Microbial culture media containing human skin-derived products and mixed microbial culture solutions cultured using them for skin improvement applications

Abstract

The present invention relates to a culture medium composition containing sugars and amino acids derived from human skin, microorganisms of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, and Rothia, and a cosmetic composition containing their lysate or culture medium. The mixed culture medium of human skin-derived microorganisms contained in the cosmetic composition is cultured in the culture medium composition and exhibits a significantly superior skin-improving effect compared to each of the individual culture mediums.

Description

Technical Field

[0001] The present invention relates to a medium composition containing saccharides and amino acids derived from human skin, a cosmetic composition containing microorganisms belonging to the genus Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, and Rothia, a disrupted liquid or a culture solution thereof, and the like.

[0002] (Blank)

Background Art

[0003] As general skin characteristics, its surface is cold, weakly acidic, and maintains a dry state. Structurally, the epidermis forms a skin barrier, plays an important role in blocking the invasion of microorganisms and toxins, and sweating to maintain moisture and body temperature. The outermost layer of the epidermis is composed of the stratum corneum. The epidermis shows a form also called the "brick and mortar structure", but the skin tissue undergoes a continuous self-renewal process, and the scales that have undergone the final process of differentiation are continuously shed from the skin tissue.

[0004] The skin barrier is composed of dead keratinocytes and intercellular lipids, protects the skin from external stimuli, and serves as a protective film that prevents the evaporation of moisture from the skin, playing a core function in skin health. That is, it prevents the excessive release of moisture from the body and the invasion of harmful substances such as chemicals and microorganisms into the body. In addition, the keratinocyte envelope that constitutes the surface of dead keratinocytes plays an important role in the stability of intercellular lipids. Keratinocytes form the skin barrier through the processes of differentiation and keratinization. The skin barrier function can be damaged by aging or external factors, and skin barrier damage can cause a decrease in skin moisture content and the formation of wrinkles.

[0005] On the other hand, the skin produces sweat, keratinocytes, lipid cells, and other substances through the processes described above, which also contain a variety of other substances, including sugars and amino acids. Such substances can have a tremendous impact on the growth of microorganisms present on the skin and their role in improving skin condition. Nevertheless, research on these substances, as well as research on microorganisms cultured using them and their efficacy, remains inadequate.

[0006] Therefore, the inventors analyzed sugars and amino acids derived from human skin, manufactured a microbial culture medium containing these, analyzed useful microorganisms derived from human skin, and found that the culture produced by mixing and culturing them in the aforementioned culture medium exhibits excellent skin-improving effects, thus completing the present invention.

[0007] (blank) [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] One embodiment provides a culture medium composition for improving the growth of human skin-derived microorganisms, comprising sugars and amino acids derived from human skin.

[0009] Another embodiment provides a method for producing a cosmetic composition for improving skin, comprising the steps of: inoculating and culturing microorganisms of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, or Rothia into the culture medium composition to produce a culture medium; centrifuging and filtering each of the culture media to collect the supernatant; and mixing each of the supernatants.

[0010] Another embodiment is to provide a skin-improving cosmetic composition produced by the above-described manufacturing method.

[0011] Further embodiments provide uses for microorganisms of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, or Rothia; culture solutions obtained by culturing the microorganisms, their concentrates, frozen structures, supernatant liquids and mixtures thereof from which bacterial strains have been removed from the culture solution; or the cosmetic compositions.

[0012] (blank) [Means for solving the problem]

[0013] One embodiment provides a culture medium composition for improving the growth of human skin-derived microorganisms, comprising sugars and amino acids derived from human skin. In this specification, the term "culture medium composition" may be used interchangeably with novel culture medium, improved culture medium, etc. Specifically, the sugars are glucose, fructose, and rhamnose; the amino acids are threonine, tyrosine, arginine, alanine, proline, histidine, isoleucine, leucine, phenylalanine, tryptophan, valine, glutamic acid, aspartic acid, serine, and glycine. The sugars, amino acids, and microorganisms are also characterized by being derived from human skin.

[0014] More specifically, the glucose is present in amounts of 0.01-100g, 0.01-10g, 0.01-5g, 0.1-100g, 0.1-10g, 0.1-5g, 1.0-100g, 1.0-10g, and 1.0-5g per liter of the culture medium composition, while the fructose and rhamnose are present in amounts of 0.01-100g, 0.01-10g, 0.01-5g, 0.1-100g, 0.1-10g, and 0.1-5g. The aforementioned threonine, tyrosine, arginine, alanine, proline, histidine, isoleucine, leucine, tryptophan, glutamic acid, aspartic acid, serine, and glycine are present in amounts of 0.1 mg to 5.0 g, 0.1 mg to 1.0 g, 0.1 mg to 100 mg, and 0.1 mg to 50 mg, respectively, and the aforementioned phenylalanine is present in amounts of 0.1 mg to 5.0 g, 0.1 mg to 1.0 g, 0.1 mg to 100 mg, 0.1 mg to 50 mg, and 0.1 mg to 10 mg.

[0015] In one example, samples were obtained by washing the facial area of ​​subjects in their teens and twenties with sterile distilled water, and the sugars, amino acids, and microbiome constituting the samples were analyzed. As a result, glucose, fructose, and rhamnose were detected as sugar components constituting the human skin-derived samples, and threonine, tyrosine, arginine, alanine, proline, histidine, isoleucine, leucine, phenylalanine, tryptophan, valine, glutamic acid, aspartic acid, serine, and glycine were detected as amino acids.

[0016] The culture medium composition is for improving the growth of human skin-derived microorganisms, and specifically, the human skin-derived microorganisms are one or more selected from the group consisting of Staphylococcus epidermidis, Streptococcus thermophilus, Cutibacterium acnes, Corynebacterium amycolatum, and Rothia kristinae.

[0017] Specifically, the Staphylococcus genus microorganism is Staphylococcus epidermidis KCCM12551P, the Streptococcus genus microorganism is Streptococcus thermophilus KCCM12004P, the Cutibacterium genus microorganism is Cutibacterium acnes KCCM12680P, the Corynebacterium genus microorganism is Corynebacterium amycolatum KCCM12689P, and the Rothia genus microorganism is Rothia kristinae KCCM12685P.

[0018] The culture medium composition may further include one or more selected from the group consisting of glycerol, magnesium sulfate (MgSO4), potassium phosphate (K2HPO4), sodium chloride (NaCl), and yeast extract, but is not particularly limited thereto, as long as it is included in the culture medium composition as an ingredient for culturing or promoting the growth of the bacterial strain.

[0019] (blank)

[0020] Another embodiment provides a method for producing a cosmetic composition for skin improvement, comprising the steps of: inoculating and culturing microorganisms of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, or Rothia into the culture medium composition to produce a culture medium; centrifuging and filtering each of the culture media to collect the supernatant; and mixing each of the supernatants. The same parts as described above also apply to the production method.

[0021] Specifically, the Staphylococcus genus microorganism is Staphylococcus epidermidis KCCM12551P, the Streptococcus genus microorganism is Streptococcus thermophilus KCCM12004P, the Cutibacterium genus microorganism is Cutibacterium acnes KCCM12680P, the Corynebacterium genus microorganism is Corynebacterium amycolatum KCCM12689P, and the Rothia genus microorganism is Rothia kristinae KCCM12685P.

[0022] In the step of preparing the culture medium, the culture temperature is preferably 25°C to 37°C, and the culture time is 24 hours to 120 hours. In the step of centrifugation and filtration to collect the supernatant, centrifugation is performed at 4,000 rpm to 8,000 rpm for 10 to 30 minutes.

[0023] In one embodiment, in the step of producing the culture solution, the microorganisms were each inoculated into the medium composition and cultured at 30 °C for 72 hours to produce a culture solution. Thereafter, each culture solution was centrifuged at 6,000 rpm for 30 minutes, and then it was filtered using a 0.45 μm filter to recover the supernatant. Also, in order to confirm the synergistic effect of the supernatant mixture of the strain culture solutions, the supernatants of the 5 types of microorganism culture solutions were mixed at 1:1:1:1:1 (v / v) to produce a mixed solution.

[0024] As used herein, the term "culture solution" means the entire culture medium containing the strain obtained by culturing in a medium capable of supplying nutrients so that the microorganism can survive and grow in a test tube, its metabolites, excess nutrients, etc., and the culture solution may include any of the culture solution itself obtained by culturing the microorganism, its concentrate or frozen construct.

[0025] As used herein, the term "supernatant" means the liquid from which the strain has been removed from the culture solution. Specifically, the culture solution is allowed to stand for a certain period of time, and only the upper layer liquid excluding the part that has sunk to the lower layer is taken, or the strain is removed through filtration, or the culture solution is centrifuged to remove the lower precipitate and the strain and only the upper liquid is taken to obtain it.

[0026] <000...90>In the method for producing the cosmetic composition for skin improvement, the skin improvement includes skin moisturization, skin barrier strengthening, skin whitening, wrinkle improvement, skin texture improvement, pore improvement, skin tone improvement, skin elasticity improvement, or skin density improvement, but is not particularly limited thereto.

[0027] As used herein, the term "skin moisturization" means all actions that retain moisture in the skin or prevent moisture loss, and "skin barrier strengthening" may mean all actions that improve the function of the skin barrier located on the outermost layer of the skin to prevent loss of moisture and the like. The "skin whitening" means not only brightening the skin tone by inhibiting the synthesis of melanin pigments in the skin, but also improving skin hyperpigmentation such as stains and freckles caused by ultraviolet rays, hormones, or genetics. The "wrinkle improvement" means all actions that eliminate skin sagging or wrinkles due to decreased skin elasticity. The "skin texture improvement" means all actions that reduce the collection of hard or soft parts of the skin tissue or the occurrence of unevenness. The "pore improvement" means all actions that reduce the area and number of pores observable with the naked eye on the skin. The "skin tone improvement" means all actions that make the skin color lighter and brighter and increase the ITA (Individual Typology Angle) value. The "skin elasticity improvement or skin density improvement" means all actions that restore the elasticity of the skin by enhancing the ability of collagen, a connective fiber in the dermis layer, and mucinous mucopolysaccharides to bind to water.

[0028] (Blank)

[0029] Another aspect further provides a cosmetic composition for skin improvement produced by the above manufacturing method. The same parts as those described above are also applied to the cosmetic composition.

[0030] Specifically, the cosmetic composition may also contain microorganisms of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, and Rothia, their culture solution, or the supernatant of the culture solution, wherein the Staphylococcus microorganism is Staphylococcus epidermidis KCCM12551P, the Streptococcus microorganism is Streptococcus thermophilus KCCM12004P, and the Cutibacterium microorganism is Cutibacterium acnes. KCCM12680P, the Corynebacterium genus microorganism is also Corynebacterium amycolatum KCCM12689P, and the Rothia genus microorganism is also Rothia kristinae KCCM12685P. Furthermore, the cosmetic composition may include the microbial culture solution produced by culturing the microorganism in a culture medium composition containing sugars and amino acids derived from human skin.

[0031] Specifically, the composition may contain as active ingredients the microorganism, its lysate, the culture medium obtained by culturing the microorganism, its concentrate and extract, a frozen structure, the supernatant liquid from which the microorganism has been removed from the culture medium, and a mixture thereof.

[0032] The aforementioned skin improvements include, but are not limited to, skin moisturizing, strengthening of the skin barrier, skin whitening, wrinkle reduction, improvement of skin texture, improvement of pores, improvement of skin tone, improvement of skin elasticity, or improvement of skin density.

[0033] In the aforementioned cosmetic composition, the Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, and Russius microorganisms, their culture solution, or the supernatant of the culture solution may be mixed in a ratio of 1-10:1-10:1-10:1-10:1-10 (v / v), specifically, in the same volume ratio, i.e., 1:1:1:1:1 (v / v).

[0034] The cosmetic composition may further include functional additives and components commonly found in cosmetic compositions, in addition to the components disclosed herein, and may further include common auxiliary components and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. For example, the cosmetic composition may further include auxiliary components such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopherol acetate, citric acid, squalane, sodium citrate, and allantoin, and solvents such as hexanediol and purified water may be included. Furthermore, the functional additives may include components selected from the group consisting of water-soluble vitamins, fat-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts. Other ingredients that may be included include, but are not limited to, oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, dyes, fragrances, blood circulation promoters, cooling agents, antiperspirants, and purified water.

[0035] (blank)

[0036] Further embodiments provide uses for microorganisms of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, or Rothia; culture solutions obtained by culturing the microorganisms, their concentrates, frozen structures, supernatant liquids and mixtures thereof from which bacterial strains have been removed from the culture solution, or the cosmetic compositions.

[0037] Specifically, the Staphylococcus genus microorganism is Staphylococcus epidermidis KCCM12551P, the Streptococcus genus microorganism is Streptococcus thermophilus KCCM12004P, the Cutibacterium genus microorganism is Cutibacterium acnes KCCM12680P, the Corynebacterium genus microorganism is Corynebacterium amycolatum KCCM12689P, and the Rothia genus microorganism is Rothia kristinae KCCM12685P.

[0038] The aforementioned uses may include improving skin condition, moisturizing skin, strengthening the skin barrier, whitening skin, reducing wrinkles, improving skin texture, improving pores, improving skin tone, improving skin elasticity, improving skin density, or preventing, improving, or treating skin diseases.

[0039] Furthermore, the aforementioned uses may include compositions for improving skin; compositions for moisturizing skin, strengthening skin barriers, whitening skin, improving wrinkles, improving skin texture, improving pores, improving skin tone, improving skin elasticity, or improving skin density; or compositions for preventing, improving, or treating skin diseases.

[0040] (blank) [Effects of the Invention]

[0041] The mixed culture solution of human skin-derived microorganisms contained in the cosmetic composition of the present invention is cultured in the aforementioned culture medium composition and exhibits a significantly superior skin-improving effect compared to each culture solution individually.

[0042] (blank) [Brief explanation of the drawing]

[0043] [Figure 1]Figure 1 shows the results of analyzing the sugar components that make up a skin-derived product obtained by washing the facial skin of a subject according to one embodiment.

[0044] [Figure 2] Figure 2 shows the results of analyzing the amino acid components of a skin-derived product obtained by washing the facial skin of a subject according to one embodiment.

[0045] [Figure 3] Figure 3 shows the distribution of skin-derived microorganisms in a skin-derived product obtained by washing the facial skin of a subject according to one embodiment.

[0046] [Figure 4] Figures 4 and 5 show the results of evaluating the moisturizing effect of skin-derived microorganisms cultured in a newly developed improved culture medium. [Figure 5] Same as above.

[0047] [Figure 6] Figure 6 shows the results of evaluating the barrier-improving effect of skin-derived microorganisms cultured in the newly developed improved culture medium.

[0048] [Figure 7] Figures 7 and 8 show the results of comparing the moisturizing effects of skin-derived microorganisms cultured in a newly developed improved culture medium and mixtures thereof. [Figure 8] Same as above.

[0049] [Figure 9] Figure 9 shows the results of comparing the barrier-improving effect on skin-derived microorganisms cultured in the newly developed improved culture medium and on mixtures thereof.

[0050] [Figure 10] Figure 10 shows the results of evaluating the skin-whitening effect of skin-derived microorganisms cultured in a newly developed improved culture medium.

[0051] [Figure 11] Figures 11 and 12 show the results of evaluating the anti-aging and wrinkle-improving effects of skin-derived microorganisms cultured in a newly developed improved culture medium. [Figure 12] Same as above.

[0052] [Figure 13] Figures 13 and 14 show the results of comparing the anti-aging and wrinkle-improving effects of skin-derived microorganisms cultured in a newly developed improved culture medium and their mixtures. [Figure 14] Same as above.

[0053] [Figure 15] Figures 15 and 16 show the results of a clinical evaluation of the improvement of wrinkles around the eyes in formulations containing five types of microbial culture solutions (Ra: arithmetic mean roughness, Rz: 10-point mean roughness). [Figure 16] Same as above.

[0054] [Figure 17] Figures 17 and 18 show the results of a clinical evaluation of skin texture improvement for formulations containing five types of microbial culture solutions (Ra: average roughness, Rmax: maximum roughness, Rz: 10-point average roughness; difference between the average length of the five highest peaks and the average length of the five lowest valleys on the mean line, Rp: maximum cross-sectional peak height; height value of the highest cross-sectional peak from the center line of cross-sectional roughness, Rv: maximum cross-sectional valley height, depth value of the lowest cross-sectional valley from the center line of cross-sectional roughness). [Figure 18] Same as above.

[0055] [Figure 19] Figures 19 and 20 show the results of a clinical evaluation of the improvement in skin moisture content of formulations containing five types of microbial culture solutions. [Figure 20] Same as above.

[0056] [Figure 21] Figures 21 and 22 show the results of a clinical evaluation of skin pore improvement using a formulation containing five types of microbial culture solutions. [Figure 22] Same as above.

[0057] [Figure 23] Figures 23 and 24 show the results of a clinical evaluation of skin tone improvement in formulations containing five types of microbial culture solutions. [Figure 24] Same as above.

[0058] [Figure 25] Figure 25 shows the results of a clinical evaluation of skin elasticity improvement for formulations containing five types of microbial culture solutions (R2(Ua / Uf): recovery ability after resisting physical force (%), R5(Ua / Ue): ratio of suction elasticity to relaxation elasticity (%), R7(Ur / Uf): ratio of elasticity to the overall curve (%)).

[0059] [Figure 26] Figures 26 and 27 show the results of a clinical evaluation of skin density improvement in formulations containing five types of microbial culture solutions. [Figure 27] Same as above.

[0060] (blank) [Modes for carrying out the invention]

[0061] The invention will be described in more detail below through the examples provided. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to them.

[0062] (blank) [Examples]

[0063] Example 1. Sampling of skin-derived products and microbiome

[0064] Participants were recruited from individuals aged 10-20. After washing their faces, participants rested for at least 30 minutes in a constant temperature and humidity chamber (22±2℃, 50±5%), and then washed their faces with sterile distilled water to collect samples. These samples were used for the analysis of constituent sugars, amino acids, and microbiome.

[0065] (blank)

[0066] Example 2. Analysis of constituent sugars of skin-derived products

[0067] The sample obtained in Example 1 was powdered through a freeze-drying process. The powdered sample was then analyzed for constituent sugars using BIO-LC equipment, and the results are shown in Figure 1. As a result, it was confirmed that glucose, fructose, and rhamnose were detected in the skin as sugars that microorganisms can use as an energy source.

[0068] (blank)

[0069] Example 3. Analysis of amino acids in skin-derived products

[0070] The sample obtained in Example 1 was powdered through a freeze-drying process. The powdered sample was subjected to free amino acid analysis using an amino acid analyzer, and the results are shown in Figure 2. As a result, it was confirmed that the following amino acids were detected in the skin as amino acids that microorganisms can use as a nitrogen source: threonine, tyrosine, arginine, alanine, proline, histidine, isoleucine, leucine, phenylalanine, tryptophan, valine, glutamic acid, aspartic acid, serine, and glycine.

[0071] (blank)

[0072] Example 4. Analysis of skin microorganisms via Next Generation Sequencing (NGS)

[0073] The sample obtained in Example 1 was filtered using a 0.45 μm filter, and only the filter membrane was recovered to extract metagenomic DNA. To proceed with NGS analysis, the V3-V4 region of the 16S rRNA gene region of the extracted DNA was amplified to create an ampiconic DNA library, and then impurities other than DNA were removed through a purification process. The prepared DNA library was analyzed using Miseq equipment (Illumina, Inc.), and the obtained data was analyzed using the Qiime2 application and is shown in Figure 3.

[0074] As a result of this analysis, it was confirmed that strains of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, and Rothia are present on the skin. Specifically, the Staphylococcus microorganism was identified as Staphylococcus epidermidis, the Streptococcus microorganism as Streptococcus thermophilus, the Cutibacterium microorganism as Cutibacterium acnes, and the Rothia microorganism as Rothia kristinae.

[0075] (blank)

[0076] Example 5. Production of a novel microbial culture medium

[0077] Based on the skin-derived sugars and amino acid components confirmed in Examples 2 and 3, a novel microbial culture medium containing the components and their contents shown in Table 1 below was prepared.

[0078] [Table 1]

[0079] (blank)

[0080] Example 6. Production of bacterial strain culture solution using a culture medium containing skin-derived components.

[0081] Based on the components shown in Table 1, each strain of Staphylococcus epidermidis, Streptococcus thermophilus, Cutibacterium acnes, Corynebacterium amycolatum, and Rothia kristinae, belonging to the strains confirmed in Example 4, was inoculated into liquid medium and cultured at 30°C for 72 hours to produce a culture medium. Subsequently, to collect only the supernatant, centrifugation was carried out at 6,000 rpm for 30 minutes, and then the supernatant was collected after being filtered using a 0.45 μm filter to completely remove sterilization.

[0082] (blank)

[0083] Example 7. Preparation of bacterial strain culture solution using commercially available culture medium.

[0084] Staphylococcus epidermidis, Corynebacterium amycolatum, and Rothia kristinae strains were inoculated into R2A medium, while Streptococcus thermophilus and Cutibacterium acnes were inoculated into TSB medium. The cultures were incubated at 30°C for 72 hours to prepare the culture medium. Subsequently, to collect only the culture supernatant, centrifugation was carried out at 6,000 rpm for 30 minutes, and then the supernatant was collected after being filtered through a 0.45 μm filter to completely remove sterilization.

[0085] (blank)

[0086] Example 8. Preparation of a mixture of supernatant liquid from bacterial strain cultures using a culture medium containing skin-derived components.

[0087] Based on the components shown in Table 1, each strain belonging to the bacterial strains confirmed in Example 4—Staphylococcus epidermidis, Streptococcus thermophilus, Cutibacterium acnes, Corynebacterium amycolatum, and Rothia kristinae—was inoculated into liquid medium and cultured at 30°C for 72 hours to prepare a culture medium. To collect only the culture supernatant, centrifugation was carried out at 6,000 rpm for 30 minutes, and then filtered using a 0.45 μm filter to collect only the supernatant that had been completely sterilized. Furthermore, in order to confirm the synergistic effect of the mixture of these bacterial strain supernatants, a mixture was prepared by mixing the five types of supernatants in a ratio of 1:1:1:1:1.

[0088] (blank)

[0089] Example 9. Comparative analysis of skin moisturizing / barrier strengthening efficacy

[0090] The effects of the culture medium or mixed culture medium prepared in Examples 6-8 on strengthening the skin barrier and moisturizing were analyzed.

[0091] Specifically, human keratinocyte HaCaT cells were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovine serum. All cultures were performed at 37°C in a 5% CO2 incubator. The cultured cell lines were then treated with the culture medium (1% w / w) and mixed solution (1% w / w) of the prepared bacterial strain, and cultured for an additional 24 hours.

[0092] Subsequently, RNA was isolated from the cells of each sample using trizole (RNA iso, DAKARA, Japan), and then quantified at 260 nm using a nano drop. cDNA was then synthesized using an amplifier with 2 μg of RNA from each sample (C1000 Thermal Cycler, Bio-Rad, USA). The synthesized cDNA was used as a template and added along with primers for the target genes HAS3, AQP3, and FLG (Filaggrin), factors involved in skin barrier strengthening and moisturizing. Real-time polymerase chain reaction was then performed using a real-time PCR machine (Step One Plus, Applied Biosystems, USA). Gene expression levels were finally analyzed after correction for the β-actin gene. Retinoic acid (RA) was used as a positive control group, and the results are shown in Figures 4 to 6.

[0093] As a result, it was confirmed that among the aforementioned strains, Streptococcus thermophilus, Staphylococcus epidermidis, Staphylococcus russiae, Corynebacterium amycolatum, and Cutibacterium acnes increased HAS3 and AQP3 expression, contributing to moisturizing (Figures 4 and 5), and that Staphylococcus epidermidis, Staphylococcus russiae, Corynebacterium amycolatum, and Cutibacterium acnes increased FLG expression, contributing to strengthening the skin barrier (Figure 6). Furthermore, the skin moisturizing / barrier strengthening efficacy of each strain was generally shown to be even higher when cultured in the novel medium (improved medium) compared to the commercially available medium.

[0094] Furthermore, as confirmed in Figures 7 to 9, it was found that a mixture of Streptococcus thermophilus, Staphylococcus epidermidis, Russia cristinae, Corynebacterium amycolatum, and Cutibacterium acnes in a ratio of 1:1:1:1 (experimental example) was more effective in strengthening the skin barrier and moisturizing than the individual fermented supernatants of these bacteria.

[0095] (blank)

[0096] Example 10. Comparative analysis of skin whitening efficacy

[0097] The effect of the culture medium or mixed culture medium prepared in Examples 6-8 on skin whitening activity was analyzed.

[0098] Specifically, mouse pigment cells, B16-F10 (Murine Melanoma) cells (ATCC), were suspended in 2 ml of DMEM medium containing 10% FBS (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) and 1x10⁶ cells were used. 5 After inoculating cells / well into a 6-well plate, the cells were cultured at 37°C in a 5% CO2 incubator for 24 hours until 40-50% of the cells adhered to the bottom of the wells. Subsequently, α-MSH was treated at a concentration of 100 nM to induce melanin production. Next, the culture medium of the bacterial strain (1% (w / w)) was treated and cultured for an additional 3 days. After that, the culture medium was collected and centrifuged at 1,000 rpm for 10 minutes to obtain the supernatant. The absorbance of the obtained supernatant was measured three times at 490 nm using a microplate reader (Victor 3) to evaluate the melanin secretion inhibitory ability. The evaluation was performed using the following formula, and the results are shown in Figure 7. Arbutin (100 ppm, Sigma aldrich) was used as the positive control group.

[0099] [Mathematics 1]

[0100] Melanin content (%) = (Average absorbance of each sample treatment group) / (Average absorbance of α-MSH treatment group) x 100 (%)

[0101] (blank)

[0102] As a result, it was confirmed that the Corynebacterium amycolatum strain significantly reduced melanin, which had been increased by α-MSH, compared to an untreated control group. Furthermore, when cultured in a novel medium (improved medium) compared to a commercially available medium, the melanin-reducing effect was shown to be even greater (Figure 10). Therefore, the strain can be usefully used for skin whitening.

[0103] (blank)

[0104] Example 11. Comparative analysis of anti-aging activity

[0105] The culture solutions of the bacterial strains produced in Examples 6-8 were examined for their skin aging and wrinkle-improving activity.

[0106] Specifically, human dermal fibroblast (Hs68) cells were placed in a 6-well plate in a 3.5 x 10⁶ size. 5 After dispensing, the mixture was incubated for 24 hours in an incubator at 37°C and 5% CO2. Subsequently, the culture medium was removed, DPBS was added, and a 20 mJ / cm³ solution was added. 2The culture medium was either irradiated with UVB or not. Immediately after UVB irradiation, DPBS was removed and replaced with a medium without FBS. Then, the culture medium of the bacterial strains prepared in Examples 6-8 was treated with 1%, and cultured for an additional 24 hours. The group not treated with UV light or bacterial culture medium was used as the negative control group, and EGCG (epigallocatechin gallate) was used as the positive control group. Subsequently, RNA was separated from the cells of each sample using Trizole (RNA iso, DAKARA, Japan), and after quantifying the RNA at 260 nm using a nano drop, cDNA was synthesized using an amplifier with 2 μg of RNA (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, SYBR Green (SYBR Green supermix, Applied Biosystems, USA) was added to the target genes COL1A1 and MMP1 along with primers and cDNA, and a real-time polymerization chain reaction was performed using a real-time PCR machine (Step One Plus, Applied Biosystems, USA). Gene expression levels were ultimately analyzed after correction for the β-actin gene.

[0107] As a result, it was confirmed that among the aforementioned bacterial strains, Streptococcus thermophilus, Staphylococcus epidermidis, Russia chrystinae, and Corynebacterium amycoratum increased COL1A1 expression, and Corynebacterium amycoratum decreased MMP-1 expression, thus contributing to anti-aging and wrinkle improvement (Figures 11 and 12). Therefore, these bacterial strains can be usefully used for anti-aging and wrinkle improvement of the skin.

[0108] On the other hand, as confirmed in Figures 13 and 14, the mixture obtained by mixing the supernatants of Streptococcus thermophilus, Staphylococcus epidermidis, Russia cristinae, Corynebacterium amicolatum, and Cutibacterium acnes in a ratio of 1:1:1:1:1 further increased COL1A1 expression and significantly decreased MMP-1 expression induced by UVB irradiation, thus confirming that it is even more effective for anti-aging and wrinkle improvement than the supernatants obtained by the individual fermentation of Streptococcus thermophilus, Staphylococcus epidermidis, Russia cristinae, Corynebacterium amicolatum, and Cutibacterium acnes.

[0109] (blank)

[0110] Example 12. Production of a preparation containing bacterial strain culture solution

[0111] Using the five microbial culture solutions prepared in Example 6, formulations for clinical evaluation were created, and their compositions and contents are shown in Table 2 below.

[0112] [Table 2]

[0113] (blank)

[0114] Example 13. Recruitment of subjects and experimental method for formulation evaluation.

[0115] A total of 23 subjects aged 40-55 years (average age 48.78 ± 2.24 years) who met the selection criteria were recruited through the H&BIO Skin Clinical Research Center. The formulation manufactured in Example 12, i.e., the test product, was applied to the designated facial area of ​​the subjects twice a day (morning and evening) for 4 weeks. Wrinkles around the eyes, skin pores, elasticity, and density were measured before product use and 4 weeks after product use. Skin texture, moisture content, and skin tone were measured before product use, 1 time after product use, 3 days after product use, and 4 weeks after product use. In all studies, measurements were taken after the subjects washed the test area and rested for at least 30 minutes in a constant temperature and humidity chamber (22 ± 2°C, 50 ± 5%).

[0116] (blank)

[0117] Example 14. Evaluation of the efficacy of the formulation in improving wrinkles and skin texture (roughness) around the eyes.

[0118] To evaluate the efficacy of the formulation manufactured in Example 12 in improving wrinkles around the eyes and skin texture, selected eye areas were photographed before product use and 4 weeks after product use. 3D images of the cheek area were also taken before product use, 1 time after product use, 3 days after product use, and 4 weeks after product use, and the wrinkle and skin texture parameter values ​​around the eyes were analyzed using an analysis program. The measurement of wrinkles around the eyes and skin texture was performed using the PRIMOSCR 3D imaging system with a high-resolution sensor, as shown in Figures 15 to 18.

[0119] As a result, as shown in Figures 15 and 16, the formulation was found to be effective in improving wrinkles by reducing the average depth, size, number, length, and roughness (Rz) of wrinkles around the eyes, and as shown in Figures 17 and 18, it was also found to be effective in improving skin texture by reducing the average / maximum roughness parameters of the skin.

[0120] (blank)

[0121] Example 15. Efficacy for improving skin moisture content

[0122] To evaluate the efficacy of the formulation prepared in Example 12 in improving skin moisture content, the moisture content of selected cheek areas was measured three times each using a Corneometer CM 825 before product use, after one use, three days after product use, and four weeks after product use. The average values ​​were then analyzed, and selected forearm areas were photographed using an Epsion Model E100, as shown in Figures 19 and 20.

[0123] As a result, as shown in Figures 19 and 20, the formulation was confirmed to improve skin moisture content compared to before use.

[0124] (blank)

[0125] Example 16. Efficacy of improving (reducing) skin pores.

[0126] To evaluate the efficacy of the formulation produced in Example 12 in improving (reducing) skin pores, optical images taken before product use and 4 weeks after product use were analyzed using Image-Pro 10 by measuring the percentage of pore spot area (%) in the analysis area of ​​the cheek region, as shown in Figures 21 and 22.

[0127] As a result, as shown in Figures 21 and 22, the formulation was found to have a skin pore improvement effect by reducing the area and number of skin pores compared to before use.

[0128] (blank)

[0129] Example 17. Skin tone improvement effect

[0130] To evaluate the skin tone improvement efficacy of the formulation produced in Example 12, the skin tone (Individual Typology Angle, ITA°) of selected cheek areas was measured three times using the following formula before product use, after one use, three days after product use, and four weeks after product use, and the average value was analyzed, as shown in Figures 23 and 24.

[0131] [Math 2]

[0132] ITA = [Arc Tangent {L*-50} / b*]] 180 / 3.14159

[0133] (blank)

[0134] Specifically, the optical system of the Spectrophotometer CM26dG, which measures skin tone, uses a diffuse illumination 8-detection method equipped with an integrating sphere to measure the spectral reflectance and Tristmulus values ​​of the sample, according to the CIE L*a*b* color system. In the L*a*b* color system, lightness is expressed as L* (brightness), and a* and b* indicate the direction of the color as hue and saturation (a*: red, -a*: green, b*: yellow, -b*: blue).

[0135] As a result, as shown in Figures 23 and 24, it was confirmed that the formulation had an effect of improving skin tone compared to before use.

[0136] (blank)

[0137] Example 18. Efficacy for improving skin elasticity

[0138] To evaluate the skin tone-improving efficacy of the formulation prepared in Example 12, skin elasticity of selected cheek areas was measured using a Cutometer MPA580 before product use and 4 weeks after product use. The R2, R5, and R7 parameters were analyzed and are shown in Figure 25.

[0139] As a result, as shown in Figure 25, the formulation was found to have an effect on improving skin elasticity by increasing the skin's recovery ability, the ratio of relaxation elasticity to suction elasticity, and the ratio of elasticity to the overall curve compared to before use.

[0140] (blank)

[0141] Example 19. Efficacy for improving skin density

[0142] To evaluate the skin tone-improving efficacy of the formulation prepared in Example 12, skin density of a selected cheek area was measured using the DermaLab Series SkinLab Combo measuring device before product use and 4 weeks after product use. The measuring device is an ultrasound imaging device that projects acoustic pulses onto the skin to measure its reactivity, and the signal intensity is output as a dark hue for low density and a light hue for high density, as shown in Figures 26 and 27.

[0143] As a result, as shown in Figures 26 and 27, it was confirmed that the formulation had the effect of increasing skin density compared to before use.

[0144] (blank)

[0145] The above-mentioned description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, the above-mentioned embodiments should be understood in all respects as illustrative and not limiting.

[0146] (blank)

[0147] [Accession Number]

[0148] Depository name: Korea Center for Microbial Conservation (KCCM)

[0149] Accession number: KCCM12551P

[0150] Date of acceptance: 20190611

[0151] [Table 3]

[0152] Depository name: Korea Center for Microbial Conservation (KCCM)

[0153] Accession number: KCCM12004P

[0154] Date of acceptance: 20170403

[0155] [Table 4]

[0156] Depository name: Korea Center for Microbial Conservation (KCCM)

[0157] Accession number: KCCM12680P

[0158] Date of acceptance: 20200327

[0159] [Table 5]

[0160] Depository name: Korea Center for Microbial Conservation (KCCM)

[0161] Accession number: KCCM12689P

[0162] Date of acceptance: 20200409

[0163] [Table 6]

[0164] Depository name: Korea Center for Microbial Conservation (KCCM)

[0165] Accession number: KCCM12685P

[0166] Date of acceptance: 20200327

[0167] [Table 7]

Claims

1. A culture medium composition for improving microbial growth, comprising sugars such as glucose, fructose, and rhamnose; and amino acids such as threonine, tyrosine, arginine, alanine, proline, histidine, isoleucine, leucine, phenylalanine, tryptophan, valine, glutamic acid, aspartic acid, serine, and glycine.

2. The culture medium composition according to claim 1, characterized in that the sugars, amino acids, and microorganisms are derived from human skin.

3. The culture medium composition according to claim 2, characterized in that the microorganism is one or more selected from the group consisting of Staphylococcus epidermidis KCCM12551P, Streptococcus thermophilus KCCM12004P, Cutibacterium acnes KCCM12680P, Corynebacterium amycolatum KCCM12689P, and Rothia kristinae KCCM12685P.

4. The culture medium composition contains glycerol, magnesium sulfate (MgSO4). 4 ), potassium phosphate (Potassium phosphate, K 2 Hpo 4 The culture medium composition according to claim 1, further comprising ), sodium chloride (NaCl) and yeast extract.

5. A step of preparing a culture medium by inoculating and culturing microorganisms of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, or Rothia, respectively, in the culture medium composition described in claim 1, The steps include: centrifuging and filtering each of the culture solutions to collect the supernatant liquid; A method for producing a cosmetic composition for improving skin, comprising the step of mixing the respective supernatant liquids.

6. The manufacturing method according to claim 5, characterized in that the skin improvement is skin moisturizing, strengthening of the skin barrier, skin whitening, wrinkle improvement, skin texture improvement, pore improvement, skin tone improvement, skin elasticity improvement, or skin density improvement.

7. The method for producing a product according to claim 5, characterized in that the Staphylococcus microorganism is Staphylococcus epidermidis KCCM12551P, the Streptococcus microorganism is Streptococcus thermophilus KCCM12004P, the Cutibacterium microorganism is Cutibacterium acnes KCCM12680P, the Corynebacterium microorganism is Corynebacterium amycolatum KCCM12689P, and the Rothia microorganism is Rothia kristinae KCCM12685P.

8. A cosmetic composition for improving skin, characterized by being manufactured by the manufacturing method described in any one of claims 5 to 7.

9. The cosmetic composition according to claim 8, wherein the cosmetic composition comprises microorganisms of the genera Staphylococcus, Streptococcus, Cutibacterium, Corynebacterium, and Rothia, their culture solution, or the supernatant of the culture solution.

10. The cosmetic composition according to claim 9, characterized in that the Staphylococcus microorganism is Staphylococcus epidermidis KCCM12551P, the Streptococcus microorganism is Streptococcus thermophilus KCCM12004P, the Cutibacterium microorganism is Cutibacterium acnes KCCM12680P, the Corynebacterium microorganism is Corynebacterium amycolatum KCCM12689P, and the Rothia microorganism is Rothia kristinae KCCM12685P.

11. The cosmetic composition according to claim 8, characterized in that the skin improvement is skin moisturizing, strengthening of the skin barrier, skin whitening, wrinkle improvement, skin texture improvement, pore improvement, skin tone improvement, skin elasticity improvement, or skin density improvement.

12. The cosmetic composition according to claim 9, characterized in that the culture medium is cultured in the culture medium composition according to any one of claims 1 to 4.

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