Mycomonas rosae CCSM007 separated from human skin and capable of improving skin health and metagen of mycomonas rosae CCSM007

Abstract

The invention discloses a Mycomonas rosae CCSM007 strain separated from human skin and capable of improving skin health and a metagen of the Mycomonas rosae CCSM007 strain, and belongs to the field of microbial technologies and medicines. The myxomonas rosae CCSM007 provided by the invention has a good barrier function repairing effect and application prospect in the aspect of external use, and the application prospect is specifically shown as follows: the activity of damaged HaCaT cells is improved in vitro, the expression of gene levels and protein levels in the aspects of functional proteins and structural proteins after damage is improved, and meanwhile, the expression of sphingomyelinase 1 (SMPD1) is improved. The metaplast of the myxomonas rosae has a huge application prospect in preparation of cosmetics or medicines for repairing skin barriers.

Description

Technical Field

[0001] This invention relates to a strain of *Myxomonas rosea* CCSM007 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 body's first line of defense and is crucial for health. It not only prevents the invasion of harmful external substances such as pathogens, chemicals, and pollutants, but also effectively prevents the loss of body fluids, maintaining the skin's hydration. The skin barrier is composed of the stratum corneum, lipid bilayer, and natural moisturizing factors, which work together to maintain the skin's physical, chemical, and immune barrier functions. When the skin barrier is damaged, the skin becomes dry and sensitive, and is more prone to inflammation, infection, or chronic skin diseases such as eczema and psoriasis. Therefore, maintaining the integrity of the skin barrier is essential for protecting the body from external stimuli and maintaining healthy skin.

[0003] The skin's microbiome interacts closely with skin cells, working together to maintain the skin's ecological balance. A healthy microbiome helps prevent the invasion of harmful pathogens, protecting the skin from infection through competitive rejection and the production of antimicrobial substances. Furthermore, microorganisms secrete metabolites and signaling molecules that promote skin cell growth, differentiation, and repair, enhancing the skin barrier's function. While the skin microbiome plays a crucial role in maintaining barrier homeostasis through competitive rejection and metabolite secretion, existing known strains suffer from limitations such as limited functionality, weak activity, significant individual variability, and insufficient stability. For example, most common skin commensal bacteria have limited ability to regulate inflammation, promote lipid production, or accelerate barrier repair, and are easily affected by environmental factors, age, and skin condition, leading to loss of activity. Therefore, it is necessary to screen for novel functional strains from the skin with stronger homeostatic regulatory effects, clearer mechanisms, and higher application potential to meet the demand for efficient skin barrier repair. Summary of the Invention

[0004] This invention discovers a metagene of a strain of Myxomonas rosea CCSM007, which, through its bacterial lysate and other active substances, can comprehensively improve the barrier function of the skin after injury when applied topically.

[0005] This invention provides a strain of *Rosemycosis* (… Roseomonas mucosa Accession number CCSM007 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 22, 2024, with accession number GDMCC NO: 64901.

[0006] In one embodiment, the *Myxomonas rosea* CCSM007 is derived from the skin of a healthy person; the colonies of *Myxomonas rosea* CCSM007 on R2A solid medium are raised, pink, smooth, and round.

[0007] The present application also provides a postbiotic prepared from the said B. roseus CCSM007.

[0008] In one embodiment, the postbiotic comprises oleic acid amide.

[0009] In one embodiment, the postbiotic comprises cell lysate, inactivated or deactivated cells, fermentation supernatant, or a powder prepared by drying any of the above. In one embodiment, the inactivated or deactivated cells are prepared by culturing the B. roseus CCSM007 in a culture medium for a period of time, collecting the cell bodies in the cell culture solution, and obtaining the inactivated cell bodies by heat treatment. In one embodiment, the heat treatment is performed at 62-65°C for 30 min.

[0010] In one embodiment, the cell lysate is prepared by culturing the B. roseus CCSM007 in a culture medium for a period of time, collecting the cell bodies, high-pressure homogenization, and obtaining the cell lysate by centrifugation of the supernatant.

[0011] In one embodiment, the fermentation supernatant is the supernatant after centrifugation of the cell culture solution obtained by culturing the B. roseus CCSM007 in a culture medium for a period of time.

[0012] The present application also provides a composition comprising the B. roseus CCSM007 and / or the postbiotic thereof.

[0013] In one embodiment, the composition comprises, but is not limited to, a pharmaceutical or a daily chemical product.

[0014] In one embodiment, the pharmaceutical comprises the composition and conventional adjuvants.

[0015] In one embodiment, the conventional adjuvants comprise 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 probiotic powder is a solid powder prepared by drying the cell lysate of the B. roseus CCSM007.

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

[0019] The application also provides the use of the composition in the preparation of a medicine or cosmetic for repairing skin barrier.

[0020] In one embodiment, the medicine comprises at least one effect as follows: (1) improving cell viability of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (2) improving the expression of barrier function protein genes of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (3) improving the expression of barrier structure protein genes of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (4) improving the expression of barrier function proteins of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (5) reducing the expression of structural proteins of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (6) improving the expression of SMPD1 of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro.

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

[0022] In one embodiment, the amount of the M. roseburyi CCSM007 probiotic in the medicine is not less than 1×10 7 CFU / mL corresponding to the number of viable bacteria.

[0023] In one embodiment, the medicine comprises the M. roseburyi CCSM007, a pharmaceutical carrier and / or a pharmaceutical excipient.

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

[0025] In one embodiment, the pharmaceutical excipient comprises solvents, propellants, solubilizers, co-solvents, emulsifiers, coloring agents, binding agents, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filtration aids or release retardants.

[0026] In one embodiment, the cosmetic contains the M. roseburyi CCSM007 probiotic, matrix raw materials and / or conventional excipients.

[0027] In an embodiment, the matrix raw material includes oil and fat raw material, wax raw material, synthetic oil and fat raw material, powder raw material, gum raw material, coagulant, surfactant.

[0028] In an embodiment, the conventional adjuvant includes one or more of moisturizing agent, whitening agent, flavoring agent, adhesive, lubricant, preservative, film agent, antioxidant, emulsifying agent, and cosmetic nutrient additive.

[0029] The application also provides use of the S. roseo-flavus CCSM007 in preparation of oleic acid amide or oleic acid amide-containing product.

[0030] Beneficial effects: The S. roseo-flavus CCSM007 of the application has the ability of relieving skin barrier damage and improving expression of related proteins in the preparation of postbiotics for external use, which is embodied in the following aspects: Roseomonas mucosa (1) improving cell activity of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage; (2) improving mRNA expression of skin keratinocyte (HaCaT) barrier function protein in vitro after sodium dodecyl sulfate (SDS) damage; (3) improving mRNA expression of skin keratinocyte (HaCaT) barrier structure protein in vitro after sodium dodecyl sulfate (SDS) damage; (4) improving skin keratinocyte (HaCaT) barrier function protein expression in vitro after sodium dodecyl sulfate (SDS) damage; (5) reducing skin keratinocyte (HaCaT) barrier structure protein expression in vitro after sodium dodecyl sulfate (SDS) damage; (6) improving skin keratinocyte (HaCaT) SMPD1 expression in vitro after sodium dodecyl sulfate (SDS) damage; (7) regulating AHR-OVOL1 pathway receptors to regulate expression of related proteins to restore skin barrier function. Therefore, the postbiotics prepared from the S. roseo-flavus CCSM007 has great application prospect in products for relieving host skin barrier damage.

[0031] Roseomonas mucosa Biological material preservation The S. roseo-flavus CCSM007 is taxonomically named as S. roseo-flavus.

[0032] The S. roseo-flavus CCSM007 is taxonomically named as S. roseo-flavus. Roseomonas mucosa Roseomonas mucosa ​​The strain was deposited in the Guangdong Microbial Culture Collection Center on July 22, 2024, and the deposit number is GDMCC No: 64901. The deposit address is No. 59, Building, Guangzhou, Guangdong Province. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The effect of different concentrations of SDS on HaCaT cell damage was evaluated.

[0034] Figure 2 The effect of postbiotics on HaCaT cell proliferation.

[0035] Figure 3 The effect of postbiotics on HaCaT cell vitality after SDS damage.

[0036] Figure 4 The gene expression of HaCaT cell barrier function protein after postbiotics repair of SDS damage.

[0037] Figure 5 The gene expression of HaCaT cell barrier structure protein after postbiotics repair of SDS damage.

[0038] Figure 6 The expression of HaCaT cell barrier function protein after postbiotics repair of SDS damage.

[0039] Figure 7 The expression of HaCaT cell barrier structure protein after postbiotics repair of SDS damage.

[0040] Figure 8 The expression of HaCaT cell SMPD1 after postbiotics repair of SDS damage.

[0041] Figure 9 HaCaT cells after postbiotics repair of SDS damage through AHR-OVOL1.

[0042] Figure 10 Liquid chromatogram of oleic acid amide standard.

[0043] Figure 11 Liquid chromatogram of oleic acid amide content in fermentation supernatant sample.

[0044] Figure 12 Liquid chromatogram of oleic acid amide content in bacterial sample.

[0045] Figure 13 Retention time control diagram of oleic acid amide standard and bacterial sample. DETAILED DESCRIPTION

[0046] The present application will be further described below in conjunction with specific examples.

[0047] The human keratinocyte cell (HaCaT) involved in the following examples was purchased from Shanghai Cell Bank.

[0048] The culture medium involved in the following examples is as follows: R2A liquid medium: yeast extract powder 0.5 g, protein peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, soluble starch 0.5 g, potassium phosphate dibasic 0.3 g, anhydrous magnesium sulfate 0.024 g, and sodium pyruvate 0.3 g, with a final pH value of 7.3±0.2.

[0049] R2A solid medium: yeast extract powder 0.5 g, protein peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, soluble starch 0.5 g, potassium phosphate dibasic 0.3 g, anhydrous magnesium sulfate 0.024 g, sodium pyruvate 0.3 g, and agar 20 g / L, with a final pH value of 7.3±0.2.

[0050] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100x penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL in the mixed solution).

[0051] Example 1: Cell resuscitation and culture First, take the frozen human keratinocyte cell strain (HaCaT) out of the -80°C refrigerator, quickly melt it in a 37°C water bath, then add 5 mL of cell culture medium and centrifuge at 1000 r / min for 3 min, discard the supernatant. Add 1 mL of cell culture medium to resuspend the cells, then place them in a culture dish and put them in a 37°C incubator containing 5% CO2 for culture. When the cells grow and recover vitality and grow to 70%~80% confluence, perform cell passage.

[0052] Example 2: Isolation and screening of Roseomonas mucosa The strain sample was derived from healthy human skin, and a sterile swab was used to pick up physiological saline and then repeatedly applied to the back of the hand three times. Direct streaking was performed on R2A solid medium, and incubation was carried out at 37°C for 48 h. Pink round colonies were picked from the grown colonies and enriched in R2A liquid medium for 16 h. Then, a loop was used to pick the bacterial liquid and purify it on R2A solid medium for 48 h. Finally, single colonies were picked and transferred to R2A liquid medium for enrichment, and 30% glycerol was used for preservation to obtain the strain. The strain genome was extracted for 16S rDNA amplification and sequencing (performed by Suzhou Jinyuizhi Biological Technology Co., Ltd.). The results were determined to be Roseomonas mucosa by NCBI sequence alignment, and the strain was named Roseomonas mucosa CCSM007.

[0053] Example 3: Preparation of Roseomonospora rosea CCSM007 postbiotic Roseomonospora rosea CCSM007 was streaked from the stock tube and cultured on R2A solid medium at 37 °C in a water-jacketed incubator for 48 h to obtain single colonies; the single colonies were inoculated into R2A liquid medium and cultured at 37 °C for 12-18 h to obtain culture solution 1; Culture solution 1 was inoculated into R2A liquid medium at a 2% (v / v) inoculation amount and cultured at 37 °C for 12 h to obtain seed solution; The seed solution was inoculated into R2A liquid medium at a 2% (v / v) inoculation amount for expansion culture, and cultured at 37 °C for 18 h to record the viable cell count and obtain bacterial solution a.

[0054] Bacterial solution a was centrifuged at 8000 r / min for 30 min, and the supernatant and bacterial slurry were collected. The supernatant was heat-treated (65 °C, 30 min) and recorded as CCSM007-Q. The bacterial slurry was resuspended with double-distilled water at 75% of the original volume, and the resuspension was heat-treated (65 °C, 30 min). Then, the resuspension was subjected to high-pressure homogenization (1000 MPa, 10 P times) in a high-pressure homogenizer. After homogenization, the supernatant was obtained by centrifugation at 8000 r / min for 30 min to obtain the bacterial lysate, which was recorded as CCSM007-S.

[0055] Alternatively, the supernatant or lysate prepared above was freeze-dried at -10 °C to -50 °C to obtain Roseomonospora rosea CCSM007 postbiotic freeze-dried powder.

[0056] Example 4: Establishment of an in vitro sodium dodecyl sulfate (SDS) damaged keratinocyte (HaCaT) model (1) 100 μL of HaCaT cells in the logarithmic growth phase were inoculated into a 96-well plate at a concentration of 1.2×10 4 cells / well, and the outermost circle was filled with PBS solution to prevent edge effects. After 36 h of culture for cell adhesion, blank, control, and SDS treatment groups were set up; Blank group: only containing cell culture medium without HaCaT cells; Control group: containing cell culture medium and HaCaT cells without SDS; Treatment group: containing cell culture medium, HaCaT cells, and SDS at a final concentration of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively.

[0057] (2) The above-mentioned hole plate was incubated at a temperature of 37 °C for 6 h, 12 h and 24 h, respectively, 10 μL of CCK8 solution was added to each hole after incubation for 1.5 h to measure the absorbance value (OD) at 450 nm.

[0058] The cell viability was calculated according to 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) x 100%.

[0059] (3) After nonlinear regression fitting, the effect on cell viability was as shown in Figure 1 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.

[0060] Example 5: Safety verification of rose mucosa CCSM007 prepared metagenome (1) Take 100 μL of HaCaT cells in logarithmic growth phase at a concentration of 1.2 x 10 4 Cells / well, fill the outermost circle with PBS solution to prevent edge effect, culture for 36 h until they adhere, and set up blank group, control group and metagenome treatment group; Blank group: only containing cell culture medium without HaCaT cells; Control group: containing cell culture medium and HaCaT cells without metagenome; Metagenome treatment group: containing cell culture medium and HaCaT cells, and containing metagenome.

[0061] Resuspend the metagenome with cell culture medium (the amount of resuspended metagenome is equivalent to the amount of metagenome prepared from bacteria with a concentration of 1.0 x 10 7 CFU / ml), add 100 μL of metagenome prepared from rose mucosa CCSM007.

[0062] (2) The above-mentioned hole plate was incubated at a temperature of 37 °C for 24 h, 10 μL of CCK8 solution was added to each hole after incubation for 1.5 h to measure the absorbance value (OD) at 450 nm.

[0063] The cell viability was calculated according to 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) x 100% The effect on cell viability was as shown in Figure 2As shown, compared with the control group (cell proliferation rate 101.02%), the addition of post-genetic agents CCSM007-Q or CCSM007-S of *Myxomonas rosea* CCSM007 at an inactivated cell concentration of 1×10⁻⁶ showed a significant increase in cell proliferation. 7 The cell proliferation rates at CFU / ml were 100.36% and 101.71%, respectively.

[0064] 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.

[0065] Example 6: Effect of metabiotic prepared from Myxomonas rosea CCSM007 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: 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.

[0066] (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.

[0067] 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. 7The amount of metabiotics prepared from bacterial cultures of CFU / ml was equivalent, and 100 μL of metabiotics CCSM007-S and CCSM007-Q prepared from *Myxomonas rosea* CCSM007 were added respectively.

[0068] (4) Add 10 μL of CCK8 solution to each well and incubate for 1.5 h to measure the absorbance (OD) at 450 nm.

[0069] 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%.

[0070] 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 104.35%), the cell viability of the model group was 66.76%, indicating that SDS modeling caused significant damage to HaCaT cells.

[0071] After adding CCSM007-S or CCSM007-Q to the treatment groups, the cell viability was 88.81% and 72.44%, respectively. Compared with the model group (66.76%), CCSM007-S can improve the viability of HaCaT cells, indicating that the metagener (cell lysate) of Myxomonas rosea CCSM007 can effectively alleviate the damage caused by SDS to HaCaT cells.

[0072] Example 7: Effect of metabiotic prepared by Myxomonas rosea CCSM007 on the expression of barrier function protein genes in SDS-damaged HaCaT cells.

[0073] (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: After changing the medium of cells in step (1), the cells contained cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent; Model group: The cells from step (1) 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.

[0074] (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.

[0075] 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 1×10⁻⁶. 7 The amount of metabiotic prepared from the bacterial culture of CFU / ml was equivalent to that of CCSM007-S prepared from Myxomonas rosea CCSM007.

[0076] (4) After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△Ct Formula calculation FLG, IVL and LOR 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.

[0077] Table 1 Primer Sequences

[0078] 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 goal. 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 0.21 after SDS intervention. The metabiotic (cell lysate) prepared from *Myxomonas rosea* CCSM007 significantly upregulated the expression level of FLG mRNA to 1.74.

[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 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 bacterial lysate of Myxomonas rosea CCSM007 can increase the expression level of IVL to 6.17.

[0080] 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 0.22 after SDS intervention. The LOR expression level could be increased to 8.64 by the lysate of Myxomonas rosea CCSM007.

[0081] Example 8: Effect of metabiotic prepared by Myxomonas rosea CCSM007 on the expression of barrier structural protein genes in SDS-damaged HaCaT cells.

[0082] (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: After changing the medium of cells in step (1), the cells contained cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent; Model group: The cells from step (1) 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.

[0083] (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.

[0084] 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 metabiotic prepared from the bacterial culture of CFU / ml is equivalent to that of CCSM007-S prepared from Myxomonas rosea CCSM007.

[0085] (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 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.

[0086] Table 2 Primer Sequences

[0087] 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, 0.34 and 0.65, respectively. However, after repair by the lysate of Myxomonas rosea CCSM007, the expression of these three proteins could be upregulated to varying degrees, up to 2.5, 2.31 and 2.04, respectively.

[0088] Example 9: Effect of metabiotic prepared by Myxomonas rosea CCSM007 on the expression of barrier function proteins in SDS-damaged HaCaT cells.

[0089] 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.

[0090] 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 metabiotic prepared from the bacterial culture of CFU / ml is equivalent to that of CCSM007-S prepared from Myxomonas rosea CCSM007.

[0091] 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.

[0092] The FLG protein content in the control group was 6610.62 pg / mL, while the content in the model group decreased significantly to 5548.12 pg / mL. After treatment with CCSM007-S, the content increased to 6439.79 pg / mL. The IVL protein content in the control group was 1242.91 pg / mL, while the content in the model group decreased significantly to 1036.71 pg / mL. After treatment with CCSM007-S, the content increased to 1117.54 pg / mL. The LOR protein content in the control group was 833.9 μg / mL, while the content in the model group decreased significantly to 736.62 μg / mL. After treatment with CCSM007-S, the content increased to 818.59 μg / mL.

[0093] Example 10: Effect of metabiotic prepared by Myxomonas rosea CCSM007 on the expression of barrier structural proteins in SDS-damaged HaCaT cells.

[0094] 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.

[0095] 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 metabiotic prepared from the bacterial culture of CFU / ml is equivalent to that of CCSM007-S prepared from Myxomonas rosea CCSM007.

[0096] 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.

[0097] Among them, the content of ZO-1 protein in the control group was 96.9 pg / mL, the content in the model group decreased significantly to 78.7 pg / mL, and the content increased to 94.82 pg / mL after CCSM007-S treatment, which was 1.2 times higher than that in the model group.

[0098] Example 11: Effect of metabiotic prepared by Myxomonas rosea CCSM007 on the repair of SMPD1 expression in SDS-damaged HaCaT cells.

[0099] 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.

[0100] 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 metabiotic prepared from the bacterial culture of CFU / ml is equivalent to that of CCSM007-S prepared from Myxomonas rosea CCSM007.

[0101] 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.

[0102] 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 this study, the SMPD1 level in the control group was 264.27 μg / mL, while in the model group it significantly decreased to 208.38 μg / mL. Treatment with CCSM007-S increased the level to 280.08 μg / mL, a 1.34-fold increase compared to the model group.

[0103] Example 12: Effect of metagenetic agents prepared from *Myxomonas rosea* CCSM007 on the expression of AHR and OVOL1 receptors in SDS-damaged HaCaT cells.

[0104] 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. The cells are incubated again for 24 hours.

[0105] 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 metabiotic prepared from the bacterial culture of CFU / ml is equivalent to that of CCSM007-S prepared from Myxomonas rosea CCSM007.

[0106] 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 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 9 As shown.

[0107] Table 3 Primer Sequences

[0108] 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 CCSM007 can significantly upregulate the gene expression of AHR and OVOL1, upregulating to 1.23 and 1.76, respectively, thus activating the expression of this pathway.

[0109] Example 13: Effective substance analysis of fermentation supernatant of *Myxomonas rosea* CCSM007 1. Substance identification based on non-targeted metabolomics: metabolomics analysis of CCSM007 fermentation supernatant: (1) After culturing the strain in R2A liquid culture at 37℃ for 12 h, take 1 mL of bacterial solution and incubate at 4℃ 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).

[0110] 2. Metabolomics analysis of CCSM007 bacterial cell structure: 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).

[0111] 3. Analysis of active ingredients in CCSM007: 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.

[0112] Literature review was conducted to collect the chemical formulas, molecular formulas, and molecular weights of potential effective substances in CCSM002. Based on non-target metabolomics, and according to substances with a P-value < 0.05 and differences compared to those before fermentation, as well as research on skin barrier repair, potential effective substances were screened. The substances are shown in Table 4.

[0113] Table 4 Potentially effective substances related to the skin barrier in CCSM007

[0114] Example 15: Quantitative detection of potential active substances in Staphylococcus aureus CCSM006 based on non-target metabolomics results Liquid chromatography-mass spectrometry (LC-MS) was used to detect substances in CCSM006. Sample preparation and detection procedures were the same as in Example 14. Based on the results of non-targeted metabolomics, oleic acid amide was detected in the sample after targeted quantitative detection. Figure 10-13 As shown, the oleamide content in the bacterial cells and fermentation supernatant was 15.6 ppb and 11.3 ppb, respectively. As an endogenous fatty amide, oleamide can directly act on the skin's lipid metabolism pathway, significantly promoting the synthesis of key barrier lipids such as ceramides and free fatty acids, thereby enhancing the structural integrity of the stratum corneum from its source. Furthermore, oleamide can also regulate inflammation and immune homeostasis by activating the PPAR pathway and CB2 receptor, effectively inhibiting inflammatory factors that damage the skin barrier, such as TNF-α and IL-1β, enabling damaged skin to repair more quickly in a low-inflammatory environment. Compared to traditional exogenous lipid supplementation, oleamide has a dual effect of promoting endogenous lipid production and alleviating inflammation, exhibiting higher bioavailability and a more lasting repair effect in restoring skin barrier function.

[0115] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person 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. Myxomonas rosea ( Roseomonas mucosa Accession number CCSM007 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 22, 2024, with accession number GDMCC NO: 64901.

2. The metabiotic prepared using the *Rosa spp.* CCSM007 as described in claim 1.

3. The epigenetic agent according to claim 2, characterized in that, The post-gene contains oleic acid amide.

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 *Myxomonas rosea* CCSM007 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 *Myxomonas rosea* CCSM007 is cultured in a culture medium for a period of time, bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate.

7. A composition containing *Rhizopus roseus* CCSM007 as described in claim 1 and / or its metagenes, 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 drug 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 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 SMPD1 in keratinocytes of the skin after injury.

10. The use of the *Rhodopseudomonas rosea* CCSM007 as described in claim 1 in the preparation of oleamide or oleamide-containing products.

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