Agent for inhibiting the enzymes of biofilm-producing oral microorganisms and a method for its production

Abstract

The present invention relates to an agent for inhibiting the enzymes of biofilm- producing oral microorganisms and a method for its production, and will find application in the field of dentistry, in particular for the prevention of plaque-related diseases in the oral cavity such as dental caries and periodontal diseases The created agent for inhibiting the enzymes of biofilm-producing oral microorganisms contains the following components in the corresponding weight percentages: vegetable oil from the kernel of a plant of the genus Prunus spp. from 12 to 13%; liquid enzyme preparation of microbial origin from 0.10 to 0.13%; sodium hydroxide from 5 to 6%; hexane from 9 to 10%; hydrochloric acid from 5 to 6.5%; anhydrous sodium sulfate from 3 to 4.4%; methyl orange from 0.004 to 0.008%; alcohol from 0.60 to 0.90%; water from 60 to 69% and tocopherol from 0 to 1%. The vegetable oil is from the kernels of plum - Prunus domestica, apricot - Prunus armeniaca or almond - Prunus dulcis.

Description

[0001] Agent for inhibiting the enzymes of biofilm-producing oral microorganisms and a method for its production

[0002] Technical field

[0003] The present invention relates to an agent for inhibiting the enzymes of the biofilmproducing oral microorganisms and a method for its production, and will find application in the field of dentistry, for the prevention of plaque-related diseases in the oral cavity such as dental caries and periodontal diseases.

[0004] Background of the invention

[0005] The oral microbiome is a complex community of microorganisms that play a crucial role in maintaining both oral and systemic health. The relation between periodontal diseases and systemic health conditions — such as cardiovascular disease, diabetes, respiratory illnesses, and adverse pregnancy outcomes — is well-documented, with the mechanisms linking oral and systemic conditions being mediated through infectious and inflammatory pathways.

[0006] Excessive use of broad- spectrum antimicrobial agents included in oral hygiene products can eliminate beneficial bacteria, predisposing individuals to a state of dysbiosis or an imbalanced microbial flora. As a result of dysbiosis, conditions may arise that promote the development of local diseases such as caries, plaque-induced gingival inflammation, as well as an increased risk of systemic infections. There is a need for the development of new strategies and oral care products that support microbial balance and reduce the risk of systemic disease.

[0007] Classical active ingredients used in oral hygiene are traditionally antibacterial with bactericidal effects and low selectivity, leading to the destruction of both pathogenic and beneficial commensal microorganisms in the mouth. Overuse of strongly bactericidal antibacterial agents in oral care products may have profound implications for overall health, associated with emerging oral dysbiosis and microbial imbalance. There is no known method for suppressing the cariogenic activity of the mutans streptococci group of bacteria — associated with the accumulation of dental biofilm (plaque) and acid production within the biofilm — through the application of a mixture of free fatty acids.

[0008] Summary of the invention

[0009] The task of the invention is to create an agent for inhibiting the enzymes of biofilmproducing oral microorganisms and a method for its production by hydrolysis of natural vegetable oils, the resulting agent having a high concentration of free fatty acids, which suppress the formation of biofilm and the secretion of acids in it, reducing the risk of dental caries and plaque-induced periodontal diseases through optimal combination of microbial lipases and organic substances of plant origin.

[0010] The task is solved by creating a method by which an agent for inhibiting the enzymes of biofilm-producing oral microorganisms is obtained. The created method includes the following sequential operations:

[0011] - charging oil of vegetable origin from the kernel of a plant of the genus Prunus spp. (stone fruit) into a laboratory vessel and adding water to the oil;

[0012] - homogenization of the solution and addition of a liquid enzyme preparation of microbial origin - lipase - to the solution;

[0013] - re-homogenization of the solution and placement of the resulting emulsion in a shaker at a temperature of 30 to 60°C for 6 to 48 hours;

[0014] - sampling of the emulsion every 3 hours to check the pH value;

[0015] - adjustment of the pH of the emulsion with a sodium base to a value of 5-6 until the pH value remains constant and hydrolysis is complete.

[0016] - acidification of the hydrolysed solution with hydrochloric acid to a pH value of 3 - 4 in the presence of methyl orange indicator;

[0017] - addition of hexane to the acidified hydrolysed solution;

[0018] - carrying out several extractions of the acidified hydrolysed solution; - addition of alcohol for additional phase separation and subsequent separation of the aqueous phase;

[0019] - washing of the obtained extracts by adding water, shaking and separation of water in the presence of methyl orange indicator until a neutral reaction is obtained;

[0020] - filtration of the neutralized extract with anhydrous sodium sulfate;

[0021] - distillation of the solvent with a vacuum evaporator;

[0022] - mixing the obtained extract with tocopherol.

[0023] The agent for inhibiting the enzymes of the oral microbiome, obtained by the described method, contains the following components in the respective weight percentages: oil from vegetable origin from the kernel of the plum from species Prunus spp. from 12 to 13%; liquid enzyme agent from microbial origin from 0,10 to 0,13%; sodium base from 5 to 6%; hexane from 9 to 10%; hydrochloric acid from 5 to 6,5%; anhydrous sodium sulfate from 3 to 4,4%; methyl orange indicator from 0,004 to 0,008%; alcohol from 0,60 to 0,90%; water from 60 to 69%, and tocopherol from 0 to 1%.

[0024] In a preferred embodiment of the created agent for inhibiting the enzymes of the oral microbiome, its components are in the following weight percentage ratios: the oil of plant origin from the kernel of a plant of the genus Prunus spp. is 12.46%; the liquid enzyme preparation of microbial origin is 0.12%; sodium hydroxide is 5.1%; hexane is 9.61%; hydrochloric acid is 5.77%; anhydrous sodium sulfate is 3.65%; methyl orange is 0.006%; alcohol is 0.77%; tocopherol is 0.1% and water is up to 100%.

[0025] Stone fruit plants of the genus Prunus spp. include various fruit trees and shrubs such as: plum - Prunus domestical apricot - Prunus armeniaca; almond - Prunus dulcis; peach - Prunus persica; cherry - Prunus avium; Japanese plum - Prunus salicina; and cherry plum - Prunus cerasifera.

[0026] In preferred embodiments of the developed agent, the plant-derived oil is obtained from the kernels of plum - Prunus domestica, or apricot - Prunus armeniaca, or almond - Prunus dulcis. Liquid enzyme preparations of microbial origin are biological products produced by fermentation of microorganisms that contain specific enzymes. These enzymes are biocatalysts that accelerate chemical reactions in various industrial and biological processes.

[0027] In a preferred embodiment of the created agent, the liquid enzyme preparation of microbial origin is a lipase from Aspergillus oryzae.

[0028] An advantage of the invention is that when using the agent for inhibiting the enzymes of biofilm-producing oral microorganisms, the formation of dental plaque and the risk of developing dental caries is reduced, as well as the risk of developing plaque-induced inflammation of the periodontal tissues. The anti-inflammatory and antioxidant effect of the obtained free fatty acids reduces inflammation and supports the restoration of the tissues of the oral mucosa and gums, reduces the risk of oral diseases as a result of dysbiosis, and reduces the risk of systemic diseases and conditions related to oral health, such as cardiovascular and autoimmune diseases, diabetes, and others. Another advantage is that the use of biotechnologically processed natural ingredients such as enzyme-activated vegetable oils offers a safe and effective way to maintain oral health without disrupting the oral microbiome.

[0029] Examples of embodiment of the invention

[0030] The invention relates to an agent for inhibiting the enzymes of biofilm-producing oral microorganisms and a method for its production for suppressing the cariogenic effect associated with the accumulation of dental biofilm (dental plaque) and the production of acids in the biofilm, by applying a mixture of free fatty acids obtained as a result of enzymatic hydrolysis of vegetable oils. The group of bacteria mutans streptococci (Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii) have a pronounced cariogenic effect by using the enzyme glucosyltransferase (GTF) for the synthesis of insoluble glucans from sucrose, which are of crucial importance for the formation of biofilm and the development of dental caries. Another end product of the metabolism of mutans streptococci, for which the enzyme glucosyltransferase is key, are acids, which lead to demineralization of hard dental tissues and the development of dental caries.

[0031] Inhibition of the enzyme glucosyltransferase by free fatty acids helps reduce biofilm formation and acid production in the biofilm, helping to maintain oral health and avoid the risk of plaque-related oral diseases.

[0032] In one example embodiment, the created agent for inhibiting the enzymes of biofilm-producing oral microorganisms contains the following components in weight percentages: vegetable oil from the kernel of a plant of the genus Prunus spp. is 12.46%; liquid enzyme preparation of microbial origin is 0.12%; sodium hydroxide is 5.1%; hexane is 9.61%; hydrochloric acid is 5.77%; anhydrous sodium sulfate is 3.65%; methyl orange is 0.006%; alcohol is 0.77%; tocopherol is 0.1% and water is up to 100%.

[0033] Stone fruit plants of the genus Prunus spp. are various fruit trees and shrubs such as: plum - Prunus domestica; apricot - Prunus armeniaca; almond - Prunus dulcis; peach - Prunus persica; cherry - Prunus avium; Japanese plum - Prunus salicina and cherry plum - Prunus ceracifera);

[0034] In preferred embodiments, the created agent is vegetable oil from the kernels of plum - Prunus domestica, apricot - Prunus armeniaca or almond - Prunus dulcis.

[0035] Liquid enzyme preparations of microbial origin are biological products produced by fermentation of microorganisms that contain specific enzymes. These enzymes are biocatalysts that accelerate chemical reactions in various industrial and biological processes.

[0036] In a preferred embodiment of the created agent, the liquid enzyme preparation of microbial origin is lipase from Aspergillus oryzae.

[0037] The created agent for inhibiting the enzymes of the oral microbiome, respectively the prevention of caries and periodontal diseases, have the following fatty acid composition: lauric acid (C12:0) from 0 to 0.15%; myristic acid (C14:0) from 0 to 3%; pentadecylic acid (C15:0) from 0 to 0.45%; palmitic acid (C16:0) from 3 to 15%; palmitoleic acid (C16:l) from 0 to 3%; stearic acid (C18:0) from 0.5 to 3%; oleic acid (C18:ln-9) from 40 to 84%; linoleic acid (C18:2n-6) from 6 to 35%; linolenic acid (C18:3n-3) from 0 to 1% and arachidonic acid (C20:0) from 0 to 0.04% by weight.

[0038] The created method for the production of an agent for inhibiting the enzymes of the oral microbiome is expressed as follows:

[0039] A vegetable oil from the kernel of a plant of the genus Prunus spp., for example from a plum - Prunus domestica, is poured into a laboratory vessel and water is added to it in a ratio of: 0.4 parts of water to 1 part of oil. The resulting solution is homogenized and a liquid enzyme preparation of microbial origin is added to it - lipase from Aspergillus oryzae in a ratio of: 0.2 parts of lipase to 1 part of hydrolysate. The solution is homogenized again, after which the resulting emulsion is placed in a shaker at a temperature of 30 to 60°C for 6 to 48 hours. Every 3 hours, a sample is taken from the shaker to check the pH. The pH of the emulsion is adjusted with 0.4% sodium hydroxide to a value of 5 - 6 in the ratio: 16 parts sodium hydroxide to 51 parts hydrolysate, until the pH of the emulsion remains constant and the hydrolysis is completed. The hydrolysed solution is then acidified with 36% hydrochloric acid to a pH of 3 - 4 in the presence of 0.5% methyl orange indicator. Hexane is added to the acidified fatty acid solution in the separatory funnel in the ratio of one part alcohol to 3 parts hydrolysate and the fatty acids are extracted vigorously and repeatedly. After the extraction, 95% alcohol is added for additional phase separation in the ratio of 1 part alcohol to 25 parts hydrolysate. The additional phase separation is waited for and the aqueous phase is subsequently separated. The obtained extracts are washed with water in a ratio of 1 part water to 3 parts hydrolysate, shaken and the water is separated in the presence of a 0.5% methyl orange indicator until a neutral reaction is obtained. The neutralized extract is filtered with anhydrous sodium sulfate, and the solvent is distilled off with a vacuum evaporator. The obtained extract is mixed with tocopherol in a ratio of 1 part tocopherol to 100 parts extract.

[0040] The created agent for the prevention of caries and periodontal diseases are superior in biological activity to natural vegetable oil, as they have a high concentration of free fatty acids, for example oleic, including anti-inflammatory, antioxidant, hypocholesterolemic and neuroprotective effects. The presence of free fatty acids allows for more efficient suppression of the enzyme glucosyltransferase (GTF) and, accordingly, improved control over the glucans produced by microorganisms from the mutans group, key to the accumulation of dental biofilm and acid production, thus achieving a reduced pathogenic function of microorganisms. The binding of free fatty acids, in particular oleic acid to GTF has been identified as a non-competitive inhibition of GTF, i.e. it binds only to the enzymesubstrate complex, preventing the enzyme from converting substrate to product. Unlike other inhibitors, here is a targeted way to bind only to the enzyme-substrate complex, not to the free enzyme, preventing the formation of cariogenic biofilms and reducing the overall bacterial load in the oral cavity. This precise targeting is an advantage as it reduces the virulence of S. mutans without affecting its survival or that of other species in the oral cavity, thus helping to maintain microbial balance.

Claims

1. CLAIMS1. A method for producing an agent for inhibiting the enzymes of biofilmproducing oral microorganisms, characterized in that it comprises the following sequence of operations:- pouring vegetable oil from the kernel of a plant of the genus Prunus spp. (stone fruit) into a laboratory vessel and adding water to the oil;- homogenization of the solution and addition of a liquid enzyme preparation of microbial origin - lipase - to the solution;- re-homogenization of the solution and placing the resulting emulsion in a shaker at a temperature of 30 to 60°C for 6 to 48 hours;- sampling the emulsion every 3 hours to check the pH value;- adjustment of the pH of the emulsion with sodium hydroxide to a value of 5 - 6 until the pH value remains constant and the hydrolysis is completed;- acidification of the hydrolysed solution with hydrochloric acid to a value of pH 3 - 4 in the presence of a methyl orange indicator;- addition of hexane to the acidified hydrolysed solution;- performing several extractions of the acidified hydrolysed solution;- addition of alcohol for additional phase separation and subsequent separation of the aqueous phase;- washing of the obtained extracts by adding water, shaking and separation of the water in the presence of methyl orange indicator until a neutral reaction is obtained;- filtration of the neutralized extract with anhydrous sodium sulfate;- distillation of the solvent with a vacuum evaporator;- mixing the obtained extract with tocopherol.

2. An agent for inhibiting the enzymes of biofilm-producing oral microorganisms, obtained by the method described in claim 1, characterized in that it contains the following components in the corresponding percentages: oil of plant origin from the kernel of a plant of the genus Prunus spp. from 12 to 13%; liquid enzymepreparation of microbial origin from 0.10 to 0.13%; sodium hydroxide from 5 to 6%; hexane from 9 to 10%; hydrochloric acid from 5 to 6.5%; anhydrous sodium sulfate from 3 to 4.4%; methyl orange from 0.004 to 0.008%; alcohol from 0.60 to 0.90%; water from 60 to 69% and tocopherol from 0 to 1%.

3. An agent for inhibiting the enzymes of biofilm-producing oral microorganisms, according to claim 2, characterized in that its components are in the following weight percentage ratios: the oil of plant origin from the kernel of a plant of the genus Prunus spp. is 12.46%; the liquid enzyme preparation of microbial origin is 0.12%; the sodium hydroxide is 5.1%; the hexane is 9.61%; the hydrochloric acid is 5.77%; the anhydrous sodium sulfate is 3.65%; the methyl orange is 0.006%; the alcohol is 0.77%; the tocopherol is 0.1% and the water is up to 100%.

4. An agent for inhibiting the enzymes of biofilm-producing oral microorganisms according to claims 1, 2 and 3, characterized in that the oil of plant origin is from the kernels of plum - Prunus domestica or apricot - Prunus armeniaca or almond - Prunus dulcis.

5. An agent for inhibiting the enzymes of biofilm-producing oral microorganisms according to claims 1, 2 and 3, characterized in that the liquid enzyme preparation of microbial origin is lipase from Aspergillus oryzae.