results were processed using the IBM SPSS Statistics 22.0 software (StatSoft, Russia). The likelihood of differences in mean indices in the groups was determined using the Mann-Whitney test. Differences were considered significant at a significance level of p <0.05.

Results.

6 weeks after the start of the study, pronounced changes in the estimated parameters were observed in the study groups. In group 1, which used the composition with a complex containing bifidobacteria lysate and bromelain, the GBI value at the end of the study was 0.04 ± 0.02, which is less than the initial values (0.16 ± 0.03; P = 0.014 ). In group 2 and 3, which used the composition with lysate of bifidobacteria and lactobacilli, respectively, a less pronounced change in GBI was noted; at the end of the study, its value was 0.10 ± 0.03 and 0.09 ± 0.04 compared to the initial values (0 , 15 ± 0.03; P = 0.023), while in group 4, which used the composition with bromelain, such changes were not observed - GBI at the beginning of the study was 0.16 ± 0.02, at the end of the study - 0.12 ± 0.04; P = 0.43.

Thus, the use of a paste with a complex containing bromelain and lysate of bifidobacteria or bromelain and lysate of Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus bulgaricus and Str. Thermophilus led to a decrease in the severity of gingivitis, and this effect was more pronounced than when using the lysate of bifidobacteria and bromelain separately, which indicates the synergism of the action of both components.

The use of the paste with the claimed complex also led to a decrease in the concentration of defensins in the gingival fluid, which indicates the normalization of the local immune response.

Since bromelain is a protease, the effect obtained can be explained by the fact that under its action new epitopes of protein antigens that are part of the lysate of bifidobacteria, lysate of Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus bulgaricus and Str. Thermophilus or Lactobacillus casei lysate, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans, which additionally stimulates immune cells that carry out regulatory responses to the inflammatory process.

Example 2.

In the experimental groups, the patients used the following compositions, which included bromelain and lysate of Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans as active ingredients in a percentage of 0.005 and 0.05% (Table 5).

As a basis for the inclusion of components used toothpaste with the composition shown in Table No. 2 (Example 1).

A prospective, randomized, double-blind study on 150 subjects (74 men and 76 women), randomized into three equal age and gender groups. The patients applied the toothpaste compositions 2 times a day for 6 weeks.

The parameter for assessing the effectiveness of the drugs was the average gum bleeding index (GBI), determined by the dentist. In addition, the composition of the microbiota in the gingival pocket was determined.

For all quantitative data, the group arithmetic mean (M), median (Me), standard deviation (SD) and standard error of the mean (SEM), interquartile ranges, 90% CI were calculated. The results were processed using the IBM SPSS Statistics 22.0 software (StatSoft, Russia). The likelihood of differences in mean indices in the groups was determined using the Mann-Whitney test. Differences were considered significant at a significance level of p <0.05.

Results.

6 weeks after the start of the study, pronounced changes in the estimated parameters were observed in the study groups. In group 1, which used the composition with the lysate complex of Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans and bromelain, the GBI value at the end of the study was 0.03 ± 0.02, which is lower than the initial values ( 0.17 ± 0.03; P = 0.01). In group 2, which used the composition with the lysate of Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans, a less pronounced change in GBI was noted, at the end of the study its value was 0.09 ± 0.03 compared with the initial values (0 , 17 ± 0.03; P = 0.03), while in group 3, which used the composition with bromelain, such changes were not observed - GBI at the beginning of the study was 0.16 ± 0.04, at the end of the study - 0 .13 ± 0.02; P = 0.18.

Thus, the use of a paste with a complex of bromelain and lysate of Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans led to a decrease in the severity of gingivitis, and this effect was more pronounced than when using lysate of bifidobacteria and bromelain separately, which speaks for synergistic action of both components.

The analysis of the composition of the microbiota revealed the following features.

After the application of a toothpaste with a complex of bromelain and lysate of Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans, a decrease in the diversity of the microbiota in the gingival fluid was noted. This indicates an improvement in the restraining factors of immunity and the establishment of a microenvironment unfavorable for colonization by pathogenic microorganisms. In particular, the representation of the genera Anaeroglobus, Bulleidia, Desulfobulbus, Filifactor, Mogibacterium, Phocaeicola, Schwartzia or TM7, associated with the development of gingivitis and periodontitis, decreased (Fig. 1).

In comparison with groups 2 and 3, in group 1, which used the composition with the bromelain complex and lysate of Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans, at the end of the study, an increase in the representation of the genera Capnocytophaga, Corynebacterium, Gemella, Lemella, , Streptococcus and Veillonella associated with a healthy oral cavity, in particular, the species Capnocytophaga ochracea, Eubacterium saburreum, Fusobacterium nucleatum, Haemophilus egyptius, Haemophilus infuenzae, Kingella oralis, Mogibacterium salivarium, Myciscopterium cannabis, Mogibacterium vescivarium, Mycopterium acnes, Propionivibrio pelophilus, Rothia dentocariosa, Streptococcus sanguinis, Treponema socranskii, which are known to be 20% higher in healthy individuals than in those with periodontitis (Fig. 2).

Thus, the use of a complex of bromelain and lysate of Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, and Candida albicans makes it possible to normalize the composition of the oral microbiota with a shift in the species abundance in the direction characteristic of a healthy oral cavity. This is of particular importance due to the fact that under the action of the complex, the representation of species decreases, the presence of which in the inflammatory process in the oral cavity (gingivitis, periodontitis) is associated with the development of pathology of the cardiovascular system (atherosclerosis, coronary heart disease, myocardial infarction, acute coronary syndrome) , which suggests a potential reduction in the risk of developing these diseases when using the complex.

Example 3.

A prospective, randomized, double-blind study on 100 children 6-9 years old (57 boys and 43 girls), randomized into two equal age and gender groups. For 52 weeks, Group A brushed its teeth twice a day with a toothpaste containing bromelain and Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis lysate according to the invention. Group B brushed its teeth twice a day with a toothpaste containing bromelain and Lactobacillus casei lysate, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans according to the invention. Group B brushed its teeth twice a day with a toothpaste containing bromelain and Lactobacillus casei lysate, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans according to the invention. Group D brushed their teeth twice a day with a toothpaste similar in composition to that used by group A, B, C, but not containing lysate and bromelain. The risk of an increase in the number of carious changes in the groups was assessed before and after the use of toothpastes. In group A, there was a decrease in the relative risk of an increase in carious lesions to a value of 0.87 compared with group D. In group B, a decrease in the relative risk of an increase in carious lesions to a value of 0.79 was noted compared to group D. In group C, a decrease was noted the relative risk of an increase in carious lesions to 0.83 compared with group G. Thus, the use of a paste with lysate and bromelain led to a decrease in the risk of caries development.

Example 4.

A prospective, randomized, double-blind study on 75 older subjects with xerostomia. For 4 weeks, group A (25 people) brushed their teeth twice a day with a toothpaste containing bromelain and lysate of Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis according to the invention. Group B (25 people) brushed their teeth twice a day with a toothpaste containing bromelain and Lactobacillus casei lysate, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans according to the invention. Group B (25 people) brushed their teeth twice a day with a toothpaste containing bromelain and Lactobacillus casei lysate, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans according to the invention. Group D (25 people) brushed their teeth twice a day with a toothpaste similar in composition to that used by group A, B, C, but not containing lysate and bromelain. The results were recorded according to a standard questionnaire for persons suffering from xerostomia. At the end of the study period, improved oral health was noted by 13 people from group A, 14 people from group B, and 13 people from group C, compared with 6 people from group D. Thus, the use of a paste with lysates contributed to a decrease in symptoms associated with xerostomia.

Example 5.

A prospective, randomized, double-blind study on 60 subjects (33 men and 27 women), randomized into three equal age and gender groups. For six weeks, Group A brushed its teeth twice a day with a toothpaste containing bromelain and Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis lysate according to the invention. Group B brushed its teeth twice a day with a toothpaste containing bromelain and Lactobacillus casei lysate, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans according to the invention. Group B brushed their teeth twice a day with a toothpaste containing bromelain. Group D brushed their teeth twice a day with a toothpaste containing Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis lysate. Group D brushed its teeth twice a day with a toothpaste containing bromelain and Lactobacillus casei lysate, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula, Candida albicans according to the invention. The mean gum bleeding index (GBI) was assessed in the groups before and after the use of toothpastes. The use of all pastes led to a decrease in the severity of gingivitis, but the effectiveness in groups A, B and D was significantly higher than in groups C and D.

Example 6.

A prospective, randomized, double-blind study on 100 subjects (52 men and 48 women), randomized into three equal age and gender groups. For six weeks, Group A brushed its teeth twice a day with a toothpaste containing bromelain, Bifidobacterium longum lysate, Bifidobacterium bifidum, Bifidobacterium adolescentis and polyvinylpyrrolidone according to the invention. Group B brushed their teeth twice a day with a toothpaste containing bromelain and polyvinylpyrrolidone. Group B brushed their teeth twice a day with a toothpaste containing Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis and polyvinylpyrrolidone lysate. Group D brushed their teeth twice a day with a toothpaste containing bromelain and Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis lysate according to the invention. The mean gum bleeding index (GBI) was assessed in the groups before and after the use of toothpastes. The use of all pastes led to a decrease in the severity of gingivitis, however, the effectiveness in group A was higher than in group D, and generally higher than in groups B and C.

Example 7.

A prospective, randomized, double-blind study on 60 subjects (30 men and 30 women), randomized into three equal age and gender groups. For six weeks, group A daily after each meal and brushing the teeth used a rinse containing a complex containing a lysate of Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis. Group B used a mouthwash containing bromelain and polyvinylpyrrolidone. Group B used a mouthwash containing Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis lysate and polyvinylpyrrolidone. The mean gum bleeding index (GBI) was assessed in the groups before and after using the mouthwash. The use of all mouth rinses resulted in a decrease in the severity of gingivitis, but the effectiveness in group A was higher than in groups B and C.

Example 8

The method for preparing the composition according to the invention includes the following steps.

1) Getting bromelain:

To two samples of 250 g of crushed mass of pineapple tops add 500 ml of water or 0.05 M ammonium bicarbonate solution and carry out further grinding and extraction in a homogenizer. The resulting mass is squeezed through several layers of gauze, centrifuged at 5500 rpm and the supernatant is lyophilized. 4 g of bromelain preparation with activity of 1.1 and 1.2 units / mg of protein are obtained using water and 0.05 M ammonium bicarbonate solution as extractant, respectively.

2) Obtaining lysates:

КОЕ/мл. Культуры отмывают трехкратно центрифугированием при 3000 об/мин в течение 10 минут и концентрируют в 10 раз. Проводят термоинактивацию микроорганизмов проводят при 80°С в течение 30 минут. Для дезинтеграции и получения компонентов клеточной стенки и органелл микроорганизмы разрушают с помощью ультразвука (четырехкратное озвучивание, время озвучивания - 5 мин, время паузы между импульсами - 60 с, амплитуда - 9 мкм). После разрушения лизат осветляют центрифугированием в течение 20 минут при 3000 об/мин на холоде, компоненты бактериальных клеток осаждают центрифугированием при 20000 g в течение 1 часа при охлаждении. Осадок промывают в том же режиме и ресуспендируют в стерильном физиологическом растворе.The bacteria Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and the fungus Candida albicans are cultured under standard conditions until a concentration is obtained.

CFU / ml. The cultures are washed three times by centrifugation at 3000 rpm for 10 minutes and concentrated 10 times. Thermal inactivation of microorganisms is carried out at 80 ° C for 30 minutes. To disintegrate and obtain components of the cell wall and organelles, microorganisms are destroyed using ultrasound (fourfold sonication, sonication time - 5 min, pause time between pulses - 60 s, amplitude - 9 μm). After destruction, the lysate is clarified by centrifugation for 20 minutes at 3000 rpm in the cold, the components of bacterial cells are precipitated by centrifugation at 20,000 g for 1 hour with cooling. The precipitate is washed in the same mode and resuspended in sterile saline.

The resulting bromelain and lysates are introduced into the composition in accordance with the production flow chart.

Thus, the application materials confirmed the achievement of all expected results, demonstrated the high efficiency of the claimed agent and its industrial applicability.

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Claims (10)

1. Composition for use in the oral cavity, which has an anti-inflammatory effect, reduces the severity of gingivitis, periodontitis, normalizes the composition of the oral microbiota, reduces the risk of caries and symptoms associated with xerostomia, containing a combination of 0.005-0.2% wt. proteolytic enzyme bromelain and 0.05-1.5% wt. lysate of Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis; or a lysate of Lactobacillus casei, Streptococcus mutans, Streptococcus sanguinis, Veillonella parvula and Candida albicans; or lysate of Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus bulgaricus and Streptococcus Thermophilus. 2. A composition according to claim 1, further comprising a cleansing enhancer. 3. Composition according to claim 2, in which the cleaning power enhancer is selected from the group: silica (silicon dioxide), calcium carbonate, dicalcium phosphate dihydrate, calcium pyrophosphate, aluminum oxide, perlite, sodium bicarbonate, charcoal, hydrogen peroxide, calcium peroxide, sodium citrate, sodium pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate, calcium peroxide. 4. The composition of claim 1, further comprising hydroxyapatite. 5. The composition according to claim 4, wherein the hydroxyapatite is enriched with elements selected from the group: calcium, magnesium, zinc. 6. The composition of claim 1, further comprising a film-forming component. 7. A composition according to claim 6, in which the film-forming component is selected from the group: polyvinylpyrrolidone, polyvinyl alcohol, wax-like substances and mixtures thereof. 8. Composition according to claim 1, additionally containing a film former, an enhancer of cleaning ability, hydroxyapatite and auxiliary substances, wt%: proteolytic enzyme bromelain 0.005-0.2 bacteria lysate 0.05-1.5 film former 0.3-1.0 cleaning power enhancer 2.5-5.0 hydroxyapatite 0.1-10.0 Excipients up to 100 9. A composition according to claim 1, in the form of a toothpaste, gel, spray, chewing gum, foam, elixir, ointment, balm or rinse.

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