Composition for suppressing oral biofilm formation

Abstract

An object of the present invention is to provide a composition comprising, as an active ingredient, a component having an effect of suppressing oral biofilm formation without excessively affecting the growth of oral bacteria although the component is derived from a natural product.SOLUTION: The above object can be achieved by a composition for suppressing oral biofilm formation comprising enzyme-treated royal jelly as an active ingredient, and the composition for suppressing oral biofilm formation which is a cosmetic, a food or drink, a quasi-drug, or a pharmaceutical used for oral care of humans.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition for inhibiting oral biofilm formation. [Background technology]

[0002] Biofilms are three-dimensional structures consisting of a group of microorganisms attached to the surface of a material in contact with water and the extracellular polysaccharides they produce. The extracellular polysaccharides produced by the microorganisms protect the microorganisms and contribute to the formation of microbial microcolonies. A biofilm is formed when a diverse group of bacteria enters and multiplies within these microcolonies. Typical examples of biofilms include slime that forms in wet areas such as kitchens, bathrooms, and washrooms, and plaque that forms in the oral cavity due to bacteria associated with periodontal disease.

[0003] Hundreds of species of bacteria exist in the oral cavity, and these bacteria combine to form complex oral biofilms. Among these oral bacteria, oral pathogenic bacteria such as oral streptococci, including periodontal pathogens and cariogenic bacteria, are known to cause oral diseases. Oral pathogenic bacteria are known to not only cause oral diseases but also serious systemic diseases such as aspiration pneumonia, infective endocarditis, Alzheimer's disease, and diabetes.

[0004] In order to prevent diseases caused by oral pathogenic bacteria, materials that inhibit the growth of oral pathogenic bacteria are being searched for. However, the pathogenicity of oral pathogenic bacteria is not immediately manifested by bacterial proliferation in the oral cavity. Oral pathogenic bacteria can exert their pathogenicity by colonizing the oral cavity, i.e., by forming an oral biofilm as dental plaque on the tooth surface, thereby escaping the oral cavity's self-cleaning function by saliva. Therefore, in order to prevent diseases caused by oral pathogenic bacteria, it is essential to inhibit the formation of oral biofilms by oral pathogenic bacteria.

[0005] Because oral bacteria can cause systemic diseases, interest in oral flora, similar to that of intestinal flora, has grown in recent years. Just as regulating the intestinal flora improves not only the health of the intestine but also the overall health of the body, it is desirable to maintain the oral flora in an appropriate state. Therefore, in order to maintain the oral flora in an appropriate state, it is desirable to avoid excessive killing of oral bacteria as a whole.

[0006] To date, several materials are known to have antibacterial properties against oral pathogenic bacteria. For example, a composition containing hydroxyapatite in combination with royal jelly or its extract has been shown to be effective against Porphyromonas gingivalis ( Porphyromonas gingivalis ) and is known to exhibit a sterilization rate of 80% or more (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 is widely consumed. However, Non-Patent Document 1 states that royal jelly is effective against periodontal pathogens such as Actinomyces actinomycetemcomitans ( Actinomyces actinomycetemcomitans It has been reported that the minimum inhibitory concentration (MIC) of royal jelly against Actinomyces actinomycetemcomitans and Porphyromonas gingivalis is higher than that of the common antibacterial agent chlorhexidine. 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 Application Laid-Open 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. Summary of the Invention [Problem to be solved by the invention]

[0010] Patent Document 1 describes that combining hydroxyapatite with royal jelly or an extract thereof enhances the antibacterial activity of hydroxyapatite against Porphyromonas gingivalis, but also describes that the antibacterial activity is inferior when royal jelly or an extract thereof is used alone.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, Patent Document 1 and Non-Patent Document 1 do not describe natural materials that suppress the formation of oral biofilms by oral bacteria. In particular, they do not describe 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 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. [Means for solving the problem]

[0013] In order to achieve the above object, the present inventors have conducted extensive research and attempted to find, from among the many naturally occurring substances, substances that have the effect of inhibiting oral biofilm formation, and have 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 subjecting royal jelly to enzyme treatment, would have the same weak antibacterial effect as royal jelly and exhibit almost no biofilm formation inhibitory effect. However, surprisingly, the inventors discovered that enzyme-treated royal jelly not only exhibits a weak antibacterial effect against oral pathogenic bacteria, but also exhibits excellent biofilm formation inhibitory effect. Even more surprisingly, the concentration at which enzyme-treated royal jelly first exhibited biofilm formation inhibitory effect was lower than the concentration at which antibacterial effect was first exhibited, demonstrating superior biofilm formation inhibitory effect compared to its antibacterial effect. These findings demonstrate that enzyme-treated royal jelly is a novel, naturally derived material that can selectively inhibit oral bacterial biofilm formation.

[0015] Based on these findings, the present inventors have succeeded in creating a composition that contains, as an active ingredient, enzyme-treated royal jelly, which is a naturally occurring ingredient and has the effect of inhibiting oral biofilm formation, in order to solve the problems of the present invention. The present invention was completed based on these findings and the first successful examples discovered by the present inventors.

[0016] Thus, the following embodiments are provided from each aspect of the present invention: [1] A composition for inhibiting oral biofilm formation, comprising enzyme-treated royal jelly as an active ingredient. [2] Aggregatibacter actinomycetemcomitans ( Aggregatibacter actinomycetemcomitans ), Streptococcus mutans ( Streptococcus mutans ), Streptococcus sobrinus ( Streptococcus sobrinus ) and Streptococcus sanguinis ( Streptococcus sanguinis) ) ) ) ) ) ). [3] A composition for inhibiting oral biofilm formation described in [1] or [2], which is a cosmetic, food or beverage, quasi-drug, or pharmaceutical used for human oral care. [Effects of the Invention]

[0017] Since enzyme-treated royal jelly has an excellent inhibitory effect on oral biofilm formation, it is useful as an active ingredient of a composition for inhibiting oral biofilm formation. The composition for inhibiting oral biofilm formation of one embodiment of the present invention can inhibit the formation of oral biofilms without excessively affecting the oral bacterial flora.

[0018] Furthermore, the oral biofilm formation-inhibiting composition of one embodiment of the present invention has a high overall safety because the active ingredient is naturally derived and is enzyme-treated royal jelly, which is a commonly eaten food. Therefore, the oral biofilm formation-inhibiting composition of one embodiment of the present invention is expected to be used to inhibit oral biofilm formation in various forms, such as cosmetics, foods and beverages, pharmaceuticals, and quasi-drugs. [Brief explanation of the drawings]

[0019]

Figure 1

Figure 2

[0020] Hereinafter, the embodiments for carrying out the present invention will be described in detail, 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 a component to the total amount of the composition. However, the total amount of the components does not exceed 100%. When a commercially available product is used, the content of the component is preferably the amount of the component contained in the commercially available product, but may also be the amount of the commercially available product itself. "Food and beverage" is a generic term meaning food, beverages, or both. "Comprise," "contain," and "include" mean that elements other than those explicitly stated as included may be added (same meaning as "comprise at least"), but also encompass "consist of" and "essentially consist of." That is, "comprise" can mean including the explicitly stated elements and any one or more elements, consisting of the explicitly stated elements, or essentially consisting of the explicitly stated elements. Elements include limitations such as ingredients, steps, conditions, and parameters. The "to" in a numerical range means a range that includes both the preceding and following values, and also includes a range excluding one of the included limits. For example, "0% to 100%" means that the range may be greater than or equal to 0% and less than or equal to 100%.

[0022] The "oral biofilm formation inhibitory effect" of the composition for inhibiting oral biofilm formation refers to the effect of inhibiting oral bacteria from forming biofilms on the surface of the teeth (tooth surface), and may be an effect of reducing the amount of biofilm formed compared to the same conditions except for the use of the composition for inhibiting oral biofilm formation, an effect of preventing biofilm formation, or an effect of decomposing biofilms that have already formed on the tooth surface.

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

[0024] In the examples described below, it has been confirmed that enzyme-treated royal jelly inhibits the formation of oral biofilms. Therefore, it is believed that the biofilms targeted by the composition for inhibiting oral biofilm formation are those formed by Aggregatibacter actinomycetemcomitans ( 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 species of oral bacteria. Aggregatibacter 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 infective endocarditis (see, for example, the "Guidelines for the Prevention and Treatment of Infective Endocarditis (2017 Revised Edition)"). The composition for inhibiting oral biofilm formation according to one embodiment of the present invention may be used to inhibit oral biofilm formation, thereby preventing or treating diseases such as periodontal disease, dental caries, and infective endocarditis.

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

[0026] Royal jelly in the present invention includes fresh royal jelly, dried royal jelly, dried royal jelly powder, royal jelly extract, royal jelly fermentation product, enzyme-treated royal jelly, etc. Royal jelly may be produced in any of Europe, Oceania, America, Brazil, Japan, China, other Asian countries, etc.

[0027] Dried royal jelly powder can be obtained by subjecting fresh royal jelly to a drying treatment to pulverize it. Drying methods include natural drying such as ventilation drying or sun drying, forced drying using heat such as electricity, and freeze-drying, which are methods commonly used in food processing, with freeze-drying being preferred.

[0028] Royal jelly extract is obtained by subjecting royal jelly (including raw, dried and crushed royal jelly) to extraction treatment with an extraction solvent such as water or aqueous ethanol.

[0029] The royal jelly fermented product can be produced by subjecting royal jelly (including raw, dried and pulverized royal jelly) to a conventional fermentation treatment 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 the royal jelly can be decomposed. The enzyme-treated royal jelly is preferably hypoallergenic enzyme-treated royal jelly, which suppresses the induction of allergic reactions. In addition to protease decomposition products of proteins contained in royal jelly, the enzyme-treated royal jelly may contain organic acids such as the aforementioned saturated fatty acids and unsaturated fatty acids, lipids, sugars, vitamins, various minerals, and the like.

[0031] The royal jelly used to produce enzyme-treated royal jelly is not particularly limited, and examples include raw royal jelly, dried royal jelly powder obtained by drying and powdering raw royal jelly, and royal jelly extract obtained by extracting raw royal jelly with water or aqueous ethanol, etc. Enzyme-treated royal jelly can be produced by treating royal jelly raw materials 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 having at least endopeptidase activity include endopeptidases derived from animals (e.g., trypsin, chymotrypsin, etc.), plants (e.g., papain, etc.), and microorganisms (e.g., lactic acid bacteria, yeast, mold, Bacillus subtilis, actinomycetes, etc.).

[0033] Examples of proteolytic enzymes having at least exopeptidase activity include carboxypeptidase, aminopeptidase, exopeptidases derived from microorganisms (e.g., lactic acid bacteria, Aspergillus, Rhizopus, etc.), and pancreatin and pepsin, which also have endopeptidase activity.

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

[0035] Specific preferred examples of enzymes having exoprotease activity include peptidases produced by Aspergillus oryzae (trade names: Umamizyme G, Promod 192P, Promod 194P, Sumizyme FLAP), peptidases produced by Aspergillus sojae (trade name: Sternzyme B15024), peptidases produced by the genus Aspergillus (trade name: Coclase P), and peptidases produced by Rhizopus oryzae (trade name: Peptidase R).

[0036] Preferred examples of enzymes having endoprotease activity include peptidases produced by Bacillus subtilis (trade names: Orientase 22BF, Nucleicin), peptidases produced by Bacillus licheniformis (trade name: Alcalase), peptidases produced by Bacillus stearothermophilus (trade name: Protease S), peptidases produced by Bacillus amyloliquefaciens (trade name: Neutrase), and peptidases produced by the genus Bacillus (trade name: Protamex).

[0037] Enzyme treatment of royal jelly can be carried out according to the descriptions in, for example, JP-A No. 2007-295919 (particularly Examples 1 to 3), JP-A No. 2007-295920 (particularly Examples 1 to 20), and Japanese Patent No. 7218961 (particularly Example 1).

[0038] Commercially available enzyme-treated royal jelly may be used. Specific examples of commercially available royal jelly include "Enzyme-treated Royal Jelly King" (manufactured by Yamada Bee Farm).

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

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

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

[0042] The composition for inhibiting oral biofilm formation is administered to, for example, a mammal, preferably a human, to inhibit biofilm formation in the oral cavity. The composition for inhibiting oral biofilm formation is preferably administered orally.

[0043] The dosage of the oral biofilm formation-inhibiting composition for oral administration can be determined as appropriate depending on the active ingredient, the form of the composition, and the application method and amount, but for example, for an adult weighing 60 kg, the daily dose, calculated on a dry solids basis, is preferably 10 mg to 30,000 mg, 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 even more preferably 2,400 mg to 8,000 mg. However, these dosages can be increased or decreased as appropriate depending on factors such as the health condition of the subject to be ingested, the administration method, the type of active ingredient, and the combination with other compositions.

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

[0045] As long as the daily dose is within the above-mentioned range, the oral biofilm formation-inhibiting composition may be administered once a day or in divided doses such as twice or three times a day. Although the oral biofilm formation-inhibiting composition is expected to be effective in a relatively short period of time after administration, continuous administration for one to four weeks, one month or more, six months or more, or one year or more is expected to further sustain or enhance the effect, and therefore continuous administration is preferred.

[0046] The composition for inhibiting oral biofilm formation can be used as, or as a part of, cosmetics, foods and beverages, quasi-drugs, or pharmaceuticals, and is preferably used as, or as a part of, cosmetics, foods and beverages, quasi-drugs, or pharmaceuticals for human oral care. However, it is preferable that the content of the active ingredient in these and the dosage of the composition for inhibiting oral biofilm formation are within the ranges described above.

[0047] As described above, the composition for inhibiting oral biofilm formation can be used as cosmetics, food and beverages, preferably food and beverages intended for health, health maintenance, or promotion, more preferably health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, nutrient functional foods and foods with functional claims, quasi-drugs, pharmaceuticals, etc. The composition for inhibiting oral biofilm formation can also be used as an additive that imparts an inhibitory effect on oral biofilm formation.

[0048] Cosmetics, foods and beverages, quasi-drugs, and pharmaceuticals that contain the composition for inhibiting oral biofilm formation as one of their ingredients can be produced, for example, by adding the composition for inhibiting oral biofilm formation to intermediate products in the manufacturing process of these products.

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

[0050] The cosmetic product may be used for oral care, preferably for human oral care, and examples thereof include (paste or liquid) dentifrices and mouthwashes.

[0051] In addition to the active ingredient, foods and beverages contain other 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, solubilizers, and humectants.

[0052] The food and drink products include any food and drink products that can be ingested by mammals, preferably humans. The types of food and drink products may be any products intended for oral care, preferably human oral care, such as dairy products, fermented foods (yogurt, etc.), beverages (soft drinks such as coffee, juice, and tea drinks, dairy drinks, lactic acid bacteria drinks, lactic acid bacteria-containing drinks, yogurt drinks, carbonated drinks, sake, Western liquor, and alcoholic beverages such as fruit wine, etc.), spreads (custard cream, etc.), pastes (fruit paste, etc.), Western confectionery products (chocolate, donuts, pies, cream puffs, gum, gummies, jellies, candies, cookies, cakes, puddings, etc.), Japanese confectionery products (daifuku, mochi, manju, castella, anmitsu, yokan, etc.), frozen desserts (ice cream, popsicles, sorbets, etc.), foods (curry, beef bowls, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.), and seasonings (dressings, sprinkles, umami seasonings, soup bases, etc.).

[0053] The method for producing cosmetics and foods and beverages is not particularly limited as long as they contain an active ingredient and / or a composition for inhibiting oral biofilm formation, and can be any known method as appropriate.

[0054] When using the composition for inhibiting oral biofilm formation as a supplement, the dosage unit form 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, herbal medicines, etc. The active ingredient and other ingredients can also be mixed with other pharmaceutical ingredients listed in the Japanese Pharmacopoeia before use.

[0056] When preparing a composition for inhibiting oral biofilm formation as a pharmaceutical, the active ingredient, together with ingredients acceptable for pharmaceuticals, can be prepared in the form of tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (dispersed medicines), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, injections (including cases where the composition is mixed with distilled water or infusions such as amino acid infusions or electrolyte infusions at the time of use to prepare a liquid), or the like, to form a pharmaceutical formulation intended for oral care of the target, preferably for human oral care.

[0057] The pharmaceutical agent may be administered topically, preferably orally.

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

[0059] The composition for inhibiting oral biofilm formation may also be used as a quasi-drug.

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

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

[0062] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0063] <Method> The periodontal pathogen Aggregatibacter actinomycetemcomitans ( Aggregatibacter actinomycetemcomitans ), and Streptococcus mutans ( Streptococcus mutans ) and Streptococcus sobrinus ( S. sobrinus ), and Streptococcus sanguinis ( S. sanguinis ) was used as the target bacteria, and the antibacterial activity (growth inhibitory activity) and biofilm formation inhibitory activity of enzyme-treated royal jelly were evaluated as follows.

[0064] Brain heart infusion (BHI) medium (BD) with or without sucrose depending on the target bacterial species was dissolved in a serial 2-fold dilution series of enzyme-treated royal jelly (Yamada Bee Farm; produced according to the method described in Example 1 of Japanese Patent No. 7218961) at concentrations of 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, with a maximum concentration of 40 mg / mL. The media were prepared as shown in Table 1. The target bacteria were seeded and cultured in 96-well plates using the media (0.2 mL) and under the culture conditions shown in Table 1.

[0065] [Table 1]

[0066] After 72 hours of incubation, the effect of the enzyme-treated royal jelly on the growth inhibition and biofilm formation of the target bacteria was evaluated.

[0067] The bacterial load was assessed by measuring the turbidity (absorbance at 620 nm), which indicates the amount of target bacteria in the well. The amount of biofilm formation was assessed by adding 0.1% crystal violet to the well after removing the culture medium, leaving it to stand for 20 minutes, staining with crystal violet, washing the well with 0.2 mL of purified water, adding 0.2 mL of 95% ethanol, and then measuring the absorbance at 540 nm. Both absorbances were measured using a "Multiskan FC" (Thermo).

[0068] In addition, the state of biofilm formation by the target bacteria when enzyme-treated royal jelly was added at 10 mg / mL and when it was not added was examined by staining using SYTO9 (Thermo), and observing using a ``BZ-X800'' (Keyence) to obtain fluorescent images.

[0069] <Result> The evaluation results for bacterial abundance and biofilm formation are shown in Figures 1 and 2 and Table 2. Figure 1 shows the bacterial abundance (Figure 1(A)-(D)) and biofilm formation (Figure 1(E)-(H)) of each concentration of enzyme-treated royal jelly against Aggregatibacter actinomycetemcomitans, Streptococcus mutans, Streptococcus sobrinus, and Streptococcus sanguinis. Table 2 summarizes the results of Figure 1. The minimum concentration of enzyme-treated royal jelly at which the bacterial abundance was reduced to 80% or less compared to the concentration without enzyme-treated royal jelly (0 mg / mL). The minimum biofilm formation inhibitory concentration (MINI) was also calculated as the minimum concentration of enzyme-treated royal jelly at which the biofilm formation was reduced to 80% or less.

[0070] [Table 2]

[0071] As shown in Figure 1, for all oral bacteria evaluated, enzyme-treated royal jelly at a given concentration reduced the amount of bacteria and biofilm formation compared to when enzyme-treated royal jelly was not added, demonstrating its effects of inhibiting proliferation and biofilm formation. Furthermore, as shown in Table 2, for all oral bacteria evaluated for enzyme treatment, the minimum biofilm formation inhibitory concentration was lower than the minimum inhibitory concentration.

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

[0073] As shown in Figure 2, when enzyme-treated royal jelly was used at 10 mg / mL, it was found that biofilm formation could be efficiently suppressed for all oral bacteria without excessively reducing the bacterial load.

[0074] These results demonstrate that enzyme-treated royal jelly has the effect of inhibiting biofilm formation in oral bacteria. Furthermore, the minimum biofilm formation inhibitory concentration of enzyme-treated royal jelly was found to be lower than its growth inhibitory effect. These results demonstrate that the use of enzyme-treated royal jelly can efficiently inhibit biofilm formation without excessively killing oral bacteria. [Industrial Applicability]

[0075] The composition for inhibiting oral biofilm formation of one embodiment of the present invention is capable of inhibiting the formation of biofilms in the oral cavity of a living body by using an active ingredient that is naturally derived and recognized as safe, thereby making it possible to prevent or treat diseases caused by biofilm formation. Furthermore, the composition for inhibiting oral biofilm formation of one embodiment of the present invention can be used in various forms such as cosmetics, foods and beverages, pharmaceuticals, and quasi-drugs for the purpose of human oral care.

Claims

1. A composition for inhibiting oral biofilm formation, comprising enzyme-treated royal jelly as an active ingredient.

2. 2. The composition for inhibiting oral biofilm formation according to claim 1, which is targeted at oral biofilms formed on tooth surfaces by at least one oral bacterium selected from the group consisting of Aggregatibacter actinomycetemcomitans, Streptococcus mutans, Streptococcus sobrinus, and Streptococcus sanguinis.

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

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