Microbiota improving agent

Abstract

A microbiota improving agent for improving mucosal (except the skin) microbiota and containing at least one substance (X) selected from the group consisting of compounds (X1) having two or more hydroxy groups included in an oxoacid group, and salts thereof, wherein the number average molecular weight of the substance (X) is 560 or more.

Description

Bacterial flora improver

[0001] The present invention relates to a bacterial flora improver.

[0002] Various bacteria coexist in the human body (mucous membranes (in the oral cavity, etc.)), forming a bacterial community called the microbiota. This microbiota is a mixture of so-called non-pathogenic resident bacteria and pathogenic bacteria. Normally, a balance of the microbiota is maintained, which is known as a healthy state, and diseases caused by pathogenic bacteria are suppressed.

[0003] However, if the balance of the bacterial flora is disrupted due to factors such as diet, antibiotic intake, stress, aging, or poor cleaning, pathogenic pathogens and opportunistic bacteria will increase, leading to disease. Therefore, to control disease, it is important not only to suppress pathogenic bacteria but also to maintain a healthy balance of the bacterial flora with non-pathogenic resident bacteria.

[0004] Incidentally, there are known techniques relating to inhibitors of virulence factor production that exhibit a function of acting on specific harmful bacteria, such as the technique described in Patent Document 1.

[0005] Patent No. 5716173

[0006] However, as a result of investigation, it was found that when the pathogenic factor production inhibitor described in Patent Document 1 is used as is, it not only suppresses the action of pathogenic bacteria, but also has a considerable adverse effect on non-pathogenic resident bacteria, and it is difficult to sustainably suppress the action of pathogenic bacteria at the application site, and that measures to improve the balance of the bacterial flora are needed to maintain a sustained healthy state.

[0007] Therefore, an object of the present invention is to provide a bacterial flora improver for improving the bacterial flora of mucous membranes (excluding skin), which exhibits a high inhibitory effect on pathogenic bacteria while maintaining or promoting the growth of non-pathogenic resident bacteria, thereby enabling sustained improvement of the bacterial flora balance.

[0008] The present inventors have conducted extensive research to solve the above-mentioned problems and have arrived at the present invention. Specifically, the present invention relates to a bacterial flora improver for improving the bacterial flora of mucous membranes (excluding skin), which contains at least one substance (X) selected from the group consisting of compounds (X1) having two or more hydroxy groups in an oxo acid group and salts thereof, wherein the substance (X) has a number-average molecular weight of 560 or more.

[0009] According to the present invention, it is possible to provide a bacterial flora improver for improving the bacterial flora of mucous membranes (excluding skin), which exhibits a high inhibitory effect on pathogenic bacteria while maintaining or promoting the growth of non-pathogenic resident bacteria, thereby enabling sustained improvement of the bacterial flora balance.

[0010] The bacterial flora improver of the present invention for improving the bacterial flora of mucous membranes (excluding skin) (hereinafter also referred to as "the bacterial flora improver of the present invention") contains at least one substance (X) selected from the group consisting of compounds (X1) having two or more hydroxy groups in an oxo acid group and salts thereof. The number-average molecular weight of the substance (X) is 560 or more. In the present invention, "mucous membrane" does not include skin.

[0011] The bacterial flora improver of the present invention exhibits a high inhibitory effect on pathogenic bacteria while maintaining or promoting the growth of non-pathogenic resident bacteria, thereby improving the balance of the bacterial flora. Note that the "high inhibitory effect on pathogenic bacteria" means at least one of killing harmful bacteria, inactivating harmful bacteria to inhibit their growth, and inhibiting the production of toxic substances (pathogenic factors) of harmful bacteria.

[0012] As used herein, "pathogenic bacteria" refers to bacteria, fungi, and the like that are highly pathogenic and cause various infectious diseases through proliferation. Specific examples include Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Candida albicans, Candida glabrata, and the like, which are known to be pathogenic bacteria that can occur in the oral cavity. Furthermore, "non-pathogenic resident bacteria" refers to resident bacteria that have the effect of inhibiting the proliferation of the above-mentioned pathogenic bacteria and maintaining health. Specific examples include Staphylococcus epidermidis, Streptococcus salivarius, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus gordonii, and the like, which are known to be normal inhabitants of the oral cavity.

[0013] The bacterial flora improver of the present invention can selectively inhibit the quorum-sensing mechanism (i.e., the mechanism that produces autoinducers) of pathogenic bacteria, and has a high inhibitory effect on pathogenic bacteria while maintaining or promoting the growth of non-pathogenic resident bacteria.

[0014] [Substance (X)] The substance (X) is at least one selected from the group consisting of compounds (X1) having two or more hydroxy groups contained in an oxo acid group and salts thereof.

[0015] Compound (X1) has two or more hydroxy groups contained in the oxo acid group. Compound (X1) may have two or more oxo acid groups. In the present invention, "oxo acid group" means a group having a structure in which a hydroxy group (-OH) and an oxo group (=O) are bonded to one central atom (carbon atom, phosphorus atom, sulfur atom, tungsten atom, etc.). Two or more hydroxy groups and two or more oxo groups may be contained in one oxo acid group. For example, in a linear polyphosphate having a degree of polymerization (chain length) of n, The oxo acid groups contained in compound (X1) are of two types: -P(=O)(OH)2 at both ends of the molecule and -P(=O)(OH)- in the center, and the oxo acid groups at both ends contain two hydroxy groups. Compound (X1) has an oxo acid group as an essential group, but the hydroxy groups not contained in the oxo acid group are not essential and can be optionally contained.

[0016] Examples of the compound (X1) include polymerized phosphoric acid (condensed phosphoric acid) and amino acid polymers (polyglutamic acid, polyaspartic acid, etc.) having a number average molecular weight of 560 or more. Examples of the polymerized phosphoric acid include polyphosphoric acid (chain compound) and metaphosphoric acid (cyclic compound). Examples of the polyphosphoric acid include polyphosphoric acid having a degree of polymerization of 5 to 150.

[0017] The salt of compound (X1) is not particularly limited, and examples thereof include inorganic acid salts such as hydrochloride, sulfate, nitrate, and phosphate of compound (X1); organic acid salts such as acetate, citrate, maleate, malate, oxalate, lactate, succinate, fumarate, and propionate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; ammonium salt; salts with organic bases such as triethylamine, triethanolamine, and pyridine; and amino acid salts with basic or acidic amino acids such as arginine and glutamic acid. Among these, sodium salt and potassium salt are preferred.

[0018] Examples of the compound (X1) and its salts include pentapolyphosphoric acid, short-chain, medium-chain or long-chain (average chain length: 14, 60, 130, etc.) polyphosphates, sodium salts or potassium salts thereof, sodium hexametaphosphate [a heat condensation product of sodium dihydrogen phosphate, also known as Graham salt], (NaPO ３ ) ｍ (m is 10 to 23)], polyglutamic acid, polyaspartic acid, and sodium and potassium salts thereof.

[0019] From the viewpoint of the effect of the bacterial flora improver, the number-average molecular weight of the substance (X) is 560 or more. When the bacterial flora improver contains multiple compounds as the substance (X), the number-average molecular weight of each molecule is 560 or more. The number-average molecular weight of the substance (X) is preferably 565 or more, more preferably 570 or more, and preferably 20,000 or less, more preferably 15,000 or less. The number-average molecular weight of the substance (X) is preferably 560 to 20,000, more preferably 560 to 15,000, even more preferably 565 to 15,000, and particularly preferably 570 to 15,000. In the present invention, the number-average molecular weight means the chemical formula weight of the compound in the case of a single compound, and can be calculated from the structural formula. In the case of a compound having a molecular weight distribution, such as a high-molecular-weight polymer, the number-average molecular weight is a value calculated from the average degree of polymerization.

[0020] The substance (X) is preferably at least one selected from the group consisting of compounds having two or more hydroxy groups contained in an oxo acid group whose central atom is a phosphorus atom and salts thereof, and more preferably at least one selected from the group consisting of polymerized phosphoric acid and salts thereof having a number-average molecular weight of 560 to 15,000. Specifically, the aforementioned sodium hexametaphosphate, sodium pentapolyphosphate, and sodium salts of polyphosphates having an average chain length of 14, 60, or 130 are preferred.

[0021] [Base (Y)] The bacterial flora improving agent of the present invention may contain a base (Y) containing a non-volatile component. Note that the substance (X) does not fall under the category of components contained in the base (Y).

[0022] The "non-volatile component" in the present invention refers to the residue remaining after a sample is heated and dried in an uncovered glass dish at 130°C for 45 minutes in a circulating air dryer.

[0023] From the viewpoint of the effects of the bacterial flora improver, the content (weight percentage) of the nonvolatile components is preferably 0 to 80 wt %, more preferably 0.1 to 80 wt %, and even more preferably 1 to 70 wt %, based on the weight of the bacterial flora improver. Also, from the viewpoint of the effects of the bacterial flora improver, the content (weight percentage) of the nonvolatile components is preferably 0 to 6,000 wt %, more preferably 30 to 6,000 wt %, and even more preferably 50 to 5,000 wt %, based on the weight of the substance (X).

[0024] Examples of non-volatile components in the base (Y) include carboxyalkyl cellulose and salts thereof (such as sodium carboxymethyl cellulose), carboxyalkyl alkyl cellulose and salts thereof (such as carboxymethyl ethyl cellulose), alginic acid and salts thereof (such as sodium alginate), xanthan gum, carrageenan, starch-sodium acrylate graft copolymer, polyalkylene glycol (such as polyethylene glycols such as PEG-20000), alkyl cellulose and salts thereof (such as methyl cellulose), hydroxyalkyl alkyl cellulose (such as hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose), glycerin, polyvinylpyrrolidone, etc. These may be used alone or in combination of two or more.

[0025] The non-volatile component preferably has a weight-average molecular weight of 20,000 or more. The weight-average molecular weight can be measured using gel permeation chromatography (GPC) under the following conditions. <GPC measurement conditions> [1] Apparatus: Gel permeation chromatography [Model number "HLC-8120GPC", manufactured by Tosoh Corporation] [2] Column: "TSKgel G6000PWxL" and "TSKgel G3000PWxL" [both manufactured by Tosoh Corporation] connected in series. [3] Eluent: Methanol / water = 30 / 70 (volume ratio) with 0.5 wt% sodium acetate dissolved therein. [4] Reference substance: Polyethylene glycol (hereinafter abbreviated as PEG) [5] Injection conditions: Sample concentration 0.25 wt%, column temperature 40°C

[0026] The base (Y) may contain only nonvolatile components, or may contain volatile components in addition to nonvolatile components. The viscosity of the base (Y) at 20°C is preferably 10 to 100,000 mPa s, more preferably 100 to 10,000 mPa s. In this specification, the viscosity is a value measured in accordance with the measurement method using a single-cylindrical rotational viscometer as specified in Viscosity Measurement Method 2.

[0027] When the bacterial flora-improving agent of the present invention is used to improve the balance of oral bacterial flora, from the viewpoint of the durability of the effect, it is preferable that the non-volatile component is at least one selected from the group consisting of carboxymethylethylcellulose and salts thereof, carboxymethylcellulose and salts thereof, sodium alginate, xanthan gum, carrageenan, starch-sodium acrylate graft copolymer, polyethylene glycol, methylcellulose and salts thereof, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, glycerin, and polyvinylpyrrolidone.

[0028] [Other Components] The bacterial flora improver of the present invention may contain components (additives) other than the substance (X) and the non-volatile components. The additives may be added separately from the base (Y) or may be appropriately contained in the base (Y).

[0029] Examples of the additives include lubricants such as wax, antioxidants, ultraviolet absorbers, antifoaming agents, preservatives, fragrances, pH adjusters (citric acid, sodium citrate, etc.), solvents (water, organic solvents (ethanol, etc.), etc.), and the like.

[0030] The bacterial flora improver preferably does not contain a bactericidal component. If it contains a bactericidal component, the total weight of the bactericidal component is preferably 2 wt% or less relative to the total weight of the substance (X), from the viewpoint of the effect of the bacterial flora improver. The bactericidal component is one or more selected from the group consisting of glycyrrhizinic acid (or a salt thereof), β-glycyrrhetinic acid, isopropylmethylphenol, cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, alkyldiaminoethylglycine chloride solution, chlorhexidine hydrochloride, triclosan, lysozyme chloride, hinokitiol, sodium lauroyl sarcosinate, and sodium lauryl sulfate.

[0031] The content of the solvent in the bacterial flora improver is not particularly limited, but is preferably 5 to 99 wt % relative to the total weight of the bacterial flora improver. Furthermore, from the viewpoints of spreadability and the effect of improving the bacterial flora balance, the content of water in the bacterial flora improver is preferably 5 to 99 wt %, and more preferably 30 to 99 wt %, relative to the total weight of the bacterial flora improver. When the bacterial flora improver contains water, it is preferable that the bacterial flora improver, after being produced by the method described below, and then allowed to stand at 25°C for 10 minutes, has a visually uniform appearance (not separated into two or more phases).

[0032] The upper limit of the content of additives other than the solvent in the bacterial flora improver is preferably 5 wt % or less, more preferably 3 wt % or less, and particularly preferably 1 wt % or less, based on the weight of the bacterial flora improver. The lower limit of the content of additives other than the solvent in the bacterial flora improver is not particularly limited, but examples include 0.01 wt % or more, based on the weight of the bacterial flora improver. The content of additives other than the solvent in the bacterial flora improver may be 0 to 5 wt %, 0 to 3 wt %, or 0.01 to 1 wt %, based on the weight of the bacterial flora improver.

[0033] When the bacterial flora improver contains a base (Y), the upper limit of the content of additives other than the solvent in the bacterial flora improver is preferably 10 wt % or less, more preferably 8 wt % or less, and particularly preferably 1 wt % or less, based on the weight of the non-volatile components in the bacterial flora improver. The lower limit of the content of additives other than the solvent in the bacterial flora improver is not particularly limited, but examples include 0.1 wt % or more, based on the weight of the non-volatile components in the bacterial flora improver. When the bacterial flora improver contains a base (Y), the content of additives other than the solvent in the bacterial flora improver may be 0 to 10 wt %, 0 to 8 wt %, or 0.1 to 1 wt %, based on the weight of the non-volatile components in the bacterial flora improver.

[0034] When the bacterial flora improver is used as a food product, it may contain a component that serves as a base material for the food product. The base material can be selected appropriately depending on the form of the food product. For example, if the food product is a candy, it may contain starch syrup or the like as the base material. Examples of the form of the food product include candy, gummies, chewing gum, chocolate, tablet sweets (such as chewing soft candy), and jelly (solid at 25°C).

[0035] [Bacterial flora improver]

[0036] The content (weight percentage) of substance (X) is preferably 0.01 to 5 wt %, and more preferably 0.05 to 3 wt %, based on the weight of the bacterial flora improver. From the viewpoint of suitably imparting a high pathogenic bacterium-inhibiting effect, the content (weight percentage) of substance (X) preferably has a lower limit of 0.01 wt %, and more preferably 0.05 wt %, based on the weight of the bacterial flora improver of the present invention. Furthermore, from the viewpoint of suitably maintaining or promoting the growth of non-pathogenic resident bacteria, the content of substance (X) is preferably an upper limit of 5 wt %, and more preferably 3 wt %, based on the weight of the bacterial flora improver of the present invention.

[0037] When the bacterial flora improver contains base (Y), the content of substance (X) preferably has a lower limit of 1 wt %, more preferably 2 wt %, based on the weight of the non-volatile components in the bacterial flora improver. Furthermore, the content of substance (X) preferably has an upper limit of 200 wt %, more preferably 150 wt %, based on the weight of the non-volatile components in the bacterial flora improver. The content of substance (X) is preferably 1 to 200 wt %, more preferably 2 to 150 wt %, based on the weight of the non-volatile components in the bacterial flora improver. By including substance (X) in the above range, it is possible to advantageously achieve the effects of maintaining or promoting the growth of non-pathogenic resident bacteria while exhibiting a high inhibitory effect on pathogenic bacteria and maintaining the above performance for a long period of time.

[0038] The mechanism by which the bacterial flora improver of the present invention exerts the above-mentioned effects is presumed to be as follows: Magnesium ions are involved in the inhibition of the growth of pathogenic and non-pathogenic bacteria, and substance (X) contained in the bacterial flora improver of the present invention binds to magnesium ions, thereby inhibiting the growth of pathogenic bacteria. The level of magnesium ions required to inhibit the growth of pathogenic bacteria differs from the level required to inhibit the growth of non-pathogenic bacteria, and non-pathogenic bacteria can maintain or grow even at magnesium ion levels that significantly inhibit the growth of pathogenic bacteria. In particular, substance (X) contained in the bacterial flora improver of the present invention is thought to milder the effect of magnesium ions on non-pathogenic bacteria than substances with lower molecular weights.

[0039] The pH of an aqueous solution containing 1 wt % of the bacterial flora improver of the present invention is preferably 6 to 10, more preferably 7 to 9, from the viewpoint of the effect of improving the bacterial flora balance. The aqueous solution can be prepared by diluting with water such as ion-exchanged water so that the concentration of the bacterial flora improver becomes 1 wt %. The pH may be appropriately adjusted by adding a pH adjuster. The pH can be measured at 25°C using a pH meter (method in accordance with JIS Z 8802:2011).

[0040] The bacterial flora improver of the present invention can be produced by blending the components and mixing them uniformly at room temperature or, if necessary, by heating (e.g., 30 to 70° C.) The order and method of blending the components are not particularly limited.

[0041] [Method of Use] The bacterial flora improver of the present invention is a bacterial flora improver for improving the bacterial flora of mucous membranes (excluding skin). It can be used by applying the bacterial flora improver to the affected area [mucous membranes (oral cavity (tongue, gums, gingiva, etc.), intestines, eyes, etc.)] or the surrounding area with a finger or other means, and application to mucous membranes is particularly preferred. For example, when the bacterial flora improver of the present invention is applied to oral mucous membranes (tongue, gums, gingiva, etc.), it is expected to be effective in preventing periodontal disease, which progresses through the stages of gingivitis and periodontitis. By using a nonvolatile component appropriate for the application, the bacterial flora improver of the present invention can also be used as a pharmaceutical, medical device (medical adhesive, wound healing material, etc.), quasi-drug, cosmetic, food, etc., and in both of these applications, it can improve the balance of the bacterial flora at the application site. Specific examples of the above applications include toothpastes, mouthwashes, oral gels, oral care foods, supplements, pet care products, etc.

[0042] The present specification discloses the following:

[0043] The present disclosure (1) is a bacterial flora improver for improving the bacterial flora of mucous membranes (excluding skin), which contains at least one substance (X) selected from the group consisting of compounds (X1) having two or more hydroxy groups contained in an oxo acid group and salts thereof, wherein the substance (X) has a number-average molecular weight of 560 or more.

[0044] The present disclosure (2) is the bacterial flora improver according to the present disclosure (1), further comprising a base (Y) containing a non-volatile component.

[0045] The present disclosure (3) is the bacterial flora improver according to the present disclosure (1) or (2), wherein the substance (X) is at least one selected from the group consisting of polymerized phosphoric acid and salts thereof having a number average molecular weight of 560 to 15,000.

[0046] The present disclosure (4) is the bacterial flora improver according to any one of the present disclosures (1) to (3), wherein the non-volatile component is at least one selected from the group consisting of carboxyalkyl cellulose and salts thereof, carboxyalkyl alkyl cellulose and salts thereof, alginic acid and salts thereof, xanthan gum, carrageenan, starch-sodium acrylate graft copolymer, polyalkylene glycol, alkyl cellulose and salts thereof, hydroxypropyl alkyl cellulose, glycerin, and polyvinylpyrrolidone.

[0047] The present disclosure (5) is the bacterial flora improver according to any one of the present disclosures (1) to (4), wherein an aqueous solution containing 1% by weight of the bacterial flora improver has a pH of 6 to 10.

[0048] The present disclosure (6) is the bacterial flora improver according to any one of the present disclosures (1) to (5), wherein the weight proportion of the substance (X) is 0.01 to 5 wt % based on the weight of the bacterial flora improver.

[0049] The present invention will be further explained below with reference to examples and comparative examples, but the present invention is not limited thereto. Unless otherwise specified, parts mean parts by weight and % means % by weight.

[0050] <Examples 1 to 32 and Comparative Examples 1 and 2: Production of bacterial flora improvers> Substance (X), base (Y), pH adjuster, and ion-exchanged water were mixed at 25°C to achieve the concentrations (% by weight) shown in Tables 1-1 to 1-7. The pH of aqueous solutions diluted with ion-exchanged water to give a concentration of 1% by weight of each bacterial flora improver is also shown in Tables 1-1 to 1-7.

[0051] <Explanation of Each Component> The raw materials corresponding to each component used in the Examples and Comparative Examples are as follows: [Substance (X)] (X-1): Sodium hexametaphosphate [heat condensation product of sodium dihydrogen phosphate, (NaPO ３ ) ｍ , m: 10 to 23, a mixture of compounds having a molecular weight of 1019.6 to 2345.1, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] (X-2): sodium pentapolyphosphate [Na ７ P ５ O １６, molecular weight 571.78, manufactured by Taihei Chemical Industry Co., Ltd.] (X-3): Short-chain sodium polyphosphate [number average molecular weight 1489.4, average chain length 14, manufactured by Regentis Co., Ltd.] (X-4): Medium-chain sodium polyphosphate [number average molecular weight 6179.7, average chain length 60, manufactured by Regentis Co., Ltd.] (X-5): Long-chain sodium polyphosphate [number average molecular weight 13317.0, average chain length 130, manufactured by Regentis Co., Ltd.] [Comparative substance X'] (X-6): Sodium polyglutamate [number average molecular weight 180,000 or more, manufactured by Fujifilm Wako Pure Chemical Industries Co., Ltd.] (X-7): Sodium polyaspartate [number average molecular weight approximately 3,500, manufactured by Sigma-Aldrich] (X'-1): Sodium tripolyphosphate [number average molecular weight 367.86, manufactured by Fujifilm Wako Pure Chemical Industries Co., Ltd.] [Base (Y)] (Y-1): Carboxymethylethylcellulose [product name: CMEC, manufactured by Sanyo Chemical Industries, Ltd., weight-average molecular weight: 49,000] (Y-2): Sodium carboxymethylcellulose [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., weight-average molecular weight: 200,000] (Y-3): Sodium alginate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., weight-average molecular weight: 100,000] (Y-4): Xanthan gum [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., weight-average molecular weight: 2,000,000] (Y-5): Starch-sodium acrylate graft copolymer [product name: Sunfresh ST100, manufactured by Sanyo Chemical Industries, Ltd., weight-average molecular weight: 1,000,000] (Y-6): PEG-20000 [manufactured by Sanyo Chemical Industries, Ltd., number-average molecular weight: 20,000] (Y-7): Methylcellulose [manufactured by Shin-Etsu Chemical Co., Ltd., degree of methoxy group substitution 1.8, viscosity of a 2% aqueous solution at 20°C of 4,000 mPa·s] (Y-8): Hydroxypropyl methylcellulose 60SH [manufactured by Shin-Etsu Chemical Co., Ltd., degree of methoxy group substitution 1.9, number of moles of hydroxypropyl group substitution 0.25 moles, viscosity of a 2% aqueous solution at 20°C of 4,000 mPa·s] (Y-9): Hydroxypropyl methylcellulose 65SH [manufactured by Shin-Etsu Chemical Co., Ltd., degree of methoxy group substitution 1.8, number of moles of hydroxypropyl group substitution 0.15 moles, viscosity of a 2% aqueous solution at 20°C of 4,000 mPa s] (Y-10): Hydroxypropyl methylcellulose 90SH [Shin-Etsu Chemical Co., Ltd., methoxy group substitution degree 1.4, hydroxypropyl group substitution molar number 0.2 moles, viscosity of 2% aqueous solution at 20 ° C. 4,000 mPa s] (Y-11): Hydroxyethyl methylcellulose SEB [Shin-Etsu Chemical Co., Ltd., methoxy group substitution degree 1.5, hydroxyethyl group substitution molar number 0.2 moles, viscosity of 2% aqueous solution at 20 ° C. 4,000 mPa s] (Y-12): Polyvinylpyrrolidone K30 [Mw 40,000, Tokyo Chemical Industry Co., Ltd.] (Y-13): Carrageenan [λ-carrageenan, Fujifilm Wako Pure Chemical Industries, Ltd.] (Y-14): Glycerin [Fujifilm Wako Pure Chemical Industries, Ltd.]

[0052] <Test to confirm selective bacterial growth inhibition> In the test, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Staphylococcus aureus, and Candida albicans were used as pathogenic bacteria, and Streptococcus salivarius, Staphylococcus epidermidis, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii were used as non-pathogenic bacteria. The media used to culture each bacterium were: modified GAM bouillon "Nissui" (Shimadzu Diagnostics Co., Ltd.) for Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius; brain heart infusion medium (Becton Dickinson) for Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, and Streptococcus gordonii; and Staphylococcus aureus (Becton Dickinson Co., Ltd.) for Staphylococcus aureus. LB medium (manufactured by Becton Dickinson) was used for S. aureus and Staphylococcus epidermidis, and YM medium (manufactured by Sigma) was used for Candida albicans. Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius were cultured under anaerobic conditions, while Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans were cultured under aerobic conditions.50 mg of each bacterial flora improver prepared with the composition shown in Tables 1-1 to 1-7 was dispensed into each well of a 24-well plate. For bacteria to be cultured under anaerobic conditions, each strain was cultured overnight at 37°C under anaerobic conditions using the above-mentioned medium, diluted with the appropriate culture medium to give an absorbance at 600 nm of 0.15, and 500 μL of each solution was dispensed into 24-well plates containing the bacterial flora improver. Then, static culture was performed at 37°C under anaerobic conditions using Anaeropack Kenki 10% (Mitsubishi Gas Chemical Company, Inc.). For bacteria to be cultured under aerobic conditions, each strain was cultured overnight with shaking at 37°C under aerobic conditions using the above-mentioned medium, diluted with the appropriate culture medium to give an absorbance at 600 nm of 0.10, and 500 μL of each solution was dispensed into 24-well plates containing the bacterial flora improver, followed by static culture at 37°C. The plates were collected after 24 hours, and the growth of each bacterium was evaluated by measuring the absorbance at 600 nm. The growth inhibitory effect of each example was expressed as a relative value when the turbidity (absorbance at 600 nm) of Comparative Example 1 was set to 1.0. The results are shown in Tables 1-1 to 1-7.

[0053] When the bacterial flora improving agent of the present invention is applied to the oral cavity, for example, if the growth of pathogenic bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia is suppressed compared to when the bacterial flora improving agent is not added, and if the growth of non-pathogenic resident bacteria such as Streptococcus salivarius remains unchanged compared to when the bacterial flora improving agent is not added, then the growth of bacteria is selectively suppressed. For example, embodiments in which the above relative values ​​fall within the following ranges can be mentioned. An embodiment in which the test result for Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia is 0.10 to 0.80 (preferably 0.10 to 0.50), and the test result for Streptococcus salivarius is 0.8 to 1.2.

[0054] <Test for inhibiting protease production by pathogenic bacteria> Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia were used as pathogenic bacteria in the test. The modified GAM bouillon "Nissui" described above was used as the medium for culturing each bacterium. 50 mg of each bacterial flora improver prepared with the composition listed in Tables 1-1 to 1-7 was dispensed into each well of a 24-well plate. Each bacterial strain was cultured overnight at 37°C under anaerobic conditions using the above medium, and diluted with the appropriate culture medium to achieve an absorbance at 600 nm of 0.15. 500 μL of each solution was dispensed into a 24-well plate containing the bacterial flora improver, followed by static culture at 37°C under anaerobic conditions using Anaeropack Kenki 10% (manufactured by Mitsubishi Gas Chemical Company, Inc.). After 48 hours, the plates were collected and centrifuged at 4 ° C and 10,000 rpm for 5 minutes to collect each culture supernatant. The activity of the protease contained in the culture supernatant was measured using an Amplite protease activity measurement kit (AAT Bioquest). The protease production effect of each example was expressed as a relative value when the protease activity value of Comparative Example 1 was set to 1.0. The results are shown in Tables 1-1 to 1-7. Here, protease is known to be one of the toxin components produced by pathogenic bacteria, and the lower the protease activity value, the more inhibited the protease production.

[0055] <Evaluation of durability of effect by halo test> [Preparation of agar medium for halo test] The test was conducted using pathogenic bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Staphylococcus aureus, and Candida albicans, and non-pathogenic bacteria such as Streptococcus salivarius, Staphylococcus epidermidis, Streptococcus mitis, Streptococcus oralis, and Streptococcus salivarius. sanguinis and Streptococcus gordonii were used. The media used to culture each bacterium were the modified GAM bouillon "Nissui" for Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius, the brain heart infusion medium for Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, and Streptococcus gordonii, and the serovar ... The above-mentioned LB medium was used for S. epidermidis, and the above-mentioned YM medium was used for Candida albicans. Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius were cultured under anaerobic conditions, while Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans were cultured under aerobic conditions. Each strain was cultured overnight at 37°C using the above medium, and 1.0 x 10 ５After preparing the bacterial solution to achieve a CFU / ml concentration, 100 μL of the bacterial solution was inoculated onto the agar medium containing each medium component and uniformly spread with a cone-like stick. [Halo Test] 200 mg of each bacterial flora improver prepared with the composition shown in Tables 1-1 to 1-7 was inoculated onto the agar medium containing the bacteria as a test sample, and the test was conducted. Bacteria cultured under anaerobic conditions were cultured at 37°C for 24 hours using Anaeropack Kenki 10% (manufactured by Mitsubishi Gas Chemical Company, Inc.). Bacteria cultured under aerobic conditions were cultured at 37°C for 24 hours. To evaluate the sustainability of the growth inhibitory effect against pathogenic bacteria, if no halo was observed after 24 hours of culture (evaluated as ×), the culture of the pathogenic bacteria was terminated, and for the rest, culture at 37°C was continued for one week. For none of the samples in which the halo disappeared after one week of culture (evaluated as △), culture at 37°C was continued for another two weeks. In the evaluation of the persistence of the effect against non-pathogenic bacteria, the effect continued for all bacteria up to two weeks after cultivation. The halo was determined by measuring the length from the end of the test sample application area to the end of the halo. If it was greater than 0 mm, it was determined that the halo was confirmed, and if it was 0 mm, it was determined that the halo was not confirmed. In the evaluation of the growth inhibitory effect against pathogenic bacteria, if a halo was confirmed, it meant that there was a growth inhibitory effect, and if no halo was confirmed, it meant that there was no growth inhibitory effect. In addition, in the evaluation of the effect against non-pathogenic bacteria, if no halo was confirmed, it meant that growth was not inhibited (growth was not affected), and if a halo was confirmed, it meant that growth was inhibited. Therefore, the evaluation of the persistence of the growth inhibitory effect against pathogenic bacteria ("persistence evaluation (pathogenic bacteria)") and the evaluation of the persistence of the effect against non-pathogenic bacteria ("persistence evaluation (non-pathogenic bacteria)") were performed according to the following criteria. The evaluation results are shown in Tables 1-1 to 1-7.<Persistence evaluation (pathogenic bacteria)> ◎: Halo was confirmed in the evaluations 24 hours, 1 week, and 2 weeks after incubation. ○: Halo was confirmed in the evaluations 24 hours and 1 week after incubation, but not confirmed (had disappeared) in the evaluation 2 weeks after incubation. △: Halo was confirmed in the evaluation 24 hours after incubation, but had disappeared in the evaluation 1 week after incubation. ×: Halo was not confirmed in the evaluation 24 hours after incubation. <Persistence evaluation (non-pathogenic bacteria)> ◎: No halo was confirmed in the evaluations 24 hours, 1 week, and 2 weeks after incubation. ○: Halo was confirmed in the evaluation 24 hours after incubation, but had disappeared in the evaluations 1 week and 2 weeks after incubation. △: Halo was confirmed in the evaluations 24 hours after incubation and 1 week after incubation, but had disappeared in the evaluation 2 weeks after incubation. ×: Halo was confirmed in the evaluations 24 hours after incubation, 1 week, and 2 weeks after incubation.

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Examples 33 to 64 and Comparative Examples 3 and 4: Production of bacterial flora improvers Substance (X), base (Y), pH adjuster, ethanol, and ion-exchanged water were mixed at 25°C to achieve the concentrations (wt%) shown in Tables 2-1 to 2-7. The pH of aqueous solutions diluted with ion-exchanged water to give a concentration of 1 wt% of each bacterial flora improver is also shown in Tables 2-1 to 2-7.

[0064] <Test to confirm selective bacterial growth inhibition> In the test, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Staphylococcus aureus, and Candida albicans were used as pathogenic bacteria, and Streptococcus salivarius, Staphylococcus epidermidis, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii were used as non-pathogenic bacteria. The media used to culture each bacterium were the modified GAM bouillon "Nissui" for Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius, the brain heart infusion medium for Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, and Streptococcus gordonii, and the serovar ... The above-mentioned LB medium was used for S. epidermidis, and the above-mentioned YM medium was used for Candida albicans. Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius were cultured under anaerobic conditions, while Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans were cultured under aerobic conditions. Each bacterial flora improver prepared with the composition shown in Tables 2-1 to 2-7 was dispensed in 50 μL portions into each well of a 24-well plate.For bacteria cultured under anaerobic conditions, each strain was cultured overnight at 37°C under anaerobic conditions using the above medium, diluted with the appropriate culture medium to give an absorbance at 600 nm of 0.15, and 500 μL of each solution was dispensed into 24-well plates containing a bacterial flora improver. Anaeropack Kenki 10% (Mitsubishi Gas Chemical Company, Inc.) was then used for static culture at 37°C under anaerobic conditions. For bacteria cultured under aerobic conditions, each strain was cultured overnight with shaking using the above medium at 37°C under aerobic conditions, diluted with the appropriate culture medium to give an absorbance at 600 nm of 0.10, and 500 μL of each solution was dispensed into 24-well plates containing a bacterial flora improver. The plates were then recovered after 24 hours, and the growth of each strain was evaluated by measuring the absorbance at 600 nm. The growth inhibitory effect of each example was expressed as a relative value when the turbidity (absorbance at 600 nm) of Comparative Example 3 was set to 1.0. The results are shown in Tables 2-1 to 2-7.

[0065] When the bacterial flora improving agent of the present invention is applied to the oral cavity, if the growth of pathogenic bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia is suppressed compared to when the bacterial flora improving agent is not added, and the growth of non-pathogenic resident bacteria such as Streptococcus salivarius remains unchanged compared to when the bacterial flora improving agent is not added, then the growth of bacteria is selectively suppressed. For example, embodiments in which the above relative values ​​fall within the following ranges can be mentioned. An embodiment in which the test result for Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia is 0.10 to 0.80 (preferably 0.10 to 0.40), and the test result for Streptococcus salivarius is 0.8 to 1.2.

[0066] <Test for inhibiting protease production by pathogenic bacteria> Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia were used as pathogenic bacteria in the test. The modified GAM bouillon "Nissui" described above was used as the medium for culturing each bacterium. 50 μL of each bacterial flora improver prepared with the composition listed in Tables 2-1 to 2-7 was dispensed into each well of a 24-well plate. Each bacterial strain was cultured overnight at 37°C under anaerobic conditions using the above medium, and diluted with the appropriate culture medium to achieve an absorbance at 600 nm of 0.15. 500 μL of each solution was dispensed into a 24-well plate containing the bacterial flora improver, followed by static culture at 37°C under anaerobic conditions using Anaeropack Kenki 10% (manufactured by Mitsubishi Gas Chemical Company, Inc.). After 48 hours, the plates were collected and centrifuged at 4 ° C. and 10,000 rpm for 5 minutes to collect each culture supernatant. The activity of the protease contained in the culture supernatant was measured using an Amplite protease activity measurement kit (AAT Bioquest). The protease production effect of each example was expressed as a relative value when the protease activity value of Comparative Example 3 was set to 1.0. The results are shown in Tables 2-1 to 2-7. Here, protease is known to be one of the toxin components produced by pathogenic bacteria, and the lower the protease activity value, the more inhibited the protease production.

[0067] <Evaluation of durability of effect by halo test> [Preparation of agar medium for halo test] The test was conducted using pathogenic bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Staphylococcus aureus, and Candida albicans, and non-pathogenic bacteria such as Streptococcus salivarius, Staphylococcus epidermidis, Streptococcus mitis, Streptococcus oralis, and Streptococcus salivarius. sanguinis and Streptococcus gordonii were used. The media used to culture each bacterium were the modified GAM bouillon "Nissui" for Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius, the brain heart infusion medium for Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, and Streptococcus gordonii, and the serovar ... The above-mentioned LB medium was used for S. epidermidis, and the above-mentioned YM medium was used for Candida albicans. Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius were cultured under anaerobic conditions, while Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans were cultured under aerobic conditions.Each strain was cultured overnight at 37°C using the above medium, and a bacterial solution was prepared to a concentration of 1.0 x 10 CFU / ml. 100 μL of the bacterial solution was then inoculated onto an agar medium containing each medium component and uniformly spread using a cone-like stick. [Halo Test] 100 μL of each bacterial flora improver prepared with the composition listed in Tables 2-1 to 2-7 was inoculated onto the agar medium containing the bacteria as a test sample, and the test was conducted. Bacteria cultured under anaerobic conditions were cultured at 37°C for 24 hours using Anaeropack Kenki 10% (manufactured by Mitsubishi Gas Chemical Company, Inc.). Bacteria cultured under aerobic conditions were cultured at 37°C for 24 hours. To evaluate the sustainability of the growth inhibitory effect against pathogenic bacteria, if no halo was observed after 24 hours of culture (evaluated as ×), the culture of the pathogenic bacteria was terminated. For other samples, culture at 37°C was continued for up to one week. In none of the samples did the halo disappear after one week of incubation (evaluation: △), and incubation at 37°C was continued for another two weeks. Evaluation of the persistence of the effect against non-pathogenic bacteria continued for two weeks for all bacteria. The length of the halo was measured from the end of the test sample application area to the end of the halo. If it was greater than 0 mm, it was determined that the halo was confirmed, and if it was 0 mm, it was determined that the halo was not confirmed. In evaluating the growth inhibitory effect against pathogenic bacteria, if a halo was confirmed, it meant that there was a growth inhibitory effect, and if no halo was confirmed, it meant that there was no growth inhibitory effect. In addition, in evaluating the effect against non-pathogenic bacteria, if no halo was confirmed, it meant that growth was not inhibited (growth was not affected), and if a halo was confirmed, it meant that growth was inhibited. Therefore, the evaluation of the persistence of the growth inhibitory effect against pathogenic bacteria ("persistence evaluation (pathogenic bacteria)") and the evaluation of the persistence of the effect against non-pathogenic bacteria ("persistence evaluation (non-pathogenic bacteria)") were performed according to the following criteria. The evaluation results are shown in Tables 2-1 to 2-7.<Persistence evaluation (pathogenic bacteria)> ◎: Halo was confirmed in the evaluations 24 hours, 1 week, and 2 weeks after incubation. ○: Halo was confirmed in the evaluations 24 hours and 1 week after incubation, but not confirmed (had disappeared) in the evaluation 2 weeks after incubation. △: Halo was confirmed in the evaluation 24 hours after incubation, but had disappeared in the evaluation 1 week after incubation. ×: Halo was not confirmed in the evaluation 24 hours after incubation. <Persistence evaluation (non-pathogenic bacteria)> ◎: No halo was confirmed in the evaluations 24 hours, 1 week, and 2 weeks after incubation. ○: Halo was confirmed in the evaluation 24 hours after incubation, but had disappeared in the evaluations 1 week and 2 weeks after incubation. △: Halo was confirmed in the evaluations 24 hours after incubation and 1 week after incubation, but had disappeared in the evaluation 2 weeks after incubation. ×: Halo was confirmed in the evaluations 24 hours after incubation, 1 week, and 2 weeks after incubation.

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Examples 65-85 and Comparative Examples 5-6: Production of Oral Care Foods Reduced maltose starch syrup (Marumi Co., Ltd.) was heated in a microwave oven to 140-150°C. After cooling to 120°C, substance (X) and base (Y) were added and mixed at room temperature to achieve the concentrations (wt%) listed in Tables 3-1 to 3-5. After dissolution, the mixture was allowed to cool and solidify at room temperature to produce candy (food) weighing approximately 3 g per piece. Aqueous solutions containing 1 wt% of each candy were prepared, and the pH was measured. The results are shown in Tables 3-1 to 3-5.

[0076] <Evaluation Test 1: In Vitro Mycelium Formation Inhibition Test of Candida Fungi> (1) Candida albicans was used as the Candida fungus. RPMI 1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 2.5% fetal bovine serum (Biosera) was used as the medium. Each candy prepared according to the composition listed in Tables 3-1 to 3-5 was added to 1 mL of the medium and stirred at 500 rpm using a stirrer at 37°C for 1 minute. The resulting supernatant was used as the sample solution for the test. Strains cultured overnight at 37°C were diluted with each sample solution to an absorbance of 0.15 at 600 nm, dispensed in 200 μL aliquots into 96-well plates, and then statically cultured at 37°C under aerobic conditions. After 48 hours, the plates were collected, and mycelium was stained using the following method.

[0077] (2) Mycelial Staining of Candida albicans The inhibitory activity against the mycelial growth of Candida albicans was evaluated by the crystal staining method. This method uses the amount of staining obtained when mycelial-grown Candida albicans is stained with an aqueous solution of crystal violet (Fujifilm Wako Pure Chemical Industries, Ltd.) as an indicator of the degree of mycelial growth of Candida albicans, based on the correlation between the number of growing colonies or the amount of [H3]-glucose uptake into the cells. After incubation, the supernatant culture medium was removed from the plate recovered, and 200 μL of 70 vol% aqueous ethanol solution was added, followed by washing twice with 200 μL of ultrapure water. After removing the supernatant, 200 μL of 0.01% aqueous crystal violet solution was added and staining was continued for 20 minutes. After staining, the samples were washed twice with 200 μL of ultrapure water, the supernatant removed, and then allowed to dry at room temperature for 10 minutes. 150 μL of 0.04 N HCl-containing isopropyl alcohol and 50 μL of 0.25% sodium dodecyl sulfate aqueous solution were added, stirred for 5 minutes, and the amount of Candida albicans mycelium formation was evaluated by measuring the absorbance at 620 nm. The mycelium formation inhibitory effect was expressed as a relative value, assuming the absorbance of 100 when the same weight of base (Y) and reduced maltose syrup alone (composition obtained by removing substance (X) from each candy) was used instead of each candy in Examples 66 to 72 (Comparative Example 5). The results are shown in Tables 3-1 to 3-5.

[0078] <Evaluation Test 2: In Vitro Growth Inhibition Test of Candida> Candida albicans was used as the Candida fungus. The above-mentioned YM medium was used as the medium. Each candy prepared with the composition listed in Tables 3-1 to 3-5 was added to 1 mL of the above medium and stirred at 500 rpm using a stirrer for 1 minute at 37°C. The supernatant was used as the sample solution for the test. Strains cultured overnight at 30°C were diluted with each sample solution so that the absorbance at 600 nm was 0.00015, and 200 μL of each solution was dispensed into a 96-well plate and statically cultured at 24°C under aerobic conditions. After 48 hours, the plates were recovered, and the absorbance at 600 nm was measured to evaluate the growth of each bacterium. The growth inhibitory effect was expressed as a relative value when the absorbance of the mixture of the same weight of base (Y) and reduced maltose syrup only [composition obtained by removing substance (X) from each candy] (Comparative Example 5) was set at 100 instead of each candy of Examples 66 to 72. The results are shown in Tables 3-1 to 3-5.

[0079] <Evaluation Test 3: Evaluation of Oral Hygiene Improvement Effects in Volunteer Tests> Five volunteers (Subjects 1-5) were recruited and asked to consume each candy prepared with the compositions listed in Tables 3-1 to 3-5 for four days. The oral hygiene improvement effects were evaluated based on the tongue coating scores and viable Candida albicans counts before and after ingestion. Tongue coating scores were calculated according to the method of Shimizu et al., which divides the dorsum of the tongue into nine sections and evaluates tongue coating adhesion. The tongue coating scores were evaluated immediately upon waking on the first morning of the test (Day 1). The tongues were then scraped three times with a commercially available tongue brush and collected in a tube containing physiological saline. The collected samples were transported refrigerated, centrifuged, and the precipitate was collected, thoroughly dispersed, and resuspended in RPMI 1640 medium. The samples were then applied to a Candida selective medium and incubated at 37°C for 48 hours, after which the viable Candida albicans count was measured. On the morning of the fifth day, the tongue coating score and the viable Candida albicans count were measured using the same method. The tongue coating collected on the morning of the fifth day was used as the tongue coating before the test. From the fifth day, the volunteers were asked to take approximately 3 g (1 piece) of candy three times a day after each meal for four days. On the morning of the ninth day from the start of the test, the tongue coating score and the viable Candida albicans count were measured using the same method. The tongue coating collected on the morning of the 9th day was used as the tongue coating after the test. The viable Candida albicans count on the morning of the 9th day was shown as a relative value when the viable Candida albicans count on the morning of the 5th day was set to 100. The results are shown in Tables 3-1 to 3-5.

[0080] <Evaluation Test 4: Test to confirm selective bacterial growth inhibition> In the test, Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia were used as pathogenic bacteria, and Streptococcus salivarius, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii were used as non-pathogenic bacteria. Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius were cultured under anaerobic conditions, while Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, and Streptococcus gordonii were cultured under aerobic conditions. The modified GAM bouillon "Nissui" was used as the medium for culturing each bacterium. Each candy prepared with the composition listed in Tables 3-1 to 3-5 was added to 1 mL of modified GAM medium and stirred at 37 °C for 1 minute at 500 rpm using a stirrer. The supernatant was used as the sample solution for the test. Each strain was cultured overnight at 37°C and diluted with each sample solution to an absorbance of 0.15 at 600 nm. 200 μL of each solution was dispensed into a 96-well plate. Bacteria cultured under anaerobic conditions were statically cultured at 37°C under anaerobic conditions using Anaeropack Kenki 10% (Mitsubishi Gas Chemical Co., Inc.). Bacteria cultured under aerobic conditions were cultured at 37°C for 24 hours. After 24 hours, the plates were collected and the absorbance at 600 nm was measured to evaluate the growth of each bacterium. The growth inhibitory effect was expressed as a relative value, with the absorbance of the mixture (Comparative Example 5) containing the same weight of base (Y) and reduced maltose syrup alone [composition in which substance (X) was removed from each candy] used instead of each of the candies in Examples 66 to 72 (100). The results are shown in Tables 3-1 to 3-5.

[0081] When the oral care food of the present invention is applied to the oral cavity, if the proliferation of Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia is suppressed compared to Comparative Example 5, and if the proliferation of Streptococcus salivarius, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii shows little change compared to Comparative Example 5, then the proliferation of bacteria is selectively suppressed. For example, embodiments in which the above relative values ​​fall within the following ranges can be mentioned. An embodiment in which the test results for Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia are all 0 to 40, and the test results for Streptococcus salivarius, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii are 50 to 100.

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] As shown in Tables 1-1 to 1-7, Tables 2-1 to 2-7, and Tables 3-1 to 3-5, in Examples 1 to 85 containing substance (X), the growth of pathogenic bacteria was selectively suppressed while the growth of non-pathogenic bacteria was not suppressed, compared to Comparative Examples 1 to 6, thereby achieving an ideal bacterial flora balance, confirming the bacterial flora improving effect. Furthermore, in Examples 1 to 64, the effect of suppressing the toxin (protease) production of pathogenic bacteria was also confirmed. Furthermore, the long-lasting bacterial flora improving effect was confirmed. Therefore, it can be seen that the bacterial flora improver of the present invention has the effect of selectively suppressing the growth and toxin production of pathogenic bacteria without affecting non-pathogenic bacteria, and that this effect can be maintained for a long period of time. Since the bacterial flora improver of the present invention can exert the effect of improving the bacterial flora balance for a long period of time after a single use, it can be seen that it is particularly useful as a bacterial flora improver.

Claims

1. A bacterial flora improver for improving the bacterial flora of mucous membranes (excluding skin), comprising at least one substance (X) selected from the group consisting of compounds (X1) having two or more hydroxy groups in an oxo acid group and salts thereof, wherein the number-average molecular weight of the substance (X) is 560 or more.

2. The bacterial flora improver according to claim 1, further comprising a base (Y) containing a non-volatile component.

3. The bacterial flora improver according to claim 1 or 2, wherein the substance (X) is at least one selected from the group consisting of polymerized phosphoric acid and salts thereof having a number average molecular weight of 560 to 15,000.

4. The bacterial flora improver according to claim 2, wherein the non-volatile component is at least one selected from the group consisting of carboxyalkyl cellulose and its salts, carboxyalkyl alkyl cellulose and its salts, alginic acid and its salts, xanthan gum, carrageenan, starch-sodium acrylate graft copolymer, polyalkylene glycol, alkyl cellulose and its salts, hydroxyalkyl alkyl cellulose, glycerin, and polyvinylpyrrolidone.

5. The bacterial flora improver according to claim 1 or 2, wherein the pH of an aqueous solution containing 1% by weight of the bacterial flora improver is 6 to 10.

6. The bacterial flora improver according to claim 1 or 2, wherein the weight proportion of the substance (X) is 0.01 to 5% by weight based on the weight of the bacterial flora improver.

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