WO2025173928A1 - Composition for scalp improvement using pseudomonas and micrococcus and method for preparing same - Google Patents

Abstract

The present invention relates to a composition for scalp improvement, the composition being prepared by dually culturing Pseudomonas sp. microorganisms and Micrococcus sp. microorganisms in a medium composition containing saccharides and amino acids. The composition for scalp improvement has excellent scalp improvement effects such as hair growth promotion, hair loss prevention, hair root strengthening, and the like, and thus can be effectively used in cosmetics, medicines, and the like.

Description

Composition for improving scalp using Pseudomonas and Micrococcus and method for preparing the same

The present invention relates to a composition for improving the scalp using microorganisms and a method for producing the same.

Human hair undergoes a cyclical cycle: a growth phase, during which hair growth ceases, a catagen phase, during which the hair bulb degenerates, and a resting phase, during which the hair follicle ceases activity. This cycle is then regulated by hormones and various growth factors. However, if hair transitions prematurely from the catagen phase to the resting phase, severe hair loss (alopecia) can occur. This hair loss can become dry, brittle, and thin. The causes of hair loss include hormonal imbalances, sebum-induced scalp ailments, and nutritional imbalances. Recently, the role of sebum has been highlighted as a major cause of hair loss. During the growth phase, sebum secreted from hair follicles to the scalp hardens, producing lipid peroxides in the scalp layer due to exposure to reactive oxygen species and ultraviolet rays. This hardened sebum clogs pores and impedes normal hair growth. Testosterone, a hormone secreted within the body, is converted into dihydrotestosterone (DHT) by 5α-reductase. When DHT binds to androgen receptors, it activates the proliferation and differentiation of sebum cells, leading to excessive sebum secretion and blockage of the sebaceous glands. This ultimately leads to excessive dandruff, scalp irritation, and hair loss.

In women, the female hormones estradiol and estrogen maintain hair growth. Unlike men, women have aromatase, an enzyme that suppresses hair loss, distributed along the front of their scalp, preventing hair loss there. Hair loss in women tends to worsen after menopause and progresses slowly and steadily.

When it comes to hair-related issues, scalp health is paramount. Therefore, research and development are needed to prevent hair loss by reducing scalp irritation and improving scalp condition.

Vascular endothelial growth factor (VEGF) is a glycoprotein that promotes angiogenesis by inducing the growth of vascular endothelial cells. VEGF binds to VEGF receptor-2 (VEGFR-2) present in the dermal papilla and induces the VEGF-2/ERK pathway, thereby inducing the proliferation of dermal papilla cells. Furthermore, fibroblast growth factor 7 (FGF7), also known as keratinocyte growth factor (KGF), is a proliferation factor that regulates the hair growth cycle by participating in the growth of the outer part of the hair follicle and the part of the hair exposed to the scalp surface. Furthermore, hepatocyte growth factor (HGF) is a proliferation factor secreted by dermal papilla cells and is involved in the formation of new hair follicles. Therefore, increasing the gene expression of VEGF, FGF7, and HGF in dermal papilla cells can be of great help in inducing hair growth.

Probiotics are a general term for microorganisms that have a beneficial effect on the human body. Most known probiotics are lactic acid bacteria. While probiotics have been reported to be effective in various ways, research on the relationship between scalp flora and the scalp is limited.

Accordingly, the inventors of the present invention have developed a probiotic hair composition for promoting scalp health that has not only scalp cell regenerative power but also hair growth efficacy, thereby completing the present invention.

One aspect provides a method for preparing a composition for improving the scalp, comprising the steps of: preparing a primary culture by culturing a Pseudomonas sp. microorganism in a medium composition containing sugars and amino acids; and preparing a secondary culture by culturing a Micrococcus sp. microorganism in the primary culture.

Another aspect is to provide a composition for improving the scalp prepared by the above method.

Another aspect provides a cosmetic composition, a composition for external use in the skin, a pharmaceutical composition, or a food composition comprising the composition for improving the scalp.

Another aspect provides a method of preventing, improving, or treating a condition of a subject comprising administering to the subject an effective amount of the above composition.

Another aspect provides a method for improving the scalp comprising administering an effective amount of the above composition to a subject in need thereof.

Another aspect provides a use of a scalp improvement composition prepared by the above method for preparing a scalp improvement cosmetic composition.

Another aspect provides a use of a scalp improvement composition manufactured by the above method for manufacturing a composition for external application to improve the scalp.

Another aspect provides a use of a scalp improvement composition manufactured by the above method for manufacturing a scalp improvement food composition.

Another aspect provides a use of a scalp improvement composition prepared by the above method for preparing a drug for promoting hair growth or preventing hair loss.

One aspect provides a method for preparing a composition for improving the scalp.

Specifically, the method comprises the steps of culturing a Pseudomonas sp. microorganism in a medium composition containing sugars and amino acids to prepare a primary culture; and the steps of culturing a Micrococcus sp. microorganism in the primary culture to prepare a secondary culture.

Accordingly, the above method may be a method for producing a dual culture solution using microorganisms of the genus Pseudomonas and microorganisms of the genus Micrococcus. Specifically, the above method may be a method for producing a dual culture solution of microorganisms of the genus Pseudomonas and microorganisms of the genus Micrococcus in a scalp environment-simulating medium.

The above sugar may include at least one selected from the group consisting of glucose, rhamnose, and fructose. The above sugar may include glucose, rhamnose, and fructose.

The glucose, rhamnose, and fructose may be included in an amount of 0.01 g to 100 g, 0.01 g to 50 g, 0.01 g to 20 g, 0.01 g to 10 g, 0.01 g to 5 g, 0.1 g to 100 g, 0.1 g to 50 g, 0.1 g to 20 g, 0.1 g to 10 g, 0.1 g to 5 g, 1.0 g to 100 g, 1.0 g to 50 g, 1.0 g to 20 g, 1.0 g to 10 g, or 1.0 g to 5 g per 1 L of the medium composition, respectively.

The amino acid may include at least one selected from the group consisting of Threonine, Tyrosine, Arginine, Alanine, Proline, Histidine, and Iso-Leucine, Tryptophan, Valine, Glutamic acid, Asparaginic acid, Serine, and Glycine. The amino acid may include at least one selected from the group consisting of Threonine, Tyrosine, Arginine, Alanine, Proline, Histidine, and Isoleucine. The amino acid may include Threonine, Tyrosine, Arginine, Alanine, Proline, Histidine, and Isoleucine.

The above threonine, tyrosine, arginine, alanine, proline, histidine, and isoleucine may be included in an amount of 0.1 mg to 5.0 g, 0.1 mg to 1.0 g, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 10 mg, 0.1 mg to 5 mg, 1 mg to 5.0 g, 1 mg to 1.0 g, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 10 mg, or 1 mg to 5 mg per 1 L of the medium composition, respectively.

The above-mentioned badge composition may additionally contain glycerol.

The glycerol may be included in an amount of 0.01 g to 50 g, 0.01 g to 20 g, 0.01 g to 10 g, 0.01 g to 5 g, 0.01 g to 1 g, 0.1 g to 50 g, 0.1 g to 20 g, 0.1 g to 10 g, 0.1 g to 5 g, or 0.1 g to 1 g per 1 L of the medium composition.

The above medium composition may additionally include at least one of MgSO ₄ , K ₂ HPO ₄ , NaCl, and yeast extract.

The above MgSO ₄ , K ₂ HPO ₄ , NaCl, and yeast extract may be included in an amount of 0.01 g to 100 g, 0.01 g to 50 g, 0.01 g to 20 g, 0.01 g to 10 g, 0.01 g to 5 g, 0.01 g to 1 g, 0.01 g to 0.5 g, 0.01 g to 0.1 g, 0.1 g to 100 g, 0.1 g to 50 g, 0.1 g to 20 g, 0.1 g to 10 g, 0.1 g to 5 g, 0.1 g to 1 g, or 0.1 g to 0.5 g per 1 L of the medium composition.

The above-mentioned badge composition may have a composition similar to the human scalp environment by including human scalp-derived sugars and human scalp-derived amino acids.

The above Pseudomonas genus ( Pseudomonas sp.) microorganisms are not limited to a specific species. The above Pseudomonas genus microorganisms are Pseudomonas azotoformans , Pseudomonas jessenii , Pseudomonas rhodesiae , Pseudomonas corrugate , Pseudomonas glycinae , Pseudomonas syringae , Pseudomonas fluorescens , Pseudomonas kilonensis , Pseudomonas lini , Pseudomonas moraviensis . moraviensis ) , Pseudomonas putida , and Pseudomonas protegens It can be selected from, but is not limited to. In one specific example, the Pseudomonas genus microorganism may be Pseudomonas azotoformans . In a specific specific example, the Pseudomonas genus microorganism may be Pseudomonas azotoformans B15 strain deposited under accession number KCCM13450P.

In the step of preparing the above primary culture solution, the culture may be performed under culture conditions suitable for culturing Pseudomonas microorganisms. Those skilled in the art may appropriately select and modify these culture conditions. For example, the culture may be performed at an appropriate temperature (e.g., 10 to 40°C) for a predetermined period of time (e.g., 12 to 168 hours).

After the step of preparing the primary culture solution, a step of obtaining a supernatant from the primary culture solution may be additionally included. The supernatant may be obtained by a step of removing bacterial cells from the primary culture solution. Therefore, in the step of preparing the secondary culture solution, the primary culture solution may refer to the supernatant of the primary culture solution. The primary culture solution may be one from which bacterial cells, specifically, Pseudomonas microbial cells, have been removed. The supernatant may be obtained by allowing the culture solution to stand still for a certain period of time and taking only the liquid in the upper layer excluding the portion that has settled to the lower layer, removing bacterial cells through filtration, or centrifuging the culture solution to remove the sediment at the lower layer and taking only the liquid in the upper layer.

The above Micrococcus sp. microorganisms are not limited to a specific species. The above Micrococcus genus microorganisms are Micrococcus luteus, Micrococcus lylae , Micrococcus cohnii, Micrococcus endophyticus, Micrococcus flavus , Micrococcus terreus , Micrococcus yunnanensis , Micrococcus porci , Micrococcus agilis , Micrococcus aloeverae , Micrococcus antarcticus , Micrococcus halobius , Micrococcus christinae. The microorganism may be selected from, but is not limited to, Micrococcus kristinae , Micrococcus lactis , Micrococcus nishinomiyaensis , Micrococcus roseus , Micrococcus sedentarius , Micrococcus varians , Neomicrococcus aestuarii , Neomicrococcus lactis , and Halomicrococcus hydrotolerans . In one specific example, the Micrococcus microorganism may be Micrococcus luteus. In a specific embodiment, the microorganism of the genus Micrococcus may be the strain Micrococcus luteus SP-12 deposited under accession number KCCM12826P.

In the step of preparing the secondary culture medium, the culture may be performed under culture conditions suitable for culturing microorganisms of the genus Micrococcus. Those skilled in the art may appropriately select and modify these culture conditions. For example, the culture may be performed at an appropriate temperature (e.g., 10 to 40°C) for a predetermined period of time (e.g., 12 to 168 hours).

After the step of preparing the secondary culture medium, a step of obtaining a supernatant from the secondary culture medium may be additionally included. The supernatant may be obtained by a step of removing bacterial cells from the secondary culture medium. Accordingly, the secondary culture medium may refer to a supernatant of the secondary culture medium. The secondary culture medium may be a liquid from which bacterial cells, specifically, microbial cells of the genus Micrococcus, have been removed. The supernatant may be obtained by allowing the culture medium to stand still for a certain period of time and taking only the liquid in the upper layer excluding the portion that has settled to the lower layer, removing bacterial cells through filtration, or centrifuging the culture medium to remove the sediment at the lower layer and taking only the liquid in the upper layer.

In this specification, the term "secondary culture" may be used interchangeably with the term "double culture".

In the above method, the “culture medium” may refer to the entire medium containing metabolites, extra nutrients, etc. of the strain obtained by culturing the strain in the medium for a certain period of time. The culture medium may include a concentrate or lyophilized product of the culture medium.

Another aspect is to provide a composition for improving the scalp manufactured by a method according to the above aspect.

The composition may have a scalp improvement effect or scalp health improvement effect. The scalp improvement or scalp health improvement may include hair growth promotion, hair loss prevention, and hair root strengthening. Therefore, the scalp improvement or scalp health improvement also encompasses hair improvement or hair health improvement.

As used herein, the term “hair growth” means the production and growth of hair.

The term "hair growth promotion" is used interchangeably with "hair growth promotion" herein, and refers to promoting hair growth and growth, thereby increasing the proportion of hair in the anagen phase among all hair types. This includes not only promoting new hair growth, but also promoting the healthy growth of existing hair.

As used herein, the term "alopecia" refers to the phenomenon of hair falling out from the scalp or a condition in which hair becomes thicker or thinner. Hair loss can be caused by a decrease in the proportion of hair in the growth phase. Hair loss can be caused by exogenous or endogenous factors. Exogenous factors include various external factors, such as ultraviolet rays. Endogenous factors include factors that occur over time, such as cell death of hair papilla cells due to telomeres and oxidative stress resulting from a decline in cell function. The above hair loss may be one or more selected from alopecia areata, androgenetic alopecia, telogen effluvium, traumatic alopecia, hair loss due to trichotillomania, pressure alopecia, anagen alopecia, pityriasis alopecia, alopecia syphlltiac, alopecia seborrhecia, symptomatic alopecia, cicatricial alopecia, and congenital alopecia, but is not limited thereto.

As used herein, the term "hair loss prevention" refers to preventing or suppressing hair loss. Therefore, "hair loss prevention" may encompass hair loss improvement, hair loss prevention, hair loss treatment, and the like.

As used herein, the term "hair root strengthening" refers to any action that strengthens and strengthens the hair root. For example, hair root strengthening can be achieved by increasing the proliferation of hair papilla cells, promoting nutrient delivery to hair papilla cells, and increasing the expression of growth factors in hair papilla cells.

The term "prevention" encompasses suppressing the onset of a disease. The term "treatment" encompasses suppressing, alleviating, or eliminating the development of a disease. The term "improvement" can refer to any action that at least reduces the severity of symptoms, such as parameters associated with alleviating or curing a condition.

In one specific example, the composition for improving the scalp can increase the expression of at least one of hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7), and vascular endothelial growth factor (VEGF). By increasing the expression of growth factors such as VEGF, FGF7, and HGF, nutrient delivery to hair papilla cells is promoted and the growth of hair root cells is induced, thereby not only strengthening hair roots but also preventing hair loss and promoting hair growth. Therefore, the composition can exhibit hair root strengthening effects, hair loss prevention effects, and hair growth promotion effects.

In one specific example, the scalp improvement composition can proliferate hair follicle cells. Accordingly, the composition can exhibit hair root strengthening, hair loss prevention, and hair growth promotion effects.

In one embodiment, the composition for improving the scalp was confirmed to significantly increase the expression of HGF, FGF7, and VEGF compared to a culture of only Pseudomonas microorganisms or a culture of only Micrococcus microorganisms by using a dual culture method using Pseudomonas microorganisms and Micrococcus microorganisms.

In one embodiment, the composition for improving the scalp was confirmed to significantly increase the expression of growth factors and significantly increase the proliferation of hair follicle cells by using a scalp environment-simulating medium, compared to when using a general culture medium (e.g., R2A medium).

The above scalp improvement composition can be used together with other substances having a scalp improvement effect to produce a synergistic effect.

The above scalp improvement composition may be in a liquid or dry state. In one specific example, the scalp improvement composition may be in the form of a dry powder.

The drying method for preparing the above scalp improvement composition in a dry state can be any method commonly used in the art and is not particularly limited. Non-limiting examples of the drying method include air drying, natural drying, spray drying, and freeze drying. These methods may be used alone or in combination of at least two methods.

The above scalp improvement composition may further include a cosmetically, pharmaceutically, or food-wise acceptable carrier. The scalp improvement composition may be formulated with the carrier and provided as a cosmetic, pharmaceutical, or food additive.

Another aspect provides a cosmetic composition, a composition for external use in the skin, a pharmaceutical composition, or a food composition comprising a composition for improving the scalp according to the above aspect.

The above cosmetic composition, composition for external skin application, pharmaceutical composition, or food composition may be used for scalp improvement or scalp health improvement. The scalp improvement or scalp health improvement may include hair growth promotion, hair loss prevention, and hair root strengthening.

The above cosmetic composition, composition for external skin application, pharmaceutical composition, or food composition may include a composition for improving the scalp according to one aspect as an active ingredient.

The above “included as an effective ingredient” means that a composition for improving the scalp according to one aspect is added to an extent that it can exhibit the effects mentioned in this specification, and includes formulation in various forms by adding various ingredients as auxiliary ingredients for drug delivery and stabilization, etc.

The composition for improving the scalp is present in an amount of 0.0001 wt% to 99.9999 wt%, for example, 0.001 wt% to 80 wt%, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, It may be included in an amount of 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt%, but is not particularly limited thereto. The content of the active ingredient may be selected as an appropriate content that can be formulated within a content range that can exhibit the effects mentioned herein by a person skilled in the art.

The above cosmetic composition may additionally include, in addition to the effective ingredients disclosed herein, components commonly used in cosmetic compositions, functional additives, etc., and may include, for example, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, surfactants, dispersants, emulsifiers, preservatives, vitamins, pigments, fragrances, etc., and carriers.

The above cosmetic composition is not particularly limited to a specific formulation, and the formulation can be appropriately selected depending on the purpose. The cosmetic composition may have, for example, a solubilized formulation, an emulsified formulation, or a dispersion formulation. The cosmetic composition may have, for example, a toner, a cream, an essence, a cleansing foam, cleansing water, a pack, an ampoule, a body lotion, a body oil, a body gel, a shampoo, a hair conditioner, a hair gel, a foundation, a lipstick, a mascara, a makeup base, or a skin-adhesive type cosmetic formulation.

The above cosmetic composition may be a formulation that can be used on the hair or scalp. The above cosmetic composition may be, for example, a formulation of a hair tonic, a hair conditioner, a hair essence, a hair lotion, a hair nourishing lotion, a hair shampoo, a hair rinse, a hair treatment, a hair cream, a hair nourishing cream, a hair moisturizing cream, a hair massage cream, a hair wax, a hair aerosol, a hair pack, a hair nourishing pack, a hair soap, a hair cleansing foam, a hair oil, a hair drying agent, a hair preservative, a hair dye, a hair waving agent, a hair bleaching agent, a hair gel, a hair glaze, a hair dressing agent, a hair lacquer, a hair moisturizer, a hair mousse, or a hair spray, and specifically, may be a formulation of a shampoo, a cleanser, a conditioner, a treatment, a hair pack, a rinse, a scalp pack, a tonic, an essence, a lotion, a cream, an oil, a mist, a spray, or a hair gel.

The above-mentioned external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The above-mentioned external preparation for skin may be appropriately mixed with ingredients commonly used in external preparations for skin such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, metal ion sequestrants, sugars, or a combination thereof, as needed.

The pharmaceutical composition may additionally comprise 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 glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium dihydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The glidant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The above pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may include granules, powders, liquids, tablets, capsules, dry syrups, etc. Parenteral dosage forms may include injections, ointments, etc.

The above composition may be a food composition. The above composition may be a health functional food composition. In this case, it may be formulated into a conventional health functional food formulation known in the art.

The above food composition can be used alone as a scalp improvement composition according to one aspect, or can be used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). There is no particular limitation on the type of the above health functional food. Among the types of health functional foods, the beverage composition can contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame can be used. The food composition may also contain nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, or combinations thereof. The food composition may also contain fruit pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, or combinations thereof.

Another aspect provides a method of preventing, improving, or treating a condition of a subject comprising administering to the subject an effective amount of the above composition.

The condition of the above object may be a condition related to hair or scalp, and specifically, a condition related to hair loss.

The term "effective amount" means an amount effective enough to produce the effect mentioned above.

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

Administration may be by any method known in the art. The route of administration, frequency of administration, and other administration methods can be appropriately selected by those skilled in the art. Administration may be administered directly to a subject by any means, including intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. Administration may be systemic or local. Administration may include application to the skin (e.g., scalp).

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 the condition of the hair or scalp, such as an individual in need of hair growth promotion, hair loss prevention, or hair root strengthening effects.

The above administration may be 0.1 mg to 1,000 mg of the composition according to one specific example per subject per day, 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. However, the dosage may vary depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and those skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or twice or more within the range of clinically acceptable side effects. Regarding the administration site, administration may be done in one or more sites, and the total number of administration days may be from 1 to 30 days per treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage per kg as for humans may be used, or the above dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (e.g., heart) of the target animal and the human.

Another aspect provides a method for improving the scalp comprising administering an effective amount of the above composition to a subject in need thereof.

Another aspect provides a use of a scalp improvement composition prepared by the above method for preparing a scalp improvement cosmetic composition.

Another aspect provides a use of a scalp improvement composition manufactured by the above method for manufacturing a composition for external application to improve the scalp.

Another aspect provides a use of a scalp improvement composition manufactured by the above method for manufacturing a scalp improvement food composition.

Another aspect provides a use of a scalp improvement composition prepared by the above method for preparing a drug for promoting hair growth or preventing hair loss.

Duplicate content is omitted in consideration of the complexity of this specification, and terms not otherwise defined herein have the meanings commonly used in the technical field to which the present invention belongs.

A composition for improving the scalp according to one aspect is obtained by dual-culturing Pseudomonas and Micrococcus microorganisms in a scalp environment-simulating medium, and has excellent scalp improvement effects such as promoting hair growth, preventing hair loss, and strengthening hair roots. Therefore, the composition can be usefully used in cosmetics, pharmaceuticals, etc.

Figure 1 is a graph showing the relative expression level of HGF according to strain type.

Figure 2 is a graph showing the relative expression level of FGF7 according to strain type.

Figure 3 is a graph showing the relative expression level of VEGF according to strain type.

Figure 4 is a graph showing the relative expression level of VEGF according to the type of medium.

Figure 5 is a graph showing the proliferation ability of mammary papilla cells according to the type of medium as cell viability (%).

Figure 6 is a graph showing the relative expression level of SREBP1 according to concentration-specific treatment of the double culture solution of Example 2.

Figure 7 is a graph showing the relative expression level of Ki67 according to concentration-specific treatment of the double culture medium of Example 2.

The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1: Production of a scalp environment-simulating medium

A scalp environment simulating medium was produced with the composition shown in Table 1 below.

ingredient Content range Glucose 0.01 g - 100 g Rhamnose 0.01 g - 100 g Fructose 0.01 g - 100 g Glycerol 0.01 g - 50 g Threonine 0.1 mg- 5.0 g Tyrosine 0.1 mg- 5.0 g Arginine 0.1 mg- 5.0 g Alanine 0.1 mg- 5.0 g Proline 0.1 mg- 5.0 g Histidine 0.1 mg- 5.0 g Isoleucine 0.1 mg- 5.0 g MgSO ₄ 0.01 g - 100 g K ₂ HPO ₄ 0.01 g - 100 g NaCl 0.01 g - 100 g Yeast extract 0.01 g - 100 g

Example 2: Production of dual cultures of Pseudomonas and Micrococcus using a scalp environment-simulating medium.

In Example 1, a Pseudomonas spp. strain was first cultured in a scalp environment simulating medium, and then a Micrococcus spp. strain was secondarily cultured to prepare a double culture solution.

In this example, the Pseudomonas strain was exemplarily used as the Pseudomonas strain, Pseudomonas azotoformans B15 strain (accession number: KCCM13450P). The B15 strain was isolated from human scalp and cultured in pure form, and the 16S rRNA sequence analysis result confirmed that it had 97.77% homology with Pseudomonas azotoformans in the BLAST of the National Center for Biotechnology Information (NCBI) in the United States. The B15 strain was deposited at the Korean Culture Center of Microorganisms (KCCM) on January 2, 2024 and assigned the accession number KCCM13450P. The B15 strain has a 16S rRNA sequence of sequence number 1 (complementary DNA).

In addition, as an example of the above Micrococcus genus strain, Micrococcus luteus SP-12 strain (accession number: KCCM12826P) was used.

The specific method for manufacturing the above double culture solution is as follows.

The Pseudomonas azotoformans strain was inoculated into the scalp environment simulating medium prepared in Example 1. After stirring and culturing in a 30°C incubator for 72 hours, precipitates and inactive substances were removed using a 0.45 um syringe filter to prepare a primary culture solution.

The Micrococcus luteus strain was inoculated into the primary culture medium above for a second time, and cultured with stirring in a 30°C incubator for 72 hours, followed by filtration to prepare a secondary culture medium.

Comparative Example 1: Preparation of Micrococcus Solo Culture Solution

A micrococcus single culture was prepared in the same manner as in Example 2, except that the cultivation step of the Pseudomonas azotoformans strain was omitted.

Comparative Example 2: Preparation of Pseudomonas Solo Culture Solution

A Pseudomonas single culture was prepared in the same manner as in Example 2, except that the culturing step of the Micrococcus luteus strain was omitted.

Comparative Example 3: Production of a Dual Culture of Pseudomonas and Micrococcus Using R2A Medium

A dual culture of Pseudomonas and Micrococcus was prepared in the same manner as in Example 2, except that R2A (Reasoner's 2A) medium was used instead of the scalp environment simulating medium.

Experimental Example 1: Evaluation of the efficacy of increasing the expression of growth factors (HGF, FGF7, VEGF) in breast papilla cells.

HGF, FGF7, and VEGF are growth factors that induce hair growth and maintain scalp health. To assess whether Example 2 can increase the expression of these growth factors, real-time quantitative polymerase chain reaction (qRT-PCR) was performed.

Specifically, human follicle dermal papilla cells (HFDPC) were seeded at ^3x105 cells/well in a 6-well plate and cultured for 24 hours under cell culture conditions. After 24 hours, the medium was discarded, washed with phosphate buffered saline (PBS), and the cells were starved using a medium that did not contain fetal bovine serum (FBS). The next day, the medium was replaced with a medium containing 0.1% (w/w), 1% (w/w), or 10% (w/w) of the culture solution of Example 2, Comparative Example 1, or Comparative Example 2; or a medium containing 1% (w/w) of the culture solution of Example 2 or Comparative Example 3, and then cultured for an additional 24 hours. A group containing 0.1 ppm of minoxidil (MXD, 6-piperidin-1-ylpyrimidine-2,4-diamine 3-oxide), a hair growth promoter, instead of the culture medium was set as a positive control group. In addition, a group containing 0.25 mM AS2P1 (ascorbic acid-2-phosphate) was set as an additional positive control group. The group without culture medium (None) was set as the control group.

After that, RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen), and then the RNA was quantified at 260 nm using a nanodrop. Then, 2 μg of RNA each was used to synthesize cDNA in an amplifier (C1000 Thermal Cycler, Bio-Rad, USA). The synthesized cDNA was mixed with primers for VEGF, FGF7, or HGF and the cyanine dye CyberGreen (SYBR Green supermis, Applied Biosystems, USA), and real-time polymerase chain reaction was performed in a real-time PCR machine to evaluate the expression levels of the VEGF, FGF7, and HGF genes. The sequences of the primers and reaction conditions used are as shown in Table 2 below, and the expression levels of the genes were finally analyzed through correction for the β-actin gene.

Primer name direction order Sequence number Reaction conditions VEGF F 5'-TCGGGCCTCCGAAACCATGA-3' 2 After polymerization enzyme activation at 94°C for 5 minutes, polymerization reaction was performed for 40 cycles under the conditions of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. R 5'-CCTGGTGAGAGATCTGGTTC-3' 3 FGF7 F 5'-TGGATCCTGCCAACTTTGCT-3' 4 R 5'-TTCTTGTGTGTCGCTCAGGG-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

Figure 1 is a graph showing the relative expression level of HGF according to strain type.

Figure 2 is a graph showing the relative expression level of FGF7 according to strain type.

Figure 3 is a graph showing the relative expression level of VEGF according to strain type.

Figure 4 is a graph showing the relative expression level of VEGF according to the type of medium.

As a result, as shown in FIGS. 1 to 3, it was confirmed that Example 2, which used both Pseudomonas genus microorganisms and Micrococcus genus microorganisms, significantly increased the expression of HGF, FGF7, and VEGF compared to Comparative Example 1, which used only Micrococcus genus microorganisms, and Comparative Example 2, which used only Pseudomonas genus microorganisms.

In addition, as shown in Fig. 4, it was confirmed that Example 2 using a scalp environment simulating medium significantly increased VEGF expression compared to Comparative Example 3 using a general culture medium, R2A medium.

Therefore, it was confirmed that the dual culture of Pseudomonas and Micrococcus using a scalp environment-simulating medium can be used for hair growth promotion, hair loss prevention, hair root strengthening, and scalp health.

Experimental Example 2: Evaluation of the efficacy of increasing the proliferation capacity of breast papilla cells.

Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, it was evaluated whether Example 2 induces proliferation of mammary papilla cells.

First, human hair follicle dermal papilla cells (HFDPC) were seeded at ^2x104 cells/well in a 96-well plate and cultured for 24 hours in an incubator at 37°C and 5% _CO2 . Afterwards, the medium was replaced with a medium containing nothing, and 1% (w/w) of the culture medium of Example 2 or Comparative Example 3 was added and cultured for an additional 24 hours. The group with 0.1 ppm of minoxidil (MXD) added and the group with 0.25 mM of AS2P1 added were each set as positive controls, and the group without culture medium (None) was also set as a control group. After 24 hours, the medium in each well was removed and washed once with DPBS (Dulbecco's phosphate-buffered saline). Then, each well was replaced with medium containing 500 μl of 0.5 mg/mL MTT (Sigma-aldrich, St. Louis, USA) and incubated at 37°C for 4 hours. Afterwards, 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 this can be used to confirm the proliferation ability of human hairy podocytes (HFDPCs).

Figure 5 is a graph showing the proliferation ability of mammary papilla cells according to the type of medium as cell viability (%).

As a result, as shown in Fig. 5, it was confirmed that Example 2 using a scalp environment simulating medium significantly increased the proliferation capacity of hair follicle cells compared to Comparative Example 3 using a general culture medium, R2A medium.

Therefore, it was confirmed that the dual culture of Pseudomonas and Micrococcus using a scalp environment-simulating medium can be used to promote hair growth, prevent hair loss, and strengthen hair roots.

Experimental Example 3: Confirmation of the effect of reducing the expression of lipid-producing genes and cell proliferation genes.

Experimental Example 3-1: Confirmation of the efficacy of inhibiting the expression of lipid synthesis inducer (SREBP1) in sebaceous gland cells.

To evaluate whether the expression of SREBP1, a factor that induces lipid synthesis in sebaceous cells (SZ95), could be reduced by the Pseudomonas and Micrococcus dual culture prepared in Example 2, quantitative real-time polymerase chain reaction (qRT-PCR) was performed.

Specifically, sebaceous gland cells (SZ95) were seeded at 3 × 10 ⁵ cells/well in a 6-well plate and cultured for 24 hours under cell culture conditions. After 24 hours of culture, the medium was discarded, washed with PBS, and the cells were starved using medium that did not contain medium supplements. Then, the medium containing 50 ng/ml of Insulin Growth Factor-1 (IGF1) (negative control) and the medium containing the Pseudomonas and Micrococcus dual culture of Example 2 were replaced, and the cells were further cultured for 48 hours. Afterwards, RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen), and the RNA was quantified at 260 nm using a nanodrop. Then, 2 μg of RNA each was used to synthesize cDNA in an amplifier (C1000 Thermal Cycler, Bio-Rad, USA). The expression level of the SREBP1 gene was finally evaluated by performing real-time polymerase chain reaction on a real-time PCR machine using a mixture of the synthesized cDNA, the target protein SREBP1 primer, and the cyanine dye CyberGreen (SYBR Green supermis, Applied Biosystems, USA). The sequences of the primers and reaction conditions used are shown in Table 3 below, and the expression level of each gene was finally analyzed through correction for the β-actin gene.

Figure 6 is a graph showing the relative expression level of SREBP1 according to concentration-specific treatment of the double culture solution of Example 2.

As a result, as shown in Fig. 6, it was confirmed that the double culture solution of Example 2 could suppress excessive lipid production by reducing the expression of lipid production factors.

Therefore, it was confirmed that the dual culture of Pseudomonas and Micrococcus using a scalp environment-simulating medium can be used to improve excessive sebum on the scalp, thereby promoting hair growth, preventing hair loss, strengthening hair roots, and improving scalp health.

Experimental Example 3-2: Confirmation of the efficacy of suppressing the expression of hyperkeratotic factor (Ki67) in human skin keratinocytes.

To evaluate whether the expression of Ki67, a hyperkeratotic factor in the epidermis, could be reduced by the dual culture of Pseudomonas and Micrococcus prepared in Example 2, quantitative real-time polymerase chain reaction (qRT-PCR) was performed.

Specifically, after culturing the SZ95 cells used in Experimental Example 3-1, the culture medium supernatant of the cultured SZ95 cells was harvested before RNA extraction. Human skin keratinocytes (HaCaT cells) were seeded at 3 × 10 ⁵ cells/well in a 6-well plate and cultured for 24 hours under cell culture conditions. After 24 hours of culture, the medium was discarded, washed with PBS, and the cells were starved using medium that did not contain medium supplements. Then, the SZ95 cell culture medium supernatant obtained in Experimental Example 3-1 was replaced with DMEM medium in a 1:1 ratio, and then cultured for an additional 24 hours. After that, RNA was isolated from the cells using RNeasy Mini Kit (Qiagen), and then RNA was quantified at 260 nm using nanodrop. Then, cDNA was synthesized using 2 μg of RNA each in an amplifier (C1000 Thermal Cycler, Bio-Rad, USA). The expression level of the Ki67 gene was ultimately evaluated by performing real-time polymerase chain reaction on a real-time PCR machine using a mixture of synthesized cDNA, target protein Ki67 primers, and cyanine dye CyberGreen (SYBR Green supermis, Applied Biosystems, USA). The sequences of the primers and reaction conditions used are shown in Table 3 below, and the expression level of each gene was finally analyzed through correction for the β-actin gene.

Primer sequence Sequence number order Reaction conditions SREBP1 F 10 5'- ACAGTGACTTCCCTGGCCTAT-3' After polymerization enzyme activation at 94℃ for 5 minutes, polymerization reaction was performed for 40 cycles under the conditions of 95℃ for 30 seconds, 60℃ for 30 seconds, and 72℃ for 30 seconds. R 11 5'- GCATGGACGGGTACATCTTCAA-3' KI67 F 12 5'- ACGCCTGGTTACTATCAAAAGG-3' R 13 5'-CAGACCCATTTACTTGTGTTGGA-3' β-actin F 14 5'- GGCCATCTCTTGCTCGAAGT-3' R 15 5'-GAGACCTTCAACACCCCAGC-3'

Figure 7 is a graph showing the relative expression level of Ki67 according to the concentration-specific treatment of the double culture medium of Example 2. As a result, as shown in Figure 7, it was confirmed that the double culture medium of Example 2 reduces the expression of factors related to hyperkeratosis.

Therefore, it was confirmed that the dual culture of Pseudomonas and Micrococcus using a scalp environment simulating medium can be used to improve scalp hyperkeratosis, promote hair growth, prevent hair loss, strengthen hair roots, and improve scalp health.

[Accession number]

Name of depositor: Korea Center for Microbiological Conservation (KCCM)

Accession number: KCCM13450P

Date of acceptance: 20240102

Claims (13)

A step of preparing a primary culture solution by culturing Pseudomonas sp. microorganisms in a medium composition containing sugars and amino acids; and A method for producing a composition for improving the scalp, comprising a step of producing a secondary culture by culturing a Micrococcus sp. microorganism in the primary culture. A method for producing a composition for improving the scalp, wherein the sugar according to claim 1 comprises at least one selected from the group consisting of glucose, rhamnose, and fructose. A method for producing a composition for improving the scalp, wherein the amino acid comprises at least one selected from the group consisting of threonine, tyrosine, arginine, alanine, proline, histidine, and isoleucine. A method for producing a composition for improving the scalp, wherein the composition further comprises glycerol according to claim 1. A method for producing a scalp improvement composition according to claim 1, wherein the Pseudomonas sp. microorganism is Pseudomonas azotoformans. A method for producing a scalp improvement composition according to claim 1, wherein the Micrococcus sp. microorganism is Micrococcus luteus. A cosmetic composition for improving the scalp, comprising a composition for improving the scalp prepared by the method of any one of claims 1 to 6. A cosmetic composition according to claim 7, wherein the scalp improvement is hair growth promotion, hair loss prevention, or hair root strengthening. A cosmetic composition according to claim 7, which increases the expression of at least one of hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7), and vascular endothelial growth factor (VEGF); or proliferates hair follicle cells. A pharmaceutical composition for promoting hair growth or preventing hair loss, comprising a composition for improving the scalp prepared by the method of any one of claims 1 to 6. Use of a scalp improvement composition manufactured by the method of any one of claims 1 to 6 for manufacturing a scalp improvement cosmetic composition. Use of a scalp improvement composition manufactured by the method of any one of claims 1 to 6 for manufacturing a drug for promoting hair growth or preventing hair loss. A method for improving the scalp, comprising the step of administering to a subject in need thereof a composition for improving the scalp prepared by the method of any one of claims 1 to 6.