Composition for inhibiting oral biofilm formation

Abstract

The present invention aims to provide a composition containing as an active ingredient a naturally occurring component that has the effect of suppressing the formation of oral biofilms without excessively affecting the growth of bacteria in the oral cavity. [Solution] The above objective can be achieved by a composition for inhibiting oral biofilm formation, which contains enzyme-treated royal jelly as an active ingredient, and by the composition for inhibiting oral biofilm formation, which is a cosmetic, food or beverage, quasi-drug, or pharmaceutical used for human oral care.

Description

Technical Field

[0001] The present invention relates to a composition for suppressing the formation of oral biofilm.

Background Art

[0002] Biofilm is a three-dimensional structure composed of a microbial population adhering to the surface of a substance in contact with water and extracellular polysaccharides produced by the microorganisms. The extracellular polysaccharides produced by the microorganisms play a role in protecting the microorganisms and contribute to the formation of microcolonies of the microorganisms. A variety of bacterial groups enter and multiply in this microcolony, thereby forming a biofilm. Representative examples of biofilms include slime generated in wet areas such as kitchens, bathrooms, and washrooms, and plaque (dental plaque) formed in the oral cavity by periodontal disease-related bacteria and the like.

[0003] There are hundreds of types of bacteria in the oral cavity, and these bacteria combine with each other to form a complex oral biofilm. Among these oral bacteria, oral pathogenic bacteria such as oral streptococci including periodontal pathogenic bacteria and cariogenic bacteria are known to cause oral diseases. Oral pathogenic bacteria are known to cause not only oral diseases but also serious systemic diseases such as aspiration pneumonia, infective endocarditis, Alzheimer's type dementia, and diabetes.

[0004] In order to prevent diseases caused by oral pathogenic bacteria, the search for materials that suppress the growth of oral pathogenic bacteria has been underway. However, the pathogenicity of oral pathogenic bacteria is not immediately exerted by the growth of bacteria in the oral cavity. Oral pathogenic bacteria can exert their pathogenicity by colonizing in the oral cavity, that is, forming an oral biofilm as dental plaque on the tooth surface and escaping from the self-cleaning action in the oral cavity by saliva. Therefore, it is important to suppress the formation of oral biofilm by oral pathogenic bacteria for the prevention of diseases caused by oral pathogenic bacteria.

[0005] Because oral bacteria can sometimes cause systemic diseases, interest in the oral microbiota has been growing in recent years, similar to that of the gut microbiota. Just as regulating the gut microbiota improves not only gut health but also overall health, it is desirable to maintain the oral microbiota in an appropriate state. Therefore, in order to maintain the oral microbiota in an appropriate state, it is desirable to avoid excessively killing all oral bacteria.

[0006] Several materials that exhibit antibacterial activity against oral pathogenic bacteria are known to date. For example, compositions containing hydroxyapatite in combination with royal jelly or its extract are known to be effective against Porphyromonas gingivalis. Porphyromonas gingivalis It is known to exhibit a sterilization rate of 80% or more against (see, for example, Patent Document 1).

[0007] The royal jelly used in Patent Document 1 is a natural material that contains a wide variety of nutrients and has a long history of being consumed. However, Non-Patent Document 1 states that royal jelly is used to kill Actinomyces actinomycetemcomitans, a periodontal pathogenic bacterium. Actinomyces actinomycetemcomitans It has been reported that the minimum inhibitory concentration (MIC) for Actinomyces actinomycetemcomitans and Porphyromonas gingivalis is higher than that of chlorhexidine, a common antibacterial agent. This indicates that royal jelly has inferior antibacterial activity against Actinomyces actinomycetemcomitans and Porphyromonas gingivalis. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2005-314266 [Non-patent literature]

[0009] [Non-Patent Document 1] Otreba M et al., “Bee Venom, Honey, and Royal Jelly in the Treatment of Bacterial Infections of the Oral Cavity: A Review.”, Life (Basel). 2021 Nov 28;11(12):1311. [Overview of the project] [Problems that the invention aims to solve]

[0010] Patent Document 1 describes that combining hydroxyapatite with royal jelly or its extract enhances the antibacterial activity of hydroxyapatite against Porphyromonas gingivalis, but also states that the antibacterial activity of royal jelly or its extract alone is inferior. Similarly, Non-Patent Document 1 describes that royal jelly has inferior antibacterial activity not only against Porphyromonas gingivalis but also against Actinomyces actinomycetemcomitans compared to the antibacterial agent chlorhexidine.

[0011] Furthermore, neither Patent Document 1 nor Non-Patent Document 1 contains any description of natural materials that suppress the formation of oral biofilms by oral bacteria. In particular, there is no description of natural materials that suppress the formation of oral biofilms without excessively affecting the growth of oral bacteria.

[0012] Therefore, the present invention aims to provide a composition containing, as an active ingredient, a component that is naturally derived and has the effect of suppressing the formation of oral biofilms without excessively affecting the growth of bacteria in the oral cavity. [Means for solving the problem]

[0013] To achieve the above objective, the inventors diligently conducted research and attempted to find a naturally derived substance that inhibits biofilm formation in the oral cavity from among many other substances. In this process, they focused on enzyme-treated royal jelly.

[0014] Royal jelly has a weak antibacterial effect against oral pathogenic bacteria such as Actinomyces actinomycetemcomitans and Porphyromonas gingivalis. Therefore, it was assumed that enzyme-treated royal jelly, obtained by enzymatic treatment of royal jelly, would have a weak antibacterial effect similar to that of royal jelly and would show almost no biofilm formation inhibitory effect. However, surprisingly, the inventors found that enzyme-treated royal jelly not only has a weak antibacterial effect against oral pathogenic bacteria, but also exhibits excellent biofilm formation inhibitory effect. Even more surprisingly, it was found that the concentration at which biofilm formation inhibitory effect was first observed in enzyme-treated royal jelly was lower than the concentration at which antibacterial effect was first observed, indicating that it has a superior biofilm formation inhibitory effect compared to its antibacterial effect. As a result, it was found that enzyme-treated royal jelly is a novel natural material that can selectively suppress the formation of biofilms by oral bacteria.

[0015] Based on these findings, the inventors have succeeded in creating a composition containing enzyme-treated royal jelly as an active ingredient, which is a naturally derived component but has an inhibitory effect on oral biofilm formation, in order to solve the problems of the present invention. The present invention is completed based on these first-of-its-kind findings and first successful examples achieved by the inventors.

[0016] Therefore, from each aspect of the present invention, the following embodiments are provided: [1] A composition for inhibiting oral biofilm formation, comprising enzyme-treated royal jelly as an active ingredient. [2] Aggregatebacter actinomycetemucomitans ( Aggregatibacter actinomycetemcomitans ), Streptococcus mutans ( Streptococcus mutans ), Streptococcus sobrinus ( Streptococcus sobrinus ) and Streptococcus sanguinis ( Streptococcus sanguinisThe composition for inhibiting the formation of oral biofilm according to [1], which targets the oral biofilm formed on the tooth surface by at least one kind of oral bacterium selected from the group consisting of [3] The composition for inhibiting the formation of oral biofilm according to [1] or [2], which is a cosmetic, food or drink, quasi-drug or pharmaceutical used for oral care of humans.

Effects of the Invention

[0017] Enzyme-treated royal jelly has an excellent effect of inhibiting the formation of oral biofilm, and thus is useful as an active ingredient of a composition for inhibiting the formation of oral biofilm. The composition for inhibiting the formation of oral biofilm according to one aspect of the present invention can inhibit the formation of biofilm in the oral cavity without unduly affecting the oral flora.

[0018] In addition, since the active ingredient of the composition for inhibiting the formation of oral biofilm according to one aspect of the present invention is a natural-derived enzyme-treated royal jelly with food experience, the overall safety can be high. Therefore, the composition for inhibiting the formation of oral biofilm according to one aspect of the present invention can be expected to be used in various forms such as cosmetics, food and drink, pharmaceuticals, quasi-drugs, etc. to inhibit the formation of biofilm in the oral cavity.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

[0020] The following describes in detail embodiments for carrying out the present invention, but the present invention is not limited to the following embodiments.

[0021] "Content" is synonymous with concentration and amount added (amount used), and refers to the ratio of the amount of the component to the total amount of the composition. However, the total amount of the component content shall not exceed 100%. When using a commercially available product, the component content is preferably the amount of the component contained in the commercially available product, but it may also be the amount of the commercially available product itself. "Food and beverages" is a comprehensive term that refers to food products, beverages, or both. "Comprise" (or "contain") means that elements other than those explicitly stated as being included can be added (it is synonymous with "at least include"), but it also encompasses "consist of" and "essentially consist of." In other words, "comprise" can mean that it includes the explicitly stated elements and any one or more of those elements, that it consists of the explicitly stated elements, or that it essentially consists of the explicitly stated elements. Examples of elements include components, processes, conditions, parameters, and other limitations. The "~" in a numerical range indicates a range that includes the numbers before and after it, and also includes the range excluding one of the limit values ​​that include those numbers. For example, "0%~100%" means that the range can be 0% or greater and 100% or less.

[0022] The "oral biofilm formation inhibitory effect" of an oral biofilm formation inhibitory composition refers to the effect of inhibiting oral bacteria from forming a biofilm on the tooth surface. Compared to the same conditions except for the use of the oral biofilm formation inhibitory composition, this effect may be to reduce the amount of biofilm formed, to prevent biofilm formation altogether, or to decompose a biofilm that has already formed on the tooth surface.

[0023] A composition for inhibiting oral biofilm formation according to one aspect of the present invention contains enzyme-treated royal jelly as an active ingredient. Enzyme-treated royal jelly has the effect of inhibiting the formation of biofilms by oral bacteria. By containing enzyme-treated royal jelly, the composition for inhibiting oral biofilm formation according to one aspect of the present invention can inhibit the formation of biofilms by oral bacteria.

[0024] In the examples described later, it has been observed that enzyme-treated royal jelly inhibits the formation of oral biofilms. Therefore, the biofilm targeted by the oral biofilm formation inhibitory composition is Aggregatibacter actinomycetemucomitans. Aggregatibacter actinomycetemcomitans ), Streptococcus mutans ( Streptococcus mutans ), Streptococcus sobrinus ( Streptococcus sobrinus ) and Streptococcus sanguinis ( Streptococcus sanguinisPreferably, the oral biofilm is formed on the tooth surface by one, two, three, or all four of the following oral bacteria. Aggregatebacter actinomycetemcomitans is known as a pathogen of periodontal disease. Streptococcus mutans and Streptococcus sobrinus are known as pathogens of dental caries. Streptococcus mutans and Streptococcus sanguinis are known as pathogens of infectious endocarditis (see, for example, the "Guidelines for the Prevention and Treatment of Infectious Endocarditis (2017 Revised Edition)"). An oral biofilm formation inhibitory composition according to one aspect of the present invention may be used to inhibit the formation of oral biofilms, thereby preventing or treating diseases such as periodontal disease, dental caries, and infectious endocarditis.

[0025] [Enzyme-treated royal jelly] Royal jelly is a milky white, jelly-like substance produced by mixing secretions from the hypopharyngeal and mandibular glands of worker bees aged 3 to 12 days. The main physiologically active components in royal jelly include, for example, organic acids such as saturated and unsaturated fatty acids unique to royal jelly, as well as proteins, amino acids, peptides, lipids, sugars, vitamins such as B vitamins, folic acid, nicotinic acid, and pantothenic acid, and various minerals.

[0026] The royal jelly used in this invention includes fresh royal jelly, dried royal jelly, dried royal jelly powder, royal jelly extract, fermented royal jelly, enzyme-treated royal jelly, and the like. The royal jelly may be sourced from European countries, Oceania countries, the United States, Brazil, Japan, China, and other Asian countries, and any of these may be used.

[0027] Dried royal jelly powder is obtained by drying raw royal jelly to produce a powder. Drying methods include natural drying such as air drying and sun drying, forced drying by heating with electricity, and freeze-drying, which are methods used in general food processing, and freeze-drying is preferred.

[0028] Royal jelly extract is obtained by subjecting royal jelly (including raw, dried, and pulverized products) to an extraction process using water and an extraction solvent such as aqueous ethanol.

[0029] Royal jelly fermented products can be produced by subjecting royal jelly (including raw, dried, and pulverized products) to a conventional fermentation process using microorganisms such as yeast and lactic acid bacteria.

[0030] Enzyme-treated royal jelly is obtained by treating royal jelly with a protease. By subjecting royal jelly to protease treatment, the proteins (allergens) contained in royal jelly can be broken down. Enzyme-treated royal jelly is preferably low-allergen enzyme-treated royal jelly, as it suppresses the induction of allergic reactions. In addition to the protease breakdown products of proteins contained in royal jelly, enzyme-treated royal jelly may contain organic acids such as saturated and unsaturated fatty acids, lipids, sugars, vitamins, and various minerals.

[0031] The royal jelly used in the production of enzyme-treated royal jelly is not particularly limited and includes, for example, raw royal jelly, dried royal jelly powder obtained by drying and pulverizing raw royal jelly, or royal jelly extract obtained by extracting raw royal jelly with water or aqueous ethanol. Enzyme-treated royal jelly can be produced by treating royal jelly raw material with an enzyme having at least endopeptidase activity, an enzyme having at least exopeptidase activity, and / or an enzyme having both endopeptidase activity and exopeptidase activity.

[0032] Examples of proteolytic enzymes that possess at least endopeptidase activity include endopeptidases derived from animals (e.g., trypsin, chymotrypsin), plants (e.g., papain), and microorganisms (e.g., lactic acid bacteria, yeast, mold, Bacillus subtilis, actinomycetes, etc.).

[0033] Examples of proteolytic enzymes that possess at least exopeptidase activity include carboxypeptidases, aminopeptidases, exopeptidases derived from microorganisms (e.g., lactic acid bacteria, Aspergillus species, Rhizopus species, etc.), and pancreatin and pepsin, which also possess endopeptidase activity.

[0034] Among these, preferred specific examples of enzymes that possess both exopeptidase and endopeptidase activity include Streptomyces griseus-produced peptidase (trade name: Actinase AS), Aspergillus oryzae-produced peptidase (trade names: Protease A, Flavorzyme, Proteax), and Aspergillus melleus-produced peptidase (trade name: Protease P).

[0035] Preferred examples of enzymes with exoprotease activity include Aspergillus oryzae-produced peptidase (product names: Umamizyme G, Promod 192P, Promod 194P, Sumizyme FLAP), Aspergillus sojae-produced peptidase (product name: Sternzyme B15024), Aspergillus-produced peptidase (product name: Cokurase P), and Rhizopus oryzae-produced peptidase (product name: Peptidase R).

[0036] Preferred examples of enzymes with endoprotease activity include peptidase produced by Bacillus subtilis (trade names: Orientase 22BF, Nucleicin), peptidase produced by Bacillus licheniformis (trade name: Alcalase), peptidase produced by Bacillus stearothermophilus (trade name: Protease S), peptidase produced by Bacillus amyloliquefaciens (trade name: Neutralase), and peptidases produced by other Bacillus species (trade name: Protamex).

[0037] Enzymatic treatment of royal jelly can be carried out, for example, in accordance with the descriptions in Japanese Patent Publication No. 2007-295919 (especially Examples 1 to 3), Japanese Patent Publication No. 2007-295920 (especially Examples 1 to 20), and Japanese Patent No. 7218961 (especially Example 1).

[0038] Commercially available enzyme-treated royal jelly can be used. Specific examples of commercially available royal jelly include, for instance, "Enzyme-Treated Royal Jelly King" (manufactured by Yamada Bee Farm Co., Ltd.).

[0039] [Composition for inhibiting oral biofilm formation] A composition for inhibiting oral biofilm formation according to one aspect of the present invention contains enzyme-treated royal jelly as an active ingredient.

[0040] The amount of enzyme-treated royal jelly should be sufficient to suppress the formation of biofilm by oral bacteria, and can be adjusted as appropriate depending on the final form (dosage form) of the composition. However, the lower limit is, as a solid content relative to the total amount of the composition, for example, 0.1% or more, 1% or more, 3% or more, 3.5% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or more, 99% or more, or less than 100% by mass.

[0041] The upper limit for the content of enzyme-treated royal jelly is, as solid content relative to the total amount of the composition, for example, less than 100% by mass, 99% or less by mass, 98% or less by mass, 95% or less by mass, 93% or less by mass, 90% or less by mass, 85% or less by mass, 80% or less by mass, 75% or less by mass, 70% or less by mass, 65% or less by mass, 60% or less by mass, 55% or less by mass, 50% or less by mass, 45% or less by mass, 40% or less by mass, 35% or less by mass, 30% or less by mass, 25% or less by mass, 20% or less by mass, 15% or less by mass, 10% or less by mass, 8% or less by mass, 5% or less by mass, 3% or less by mass, or 1% or less by mass.

[0042] Compositions for inhibiting oral biofilm formation are administered to mammals, preferably humans, for example, to suppress biofilm formation in the oral cavity. Oral administration is preferred for oral biofilm formation inhibitory compositions.

[0043] The dosage of the oral biofilm formation inhibitory composition when administered orally can be appropriately determined depending on the active ingredient, the form of the composition, and the method and amount of application. For example, for an adult weighing 60 kg, the daily dose can preferably be 10 mg to 30,000 mg in terms of dry solids, more preferably 600 mg to 12,000 mg, 100 mg to 20,000 mg, or 150 mg to 15,000 mg, even more preferably 1,200 mg to 10,000 mg, and still more preferably 2,400 mg to 8,000 mg. However, these dosages can be appropriately increased or decreased depending on factors such as the health condition of the target individual, the method of administration, the type of active ingredient, and combinations with other compositions.

[0044] The oral biofilm formation inhibitory composition can suppress the formation of biofilms by oral bacteria, and preferably can suppress the formation of biofilms by oral bacteria such as Aggregatibacter actinomycetemcomitans, Streptococcus mutans, Streptococcus sobrinus, and Streptococcus sanguinis.

[0045] The oral biofilm formation inhibitory composition may be administered once a day, or divided into multiple doses such as two or three times a day, as long as the daily dose is within the range described above. While the oral biofilm formation inhibitory composition can be expected to show effects in a relatively short period after administration, it is preferable to administer it continuously for 1 to 4 weeks, or for 1 month or more, 6 months or more, or 1 year or more, as this is expected to prolong or enhance the effect.

[0046] The oral biofilm formation inhibitory composition can be used as a cosmetic, food or beverage, quasi-drug, or pharmaceutical, or as part thereof, preferably as a cosmetic, food or beverage, quasi-drug, or pharmaceutical used for human oral care, or as part thereof. However, the content of the active ingredient and the dosage of the oral biofilm formation inhibitory composition in these products are preferably within the range of the amounts described above.

[0047] As described above, the oral biofilm formation inhibitory composition can be used as a cosmetic, food and beverage, preferably food and beverage intended for health, health maintenance, or promotion, more preferably health food, functional food, nutritional composition, nutritional supplement, supplement, health food, food for specified health uses, nutrient function food and functional food, quasi-drug, pharmaceutical, etc. Furthermore, the oral biofilm formation inhibitory composition can also be used as an additive that imparts an oral biofilm formation inhibitory effect.

[0048] Cosmetics, foods and beverages, quasi-drugs, and pharmaceuticals containing a composition for inhibiting oral biofilm formation as one component can be manufactured, for example, by adding the oral biofilm formation inhibiting composition to an intermediate product in the manufacturing process of these products.

[0049] In addition to the active ingredient enzyme-treated royal jelly, cosmetics may contain other ingredients commonly used in cosmetics, such as disinfectants, preservatives, surfactants, alcohols, aqueous components, water, colorants, pH adjusters, solubilizers, abrasives, foaming agents, humectants, enzymes, flavoring agents, chelating agents, thickeners, and cleaning agents (lactic acid bacteria), as needed.

[0050] The uses of cosmetics may be limited to oral care, preferably for human oral care, and examples include (paste or liquid) toothpaste and mouthwash.

[0051] Food and beverages contain other ingredients in addition to the active ingredients, such as minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solvents, and humectants.

[0052] Food and beverages include all foods and beverages that can be consumed by mammals, preferably humans. The types of food and beverages can be those intended for oral care, preferably human oral care, and include, for example, dairy products; fermented foods (yogurt, etc.); beverages (soft drinks such as coffee, juice, and tea, milk drinks, lactic acid bacteria drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, sake, Western liquors, fruit wines, etc.); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western confectionery (chocolate, donuts, pies, cream puffs, gum, gummies, jelly, candy, cookies, cakes, puddings, etc.); Japanese confectionery (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, ice pops, sherbet, etc.); food products (curry, beef bowl, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

[0053] The manufacturing methods for cosmetics and food and beverages are not particularly limited as long as they contain an active ingredient and / or a composition for inhibiting oral biofilm formation, and can be carried out according to known methods as appropriate.

[0054] The dosage form of the oral biofilm formation inhibitory composition used as a supplement is not particularly limited and can be selected as appropriate. Examples include chewable tablets, lozenges, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

[0055] Pharmaceuticals contain other ingredients in addition to the active ingredient, such as vitamins and herbal medicines. The active ingredient and other ingredients can also be used in combination with other pharmaceutical ingredients listed in the Japanese Pharmacopoeia.

[0056] When preparing a composition for inhibiting oral biofilm formation as a pharmaceutical product, the active ingredient may be prepared together with other pharmaceutical ingredients in the form of tablets (including uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders, granules, liquids, suspensions, emulsions, syrups, pastes, or injections (including cases where the composition is mixed with distilled water or intravenous solutions such as amino acid solutions or electrolyte solutions at the time of use to prepare a liquid formulation), and this formulation may be used as a pharmaceutical product for the purpose of oral care, preferably for human oral care.

[0057] The drug may be administered locally. Oral administration is preferred.

[0058] Pharmaceuticals may contain, in addition to the active ingredient, pharmaceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, stabilizers, surfactants, sweeteners, flavoring agents, and fragrances, as appropriate.

[0059] Furthermore, compositions for inhibiting oral biofilm formation may also be used as quasi-drugs.

[0060] A composition for inhibiting intraoral biofilm formation according to one aspect of the present invention is applied to oral care for mammals, including humans (preferably humans).

[0061] Furthermore, since one embodiment of the present invention's composition for inhibiting oral biofilm formation uses enzyme-treated royal jelly, which has been conventionally used as a food ingredient, as an active ingredient, it can be used as a highly safe product overall. [Examples]

[0062] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0063] <Method> Aggregatibacter actinomycetemcomitans, a periodontal pathogenic bacterium Aggregatibacter actinomycetemcomitans ), Streptococcus mutans, an oral streptococcus considered to be a cariogenic bacterium ( Streptococcus mutans ) and Streptococcus sobrinus ( S. sobrinus ), as well as Streptococcus sanguinis, the causative agent of infectious endocarditis ( S. sanguinis The antibacterial activity (growth inhibition) and biofilm formation inhibition activity of enzyme-treated royal jelly were evaluated as follows, using the target bacteria () as the target bacteria.

[0064] Depending on the target bacterial species, Brain Heart Infusion (BHI) medium (BD) containing or not containing sucrose was prepared by dissolving enzyme-treated royal jelly (manufactured by Yamada Bee Farm Co., Ltd.; manufactured according to the method described in Example 1 of Patent No. 7218961) in a series of 2-fold dilutions with a maximum concentration of 40 mg / mL (40 mg / mL, 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL, 0.156 mg / mL, 0.0781 mg / mL, and 0.0391 mg / mL) in the Brain Heart Infusion (BHI) medium (manufactured by BD) with or without sucrose, as shown in Table 1. The target bacteria were seeded and cultured in 96-well plates using the medium (0.2 mL) and culture environment shown in Table 1.

[0065] [Table 1]

[0066] After 72 hours of incubation, the bacterial load (growth inhibition) and biofilm formation (biofilm inhibition) of the target bacteria were evaluated using enzyme-treated royal jelly.

[0067] Bacterial quantity was evaluated by measuring turbidity (absorbance at 620 nm), which represents the amount of target bacteria in the well. Biofilm formation was evaluated by adding 0.1% crystal violet to the well after removing the culture medium and allowing it to stand for 20 minutes to perform crystal violet staining. Then, the well was washed with 0.2 mL of purified water, followed by adding 0.2 mL of 95% ethanol, and then measuring the absorbance of the well at 540 nm. All absorbances were measured using "Multiskan FC" (Thermo).

[0068] Furthermore, the biofilm formation of the target bacteria was observed using SYTO9 (Thermo Corporation) and fluorescent images were obtained by observing the samples with and without the addition of enzyme-treated royal jelly at a concentration of 10 mg / mL using a "BZ-X800" (Keyence Corporation).

[0069] <Result> The evaluation results for bacterial load and biofilm formation are shown in Figures 1 and 2, and Table 2. Figure 1 shows the bacterial load (Figures 1(A) to (D)) and biofilm formation (Figures 1(E) to (H)) for each concentration of enzyme-treated royal jelly against Aggregatibacter actinomycetemucomitans, Streptococcus mutans, Streptococcus sobrinus, and Streptococcus sanguinis. Furthermore, based on the results in Figure 1, for each target bacterium, the minimum concentration of enzyme-treated royal jelly at which the bacterial load was reduced to 80% or less compared to the case without enzyme-treated royal jelly (concentration of 0 mg / mL) was defined as the minimum growth inhibition concentration, and the minimum concentration of enzyme-treated royal jelly at which the biofilm formation was reduced to 80% or less was defined as the minimum biofilm formation inhibition concentration, and these results are summarized in Table 2.

[0070] [Table 2]

[0071] As shown in Figure 1, enzyme-treated royal jelly at a predetermined concentration reduced bacterial count and biofilm formation compared to the control group without enzyme-treated royal jelly, demonstrating both growth inhibitory and biofilm formation inhibitory effects on all oral bacteria tested. Furthermore, as shown in Table 2, the minimum biofilm formation inhibitory concentration was lower than the minimum growth inhibitory concentration for all oral bacteria tested with enzyme treatment.

[0072] Figure 2 shows the results of comparing Aggregatibacter actinomycetemcomitans and Streptococcus sanguinis with and without the addition of 10 mg / mL of enzyme-treated royal jelly.

[0073] As shown in Figure 2, it was found that when enzyme-treated royal jelly was used at a concentration of 10 mg / mL, biofilm formation could be efficiently suppressed in all oral bacteria without causing an excessive reduction in bacterial load.

[0074] From these results, it was found that enzyme-treated royal jelly has an inhibitory effect on biofilm formation against oral bacteria. Furthermore, it was found that the minimum concentration of enzyme-treated royal jelly required to inhibit biofilm formation was lower than the minimum concentration required to inhibit bacterial growth. Based on these results, it can be concluded that by using enzyme-treated royal jelly, biofilm formation can be efficiently suppressed without excessively killing oral bacteria. [Industrial applicability]

[0075] One embodiment of the present invention provides an oral biofilm formation inhibitory composition that contains naturally derived and safe active ingredients, which can suppress the formation of biofilms in the oral cavity of living organisms, thereby preventing or treating diseases caused by biofilm formation. Furthermore, one embodiment of the present invention provides an oral biofilm formation inhibitory composition that can be used in various forms, such as cosmetics, foods and beverages, pharmaceuticals, and quasi-drugs, for the purpose of oral care for humans.

Claims

1. A composition for inhibiting oral biofilm formation, containing enzyme-treated royal jelly as an active ingredient, targeting oral biofilms formed on tooth surfaces by Aggregatilacter actinomicetemucomitans.

2. The oral biofilm formation inhibitory composition according to claim 1, which is a cosmetic, food or beverage, quasi-drug or pharmaceutical product used for human oral care.

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