Lactobacillus reuteri CCFM1502 with effects of up-regulating skin ceramide synthesis and reducing skin inflammation and metagen of lactobacillus reuteri CCFM1502

Abstract

The invention discloses lactobacillus reuteri CCFM1502 capable of up-regulating skin ceramide synthesis and reducing skin inflammation and a metagen of the lactobacillus reuteri CCFM1502, and belongs to the technical field of microorganisms and medicines. The lactobacillus reuteri CCFM1502 provided by the invention can remarkably promote synthesis of ceramide when being externally used and orally taken, and can reduce skin inflammation response of mice after being orally taken. Specifically, the strain lysate and the fermentation supernatant both can promote HaCaT cells to secrete ceramide; the total amount of ceramide in skin can be up-regulated by oral administration of viable bacteria and metabiotics of the lactobacillus reuteri, apparent indexes of the skin can be regulated and controlled, inflammatory reactions in vivo and tissues can be reduced, and particularly, the effect of fermentation supernate of the lactobacillus reuteri is optimal. And the thallus components and viable bacteria have obvious effects in promoting the expression of anti-inflammatory factors in skin tissues. Therefore, the lactobacillus reuteri has a huge application prospect in preparation of a product for promoting synthesis of the natural active ingredient ceramide of the skin.

Description

Technical Field

[0001] This invention relates to Lactobacillus reuteri CCFM1502 and its post-biotics, which upregulate skin ceramide synthesis and reduce skin inflammation, and belongs to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The skin is an important physical, chemical, and immune barrier for the body, effectively resisting external environmental stimuli, pathogenic microorganisms, and preventing the loss of body fluids. The integrity of the skin barrier function mainly depends on the stratum corneum, the outermost layer of the epidermis. This structure is often likened to a "brick wall model," with keratinocytes acting as the "bricks" and intercellular lipids filling the gaps like "mortar," working together to maintain the structural stability and functional integrity of the skin barrier. Ceramides account for approximately 50% of intercellular lipids, making them the most abundant and functionally crucial type of lipid molecule. They not only function as a structural component in the physical barrier process but also actively participate in cell signal transduction, regulating the proliferation, differentiation, and apoptosis of keratinocytes. Therefore, the balance of ceramide content and composition is of paramount importance for maintaining healthy skin.

[0003] Studies have shown that insufficient ceramide synthesis or an imbalance in its ratio is a common pathological feature of various inflammatory skin diseases, such as atopic dermatitis (AD), psoriasis, and dry skin. Ceramide deficiency directly weakens the skin barrier function, leading to increased transepidermal water loss and decreased stratum corneum moisture content, making it easier for external allergens, irritants, and pathogens to invade. This process further triggers and continuously exacerbates the skin inflammatory response, forming a vicious cycle of "barrier damage—inflammation exacerbation—barrier repair impairment." Therefore, promoting endogenous ceramide synthesis to enhance skin barrier function at its source has become an important strategy for breaking this cycle and effectively inhibiting skin inflammation. However, current methods for exogenous ceramide supplementation (such as topical preparations) have limitations, including low transdermal absorption efficiency, high cost, and the fact that they only achieve passive supplementation rather than active regulation of the body's synthesis.

[0004] Currently, there are few inventions targeting the regulation of endogenous ceramide synthesis in the skin. Most focus on the direct application of artificially synthesized ceramides or the addition of ceramide synthesis raw materials to promote their synthesis. For example, CN118910187B discloses that a biopolysaccharide prepared from yam and Solomon's seal can promote the expression of ceramide synthase; CN110151672A discloses a composition of Lactobacillus plantarum GMNL-6 that can promote collagen secretion or the expression of ceramide synthase. While using topical synthetic biopolysaccharides or probiotic compositions can increase the expression of related enzymes, the effect on ceramide content is unclear. Furthermore, topical biomolecules have low skin penetration, making it difficult to penetrate deep into the skin to regulate or supplement ceramides, resulting in short-term effects and hindering long-term regulation. This invention screens and isolates a strain of Lactobacillus reuteri CCFM1502. Its live bacteria and metabiotics significantly promote ceramide synthesis in MC903-constructed mouse models, demonstrating a good skin barrier repair effect. According to existing reports, the primary model of skin barrier damage is the mouse Alzheimer's disease (AD) model, with mainstream construction methods including DNFB and MC903 induction. The AD model constructed using MC903 is predominantly based on the Th2 / Th17 axis, simulating endogenous AD; however, DNFB exhibits a mixed Th1-Th2-Th17 pattern, simulating exogenous AD, requiring sensitization and stimulation. The combination of MC903 and repeated peeling with 3M tape simultaneously amplifies physical barrier damage, further amplifying ceramide loss and the inflammatory cascade, making it more conducive to exploring the correlation between ceramide loss and skin health. However, no effective treatment for skin damage in the MC903 combined with repeated 3M tape peeling model has been reported. Summary of the Invention

[0005] This invention provides a *Lactobacillus reuteri* ( Limosilactobacillus reuteri CCFM1502 and its precursors are used in the preparation of products that regulate the synthesis of skin ceramides and relieve skin inflammation.

[0006] This invention provides a strain of *Lactobacillus reuteri* (… Limosilactobacillus reuteri The *Lactobacillus reuteri* strain described in CCFM1502 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo: 66740.

[0007] The *Lactobacillus reuteri* CCFM1502 strain 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 genus *Lactobacillus reuteri*, and it was named *Lactobacillus reuteri* CCFM1502.

[0008] The colonies of *Lactobacillus reuteri* CCFM1502 on MRS solid medium were white, moist, raised, and had regular edges.

[0009] The present invention also provides a microbial preparation containing *Lactobacillus reuteri* CCFM1502, wherein the content of *Lactobacillus reuteri* CCFM1502 in the microbial preparation is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0010] The present invention also provides a metabiotic prepared using the aforementioned *Lactobacillus reuteri* CCFM1502.

[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 deactivated cells are prepared by culturing *Lactobacillus soreii* CCFM1502 in a culture medium for a period of time, collecting bacterial cells from 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: after culturing the Lactobacillus reuteri CCFM1502 in a culture medium for a period of time, the bacterial cells are collected, and the supernatant obtained by high-pressure homogenization and centrifugation is the bacterial lysate. In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging *Lactobacillus reuteri* CCFM1502 after culturing it in a culture medium for a certain period of time. The present invention also provides a composition containing the aforementioned *Lactobacillus reuteri* CCFM1502 and / or its postgenes.

[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 medicines include topical medicines or oral medicines.

[0015] In one embodiment, the daily chemical product does not contain live bacteria.

[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 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.

[0018] In one embodiment, the health product includes the above-described composition and conventional excipients.

[0019] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0020] In one embodiment, the composition is a probiotic powder.

[0021] In one embodiment, the bacterial powder is a solid powder of Lactobacillus reuteri CCFM1502 prepared by drying the liquid postbiotic.

[0022] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0023] The present invention also provides the use of the composition in products that promote the synthesis of ceramides in the skin and reduce skin inflammation.

[0024] 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) Increase the water content of the stratum corneum in mouse skin tissue; (3) Reduces transepidermal water loss (TEWL) in mouse skin tissue; (4) Promotes the synthesis of ceramides in mouse skin tissue; (5) Alleviates inflammatory response in mice; (6) Relieves the inflammatory response in mouse skin tissue.

[0025] In one embodiment, the stratum corneum water content is an important indicator used to measure the water content in mouse skin. A decrease in its content indicates that there is some damage to the skin barrier, which can easily trigger an inflammatory response.

[0026] In one embodiment, transepidermal water loss is the most direct and critical indicator for assessing the failure of skin barrier function caused by a decrease in the content of ceramides in the skin. An increase in its value directly represents skin barrier damage, and the skin may have a certain inflammatory response.

[0027] In one embodiment, the application can be either topical or oral.

[0028] In one embodiment, the product contains at least 1 × 10⁻⁶ Lactobacillus reuteri CCFM1502. 6 CFU / mL.

[0029] In one embodiment, the product contains a dose of postbiotic prepared from *Lactobacillus reuteri* CCFM1502 at a dose equal to 5 times the number of live bacteria.

[0030] In one embodiment, the product is a pharmaceutical or cosmetic product.

[0031] In one embodiment, the pharmaceutical product comprises the *Lactobacillus reuteri* CCFM1502, a drug carrier, and / or pharmaceutical excipients.

[0032] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0033] 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.

[0034] In one embodiment, the cosmetic contains a postbiotic prepared from Lactobacillus reuteri CCFM1502, matrix ingredients, and / or conventional excipients.

[0035] 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.

[0036] 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.

[0037] The present invention also provides the application of the aforementioned *Lactobacillus reuteri* CCFM1502 in food production.

[0038] The present invention also provides the use of the Lactobacillus reuteri CCFM1502 or the microbial preparation, or the post-biotic, in the preparation of daily chemical products that enhance skin ceramide synthesis and / or improve skin's water retention capacity.

[0039] The present invention also provides the use of the Lactobacillus reuteri CCFM1502 or the microbial preparation, or the postbiotic in the preparation of ceramides or products containing ceramides.

[0040] Beneficial effects: The present invention contains *Lactobacillus reuteri* ( Limosilactobacillus reuteri CCFM1502 and its prepared post-biotics can effectively promote the synthesis of ceramides in both topical and oral applications; at the same time, after oral administration, it can also alleviate individual and skin inflammatory responses, specifically manifested in: (1) Promotes the secretion of ceramides by keratinocytes (HaCaT) in vitro; (2) Increase the water content of the stratum corneum in mouse skin tissue; (3) Reduces transepidermal water loss (TEWL) in mouse skin tissue; (4) Promotes the synthesis of ceramides in mouse skin tissue; (5) Alleviates inflammatory response in mice; (6) Relieves the inflammatory response in mouse skin tissue.

[0041] Therefore, *Lactobacillus reuteri* ( Limosilactobacillus reuteri The post-biotic prepared by CCFM1502 has great application potential in products that promote ceramide synthesis and alleviate inflammatory responses.

[0042] Preservation of biological materials Lactobacillus reuteri ( Limosilactobacillus reuteri CCFM1502, taxonomically named Limosilactobacillus reuteri It was deposited on July 24, 2025, at the Guangdong Provincial Center for the Preservation of Microbial Cultures, with accession number GDMCC No: 66740, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0043] Figure 1 Evaluation of the effects of different post-adrenergic stimulants on promoting the secretion of ceramides by keratinocytes (HaCaT); Figure 2 Animal experimentation procedures; Figure 3 Effects of Lactobacillus reuteri CCFM1502 and its prepared metabiotic on the stratum corneum water content in mouse skin tissue; Figure 4 Effects of Lactobacillus reuteri CCFM1502 and its prepared metabiotic on transepidermal water loss in mouse skin tissue; Figure 5 Effects of Lactobacillus reuteri CCFM1502 and its prepared metabiotic on ceramide content in mouse skin tissue; Figure 6 Effects of Lactobacillus reuteri CCFM1502 and its prepared metabiotic on IgE levels in mice; Figure 7Effects of Lactobacillus reuteri CCFM1502 and its prepared metabiotic on the anti-inflammatory factor IL-10 in mouse skin tissue; Figure 8 Effects of Lactobacillus reuteri CCFM1502 and its prepared metabiotic on inflammatory responses 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

[0044] The present invention will be further described below with reference to specific embodiments.

[0045] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0046] The BALB / c mice used in the following examples were purchased from Vital Rivers.

[0047] The *Lactobacillus reuteri* FYNLJ83L8, *Lactobacillus reuteri* C57BLA52-S, *Lactobacillus reuteri* HeN4M1-S, *Lactobacillus reuteri* CCFM1502, and *Lactobacillus reuteri* MRJSWX25L3 strains involved in the following examples were self-screened strains from the Food Biotechnology Center of Jiangnan University.

[0048] 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, and Tween-80 1 mL / L, pH 6.2~6.4.

[0049] 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 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, and agar 18.0 g / L, pH 6.2–6.4.

[0050] 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).

[0051] 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. Using a sterile pipette, 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.

[0052] Example 1: Screening of *Lactobacillus reuteri* and preparation of metabiotics 1. Screening and identification of *Lactobacillus reuteri* 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 incubation at 37°C for 48 h, a suitable dilution was selected, and single colonies were streaked onto MRS solid medium for purification. After incubation at 37°C for 48 h, typical colonies of *Lactobacillus reuteri* were streaked onto MRS solid medium for purification. After incubation at 37°C for 36–48 h, single colonies were picked and enriched in MRS liquid medium. After incubation for 24 h, the medium 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 *Lactobacillus reuteri*, and named *Lactobacillus reuteri* FYNLJ83L8, *Lactobacillus reuteri* C57BLA52-S, *Lactobacillus reuteri* HeN4M1-S, *Lactobacillus reuteri* CCFM1502, and *Lactobacillus reuteri* MRJSWX25L3, respectively. *Lactobacillus reuteri* CCFM1502 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66740.

[0053] 2. Preparation of *Lactobacillus reuteri* CCFM1502 and its postbiotics (1) The frozen Lactobacillus reuteri CCFM1502 was streaked on a modified MRS solid medium for revival and placed in a water-jacketed incubator at 37°C 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 Lactobacillus reuteri fermentation supernatant CCFM1502 (denoted as CCFM1502-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 lysate (denoted as CCFM1502-J).

[0054] The preparation method of live bacteria of *Lactobacillus reuteri* CCFM1502 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 bacteria of *Lactobacillus reuteri* CCFM1502 in glycerol tubes, which are denoted as CCFM1502-H.

[0055] (2) Following the method in step (1), the metagenes (cell lysate and fermentation supernatant) of *Lactobacillus reuteri* FYNLJ83L8, *Lactobacillus reuteri* C57BLA52, *Lactobacillus reuteri* HeN4M1, *Lactobacillus reuteri* CCFM1502 and *Lactobacillus reuteri* MRJSWX25L3 were prepared simultaneously.

[0056] Example 2: In vitro promotion of ceramide secretion from keratinocytes (HaCaT) (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.

[0057] (2) After the cells adhered to the wall, a control group and an experimental group were set up for 12 h of incubation.

[0058] Control group: After 24 h of culture in (1), the original cell culture medium was removed and 2 mL of cell culture medium 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 Lactobacillus reuteri FYNLJ83L8, Lactobacillus reuteri C57BLA52, Lactobacillus reuteri HeN4M1, Lactobacillus reuteri CCFM1502 and Lactobacillus reuteri MRJSWX25L3, respectively).

[0059] 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 amounts of postbiotics prepared from bacterial cultures of CFU / mL were equivalent. 2 mL of resuspended postbiotics prepared from *Lactobacillus reuteri* FYNLJ83L8 (FYNLJ83L8-S and FYNLJ83L8-J), *Lactobacillus reuteri* C57BLA52 (C57BLA52-S and C57BLA52-J), *Lactobacillus reuteri* HeN4M1 (HeN4M1-S and HeN4M1-J), *Lactobacillus reuteri* CCFM1502 (CCFM1502-S and CCFM1502-J), and *Lactobacillus reuteri* MRJSWX25L3 (MRJSWX25L3-S and MRJSWX25L3-J) were added to each culture.

[0060] (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 secreted ceramide 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.

[0061] Normalization of the data revealed that, compared with the control group (100%), C57BLA52-S, HeN4M1-J, CCFM1502-S, and CCFM1502-J significantly increased the ceramide content in HaCaT, reaching 117.99%, 114.23%, 117.44%, and 124.26%, respectively. Among them, the post-biotic prepared by Lactobacillus reuteri CCFM1502 showed the best effect, while the other experimental groups did not have a significant effect on promoting HaCaT secretion.

[0062] Example 3: Effects of Lactobacillus reuteri CCFM1502 and its postbiotics on increasing the stratum corneum moisture content of mouse skin tissue In the implementation plan, the preparation method of live Lactobacillus reuteri CCFM1502 and its postbiotics (CCFM1502-S and CCFM1502-J) is the same as in Example 1. The difference is that in the preparation method of the postbiotic cells (CCFM1502-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 minutes to obtain inactivated bacterial cells.

[0063] 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 (CCFM1502-S), the metagenic bacterial cell group (CCFM1502-J), and the live bacteria group (CCFM1502-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 sample for gavage. 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.

[0064] CCFM1502-H group: The gavage sample was live Lactobacillus reuteri CCFM1502, at a dose of 1×10⁻⁶. 9 CFU / mouse / day; mouse modeling method is the same as that of the model group.

[0065] CCFM1502-J group: The gavage sample was postbiotic (inactivated bacteria), and the dosage was: 5 × 10⁶ live bacteria count. 9 CFU-prepared post-biotic inactivated bacterial cells; mouse modeling method was the same as the model group.

[0066] CCFM1502-S group: The gavage sample was postbiotic (supernatant group), and the dosage was: viable bacteria count 5×10⁻⁶. 9 The supernatant was obtained from the fermentation broth of *Lactobacillus reuteri* CCFM1502 containing CFU; the mouse modeling method was the same as that of the model group.

[0067] 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 2 As shown. After the experiment, mice were briefly anesthetized via isoflurane inhalation, and the stratum corneum moisture content of the mouse skin was measured using a Cutometer DUAL MPA580 equipped with a CM825 probe for stratum corneum moisture content. The experimental results are as follows: Figure 3 The determination of stratum corneum water content in mouse skin revealed that the stratum corneum water content in the control group was 68.08%, while in the model group, the stratum corneum water content decreased to 47.77%. After gavage administration of *Lactobacillus reuteri* CCFM1502-S, CCFM1502-J, and CCFM1502-H, the stratum corneum water content of mice increased to 58.03%, 56.61%, and 54.93%, respectively. Among them, CCFM1502-S and CCFM1502-J were able to effectively increase the stratum corneum water content of mouse skin.

[0068] In summary, the experimental results show that the post-biotics (CCFM1502-S and CCFM1502-J) prepared from Lactobacillus reuteri CCFM1502 can effectively enhance the water-locking ability of mouse skin, thereby delaying the development of inflammation to a certain extent. The CCFM1502-S group showed a more significant effect.

[0069] Example 4: Effects of *Lactobacillus reuteri* CCFM1502 and its postbiotics on reducing transepidermal water loss (TEWL) in mouse skin tissue. The animal experiment design and experimental grouping were the same as in Example 3. After the experiment, mice were briefly anesthetized via isoflurane inhalation, and transepidermal water loss (TEWL) on the mouse skin surface was measured using a Cutometer DUAL MPA580 equipped with a TEWL probe. Transepidermal water loss (TEWL) is a key indicator of skin barrier damage and can represent skin condition and inflammation. The experimental results are as follows: Figure 4 Data showed that the 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 When mice were administered *Lactobacillus reuteri* CCFM1502-S, CCFM1502-J, and CCFM1502-H via gavage, respectively, transepidermal water loss was significantly reduced compared to the model group, decreasing to 16.84 gm. -2 h -1 20.06 gm -2 h -1 24.07 gm -2 h -1 Among them, the CCFM1502-S group showed a reduction effect close to that of the control group, effectively repairing skin barrier damage and reducing inflammatory response; CCFM1502-J also showed a significant effect, but lower than that of the CCFM1502-S group, while the CCFM1502-H group did not show a good regulatory effect.

[0070] Based on the combined results of Examples 3 and 4, it was found that the live bacteria (CCFM1502-H) did not have a significant effect on improving the skin appearance indicators of mice, while the post-biotic groups (CCFM1502-S and CCFM1502-J) showed a better positive regulatory effect on the skin appearance indicators.

[0071] Example 5: Effects of Lactobacillus reuteri CCFM1502 and postbiotics on ceramide content in mouse skin tissue The animal experiment design and experimental grouping were the same as in Example 3. After completing the determination of the skin appearance index of mice, the mice were sacrificed, and the skin tissue on 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.

[0072] The content of ceramides in mouse skin was determined using the ELISA method, such as... Figure 5As shown in the data, the ceramide content in the skin of mice in the control group was 32.59 µmol / L. In the model group, the ceramide content decreased to 27.97 µmol / L. The ceramide content in the CCFM1502-S group was 31.76 µmol / L, the CCFM1502-J group was 28.03 µmol / L, and the CCFM1502-H group was 29.60 µmol / L. The results showed that the CCFM1502-S group had a significant promoting effect on skin ceramide synthesis, while the CCFM1502-J and CCFM1502-H groups did not have a significant upregulation effect.

[0073] Example 6: Effects of Lactobacillus reuteri CCFM1502 and postbiotics on alleviating inflammatory responses in mice; The animal experimental design and experimental grouping involved in the following examples are the same as in Example 3. After the skin appearance index was measured, mouse eye blood was taken into a 1.5 mL centrifuge tube, left to stand for 40 min, and then centrifuged at 3000 r / min for 20 min. The blood supernatant was used for subsequent experimental measurements.

[0074] The level of IgE in mouse serum was determined using the ELISA method. IgE is an immunoglobulin, and its level increases significantly in mouse serum when the skin barrier is damaged or inflammation is present. The experimental results are as follows: Figure 6 As shown, compared with the model group (3007.4 ng / mL), the IgE content in the control group (2755.7 ng / mL) was significantly lower than that in the model group. After oral administration of *Lactobacillus reuteri* CCFM1502 and its prepared metabiotics, the serum IgE content in the CCFM1502-S group decreased to 2772.76 ng / mL, and the IgE content in the CCFM1502-J group decreased to 2676.11 ng / mL, while CCFM1502-H did not show a significant downregulation (2868.9 ng / mL). Data comparison revealed that oral administration of the metabiotics prepared from *Lactobacillus reuteri* CCFM1502 (CCFM1502-S and CCFM1502-J) can reduce the inflammatory response in mice and has a good anti-inflammatory effect.

[0075] Example 7: Effects of Lactobacillus reuteri CCFM1502 and postbiotics on alleviating inflammatory responses in mouse skin tissue The animal experimental design and experimental grouping involved in the following examples are the same as in Example 3, and the sample processing and preparation methods are the same as in Example 5.

[0076] (1) Effects of Lactobacillus reuteri CCFM1502 and its postbiotics on the content of anti-inflammatory factor IL-10 in mouse skin tissue The sample preparation method was the same as in Example 5. In skin tissue, IL-10 is a key anti-inflammatory cytokine with potent anti-inflammatory effects. Its balance can suppress excessive immune responses, prevent tissue damage, and promote tissue repair. When IL-10 levels in skin tissue decrease, it can lead to uncontrolled inflammatory responses, causing severe skin inflammation and disease. The anti-inflammatory factor IL-10 in mouse skin tissue was measured using an ELISA method. The experimental results are as follows: Figure 7 As shown in the data, the IL-10 level in the model group was 4.05 ng / mg, which was significantly lower than that in the control group (5.59 ng / mg). After oral administration of *Lactobacillus reuteri* CCFM1502 and its postbiotics (CCFM1502-S, CCFM1502-J, and CCFM1502-H), the IL-10 levels increased to 4.64 ng / mg, 5.39 ng / mg, and 5.59 ng / mg, respectively. The oral administration of CCFM1502-J and CCFM1502-H significantly upregulated IL-10 expression, thereby reducing skin tissue inflammation and repairing tissue damage.

[0077] (2) Effects of Lactobacillus reuteri CCFM1502 and its postbiotics on the content of pro-inflammatory factor TNF-α in mouse skin tissue The sample preparation method was the same as in Example 5. TNF-α is the "core commander" of inflammation in mouse skin tissue and one of the initiating signals of the inflammatory response. It promotes the production of other pro-inflammatory factors, and its persistently significant increase can cause a huge inflammatory response in the skin tissue, resulting in tissue damage. The inflammatory factor TNF-α in mouse skin tissue was measured using an ELISA method. The experimental results are as follows: Figure 8 As shown in the figure. Experimental results showed that after gavage administration of *Lactobacillus reuteri* CCFM1502 and its post-biotic, the TNF-α levels in mouse skin tissue were 20.48 pg / mg, 23.46 pg / mg, and 20.90 pg / mg, respectively, all lower than the model group (27.03 pg / mg). Among them, the CCFM1502-J group did not show a good regulatory effect on TNF-α levels. However, the CCFM1502-S and CCFM1502-H groups were able to regulate TNF-α levels, reduce skin inflammation, maintain the skin barrier, and reduce the production of other pro-inflammatory factors in mouse skin tissue inflammation.

[0078] In summary, all the above examples show that *Lactobacillus reuteri* CCFM1502 and its prepared metabiotics (supernatant group CCFM1502-S and cell group CCFM1502-J) have a positive regulatory effect on promoting skin ceramide synthesis and reducing inflammation in mice and their skin tissues. CCFM1502-S showed a more prominent regulatory effect, significantly upregulating skin ceramides, increasing stratum corneum water content, reducing transepidermal water loss, reducing serum immunoglobulin IgE, and reducing tissue TNF-α levels. CCFM1502-J and CCFM1502-H groups showed good effects in promoting the synthesis of the anti-inflammatory factor IL-10 in the skin. Therefore, *Lactobacillus reuteri* CCFM1502 and its metabiotics can significantly upregulate the ceramide content in the skin, positively regulate skin epigenetic indicators, and significantly reduce inflammatory responses in mice and their skin tissues.

[0079] 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 *Lactobacillus reuteri* ( Limosilactobacillus reuteri CCFM1502, the Lactobacillus reuteri CCFM1502, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66740.

2. A microbial preparation containing the *Lactobacillus reuteri* CCFM1502 as described in claim 1.

3. The metabiotic prepared from *Lactobacillus reuteri* CCFM1502 as described in claim 1, characterized in that, The postgenetic is any one of (a) to (c): (a) After fermenting the Lactobacillus reuteri CCFM1502, collect the fermentation supernatant; (b) Heat-treat the cells of the *Lactobacillus reuteri* CCFM1502 to obtain inactivated cells; (c) The bacterial cells of the Lactobacillus reuteri CCFM1502 were resuspended and homogenized under high pressure. The supernatant was collected by centrifugation and then heat-treated to obtain the bacterial cell lysate. The fermentation supernatant or cell lysate is either a liquid or a powder obtained after freeze-drying.

4. A product containing *Lactobacillus reuteri* CCFM1502 as described in claim 1, or the microbial preparation as described in claim 2, or the postbiotic as described in 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 *Lactobacillus reuteri* CCFM1502 as described in claim 1, or the microbial preparation as described in claim 2, or the postbiotic as described in 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 Lactobacillus reuteri CCFM1502 of claim 1, or the microbial preparation of claim 2, or the postbiotic of claim 3 in the preparation of ceramides or ceramide-containing products.

Images