Bacterium, gene expression enhancer, and formulation

Abstract

To provide a formulation including components contributing to epidermal and dermal health.SOLUTION: The present invention provides a bacterium belonging to Staphylococcus epidermidis species, strain BG-S174, deposited as accession number NITE P-04075, which enhances expression of the ceramide synthase 3 gene and also enhances expression of the hyaluronan synthase 3 gene. A formulation prepared using this strain can enhance expression of the ceramide synthase 3 gene and also enhance expression of the hyaluronan synthase 3 gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a bacterium belonging to the species Staphylococcus epidermidis, a gene expression enhancer that uses the bacterium to promote ceramide synthesis and hyaluronic acid synthesis, and a preparation such as a topical skin preparation that uses the bacterium. [Background technology]

[0002] The skin has a sensory function as a sensory organ, a thermoregulatory function, and a barrier or protective function that prevents moisture from evaporating from the body while also preventing external stimuli and the intrusion of foreign substances. The skin, which performs these functions, is composed of three layers: the epidermis, dermis, and subcutaneous tissue. The epidermis, the outermost layer of the skin, has a major impact on the skin's barrier function, the dermis, which is located in the middle, is a thick layer that gives the skin flexibility and durability, and the subcutaneous tissue, the innermost layer, plays a role in absorbing external shocks and storing energy.

[0003] If the properties of skin, which has various functions, deteriorate, not only will the skin's beauty be compromised, but it may also cause various other problems, such as skin diseases and mental disorders. Therefore, various techniques have been proposed to maintain healthy skin.

[0004] For example, Japanese Patent Application Laid-Open No. 2022-077501 (Patent Document 1) describes an invention relating to a topical skin composition containing a component that promotes ceramide synthesis. Japanese Patent Application Laid-Open No. 2016-169238 (Patent Document 2) describes an invention relating to a substance that inhibits fine wrinkle formation in the skin. Japanese Patent Application Laid-Open No. 2021-050179 (Patent Document 3) describes an invention relating to a hyaluronic acid production promoter containing acetylspermine or the like as an active ingredient. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-077501 [Patent Document 2] JP 2016-169238 A [Patent Document 3] Patent Publication No. 2021-050179 Summary of the Invention [Problem to be solved by the invention]

[0006] In addition to the above technologies, various studies have been conducted to develop substances that can be used as ingredients in topical skin preparations to maintain good skin condition. However, it is time-consuming and expensive to identify the causes of adverse effects on the skin and to consider ways to eliminate those causes, and it is also a difficult task to ensure the safety of the developed substances.

[0007] Meanwhile, the present inventors have attempted to develop ingredients that contribute to the health of the epidermis and dermis, taking into consideration the fact that dry skin shows a decrease in ceramide, an intercellular lipid present in the stratum corneum (horny layer) of the epidermis that has a significant impact on the barrier function, and that the dermis, which contains nerve fibers related to sensory functions as sensors and has a significant impact on skin elasticity, contains a large amount of hyaluronic acid.The present invention was born as a result of such research. [Means for solving the problem]

[0008] A first aspect of the present disclosure relates to Staphylococcus epidermidis ( ), deposited under Accession Number: NITE P-04075, which increases ceramide synthase 3 gene expression and increases hyaluronan synthase 3 gene expression. Staphylococcus epidermidis The bacterium is strain BG-S174, which belongs to the Bacillus species.

[0009] A first aspect of the present disclosure relates to Staphylococcus epidermidis ( ), deposited under Accession Number: NITE P-04075, which increases ceramide synthase 3 gene expression and increases hyaluronan synthase 3 gene expression. Staphylococcus epidermidis The bacteria used was the BG-S174 strain belonging to the Staphylococcus epidermidis species, which can be obtained from human skin. This shows that bacteria belonging to the species Staphylococcus epidermidis can increase the expression of the ceramide synthase 3 gene and the hyaluronan synthase 3 gene.

[0010] A second aspect of the present disclosure is a gene expression enhancer that increases ceramide synthase 3 gene expression and increases hyaluronic acid synthase 3 gene expression, the gene expression enhancing agent comprising the culture supernatant of the above-mentioned bacterium as a main component.

[0011] The second aspect of the present disclosure is a gene expression enhancer that contains the culture supernatant of the above-mentioned bacteria as its main component and increases the expression of the ceramide synthase 3 gene and increases the expression of the hyaluronic acid synthase 3 gene, thereby increasing the expression of the ceramide synthase 3 gene and increasing the expression of the hyaluronic acid synthase 3 gene.

[0012] A third aspect of the present disclosure is a gene expression enhancer that contains the bacterium as an active ingredient and that enhances ceramide synthase 3 gene expression and thereby enhances hyaluronic acid synthase 3 gene expression.

[0013] The third aspect of the present disclosure is a gene expression enhancer that contains the bacterium as an active ingredient and increases the expression of the ceramide synthase 3 gene and increases the expression of the hyaluronic acid synthase 3 gene, thereby increasing the expression of the ceramide synthase 3 gene and increasing the expression of the hyaluronic acid synthase 3 gene.

[0014] A fourth aspect of the present disclosure is a preparation containing the bacterium or the gene expression enhancer as an active ingredient. In the fourth aspect of the present disclosure, the bacterium or the gene expression enhancer is used as a preparation to enhance the expression of the ceramide synthase 3 gene and the hyaluronic acid synthase 3 gene, which can be used in, for example, pharmaceutical preparations, cosmetics, etc.

[0015] A fifth aspect of the present disclosure is the preparation, which is an external preparation for skin. In the fifth aspect of the present disclosure, the formulation is an external preparation for the skin, and therefore can be used as basic cosmetics, cream products such as skin care products, and cosmetic liquids that maintain and improve moisture retention and barrier function of the epidermis.

[0016] The preparation according to any of the above embodiments can take various forms such as liquid, solid, powder, jelly, gel, etc. [Effects of the Invention]

[0017] According to one embodiment of the present disclosure, the expression of the ceramide synthase 3 gene can be increased, and the expression of the hyaluronic acid synthase 3 gene can be increased. DETAILED DESCRIPTION OF THE INVENTION

[0018] One aspect of the present disclosure will be described based on an exemplary embodiment. The following embodiment does not unduly limit the content of the present invention described in the claims. Furthermore, not all of the configurations described in this embodiment are necessarily essential as a solution to the present invention.

[0019] All embodiments and optional embodiments included in this disclosure may be combined with each other to form new embodiments, and all technical features and optional technical features included in this disclosure may be combined with each other to form new technical features.

[0020] The term "or" used in this disclosure is used as an inclusive term. For example, "A or B" means "A, B, or both A and B." "A," "B," and "both A and B" all respectively satisfy "A or B."

[0021] In this specification and claims, when "first," "second," and "nth (where n is a natural number)" are used to distinguish between different elements, they are not intended to indicate a particular order, superiority, or inferiority. Configurations that are common to each embodiment are assigned the same reference numerals and redundant explanations will be omitted.

[0022] The directions or positional relationships indicated by terms such as "upper," "lower," "front," "rear," "left," and "right" and terms including these terms used in this specification and claims are based on the drawings and are merely for the purpose of conveniently and simply describing the embodiments. Therefore, unless expressly defined or limited, it is not intended to expressly or imply that a particular element or its use is configured in a particular direction, and it should be understood that it does not limit the scope of the claims and the embodiments.

[0023] Numerical values ​​or elements modified by the terms "about," "approximately," "nearly," or "substantially" as used in this specification and claims are understood to include the numerical value and any surrounding numerical values, and to include the element and anything that can be considered the same as the element. For example, when describing "about 3," "3" and any subsequent numerical values ​​can be included in "about 3" as long as the technical features and technical significance claimed in this disclosure are not different. Furthermore, when describing "B that is substantially identical to A," B can be completely identical to A, or even if there are differences, they can be included in the scope of "substantially" as long as the technical features and technical significance claimed in this disclosure are the same.

[0024] Unless otherwise specified, the symbol "~" used in this specification and claims is understood to include the lower limit and upper limit, and also to include any value between the lower limit and upper limit. For example, "3 to 5" means "3 to 5," and "2.5 to 6 g" means "2.5 g to 6 g."

[0025] The technology related to the bacteria that has the effect of promoting ceramide synthesis and hyaluronic acid synthesis will be explained in detail. More specifically, the technology is the bacteria that promotes ceramide synthesis and hyaluronic acid synthesis, the supernatant obtained from this bacteria, the mixture that contains this bacteria, the ceramide and hyaluronic acid synthesis promoter that uses these, and the preparation of the skin external preparation that uses these.

[0026] In a preferred embodiment, the bacterium that promotes ceramide synthesis and hyaluronic acid synthesis is Staphylococcus epidermidis ( Staphylococcus epidermidis ), with strain BG-S174 being particularly preferred. This strain BG-S174 has been deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under accession number NITE P-04075 and date of deposit: February 2, 2024. The 16S rRNA base sequence of this strain is shown in the sequence listing as SEQ ID NO: 1.

[0027] Strain BG-S174 was identified by a homology search of its 16S rRNA gene sequence against sequences in a database, and was found to belong to the species Staphylococcus epidermidis, with a homology of over 98.52%. The mycological characteristics of strain BG-S174 are the same as those of Staphylococcus epidermidis.

[0028] The cells of the BG-S174 strain can be cultured by the method described below, and the cells and supernatant separated and purified from the culture by means of centrifugation or the like can be used. Furthermore, the BG-S174 strain cells or a supernatant prepared from the cells can be appropriately mixed with excipients, disintegrants, binders, stabilizers, humectants, etc. to prepare a formulation containing BG-S174 (bacteria) as an active ingredient or a culture supernatant of BG-S174 (bacteria) as a main ingredient. This formulation can be made into a skin topical agent or the like in the form of tablets, granules, powders, capsules, solutions, emulsions, creams, etc.

[0029] Furthermore, those containing the BG-S174 strain (bacteria) as an active ingredient, or those containing the culture supernatant of BG-S174 (bacteria) as a main ingredient, can be used as a gene expression enhancer that increases ceramide synthase 3 gene expression and increases hyaluronic acid synthase 3 gene expression, or as a ceramide synthesis promoter or hyaluronic acid synthesis promoter, and preparations containing this gene expression enhancer, ceramide synthesis promoter, or hyaluronic acid synthesis promoter as an active ingredient can be used to enhance ceramide synthesis and hyaluronic acid synthesis, and to maintain and improve epidermal moisture retention and barrier function. [Example]

[0030] Experiment 1: <Acquisition of bacteria> 90 mL of distilled water was mixed with 0.45 g of KH2PO4, 0.6 g of Na2HPO4, 0.1 g of agar, 50 μL of Tween 80 (Biomedical Sciences), and 50 μL of glycerol, then diluted to 100 mL with distilled water. The solution was autoclaved at 121°C for 15 minutes to prepare an AG solution. Bacteria collected from human faces using a sterile cotton swab were suspended in this solution. This solution was serially diluted 1x, 10x, 100x, and 1000x with PBS(-), plated on TS agar plates, and incubated at 37°C for 48 hours. Several colonies representing Staphylococcus epidermidis were selected from the surface of the plates, dissolved in 50 μL of dH2O, and used as a template for real-time PCR detection of Staphylococcus epidermidis. The primer sequences used for detection of Staphylococcus epidermidis were sense 5'-TCAGCAGTTGAAGGGACAGAT-3' and antisense 5'-CCAGAACAATGAATGGTTAAGG-3', and real-time PCR was performed using KOD SYBR qPCR Mix (TOYOBO).

[0031] Colonies identified as Staphylococcus epidermidis were dissolved in PBS and tested for drug resistance. Specifically, the colony suspension was plated on Mueller-Hinton agar, and a BD Sensi-Disc Cefoxitin 30 (Becton Dickinson Japan) plated on the center and incubated at 35°C for 24 hours. Staphylococcus epidermidis with an inhibition zone of 2.1 cm or greater was considered non-resistant. Staphylococcus epidermidis strains not identified as drug-resistant were plated on TS agar (BD) and incubated at 37°C for 48 hours. Formed colonies (1 mm in diameter) were picked and incubated in TS liquid medium (BD) at 37°C for 24 hours, then stored in 10% glycerol at -80°C.

[0032] DNA was extracted from the stocked bacteria in this way and subjected to sequence analysis. More specifically, colonies formed from each stocked strain were scraped with a pipette tip and suspended in 50 μL of dH2O. 50 μL of 100 mM NaOH was added, mixed, and then treated at 95°C for 15 minutes. 11 μL of 1 M Tris-HCl (pH 7.0) was then added, and the mixture was centrifuged. The supernatant was collected and used as the DNA solution. The resulting DNA solution was used as a template to obtain PCR amplification products for sequence analysis using a Bacterial 16S rDNA PCR Kit (Takara Bio Inc.) according to the manufacturer's protocol.

[0033] A portion of the PCR amplified product was subjected to agarose gel electrophoresis (100 V, 30 minutes) to confirm whether the desired amplified product had been obtained. The PCR amplified product was then purified from the PCR reaction mixture using a QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer's protocol.

[0034] The sequences obtained from PCR amplification products were edited using the sequence editing software AqE, and the 16S rRNA gene sequences of each colony were compared with sequences in a database to identify the species. NCBI was used as the database. The results indicated that the bacteria belonged to the species Staphylococcus epidermidis. The resulting strains were stored frozen in glycerol at -80°C.

[0035] Experiment 2: <Identification of Staphylococcus epidermidis strains that promote ceramide synthesis> 2-1: Preparation of Staphylococcus epidermidis fermentation broth Staphylococcus epidermidis fermentation broth was prepared according to the following procedure. Each of the 100 strains of Staphylococcus epidermidis obtained and frozen in Experiment 1 was thawed and isolated and cultured at 37°C on TS plates containing 5% sheep blood. Formed single colonies were inoculated onto TS plates and cultured at 37°C for 48 hours. A single colony approximately 1 mm in diameter was picked and suspended in 10 mL of soy milk-like medium for 24 hours. The resulting Staphylococcus epidermidis soy milk fermentation product was sterilized in an autoclave at 121°C for 15 minutes. The fermentation product was then centrifuged (14,000 g for 15 minutes), and the supernatant was collected and sterilized by filtration through a 0.22 μm filter. Fermentation broths for each of the 100 strains of Staphylococcus epidermidis were thus obtained. These fermentation broths were stored at 4°C.

[0036] 2-2: Cultivation of PSVK-1 cells and treatment with Staphylococcus epidermidis fermentation broth This experiment was carried out using human epidermis-derived immortalized cells PSVK-1 purchased from the National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN). PSVK-1 cells were cultured in serum-free medium prepared by adding insulin, hydrocortisone, transferrin, phosphorylethanolamine, ethanolamine, epidermal growth factor (EGF), and bovine pituitary extract (BPE) to MCDB153 medium, resulting in concentrations of 5 μg / mL insulin, 0.5 μg / mL hydrocortisone, 10 μg / mL transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 10 μg / L EGF, and 50 μg / mL bovine pituitary extract.

[0037] The cultured PSVK-1 cells were seeded onto 24-well plates and further cultured until they reached 80% confluency. After replacing the culture medium with fresh medium, the Staphylococcus epidermidis fermentation liquid of each of the 100 strains obtained in 2-1 above was added to the plate to a final concentration of 10% (v / v), and the cells were exposed for 24 hours.

[0038] 2-3: RNA extraction and reverse transcription After discarding the PSVK-1 cell culture medium obtained in 2-2 above, total RNA was extracted from the PSVK-1 cells. RNA was extracted using an RNA extraction reagent (ISOGEN II; Nippon Gene) according to the manufacturer's protocol. The extracted RNA was dissolved in dH2O, and genomic DNA was removed with DNase I (Sigma). This was then used as a template for reverse transcription to synthesize cDNA using a real-time PCR reverse transcription kit (ReverTra™ Ace qPCR RT Master Mix; Toyobo).

[0039] 2-4: Gene expression analysis Quantitative gene expression analysis was performed using a reagent (THUNDERBURD™ SYBR qPCR Mix; manufactured by TOYOBO) with a real-time PCR detection system (CFX Connect™ Real-Time PCR Detection System; manufactured by Bio-Rad). The primer sequences used to detect the ceramide synthase 3 gene (CERS3 gene) are: sense 5'-CATGATCTTGCAGGTCCTTCACC-3' antisense 5'-CTCGTCATCACTCCTCACATCC-3' The reference gene is beta-actin (sense 5'-CACCATTGGCAATGAGCGGTTC-3' and The antisense 5'-AGGTCTTTGCGGATGTCCACGT-3' was used. Gene expression levels were evaluated using this ΔΔCt method.

[0040] 2-5:Results As described above, changes in ceramide synthase 3 gene expression were examined using quantitative real-time PCR when fermentation broth from 100 strains of Staphylococcus epidermidis was exposed to PSVK-1 cells. Results showed that the behavior of each strain varied, ranging from decreasing to increasing ceramide synthase 3 gene expression. Approximately half of the 100 strains decreased ceramide synthase 3 gene expression compared to the untreated control, but increases of ceramide synthase 3 gene expression of 2.0-fold or more compared to the untreated control were considered significant. Of the 100 strains, 11 were considered significant. Among these 11 strains, strains BG-S174 and BG-PG180 were particularly superior, with ceramide synthase 3 gene expression of 4.0-fold or more.

[0041] Experiment 3: <Identification of Staphylococcus epidermidis strains that promote hyaluronic acid synthesis> 3-1: Gene expression analysis The fermentation broths obtained from the 11 strains of Staphylococcus epidermidis obtained in Experiment 2 were used to examine the changes in hyaluronic acid synthase 3 gene (HAS3 gene) expression when exposed to PSVK-1 cells using quantitative real-time PCR in the same manner as in Experiment 2. The primer sequences used to detect the hyaluronic acid synthase 3 gene (HAS3 gene) were: sense 5'-AGCACCTTCTCGTGCATCATGC-3' The antisense primer was 5'-TCCTCCAGGACTCGAAGCATCT-3'.

[0042] 3-2:Results In the case of the fermentation liquids of all of the above 11 strains of Staphylococcus epidermidis, it was observed that the expression of the hyaluronan synthase 3 gene was increased. However, when comparing the median values ​​of the six experiments, the BG-S174 strain was particularly superior, increasing the expression of the hyaluronan synthase 3 gene by 4.5 times.

Claims

1. A bacterium that is the BG-S174 strain belonging to the species Staphylococcus epidermidis deposited under accession number: NITE P-04075, which increases the expression of the ceramide synthase 3 gene and increases the expression of the hyaluronic acid synthase 3 gene.

2. A gene expression enhancer that increases ceramide synthase 3 gene expression and increases hyaluronic acid synthase 3 gene expression, comprising the culture supernatant of the bacterium described in claim 1 as its main component.

3. A gene expression enhancer that increases ceramide synthase 3 gene expression and increases hyaluronic acid synthase 3 gene expression, comprising the bacterium according to claim 1 as an active ingredient.

4. A preparation comprising the bacterium according to claim 1 or the gene expression enhancer according to claim 2 or 3 as an active ingredient.

5. 5. The preparation according to claim 4, which is an external preparation for skin.