Composition for suppressing biofilm formation by bacteria

Abstract

Disclosed is a composition for preventing and / or ameliorating caries, the composition comprising N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof as an active ingredient. This composition can suppress biofilm formation by bacteria.

Description

Composition for inhibiting bacterial biofilm formation

[0001] The present invention relates to a composition that inhibits bacterial biofilm formation.

[0002] Biofilms are membrane-like structures containing an extracellular matrix of polysaccharides, proteins, and other substances produced by microorganisms. By forming biofilms, microorganisms can grow at high densities while protecting themselves from external factors such as antibiotics and immune cells. This allows microorganisms to locally accumulate metabolites and produce toxins, which are known to have adverse effects on humans (Non-Patent Document 8).

[0003] Biofilms formed in the oral cavity of humans and other organisms are also called dental plaque and are mainly formed by Streptococcus mutans (hereinafter also referred to as "S. mutans"). It is known that as biofilms form and mature, Streptococcus mutans locally accumulates acid, which erodes the tooth surface and causes dental caries (cavities). Therefore, it is expected that inhibiting biofilm formation in the oral cavity can prevent and / or ameliorate the accumulation of the above-mentioned acid, thereby preventing and / or ameliorating dental caries.

[0004] In addition, in addition to S. mutans, several types of microorganisms have been detected in oral biofilms, including Fusobacterium nucleatum, which has been reported to be associated with colorectal cancer, and the presence of SARS-CoV2, which causes respiratory infections, has also been reported (Non-Patent Documents 1 and 2). Because oral biofilm formation is thought to protect the above-mentioned pathogens from antibiotics, immune cells, and the like (Non-Patent Document 3), oral biofilm formation may cause not only oral diseases but also systemic diseases. Therefore, suppressing oral biofilm formation is important from the perspective of maintaining human health.

[0005] Human milk oligosaccharides (HMOs) are a collective term for oligosaccharides found in breast milk, and are the third most abundant solid component in breast milk after lactose and lipids. The structure of HMOs varies depending on the type, but the main known structures are those in which fucose is bound to lactose (fucosyllactose) and those in which sialic acid is bound to lactose (sialyllactose). Representative HMOs include 2'-fucosyllactose (O-α-L-fucopyranosyl-(1→2)-O-β-D-galactopyranosyl-(1→4)-D-glucose, hereinafter also referred to as "2'-FL"), which is the most abundant in breast milk, and 3'-sialyllactose (O-(N-acetyl-α-neuramino)-D-glucose), which is the major acidic HMO. syl)-(2→3)-O-β-D-galactopyranosyl-(1→4)-D-glucose, hereinafter also referred to as "3'-SL"); and 6'-sialyllactose (6'-sialyllactose, O-(N-acetyl-α-neuraminosyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose, hereinafter also referred to as "6'-SL"), which is also a major acidic HMO.

[0006] Previous studies have reported that a mixture of HMOs extracted from human breast milk inhibits biofilm formation against Streptococcus agalactiae, a normal vaginal flora (Non-Patent Documents 4 and 5). On the other hand, it has been reported that N-acetylneuraminic acid (Neu5Ac), a type of sialic acid, promotes biofilm formation by Streptococcus pneumoniae (Non-Patent Document 7).

[0007] Knowledge about the effects of HMO and sialic acids such as Neu5Ac on oral bacteria is limited, but for example, Patent Document 1 shows that the oral bacterial flora can be improved by combining an antibacterial component containing sialic acid with an oligosaccharide component containing HMO. However, this document does not disclose the effects of sialic acid, HMO, etc. on bacterial biofilm formation. Furthermore, Non-Patent Document 6 reports that, among HMOs, 2'-FL has the effect of suppressing the adhesion of S. mutans to glass surfaces, etc. However, knowledge about the effects of sialic acids such as Neu5Ac and 6'-SL on oral bacterial biofilm formation is unknown.

[0008] Japanese Patent Application No. 2020-515564 Japanese Patent Application No. 2014-504864

[0009] Tuominen et al., “Oral microbiota and cancer development.” Pathobiology 88.2 (2021): 116-126.Gomes et al., “Dental biofilm of symptomatic COVID‐19 patients harbours SARS‐CoV‐2.” Journal of clinical periodontology 48.7 (2021): 880‐885.Ray, “Role of Virus on Oral Biofilm: Inducer or Eradicator?” Applied Biochemistry and Biotechnology 196.3 (2024): 1652‐1668.Craft et al., “Interrogation of human milk oligosaccharide fucosylation patterns for antimicrobial and antibiofilm trends in group B Streptococcus.” ACS Infectious Diseases 4.12 (2018): 1755‐1765.Craft et al., “Sialylated variants of lacto‐N‐tetraose exhibit antimicrobial activity against Group B Streptococcus.” Organic & Biomolecular Chemistry 17.7 (2019): 1893‐1900.Salli et al., “Influence of 2'‐fucosyllactose and galacto‐oligosaccharides on the growth and adhesion of Streptococcus mutans.” British Journal of Nutrition 124.8 (2020): 824‐831.Trappetti et al., “Sialic acid: a preventable signal for pneumococcal biofilm formation, colonization, and invasion of the host.” The Journal of infectious diseases 199.10 (2009): 1497‐1505. Zhao et al., “Understanding bacterial biofilms: From definition to treatment strategies.” Frontiers in cellular and infection microbiology 13 (2023): 1137947.

[0010] An object of the present invention is to provide a composition that inhibits biofilm formation by bacteria.

[0011] The present inventors have found that Neu5Ac, 6'-SL, or a salt thereof inhibits biofilm formation by bacteria.

[0012] The present disclosure provides, for example, the inventions described in the following [1] to

[29] . [1] A composition for preventing and / or improving dental caries, comprising N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof as an active ingredient. [2] A composition for inhibiting oral biofilm formation by oral bacteria, comprising N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof as an active ingredient. [3] A composition for inhibiting bacterial biofilm formation on a solid surface, comprising N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof as an active ingredient. [4] The composition described in [3], wherein the bacterium is an oral bacterium. [5] The composition described in [2] or [4], wherein the oral bacterium is Streptococcus mutans. [6] An oral care composition containing N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof as an active ingredient (however, excluding an oral flora improving agent containing (A) at least one selected from the group consisting of spermine, spermidine, sialic acid, and lactoferrin, and (B) at least one selected from the group consisting of lactulose, oligosaccharides, and sialic acid-containing disaccharides). [7] A non-cariogenic composition containing N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof. [8] The composition according to any of [1] to [6], wherein the composition is a drug or quasi-drug. [9] The composition according to [1] to [7], wherein the composition is a food or drink.

[10] A method for preventing and / or ameliorating dental caries, comprising having a subject in need thereof ingest or administer N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof.

[11] A method for inhibiting oral biofilm formation by oral bacteria, comprising administering or having a subject in need thereof ingest N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof.

[12] A method for inhibiting bacterial biofilm formation on a solid surface, comprising administering or having a subject in need thereof ingest N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof.

[13] A method for maintaining or improving oral health, comprising ingesting or administering N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof to a subject in need thereof.

[14] N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for use in a therapeutic method for preventing and / or ameliorating dental caries.

[15] N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for use in a therapeutic method for inhibiting intraoral biofilm formation by oral bacteria.

[16] N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for use in a therapeutic method for inhibiting bacterial biofilm formation on a solid surface.

[17] N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for use in a therapeutic method for maintaining or improving oral health.

[18] Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method for preventing and / or ameliorating dental caries.

[19] Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method for suppressing oral biofilm formation by oral bacteria.

[20] Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method for suppressing bacterial biofilm formation on a solid surface.

[21] Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method for maintaining or improving oral health.

[22] Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the manufacture of a composition for preventing and / or improving dental caries.

[23] Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the manufacture of a composition for suppressing oral biofilm formation by oral bacteria.

[24] Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the manufacture of a composition for suppressing bacterial biofilm formation on a solid surface.

[25] Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the manufacture of an oral care composition.

[26] The method, N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof, or use according to

[12] ,

[16] ,

[20] , or

[24] , wherein the bacterium is an oral bacterium.

[27] The method, N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof, or use according to

[11] ,

[15] ,

[19] ,

[23] , or

[26] , wherein the oral bacterium is Streptococcus mutans.

[28] The use according to any of

[22] to

[27] , wherein the composition is a pharmaceutical or quasi-drug.

[29] The use according to any of

[22] to

[27] , wherein the composition is a food or drink.

[0013] According to the present invention, a composition that inhibits biofilm formation by bacteria can be provided.

[0014] 1 shows the results of measuring biofilms when S. mutans was cultured in a medium containing xylitol, 2'-FL, 3'-SL, 6'-SL, or sodium N-acetylneuraminic acid (hereinafter also referred to as "SIA"). 2 shows the growth curve of S. mutans when cultured in a medium containing xylitol, 2'-FL, 3'-SL, 6'-SL, or SIA.

[0015] Hereinafter, embodiments of the present disclosure will be described in detail.

[0016] [A: Compositions containing Neu5Ac, 6'-SL, or a salt thereof as an active ingredient] The composition for preventing and / or ameliorating dental caries according to the present disclosure, the composition for inhibiting intraoral biofilm formation by oral bacteria according to the present disclosure, the composition for inhibiting biofilm formation on a solid surface by bacteria according to the present disclosure, the oral care composition according to the present disclosure, and the non-cariogenic composition according to the present disclosure (hereinafter, these five compositions may be collectively referred to as the "compositions according to the present disclosure") contain Neu5Ac, 6'-SL, or a salt thereof as an active ingredient. The composition according to the present disclosure may contain at least one selected from the group consisting of Neu5Ac, 6'-SL, and a salt thereof as an active ingredient.

[0017] The composition according to the present disclosure, by containing Neu5Ac, 6'-SL, or a salt thereof, can inhibit bacterial biofilm formation. The inhibition of bacterial biofilm formation may be achieved by inhibiting the adhesion of bacteria to a solid surface, or by inhibiting the growth of a biofilm that occurs after the bacteria have attached to the solid surface. Furthermore, the inhibition of biofilm growth may be achieved by inhibiting the production or secretion of biofilm components (e.g., extracellular polymeric substances, etc.) by the attached bacteria.

[0018] The inhibition of bacterial biofilm formation by the composition of the present disclosure can be confirmed by measuring the inhibition of biofilm formation by bacteria (e.g., S. mutans) on solid surfaces by the composition of the present disclosure. Specifically, if the amount of biofilm formed on the bottom surface of a cell culture plate containing the composition and medium, measured by the method described below, is less than the amount of biofilm formed under the same conditions using a cell culture plate containing only medium without the composition, the composition can be determined to have inhibited bacterial biofilm formation. For example, if the amount of biofilm formed in the former case is 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, 0.5-fold or less, 0.4-fold or less, 0.3-fold or less, or 0.2-fold or less than the amount of biofilm formed in the latter case, the composition can be determined to have inhibited bacterial biofilm formation.

[0019] The amount of biofilm can be measured using a known crystal violet staining method, etc. For example, it can be measured by the following method. First, the cell culture plate is washed with pure water. This removes floating bacteria. Next, crystal violet is added to the cell culture plate and allowed to stand to stain the biofilm. Thereafter, the cell culture plate is washed with pure water, and then ethanol is added and allowed to stand to extract the crystal violet. The amount of biofilm is measured by measuring the absorbance at 590 nm of the crystal violet extract obtained.

[0020] Substances with antibacterial activity (a collective term for the activity of killing bacteria and the activity of inhibiting bacterial growth; hereinafter, the latter activity is also referred to as "growth-inhibitory activity") are known to inhibit bacterial biofilm formation through this activity. For example, xylitol can inhibit bacterial biofilm formation through its growth-inhibitory activity (Loimaranta, Vuokko, et al. "Xylitol and erythritol inhibit real-time biofilm formation of Streptococcus mutans," BMC microbiology 20 (2020): 1-9). On the other hand, compositions according to the present disclosure containing Neu5Ac, 6'-SL, or salts thereof can inhibit bacterial biofilm formation through a mechanism different from that of biofilm formation suppressed by antibacterial activity. For example, Neu5Ac, 6'-SL, or salts thereof inhibit S. mutans growth less strongly than xylitol, but more strongly inhibits biofilm formation by S. mutans than xylitol. Substances that inhibit bacterial biofilm formation based on their antibacterial activity are prone to generating resistant bacteria, which can be a concern as it significantly reduces the biofilm formation inhibitory effect. However, the composition disclosed herein is less likely to generate resistant bacteria, and is expected to have a highly durable biofilm formation inhibitory effect.

[0021] The composition according to the present disclosure contains Neu5Ac, 6'-SL, or a salt thereof, and thereby can inhibit the formation of oral biofilms by oral bacteria such as S. mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis, with S. mutans being preferred.

[0022] The formation of a biofilm in the oral cavity by the oral bacteria is not particularly limited as long as it is on the surface of a solid or soft tissue in the oral cavity. Examples of the solid in the oral cavity include teeth or dentures, and preferably teeth. Examples of the soft tissue in the oral cavity include gums, tongue, and oral mucosa. As used herein, "solid" refers to something that has a definite shape and volume. As used herein, "soft tissue" refers to tissue in a living body other than the skeleton.

[0023] As described above, the composition according to the present disclosure can inhibit biofilm formation by oral bacteria, thereby inhibiting biofilm protection of oral bacteria from antibiotics, immune cells, etc., and inhibiting high-density proliferation of oral bacteria. This can inhibit local accumulation of metabolites and production of toxins by oral bacteria, making it suitable for oral care. Oral care refers to maintaining or improving oral health or oral function. Oral care can be achieved by inhibiting biofilm formation, inhibiting local accumulation of metabolites by oral bacteria, or inhibiting toxin production by oral bacteria.

[0024] The oral care composition according to the present disclosure may exclude an oral flora improving agent containing (A) at least one selected from the group consisting of spermine, spermidine, sialic acid, and lactoferrin, and (B) at least one selected from the group consisting of lactulose, oligosaccharides, and sialic acid-containing disaccharides.

[0025] In the oral flora improving agent, the sialic acid is a compound in which the amino group and / or hydroxy group of neuraminic acid is substituted, and may be Neu5Ac or N-glycolylneuraminic acid.

[0026] In the oral flora improving agent, a disaccharide is a saccharide formed by the bonding of two monosaccharides, and an oligosaccharide is a saccharide formed by the bonding of 3 to 10 monosaccharides. Examples of combinations of monosaccharides constituting a disaccharide or oligosaccharide include combinations of monosaccharides such as glucose (grape sugar), fructose, galactose, mannose, idose, altose, gulose, talose, allose, xylose, arabinose, lyxose, ribose, threose, erythrose, erythrulose, xylulose, ribulose, psicose, sorbose, tagatose, D-2-deoxyribose, L-rhamnose, L-fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, glucuronic acid, mannuronic acid, galacturonic acid, iduronic acid, D-glucitol, D-mannitol, ribitol, and galactitol.

[0027] The composition according to the present disclosure can inhibit biofilm formation by bacteria on the surface of a solid or soft tissue. The solid can be any solid, whether artificial or natural, on whose surface a biofilm can be formed, and examples thereof include teeth, dentures, medical instruments, and plumbing equipment. The soft tissue can be any soft tissue, whether human or non-human, on whose surface a biofilm can be formed, and examples thereof include gums, tongue, skin, oral mucosa, intestinal mucosa, and vagina. The bacteria can be any bacteria that can adhere to the surface of a solid or soft tissue to form a biofilm, and examples thereof include S. Examples of the bacterium include oral bacteria such as S. mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis, as well as Escherichia coli and Staphylococcus aureus, with oral bacteria being preferred and S. mutans being more preferred.

[0028] The composition for inhibiting bacterial biofilm formation on solid surfaces according to the present disclosure may be used in the oral cavity, skin, intestines, vagina, medical instrument cleaning, water pipes, drainage pipes, and other plumbing, with use in the oral cavity, medical instrument cleaning, water pipes, drainage pipes, and other plumbing being preferred. When used in the oral cavity, the composition can inhibit bacterial biofilm formation on, for example, teeth, dentures, or oral medical instruments. When used on the skin, the composition can inhibit bacterial biofilm formation on, for example, medical instruments in contact with the skin. When used in the intestines or vagina, the composition can inhibit bacterial biofilm formation on, for example, intestinal or vaginal medical instruments.

[0029] Furthermore, the composition according to the present disclosure contains Neu5Ac, 6'-SL, or a salt thereof, and thus can inhibit the formation of oral biofilms caused by oral bacteria, particularly S. mutans, and can therefore prevent and / or ameliorate dental caries.

[0030] As described above, the composition according to the present disclosure can inhibit biofilm formation by S. mutans, and therefore can inhibit local acid accumulation in biofilms caused by S. mutans, thereby inhibiting tooth surface erosion by the acid. Therefore, although the composition may inhibit caries, it does not promote caries, and therefore the composition is non-cariogenic. Non-cariogenicity refers to the property of not promoting caries.

[0031] The salt of Neu5Ac or 6'-SL may be a pharmaceutically acceptable salt. Examples of the pharmaceutically acceptable salt include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid; salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with amino acids such as arginine.

[0032] Neu5Ac, 6'-SL, or a salt thereof may exist in the form of crystalline polymorphs, but is not limited to any particular type, and may be a single crystalline form or a mixture of crystalline forms. Neu5Ac, 6'-SL, or a salt thereof also includes amorphous forms. Furthermore, Neu5Ac, 6'-SL, or a salt thereof includes anhydrous forms and solvates (particularly hydrates).

[0033] As Neu5Ac, 6'-SL or a salt thereof, 6'-SL, sodium salt of 6'-SL, Neu5Ac or sodium salt of Neu5Ac (SIA) are preferred.

[0034] 6'-SL or a salt thereof can be produced by a known method; for example, the sodium salt of 6'-SL can be produced by the method described in WO 2017 / 086443. Neu5Ac or a salt thereof can be produced by a known method, for example, the known method described in Yamaguchi, Shinya, et al. "Simple and large-scale production of N-acetylneuraminic acid and N-acetyl-D-mannosamine," Trends in Glycoscience and Glycotechnology 18.102 (2006): 245-252. Alternatively, commercially available Neu5Ac, 6'-SL, or a salt thereof can be used; for example, the sodium salt of 6'-SL or Neu5Ac available from Tokyo Chemical Industry Co., Ltd. can also be used.

[0035] The total content of Neu5Ac, 6'-SL, and salts thereof in the composition according to the present disclosure is not particularly limited, based on the total amount of the composition, but can be, for example, preferably 0.01% by mass to 100% by mass, more preferably 0.1% by mass to 90% by mass, and particularly preferably 1% by mass to 80% by mass.

[0036] From the viewpoint of further enhancing the effect of inhibiting bacterial biofilm formation, the composition according to the present disclosure may further contain at least one selected from the group consisting of xylitol, 2'-FL, and 3'-SL. Xylitol, 2'-FL, and 3'-SL can inhibit bacterial biofilm formation.

[0037] Xylitol, 2'-FL, and 3'-SL may exist in the form of crystalline polymorphs, but are not limited to any particular type, and may be a single crystalline form or a mixture of crystalline forms. Xylitol, 2'-FL, and 3'-SL also include amorphous forms. Xylitol, 2'-FL, and 3'-SL also include anhydrous forms and solvates (especially hydrates).

[0038] When the composition according to the present disclosure contains at least one selected from the group consisting of xylitol, 2'-FL, and 3'-SL, the total content of Neu5Ac, 6'-SL and salts thereof, and xylitol, 2'-FL, and 3'-SL in the composition is not particularly limited, but may be, for example, preferably 0.01% to 90% by mass, more preferably 0.1% to 80% by mass, based on the total amount of the composition. Furthermore, the ratio of the total content of Neu5Ac, 6'-SL and salts thereof to the total content of xylitol, 2'-FL, and 3'-SL in the composition is not particularly limited, but may be 0.01:99.9 to 99.9:0.01.

[0039] The composition according to the present disclosure (excluding the non-cariogenic composition according to the present disclosure) may be a food or drink, a food additive, a pharmaceutical product, or a quasi-drug. The non-cariogenic composition according to the present disclosure may be a food or drink, or a food additive. Subjects to which the composition according to the present disclosure can be applied include humans and non-human mammals, with humans being preferred. Examples of non-human mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, etc., and the composition according to the present disclosure can also be used as feed, which is a form of food. The human may be an adult, elderly person, child, infant, or newborn.

[0040] The composition according to the present disclosure, which is a food or drink or a food additive, is orally ingested, and the composition according to the present disclosure that passes through or remains in the oral cavity during this process can inhibit the formation of oral biofilms by bacteria in the oral cavity (for example, the formation of biofilms on the surfaces of teeth by bacteria).

[0041] The compositions according to the present disclosure, which are pharmaceuticals or quasi-drugs (excluding the non-cariogenic compositions according to the present disclosure), can be ingested or administered to the oral cavity. By ingesting or administering the compositions according to the present disclosure to the oral cavity, they can suppress intraoral biofilm formation by bacteria (e.g., biofilm formation on tooth surfaces by bacteria).

[0042] The dosage or intake of the composition according to the present disclosure is not particularly limited. For example, for a subject weighing 60 kg, the dosage or intake of Neu5Ac, 6'-SL, or a salt thereof per day can be preferably 50 mg to 30 g, and more preferably 100 mg to 10 g.

[0043] The number of times the composition according to the present disclosure is administered (ingested) is not particularly limited, but can be, for example, once to three times a day.

[0044] The food and drink products, food additives, pharmaceuticals, and quasi-drugs can be produced according to standard methods. The content of Neu5Ac, 6'-SL, or a salt thereof in the food and drink products, food additives, pharmaceuticals, and quasi-drugs is not particularly limited and can be freely set depending on the purpose.

[0045] When the composition according to the present disclosure is a food or beverage, a food additive, a drug, or a quasi-drug, the food or beverage, food additive, drug, or quasi-drug may contain ingredients that are typically used in food or beverage, food additive, drug, or quasi-drug, respectively. The food or beverage, food additive, drug, or quasi-drug according to the present disclosure may contain bases, carriers, or additives that are typically used in food or beverage, food additive, drug, or quasi-drug, respectively. Examples of additives include excipients, oils, powders, buffers, solubilizers, antioxidants, surfactants, thickeners, preservatives, pH adjusters, chelating agents, stabilizers, irritation reducers, antiseptics, pigments, colorants, fragrances, gloss-imparting agents, gelling agents, alcohols, water-soluble polymers, film-forming agents, or resins. The bases, carriers, and various additives described above may be used singly or in combination as needed.

[0046] When the composition according to the present disclosure is a food or drink, the food or drink may be a general food or drink, a food for specified health uses, a food with nutrient functions, a food with functional claims, a food for hospital patients, a supplement, etc. When the composition according to the present disclosure is a food for specified health uses, a food with nutrient functions, or a food with functional claims, the composition may be in the form of a capsule, a tablet, a granule, a powder, a lozenge, etc. When the composition according to the present disclosure is a food additive, the food additive can be contained in these foods or drinks.

[0047] When the composition according to the present disclosure is a food or beverage, the type of the food or beverage is not particularly limited as long as the caries prevention and / or amelioration effect, the effect of inhibiting biofilm formation by oral bacteria, the effect of inhibiting biofilm formation by bacteria on solid surfaces, the oral care effect, or the non-cariogenicity is not lost. Examples of foods and beverages include seasonings, processed meat products, processed agricultural products, beverages (lactic acid bacteria drinks, soft drinks, alcoholic drinks, carbonated drinks, dairy drinks, fruit juice drinks, tea, coffee, energy drinks, etc.), powdered drinks (powdered juice, powdered soup, powdered milk, etc.), concentrated beverages, confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candies, chewable tablets, tablets, chocolate, etc.), bread, cereal, etc.

[0048] When the composition according to the present disclosure is a food additive, examples of foods and beverages to which the food additive can be added include seasonings, processed meat products, processed agricultural products, beverages (lactic acid bacteria drinks, soft drinks, alcoholic drinks, carbonated drinks, dairy drinks, fruit juice drinks, tea, coffee, energy drinks, etc.), powdered drinks (powdered juice, powdered soup, powdered milk, etc.), concentrated beverages, confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candies, chewable tablets, tablets, chocolate, etc.), bread, cereal, etc.

[0049] When the composition according to the present disclosure (excluding the non-cariogenic composition according to the present disclosure) is a pharmaceutical or quasi-drug, the dosage form of the pharmaceutical or quasi-drug may be, for example, a liquid, suspension, paste, lubricant, powder, capsule, granule, pill, scatter, tablet, lozenge, etc. Indications for use as a pharmaceutical include dental caries, periodontal disease, gingivitis, otitis media, bacterial vaginosis, cystitis, urinary tract infection, pneumonia, etc.

[0050] When the composition according to the present disclosure (excluding the non-cariogenic composition according to the present disclosure) is a pharmaceutical or quasi-drug, examples of the quasi-drug include dentifrices, mouthwashes, oral fresheners, anti-halitosis agents, skin creams, and vaginal cleansers.

[0051] When the composition for inhibiting biofilm formation on a solid surface by bacteria according to the present disclosure is used for cleaning pipes such as water pipes and drainage pipes, or medical instruments, the formulation of the composition may be, for example, a liquid, suspension, powder, etc.

[0052] [B: Methods Comprising Ingestion or Administration of N-acetylneuraminic Acid, 6'-sialyllactose, or a Salt Thereof to a Subject in Need Thereof] One aspect of the present disclosure relates to a method for preventing and / or ameliorating dental caries, comprising ingesting or administering N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof to a subject in need thereof (hereinafter also referred to as "Method 1 of the present disclosure"). One aspect of the present disclosure relates to a method for inhibiting oral biofilm formation by oral bacteria, comprising ingesting or administering N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof to a subject in need thereof (hereinafter also referred to as "Method 2 of the present disclosure"). One aspect of the present disclosure relates to a method for inhibiting bacterial biofilm formation on a solid surface, comprising ingesting or administering N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof to a subject in need thereof (hereinafter also referred to as "Method 3 of the present disclosure"). One aspect of the present disclosure relates to a method for maintaining or improving oral health (hereinafter also referred to as "Method 4 of the present disclosure"), which comprises ingesting or administering N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof to a subject in need thereof.

[0053] In Methods 1 to 4 according to the present disclosure, the administration (or ingestion) conditions (e.g., the subject to be administered (or ingested), the amount to be administered (or ingested), the number of times to be administered (or ingested), etc.) can be the same as those described above in [A: Composition containing Neu5Ac, 6′-SL, or a salt thereof as an active ingredient] as the administration (or ingestion) conditions of the composition according to the present disclosure, without any restrictions.

[0054] N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof may be administered (or ingested) to a subject as is, or may be prepared as a composition according to the present disclosure described above in [A: Composition containing Neu5Ac, 6'-SL, or a salt thereof as an active ingredient] and administered (or ingested) to a subject. In the former case, N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof may be administered (or ingested) alone, or may be administered (or ingested) in combination with an additional component.

[0055] [C: N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for use in a therapeutic method] One aspect of the present disclosure relates to N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for use in a therapeutic method for preventing and / or ameliorating dental caries (hereinafter also referred to as "N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof 1 of the present disclosure"). One aspect of the present disclosure relates to N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for use in a therapeutic method for suppressing oral biofilm formation by oral bacteria (hereinafter also referred to as "N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof 2 of the present disclosure"). One aspect of the present disclosure relates to N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof (hereinafter also referred to as "N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof 3 of the present disclosure") for use in a therapeutic method for inhibiting bacterial biofilm formation on a solid surface. One aspect of the present disclosure relates to N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof (hereinafter also referred to as "N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof 4 of the present disclosure") for use in a therapeutic method for maintaining or improving oral health.

[0056] As specific embodiments of N-acetylneuraminic acid, 6'-sialyllactose, or salts thereof 1 to 4 according to the present disclosure, the embodiments relating to administration described above in [B: Method comprising administering or ingesting N-acetylneuraminic acid, 6'-sialyllactose, or salts thereof to a subject in need thereof] can be applied without limitation.

[0057] As used herein, the term "therapeutic method" refers to a method of treating a mammalian body. For example, the method may be a method aimed at or involving medical treatment. More specifically, the method may involve, for example, a medical professional administering a substance to a mammalian body or instructing a medical professional to administer a substance to a mammalian body.

[0058] [D: Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method] One aspect of the present disclosure relates to the use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method for preventing and / or ameliorating dental caries (hereinafter also referred to as "Use 1 of the present disclosure"). One aspect of the present disclosure relates to the use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method for inhibiting oral biofilm formation by oral bacteria (hereinafter also referred to as "Use 2 of the present disclosure"). One aspect of the present disclosure relates to the use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method for inhibiting bacterial biofilm formation on a solid surface (hereinafter also referred to as "Use 3 of the present disclosure"). One aspect of the present disclosure relates to the use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof in a non-therapeutic method for maintaining or improving oral health (hereinafter also referred to as "Use 4 of the present disclosure").

[0059] As specific embodiments of Uses 1 to 4 according to the present disclosure, the embodiments relating to ingestion described above in [B: A method comprising ingestion or administration of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof to a subject in need thereof] can be applied without limitation.

[0060] As used herein, the term "non-therapeutic method" refers to a method that does not fall under the category of a therapeutic method. For example, it may be a method that is not intended for and / or does not involve medical treatment. More specifically, it may be a method that does not involve a medical professional administering a substance to a mammal and / or instructing a mammal to administer a substance. Furthermore, a non-therapeutic method may be, for example, a method for the purpose of promoting health.

[0061] [E: Use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the manufacture of a composition] One aspect of the present disclosure relates to the use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the manufacture of a composition for preventing and / or ameliorating dental caries (hereinafter also referred to as "Use 1 for the manufacture according to the present disclosure"). One aspect of the present disclosure relates to the use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the manufacture of a composition for inhibiting intraoral biofilm formation by oral bacteria (hereinafter also referred to as "Use 2 for the manufacture according to the present disclosure"). One aspect of the present disclosure relates to the use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the manufacture of a composition for inhibiting bacterial biofilm formation on a solid surface (hereinafter also referred to as "Use 3 for the manufacture according to the present disclosure"). One aspect of the present disclosure relates to the use of N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof for the production of an oral care composition (hereinafter also referred to as "Use 4 for the production according to the present disclosure").

[0062] Specific embodiments of Uses 1 to 4 for manufacturing according to the present disclosure include, without limitation, the embodiments described above in [A: A composition containing Neu5Ac, 6'-SL, or a salt thereof as an active ingredient] and [B: A method comprising ingesting or administering N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof to a subject in need thereof].

[0063] The present invention will be described in more detail below with reference to examples.

[0064] The following evaluation materials and cultured bacterial cells were used in the evaluation of the biofilm formation inhibitory effect and the proliferation inhibitory effect described below.

[0065] (Evaluation Materials) The evaluation materials are as follows: Xylitol: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. 2'-FL: manufactured by Kyowa Hakko Bio Co., Ltd. 3'-SL: manufactured by Kyowa Hakko Bio Co., Ltd. 6'-SL: manufactured by Kyowa Hakko Bio Co., Ltd. SIA: manufactured by Kyowa Hakko Bio Co., Ltd.

[0066] (Cultured Bacteria) A frozen solution of S. mutans (ATCC700610, manufactured by American Type Culture Collection) was inoculated into Brain Heart Infusion (hereinafter also referred to as "BHI"; manufactured by Sigma-Aldrich) agar medium using a platinum loop and cultured at 37 ° C. for 24 hours under 5% by volume carbon dioxide conditions. Subsequently, the grown colonies were suspended in BHI dispensed in 800 μL aliquots into a 24-well multi-plate (manufactured by AGC Technoglass Co., Ltd.), and cultured at 37 ° C. for 24 hours under 5% by volume carbon dioxide conditions. The culture solution after culture was recovered, and an S. mutans culture solution was obtained.

[0067] Example 1 Evaluation of Biofilm Formation Inhibitory Effect (Preparation of Evaluation Samples) Each of the above evaluation materials was weighed and dissolved in BHI (hereinafter also referred to as "BHIs") containing 1% by mass of sucrose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a solution containing 200 mM of each evaluation material. Each solution was then filter-sterilized using a 0.2 μm pore sterilizing membrane filter (manufactured by Kurabo Industries, Ltd.), and the resulting solution was used as a BHIs solution of each evaluation material.

[0068] (Biofilm formation culture) BHIs was added to the S. mutans culture solution to prepare a bacterial solution so that the absorbance (600 nm) was 0.02. 90 μL of the BHIs solution of each of the above evaluation materials was dispensed into a 96-well multi-plate (manufactured by Violamo). As a control, 90 μL of BHIs containing no evaluation material was added to the 96-well multi-plate. Next, 90 μL of the bacterial solution was inoculated into the 96-well multi-plate, and the final evaluation material in each well was adjusted to 100 mM and the absorbance (600 nm) to 0.01. Then, the culture was incubated at 37 ° C. for 6 hours under 5% carbon dioxide by volume, and a biofilm was formed on the bottom of the plate.

[0069] (Biofilm Measurement) After biofilm formation, each well of the 96-well multi-plate was washed twice with 200 μL of pure water to remove floating bacteria. Subsequently, 200 μL of 0.1% by mass crystal violet (Fujifilm Wako Pure Chemical Industries, Ltd.) solution was added to each well and allowed to stand for 15 minutes to stain the biofilm. After that, the wells were washed twice with pure water, and 200 μL of 99.5% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well and allowed to stand for 1 hour to extract the crystal violet. The absorbance (590 nm) of each resulting extract was measured using a plate reader to measure the biofilm amount. All experiments were performed with a sample size of N = 3. Statistical testing was performed using one-way analysis, and if significant differences were observed, comparisons between each material were performed using Tukey's multiple regression analysis. A p value of less than 0.05 was considered significant. Figure 1 shows the biofilm measurement results for each evaluation material, with the control absorbance set to 1. "**" in the figure indicates that the p-value is less than 0.01.

[0070] As shown in Figure 1, it was confirmed that each of the evaluated materials (xylitol, 2'-FL, 3'-SL, 6'-SL, and SIA) inhibited biofilm formation. When comparing the evaluated materials, 6'-SL and SIA had significantly lower absorbance than xylitol, 2'-FL, and 3'-SL, demonstrating their particularly strong inhibitory effect on biofilm formation.

[0071] [Example 2 - Evaluation of proliferation inhibitory effect] (Preparation of evaluation samples) Each of the above evaluation materials was weighed and dissolved in BHI to prepare a solution containing 200 mM of each evaluation material. Subsequently, each solution was filter-sterilized using a 0.2 μm pore sterilizing membrane filter (manufactured by Kurabo Industries, Ltd.), and the resulting solution was used as a BHIs solution of each evaluation material.

[0072] (Measurement of Growth Curve) A bacterial solution was prepared by adding BHIs to the S. mutans culture solution so that the absorbance (600 nm) was 0.02. 90 μL of the BHI solution of each evaluation material was dispensed into a 96-well multi-plate (manufactured by Violamo). As a control, 90 μL of BHI containing no evaluation material was added to the 96-well multi-plate. Next, 90 μL of the bacterial solution was inoculated into the 96-well multi-plate, and the final evaluation material concentration in each well was adjusted to 100 mM and the absorbance (600 nm) to 0.01. The 96-well multi-plate was then placed in a Stratus absorbance plate reader (manufactured by Cerillo), and the absorbance (600 nm) was measured over time while culturing for 24 hours at 37 °C under 5% carbon dioxide by volume. From the obtained data, a growth curve was created with time (hours) on the horizontal axis and absorbance (600 nm) on the vertical axis. All experiments were performed with a sample number of N=3. Figure 2 shows the growth curves for each evaluation material and the control. In Figure 2, "Blank" represents a sample in which 180 μL of BHIs solution containing no evaluation material was added to the above-mentioned 96-well multi-plate.

[0073] As shown in Figure 2, the addition of each evaluation material increased the incubation time to reach stationary phase compared to the control, and a delay in growth was observed. Xylitol had the longest incubation time to reach stationary phase and a small slope of the growth curve during the logarithmic growth phase, demonstrating the strongest inhibitory effect on S. mutans growth. SIA had a shorter incubation time to reach stationary phase compared to xylitol, and a larger slope during the logarithmic growth phase, demonstrating that its inhibitory effect on S. mutans growth was weaker than xylitol. Furthermore, 3'-SL, 6'-SL, and 2'-FL had shorter incubation times to reach stationary phase compared to xylitol and SIA, demonstrating that their inhibitory effects were weaker than those of xylitol and SIA.

[0074] Taking into account the biofilm formation inhibitory effect evaluated in Example 1 and the proliferation inhibitory effect evaluated in Example 2, it was revealed that 6'-SL and SIA are materials that have a stronger biofilm formation inhibitory effect than xylitol, but a weaker proliferation inhibitory effect than xylitol. Therefore, it was shown that 6'-SL and SIA can inhibit biofilm formation by a mechanism different from that of materials such as xylitol, which inhibit biofilm formation through conventional antibacterial activity.

Claims

1. A composition for preventing and / or improving dental caries, which contains N-acetylneuraminic acid, 6'-sialyllactose or a salt thereof as an active ingredient.

2. A composition for inhibiting the formation of oral biofilms caused by oral bacteria, which contains N-acetylneuraminic acid, 6'-sialyllactose or a salt thereof as an active ingredient.

3. A composition for inhibiting bacterial biofilm formation on a solid surface, which contains N-acetylneuraminic acid, 6'-sialyllactose or a salt thereof as an active ingredient.

4. The composition of claim 3, wherein the bacteria is an oral bacterium.

5. The composition according to claim 2 or 4, wherein the oral bacterium is Streptococcus mutans.

6. The composition according to claim 2 or 3, wherein the composition is a pharmaceutical or quasi-drug.

7. The composition according to claim 2 or 3, wherein the composition is a food or beverage.

8. An oral care composition containing N-acetylneuraminic acid, 6'-sialyllactose, or a salt thereof as an active ingredient (however, excluding oral flora improvers containing (A) at least one selected from the group consisting of spermine, spermidine, sialic acid, and lactoferrin, and (B) at least one selected from the group consisting of lactulose, oligosaccharides, and sialic acid-containing disaccharides).

9. A non-cariogenic composition containing N-acetylneuraminic acid, 6'-sialyllactose or a salt thereof.

Images