Metagen prepared from bifidobacterium longum CCFM1508 and capable of regulating skin ceramide de novo synthesis and improving skin health

Abstract

The invention discloses a metagen which is prepared from bifidobacterium longum CCFM1508 and has the effects of regulating skin ceramide de novo synthesis and improving skin health, and belongs to the technical field of microorganisms and medicines. The bifidobacterium longum CCFM1508 and the metagen prepared from the bifidobacterium longum CCFM1508, provided by the invention, have the effects of promoting ceramide synthesis and improving skin health by external use and oral administration, and are specifically shown as follows: a thallus lysate and a fermented supernatant of the bifidobacterium longum CCFM1508 can promote HaCaT cells to secrete ceramide in the aspect of external use, and the thallus lysate can remarkably improve the activity of the HaCaT cells. And the viable bacteria and the postbiotics of the bifidobacterium longum CCFM1508 which are orally taken have obvious effects in skin apparent indexes, the content of cerayl and proinflammatory factors. Therefore, the bifidobacterium longum has huge application prospects in preparation of external or oral medicines for promoting synthesis of skin ceramide and improving skin health and preparation of food, health care products or cosmetics.

Description

Technical Field

[0001] This invention relates to a metabiotic prepared from a strain of Bifidobacterium longum CCFM1508, which has the function of regulating the de novo synthesis of skin ceramides and improving skin health, and belongs to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The skin, the largest organ in the human body, is the first physical barrier against external environmental stimuli and to prevent moisture loss. The integrity of its barrier function is crucial, and its impairment is associated with various skin problems such as dryness, itching, sensitivity, and atopic dermatitis. Ceramides, which fill the intercellular spaces of the stratum corneum, play an irreplaceable core role in the skin barrier function, accounting for more than 50% of intercellular lipids. Ceramides, by arranging themselves in an orderly manner with cholesterol and free fatty acids, form a dense lipid bilayer structure, effectively locking in moisture, maintaining skin homeostasis, and resisting external aggressors. The synthesis of skin ceramides mainly occurs through de novo synthesis, the sphingomyelinase pathway, and the salvage pathway, with de novo synthesis being the dominant pathway and the primary means of maintaining skin ceramide balance. Studies have shown that the content and composition ratio of ceramides in the epidermis of patients with dry skin, aging, and various dermatitis diseases are significantly abnormal. Therefore, timely and effective supplementation of skin ceramides can effectively improve skin health and prevent the development of related skin inflammations.

[0003] Currently, strategies for supplementing ceramides to improve skin problems are mainly divided into two categories: (1) Exogenous supplementation: that is, adding ceramides directly to cosmetics or topical preparations. This method has significant limitations. Most exogenously added ceramides are artificially synthesized, and their efficacy is questionable. Moreover, their transdermal absorption efficiency is low, making it difficult to effectively penetrate deep into the stratum corneum of the skin; (2) Endogenous promotion: stimulating skin cells to synthesize ceramides themselves through active ingredients. Common methods include using some plant extracts (such as sphingolipids), vitamins (such as niacinamide), etc. However, the effects of these ingredients vary, their mechanisms of action are unclear, and they may irritate some people with sensitive skin. Therefore, promoting the synthesis of endogenous ceramides in the skin, especially the de novo synthesis pathway, is considered one of the key strategies for maintaining the skin barrier and improving skin health.

[0004] In recent years, the concept of epigenetics has attracted widespread attention in the field of skin health. Epigenetics refers to preparations of non-living microorganisms and / or their components that are beneficial to the host's health, including bacterial components, metabolites, and cell lysates. Some studies have found that specific lactic acid bacteria or bifidobacteria fermentation lysates have the potential to improve skin hydration and alleviate inflammation. However, most existing research focuses on macroscopic effects (such as moisturizing and anti-inflammation), and their mechanisms of action are often unclear, especially in regulating skin ceramide synthesis. Related reports are scarce, and most are topical lotions containing epigenetic components. Their absorption and effects remain superficial. Furthermore, no research has yet reported that an epigenetic prepared from a specific strain of Bifidobacterium longum can endogenously regulate skin ceramide synthesis and improve skin health by modulating the expression of key enzymes in the de novo ceramide synthesis pathway (such as serine palmitoyltransferase SPT and ceramide synthase CerS). Therefore, there is an urgent need in this field to develop a post-genetic raw material with clear efficacy, well-defined mechanism, and stable safety. This raw material should be able to effectively activate the endogenous biosynthetic pathway of skin ceramides, providing new solutions for developing functional cosmetics, skin care products, or medical adjunctive products that improve dry skin, damaged skin barrier, and related skin problems.

[0005] Currently, there are few studies exploring the endogenous regulation of natural ceramide synthesis and its regulatory pathways through oral administration of probiotics or postbiotics. For example, CN115141783A discloses that a strain of Lactobacillus sakei subsp. sakei can maintain and repair the skin barrier and regulate related inflammatory factors to improve skin health, but there is no relevant research on ceramides, which are closely related to the skin barrier and health. CN116083301B discloses that the fermentation supernatant of a strain of Bifidobacterium bifidum can promote the synthesis of ceramides by epidermal cells, but the amount of ceramide synthesized is low, making it difficult to effectively regulate the skin health status. The main model for skin barrier damage is the mouse Alzheimer's disease (AD) model. The mainstream construction methods are DNFB and MC903 induction. DNFB is a Th1-Th2-Th17 mixed model, simulating exogenous AD, and requires sensitization and stimulation. The AD model constructed by MC903 is mainly based on the Th2 / Th17 axis, simulating endogenous AD. The combination of MC903 and 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. Currently, there is no reported effective method for treating endogenous AD combined with physical barrier damage. Summary of the Invention

[0006] This invention provides a long Bifidobacterium ( Bifidobacterium longum subsp. longum CCFM1508 and its derivatives are used in the preparation of products that promote the synthesis of skin ceramides and maintain skin homeostasis.

[0007] This invention provides a strain of Bifidobacterium longum ( Bifidobacterium longum subsp. longum The *Bifidobacterium longum* described in CCFM1508 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65087.

[0008] The *Bifidobacterium longum* CCFM1508 was derived from the feces of healthy individuals. The strain was sequenced and analyzed, and the obtained sequence was compared with the nucleic acid sequence in NCBI. The results showed that it belonged to the genus *Bifidobacterium*, *Bifidobacterium longum*, and it was named *Bifidobacterium longum* CCFM1508.

[0009] The colonies of Bifidobacterium longum CCFM1508 on MRS solid medium are raised, white, smooth, and round.

[0010] The present invention also provides a microbial preparation containing the aforementioned Bifidobacterium longum CCFM1508, wherein the content of Bifidobacterium longum CCFM1508 in the microbial preparation is ≥1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0011] The present invention also provides a metabiotic prepared using the aforementioned Bifidobacterium longum CCFM1508.

[0012] In one embodiment, the metabiotic includes cell lysate, inactivated or dead cells, autofermentation supernatant, or any of the above-mentioned powders prepared by drying. In one embodiment, the inactivated or dead cells are prepared by culturing Bifidobacterium CCFM1508 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.

[0013] In one embodiment, the method for preparing the bacterial lysate is as follows: the Bifidobacterium longum CCFM1508 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. In one embodiment, the fermentation supernatant is obtained by culturing Bifidobacterium longum CCFM1508 in a culture medium for a period of time, centrifuging it, and then taking its supernatant. The present invention also provides compositions containing the aforementioned Bifidobacterium longum CCFM1508 and / or its postbiotic.

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

[0015] In one embodiment, the daily chemical products include skin care products, cosmetics, or toiletries; the medicines include topical medicines or oral medicines.

[0016] In one embodiment, the dosage form of the daily chemical product includes powder, gel, emulsion, ointment, or solid dosage form.

[0017] In one embodiment, the daily chemical product does not contain live bacteria.

[0018] 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.

[0019] In one embodiment, the health product includes the above-described composition and conventional excipients.

[0020] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0021] In one embodiment, the composition is a probiotic powder.

[0022] In one embodiment, the bacterial powder is a solid powder of Bifidobacterium longum CCFM1508 prepared by drying the liquid metabiotic.

[0023] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0024] The present invention also provides the use of the composition in products that promote the synthesis of ceramides in the skin and maintain skin homeostasis.

[0025] In one embodiment, the product includes at least one of the following functions: (1) Promotes the synthesis of ceramides in keratinocytes (HaCaT) in vitro; (2) Promotes the cell activity of skin keratinocytes (HaCaT) in vitro; (3) Reduces transepidermal water loss (TEWL) in mouse skin tissue; (4) Increase the water content of the stratum corneum of mouse skin tissue (5) Increase the content of ceramides in mouse skin tissue; (6) Promotes the expression of key enzymes in the de novo synthesis of ceramides in mouse skin tissue; (7) Reduces inflammatory response in mouse skin tissue.

[0026] In one embodiment, the direct apparent indicators of the decreased skin ceramide levels are transepidermal water loss and stratum corneum water content.

[0027] In one embodiment, the key enzymes in the de novo synthesis of ceramide include SPT and CerS4.

[0028] In one embodiment, the SPT is serine palmitoyltransferase, the rate-limiting enzyme in the de novo synthesis of ceramide.

[0029] In one embodiment, the CerS4 ceramide synthase synthesizes ceramides.

[0030] In one embodiment, the application can be either topical or oral.

[0031] In one embodiment, the dose of the postbiotic prepared from Bifidobacterium longum CCFM1508 in the product is not less than 1500 μg / kg body weight.

[0032] In one embodiment, the product is a pharmaceutical or cosmetic product.

[0033] In one embodiment, the drug comprises the Bifidobacterium longum CCFM1508, a drug carrier, and / or pharmaceutical excipients.

[0034] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0035] 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.

[0036] In one embodiment, the cosmetic contains postbiotics prepared from Bifidobacterium longum CCFM1508, matrix raw materials, and / or conventional excipients; the cosmetic does not contain live bacteria.

[0037] 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.

[0038] 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.

[0039] The present invention also provides the application of the aforementioned Bifidobacterium longum CCFM1508 in food production.

[0040] The present invention also provides the application of the Bifidobacterium longum CCFM1508 or the microbial preparation or the postbiotic in the preparation of daily chemical products that improve skin amide content and enhance skin water retention capacity.

[0041] The present invention also provides the use of the Bifidobacterium longum CCFM1508 or the microbial preparation, or the postbiotic in the preparation of ceramide or ceramide-containing products.

[0042] Beneficial effects: The present invention contains Bifidobacterium longum ( Bifidobacterium longum subsp. longum CCFM1508 and its prepared post-biotics can increase the content of ceramides in the skin, maintain skin homeostasis, and enhance the skin's moisturizing ability. Specifically, this is reflected in the following aspects: (1) Increase the content of ceramide secreted by keratinocytes (HaCaT) in vitro; (2) Enhance the cell vitality of skin keratinocytes (HaCaT); (3) Improve the skin health of mice with skin barrier damage (reduce transepidermal water loss and increase the water content of the stratum corneum of mouse skin). (4) Increase the ceramide content in mouse skin; (5) Promotes the expression of key enzymes (SPT, CerS4) in the de novo synthesis of ceramides in mouse skin; (6) Relieves inflammatory response caused by decreased ceramide content in the skin.

[0043] Therefore, Bifidobacterium longum ( Bifidobacterium longum The post-genetic agents prepared by CCFM1508 have great application potential in products that increase skin ceramide content, maintain skin barrier homeostasis, and improve skin health.

[0044] Preservation of biological materials Bifidobacterium longum ( Bifidobacterium longum subsp. longum CCFM1508, taxonomically named Bifidobacterium longum subsp. longumIt was deposited on July 24, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66744, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0045] Figure 1 Evaluation of the role of different post-genetic agents in promoting ceramide synthesis in HaCaT cells; Figure 2 Effects of Bifidobacterium longum CCFM1508 and its prepared metabiotics on HaCaT cell viability; Figure 3 Animal experimentation procedures; Figure 4 Effects of Bifidobacterium longum CCFM1508 and its prepared metabiotic on transepidermal water loss (TEWL) in mouse skin tissue; Figure 5 Effects of Bifidobacterium longum CCFM1508 and its prepared metabiotics on the water content of the stratum corneum of mouse skin; Figure 6 Effects of Bifidobacterium longum CCFM1508 and its preparation on ceramide content in postbiotic mouse skin tissue; Figure 7 Effects of Bifidobacterium longum CCFM1508 and its prepared postbiotic on the rate-limiting enzyme (SPT) in ceramide synthesis in mouse skin tissue; Figure 8 The effects of Bifidobacterium longum CCFM1508 and its prepared metabiotic on the key enzyme (CerS4) in ceramide synthesis in mouse skin tissue; Figure 9 Effects of Bifidobacterium longum CCFM1508 and its prepared postbiotics on inflammation 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

[0046] The present invention will be further described below with reference to specific embodiments.

[0047] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0048] The BALB / c mice used in the following examples were purchased from Vital Rivers.

[0049] The *Bifidobacterium longum* strains FSCREG6M5-3, 1X3, B38X1, B23D8, and CCFM1508 involved in the following examples were self-screened strains from the Food Biotechnology Center of Jiangnan University.

[0050] 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 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, cysteine ​​1 g / L, and Tween-80 1 mL / L, pH 6.2–6.4.

[0051] Modified MRS solid 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 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, cysteine ​​1 g / L, Tween-80 1 mL / L, and agar 18.0 g / L, pH 6.2–6.4.

[0052] 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).

[0053] 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.

[0054] Example 1: Screening of Bifidobacterium longum and preparation of postbiotics 1. Screening and identification of Bifidobacterium longum The bacterial strain samples were obtained from the feces of healthy individuals. After pretreatment, the samples were stored in 30% glycerol at -80°C. After thawing, the samples were mixed, and 0.5 mL of the sample was added to 4.5 mL of physiological saline. Serial dilutions were performed using physiological saline, and appropriate serial dilutions were plated onto MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Bifidobacterium longum* were picked and streaked onto MRS solid medium for purification. After incubation at 37°C for 48 h, single colonies were picked and transferred to MRS liquid medium for enrichment. The culture was incubated at 37°C for 12-18 h. 1 mL of the mixed culture was centrifuged, washed, and the supernatant was discarded. 30% glycerol was added, and the mixture was vortexed and stored at -80°C to obtain the bacterial strain. Gene amplification and sequencing of the obtained strain (performed by Suzhou Genewiz Biotechnology Co., Ltd.) were performed. The sequencing results, confirmed by NCBI sequence alignment, identified the strain as *Bifidobacterium longum*, and it was named *Bifidobacterium longum*. Bifidobacterium longum subsp. longum CCFM1508, the Bifidobacterium longum CCFM1508, has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66744.

[0055] 2. Preparation of postbiotics from Bifidobacterium longum CCFM1508 (1) The frozen Bifidobacterium longum CCFM1508 was streaked on modified MRS solid medium for revival and placed in a 37°C water-jacketed constant temperature incubator for 36-48 h. Single colonies were picked and transferred to 5 mL of modified MRS liquid medium and cultured at 37°C for 16-22 h. Then, 2% (v / v) inoculum was transferred to a new 5 mL of modified MRS liquid medium and cultured at 37°C for 18 h. Then, 2% (v / v) inoculum was transferred to 200 mL of modified MRS liquid medium for expansion culture and cultured at 37°C 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 adjusted to neutral pH, filtered through a membrane (0.22 µm aqueous filter membrane) for sterilization, and then freeze-dried to obtain the freeze-dried powder of Bifidobacterium longum CCFM1508 fermentation supernatant (denoted as CCFM1508-S) for later use. The bacterial sludge is resuspended in sterile water at a wet weight ratio of 1:50 (m / v). The resulting resuspended solution is homogenized under high pressure (1200 MPa, 12 times). After homogenization, centrifuge at 8000 g for 25 min to collect the supernatant, and then heat-treated (85℃, 15 min) to obtain the bacterial cell lysate (denoted as CCFM1508-J).

[0056] The preparation method of live Bifidobacterium longum CCFM1508 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 live Bifidobacterium longum CCFM1508 glycerol tube, which is denoted as CCFM1508-H.

[0057] (2) The postgenetic agents (cell lysate and fermentation supernatant) of Bifidobacterium longum FSCREG6M5-3, Bifidobacterium longum 1X3, Bifidobacterium longum B38X1 and Bifidobacterium longum B23D8 were prepared simultaneously according to the method in step (1).

[0058] Example 2: Metabiotics prepared from different Bifidobacterium longum increase the ceramide content secreted by 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.

[0059] (2) After the cells adhered to the wall, a control group and an experimental group were set up for 12 h of incubation.

[0060] 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 24 h of culture 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 longum CCFM1508, Bifidobacterium longum FSCREG6M5-3, Bifidobacterium longum 1X3, Bifidobacterium longum B38X1 and Bifidobacterium longum B23D8, respectively).

[0061] 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 longum CCFM1508 (CCFM1508-S and CCFM1508-J), Bifidobacterium longum FSCREG6M5-3 (FSCREG6M5-3-S and FSCREG6M5-3-J), Bifidobacterium longum 1X3 (1X3-S and 1X3-J), Bifidobacterium longum B38X1 (B38X1-S and B38X1-J), and Bifidobacterium longum B23D8 (B23D8-S and B23D8-J) were added respectively.

[0062] (4) 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 is described in the kit instructions. The experimental results are as follows: Figure 1 As shown.

[0063] Experiments showed that, compared to the control group (100%), the postbiotic prepared from *Bifidobacterium longum* B23D8 (cell group B23D8-J) promoted ceramide secretion in HaCaT, increasing it to 134.996%; the postbiotic prepared from *Bifidobacterium longum* 1X3 (supernatant group 1X3-S) increased it to 149.762%; and the postbiotic prepared from *Bifidobacterium longum* CCFM1508 (cell lysate group CCFM1508-J and supernatant group CCFM1508-S) both promoted ceramide secretion in HaCaT, increasing it to 176.863% and 159.189%, respectively. Other experimental groups showed no significant promoting effect on ceramide secretion in HaCaT. Based on these results, the postbiotic prepared from *Bifidobacterium longum* CCFM1508 (cell and supernatant groups) showed the best effect in promoting ceramide synthesis in HaCaT.

[0064] Example 3: Effect of postbiotic prepared from Bifidobacterium longum CCFM1508 on HaCaT cell activity (1) HaCaT cells in the logarithmic growth phase were taken at 1.0 × 10⁻⁶. 4100 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.

[0065] 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 the bacterial culture at CFU / mL is equivalent to that prepared from the bacteria. 100 μL of resuspended postbiotic prepared from Bifidobacterium longum CCFM1508 was added.

[0066] (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.

[0067] 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.

[0068] HaCaT cells are core cells for the synthesis, secretion, and homeostasis of ceramides in the skin. When the proliferation, differentiation, or metabolic function of keratinocytes is impaired, abnormal ceramide synthesis inevitably occurs, leading to skin barrier dysfunction. Therefore, promoting keratinocyte vitality can indirectly promote the synthesis of ceramides in the skin. The effects of Bifidobacterium longum CCFM1508 on cell activity are as follows... Figure 2 As shown, compared with the control group (cell proliferation rate 100%), the addition of postbiotics prepared from Bifidobacterium longum CCFM1508 (CCFM1508-S and CCFM1508-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 105.089% and 144.55%, respectively. The experimental results showed that the postbiotics (CCFM1508-S and CCFM1508-J) prepared from Bifidobacterium longum CCFM1508 could both enhance HaCaT activity, with CCFM1508-J showing the best effect.

[0069] Based on Examples 2 and 3, it was determined that Bifidobacterium longum CCFM1508 has the greatest potential to promote the synthesis of skin ceramides. Therefore, it was selected for in vivo experimental verification.

[0070] Example 4: Metabiotics prepared from Bifidobacterium longum CCFM1508 improve skin health in mice with skin barrier damage (reducing transepidermal water loss and increasing stratum corneum moisture content). In the implementation plan, the preparation method of live Bifidobacterium longum CCFM1508 and its postbiotics (CCFM1508-S and CCFM1508-J) is the same as in Example 1. The difference is that in the preparation method of the postbiotic cells (CCFM1508-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.

[0071] Thirty healthy male BALB / c mice aged 7 weeks from Vital River 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 (CCFM1508-S), the metagenic bacterial cell group (CCFM1508-J), and the live bacteria group (CCFM1508-H)). The details are as follows: Control group: Using physiological saline as a control for the gavage sample.

[0072] 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 was applied for each modeling.

[0073] CCFM1508-H group: The gavage sample contained live Bifidobacterium longum CCFM1508, at a dose of 1×10⁻⁶. 9 CFU / mouse / day; mouse modeling method is the same as that of the model group.

[0074] Group CCFM1508-J: The gavage sample was postbiotics (inactivated bacteria), 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.

[0075] CCFM1508-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 *Bifidobacterium longum* CCFM1508 containing CFU; the mouse modeling method was the same as that of the model group.

[0076] 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.

[0077] 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 the mice's skin surface parameters (transepidermal water loss (TEWL)) and stratum corneum moisture content, respectively. The experimental results are as follows: Figure 4 The test results showed that, compared to the blank group (15.27 gm), -2 h -1 In the model group, transepidermal water loss increased to 28.00 gm. -2 h -1 Compared to the model group, oral administration of Bifidobacterium longum CCFM1508-S, CCFM1508-J, and CCFM1508-H significantly reduced transepidermal water loss in mice, decreasing it to 19.02 gm. -2 h -1 13.32 gm -2 h -1 18.77 gm -2 h -1 The CCFM1508-J group showed the best results. The results of the mouse skin stratum corneum water content measurement are as follows: Figure 5 As shown, the stratum corneum water content in the control group was 66.08%, while in the model group, the stratum corneum water content of mice was as low as 47.77%. The stratum corneum water content of mice was increased to 52.98%, 63.47%, and 52.43% respectively by gavage with Bifidobacterium longum CCFM1508-S, CCFM1508-J, and CCFM1508-H groups, respectively. Among them, the effect of CCFM1508-J group was better than that of CCFM1508-S and CCFM1508-H.

[0078] In summary, the experimental results show that Bifidobacterium longum CCFM1508 and its prepared metabiotic can significantly improve the skin appearance indicators of mice, reflecting that Bifidobacterium longum CCFM1508 and its prepared metabiotic can effectively enhance the water-locking capacity of mouse skin and improve skin health in mice with skin barrier damage, with the CCFM1508-J group showing the most significant effect.

[0079] Example 5: Effects of Bifidobacterium longum CCFM1508 and its prepared postbiotic on ceramide content in mouse skin tissue.

[0080] 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.

[0081] The content of ceramides in mouse skin was determined using the ELISA method, such as... Figure 6 As shown in the figure. The results showed that the ceramide content in the skin of mice in the control group was 32.59 μmol / L, while in the model group, the ceramide content decreased to 27.97 μmol / L. The ceramide content in the CCFM1508-S group was 27.63 μmol / L, the CCFM1508-J group was 33.07 μmol / L, and the CCFM1508-H group was 30.07 μmol / L. Except for the CCFM1508-S group, both the CCFM1508-J and CCFM1508-H groups promoted the synthesis of ceramide in mouse skin tissue, with the CCFM1508-J group showing the best promoting effect.

[0082] Example 6: Bifidobacterium longum CCFM1508 and its prepared metabiotic promote the expression of key enzymes (SPT, CerS4) in the de novo synthesis of ceramides in mouse skin. 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.

[0083] (1) Effects of Bifidobacterium longum CCFM1508 and its postbiotic on the expression of rate-limiting enzyme (SPT) during de novo synthesis of ceramides in mouse skin; The sample preparation method was the same as in Example 5. The ELISA method was used to determine the content of the rate-limiting enzyme SPT in mouse skin tissue. The results are as follows: Figure 7Data 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 longum CCFM1508 and its post-biotic, the SPT content in the CCFM1508-S group slightly increased to 31.03 ng / mg, and in the CCFM1508-J group, the SPT content in the skin was 36.03 ng / mg, significantly higher than that in the model group. In the CCFM1508-H group, the SPT content was 32.46 ng / mg. These results indicate that the post-biotic prepared from Bifidobacterium longum CCFM1508 (CCFM1508-J) can effectively promote the synthesis of the rate-limiting enzyme in the de novo synthesis of ceramides in the skin.

[0084] (2) Effects of Bifidobacterium longum CCFM1508 and its postbiotic on gene expression of CerS4, a key enzyme in the de novo synthesis of ceramides in mouse skin; The sample collection method was the same as in Example 5. 30 mg of the obtained mouse skin tissue was weighed, added to the lysis buffer (the lysis buffer from the RNA extraction kit), and homogenized to obtain a tissue slurry. RNA was extracted from the skin tissue according to the kit instructions, and cDNA was obtained by reverse transcription. The expression level of the ceramide synthase (CerS4) gene in the mouse skin tissue was measured using RT-qPCR. The results are as follows: Figure 8 The primer sequences used for the CerS4 gene are shown in Table 1.

[0085] Table 1: Primer sequences

[0086] The relative expression levels of ceramide synthase 4 (CerS4) were measured by oral administration of CCFM1508-S, CCFM1508-J, and CCFM1508-H. The relative expression levels of CerS4 mRNA were 0.21, 0.66, and 0.45, respectively. Compared with the model group (0.10), CCFM1508-J and CCFM1508-H significantly upregulated the expression level of CerS4, while CCFM1508-S had no significant effect.

[0087] In summary, the results indicate that Bifidobacterium longum CCFM1508 and its postbiotics can promote the synthesis of neuroceramide in mouse skin tissue by promoting the expression of key enzymes (SPT, CerS4) in the de novo ceramide synthesis pathway.

[0088] Example 7: Bifidobacterium longum CCFM1508 and its prepared postbiotics reduce skin inflammatory responses caused by decreased skin ceramide content. 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.

[0089] (1) Effects of Bifidobacterium longum CCFM1508 and its postbiotics on the content of pro-inflammatory factor IL-1 in mouse skin tissue The sample preparation method was the same as in Example 5. The ELISA method was used to measure the inflammatory factor IL-1, and the experimental results are as follows: Figure 9 As shown, interleukin-1 (IL-1) is a key pro-inflammatory cytokine in skin tissue, and its excessive release is a core driver of various skin inflammations. In mice, IL-1 levels significantly increased after skin barrier damage, but decreased after gavage administration of *Bifidobacterium longum* CCFM1508 and its postbiotics. In the model group, the IL-1 level was 15.14 pg / mg, significantly higher than the control group (11.75 pg / mg). After oral administration of *Bifidobacterium longum* CCFM1508 and its postbiotics (CCFM1508-S, CCFM1508-J, CCFM1508-H), the IL-1 levels were 13.45 pg / mg, 11.80 pg / mg, and 12.21 pg / mg, respectively, all lower than the model group. The postbiotic group (CCFM1508-J) showed the best effect, effectively reducing inflammation and maintaining skin homeostasis.

[0090] (2) Effects of Bifidobacterium longum CCFM1508 and its postbiotics on the content of pro-inflammatory factor IL-4 in mouse skin tissue The sample preparation method was the same as in Example 5. The ELISA method was used to measure the inflammatory factor IL-4, and the experimental results are as follows: Figure 9 As shown. Interleukin-4 (IL-4) is a pluripotent cytokine mainly secreted by activated Th2 T cells, mast cells, and basophils. It plays a crucial role in skin immune regulation and inflammatory responses. Its function is bidirectional: low levels of IL-4 participate in regulating the differentiation balance of keratinocytes, while excessive IL-4 is an important cause of skin barrier damage. It can reduce the integrity and water retention capacity of the stratum corneum by inhibiting the synthesis of barrier-related components (such as ceramides) by keratinocytes, leading to increased skin permeability (making it easier for external allergens and pathogens to invade). Experimental results showed that after oral administration of Bifidobacterium longum CCFM1508 and its post-biotic, its content decreased to 3.079 pg / mg, 2.578 pg / mg, and 2.371 pg / mg, respectively, which were significantly lower than those in the model group (3.646 pg / mg), effectively maintaining the integrity of the skin barrier. Compared to the control group (2.583 pg / mg), the CCFM1508-J group was able to maintain the skin barrier and regulate the differentiation balance of keratinocytes at an appropriate low level.

[0091] In summary, all the above examples show that the postbiotic prepared from *Bifidobacterium longum* CCFM1508 (cell group CCFM1508-J) exhibits significant positive regulatory effects in promoting skin ceramide synthesis, increasing the expression of housekeeping enzymes (SPT) during de novo ceramide synthesis, reducing pro-inflammatory factors (IL-1, IL-4), reducing transepidermal water loss, and increasing stratum corneum moisture content. Therefore, the postbiotic prepared from *Bifidobacterium longum* CCFM1508 can effectively maintain skin health by promoting skin ceramide synthesis.

[0092] 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. A strain of Bifidobacterium longum ( Bifidobacterium longum subsp. longum CCFM1508, the Bifidobacterium longum CCFM1508, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCCNo: 66744.

2. A microbial preparation containing the Bifidobacterium longum CCFM1508 as described in claim 1.

3. The metabiotic prepared from *Bifidobacterium longum* CCFM1508 as described in claim 1, characterized in that, The postgenetic is any one of (a) to (c): (a) After fermenting the Bifidobacterium longum CCFM1508, the fermentation supernatant was collected; (b) Heat-treat the cells of Bifidobacterium longum CCFM1508 to obtain inactivated cells; (c) The bacterial cells of Bifidobacterium longum CCFM1508 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 longum CCFM1508 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 longum* CCFM1508 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 longum CCFM1508 of claim 1, the microbial preparation of claim 2, or the postbiotic of claim 3 in the preparation of ceramides or products containing ceramides.

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