Strain of staphylococcus warneri CCSM005 separated from human skin and capable of improving skin health and metagen of staphylococcus warneri CCSM005

Abstract

The invention discloses staphylococcus warneri CCSM005 separated from human skin and capable of improving skin health and a metagen thereof, and belongs to the technical field of microorganisms and medicines. The staphylococcus warneri CCSM005 provided by the invention can be fermented to produce the L-glycine-valine dipeptide, and a fermentation supernatant of the staphylococcus warneri CCSM005 has a good effect of repairing a skin 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, ZO-1, CLDN and Occludin can be improved at the same time after the damage. Therefore, the staphylococcus warneri CCSM005 has a huge application prospect in preparation of external daily chemical products or medicines for repairing the skin barrier.

Description

Technical Field

[0001] This invention relates to a strain of Staphylococcus wartii CCSM005 isolated from human skin that has the effect of improving skin health, and its post-biotics, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The skin barrier, the outer protective layer of the skin, is mainly composed of the stratum corneum, sebum, and natural moisturizing factors. It plays a crucial role in preventing moisture loss and resisting the invasion of harmful external substances. A healthy skin barrier maintains the skin's moisture balance, preventing excessive dryness or sensitivity. When the barrier is damaged, the skin is prone to dryness, sensitivity, and even inflammation or infection. Furthermore, the condition of the skin can reveal information about the body's immune function, nutrient absorption, and hormonal balance. When the skin experiences dryness, inflammation, or allergies, it often indicates potential health problems such as vitamin deficiencies, endocrine disorders, or weakened immunity. Therefore, maintaining healthy skin is essential for overall health.

[0003] Current methods for repairing the skin barrier mainly revolve around lipid replenishment, moisturizing, and anti-inflammation, but they still have significant limitations. While exogenous lipids (such as ceramides and cholesterol) can fill the skin's lipid deficit in the short term, they cannot truly promote the synthesis of endogenous lipids, and barrier reconstruction often lacks sustainability. Moisturizers, although reducing TEWL (transient endothelial protein) levels, do not have the ability to regulate immunity and cellular metabolism. Anti-inflammatory agents and antioxidants mostly act on the symptom level, and long-term use may lead to irritation or tolerance problems. Therefore, these methods can usually only "passively repair" the structure and are difficult to fundamentally restore the skin's biological homeostasis.

[0004] In contrast, microbial postbiotics have irreplaceable advantages: their active molecules can directly regulate keratinocyte metabolism, promote the endogenous synthesis of key barrier lipids such as ceramides and free fatty acids, while simultaneously regulating immunity, inhibiting inflammation, and restoring the balance of the skin's microecology, thus achieving a shift from "structural replacement" to "functional self-repair." This multi-target bioregulation of the skin barrier is difficult to achieve with traditional chemical components. Summary of the Invention

[0005] This invention provides a Staphylococcus warwick ( Staphylococcus warneri The application of CCSM005 and its post-genes in the preparation of products that repair the skin barrier.

[0006] This invention provides a strain of Staphylococcus wartii ( Staphylococcus warneri The *Staphylococcus wartii* strain described in CCSM005 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GMDCC NO: 64899.

[0007] The *Staphylococcus wartii* CCSM005 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* CCSM005.

[0008] The colonies of *Staphylococcus wartii* CCSM005 on TSB solid medium are raised, white, smooth, and round.

[0009] The present invention also provides a metabiotic prepared using the aforementioned Staphylococcus wartii CCSM005.

[0010] In one embodiment, the metabiotic includes cell lysate, inactivated or dead cells, fermentation supernatant, or any of the above-mentioned powders prepared by drying. In one embodiment, the inactivated or dead cells are prepared by culturing the Staphylococcus warwick CCSM005 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: 62-65°C for 30 minutes.

[0011] In one embodiment, the method for preparing the bacterial lysate is as follows: Staphylococcus warwick CCSM005 is cultured in a culture medium for a period of time, bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate.

[0012] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging Staphylococcus aureus CCSM005 after culturing it in a culture medium for a period of time.

[0013] The present invention also provides a composition containing the aforementioned Staphylococcus wartii CCSM005 and / or its postgenetic agent.

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

[0015] In one embodiment, the dosage form of the composition includes powder, tablet, block, liquid formulation, ointment, film, or gel.

[0016] In one embodiment, the composition further includes conventional excipients.

[0017] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0018] In one embodiment, the composition is a probiotic powder.

[0019] In one embodiment, the bacterial powder is a solid powdered postbiotic prepared by drying the prepared liquid postbiotic of Staphylococcus warwick CCSM005.

[0020] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0021] The present invention also provides the use of the composition in the preparation of products that repair the skin barrier.

[0022] In one embodiment, the skin barrier repair includes repairing the stratum corneum and / or regulating the expression of skin barrier-related genes.

[0023] In one embodiment, the repair of the stratum corneum involves improving the cellular activity of keratinocytes after damage to the skin barrier.

[0024] In one embodiment, the regulation of skin barrier-related gene expression includes increasing the expression of filaggrin gene, ZO-1 protein gene, CLDN protein gene, and / or OCCLUDIN protein gene in keratinocytes after skin barrier damage.

[0025] In one embodiment, the product is applied topically.

[0026] In one embodiment, the amount of Staphylococcus wartii CCSM005 and the prepared postbiotic in the product is not less than 1 × 10⁻⁶ of the corresponding viable count. 7 CFU / mL.

[0027] In one embodiment, the product is a pharmaceutical product or a daily chemical product.

[0028] In one embodiment, the pharmaceutical product comprises the Staphylococcus wartii CCSM005, a drug carrier, and / or pharmaceutical excipients.

[0029] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0030] 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.

[0031] In one embodiment, the daily chemical products include skin care products or washing and care products.

[0032] In one embodiment, the daily chemical product contains Staphylococcus wartii CCSM005, matrix raw materials, and / or conventional excipients.

[0033] 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.

[0034] 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.

[0035] The present invention also provides the use of the aforementioned Staphylococcus wartii CCSM005 in the preparation of L-glycine-valley dipeptide or products containing L-glycine-valley dipeptide.

[0036] Beneficial effects: Staphylococcus warwick of the present invention ( staphylococcus warneri CCSM005 can be fermented to produce L-glycine-valley dipeptide. The metabiotic prepared from the *Staphylococcus wartii* CCSM005 has the ability to alleviate skin barrier damage and improve 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, ZO-1, CLDN and Occludin in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage; Therefore, Staphylococcus warwick ( Staphylococcus warneri The post-biotic prepared by CCSM005 has great application potential in products that alleviate damage to the host skin barrier.

[0037] Preservation of biological materials Staphylococcus wartii ( Staphylococcus warneri CCSM005, taxonomically named Staphylococcus warneri It was deposited on July 22, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64899, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0038] 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 2The 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 ZO-1 mRNA expression in HaCaT; Figure 6 The effect of post-genetic repair of SDS-damaged CLDN mRNA expression in HaCaT; Figure 7 The effect of post-genetic repair of SDS damage on Occludin mRNA expression in HaCaT; 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. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments.

[0040] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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).

[0045] 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.

[0046] Example 2: Isolation and screening of Staphylococcus warwick The bacterial strain was screened from healthy human skin. A sterile swab was 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 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 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 it was named *Staphylococcus warwickii*. Staphylococcus warneri The strain CCSM005 is described in the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 64899).

[0047] Example 3: Preparation of postbiotics from Staphylococcus warwick CCSM005 (1) Staphylococcus warwick CCSM005 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 18 h. The number of viable bacteria was recorded and bacterial culture a was obtained.

[0048] 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 CCSM005-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 CCSM005-S).

[0049] The following were prepared by the above methods: Staphylococcus warwick CCSM005 postbiotic (cell lysate CCSM005-S and lyophilized fermentation supernatant CCSM005-Q).

[0050] 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 96-well plates, 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.

[0051] (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 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.

[0052] 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%.

[0053] (3) The effect on cell activity after nonlinear regression fitting is as follows: Figure 1 As 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.

[0054] Example 5: Safety verification of postbiotics prepared from Staphylococcus warwick CCSM005 on HaCaT cells (1) Take 100 μL of HaCaT cells in the logarithmic growth phase and use 1.2 × 10⁻⁶ ppm. 4 Cells per well were seeded at a concentration of 100 cells / well in 96-well plates, 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 post-genetic 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 post-genetics; The post-genetic treatment group contained cell culture medium and HaCaT cells, as well as post-genetic agents.

[0055] The metabiotic was resuspended in cell culture medium to obtain a metabiotic solution (the amount of resuspended metabiotic was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 (The amount of postbiotic prepared from bacterial culture of CFU / ml is equivalent), add 100 μL of postbiotic solution prepared from Staphylococcus warwick CCSM005.

[0056] (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.

[0057] Cell viability was calculated using the following formula: Cell viability (%) = (OD value of treated group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100% Effects on cell viability, such as Figure 2 As shown, compared with the control group (cell proliferation rate 100.79%), the addition of postbiotics composed of Staphylococcus warwick CCSM005 (CCSM005-Q and CCSM005-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 101.38% and 101.24%, respectively.

[0058] 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. 7 HaCaT 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.

[0059] Example 6: Effect of post-biotic prepared from Staphylococcus warwick CCSM005 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 A concentration of cells / well was seeded into 96-well plates, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 36 h to allow the cells 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 °C 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 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.

[0060] (3) After incubation, discard the original culture medium and wash once with PBS. Add the corresponding metagenic solution to the metagenic treatment group, and add an equal volume of cell culture medium to the control group and the model group. Incubate again for 24 hours.

[0061] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium to obtain a metabiotic solution (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of postbiotic prepared from bacterial culture of CFU / ml is equivalent, and 100 μL of postbiotic solution prepared from Staphylococcus warwick CCSM005 is added to each solution.

[0062] (4) Add 10 μL of CCK8 solution to each well and incubate for 1.5 h to measure the absorbance (OD) at 450 nm.

[0063] 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%.

[0064] The results of the effect of post-genetic agents on SDS-induced HaCaT cell damage are as follows: Figure 3Compared with the control group (cell viability 96.94%), the cell viability of the model group was 66.52%, indicating that SDS modeling caused damage to HaCaT cells.

[0065] After adding CCSM005-S and CCSM005-Q to the treatment groups, the cell viability was 80.65% and 74.36%, respectively. Compared with the model group (66.52%), CCSM005-S can improve the viability of HaCaT cells, indicating that the metagenetic product (cell lysate) of Staphylococcus warwickii CCSM005 can effectively alleviate the damage caused by SDS to HaCaT cells.

[0066] Example 7: Effect of post-genetic agents prepared from Staphylococcus warwick CCSM005 on FLG gene expression in HaCaT cells damaged by SDS. (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.

[0067] (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.

[0068] (3) After incubation, discard the original culture medium and wash once with PBS. Add the corresponding metagenic solution to the metagenic treatment group, and add an equal volume of cell culture medium to the control group and the model group. Incubate again for 24 hours.

[0069] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium to obtain a metabiotic solution (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of postbiotic prepared from the bacterial culture of CFU / ml is equivalent), and 2 mL of postbiotic solution prepared by Staphylococcus warwick CCSM005 (CCSM005-S) is added to each.

[0070] (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.

[0071] Table 1 Primer Sequences

[0072] 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 the expression level of FLG mRNA in the control group was 1, while the expression level in the model group decreased to 0.8 after SDS intervention. The postbiotic (cell lysate) prepared by Staphylococcus warwick CCSM005 significantly upregulated the expression level of FLG mRNA to 1.27.

[0073] Example 8: Effect of post-genetic agents prepared from Staphylococcus warwick CCSM005 on ZO-1 protein gene expression in HaCaT cells damaged by SDS. For specific implementation details, refer to Example 7. 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 ZO-1 , Occludin and CLDN The 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.

[0074] Table 2 Primer Sequences

[0075] ZO-1 (Zonula Occludens-1) is a tight junction protein that plays a crucial role in maintaining the integrity of the skin barrier. The skin barrier is an important line of defense against the invasion of harmful external substances and water loss. Tight junctions are specialized structures between epithelial cells that seal intercellular spaces, restricting the passage of water, ions, and pathogens. ZO-1 enhances the skin barrier's shielding function by connecting the cytoskeleton with other tight junction proteins. Specifically, ZO-1 expression is mediated by… Figure 5 As shown, the mRNA expression of ZO-1 protein in the model group decreased to 0.47, while the expression of ZO-1 protein was upregulated to 1.97 after partial repair by Staphylococcus warwickiana CCSM005 cell lysate.

[0076] Example 9: Effect of post-genetic agents prepared from Staphylococcus warwick CCSM005 on CLDN protein gene expression in HaCaT cells damaged by SDS. For specific implementation details, refer to Example 7. 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 CLDN 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.

[0077] Table 3 Primer Sequences

[0078] Claudins (CLDNs) are important members of the tight junction protein family and play a crucial role in maintaining skin barrier function. Tight junctions are a form of intercellular connection that controls the permeation of substances between cells, preventing water loss and the invasion of harmful external substances. CLDNs ensure the stability and selective permeability of the skin barrier by regulating intercellular permeability. Different types of CLDN proteins (such as CLDN-1 and CLDN-4) are expressed in the skin, contributing to maintaining skin hydration and protection. CLDN expression is influenced by… Figure 6 As shown, the mRNA expression of CLDN protein in the model group decreased to 0.65, while the expression of CLDN protein was upregulated to 1.52 after partial repair by Staphylococcus warwickiana CCSM005 cell lysate.

[0079] Example 10: Effect of post-genetic agents prepared from Staphylococcus warwick CCSM005 on the expression of Occludin protein gene in HaCaT cells damaged by SDS. For specific implementation details, refer to Example 7. 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 Occludin' 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.

[0080] Table 4 Primer Sequences

[0081] Occludin is an important transmembrane protein in tight junctions, playing a crucial role in maintaining the integrity of the skin barrier. Tight junctions regulate intercellular permeability by sealing intercellular spaces, protecting the skin from external stimuli and pathogens. Occludin interacts with other tight junction proteins, such as ZO-1 and Claudins, to help form and maintain tight junctions between cells, ensuring the structural and functional integrity of the skin barrier. Occludin expression is regulated by… Figure 7 As shown, the mRNA expression of Occludin protein in the model group decreased to 0.34, while after partial repair by Staphylococcus wartii CCSM005 cell lysate, the Occludin protein expression was upregulated to 1.69.

[0082] Example 11: Effective substance analysis of fermentation supernatant of Staphylococcus warwick CCSM005 1. Substance identification based on non-targeted metabolomics: Sample preparation and detection before CCSM005 fermentation supernatant metabolomics analysis: (1) After culturing Staphylococcus warwick CCSM005 for 12 h, take 1 mL of bacterial solution (the viable count is 1 x 10⁻⁶). 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-cooled 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).

[0083] 2. Sample preparation and detection before CCSM005 cell structure and metabolomics analysis: After culturing *Staphylococcus warwick* CCSM005 for 12 hours as described in step 1, take 1 mL of the bacterial culture and incubate at 4°C for 10,000 hours. 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).

[0084] 3. Analysis of active ingredients in CCSM005: 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.

[0085] 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.

[0086] Table 5 Potentially effective substances related to the skin barrier in CCSM003

[0087] Example 12: Quantitative detection of potential active substances in Staphylococcus warwick CCSM005 based on non-target metabolomics results The substances in CCSM005 were detected by liquid chromatography-mass spectrometry, and the detection method was the same as in Example 11.

[0088] Based on the results of non-targeted metabolomics, L-glycine-valla dipeptide was detected in the sample after targeted quantitative detection. Figures 8-10 As shown, this substance was detected only in the fermentation supernatant, at a concentration of 0.82 ppm. L-Glutava dipeptide is a small molecule dipeptide with significant barrier regulatory potential, capable of promoting skin barrier recovery through multiple mechanisms. On one hand, this dipeptide can effectively reduce the activation of signals such as NF-κB, IL-1β, and TNF-α in keratinocytes under inflammatory stimulation, alleviating barrier damage caused by inflammation. On the other hand, L-Glutava dipeptide can promote keratinocyte differentiation and the expression of structural proteins (such as FLG, LOR, and IVL), while enhancing the production of barrier lipids such as ceramides, thereby restoring the density and integrity of the stratum corneum. In addition, its regulation of cellular energy metabolism and oxidative stress pathways also helps improve the skin's self-repair ability after injury, making the barrier more stable and more resistant to external stimuli.

[0089] 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. A strain of Staphylococcus warwick ( Staphylococcus warneri The *Staphylococcus wartii* CCSM005 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GMDCC No: 64899.

2. The metabiotic prepared using *Staphylococcus wartii* CCSM005 as described in claim 1, characterized in that, The postgenetic includes (a) or (b): (a) The Staphylococcus wartii CCSM005 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 CCSM005 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.

3. A composition containing the postgenetic agent as described in claim 2.

4. The composition according to claim 3, characterized in that, The composition includes pharmaceuticals or daily chemical products.

5. The composition according to claim 4, characterized in that, The dosage forms of the composition include powders, tablets, blocks, liquid formulations, ointments, films, or gels.

6. The composition according to claim 5, characterized in that, The composition further includes conventional excipients, which include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

7. The composition according to claim 6, characterized in that, Daily chemical products include skin care products or washing and care products.

8. The use of Staphylococcus wartii CCSM005 and / or its postbiotics as described in claim 1 in the preparation of products for repairing the skin barrier.

9. The application as described in claim 8, characterized in that... The repair of the skin barrier includes repairing the stratum corneum and / or regulating the expression of skin barrier-related genes.

10. The use of Staphylococcus warwick CCSM005 as described in claim 1 in the preparation of L-glycine-valley dipeptide or products containing L-glycine-valley dipeptide.

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