Skin-derived staphylococcus warneri CCSM004 prepared metagen with skin barrier repairing effect

Abstract

The invention discloses a skin-derived staphylococcus warneri CCSM004 prepared metagen with a skin barrier repairing effect, and belongs to the technical field of microorganisms and the technical field of medicines. The staphylococcus warneri CCSM004 provided by the invention can generate L-glycine-valine dipeptide and oleamide, and a fermentation supernatant prepared from the staphylococcus warneri CCSM004 has a good effect of repairing a barrier function, which is specifically shown in that the activity of damaged HaCaT cells is improved in vitro, and the gene level expression of FLG, IVL, AQP3, ZO-1, CLDN and Occludin can be improved at the same time after the damage. Therefore, the staphylococcus warneri has a huge application prospect in preparation of external cosmetics or medicines for repairing the skin barrier.

Description

Technical Field

[0001] This invention relates to a post-biotic prepared from a skin-derived Staphylococcus wartii strain CCSM004, which has the function of repairing the skin barrier, and belongs to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The skin barrier is the protective system formed by the outermost layer of the skin—the stratum corneum—and its related structures. Its main function is to protect the body from external physical, chemical, and microbial attacks, while maintaining the body's water and electrolyte balance. The skin barrier is composed of keratinocytes and intercellular lipids. Keratinocytes act as the "building blocks," while intercellular lipids (including ceramides, cholesterol, and free fatty acids) act as the "mortar," together constructing a strong defense. Under normal circumstances, the skin barrier effectively prevents the penetration of external irritants and regulates water evaporation. However, when the barrier function is impaired, it can lead to increased water loss, dry skin, and increased sensitivity to external irritants, subsequently triggering a series of skin problems such as inflammation, infection, and allergic reactions.

[0003] Currently, methods for repairing the skin barrier mainly focus on restoring the integrity of skin structure and function to enhance its defense capabilities. Firstly, moisturizers containing ceramides, cholesterol, and free fatty acids play a crucial role in barrier repair because they can mimic the skin's natural lipid structure, helping to rebuild the damaged lipid bilayer. Secondly, barrier replacement therapy is widely used, promoting skin barrier repair by supplementing exogenous lipids, especially ceramides, cholesterol, and fatty acids. Topical application of anti-inflammatory agents (such as steroids or non-steroidal anti-inflammatory drugs) can also reduce skin inflammation, thereby indirectly improving skin barrier function. Simultaneously, antioxidants (such as vitamins C and E) assist in barrier repair by reducing free radical damage. Although traditional methods such as ceramide supplementation, lipid replacement, anti-inflammatory and antioxidant treatments can alleviate barrier damage, they mostly only passively replenish components and are difficult to actively regulate the skin's own barrier synthesis and immune homeostasis. In contrast, microbial products, through multi-dimensional mechanisms such as activating the skin's inherent metabolism, enhancing lipid production, regulating inflammatory responses, and restoring microecological balance, can achieve active, systemic barrier repair that traditional exogenous ingredients cannot, possessing irreplaceable biological advantages. Therefore, screening microbial products with active skin barrier repair function has great application value. Summary of the Invention

[0004] This invention provides a Staphylococcus warwick ( Staphylococcus warneri The application of CCSM004 and its post-genes in the preparation of products that repair the skin barrier.

[0005] This invention provides a strain of Staphylococcus wartii ( Staphylococcus warneriThe *Staphylococcus wartii* strain described in CCSM004 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GMDCC No: 64898.

[0006] The *Staphylococcus wartii* CCSM004 strain was derived from the skin of healthy individuals. The strain was sequenced and the resulting sequence was compared with the nucleic acid sequence in NCBI. The result showed that it belonged to *Staphylococcus wartii* of the genus *Streptococcus*, and it was named *Staphylococcus wartii* CCSM004.

[0007] The colonies of *Staphylococcus wartii* CCSM004 on TSB solid medium are raised, white, smooth, and round.

[0008] The present invention also provides a postbiotic prepared using the aforementioned Staphylococcus wartii CCSM004.

[0009] In one embodiment, the metabiotic includes cell lysate and / or fermentation supernatant, or any of the above-mentioned powders prepared by drying. In one embodiment, the inactivated or deadened cells are prepared by culturing the *Staphylococcus wartii* CCSM004 in a culture medium for a period of time, collecting the bacterial cells in the cell culture medium, and obtaining inactivated bacterial cells after heat treatment. In one embodiment, the heat treatment conditions are: 65°C for 30 minutes.

[0010] In one embodiment, the method for preparing the bacterial lysate is as follows: Staphylococcus warwick CCSM004 is cultured in a culture medium for a period of time, bacterial cells are collected and resuspended, inactivated by heat treatment, homogenized under high pressure, and the supernatant is collected after centrifugation to obtain the bacterial lysate.

[0011] In one embodiment, the fermentation supernatant is obtained by culturing Staphylococcus aureus CCSM004 in a culture medium for a period of time, collecting the supernatant after centrifugation, and then inactivating it by heat treatment.

[0012] In one embodiment, the post-genetic agent is dried to obtain a powder.

[0013] The present invention also provides compositions containing the aforementioned Staphylococcus wartii CCSM004 and / or its postgenes.

[0014] In one embodiment, the composition includes, but is not limited to, pharmaceuticals or daily chemical products.

[0015] In one embodiment, the composition is a microbial preparation.

[0016] In one embodiment, the microbial preparation is a powder.

[0017] In one embodiment, the powder is prepared by drying the culture medium of Staphylococcus wartii CCSM004; or by drying the prepared liquid post-biotic of Staphylococcus wartii CCSM004 to obtain a solid powder post-biotic.

[0018] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0019] The present invention also provides products containing the aforementioned Staphylococcus wartii CCSM004, and / or its postbiotic, and / or its microbial preparations, said products including pharmaceuticals or daily chemical products.

[0020] In one embodiment, the dosage form of the product includes powder, tablet, block, liquid formulation, ointment, film, or gel.

[0021] In one embodiment, the drug contains Staphylococcus wartii CCSM004, pharmaceutical excipients, and / or a pharmaceutical carrier.

[0022] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0023] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0024] In one embodiment, the daily chemical product contains Staphylococcus wartii CCSM004, matrix raw materials, and / or conventional excipients.

[0025] In one embodiment, the matrix raw materials include oil-based raw materials, wax-based raw materials, synthetic oil-based raw materials, powder-based raw materials, gel-based raw materials, coagulants, and surfactants.

[0026] In one embodiment, the conventional excipients include one or more of the following: moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0027] In one embodiment, the daily chemical products include skin care products or washing and care products.

[0028] In one embodiment, the amount of Staphylococcus wartii CCSM004 and the prepared postbiotic in the product is not less than 1 × 10⁻⁶ of the corresponding viable count.7 CFU / mL.

[0029] The present invention also provides the use of the aforementioned Staphylococcus wartii CCSM004 and / or its postbiotics in the preparation of products for repairing the skin barrier.

[0030] In one embodiment, the preparation and repair of the skin barrier includes at least one of the following functions: (1) Enhance the cell activity of keratinocytes in the skin after skin barrier damage; (2) Increase the expression of filaggrin gene, aquaporin gene, inner lining protein gene, and tight junction protein (ZO-1, OCCLUDIN and CLDN) genes in keratinocytes after skin barrier damage.

[0031] The present invention also provides the use of the aforementioned Staphylococcus wartii CCSM004 in the preparation of L-glycine-valli dipeptide and / or oleamide.

[0032] Beneficial effects: Staphylococcus warwick of the present invention ( Staphylococcus warneri CCSM004 can produce L-glycine-valley dipeptide and oleamide. Metabiotics prepared from Staphylococcus warwickii CCSM004 have the ability to alleviate skin barrier damage and increase the expression of related proteins when applied topically, specifically in the following ways: (1) Enhance the cell activity of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage; (2) Increase the mRNA expression of FLG, IVL, AQP3 and tight junction protein in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage; Therefore, Staphylococcus warwick ( Staphylococcus warneri The post-biotic prepared by CCSM004 has great application potential in products that alleviate damage to the host skin barrier.

[0033] Preservation of biological materials Staphylococcus wartii ( Staphylococcus warneri CCSM004, taxonomically named Staphylococcus warneri It was deposited on July 22, 2024, at the Guangdong Provincial Center for the Preservation of Microbial Cultures, with accession number GDMCC No: 64898, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0034] Figure 1 Evaluation of the effects of different concentrations of SDS on HaCaT cell damage; AC: fitted curves of cell viability at different incubation times; D: fitted data of the effect of different SDS concentrations on cell viability under 6h incubation conditions. Figure 2 The effect of post-genetics on HaCaT cell proliferation; Figure 3 The effect of post-genetic repair on HaCaT cell viability after SDS damage; Figure 4 The effect of post-genetic repair of SDS damage on FLG mRNA expression in HaCaT; Figure 5 The effect of post-genetic repair of SDS-damaged cells on IVL mRNA expression in HaCaT; Figure 6 The effect of post-genetic repair of SDS damage on AQP3 mRNA expression in HaCaT; Figure 7 The effect of post-genetic repair of SDS-damaged HaCaT tight junction mRNA expression; Figure 8 L-glycine-valine dipeptide standard liquid chromatography-mass spectrometry; Figure 9 Liquid chromatography-mass spectrometry (LC-MS) of L-glycine-valla dipeptide content in fermentation supernatant samples; Figure 10 A comparison chart of retention times for L-gamma-valpy dipeptide standard, fermentation supernatant, and cell samples; Figure 11 LC-MS / MS of oleamide standard; Figure 12 Liquid chromatography-mass spectrometry (LC-MS) of oleic acid amide content in fermentation supernatant samples; Figure 13 A comparison chart of retention times for oleamide standards, fermentation supernatant, and cell samples. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments.

[0036] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0037] The culture media involved in the following examples are as follows: TSB liquid medium: tryptone: 17.0 g / L; soybean peptone: 3.0 g / L; sodium chloride: 5.0 g / L; potassium dihydrogen phosphate: 2.5 g / L and glucose: 2.5 g / L.

[0038] TSB solid medium: tryptone: 17.0 g / L; soybean peptone: 3.0 g / L; sodium chloride: 5.0 g / L; potassium dihydrogen phosphate: 2.5 g / L; glucose: 2.5 g / L and agar powder 20 g / L.

[0039] Modified BHI screening solid culture medium: bovine brain extract: 4.0 g / L, bovine heart extract: 4.0 g / L, peptone: 5.0 g / L, casein peptone: 16.0 g / L, sodium chloride: 5.0 g / L, glucose: 2.0 g / L, disodium hydrogen phosphate: 2.5 g / L, nicotinic acid: 0.3 g / L, neomycin: 0.4 g / L, and agar: 20 g / L.

[0040] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL).

[0041] Example 1: Cell resuscitation and culture First, remove the frozen human keratinocyte cell line (HaCaT) from the -80℃ freezer, thaw it rapidly in a 37℃ water bath, then add 5 mL of cell culture medium and centrifuge at 1000 r / min for 3 min, discarding the supernatant. Resuspend the cells in 1 mL of cell culture medium and place them in a culture dish, then incubate in a 37℃ incubator containing 5% CO2. When the cells regain viability and reach 70%-80% confluence after 1-2 days, passage the cells.

[0042] Example 2: Isolation and screening of Staphylococcus warwick The bacterial strain samples were selected from healthy human skin. Sterile swabs were applied to the back of the hand three times with physiological saline, and then streaked directly onto a modified BHI screening solid medium. The culture was incubated at 37°C for 48 hours. Large, pale yellow, round colonies were selected from the grown colonies and enriched in BHI liquid medium for 16 hours. The bacterial culture was then transferred to BHI solid plates for purification and incubation for 48 hours. Finally, single colonies were selected and transferred to BHI liquid medium for enrichment. The strain was preserved in 30% glycerol. The genome of the strain was extracted, and 16S rDNA was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The results, confirmed by NCBI sequence alignment, identified it as *Staphylococcus warwickii*, and named *Staphylococcus warwickii* CCSM004.

[0043] Example 3: Preparation of postbiotics from Staphylococcus warwick CCSM004 (1) Staphylococcus wartii CCSM004 was streaked from the preservation tube and revived. It was then cultured in a water-jacketed incubator at 37°C for 48 h on BHI solid medium to obtain single colonies. Single colonies were picked and inoculated into BHI liquid medium and cultured at 37°C for 12-18 h to obtain culture solution 1. Culture solution 1 was inoculated into TSB liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 12 h to obtain seed culture; The seed culture was inoculated into TSB liquid medium at 2% (v / v) for expansion culture, and cultured at 37℃ for 18h. The number of viable bacteria was recorded and bacterial culture a was obtained.

[0044] The bacterial culture a was centrifuged at 8000 r / min for 30 min, and the supernatant and bacterial sludge were collected. The supernatant was heat-treated (65℃, 30 min) and freeze-dried to obtain powder for later use, thus preparing the freeze-dried powder of Staphylococcus warwick fermentation supernatant (denoted as CCSM004-Q). The bacterial sludge was resuspended in 75% of the original bacterial culture volume of double-distilled water, and the resuspended liquid was heat-treated (65℃, 30 min), and then homogenized under high pressure (1000 MPa, 10 times) using a high-pressure homogenizer. After homogenization, the supernatant was collected by centrifugation at 8000 r / min for 30 min to obtain the bacterial cell lysate (denoted as CCSM004-S).

[0045] The following were prepared by means of the above method: Staphylococcus warwick CCSM004 postbiotic (cell lysate CCSM004-S and lyophilized fermentation supernatant CCSM004-Q).

[0046] Example 4: Establishment of an in vitro model of keratinocyte damage caused by sodium dodecyl sulfate (SDS) (HaCaT) (1) Take 100 μL of HaCaT cells in the logarithmic growth phase and use 1.2 × 10⁻⁶ ppm. 4 Cells were seeded at a concentration of 100 cells / well in a 96-well plate, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 36 h to allow the cells to adhere, blank control, control and SDS treatment groups were set up. Control group: Contains only cell culture medium and does not contain HaCaT cells; Control group: Contains cell culture medium and HaCaT cells, but does not contain SDS; Treatment groups: containing cell culture medium, HaCaT cells, and SDS at final concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively.

[0047] (2) The above well plates were incubated in an incubator at a temperature of 37 °C for 6 h, 12 h and 24 h respectively. After the incubation, 10 μL of CCK8 solution was added to each well and incubated for 1.5 h to measure the absorbance (OD) at 450 nm.

[0048] Cell viability is calculated using the following formula: Cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0049] (3) The effect on cell activity after nonlinear regression fitting is as follows: Figure 1As shown, compared with the control group (cell proliferation rate 100%), the cell viability of the model group treated with 15 μg / mL SDS for 6 h was 60.6%, and SDS modeling caused significant damage to HaCaT cells.

[0050] Example 5: Safety verification of postbiotics prepared from Staphylococcus warwick CCSM004 on HaCaT cells (1) Take 100 μL of HaCaT cells in the logarithmic growth phase and use 1.2 × 10⁻⁶ ppm. 4 Cells were seeded at a concentration of 100 cells / well in a 96-well plate, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 36 h to allow the cells to adhere, blank control group, control group, and post-genetic treatment group were set up. Control group: Contains only cell culture medium and does not contain HaCaT cells; Control group: Contains cell culture medium and HaCaT cells, but does not contain post-genetics; The post-genetic treatment group contained cell culture medium and HaCaT cells, as well as post-genetic agents.

[0051] The metabiotic was resuspended in cell culture medium (the amount of resuspended metabiotic was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of metabiotic prepared from bacterial culture at CFU / ml was equivalent. In the metabiotic treatment group, the culture medium was replaced with 100 μL of Staphylococcus warwick CCSM004 metabiotic resuspended in cell culture medium; in other groups, the culture medium was replaced with 100 μL of fresh culture medium.

[0052] (2) The above well plates were incubated in an incubator at 37°C for 24 h. After incubation, 10 μL of CCK8 solution was added to each well and incubated for 1.5 h. The absorbance (OD) at 450 nm was measured.

[0053] Cell viability is calculated using the following formula: Cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0054] Effects on cell viability, such as Figure 2 As shown, compared with the control group (cell proliferation rate 101.29%), the addition of postbiotics composed of Staphylococcus warwick CCSM004 (CCSM004-Q and CCSM004-S) at an inactivated cell concentration of 1.0 × 10⁻⁶ cells significantly improved cell proliferation. 7 The cell proliferation rates at CFU / ml were 101.51% and 102.15%, respectively.

[0055] According to the ISO 10993-5:2009 toxicity classification evaluation method, cells with a viability greater than 70% can be considered non-toxic. The above results indicate that an inactivated bacterial concentration of 1.0 × 10⁻⁶ is appropriate. 7HaCaT cells at a postbiotic concentration of CFU / ml showed high viability (over 95%). Considering their lack of cytotoxicity, an inactivated cell concentration of 1.0 × 10⁻⁶ was selected. 7 CFU / ml is a suitable postbiotic concentration for subsequent cell experiments.

[0056] Example 6: Effect of post-biotic prepared from Staphylococcus warwick CCSM004 on SDS-induced damage to HaCaT cells (1) Take 100 μL of HaCaT cells in the logarithmic growth phase and use 1.2 × 10⁻⁶ ppm. 4 Cells were seeded at a concentration of 100 cells / well in a 96-well plate, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 36 h to allow them to adhere, control group 1 was obtained. (2) Set up control group, model group and treatment group respectively for the well plates prepared in step (1), and then incubate them in an incubator at 37℃ for 6 h: Control group: After changing the medium of control group 1 in step (1), it contains cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent; Model group: The control group 1 in step (1) was replaced with a cell culture medium containing 15 μg / mL SDS, containing the original HaCaT cells, without post-genetic treatment; the cell culture medium containing SDS modeling agent was prepared by mixing SDS evenly in the cell culture medium and passing it through a 0.22 μm aqueous filter membrane for sterilization, so that the final concentration of SDS in the cell culture medium was 15 μg / mL; Postgenetic treatment group: The treatment method is the same as that of the model group.

[0057] (3) After incubation, discard the original culture medium and wash once with PBS. Add the corresponding metagenic sample to the metagenic treatment group, and add an equal amount of cell culture medium to the control group and the model group. Incubate again for 24 hours.

[0058] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of metabiotic prepared from the bacterial culture at CFU / ml was equivalent, and 100 μL of Staphylococcus warwick CCSM004 metabiotic resuspended in cell culture medium was added to each culture.

[0059] (4) After 24 hours of incubation, add 10 μL of CCK8 solution to each well and incubate for 1.5 hours to measure the absorbance (OD) at 450 nm.

[0060] Cell viability was calculated using the following formulas: Model group cell viability (%) = (Model group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × 100%; Treatment group cell viability (%) = (Treatment group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × 100%.

[0061] The results of the effect of post-genetic agents on SDS-induced HaCaT cell damage are as follows: Figure 3 Compared with the control group (cell viability 96.94%), the cell viability of the model group was 66.76%, indicating that SDS modeling caused damage to HaCaT cells.

[0062] After adding CCSM004-S and CCSM004-Q to the treatment groups, the cell viability was 76.11% and 85.22%, respectively. Compared with the model group (66.76%), CCSM004-Q could increase the viability of HaCaT cells by 1.28 times, indicating that the metagenetic agent of Staphylococcus warwickii CCSM004 (fermentation supernatant) can effectively alleviate the damage to HaCaT cells caused by SDS.

[0063] Example 7: Effect of post-biotic repair of SDS-damaged HaCaT cells prepared with Staphylococcus warwick CCSM004 on FLG gene expression (1) HaCaT cells were injected at a concentration of 1.8 × 10⁻⁶. 4 One cell per well was seeded into a 6-well plate and cultured for 36 hours until the cells adhered to the plate, thus obtaining control group 1.

[0064] (2) Set up control group, model group and treatment group respectively for the well plates prepared in step (1), and then incubate them in an incubator at 37 ℃ for 6 h: Control group: After changing the medium of control group 1 in step (1), it contains cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent; Model group: The control group 1 in step (1) was replaced with a cell culture medium containing 15 μg / mL SDS, containing the original HaCaT cells, without post-genetic treatment; the cell culture medium containing SDS modeling agent was prepared by mixing SDS evenly in the cell culture medium and passing it through a 0.22 μm aqueous filter membrane for sterilization, so that the final concentration of SDS in the cell culture medium was 15 μg / mL; Postgenetic treatment group: The treatment method is the same as that of the model group.

[0065] (3) After incubation, discard the original culture medium and wash once with PBS. Add the corresponding metagenic sample to the metagenic treatment group, and add an equal amount of cell culture medium to the control group and the model group. Incubate again for 24 hours.

[0066] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of metabiotic prepared from the bacterial culture at CFU / ml was equivalent, and 2 mL of Staphylococcus warwick CCSM004 (CCSM004-Q) metabiotic resuspended in cell culture medium was added to each culture.

[0067] (4) After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△Ct Formula calculation FLG The mRNA expression levels were measured, with GAPDH as the internal reference. The primers are described in Table 1 below, and the results are as follows: Figure 4 As shown.

[0068] Table 1 Primer Sequences

[0069] Filamentin (FLG) plays a crucial role in the skin barrier function. It is an important molecule connecting keratin fibers in the stratum corneum, forming a robust physical barrier on the outermost layer of the epidermis by helping keratin fibers aggregate in an orderly manner, preventing moisture loss and blocking the invasion of external allergens. FLG deficiency is closely associated with various skin diseases, such as eczema (atopic dermatitis) and ichthyosis. In these diseases, FLG deficiency or absence weakens the skin barrier function, making the skin more sensitive to external stimuli. Therefore, reducing SDS-induced damage to keratinocytes by increasing FLG gene expression has become a therapeutic target. FLG expression results are obtained from… Figure 4 It can be seen that, with the control group's FLG mRNA expression level as 1, the expression level in the model group decreased to 0.8 after SDS intervention, and the post-biotic prepared by Staphylococcus wartii CCSM004 (fermentation supernatant) significantly upregulated the FLG mRNA expression level to 2.49.

[0070] Example 8: Effect of post-genetic agents prepared from Staphylococcus warwick CCSM004 on IVL gene expression in HaCaT cells damaged by SDS. The specific implementation method is as described in Example 7. After incubation, the culture supernatant was discarded, and each well was quickly washed three times with PBS. 1 mL of cell lysis buffer was added to each well, and the cells were repeatedly pipetted. RNA was extracted from the cell lysis buffer and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. Gene expression in HaCaT cells was detected using real-time quantitative PCR. -△△Ct Formula calculation IVLThe mRNA expression levels were measured, with GAPDH as the internal reference. The primers are described in Table 2 below, and the results are as follows: Figure 5 As shown.

[0071] Table 2 Primer Sequences

[0072] In keratinocytes, inner lamina protein (IVL) cross-links with laminarin via transglutaminase catalysis, forming an insoluble keratinized capsule that constitutes the unique stratum corneum barrier structure of the epidermis. Simultaneously, it interacts with other keratinocyte differentiation proteins such as filaggrin (FLG) to jointly maintain the structure and function of the skin barrier. IVL expression results are derived from… Figure 5 It can be seen that, with the expression level of IVL mRNA in the control group as 1, the expression level in the model group decreased to 0.68 after SDS intervention, and the supernatant of Staphylococcus wartii CCSM004 fermentation could increase the expression level of IVL to 1.46.

[0073] Example 9: Effect of post-genetic agents prepared from Staphylococcus warwick CCSM004 on AQP3 gene expression in HaCaT cells damaged by SDS. The specific implementation method is as described in Example 7. After incubation, the culture supernatant was discarded, and each well was quickly washed three times with PBS. 1 mL of cell lysis buffer was added to each well, and the cells were repeatedly pipetted. RNA was extracted from the cell lysis buffer and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. Gene expression in HaCaT cells was detected using real-time quantitative PCR. -△△Ct Formula calculation AQP3 The mRNA expression levels were measured, with GAPDH as the internal reference. The primers are described in Table 3 below, and the results are as follows: Figure 6 As shown.

[0074] Table 3 Primer Sequences

[0075] Aquaporins (AQPs) play a crucial role in skin barrier function, especially AQP3. AQPs regulate the transport and distribution of water in the skin, maintaining skin hydration and elasticity by controlling the water content within the stratum corneum. AQP3 not only promotes water transport but also regulates glycerol transport, playing a vital role in maintaining skin suppleness and barrier function. The expression of AQP3 is determined by… Figure 6 It can be seen that the mRNA expression level of AQP3 in the control group was about 1, while the expression level in the model group decreased to 0.54 after SDS intervention. After repair by the supernatant of Staphylococcus wartii CCSM004 fermentation, the expression level of AQP3 was restored to 2.46.

[0076] Example 10: Effect of post-genetic agents prepared from Staphylococcus warwick CCSM004 on the expression of tight junction-related protein genes in HaCaT cells damaged by SDS. The specific implementation method is as described in Example 7. After incubation, the culture supernatant was discarded, and each well was quickly washed three times with PBS. 1 mL of cell lysis buffer was added to each well, and the cells were repeatedly pipetted. RNA was extracted from the cell lysis buffer and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. Gene expression in HaCaT cells was detected using real-time quantitative PCR. -△△Ct Formula calculation ZO-1 , Occludin and CLDN The mRNA expression levels were measured, with GAPDH as the internal reference. The primers are described in Table 4 below, and the results are as follows: Figure 7 As shown.

[0077] Table 4 Primer Sequences

[0078] Tight junction proteins are essential components for maintaining skin barrier function. Located between keratinocytes, they form sealed junctions that prevent the permeation of external substances and water through the intercellular spaces, thus maintaining the integrity of the skin barrier. Zygomatic band occlusion protein (ZO-1) and occlusive junction protein (OCCLUDIN) are core components of tight junctions and important structural proteins of the skin barrier. They play a crucial role in regulating the transport of substances through the intercellular spaces, maintaining the selective permeability of the skin barrier. Furthermore, these proteins not only participate in physical cell-cell connections but also function in intracellular signal transduction, influencing cell proliferation, differentiation, and migration. The expression of ZO-1, Occludin, and CLDN is... Figure 7 As shown, the mRNA expression of these three proteins in the model group decreased to 0.47, 0.34 and 0.65, respectively. However, after repair by Staphylococcus warwick fermentation supernatant, the expression of these three proteins could be upregulated to varying degrees, reaching 2.72, 2.64 and 2.24, respectively.

[0079] Example 11: Effective substance analysis of fermentation supernatant of Staphylococcus warwick CCSM004 1. Substance identification based on non-targeted metabolomics: Metabolomics analysis of CCSM004 fermentation supernatant.

[0080] Sample preparation and testing before testing: (1) Following the method in Example 3, Staphylococcus warwick CCSM004 was cultured in TSB liquid medium for 12 h, and then 1 mL of bacterial suspension (with a viable count of 1 x 10⁻⁶) was taken.7 CFU / mL), 4℃, 10000 g Centrifuge for 5 minutes and collect the supernatant. (2) Transfer 100 μL of supernatant into a 1.5 mL centrifuge tube; (3) Add 400 μL of methanol:acetonitrile = (1:1, v / v) (pre-cool at -20℃ in advance) to precipitate the protein; (4) Vortex for 30 s, followed by ice bath ultrasound for 10 min; (5) Place the sample in a -20℃ refrigerator for 1 h to increase the protein precipitation rate (secondary precipitation removes protein). (6) Centrifuge at 15000 rpm for 15 min at 4℃. (7) Take the supernatant and concentrate it under vacuum; (8) Redissolve by adding 100 μL of acetonitrile:water (1:1) and vortexing for 30 s; (9) Centrifuge at 15,000 rpm for 15 min at 4℃, take the supernatant, transfer the appropriate volume into a vial for instrument testing; (10) For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column for chromatographic separation of the target compounds. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B was acetonitrile. Sample tray temperature: 4℃, injection volume: 2 μL. The Orbitrap Exploris 120 mass spectrometer was able to acquire primary and secondary mass spectrometry data under the control of the control software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheath gasflow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0081] 2. Sample preparation and detection before CCSM004 bacterial cell structure metabolomics analysis: Following the method in step 1, culture *Staphylococcus warwick* CCSM004 for 12 h, then take 1 mL of the bacterial suspension and incubate at 4°C for 10,000 minutes. g Centrifuge for 5 minutes to collect the bacterial sludge; (1) Wash once with pre-cooled 0.9% physiological saline to remove residual culture medium; (2) After centrifuging to remove physiological saline, 1.5 mL centrifuge tubes containing bacterial sludge are placed into liquid nitrogen for quenching to stop metabolic activity; (3) Remove the sample from the liquid nitrogen and add 500 μL of methanol:acetonitrile = (1:1, v / v) (pre-cooled at -20℃ in advance); (4) After vortexing for 30 seconds, the cells were repeatedly frozen and thawed three times with liquid nitrogen to lyse the cells and release intracellular metabolites. (5) Place the sample in a -20℃ refrigerator for 1 h to increase the protein precipitation rate (secondary precipitation removes protein). (6) Centrifuge at 15,000 rpm for 15 min at 4℃; (7) Take the supernatant and evaporate it to dryness using a rotary evaporator; (8) Redissolve by adding 100 μL of acetonitrile:water (1:1) and vortexing for 30 s; (9) Centrifuge at 15,000 rpm for 15 min at 4℃, take the supernatant, transfer an appropriate volume into a vial for instrument testing; (10) For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column for chromatographic separation of the target compounds. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B was acetonitrile. Sample tray temperature: 4℃, injection volume: 2 μL. The Orbitrap Exploris 120 mass spectrometer was able to acquire primary and secondary mass spectrometry data under the control of the control software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheath gasflow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0082] 3. Analysis of active ingredients in CCSM004: The raw data was converted into mzXML format using ProteoWizard software, and then metabolite identification was performed using a collaboratively developed R package. The database used was BiotreeDB (V3.0), and then visualization analysis was performed using a self-developed R package.

[0083] Literature review was conducted to collect the chemical formulas, molecular formulas, and molecular weights of potential effective substances in CCSM002. Based on non-target metabolomics, and according to substances with a P-value < 0.05 and differences compared to those before fermentation, as well as research on skin barrier repair, potential effective substances were screened. The substances are shown in Table 5.

[0084] Table 5. Potentially effective substances related to the skin barrier in CCSM004

[0085] Example 12: Quantitative detection of potential active substances in Staphylococcus warwick CCSM004 based on non-target metabolomics results The substances in CCSM004 were detected by liquid chromatography-mass spectrometry, and the detection method was the same as in Example 11.

[0086] Based on the results of non-targeted metabolomics, the following substances were detected in the sample after targeted quantitative detection: (1) L-gamma-valpy dipeptide like Figures 8-10 As shown, this substance was detected only in the fermentation supernatant, at a concentration of 1.63 ppm. L-Glutava dipeptide is a bioactive dipeptide with a reliable source and simple structure, exhibiting significant cellular regulatory potential in skin barrier repair. Studies have shown that this dipeptide can promote the proliferation and differentiation of keratinocytes, enhance the expression of key barrier proteins (such as FLG, LOR, and IVL), and strengthen the structural integrity of the stratum corneum from the source. Simultaneously, it can inhibit the release of factors such as TNF-α and IL-6 under inflammatory stimulation, reducing the amplified inflammatory response after barrier damage. Furthermore, L-Glutava dipeptide can activate signaling pathways related to skin energy metabolism and repair, improve cellular tolerance to oxidation and external stimuli, thereby accelerating the recovery of the damaged barrier and improving overall skin homeostasis.

[0087] (2) Oleamide like Figures 11-13 As shown, this substance was detected only in bacterial cells, at a concentration of 1.3 ppm. Oleamide is an endogenous fatty amide signaling molecule with multiple biological effects promoting skin barrier reconstruction. On the one hand, oleamide can regulate the PPAR pathway and CB2 receptor in keratinocytes, significantly reducing the production of inflammatory factors such as TNF-α and IL-1β, thus alleviating barrier damage caused by inflammation at its source. On the other hand, it can promote the synthesis and metabolism of key barrier lipids (such as ceramides, free fatty acids, and cholesterol), enhancing the integrity of the lipid bilayer structure. In addition, oleamide can improve the composition of the sebum film, increase the flexibility and water-locking capacity of the stratum corneum, comprehensively enhance the skin's resistance to external stimuli, and promote the rapid recovery of the damaged barrier.

[0088] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Staphylococcus warwick ( Staphylococcus warneri Accession number CCSM004 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 22, 2024, with accession number GMDCC No: 64898.

2. The metabiotic prepared using *Staphylococcus wartii* CCSM004 as described in claim 1, characterized in that, The metabiotic includes the fermentation supernatant and / or cell lysate of Staphylococcus warwick CCSM004 as described in claim 1.

3. The method for preparing the postgenetic agent according to claim 2, characterized in that, Including (a) or (b): (a) The Staphylococcus wartii CCSM004 of claim 1 is cultured to the logarithmic growth phase, the fermentation supernatant is collected, and the supernatant is inactivated by heat treatment to obtain the metagene; (b) The Staphylococcus wartii CCSM004 of claim 1 is cultured to the logarithmic growth phase, the bacterial cells are collected, inactivated by heat treatment, and then homogenized under high pressure. The supernatant is collected to obtain the metagene.

4. A microbial preparation containing the Staphylococcus wartii CCSM004 as described in claim 1 and / or its postbiotic.

5. A product containing *Staphylococcus warwickii* CCSM004 as described in claim 1, and / or its metabiotic, and / or its microbial preparation, characterized in that, The products include pharmaceuticals or daily chemical products.

6. The product as described in claim 5, characterized in that, The dosage forms of the products include powders, tablets, blocks, liquid preparations, ointments, films, or gels.

7. The product as described in claim 6, characterized in that, The daily chemical products mentioned include skin care products or washing and care products.

8. The product as described in claim 7, characterized in that, The drug also contains conventional pharmaceutical excipients and / or pharmaceutical carriers.

9. The use of Staphylococcus wartii CCSM004 and / or its post-biotics as described in claim 1 in the preparation of products for repairing the skin barrier, characterized in that, The repair of the skin barrier includes repairing the stratum corneum and / or regulating the levels of barrier-related genes.

10. The use of Staphylococcus warwick CCSM004 as described in claim 1 in the preparation of L-glycine-valli dipeptide and / or oleamide.

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