Staphylococcus follicularis CCSM014 prepared metagen with multi-target synergistic hair loss prevention and hair care effects

Abstract

The invention discloses a metagen which is prepared from staphylococcus follicularis CCSM014 and has a multi-target synergistic anti-hair loss and hair care effect, and belongs to the technical field of microorganisms and medicines. The strain and the metagen thereof can regulate and control the hair follicle development process and inhibit hair follicle cell apoptosis, so that the anti-hair-loss and hair-promoting effects are achieved. The metagen can promote secretion of Wnt10b and VEGF in hair follicle dermal papilla cells and up-regulate expression of hair follicle growth related genes such as beta-catenin and Gli1; the expression level of TGF-beta1 in the back skin of the mouse is reduced, and meanwhile, the expression of FGF-2 and FGF-7 is up-regulated. In conclusion, the staphylococcus capitis CCSM014 and the metaplasm thereof can promote hair follicle growth and prevent and / or improve alopecia, are suitable for development of anti-alopecia hair care drugs, functional skin care products and related preparations, and have good industrial application prospects.

Description

Technical Field

[0001] This invention relates to a post-genetic agent prepared from a strain of Staphylococcus aureus CCSM014, which has a multi-target synergistic effect in preventing hair loss and protecting hair, and belongs to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Hair loss is a common health problem affecting people of all ages, and its occurrence is related to a variety of factors, including aging, stress, endocrine disorders, nutritional status, and lifestyle. With the accelerated pace of life and changes in dietary structure, the incidence of thinning hair and baldness continues to rise, becoming a significant issue affecting appearance and mental health. Based on clinical characteristics, hair loss can be divided into two categories: scarring and non-scarring. The latter mainly includes androgenetic alopecia, alopecia areata, and telogen effluvium. The occurrence of hair loss is closely related to the disruption of the hair follicle growth cycle; various endogenous and exogenous factors can interfere with the regulation of the hair follicle cycle, leading to hair loss.

[0003] The hair follicle growth cycle consists of three phases: anagen (growth phase), catagen (regression phase), and telogen (resting phase), regulated by multiple signaling pathways. The anagen phase is the active proliferation phase of the hair follicle, during which the hair shaft continuously forms and elongates. It is primarily regulated by the Wnt / β-catenin signaling pathway, with Wnt10b promoting hair follicle regeneration by activating the classical Wnt pathway. β-catenin, as a key effector molecule, is closely related to hair follicle formation and regeneration. The catagen phase is a brief transitional phase dominated by apoptosis, mediated by transforming growth factor β (TGF-β) signaling, leading to hair follicle structural degeneration. The telogen phase is the static phase of the hair follicle, preparing it for the next anagen phase. Furthermore, the transcription factor GLI1 in the Hedgehog signaling pathway plays a role in hair follicle cell proliferation and differentiation. When Shh signaling is activated, GLI1 upregulation promotes hair follicle regeneration and tissue repair. Dermal papilla cells, as the signaling center of the hair follicle, secrete signaling molecules such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and TGF-β, co-regulating hair follicle development and cycle transitions with epithelial cells. The research on the above mechanisms provides a theoretical basis for screening and developing bioactive ingredients that promote hair growth. Hair follicle growth and development depend on the synergistic regulation of multiple signaling factors and cell types. Among them, dermal papillary cells (HDPCs) are the core cells in hair follicle cycle regulation and play a decisive role in the entry of hair follicles into the anagen phase. The proliferation and activity levels of HDPCs can reflect the functional state of hair follicles and are therefore often used as key evaluation indicators in hair growth research. With increasing attention being paid to the role of scalp microecology in hair follicle health, fermentation products and metabiotics derived from scalp symbiotic bacteria are considered to potentially participate in hair follicle regulation. Using an in vitro proliferation model of HDPCs, preliminary screening of these microbial-derived substances can be conducted to quickly determine their potential to promote hair follicle activity, thus providing a scientific basis for subsequent animal experiments and in-depth mechanistic studies. Therefore, HDPC proliferation experiments, as a preliminary screening step, have clear necessity and evaluative value.

[0004] Although the molecular regulatory mechanisms of the hair follicle cycle have been extensively studied, current interventions for hair loss still primarily rely on drug therapy. Commonly used clinical drugs include minoxidil and finasteride. Minoxidil, as a potassium channel opener, promotes hair growth by improving local blood circulation in the hair follicle; finasteride lowers dihydrotestosterone levels by inhibiting 5α-reductase activity. However, both drugs have limitations, potentially causing adverse reactions such as scalp irritation and sexual dysfunction, and their efficacy varies among individuals. Therefore, the industry and research fields continue to focus on multi-source, multi-mechanism hair loss prevention and care intervention strategies and the development of active substances.

[0005] However, from the perspective of existing research and application practices, hair loss prevention and hair care technologies still face several common problems: On the one hand, the occurrence of hair loss and the regulation of the hair follicle cycle have significant multi-factor and multi-pathway synergistic characteristics. Existing research and intervention strategies are mostly focused on a single signaling pathway or a single target, which makes it difficult to fully reflect the overall process of hair follicle growth regulation. On the other hand, for candidate technologies from different sources and of different types, there is still a lack of systematic and comparable in vitro and in vivo verification of their modes of action and effects, making it difficult to form a clear and reproducible technical understanding of the relevant research results.

[0006] Therefore, it is necessary to further explore novel hair follicle regulation technologies with clear sources and research value based on existing research, and to systematically characterize their effects through in vitro and in vivo experimental systems, so as to enrich the technological options in the field of hair loss prevention and hair care and make up for the deficiencies in existing research. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a Staphylococcus aureus strain that can regulate multiple targets to achieve anti-hair loss and hair care effects. 头葡萄球菌 This invention aims to address the technical problem of numerous side effects associated with existing hair loss treatments such as minoxidil and finasteride. The invention first uses HDPC proliferation level as a preliminary screening indicator to evaluate the hair follicle activity of *Staphylococcus aureus* CCSM014 and its fermentation products, and selects representative components for subsequent mechanism studies. Simultaneously, animal experiments were conducted to verify the hair growth-promoting effects of the live bacteria strain and different types of epigenetics.

[0008] The first technical solution provided by this invention is a strain of Staphylococcus aureus CCSM014 ( 葡萄球菌属 头的;头部的 ), the cephalosporin ( 头葡萄球菌 CCSM014 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 6, 2025, with accession number GDMCC No: 67229. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The cephalosporin ( 头葡萄球菌 CCSM014 was isolated from healthy human hair follicles and has the following characteristics: The cephalosporin ( 头葡萄球菌 CCSM014 was inoculated onto TSA solid medium and cultured at 37°C for 48 h. The colonies were round, raised, smooth and moist with neat edges, and opaque white or milky white in color.

[0010] The cephalosporin ( 头葡萄球菌CCSM014 is a Gram-positive coccus arranged in grape-like clusters. It is facultatively anaerobic, with an optimal growth temperature of 30-37℃ and an optimal growth pH of 7.0-7.5.

[0011] The second technical solution provided by the present invention is a microbial preparation containing the Staphylococcus aureus CCSM014 described in the first technical solution.

[0012] In one embodiment, the content of *Staphylococcus aureus* CCSM014 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0013] The third technical solution provided by the present invention is a metabiotic, which includes the fermentation supernatant, inactivated cells and / or cell lysates of the Staphylococcus aureus CCSM014 described in the first technical solution or the microbial preparation described in the second technical solution.

[0014] In one embodiment, the metabiotic is obtained by inoculating Staphylococcus aureus CCSM014 into TSB medium to obtain fermentation broth, centrifuging to obtain bacterial cells, and then heat-treating them.

[0015] In one embodiment, the heat treatment is performed at 60-70°C for 25-35 minutes.

[0016] In one embodiment, the fermentation supernatant is obtained by centrifuging the fermentation broth of Staphylococcus aureus CCSM014.

[0017] In one embodiment, the bacterial lysate is obtained by high-pressure homogenization of heat-inactivated bacterial cells followed by centrifugation to obtain the supernatant.

[0018] In one embodiment, the post-generic agent can be dried into powder or used directly by conventional drying methods such as vacuum drying, spray drying, vacuum freeze drying, and fluidized bed drying.

[0019] The fourth technical solution provided by the present invention is a composition containing the Staphylococcus aureus CCSM014 of the first technical solution or the microbial preparation of the second technical solution, or the metabiotic of the third technical solution.

[0020] In one embodiment, the composition may also comprise a live bacterial preparation made from Staphylococcus aureus CCSM014, having a live bacterial count of not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0021] The fifth technical solution provided by the present invention is the application of the Staphylococcus aureus CCSM014 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotic described in the third technical solution, or the composition described in the fourth technical solution in the preparation of hair care, hair loss prevention and / or hair growth promotion products.

[0022] In one embodiment, the product includes cosmetics and pharmaceuticals.

[0023] In one implementation, the application includes at least one of the following functions: (1) Increase the content of Wnt10b in HDPC; (2) Increase the level of VEGF secreted by HDPC cells; (3) Upregulate the expression level of β-catenin in HDPC; (4) Upregulates the expression level of Gli1; (5) Promotes individual hair regeneration and increases hair coverage; (6) Downregulates the expression level of TGF-β1 in individual skin tissue; (7) Upregulates the expression level of FGF-2 in individual skin tissue; (8) Promotes the intranuclear translocation of individual β-catenin in hair follicle cells and enhances the activation of its downstream growth-promoting signals.

[0024] In one embodiment, the cosmetic comprises a postbiotic, matrix ingredient, and / or conventional excipients prepared from the Staphylococcus aureus CCSM014.

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

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

[0027] In one embodiment, the pharmaceutical product comprises a metabiotic, a drug carrier, and / or pharmaceutical excipients prepared from the Staphylococcus aureus CCSM014.

[0028] 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.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention screened and obtained a strain of Staphylococcus aureus ( 头葡萄球菌 CCSM014, whose precursor enzymes have been verified in in vitro and in vivo experiments to have significant effects on promoting hair growth and preventing hair loss, specifically manifested as follows: (1) Significantly increases the content of Wnt10b in HDPC; (2) Increase the level of VEGF secreted by HDPC cells; (3) Upregulate the expression level of β-catenin in HDPC; (4) Upregulates the expression level of Gli1; (5) In mouse in vivo experiments, it significantly promoted hair regeneration and increased hair coverage; (6) Downregulates the expression level of TGF-β1 in skin tissue; (7) Upregulates the expression level of FGF-2 in skin tissue; (8) Promotes the intranuclear translocation of β-catenin in hair follicle cells and enhances the activation of its downstream growth-promoting signals.

[0030] Therefore, Staphylococcus aureus ( 头葡萄球菌 CCSM014 and its derivatives have promising applications in anti-hair loss and hair growth-promoting biological preparations, functional hair care products, cosmetic products, and topical medications.

[0031] Preservation of biological materials A strain of Staphylococcus aureus ( 头葡萄球菌 CCSM014, taxonomically named 头葡萄球菌 It was deposited on November 6, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67229, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0032] Figure 1 The effect of Staphylococcus aureus CCSM014 and its postbiotics on the proliferation activity of HDPC cells; Figure 2The effects of Staphylococcus aureus CCSM014 and its postbiotics on the expression levels of β-catenin and Gli1 genes in HDPC cells; Figure 3 The effects of Staphylococcus aureus CCSM014 and its postbiotics on the levels of Wnt10b and VEGF in HDPC cells; Figure 4 The effects of Staphylococcus aureus CCSM014 and its post-genes on hair growth in mice; Figure 5 The effects of Staphylococcus aureus CCSM014 and its post-biotic on mouse hair follicles; Figure 6 The effects of Staphylococcus aureus CCSM014 and its postbiotic on the expression levels of TGF-β1 (A), FGF-2 (B) and FGF-7 (C) in mouse skin; "*" indicates a statistically significant difference from the control group (P<0.05), "**" indicates a statistically significant difference from the control group (P<0.01), and "***" indicates a highly statistically significant difference from the control group (P<0.001). Detailed Implementation

[0033] The following describes preferred embodiments of the present invention. It should be understood that these embodiments are for better explanation of the present invention and are not intended to limit the present invention. The Staphylococcus aureus strain SJSWX1B4 (a negative control strain) involved in the following embodiments was a self-screened strain from the Food Biotechnology Center Laboratory of Jiangnan University. Like Staphylococcus aureus CCSM014, it was isolated from hair follicles of healthy individuals, but from different individuals. It was used as a control strain in the same source system to demonstrate the differences between the screened strains and strains from the same source.

[0034] Raw materials used in the examples: DMEM culture medium was purchased from Thermo Fisher Scientific, USA; fetal bovine serum, trypsin, and penicillin-streptomycin were purchased from Gibco, USA; and other routine reagents and culture medium components were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0035] PBS buffer solution ( / L): Sodium chloride 8.0 g, potassium chloride 0.2 g, disodium hydrogen phosphate 1.44 g, potassium dihydrogen phosphate 0.24 g, adjust pH to 7.4. Autoclave at 115℃ for 20 min.

[0036] TSB liquid medium ( / L): tryptone 17.0 g, soybean peptone 3.0 g, sodium chloride 5.0 g, dipotassium hydrogen phosphate 2.5 g, glucose 2.5 g. Autoclave at 115℃ for 20 min.

[0037] TSA solid medium: Based on the above TSB liquid medium components, add 15.0 g / L agar powder and autoclave at 115℃ for 20 minutes.

[0038] Cell culture medium: 90% (v / v) DMEM medium, 10% fetal bovine serum, 100 U / mL penicillin, 100 mg / L streptomycin.

[0039] Example 1: Screening and identification of Staphylococcus aureus 1. Screening The strain was isolated from healthy human hair follicle samples. After pretreatment, it was stored in 30% glycerol. 80℃. After thawing, take 0.5 mL of the sample and add it to 4.5 mL of sterile physiological saline to obtain 10. -1 Dilute the solution, then take 0.5 mL of 10 -1 The diluent was diluted in 4.5 mL of physiological saline to obtain 10. -2 Diluents were prepared sequentially to obtain different concentrations. Suitable gradient dilutions were then plated onto TSA solid medium and incubated at 37°C for 48 h. Typical colonies exhibiting morphological characteristics consistent with Staphylococcus aureus were selected for purification. After multiple streak purifications, a stable strain, designated CCSM014, was obtained. Single colonies were transferred to TSB liquid medium and incubated at 37°C for 18 h. The colonies were then preserved in 30% glycerol to obtain Staphylococcus aureus CCSM014. Typical colony characteristics included round, raised, smooth, moist surface, regular edges, and an opaque white or milky white color.

[0040] 2. Identification Genomic DNA was extracted from strain CCSM014, and its 16S rDNA sequence was amplified and sequenced (performed by Sangon Biotech (Shanghai) Co., Ltd.). Nucleic acid sequence alignment with the NCBI database confirmed that this strain is *Staphylococcus capsulatum*. 头葡萄球菌 It was named Cephalosporin CCSM014.

[0041] Example 2: Preparation of Staphylococcus aureus CCSM014 and its postbiotics All strains were preserved at -80℃ in 30% glycerol before use. A small amount of *Staphylococcus aureus* CCSM014 culture was picked up with a sterile inoculation loop and streaked onto TSA solid medium for 48 h (37℃). Single colonies were then inoculated into TSB liquid medium and incubated at 37℃ for 18 h. Finally, a 2% inoculum was transferred to 200 mL of TSB medium and incubated for another 18 h, yielding approximately 2.5 × 10⁻⁶ colonies. 8 CFU / mL bacterial suspension.

[0042] The method for preparing live Staphylococcus aureus CCSM014 is as follows: After preparing the bacterial suspension according to the above method, centrifuge to obtain bacterial sludge, resuspend in sterile physiological saline and adjust the concentration to 2.5 × 10⁻⁶. 8 CFU / mL, used as a live bacterial suspension for animal experiments (CCSM014-W). For long-term preservation of the strain, the bacterial sludge can be prepared into cryopreservation tubes at a ratio of 1 g: 2 mL glycerol.

[0043] Preparation method of Staphylococcus aureus CCSM014 fermentation metabolites and cell lysates: The above bacterial solution was centrifuged at 8000 r / min, 4℃ for 30 min, and the supernatant was lyophilized to obtain Staphylococcus aureus CCSM014 fermentation supernatant lyophilized powder (denoted as CCSM014-F). The obtained bacterial sludge was resuspended in double-distilled water at 75% of the original bacterial solution volume, heated at 65℃ for 30 min, and then homogenized under high pressure (1000 bar, 10 passes). Subsequently, it was centrifuged at 8000 r / min for 30 min, and the supernatant was collected to obtain cell lysates (denoted as CCSM014-L). The preparation method of Staphylococcus aureus SJSWX1B4 (negative control) supernatant lyophilized powder (SJSWX1B4-F) and cell lysate lyophilized powder (SJSWX1B4-L) was the same as above.

[0044] The preparation method of inactivated Staphylococcus aureus CCSM014 cells is as follows: After culturing the bacterial solution according to the above method, centrifuge at 8000 r / min for 30 min, collect the bacterial sludge, add double-distilled water at 75% of the original bacterial solution volume to resuspend, and treat at 65℃ for 30 min without high-pressure homogenization.

[0045] Example 3: Effect of Staphylococcus aureus CCSM014 on HDPC cell proliferation Cryopreserved human dermal papillary cells (HDPCs) (a gift from the School of Chemistry and Materials Engineering, Jiangnan University) were rapidly thawed in a 37°C water bath, centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. The precipitated cells were resuspended in DMEM high-glucose medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / L streptomycin, and cultured in a 37°C, 5% CO2 incubator. When the cells adhered and grew to approximately 80% confluence, they were digested and passaged using trypsin containing 0.25% EDTA, and cells in the logarithmic growth phase were used for subsequent experiments.

[0046] Logarithmic growth phase HDPC cells were digested, counted, and the cell density was adjusted to 1×10⁻⁶. 5100 μL was inoculated into each well of a 96-well plate at a concentration of / mL. To avoid edge effects, PBS solution was added to the outer wells. The plates were incubated at 37°C and 5% CO2 for 24 h. The experiment included a blank control group, a control group, a probiotic group, and a treatment group. Since live bacteria may proliferate in the cell system and interfere with detection, this example only evaluated the interaction between the fermentation supernatant and the cell lysate.

[0047] Control group: Contains only culture medium, without cells; Control group: Contains cells and culture medium, without added post-biotics; Yangshen group: containing cells and culture medium, with 0.1 μM minoxidil added; Treatment group: containing cells and culture medium, with the addition of various metabiotics. The supernatants of Staphylococcus aureus CCSM014 and SJSWX1B4 were labeled CCSM014-F and SJSWX1B4-F, respectively, and the cell lysate groups were labeled CCSM014-L and SJSWX1B4-L, respectively.

[0048] In cell experiments, the lyophilized powder was resuspended in fresh culture medium. The amount of fermentation supernatant lyophilized powder added to the supernatant groups (CCSM014-F and SJSWX1B4-F) was equivalent to that added to a viable cell concentration of 1.3 × 10⁻⁶. 7 The amount of metabolites produced by fermentation broth with FU / mL; the cell lysate group (CCSM014-L and SJSWX1B4-L) corresponds to 7.8 × 10⁻⁶. 6 The postbiotic equivalent concentration of FU / mL.

[0049] After 24 h of incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37℃ for 1.5 h before measuring the absorbance (OD) at 450 nm. The cell proliferation rate was calculated using the following formula: Cell proliferation rate (%) = [(OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%.

[0050] In the treatment concentration and detection time set in this embodiment, no decrease in HDPC cell viability was observed in any treatment group.

[0051] Experimental results are as follows Figure 1 As shown in the figure. Compared with the control group, both CCSM014-F and CCSM014-L significantly promoted HDPC cell proliferation, with cell proliferation rates increasing by 56.42% and 95.18%, respectively (***P<0.001). Among them, the CCSM014-L group showed the most significant effect, which was higher than that of the CCSM014-F group and the corresponding treatment groups of the negative control strain.

[0052] Preliminary screening results showed that both CCSM014-F and CCSM014-L had the ability to enhance the viability of dermal papilla cells in hair follicles, with the bacterial lysate showing a more significant effect. Therefore, CCSM014-L was selected for subsequent cellular-level mechanism studies. Furthermore, the proliferation level of HDPC cells was used as a preliminary screening criterion in this invention, which can be used to rapidly assess the hair follicle activity potential of the test strains and their fermentation products, providing a scientific basis for subsequent mechanism studies and animal experiments.

[0053] Example 4: Staphylococcus aureus CCSM014 promotes the expression of anti-hair loss and hair care-related genes and proteins in HDPC cells. 1. Effects of metabiotics prepared from Staphylococcus aureus CCSM014 on the expression of β-catenin and Gli1 in HDPC cells. The preparation method and concentration of the postbiotic for Staphylococcus aureus CCSM014 are the same as in Example 2.

[0054] HDPC cells in logarithmic growth phase were digested and counted, and the cell concentration was adjusted to 1.5 × 10⁻⁶. 5 Cells were cultured at a density of 2 mL / well in a 6-well plate at 5% CO2 and 37 °C overnight until adherence. The culture medium was discarded, and the cells were washed three times with PBS. Control and treatment groups were set up as in Example 23.

[0055] Add 2 mL of CCSM014-L to each well of the treatment group and incubate overnight at 37 ℃. After incubation, discard the supernatant, wash three times with PBS, and add 1 mL of lysis buffer to each well to fully lyse the cells. Collect the lysis buffer to extract total RNA, reverse transcribe it into cDNA using RT-PCR, and detect the mRNA expression levels of β-catenin and Gli1 in HDPC cells by real-time quantitative PCR. Using GAPDH as an internal control, utilize 2 -△△Ct The relative expression level was calculated using the method shown in Table 1. Table 1 Primer sequences (5'-3')

[0056] The results are as follows Figure 2As shown, with the expression level of β-catenin mRNA in the control group as 1, the post-biotic prepared from *Staphylococcus aureus* CCSM014 (CCSM014-L) significantly promoted the expression of β-catenin mRNA in HDPC cells, increasing the expression level by 3.32-fold (***P<0.001); while the expression level of β-catenin mRNA in the *Staphylococcus aureus* group only increased by 2.25-fold after intervention (***P<0.001). Simultaneously, CCSM014-L also significantly promoted the expression of Gli1 mRNA in HDPC cells, increasing it by approximately 2.49-fold compared to the control group (***P<0.001), which was higher than that in the *Staphylococcus aureus* group (1.35-fold, ***P<0.001).

[0057] β-catenin is a key regulator in the Wnt / β-catenin signaling pathway. Its activation promotes the proliferation of hair follicle stem cells and the activation of dermal papilla cells, thereby inducing hair follicles to enter the anagen phase and prolonging its duration. Gli1 is a major downstream transcription factor of the Shh signaling pathway and an important marker molecule for hair follicle regeneration. Shh signaling promotes the proliferation and differentiation of hair follicle stem cells by activating Gli1 and synergistically enhances β-catenin signaling activity. The upregulation of both stimulates hair follicle growth signals, promotes hair follicle regeneration, and improves thinning hair.

[0058] Experimental results show that, under the HDPC cell model and detection conditions used in this embodiment, CCSM014-L treatment can significantly upregulate the mRNA expression levels of β-catenin and Gli1 (by 3.32-fold and 2.49-fold, respectively, ***P<0.001), indicating that it exhibits clear regulatory characteristics at the level of signaling pathway indicators related to hair follicle growth.

[0059] 2. Effects of metabiotics prepared from Staphylococcus aureus CCSM014 on the expression of Wnt10b and VEGF in HDPC cells. The preparation and concentration of the postbiotic for Staphylococcus aureus CCSM014 were carried out in accordance with Example 2.

[0060] HDPC cells in the logarithmic growth phase were digested with trypsin and then counted. The cell density was adjusted to 1.5 × 10⁻⁶. 5 Cells were inoculated at a rate of 2 mL / mL into each well of a 6-well plate and cultured overnight at 37°C with 5% CO2. After cell attachment, the old culture medium was discarded. Cells were washed three times with PBS, and treatment and control groups were set up according to the grouping method in Example 23. Then, 2 mL of CCSM014-L sample was added to each treatment well, and the cells were cultured overnight under the same conditions. After incubation, the levels of Wnt10b and VEGF in the culture supernatant were determined according to the ELISA kit instructions.

[0061] Experimental results are as followsFigure 3 As shown in the figure, compared with the control group, CCSM014-L treatment significantly increased the secretion level of Wnt10b in HDPC cells by approximately 53.88% (***P<0.001), which was higher than the approximately 38.28% increase in the Yangshen group (**P<0.01). Simultaneously, CCSM014-L treatment also significantly promoted VEGF secretion, increasing it by approximately 71.25% compared with the control group (***P<0.001), while increasing it by approximately 14.27% in the Yangshen group (**P<0.01).

[0062] Wnt10b is an important ligand in the Wnt / β-catenin signaling pathway. Its upregulation can promote the nuclear translocation of β-catenin and activate the expression of downstream genes, thereby stimulating the proliferation of dermal papilla cells and hair follicle regeneration. VEGF, as a key angiogenic factor, promotes angiogenesis around hair follicles by activating the PI3K-AKT signaling pathway, improving nutrient supply and microenvironment stability. Both may have a synergistic effect in hair follicle development, jointly enhancing hair follicle activity and regenerative potential, providing a molecular basis for preventing hair loss and promoting hair growth.

[0063] Experimental results showed that in the HDPC cell culture system of this embodiment, the secretion levels of Wnt10b and VEGF in the culture supernatant of the CCSM014-L treatment group were significantly increased compared with the control group, by approximately 53.88% and 71.25%, respectively, and the differences were statistically significant (***P<0.001). These results suggest that CCSM014-L has a quantifiable role in hair follicle-related secretory factor indicators.

[0064] Example 5: Effects of Cephalosporin CCSM014 and its post-genetics on hair growth in mice The preparation method of the live cephalosporin CCSM014 (CCSM014-W) and its post-biotics (CCSM014-F and CCSM014-S) used in this embodiment is the same as in Example 2.

[0065] Literature review indicates that melanocytes in C57BL / 6J mice are located only within hair follicles, and their melanin synthesis process is closely related to the hair growth cycle. The hair follicles of 7-week-old C57BL / 6J mice are in a synchronized resting phase, serving as a standard model for evaluating the initiation of hair growth by exogenous substances. Therefore, observing the gradual change in skin color from pink (resting phase) to black (anagen phase) on the mouse's back reflects the hair growth status; this method is commonly used to evaluate the effectiveness of hair-promoting products.

[0066] Thirty 6-week-old male C57BL / 6J mice, weighing approximately 20 g, were purchased from Spiefol (Beijing) Biotechnology Co., Ltd., and the experiment began after a week of acclimatization. First, the hair on the back of the mice was removed using an animal-specific shaver, and then depilatory cream (purchased from the Veet official flagship store) was applied evenly to thoroughly remove any remaining hair. The treated area was approximately 2 cm × 3 cm. At this point, the skin on the back of the mice was pink, indicating that the hair follicles were in the resting phase, thus successfully establishing the C57BL / 6J mouse telogen effluvium model.

[0067] After hair removal treatment, the mice were left to rest for 24 hours to stabilize their skin condition. Then, they were randomly divided into 5 groups of 6 mice each, as follows: Control group: Apply saline solution locally to the back; Yangshen group (Minoxidil): used 5% minoxidil solution; CCSM014-W group: Administered live cephalosporin CCSM014 at a dose of 1×10⁻⁶. 9 CFU / kg mouse body weight; CCSM014-F group: The mice were given a 100 mg / kg body weight of Staphylococcus aureus fermentation metabolite. CCSM014-S group: Staphylococcus aureus CCSM014 postbiotic (inactivated bacterial cells), administered at a dose of 1×10⁻⁶. 9 The dose of CFU live bacteria is equivalent to the amount of inactivated bacteria, and the dosage is 1×10⁻⁶. 8 CFU / kg body weight.

[0068] Each mouse was treated daily with 0.2 mL of the corresponding sample applied evenly to the back of the hair-removed area for 3 consecutive weeks. In each group, inactivated bacteria or metabolites were measured using 1×10⁻⁶ ppm. 9 Inactivated bacteria or metabolites were prepared from a bacterial suspension containing an equal amount of live CFU. Hair growth was recorded by photographing on days 7, 14, and 21 after administration (e.g.,...). Figure 4 (As shown).

[0069] The results showed that after 7 days of administration, the skin on the back of the control group was still pink, while some mice in the CCSM014-F and CCSM014-S groups had gray areas on their backs; on the 14th day, the back areas of some mice in the control group and CCSM014-W group began to turn gray and sparse hair appeared, while the black area on the back of the Minoxidil group, CCSM014-F group and CCSM014-S group was significantly expanded and the hair shafts were clearly visible; by the 21st day, all mice in each group had entered the hair growth phase and their skin had completely turned black.

[0070] Experimental observation results showed that, compared with the blank control group, mice in the CCSM014-F group and CCSM014-S group showed premature skin pigmentation on the 7th and 14th days after drug administration, and the appearance of new hair shafts was earlier than that in the control group; by the 21st day, all treatment groups had entered the hair growth stage.

[0071] Example 6: Effects of Staphylococcus aureus CCSM014 and its post-biotics on mouse hair follicles At the end of day 21, mice in each group were sacrificed, and skin tissue samples from their backs were collected. These samples were fixed in 4% (v / v) paraformaldehyde solution, followed by routine dehydration and paraffin embedding before sectioning. Hematoxylin and eosin (H&E) staining was then performed for observation. The staining results are as follows: Figure 5 As shown.

[0072] The results are as follows Figure 5 As shown in the figure. Compared with the blank control group, mice treated with topical Staphylococcus aureus CCSM014-F and CCSM014-S showed more intact hair follicle structure and significantly improved morphology, with hair growth-promoting effects similar to those of the minoxidil positive control group. Specifically, the hair follicle density in the dorsal skin of mice in the CCSM014-F and CCSM014-S groups was significantly increased, and the dermal layer thickness was significantly thicker than that of the control group. The hair shafts were able to penetrate the dermis and break through the epidermis to grow out, indicating that these two treatments can promote hair follicles to enter the growth phase and accelerate the hair regeneration process, while CCSM014-W showed a hair growth state similar to the blank group.

[0073] Example 7: Effects of Staphylococcus aureus CCSM014 and its postbiotics on the expression of TGF-β1 and FGF-2 in mouse skin Mice were sacrificed on day 21, and skin tissue was harvested from the back of the mice to detect the gene expression levels of TGF-β1 and FGF-2 in the skin tissue. Mouse GAPDH was used as the internal control. The primer sequences are shown in Table 2.

[0074] Table 2 Primer sequences (5'-3')

[0075] This invention further examined the mRNA expression levels of TGF-β1, FGF-2, and FGF-7, regulatory factors closely related to hair follicle growth. The results showed ( Figure 6In the blank control group, the relative expression level of TGF-β1 was 1.00. In contrast, after treatment with CCSM014-W, CCSM014-F, and CCSM014-S, the TGF-β1 mRNA level in mouse skin decreased significantly to 0.20, 0.58, and 0.48, respectively (***P<0.001), indicating that both Cephalosporin CCSM014 and its subsequent biotic treatment effectively inhibited TGF-β1 expression. In contrast, the positive control group (5% minoxidil) showed the strongest inhibitory effect, with TGF-β1 expression downregulated to 0.12 (***P<0.001).

[0076] FGF-2 mRNA expression status ( Figure 6 B), the CCSM014-W, CCSM014-F, and CCSM014-S treatments significantly upregulated FGF-2 mRNA levels, reaching 1.62 (*P<0.05), 2.78 (***P<0.001), and 1.86 (**P<0.01), respectively, which were significantly higher than the control group (1.00). This indicates that CCSM014 and its precursors can promote the activation of FGF-2 signaling related to hair follicle growth, thereby enhancing hair follicle regeneration. The FGF-7 mRNA expression level is shown below. Figure 6 Treatment with CCSM014-C, CCSM014-W, CCSM014-F, and CCSM014-S significantly upregulated FGF-7 mRNA levels, reaching 2.52, 3.12, and 3.81, respectively (***P<0.001), which was significantly higher than the control group (1.02). However, the effect was not as good as the positive control group (5% minoxidil), with an upregulation level of 4.38 (***P<0.001). Nevertheless, this still demonstrates that CCSM014 and its precursors can promote the activation of FGF-7 signaling related to hair follicle growth, thereby enhancing hair follicle regeneration capacity.

[0077] TGF-β1 is a crucial regulator of the hair follicle regression phase. Increased TGF-β1 expression can induce apoptosis in hair follicle epithelial cells, promoting the transition of hair follicles from the anagen (growth) to the catagen (regression) phase, leading to follicle miniaturization and hair loss. Conversely, downregulation of TGF-β1 helps prolong the anagen phase and inhibits follicle regression. FGF-2 and FGF-7 are key growth factors promoting hair follicle regeneration, stimulating the proliferation and differentiation of dermal papilla cells and hair follicle stem cells through the PI3K-AKT pathway. These two factors exhibit an antagonistic relationship during the hair follicle cycle, and their balance is essential for the dynamic homeostasis of hair follicles.

[0078] The above-mentioned index measurements revealed that in the mouse alopecia model used in this embodiment, the TGF-β1 mRNA expression levels in the CCSM014 and its post-biotic treatment groups showed a decreasing trend compared to the control group. Simultaneously, the mRNA expression levels of FGF-2 and FGF-7 were upregulated to varying degrees, indicating their regulatory characteristics at the level of molecular indicators related to the hair follicle cycle. In summary, this invention, through a systematic study of Staphylococcus aureus CCSM014 and its post-biotic, verified its mechanism of action in promoting hair follicle cell activation, accelerating hair growth, and prolonging the growth phase. It has potential value for application in anti-hair loss and hair growth-promoting biological preparations, functional hair care products, and medical cosmetics.

[0079] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Staphylococcus aureus ( Staphylococcus capitis CCSM014, characterized in that, The aforementioned Staphylococcus aureus CCSM014 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 6, 2025, with accession number GDMCC No: 67229.

2. A microbial preparation, characterized in that, The microbial preparation contains the Staphylococcus aureus CCSM014 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The content of *Staphylococcus aureus* CCSM014 in the microbial preparation is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. An epigenetic agent, characterized in that, The metabiotic includes the fermentation supernatant, inactivated cells and / or cell lysates of the Staphylococcus aureus CCSM014 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 3.

5. The epigenetic agent according to claim 4, characterized in that, The metabiotic is obtained by culturing Staphylococcus aureus CCSM014 to obtain fermentation broth, centrifuging to obtain bacterial cells, and then heat-treating the bacterial cells.

6. The epigenetic agent according to claim 4, characterized in that, The fermentation supernatant was obtained by centrifugation of the Staphylococcus aureus CCSM014 fermentation broth.

7. The epigenetic agent according to claim 4, characterized in that, The bacterial lysate was obtained by high-pressure homogenization of heat-inactivated bacterial cells followed by centrifugation to obtain the supernatant.

8. A composition, characterized in that, The composition contains Staphylococcus aureus CCSM014 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 3, or contains a metabiotic as described in any one of claims 5 to 7.

9. The use of the *Staphylococcus aureus* CCSM014 according to claim 1, or the microbial preparation according to any one of claims 2-3, or the metabiotic according to any one of claims 5-7, or the composition according to claim 8, in the preparation of hair care, hair loss prevention, and / or hair growth promotion products, characterized in that, The products include cosmetics and pharmaceuticals.

10. The application according to claim 9, characterized in that, The application includes at least one of the following functions: (1) Increase the content of Wnt10b in HDPC; (2) Increase the level of VEGF secreted by HDPC cells; (3) Upregulate the expression level of β-catenin in HDPC; (4) Upregulates the expression level of Gli1; (5) Promotes individual hair regeneration and increases hair coverage; (6) Downregulates the expression level of TGF-β1 in individual skin tissue; (7) Upregulates the expression level of FGF-2 in individual skin tissue; (8) Promotes the intranuclear translocation of individual β-catenin in hair follicle cells and enhances the activation of its downstream growth-promoting signals.

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