Skin-sourced staphylococcus coriolis CCSM006 for improving skin health based on multiple target points and metagen of staphylococcus coriolis CCSM006

Abstract

The invention discloses a skin-derived staphylococcus coriolis CCSM006 for improving skin health based on multiple targets and a metagen thereof, and belongs to the field of microbial technologies and medicines. The staphylococcus coriolis CCSM006 provided by the invention shows a remarkable barrier repair effect in the aspect of external use, and the remarkable barrier repair effect is specifically shown as follows: in an in-vitro experiment, the activity of damaged HaCaT cells can be promoted, the gene and protein expression levels of functional proteins and structural proteins after damage are improved, and meanwhile, the expression of sphingomyelinase 1 (SMPD1) and tissue kinin enzyme 7 (KLK7) is remarkably improved. Research finds that the strain regulates the expression of skin barrier related proteins and enzymes through an AHR-OVOL1 pathway. The staphylococcus coriolis metagen has a wide application prospect in the field of cosmetics or medicines.

Description

Technical Field

[0001] This invention relates to a skin-derived Staphylococcus aureus strain CCSM006 that improves skin health based on multiple targets and its metabiotics, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Maintaining the skin barrier is crucial for human health. The skin barrier is the first line of defense against the external environment, preventing the invasion of harmful substances, pathogens, and allergens, and maintaining the body's water balance to prevent excessive water loss. A healthy skin barrier effectively protects the body from external stimuli, reducing the risk of infection and inflammation. Conversely, when the skin barrier is damaged, the skin becomes dry and sensitive, more susceptible to bacteria and other harmful substances, leading to skin problems such as eczema, dermatitis, and allergies. Furthermore, the integrity of the skin barrier is closely related to the health of the overall immune system; maintaining a healthy skin barrier not only contributes to the skin's appearance and comfort but is also an important aspect of maintaining overall health.

[0003] The skin's surface microbiome, including bacteria, fungi, and viruses, constitutes the skin's micro-ecosystem. These beneficial microorganisms, through synergistic interactions with skin cells, help maintain and repair the skin barrier. While the skin microecology plays a crucial role in maintaining skin barrier homeostasis, existing skin symbiotic strains isolated or applied in research still suffer from technical limitations such as limited functionality and unstable effects. On the one hand, many strains traditionally studied (such as Staphylococcus epidermidis and Propionibacterium) have limited bioactivity in regulating inflammation, promoting barrier lipid synthesis, and enhancing cell repair capabilities. Furthermore, their activity varies significantly among individuals and is greatly influenced by skin condition and environmental factors, making it difficult to guarantee stable barrier repair effects. On the other hand, some strains lack sufficient evidence in key areas such as responding to oxidative stress, repairing structural proteins, and promoting ceramide production, and their mechanisms of action are unclear, failing to meet the demands of cosmetics and skin health products for highly effective, safe, and controllable functional activities. In addition, many known strains have relatively simple metabolite profiles, failing to provide multi-dimensional support for barrier repair.

[0004] Therefore, it is necessary to continue screening for novel skin symbiotic strains with stronger barrier regulation capabilities, higher metabolic activity, and more stable ecological adaptability based on in-depth research on skin microbiota, in order to make up for the limitations of existing strains and promote the development of skin barrier repair technology. Summary of the Invention

[0005] This invention provides a Staphylococcus aureus ( Staphylococcus cohnii The application of CCSM006 and its post-genes in the preparation of products that repair the skin barrier.

[0006] This invention provides a strain of Staphylococcus aureus ( Staphylococcus cohniiThe *Staphylococcus coli* strain described in CCSM006 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 64900.

[0007] In one embodiment, the Staphylococcus coli CCSM006 is derived from the skin of a healthy person; the colonies of the Staphylococcus coli CCSM006 on TSB solid medium are raised, pale yellow, smooth, and round.

[0008] The present invention also provides a metabiotic prepared using the aforementioned Staphylococcus coli CCSM006.

[0009] In one embodiment, the metabiotic contains oleamide and / or L-glycine-valpy dipeptide.

[0010] In one embodiment, the metabiotic includes bacterial lysate, inactivated or dead cells, fermentation supernatant, or a powder prepared by drying one or more of the above components. In one embodiment, the inactivated or deadened cells are prepared by culturing the Staphylococcus aureus CCSM006 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℃, 30min.

[0011] In one embodiment, the method for preparing the bacterial lysate is as follows: Staphylococcus aureus CCSM006 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 the bacterial lysate.

[0012] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging Staphylococcus aureus CCSM006 after culturing it in a culture medium for a period of time.

[0013] The present invention also provides compositions containing the aforementioned Staphylococcus coli CCSM006 and / or its postbiotic.

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

[0015] In one embodiment, the drug includes the postbiotic and conventional excipients.

[0016] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0017] In one embodiment, the composition is a probiotic powder.

[0018] In one embodiment, the bacterial powder is a solid powder prepared by drying the prepared liquid postbiotic of Staphylococcus coli CCSM006.

[0019] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0020] The present invention also provides the use of the composition in the preparation of pharmaceuticals or cosmetics for repairing the skin barrier.

[0021] In one embodiment, the drug or cosmetic has at least one of the following effects: (1) Enhance the cell activity of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (2) In vitro, the expression of barrier function protein genes in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage was increased; (3) In vitro, the expression of barrier structure protein genes in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage was increased; (4) Increase the expression of barrier function proteins in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (5) Reduced the expression of structural proteins in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (6) Increase the expression of sphingomyelinase 1 (SMPD1) in keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (7) Reduced tissue kallikrein 7 (KLK7) expression in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro.

[0022] In one implementation, the application method is local external application.

[0023] In one embodiment, the amount of Staphylococcus aureus CCSM006 and the prepared postbiotic in the drug or cosmetic is not less than 1 × 10⁻⁶ of the corresponding viable bacterial count. 7 CFU / mL.

[0024] In one embodiment, the drug comprises the Staphylococcus aureus CCSM006, a drug carrier, and / or pharmaceutical excipients.

[0025] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0026] 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-flocculators, filter aids, or release inhibitors.

[0027] In one embodiment, the cosmetic contains Staphylococcus aureus CCSM006, matrix ingredients, and / or conventional excipients.

[0028] 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.

[0029] 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.

[0030] The present invention also provides the use of the aforementioned Staphylococcus aureus CCSM006 in the preparation of oleic acid amide and / or L-glycine-valley dipeptide.

[0031] Beneficial effects: Staphylococcus coli of the present invention ( Staphylococcus cohnii The post-biotic prepared by CCSM006 has 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 barrier function proteins in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (3) Increase the mRNA expression of barrier structural proteins in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (4) Increase the expression of barrier function proteins in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (5); In vitro reduction of barrier structural protein expression in keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage. (6) Increase the expression of SMPD1 in keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) injury in vitro; (7) Reduced KLK7 expression in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (8) Regulate the AHR-OVOL1 pathway receptor to regulate the expression of related proteins and thus restore skin barrier function.

[0032] Therefore, Staphylococcus coli ( Staphylococcus cohnii The post-biotic prepared by CCSM006 has great application potential in products that alleviate damage to the host skin barrier.

[0033] Preservation of biological materials Staphylococcus coli ( Staphylococcus cohnii CCSM006, taxonomically named Staphylococcus cohnii It was deposited on July 22, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64900, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0034] Figure 1 To evaluate the effects of different concentrations of SDS on HaCaT cell damage.

[0035] Figure 2 The effect of post-genetics on HaCaT cell proliferation.

[0036] Figure 3 The effect of post-genetic repair on HaCaT cell viability after SDS damage.

[0037] Figure 4 Gene expression of HaCaT cell barrier function proteins that promote post-serogenetic repair of SDS-damaged cells.

[0038] Figure 5 Gene expression of HaCaT cell barrier structural proteins that promote the repair of SDS damage by metagenes.

[0039] Figure 6 The expression of HaCaT cell barrier function proteins for post-genetic repair of SDS-damaged cells.

[0040] Figure 7 The expression of barrier structural proteins in HaCaT cells after SDS damage is improved by metagenesis.

[0041] Figure 8 To promote the expression of SMPD1 in HaCaT cells after SDS damage by metagenesis.

[0042] Figure 9 To promote the repair of KLK7 expression in HaCaT cells after SDS damage by metagenes.

[0043] Figure 10The metagener used AHR-OVOL1 to repair HaCaT cells damaged by SDS.

[0044] Figure 11 This is a liquid chromatography-mass spectrometry (LC-MS) diagram of oleamide standard.

[0045] Figure 12 This is a liquid chromatography-mass spectrometry (LC-MS) graph showing the oleic acid amide content in the bacterial cell sample.

[0046] Figure 13 This is a comparison chart of retention times for oleic acid amide standards and bacterial samples.

[0047] Figure 14 This is a liquid chromatography-mass spectrometry (LC-MS) image of the L-glycine-valine dipeptide standard.

[0048] Figure 15 Liquid chromatography-mass spectrometry (LC-MS) plot of L-glycine-valve dipeptide content in fermentation supernatant sample.

[0049] Figure 16 A comparison chart showing the retention times of L-glycine-valine standard and fermentation supernatant samples. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments.

[0051] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (the mixed solution contains 10,000 U / mL penicillin and 10 mg / mL streptomycin).

[0056] 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 °C incubator containing 5% CO2. When the cells regain viability and reach 70%-80% confluence after 1-2 days, passage the cells.

[0057] Example 2: Isolation and screening of Staphylococcus coli 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 solid medium. The culture was incubated at 37°C for 48 hours. Pale yellow, round colonies were picked 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 picked 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 coli*, and named *Staphylococcus coli* CCSM006.

[0058] Example 3: Preparation of Staphylococcus coli metabiotic Staphylococcus coli CCSM006 was streaked from the preservation tube and revived. It was then cultured on TSB solid medium in a 37°C water-jacketed incubator for 48 h to obtain single colonies. Single colonies were picked and inoculated into TSB liquid medium and cultured at 37°C for 12-18 h to obtain culture medium 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.

[0059] Centrifuge bacterial suspension a at 8000 r / min for 30 min and collect the supernatant, which is designated CCSM006-Q. Collect bacterial sludge and resuspend it in double-distilled water at 75% of the original bacterial suspension volume. Heat-treat the resuspended suspension (65 ℃, 30 min) and then homogenize it under high pressure (1000 MPa, 10 times). After homogenization, centrifuge at 8000 r / min for 30 min and collect the supernatant to obtain bacterial cell lysate, which is designated CCSM006-S.

[0060] Optionally, the supernatant or lysis buffer prepared above is freeze-dried at -10℃ to -50℃ to prepare post-biotic freeze-dried powder of Staphylococcus coli CCSM006.

[0061] 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.

[0062] (2) The above well plates were incubated in an incubator at 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.

[0063] 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%.

[0064] (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.

[0065] Example 5: Safety verification of postbiotics prepared from Staphylococcus aureus in 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 them 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.

[0066] 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 of CFU / ml is equivalent to that prepared from bacteria of Staphylococcus aureus CCSM006. 100 μL of metabiotic prepared from Staphylococcus aureus CCSM006 was added.

[0067] (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.

[0068] 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 99.13%), the addition of post-genetic agents CCSM006-S or CCSM006-Q of Staphylococcus aureus CCSM006 at an inactivated bacterial concentration of 1×10⁻⁶ significantly improved cell proliferation. 7 The cell proliferation rates at CFU / ml were 99.4% and 99.5%, respectively.

[0069] According to the ISO 10993-5:2006 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 cell viability was above 95% at postbiotic concentrations of CFU / ml. Considering its non-cytotoxicity, an inactivated cell concentration of 1.0 × 10⁻⁶ was selected. 7 CFU / ml is a suitable postbiotic concentration for subsequent cell experiments.

[0070] Example 6: Effect of post-biotics prepared from Staphylococcus aureus 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 10 cells / well was seeded into a 96-well plate, with the outermost ring filled with PBS solution to prevent edge effects, and cultured for 36 h to allow the cells to adhere. (2) The well plates prepared in step (1) were incubated in an incubator at 37 °C for 6 h, and control group, model group and treatment group were set up respectively: Control group: Contains cell culture medium and HaCaT cells, without post-genetic treatment, and without SDS modeling agent; Model group: The cell medium prepared in step (1) was changed to 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.

[0071] (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 cell culture medium to the control group and model group. Incubate again for 24 hours.

[0072] In the metabiotic treatment group, the metabiotics CCSM006-S and CCSM006-Q were resuspended in cell culture medium, and the amount of resuspended metabiotics was adjusted to a fermentation concentration of 1.0 × 10⁻⁶. 7 The amounts of postbiotics prepared from bacterial cultures of CFU / ml were equivalent, and 100 μL of postbiotics CCSM006-S and CCSM006-Q prepared from Staphylococcus aureus were added respectively.

[0073] (4) Add 10 μL of CCK8 solution to each well and incubate for 1.5 h to measure the absorbance (OD) at 450 nm.

[0074] 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%.

[0075] 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 102.28%), the cell viability of the model group was 66.53%, indicating that SDS modeling caused significant damage to HaCaT cells.

[0076] After adding CCSM006-S and CCSM006-Q to the treatment groups, the cell viability was 82.16% and 68.4%, respectively. Compared with the model group (66.53%), CCSM006-S could improve the viability of HaCaT cells, indicating that Staphylococcus aureus CCSM006 (cell lysate fraction) can effectively alleviate the damage to HaCaT cells caused by SDS. Therefore, subsequent investigations will focus on the efficacy of Staphylococcus aureus CCSM006-S.

[0077] Example 7: Effect of postbiotics prepared from Staphylococcus aureus CCSM006 on the expression of barrier function protein genes in SDS-damaged HaCaT cells.

[0078] The specific implementation method is the same as in Example 6; the difference is that after step (3) incubation, the original culture medium is discarded and washed once with PBS. The corresponding metagenic sample is added to the metagenic treatment group, and cell culture medium is added to the control group and the model group. The cells are incubated again for 24 hours.

[0079] In the epigenetic treatment group, the epigenetic elements were resuspended in cell culture medium (the amount of resuspended epigenetic elements was the same as that fermented to a concentration of 1×10⁻⁶). 7 The amount of metabiotic prepared from the bacterial culture at CFU / ml was equivalent, and 2 mL of metabiotic prepared from Staphylococcus coli (CCSM006-S) was added to each culture.

[0080] 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. Gene expression in HaCaT cells is detected using real-time quantitative PCR. -△△Ct Formula calculation FLG, IVL and AQP3 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.

[0081] Table 1 Primer Sequences

[0082] 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 0.22 after SDS intervention. The postbiotic (cell lysate) prepared by Staphylococcus coli CCSM006 significantly upregulated the expression level of FLG mRNA to 2.37.

[0083] 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 4 It can be seen that the expression level of IVL mRNA in the control group was about 1, while the expression level in the model group decreased to 0.17 after SDS intervention. The lysate of Staphylococcus aureus CCSM006 can increase the expression level of IVL to 6.53.

[0084] LOR (Leydigin of Ochratoxin) plays a crucial role in keratinization, helping to form a tough, water-insoluble outer membrane—the keratin capsule—which is essential for maintaining the integrity of the skin barrier. Furthermore, it helps maintain skin hydration, keeping the skin soft and elastic through synergistic effects with other keratinocyte differentiation proteins. If LOR protein is defective or absent, the skin barrier function is weakened, making the skin more susceptible to external stimuli. Aberrant expression or loss of function of LOR is associated with several skin diseases, such as ichthyosis and certain types of keratosis. LOR expression is determined by… Figure 4 It can be seen that the expression level of LOR mRNA in the control group was about 1, while the expression level in the model group decreased to 0.54 after SDS intervention. The lysate of Staphylococcus aureus CCSM006 cells could increase the expression level of LOR to 8.63.

[0085] Example 8: Effect of postbiotics prepared from Staphylococcus aureus CCSM006 on the expression of barrier structural protein genes in SDS-damaged HaCaT cells.

[0086] The specific implementation method is the same as in Example 6, except that after step (3) incubation, the original culture medium is discarded and washed once with PBS. The corresponding metagenic sample is added to the metagenic treatment group, and cell culture medium is added to the control group and the model group. The cells are incubated again for 24 hours.

[0087] In the epigenetic treatment group, the epigenetic elements were resuspended in cell culture medium (the amount of resuspended epigenetic elements was the same as that fermented to a concentration of 1×10⁻⁶). 7 The amount of metabiotic prepared from the bacterial culture at CFU / ml was equivalent, and 2 mL of metabiotic CCSM006-S prepared from Staphylococcus coli (CCSM006-S) was added to each culture.

[0088] 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. Gene expression in HaCaT cells is 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 2 below, and the results are as follows: Figure 5 As shown.

[0089] Table 2 Primer Sequences

[0090] 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. ZO-1, Occludin, and CLDN are core components of tight junctions and are 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, CLDN, and Occludin is influenced by… Figure 5 As shown, the mRNA expression of these three proteins in the model group decreased to 0.48, 0.50 and 0.34, respectively. After repair by Staphylococcus aureus CCSM006 bacterial lysate, the expression of these three proteins could be upregulated to different degrees, to 2.67, 3.25 and 1.86, respectively.

[0091] Example 9: Effect of post-genetic agents prepared from Staphylococcus aureus CCSM006 on the expression of barrier function proteins in SDS-damaged HaCaT cells.

[0092] The specific implementation method is the same as in Example 6, except that after step (3) incubation, the original culture medium is discarded and washed once with PBS. The corresponding metagenic sample is added to the metagenic treatment group, and cell culture medium is added to the control group and the model group. The cells are incubated again for 24 hours.

[0093] In the epigenetic treatment group, the epigenetic elements were resuspended in cell culture medium (the amount of resuspended epigenetic elements was the same as that fermented to a concentration of 1×10⁻⁶). 7 (The amount of postbiotic prepared from bacterial culture of CFU / ml is equivalent), and 2 mL of postbiotic CCSM006-S prepared from Staphylococcus coli CCSM006 was added to each.

[0094] After incubation, the culture supernatant was collected, and the expression levels of FLG, IVL, and LOR were calculated using the Sempercap ELISA kit via enzyme-linked adsorption reaction. The results are as follows: Figure 6 As shown.

[0095] The levels of FLG protein in the control group were 6610.62 pg / mL, while those in the model group were significantly reduced to 5548.12 pg / mL. After treatment with CCSM006-S, the levels increased to 6176.04 pg / mL. The levels of IVL protein in the control group were 1242.91 pg / mL, while those in the model group were significantly reduced to 1036.71 pg / mL. After treatment with CCSM006-S, the levels increased to 1144.77 pg / mL. The levels of LOR protein in the control group were 833.9 μg / mL, while those in the model group were significantly reduced to 736.62 μg / mL. After treatment with CCSM006-S, the levels increased to 814.51 μg / mL.

[0096] Example 10: Effect of post-genetic agents prepared from Staphylococcus aureus CCSM006 on the expression of barrier structural proteins in SDS-damaged HaCaT cells.

[0097] The specific implementation method is the same as in Example 6, except that after step (3) incubation, the original culture medium is discarded and washed once with PBS. The corresponding metagenic sample is added to the metagenic treatment group, and cell culture medium is added to the control group and the model group. The cells are incubated again for 24 hours.

[0098] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium, and the amount of resuspended metabiotics was adjusted to a concentration of 1×10⁻⁶ after fermentation. 7 The amount of postbiotic prepared from the bacterial culture of CFU / ml is equivalent to that of 2 mL of postbiotic CCSM006-S prepared from Staphylococcus aureus CCSM006.

[0099] After incubation, the culture supernatant was collected, and the expression level of ZO-1 was calculated using the Sempercapto ELISA kit via enzyme-linked adsorption reaction. The results are as follows: Figure 7 As shown.

[0100] The ZO-1 protein content in the control group was 96.9 pg / mL, while the content in the model group decreased significantly to 78.7 pg / mL. After treatment with CCSM006-S, the content increased to 90.77 pg / mL, which was 1.02 times higher than that in the model group.

[0101] Example 11: Effect of postbiotic prepared from Staphylococcus aureus CCSM006 on the repair of SMPD1 expression in SDS-damaged HaCaT cells.

[0102] The specific implementation method is the same as in Example 6, except that after step (3) incubation, the original culture medium is discarded and washed once with PBS. The corresponding metagenic sample is added to the metagenic treatment group, and cell culture medium is added to the control group and the model group. The cells are incubated again for 24 hours.

[0103] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium, and the amount of resuspended metabiotics was adjusted to a concentration of 1×10⁻⁶ after fermentation. 7 The amount of postbiotic prepared from the bacterial culture of CFU / ml is equivalent to that of 2 mL of postbiotic CCSM006-S prepared from Staphylococcus aureus CCSM006.

[0104] After incubation, the culture supernatant was collected, and the expression level of SMPD1 was calculated using the Smbega ELISA kit via enzyme-linked adsorption reaction. The results are as follows: Figure 8 As shown.

[0105] Sphingomyelinase 1 (SMPD1) plays a crucial role in maintaining skin barrier function. It breaks down sphingomyelin to generate ceramides, a lipid molecule that functions vitally in the skin barrier. In the control group, the SMPD1 level was 264.27 μg / mL, while in the model group it significantly decreased to 208.38 μg / mL. Treatment with CCSM006-S increased the level to 233.93 μg / mL.

[0106] Example 12: Effect of post-genetic repair of KLK7 expression in HaCaT cells damaged by SDS using Staphylococcus aureus CCSM006.

[0107] (1) HaCaT cells were injected at a concentration of 1.8 × 10⁻⁶. 4 10 cells / well were seeded into a 6-well plate and cultured for 36 hours until the cells adhered. (2) The well plates prepared in step (1) were incubated in an incubator at 37 °C for 6 h, and control group, model group and treatment group were set up respectively: Control group: The cells prepared in step (1) were changed in medium and contained cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent; Model group: The cell medium prepared in step (1) was changed to 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.

[0108] (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 cell culture medium to the control group and model group. Incubate again for 24 hours.

[0109] In the epigenetic treatment group, the epigenetic elements were resuspended in cell culture medium (the amount of resuspended epigenetic elements was the same as that fermented to a concentration of 1×10⁻⁶). 7 (The amount of postbiotic prepared from bacterial culture of CFU / ml is equivalent), and 2 mL of postbiotic CCSM006-S prepared from Staphylococcus coli CCSM006 was added to each.

[0110] (4) After incubation, the culture supernatant was collected, and the expression level of SMPD1 was calculated by enzyme-linked adsorption reaction using the Smbega ELISA kit. The results are as follows: Figure 9 As shown.

[0111] Tissue kallikrein 7 (KLK7) is a serine protease that plays a crucial role in skin barrier function. It primarily participates in the degradation of intercellular junctions in the stratum corneum, a process essential for stratum corneum shedding and renewal. Excessive KLK7 activity leads to excessive stratum corneum degradation, weakening the skin barrier and resulting in increased skin moisture loss and sensitivity to external stimuli. In this study, the KLK7 level in the control group was 16.02 ng / mL, while the level in the model group significantly increased to 19.27 ng / mL. Treatment with CCSM006-S reduced the level to 15.78 g / mL, a 1.22-fold increase compared to the model group.

[0112] Example 13: Effect of postgenetic agents prepared from Staphylococcus aureus CCSM006 on the expression of AHR and OVOL1 receptors in SDS-damaged HaCaT cells.

[0113] (1) HaCaT cells were injected at a concentration of 1.8 × 10⁻⁶. 4 10 cells / well were seeded into a 6-well plate and cultured for 36 hours until the cells adhered. (2) The well plates prepared in step (1) were incubated in an incubator at 37 °C for 6 h, and control and treatment groups were set up respectively: Control group: After changing the medium, the cells prepared in step (1) contained cell culture medium and HaCaT cells. Post-genetic treatment group: After changing the medium of the cells prepared in step (1), the culture medium containing cell culture and HaCaT cells was added. (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 cell culture medium to the control group. Incubate again for 24 hours.

[0114] In the epigenetic treatment group, the epigenetic elements were resuspended in cell culture medium (the amount of resuspended epigenetic elements was the same as that fermented to a concentration of 1×10⁻⁶). 7 The amount of metabiotic prepared from bacterial culture with CFU / ml was equivalent, and 2 mL of metabiotic prepared from Staphylococcus coli CCSM006 (CCSM006-S) was added to each culture.

[0115] (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 AHR and OVOL1 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 10 As shown.

[0116] Table 3: Primer sequences

[0117] The AHR signaling pathway plays a crucial role in maintaining the integrity of the skin barrier. Decreased AHR expression levels increase skin barrier permeability, leading to a higher risk of infection and more pronounced pathological features in atopic dermatitis models. OVOL1 is a transcription factor responsible for regulating the expression of genes related to skin barrier function. Studies have shown that OVOL1 is a direct transcriptional target of AHR, and its expression affects AHR's ability to regulate related genes in keratinocytes. The absence of OVOL1 in keratinocytes weakens the promoting effect of AHR on skin barrier function. Figure 9 The results show that the bacterial lysate of CCSM006 can significantly upregulate the gene expression of AHR and OVOL1, upregulating to 1.85 and 1.68, respectively, thus activating the expression of this pathway.

[0118] Example 14: Effective substance analysis of fermentation supernatant of Staphylococcus aureus CCSM006 1. Substance identification based on non-targeted metabolomics: Metabolomics analysis of CCSM006 fermentation supernatant: (1) After culturing the strain in TSB liquid medium at 37°C for 12 h, take 1 mL of the bacterial solution and incubate at 4°C for 10,000 hours. 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).

[0119] 2. Sample preparation and detection before CCSM006 bacterial cell structure and metabolomics analysis: After culturing the strain for 12 h using the method described above, take 1 mL of bacterial solution and incubate at 4℃ 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).

[0120] 3. Analysis of active ingredients in CCSM006: 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.

[0121] Literature review was conducted to collect the chemical formulas, molecular formulas, and molecular weights of potential effective substances in CCSM008. 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 4.

[0122] Table 4. Potentially effective substances related to the skin barrier in CCSM006

[0123] Example 15: Quantitative detection of potential active substances in Staphylococcus aureus CCSM006 based on non-target metabolomics results The substances in CCSM006 were detected using liquid chromatography-mass spectrometry (LC-MS), and the detection method was the same as in Example 14. Based on the results of non-targeted metabolomics, the following substances were detected in the sample after targeted quantitative detection: (1) Oleamide like Figures 11-13 As shown, this substance was detected only in bacterial cells, at a concentration of 90.3 ppb. Oleamide is a class of endogenous fatty amide molecules, which has recently been considered a potential active ingredient for regulating skin barrier homeostasis. In vitro studies in primary human keratinocyte and immune cell models have confirmed that oleamide can significantly inhibit the production of inflammatory factors such as TNF-α and IL-1β, thereby reducing the damage to the skin barrier caused by the inflammatory microenvironment. Simultaneously, oleamide can exert immunomodulatory and homeostatic effects by regulating the PPAR signaling pathway and CB2 receptor.

[0124] In terms of barrier repair, oleamides have been shown to promote the production of key barrier lipids in keratinocytes, including ceramides, which helps restore and optimize the lamellar structure of the stratum corneum lipids, enhancing the integrity and stability of the sebum film. By improving the skin's resistance to external stimuli (such as dryness, microbial invasion, and inflammatory factors), oleamides demonstrate significant application potential in skin health maintenance and barrier function repair.

[0125] (2) L-gamma-valpy dipeptide like Figures 14-16 As shown, this substance was detected only in the fermentation supernatant, at a concentration of 0.81 ppm. L-Glucotrol is a class of bioactive functional dipeptides that can promote the repair and homeostasis of the skin barrier through multiple pathways. Studies have shown that this dipeptide can significantly enhance the vitality of keratinocytes and promote the expression of key barrier structural proteins (such as filaggrin, loricrin, and involucrin), thereby enhancing the structural integrity of the stratum corneum from its source. In addition, L-Glucotrol can regulate cellular inflammatory responses and inhibit the production of inflammatory factors such as TNF-α and IL-6, thereby reducing persistent inflammatory stimulation caused by barrier damage. It can also promote the synthesis of barrier lipids such as ceramides, strengthen the skin lipid matrix, reduce transepidermal water loss (TEWL), and improve skin tolerance and repair capacity. Overall, L-Glucotrol has a significant effect on improving damaged skin barriers through a multidimensional mechanism of "enhancing structure, regulating inflammation, and strengthening lipids."

[0126] 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 aureus ( Staphylococcus cohnii Accession number CCSM006 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 22, 2024, with accession number GDMCC NO: 64900.

2. The postbiotic prepared using the Staphylococcus coli CCSM006 as described in claim 1.

3. The epigenetic agent according to claim 2, characterized in that, It contains oleamide and / or L-glycine-valpy dipeptide.

4. The epigenetic agent according to claim 2 or 3, characterized in that, The metabiotic includes bacterial lysate, inactivated or dead cells, fermentation supernatant, or powder prepared by drying one or more of the above components.

5. The epigenetic agent according to claim 4, characterized in that, The inactivated or deactivated cells are prepared as follows: Staphylococcus aureus CCSM006 is cultured in a culture medium for a period of time, bacterial cells in the cell culture medium are collected, and inactivated bacterial cells are obtained after heat treatment.

6. The epigenetic agent according to claim 4, characterized in that, The method for preparing the bacterial lysate is as follows: Staphylococcus aureus CCSM006 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 the bacterial lysate.

7. A composition containing Staphylococcus coli CCSM006 as described in claim 1 and / or its postbiotic, characterized in that, The composition includes, but is not limited to, pharmaceuticals or daily chemical products.

8. The use of the post-genetic agent according to any one of claims 2 to 6 in the preparation of a medicament or cosmetic for repairing the skin barrier.

9. The application according to claim 8, characterized in that, The application includes at least one of the following functions: (1) Enhance the cell activity of keratinocytes in the skin after injury; (2) Increase the expression of barrier function protein genes in keratinocytes of the skin after injury; (3) Increase the expression of barrier structure protein genes in keratinocytes of the skin after injury; (4) Increase the expression of barrier function proteins in keratinocytes of the skin after injury; (5) Reduces the expression of structural proteins in keratinocytes of the skin after injury; (6) Increase the expression of sphingomyelinase 1 in keratinocytes of damaged skin; (7) Reduces tissue kininase 7 expression in keratinocytes of the skin after injury.

10. The use of Staphylococcus aureus CCSM006 as described in claim 1 in the preparation of oleic acid amide and / or L-glycine-valley dipeptide.

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