WO2023277367A1 - Cosmetic composition comprising antarctic microorganism culture medium - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising, as active ingredients: a Pseudozyma antarctica strain, a fermentation solution thereof, a lysate thereof, an extract thereof or a culture medium thereof; and a Pseudoalteromonas neustonica strain, a fermentation solution thereof, a lysate thereof, an extract thereof or a culture medium thereof.

Description

Cosmetic Composition Containing Antarctic Microbial Culture

The present invention relates to a cosmetic containing an Antarctic-derived microorganism or a culture solution thereof.

The skin is the most basic defense organ that protects the body against stimuli in contact with the external environment, and is an important organ that expresses external beauty.

Since external beauty is not only a criterion for judgment that symbolizes youth and health, but also a competitive edge that affects work ability by expressing confidence, the tendency to manage skin in modern society is increasing.

However, with age, the secretion of various hormones that regulate metabolism decreases, and the function of immune cells and the activity of cells decrease, resulting in a decrease in the biosynthesis of immune proteins and biocomponent proteins necessary for the body, and externally destroying the ozone layer. Due to physical and chemical stimuli and stress caused by an increase in ultraviolet rays, free radicals, and active oxygen caused by environmental pollution such as, the normal function of the skin is weakened, aging is accelerated, complexion deteriorates, and the skin wrinkles.

On the other hand, melanin serves to protect the skin from ultraviolet rays, but when produced excessively, pigment is deposited on the skin to form spots and freckles, and in severe cases, it may cause skin cancer.

Therefore, in order to prevent pigmentation of the skin, it is necessary to reduce melanin production by inhibiting a part of the melanin production process.

Melanin is a high-molecular natural pigment of phenols widely present in animals, plants, and microorganisms, and is synthesized in melanosomes in melanocytes present in the basal layer of the epidermis.

Melanin is synthesized by a continuous oxidation reaction of L-tyrosine. L-tyrosine is converted into L-DOPA (3,4-dihydrozyphenylalanine) by tyrosinase, and L-DOPA is phenyl It is oxidized to anin-3,4-quinone, and finally becomes melanin through intermediate metabolites.

Therefore, in order to develop a material for functional cosmetics related to skin whitening, it is important whether the material inhibits the activity of tyrosinase.

In addition, keratinocytes in normal skin express structural proteins important for maintaining the skin barrier function, such as keratin or filaggrin, through a differentiation process to protect the skin from water loss and various environmental aggressions.

Moisturizing and skin barriers can change due to changes in the external environment and lifestyle due to sudden changes in temperature, irritation from environmental pollution, various stresses, excessive washing of the face, and natural aging due to age increase.

In order to prevent this phenomenon and maintain a bright skin tone, we are trying to improve the skin by adding physiologically active substances obtained from various known animals, plants, microorganisms, etc. to cosmetics to prevent skin aging due to oxidation. , In particular, the cosmetics industry is developing a number of products using natural products to reduce skin irritation caused by various chemicals.

Natural materials not only have few side effects on the skin, but also the value of development as a raw material for cosmetics is gradually increasing as consumers' response to cosmetics using natural materials has recently increased.

In the current situation, fermented products and bio-conversion technology using microorganisms are actively used in the field of cosmetic materials, and through this, efforts to find microorganism-based cosmetic materials that not only effectively suppress melanin production but also immediately affect skin moisturizing this is being requested

The present invention is to solve the problems of the prior art described above, and an object of the present invention is to provide an eco-friendly cosmetic raw material with excellent skin improvement activity as well as biosafety.

According to one aspect of the present invention, the pseudojima antarctica ( Pseudozyma antarctica ) strain, its fermentation broth, lysate, extract or culture solution; And Pseudo Alteromonas New Stonica ( Pseudoalteromonas neustonica ) strain, its fermentation broth, lysate, extract or culture; a cosmetic composition containing as an active ingredient is provided.

In one embodiment, the Pseudozyma antarctica strain is Pseudozyma antarctica LAB-10 (accession number: KCTC 14550BP).

In one embodiment, Pseudo Alteromonas New Stonica ( Pseudoalteromonas neustonica ) The strain may be Pseudoalteromonas New Stonica LAB-08 (accession number: KCTC 14549BP).

In one embodiment, the culture solution is Pseudozyma antarctica ( Pseudozyma antarctica ) strain or Pseudo Alteromonas New Stonica ( Pseudoalteromonas neustonica ) The culture medium itself obtained by culturing the strain, or the culture supernatant obtained by removing the strain therefrom , or a concentrate or lyophilizate of the culture supernatant.

In one embodiment, the cosmetic composition may be for skin moisturizing or skin barrier strengthening.

In one embodiment, the cosmetic composition may be used for skin whitening or improving skin tone.

In one embodiment, the cosmetic composition softening lotion, nutrient lotion, moisture cream, nutrient cream, massage cream, nutrient lotion, essence, ampoule, gel, eye cream, oil, foundation, cleansing cream, cleansing foam, cleansing water, mask It may consist of one formulation selected from the group consisting of packs, sprays and powders.

According to another aspect of the present invention, Pseudojima antarctica ( Pseudozyma antarctica ) strain, its fermentation broth, lysate, extract or culture solution; And Pseudoalteromonas New Tonica ( Pseudoalteromonas neustonica ) strain, its fermentation broth, lysate, extract or culture; there is provided a composition for external application for skin containing as an active ingredient.

According to the present invention, the Antarctic-derived microorganisms produce a large amount of ingredients beneficial to the skin during the fermentation process, effectively inhibit melanin production and have an excellent skin moisturizing effect.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

1 is a result of evaluating the melanin production inhibitory effect of the strain culture medium and extract according to an embodiment of the present invention.

Figures 2 and 3 are the results of evaluating the expression level of AQP3 and HAS3 according to the strain culture and extract treatment according to an embodiment of the present invention.

Figure 4 is a result of measuring the skin tone improvement effect according to the strain culture medium and extract treatment according to an embodiment of the present invention.

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies.

In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, embodiments of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

One aspect of the present invention is Pseudojima Antarctica ( Pseudozyma antarctica ) strain, its fermentation broth, lysate, extract or culture medium; And Pseudo Alteromonas New Stonica ( Pseudoalteromonas neustonica ) strain, its fermentation broth, lysate, extract or culture; provides a cosmetic composition containing as an active ingredient.

The present inventors confirmed the excellent skin improvement effect of the culture solution and extract of the Pseudojima antarctica strain and the Pseudoalteromonas newstonica strain, and through this, it was found that it can be used as a cosmetic raw material for skin whitening and skin moisturizing.

The " Pseudozyma antarctica " is a microorganism isolated and identified in Antarctica that can produce an emulsified material, and produces a large amount of viscous polysaccharide (EPS) having whitening activity during cultivation and fermentation, resulting in skin whitening and skin tone. It can be used for improvement purposes.

The “ Pseudoalteromonas neustonica ” strain is a microorganism isolated and identified from the surface microlayer of the Antarctic Ross Sea, and contains a large amount of GG (GlycerylGlucoside) having skin moisturizing activity during cultivation and fermentation. It can be produced and used for skin moisturizing and skin barrier strengthening.

In particular, the present inventors confirmed that the fermentation broth, extract, and culture broth of the strain can exhibit better functionality through mutual synergistic activity when acting simultaneously.

The Pseudozyma antarctica strain may be Pseudozyma antarctica LAB-10 (accession number: KCTC 14550BP), and the Pseudoalteromonas Newstonica strain may be Pseudoalteromonas newstonica LAB-10. 08 (accession number: KCTC 14549BP).

As a result of identifying the microorganisms obtained from a region in Korea (Mt. Jiri Eumeumgol), the present inventors confirmed that they were Pseudojima antarctica strains and Pseudoalteromonas newtonica strains derived from Antarctica, and compared to conventional Antarctic microorganisms, they showed better skin quality. It was confirmed that it can exhibit improvement activity.

The culture solution is Pseudozyma antarctica strain or Pseudo Alteromonas New Stonica strain ( Pseudoalteromonas neustonica ) The culture medium itself obtained by culturing the strain, or the culture supernatant obtained by removing the strain therefrom, or the culture supernatant It may be a concentrate or lyophilizate.

The culture broth is a Pseudojima anthotica strain or Pseudoalteromonas newtonica strain at least one selected from the group consisting of glucose, fructose, mannose, galactose, xylose, ribose, maltose, sucrose, dextrin, glycerin, and inulin carbon source; And it may be obtained by culturing the strain in a medium containing at least one nitrogen source selected from the group consisting of potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium oxalate, and inulin diammonium phosphate.

The carbon source or nitrogen source used in the culture medium is, for example, about 0.1 to 30 w/v%, 1 to 25 w/v%, 1 to 20 w/v%, 1 to 10 w/v%, 1 to 5 w/v %, or 1 to 1.5 w/v%.

The culture medium may include a salt commonly used in the culture medium of the strain, and the salt includes, for example, at least one salt selected from the group consisting of potassium nitrate, potassium sulfate, magnesium carbonate, magnesium sulfate, and magnesium nitrate. The salt may be included in, for example, about 0.01 to 10 w/v%, 0.1 to 7 w/v%, or 0.5 to 5 w/v%.

The culture medium may include about 0.01 to 10 w/v%, 0.05 to 5 w/v%, or 0.1 to 2 w/v% of yeast autolysate, and the culture medium may have a pH of about 2.0 to 7.0. but not limited thereto.

The culture solution is the Pseudojima anthotica strain or Pseudoalteromonas newstonica strain, for example, about 20 to 42 ℃, about 25 to 35 ℃, or about 27 to 30 ℃ under temperature conditions, such as about 24 to 144 hours, 48 to 120 hours, or may be obtained by culturing for 15 to 35 hours.

The culture conditions of the strain may include adding the strain in a medium and fermenting it, and the fermenting step may use a commonly used fermentation method, for example, the method may use fed batch fermentation. can

The extract may be obtained by extracting the separated culture solution of the Pseudojima antarctica strain or Pseudoalteromonas newstonica strain with various organic solvents.

The lysate is separated from the culture solution of the Pseudojima antarctica strain or Pseudoalteromonas newstonica strain, and then breaks the cell wall or cell membrane through mechanical methods or nonmechanical methods It may be obtained by releasing intracellular products.

The fermented broth may be obtained by culturing the Pseudojima antarica strain or the Pseudoalteromonas newstonica strain and extracting a fat-soluble component from a liquid medium in which the fermentation process is completed or therefrom.

The cosmetic composition may include, for example, 1 to 99.99% by weight, for example, 1.5 to 99.99% by weight, or 2 to 99.99% by weight of the Pseudojima antarctica strain and the Pseudoalteromonas newstonica strain, based on the total weight of the composition.

The Pseudojima antarctica strain and Pseudoalteromonas newtonica strain, their fermentation broth, lysate, extract or culture medium significantly reduce the amount of melanin production and at the same time exhibit skin moisturizing activity 2 with respect to the total weight of the cosmetic composition % (w/w) or more may be included.

The cosmetic composition may be used for moisturizing the skin or strengthening the skin barrier.

The "moisturizing the skin" refers to all actions to maintain the homeostasis of the skin tissue by properly controlling the loss of moisture (moisture evaporation) of the skin. The skin moisturizing effect may be accompanied by additional skin improving effects in various aspects, such as an exfoliating effect and a skin irritation reducing effect.

The skin structure of the "skin barrier" is divided into the externally exposed epidermis and the inner dermis, which corresponds to the outermost stratum corneum in the epidermis. The role of the skin barrier is to protect external harmful elements from invading the skin while protecting internal moisture from escaping.

The term "strengthening the skin barrier" means that when the skin barrier is weakened, it becomes vulnerable to external stimuli and easily loses moisture, which increases the possibility that the skin becomes dry and sensitive.

The cosmetic composition may be used for skin whitening or skin tone improvement.

The "skin whitening" relieves skin pigmentation phenomena such as blemishes, freckles, and melasma by suppressing the production of various biomolecules such as melanin, oxidation-reduction hemoglobin, carotene, and melanoids that affect skin color, skin yellowing, It means the effect of improving skin brightness and uniformity by reducing redness.

The above “improvement of skin tone” means brightening the color of the skin and softening the skin texture. Since the skin tone is affected by melanin pigment, the skin tone improving effect may be accompanied by a skin whitening effect.

The cosmetic composition softening lotion, nutrient lotion, moisture cream, nutrient cream, massage cream, nutrient lotion, essence, ampoule, gel, eye cream, oil, foundation, cleansing cream, cleansing foam, cleansing water, mask pack, spray and powder It may consist of one formulation selected from the group consisting of, but is not limited thereto.

According to another aspect of the present invention, Pseudojima antarctica ( Pseudozyma antarctica ) strain, its fermentation broth, lysate, extract or culture solution; And Pseudoalteromonas New Tonica ( Pseudoalteromonas neustonica ) strain, its fermentation broth, lysate, extract or culture; there is provided a composition for external application for skin containing as an active ingredient.

The composition for external application for skin may be formulated with one or more selected from the group consisting of creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, pasta agents, and cataplasma agents, but is not limited thereto.

The cosmetic composition may be formulated by conventional methods. In the formulation of the cosmetic composition, for example, the International cosmetic ingredient dictionary, 6th ed (The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995) can be referred to, and the formulation of the skin external application Reference may be made to content disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

Specifically, the cosmetic composition may be prepared in a general emulsified formulation and solubilized formulation. For example, cosmetic lotion, such as softening lotion or nourishing lotion; emulsions such as facial lotion and body lotion; creams such as nourishing creams, moisture creams, and eye creams; essence; cosmetic ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleanser such as a cleansing foam, soap, body wash, etc., but is not limited thereto.

In addition to the essential components in each formulation, the cosmetic composition and the composition for external application for skin may be appropriately mixed with other components within a range that does not impair the purpose of the present invention depending on the type of formulation or purpose of use.

The cosmetic composition and composition for external application for skin may include a generally acceptable carrier, and for example, oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately blended. It is not limited to this.

The acceptable carrier may vary depending on the formulation. For example, animal oil, vegetable oil, wax, paraffin, starch, tracanthate, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or any of these as a carrier component when formulated into an ointment, paste, cream or gel Mixtures may be used.

When the cosmetic composition and composition for external application for skin are formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and in the case of a spray It may further contain a propellant such as chlorofluorohydrocarbon, propane, butane or dimethyl ether.

When the cosmetic composition and composition for external application for skin are formulated as a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate , propylene glycol, 1,3-butylglycol oil can be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan can be used. can be used

When the cosmetic composition and the composition for external application for skin are formulated as a suspension, water, a liquid diluent such as ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester and Such suspending agents, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

When the cosmetic composition and the composition for external application for skin are formulated with soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol as carrier components , sugar, etc. may be used.

The cosmetic composition and composition for external application for skin may include fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, and foaming agents commonly used in the industry depending on the quality or function of the final product. agent), fragrance, surfactant, water, ionic or nonionic emulsifier, filler, sequestering agent, chelating agent, preservative, barrier agent, wetting agent, essential oil, dye, pigment, hydrophilic or lipophilic active agent, It may additionally contain adjuvants commonly used in the field of cosmetology or dermatology, such as any other commonly used ingredients.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect desirable properties of the cosmetic composition according to the present invention.

Through the following examples, the present invention is further detailed, but it is obvious that the present invention is not limited by the following examples.

Preparation Example 1-1: Microorganisms ( Pseudozyma antarctica ) isolation and identification

Samples were taken from Jirisan ice bone, crushed, and homogenized. Then, 1.0 g of the sample was suspended in 9.0 mL of sterilized physiological saline (0.9% NaCl), diluted in stages, and smeared on YM growth medium, respectively.

After culturing at 25 ° C. for 24 to 48 hours, strains having different colony types were selected and purified through several pure culture processes.

The pure isolated strain was spread on YM solid medium and cultured at 25 ° C for 24 hours, and the pure isolated colony was cultured on YM liquid medium and mixed with 20% (v / v) glycerol at a ratio of 1: 1 and stored in a -80 ° C cryogenic freezer.

In order to identify the strain, the internal transcribed spacer (ITS) nucleotide sequence and 26S rRNA nucleotide sequence were analyzed, and the strain was identified by searching NCBI blast.

As a result of sequencing, the isolated strain was identified as Pseudozyma antarctica.

The above identification bacteria was named Pseudojima Antarctica LAB-10, and it was deposited at the Biological Resource Center of the Jeonbuk Branch of the Korea Research Institute of Bioscience and Biotechnology on April 26, 2021, and was given the accession number KCTC 14550BP.

Preparation Example 1-2: Microorganisms ( Pseudozyma antarctica ) Preparation of culture medium and extract

Since the identified microorganism produces exopolysaccharide (EPS) through fermentation and secretes it extracellularly, the microorganism was cultured and a culture solution was obtained therefrom.

The appropriate culture conditions for exopolysaccharide (EPS) production of the identified microorganisms are 1 to 1.5 w/v% of dextrose, 0.2 to 0.4 w/v% of yeast extract, 0.2 to 0.4 w/v% of malt extract, and 0.4 to 0.7 w/v% of peptone in the medium. v%, containing 1 to 1.5 w/v% of each inorganic salt, after culturing at 25 ° C. at 200 rpm for 36 hours, and then culturing at 12 ° C. at 200 rpm for 12 hours, EPS derived from microorganisms under the above culture conditions The content was measured as 0.01% in the culture medium.

In order to analyze the efficacy of the culture medium, the strain was cultured with the medium composition, and the resulting culture medium was centrifuged at 5000 x g (10 minutes) to recover only the supernatant.

After filtering the supernatant using a 0.2 μm filter for sterilization, the final culture solution was obtained.

In order to analyze the efficacy of the extract, the strain was cultured with the medium composition, the resulting culture was extracted for 3 hours through ultrasonic extraction, and only the supernatant was recovered by centrifugation at 5000 x g (10 minutes).

After filtering the supernatant using a 0.2 μm filter for sterilization, the final extract was obtained.

Preparation Example 2-1: Microorganisms ( Pseudoalteromonas neustonica ) Isolation and identification of

Samples were collected from Jirisan ice bone, crushed, and homogenized. Then, 1.0 g of the sample was suspended in 9.0 mL of sterilized physiological saline (0.9% NaCl), diluted in stages, and smeared on a microbial growth medium (Difco Nutrient agar), respectively.

After culturing at 25° C. for 24 to 48 hours, strains having different colony types were selected and purified through several pure culture processes.

The pure isolated strain was spread on a microbial solid medium and cultured at 25 ° C for 24 hours, and the pure isolated colony was cultured on NB liquid medium and mixed with 20% (v / v) glycerol at a ratio of 1: 1 and stored in a -80 ° C cryogenic freezer.

After analyzing the base sequence of 16S rRNA to identify the strain, the strain was identified by searching NCBI blast.

As a result of sequencing, the isolated strain was identified as Pseudoalteromonas neustonica.

The identification bacteria was named Pseudoalteromonas Newstonica LAB-08, and it was deposited at the Korea Research Institute of Bioscience and Biotechnology, Jeonbuk Branch Bioresource Center on April 26, 2021, and was given the accession number KCTC 14549BP.

Preparation Example 2-2: Microorganisms ( Pseudoalteromonas neustonica ) Preparation of culture medium and extract

The identified microorganism can produce intracellular glyceryl glucoside (GG) through fermentation, and an extract culture solution containing GG extracted from the microorganism was obtained by culturing the microorganism.

The appropriate culture conditions for producing glyceryl glucoside (GG) of the identified microorganisms are 0.5 to 1.5 w/v% of tryptone, 0.2 to 0.4 w/v% of yeast extract, 0.5 to 3 w/v% of NaCl, and 0.05 to 0.1 w/v% of KCl in the medium. v%, CaCl ₂ 0.1 to 0.2 w/v%, MgCl ₂ 6H ₂ O 0.3 to 0.7 w/v%, NaHCO3 0.01 to 0.02 w/v%, MgSO ₄ 7H ₂ O 0.2 to 0.4 w/v% Including, it was confirmed when cultured at 200 rpm at 25 ° C. for 36 hours and then cultured at 12 ° C. at 200 rpm for 12 hours.

In order to analyze the efficacy of the culture medium, the strain was cultured with the medium composition, and the resulting culture medium was centrifuged at 5000 x g (10 minutes) to recover only the supernatant.

After filtering the supernatant using a 0.2 μm filter for sterilization, the final culture solution was obtained.

In order to analyze the efficacy of the extract, the strain was cultured with the medium composition, the resulting culture was extracted for 3 hours through ultrasonic extraction, and only the supernatant was recovered by centrifugation at 5000 x g (10 minutes).

After filtering the supernatant using a 0.2 μm filter for sterilization, the final extract was obtained.

Examples and Comparative Examples

In order to verify the skin improvement effect of the strain culture medium or extract of the present invention, Examples and Comparative Examples were set as follows.

The activities of Pseudozyma antarctica LAB-10 and Pseudoalteromonas neustonica LAB-08 strains isolated by the present inventors were compared with conventional Antarctic microorganisms ( Pseudozyma antarctica ATCC34888, Pseudoalteromonas sp. PAMC28425), and the synergistic activity by the two strain cultures was verified.

A culture solution of 10% concentration was used, and the extract was 1 mg/mL concentration.

[Amount (parts by weight)]

division	Example						comparative example
	One	2	3	4	5	6	One	2	3	4	5	6	7	8
Pseudozyma antarctica LAB-10 culture medium	10	-	-	-	5	-	20	-	-	-	-	-	-	-
Pseudoalteromonas neustonica LAB-08 culture medium	10	-	-	-	5	-	-	20	-	-	-	-	-	-
Pseudozyma antarctica LAB-10 Extract	-	10	-	-	5	-	-	-	20	-	-	-	-	-
Pseudoalteromonas neustonica LAB-08 Extract	-	10	-	-	5	-	-	-	-	20	-	-	-	-
Pseudozyma antarctica ATCC34888 culture medium (KCCM 11472)	-	-	10	-	-	5	-	-	-	-	20	-	-	-
Pseudoalteromonas sp. PAMC28425 culture medium (KCCM 43192)	-	-	10	-	-	5	-	-	-	-	-	20	-	-
Pseudozyma antarctica ATCC34888 extract (KCCM 11472)	-	-	-	10	-	5	-	-	-	-	-	-	20	-
Pseudoalteromonas sp. PAMC28425 extract (KCCM 43192)	-	-	-	10	-	5	-	-	-	-	-	-	-	20

Experimental Example 1: Evaluation of melanin production

Melamine content analysis (melanin contents assay) was evaluated for melanogenesis inhibition by the samples of Examples and Comparative Examples.

The B16F10 cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1X penicillin/streptomycin (P/S), and the cells were counted.

Diluted to 2 x 10 ⁴ cell/well in a 24-well plate, seeded (seeding), and cultured for 24 hours in a 5% CO ₂ , 37°C incubator.

After culturing, α-msh (α-Melanocyte-stimulating hormone) was treated with 100 nM in a medium containing 10% FBS, and samples were diluted by concentration using the medium.

As a positive control, 200ppm arbutin was used, and the sample was cultured for 72 hours in a 5% CO ₂ , 37°C incubator, and then the medium was removed and the cells were washed with DPBS.

120 μL of 1N NaOH (sodium hydroxide) was added and intracellular melanin was dissolved in a 5% CO ₂ , 37°C incubator for 30 minutes.

100 μL of completely dissolved intracellular melanin was transferred to a 96 well plate, and absorbance was measured at 450 nm using an absorbance measuring device (Multikan Go, Thermo Fisher Scientific) (Fig. 1, Table 1).

The group treated with α-msh was used as a control and the results were summarized.

division	Amount of melanin produced (absorbance)
Example 1	44.2
Example 2	46.5
Example 3	65.4
Example 4	67.2
Example 5	36.9
Example 6	60.5
Comparative Example 1	60.0
Comparative Example 2	77.9
Comparative Example 3	51.1
Comparative Example 4	57.9
Comparative Example 5	89.3
Comparative Example 6	90.2
Comparative Example 7	86.1
Comparative Example 8	91.2
control group	100
positive control	47.9

Referring to Table 2, Pseudojima Antarctica ( Pseudozyma antarctica ) Strains and Pseudo Alteromonas New Stonica ( Pseudoalteromonas neustonica ) Examples 1 to 6 containing the strain culture or extract at the same time melanin more effectively than each single strain culture or extract production was inhibited.

In particular, in the case of Examples 5 and 6 containing the respective strain culture and extract at the same time, the melanin inhibitory activity was further enhanced.

In addition, even when comparing single strains, the Pseudozyma antarctica LAB-10 or Pseudoalteromonas neustonica LAB-08 strain culture solution or extract (Comparative Examples 1 to 4) isolated by the present inventors is a conventional Antarctic microbial culture solution or extract (Comparative Examples 5 to 8 ) was evaluated as superior in melanin production inhibitory ability.

The above results suggest that the culture solutions or extracts of Pseudozyma antarctica LAB-10 and Pseudoalteromonas neustonica LAB-08 strains have better skin improvement activity than conventional Antarctic microorganism culture solutions or extracts, and can exhibit mutual synergistic activity when applied simultaneously.

Experimental Example 2: Evaluation of skin moisturizing activity

The skin moisturizing effect was evaluated using the samples of Examples and Comparative Examples.

The HaCaT cell line was cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1X penicillin/streptomycin (P/S), and after counting the cells, the number was 5 x 10 ⁵ cells/well in a 6-well plate. Diluted and seeded, 5% CO ₂ , and cultured for 24 hours in an incubator at 37°C.

After culturing, it was replaced with a serum-free medium containing 1% FBS, and then cultured for 24 hours by starvation.

Samples were diluted and treated according to concentration, and 10M retinol (Retinoic Acid) was used as a positive control.

10M retinol was incubated with the sample in a 5% CO ₂ , 37°C incubator for 24 hours, and then the medium was removed and washed with DPBS.

RNA was isolated from the cells of each sample using an RNA Extraction Kit (TaKaRa Mini BEST Universal kit), RNA was quantified with a Qubit Fluorometer, and cDNA was synthesized using an amplifier using 1 μg of RNA each (Step One Plus, Applied Biosystems).

Taqman primers AQP3, HAS3, and GAPDH were purchased from life technologies and used. Using the Taqman master mix, the desired gene was amplified using Real-time PCR equipment Step One Plus Real-Time PCR (Applied Biosystem) to confirm changes in gene expression (Table 3).

The expression level of the gene was finally analyzed through correction for the GAPDH gene.

[Relative AQP3, HAS3/GAPDH mRNA]

division	AQP3 expression level	HAS3 expression level
Example 1	2.86	3.92
Example 2	2.59	4.32
Example 3	1.63	1.96
Example 4	1.72	1.87
Example 5	3.21	6.73
Example 6	2.02	2.82
Comparative Example 1	0.88	1.26
Comparative Example 2	1.42	1.48
Comparative Example 3	0.67	1.46
Comparative Example 4	1.54	3.44
Comparative Example 5	0.96	1.05
Comparative Example 6	0.74	0.97
Comparative Example 7	0.53	0.74
Comparative Example 8	0.67	1.46
control group	1.01	1.02
positive control	4.00	3.82

Referring to Table 3, Pseudozyma antarctica ( Pseudozyma antarctica ) Strains and Pseudo Alteromonas New Stonica ( Pseudoalteromonas neustonica ) Examples 1 to 6 containing strain cultures or extracts at the same time are each single strain cultures or extracts compared to AQP3 and HAS3 expression was effectively promoted.

In particular, the expressions of AQP3 and HAS3 were more effectively promoted in the case of Examples 5 and 6 containing the respective strain culture and extract at the same time.

In addition, even when comparing single strains, the Pseudozyma antarctica LAB-10 or Pseudoalteromonas neustonica LAB-08 strain culture solution or extract (Comparative Examples 1 to 4) isolated by the present inventors is a conventional Antarctic microbial culture solution or extract (Comparative Examples 5 to 8 ) was evaluated as excellent in skin moisturizing activity.

The above results show that the culture broth or extract of Pseudozyma antarctica LAB-10 and Pseudoalteromonas neustonica LAB-08 strains promotes the expression of aquaporin protein and the synthesis of hyaluronic acid more effectively than the conventional Antarctic microorganism culture broth or extract, and synergistic activity when applied simultaneously. indicates that it can be expressed

Experimental Example 3: Evaluation of skin tone improvement activity

The sample of Example 5 was formulated into a lotion type, and after applying to 20 subjects, the change in skin brightness was confirmed.

After washing your face using the standard face wash provided by the tester, pat dry with a paper towel, and then let it rest in constant temperature and humidity conditions (20 to 24 ℃, 40 to 60% H) for 30 minutes, then MARK Vu (PSI PLUS, Korea) was used to take pictures of the facial part.

The measurement site was partitioned so that measurements could be made at the same site at each evaluation, and Example 5 and the control group were applied to the partitioned site, respectively.

Chromameter (CR-400, Konica-Minolta, Japan) was measured 5 times each, and then the average value of the measured values was recorded as the measured value.

Before the test, 2 weeks after the test, and 4 weeks after the test, skin brightness (L-value) was measured using a chromameter, and the skin brightness improvement rate was calculated (Table 4, FIG. 4).

division	Example 5		control group
	measured value (AU)	improvement rate	measured value (AU)	improvement rate
before exam	65.71	-	65.76	-
After two weeks	66.47	1.18%	65.40	-0.57%
4 weeks later	68.14	3.71%	65.42	-0.53%

Referring to Table 4, the test site using the sample of Example 5 increased skin brightness (L-value) at a statistically significant level (p <0.05) from 2 weeks after the test compared to before the test.

In addition, it was confirmed that the sample of Example 5 can effectively improve skin tone by showing a statistically significant difference (p<0.05) in comparison with the control area using the control group.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14550BP

Entrusted date: 2021419

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14549BP

Entrusted date: 2021419

　

　

Claims (8)

1. Pseudozyma antarctica strain, its fermentation broth, lysate, extract or culture medium; and

   Pseudo Alteromonas New Tonica ( Pseudoalteromonas neustonica ) strain, its fermentation broth, lysate, extract or culture; a cosmetic composition containing as an active ingredient.
2. According to claim 1,

   The Pseudozyma antarctica strain is Pseudozyma antarctica LAB-10 (accession number: KCTC 14550BP ) phosphorus, cosmetic composition.
3. According to claim 1,

   The pseudoalteromonas new tonica ( Pseudoalteromonas neustonica ) The strain is pseudoalteromonas new tonica LAB-08 (accession number: KCTC 14549BP), a cosmetic composition.
4. According to claim 1,

   The culture solution is Pseudozyma antarctica strain or Pseudo Alteromonas New Stonica strain ( Pseudoalteromonas neustonica ) The culture medium itself obtained by culturing the strain, or the culture supernatant obtained by removing the strain therefrom, or the culture supernatant Concentrate or lyophilisate, a cosmetic composition.
5. According to any one of claims 1 to 4,

   A cosmetic composition for skin moisturizing or skin barrier enhancement.
6. According to any one of claims 1 to 4,

   A cosmetic composition for skin whitening or skin tone improvement.
7. According to any one of claims 1 to 4,

   In the group consisting of softening lotion, nutrient lotion, moisturizing cream, nutrient cream, massage cream, nutrient lotion, essence, ampoule, gel, eye cream, oil, foundation, cleansing cream, cleansing foam, cleansing water, mask pack, spray and powder Consisting of one selected formulation, a cosmetic composition.
8. Pseudozyma antarctica strain, its fermentation broth, lysate, extract or culture medium; and

   Pseudoalteromonas new tonica ( Pseudoalteromonas neustonica ) strain, its fermentation broth, lysate, extract or culture; a composition for external application for skin containing as an active ingredient.

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