WO2025048553A1 - Novel bacillus subtilis strain and use thereof - Google Patents

Abstract

The present invention relates to: a Bacillus subtilis HKtulip001 (accession no. KCTC15331BP) strain, a culture thereof, a lysate thereof, or an extract thereof; and a use thereof. The strain according to the present invention has excellent skin barrier improvement, pruritus amelioration, moisturization, and wrinkle amelioration effects, and also has a high total polyphenol content, and thus can be effectively utilized for skin condition improvement purposes.

Description

Novel Bacillus subtilis strains and their uses

The present invention relates to a novel Bacillus Subtilis HKtulip001 strain and uses thereof.

The skin barrier prevents moisture and electrolytes from being lost to the outside through the skin, and protects against the invasion of harmful chemicals or microorganisms. In particular, the stratum corneum, the outermost layer of the epidermis, is responsible for the barrier function of preventing damage to the skin barrier caused by external factors such as external humidity, temperature changes, ultraviolet rays, and microorganisms, and preventing moisture inside the skin from evaporating to the outside.

The skin barrier is divided into the stratum corneum barrier and the tight junction that prevents moisture evaporation through adhesion between cells below the stratum corneum. The research results of atopic dermatitis, which are mainly caused by damage to the skin barrier function and the related immune response, have been explained in many review articles (Kim et al. Allergy Asthma Immunol Res 2012;4:12-6; Wolf et al. Clin Dermatol 2012;30:329-34.). It is known that the decrease in skin moisture in atopic patients is related to the decrease in intercellular lipids in the stratum corneum due to epidermal differentiation abnormality and the weakening of tight junctions due to the decrease in claudin (J Allergy Clin Immunol. 2011 Mar;127(3):773-86). In addition, it has been reported that when the expression of Claudin1 protein is suppressed by knocking down the claudin1 gene, the amount of moisture loss in the skin increases and filaggrin is not decomposed. Furthermore, it has been reported that the infiltration of immune cells greatly increases in the area where atopic dermatitis occurs, and the expression level of the claudin1 gene decreases in the skin of atopic dermatitis patients due to cytokines such as IL-33 secreted by the immune cells (Tomoko Sugawara et al., Journal of Dermatological Science. 2013, 70, 12-18).

Meanwhile, water makes up about 70% of the human body and helps maintain moisture and gloss in the skin. The stratum corneum of a healthy epidermis contains 15-20% of water, and when the value drops below 10%, the skin barrier becomes abnormal, the skin becomes dry, and wrinkles increase. The water content of the stratum corneum is determined by the sebum film, a lipid mixture produced and secreted by the epidermis, and the natural moisturizing factor (NMF), a water-soluble component present in the stratum corneum. Hyaluronic acid, one of the natural moisturizing factors, is formed by fibroblasts and keratinocytes and is a component of viscoelastic polysaccharides, so it contains a lot of polysaccharides and has the characteristic of being able to contain a significant amount of water. The cycle is 2-4.5 days, and it combines with water that weighs 1,000 times its own weight to regulate the skin barrier function and hydrate the extracellular matrix, maintaining homeostasis of moisture in the tissue. Therefore, when the moisture content in the skin decreases and the amount of hyaluronic acid decreases, it directly leads to skin aging.

Aquaporin (AQP) is a membrane pore protein that acts as a water channel that selectively induces the entry and exit of water molecules while blocking the passage of other ions or solutes. Among them, AQP-3 allows glycerol to pass together with water, so it is also called aquaglyceroporin. It exists in human keratinocytes and prevents the loss of water molecules, thereby performing functions such as skin hydration, wound healing, and homeostasis. Therefore, the moisturizing effect of the raw material can be confirmed through the increase in the expression of AQP-3. In addition, it is reported that the amount of hyaluronic acid in human skin decreases with aging, and this is thought to be one of the direct causes of decreased skin elasticity, wrinkles, and decreased moisture content due to aging. The synthesis of hyaluronic acid is carried out by HAS (Hyaluronic Acid Synthase), and the moisturizing effect of the raw material can be confirmed through increased expression of HAS-2 (Journal of Drugs in Dematology, 6, s20-4, 2007; Experimental Dermatology, 18(12), 1028-1035, 2009).

It is known that skin wrinkles are caused by a decrease in collagen and elastic fibers, which are major components of the extracellular matrix. Collagen is a major matrix protein produced by skin fibroblasts and accounts for approximately 30% of the total weight of biological proteins. Elastic fibers account for 3% of the skin's dry weight and provide elasticity to the skin. Elastic fibers can be divided into three types: oxytalan, elanin, and elastic fibers, and the type is determined by the combination of fibrillin and elastin. These collagen and elastic fibers decrease due to age and photoaging caused by ultraviolet irradiation, and it is known that this is closely related to the formation of wrinkles on the skin.

Elastin fibers form cross-links with collagen and are an important skin component involved in wrinkle formation and skin elasticity. Deficiency and aggregation of elastin fibers and a significant increase in the activity of elastase, an elastin-decomposing enzyme, have been identified as one of the factors causing wrinkle formation. Elastase is the only enzyme that can decompose elastin, and inhibition of it is known to fundamentally reduce the improvement of skin wrinkles.

Against this backdrop, the inventors of the present invention have conducted extensive research efforts to find a strain that exhibits excellent effects in improving skin conditions, such as improving skin wrinkles and moisturizing, and have thus completed the present invention by confirming that the Bacillus Subtilis HKtulip001 strain has skin moisturizing, wrinkle improvement, and skin barrier improvement effects.

Accordingly, the purpose of the present invention is to provide a Bacillus Subtilis HKtulip001 strain having the accession number KCTC15331BP, a use thereof, and a composition containing the same as an effective ingredient.

In order to achieve the above-mentioned purpose, as a result of extensive research efforts, the inventors of the present invention have confirmed that the Bacillus subtilis HKtulip001 strain has excellent effects in improving skin barrier, alleviating itching, moisturizing, and improving wrinkles, thereby completing the present invention.

The present invention provides a Bacillus Subtilis HKtulip001 strain.

In the present invention, the strain may be isolated. The term "isolated" as used herein means something that is not present in nature but is artificially isolated and available. For example, the strain may be isolated from a tulip, but the acquisition route thereof is not limited thereto. In the present invention, "Bacillus subtilis isolated from a tulip" may be used interchangeably with "Bacillus subtilis derived from a tulip."

The Bacillus subtilis HKtulip001 strain of the present invention was confirmed to have a 16s rRNA base sequence of sequence number 1 during the isolation and identification process, and was deposited with the Biological Resource Center on February 22, 2023 (Accession number KCTC15331BP).

The term "sequence identity" used in the present invention refers to the degree of identical amino acid residues or bases between sequences after aligning the two sequences to be as identical 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 within the comparison region may be added or deleted compared to the reference sequence. The percentage of sequence identity can be calculated, for example, by the steps of comparing two optimally aligned sequences in the entire comparison region, determining the number of positions at which the same amino acid or nucleic acid appears in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison range (i.e., range size), and multiplying the result by 100 to obtain the percentage of sequence identity. The percent sequence identity can be determined using known sequence comparison programs, including, but not limited to, BLASTN (NCBI), CLC Main Workbench (CLC bio), and MegAlignTM (DNASTAR Inc).

In the present invention, the strain may include 16S rRNA having a sequence identity of 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 with sequence number 1. The strain may include 16S rRNA of sequence number 1.

It was experimentally confirmed that the strain of the present invention has superior effects on skin moisturizing, skin barrier improvement, and skin wrinkle improvement compared to the Bacillus subtilis strain of the same genus and species, and has a high polyphenol content. Specifically, it was confirmed that the strain increases the expression of genes related to skin moisturizing and skin barrier; or reduces the activity of enzymes that induce skin wrinkle formation.

In addition, the present invention provides Bacillus subtilis HKtulip001 strain, a culture thereof, a lysate thereof or an extract thereof; or a use thereof. In a specific embodiment of the present invention, a composition comprising Bacillus subtilis HKtulip001 strain, a culture thereof, a lysate thereof or an extract thereof is provided.

The Bacillus subtilis HKtulip001 strain included in the composition according to the present invention may exist as a live cell or a dead cell, and may also exist in a dried or lyophilized form. The form of a beneficial bacteria suitable for inclusion in various compositions and a method for preparing the same are well known to those skilled in the art. For example, the Bacillus subtilis HKtulip001 strain may be prepared in the form of a culture obtained by culturing in a known liquid medium or solid medium, a concentrate of the culture, a dried product of the culture, a fermented product obtained by culturing the strain together with an additional component, an extract obtained by extracting the strain with an organic solvent, a lysate (or lysate) obtained by dissolving, crushing, or homogenizing the cell membrane of the strain, but is not limited thereto.

In the present invention, the "culture" means the entire medium including the strain, its metabolites, extra nutrients, etc., obtained by culturing the strain in a medium capable of supplying nutrients so that the Bacillus subtilis HKtulip001 strain can grow and survive in a test tube for a certain period of time, but also includes the supernatant from which the strain is removed after culturing the strain, and extracts and fractions thereof. The liquid from which the bacterial cells are removed from the culture solution is also called the "supernatant" or "cell-free supernatant", and can be obtained by allowing the culture solution to stand still for a certain period of time and taking only the liquid from the upper layer excluding the portion that has settled to the lower layer, removing the bacterial cells through filtration, or centrifuging the culture solution to remove the sediment at the lower layer and taking only the liquid at the upper layer. In addition, the culture solution may include a fermentation product. In one example, the culture solution may be a fermentation product obtained by culturing the strain in a medium. The "fermentation product" means the result of enzymatic or metabolic decomposition of an organic substance using a microorganism. In the present invention, "fermentation" may mean any activity or process that includes enzymatic or metabolic decomposition of organic substances using microorganisms, but is not a putrefaction reaction. The culture medium may be in the form of a liquid medium as well as a dried product thereof, for example, a freeze-dried product of the culture medium.

In the present invention, the composition may be a composition comprising Bacillus subtilis HKtulip001 strain existing as a live or dead cell.

In addition, the composition according to the present invention may include a culture filtrate of the Bacillus subtilis HKtulip001 strain. That is, the culture of the Bacillus subtilis HKtulip001 strain may include a culture filtrate from which the Bacillus subtilis HKtulip001 strain has been removed. For example, the composition according to the present invention may include a lyophilized product of a culture filtrate of the Bacillus subtilis HKtulip001 strain.

The composition is present in an amount of 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 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%, 0.1 It may comprise from 0.1 wt % to 30 wt %, from 0.1 wt % to 20 wt %, from 0.1 wt % to 10 wt %, or from 0.1 wt % to 5 wt % of the strain, a culture thereof, a lysate thereof, or an extract thereof.

The present invention provides a cosmetic composition for improving skin condition, comprising the Bacillus subtilis HKtulip001 strain of the present invention, a culture thereof, a pulverized product thereof, or an extract thereof as an effective ingredient.

In addition, the present invention provides a use of a cosmetic composition comprising Bacillus subtilis HKtulip001 strain, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient for manufacturing a product for improving skin condition; and a method for improving skin condition, comprising a step of applying a cosmetic composition comprising Bacillus subtilis HKtulip001 strain, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient to the skin of a subject.

In the present invention, the term "improvement" means any action that at least reduces the degree of a symptom, for example a parameter related to alleviation or treatment of a condition.

In the present invention, “improving skin condition” refers to reducing the degree of symptoms related to worsening skin condition, and the skin condition may be parameters related to skin moisturizing, wrinkle improvement, skin barrier improvement, and itching improvement.

In the present invention, "improvement of skin barrier" includes both improvement of skin barrier and protection function, and may mean all actions that enhance the function of the skin barrier, which is located at the outermost part of the skin and prevents moisture and nutrient loss. In the embodiment of the present invention, the effect of improving the skin barrier was confirmed by increasing the expression of claudin or filaggrin.

In the present invention, "itchiness of the skin" means an unpleasant sensation that causes a desire to scratch or rub the skin, and examples thereof include itching due to dry skin; itching due to hives; itching due to insect bites; itching caused by heat rash, acne, scabies, frostbite, contact dermatitis, seborrheic dermatitis, or psoriasis; itching due to various skin diseases; itching of the scalp, etc. "Improvement of itching" in the present specification means helping to improve itching symptoms by restoring the function of the skin barrier.

In the present invention, "skin moisturizing" means increasing moisture in the skin and maintaining a moist state by suppressing moisture loss. In an embodiment of the present invention, a skin moisturizing effect was confirmed by increasing the expression of HAS-2 (Hyaluronan Synthase 2) or AQP-3 (Aquaporin 3).

In addition, "wrinkle improvement" means preventing, suppressing, or inhibiting the formation of wrinkles on the skin, or alleviating wrinkles that have already formed. In the embodiment of the present invention, the effect of improving skin wrinkles was confirmed by reducing the activity of elastase.

The above cosmetic composition is not particularly limited to a specific formulation, and the formulation can be appropriately selected depending on the purpose. For example, the above cosmetic composition can be manufactured into a formulation including a toner (skin lotion), skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, hand sanitizer, foundation, essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, suspension, gel, powder, paste, mask pack, and sheet. The composition of such a formulation can be manufactured according to a conventional method in the relevant field.

In addition to the effective ingredients disclosed in this specification, the cosmetic composition may further contain functional additives and components included in general cosmetic compositions, and may further contain purified water, a thickener, a preservative, a stabilizer, a solubilizer, a surfactant, a carrier, a fragrance, or a combination thereof, which are commonly used. The functional additives may include components selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular polysaccharides, sphingolipids, and seaweed extracts. The carriers may include, for example, alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, colorants, fragrances, and the like. The compounds/compositions that can be used as the above alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, colorants, fragrances, etc. are already known in the art, and therefore, a person skilled in the art can select and use the appropriate corresponding substances/compositions. In addition, the cosmetic composition may further contain, as necessary, ingredients such as sunscreens, antioxidants (butylated hydroxyanisole, propyl gallate, ellisorbic acid, tocopheryl acetate, butylated hydroxytoluene, etc.), preservatives (methylparaben, butylparaben, propylparaben, phenoxyethanol, imidazolidinyl urea, chlorphenesin, etc.), colorants, pH adjusters (triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, formaldehyde, sodium formaldehyde, succinic acid, sodium succinate, sodium hydroxide, sodium hydrogen phosphate, etc.), moisturizers (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glycereth-26, methylgluceth-20, etc.), and lubricants.

The present invention also provides a food composition for improving skin condition, comprising Bacillus subtilis HKtulip001 strain, a culture thereof, a pulverized substance thereof, or an extract thereof as an effective ingredient.

In the above food composition, all of the contents described above regarding the Bacillus subtilis strain and its use can be applied as is.

In the present invention, the “improvement in skin condition” may be a parameter related to skin moisturizing, wrinkle improvement, skin barrier improvement, and itching improvement.

The "food" of the present invention includes raw materials and all processed materials consumed for maintaining health and growth and development, and includes not only general foods meaning general food or processed foods, but also functional foods, health functional foods, and medical foods.

For example, the food may be a functional food. In the present invention, the "functional food" means a food having a physiological function or activity, and means a food containing a biologically active ingredient at a level that can provide a specific health effect. In this respect, the present invention provides a functional food composition for improving skin condition, which contains Bacillus subtilis HKtulip001, a culture thereof, a pulverized substance thereof, or an extract thereof as an effective ingredient.

For example, the food may be a health functional food. In the present invention, the "health functional food" refers to a food manufactured using specific nutrients or raw materials or ingredients having functions useful to the human body, that is, functional raw materials, and can help human health by maintaining normal functions of the human body or activating physiological functions. In this respect, the present invention provides a health functional food composition for improving skin condition, comprising Bacillus subtilis HKtulip001, a culture thereof, a pulverized substance thereof, or an extract thereof as an effective ingredient.

When the composition of the present invention is utilized as a food composition, the food composition may include a form of a health functional food or a seasoning, a beverage, a bar, etc. In addition, the food composition containing the strain as an active ingredient may include a beverage such as fermented milk, and the food composition containing the strain as an active ingredient may be ingested by granulating, encapsulating, or powdering.

The food composition of the present invention can be manufactured using, in addition to the above-mentioned effective ingredient, a food-related suitable and physiologically acceptable auxiliary agent, and the auxiliary agent can be an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a glidant, or a flavoring agent.

The above food composition may be preferably formulated as a food composition by additionally including at least one food-wise acceptable carrier in addition to the above-described effective ingredient for administration.

For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an orally, non-toxic, food-wise acceptable, inert carrier such as ethanol, glycerol, water, and the like. In addition, suitable binders, lubricants, disintegrants, and coloring agents, if desired or necessary, may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, natural and synthetic gums such as corn sweetener, acacia, tracheacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. When formulated as a liquid solution, it includes suspensions, solutions, emulsions, syrups, etc., and diluents such as water and liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, etc., but are not limited thereto.

The food composition according to the present invention can be added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, vitamin complexes, health supplements, etc.

The food composition of the present invention may include components commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates described above include monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc., and common sugars and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. In addition, vitamins, such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3, and vitamin E, can be included. For example, when the food composition of the present invention is manufactured into a drink or beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included.

The advantages and features of the present invention, and the method for achieving them, will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and these embodiments are provided only to make the disclosure of the present invention complete and to fully inform a person having ordinary skill in the art to which the present invention belongs of the scope of the invention, and the present invention is defined only by the scope of the claims.

The Bacillus subtilis HKtulip001 strain according to the present invention is effective in increasing the expression of Claudin1, Fliaggrin, HAS-2 or AQP3 and inhibiting elastase activity, and has a high polyphenol content, so it can be effectively used for improving skin barrier, improving itching, moisturizing skin or improving wrinkles.

Figure 1 shows the results of testing the cytotoxicity of a lyophilized product of a tulip-derived Bacillus subtilis strain culture using an MTT assay on HaCaT cells.

Figure 2 shows the results of testing the cytotoxicity of a lyophilized culture of a tulip-derived Bacillus subtilis strain using an MTT assay on human fibroblasts.

Figure 3 shows the results of a test to determine whether the expression of the Claudin1 gene increases with treatment of a lyophilized culture of a tulip-derived Bacillus subtilis strain.

Figure 4 shows the results of a test to determine whether the expression of the Fliaggrin gene increases with treatment of a lyophilized culture of a tulip-derived Bacillus subtilis strain.

Figure 5 shows the results of a test to determine whether the expression of the HAS-2 gene increases with treatment of a lyophilized culture of a tulip-derived Bacillus subtilis strain.

Figure 6 shows the results of a test to determine whether the expression of the AQP-3 gene increases with treatment of a lyophilized culture of a tulip-derived Bacillus subtilis strain.

Figure 7 shows the results of a test for inhibition of elastase activity according to treatment of a lyophilized culture solution of a tulip-derived Bacillus subtilis strain.

Figure 8 shows the results of measuring the total polyphenol content of a freeze-dried product of a tulip-derived Bacillus subtilis culture.

Hereinafter, the present invention will be described in detail through examples. The following examples are only intended to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

[Example]

Example 1: Isolation of Bacillus subtilis strains derived from tulips

Tulip was homogenized by adding 10 times the amount of saline solution. 100 uL of the homogenized sample was plated on Tryptic Soy Agar (TSA), and the plate was cultured in an incubator at 35°C for 48 hours. Each colony formed was inoculated into Tryptic Soy Broth (TSB) and cultured with shaking at 35°C and 150 rpm for 24 hours. The culture was streaked on TSA and cultured at 35°C for 24 hours, and colonies showing light-colored colonies were collected. The above procedure was repeated to purely isolate a single strain.

The purely isolated strains above were analyzed for homology using 16s rRNA analysis and the BLAST program (NCBI Database) by requesting Macrogen Co., Ltd.

16s rRNA gene amplification was performed using two universal primers, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), and then sequencing analysis of the amplified 16s rRNA gene was performed. Using the analyzed 16s rRNA sequence data and the BLAST program (NCBI Database), one strain was isolated and identified, named Bacillus subtilis HKtulip001, and deposited in the National Institute of Biological Resources on February 22, 2023 (Accession No. KCTC15331BP).

Bacillus subtilis HKtulip001 strain was analyzed to have a 16S rRNA base sequence of sequence number 1.

Example 2. Preparation of freeze-dried product of tulip-derived Bacillus subtilis culture solution

A freeze-dried product of the culture solution of the strain ( Bacillus subtilis HKtulip001) isolated in the above Example 1 was prepared and inoculated into TSB, and cultured for 24 hours at 35°C and 150 rpm. After centrifugation at 10,000 rpm for 10 minutes, the culture solution was obtained by removing the bacterial cells by filtering through a 0.45 μm membrane filter. After that, it was frozen at -80°C for 12 hours or more and then freeze-dried to prepare a freeze-dried product powder of the culture solution of the tulip-derived Bacillus subtilis strain.

Experimental Example 1. Cytotoxicity Evaluation

(1) MTT assay for HaCaT cells

To determine whether the tulip-derived Bacillus subtilis strain has cytotoxicity, the following experiment was performed. HaCaT, a keratinocyte cell line, was cultured in DMEM medium containing 10% FBS and seeded in a 96-well plate at a cell density of ^2X104 and cultured in an incubator at 37℃ and 5% _CO2 for 24 hours. After culture, the medium was removed, and the cells were treated with a diluted solution prepared by diluting the lyophilized tulip-derived Bacillus subtilis culture of Example 2 in dimethyl sulfoxide to a concentration of 5, 10, or 25 ppm, and cultured for 24 hours. Then, 20 μl of MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazoliumboromide, Sigma) solution dissolved at a concentration of 5 mg/ml was added to each well and reacted for 2 hours. After removing the supernatant, 150 ㎕ of dimethyl sulfoxide was added, and the mixture was shaken for 30 minutes to dissolve the produced formazan, and the absorbance was measured at 540 nm using a multi-microplate reader (Molecular device Spectra max190). The cell viability was calculated according to the following mathematical formula 1, and the results are shown in Table 1 and Figure 1.

[Mathematical formula 1]

Cell viability (%) = Absorbance of sample addition group / Absorbance of control group * 100

　 Control HKtulip001 (ppm) 5 10 25 Cell viability (%) 100.00 107.34 108.35 111.05 Standard deviation 1.65 4.37 2.27 3.61

As a result of the test, it was confirmed that the lyophilized product of tulip-derived Bacillus subtilis culture did not exhibit cytotoxicity against HaCaT cells, a keratinocyte cell line, and thus there were no safety issues.

(2) MTT assay for fibroblasts

To determine whether the tulip-derived Bacillus subtilis strain exhibits cytotoxicity, human dermal fibroblasts (HDF) were cultured in DMEM/F12 medium containing 10% FBS and seeded at a cell concentration of ^2X104 in a 96-well plate and cultured in an incubator at 37°C and 5% _CO2 for 24 hours. After culture, the medium was removed, and the cells were treated with a diluted solution prepared by diluting the lyophilized tulip-derived Bacillus subtilis culture of Example 2 in dimethyl sulfoxide to a concentration of 10, 25, 50, or 100 ppm, and cultured for 24 hours. Then, 20 μl of an MTT solution dissolved at a concentration of 5 mg/ml was added to each well and reacted for 2 hours. After removing the supernatant, 150 ㎕ of dimethyl sulfoxide was added, and the mixture was shaken for 30 minutes to dissolve the produced formazan, and the absorbance was measured at 540 nm using a multi-microplate reader (Molecular device Spectra max190). The cell viability was calculated according to the above mathematical formula 1, and the results are shown in Table 2 and Figure 2 below.

　 Control HKtulip001 (ppm) 10 25 50 100 Cell viability (%) 100.00 96.72 95.96 98.18 96.53 Standard deviation 1.13 3.60 6.61 4.22 1.93

The test results showed that the lyophilized tulip-derived Bacillus subtilis culture did not exhibit cytotoxicity toward human fibroblast cells, indicating that there were no safety issues.

Experimental Example 2. Confirmation of the skin barrier improvement effect of Bacillus subtilis HKtulip001 strain

In order to confirm the skin barrier improvement efficacy of the tulip-derived Bacillus subtilis strain, the expression of skin barrier-related genes according to the treatment of the freeze-dried product of the culture solution of the Bacillus subtilis strain was confirmed using the following method.

HaCaT cells (2.5 x 10 ⁵ ) were seeded into 60 mm culture dishes and cultured for 18 hours. After replacing the cultured HaCaT cells with serum-free DMEM medium, the lyophilized tulip-derived Bacillus subtilis culture of Example 2 was treated at a concentration of 5, 10, or 25 ppm for 24 hours. The cells were harvested, and RNA was isolated using NucleoZOL lysis buffer (MACHEREY-NAGEL, 740404) according to the manufacturer's protocol. The isolated RNA was quantified using a microplate reader, and cDNA was synthesized using HiSenScript™ RH(-) RT PreMix Kit (intron, 25087). Real-time polymerase chain reaction was analyzed after amplifying the gene using 2X Real-Time PCR Master mix (Biofact, DQ385-40h).

As a result, it was confirmed that Bacillus subtilis HKtulip001 dose-dependently increases the gene expression of claudin1, a protein constituting tight junctions, and filaggrin, one of the structural proteins expressed in the differentiation stage of keratinocytes. Specifically, in the group treated with the freeze-dried product of the culture solution of Bacillus subtilis HKtulip001, Claudin1 expression was up to 164% compared to the untreated control group (Table 3 and Fig. 3), and Fliaggrin expression was up to 115% compared to the untreated control group (Table 4 and Fig. 4).

　 Control HKtulip001 (ppm) 5 10 25 Relative mRNA expression
(% of control) 100.00 115.21 128.72 164.08 Standard deviation 0.62 0.86 0.01 3.42

　 Control HKtulip001 (ppm) 5 10 25 Relative mRNA expression
(% of control) 100.00 114.91 114.71 110.93 Standard deviation 0.08 0.15 0.55 2.91

Experimental Example 3. Confirmation of skin moisturizing effect of Bacillus subtilis HKtulip001 strain. In order to confirm the skin moisturizing effect of tulip-derived Bacillus subtilis strain, the expression of skin moisturizing-related genes according to the treatment of freeze-dried product of culture solution of Bacillus subtilis strain was confirmed using the following method.

HaCaT cells (2.5 x 10 ⁵ ) were seeded into 60 mm culture dishes and cultured for 18 hours. After replacing the cultured HaCaT cells with serum-free DMEM medium, the lyophilized tulip-derived Bacillus subtilis culture of Example 2 was treated at a concentration of 5, 10, or 25 ppm for 24 hours. In addition, retinoic acid was treated at a concentration of 0.3 ppm as a positive control. Cells were harvested, and RNA was isolated using NucleoZOL lysis buffer (MACHEREY-NAGEL, 740404) according to the manufacturer's protocol. The isolated RNA was quantified using a microplate reader, and cDNA was synthesized using HiSenScript™ RH(-) RT PreMix Kit (intron, 25087). Real-time polymerase chain reaction was performed using 2X Real-Time PCR Master mix (Biofact, DQ385-40h) to amplify the genes and then analyze them.

As a result, it was confirmed that the freeze-dried Bacillus subtilis HKtulip001 culture solution concentration-dependently increased the expression of HAS-2, a hyaluronic acid synthase, and AQP-3, a transmembrane protein that regulates the movement of water and glycerol. Specifically, the freeze-dried Bacillus subtilis HKtulip001 culture solution showed a maximum of 415% in HAS-2 expression and a maximum of 155% in AQP-3 expression compared to the untreated control group (Table 5 and Fig. 5).

　 Control HKtulip001 (ppm) Retinoic acid
(ppm) 5 10 25 0.3 Relative mRNA expression
(% of control) 100.84 102.37 198.23 415.20 289.33 Standard deviation 13.00 61.71 2.77 95.98 53.01

　 Control HKtulip001 (ppm) Retinoic acid
(ppm) 5 10 25 0.3 Relative mRNA expression
(% of control) 100.72 101.11 97.78 154.69 171.02 Standard deviation 11.98 0.31 16.83 3.13 3.84

Experimental Example 4. Confirmation of the skin wrinkle alleviation effect of Bacillus subtilis HKtulip001 strain

In order to confirm the skin wrinkle alleviation effect of the tulip-derived Bacillus subtilis strain, the elastase inhibitory activity of the lyophilized culture solution of the tulip-derived Bacillus subtilis strain was confirmed by the following method.

1% Triton X-100 was added to the human fibroblast pellet, dissolved, and then frozen in liquid nitrogen, which was repeated three times. The solution was centrifuged at 12,000 rpm at 4°C for 5 min, and the supernatant was prepared into an enzyme solution containing fibroblast elastase. The enzyme solution was quantified using the Bradford method, and an amount containing 80 ug of protein per well was added to 96 wells. 0.1 M Tris-HCl buffer was added to make 88 μl, and 10 μl of the sample was added to each well. 2 μl of the substrate STANA (N-succinyl-tri-alanyl-p-nitroanilide, 50 mM) solution was added to each well, and the mixture was incubated at 37°C. After 90 min, the absorbance was measured at 405 nm using a spectrophotometer. Elastase inhibition activity was calculated using the following mathematical formula 2, with the case in which no sample was added serving as the control group.

[Mathematical formula 2]

Elastase inhibition activity (%) = 100 - (absorbance of the group treated with the sample / absorbance of the control group not treated with the sample X 100)

As a result, it was confirmed that Bacillus subtilis HKtulip001 reduced elastase activity by up to 58% (Table 7 and Figure 7).

　 Control HKtulip001 (ppm) Phosphoramidon
(μM) 10 25 50 100 10 Elastase activity (%) 100.00 82.79 73.13 59.03 42.20 11.37 Standard deviation 1.30 2.23 0.67 0.23 0.71 1.21

Experimental Example 5. Analysis of polyphenol content

The total polyphenol content of the lyophilized product of a tulip-derived Bacillus subtilis strain culture was measured.

The total polyphenol content was determined according to the well-known Folin-denis (1912) method. 20 μl of the sample was added with 40 μl of Folin-denis reagent (Sigma, USA) and 400 μl of distilled water. The mixture was reacted at room temperature for 3 minutes, then 400 μl of a 20% Na ₂ CO ₃ solution was added and reacted at room temperature for 1 hour. After the reaction was completed, the absorbance was measured at 765 nm. The total polyphenol content was calculated as gallic acid equivalent (GAE) by substituting the absorbance of the sample into a standard curve previously prepared using a gallic acid standard in the same manner. The total polyphenol content is shown in Table 8 and Fig. 8.

Sample name Total polyphenol content (%) HKtulip001 1.550

In summary of the above experimental results, the Bacillus subtilis HKtulip001 strain was found to have excellent efficacy in all aspects, including improving the skin barrier, moisturizing, and improving wrinkles, and also to have a high total polyphenol content.

[Acceptance number]

Name of depository institution: Biological Resource Center

Accession number: KCTC15331BP

Date of acceptance: 20230222

Claims (9)

Bacillus subtilis HKtulip001 strain with accession number KCTC15331BP. A cosmetic composition for improving skin condition, comprising as an effective ingredient a Bacillus Subtilis HKtulip001 strain having the accession number KCTC15331BP, a culture thereof, a pulverized product thereof, or an extract thereof. In the second paragraph. A cosmetic composition, wherein the improvement in skin condition is at least one selected from the group consisting of skin moisturizing, wrinkle improvement, skin barrier improvement, and itching improvement. In the second paragraph, The composition is a cosmetic composition having an effect of increasing the expression of claudin or filaggrin. In the second paragraph, The composition above is a cosmetic composition having an effect of increasing the expression of HAS-2 or AQP3. In the second paragraph, The composition is a cosmetic composition having an elastase activity reducing effect. In the second paragraph, The composition above is a cosmetic composition in the form of a toner (skin lotion), skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, hand sanitizer, foundation, essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, suspension, gel, powder, paste, mask pack, and sheet. A food composition for improving skin condition, comprising as an effective ingredient a Bacillus Subtilis HKtulip001 strain having the accession number KCTC15331BP, a culture thereof, a pulverized product thereof, or an extract thereof. In Article 8, A food composition, wherein the improvement in the skin condition is at least one selected from the group consisting of skin moisturizing, wrinkle improvement, skin barrier improvement, and itching improvement.

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