Bifidobacterium breve CCFM1505 capable of regulating and controlling skin ceramide de novo synthesis and improving skin health and metagen of bifidobacterium breve CCFM1505

Abstract

The invention discloses bifidobacterium breve CCFM1505 capable of regulating skin ceramide de novo synthesis and improving skin health and a metagen thereof, and belongs to the technical field of microorganisms and medicines. The bifidobacterium breve CCFM1505 obtained through screening shows the effect of remarkably promoting skin ceramide synthesis in the aspects of external use and oral administration. The viable bacteria of the bifidobacterium breve CCFM1505 and the metabiotics thereof, especially the thallus lysate of the bifidobacterium breve, are orally taken, so that the content of ceramide in the skin tissue is remarkably increased, and meanwhile, the effects of increasing of transpedermis water loss, reducing of the water content of cuticle and inflammatory response caused by skin injury and reducing of the ceramide content can be effectively regulated and controlled, and the skin healing effect is improved. Therefore, the skin health is improved. Therefore, the bifidobacterium breve has a huge application prospect in preparation of external or oral medicines for promoting synthesis of skin ceramide and improving skin health, as well as in preparation of foods, health-care products or cosmetics.

Description

Technical Field

[0001] This invention relates to a short bifidobacterium strain CCFM1505 that regulates the de novo synthesis of skin ceramides and improves skin health, and its post-genes, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] As the outermost protective organ of the human body, the skin is not only the first line of defense against external physical, chemical, and biological aggressors, but also plays a vital role in maintaining physiological homeostasis, regulating body temperature, and sensing external stimuli. Its health directly impacts overall health and quality of life. Among the skin's multi-layered structure, the stratum corneum of the epidermis is the core area of ​​the skin barrier function, and the lipid components within the stratum corneum, especially ceramides, are key substances determining the integrity and normal function of the skin barrier. Ceramides are sphingolipid compounds naturally found in the lipids of the stratum corneum, accounting for approximately 40%-50% of the total lipids. Through interaction with lipid components such as cholesterol and fatty acids, they arrange themselves in an orderly, layered liquid crystal structure, constructing a dense physical barrier that effectively prevents external moisture and harmful substances from penetrating the skin, while simultaneously reducing excessive evaporation of internal moisture and maintaining skin hydration. In addition, ceramides possess various bioactive functions, such as participating in cell signaling processes, regulating the proliferation and differentiation of keratinocytes, and promoting skin self-repair and renewal. Regarding the regulation of inflammatory responses, they can inhibit the release of inflammatory mediators, reduce skin inflammation, and have a positive effect on the repair of sensitive and damaged skin. However, under the combined effects of physiological aging, external environmental stress, and unhealthy lifestyles, the metabolic balance of skin ceramide synthesis is easily disrupted, leading to a decrease in skin ceramide content or structural abnormalities, which in turn damages the skin barrier function and harms skin health.

[0003] Currently, ceramide deficiency is mainly addressed through two pathways: First, exogenous supplementation, which involves applying skincare products containing synthetic ceramides or their analogues directly to the skin surface to compensate for the skin's own ceramide deficiency and temporarily repair the skin barrier. However, exogenous supplementation has significant limitations. Ceramides have a large molecular weight and poor hydrophilicity, resulting in extremely poor permeability in the stratum corneum, making it difficult to penetrate deep into the skin for sustained repair. Furthermore, exogenously synthesized ceramides or their analogues differ from naturally synthesized ceramides in structure, composition, and metabolic pathways, potentially leading to a lack of complete adaptation to the skin's physiological needs with long-term use, thus limiting the repair effect. Second, endogenous regulation, which involves regulating the de novo synthesis pathway of ceramides in the skin, activating the skin cells' self-synthetic capacity, and fundamentally increasing ceramide levels to achieve long-term repair and maintenance of the skin barrier. Therefore, finding safe and non-toxic active substances that can effectively regulate the de novo synthesis of ceramides in the skin has become a key and hot research area in the field of skin health. Recent studies have shown that postbiotics possess excellent physiological activities in regulating gut microbiota balance, improving immune function, and inhibiting inflammatory responses. Furthermore, increasing research confirms that postbiotics from specific sources can also exert positive regulatory effects on skin health through blood circulation or local skin action, such as inhibiting melanin production, reducing skin inflammation, and promoting skin cell proliferation and repair. However, current research on postbiotic regulation of de novo ceramide synthesis in the skin is still in its early stages. Existing studies mostly focus on the ameliorative effects of postbiotics on skin inflammation or oxidative stress. Postbiotics specifically targeting and empirically demonstrating their ability to directly regulate the expression of key enzymes in the "de novo ceramide synthesis" pathway in keratinocytes, thereby fundamentally increasing endogenous ceramide levels, especially those derived from *Bifidobacterium breve*, remain a gap in our understanding. Therefore, screening and obtaining an epigenetic that can efficiently and specifically regulate the de novo synthesis of skin ceramides and thus significantly improve skin health, as well as the microbial strain that produces this epigenetic, is of great theoretical significance and practical application value for developing novel and efficient skin health care products. At the same time, it can also provide new ideas and strategies for the prevention and adjuvant treatment of diseases related to impaired skin barrier function.

[0004] Currently, there are a few reports on the regulation of endogenous ceramide synthesis. For example, CN116083301B discloses that the fermentation supernatant of a strain of Bifidobacterium bifidum can promote the synthesis of ceramide in epidermal cells, but the synthesis amount is low, and the regulatory pathway is not clear, and the oral efficacy has not been verified. CN119776238B discloses that a strain of Lactobacillus plantarum 24 can regulate sphingomyelinase synthesis to promote ceramide synthesis, but the de novo synthesis process of ceramide has not been explored. According to existing reports, the main model of skin barrier damage is the mouse AD model, and the mainstream construction methods are DNFB and MC903 induction. Among them, the AD model constructed by MC903 is mainly based on the Th2 / Th17 axis, simulating endogenous AD; but DNFB is a Th1-Th2-Th17 mixed type, simulating exogenous AD, and requires sensitization and challenge. MC903 combined with repeated peeling with 3M tape simultaneously superimposes physical barrier damage, further amplifying the ceramide loss and inflammatory cascade, which is more conducive to exploring the correlation between ceramide loss and skin health. There are currently no reported probiotic products that can improve the skin barrier damage caused by repeated peeling of MC903 combined with 3M tape. Summary of the Invention

[0005] This invention provides a short-lived Bifidobacterium (Bifidobacterium breve) Bifidobacterium breve Application of CCFM1505 and its precursors in the preparation of products that regulate the synthesis of skin ceramides.

[0006] This invention provides a strain of Bifidobacterium breve ( Bifidobacterium breve CCFM1505, the Bifidobacterium shortis CCFM1505, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66743.

[0007] The Bifidobacterium breve CCFM1505 was derived from the feces of healthy individuals. The strain was sequenced and the obtained sequence was compared with the nucleic acid sequence in NCBI. The results showed that it belonged to the Bifidobacterium genus Bifidobacterium breve, and it was named Bifidobacterium breve CCFM1505.

[0008] The *Bifidobacterium breve* CCFM1505 colonies on MRS solid medium are milky white, with regular and neat edges, smooth and glossy surfaces, opaque, and round in shape.

[0009] The present invention also provides a microbial preparation containing the aforementioned Bifidobacterium breve CCFM1505, wherein the content of Bifidobacterium breve CCFM1505 in the microbial preparation is ≥1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] The present invention also provides a metabiotic prepared using the aforementioned Bifidobacterium breve CCFM1505.

[0011] In one embodiment, the metabiotic includes cell lysate, inactivated or dead cells, fermentation supernatant, or any of the above-mentioned powders prepared by drying. In one embodiment, the inactivated or dead cells are prepared by culturing the Bifidobacterium breve CCFM1505 in a culture medium for a period of time, collecting the bacterial cells in the bacterial culture medium, and obtaining inactivated bacterial cells after heat treatment. In one embodiment, the heat treatment conditions are: 85°C for 15 min.

[0012] In one embodiment, the method for preparing the bacterial lysate is as follows: the Bifidobacterium breve CCFM1505 is cultured in a culture medium for a period of time, the bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate. In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging Bifidobacterium shortienes CCFM1505 after culturing it in a culture medium for a period of time. The present invention also provides compositions containing the aforementioned Bifidobacterium breve CCFM1505 and / or its postbiotic.

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

[0014] In one embodiment, the daily chemical products include skin care products, cosmetics, or toiletries; the daily chemical products do not contain live bacteria; the medicines include topical medicines or oral medicines.

[0015] In one embodiment, the dosage form of the daily chemical product includes powder, gel, emulsion, ointment, or solid dosage form.

[0016] In one embodiment, the food product includes the above-described composition and conventional excipients. In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0017] In one embodiment, the health product includes the above-described composition and conventional excipients.

[0018] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0019] In one embodiment, the composition is a probiotic powder.

[0020] In one embodiment, the bacterial powder is a solid powder of Bifidobacterium breve CCFM1505 prepared by drying the liquid metabiotic.

[0021] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0022] The present invention also provides the use of the composition in the preparation of products that upregulate skin ceramides and improve skin health.

[0023] In one embodiment, the product includes at least one of the following functions: (1) Promotes the secretion of ceramides by keratinocytes (HaCaT) in vitro; (2) Promotes the activity of skin keratinocytes (HaCaT) in vitro; (3) Increase the content of ceramides in mouse skin tissue; (4) Promotes the expression of the rate-limiting enzyme (SPT) in mouse skin tissue that promotes de novo synthesis of ceramides; (5) Reduces transepidermal water loss in mouse skin, increases stratum corneum moisture content, and repairs skin barrier; (6) It alleviates the inflammatory response of mouse skin tissue and improves skin health.

[0024] In one embodiment, the application can be either topical or oral.

[0025] In one embodiment, the dose of the postbiotic prepared from Bifidobacterium breve CCFM1505 in the product is not less than 1500 μg / kg body weight.

[0026] In one embodiment, the product is a pharmaceutical or cosmetic product; the cosmetic product does not contain live bacteria.

[0027] In one embodiment, the drug comprises the Bifidobacterium breve CCFM1505, a drug carrier, and / or pharmaceutical excipients.

[0028] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0029] 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.

[0030] In one embodiment, the cosmetic contains Bifidobacterium breve CCFM1505, matrix ingredients, and / or conventional excipients. 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.

[0031] 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.

[0032] The present invention also provides the application of the aforementioned Bifidobacterium breve CCFM1505 in food production.

[0033] The present invention also provides the application of the aforementioned Bifidobacterium breve CCFM1505 or the aforementioned microbial preparation, or the aforementioned postbiotic, in the preparation of daily chemical products that improve skin hydration.

[0034] The present invention also provides the use of the aforementioned Bifidobacterium breve CCFM1505 or the aforementioned microbial preparation, or the aforementioned postbiotic in the preparation of ceramides or products containing ceramides.

[0035] Beneficial effects: The present invention contains Bifidobacterium breve ( Bifidobacterium breve CCFM1505 and its prepared post-biotic formulations, both topical and oral, have the ability to alleviate skin barrier damage and increase the expression of related proteins, specifically manifested in: (1) Promotes the secretion of ceramides by keratinocytes (HaCaT) in vitro; (2) Promotes cell viability of skin keratinocytes (HaCaT) in vitro; (3) Reduce transepidermal water loss in mouse skin, increase stratum corneum moisture content, and repair skin barrier; (4) Increase the content of ceramides in mouse skin tissue; (5) Promotes the expression of the rate-limiting enzyme (SPT) in mouse skin tissue that promotes de novo synthesis of ceramides; (6) It alleviates the inflammatory response of mouse skin tissue and improves skin health.

[0036] Therefore, Bifidobacterium breve ( Bifidobacterium breve CCFM1505 and its prepared post-genetic derivatives have great application potential in products that upregulate skin ceramides and improve skin health.

[0037] Preservation of biological materials Bifidobacterium breve ( Bifidobacterium breve CCFM1505, taxonomically named Bifidobacterium breve It was deposited on July 24, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66743, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0038] Figure 1 Evaluation of the effects of different preparations of Bifidobacterium breve on promoting the secretion of ceramides by HaCaT cells; Figure 2 Effects of metabiotics prepared from Bifidobacterium breve CCFM1505 on HaCaT cell viability; Figure 3 Animal experimentation procedures; Figure 4 Effects of Bifidobacterium breve CCFM1505 and its prepared metabiotic on transepidermal water loss and stratum corneum water content in mouse skin; Figure 5 Effects of Bifidobacterium breve CCFM1505 and its prepared postbiotic on ceramide content in mouse skin tissue; Figure 6 Effects of Bifidobacterium breve CCFM1505 and its prepared metabiotics on the rate-limiting enzyme SPT in the de novo synthesis of ceramide; Figure 7 Effects of Bifidobacterium breve CCFM1505 and its prepared postbiotic on inflammatory markers (IL-13, TSLP) in mouse skin tissue; "*" indicates a statistically significant difference from the Model group (P<0.05), "**" indicates a statistically significant difference from the Model group (P<0.01), "***" indicates an extremely statistically significant difference from the Model group (P<0.001), and "****" indicates an extremely statistically significant difference from the Model group (P<0.0001). Detailed Implementation

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

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

[0041] The BALB / c mice used in the following examples were purchased from Vital Rivers.

[0042] The *Bifidobacterium breve* FSHMX3M2, FZJHZ3M8, FSDWF2M3, FZJHZ13M8, and CCFM1505 involved in the following examples were self-screened strains from the Food Biotechnology Center of Jiangnan University.

[0043] The culture media involved in the following examples are as follows: Modified MRS liquid culture medium: yeast extract 5.0 g / L, peptone 10.0 g / L, glucose 15.0 g / L, disodium hydrogen phosphate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, cysteine ​​1 g / L, and Tween-80 1 mL / L, pH 6.2–6.4.

[0044] Modified MRS solid medium: yeast extract 5.0 g / L, peptone 10.0 g / L, glucose 15.0 g / L, disodium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, cysteine ​​1 g / L, and agar 18.0 g / L, pH 6.2–6.4.

[0045] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (the mixed solution contains 10,000 U / mL penicillin and 10 mg / mL streptomycin).

[0046] The cell resuscitation and culture methods involved in the following examples are as follows: Preheat the cell culture medium to a suitable temperature in a water bath. In a clean bench, prepare a 15 mL centrifuge tube and a cell culture dish, adding 9 mL of preheated cell culture medium to each. Then, quickly remove the frozen human keratinocytes (HaCaT) from the -80°C freezer and immediately place them in a 37°C water bath for rapid thawing until the cell suspension is completely thawed. Use a sterile pipette to transfer the thawed cell suspension to the prepared 15 mL centrifuge tube and centrifuge at 1000 r / min for 5 min. After centrifugation, carefully discard the supernatant, add 1 mL of cell culture medium to the cell pellet, and gently pipette to resuspend the cells. Transfer the resuspended cell suspension entirely to a cell culture dish containing culture medium, gently agitating in a "cross" motion to distribute the cells evenly. Observe the cell seeding and distribution uniformity under a microscope. Place the culture dish in a 37°C incubator containing 5% CO2 for further culture. Change the medium after 24 hours. After culturing for 1-2 days, when the cell confluence reaches 70%-80%, the cells can be passaged.

[0047] Example 1: Screening of Bifidobacterium breve and preparation of postbiotics 1. Screening and identification of Bifidobacterium breve The screening samples for the strains were obtained from the feces of healthy individuals. 30% glycerol was added to the collected samples, which were then stored at -80°C and thawed. The original samples were serially diluted and plated. After anaerobic incubation at 37°C for 48 h, a suitable dilution was selected, and single colonies were streaked onto MRS solid medium for purification. After anaerobic incubation at 37°C for 48 h, typical colonies of *Bifidobacterium breve* were streaked onto MRS solid medium for purification. After anaerobic incubation at 37°C for 36–48 h, single colonies were picked and enriched in MRS liquid medium. After incubation for 24 h, the culture was centrifuged at 6000 r / min for 3 min to obtain bacterial sludge. The sludge was washed 1–2 times with sterile water, resuspended in 30% glycerol, and stored at -80°C. The selected strains were subjected to 16S sequencing (performed by Suzhou Genewiz Biotechnology Co., Ltd.), and the results, confirmed by NCBI sequence alignment, identified them as Bifidobacterium breve, and named them Bifidobacterium breve FSHMX3M2, Bifidobacterium breve FZJHZ3M8, Bifidobacterium breve FSDWF2M3, Bifidobacterium breve FZJHZ13M8, and Bifidobacterium breve CCFM1505, respectively. Bifidobacterium breve CCFM1505 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66743.

[0048] 2. Preparation of Bifidobacterium breve CCFM1505 and its postbiotics (1) The frozen Bifidobacterium breve CCFM1502 was streaked on modified MRS solid medium for revival and placed in a water-jacketed incubator at 37℃ for 36-48 h. Single colonies were picked and transferred to 5 mL of modified MRS liquid medium and cultured at 37℃ for 16-22 h. Then, 2% (v / v) inoculation was transferred to a new 5 mL of modified MRS liquid medium and cultured at 37℃ for 18 h. Then, 2% (v / v) inoculation was transferred to 200 mL of modified MRS liquid medium for expansion culture and cultured at 37℃ for 22 h to obtain 200 mL of bacterial suspension. Centrifuge 200 mL of bacterial suspension at 8000 g for 25 min, and collect the supernatant and bacterial sludge separately. The obtained supernatant needs to be pH adjusted, filtered through a membrane (0.22 μm aqueous filter membrane) for sterilization, and then freeze-dried to obtain the freeze-dried powder of Bifidobacterium breve fermentation supernatant (denoted as CCFM1505-S) for later use. Add sterile water to the bacterial sludge at a wet weight ratio of 1:50 and resuspend. The resulting resuspended solution is homogenized under high pressure (1200 MPa, 12 times). After homogenization, centrifuge at 8000 g for 25 min, collect the supernatant, and heat-treat (85℃, 15 min) to obtain the bacterial lysate (denoted as CCFM1505-J).

[0049] The preparation method of live Bifidobacterium breve CCFM1505 is as follows: 200 mL of bacterial solution is prepared according to the same method as above. The bacterial sludge obtained by centrifugation at 8000 g for 25 min is resuspended in glycerol at a ratio of 1 g: 2 mL to obtain a glycerol tube of live Bifidobacterium breve CCFM1505, which is denoted as CCFM1505-H.

[0050] (2) The postgenes (cell lysate and fermentation supernatant) of Bifidobacterium breve FSHMX3M2, Bifidobacterium breve FZJHZ3M8, Bifidobacterium breve FSDWF2M3 and Bifidobacterium breve FZJHZ13M8 were prepared simultaneously according to the method in step (1).

[0051] Example 2: Different metagenes promote the secretion of ceramides from keratinocytes (HaCaT) in vitro. (1) HaCaT cells were fed at a rate of 3 × 10 5 One cell per well was seeded into a 6-well plate, and after 24 h of culture, the cells adhered to the plate.

[0052] (2) After the cells adhered to the wall, a control group and an experimental group were set up for 12 h of incubation.

[0053] Control group: After 24 h of culture in (1), the original cell culture medium was removed and 2 mL of cell culture medium without post-genetic agents was added; Experimental group: After adhering cells cultured for 24 h in (1), the original cell culture medium was removed, and 2 mL of cell culture medium containing 5% metagenes was added (the metagenes were prepared from Bifidobacterium breve FSHMX3M2, Bifidobacterium breve FZJHZ3M8, Bifidobacterium breve FSDWF2M3, Bifidobacterium breve FZJHZ13M8 and Bifidobacterium breve CCFM1505, respectively).

[0054] In the experimental group, the metabiotic was resuspended in cell culture medium (the amount of resuspended metabiotic was the same as that fermented to a concentration of 5.0 × 10⁻⁶). 7 The amount of metabiotics prepared from bacterial cultures of CFU / mL was equivalent. 2 mL of resuspended metabiotics prepared from Bifidobacterium breve FSHMX3M2 (FSHMX3M2-S and FSHMX3M2-J), Bifidobacterium breve FZJHZ3M8 (FZJHZ3M8-S and FZJHZ3M8-J), Bifidobacterium breve FSDWF2M3 (FSDWF2M3-S and FSDWF2M3-J), Bifidobacterium breve CCFM1505 (CCFM1505-S and CCFM1505-J), and Bifidobacterium breve FZJHZ13M8 (FZJHZ13M8-S and FZJHZ13M8-J) were added to each culture.

[0055] (3) After incubation, aspirate the cell culture supernatant into a 1.5 mL centrifuge tube, centrifuge at 3000 r / min for 20 min, collect the supernatant, and determine the content of ceramide, a secretory component, in the cell culture supernatant. The determination was performed using an ELISA kit; the determination method was as per the kit instructions. The experimental results are as follows: Figure 1 As shown.

[0056] Normalization of the data revealed that, compared with the control group (100%), the post-biotic prepared from Bifidobacterium breve FZJHZ3M8 (FZJHZ3M8-J) and the post-biotic prepared from Bifidobacterium breve CCFM1505 (CCFM1505-S and CCFM1505-J) significantly increased the ceramide content in HaCaT, increasing it to 109.45%, 112.45%, and 115.67%, respectively. Among them, Bifidobacterium breve CCFM1505 and its prepared post-biotic showed the best effect, while other experimental groups did not show a significant increase in promoting HaCaT secretion.

[0057] Example 3: Effect of postbiotic prepared from Bifidobacterium breve CCFM1505 on HaCaT cell viability (1) HaCaT cells in the logarithmic growth phase were taken at 1.0 × 10⁻⁶. 4 100 μL / well of cells were seeded into 96-well plates, with the outermost ring of the wells filled with PBS solution to prevent edge effects. After culturing for 24 h until the cells adhered, blank group, control group and post-biotic treatment group were set up. Control group: Contains only cell culture medium and does not contain HaCaT cells; Control group: Contains cell culture medium and HaCaT cells, but does not contain post-genetics; The metagenetic treatment group contained cell culture medium and HaCaT cells, as well as metagenes.

[0058] The metabiotic was resuspended in cell culture medium (the amount of resuspended metabiotic was the same as that fermented to a concentration of 5.0 × 10⁻⁶). 9 The amount of postbiotic prepared from bacterial culture of CFU / mL is equivalent to that prepared from bacteria. 100 μL of resuspended postbiotic prepared from Bifidobacterium breve CCFM1505 was added.

[0059] (2) The above well plate was incubated in an incubator at 37°C for 24 h. After the incubation, 10 μL CCK8 solution was added to each well and incubated for 1.5 h. The absorbance (OD) at 450 nm was measured.

[0060] Cell viability was calculated using the following formula: Cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. Experimental results are as follows: Figure 2 As shown.

[0061] The effect of Bifidobacterium breve CCFM1505 on cell viability, such as Figure 2 As shown, compared with the control group (cell proliferation rate 100%), the addition of postbiotics prepared from Bifidobacterium breve CCFM1505 (CCFM1505-S and CCFM1505-J) at an inactivated cell concentration of 5.0 × 10⁻⁶ cells significantly improved cell proliferation. 9 The cell proliferation rates at CFU / mL were 103.298% and 141.203%, respectively. Keratinocytes are the core functional cells for the synthesis and metabolism of ceramides in the skin, playing an irreplaceable and crucial role in maintaining the integrity and physiological function of the skin barrier. As the main "production factory" for ceramide synthesis in the skin, keratinocytes undertake almost all the synthesis tasks of ceramides in the epidermis. Keratinocytes directly determine the integrity, moisturizing ability, and defensive function of the skin barrier by precisely regulating the synthesis, transport, and functional release of ceramides. Therefore, promoting keratinocyte vitality can indirectly promote the synthesis of ceramides in the skin and improve skin health. The experimental results showed that among the post-biotics (CCFM1505-S and CCFM1505-J) prepared from Bifidobacterium breve CCFM1505, the CCFM1505-J group significantly improved the vitality of keratinocytes, while the CCFM1505-S group had no significant effect.

[0062] Based on Examples 2 and 3, it was determined that *Bifidobacterium breve* CCFM1505 has the greatest potential to promote the synthesis of ceramides in the skin, maintaining skin integrity and improving skin health by regulating the balance of ceramides. Therefore, *Bifidobacterium breve* CCFM1505 was selected for in vivo experiments to verify its efficacy.

[0063] Example 4: Effects of Bifidobacterium breve CCFM1505 and its prepared metabiotic on transepidermal water loss and stratum corneum water content in mouse skin In the implementation plan, the preparation method of live Bifidobacterium breve CCFM1505 and its postbiotics (CCFM1505-S and CCFM1505-J) is the same as in Example 1. The difference is that in the preparation method of the postbiotic cells (CCFM1505-J), after centrifugation to obtain bacterial sludge, high-pressure homogenization is not required. After resuspending the obtained bacterial sludge at the required concentration, it is heat-treated at 85°C for 15 min to obtain inactivated bacterial cells.

[0064] Thirty healthy male BALB / c mice aged 7 weeks from Vitallii were purchased and randomly divided into 5 cages with 6 mice per cage. The 5 cages were: 1 cage for the model group and 1 cage for the control group, and 3 cages for the experimental groups (namely, the metagenic supernatant group (CCFM1505-S), the metagenic bacterial cell group (CCFM1505-J), and the live bacteria group (CCFM1505-H)). The details are as follows: Control group: Using physiological saline as a control for the gavage samples; Model group: Physiological saline was used as a control for gavage samples. The mouse model was a skin barrier damage model. The modeling method was: 3M tape and MC903 combined modeling method. Except for the blank group, the other groups used 3M tape to stick and peel 3 times and applied 10 μM MC903. 200 μL of MC903 was applied for each modeling.

[0065] CCFM1505-H group: The gavage sample was live Bifidobacterium breve CCFM1505, at a dose of 1×10⁻⁶. 9 CFU / mouse / day; mouse modeling method is the same as that of the model group.

[0066] CCFM1505-J group: The gavage sample was postbiotic (inactivated bacterial cells), and the dosage was: 5 × 10⁶ live bacteria per day via gavage. 9 CFU-prepared post-biotic inactivated bacterial cells; mouse modeling method was the same as the model group.

[0067] CCFM1505-S group: The gavage sample was postbiotic (supernatant group), and the dosage was: 5 × 10⁶ live bacteria per day via gavage. 9 The supernatant was obtained from the fermentation broth of CFU-containing Bifidobacterium breve CCFM1505; the mouse modeling method was the same as that of the model group.

[0068] The experiment lasted 28 days. Days 1 to 7 were the acclimatization period for mice. On day 6, the skin on the backs of the mice was shaved using a razor and depilatory cream, measuring 3 cm long x 2 cm wide. Modeling began on day 8, and mice were administered the treatment via gavage daily from day 8 to day 28. A combined 3M tape and MC903 application method was used for modeling, twice a week. The control group mice received an equal volume of physiological saline. Specific animal experimental methods are as follows... Figure 3 As shown.

[0069] After the experiment, mice were briefly anesthetized via isoflurane inhalation. A Cutometer DUAL MPA580, equipped with a TEWL probe and a CM825 probe for stratum corneum moisture content, was used to measure transepidermal water loss and stratum corneum moisture content in the mice. The experimental results are as follows: Figure 4 TEWL data showed that transepidermal water loss from the skin surface of control mice was 15.27 gm. -2 h -1 In the model group mice, transepidermal water loss increased to 28.00 gm. -2 h -1 After oral administration of Bifidobacterium breve CCFM1505-S, CCFM1505-J, and CCFM1505-H, respectively, transepidermal water loss from mouse skin was significantly reduced compared to the model group, decreasing to 12.88 gm. -2 h -1 12.39 gm -2 h -1 16.51 gm -2 h -1 All three experimental groups significantly reduced transepidermal water loss in mouse skin, demonstrating a significant effect on maintaining the stability of the mouse skin barrier to a certain extent.

[0070] The determination of stratum corneum water content in mouse skin revealed that the stratum corneum water content in the control group was 66.08%, while in the model group, the stratum corneum water content decreased to 47.77%. After gavage administration of Bifidobacterium breve CCFM1505-S, CCFM1505-J, and CCFM1505-H, the stratum corneum water content of mice increased to 67.47%, 66.18%, and 65.57%, respectively. The intervention by Bifidobacterium breve CCFM1505 and its prepared postbiotic significantly increased the stratum corneum water content of mouse skin, indirectly reflecting the skin's health level and significantly enhancing the skin's water-locking ability and barrier function.

[0071] In summary, the experimental results show that Bifidobacterium breve CCFM1505 and its prepared postbiotics (CCFM1505-S and CCFM1505-J) can positively regulate the epigenetic indicators of mouse skin, enhance its barrier function, improve skin health, effectively reduce the invasion of harmful substances, and reduce the probability of inflammatory response.

[0072] Example 5: Effects of Bifidobacterium breve CCFM1505 and its prepared metabiotic on ceramide content in mouse skin tissue The animal experiment design and experimental grouping were the same as in Example 4. After completing the determination of mouse skin appearance index, the mice were sacrificed, and skin tissue from the back was cut off. An appropriate weight of skin tissue was cut off, and pre-cooled PBS (1:10, tissue:PBS) was added. The tissue was homogenized and centrifuged at 3000 r / min for 20 min at 4℃. The supernatant was collected for later use.

[0073] The content of ceramides in mouse skin was determined using the ELISA method, such as... Figure 5 As shown in the data, the ceramide content in the skin of mice in the control group was 32.59 μmol / L, while in the model group it decreased to 27.97 μmol / L. The ceramide content in the CCFM1505-S group was 28.77 μmol / L, the CCFM1505-J group was 31.93 μmol / L, and the CCFM1505-H group was 32.99 μmol / L. The results indicate that both the CCFM1505-J and CCFM1505-H groups significantly increased the ceramide content in mouse skin tissue in promoting skin ceramide synthesis.

[0074] Example 6: Effect of Bifidobacterium breve CCFM1505 and its prepared metagenes on the rate-limiting enzyme SPT in the de novo synthesis of ceramide; The animal experimental design and experimental grouping involved in the following examples are the same as in Example 4, and the sample processing and preparation methods are the same as in Example 5.

[0075] Serine palmitoyltransferase (SPT) is a key rate-limiting enzyme in the skin ceramide synthesis pathway, serving as the initiating step in de novo ceramide synthesis. The content and activity of SPT directly determine the initiation efficiency of subsequent enzymatic reactions and the overall synthesis rate. Therefore, it is a core molecular target for regulating the total amount of skin ceramides, maintaining the integrity of the stratum corneum lipid barrier, and enhancing skin moisturizing function. The SPT content in mouse skin tissue was measured using ELISA, and the results are as follows... Figure 6Data showed that the SPT content in the skin tissue of the control group was 36.25 ng / mg, while in the model group, the SPT content decreased to 29.12 ng / mg. After gavage administration of Bifidobacterium breve CCFM1505 and subsequent biogener, the SPT content in the CCFM1505-S group was 36.46 ng / mg, in the CCFM1505-J group it was 40.49 ng / mg, and in the CCFM1505-H group it was 30.30 ng / mg. Compared with the model group, the CCFM1505-S and CCFM1505-J groups significantly increased the content of SPT, the rate-limiting enzyme in the de novo synthesis of ceramide.

[0076] Example 7: Effects of Bifidobacterium breve CCFM1505 and its prepared metabiotic on inflammatory markers (IL-13, TSLP) in mouse skin tissue; The animal experimental design and experimental grouping involved in the following examples are the same as in Example 4, and the sample processing and preparation methods are the same as in Example 5.

[0077] (1) Effects of Bifidobacterium breve CCFM1505 and its postbiotics on the content of pro-inflammatory factor IL-13 in mouse skin tissue The sample preparation method was the same as in Example 5. In mouse skin tissue, IL-13 is a key cytokine regulating skin immunity and barrier function, primarily involved in the regulation of inflammatory responses and the maintenance of skin homeostasis. When the skin barrier is damaged, the expression level of IL-13 increases. Elevated IL-13 further acts on keratinocytes, inhibiting the synthesis of barrier lipids such as ceramides, interfering with cell differentiation, promoting the release of other pro-inflammatory factors, exacerbating barrier damage and inflammatory responses, leading to skin health problems such as dryness and sensitivity. The inflammatory factor IL-13 was measured using ELISA, and the experimental results are as follows: Figure 7 As shown, after gavage administration of *Bifidobacterium breve* CCFM1505 and its post-biotic, IL-13 levels were significantly reduced. In the model group, the IL-13 level was 3.19 pg / mg, significantly higher than that in the control group (2.45 pg / mg). After oral administration of *Bifidobacterium breve* CCFM1505 and its post-biotics (CCFM1505-S, CCFM1505-J, CCFM1505-H), the IL-13 levels were 2.39 pg / mg, 2.29 pg / mg, and 2.36 pg / mg, respectively, all significantly lower than those in the model group. Therefore, *Bifidobacterium breve* CCFM1505 and its prepared post-biotics have a good effect in reducing skin inflammation.

[0078] (2) Effects of Bifidobacterium breve CCFM1505 and its postbiotics on the pro-inflammatory factor TSLP in mouse skin tissue The sample preparation method was the same as in Example 5. TSLP (thymic stromal lymphopoietin) is a key cytokine regulating skin immune homeostasis and barrier function, and a crucial "alarm" produced by keratinocytes in response to damage. It primarily acts as a bridge between skin barrier damage and immune abnormalities. When the skin barrier is damaged, it rapidly induces keratinocytes to secrete TSLP, causing a sharp increase in its expression level. Simultaneously, the elevated TSLP activates immune cells such as Langerhans cells and mast cells, promotes the release of Th2 inflammatory factors, and inhibits the synthesis of barrier lipids such as ceramides by keratinocytes, ultimately exacerbating skin inflammation. The inflammatory factor TSLP was measured using ELISA, and the experimental results are as follows: Figure 7 As shown in the figure. The experimental results showed that after oral administration of Bifidobacterium breve CCFM1505 and its postbiotic, its content decreased to 2.56 pg / mg, 2.14 pg / mg, and 1.98 pg / mg, respectively. Compared with the model group (2.93 pg / mg), the CCFM1505-J and CCFM1505-H groups could significantly inhibit the expression of TSLP, thereby inhibiting the inflammatory response of the skin, improving skin health, and repairing skin damage.

[0079] In summary, all the above examples show that topical or oral administration of the post-biotic prepared from *Bifidobacterium breve* CCFM1505 can effectively promote ceramide synthesis. The CCFM1505-J group showed significant effects in promoting ceramide synthesis, increasing the expression of the rate-limiting enzyme (SPT) during de novo synthesis, positively regulating skin epigenetic indicators (transepidermal water loss, stratum corneum water content), and reducing the expression of pro-inflammatory factors (IL-13, TSLP). CCFM1505-H also exhibits a certain positive regulatory effect in increasing ceramide content in tissues, alleviating skin inflammation, and improving skin epigenetic indicators. *Bifidobacterium breve* CCFM1505 and its prepared post-biotic can improve skin health by promoting de novo ceramide synthesis, thereby increasing ceramide content, improving skin epigenetic indicators, and inhibiting inflammatory responses.

[0080] 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 Bifidobacterium breve ( Bifidobacterium breve CCFM1505, the Bifidobacterium shortis CCFM1505, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66743.

2. A microbial preparation containing the Bifidobacterium breve CCFM1505 as described in claim 1.

3. The metabiotic prepared from *Bifidobacterium breve* CCFM1505 as described in claim 1, characterized in that, The postgenetic is any one of (a) to (c): (a) After fermenting the Bifidobacterium breve CCFM1505, collect the fermentation supernatant; (b) Heat-treat the cells of the said Bifidobacterium breve CCFM1505 to obtain inactivated cells; (c) The bacterial cells of the said Bifidobacterium breve CCFM1505 were resuspended and homogenized under high pressure. After centrifugation, the supernatant was collected and heat-treated to obtain bacterial cell lysate. The fermentation supernatant or cell lysate is either a liquid or a powder obtained after freeze-drying.

4. A product containing the Bifidobacterium breve CCFM1505 of claim 1, the microbial preparation of claim 2, or the postbiotic of claim 3.

5. The product as described in claim 4, characterized in that, The products include food, medicine, health products, or daily chemical products.

6. The product as described in claim 5, characterized in that, The daily chemical products include skin care products, cosmetics, or toiletries; the daily chemical products do not contain live bacteria.

7. The product as described in claim 5, characterized in that, The dosage forms of the daily chemical products include powders, gels, emulsions, ointments, or solid preparations.

8. The product as described in claim 5, characterized in that, The medicine mentioned is either for internal use or for external use.

9. The use of the *Bifidobacterium breve* CCFM1505 of claim 1, the microbial preparation of claim 2, or the postbiotic of claim 3 in the preparation of pharmaceuticals or daily chemical products with skin-care effects, characterized in that... The skin care effect refers to increasing the skin's ceramide content and / or enhancing the skin's water retention capacity.

10. The use of the Bifidobacterium breve CCFM1505 of claim 1, the microbial preparation of claim 2, or the postbiotic of claim 3 in the preparation of ceramides or ceramide-containing products.

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