Cosmetic composition containing lactoferrin, and its uses

Abstract

Providing a cosmetic composition containing lactoferrin. [Solution] (A) At least one selected from the group consisting of lactoferrin and its salts, (B) Sophorolipid, and (C) Glycyrrhizinate A cosmetic composition containing a pH of 5 to 7, The ratio of (B) to the total amount of (A) (100 parts by mass) is 50 to 1000 parts by mass. The ratio of (C) to the total amount of (A) (100 parts by mass) is 80 to 120 parts by mass. A cosmetic composition.

Description

Technical Field

[0001] The present disclosure relates to a lactoferrin-containing cosmetic composition and its uses.

Background Art

[0002] Lactoferrin is an iron-binding glycoprotein with a molecular weight of approximately 80 kDa found in bovine milk and is contained in mammalian exocrine fluids such as tears, saliva, and breast milk. Lactoferrin has many functions, and one of the important functions is its action against bacteria. Specifically, lactoferrin has broad antibacterial activity due to its ability to sequester iron required for bacterial growth.

[0003] In addition, functions of lactoferrin on skin cells have also been reported. For example, with respect to skin fibroblasts, it has an effect of promoting the production of hyaluronic acid (Non-Patent Document 1), an effect of promoting the expression of tropoelastin, which is a precursor of elastin, by activating the PI3K / Akt1 pathway via LRP-1 (Non-Patent Document 2), an effect of suppressing melanogenesis by reducing melanocyte MITF gene expression and its protein synthesis (Non-Patent Document 3), etc. have been reported.

[0004] Furthermore, it has been reported that lactoferrin also exhibits many functions when orally ingested as a supplement. For example, it has been found that ingesting fermented milk and lactoferrin daily reduces sebum production and improves acne (Non-Patent Document 4), that co-administering lactoferrin, vitamin E, and zinc to patients with mild to moderate acne vulgaris reduces acne lesion counts (Non-Patent Document 5), that oral ingestion of lactoferrin may improve skin moisture loss and texture in winter (Non-Patent Document 6), etc. are known.

[0005] The microbiome (microbial flora, bacterial flora) is a population composed of a variety of microorganisms present in the earth's environment and on the body surfaces and intestinal tracts of mammals such as humans. In recent years, the advent of next-generation sequencers has made comprehensive analysis of the microbiome possible, revealing that a wide variety of microorganisms inhabit the gut, oral cavity, and skin, performing beneficial functions for the host. These microorganisms compete for habitat and resources, or coexist with one another, maintaining a balance. This balance in the microbial ecosystem is called eubiosis. On the other hand, when the balance among microorganisms is disrupted and some microorganisms become dominant, resulting in a decrease in the diversity of the microbiome, this is called dysbiosis. Dysbiosis in the microbiome can alter the body's condition (health) and potentially cause disease. Extensive research on dysbiosis in the gut microbiota has reported that inflammatory diseases such as asthma, inflammatory bowel disease (IBD), non-alcoholic fatty liver disease (NAFLD), rheumatoid arthritis (RA), and type 2 diabetes mellitus (T2DM) are caused by abnormalities in the gut microbiota (Non-Patent Literature 7).

[0006] In the human skin microbiome, mutualism has been reported, where the host provides nutrients to microorganisms, while the microorganisms enhance the host skin's barrier function and stimulate the immune system through their metabolites (Non-Patent Literature 8). Skin dysbiosis is associated with psoriasis, eczema, acne, atopic dermatitis, and other similar diseases (Non-Patent Literature 9-13). Crosstalk between the gut microbiota and the skin microbiota has also been reported (Non-Patent Literature 14).

[0007] In recent years, there has been an increase in research on the relationship between changes in the skin microbiota due to the use of cosmetics containing specific ingredients and skin condition. For example, there are reports that continuous use of lotion improves dryness scores and strengthens the skin microbiota network (Non-Patent Literature 15). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Saito , S. , Takayama , Y. , Mizumachi , K. et al. Lactoferrin promotes hyaluronan synthesis in human dermal fibroblasts. Biotechnol Lett 33, 33–39. https: / / doi.org / 10.1007 / s10529-010-0389-3

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[0009] An object of the present disclosure is to provide a lactoferrin-containing cosmetic composition and its use. Means for Solving the Problems

[0010] The inventors of the present invention repeated studies to solve the above problems. As a result, they found that when lactoferrin is combined with sophorolipid and glycyrrhizinate and formulated into a cosmetic composition, and this is continuously applied (used in combination) to the skin, the skin flora changes, the amount of skin surface lipids (sebum amount) tends to decrease, and / or the skin tends to brighten. In addition, these changes in skin flora and / or changes in skin quality (improvement effects) can be significantly obtained by combining lactoferrin with sophorolipid and glycyrrhizinate, and it was confirmed that the desired effects cannot be obtained when only sophorolipid is formulated.

[0011] The present disclosure has been completed based on these findings and includes the following embodiments. In the following specification, lactoferrin may be abbreviated as "Lf", sophorolipid as "SL", and glycyrrhizinate as "SGr". Item 1. A cosmetic composition having a pH of 5 to 7, containing (A) at least one selected from the group consisting of lactoferrin and its salts, (B) sophorolipid, and (C) glycyrrhizinate, where the ratio of (B) to 100 parts by mass of the total amount of (A) is 50 to 1000 parts by mass, and the ratio of (C) to 100 parts by mass of the total amount of (A) is 80 to 120 parts by mass is a cosmetic composition. Item 2. The cosmetic composition according to claim 1, further comprising (D) an anionic compound and / or (E) a phospholipid. Item 3. A cosmetic composition according to item 1 or 2, wherein the SL is an acid-type SL. Item 4. A cosmetic composition according to item 1 or 2, used to increase the proportion of Halomonas titanicae in the skin's normal flora of human skin. Item 5. A cosmetic composition according to item 1 or 2, used to enhance the α- and β-diversity of the resident skin microbiota in human skin. Item 6. A cosmetic composition according to item 1 or 2, characterized by reducing the amount of surface lipids in human skin and / or brightening the skin. [Effects of the Invention]

[0012] This disclosure provides Lf-containing cosmetic compositions and their uses. More specifically, this disclosure provides cosmetic compositions containing Lf, SL, and SGr and their uses.

[0013] The cosmetic composition of this disclosure, when applied to the skin continuously, can suitably alter the skin microbiota (increase in diversity, particularly increase in α-diversity and β-diversity). Furthermore, the cosmetic composition of this disclosure, when applied to the skin continuously, can reduce the amount of surface lipids (sebum). Moreover, the cosmetic composition of this disclosure, when applied to the skin continuously, can brighten the skin.

[0014] The cosmetic composition disclosed herein maintains uniform stability over time. Furthermore, since the cosmetic composition disclosed herein maintains this uniform stability even when anionic compounds (particularly anionic polymer thickeners) are used in combination with Lf, SL, and SGr, it is possible to prepare it into various dosage forms with different viscosities, such as lotions, gels, emulsions, ointments, and creams, which are commonly used in cosmetics. [Brief explanation of the drawing]

[0015] [Figure 1] Figures (a) to (d) compare alpha diversity between the Lf continuous use group (after 1 month) and the placebo group (after 1 month) using each indicator ((a): ASV Number, (b): Species Number, (c): ACE Index, (d): Shannon index). [Figure 2] This figure compares the placement of β diversity in the Lf continuous use group (after 1 month) using principal coordinate analysis. [Figure 3] This figure compares the placement of β diversity in the placebo group (after 1 month) using principal coordinate analysis. [Figure 4] The PLS analysis results (relationship between Lf application amount and bacterial flora) are shown. [Figure 5] The PLS analysis results (visualization of factor loadings) are shown. [Figure 6] Figures 6(a) and (b) show the changes in skin epidermal sebum levels over time in the Lf continuous use group and the placebo group, respectively. [Figure 7] Figure 7(a) shows the analysis results of the skin condition measurement (regression analysis using a mixed-effects model), and Figure 7(b) shows the analysis results of the regression line for the Lf continuous use group. [Figure 8] This graph shows the percentage (vertical axis) of each subject (Lf continuous use group, placebo group) who selected each option "1 (very dull), 2 (dull), 3 (no change), 4 (brightened), and 5 (very brightened)" regarding skin brightness, against the usage period (September to November) (horizontal axis). [Modes for carrying out the invention]

[0016] (I) Cosmetic composition The cosmetic composition of this disclosure contains (A) at least one selected from the group consisting of Lf and its salts, (B) SL, and (C) SGr. (A) Lactoferrin (Lf) and its salts As Lf, Lf can be isolated by ion exchange chromatography or the like from colostrum, transitional milk, normal milk, late-stage milk, or processed products of these milks such as skim milk and whey from mammals such as cattle, goats, sheep, and humans, or Lf that has been subsequently concentrated / powdered. Genetically modified Lf produced by genetic engineering techniques can also be used. Genetically modified Lf includes not only recombinant Lf produced based on modified Lf genes, but also functional equivalents such as Lf secreted by transgenic animals and Lf active fragments. Furthermore, Lf may be treated with existing technologies to enhance the stability of its physiological activity. In addition, Lf may be a mixture of multiple Lf species blended in any proportion.

[0017] Lf salts are physiologically acceptable salts of Lf, and include, for example, alkali metal salts such as sodium salts and potassium salts, sulfates, and phosphates.

[0018] Lf or its salt may be commercially available as a reagent or ingredient in food and beverages. For example, commercially available Lf from dairy companies such as Morinaga Milk Industry Co., Ltd. can be used.

[0019] The form of Lf may be the commercially available product as is, or it may be enterically coated, or its three-dimensional structure may be partially modified by surfactants (such as sucrose fatty acid esters, glycerin fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, lecithin, saponins, sodium caseinate, oxyethylene fatty acid alcohol, sodium oleate, polyoxyethylene higher fatty acid alcohol, sophorose lipid, etc.). Alternatively, Lf may be a mixture or dispersion of oils and fats (medium-chain fatty acids, palm oil, coconut oil, rapeseed oil, sesame oil, perilla oil) and surfactants.

[0020] As long as the effects of the present invention are achieved, the proportion of component (A) contained in 100% by mass of the cosmetic composition is not limited, but can be selected from the range of 0.05 to 5% by mass. Preferably it is 0.1 to 3% by mass, and more preferably 0.1 to 2% by mass.

[0021] (B) Sophorolipid (SL) SL is a glycolipid composed of sophorose or sophorose with a partially acetylated hydroxyl group, and a hydroxyl fatty acid. Sophorose is a sugar consisting of two glucose molecules linked by a β1→2 bond. A hydroxyl fatty acid is a fatty acid containing a hydroxyl group.

[0022] SLs are broadly classified into acidic SLs, in which the carboxyl group of a hydroxyl fatty acid represented by the following chemical formula (1) is liberated, and lactone SLs, in which intramolecular sophorose is bonded, represented by the following chemical formula (2).

[0023] [ka]

[0024] [ka]

[0025] In the above chemical formula, R 0 R is either a hydrogen atom or a methyl group. 1 and R 2 Each of these is independently either a hydrogen atom or an acetyl group. 3The SL is composed of saturated aliphatic hydrocarbons or unsaturated aliphatic hydrocarbons having at least one double bond. Here, the number of carbon atoms in the aliphatic hydrocarbon is usually 11 to 20, preferably 13 to 17, and more preferably 14 to 16. The SL targeted in this invention also includes salts of the acid-type SL represented by the above chemical formula (1). Examples of such salts include alkali metal salts such as potassium and sodium, alkaline earth metal salts such as magnesium and calcium, and ammonium salts. Preferably, alkali metal salts are used, and more preferably potassium salts and sodium salts.

[0026] In this specification, the term "sophorose lipid (SL)" refers without distinction to lactone-type SL, acid-type SL, and their salts. In this case, it may be lactone-type SL, acid-type SL, or a salt of acid-type SL, or it may contain a mixture of two or more of these. When referring to any of these, it will be called "acid-type SL" or "lactone-type SL." Unless otherwise specified, the meaning of the term "acid-type SL" includes both free and salt forms of acid-type SL. Note that in the above formula, acid-type SL and lactone-type SL are R 0 ~R 3 It may be a single compound consisting of a specific atom or substituent, or various R 0 ~R 3 It may also be a mixture (composition) of multiple compounds having the same properties.

[0027] Specifically, SL is obtained by culturing microorganisms, particularly yeast. SL obtained by yeast fermentation includes both the acidic and lactoneic forms of SL described above.

[0028] Examples of SL-producing yeasts include Starmerella (Candida) bombicola, C. apicola, C. petrophilum, and Rhodotorula (Candida) bogoriensis. SL can be produced and accumulated in large quantities (100-150 g / L) in a culture medium containing high concentrations of sugar and an oily substrate. The SL can be separated from this culture by purification processes such as centrifugation, decantation, and ethyl acetate extraction, and further washed with hexane to obtain a brownish, syrupy substance. Since SL has a higher specific gravity than water, after cultivation, it can be easily separated by settling to the bottom layer by allowing the culture to stand. The resulting SL is a watery SL product containing approximately 50% water by mass. Furthermore, this SL is a mixture of multiple compounds represented by the above chemical formulas (1) or (2), including acidic SL and lactoneic SL.

[0029] In this disclosure, SL may be either acidic SL or lactoneic SL, or a mixture of both. From the viewpoint of product stability (pH stability, suppression of appearance changes (such as precipitation) due to pH fluctuations, etc.), acidic SL is preferred.

[0030] Acid-type SL can be prepared by alkaline treatment of a mixture of acid-type SL and lactone-type SL obtained by the above method, thereby hydrolyzing the ester bonds in the lactone-type SL. Examples of alkaline treatment methods include alkaline reflux (see, for example, Japanese Patent Application Publication No. 2006-070231), but the method is not limited to this, and any known alkaline treatment method can be used. Acid-type SL is readily available commercially; for example, it is sold by Saraya Co., Ltd. under product names such as "SOFORO(registered trademark) AC-30" and "SOFORO(registered trademark) AC-100". The former product is an aqueous acid-type SL containing 70% by mass of water. The latter product is high-purity SL consisting of 100% acid-type SL.

[0031] The proportion of component (B) (SL) in the cosmetic composition is preferably adjusted so that the ratio of SL to 100 parts by mass of component (A) in the cosmetic composition is 50 to 1000 parts by mass. (A) The lower limit of the ratio of SL to 100 parts by mass of component is not particularly limited as long as it does not hinder the effects of this disclosure, but is preferably 50 parts by mass or more, preferably 80 parts by mass or more, and more preferably 100 parts by mass or more. The upper limit of the ratio of SL is not particularly limited as long as it does not hinder the effects of this disclosure, but can be said to be 1000 parts by mass or less, preferably 500 parts by mass or less, more preferably 200 parts by mass or less, and particularly preferably 100 parts by mass or less. These lower and upper limits can be arbitrarily selected and combined. For example, the ranges of 50 to 500 parts by mass, 50 to 200 parts by mass, 50 to 120 parts by mass, 80 to 120 parts by mass, and 80 to 100 parts by mass can be selected.

[0032] As long as the effects of the present invention are achieved, the proportion of component (B) contained in 100% by mass of the cosmetic composition is not limited, but can be selected from the range of 0.1 to 5% by mass. Preferably, it is 0.1 to 3% by mass, and more preferably 0.1 to 1% by mass.

[0033] (C) Glycyrrhizinate (SGr) Examples of SGr include trisodium glycyrrhizinate, dipotassium glycyrrhizinate, and monoammonium glycyrrhizinate. These may be used individually or in any combination of two or more. Dipotassium glycyrrhizinate is preferred. Any commercially available SGr can be used.

[0034] The proportion of component (C) (SGr) in the cosmetic composition is preferably adjusted so that the ratio of SGr to 100 parts by mass of component (A) in the cosmetic composition is 80 to 120 parts by mass. (A) The lower limit of the ratio of SGr to 100 parts by mass of component is not particularly limited as long as it does not hinder the effects of this disclosure, but is 80 parts by mass or more, preferably 85 parts by mass or more, and more preferably 95 parts by mass or more. The upper limit of the ratio of SGr is not particularly limited as long as it does not hinder the effects of this disclosure, but can be 120 parts by mass or less, preferably 110 parts by mass or less, and more preferably 105 parts by mass or less. These lower and upper limits can be arbitrarily selected and combined. For example, the ranges of 85 to 115 parts by mass, 90 to 110 parts by mass, and 95 to 105 parts by mass can be selected.

[0035] (D) Anionic compounds The cosmetic composition of this disclosure may contain anionic compounds. Examples of anionic compounds include polymeric thickeners such as carboxymethylcellulose, hyaluronic acid or its salts, alginic acid or its salts, xanthan gum, carrageenan, gum arabic, guar gum, carboxyvinyl polymer, acrylic acid / alkyl methacrylate copolymer, and sodium polyacrylate. These may be used individually or in any combination of two or more. Acrylic acid / alkyl methacrylate copolymer is preferred. Any commercially available materials can be used.

[0036] Component (D) in the cosmetic composition is an optional component that is added when preparing the cosmetic composition in the form of a gel, cream, ointment, etc. In this case, it is preferable to adjust the ratio of component (D) to 100 parts by mass of component (A) in the cosmetic composition to 20 to 1000 parts by mass. Although not limited, for example, ranges of 50 to 500 parts by mass and 50 to 200 parts by mass can be selected.

[0037] As long as the effects of the present invention are achieved, the proportion of component (C) contained in 100% by mass of the cosmetic composition is not limited, but can be selected from the range of 0.1 to 2% by mass. Preferably it is 0.1 to 1% by mass, and more preferably 0.5 to 1% by mass.

[0038] (E) Phospholipids The cosmetic composition of this disclosure may contain phospholipids. Examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, egg yolk lecithin, hydrogenated egg yolk lecithin, soy lecithin, hydrogenated soy lecithin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, alkynyl ether phospholipids, choline plasmidsmalogen, and ethanolamine plasmidsmalogen. These may be used individually or in any combination of two or more. Preferably, egg yolk lecithin, hydrogenated egg yolk lecithin, soy lecithin, and hydrogenated soy lecithin are used. Any commercially available phospholipids can be used.

[0039] Component (E) in the cosmetic composition is an optional component that is added when preparing the cosmetic composition in the form of a gel, cream, ointment, etc. In this case, it is preferable to adjust the ratio of component (E) to 100 parts by mass of component (A) in the cosmetic composition so that it is between 1 and 100 parts by mass. Although not limited, for example, ranges of 1 to 50 parts by mass and 1 to 20 parts by mass can be selected.

[0040] As long as the effects of the present invention are achieved, the proportion of component (E) contained in 100% by mass of the cosmetic composition is not limited, but can be selected from the range of 0.005 to 0.5% by mass. Preferably it is 0.01 to 0.2% by mass, and more preferably 0.05 to 0.1% by mass.

[0041] (F) Other ingredients The cosmetic compositions of this disclosure may further contain any other cosmetically acceptable ingredients, as they do not interfere with the effects of this disclosure. Examples of other ingredients, without limiting this disclosure, include bases, pH adjusters, humectants, oily components, thickeners, emulsifiers, surfactants, solubilizers, antioxidants, preservatives, stabilizers, chelating agents, powder components, fragrances, colorants, herbal medicines, plant extracts, vitamins, etc. These other ingredients may be selected as appropriate depending on the purpose, and may be used individually or in combination of two or more, with their proportions determined as appropriate.

[0042] Without limiting the present disclosure, an example of such other components is a base, which can be water. The water is not particularly limited, and examples include purified water, distilled water, deionized water, ultrapure water, sterile water, etc., with purified water being preferred. When the composition of this disclosure contains water, the water content can be appropriately set depending on the formulation form of the composition, but 95% by mass or less is exemplified, and if the composition is in liquid or semi-solid form, more preferably 15 to 90% by mass, and more preferably 20 to 80% by mass is exemplified.

[0043] Furthermore, without limiting the disclosure, examples of other components include lower alcohols having 1 to 6 carbon atoms as a base other than water, such as ethanol, isopropanol, 1,3-butylene glycol, ethylene glycol, propylene glycol, isoprene glycol, diethylene glycol, and dipropylene glycol. Ethanol is preferably exemplified as a lower alcohol having 1 to 6 carbon atoms. These may be used individually or in combination of two or more. If the composition of the disclosure contains lower alcohols having 1 to 6 carbon atoms, the content thereof is as follows: The amount may be set appropriately depending on the formulation form, etc., but examples include 5% by mass or more in the composition, preferably 10 to 30% by mass, more preferably 10 to 20% by mass, etc. In addition, polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, diglycerin, 1,2-pentanediol, 2,4-hexylene glycol, 1,2-hexanediol, 1,2-octanediol, etc. may also be included.

[0044] Furthermore, although not limiting to this disclosure, examples of other components besides water and lower alcohols having 1 to 6 carbon atoms include hydrocarbons (paraffin, isoparaffin, petrolatum, squalane, etc.) and aliphatic monocarboxylic acid esters (isopropyl myristate, isopropyl palmitate, etc.). These components may be used individually or in combination of two or more.

[0045] Without limiting the scope of this disclosure, examples of other components, such as pH adjusters, include organic acids and their salts, such as citric acid, sodium citrate, anhydrous citric acid, malic acid, tartaric acid, lactic acid, calcium lactate, and sodium lactate; inorganic acids and their salts, such as hydrochloric acid, phosphoric acid, and sodium hydrogen phosphate; alkali hydroxides, such as sodium hydroxide and potassium hydroxide; and amines, such as triethanolamine, diethanolamine, and diisopropanolamine. The cosmetic compositions of this disclosure have a pH in the range of 5 to 7, preferably 6 to 7. To achieve this pH range, one or more pH adjusters may be used alone or in combination.

[0046] Without limiting this disclosure, examples of oily components include olive oil, sesame oil, castor oil, safflower oil, cottonseed oil, jojoba seed oil, coconut oil, palm oil, macadamia nut oil, waxes, liquid paraffin, squalane, pristane, paraffin, ceresin, and the like.

[0047] The form of the cosmetic composition disclosed herein is not limited and may be set as appropriate depending on the purpose, and may be either a liquid form (liquid, emulsion, etc.) or a semi-solid form (gel, cream, paste, mousse, etc.). Thus, the composition may be in any dosage form, such as a liquid (lotion, liniment, emulsion, etc.), a spray, aerosol, ointment, gel, cream, foam, etc., and may be used by being supported on a carrier such as a sheet. Examples of preferred dosage forms include liquids (lotions), gels, creams, ointments, etc.

[0048] The cosmetic compositions of this disclosure can be manufactured by appropriately mixing components (A), (B), and (C), components (D) and / or (E) as needed, and other components as needed, according to procedures conventionally known in the art, depending on the formulation form, route of application, etc. As an example of the manufacturing method, the procedure described in the following examples is provided, but this does not limit the disclosure, and the product may be manufactured by known methods, such as those described in the General Provisions for Preparations of the Seventeenth Edition of the Japanese Pharmacopoeia.

[0049] The cosmetic compositions of this disclosure are typically housed in containers (including packaging), and the material, shape, etc., of the container are not particularly limited as long as they can contain the composition, and may be appropriately determined according to the formulation form, application route, etc., of the composition. Although this does not limit this disclosure, if we take the case where the composition is in liquid form as an example, the container is not limited as long as it is a known container that can contain the composition. Examples of such containers include bottle containers, pump spray containers, aerosol containers, etc., in order to allow the composition to be easily applied (applied, etc.) directly to the skin from the container. In the case of bottle containers, bottle containers equipped with a sponge-like application member (head), roll-on type bottle containers, etc., are preferred examples. Commercially available containers may be used.

[0050] The amount of the cosmetic composition disclosed herein is not limited and may be set appropriately according to the age of the subject (human), skin condition, formulation form, intended use, and expected degree of effect. Although not limited to this disclosure, the number of times the composition is applied per day may be a single application (1 time) or multiple applications (for example, twice a day, in the morning and evening, or 3 to 6 times). When applied to the entire face, the amount applied per application depends on the form of the cosmetic composition, but an example would be about 2 to 4 g, which is roughly the size of a cherry or muscat grape when dispensed into the palm of the hand. Furthermore, it is preferable to use the cosmetic composition disclosed herein continuously every day. Since the skin turnover cycle is 28 days, it is preferable to use it continuously for one month or more, preferably two months or more.

[0051] As described later, the present inventors have found that continuous application of a cosmetic composition containing components (A) to (C) (the present composition) to the skin increases the proportion of Halomonas titanicae (hereinafter, H. titanicae) present in the skin's commensal microbiota, and also increases the diversity of the skin's commensal microbiota (α diversity and β diversity), compared to continuous application of a cosmetic composition that does not contain component (A) of the present composition (comparative composition). Therefore, the cosmetic composition of this disclosure is used to increase the proportion of H. titanicae present in the skin's commensal microbiota (H. titanicae increasing agent in the skin's commensal microbiota). H. titanicae is a type of skin commensal bacterium known to produce pio melanin. Pio melanin has DPPH antioxidant activity and is known to remove reactive oxygen species from human keratinocytes that have been irradiated with light. Therefore, the presence of H. titanicae in the skin's resident bacterial flora (or the amount of H. titanicae present) can produce biomelanin and remove reactive oxygen species, thereby indirectly contributing to the suppression of melanin formation. Furthermore, the cosmetic composition of this disclosure is used to increase the diversity of the skin's resident microbiota (a skin resident microbiota diversity enhancer).

[0052] The proportion and diversity of H. titanicae in the skin's commensal microbiota can be evaluated based on the examples described below. The proportion and diversity index of H. titanicae in the skin's commensal microbiota can be calculated and compared before application and after continuous use for evaluation. Specifically, the proportion and diversity index of H. titanicae in the skin's commensal microbiota are calculated using a microbiota analysis method based on amplification of the 16S rRNA gene, using samples taken from the skin before (before application) and after (after continuous use) application of the cosmetic composition containing components (A) to (C) (this composition), and evaluated by comparing before application and after continuous use.

[0053] The bacterial flora analysis methods used to evaluate the proportion and diversity of H. titanicae can be conventional methods, such as 16S amplicon sequencing, shotgun sequencing, 16S full-length amplicon sequencing, and equivalent bacterial flora analysis methods. An index for diversity increase can be, for example, the Shannon Diversity Index for α-diversity. By comparing bacterial flora analysis methods based on 16S rRNA gene amplification with samples taken from the skin after repeated use of this composition, if the Shannon Diversity Index increases after repeated use, diversity can be determined to have increased.

[0054] As described later, the present inventors have found that continuous application of a cosmetic composition containing components (A) to (C) (the present composition) to the skin results in a moderate reduction in the amount of lipids (sebum) in the skin and brighter skin, compared to continuous application of a cosmetic composition that does not contain component (A) of the present composition (comparative composition). Therefore, the cosmetic composition of this disclosure is used to suppress sebum secretion (sebum secretion inhibitor). The cosmetic composition of this disclosure is also used to brighten the skin (skin brightening agent). The amount of sebum and the brightness of the skin can be evaluated based on the examples described later. Specifically, the amount of sebum and the brightness of the skin can be evaluated by comparing them before application and after continuous application.

[0055] From the above, it can also be said that this disclosure provides a method for imparting at least one effect selected from the group consisting of (1) to (4) below to a composition containing component (A) by incorporating components (B) and (C). (1) Increases the proportion of H. titanicae in the skin's normal flora. (2) Effect of increasing the diversity of the skin's commensal bacterial flora (α diversity, β diversity) (3) Effect of reducing the amount of lipids in the skin (effect of suppressing the amount of lipid secretion in the skin) (4) Brightens skin tone (improves dullness of the skin).

[0056] In this specification, the terms “contains” and “includes” include the meanings of “consisting of” and “substantially consisting of.” [Examples]

[0057] The present invention will be described below using test examples to aid in understanding its structure and effects. However, the present invention is not limited in any way by these test examples. Unless otherwise specified, the following tests were conducted at room temperature (25±5℃) and under atmospheric pressure conditions. Unless otherwise specified, "%" below means "mass percent" and "parts" means "parts by mass".

[0058] Reference Manufacturing Example 1: Preparation of SL As a culture medium, a liquid medium containing 10g of hydrated glucose (manufactured by Nippon Shokuhin Kako Co., Ltd., product name: Nisshoku Hydrated Crystalline Glucose), 10g of peptone (manufactured by Oriental Yeast Co., Ltd., product name: Peptone CB90M), and 5g of yeast extract (manufactured by Asahi Food and Healthcare Co., Ltd., product name: Meast Powder N) per liter was used, and Candidabombicola ATCC22214 was cultured with shaking at 30°C for 2 days, and this was used as the pre-culture medium.

[0059] The pre-culture solution was inoculated into the main culture medium (3L) placed in a 5L capacity fermentation tank at a ratio of 4% of the total volume, and cultured and fermented at 30°C for 6 days under aeration conditions of 0.6vvm. The main culture medium used was a medium (pH 4.5-4.8 before sterilization) containing 100g of hydrated glucose, 50g of palm olein (NOF Corporation, product name: Permaly 2000), 50g of oleic acid (ACID CHEM, product name: Palmac 760), 1g of sodium chloride, 10g of monopotassium phosphate, 10g of magnesium sulfate heptahydrate, 2.5g of yeast extract (Asahi Food & Healthcare Co., Ltd., product name: Meast Powder N), and 1g of urea per liter.

[0060] On the sixth day after the start of cultivation, fermentation was stopped, the culture medium was removed from the fermenter, heated, and then allowed to stand for 2-3 days. This separated the mixture into three layers from bottom to top: a liquid brown precipitate layer, a milky white solid layer mainly consisting of microbial cells, and a supernatant. After removing the supernatant, an equal amount of industrial water or groundwater was added. While stirring, a 48% by mass sodium hydroxide solution was gradually added to adjust the pH to 6.5-6.9, solubilizing the SL contained in the culture medium. This was then centrifuged using a benchtop centrifuge (Westphalia: Westphalia Separator AG) to precipitate the milky white solid, and the supernatant was collected. While stirring the collected supernatant, 62.5% by mass sulfuric acid was gradually added to adjust the pH to 2.5-3.0, re-insolubilizing the SL. After standing for 2 days, the supernatant was removed as much as possible by decantation, and the residue was obtained as "natural SL" (approximately 50% water content).

[0061] Reference Manufacturing Example 2: Preparation of Acid-Type SL In the aforementioned Reference Production Example 1, a sodium hydroxide aqueous solution was added to the naturally occurring SL to adjust the pH to 14, and hydrolysis (alkaline hydrolysis) was carried out by treating it at 80°C for 2 hours. Next, after returning it to room temperature, the pH was adjusted to 8 using sulfuric acid (9.8M aqueous solution), and the generated insoluble matter was filtered off to obtain an aqueous solution containing SL. This aqueous solution containing SL contains acidic SL at a rate of 30% by mass. Using this as a raw material, SL containing 100% by mass of acidic SL can be prepared.

[0062] Examples A1-A8 and Comparative Examples A1-A6: Preparation of test lotions The test lotions (Lf&SL&SGr-containing lotions (Examples A1-A8) and comparative lotions (Comparative Examples A1-A6)) were prepared by mixing each component except the pH adjuster in the proportions shown in Table 1, and finally adjusting the pH to 6.6 with the pH adjuster.

[0063] [Table 1]

[0064] Example B1 and Comparative Example 2: Preparation of the test gel The components other than the pH adjuster were mixed in the proportions shown in Table 2, and finally the pH was adjusted to 6.3 with the pH adjuster to prepare the test gels (Lf&SL&SGr-containing gels (Examples B1-B6) and comparative gels (Comparative Examples B1-B7)).

[0065] [Table 2]

[0066] Test Example 1 The following clinical trial was conducted in compliance with the "Declaration of Helsinki" and the "Ethical Guidelines for Life Science and Medical Research Involving Human Subjects" (announced March 23, 2021, effective June 30, 2021), and was approved by Saraya Co., Ltd.'s internal ethics committee before the start of the study on September 14, 2022 (approval number: 2022-01). All subjects were fully informed about this clinical trial, and all subjects provided signed informed consent. This trial was conducted from September 14, 2022 to November 25, 2022. This trial was planned as a randomized controlled trial.

[0067] Test method 1. Selection Criteria for Subjects The selection and exclusion criteria for subjects are as follows: [Selection Criteria] (1) Women (2) Resident of Osaka Prefecture (3) At the time of obtaining consent, the person is between 30 and 49 years old (4) Wearing a mask on a daily basis (5) Those who have not used lactoferrin-containing cosmetics (at least one month has passed since the first day of the experiment). (6) Do not have any hobbies that involve sun exposure (Examples of hobbies: tanning salons, surfing, camping / barbecues, mountain climbing, walking, jogging, outdoor festivals, golf, etc.) (7) Persons who have received a full explanation regarding participation in this experiment, fully understood the explanation, and given written consent of their own free will. [Exclusion criteria] (a) Persons currently using cosmetics containing lactoferrin (b) Anyone who has had a dermatological illness caused by the use of skincare products. (c) Persons with a history of allergies to cosmetics (d) Pregnant or breastfeeding women (e) Individuals with prior experience in cosmetic medical procedures that affect the area being examined.

[0068] 2. Assignment of subjects (grouping) Twenty-nine subjects were divided into two groups: a lactoferrin continuous use group (hereinafter referred to as the "Lf continuous use group") (n=15) and a placebo group (n=14). Subjects were assigned to the Lf continuous use group and the placebo group using a random number table in Excel software. Age and self-reported skin type were used as assignment factors.

[0069] 3. Clinical Trials Various measurements were taken on the test start date, one month after the start of the test, and two months after the start of the test. On the first day of the trial, the Lf continuous use group was provided with two test samples (test lotion and test gel): a lotion containing all three components Lf, SL, and SGr (Lf&SL&SGr lotion) (Example A2) and a gel containing Lf&SL&SGr (Lf&SL&SGr gel) (Example B3). The placebo group was provided with two comparative samples (comparative lotion and comparative gel): an SL&SGr lotion without Lf (Comparative Example A6) and an SL&SGr gel (Comparative Example B7). However, the ingredients of the lotion and gel provided to each subject were not disclosed, nor were subjects informed whether they belonged to the Lf continuous use group or the placebo group.

[0070] During the study period (2 months), the subjects (Lf continuous use group and placebo group) used either the test sample or the comparison sample, respectively, according to the following method. There were no restrictions on facial cleansing or makeup removal; subjects were allowed to use the products they normally use. Morning: After washing your face, apply the test lotion and test gel (the placebo group uses the comparison lotion and comparison gel) to your entire face. Night: After removing makeup and washing the face, apply the test lotion and test gel (the placebo group uses the comparison lotion and comparison gel) to the entire face.

[0071] It was applied to the skin according to the following instructions. [lotion] Take an amount about the size of a muscat grape (approximately 3.5g) onto a cotton pad or your palm and apply it to your entire face, working from the inside outwards. Gently pat the lotion into your skin by cupping your face with your palms. [Gel] In the morning, take an amount the size of a cherry (about 2.5g), and in the evening, take an amount the size of a muscat grape (about 3.5g), and divide it into five areas: both cheeks, forehead, nose, and chin. Using your fingertips, gently spread and blend the gel over your entire face, working from the inside out and upwards from bottom to top. Gently press the gel into your skin by cupping your face with your palms.

[0072] On the first day of the trial, participants (Lf continuous use group, placebo group) were given an explanatory document and the principal investigator explained the trial. After carefully reading the document, participants signed and submitted a consent form. Subsequently, participants were asked to complete a questionnaire about themselves. At the first and second months of the trial, participants were asked to complete questionnaires regarding the use of the test sample or the control sample.

[0073] Participants (Lf continuous use group, placebo group) were instructed to use the test sample (comparison sample for the placebo group) on the morning of the measurement day (one month and two months after the start of the trial), then to gather at the measurement site wearing a mask without applying any makeup to their cheeks. Before the skin condition measurement, participants were instructed to refrain from excessive exercise, sit in a measurement room adjusted to a room temperature of 20-25°C and humidity of 40-60%, remove their masks, and rest quietly for 30 minutes.

[0074] 4. Questionnaire (1) Questionnaire on the first day of the exam The subjects were asked to fill out a questionnaire about themselves, including their work style (time of day, whether they work indoors or outdoors), race (whether they are of East Asian descent; all were of East Asian descent), self-identified skin type (normal, oily, dry, combination), skin concerns (none in particular, dryness, dullness, uneven skin tone / redness, acne / pimples, freckles / spots, wrinkles, sagging, enlarged pores, other), presence or absence of skin diseases (atopic dermatitis, allergic dermatitis, acne / acne vulgaris, pruritus, cutaneous mycosis), medication use, smoking habits, drinking habits, sun protection measures for their face (frequency of using base makeup with UV protection), and their usual skincare routine (use of facial cleanser, lotion, serum, emulsion, cream, and other cosmetics).

[0075] (2) Questionnaires from the first month and the second month after the start of the trial. The questionnaire after using the test sample (or comparison sample) included the following: Sensitivity (5-point scale: very good, good, average (no change), poor, very bad), moisture / dryness (5-point scale: very moisturized, moisturized, no change, dry, very dry), skin brightness (5-point scale: much brighter, brighter, no change, dull, very dull), redness (3-point scale: improved, no change, redness appeared), acne / pimples (3-point scale: improved, no change, acne / pimples appeared), Participants were asked questions about blemishes / freckles (3-point scale: improved, no change, blemishes / freckles became darker), wrinkles (3-point scale: improved, no change, wrinkles became deeper), sagging (3-point scale: improved, no change, sagging increased), skin condition (multiple selections allowed: no particular change, became smoother, became firmer, became more radiant, became finer in texture, makeup applied better, makeup lasted longer, skin condition worsened), and overall evaluation (3-point scale: improved, no change, worsened), and were asked to fill in their answers.

[0076] 5. Method for analyzing questionnaire results (ordinal logistic regression) In this study, to consider the effect of Lf on the subjects' skin obtained from the questionnaire results, a 5-point scale (1: very bad, 2: bad, 3: no change, 4: better, 5: very good) or a 3-point scale (1: bad, 2: no change, 3: good) was used as the response item for the questions. Since the above response items are ordinal scale data, an ordinal logistic regression model was used for analysis. The polr package in R was used for the analysis. The questionnaire results were set with the result at the start of the study as the baseline (3-point scale: 2, 5-point scale: 3), and the evaluation values ​​for each subject after approximately 1 month and approximately 2 months of continuous use were taken as a comparison to this baseline.

[0077] 6. Method for collecting DNA samples from the subject's cheek DNA samples were collected from the subjects' cheeks using the tape stripping method. Specifically, 30 minutes after the subjects removed their masks, a piece of tape (Tegaderm® Transparent Dressing (3M)) cut to approximately 2.2cm x 2.2cm was applied to the intersection of the imaginary lines extending from the pupil to the nostril on the right side of the face, and left for 1-2 minutes. After that, it was peeled off with tweezers (KFI Tweezers K-16, stainless steel / alcohol disinfected), and placed in a 1.5mL sterile tube (WATSON Hypermicrotube, sterile) while ensuring that the adhesive side did not curl, and stored in a refrigerator (4°C) (after the test, it was stored in a freezer (-80°C)). After the stored tape strip samples were left in the refrigerator in their tubes for several hours, DNA extraction was performed using the QIAamp DNA Mini Kit (QIAGEN).

[0078] 7. Amplification and sequencing of DNA samples The region encoding v3-v of the 16S rRNA in DNA collected from the subject's cheek was amplified by PCR. After reading the DNA sequence using a MiSeq sequencer, the base sequence was analyzed using QIIME2 to obtain an ampicon sequence variants (ASVs) table.

[0079] 8. Diversity Index (modified based on Non-Patent Documents 17-20) From the ASVs table, α-diversity (Shannon index) and β-diversity were calculated for each subject (Lf continuous use group, placebo group). The Shannon index of both groups was tested using the Wilcoxon test, and p-values ​​for statistical significance were calculated. For β-diversity, the Aitchison distance and Canberra distance were calculated between all samples, and the generated distance matrix was plotted on a 2D plane using principal coordinate analysis for visualization. Furthermore, the statistical significance of the difference between the two groups was evaluated using the p-value by the PERMANOVA test. The analysis mainly used the diversity and adonis2 functions of the vegan package in R.

[0080] 9. PLS analysis (Partial least squares [PLS] analysis of Lf application amount and bacterial flora) The amount of Lf applied by the subjects ("amount of test gel used by the subject in one month" × "amount of Lf in the test gel (0.5%)" + "amount of test lotion used by the subject in one month" × "amount of gel Lf in the test lotion (0.1%)") was used as the dependent variable. [formula] Lf application amount (mg) = {(Amount of gel used (g) × Lf content 0.5%) + (Amount of lotion used (g) × Lf content 0.1%)} × 1000

[0081] The subjects' skin microbiome data (approximately one month after the start of the study) was used as an explanatory variable. The PLS model was constructed using the opls package in R, and the relationship between the amount of Lf applied to the skin and the microbiome was evaluated.

[0082] 10. Skin condition measurement After collecting DNA from the subject's cheek, skin condition was measured on the left side of the face at the intersection of the imaginary lines extending from the pupil to the nostril. The measured parameters were transepidermal water loss (TEWL), stratum corneum water content, (surface) sebum content, and pH, measured in this order using a skin viscoelasticity analyzer [device + viscoelastic probe] (product name: Cutometer DUAL MPA580, manufactured by Integral Co., Ltd.).

[0083] 11. Analysis method for skin condition measurement (regression analysis using a mixed-effects model) In this study, a linear regression mixed-effects model was used to analyze the data and account for the effect of Lf on physicochemical measurements of the skin. The effect of Lf was the dependent variable, and fixed effects were estimated using the amount of gel and lotion used per month and the presence or absence of Lf (0 or 1) as independent variables. Differences between subjects were treated as random effects. The lme4 package in R was used for estimation of the mixed-effects model.

[0084] Test results 1. Diversity Index The results are shown in Figures 1-3. Figures 1(a) to 1(d) compare alpha diversity between the Lf continuous use group (after 1 month) and the placebo group (after 1 month) using each indicator ((a): ASV Number, (b): Species Number, (c): ACE Index, (d): Shannon Index). As can be seen from these figures, the Lf continuous use group (after 1 month) showed higher alpha diversity compared to the placebo group for all indicators. In particular, the Lf continuous use group showed significantly higher results for ASV (sequence level) and the ACE index. Figures 2 and 3 compare the assignment of β diversity between the Lf continuous use group (after 1 month) and the placebo group (after 1 month) using principal coordinate analysis. Of the distances used for β diversity, the Canberra distance (Figure 2) and Aitchison distance (Figure 3) were adopted. In each figure, each plot represents each subject, and the ellipse occupies the 95% highest density of observation sites. It was confirmed that β diversity was higher for both the Canberra distance and the Aitchison distance. The PERMANOVA test results were p=0.006 and p=0.037, respectively.

[0085] 2. PLS analysis (Partial least squares [PLS] analysis of Lf application amount and bacterial flora) The results of the PLS analysis are shown in Figure 4 (relationship between Lf application amount and microbiota) and Figure 5 (visualization of factor loadings). The numbers plotted in Figure 4 represent subject IDs, the color indicates the amount of Lf applied, and the location of the plot is assigned according to the similarity of the bacterial flora. From these results, it was confirmed that the bacterial flora of subjects with 0g of Lf applied was distributed in the second and third quadrants, and subjects with a large amount of Lf applied were distributed in the first and fourth quadrants. The numbers plotted in Figure 5 label the bacteria identified from the subjects' skin (to allow for matching during analysis). Figure 5 visualizes the strength of the influence of the subject ID in Figure 4 on the placement of bacteria as a weighting. The majority of bacteria are concentrated near the origin, but bacteria weighted along the X-axis are placed. The bacteria shown second from the top in Table 3 are those weighted according to the amount of Lf applied.

[0086] [Table 3]

[0087] This PLS analysis confirmed that the continuous use of Lf and SL increased the amount of Halomons titanicae in the skin's commensal microbiota. This result indicates that Halomons titanicae is an important bacterial species among the skin's commensal bacteria based on the application of Lf and SL.

[0088] 3. Analysis of skin condition measurements (regression analysis using a mixed-effects model) (1) Skin surface sebum amount Figures 6(a) and (b) show the changes in skin sebum levels over time in the Lf continuous use group and the placebo group. In both groups, a tendency for skin sebum levels to decrease with continuous use of the test sample or the comparison sample was observed compared to before the test. However, the decrease was larger in the Lf continuous use group, which used a test sample containing both SL and Lf, compared to the placebo group, which used a comparison sample containing SL, and a statistically significant difference was observed.

[0089] (2) Regression analysis using a mixed-effects model The results are shown in Figures 7(a) and 7(b). Figure 7(a) shows the regression line for the Lf continuous treatment group, and Figure 7(b) shows the regression line for the placebo group. Table 4 shows the analysis results of the regression lines.

[0090] [Table 4]

[0091] Table 4 shows that the p-value for the interaction term (GLperMon × Group_cat3Lfplus: "sum of gel and lotion usage per month" × presence or absence of Lf) is 0.076, indicating that the difference in the slopes of the two lines, "-0.11", is statistically significant. From these results, it became clear that, regarding the measured skin condition (surface lipids) of the subjects, continuous use of Lf and SL significantly reduced the amount of surface lipids. Incidentally, while a p-value of 0.05 or less is generally considered statistically significant, the power of the p-value for interaction terms is low, so in medical statistics papers, a p-value of 0.1 is sometimes considered statistically significant (Non-Patent Literature 15).

[0092] 4. Analysis of the questionnaire (ordinal logistic regression analysis) The results of the survey analysis are shown in Figure 8. Figure 8 shows the percentage (vertical axis) of each subject (Lf continuous use group, placebo group) who chose each option "1 (very dull), 2 (dull), 3 (no change), 4 (brightened), and 5 (very brightened)" regarding skin brightness, against the usage period (September to November) (horizontal axis). In September, which is the baseline month (start month of the trial), everyone chose "3," but as the usage period lengthened, the percentage of those choosing "3" in the Lf continuous use group decreased, while the percentage choosing "4" increased. On the other hand, in the placebo group, the percentage of those choosing "3" remained constant regardless of the usage period. Table 5 shows the results of the ordinal logistic regression analysis.

[0093] [Table 5]

[0094] The results in Table 5 show that the slopes of the curves for each group (Lf continuous use group, placebo group) were statistically significant, with the p-value of the interaction term (group_cat × TIME: Lf presence / absence × duration of use) being <0.001. This indicates that subjects could perceive an improvement in skin brightness by continuously using Lf and SL.

[0095] Test Example 2: Evaluation of Uniform Steady-State (Stability) The uniform stability (immediately after preparation and after stress testing) was evaluated for each test lotion (Examples A1-A8, Comparative Examples A1-A6) and test gel (Examples B1-B6, Comparative Examples B1-B6). Uniform stability was evaluated visually according to the following criteria. [Immediately after preparation] ○: No aggregation was observed, and the overall consistency is uniform. ×: Aggregation, separation, or precipitation is observed. [After rigorous testing] Each test lotion and test gel was evaluated after being left to stand in a dark room at 50°C for one month in its container. ○: No changes in appearance were observed compared to before storage. △: Slight aggregation, separation, or precipitation is observed. ×: Aggregation, separation, or precipitation is clearly observed.

[0096] The results are shown in Table 6.

[0097] [Table 6]

[0098] As shown in Table 6, although the inclusion of Lf makes the uniform stability of cosmetic compositions such as lotions and gels unstable, by incorporating SL and SGr in specific proportions in addition to Lf, no changes in appearance such as aggregation were observed immediately after preparation or after standing at 50°C for one month. From this, it was confirmed that cosmetic compositions with good uniform stability can be provided by incorporating Lf, SL, and SGr in specific proportions.

Claims

1. (A) At least one selected from the group consisting of lactoferrin and its salts, (B) Sophorolipid, and (C) Glycyrrhizinate A cosmetic composition having a pH of 5 to 7, containing the following: The ratio of (B) to the total amount of (A) is 50 to 1000 parts by mass. The ratio of (C) to the total amount of (A) (100 parts by mass) is 80 to 120 parts by mass. A cosmetic composition.

2. The cosmetic composition according to claim 1, further comprising (D) an anionic compound and / or (E) a phospholipid.

3. The cosmetic composition according to claim 1 or 2, wherein the sophorolipid is an acid-type sophorolipid.

4. A cosmetic composition according to claim 1 or 2, used to increase the proportion of Halomonas titanicae in the skin's commensal bacterial flora in human skin.

5. A cosmetic composition according to claim 1 or 2, used to enhance the α-diversity and β-diversity of the commensal bacterial flora in human skin.

6. A cosmetic composition according to claim 1 or 2, characterized by reducing the amount of surface lipids in human skin and / or brightening the skin.

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