KR20220096260A - Cutibacterium granulosum strain and its use for improving hair or scalp condition - Google Patents

Abstract

The present invention relates to a novel microorganism, and a lysate, culture medium, extract, fermented extract thereof, and a use thereof for improving hair condition or scalp condition. A novel Cutibacterium granulosum strain according to one aspect of the present invention is effective in promoting hair growth, preventing hair loss, strengthening hair roots, suppressing the production of scalp lipids, preventing dandruff, and inhibiting scalp inflammation, so that it can be widely applied for improving, preventing, or treating hair condition or scalp condition.

Description

Cutibacterium granulosum strain and its use for improving hair or scalp condition {Cutibacterium granulosum strain and its use for improving hair or scalp condition}

It relates to novel microorganisms, their lysate, culture solution, extract, fermented solution, and their use for improving hair or scalp condition.

In the human body, hair grows from hair follicles on the scalp, and cycles such as anagen, catagen, and telogen are continuously repeated throughout life, and they go through generation, growth, and loss (Paus et al. , J Invest Dermatol. 113, pp. 523-532, 1999). In the degenerative phase of the hair follicle, the growth of the hair follicle is stopped, apoptosis and the reorganization of the extracellular matrix occur. As the number of hair follicle cells in the degenerative and resting state of the hair follicle increases, the hair falls out and does not grow back. .

Hair is divided into visible and invisible hair roots, and the root from which hair grows is called the hair bulb, and the recessed part at the bottom is called the dermal papilla (Fig. One). The dermal papilla is the most important part in the development of hair and has blood vessels and nerves to deliver nutrients and growth factors to promote hair growth. Since the number of hairs decreases due to apoptosis and reduction in growth of dermal papilla cells, proliferation of dermal papilla cells is required for maintenance and increase of hair.

Vascular endothelial growth factor (VEGF) is a glycoprotein that promotes angiogenesis by inducing the growth of vascular endothelial cells. VEGF induces proliferation of dermal papilla cells by inducing the VEGF-2/ERK pathway by binding to VEGF receptor-2 (VEGFR-2) present in the dermal papilla (Wei Li et al., Exp Cell Res. 2012 Aug 15;318 ( 14):1633-40). In addition, Fibroblast growth factor 7 (FGF7) is also called keratinocyte growth factor (KGF), and regulates the hair generation cycle by participating in the growth of the hair part exposed to the outside of the hair follicle and the scalp surface. (Genes Dev. 1996;10(2):165-175). In addition, hepatocyte growth factor (HGF) is a growth factor secreted from dermal papilla cells and involved in the formation of new hair follicles (Journal of dermatological science, 2001;25(2):156-163). Therefore, an increase in the gene expression of VEGF, FGF7, and HGF in dermal papilla cells can be of great help in inducing hair growth.

In addition, in the case of seborrheic scalpitis in which scalp lipids are excessively secreted, sebum secreted from the hair follicles sebaceous glands is increased by the action of androgen hormones, and as the sebum increases, the amount of microorganisms present in the scalp increases. These scalp microorganisms can generate waste products on the scalp and cause hair loss accompanied by itching and pain (Journal of the Korean Society of Aesthetics, Vol. 18 No. 2 (2012), pp. 279-289).

Recently, as environmental pollution and urban stress become serious, the number of modern people with alopecia increases, and interest in materials that can improve it is increasing. Therefore, it was attempted to invent a material that can help to strengthen the hair root and prevent hair loss by inducing hair growth by increasing the expression of the growth factor of the dermal papilla cells responsible for the initiation of hair generation among the hair roots. In addition, it was attempted to invent a material that solves inflammation and itchiness caused by sebum by using microbial cells and strain cultures that inhibit scalp lipids, and further prevents hair loss symptoms.

In this regard, Korean Patent No. 10-2102578 discloses a cosmetic composition for promoting hair growth or hair growth containing a plurality of compounds. However, there are not many studies on microorganisms that can improve the condition of hair or scalp.

Probiotics are microorganisms that have beneficial effects on the human body. Most probiotics known to date are known as lactic acid bacteria. Although probiotics have been reported to produce effective efficacy through various beneficial effects on the human body, studies on the interaction between the scalp flora and the scalp are scarce.

Accordingly, there is a need for the development of flora on the scalp that can be usefully used for scalp-related conditions.

One aspect is to provide a cutibacterium granulosum ( Cutibacterium granulosum ) strain.

Another aspect is to provide a lysate of the strain, a culture solution, or an extract of the culture solution.

Another aspect is to provide a composition comprising a Cutibacterium granulosum strain, a lysate thereof, a culture solution, or an extract of the culture solution.

Another aspect provides a method of preventing, ameliorating, or treating a condition in a subject comprising treating or administering to the subject in need thereof an effective amount of the composition described above.

One aspect provides a cutibacterium granulosum ( Cutibacterium granulosum ) strain.

The strain may be a strain belonging to the genus Cutibacterium sp .

The strain comprises a 16S rRNA having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to SEQ ID NO: 1. may be doing The strain may include the 16S rRNA of SEQ ID NO: 1.

As used herein, the term "sequence identity" refers to the degree of the same degree of amino acid residues or bases between sequences after aligning both sequences to be as consistent as possible in a specific comparison region. Sequence identity is a value measured by optimally aligning and comparing two sequences in a specific comparison region, and a portion of the sequence in the comparison region may be added or deleted compared to a reference sequence. Percent sequence identity can be determined by, for example, comparing two optimally aligned sequences throughout the comparison region, determining the number of positions in both sequences where the same amino acid or nucleic acid appears, to obtain the number of matched positions. , dividing the number of matched positions by the total number of positions within the comparison range (ie, range size), and multiplying the result by 100 to obtain a percentage of sequence identity. The percent sequence identity may be determined using a known sequence comparison program, and examples thereof include BLASTN (NCBI), CLC Main Workbench (CLC bio), MegAlign™ (DNASTAR Inc), and the like.

The Cutibacterium granulosum strain may be a strain deposited with accession number KCCM12825P.

The strain may have an effect of improving hair or scalp condition. For example, the improvement of the hair or scalp condition may be at least one of promoting hair growth, preventing hair loss, strengthening hair roots, suppressing scalp lipids, preventing dandruff, and suppressing scalp inflammation.

The strain may be isolated. As used herein, the term “isolated” refers to an artificially isolated and usable one that does not exist in nature. The strain was artificially isolated and deposited with the accession number. The strain may be isolated from human skin. The strain may be isolated from human scalp.

Another aspect provides a lysate of the strain, a culture solution, or an extract of the culture solution.

Specific details of the strain are the same as described above.

As used herein, the term “lysate” may refer to a product obtained by crushing the cell wall of the strain by applying a chemical or physical force to the strain itself.

As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and can be used to supply nutrients so that the Cutibacterium granulosum strain can grow and survive in vitro. It may mean the entire medium including the strain obtained by culturing the strain for a certain period of time, its metabolites, and extra nutrients in a medium that can. In addition, the culture solution may refer to a culture solution obtained by culturing the strain in which the cells are removed from the culture solution. On the other hand, the liquid from which the cells are removed from the culture medium is also called "supernatant", and the culture solution is left for a certain period of time to take only the liquid from the upper layer except for the part that has sunk to the lower layer, or to remove the cells through filtration, or centrifuge the culture solution to the lower part. It can be obtained by removing the precipitation of and taking only the liquid at the top. The "cell" refers to the strain itself of the present invention, and includes the strain itself selected by separating it from a skin sample, or a strain isolated from the culture solution by culturing the strain. The cells can be obtained by centrifuging the culture solution to take the part that has sunk to the lower layer, or it can be obtained by removing the upper liquid after leaving it for a certain period of time because it sinks to the lower layer of the culture solution by gravity.

The culture medium may include the culture medium itself obtained by culturing the strain, its concentrate or lyophilisate, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate or lyophilisate.

The culture medium may be obtained by culturing a Cutibacterium granulosum strain in a medium (eg, Reinforced Clostridium Medium) at any temperature of more than 10 ° C or less than 40 ° C for a certain time, for example, 4 to 50 hours. have.

In one embodiment, the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture to remove the strain.

In another embodiment, the concentrate may be obtained by concentrating the supernatant obtained after filtering the strain culture solution itself, or the culture solution using centrifugation or a filter.

The culture medium and culture conditions for culturing the Cutibacterium granulosum strain may be appropriately selected or modified by those of ordinary skill in the art.

As used herein, the term "culture solution extract" means extraction from the culture solution or its concentrate, and may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these prepared or purified products, and a fraction obtained by fractionating it. can

Another aspect provides the use of a Cutibacterium granulosum strain, a lysate of the strain, a culture solution, or an extract of the culture solution. Specifically, it provides a composition comprising a Cutibacterium granulosum strain, a lysate thereof, a culture solution or an extract of the culture solution.

In the above use or composition, the Cutibacterium granulosum strain may be a Cutibacterium granulosum strain according to the aspect. In one embodiment, the strain has about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.9% or more sequence identity to SEQ ID NO: 1 It may be one containing 16S rRNA with In another embodiment, the strain may include 16S rRNA of SEQ ID NO: 1. In another embodiment, the strain may be a strain deposited with accession number KCCM12825P.

Uses of the strain may include improving the condition of hair or scalp.

The hair or scalp condition improvement may be one or more of hair growth promotion, hair loss prevention, hair root strengthening, scalp lipid inhibition, dandruff prevention, and scalp inflammation inhibition.

As used herein, the term "hair growth" refers to the generation and growth of hair.

As used herein, the term "promoting hair growth" is used interchangeably with "promoting hair growth", and refers to increasing the proportion of hair growth phase in total hair by promoting the generation and growth of hair. This includes not only promoting the generation of new hair, but also making the existing hair healthy.

As used herein, the term "hair loss (alopecia)" refers to a phenomenon in which hair is removed from the scalp or a state in which the hair is sparse or thin. The hair loss may be induced as the specific gravity of the hair growth phase decreases. The hair loss may be caused by extrinsic factors or intrinsic factors. The extrinsic factor refers to various external factors, such as ultraviolet rays, and the intrinsic factor is a factor that occurs over time, and includes telomere-induced cell death of dermal papilla cells and oxidative stress caused by cell function decline. The hair loss is alopecia areata, hereditary androgenetic alopecia, telogen effluvium, traumatic alopecia, hair loss due to trichotillomania, pressure alopecia, genital alopecia, pityriasis It may be at least one selected from alopecia, alopecia syphlltiac, alopecia seborrhecia, symptomatic alopecia, alopecia scar, and alopecia congenital, but is not limited thereto.

As used herein, the term "prevention of hair loss" means preventing or inhibiting hair loss. Therefore, the prevention of hair loss may mean including improvement of hair loss, prevention of hair loss, treatment of hair loss, and the like.

As used herein, the term “strengthening the hair root” refers to any action that strengthens and strengthens the hair root part of the hair. For example, hair roots can be strengthened by proliferation of dermal papilla cells, promotion of nutrient delivery to dermal papilla cells, and increased expression of growth factors in dermal papilla cells.

As used herein, the term “scalp lipid” refers to lipids produced in the scalp. Excessive accumulation of lipids on the scalp causes problems such as oiliness, dandruff, itching, and itching. When sebum is secreted excessively, a lipid film is formed on the scalp. When the lipid film is excessively formed on the scalp, the growth of anaerobic bacteria and the production of fatty acids increase, which can cause various troubles, seborrheic scalpitis, odor, hair loss, and the like. The lipid may refer to a lipid peroxide.

As used herein, the term “scalp lipid inhibition” refers to any action that reduces or inhibits the production of lipids in the scalp. Through suppression of scalp lipids, it is possible to obtain effects of preventing dandruff, improving or suppressing scalp inflammation and itching caused by sebum, and preventing hair loss due to lipid peroxide.

As used herein, the term “dandruff” refers to a phenomenon in which epidermal exfoliation occurs on the scalp and keratin appears conspicuously. Dandruff can be accompanied by itching.

As used herein, the term “anti-dandruff” means preventing or inhibiting the generation of dandruff. Accordingly, the prevention of dandruff may include dandruff improvement, dandruff prevention, and the like.

As used herein, the term “scalp inflammation” refers to inflammation occurring on the scalp. The scalp inflammation may include any one or more of seborrheic dermatitis, pruritus, dry eczema, erythema, urticaria, psoriasis, weak rash, and scalp acne.

As used herein, the term "scalp inflammation inhibition" refers to any action that reduces or improves scalp inflammation, or prevents or inhibits the occurrence of scalp inflammation.

As used herein, the term “prevention” includes inhibiting the development of a disease.

As used herein, the term “treatment” includes inhibiting, alleviating, or eliminating the development of a disease.

The composition according to one aspect can proliferate human follicular dermal papilla cells (HFDPC) by including a Cutibacterium granulosum strain, a lysate thereof, a culture medium, or an extract of the culture medium. Accordingly, the composition may exhibit a hair root strengthening effect, a hair loss prevention effect, and a hair growth promoting effect.

The composition according to an aspect is a vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF7), and hepatocyte growth factor (HGF) by including an extract of the Cutibacterium granulosum strain, its lysate, culture solution or culture solution. One or more expression may be increased. By increasing the expression of growth factors such as VEGF, FGF7, and HGF, it is possible to promote the delivery of nutrients to the dermal papilla cells and to induce the growth of hair root cells, thereby preventing hair loss and promoting hair growth as well as strengthening the hair roots. Accordingly, the composition may exhibit a hair root strengthening effect, a hair loss prevention effect, and a hair growth promoting effect.

The composition according to one aspect can inhibit scalp lipids by including the Burkholderia vietnamiensis strain, its lysate, culture solution or extract of the culture solution. Therefore, the composition can exhibit the effect of preventing dandruff, improving or suppressing scalp inflammation and itching due to sebum, and preventing hair loss due to lipid peroxide through suppression of scalp lipids.

In another embodiment, the strain may exhibit a synergistic effect by being used with another strain having an effect of improving hair or scalp conditions, for example, a strain belonging to the genus Cutibacterium. The strains belonging to the genus Cutibacterium are, for example, Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, Cutibacterium namnetense, Cutibacterium acnes subsp. acnes, Cutibacterium acnes subsp. defendens, or Cutibacterium acnes subsp. may contain elongatum .

The composition comprises 0.001 wt% to 80 wt%, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, based on the total weight of the composition %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10% by weight, or 0.1% to 5% by weight of the strain, a lysate thereof, a culture medium, or an extract of the culture medium.

As used herein, the term "included as an active ingredient" means that the strain of the present specification, a lysate thereof, a culture medium, or an extract of the culture medium is added to the extent that the above-mentioned effect can be exhibited, and drug delivery and stabilization, etc. For this purpose, it is meant to include being formulated in various forms by adding various components as sub-components.

The composition may be in a liquid state or a dry state. In one embodiment, the composition may be in the form of a dry powder.

The drying method for preparing the composition in a dry state may use a method generally used in the art, and is not particularly limited. Non-limiting examples of the drying method include an air drying method, a natural drying method, a spray drying method, a freeze drying method, and the like. These methods can be used alone or at least two methods can be used together.

The composition may include an additive in an effective amount sufficient to reduce deterioration of the strain, its lysate, culture solution, or extract of the culture solution. The additive may be, for example, a binder, but is not limited thereto.

The composition may further include a cosmetically, pharmaceutically or food-acceptable carrier. The composition may be formulated with a carrier and provided as a cosmetic, drug, food additive, and the like.

The composition may be a cosmetic composition.

The cosmetic composition may further include components commonly used in cosmetic compositions, functional additives, etc. in addition to the active ingredients disclosed herein, for example, antioxidants, stabilizers, solubilizers, surfactants, dispersants, preservatives, vitamins , conventional adjuvants such as pigments, fragrances, and the like, and carriers.

The cosmetic composition is not particularly limited to a specific formulation, and the formulation may be appropriately selected according to the purpose. The cosmetic composition may have, for example, a solubilized formulation, an emulsified formulation, or a dispersed formulation. The cosmetic composition may, for example, have a cosmetic formulation of a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin adhesion type.

The cosmetic composition may be a formulation that can be used for hair or scalp. The cosmetic composition is, for example, hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair conditioner, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream, hair Wax, hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair preservative, hair dye, hair wave agent, hair bleach, hair gel, hair glaze, hair dresser, hair lacquer, It may be in the form of a hair moisturizer, hair mousse or hair spray, and specifically shampoo, cleanser, conditioner, treatment, hair pack, conditioner, scalp pack, tonic, essence, lotion, cream, oil, mist, spray, or hair gel. It may be in the form

The composition may be a composition for external application to the skin.

In the present specification, the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermally delivered patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; drugs such as caffeine, tannin, bellapamil, licorice extract, glablidine, hot water extract of kalin fruit, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and its derivatives or salts thereof; vitamin C, magnesium ascorbyl phosphate, ascorbic acid glucoside, arbutin, kojic acid; Sugars, such as glucose, fructose, and trehalose, etc. can be mix|blended suitably.

The composition may be a pharmaceutical composition.

The pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, mannitol, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be carboxymethyl cellulose calcium, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, and the like. Formulations for parenteral administration may be injections, ointments, and the like.

The composition may be a health functional food composition.

The health functional food composition may be used alone, or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment). In general, in the production of food or beverage, the composition of the present specification may be added in an amount of 50 parts by weight or less, or 15 parts by weight or less based on the raw material. There is no particular limitation on the type of the health functional food. Among the types of health functional food, the beverage composition may contain various flavoring agents or natural carbohydrates as an additional component like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin; These are sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition can also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverage, fruit flesh for the production of vegetable beverage, or a combination thereof.

Another aspect provides a method of preventing, ameliorating, or treating a condition in a subject comprising treating or administering to the subject in need thereof an effective amount of the composition described above.

The individual's condition may be a hair or scalp condition, and specifically may be a condition related to hair loss, or a condition related to scalp inflammation.

As used herein, the terms "administering", "introducing", and "implanting" are used interchangeably and are subject to a method or route that results in at least partial localization of a composition according to one embodiment to a desired site. may refer to the placement of a composition according to one embodiment into

Administration may be administered by methods known in the art. Administration can be administered directly to a subject by any means, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be systemically or locally.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of improvement in hair or scalp condition, for example, an individual in need of promoting hair growth, preventing hair loss, strengthening hair roots, suppressing scalp lipids, preventing dandruff, or inhibiting scalp inflammation.

The administration is 0.1 mg to 1,000 mg, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg , 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration may be once a day or twice or more within the range of clinically acceptable side effects, and may be administered to one or two or more sites for the administration site, and total daily or at intervals of 2 to 5 days The number of days of administration may range from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For animals other than humans, the dose is the same as that of a human per kg, or the above dose is converted, for example, by the volume ratio (for example, average value) of the target animal and the organ (heart, etc.) of the human One dose can be administered.

According to the novel Cutibacterium granulosum strain according to an aspect, there is an effect of promoting hair growth, preventing hair loss, strengthening hair roots, inhibiting scalp lipids, preventing dandruff, and inhibiting scalp inflammation, improving, preventing, or treating hair or scalp condition can be applied in various ways.

1 is a diagram showing the structure of the hair and dermal papilla cells.
2 is a result confirming the efficacy of increasing the proliferation capacity of dermal papilla cells.
3 is a graph showing the level of VEGF gene expression in dermal papilla cells.
4 is a graph showing the level of FGF7 gene expression in dermal papilla cells.
5 is a graph showing the level of HGF gene expression in dermal papilla cells.
6 shows the effect of reducing the generation of lipids secreted from keratinocytes.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Isolation and identification of strains

A sample was obtained by washing the scalp of a male in his 20s with a healthy scalp with sterile distilled water. The sample was inoculated in Reinforced Clostridium Medium (RCM) liquid medium or solid medium (manufacturer: OXOID). After culturing in a 35 ℃ incubator for 48 hours after inoculation, 100 colonies formed were separated and cultured in pure water, and cultured in a 35 ℃ incubator for 48 hours. The cultured colonies were subjected to 16S rRNA sequence identification. The primers used at this time were designed to amplify in response only to bacteria. PCR amplification was carried out in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, 75°C for 1 minute and 30 seconds each, and finally maintained at 72°C for 8 minutes and then stored at 4°C. After the PCR reaction was completed, the DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA).

When the nucleotide sequence of the 16S rRNA region determined from the isolated and cultured microbial colonies was compared with other strains registered through the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI), homology 97 % or less of novel species were selected, and among them, a novel microorganism Cutibacterium granulosum with less than 98% homology ( Cutibacterium granulosum ) strain (hereinafter referred to as "SP-11") was selected. The selected SP-11 strain was deposited with the Korea Microbial Conservation Center on October 28, 2020 and was given an accession number KCCM12825P. The SP-11 strain has a 16S rRNA sequence of SEQ ID NO: 1 (complementary DNA).

Experimental Example 1. Evaluation of human hair papilla cell (HFDPC) proliferation efficacy

In order to evaluate whether the culture medium of the SP-11 strain isolated in Example 1 significantly induces the proliferation of dermal papilla cells, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide) analysis was performed.

First, human hair papilla cells (HFDPC) were aliquoted in a 96-well plate at a rate of 2x10 ⁴ cells/well, and then cultured for 24 hours in an incubator at 37° C. and 5% CO ₂ conditions. Then, after changing to a medium containing nothing, 1% (w/w) of the culture solution of the SP-11 strain was added and further cultured for 24 hours. A group in which 10 ppm of minoxidil (Minoxidil, MXD, 6-piperidin-1-ylpyrimidine-2,4-diamine 3-oxide), known as a hair growth promoter, was added instead of the strain culture medium was set as a positive control group, and the medium not containing the strain culture medium The addition group (medium con) was set as a negative control, and the non-addition group (None) was also set as a control to confirm the efficacy of the strain culture. After 24 hours, the medium from each well was removed and washed once with DPBS (Dulbecco's phosphate buffered saline), and then 500 μl of 0.5 mg/mL MTT (Sigma-aldrich, St. Louis, USA) was included in each well. medium and reacted at 37°C for 4 hours. Then, the absorbance was measured at 570 nm using Victor 3 (Perkin-Elmer). The cell viability was expressed as a percentage of the measured average absorbance value, and through this, the proliferative capacity of human hair papilla cells (HFDPC) can be confirmed.

2 is a result confirming the efficacy of increasing the proliferation capacity of dermal papilla cells.

As shown in Figure 2, it was confirmed that the strain of Example 1 exhibits an excellent dermal papilla cell proliferation effect.

Therefore, it was confirmed that the SP-11 strain can be used for strengthening hair roots, preventing hair loss, and promoting hair growth.

Experimental Example 2. Efficacy of increasing expression of growth factors (VEGF, FGF7, and HGF) in human hair papilla cells (HFDPC)

In order to evaluate whether the culture medium of the SP-11 strain isolated in Example 1 can increase the expression of VEGF, FGF7, and HGF growth factors that induce hair growth and maintain scalp health, real-time polymerase chain reaction (Quantitative Real-Time Polymerase Chain Reaction: qRT-PCR) was performed.

Specifically, human hair papilla cells (HFDPC) were seeded in a 6-well plate at a rate of 3x10 ⁵ cells/well, and then cultured in cell culture conditions for 24 hours. After 24 hours, the medium was discarded, washed with phosphate buffered saline (PBS), and the cells were starved using a medium not containing fetal bovine serum (FBS). The next day, the culture medium of the SP-11 strain was replaced with a medium containing 1% (w/w), and further cultured for 24 hours. A group in which 10 ppm of minoxidil (MXD) was added was set as a positive control group, a medium addition group (medium con) that did not contain a culture medium was set as a negative control group, and a culture solution non-added group (None) was set as a control group. Thereafter, RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen), and RNA was quantified at 260 nm using nanodrops, and 2 μg of each RNA was used in an amplifier (C1000 Thermal Cycler, Bio -Rad, USA) synthesized cDNA. By performing a real-time polymerase chain reaction in a real-time PCR machine using a mixture of a VEGF, FGF7, or HGF primer and a cyanine dye, cybergreen (SYBR Green supermis, Applied Biosystems, USA) added to the synthesized cDNA, finally, Expression levels of VEGF, FGF7, and HGF genes were evaluated. The sequences and reaction conditions of the primers used are shown in Table 1 below, and the expression level of the gene was finally analyzed through correction for the β-actin gene.

Primer name direction order SEQ ID NO: reaction conditions VEGF F 5'-GTGCCCACTGAGGAGTTCAAC-3' 2 Polymerase activation at 94°C for 5 minutes, polymerization reaction in 40 cycles at 95°C 30 seconds, 60°C 30 seconds, 72°C 30 seconds R 5'-CCCTATGTGCTGGCCTTGAT-3' 3 FGF7 F 5'-TCCTGCCAACTTTGCTCTACA-3' 4 R 5'-CAGGGCTGGAACAGTTCACAT-3' 5 HGF F 5'-GCTATCGGGGTAAAGACCTACA-3' 6 R 5'-CGTAGCGTACCTCTGGATTGC-3' 7 β-actin F 5'-GGCCATCTCTTGCTCGAAGT-3' 8 R 5'-GAGACCTTCAACACCCCAGC-3' 9

3 is a graph showing the level of VEGF gene expression in dermal papilla cells. 4 is a graph showing the level of FGF7 gene expression in dermal papilla cells. 5 is a graph showing the level of HGF gene expression in dermal papilla cells.

3 to 5, the strain of Example 1 was confirmed to exhibit an excellent effect of increasing the expression of vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF7), and hepatocyte growth factor (HGF). .

Therefore, it was confirmed that the SP-11 strain can be used for strengthening hair roots, preventing hair loss, promoting hair growth, and improving scalp health.

Experimental Example 3. Scalp Lipid Inhibition Efficacy Evaluation

In order to evaluate whether the culture solution of the SP-11 strain isolated in Example 1 inhibits scalp lipids, lipid inhibition evaluation was performed after inducing lipid peroxide to keratinocytes.

Specifically, human keratinocytes (HaCaT) were seeded in a 6-well plate at a rate of 3x10 ⁵ cells/well, and then cultured in cell culture conditions for 24 hours. After 24 hours, the medium was discarded, washed with PBS, and then diluted with 1% (w/w) peroxidated squalene in a medium not containing FBS, and lipid peroxide was induced by adding it to the cells. Then, 1% (w/w) of the culture solution of the SP-11 strain was added and further cultured for 72 hours. A 1% (w/w) addition group (medium con, RCM control) containing no strain culture medium was set as a negative control. The medium was discarded and washed three times with PBS, and then Nile red O staining was performed to stain the lipids of the cells. Nile red solution (1 mg/ml in acetone) was diluted 1/1000 in FBS-free medium and added to the cells, and 10 minutes later, it was placed in an IncucyteZoom (Essen BioScience) instrument, photographed with a fluorescence microscope, and fluorescence intensity was quantified. did.

6 shows the effect of reducing the generation of lipids secreted from keratinocytes.

As shown in Figure 6, the strain of Example 1 was confirmed to exhibit an excellent lipid inhibitory effect.

Therefore, it was confirmed that the SP-11 strain can be used for scalp lipid suppression, prevention of dandruff, improvement or suppression of scalp inflammation and itching caused by sebum, prevention of hair loss due to lipid peroxide, and scalp health.

The above description is merely illustrative of the technical spirit of the present invention, and various modifications and variations will be possible without departing from the essential characteristics of the present invention by those skilled in the art to which the present invention pertains.

Korea Microorganism Conservation Center (Overseas) KCCM12825P 20201028

SEQUENCE LISTING <110> Cosmax, INC. <120> Cutibacterium granulosum strain and its use for improving hair or scalp condition <130> PN136755 <160> 9 <170> PatentIn version 3.2 <210> 1 <211> 1355 <212> DNA <213> Cutibacterium granulosum <400> 1 ggccctttcg ggggtacacg agtggcgaac gggtgagtaa cacgtgagta acctgcccac 60 aactttggga taacgctagg aaactggtgc taatactgga tatgtgctcc tgctgcatgg 120 tgggggttgg aaagctccgg cggttgtgga tggactcgcg gcctatcagc ttgttggtgg 180 ggtagtggcc taccaaggcg gcgacgggta gccggcctga gagggtgacc ggccacattg 240 ggactgagat acggcccaga ctcctacggg aggcagcagt ggggaatatt gcacaatggg 300 cgcaagcctg atgcagcaac gccgcgtgcg ggatgacggc cttcgggttg taaaccgctt 360 tcagcaggga cgaagctttt tgtgacggta cctgcagaag aagcaccggc taactacgtg 420 ccagcagccg cggtgatacg tagggtgcga gcgttgtccg gatttattgg gcgtaaaggg 480 ctcgtaggcg gttgatcgcg tcggaagtgg aaacttgatg cttaacgttg agcgtgcttt 540 cgatacgggt tgacttgagg aaggtagggg agaatggaat tcctggtgga gcggtggaat 600 gcgcagatat caggaggaac accagtggcg aaggcggttc tctggacctt tcctgacgct 660 gaggagcgaa agcgtgggga gcgaacaggc tagataccc tggtagtcca cgctgtaaac 720 ggtgggtact aggtgtgggg tccattccac ggattctgtg ccgtagctaa cgcattaagt 780 accccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg gggccccgca 840 caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa gaaccttacc tgggtttgac 900 atggatcggg agcttccaga gatggttgtg cctcttttgg ggtcggttca caggtggtgc 960 atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1020 tcgtccactg ttgccagcaa ttcggttggg gactcagtgg agaccgccgg ggtcaactcg 1080 gaggaaggtg gggatgacgt caagtcatca tgccccttat gtccagggct tcacgcatgc 1140 tacaatggcc ggtacagtga gttgcgacat cgtaaggtgg agcgaatctc aaaaagccgg 1200 tctcagttcg gattggggtc tgcaactcga cctcatgaag tcggagtcgc tagtaatcgc 1260 agatcagcaa cgctgcggtg aatacgttcc cggggcttgt acacaccgcc cgtcaagtca 1320 tgaaagtcgg taacactcga agccggtggc ctaac 1355 <210> 2 <211> 21 <212> DNA <213> <220> <223> VEGF forward primer <400> 2 gtgcccactg aggagttcaa c 21 <210> 3 <211> 20 <212> DNA <213> <220> <223> VEGF reverse primer <400> 3 ccctatgtgc tggccttgat 20 <210> 4 <211> 21 <212> DNA <213> <220> <223> FGF7 forward primer <400> 4 tcctgccaac tttgctctac a 21 <210> 5 <211> 21 <212> DNA <213> <220> <223> FGF7 reverse primer <400> 5 cagggctgga acagttcaca t 21 <210> 6 <211> 22 <212> DNA <213> <220> <223> HGF forward primer <400> 6 gctatcgggg taaagaccta ca 22 <210> 7 <211> 21 <212> DNA <213> <220> <223> HGF reverse primer <400> 7 cgtagcgtac ctctggattg c 21 <210> 8 <211> 20 <212> DNA <213> <220> <223> beta-actin forward primer <400> 8 ggccatctct tgctcgaagt 20 <210> 9 <211> 20 <212> DNA <213> <220> <223> beta-actin reverse primer <400> 9 gagaccttca acaccccagc 20

Claims (12)

SEQ ID NO: 1 and the 16S rRNA having a sequence identity of 95% or more, belonging to the genus Cutibacterium ( Cutibacterium sp .) Cutibacterium granulosum ( Cutibacterium granulosum ) strain. The strain according to claim 1, which has been deposited with accession number KCCM12825P. The culture solution of the strain of claim 1. A cosmetic composition comprising the strain of claim 1, a lysate thereof, a culture solution, or an extract of the culture solution. The cosmetic composition according to claim 4, which is for improving the condition of hair or scalp. The cosmetic composition of claim 5, wherein the improvement of the hair or scalp condition is at least one of promoting hair growth, preventing hair loss, strengthening hair roots, suppressing scalp lipids, preventing dandruff, and suppressing scalp inflammation. The cosmetic composition according to claim 4, which proliferates human hair papilla cells. The cosmetic composition according to claim 4, which increases the expression of one or more of vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF7), and hepatocyte growth factor (HGF). The cosmetic composition according to claim 4, which inhibits scalp lipids. Cutibacterium granulosum ( Cutibacterium granulosum ) A cosmetic composition for improving hair or scalp condition comprising an extract of a strain, its lysate, culture solution, or culture solution. Cutibacterium granulosum ( Cutibacterium granulosum ) A pharmaceutical composition for promoting hair growth or preventing hair loss, comprising as an active ingredient an extract of a strain, its lysate, culture solution, or culture solution. Cutibacterium granulosum ( Cutibacterium granulosum ) A health functional food composition for improving hair or scalp condition comprising an extract of a strain, its lysate, culture solution, or culture solution.

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