Novel bacillus subtilis strain and use thereof

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 strain and its use

[0001] The present invention relates to a novel Bacillus subtilis HKtulip001 strain and uses thereof.

[0002] The skin barrier prevents the loss of moisture and electrolytes through the skin and protects against the invasion of harmful chemicals and microorganisms. In particular, the stratum corneum, the outermost layer of the epidermis, functions as a barrier, protecting the skin from damage caused by external factors such as humidity, temperature changes, UV rays, and microorganisms, as well as preventing moisture from evaporating from the skin.

[0003] The skin barrier is divided into the stratum corneum barrier and the tight junctions that prevent moisture evaporation through intercellular adhesion below the stratum corneum. The research results of atopic dermatitis, which show that the damage to the skin barrier function and the related immune response are the main pathogenesis, 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 abnormalities 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 has occurred, and the expression of the claudin1 gene in the skin of atopic dermatitis patients is reduced by cytokines such as IL-33 secreted by the immune cells (Tomoko Sugawara et al., Journal of Dermatological Science. 2013, 70, 12-18).

[0004] Meanwhile, water makes up approximately 70% of the human body and helps maintain skin moisture and radiance. The stratum corneum of a healthy epidermis contains 15-20% water. When this falls below 10%, the skin barrier becomes damaged, drying out the skin and increasing wrinkles. The moisture 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 within the stratum corneum. Hyaluronic acid, one of the natural moisturizing factors, is formed by fibroblasts and keratinocytes. It is a component of polysaccharides and has the characteristic of containing significant amounts of water due to its high polysaccharide content. The cycle lasts 2-4.5 days, and it binds to water 1,000 times its weight in water, regulating the skin barrier function and hydrating the extracellular matrix to maintain moisture homeostasis within the tissue. Therefore, when the moisture content in the skin decreases and the amount of hyaluronic acid decreases, it directly leads to skin aging.

[0005] Aquaporin (AQP) is a membrane pore protein that acts as a water channel that selectively allows water molecules to enter and exit while blocking the passage of other ions or solutes. Among them, AQP-3 allows glycerol to pass along with water, so it is also called aquaglyceroporin. It exists in human keratinocytes and prevents the loss of water molecules, thereby functioning in skin hydration, wound healing, and homeostasis. Therefore, the moisturizing effect of the raw material can be confirmed through increased 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).

[0006] Wrinkles are known to be caused by a decrease in collagen and elastic fibers, major components of the extracellular matrix. Collagen is the main matrix protein produced by dermal fibroblasts and accounts for approximately 30% of the total body protein weight. Elastic fibers account for 3% of the skin's dry weight and provide elasticity. Elastic fibers can be classified into three types: oxytalan, elanin, and elastic fibers, and their type is determined by the combination of fibrillin and elastin. Collagen and elastic fibers decrease with age and photoaging caused by UV exposure, and this is known to be closely related to the formation of wrinkles.

[0007] Elastin fibers form cross-links with collagen, contributing to skin elasticity and wrinkle formation. Deficiency and aggregation of elastin fibers, as well as a significant increase in the activity of elastase, an enzyme that breaks down elastin, have been identified as contributing factors to wrinkle formation. Elastase is the only enzyme capable of breaking down elastin, and its inhibition is known to fundamentally reduce wrinkles.

[0008] 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 effects in moisturizing the skin, improving wrinkles, or improving the skin barrier.

[0009] Accordingly, the purpose of the present invention is to provide a Bacillus subtilis HKtulip001 strain having a deposit number of KCTC15331BP, a use thereof, and a composition comprising the same as an effective ingredient.

[0010] 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.

[0011] The present invention provides a Bacillus subtilis HKtulip001 strain.

[0012] In the present invention, the strain may be isolated. The term "isolated" as used herein refers to something that is not found in nature but is artificially isolated and available. For example, the strain may be isolated from a tulip, but its acquisition route 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."

[0013] 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 at the Biological Resource Center on February 22, 2023 (Accession number KCTC15331BP).

[0014] 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 comparing two sequences by optimally aligning them 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 where 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 percentage of sequence identity can be determined using known sequence comparison programs, such as BLASTN (NCBI), CLC Main Workbench (CLC bio), and MegAlignTM (DNASTAR Inc).

[0015] In the present invention, the strain may comprise 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 SEQ ID NO: 1. The strain may comprise 16S rRNA of SEQ ID NO: 1.

[0016] The strain of the present invention was experimentally confirmed to have superior skin moisturizing, skin barrier improvement, and wrinkle-improving effects compared to the Bacillus subtilis strain of the same genus and species, and to have a high polyphenol content. Specifically, the strain was confirmed to increase the expression of genes related to skin moisturizing and skin barrier, or to reduce the activity of enzymes that induce wrinkle formation.

[0017]

[0018] In addition, the present invention provides a Bacillus subtilis strain HKtulip001, 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 the Bacillus subtilis strain HKtulip001, a culture thereof, a lysate thereof, or an extract thereof is provided.

[0019] 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 and formulation method of a beneficial bacterium suitable for inclusion in various compositions are well known to those skilled in the art. For example, the Bacillus subtilis HKtulip001 strain may be formulated 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.

[0020] In the present invention, "culture" means the entire medium including the strain, its metabolites, extra nutrients, etc., obtained by culturing the Bacillus subtilis HKtulip001 strain for a certain period of time in a medium capable of supplying nutrients so that the strain can grow and survive in a test tube, but it is a concept that includes all of the supernatant from which the strain is removed after culturing the strain, and the extracts and fractions thereof. The liquid from the culture medium from which the bacterial cells are removed is also called "supernatant" or "cell-free supernatant", and can be obtained by allowing the culture medium to stand still for a certain period of time and taking only the liquid from the upper layer excluding the part that has settled to the lower layer, removing the bacterial cells through filtration, or centrifuging the culture medium to remove the sediment at the bottom and taking only the liquid at the upper layer. In addition, the culture medium may include a fermentation product. In one example, the culture may be a fermentation product obtained by culturing the strain in a medium. The "fermentation product" refers to the result of enzymatic or metabolic decomposition of organic substances using microorganisms. In the present invention, "fermentation" may refer to any activity or process involving the enzymatic or metabolic decomposition of organic matter using microorganisms, other than a putrefactive reaction. The culture medium may be in the form of a liquid medium or a dried product thereof, such as a freeze-dried product of the culture medium.

[0021] In the present invention, the composition may be a composition comprising Bacillus subtilis HKtulip001 strain existing as a live or dead cell.

[0022] 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.

[0023] 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, its culture, its lysate, or its extract.

[0024]

[0025] 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.

[0026]

[0027] In addition, the present invention provides a use of a cosmetic composition comprising a 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 a Bacillus subtilis HKtulip001 strain, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient to the skin of a subject.

[0028] In the present invention, the term "improvement" means any action that at least reduces the severity of a symptom, for example, a parameter related to alleviation or treatment of a condition.

[0029] In the present invention, “improving skin condition” refers to reducing the degree of symptoms associated with worsening skin condition, and the skin condition may be a parameter related to skin moisturizing, wrinkle improvement, skin barrier improvement, and itching improvement.

[0030] In the present invention, "skin barrier improvement" encompasses both skin barrier improvement and protective functions, and can refer to any action that enhances the function of the skin barrier, which is located at the outermost layer of the skin and prevents moisture and nutrient loss. In an embodiment of the present invention, the skin barrier improvement effect was confirmed by increasing the expression of claudin or filaggrin.

[0031] In the present invention, "skin itching" means an unpleasant sensation that causes a desire to scratch or rub the skin, and examples thereof include itching caused by dry skin; itching caused by hives; itching caused by insect bites; itching caused by heat rash, acne, scabies, frostbite, contact dermatitis, seborrheic dermatitis, or psoriasis; itching caused by various skin diseases; and scalp itching. In the present specification, "improvement in itching" means helping to improve itching symptoms by restoring the function of the skin barrier.

[0032] In the present invention, "skin moisturizing" means increasing moisture in the skin and maintaining a moist state by inhibiting 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).

[0033] Additionally, "wrinkle improvement" refers to preventing, suppressing, or inhibiting the formation of wrinkles on the skin, or alleviating wrinkles that have already formed. In an embodiment of the present invention, the wrinkle improvement effect was confirmed by reducing the activity of elastase.

[0034] 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, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, hand sanitizer, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, suspension, gel, powder, paste, mask pack, and sheet. A composition of such a formulation can be manufactured according to a method conventional in the art.

[0035] In addition to the effective ingredients disclosed herein, the above cosmetic composition may further include functional additives and components included in general cosmetic compositions, and may further include purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, fragrances, or combinations thereof that 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 scatterers, ultraviolet absorbers, colorants, fragrances, and the like. Compounds / compositions that can be used as the above alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, ultraviolet scatterers, ultraviolet absorbers, colorants, fragrances, etc. are already known in the art, so 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.

[0036]

[0037] The present invention also provides a food composition for improving skin condition, comprising Bacillus subtilis HKtulip001 strain, a culture thereof, a pulverized product thereof, or an extract thereof as an effective ingredient.

[0038] In the above food composition, all of the contents described above can be applied as is to the Bacillus subtilis strain and its use.

[0039] In the present invention, the above “improvement in skin condition” may be a parameter related to skin moisturizing, wrinkle improvement, skin barrier improvement, and itching improvement.

[0040] The term "food" in 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.

[0041] For example, the food may be a functional food. As used herein, the term "functional food" refers to a food having a physiological function or activity, and includes biologically active ingredients capable of providing specific health benefits. In this regard, the present invention provides a functional food composition for improving skin condition, comprising Bacillus subtilis HKtulip001, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

[0042] For example, the food may be a health functional food. In the present invention, the term "health functional food" refers to a food manufactured using specific nutrients or raw materials or ingredients with functions beneficial to the human body, i.e., functional raw materials, and is a food that can help human health by maintaining normal functions of the human body or activating physiological functions. In this regard, the present invention provides a health functional food composition for improving skin condition, comprising Bacillus subtilis HKtulip001, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

[0043] When the composition of the present invention is utilized as a food composition, the food composition may take the form of a health functional food, seasoning, beverage, 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 granulated, encapsulated, or powdered for consumption.

[0044] The food composition of the present invention can be manufactured using, in addition to the above-mentioned effective ingredient, a food-related and physiologically acceptable adjuvant, and the adjuvant may include an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a glidant, or a flavoring agent.

[0045] The above food composition may be preferably formulated as a food composition by including at least one food-related acceptable carrier in addition to the above-described effective ingredient for administration.

[0046] For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an orally, non-toxic, food-grade, and inert carrier such as ethanol, glycerol, or water. In addition, suitable binders, lubricants, disintegrants, and coloring agents may also be included in the mixture, if desired or necessary. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as 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.

[0047] 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, and health supplements.

[0048] The food composition of the present invention may include ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates mentioned 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.

[0049]

[0050] The advantages and features of the present invention, and the methods for achieving them, will become clearer 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. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims.

[0051] 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.

[0052] 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.

[0053] Figure 2 shows the results of testing the cytotoxicity of a lyophilized product of a tulip-derived Bacillus subtilis strain culture using an MTT assay on human fibroblasts.

[0054] Figure 3 shows the results of a test for increased expression of the Claudin1 gene according to treatment of a freeze-dried product of a tulip-derived Bacillus subtilis strain culture.

[0055] Figure 4 shows the results of a test for increased expression of the Fliaggrin gene according to treatment of a freeze-dried product of a tulip-derived Bacillus subtilis strain culture.

[0056] Figure 5 shows the results of a test for increased expression of the HAS-2 gene according to treatment of a freeze-dried product of a tulip-derived Bacillus subtilis strain culture.

[0057] Figure 6 shows the results of a test for increased expression of the AQP-3 gene according to treatment of a lyophilized culture solution of a tulip-derived Bacillus subtilis strain.

[0058] Figure 7 shows the results of a test for inhibition of elastase activity according to treatment of a lyophilized product of a tulip-derived Bacillus subtilis strain culture.

[0059] Figure 8 shows the results of measuring the total polyphenol content of a freeze-dried product of a tulip-derived Bacillus subtilis culture.

[0060] Hereinafter, the present invention will be described in detail through examples. The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0061]

[0062] [Example]

[0063] Example 1: Isolation of a Bacillus subtilis strain derived from tulips.

[0064] A 10-fold volume of saline solution was added to the tulip and homogenized. 100 μL of the homogenized sample was plated on Tryptic Soy Agar (TSA), and the plate was incubated in a 35°C incubator for 48 hours. Each colony formed was inoculated into Tryptic Soy Broth (TSB) and incubated with shaking at 35°C and 150 rpm for 24 hours. The culture was streaked onto TSA and incubated at 35°C for 24 hours, and colonies showing off-white colonies were collected. The above procedure was repeated to purely isolate a single strain.

[0065] The purely isolated strains above were analyzed for homology using 16s rRNA analysis and the BLAST program (NCBI Database) by requesting Macrogen Co., Ltd.

[0066] 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 subtilisHKtulip001, and deposited at the National Institute of Biological Resources on February 22, 2023 (Accession No. KCTC15331BP).

[0067] The Bacillus subtilisHKtulip001 strain was analyzed to have the 16S rRNA base sequence of sequence number 1.

[0068]

[0069] Example 2. Preparation of freeze-dried tulip-derived Bacillus subtilis culture solution

[0070] A freeze-dried culture solution of the strain (Bacillus subtilisHKtulip001) isolated in Example 1 was prepared, 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. Afterwards, it was frozen at -80°C for 12 hours or more and freeze-dried to prepare a freeze-dried powder of the culture solution of the tulip-derived Bacillus subtilis strain.

[0071]

[0072] Experimental Example 1. Cytotoxicity Evaluation

[0073] (1) MTT assay for HaCaT cells

[0074] To determine whether a tulip-derived Bacillus subtilis strain is cytotoxic, the following experiment was performed. HaCaT, a keratinocyte cell line, was cultured in DMEM medium containing 10% FBS, and 2X10 4The cells were inoculated at a cell concentration of 10 and cultured in a 37°C, 5% CO2 incubator for 24 hours. After culture, the medium was removed, and the cells were treated with a diluted solution prepared by diluting the lyophilized Bacillus subtilis culture solution from tulip 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 the reaction was performed for 2 hours. After removing the supernatant, 150 μl of dimethyl sulfoxide was added, and the mixture was shaken for 30 minutes to dissolve the produced formazan. 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.

[0075] [Mathematical Formula 1]

[0076] Cell viability (%) = Absorbance of the sample-added group / Absorbance of the control group * 100

[0077]

[0078] ControlHKtulip001 (ppm)51025Cell viability (%)100.00107.34108.35111.05Standard deviation1.654.372.273.61

[0079] As a result of the test, it was confirmed that the lyophilized product of tulip-derived Bacillus subtilis culture did not exhibit cytotoxicity toward HaCaT cells, a keratinocyte cell line, and thus there were no safety issues.

[0080] (2) MTT assay for fibroblasts

[0081] To determine whether the tulip-derived Bacillus subtilis strain exhibited cytotoxicity, human dermal fibroblasts (HDF) were cultured in DMEM / F12 medium containing 10% FBS, and 2X10 4 The cells were inoculated at a cell density of 10 and cultured in a 37℃ and 5% CO2 incubator for 24 hours. After culture, the medium was removed, and the cells were treated with a diluted solution prepared by diluting the lyophilized Bacillus subtilis culture solution from tulip 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 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 μl of dimethyl sulfoxide was added, and the formazan was dissolved by shaking for 30 minutes, 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.

[0082]

[0083] ControlHKtulip001 (ppm)102550100Cell viability (%)100.0096.7295.9698.1896.53Standard deviation1.133.606.614.221.93

[0084] The test results confirmed that the lyophilized tulip-derived Bacillus subtilis culture did not exhibit cytotoxicity toward human fibroblast cells, indicating that there were no safety issues.

[0085]

[0086] Experimental Example 2. Confirmation of the skin barrier improvement efficacy of Bacillus subtilis HKtulip001 strain.

[0087] In order to confirm the skin barrier improvement effect of the tulip-derived Bacillus subtilis strain, the expression of skin barrier-related genes was confirmed according to the treatment of the freeze-dried culture solution of the Bacillus subtilis strain using the following method.

[0088] 2.5 x 10 HaCaT cells 5 The cells were seeded in 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 performed using 2X Real-Time PCR Master mix (Biofact, DQ385-40h) to amplify the genes and then analyzed.

[0089] 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, a structural protein expressed during the differentiation stage of keratinocytes. Specifically, in the group treated with the freeze-dried 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).

[0090]

[0091] ControlHKtulip001 (ppm)51025Relative mRNA expression(% of control)100.00115.21128.72164.08Standard deviation0.620.860.013.42

[0092] ControlHKtulip001 (ppm)51025Relative mRNA expression(% of control)100.00114.91114.71110.93Standard deviation0.080.150.552.91

[0093]

[0094] Experimental Example 3. Confirmation of skin moisturizing effect of Bacillus subtilis HKtulip001 strain. In order to confirm the skin moisturizing effect of the tulip-derived Bacillus subtilis strain, the expression of skin moisturizing-related genes was confirmed according to the treatment of the freeze-dried culture solution of the Bacillus subtilis strain using the following method.

[0095] 2.5 x 10 HaCaT cells 5The cells were seeded in 60 mm culture dishes and cultured for 18 hours. After replacing the cultured HaCaT cells with serum-free DMEM medium, the lyophilized Bacillus subtilis culture solution from tulip 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 analyzed.

[0096] 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% of the expression of HAS-2 compared to the untreated control group (Table 5 and Fig. 5), and a maximum of 155% of the expression of AQP-3 (Table 6 and Fig. 6).

[0097]

[0098] ControlHKtulip001 (ppm)Retinoic acid(ppm)510250.3Relative mRNA expression(% of control)100.84102.37198.23415.20289.33Standard deviation13.0061.712.7795.9853.01

[0099] ControlHKtulip001 (ppm)Retinoic acid(ppm)510250.3Relative mRNA expression(% of control)100.72101.1197.78154.69171.02Standard deviation11.980.3116.833.133.84

[0100]

[0101] Experimental Example 4. Confirmation of the skin wrinkle alleviation effect of Bacillus subtilis HKtulip001 strain.

[0102] 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 using the following method.

[0103] 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 for 5 min at 4°C, and the supernatant was prepared as an enzyme solution containing fibroblast elastase. The enzyme solution was quantified using the Bradford method, and an amount containing 80 μg 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 incubated at 37°C. After 90 min, the absorbance was measured at 405 nm using a spectrophotometer. Elastase inhibitory activity was calculated using the following mathematical formula 2, with the case in which no sample was added serving as a control group.

[0104] [Equation 2]

[0105] Elastase inhibition activity (%) = 100 - (absorbance of the sample-treated group / absorbance of the untreated control group X 100)

[0106]

[0107] As a result, it was confirmed that Bacillus subtilis HKtulip001 reduced elastase activity by up to 58% (Table 7 and Fig. 7).

[0108] ControlHKtulip001 (ppm)Phosphoramidon(μM)10255010010Elastase activity (%)100.0082.7973.1359.0342.2011.37Standard deviation1.302.230.670.230.711.21

[0109]

[0110] Experimental Example 5. Analysis of Polyphenol Content

[0111] The total polyphenol content of the freeze-dried product of the tulip-derived Bacillus subtilis strain culture was measured.

[0112] Total polyphenol content was determined by adding 40 μl of Folin-denis reagent (Sigma, USA) and 400 μl of distilled water to 20 μl of the sample according to the well-known Folin-denis (1912) method, reacting at room temperature for 3 minutes, then adding 400 μl of 20% Na2CO3 solution and reacting 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 prepared in advance using gallic acid standards in the same manner. The total polyphenol content is shown in Table 8 and Fig. 8.

[0113] Sample name Total polyphenol content (%) HKtulip0011.550

[0114]

[0115] In summary of the above experimental results, it was confirmed that the Bacillus subtilis HKtulip001 strain has excellent efficacy in all aspects, including improving the skin barrier, moisturizing, and improving wrinkles, and also has a high total polyphenol content.

[0116]

[0117] [Accession number]

[0118] Name of depositor: Biological Resource Center

[0119] Accession number: KCTC15331BP

[0120] Date of acceptance: 20230222

[0121]

Claims

1. Bacillus subtilis HKtulip001 strain with accession number KCTC15331BP.

2. A cosmetic composition for improving skin condition, comprising as an active ingredient a Bacillus subtilis HKtulip001 strain having the accession number KCTC15331BP, a culture thereof, a pulverized product thereof, or an extract thereof.

3. In paragraph 2. 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.

4. In paragraph 2, The above composition is a cosmetic composition having an effect of increasing the expression of claudin or filaggrin.

5. In paragraph 2, The above composition is a cosmetic composition having an effect of increasing HAS-2 or AQP3 expression.

6. In paragraph 2, The above composition is a cosmetic composition having an elastase activity reducing effect.

7. In paragraph 2, The above composition is a cosmetic composition in the form of a toner (skin lotion), skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, hand sanitizer, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, suspension, gel, powder, paste, mask pack, and sheet.

8. A food composition for improving skin condition, comprising as an active ingredient a Bacillus subtilis HKtulip001 strain having the accession number KCTC15331BP, a culture thereof, a pulverized product thereof, or an extract thereof.

9. In paragraph 8, A food 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.

Images