Enzyme compositions for treating dental plaque and dental plaque bacterial biofilms and for naturally whitening tooth enamel - Patents.com

Abstract

One aspect of the present invention relates to an oral hygiene enzyme complex for use in the treatment of dental plaque and plaque bacterial biofilms and for the natural whitening of enamel, the complex comprising a combination of the following compounds: sucrase enzymes, particularly those capable of hydrolyzing the β-2,6 and / or β-2,1 bonds of sucrose; a mixture of glucanohydrolase enzymes, including mutanases having exolytic activity and obtained from fungi, and mutanases having endolytic activity and obtained from bacteria, particularly those capable of hydrolyzing (1→3)-α-D-glucans and (1→6)-α-D-glucans; glycoside hydrolase enzymes, particularly those capable of hydrolyzing the (1→6)-α-D-glycosidic bond in monosaccharide dimers and trimers; oxidoreductases, particularly those capable of producing hydrogen peroxide from glucose and monosaccharide monomers; and peroxidase enzymes, particularly those capable of converting thiocyanate to hypothiocyanate in the presence of hydrogen peroxide.

Description

[Technical Field]

[0001] The technical field of the invention is that of oral dental hygiene and cosmetics.

[0002] The present invention relates to a novel enzyme complex that can prevent the formation of plaque, caries, and tartar while naturally whitening enamel. The present invention also relates to a novel form of dental bacterial biofilm prevention and treatment using the enzyme complex. [Background technology]

[0003] Dietary sugars (more than 90% of which is sucrose) are degraded into glucans by cariogenic bacteria present in the oral cavity, resulting in the formation of bacterial biofilms on tooth enamel. Bacterial biofilms are organized structures consisting of bacterial cells encased in an extracellular matrix of variable density and composition. The main component of the dental biofilm matrix is ​​polysaccharides, accounting for up to 40% of the dry weight. In the presence of sucrose, this bacterial biofilm grows thickly on the tooth surface, first forming a soft dental plaque composed of soluble glucans [(1→6)-α-D-glucan, or dextran], which are then converted into water-insoluble glucans [(1→3)-α-D-glucan, or mutans], forming a hard dental plaque.

[0004] A high-carbohydrate diet leads to an acidic pH in dental plaque, promoting the formation of caries, whereas a meat-based diet leads to an alkaline pH in dental plaque, causing hard dental plaque to calcify and turn into tartar, which is extremely difficult to remove.

[0005] According to a 2021 World Health Organization (WHO) report, approximately 3.5 billion people worldwide suffered from dental diseases related to caries and / or tartar formation as of 2020. Additionally, data from the 2018 U.S. National Health and Nutrition Examination Survey (NHANES) showed that 59% of adolescents aged 12 to 19 and 92% of adults aged 20 to 64 had caries in their permanent teeth. Therefore, there is still a need to reduce these symptoms.

[0006] Current approaches aimed at removing dental plaque include mechanical cleaning, chemicals, fluoride applications, and broad-spectrum antibacterial antiseptics such as chlorhexidine. While all of these methods are effective in the short term, they are associated with side effects. Mechanical cleaning can cause tooth sensitivity and gingival bleeding, chemicals are known to pose health risks, and fluoride use may have significant adverse effects on human health, while its caries prevention effect is limited. Chlorhexidine may stain teeth and caries fillings. Furthermore, the use of antiseptics and chemicals has been reported to disrupt the indigenous oral flora, increase the pathogenicity and cytotoxicity of host cells, and cause other adverse effects on oral health.

[0007] Therefore, these solutions remain insufficient.

[0008] There is growing interest in more preventative treatments that combine consideration of the consumer's overall health with minimizing side effects, such as the use of probiotics, prebiotics or natural substances, but as is well known, these have limited therapeutic utility.

[0009] Among oral pathogenic bacteria, Streptococcus mutans is primarily involved in the formation of bacterial biofilms, the primary cause of dental caries. This bacterium utilizes dietary sucrose to produce lactic acid and synthesize extracellular polysaccharides, particularly (1→3) and (1→6)-α-D-glucan, which promote biofilm formation and enhance the resistance of microorganisms within the biofilm. Lactic acid causes demineralization of the tooth surface and promotes the formation of plaque and periodontitis. Acidic biofilms formed by Streptococcus mutans are highly resistant to harsh external environments, host immunity, and antimicrobial treatments (especially lysozyme). Furthermore, dental pathology studies have demonstrated that these streptococci, protected within the matrix of bacterial biofilms, are difficult to eliminate.

[0010] Therefore, there is a need to inhibit the growth of Streptococcus mutans.

[0011] Lactoperoxidase is an enzyme known to exhibit bactericidal activity, particularly against Streptococcus mutans, in the presence of hydrogen peroxide and thiocyanate. It is known that exogenous lactoperoxidase, glucose oxidase, and potassium thiocyanate can be combined to provide these three substances. However, because the amount of hydrogen peroxide produced in this type of composition is insufficient, the bactericidal activity of lactoperoxidase does not significantly affect Streptococcus mutans biofilms and inhibit caries and / or tartar formation. Furthermore, the addition of exogenous glucose to such compositions is not recommended in the field of oral hygiene. Therefore, this solution is not satisfactory in terms of inhibiting the growth of Streptococcus mutans.

[0012] In general, the greatest challenge in preventing caries and tartar and maintaining healthy, beautiful teeth is maintaining dental plaque at a physiologically neutral pH as much as possible on a daily basis. Recent research on dental diseases has shown that the best means of preventing them is to remove bacterial biofilms. To this end, it is particularly effective to prevent the formation of biofilms by Streptococcus mutans and to break down existing biofilms and replace them with physiological plaque, i.e., the natural protective biofilm (composed of saliva and the oral microflora) that covers healthy teeth.

[0013] In addition to having healthy teeth, people want to have the most beautiful teeth possible. Maintaining white tooth enamel is especially important to combat the yellowing caused by aging and the buildup of insoluble plaque.

[0014] French Patents Nos. 2651433, 2822700, 3020758, 2803199 and WO 2021 / 144211 all disclose enzyme complexes for oral hygiene, but none of them disclose a mixture of glucanohydrolase enzymes comprising a mutanase having exolytic activity and obtained from a fungus, and a mutanase having endolytic activity and obtained from a bacterium. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] French Patent Invention No. 2651433 [Patent Document 2] French Patent Invention No. 2822700 [Patent Document 3] French Patent Invention No. 3020758 [Patent Document 4] French Patent Invention No. 2803199 [Patent Document 5] International Publication No. 2021 / 144211 Summary of the Invention [Means for solving the problem]

[0016] (Summary of the Invention) The present invention provides a solution to the above-mentioned problems by providing an enzyme complex having a composition based in particular on the synergistic action of each component, which is the result of extensive research carried out by the applicant. Due to its natural origin, the enzyme complex is considerate of the overall health of the consumer and has no side effects.

[0017] One aspect of the present invention relates to an enzyme complex for use in the treatment of dental plaque and bacterial biofilms of dental plaque and for the natural whitening of tooth enamel, comprising the following compounds: - sucrase enzyme, a mixture of glucanohydrolase enzymes including dextranase, a mutanase having exolytic activity and obtained from a fungus, and a mutanase having endolytic activity and obtained from a bacterium; - glycoside hydrolase enzymes, - oxidoreductases, - Peroxidase enzyme.

[0018] According to one aspect of the invention, the mixture of glucanohydrolase enzymes comprises a mixture of chimeric glucanases comprising genetically linked dextranase and mutanase.

[0019] According to another aspect of the present invention, a mutanase having exolytic activity is obtained from the fungus Trichoderma harzianum.

[0020] According to yet another embodiment of the present invention, the mutanase having endolytic activity is obtained from Paracoccus mutanolyticus, Paenibacillus or Streptomyces bacteria.

[0021] According to one aspect of the invention, the sucrase enzyme is an isomaltase enzyme or an invertase enzyme.

[0022] According to another aspect of the present invention, the glycoside hydrolase enzyme is an amyloglucosidase enzyme, preferably amyl-α-1,6-glucosidase.

[0023] According to yet another aspect of the present invention, the oxidoreductase is a glucose oxidase enzyme.

[0024] According to one aspect of the present invention, the peroxidase enzyme is a lactoperoxidase enzyme.

[0025] According to another aspect of the invention, the enzyme complex comprises the following compounds: sucrase, preferably invertase or isomaltase, - dextranase, a mixture of mutanases comprising a mutanase having exolytic activity and obtained from a fungus, and a mutanase having endolytic activity and obtained from a bacterium; amyloglucosidase, preferably amyl-α-1,6-glucosidase, glucose oxidase, and - Lactoperoxidase.

[0026] According to yet another aspect of the present invention, the enzyme complex comprises the following compounds, the percentages given being by volume: - 10-30%, preferably 15-25% sucrase, 1 to 10%, preferably 4 to 7% dextranase, - a mixture comprising 10 to 40%, preferably 15 to 30%, of a mutanase having exolytic activity and obtained from a fungus and a mutanase having endolytic activity and obtained from a bacterium, - 20 to 60%, preferably 30 to 50%, of amyloglucosidase, preferably amyl-α-1,6-glucosidase, - 5 to 20%, preferably 7 to 15%, of glucose oxidase, and - 1-10%, preferably 4-7% lactoperoxidase.

[0027] According to one aspect of the invention, the enzyme complex further comprises lysozyme.

[0028] Another aspect of the present invention relates to oral compositions for use in the treatment of dental plaque and plaque bacterial biofilms and the natural whitening of tooth enamel, comprising the enzyme complex described above.

[0029] According to one embodiment of the present invention, the oral composition contains 1.4 to 3%, preferably 2.0 to 2.5%, of the enzyme complex.

[0030] According to another aspect of the present invention, the oral composition further comprises lactoferrin.

[0031] According to yet another aspect of the present invention, the oral composition comprises the following ingredients: 20 to 60%, preferably 30 to 50%, of sorbitol, - 1 to 20%, preferably 5 to 10% xylitol, - 0-3%, preferably 1-2% polysorbate 80, - 0-3%, preferably 1-2% polysorbate 20, - 0 to 0.5%, preferably 0.1 to 0.2%, of sodium saccharin; - 0-5%, preferably 1-2%, of colloidal silica, - 0-3%, preferably 1-2% dimethicone, - 0-5%, preferably 1-2%, of mint flavoring and / or mint essential oil, 0.1 to 0.5%, preferably 0.1 to 0.3%, of zinc citrate, - 0 to 0.5%, preferably 0.1 to 0.2%, of potassium thiocyanate, - 0 to 0.5%, preferably 0.1 to 0.2%, of a preservative, and - Enough water to make it 100%.

[0032] According to one aspect of the invention, the oral composition is in the form of a paste, liquid, gel, mouthwash, mist, spray, capsule, tablet, chewing gum, or the like.

[0033] As described in detail below, by combining various bacteriostatic and bactericidal activities against cariogenic streptococci, the glycolytic enzyme system of the present invention can significantly reduce the virulence of Streptococcus mutans. The enzyme complex not only acts on dietary sucrose to sever the bacterial substrate, but also directly degrades the cell walls of these streptococci.

[0034] The enzyme complex of the present invention converts sucrose into monosaccharide monomers, glucose and fructose, by the use of a sucrase enzyme (preferably invertase or isomaltase) that hydrolyzes the β-2,6 and / or β-2,1 bonds of sucrose, thereby advantageously reducing the amount of sucrose available in the oral cavity, thereby cutting off the supply of metabolic substrates to cariogenic bacteria and significantly suppressing the production of soluble and insoluble glucans, thereby contributing to the reduction of bacterial biofilms.

[0035] The enzyme complex of the present invention advantageously prevents bacterial biofilm formation and disintegrates existing biofilms by utilizing a mixture of glucanohydrolase enzymes that hydrolyze (1→3) and (1→6)-α-D-glucan bonds, which are the major structural and functional components of the Streptococcus mutans biofilm matrix. Disruption of bacterial biofilms further suppresses plaque formation, which produces malodor and causes yellowing of teeth under the influence of tobacco or coloring substances such as coffee or tea. Soluble glucans are converted into monosaccharide dimers or trimers by the mixture of glucanohydrolase enzymes, which are then converted back into monosaccharide monomers by the action of a glycoside hydrolase enzyme (preferably an amyloglucosidase, e.g., amyl-α-1,6-glucosidase) that hydrolyzes the (1→6)-α-D-glycosyl bonds of the monosaccharide dimers or trimers.

[0036] The combination of dextranase with mutanase, preferably with mutanase from different fungal or bacterial origin, or with chimeric glucanase (dextranase-mutanase), has been shown to be highly effective in preventing Streptococcus mutans biofilm formation and disintegrating existing biofilms within minutes at nanomolar concentrations. This combination significantly increases the susceptibility of biofilms to antibacterial and bactericidal agents, such as lysozyme, making it a very promising approach against acidic biofilm-associated dental caries.

[0037] Residual sugar derivatives resulting from the degradation of glucans in dental plaque are converted to monosaccharide monomers by glycoside hydrolases, which hydrolyze the (1→6)-α-D-sugar bond. The resulting monosaccharide monomers are then converted by oxidoreductases to D-glucono-δ-lactone and, more importantly, to hydrogen peroxide. In fact, oxidoreductases generate hydrogen peroxide from the monosaccharide monomers. The formation of hydrogen peroxide is advantageous in that it has an antiseptic effect that actively contributes to the elimination of Streptococcus mutans, and a natural enamel whitening effect that contributes to improved tooth esthetics.

[0038] The enzyme complex of the present invention provides multiple sources of hydrogen peroxide by converting sucrose, monosaccharide dimers and trimers, soluble glucans, and insoluble glucans directly into monosaccharide monomers (i.e., glucose) in the oral cavity, thereby advantageously enhancing their beneficial effects. This constant supply of glucose is converted by the glucose oxidase enzyme to produce hydrogen peroxide in a regular, gentle, and sustained manner in direct contact with the tooth surface for optimal oral hygiene and enamel whitening effects. Because glucose is converted in real time, the amount of glucose in the oral cavity at any given time is advantageously kept low compared to exogenously added glucose, resulting in significantly reduced harmful effects.

[0039] The amount of hydrogen peroxide produced in the oral cavity by the enzyme complex of the present invention is significantly less than that typically supplied exogenously for tooth whitening purposes, but it remains in the oral cavity for a long time, thereby achieving a satisfactory progressive whitening effect without the typical adverse effects associated with large amounts of exogenous hydrogen peroxide supplied, such as demineralization of hard tissues, mucosal irritation, reaction with occlusive materials, premature enamel wear accompanied by hypersensitivity, and tooth weakening.

[0040] Furthermore, in the presence of hydrogen peroxide, the peroxidase enzyme advantageously converts thiocyanate, which is naturally present in saliva, to hypothiocyanate, a potent antiseptic, further enhancing the dental hygiene effects of the enzyme complex of the present invention. Exogenous thiocyanate can also be added to enhance this effect.

[0041] The addition of sucrase (preferably invertase or isomaltase) to the enzyme complex of the present invention is advantageous in that the enzyme breaks down starch and glycogen present in the oral cavity into dimeric monosaccharides (maltose), which are then converted to monosaccharide monomers by α-amylase, which is naturally present in saliva.

[0042] Furthermore, the addition of lysozyme to the enzyme complex of the present invention is advantageous in that it hydrolyzes the polysaccharide cell walls of bacteria and exerts potent bactericidal activity in the absence of bacterial biofilms produced by Streptococcus mutans, thereby positively contributing to oral hygiene. Indeed, lysozyme acts more effectively "after" the bacterial biofilm has disappeared, enhancing the bactericidal activity of the enzyme complex of the present invention.

[0043] In summary, the enzyme complex of the present invention provides the following beneficial effects without side effects, taking into consideration the overall health of the consumer, due to the synergistic action of its constituent components: - Anti-cariosclerotic sugar action by inhibiting the acid conversion of dietary carbohydrates and the associated caries prevention effect; - Preventive effect against periodontal disease and tartar by destroying the bacterial biofilm in dental plaque, - Hygiene of the oral microflora by reducing pathogenic bacteria through stimulation of the lactoperoxidase system in saliva; - Gentle and long-lasting whitening action on tooth enamel through the natural production of hydrogen peroxide.

[0044] The addition of lactoferrin to oral compositions containing enzyme complexes advantageously results in the effective removal of Streptococcus mutans biofilms by ensuring iron chelation necessary for binding to microbial envelope polysaccharides, which are essential for the survival of cariogenic streptococci, contributing to the aforementioned synergistic effect. Indeed, lactoferrin dissolves the bacterial membranes of many microorganisms, including acidogenic streptococci, significantly strengthening the host's immune defense mechanisms.

[0045] The invention and its various applications can be more clearly understood with reference to the following description. DETAILED DESCRIPTION OF THE INVENTION

[0046] Streptococcus mutans utilizes dietary sucrose to synthesize exopolysaccharides (EPS) to promote bacterial biofilm formation. Recent studies have demonstrated that the EPS matrix, consisting of glucose polymers linked by α-(1→6) (dextran) and α-(1→3) (mutan) branching linkages, plays an important role in regulating the formation and pathogenicity of cariogenic biofilms by influencing their physical and biochemical properties. Cariogenic biofilms promote the accumulation and attachment of microorganisms and accelerate the aggregation of bacterial cells with each other and with apatite surfaces. This regulates the early stages of cariogenic biofilm development and growth and promotes the formation of mature dental plaque. Furthermore, the EPS matrix acts as a reserve energy source, protecting microorganisms from the harmful external environment, influencing the diffusion of materials into and out of the biofilm, and helping to concentrate metal ions and other physiological nutrients within the microenvironment. As a physical barrier, cariogenic biofilms prevent the diffusion of antibiotics and host-derived antimicrobial factors deep into the biofilm, thereby increasing the resistance of the biofilm microorganisms. During this process, Streptococcus mutans produces dextranase (DexA), which hydrolyzes the α-1,6 bond of dextran to generate isomaltooligosaccharides of various sizes. DexA is essential for bacterial biofilm formation and is thought to be involved in the ecology of pathogenic dental plaque. By hydrolyzing glucans, which serve as potential storage polysaccharides, DexA provides a nutrient source for bacterial metabolism, regulates the amount and composition of extracellular glucans, and enhances their adhesiveness. However, this effect of DexA can be counteracted by exogenously supplied dextranase enzymes.

[0047] In our studies to select the appropriate enzyme complex for the present invention, we focused on identifying enzymes that selectively target and degrade EPS. These studies confirmed that exogenously supplied dextranase in combination with mutanase significantly enhanced biofilm formation inhibition and pre-existing biofilm destruction. Interestingly, we also found that mutanases of different origins—those derived from fungi with exolytic activity and those derived from bacteria (e.g., streptococci) with endolytic activity—as well as chimeric glucanases (dextranase-mutanase) effectively inhibited Streptococcus mutans biofilm formation and degraded existing biofilms within minutes at nanomolar concentrations. This combination dramatically enhanced the susceptibility of biofilms to antimicrobial agents and lysozyme, suggesting that it may be a promising approach to combat acidic biofilm-associated dental caries.

[0048] The innovative action of the present invention against bacterial biofilms is achieved by combining it with other enzymes belonging to the invertase, isomaltase, or sucrase family, which degrades sucrose, cutting off the supply of metabolic substrates to cariogenic bacteria and significantly suppressing glucan production. This contributes to a significant reduction in bacterial biofilms. The sugar derivatives thus obtained are converted to monosaccharide monomers by the action of oxidoreductases, which then generate hydrogen peroxide. This hydrogen peroxide then induces the highly bactericidal lactoperoxidase reaction in saliva, resulting in the elimination of bacterial biofilms.

[0049] Furthermore, because this hydrogen peroxide is produced continuously, even in small amounts, it can naturally and slowly whiten tooth enamel and avoid problems such as tooth sensitivity.

[0050] The enzyme complex according to the present invention will be described in detail below.

[0051] As used herein, an enzyme complex refers to a construct consisting of multiple different proteins stably associated according to a predetermined structure, in which the enzyme activities of each protein contribute to the expression of a synergistic effect on a target substrate in an orderly manner.

[0052] The enzyme complex of the present invention is intended for use in the oral cavity for the purpose of improving oral hygiene and whitening tooth enamel.

[0053] The enzyme complex comprises a combination of the following components: - sucrase enzymes, in particular those capable of hydrolyzing the β-2,6 and / or β-2,1 bonds of sucrose; a mixture of glucanohydrolase enzymes, in particular those capable of hydrolyzing (1→3)-α-D-glucan and (1→6)-α-D-glucan, - glycoside hydrolase enzymes, in particular those capable of hydrolyzing the (1→6)-α-D-glycosyl bonds of monosaccharide dimers and trimers; - oxidoreductases, in particular those capable of decomposing monosaccharide monomers to produce hydrogen peroxide; - Peroxidase enzymes, especially those capable of converting thiocyanate to hypothiocyanate in the presence of hydrogen peroxide.

[0054] The sucrase having the ability to hydrolyze the β-2,6 bond and / or β-2,1 bond of sucrose preferably includes invertase or isomaltase, which decomposes sucrose into glucose and fructose.

[0055] A mixture of glucanohydrolase enzymes degrades dental plaque into monosaccharide dimers and trimers. This mixture is preferably a mixture of mutanase and dextranase. Dextranase degrades soft dental plaque by converting soluble dextran into 1,6-monosaccharide dimer- or trimer-type sugar polymers, while mutanase degrades insoluble mutan in hard dental plaque by converting it into 1,3-monosaccharide dimer- or trimer-type sugar polymers.

[0056] In the mixture of glucanohydrolase enzymes, mutanase with exolytic activity and mutanase with endolytic activity are preferred. Mutanase with exolytic activity can be obtained from fungi, particularly Trichoderma hartianum. Mutanase with endolytic activity can be obtained from bacteria, particularly Paracoccus mutanolyticus, Paenibacillus, or Streptomyces.

[0057] Furthermore, in the mixture of glucanohydrolase enzymes, a mixture of chimeric glucanases containing genetically linked dextranase and mutanase can be used instead of a mixture of mutanase and dextranase.

[0058] Glycoside hydrolases are enzymes that break down monosaccharide dimers or trimers into the glycan monomer, i.e., glucose. The enzyme is preferably amyloglucosidase, and more preferably amylo-α-1,6-glucosidase enzyme. Amyloglucosidase converts sugar derivatives into glucose.

[0059] Oxidoreductase is an enzyme that converts glucose into hydrogen peroxide. The enzyme is preferably glucose oxidase (also called glucoxidase). Glucose oxidase converts glucose into D-glucono-δ-lactone and hydrogen peroxide. D-glucono-δ-lactone is an acidity regulator. In the oral cavity, a portion of D-glucono-δ-lactone is converted into gluconic acid.

[0060] In the presence of hydrogen peroxide, the peroxidase enzyme converts thiocyanate to hypothiocyanate according to the following reaction: SCN+H2O2 → OSCN+H2O

[0061] The peroxidase enzyme is preferably lactoperoxidase.

[0062] Hypothiocyanate is a powerful antiseptic and inhibits bacterial growth in the oral cavity.

[0063] The enzyme complex according to the invention preferably has the following composition (percentages are by volume): - invertase: 10-30%, preferably 15-25%, Mutanase: 10-40%, preferably 15-30%, Dextranase: 1 to 10%, preferably 4 to 7%, amyloglucosidase: 20 to 60%, preferably 30 to 50%, - glucose oxidase: 5-20%, preferably 7-15%, - Lactoperoxidase: 1-10%, preferably 4-7%.

[0064] The table below shows an example of a control enzyme conjugate formulation (CET) containing a high amount of amyloglucosidase but no mutanase, as well as three example enzyme conjugate formulations according to the present invention (CE1-CE3). The percentages shown are by volume.

[0065] [Table 1]

[0066] The applicant conducted a six-month clinical trial on subjects under the supervision of a physician and in cooperation with a research institution. The results showed that the CET formulation did not demonstrate sufficient efficacy. Specifically, subjects who participated in the six-month study and used the formulation showed plaque and only slight (<1 shade) whitening of tooth enamel compared to the control group.

[0067] The CE1 formulation performed better than the CET formulation: subjects who participated in the study for 6 months and used this formulation experienced a reduction in oral plaque and a noticeable whitening of tooth enamel (+1 whiter shade) compared to the control group.

[0068] The CE2 formulation performed better than the CET formulation: subjects who participated in the study for 6 months and used this formulation experienced a reduction in oral plaque and a significant whitening of tooth enamel (+1 whiter shade) compared to the control group.

[0069] The CE3 formulation performed better than the CE1 and CE2 formulations: subjects who participated in the study for six months and used this formulation had virtually no plaque in their mouths and significantly whitened their tooth enamel (+2 whiter shades) compared to the control group.

[0070] The enzyme complex of the present invention may contain lysozyme.

[0071] The enzyme complex preferably contains 0.1 to 0.4 wt %, more preferably 0.2 wt %, of lysozyme, and preferably 0 to 0.4 wt % of sucrase, preferably invertase or isomaltase, more preferably 0.2 wt %.

[0072] The enzyme complex of the present invention can be used in oral compositions for oral hygiene and enamel whitening.

[0073] The oral composition may be in the form of, for example, a paste, liquid, gel, mouthwash, mist, spray, capsule, tablet, or chewing gum.

[0074] The oral composition according to the present invention preferably has the following composition (the enzyme complex is as described above and the percentages given are by weight): - enzyme complex: 1.4 to 3%, preferably 2.0 to 2.5%, - sorbitol: 20-60%, preferably 30-50%, - xylitol: 1 to 20%, preferably 5 to 10%, - Polysorbate 80: 0-3%, preferably 1-2%, - Polysorbate 20: 0-3%, preferably 1-2%, - sodium saccharin: 0 to 0.5%, preferably 0.1 to 0.2%, - colloidal silica: 0-5%, preferably 1-2%, - Dimethicone: 0-3%, preferably 1-2%, - mint flavoring and / or mint essential oil: 0-5%, preferably 1-2%, Zinc citrate: 0.1 to 0.5%, preferably 0.1 to 0.3%, - potassium thiocyanate: 0 to 0.5%, preferably 0.1 to 0.2%, - preservatives: 0-0.5%, preferably 0.1-0.2%, - Enough water to make it 100%.

[0075] The preservative is preferably citric acid.

[0076] The oral composition according to the present invention may further contain preferably 0 to 0.4% by weight, more preferably 0.1% by weight, of lactoferrin.

[0077] Furthermore, the oral composition according to the present invention may contain, for example, 0 to 0.1% by weight of hydrogen peroxide and a violet dye for achieving a rapid whitening effect.

[0078] The table below shows an example of a control formulation (COT) of an oral composition for oral dental use that does not contain an enzyme complex, and three example formulations (CO1-CO3) of oral compositions according to the present invention that contain enzyme complex CE3. The percentages shown are by weight.

[0079] [Table 2]

[0080] The efficacy of each of these preparations was tested against Streptococcus salivarius, a Gram-positive cocci belonging to the same Viridans group as Streptococcus mutans, which forms biofilms in the presence of sucrose and is known to be equally susceptible to antiseptics and antibacterial agents as Streptococcus mutans. The efficacy of these preparations was compared with a placebo preparation not containing the enzyme complex of the present invention and a mouthwash containing 0.12% chlorhexidine.

[0081] The applicant conducted an in vitro clinical trial using the enzyme complex CE3 under the supervision of a PhD in Pharmacy and with the cooperation of another analytical laboratory. The COT formulation did not demonstrate sufficient efficacy. Specifically, when a placebo formulation, COT formulation, and chlorhexidine formulation were compared, large amounts of Streptococcus salivarius were detected in the placebo formulation, and similarly large amounts were detected in the COT formulation, whereas the bacteria were not detected in the chlorhexidine formulation.

[0082] The CO1 formulation showed better results than the COT formulation. Specifically, when comparing the placebo, CO1, and chlorhexidine formulations, a large amount of Streptococcus salivarius was detected with the placebo formulation, a small amount with the CO1 formulation, and no Streptococcus salivarius was detected with the chlorhexidine formulation.

[0083] The CO2 formulation showed better results than the COT and CO1 formulations. Specifically, when comparing the placebo, CO2, and chlorhexidine formulations, a large amount of Streptococcus salivarius was detected in the placebo formulation, but the same bacteria was not detected in the CO2 and chlorhexidine formulations.

[0084] The CO3 formulation showed results as good as those of the CO2 formulation. Specifically, when comparing the placebo formulation, the CO3 formulation, and the chlorhexidine formulation, a large amount of Streptococcus salivarius was detected in the placebo formulation, but the same bacteria was not detected in the CO3 formulation or the chlorhexidine formulation.

[0085] Therefore, it has been confirmed that in compositions for oral dental use containing an enzyme complex based on the enzyme formula CE3, a certain level of clinical effect can be achieved even if the amount of the enzyme complex is less than 2% by weight, but the bactericidal effect against Streptococcus can only be fully achieved when the amount of the enzyme complex is 2% by weight or more.

Claims

1. 1. An enzyme complex for use in the treatment of dental plaque and bacterial biofilms of dental plaque and for the natural whitening of tooth enamel, comprising the following compounds: - sucrase enzyme, a mixture of glucanohydrolase enzymes including dextranase, a mutanase having exolytic activity and obtained from a fungus, and a mutanase having endolytic activity and obtained from a bacterium; - glycoside hydrolase enzymes, - oxidoreductases, - Peroxidase enzyme An enzyme complex comprising:

2. 2. The enzyme conjugate of claim 1, wherein the mixture of glucanohydrolase enzymes comprises a mixture of chimeric glucanases comprising genetically linked dextranase and mutanase.

3. 3. The enzyme complex according to claim 1, wherein the mutanase having exolytic activity is obtained from the fungus Trichoderma hartianum.

4. 4. The enzyme complex according to claim 1, wherein the mutanase having endolytic activity is obtained from Paracoccus mutanolyticus, a bacterium belonging to the genus Paenibacillus, or a bacterium belonging to the genus Streptomyces.

5. The enzyme complex according to any one of claims 1 to 4, wherein the sucrase enzyme is an isomaltase enzyme or an invertase enzyme.

6. The enzyme complex according to any one of claims 1 to 5, characterized in that the glycoside hydrolase enzyme is an amyloglucosidase enzyme, preferably amyl-α-1,6-glucosidase.

7. 7. The enzyme complex according to claim 1, wherein the oxidoreductase is glucose oxidase.

8. The enzyme complex according to any one of claims 1 to 7, characterized in that the peroxidase enzyme is lactoperoxidase.

9. The following compounds: - sucrase, preferably invertase or isomaltase, - dextranase, a mixture of mutanases comprising a mutanase having exolytic activity and obtained from a fungus, and a mutanase having endolytic activity and obtained from a bacterium; amyloglucosidase, preferably amylo-α-1,6 glucosidase, glucose oxidase, and - lactoperoxidase The enzyme complex according to any one of claims 1 to 8, comprising:

10. The following compounds: 10 to 30%, preferably 15 to 25% of sucrase, - 1 to 10%, preferably 4 to 7% of dextranase, a mixture comprising 10 to 40%, preferably 15 to 30%, of a mutanase having exolytic activity and obtained from a fungus and a mutanase having endolytic activity and obtained from a bacterium, - 20 to 60%, preferably 30 to 50%, of amyloglucosidase, preferably amyl-α-1,6-glucosidase; - 5 to 20%, preferably 7 to 15%, of glucose oxidase, and 1-10%, preferably 4-7% lactoperoxidase 9. The enzyme complex of claim 8, wherein the percentages indicated are percent by volume.

11. An oral composition for use in the treatment of dental plaque and plaque bacterial biofilms and in the natural whitening of tooth enamel, characterized in that the oral composition comprises an enzyme complex according to any one of claims 1 to 10.

12. Oral composition according to claim 11, characterized in that it contains 1.4 to 3%, preferably 2.0 to 2.5%, of an enzyme complex.

13. The oral composition according to claim 11 or 12, further comprising lactoferrin.

14. below: 20 to 60%, preferably 30 to 50%, of sorbitol, - 1 to 20%, preferably 5 to 10% xylitol, 0-3%, preferably 1-2%, of polysorbate 80, 0-3%, preferably 1-2%, of polysorbate 20, - 0 to 0.5%, preferably 0.1 to 0.2%, of sodium saccharin; 0 to 5%, preferably 1 to 2%, of colloidal silica, - 0 to 3%, preferably 1 to 2% of dimethicone, - 0-5%, preferably 1-2%, of mint flavoring and / or mint essential oil, 0.1 to 0.5%, preferably 0.1 to 0.3%, of zinc citrate, 0 to 0.5%, preferably 0.1 to 0.2%, of potassium thiocyanate, 0 to 0.5%, preferably 0.1 to 0.2%, of a preservative, and - Enough water to make it 100% The oral composition according to any one of claims 11 to 13, further comprising:

15. The oral composition according to any one of claims 11 to 14, which is in the form of a paste, liquid, gel, mouthwash, mist, spray, capsule, tablet or chewing gum.

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