Skin quality improving agent and topical skin preparation containing said skin quality improving agent

Abstract

To provide skin quality-improving agents, and to provide skin external preparations containing the skin quality-improving agent.SOLUTION: Provided is a skin quality-improving agent containing Lactobacillus rhamnosus as an active ingredient. Preferably, the skin quality-improving agent is used for at least one of the improvement of skin barrier function, the improvement of resistance to bacteria, and the suppression of TEWL. Also preferably, the Lactobacillus rhamnosus is KO3 strain (NITE BP-771).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a skin quality improving agent containing Lactobacillus rhamnosus as an active ingredient, and to an external skin preparation containing the skin quality improving agent. [Background technology]

[0002] The skin is the outermost organ of the body and is constantly regenerating. It is roughly divided into three layers: the epidermis, dermis, and subcutaneous tissue. The epidermis and dermis are connected via a basement membrane. Basal cells, which are in contact with the epidermis side of the basement membrane, are constantly dividing, and these divided cells are pushed upward one after another, undergo differentiation, and form the stratum corneum, the uppermost layer of the epidermis.

[0003] Furthermore, the skin has a skin barrier function that protects the body against external stimuli such as infection, temperature, humidity, chemical attack, and mechanical attack, and plays an important role in maintaining biological functions. A failure of the skin barrier function manifests itself as a deterioration in skin condition, such as dry skin and rough skin. Further deterioration of such skin condition increases the risk of direct damage due to the invasion of bacteria and harmful substances into the body, as well as allergic reactions.

[0004] The stratum corneum, located at the outermost layer of the skin, plays a central role in the skin's barrier function. The stratum corneum not only prevents moisture from evaporating, but also acts as a barrier to prevent the intrusion of various foreign substances such as allergens and bacteria. The stratum corneum is formed by the accumulation of cells differentiated from basal cells, and as it ages, it undergoes a series of processes in which it turns into dandruff and falls off. This series of processes is called turnover and is repeated at regular intervals. The barrier function of the stratum corneum is maintained by the normal functioning of cell metabolism through turnover, the production and transport of cellular metabolites, and enzymatic reactions within the stratum corneum. However, when the barrier function is disrupted by physiological aging, external damage, etc., it declines, causing skin problems such as dry skin and rough skin. Therefore, promoting cell proliferation and cell metabolism is thought to normalize the function of the stratum corneum and lead to improvements in moisture retention and skin barrier function.

[0005] The presence of intercellular spaces is also known to be important for the skin's barrier function. It has been reported that high concentrations of hyaluronic acid (HA), approximately several mg / mL, exist in the intercellular spaces of the epidermis. Notable physical properties of HA include its hydration capacity, viscoelasticity, and the variable mesh structure it can adopt in aqueous solution. This mesh structure, which changes dynamically depending on the concentration or molecular weight of HA, or the surrounding ions or pH, is thought to be involved in maintaining intercellular space and osmotic pressure, as well as in the retention and transport of nutrients and the inhibition of the transport of macromolecular components. It is known that the HA content in the skin decreases with physiological aging, and it has been suggested that this decrease in HA content in the skin may be involved in aging symptoms such as dryness and atrophy of the skin, loss of elasticity, and the formation of fine wrinkles. Therefore, promoting HA production may contribute to preventing skin aging and improving skin condition.

[0006] Additionally, tight junctions (TJs) are also known to play a major role in the skin barrier function. TJs are intercellular adhesion structures that exist in a belt-like shape around cells and continuously connect adjacent cells by adhering them together, thereby controlling the permeation of fluids and substances through the intercellular spaces. When TJ function weakens, intercellular adhesion and permeability control functions weaken. This weakening of intercellular adhesion and permeability control functions also weakens the skin's barrier function and moisture retention, which is thought to be the cause of skin symptoms such as dry skin, rough skin, atopic dermatitis, and various infections. Therefore, promoting the production of TJ components is thought to lead to improved skin barrier and moisture retention functions.

[0007] Furthermore, when the skin barrier function is weakened or destroyed, bacteria and other microorganisms become more susceptible to invasion into the body. It is known that innate immune mechanisms, such as antimicrobial peptides, act to prevent the invasion of bacteria and other microorganisms into the body. Antimicrobial peptides are peptides with antibacterial activity against bacteria and other microorganisms, and are produced when the body is exposed to bacteria or inflammatory cytokines. They function as a biological barrier that prevents the invasion of bacteria and other microorganisms into the body. It has been suggested that low expression levels of antimicrobial peptides may result in insufficient antibacterial activity against bacteria, etc., and may lead to the onset or worsening of atopic dermatitis. Therefore, increasing the expression levels of antimicrobial peptides to an appropriate level is thought to lead to improved resistance to bacteria, etc., and suppression of atopic dermatitis, etc.

[0008] In addition, a decline in skin barrier function is known to increase transepidermal water loss (TEWL). Water constantly evaporates from the skin surface. This water evaporation, formerly known as insensible evaporation, is now commonly referred to as TEWL. This water evaporation refers to water that evaporates through the stratum corneum and is distinct from water evaporation from sweat glands in the skin. A decline in skin barrier function and an increase in TEWL are known to cause dry skin and itching. This dryness and itching are thought to occur as a result of various stimuli, such as ultraviolet rays, affecting the internal skin environment and inhibiting skin barrier function. Furthermore, in pathological skin conditions such as atopic dermatitis, the stratum corneum's function is reduced and TEWL is elevated. In skin care, keeping the skin moisturized is a very important factor, and the barrier function against water evaporation is considered to be the most important factor. TEWL is measured at a temperature of approximately 20°C and a humidity of approximately 50%, at which sweat glands are thought to be inactive, and is widely used as an index of skin barrier function.

[0009] As described above, it has been revealed that a decrease in skin barrier function has various adverse effects on the living body. Therefore, much research has been conducted to improve skin barrier function or skin quality. However, research on materials that improve skin barrier function or skin quality is insufficient, and the development of new materials is still desired.

[0010] On the other hand, lactic acid bacteria are well-known among consumers and are known to have beneficial physiological activities such as intestinal regulation and immunostimulation. Lactic acid bacteria with such beneficial physiological activities have traditionally been used in foods, cosmetics, and other applications. For example, oral ingestion of Lactobacillus plantarum and Lactobacillus brevis cells has been reported to inhibit TEWL, improve skin barrier function, increase stratum corneum moisture content, reduce skin sebum production, and improve skin elasticity (Patent Document 1). Oral ingestion of fig fermentation products of Lactobacillus pentosus has been reported to improve skin barrier function, inhibit TEWL, and increase stratum corneum moisture content (Patent Document 2). Furthermore, oral ingestion of Lactobacillus brevis cells and processed cells has been reported to inhibit TEWL (Patent Document 3).

[0011] As mentioned above, it has been reported that oral ingestion of Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus pentosus cells or processed cells has beneficial effects such as improving skin barrier function. However, the mechanisms that occur within cells when these beneficial effects are achieved, particularly the changes in specific gene expression related to improving skin barrier function, have not been elucidated. Furthermore, the methods of use of the inventions described in Patent Documents 1 to 3 involve oral ingestion, and there is no mention of the effect of applying them to the skin to improve skin barrier function.

[0012] Among Lactobacillus lactic acid bacteria, Lactobacillus rhamnosus KO3 strain (NITE BP-771) (hereinafter referred to as KO3 strain) is a type of lactic acid bacterium isolated from human saliva. The KO3 strain has been reported to have a broad antibacterial spectrum against bacteria that cause oral diseases, such as cariogenic bacteria and periodontal disease, and is used in oral care products and foods (Patent Documents 4 to 6). Furthermore, the KO3 strain has also been used as a topical skin preparation, taking advantage of its antibacterial properties (Patent Documents 7 and 8).

[0013] To date, the KO3 strain has been primarily studied for its antibacterial properties against oral and skin bacteria. However, it was unknown whether the KO3 strain, or indeed Lactobacillus rhamnosus, could improve skin barrier function. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Patent Publication No. 2008-179601 [Patent Document 2] Patent Publication No. 2019-11316 [Patent Document 3] Patent No. 6067292 [Patent Document 4] Patent No. 5645192 [Patent Document 5] Patent Publication No. 2018-100228 [Patent Document 6] Patent Publication No. 2019-11268 [Patent Document 7] Patent No. 6746091 [Patent Document 8] Patent Publication No. 2018-2661 Summary of the Invention [Problem to be solved by the invention]

[0015] In light of the above circumstances, the present inventors have conducted extensive research and found that Lactobacillus rhamnosus bacteria, particularly the KO3 strain, are effective in improving skin barrier function, enhancing resistance to bacteria, and suppressing TEWL. The present inventors have completed a skin quality improving agent containing Lactobacillus rhamnosus as an active ingredient and a topical skin preparation containing the skin quality improving agent. Specifically, the present invention aims to provide a skin quality improving agent containing Lactobacillus rhamnosus as an active ingredient and a topical skin preparation containing the skin quality improving agent. [Means for solving the problem]

[0016] That is, the gist of the present invention is as follows. (1) Lactobacillus rhamnosus KO3 strain (NITE BP-771) The active ingredient is the fungus or an extract of the fungus. and promotes the expression of at least one of HBEGF, HAS3, CLDN1, or DEFB2 genes. Skin quality improver. (2) It is used for the purpose of improving skin barrier function, increasing resistance to bacteria, or suppressing TEWL. (1) The skin quality improving agent described in (3) (1) or (2) A topical skin preparation containing the skin quality improving agent described above. (4) The amount of the skin quality improving agent is 0.1 to 10.0% by mass. (3) The topical skin preparation described above. [Effects of the Invention]

[0017] According to an embodiment of the present invention, it is possible to provide a skin quality improving agent containing Lactobacillus rhamnosus cells as an active ingredient, and an external skin preparation containing the skin quality improving agent. [Brief explanation of the drawings]

[0018] [Figure 1] This figure shows the expression level of a gene (HBEGF) when a 1% sterilized powder (sterilized powder) and a 0.2% by mass aqueous solution of hydroxypropyl cellulose (control) were added to a three-dimensional cultured human epidermal model (RHE). The vertical axis shows the relative expression level of the sterilized powder when the gene expression level of the control is set to 1. [Figure 2] FIG. 2 shows the expression level of a gene (HAS3) when a test was carried out in the same manner as in FIG. [Figure 3] FIG. 2 shows the expression level of a gene (CLDN1) when a test was carried out in the same manner as in FIG. [Figure 4] FIG. 2 shows the expression level of a gene (DEFB2) when a test was carried out in the same manner as in FIG. [Figure 5] A cream containing antiseptic powder (antiseptic powder) or a placebo cream (placebo) was applied, and the ratio (%) of the TEWL measurement value after 2 weeks (after 2 weeks) to the TEWL measurement value at the start of the test (initial value) was calculated (initial value ratio (%)), and this is the graph showing the average of the initial value ratios (%) of 12 people. DETAILED DESCRIPTION OF THE INVENTION

[0019] An embodiment of the present invention will be described below.

[0020] Skin quality improver The skin quality improving agent of the present invention contains, as an active ingredient, the cells or extract of cells of lactic acid bacteria belonging to Lactobacillus rhamnosus. A specific example of Lactobacillus rhamnosus is the Lactobacillus rhamnosus KO3 strain, deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE BP-771 (hereinafter simply referred to as the KO3 strain).

[0021] The KO3 strain was first isolated by the present inventors from human saliva and is a human-derived lactic acid bacterium. The 16S rRNA sequence of the KO3 strain showed 100% homology with that of Lactobacillus rhamnosus strain IDCC3201 (1485 / 1485). Its microscopic appearance after Gram staining revealed a Gram-positive bacillus. Therefore, it was identified as Lactobacillus rhamnosus. The main bacteriological properties of the KO3 strain are listed below. (1) Gram-positive lactobacillus, (2) homolactic acid fermentation, (3) catalase-negative, (4) non-spore-forming, (5) can be cultivated under aerobic conditions, (6) produces exopolysaccharides.

[0022] In the present invention, the lactic acid bacteria cells are cultured according to a conventional method for culturing lactic acid bacteria, and the cells separated from the resulting culture by a collection means such as centrifugation can be used as is. In addition, a concentrated solution of the cells, or an extract of the cells, such as cytoplasm or cell wall fraction, obtained by treating the cells with enzymes or physical means can also be used. Not only live cells but also killed cells may be used. However, when incorporating the cells into a skin topical preparation to be formulated, killed cells are preferred from the standpoints of quality control and safety.

[0023] Various media can be used to culture the lactic acid bacteria of the present invention, such as fruit juice media, vegetable juice media, milk media, skim milk powder media, media containing milk components, or semi-synthetic media not containing milk components. Examples of such media include reduced skim milk media prepared by reducing and heat-sterilizing skim milk, skim milk powder media supplemented with yeast extract, MRS medium, and GAM medium. The culture method is not particularly limited as long as the conditions are such that the bacteria grow well, and may be static culture, neutral culture at a constant pH, batch culture, or continuous culture.

[0024] The term "bacterial cells" as used herein refers not only to lactic acid bacteria cells themselves but also to crushed cells.

[0025] The extract of the fungus cells in the present invention means various solvent extracts obtained by solvent extraction of the fungus cells, dilutions thereof, concentrates thereof or dried powders thereof.

[0026] The extraction solvent used to obtain the extract of the present invention can be either a polar solvent or a non-polar solvent, or a mixture of these. Examples include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as hexane, cyclohexane, and petroleum ether; aromatic hydrocarbons such as benzene and toluene; and pyridines. Of these, alcohols such as ethanol are preferred.

[0027] The extraction conditions vary depending on the solvent used. For example, it is preferable to use 1 to 10 parts by mass of solvent per 1 part by mass of culture solution containing the bacterial cells, and extract at a temperature of 0 to 50°C, preferably 25 to 37°C, for 0.5 to 3 hours.

[0028] The extract can be used as it is, or can be diluted, concentrated, or freeze-dried, and then prepared into a powder or paste as needed. It can also be used after appropriate purification by techniques such as liquid-liquid distribution.

[0029] The bacterial cells or bacterial cell extract of lactic acid bacteria belonging to Lactobacillus rhamnosus in the present invention can be used as a skin quality improving agent. The skin quality improving agent in the present invention refers to an agent that has the effect of improving skin function. Although Lactobacillus rhamnosus is a well-known bacterial species, there have been no reports to date that its application to the skin improves skin function, and this effect was first discovered by the present inventors.

[0030] In the present invention, improving skin function refers to at least one of improving skin barrier function, increasing resistance to bacteria, and suppressing TEWL. The skin quality improving agent of the present invention can also be used as an agent for improving skin barrier function, an agent for improving resistance to bacteria, and an agent for suppressing TEWL, respectively, for the purpose of improving these skin functions. Here, the skin barrier function refers to the function possessed by the skin tissue near the surface, which prevents foreign substances from entering the tissue from the outside.

[0031] Specifically, improvement of skin barrier function refers to at least one of the promotion of cell proliferation, promotion of HA production, and strengthening of TJs.

[0032] Promoting cell proliferation refers to encouraging cell proliferation through cell division and may also include promoting cell turnover. Specifically, it refers to increasing the expression of heparin-binding EGF-like growth factor, a protein involved in promoting cell proliferation, and further refers to increasing the expression of the gene encoding heparin-binding EGF-like growth factor. The gene encoding heparin-binding EGF-like growth factor is called heparin-binding EGF-like growth factor and its symbol is HBEGF. Here, "increasing the expression of the gene encoding heparin-binding EGF-like growth factor" means that the amount of mRNA present in cells transcribed from DNA encoding heparin-binding EGF-like growth factor is significantly increased compared to the amount present in the absence of Lactobacillus rhamnosus. By applying the skin quality improving agent of the present invention to the skin, HBEGF expression is promoted at the administration site, and diseases or conditions that can be improved by HBEGF expression at that site can be improved, treated, or prevented. More specifically, by applying the skin quality improving agent of the present invention to the skin, turnover can be improved, and in connection with this, moisture retention function and skin barrier function can be improved.

[0033] Promoting HA production refers to increasing the amount of HA produced. Specifically, it refers to increasing the amount of hyaluronan synthase 3, the enzyme that synthesizes HA, and further refers to increasing the expression level of the gene encoding hyaluronan synthase 3. The gene name for the gene encoding hyaluronan synthase 3 is hyaluronan synthase 3, and its gene symbol is HAS3. Here, "increasing the expression level of the gene encoding hyaluronan synthase 3" means that the amount of mRNA transcribed from DNA encoding hyaluronan synthase 3 in cells is significantly increased compared to the amount present in the absence of Lactobacillus rhamnosus. Applying the skin quality improving agent of the present invention to the skin promotes HAS3 expression at the application site, thereby improving, treating, or preventing diseases or conditions at that site that can be improved by HAS3 expression. More specifically, applying the skin quality improving agent of the present invention to the skin promotes the production of HA, which in turn helps maintain intercellular space and osmotic pressure, as well as facilitates the retention and movement of nutrients, etc., and is thought to ultimately lead to improvements in rough skin, fine wrinkles, etc.

[0034] Strengthening TJs refers to enhancing the formation of TJ strands. Specifically, it refers to increasing the expression level of claudin 1, a protein responsible for strand formation, and further refers to increasing the expression level of the gene encoding claudin 1. The gene encoding claudin 1 is called claudin 1 and its symbol is CLDN1. Here, "increasing the expression level of the gene encoding claudin 1" means that the amount of mRNA transcribed from DNA encoding claudin 1 in cells is significantly increased compared to the amount present in the absence of Lactobacillus rhamnosus. Applying the skin quality improving agent of the present invention to the skin promotes the expression of TJs at the application site, thereby improving, treating, or preventing diseases or conditions at the site that can be improved by TJ expression. More specifically, applying the skin quality improving agent of the present invention to the skin can promote the production of TJs, which in turn strengthens the formation of TJ strands and thereby improves the skin's barrier function and moisture retention function.

[0035] Improving resistance to bacteria refers to improving antibacterial activity against bacteria. Specifically, it refers to increasing the expression level of an antibacterial peptide, which is a peptide that exhibits antibacterial activity against bacteria, and further refers to increasing the expression level of a gene encoding the antibacterial peptide. The gene encoding the antibacterial peptide is called defensin beta 2 and its symbol is DEFB2. Here, "increasing the expression level of a gene encoding an antibacterial peptide" means that the amount of mRNA transcribed from DNA encoding the antibacterial peptide in cells is significantly increased compared to the amount present in the absence of Lactobacillus rhamnosus. Applying the skin quality improving agent of the present invention to the skin promotes DEFB2 expression at the application site, thereby improving, treating, or preventing diseases or conditions that can be improved by DEFB2 expression at that site. More specifically, applying the skin quality improving agent of the present invention to the skin can strengthen resistance to bacteria, and related effects such as suppression of atopic dermatitis can be expected.

[0036] Specifically, suppressing TEWL means suppressing the amount of transepidermal water loss. TEWL is used as an indicator of skin barrier function. By suppressing TEWL, it is expected that the skin barrier function will be strengthened.

[0037] Other benefits include improving skin function, improving skin texture, making makeup easier to apply, and suppressing breakouts. It also improves skin firmness, prevents skin sagging, and tightens pores (especially on the face).

[0038] Furthermore, the skin quality improving agent of the present invention not only has the effect of improving skin function when it is impaired and returning it to a normal state, but also has the effect of improving skin function from a normal state to an even better state. That is, the skin quality improving agent of the present invention can be used not only as a skin function repairing agent but also as a skin function enhancing agent. Furthermore, the skin quality improving agent of the present invention can also have the effect of preventing skin function decline. That is, by applying the agent in advance when skin function has not declined but there is a possibility of decline, decline in skin function can be suppressed.

[0039] "External skin preparations" The external skin preparation in the present invention refers to a formulation prepared by blending a skin quality improving agent with, for example, a drug, a quasi-drug, a cosmetic, or the like.

[0040] The dosage form of the external skin preparation is not particularly limited as long as it does not impair the effect of the skin quality improving agent, and any dosage form can be used. The type of dosage form is not particularly limited, but examples thereof include semi-solids, liquids, and solids such as ointments, gels, creams, packs, patches, emulsions, lotions, skin lotions, and powders.

[0041] The topical skin preparation of the present invention is not particularly limited as long as it does not impair the effects of the skin quality improving agent, and additives commonly used in the fields of pharmaceuticals, quasi-drugs, cosmetics, etc. can be appropriately blended. The additives are not particularly limited, and examples thereof include purified water, oily components, surfactants, emulsifiers, lower alcohols, higher alcohols, polyhydric alcohols, water-soluble polymers, viscosity modifiers, gelling agents, moisturizers, disinfectants, anti-inflammatory agents, analgesics, antifungal agents, stratum corneum softening and peeling agents, skin colorants, hormones, ultraviolet absorbers, sweat deodorants, vitamins, blood flow promoters (vasodilators, blood circulation promoters), herbal medicines, pH adjusters, sequestering agents, pearlizing agents, natural fragrances, synthetic fragrances, dyes, pigments, antioxidants, preservatives, and perfume oils.

[0042] The amount of skin quality improving agent to be incorporated into the topical skin preparation of the present invention is not particularly limited, but the amount of skin quality improving agent incorporated is preferably 0.1 to 10.0% by mass, more preferably 0.5 to 5.0% by mass, and even more preferably 1.0% by mass, relative to the total mass of the topical skin preparation. If the amount of skin quality improving agent incorporated exceeds 10.0% by mass, problems such as aggregation are likely to occur, which is undesirable. On the other hand, if the amount is less than 0.1% by mass, the effect of the skin quality improving agent cannot be fully exerted, which also makes the topical skin preparation undesirable.

[0043] By incorporating the skin quality improving agent according to the present invention into an external skin preparation, it is possible to achieve the effect of making the skin quality improving agent easier for the user to handle. [Example]

[0044] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to these examples.

[0045] "Evaluation of gene expression by Lactobacillus rhamnosus" Gene expression evaluation by Lactobacillus rhamnosus was performed as follows.

[0046] 1. Method 1-1 Ingredients As a test sample, Lactobacillus rhamnosus KO3 strain (NITE BP-771) was used for gene expression evaluation. The sterilized lactic acid bacteria powder was prepared by culturing the above-mentioned KO3 strain in a medium containing rice protein, then adding an excipient to the heat-sterilized bacterial cells, drying them, and pulverizing them (product name: L8020 sterilized powder, manufactured by Yaegaki Fermentation Engineering Co., Ltd.).

[0047] 1-2. Test sample A 0.2% by mass aqueous solution of hydroxypropyl cellulose was prepared by diluting hydroxypropyl cellulose with distilled water to a 0.2% by mass aqueous solution, and this aqueous solution was used as a control. The 1% sterilized powder was prepared by dissolving and dispersing the sterilized lactic acid bacteria powder in a 0.2% by mass aqueous solution of hydroxypropyl cellulose to a concentration of 1% by mass.

[0048] 1-3. Cultivation of the 3D cultured human epidermal model (RHE) and sample addition RHE was prepared using a LabCyte EPI-MODEL 24 manufactured by Japan Tissue Engineering Co., Ltd. Testing was performed according to the manufacturer's instructions. RHE obtained from the manufacturer were transferred to culture medium and cultured for 24 hours at 37°C and 5% CO2. Next, 50 μL of each sample was added to the stratum corneum side of the RHE and cultured for 1 hour at 37°C and 5% CO2. After that, each sample on the stratum corneum was removed and cultured for an additional 5 hours before use in subsequent analysis.

[0049] 1-4. Quantitative RT-PCR RHE tissue was transferred from the cell culture insert to Buffer RLT Plus (Qiagen) supplemented with mercaptoethanol. The RHE tissue was pulverized, and the supernatant was purified with a phenol-chloroform mixture. Total RNA was then purified using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific). Quantitative PCR was performed using TB Green Premix EX Taq II (Tli RNaseH Plus) (Takara Bio). GAPDH was used as an endogenous control in quantitative PCR analysis. The relative expression levels of genes compared to the control were calculated using the △△Ct method. Tests were performed three times, and results are shown as the mean ± standard deviation of triplicates. Significance of differences compared to the control was tested using Student's t-test (α = 0.05).

[0050] 2.Results Figures 1 to 4 show the relative expression levels of specific genes compared to the control 6 hours after adding 1% sterilized powder to RHE. Significant increases were observed in the relative expression levels of HBEGF, HAS3, CLDN1, and DEFB2. Although not shown in the figures, no significant changes were observed in the relative expression levels of the other genes evaluated.

[0051] "Evaluation test of continuous use of cream containing antiseptic powder" The continuous use evaluation test of the cream containing the sterilized powder was carried out in the following manner.

[0052] 1. Method 1-1. Test sample The sterilized powder-containing cream was prepared by blending the above sterilized powder into the cream. The placebo cream was a cream with the same formulation as the antiseptic powder-containing cream, except that the antiseptic powder was replaced with water.

[0053] 1-2. Test method The study was conducted on 12 people (8 women and 4 men) in their 20s to 50s. The study lasted for two weeks. After washing their faces twice a day, participants applied a cream containing antiseptic powder to one half of their face, and a placebo cream to the other half. Before and after the two-week treatment, the subjects washed their faces with face wash and allowed to acclimate to the measurement environment (room temperature 20°C ± 1°C, humidity 50% ± 5%) for 20 minutes before measuring TEWL. The measuring device used was AquaFlux (manufactured by Biox).

[0054] 2.Results The average test results of 12 subjects are shown in Figure 5. The TEWL of the face on the side where the antiseptic powder-containing cream was used for two consecutive weeks was significantly reduced compared to the TEWL measured before the start of the two-week continuous use. On the other hand, the TEWL of the face on the side where the placebo cream was used for two consecutive weeks was not significantly different from the TEWL measured before the start of the two-week continuous use. This demonstrates that the use of an external skin preparation containing the skin quality improving agent of the present invention improves skin barrier function.

[0055] Examples of formulations in which the skin quality improving agent of the present invention is applied to external skin preparations are given below. Each formulation example is prepared by a conventional method. <Formulation Example 1> Emulsion (Component name) (mass%) Skin quality improver 0.1 Squalane 5.0 Jojoba oil 0.3 Self-emulsifying glyceryl monostearate 1.0 Stearic acid 0.1 Carboxyvinyl polymer 0.1 Glycerin 1.5 Ethanol 7.0 pH adjuster (appropriate amount) Preservatives (appropriate amount) Purified water remainder

[0056] <Formulation example 2> Cosmetic cream (Component name) (mass%) Skin Quality Improver 1.0 Shea Butter 2.0 Jojoba oil 1.0 Stearyl Alcohol 5.0 Squalane 10.0 Self-emulsifying glyceryl monostearate 3.0 Polyoxyethylene cetyl ether (20E.0) 1.0 1,3 Butylene Glycol 5.0 Glycerin 5.0 pH adjuster (appropriate amount) Preservatives (appropriate amount) Purified water remainder

[0057] <Formulation example 3> Lotion (Component name) (mass%) Skin Quality Improver 5.0 Glycerin 5.0 1,3 Butylene Glycol 5.0 Trimethylglycine 3.0 Polyoxyethylene hydrogenated castor oil (60E.0) 0.5 Ethanol 2.0 pH adjuster (appropriate amount) Preservatives (appropriate amount) Purified water remainder

[0058] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their omissions, substitutions, modifications, etc. are included within the scope and spirit of the invention, as well as within the scope of the invention described in the claims and their equivalents.

Claims

1. A skin quality improving agent containing, as an active ingredient, cells or an extract of cells of Lactobacillus rhamnosus KO3 strain (NITE BP-771), which promotes the expression of at least one gene selected from HBEGF, HAS3, CLDN1, and DEFB2.

2. The skin quality improving agent according to claim 1, which is used for the purpose of improving at least one of the skin barrier function, increasing resistance to bacteria, and suppressing TEWL.

3. A topical skin preparation containing the skin quality improving agent described in claim 1 or 2.

4. The external skin preparation according to claim 3, wherein the blending amount of the skin quality improving agent is 0.1 to 10.0% by mass.

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