Sphingomonas CCSM001 separated from human skin and capable of improving skin health and metagen of sphingomonas CCSM001

Abstract

The invention discloses sphingomonas CCSM001 separated from human skin and capable of improving skin health and a metagen thereof, and belongs to the field of microbial technology and medicine. The sphingomonas CCSM001 provided by the invention has a good barrier function repairing effect and application prospect in the aspect of external use, and is specifically shown in that the activity of damaged HaCaT cells is improved in vitro, the gene level expression of FLG, IVL, AQP3, ZO-1, Occludin and CLDN is improved in the aspects of functional protein and structural protein after damage. The metagen prepared from the sphingomonas can be used for preparing cosmetics or medicines for repairing skin barriers, and has a huge application prospect.

Description

Technical Field

[0001] This invention relates to a strain of Sphingosine monocytogenes CCSM001 isolated from human skin that improves skin health and its metabiotics, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The skin barrier is the outermost protective layer of the skin, mainly composed of the stratum corneum, tight junction proteins, and the sebum film. Its primary function is to protect the body from external environmental damage, preventing the invasion of harmful substances, bacteria, and allergens, while also preventing the loss of body fluids. A healthy skin barrier maintains skin hydration and elasticity, regulates temperature, and resists external stimuli. Any damage to or dysfunction of the skin barrier can lead to dry, sensitive skin and increase the risk of infection and disease. Therefore, the skin barrier plays a crucial role in maintaining skin health and overall physiological function.

[0003] Furthermore, a healthy skin microbiome is also related to the skin barrier's repair capacity. However, existing research on skin microbes has limitations in physiological function, insufficient activity, or unclear mechanisms of action, making it difficult to effectively achieve the goals of skin barrier repair, cell regeneration promotion, and overall skin condition improvement. The stability and bioactivity of their metabolites also vary considerably. Therefore, existing microbial resources are insufficient to meet the demands for efficient, safe, and controllable skin repair applications. Summary of the Invention

[0004] This invention provides a sphingosomal bacterium (Sphingosomalmonella) Sphingomonas sp . The application of CCSM001 and its post-genes in the preparation of products that repair the skin barrier.

[0005] This invention provides a strain of Sphingosine monocytogenes (SMP) Sphingomonas sp . Accession number CCSM001 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 22, 2024, with accession number GDMCC NO: 64896.

[0006] In one embodiment, the sphingomonas CCSM001 is derived from the skin of a healthy person; the colonies of the sphingomonas CCSM001 on R2A solid medium are raised, yellow, smooth, and round.

[0007] The present invention also provides a metabiotic prepared using the aforementioned Sphingomonas CCSM001.

[0008] In one embodiment, the metagenerium contains D-erythrosine and / or oleamide.

[0009] In one embodiment, the metabiotic includes bacterial lysate, inactivated or dead cells, fermentation supernatant, or a powder prepared by drying any of the above components. In one embodiment, the inactivated or dead cells are prepared by culturing the Sphingomonas CCSM001 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.

[0010] In one embodiment, the method for preparing the bacterial lysate is as follows: the Sphingomonas CCSM001 is cultured in a culture medium for a period of time, the bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate.

[0011] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging *Sphingomonas CCSM001* after culturing it in a culture medium for a period of time.

[0012] The present invention also provides compositions containing the aforementioned Sphingomonas CCSM001 and / or its metagenes.

[0013] In one embodiment, the composition includes, but is not limited to, pharmaceuticals or daily chemical products.

[0014] In one embodiment, the drug comprises the postbiotic and conventional excipients.

[0015] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0016] In one embodiment, the composition is a probiotic powder.

[0017] In one embodiment, the bacterial powder is a solid powder prepared by drying the prepared liquid metabiotic Sphingomonas CCSM001.

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

[0019] The present invention also provides the use of the composition in the preparation of pharmaceuticals or cosmetics for repairing the skin barrier.

[0020] 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 filaggrin gene in keratinocytes (HaCaT) of the skin after sodium dodecyl sulfate (SDS) injury was increased; (3) In vitro, the expression of aquaporin gene in keratinocytes (HaCaT) of the skin after sodium dodecyl sulfate (SDS) injury was increased; (4) Increase the expression of inner lining protein gene in keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (5) In vitro, the expression of tight junction proteins (ZO-1, OCCLUDIN and CLDN) in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) injury was increased.

[0021] In one embodiment, the drug or cosmetic is applied topically.

[0022] In one embodiment, the amount of Sphingomonas CCSM001 and the prepared postbiotic in the product is not less than 1 × 10⁻⁶ of the corresponding viable bacterial count. 7 CFU / mL.

[0023] In one embodiment, the drug comprises the sphingomonas CCSM001, a drug carrier, and / or pharmaceutical excipients.

[0024] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0025] 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.

[0026] In one embodiment, the cosmetic contains Sphingomonas CCSM001 postbiotic, matrix ingredients, and / or conventional excipients.

[0027] 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.

[0028] 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.

[0029] The present invention also provides the use of the aforementioned Sphingomonas CCSM001 in the preparation of D-erythrosphine and / or oleamide.

[0030] Beneficial effects: The Sphingosine Monoclonal strain of the present invention ( Sphingomonas sp . The post-biotic prepared by CCSM001 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 in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (3) Increase the mRNA expression of IVL in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) injury in vitro; (4) Increase the mRNA expression of AQP3 in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (5) Increase the mRNA expression of tight junction protein in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; Therefore, Sphingosomalmonella ( Sphingomonas sp . The post-biotic prepared by CCSM001 has great application potential in products that alleviate damage to the host skin barrier.

[0031] Preservation of biological materials Sphingosomalidone (Sphingosomalidone) Sphingomonas sp . CCSM001, taxonomically named Sphingomonas sp . It was deposited on July 22, 2024, at the Guangdong Provincial Center for the Preservation of Microbial Cultures, with accession number GDMCC No: 64896, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0032] Figure 1 Phylogenetic tree of housekeeping genes for Sphingosine Monoclonal sphingolipids CCSM001.

[0033] Figure 2 Evaluation of the effects of different concentrations of SDS on HaCaT cell damage Figure 3 Effects of metageners on HaCaT cell proliferation Figure 4The effect of metagenes on HaCaT cell viability after SDS damage repair Figure 5 The effect of post-genetic repair of SDS-related FLG mRNA expression in HaCaT Figure 6 Effect of post-genetic repair of SDS-induced damage on IVL mRNA expression in HaCaT Figure 7 The effect of post-genetic repair of SDS-damaged AQP3 mRNA expression in HaCaT Figure 8 The effect of post-genetic repair of tight junction mRNA expression in HaCaT Figure 9 This is a liquid chromatography-mass spectrometry (LC-MS) diagram of sphingosine standard.

[0034] Figure 10 This is a liquid chromatography-mass spectrometry (LC-MS) graph showing the sphingosine content in the fermentation supernatant sample.

[0035] Figure 11 This is a liquid chromatography-mass spectrometry (LC-MS) graph showing the sphingosine content in the bacterial cell sample

[0036] Figure 12 This is a comparison chart of retention times for sphingosine standards, fermentation supernatant, and cell samples.

[0037] Figure 13 This is a liquid chromatography-mass spectrometry (LC-MS) diagram of oleamide standard.

[0038] Figure 14 The liquid chromatography-mass spectrometry (LC-MS) plot shows the oleic acid amide content in the fermentation supernatant sample.

[0039] Figure 15 This is a liquid chromatography-mass spectrometry (LC-MS) graph showing the oleic acid amide content in the bacterial cell sample

[0040] Figure 16 This is a comparison chart of retention times for oleic acid amide standards, fermentation supernatant, and cell samples. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments.

[0042] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0043] The culture media involved in the following examples are as follows: R2A liquid medium: 0.5g yeast extract, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.024g anhydrous magnesium sulfate, and 0.3g sodium pyruvate, with a final pH of 7.3±0.2.

[0044] R2A solid medium: 0.5g yeast extract, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.024g anhydrous magnesium sulfate, 0.3g sodium pyruvate, and 20g / L agar. The final pH value is 7.3±0.2.

[0045] 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).

[0046] 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.

[0047] Example 2: Isolation and screening of Sphingosine monocytogenes The bacterial strain samples were derived from healthy human skin. Using sterile swabs dipped in physiological saline, the samples were repeatedly smeared three times on the back of the subjects' hands before being streaked directly onto R2A solid medium for isolation and incubated at 37 °C for 48 h. After incubation, yellow, round colonies were picked from the grown colonies and inoculated into R2A liquid medium for enrichment culture for 16 h. Subsequently, bacterial culture was taken using an inoculation loop and streaked again onto R2A solid medium for purification culture for 48 h. Single colonies were picked and inoculated into R2A liquid medium for enrichment culture. After incubation, 30% glycerol was added for low-temperature preservation to obtain the target bacterial strain.

[0048] The purified strain CCSM001 was inoculated into R2A medium at a 2% (v / v) inoculum and continuously passaged to the third generation. Mycelial sludge was collected and sent to Shanghai Meiji Biomedical Technology Co., Ltd. for draft genome sequencing. Phylogenetic analysis based on housekeeping genes (…) Figure 1 The results showed that this strain was related to Sphingomonas sanguinis 98.3% Sphingomonas sanguinis (98.2%) Sphingomonas parapaucimobilis (97.9%) Sphingomonas excrementigallinarum (97.9%) and Sphingomonas paucimobilis(97.7%) showed a high phylogenetic relationship, indicating that CCSM001 may be a potential new species of the genus *Sphingosporium*. Genomic DNA was further extracted from this strain, and the 16S rDNA gene was amplified and sequenced (performed by Suzhou Genewise Biotechnology Co., Ltd., nucleotide sequence shown in SEQ ID NO.1). Sequence alignment analysis using the NCBI database confirmed that this strain belongs to the genus *Sphingosporium*, and it was named *Sphingosporium*. Sphingomonas sp. CCSM001.

[0049] Example 3: Preparation of postbiotics from Sphingomonas CCSM001 (1) Sphingomonas CCSM001 was streaked from the preservation tube and revived. It was then cultured in a water-jacketed incubator at 37°C for 48 h on R2A solid medium to obtain single colonies. Single colonies were picked and inoculated into R2A liquid medium and cultured at 37°C for 12-18 h to obtain culture solution 1. Culture medium 1 was inoculated into R2A 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 at 2% (v / v) into R2A liquid medium for expansion culture, and cultured at 37℃ for 18h. The number of viable bacteria was recorded and bacterial culture a was obtained.

[0050] 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 at -10℃ to -50℃ to obtain powder for later use, thus preparing the freeze-dried powder of Sphingomonas CCSM001 fermentation supernatant (denoted as CCSM001-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 CCSM001-S).

[0051] Optionally, the supernatant or lysis buffer prepared above is freeze-dried at -10℃ to -50℃ to obtain sphingosomal bacteria CCSM001 postbiotic freeze-dried powder.

[0052] 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. 4Cells 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.

[0053] (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.

[0054] 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%.

[0055] (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.

[0056] Example 5: Safety verification of the activity of the metabiotic prepared by Sphingomonas CCSM001 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.

[0057] 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 of metabiotic prepared from Sphingomonas CCSM001), add 100 μL of metabiotic prepared from Sphingomonas CCSM001.

[0058] (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.

[0059] 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 101.6%), the addition of post-genetic agents CCSM001-Q or CCSM001-S of Sphingomonas CCSM001 at an inactivated cell concentration of 5.0 × 10⁻⁶ cells significantly improved cell proliferation. 7 The cell proliferation rates at CFU / ml were 101.15% and 99.74%, respectively.

[0060] 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 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.

[0061] Example 6: Effect of metabiotic prepared from Sphingomonas CCSM001 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: After changing the cell medium in step (2), the cells contained cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent; Model group: The cells from step (2) were 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.

[0062] (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.

[0063] In the metabiotic treatment group, the metabiotics 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 amount of metabiotics prepared from bacterial cultures with CFU / ml was equivalent, and 100 μL of metabiotics CCSM001-S and CCSM001-Q prepared from Sphingomonas CCSM001 were added respectively.

[0064] (4) Add 10 μL of CCK8 solution to each well and incubate for 1.5 h to measure the absorbance (OD) at 450 nm.

[0065] 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%.

[0066] 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 (100% cell viability), the cell viability of the model group was 74.07%, and SDS modeling caused significant damage to HaCaT cells.

[0067] After adding CCSM001-S and CCSM001-Q to the treatment groups, the cell viability was 82.76% and 69.41%, respectively. Compared with the model group (74.07%), CCSM001-S can improve the viability of HaCaT cells, indicating that the metagenetic component (cell lysate) of Sphingomonas CCSM001 can effectively alleviate the damage caused by SDS to HaCaT cells.

[0068] Example 7: Effect of metagenetic agents prepared by Sphingomonas CCSM001 on FLG gene expression in HaCaT cells damaged by SDS.

[0069] 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.

[0070] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium, and the amount of resuspended metabiotics was adjusted to a fermentation concentration of 5.0 × 10⁻⁶. 7 The amount of metabiotic prepared from the bacterial culture with CFU / ml is equivalent to that of CCSM001-S prepared from Sphingomonas CCSM001. 2 mL of this metabiotic is added.

[0071] 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 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.

[0072] Table 1 Primer Sequences

[0073] 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 mRNA expression level of FLG in the control group was 1, while the expression level in the model group decreased to 0.67 after SDS intervention. The metabiotic (cell lysate) prepared by Sphingomonas CCSM001 significantly upregulated the mRNA expression level of FLG to 1.72.

[0074] Example 8: Effect of metagenetic agents prepared from Sphingomonas CCSM001 on IVL gene expression in HaCaT cells damaged by SDS.

[0075] 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.

[0076] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium, and the amount of resuspended metabiotics was adjusted to a fermentation concentration of 5.0 × 10⁻⁶. 7 The amount of metabiotic prepared from the bacterial culture with CFU / ml is equivalent to that of CCSM001-S prepared from Sphingomonas CCSM001. 2 mL of this metabiotic is added.

[0077] 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 IVL 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.

[0078] Table 2 Primer Sequences

[0079] In keratinocytes, inner lamina protein (IVL) cross-links with laminarin via transglutaminase catalysis, forming an insoluble keratinized capsule that constitutes the unique stratum corneum barrier structure of the epidermis. Simultaneously, it interacts with other keratinocyte differentiation proteins such as filaggrin (FLG) to jointly maintain the structure and function of the skin barrier. IVL expression results are derived from… Figure 5 It can be seen that the expression level of IVL mRNA in the control group was about 1, while the expression level in the model group decreased to 0.55 after SDS intervention. The lysate of Sphingomonas CCSM001 can increase the expression level of IVL to 1.6.

[0080] Example 9: Effect of metagenetic agents prepared from Sphingomonas CCSM001 on AQP3 gene expression in HaCaT cells damaged by SDS.

[0081] 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.

[0082] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium, and the amount of resuspended metabiotics was adjusted to a fermentation concentration of 5.0 × 10⁻⁶. 7 The amount of metabiotic prepared from the bacterial culture with CFU / ml is equivalent to that of CCSM001-S prepared from Sphingomonas CCSM001. 2 mL of this metabiotic is added.

[0083] 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 AQP3 The mRNA expression levels were measured, with GAPDH as the internal reference. The primers are described in Table 3 below, and the results are as follows: Figure 6 As shown.

[0084] Table 3 Primer Sequences

[0085] Aquaporins (AQPs) play a crucial role in skin barrier function, especially AQP3. AQPs regulate the transport and distribution of water in the skin, maintaining skin hydration and elasticity by controlling the water content within the stratum corneum. AQP3 not only promotes water transport but also regulates glycerol transport, playing a vital role in maintaining skin suppleness and barrier function. The expression of AQP3 is determined by… Figure 6 It can be seen that the mRNA expression level of AQP3 in the control group was about 1, while the expression level in the model group decreased to 0.44 after SDS intervention. After repair by sphingosine monocytogenes CCSM001 bacterial lysate, the expression level of AQP3 was restored to 1.03, which is close to the normal level.

[0086] Example 10: Effect of metagenetic agents prepared by Sphingomonas CCSM001 on the expression of tight junction-related protein genes in HaCaT cells damaged by SDS.

[0087] 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.

[0088] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium, and the amount of resuspended metabiotics was adjusted to a fermentation concentration of 5.0 × 10⁻⁶. 7 The amount of metabiotic prepared from the bacterial culture with CFU / ml is equivalent to that of CCSM001-S prepared from Sphingomonas CCSM001. 2 mL of this metabiotic is added.

[0089] 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 4 below, and the results are as follows: Figure 7 As shown.

[0090] Table 4 Primer Sequences

[0091] Tight junction proteins are essential components for maintaining skin barrier function. Located between keratinocytes, they form sealed junctions that prevent the permeation of external substances and water through the intercellular spaces, thus maintaining the integrity of the skin barrier. Zygomatic band occlusion protein (ZO-1), occlusive junction protein (OCCLUDIN), and cloning junction protein (CLDN) are core components of tight junctions and important structural proteins of the skin barrier. They play a crucial role in regulating the transport of substances through the intercellular spaces, maintaining the selective permeability of the skin barrier. Furthermore, these proteins not only participate in physical cell-cell connections but also function in intracellular signal transduction, influencing cell proliferation, differentiation, and migration. The expression of ZO-1, Occludin, and CLDN is controlled by… Figure 7 As shown, the mRNA expression of these three proteins in the model group decreased to 0.47, 0.34 and 0.64, respectively. However, after repair by sphingosine monocytogenes CCSM001 bacterial lysate, the expression of these three proteins could be upregulated to varying degrees, reaching 1.41, 1.31 and 1.14, respectively.

[0092] Example 11: Analysis of the effective substances of Sphingomonas CCSM001 in repairing SDS-damaged HaCaT cells based on non-target metabolomics. 1. Metabolomics analysis of CCSM001 fermentation supernatant: (1) After culturing the strain in R2A liquid medium for 12 h, take 1 mL of bacterial solution and incubate at 4℃ for 10000 hours. g Centrifuge for 5 min and collect the supernatant 1; use the same blank liquid culture medium as liquid 2. (2) Transfer 100 μL of supernatant 1 and 2 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).

[0093] 2. Metabolomics analysis of CCSM001 cells: After culturing the strain for 12 h as described above, take 1 mL of the 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).

[0094] 3. Analysis of active ingredients in CCSM001: 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.

[0095] Literature review was conducted to collect the chemical formulas, molecular formulas, and molecular weights of potential effective substances in CCFM1403. 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 3.

[0096] Table 3 Potential active ingredients related to skin barrier in CCSM001

[0097] Example 12: Quantitative Detection of Potentially Effective Substances in Sphingomonas CCSM001 Based on Non-Target Metabolomics Results Liquid chromatography-mass spectrometry was used to detect substances in the fermentation supernatant and bacterial cells of CCFM1460, and the detection method was the same as in Example 11.

[0098] Based on the results of non-targeted metabolomics, the following substances were detected in the sample after targeted quantitative detection: (1) D-erythrosine disphingosine like Figure 8-11 As shown, this substance was detected in both the fermentation supernatant and bacterial cells, with concentrations of 1.2 ppm and 7.1 ppm, respectively. Sphingosine, as an important lipid molecule on the skin surface, has a clear potential for maintaining barrier homeostasis. In vitro studies in a human keratinocyte model have confirmed that sphingosine can significantly inhibit the growth of various conditionally pathogenic bacteria on the skin, reduce TLR2 / TLR4-mediated inflammatory signaling, and alleviate the release of NF-κB downstream inflammatory factors, thereby restoring cellular homeostasis. Furthermore, sphingosine can also promote the synthesis of intrinsic skin lipids, enhance the activity of the sphingomyelin-ceramide metabolic pathway, and improve the structural integrity of keratinocytes.

[0099] (2) Oleamide like Figure 12-15As shown, this substance was detected in the fermentation supernatant and bacterial cells, with concentrations of 17.8 ppb and 123.5 ppb, respectively. Oleamide is a class of endogenous fatty amide molecules with the potential to promote skin physiological regulation, such as promoting barrier lipid 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, and can exert immune homeostasis by regulating the PPAR pathway and CB2 receptor. Simultaneously, oleamide can promote the production of key barrier lipids (such as ceramides and free fatty acids) in keratinocytes, improve the structure of the sebum membrane, and thus enhance the skin's ability to resist external stimuli.

[0100] 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. Sphingosine monocytogenes (Sphingosine monocytogenes) Sphingomonas sp . Accession number CCSM001 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 22, 2024, with accession number GDMCC NO: 64896.

2. The metabiotic prepared using the Sphingomonas CCSM001 described in claim 1.

3. The epigenetic agent according to claim 2, characterized in that, The post-biotic contains D-erythrosine and / or oleamide.

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 a powder prepared by drying any of the above components.

5. The epigenetic agent according to claim 4, characterized in that, The inactivated or deactivated cells are prepared as follows: the Sphingomonas CCSM001 is cultured in a culture medium for a period of time, the 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: the Sphingomonas CCSM001 is cultured in a culture medium for a period of time, the bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate.

7. A composition containing *Sphingomonas CCSM001* as described in any one of claims 2-6 and / or its metagenes, characterized in that, The composition includes, but is not limited to, pharmaceuticals or cosmetics.

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 drug or cosmetic has at least one of the following effects: (1) Enhance the cell activity of keratinocytes in the skin after injury; (2) Increase the expression of filaggrin gene in keratinocytes of damaged skin; (3) Increase the expression of aquaporin genes in keratinocytes of damaged skin; (4) Increase the expression of inner lining protein gene in keratinocytes of damaged skin; (5) Increase the expression of tight junction protein genes in keratinocytes of damaged skin.

10. The use of the Sphingomonas CCSM001 of claim 1 in the preparation of D-erythrosine and / or oleamide.

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