Enzyme composition for treating dental plaque and plaque bacterial biofilms, and for the natural whitening of tooth enamel

Abstract

One aspect of the invention relates to an oral health enzyme complex for use in treating dental plaque and dental plaque bacterial biofilms and for the natural whitening of enamel, the complex comprising a combination of the following compounds: - a sucrase enzyme, in particular capable of hydrolysing the β-2,6 and / or β-2,1 bonds of sucrose; - 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, in particular capable of hydrolysing (1-3)-α-D-glucans and (1-6)-α-D-glucans; - a glycoside hydrolase enzyme, in particular capable of hydrolysing the (1-6)-α-D glycosidic bonds of monosaccharide dimers and trimers; - an oxide reductase enzyme, in particular capable of forming hydrogen peroxide from glucose and monosaccharide monomers; - a peroxidase enzyme, in particular capable of converting thiocyanate into hypothiocyanate in the presence of hydrogen peroxide.

Description

ENZYMATIC COMPOSITION FOR THE TREATMENT OF DENTAL PLAQUE AND BACTERIAL BIOFILM OF PLAQUE AND FOR THE NATURAL WHITENING OF TOOTH ENAMEL TECHNICAL FIELD OF THE INVENTION

[0001] The technical field of the invention is that of oral hygiene and cosmetic products.

[0002] The present invention relates to a novel enzyme complex capable of preventing the formation of dental plaque, cavities and tartar, while achieving natural enamel whitening. The present invention also relates to a novel form of prevention and treatment of dental bacterial biofilm by said enzyme complex. TECHNOLOGICAL BACKGROUND OF THE INVENTION

[0003] Sugar from the diet, which is more than 90% sucrose, is broken down into glucans by cariogenic bacteria in the oral cavity, which form a bacterial biofilm on tooth enamel. A bacterial biofilm is an organized and structured community of bacterial cells entangled in an extracellular matrix of variable density and composition. The majority of the matrix of dental biofilms is made up of polysaccharides. Indeed, up to 40% of the dry weight of the dental biofilm is composed of polysaccharides. In the presence of sucrose, this bacterial biofilm grows thickly on the teeth, first forming soft dental plaque, composed of soluble glucans (known as (1-6)-aD-glucan or dextrans), and then hard dental plaque formed by the transformation of said dextrans into water-insoluble glucans (known as (1-3)-aD-glucan or mutans).

[0004] In the case of a diet that is predominantly sugary, the pH of dental plaque becomes acidic and promotes the formation of cavities, while in the case of a diet that is predominantly meat-based, the pH becomes basic and the hard plaque calcifies to form tartar, which becomes very difficult to remove.

[0005] According to a 2021 WHO report, in 2020, 3.5 billion people worldwide suffered from dental problems related to the formation of cavities and / or tartar. Similarly, according to data from the American National Health and Nutrition Examination Survey in 2018, 59% of adolescents aged 12 to 19 and 92% of adults aged 20 to 64 have suffered from dental caries in their permanent teeth. There is therefore still a need to reduce these phenomena.

[0006] Current approaches to reducing dental plaque include mechanical cleaning, chemicals, fluoride applications, and broad-spectrum antibacterial agents with antiseptic action such as chlorhexidine. While effective in the short term, these therapies all have side effects. Mechanical cleaning is known to cause tooth sensitivity and bleeding gums, chemicals are known to pose a health risk, fluoride use can cause major adverse human health problems while having only a modest effect on preventing dental caries, and chlorhexidine can cause tooth staining and fillings in cavities.Additionally, the use of antiseptics and chemicals has been shown to disrupt the commensal microbiota, increasing the pathogenic characteristics and cytotoxicity of host cells and causing other adverse effects on oral health.

[0007] These solutions are therefore not satisfactory.

[0008] It is increasingly preferred to use more preventive treatments that are more respectful of the consumer's overall health, with minimal side effects, such as the use of probiotics, prebiotics or natural substances, but their therapeutic usefulness is notoriously limited.

[0009] Among the pathogenic bacteria of the oral cavity, Streptococcus mutans is the main responsible for the formation of bacterial biofilm, the main cause of caries, because it uses dietary sucrose to produce lactic acid and synthesize extracellular polysaccharides, especially (1-3) and (1-6)-aD-glucans, which promote the formation of biofilms and increase the resistance of microorganisms in the biofilm. Lactic acid demineralizes the tooth surface, which promotes the formation of dental plaque and periodontitis. The acid biofilm formed by Streptococcus mutans is highly resistant to the hostile environment, host immunity and antimicrobial treatments, especially lysozyme. The latest research on dental pathology shows the difficulty of eliminating these streptococci protected within the matrix by the bacterial biofilm.

[0010] There is therefore a need to prevent the proliferation of Streptococcus mutans.

[0011] Lactoperoxidase is an enzyme that is known for its bactericidal effect in the presence of hydrogen peroxide and thiocyanate, particularly against Streptococcus mutans. It is known to provide these three substances by providing exogenous lactoperoxidase, glucose oxidase and potassium thiocyanate. In this type of composition, however, the formation of hydrogen peroxide is insufficient for the bactericidal effect of lactoperoxidase to significantly affect the Streptococcus mutans biofilm from the point of view of reducing the formation of caries and / or tartar. Furthermore, the provision of exogenous glucose in this type of composition is not recommended in the field of oral hygiene. This solution is therefore not satisfactory for preventing the proliferation of Streptococcus mutans.

[0012] Generally speaking, the major problem in the fight against cavities and tartar, and in maintaining healthy and beautiful teeth, is therefore to have dental plaque with a neutral pH and as physiological as possible every day. Recent research on dental pathologies shows that the best way to avoid them is to be able to eradicate bacterial biofilms. To this end, it is particularly important to prevent the formation of Streptococcus mutans biofilm and to disassemble existing biofilms in order to replace them with physiological plaque, that is to say the natural protective biofilm that surrounds a healthy tooth and which is made up of saliva and microbiota from the oral cavity.

[0013] In addition to having healthy teeth, it is also desirable to have the most beautiful teeth possible. There is therefore a constant need to whiten tooth enamel to combat yellowing, caused in particular by aging and the formation of insoluble dental plaque.

[0014] Documents FR 2651433, FR 2822700, FR 3020758, FR 2803199 and WO 2021 / 14421 1 each disclose an enzyme complex for oral health, but none of these documents discloses 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. SUMMARY OF THE INVENTION

[0015] The invention offers a solution to the problems mentioned above, by providing an enzyme complex whose composition, which is based in particular on a synergistic action of its various constituents, results from numerous studies carried out by the applicant. Of natural origin, this enzyme complex is respectful of the overall health of the consumer and does not present any side effects.

[0016] One aspect of the invention relates to an enzyme complex for use in the treatment of dental plaque and bacterial biofilm of plaque and for the natural whitening of tooth enamel, comprising the following compounds: a sucrase enzyme, a mixture of glucanohydrolase enzymes comprising a dextranase, a mutanase having exolytic activity and obtained from a fungus, and a mutanase having endolytic activity and obtained from a bacterium, a glycoside hydrolase enzyme, an oxidoreductase enzyme, a peroxidase enzyme.

[0017] According to one aspect of the invention, the glucanohydrolase enzyme mixture comprises a chimeric glucanase mixture comprising a dextranase and a mutanase linked by genetic engineering.

[0018] According to another aspect of the invention, the mutanase having exolytic activity is obtained from a fungus Trichoderma Harzianum.

[0019] According to a further aspect of the invention, the mutanase having endolytic activity is obtained from a bacterium Paracoccus Mutanolyticus, Paenibacillus or Streptomyces

[0020] According to one aspect of the invention, the sucrase enzyme is an isomaltase enzyme or an invertase enzyme.

[0021] According to another aspect of the invention, the glycoside hydrolase enzyme is an amyloglucosidase enzyme, preferably an amylo-α-1,6 glucosidase.

[0022] According to a further aspect of the invention, the oxidoreductase enzyme is a glucose oxidase.

[0023] According to one aspect of the invention, the peroxidase enzyme is a lactoperoxidase.

[0024] According to another aspect of the invention, the enzyme complex comprises the following compounds: a sucrase, preferably an invertase or an isomaltase, a 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, an amyloglucosidase, preferably an amylo-a-1,6 glucosidase, a glucose oxidase, and a lactoperoxidase.

[0025] According to a further aspect of the invention, the enzyme complex comprises the following compounds, the percentages indicated being percentages by volume: from 10 to 30%, preferably from 15 to 25%, of a sucrase, from 1 to 10%, preferably from 4 to 7%, of dextranase, from 10 to 40%, preferably from 15 to 30%, of a mixture comprising a mutanase having exolytic activity and obtained from a fungus and a mutanase having endolytic activity and obtained from a bacterium, from 20 to 60%, preferably from 30 to 50%, of an amyloglucosidase, preferably an amylo-α-1,6 glucosidase, from 5 to 20%, preferably from 7 to 15%, of glucose oxidase, and from 1 to 10%, preferably from 4 to 7%, of lactoperoxidase.

[0026] According to one aspect of the invention, the enzyme complex further comprises lysozyme.

[0027] Another aspect of the invention relates to an oral composition for use in the treatment of dental plaque and bacterial biofilm of plaque and the natural whitening of tooth enamel, which comprises an enzyme complex as described above.

[0028] According to one aspect of the invention, the oral composition comprises from 1.4 to 3%, preferably from 2.0 to 2.5%, of enzyme complex.

[0029] According to another aspect of the invention, the oral composition further comprises lactoferrin.

[0030] According to a further aspect of the invention, the oral composition further comprises: from 20 to 60%, preferably from 30 to 50%, of sorbitol, from 1 to 20%, preferably from 5 to 10%, of xylitol, from 0 to 3%, preferably from 1 to 2%, of polysorbate 80, from 0 to 3%, preferably from 1 to 2%, of polysorbate 20, from 0 to 0.5%, preferably from 0.1 to 0.2%, of sodium saccharin, from 0 to 5%, preferably from 1 to 2%, of colloidal silica, from 0 to 3%, preferably from 1 to 2%, of dimethicone, from 0 to 5%, preferably from 1 to 2%, of mint flavoring and / or mint essential oils, from 0.1 to 0.5%, preferably from 0.1 to 0.3% %, zinc citrate, 0 to 0.5%, preferably 0.1 to 0.2%, potassium thiocyanate, 0 to 0.5%, preferably 0.1 to 0.2%, preservative, and water, in an amount sufficient to reach 100%.

[0031] According to one aspect of the invention, the oral composition is in the form of a paste, liquid, gel, mouthwash, nebulization, spray, capsule, tablet or chewing gum.

[0032] As will be seen in detail below, by combining various bacteriostatic and bactericidal actions against cariogenic streptococci, the The glycolytic enzyme system of the invention makes it possible to significantly reduce the toxicity of Streptococcus mutans. This enzyme complex acts on food sucrose in such a way as to deprive the bacteria of their substrate but also by directly degrading the cell wall of these streptococci.

[0033] Thanks to the enzyme sucrase, preferably an invertase or an isomaltase, which hydrolyzes the [3-2,6 and / or [3-2,1] bonds of sucrose, the enzyme complex according to the invention advantageously reduces the quantity of sucrose available in the oral cavity by transforming it into monomers of oses (glucose) and into fructose, which deprives the cariogenic bacteria of metabolic substrate, greatly reduces the production of soluble and insoluble glucans, and thus contributes to the reduction of the bacterial biofilm.

[0034] Thanks to the glucanohydrolase enzyme mixture, which hydrolyzes the (1-3) and (1-6)-aD-glucan bonds, which are key structural and functional constituents of the Streptococcus mutans biofilm matrix, the enzyme complex according to the invention advantageously prevents the formation of bacterial biofilm and disassembles existing biofilms. The destruction of the bacterial biofilm further reduces the formation of dental plaque, which is malodorous and yellows teeth under the influence of tobacco or dyes such as coffee or tea. Soluble glucans are converted into sugar dimers or trimers by the mixture of glucanohydrolase enzymes, these sugar derivatives then being converted into sugar monomers by the action of the glycoside hydrolase enzyme, preferably amyloglucosidase, for example amylo-a-1,6 glucosidase, which hydrolyzes the (1-6)-aD sugar bonds of the sugar dimers and trimers.

[0035] The preferential association of a dextranase with a mutanase, more preferably from different fungi or bacteria, or a chimeric glucanase (dextranase-mutanase), shows very satisfactory effects aimed at preventing the formation of Streptococcus mutans biofilm and disassembling existing biofilms in a few minutes at nanomolar concentrations. This association significantly improves the sensitivity of the biofilm to antimicrobial and antibacterial agents, such as lysozyme, highlighting its great potential in the fight against dental caries related to acid biofilm.

[0036] The residual sugar derivatives from the degradation of glucans in dental plaque are then transformed into sugar monomers by a glycoside hydrolase enzyme that hydrolyzes the (1-6)-aD sugars, while the sugar monomers are transformed by the oxidoreductase enzyme into D-glucono-delta-lactone, but also, above all, into hydrogen peroxide. Indeed, the oxidoreductase enzyme forms hydrogen peroxide from the sugar monomers. The formation of hydrogen peroxide is advantageous because it has an antiseptic effect that actively participates in eliminating Streptococcus mutans, and a natural enamel whitening effect, which contributes to a more beautiful appearance of the teeth.

[0037] These beneficial effects are advantageously reinforced by the fact that the enzymatic complex according to the invention provides several sources of hydrogen peroxide in the oral cavity, by the in situ transformation of sucrose, saccharide dimers and trimers, soluble glucans and insoluble glucans into ose monomers, i.e. into glucose. The glucose thus regularly available, allows, through its transformation by the gluco-oxidase enzyme, to produce hydrogen peroxide directly in contact with the teeth in a regular, gentle and lasting manner, for an optimal effect with regard to oral hygiene and enamel whitening. Since the glucose is transformed in real time, the quantity of glucose present in the oral cavity at any given moment advantageously remains much lower and therefore much less harmful than with an exogenous glucose supply.

[0038] The hydrogen peroxide generated in situ thanks to the enzymatic complex according to the invention is produced in a much smaller quantity than that usually used for tooth whitening which is supplied exogenously, but it is also present in the oral cavity for a longer period, which makes it possible to provide a satisfactory progressive whitening effect, without being accompanied by the usual effects encountered with a massive and exogenous supply of hydrogen peroxide, namely: demineralization of the hard tissues of the tooth, irritation of the mucous membranes, reaction with the filling materials, premature wear of the enamel with hypersensitivity and weakening of the teeth.

[0039] Furthermore, in the presence of hydrogen peroxide, the enzyme peroxidase advantageously transforms the thiocyanate naturally present in saliva into hypothiocyanate, which is a powerful bactericide and further enhances the action of the enzyme complex of the invention with regard to dental health. To enhance this effect, it is also possible to add exogenous thiocyanate.

[0040] The addition of a sucrase, preferably an invertase or an isomaltase, to the enzyme complex according to the invention is advantageous in that this enzyme breaks down starch and glycogen possibly present in the oral cavity into ose dimers (maltose) which are then transformed into ose monomers by an alpha amylase naturally present in saliva.

[0041] The addition of lysozyme to the enzyme complex according to the invention is advantageous in that it hydrolyzes the polysaccharide cell wall of bacteria and, in the absence of the bacterial biofilm generated by Streptococcus mutans, exhibits strong bactericidal activity actively participating in oral hygiene. Indeed, the bactericidal activity of the enzyme complex according to the invention is reinforced by lysozyme which acts more effectively "after" the disappearance of the bacterial biofilm.

[0042] In summary, through the synergistic action of its components, the enzymatic complex according to the invention ensures the following beneficial effects while respecting the overall health of the consumer and without side effects: an anti-cariogenic sugar action by inhibiting the acid transformation of dietary sugars and therefore a preventive effect against cavities, an action of destructuring the bacterial biofilm of dental plaque and therefore a preventive effect against periodontal diseases and tartar, an action of sanitizing the microbiota of the oral cavity by reducing pathogenic bacteria under the effect of stimulation of the salivary lactoperoxidase system, a gentle and prolonged action of whitening of dental enamel by natural production of hydrogen peroxide.

[0043] The addition of lactoferrin to an oral composition comprising an enzyme complex, by ensuring the chelation of iron necessary for the binding of the polysaccharides of the microbial envelope, necessary for the survival of cariogenic streptococci, advantageously leads to better eradication of the Streptococcus mutans biofilm and contributes to the synergy mentioned above. Indeed, lactoferrin lyses the bacterial membrane of many organisms, including acidogenic streptococci, and thus significantly increases the body's immune defenses.

[0044] The invention and its various applications will be better understood by reading the following description. DETAILED DESCRIPTION

[0045] Streptococcus mutans utilizes dietary sucrose to synthesize extracellular polysaccharides (EPS) to promote bacterial biofilm formation. Recent studies show that the EPS matrix, consisting of an α-(1-6)-linked glucose polymer (dextran) with α-(1-3)-linked branch (mutan), plays a crucial role in regulating cariogenic biofilm formation and virulence by influencing the physical and biochemical properties of the biofilm. Cariogenic biofilm can promote the accumulation and adhesion of microorganisms and accelerate the coherence of bacterial cells with each other and with the apatite surface, thereby modulating the initial stages of cariogenic biofilm development and proliferation and facilitating the formation of mature dental plaque.In addition, as a reserve energy source, the EPS matrix protects microorganisms from hostile influences, affects the diffusion of substances into and out of the biofilm, and helps concentrate metal ions and other physiological nutrients in a microenvironment. As a physical barrier, the cariogenic biofilm hinders the diffusion of antibiotics and host-induced antimicrobial factors into the deeper layers of the biofilm, which increases the resistance of microorganisms in the biofilm. To do this, Streptococcus mutans produces a dextranase, DexA, which hydrolyzes the α-1,6 bonds of dextran and produces isomaltoligosaccharides of various sizes.Dextranase DexA is crucial in the process of bacterial biofilm formation and considered responsible for the pathogenic dental plaque ecosystem: Dextranase DexA hydrolyzes glucans as potential storage polysaccharides to provide nutrients for bacterial metabolism and controls the amount and content of extracellular glucans to make it more adhesive. However, the effect of Dextranase DexA can be competed with an externally supplied dextranase enzyme.

[0046] In the studies carried out to select the appropriate enzyme complex according to the invention, it was sought in particular to identify enzymes which selectively target and degrade EPS. In this study, it was found that the Dextranase shows much higher activity in inhibiting biofilm formation and disrupting a pre-formed biofilm when supplied exogenously in combination with a mutanase. But more interestingly, it was found that the use of a mutanase from different origins, one of fungal origin with exolytic action, the other bacterial with endolytic action like streptococcus, or a chimeric glucanase (dextranase-mutanase), prevents Streptococcus mutans biofilm formation very satisfactorily, and disassembles existing biofilms within minutes at nano molar concentrations. This combination markedly improves the sensitivity of the biofilm to antimicrobial agents and lysozyme, highlighting its great potential in combating acid biofilm-related dental caries.

[0047] This innovative action on the bacterial biofilm associated with an invertase, an isomaltase or another enzyme of the saccharase family, which degrades sucrose and thus deprives cariogenic bacteria of metabolic substrate, strongly reduces the production of glucans and thus contributes to a significant reduction of the bacterial biofilm. The saccharide derivatives thus obtained give monomers of oses which under the action of oxidoreductase enzymes produce hydrogen peroxide which triggers the salivary lactoperoxidase reaction which is highly bactericidal and leads to the disappearance of the bacterial biofilm.

[0048] Furthermore, this hydrogen peroxide, produced in small quantities but continuously, allows for the natural and gradual whitening of tooth enamel, thus avoiding any problems of tooth sensitivity.

[0049] The enzyme complex according to the invention will be described in detail below.

[0050] By enzyme complex we mean here a stable association and according to a defined structure of a number of different proteins whose various enzymatic activities will contribute in an orderly manner to the development of a synergistic activity on a target substrate.

[0051] The enzyme complex according to the invention is intended for oral use to improve oral health and whiten tooth enamel.

[0052] It comprises a combination of the following constituents: a sucrase enzyme, in particular capable of hydrolyzing the [3-2,6 and / or [3-2,1] bonds of sucrose, a mixture of glucanohydrolase enzymes, in particular capable of hydrolyzing (1-3)-aD-glucans and (1-6)-aD-glucans, a glycoside hydrolase enzyme, in particular capable of hydrolyzing the (1-6)-aD-saccharide bonds of ose dimers and trimers, an oxidoreductase enzyme, in particular capable of decomposing ose monomers to form hydrogen peroxide, a peroxidase enzyme, in particular capable of transforming thiocyanate into hypothiocyanate in the presence of hydrogen peroxide.

[0053] The sucrase enzyme capable of hydrolyzing the [3-2,6 and / or [3-2,1] bonds of sucrose is preferably an invertase or an isomaltase, degrading sucrose into glucose and fructose.

[0054] The glucanohydrolase enzyme mixture degrades dental plaque into ose dimers or trimers. This is preferentially a mixture of mutanase and dextranase. Dextranase degrades soft dental plaque by transforming soluble dextrans into 1,6-dimer or ose-trimer osidic polymers, while mutanase degrades insoluble mutans in hard plaque by transforming them into 1,3-dimer or ose-trimer osidic polymers.

[0055] In the glucanohydrolase enzyme mixture, a mutanase having exolytic activity and a mutanase having endolytic activity are preferred. As the mutanase having exolytic activity, a mutanase obtained from a fungus, especially the fungus Trichoderma Harzianum, can be used. As the mutanase having endolytic activity, a mutanase obtained from a bacterium, especially the bacterium Paracoccus Mutanolyticus, the bacterium Paenibacillus, or the bacterium Streptomyces, can be used.

[0056] In the glucanohydrolase enzyme mixture, instead of a mixture of mutanase and dextranase, a chimeric glucanase mixture comprising a dextranase and a mutanase linked by genetic engineering can also be used.

[0057] The enzyme glycoside hydrolase is an enzyme that reduces ose dimers or trimers into osediamine monomers, i.e. glucose. It is preferably an amyloglucosidase, more preferably an enzyme Amylo-a-1,6 glucosidase. Amyloglucosidase converts sugar derivatives into glucose.

[0058] The oxidoreductase enzyme is an enzyme that converts glucose into hydrogen peroxide. It is primarily a glucose oxidase, also known as glucooxidase. The glucooxidase enzyme converts glucose into D-glucono-delta-lactone and hydrogen peroxide. D-glucono-delta-lactone is an acidity regulator. In the oral cavity, it is partially converted into gluconic acid.

[0059] In the presence of hydrogen peroxide, the enzyme peroxidase converts thiocyanate into hypothiocyanate according to the following reaction:

[0060] SCN + H2O2 => OSCN + H2O

[0061] The peroxidase enzyme is preferentially a lactoperoxidase.

[0062] Hypothiocyanate, a strong bactericide, limits the growth of bacteria in the oral cavity.

[0063] The enzymatic complex according to the invention preferably has the following composition (the percentages indicated are percentages by volume): Invertase: 10 to 30%, preferably 15 to 25%, Mutanase: 10 to 40%, preferably 15 to 30%, Dextranase: from 1 to 10%, preferably from 4 to 7%, Amyloglucosidase: from 20 to 60%, preferably from 30 to 50%, Glucose oxidase: from 5 to 20%, preferably from 7 to 15%, and Lactoperoxidase: from 1 to 10%, preferably from 4 to 7%.

[0064] The table below gives an example of a control formulation CET of enzyme complex with a high amount of amyloglucosidase and without mutanase, and three examples of formulations CE1-3 for the enzyme complex according to the invention. The percentages indicated are percentages by volume.

[0065] [Table 1] 0066] In clinical trials conducted on patients for six months by the applicant with the participation of a research laboratory under the supervision of a medical doctor, the CET formulation gave unsatisfactory results. In fact, compared to control subjects, in subjects participating in the study for six months and using this formulation, the presence of dental plaque and a very slight whitening of the tooth enamel (<1 tone) was noted.

[0067] The CE1 formulation gave more satisfactory results than the CET formulation. In fact, compared to the control subjects, in the subjects participating in the study for six months and using this formulation, a lower amount of dental plaque in the oral cavity was noted, as well as a significant whitening of the tooth enamel (+1 shade of white).

[0068] The CE2 formulation gave more satisfactory results than the CET formulation. In fact, compared to the control subjects, in the subjects participating in the study for six months and using this formulation, a lower amount of dental plaque in the oral cavity was noted, as well as a significant whitening of the tooth enamel (+1 shade of white).

[0069] The CE3 formulation gave more satisfactory results than the CE1 and CE2 formulations. In fact, compared to the control subjects, in the subjects participating in the study for six months and using this formulation, there was a virtual absence of dental plaque in the oral cavity, as well as a significant whitening of the tooth enamel (+2 shades of white).

[0070] The enzyme complex according to the invention may also comprise lysozyme.

[0071] The enzyme complex preferably comprises from 0.1 to 0.4% by weight of lysozyme, more preferably 0.2%. It may also preferably comprise from 0 to 0.4% by weight of sucrase, preferably an invertase or an isomaltase, more preferably 0.2% by weight.

[0072] The enzyme complex according to the invention can be used in an oral composition for oral health and enamel whitening.

[0073] This oral composition can, for example, be presented in the form of paste, liquid, gel, mouthwash, nebulosity, spray, capsule, tablet or chewing gum.

[0074] The oral composition for oral-dental use according to the invention preferably has the following composition (the enzymatic complex is that described previously and the percentages indicated are percentages by weight): Enzyme complex: from 1.4 to 3%, preferably from 2.0 to 2.5% Sorbitol: 20 to 60%, preferably 30 to 50% Xylitol: from 1 to 20%, preferably from 5 to 10%, Polysorbate 80: from 0 to 3%, preferably from 1 to 2%, Polysorbate 20: from 0 to 3%, preferably from 1 to 2%, Sodium saccharin: from 0 to 0.5%, preferably from 0.1 to 0.2%, Colloidal silica: from 0 to 5%, preferably from 1 to 2%, Dimethicone: 0 to 3%, preferably 1 to 2%, Mint aroma and / or mint essential oils: 0 to 5%, preferably 1 to 2%, Zinc citrate: from 0.1 to 0.5%, preferably from 0.1 to 0.3%, Potassium thiocyanate: from 0 to 0.5%, preferably from 0.1 to 0.2%, Preservative: from 0 to 0.5%, preferably from 0.1 to 0.2%, Water: sufficient quantity to reach 100%.

[0075] The preservative is preferably citric acid.

[0076] The oral composition according to the invention may also comprise lactoferrin, preferably from 0 to 0.4% by weight, more preferably 0.1%.

[0077] It may also include hydrogen peroxide, for example 0 to 0.1%, and violet dye, for a more immediate whitening effect.

[0078] The table below gives an example of a control formulation COT of an oral composition for oral use with the absence of an enzyme complex, and three examples of formulations CO1-3 for the oral composition according to the invention with the enzyme complex CE3. The percentages indicated are percentages by weight.

[0079] [Table 2] 080] The effectiveness of these different formulations was tested on Streptococcus salivarius, which is from the same Viridans family as Streptococcus mutans, and a Gram-positive cocci bacteria like the latter, also forming a biofilm in the presence of sucrose, and known to have the same sensitivity to antiseptics and antibacterials as Streptococcus mutans. The efficacy of said formulations was compared to a placebo not comprising an enzyme complex according to the invention and to a mouthwash solution comprising 0.12% chlorhexidine.

[0081] In in vitro clinical trials conducted by the applicant with the participation of another analysis laboratory under the supervision of a doctor of pharmacy, carried out with the CE3 enzyme complex, the COT formulation gave unsatisfactory results. Indeed, compared to the placebo vs. COT formulation vs. chlorhexidine, a high quantity of Streptococcus salivarius was noted in the placebo, a still high quantity of Streptococcus salivarius in the COT formulation and a zero quantity in the chlorhexidine-based formulation.

[0082] The CO1 formulation gave more satisfactory results than the COT formulation. Indeed, compared to placebo vs. CO1 formulation vs. chlorhexidine, a high amount of Streptococcus salivarius was observed in the placebo, a low amount of Streptococcus salivarius in the CO1 formulation, and zero amount in the chlorhexidine-based formulation.

[0083] The CO2 formulation gave more satisfactory results than the COT formulation and the CO1 formulation. Indeed, compared to the placebo vs. CO2 formulation vs. chlorhexidine, a high amount of Streptococcus salivarius was noted in the placebo, a zero amount of Streptococcus salivarius in the CO2 formulation and a zero amount in the chlorhexidine-based formulation.

[0084] The CO3 formulation gave results as satisfactory as the CO2 formulation. Indeed, compared to the placebo vs. CO3 formulation vs. chlorhexidine, a high amount of Streptococcus salivarius was observed in the placebo, a zero amount of Streptococcus salivarius in the CO3 formulation, and a zero amount in the chlorhexidine-based formulation.

[0085] Thus, for an oral composition for oral use comprising an enzyme complex according to the enzymatic formula CE3, although clinical effects could be observed with a quantity of enzyme complex less than 2%, the bactericidal effect against Streptococcus is completely effective with an amount of enzyme complex greater than or equal to 2%.

Claims

CLAIMS

1. Enzyme complex for use in the treatment of dental plaque and bacterial biofilm of plaque and for the natural whitening of tooth enamel, characterized in that it comprises the following compounds: - a sucrase enzyme, - a mixture of glucanohydrolase enzymes comprising a dextranase, a mutanase having exolytic activity and obtained from a fungus, and a mutanase having endolytic activity and obtained from a bacterium, - a glycoside hydrolase enzyme, - an oxidoreductase enzyme, - a peroxidase enzyme.

2. Enzyme complex according to claim 1, characterized in that the mixture of glucanohydrolase enzymes comprises a mixture of chimeric glucanases comprising a dextranase and a mutanase linked by genetic engineering.

3. Enzyme complex according to claim 1 or 2, characterized in that the mutanase having exolytic activity is obtained from a fungus Trichoderma Harzianum.

4. Enzyme complex according to any one of the preceding claims, characterized in that the mutanase having endolytic activity is obtained from a Paracoccus Mutanolyticus, Paenibacillus or Streptomyces bacterium.

5. Enzyme complex according to any one of the preceding claims, characterized in that the saccharase enzyme is an isomaltase enzyme or an invertase enzyme.

6. Enzyme complex according to any one of the preceding claims, characterized in that the glycoside hydrolase enzyme is an amyloglucosidase enzyme, preferably an amylo-a-1,6 glucosidase.

7. Enzyme complex according to any one of the preceding claims, characterized in that the oxidoreductase enzyme is a glucose oxidase.

8. Enzyme complex according to any one of the preceding claims, characterized in that the peroxidase enzyme is a lactoperoxidase.

9. Enzymatic complex according to any one of the preceding claims, characterized in that it comprises the following compounds: - a sucrase, preferably an invertase or an isomaltase, - a 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, - an amyloglucosidase, preferably an amylo-a-1,6 glucosidase, - a glucose oxidase, and - a lactoperoxidase.

10. Enzymatic complex according to claim 8, characterized in that it comprises the following compounds, the percentages indicated being percentages by volume: - from 10 to 30%, preferably from 15 to 25%, of a saccharase, - from 1 to 10%, preferably from 4 to 7%, of dextranase, - from 10 to 40%, preferably from 15 to 30%, of a mixture comprising a mutanase having exolytic activity and obtained from a fungus and a mutanase having endolytic activity and obtained from a bacterium, - from 20 to 60%, preferably from 30 to 50%, of an amyloglucosidase, preferably an amylo-a-1,6 glucosidase, - from 5 to 20%, preferably from 7 to 15%, of glucose oxidase, and - from 1 to 10%, preferably from 4 to 7%, of lactoperoxidase. [Claim 1 1] Oral composition for use in the treatment of dental plaque and bacterial biofilm of plaque and the natural whitening of tooth enamel, characterized in that it comprises an enzyme complex according to any one of the preceding claims.

12. Oral composition according to claim 11, characterized in that it comprises from 1.4 to 3%, preferably from 2.0 to 2.5%, of enzymatic complex.

13. Oral composition according to claim 11 or 12, characterized in that it further comprises lactoferrin.

14. Oral composition according to any one of claims 11 to 13, characterized in that it further comprises: - from 20 to 60%, preferably from 30 to 50%, of sorbitol, - from 1 to 20%, preferably from 5 to 10%, of xylitol, - from 0 to 3%, preferably from 1 to 2%, of polysorbate 80, - from 0 to 3%, preferably from 1 to 2%, of polysorbate 20, - from 0 to 0.5%, preferably from 0.1 to 0.2%, of sodium saccharin, - from 0 to 5%, preferably from 1 to 2%, of colloidal silica, - from 0 to 3%, preferably from 1 to 2%, of dimethicone, - from 0 to 5%, preferably from 1 to 2%, of mint aroma and / or mint essential oils, - from 0.1 to 0.5%, preferably from 0.1 to 0.3%, of zinc citrate, - from 0 to 0.5%, preferably from 0.1 to 0.2%, of potassium thiocyanate, - from 0 to 0.5%, preferably from 0.1 to 0.2%, of preservative, - water, in a quantity sufficient to reach 100%.

15. An oral composition according to any one of claims 11 to 14, characterized in that it is in the form of a paste, liquid, gel, mouthwash, nebulosity, spray, capsule, tablet or chewing gum.

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