A beauty method that includes a process to increase the bacterial population of Staphylococcus hominis.

Abstract

To provide novel beauty treatments. [Solution] The method includes a step to increase the amount of Staphylococcus hominis on the skin, The aforementioned bacterial growth increasing step includes applying a Staphylococcus hominis bacterial growth promoter to the skin, or applying Staphylococcus hominis to the skin. A cosmetic method (excluding medical procedures) comprising the aforementioned bacterial growth step, which includes performing one or more selected from the following: promoting a decrease in lactic acid content in the skin, promoting an increase in short-chain fatty acid content in the skin, promoting an increase in malonic acid content in the skin, improving the TER value of the epidermis, and promoting the formation of tight junction structures.

Description

[Technical Field]

[0001] The present invention relates to a cosmetic method that includes a step to increase the bacterial count of Staphylococcus hominis. [Background technology]

[0002] In recent years, cosmetics containing bacterial cells with beneficial effects on the human body as active ingredients have been proposed (Patent Document 1). For example, Patent Document 2 discloses a therapeutic use for atopic dermatitis using Staphylococcus hominis as an active ingredient.

[0003] Furthermore, technologies that specifically control certain types of bacteria that have beneficial effects on skin condition are also attracting attention.

[0004] Here, the TER value (Transepithelial electrical resistance) is used as an indicator of barrier function, and a technology has been proposed to improve and enhance barrier function by improving the TER value (Non-Patent Literature 1).

[0005] Furthermore, adhesion proteins involved in TER values ​​include TJP1 (Tight Junction Protein 1), CLD1 (Claudin 1), OCL (Occludin), and ZO1 (Zonula Occludens Protein 1) (Non-Patent Literature 2).

[0006] Furthermore, as a prior art, for example, Patent Document 3 discloses a beauty method (excluding medical procedures) for improving skin firmness, which includes a step of massaging the face while applying a cosmetic containing collagen or the like to the face. Furthermore, Patent Document 4 discloses a beauty method that can exhibit acne-preventing effects and simultaneously performs acne prevention and cleansing. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 09-020638 [Patent Document 2] Special Publication No. 2018-515488 [Patent Document 3] Patent No. 6824850 [Patent Document 4] Patent No. 6656553 [Non-patent literature]

[0008] [Non-Patent Document 1] The Journal of Nutrition, 2011 Jan;141(1):87-94 [Non-Patent Document 2] Frontiers in Immunology, December 2015, Volume 6, Article 612 [Overview of the project] [Problems that the invention aims to solve]

[0009] Given the prior art described above, the present inventors discovered the cosmetic effect of increasing Staphylococcus hominis and completed the present invention.

[0010] In other words, the present invention aims to provide a cosmetic method (excluding medical procedures) that increases Staphylococcus hominis. [Means for solving the problem]

[0011] The cosmetic method according to the present invention (excluding medical procedures) The method is characterized by including a step to increase the bacterial count of Staphylococcus hominis.

[0012] In a preferred embodiment of the present invention, it promotes one or more selected from a decrease in the amount of lactic acid, an increase in the amount of short-chain fatty acids, and an increase in the amount of malonic acid in the skin.

[0013] In a preferred embodiment of the present invention, the short-chain fatty acid is propionic acid and / or acetic acid.

[0014] In a preferred embodiment of the present invention, it improves the TER value of the epidermis and / or promotes the formation of tight junction structures.

[0015] In a preferred embodiment of the present invention, the promotion of the formation of the tight junction structure is achieved by increasing the expression levels of CLD1 (Claudin 1) and / or OCL (Occludin).

[0016] In a preferred embodiment of the present invention, the step of increasing the amount of bacteria includes the step of applying a Staphylococcus hominis bacteria amount promoter to the skin.

[0017] In a preferred embodiment of the present invention, the Staphylococcus hominis bacteria amount promoter is a kiwifruit extract and / or a yuzu extract.

[0018] In a preferred embodiment of the present invention, the step of increasing the amount of bacteria includes applying Staphylococcus hominis to the skin.

[0019] The present invention is also a reducing agent for the amount of lactic acid in the skin, which contains Staphylococcus hominis as an active ingredient. The present invention is also an increasing agent for the amount of short-chain fatty acids in the skin, which contains Staphylococcus hominis as an active ingredient. Furthermore, the present invention is also an agent that increases the amount of malonic acid in the skin, with Staphylococcus hominis as the active ingredient. Furthermore, the present invention is also an agent for improving the TER value of the epidermis, with Staphylococcus hominis as the active ingredient. Furthermore, the present invention is also a skin tight junction structure formation promoter, with Staphylococcus hominis as the active ingredient. [Effects of the Invention]

[0020] According to the present invention, a cosmetic method (excluding medical procedures) can be provided by increasing Staphylococcus hominis. [Brief explanation of the drawing]

[0021] [Figure 1] This figure shows the results of the (1) TER value measurement test in <Test Example 1>. [Figure 2] This figure shows the evaluation results of the effect of promoting the formation of tight junction structures in (2) Test Example 1. [Figure 3] This figure shows the measurement results of lactic acid levels after culturing Staphylococcus hominis (S. hominis) in <Test Example 2> (2-1). [Figure 4] This figure shows the measurement results of the amount of propionic acid after culturing Staphylococcus hominis (S. hominis) in <Test Example 2> (2-2). [Figure 5] This figure shows the measurement results of the amount of acetic acid after culturing Staphylococcus hominis (S. hominis) in <Test Example 2> (2-3). [Figure 6] This figure shows the measurement results of malonic acid levels after culturing Staphylococcus hominis (S. hominis) in <Test Example 2> (2-4). [Figure 7]This graph shows the results of the Staphylococcus hominis (S. hominis) amount in the coating test of <Test Example 3>. [Figure 8] <Test Example 3>: This graph shows the comparison results between the 802 medium containing 1% kiwi extract and the comparative example. [Figure 9] <Test Example 3>: This graph shows the comparison results between the 802 medium containing 0.1% kiwi extract and the comparative example. [Figure 10] <Test Example 3>: This graph shows the comparison results between the 802 medium containing 1% yuzu extract and the comparative example. [Modes for carrying out the invention]

[0022] The present invention will be described in detail below, but it goes without saying that the technical scope of the present invention is not limited to the following description.

[0023] <1> beauty method The cosmetic method according to this embodiment is characterized by including a step to increase the bacterial count of Staphylococcus hominis (hereinafter simply referred to as S. hominis). Herein, the cosmetic method according to this embodiment does not include medical procedures. Furthermore, the cosmetic method according to the present invention is a non-therapeutic method. By performing the above process to increase the bacterial count, cosmetic effects can be obtained.

[0024] In the present invention, the bacterial quantity increase step refers to a step in which the amount of S. hominis after performing the means related to the bacterial quantity increase step is greater than the amount of S. hominis before performing the means related to the bacterial quantity increase step.

[0025] In a preferred embodiment of the present invention, the bacterial quantity increase step is a step of increasing the amount of S. hominis after performing the means related to the bacterial quantity increase step to preferably 1.2 times, more preferably 1.5 times, more preferably 2 times, more preferably 5 times, even more preferably 10 times, even more preferably 50 times, and particularly preferably 100 times the amount of S. hominis before performing the means related to the bacterial quantity increase step.

[0026] The effects of the beauty method of the present invention will be explained in more detail below.

[0027] As shown in the examples described later, increasing the amount of S. hominis on the skin can promote a reduction in lactic acid levels in the skin.

[0028] According to the present invention's beauty method, sensitive skin can be prevented or improved by reducing the amount of lactic acid in the skin. In this specification, sensitive skin refers to a skin condition in which even mild stimuli can cause symptoms such as itching, redness, or tingling. Furthermore, according to the beauty method of the present invention, by reducing the amount of lactic acid in the skin, it is possible to suppress and / or reduce redness, pain caused by irritation, and stinging sensations of the skin.

[0029] Furthermore, as shown in the examples described later, increasing the amount of S. hominis on the skin can increase the amount of short-chain fatty acids in the skin.

[0030] Furthermore, by promoting an increase in short-chain fatty acids in the skin, it is possible to suppress skin inflammation. It can also suppress skin redness, dryness, blemishes, wrinkles, and enlarged pores.

[0031] In a preferred embodiment of the present invention, the short-chain fatty acid is propionic acid and / or acetic acid.

[0032] Furthermore, as shown in the examples described later, increasing the amount of S. hominis on the skin can increase the amount of malonic acid in the skin.

[0033] Malonic acid is known to promote the proliferation of fibroblasts (Tsitologiia. 1993;35(2):68-70.). Furthermore, malonic acid is known to have effects such as promoting the expression of antioxidant enzymes and other genes, reducing inflammatory cytokines, and suppressing the activation of collagen-degrading enzymes and promoting collagen production in keratinocytes of the epidermis irradiated with UVB (Polymers (Basel). 2021 Mar 7;13(5):816.). In other words, by promoting an increase in malonic acid levels in the skin, it is possible to obtain effects such as promoting anti-aging through increased fibroblast proliferation, promoting antioxidant effects, suppressing skin inflammation, and inhibiting collagen breakdown while promoting collagen production.

[0034] Furthermore, as shown in the examples described later, increasing the amount of S. hominis on the skin can improve the TER value of the epidermis and / or promote the formation of tight junction structures. In this invention, "TER value" refers to the transepithelial electronic resistance. In a preferred embodiment of the present invention, this is achieved by increasing the expression levels of CLD1 (Claudin 1) and / or OCL (Occludin).

[0035] Furthermore, according to the cosmetic method of the present invention, the barrier function of the skin can be improved by increasing the TER value of the epidermis and / or promoting the formation of tight junction structures.

[0036] In other words, the cosmetic method of the present invention is particularly useful for preventing or improving skin conditions caused by a decrease in the skin's barrier function. Here, skin conditions caused by a weakened barrier function include dry skin, rough skin, wrinkles, hyperpigmentation, and sagging.

[0037] The following describes in detail the step of increasing the bacterial count in the cosmetic method of the present invention.

[0038] The bacterial growth increase step may be either the step of applying S. hominis to the skin or the step of applying an S. hominis bacterial growth enhancer to the skin.

[0039] (i) Regarding the form of the process of applying S.hominis to the skin The following provides a more detailed explanation of the process of applying S.hominis to the skin.

[0040] In this invention, S. hominis can be isolated and used from samples collected from various environments reported as habitats for S. hominis, or commercially available strains or deposited strains can be used.

[0041] As a deposited strain, Staphylococcus hominis GTC485 (hereinafter also simply referred to as S. hominis GTC485) can be used.

[0042] The S. hominis GTC485 strain is deposited at the Center for Microbial Genetic Resources Conservation (GCMR, postal code: 501-1194, address: 1-1 Yanagido, Gifu City), Gifu University Faculty of Medicine, and is available to the public.

[0043] Here, strain S. hominis GTC485 has been assigned List No. 238 by the Japanese Society for Bacteriology (postal code: 170-0003, address: Komagome TS Building, 1-43-9 Komagome, Toshima-ku, Tokyo, Japan, c / o Oral Health Association), and can be obtained through the Japanese Society for Bacteriology (see "Appendix 1 List of Bacteriological Educational Strains by Institution - Japanese Society for Bacteriology," [pdf], Japanese Society for Bacteriology, Internet <URL: http: / / jsbac.org / material / strain_kikan.pdf>).

[0044] This strain can also be obtained from the American Type Culture Collection (ATCC) under accession number ATCC 27844.

[0045] S. hominis GTC485 strain can be cultured by conventional methods. For example, it can be cultured using SCD or 802 agar medium at a temperature of 30-37°C (preferably 37°C), pH 5-9 (preferably pH 7.0), and under aerobic conditions.

[0046] In terms of application, it can be applied as a topical skin composition (applied to the skin) depending on the patient's symptoms, etc.

[0047] Examples of topical skin compositions include cosmetics, quasi-drugs, and topical medicinal products. Here, it is preferable that the topical skin composition be in the form of a cosmetic that can be used continuously. In particular, the present invention is preferably used in the form of a lotion, serum, emulsion, cream, gel, or sun care product.

[0048] The amount of S. hominis bacteria included in a topical skin composition varies depending on the dosage form of the composition, but is preferably 0.5 × 10 per application. 9 More than CFU units, more preferably 0.8 × 10 9 It can be designed to provide an application amount exceeding CFU units to the skin. Furthermore, the amount of S. hominis bacteria incorporated into the topical skin composition of the present invention is preferably 2.0 × 10⁶. 9 Less than or equal to CFU units, more preferably 1.5 × 10 9 It can be designed so that the amount applied to the skin is less than or equal to CFU units.

[0049] The amount of S. hominis bacteria incorporated into a topical skin composition varies depending on the dosage form of the composition, but is preferably 3 × 10 9 CFU / mL or higher, more preferably 4×10 9A CFU / mL or higher can be used as a guideline. Furthermore, the amount of S. hominis bacteria incorporated into the topical skin composition is preferably 8 × 10 9 CFU / mL or less, more preferably 6×10 9 A CFU / mL level of 3 or less can be used as a guideline.

[0050] The application of the topical skin composition to mammals, including humans (corresponding to the bacterial growth step), can be done once or several times per week. Furthermore, it is preferable that the application of the topical skin composition to mammals, including humans (corresponding to the bacterial growth step), be carried out in a continuous manner.

[0051] When a topical skin composition is applied continuously, the application period is preferably two weeks or more, more preferably three weeks or more, and even more preferably four weeks or more. Furthermore, when the topical skin composition is applied continuously for the above period, the frequency of application can be once or more, preferably twice or more, per week.

[0052] When provided in the form of a topical skin composition, its composition is not particularly limited and may include any commonly used components, as long as they do not impair the effects of the present invention. Such optional components can be incorporated by following the description in (ii) the step of applying the S. hominis bacterial growth promoter to the skin.

[0053] (ii) Regarding the form of the process of applying the S. hominis bacterial growth enhancer to the skin. The following provides a more detailed explanation of the process of applying the S. hominis bacterial growth enhancer to the skin.

[0054] In the present invention's cosmetic method, the amount of S. hominis can also be increased by applying a S. hominis bacterial growth promoter to the skin.

[0055] Here, with respect to the "S. hominis bacterial growth enhancer," the candidate substance can be said to be an S. hominis bacterial growth enhancer if the bacterial growth of S. hominis in the sample to which the candidate substance was added is greater than 1 times the bacterial growth of S. hominis in the comparison group without the candidate substance added.

[0056] Here, the bacterial growth promoter for S. hominis can be any compound that exhibits the above-mentioned effects, and for example, commercially available compounds (including peptides), known compounds (including peptides), compounds obtained by combinatorial chemistry technology, natural ingredients derived from plants or marine organisms, and animal tissue extracts can be used as active ingredients.

[0057] Here, "plant and animal extracts" refers not only to the animal or plant extracts themselves, but also to fractions of the extracts, purified fractions, and solvent-removed products of the extracts or fractions or purified products. Furthermore, plant-derived extracts include extracts from plants that grow wild or are cultivated, extracts from plants sold as raw materials for herbal medicines, and commercially available extracts.

[0058] As the active ingredient for the bacterial growth promoter of S. hominis, plant-derived extracts are preferred. In particular, kiwi extract and / or yuzu extract can be cited as active ingredients in S. hominis bacterial growth promoters.

[0059] The following describes a more preferred embodiment in which kiwi extract and / or yuzu extract are used as the active ingredients of the S. hominis bacterial growth promoter.

[0060] Kiwi extract can be purchased and used from commercially available kiwi extracts sold by companies that handle plant-based raw materials. Alternatively, kiwi extract can be produced in Japan by extracting the fruit of kiwis that grow wild or cultivated in Japan.

[0061] When extracting kiwi extract, it is preferable to process the kiwi fruit beforehand by crushing or finely chopping it to improve extraction efficiency.

[0062] The extract can be obtained by the following method: Add 1 to 30 parts by mass of solvent to 1 part by mass of kiwi fruit or its dried product, and immerse for several days at room temperature or for several hours at a temperature near the boiling point. After immersion, cool to room temperature, remove insoluble matter as desired, and then remove the solvent by vacuum concentration or other means. Subsequently, fractional purification can be performed using column chromatography packed with silica gel or ion exchange resin to obtain the desired extract.

[0063] As the extraction solvent, polar solvents are preferred, and suitable examples include one or more selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol, polypropylene glycol, and 1,3-butylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. Among these, 1,3-butylene glycol can be preferred as the extraction solvent.

[0064] Yuzu extract can be purchased commercially from companies that handle plant-based raw materials and used. Alternatively, yuzu extract can be produced in Japan by extracting the fruit or dried yuzu fruit that grows wild or cultivated in Japan.

[0065] Furthermore, when extracting yuzu extract, it is preferable to process the yuzu fruit or its dried form beforehand by crushing or finely chopping it to improve extraction efficiency.

[0066] The extract can be obtained by the following method: Add 1 to 30 parts by mass of solvent to 1 mass of yuzu fruit or its dried product or its dried product, and immerse for several days at room temperature or for several hours at a temperature near the boiling point. After immersion, cool to room temperature, remove insoluble matter as desired, and then remove the solvent by vacuum concentration or other means. Subsequently, fractional purification can be performed using column chromatography packed with silica gel or ion exchange resin to obtain the desired extract.

[0067] As the extraction solvent, polar solvents are preferred, and suitable examples include one or more selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol, polypropylene glycol, and 1,3-butylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. Among these, 1,3-butylene glycol can be preferred as the extraction solvent.

[0068] Here, the bacterial growth promoter can be in the form of a topical skin composition or an oral composition. In particular, the bacterial growth promoter is preferably in the form of a topical skin composition.

[0069] Suitable examples of compositions for external use on the skin include cosmetics and pharmaceuticals. In particular, it is preferable to provide a cosmetic product that can be used continuously, such as a lotion, emulsion, serum, cream, gel, or sun care product.

[0070] The content of kiwi extract and / or yuzu extract in the topical skin composition is 0.05% by mass or more, preferably 0.08% by mass or more, and more preferably 0.1% by mass or more. Furthermore, the content of kiwi extract and / or yuzu extract in the topical skin composition is preferably 2% by mass or less, preferably 1.5% by mass or less, and more preferably 1.2% by mass or less.

[0071] The topical skin composition can be applied to mammals, including humans, once or several times a week, and it is preferable to apply the bacterial growth promoter of the present invention continuously.

[0072] When a topical skin composition is applied continuously, the application period is preferably two weeks or more, more preferably three weeks or more, and even more preferably four weeks or more. Furthermore, when the topical skin composition is applied continuously for the above period, the frequency of application can be once or more, preferably twice or more, per week.

[0073] When provided in the form of a topical skin composition, its composition is not particularly limited and may include any commonly used components, as long as they do not impair the effects of the present invention.

[0074] Such optional ingredients include, for example, oils and waxes such as macadamia nut oil, avocado oil, corn oil, olive oil, rapeseed oil, sesame oil, castor oil, safflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil, liquid lanolin, hydrogenated coconut oil, hydrogenated oil, Japanese wax, hydrogenated castor oil, beeswax, candelilla wax, carnauba wax, privet wax, lanolin, reduced lanolin, hard lanolin, jojoba wax, liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, petrolatum, and mic. Hydrocarbons such as chlorocrystalline wax; higher fatty acids such as oleic acid, isostearic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, and undecylenic acid; higher alcohols such as cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol, octyldodecanol, myristyl alcohol, and cetostearyl alcohol; cetyl isooctanoate, isopropyl myristate, hexyldecyl isostearate, diisopropyl adipate, sebaci Oils such as synthetic ester oils including di-2-ethylhexyl ethanolate, cetyl lactate, diisostearyl malate, ethylene glycol di-2-ethylhexanoate, neopentyl glycol dicaprate, glycerin di-2-heptyl undecanoate, glycerin tri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentane erythritol tetra-2-ethylhexanoate, etc.; fatty acid soaps (sodium laurate, sodium palmitate) Anionic surfactants such as potassium lauryl sulfate and alkyl sulfate triethanolamine ether; cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, and laurylamine oxide; amphoteric surfactants such as imidazoline-based surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine-based surfactants (alkylbetaine, amidebetaine, sulfobetaine, etc.), and amphoteric surfactants such as acylmethyl taurine;Sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (glyceryl monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitan fatty acid esters (POE-sorbitan monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), Pluronic® type, Nonionic surfactants such as POE·POP alkyl ethers (e.g., POE·POP 2-decyltetradecyl ether), tetronics, POE castor oil and hydrogenated castor oil derivatives (e.g., POE castor oil, POE hydrogenated castor oil), sucrose fatty acid esters, and alkyl glucosides; polyhydric alcohols such as polyethylene glycol, glycerin, erythritol, sorbitol, maltitol, propylene glycol, and 2,4-hexanediol; moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid, and sodium lactate; para-aminobenzoic acid-based UV absorbers; anthranilic acid-based UV absorbers; salicylic acid-based UV absorbers; cinnamic acid-based UV absorbers; benzophenone-based UV absorbers; sugar-based UV absorbers; and UV absorbers such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane are preferred examples.

[0075] When used as an oral composition, it is preferable to use a food composition containing the active ingredient. Specifically, supplements can take the form of general foods, tablets, granules, drinks, or other dosage forms.

[0076] Here, the content and content ratio of kiwi extract and / or yuzu extract in the oral composition can be determined by following the description of the preferred embodiment of the topical skin composition mentioned above.

[0077] Furthermore, when used as an oral composition, optional components may be appropriately blended as long as they do not impair the effects of the present invention.

[0078] <2> A skin condition improving agent containing Staphylococcus hominis as the active ingredient. As shown in the examples described later, increasing the amount of S. hominis on the skin can promote a reduction in lactic acid levels in the skin. In other words, S. hominis can be used to reduce lactic acid levels in the skin. Furthermore, S. hominis can be used to suppress and / or reduce skin redness, irritation, and stinging.

[0079] Furthermore, it can be formulated as a lactic acid-reducing agent in the skin, with S. hominis as the active ingredient.

[0080] Furthermore, as shown in the examples described later, increasing the amount of S. hominis on the skin can increase the amount of short-chain fatty acids in the skin. In other words, S. hominis can be used to increase the amount of short-chain fatty acids in the skin. Furthermore, S. hominis can be used to suppress skin redness, dryness, blemishes, wrinkles, and enlarged pores.

[0081] Furthermore, it can be formulated as an agent that increases the amount of short-chain fatty acids in the skin, with S. hominis as the active ingredient.

[0082] Furthermore, as shown in the examples described later, increasing the amount of S. hominis on the skin can increase the amount of malonic acid in the skin. In other words, S. hominis can be used to increase the amount of malonic acid in the skin. Furthermore, S. hominis can be used to promote anti-aging effects by stimulating fibroblast proliferation, enhance antioxidant effects, suppress skin inflammation, and inhibit collagen breakdown and promote collagen production.

[0083] Furthermore, it can be formulated as an agent that increases the amount of malonic acid in the skin, with S. hominis as the active ingredient.

[0084] Furthermore, as shown in the examples described later, increasing the amount of S. hominis on the skin can improve the TER value of the epidermis and / or promote the formation of tight junction structures. In a preferred embodiment of the present invention, this is achieved by increasing the expression levels of CLD1 (Claudin 1) and / or OCL (Occludin).

[0085] Furthermore, S. hominis can be used to improve the TER value of the epidermis and / or to promote the formation of tight junction structures. In other words, the present invention can take the form of an agent that improves the TER value of the epidermis, with S. hominis as an active ingredient. Alternatively, the present invention can take the form of an agent that promotes the formation of tight junction structures in the skin, with S. hominis as an active ingredient.

[0086] Furthermore, the above-described content can be applied to preferred embodiments of a skin condition improving agent containing S. hominis as an active ingredient. [Examples]

[0087] <Culturing of bacterial strains> The Staphylococcus hominis GTC485 strain (hereinafter also simply referred to as S. hominis) used in the test examples described below was purchased from the Japanese Society for Bacteriology (Postal Code: 170-0003, Address: 1-43-9 Komagome, Toshima-ku, Tokyo, Komagome TS Building (Japan Dental Association Foundation)). Also, Staphylococcus epidermidis was purchased from the National Institute of Technology and Evaluation.

[0088] The purchased bacteria were cultured using agar media or liquid media suitable for each, and used as the strains in subsequent tests.

[0089] <Test Example 1: Evaluation of the Effect of Improving the Barrier Function> The effect of improving the barrier function of S. hominis was evaluated by the following method.

[0090] (1) TER value measurement test First, normal human epidermal keratinocytes (NHEK) (manufactured by Kurabo Industries Ltd.) were seeded in a 12-well Transwell plate (70,000 cells / well). Then, the seeded NHEK were cultured for 3 days in an environment of 37°C and 5% CO2. After 3 days of culture, the culture medium was replaced with a high calcium medium (1.45 mM). Here, a suspension of the S. hominis strain adjusted to a final concentration of 7,500 CFU / well with the medium was added. In addition, a sample to which a suspension of the Staphylococcus epidermidis strain was added and a sample without the strain were prepared as comparative examples.

[0091] After 6 hours of culture, the TER value was measured using Millicell ERS2 (manufactured by Millipore). The results are shown in Figure 1. In Figure 1, the vertical axis represents the difference from the initial value of the TER value (Ω·cm 2 ).

[0092] As shown in Figure 1, NHEK treated with a suspension of the S. hominis strain showed a significant increase in TER values ​​compared to the comparative example. These results demonstrate that S. hominis exhibits excellent barrier function-enhancing effects.

[0093] (2) Evaluation of the effect of promoting the formation of tight junction structures The formation of tight junction structures was visualized using the following method, and the effect of S. hominis on promoting tight junction structure formation was evaluated.

[0094] First, normal human epidermal keratinocytes (NHEK) (Kurabo Industries Ltd.) were seeded in a 4-well chamber plate (30,000 cells / well). The seeded NHEK cells were then cultured at 37°C in a 5% CO2 environment for 3 days. After 3 days of culture, the culture medium was replaced with a high-calcium medium (1.45 mM). To this, suspensions of each bacterial strain, whose concentration was adjusted in the medium to a final level of 2,300 CFU / well, were added. A sample without any bacterial strains was prepared as a control.

[0095] After 6 hours of incubation, the culture medium was removed and the cells were washed once with phosphate-buffered saline (PBS). Next, to fix the cells, they were treated with ethanol at 4°C for 15 minutes, followed by acetone treatment at room temperature for 1 minute. Then, they were treated with 0.1% Tween20 / PBS solution for 5 minutes, and further treated with 0.5% TritonX100 / PBS solution for 20 minutes. Finally, to prevent nonspecific reactions, they were treated with 10% Block Ace / PBS solution at room temperature for 30 minutes.

[0096] Subsequently, a 100-fold diluted mouse anti-Occludin antibody (Invitrogen) and a 200-fold diluted rabbit anti-Claudin-1 antibody (Invitrogen) were added to a 4-well chamber plate as primary antibodies and reacted overnight at 4°C. After washing with 0.1% Tween20 / PBS solution, a 250-fold diluted goat anti-mouse IgG antibody Alexa Fluor 488 (Invitrogen) and a 250-fold diluted goat anti-rabbit IgG antibody Alexa Fluor 555 (Invitrogen) were added to the 4-well chamber plate as secondary antibodies and reacted at room temperature for 45 minutes. After washing, the plates were mounted using Fluoromount-G (Diagnostic BioSystems). The prepared samples were observed using a confocal microscope (LSM510: Carl Zeiss).

[0097] Figure 2 shows immunohistochemical staining images of NHEK obtained by confocal microscopy. In the image, white arrowheads indicate areas where Occludin and Claudin-1 are continuously co-stained (Figure 2).

[0098] As shown in Figure 2, NHEK treated with S. hominis showed strong coloration in the intercellular spaces. Furthermore, as shown in Figure 2, we were able to confirm that Occludin and Claudin-1 were highly localized in the intercellular spaces in NHEK treated with S. hominis. Furthermore, as shown in Figure 2, in the integrated image of Occludin and Claudin-1, many areas where Occludin and Claudin-1 were continuously co-stained were observed. These results indicate that S. hominis has an excellent effect in promoting tight junction structure formation.

[0099] (3) Summary Based on these results, it was found that a cosmetic method that includes a bacterial growth process to increase the amount of S. hominis on the skin can improve the TER value of the epidermis. Furthermore, it was found that a cosmetic method that includes a bacterial growth process to increase the amount of S. hominis on the skin can promote the formation of tight junction structures.

[0100] <Test Example 2: Evaluation of decreased lactic acid levels, increased short-chain fatty acid levels, and increased malonic acid levels> The material consumption and material production capacity of S. hominis were evaluated using the following methods.

[0101] (1) Preparation of bacterial suspension First, the S. hominis GTC485 strain was cultured at 37°C under aerobic conditions using medium 802 with a pH of 7.0 (see Table 1 below). For comparison, samples containing a suspension of the Staphylococcus epidermidis strain and / or samples without the strain were prepared.

[0102] [Table 1]

[0103] (2) Measurement (2-1) About lactic acid The amount of lactate in the culture medium after 8 hours of incubation was analyzed by LC-MS / MS. The results are shown in Figure 3. As shown in Figure 3, it was found that lactic acid is consumed by S. hominis. Here, lactic acid is known as a factor that causes sensitive skin. In other words, by incorporating a cosmetic method that includes a bacterial growth process to increase the amount of S. hominis on the skin, it is possible to prevent or improve sensitive skin.

[0104] (2-2) About propionic acid After 8 hours of incubation, the amount of propionic acid in the culture medium was analyzed by LC-MS / MS. The results are shown in Figure 4. As shown in Figure 4, it was found that propionic acid is produced by S. hominis. Here, propionic acid is known to have anti-inflammatory effects. In other words, by incorporating a cosmetic method that includes a bacterial growth process to increase the amount of S. hominis on the skin, it is possible to suppress skin inflammation.

[0105] (2-3) About Acetic Acid After 8 hours of incubation, the amount of acetic acid in the culture medium was analyzed by LC-MS / MS. The results are shown in Figure 5. As shown in Figure 5, it was found that acetic acid is produced by S. hominis. Here, acetic acid is known to have anti-inflammatory effects. In other words, by incorporating a cosmetic method that includes a bacterial growth process to increase the amount of S. hominis on the skin, it is possible to suppress skin inflammation.

[0106] (2-4) About malonic acid After 8 hours of incubation, the amount of malonic acid in the culture medium was analyzed by LC-MS / MS. The results are shown in Figure 6. As shown in Figure 6, it was found that malonic acid is produced by S. hominis. Here, malonic acid is known to contribute to the promotion of anti-aging effects by promoting the proliferation of fibroblasts, the promotion of antioxidant effects, the suppression of skin inflammation, and the suppression of collagen breakdown and the promotion of collagen production. In other words, by incorporating a cosmetic method that includes a bacterial growth process to increase the amount of S. hominis on the skin, it is possible to promote the anti-aging effect of the skin, promote antioxidant effects, suppress skin inflammation, and induce the suppression of collagen breakdown and the promotion of collagen production.

[0107] (3) Summary Based on these results, it was found that a beauty treatment method that includes a step to increase the amount of bacteria that increases the amount of S. hominis on the skin can promote a decrease in lactic acid, an increase in short-chain fatty acid, and an increase in malonic acid.

[0108] <Test Example 3: Test to increase the amount of S. hominis bacteria on the skin> The following method was used to test the increase in the amount of S. hominis bacteria on the skin.

[0109] <3-1> Verification of the process of applying S.hominis to the skin The following study confirmed an increase in the amount of S. hominis on the skin after applying S. hominis to the skin.

[0110] (1) Subjects The subjects were 11 healthy Japanese women in their 30s who did not have any diseases such as atopic dermatitis.

[0111] (2) Test method (2-1) Production of cosmetics containing S. hominis (example) and cosmetics not containing S. hominis (comparative example, placebo cosmetic) The aforementioned freeze-dried S. hominis was suspended in 10 mL of distilled water to create a suspension (S. hominis content: approximately 10 mL). 10 A solution (CFU / mL) was prepared. Mix 1 mL of the prepared suspension with 1 mL of lotion (containing polyethylene glycol, dipropylene glycol, glycerin, diglycerin, potassium hydroxide, and water) to make a S. hominis-containing cosmetic (Example, S. hominis content: approximately 5 × 10⁻⁶). 9 We manufactured CFU / mL. In addition, S.hominis-free freeze-dried skim milk was used in place of S.hominis freeze-dried product, and S.hominis-free cosmetics (comparative example, placebo cosmetic) were manufactured using the same method as for S.hominis-containing cosmetics.

[0112] (2-2) Application of cosmetics The subject applied 0.2 mL of S. hominis-containing cosmetic (Example) to one cheek and 0.2 mL of S. hominis-free cosmetic (Comparative Example, Placebo) to the other cheek. (Amount of S. hominis applied per application: 10 9 CFU (units of CFU) were applied twice a week for one month. One month after the start of cosmetic application, the skin condition and S. hominis levels on the cheeks were measured using the following method.

[0113] (3) Measurement of S. hominis levels and skin condition (3-1) Measurement and evaluation of S. hominis levels in the cheek area The amount of S. hominis in each cheek was measured using the following method before the start of cosmetic application, during the cosmetic application period, and at the end of the cosmetic application test (one month after the start of cosmetic application).

[0114] Bacterial samples were collected from the cheeks of female subjects using a bacterial collection sticker or cotton swab (swab method). Next, DNA was extracted from the collected bacterial samples, and the amount of S. hominis was measured by performing OTU (Operational Taxonomic Unit) analysis and counting the number of reads belonging to each OTU.

[0115] Then, for each subject, the amount of S.hominis at each time point in the cosmetic application test was calculated by setting the number of S.hominis leads before cosmetic application to 1, and calculating the ratio of leads at 1 week, 2 weeks, 3 weeks, and 1 month after application. The results are expressed as the mean ± standard error (se) for each subject (N=11). Statistical analysis was performed based on two-way ANOVA (*P<0.05).

[0116] (4) Results The results are shown in Table 2 and Figure 7.

[0117] [Table 2]

[0118] As shown in Table 2 and Figure 7, it was found that subjects who continuously used cosmetics containing S. hominis had higher levels of S. hominis. In other words, applying S. hominis to the skin can increase the amount of S. hominis present in the skin.

[0119] <3-2> Examination of forms for applying S. hominis bacterial growth enhancers to the skin The following study investigated the increase in S. hominis levels achieved by applying a S. hominis bacterial growth enhancer to the skin. Specifically, we investigated the changes in S. hominis levels after using kiwi extract and yuzu extract.

[0120] (1) Preparation of bacterial suspension First, strain S. hominis GTC485 (hereinafter also simply referred to as S. hominis) was cultured at 37°C under aerobic conditions using medium 802 with a pH of 7.0 (see Table 1 above).

[0121] The OD660 of the cultured bacterial suspension was measured using a turbidity analyzer (NovaspecII (Amercham Pharmacia Biotech)) and the bacterial concentration was calculated.

[0122] (2) Test methods and results The bacterial suspension prepared using the method described above was added to the measurement media shown in Table 3 (Example media (Table 3 Example) and Comparative media (Table 3 Comparative Example)) so that the total volume was 5 mL. The amount of bacterial cells in the culture medium after bacterial addition is 5 × 10 7 They were cells.

[0123] Here, we have prepared the following three examples. • 802 medium containing 1% kiwi extract • 802 medium containing 0.1% kiwi extract • 802 medium containing 1% yuzu extract Here, as the kiwi extract, we used Falcorex® Kiwi B (Kiwi 1,3-butylene glycol extract, Ichimaru Falcos). Furthermore, as the yuzu extract, we used yuzu ceramide B (yuzu 1,3-butylene glycol extract, Ichimaru Falcos).

[0124] [Table 3]

[0125] After addition, the mixture was cultured with shaking under aerobic conditions at 37°C.

[0126] From the start of shaking culture, OD660 was measured every hour using a turbidity analyzer (Novaspec II (Amercham Pharmacia Biotech)). The change in OD660 value (OD660Δinitial value) was calculated by subtracting the OD660 value measured at the time of measurement from the OD660 value measured at the start of shaking culture. The results are shown in Figures 8 to 10 and Table 4.

[0127] [Table 4]

[0128] (3) Discussion The results of the examples showed that kiwi extract has a bacterial growth promoting effect on S. hominis. Furthermore, the results of the examples also showed that yuzu extract can be used to promote the bacterial growth of S. hominis.

[0129] (4) Summary Based on these results, it is possible to increase the amount of S. hominis on the skin by applying a S. hominis bacterial growth enhancer to the skin. [Industrial applicability]

[0130] This invention can be applied to beauty treatments.

Claims

1. The process includes a bacterial growth step that increases the amount of Staphylococcus hominis on the skin. The aforementioned bacterial growth increasing step includes applying a Staphylococcus hominis bacterial growth enhancer to the skin, or applying Staphylococcus hominis to the skin. A cosmetic method (excluding medical procedures) comprising the above-mentioned bacterial growth step, which includes performing one or more selected from the following: promoting a decrease in lactic acid levels in the skin, promoting an increase in short-chain fatty acid levels in the skin, promoting an increase in malonic acid levels in the skin, improving the TER value of the epidermis, and promoting the formation of tight junction structures.

2. The beauty method according to claim 1, wherein the short-chain fatty acid is propionic acid and / or acetic acid.

3. The cosmetic method according to claim 1 or 2, wherein the promotion of tight junction structure formation is achieved by increasing the expression levels of CLD1 (Claudin 1) and / or OCL (Occludin).

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