Lactobacillus paracasei CCFM1503 capable of inhibiting skin ceramide decomposition and relieving skin inflammation and metagen of lactobacillus paracasei CCFM1503

Abstract

The invention discloses a lactobacillus paracasei CCFM1503 capable of inhibiting skin ceramide decomposition and relieving skin inflammation and a metagen thereof, and belongs to the technical field of microorganisms and medicines. The lactobacillus paracasei CCFM1503 provided by the invention can be used for effectively increasing the content of ceramide secreted by keratinocytes and effectively promoting the vitality of the keratinocytes in external application. The composition can inhibit the expression of ceramide decomposition related enzymes and relieve skin inflammation during oral administration, specifically, the expression of SMS and ASAH1 enzymes is inhibited, so that the abnormal decomposition of ceramide is inhibited; the compound can inhibit the expression of related proinflammatory factors (IL-1, IL-4 and TSLP), reduce the skin inflammation reaction caused by abnormal decomposition of skin ceramide and repair skin injury, has a certain application prospect in treatment of skin inflammation and injury caused by abnormal decomposition of ceramide, and is used for preparing external or oral medicines.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus paracasei CCFM1503 that inhibits the breakdown of skin ceramides and relieves skin inflammation, and its post-biotics, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The integrity of the skin barrier function is the core foundation for maintaining healthy skin. The skin barrier is mainly composed of keratinocytes and intercellular lipids, and ceramides, as the most abundant lipid component in this structure (accounting for more than 50%), are a core component for maintaining barrier function. Ceramides, through synergistic effects with cholesterol and free fatty acids, not only effectively prevent moisture loss from the stratum corneum and reduce transepidermal water loss (TEWL), but also show a clear causal relationship between decreased ceramide levels and the occurrence of skin inflammation. When ceramide levels decline, the skin barrier function fails, stimulating keratinocytes to release pro-inflammatory cytokines, leading to inflammatory symptoms such as erythema, burning, and itching. Clinical studies have confirmed that the ceramide content in the lesions of patients with atopic dermatitis and eczema is significantly lower than that in healthy skin, and the degree of reduction is positively correlated with the severity of inflammation. After ceramide supplementation, the barrier function of these patients is effectively repaired, and the levels of inflammatory factors decrease significantly. These inflammations lead to increased expression and activity of a key hydrolytic enzyme (acid ceramidinase ASAH1). This type of acid ceramidinase specifically hydrolyzes ceramides, resulting in decreased ceramide levels in the skin, causing skin barrier damage and increased skin inflammation. Therefore, directly inhibiting the activity of acid ceramidinase in the skin and reducing excessive ceramide breakdown is an effective strategy for repairing the skin barrier and fundamentally alleviating skin inflammation.

[0003] Existing research indicates that oral or topical application of specific probiotics can improve skin barrier function and alleviate inflammation. For example, CN116083301B discloses that the fermentation supernatant of a strain of Bifidobacterium bifidum can promote the synthesis of ceramides in epidermal cells, but the regulatory pathway is not clearly defined, and its oral efficacy has not been verified. CN119776238B discloses that a strain of Lactobacillus plantarum 24 can regulate sphingomyelinase synthesis, thereby promoting ceramide synthesis, and can also regulate the expression of skin-related inflammatory markers, but there is no data showing its inhibitory effect on ceramides. However, due to the high degree of variability among probiotic strains, their functional effects are difficult to simply extrapolate. Currently, CN118360179A discloses a strain of Lactobacillus paracasei and its metabolites, which have certain effects on anti-acne, anti-inflammatory, and antibacterial effects on the skin. However, related studies are mainly based on in vitro cell experiments and focus on changes in the skin appearance (such as water content and sebum secretion) of specific populations, lacking systematic research on specific skin barrier damage models. At the same time, the specific mechanism and effect of its inflammatory repair function have not been thoroughly evaluated. Summary of the Invention

[0004] This invention discovers that a strain of Lactobacillus paracasei and its metabolites can effectively inhibit the expression of enzymes related to ceramide degradation, inhibit ceramide degradation, repair the skin barrier, and relieve skin inflammation. It has great application potential in oral or topical medicines, cosmetics, food, and health products.

[0005] This invention provides a Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1503 and its derivatives are used in the preparation of products that inhibit the decomposition of skin ceramides and relieve skin inflammation.

[0006] This invention provides a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei The *Lactobacillus paracasei* strain described in CCFM1503 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66741.

[0007] In one embodiment, the *Lactobacillus paracasei* CCFM1503 colonies on MRS solid medium are small, round, smooth, with neat edges, opaque, and milky white.

[0008] The present invention also provides a microbial preparation containing the aforementioned *Lactobacillus paracasei* CCFM1503, wherein the content of *Lactobacillus paracasei* CCFM1503 in the microbial preparation is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0009] The present invention also provides a metabiotic prepared using the aforementioned Lactobacillus paracasei CCFM1503.

[0010] 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 configured as follows: Preparation method: Lactobacillus paracasei CCFM1503 was cultured in a culture medium for a period of time, and bacterial cells in the bacterial culture medium were collected and inactivated bacterial cells were obtained after heat treatment. In one embodiment, the heat treatment conditions are: 85°C for 15 min.

[0011] In one embodiment, the method for preparing the bacterial lysate is as follows: the *Lactobacillus paracasei* CCFM1503 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 the bacterial lysate. In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging Lactobacillus paracasei CCFM1503 after culturing it in a culture medium for a period of time. The present invention also provides compositions containing the aforementioned *Lactobacillus paracasei* CCFM1503 and / or its postgenes.

[0012] In one embodiment, the composition includes, but is not limited to, pharmaceuticals or daily chemical products.

[0013] In one embodiment, the daily chemical products include skin care products, cosmetics, or toiletries; the medicine includes topical medicines or oral medicines.

[0014] In one embodiment, the composition is a probiotic powder.

[0015] In one embodiment, the bacterial powder is a solid powder of Lactobacillus paracasei CCFM1503 prepared by drying the liquid postbiotic.

[0016] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0017] The present invention also provides the use of the aforementioned Lactobacillus paracasei CCFM1503 and / or its metabiotics in the preparation of drugs or daily chemical products that increase the ceramide content in the skin.

[0018] The present invention also provides the use of the composition in the preparation of a medicament for repairing the skin barrier or skin damage.

[0019] In one embodiment, the drug has at least one of the following effects: (1) Promotes the secretion of ceramides by keratinocytes (HaCaT); (2) Promotes the vitality of keratinocytes (HaCaT); (3) Increase the moisture content of the individual's stratum corneum; (4) Relieves transepidermal water loss in individual skin; (5) Inhibits the expression of ceramide degradation-related enzymes in individual skin tissue; (6) Alleviate the inflammatory response of individual skin tissues; In one embodiment, the skin inflammatory response is characterized by decreased stratum corneum moisture content, increased transepidermal water loss, and increased expression of pro-inflammatory factors (IL-1, IL-4, TSLP).

[0020] In one embodiment, the enzymes associated with ceramide degradation include sphingomyelin synthase (SMS) and acid ceramide enzyme (ASAH1).

[0021] In one embodiment, the drug is applied topically and orally.

[0022] In one embodiment, the content of *Lactobacillus paracasei* CCFM1503 in the drug is not less than 1 × 10⁻⁶. 6 CFU / mL.

[0023] In one embodiment, the content of *Lactobacillus paracasei* CCFM1503 in the drug is not less than 5.0 × 10⁻⁶. 9 CFU / mL.

[0024] In one embodiment, the dose of the postbiotic prepared from Lactobacillus paracasei CCFM1503 in the drug is not less than 1500 μg / kg body weight.

[0025] In one embodiment, the drug comprises the *Lactobacillus paracasei* CCFM1503, a drug carrier, and / or pharmaceutical excipients.

[0026] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0027] 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.

[0028] In one embodiment, the cosmetic contains *Lactobacillus paracasei* CCFM1503, 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.

[0029] 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.

[0030] Beneficial effects: The present invention contains Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1503 and its prepared post-biotics, when applied topically, can promote ceramide synthesis, and when taken orally, can effectively inhibit ceramide decomposition and alleviate skin inflammation. Specifically: (1) Promotes the secretion of ceramides by keratinocytes (HaCaT) in vitro; (2) Promotes the viability of keratinocytes (HaCaT) in vitro; (3) Increase the water content of the stratum corneum of mouse skin; (4) Reduces transepidermal water loss in mice; (5) Inhibits the expression of ceramide degradation-related enzymes in mouse skin tissue; (6) Alleviates inflammatory response in mouse skin tissue; Therefore, Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1503 and its prepared post-biotics have great application potential in products that relieve skin inflammation and repair the skin barrier.

[0031] Preservation of biological materials Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1503, taxonomically named Lacticaseibacillus paracasei It was deposited on July 24, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66741, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0032] Figure 1 Evaluation of the effects of different metagenes on promoting ceramide secretion from keratinocytes (HaCaT); Figure 2 The effect of Lactobacillus paracasei CCFM1503 on keratinocyte (HaCaT) viability; Figure 3 Animal experimentation procedures; Figure 4 The effect of Lactobacillus paracasei CCFM1503 and its post-biotics on the water content of the stratum corneum in mouse skin; Figure 5 The effect of Lactobacillus paracasei CCFM1503 and its postbiotics on transepidermal water loss in mouse skin; Figure 6 The effect of Lactobacillus paracasei CCFM1503 and its postbiotics on the expression of ceramide degradation-related enzymes in mouse skin tissue; Figure 7 The effects of Lactobacillus paracasei CCFM1503 and its postbiotics on inflammatory responses in mouse skin tissue; Figure 8 Visual representation of Lactobacillus paracasei CCFM1503 and its postbiotics in alleviating skin inflammation in mice in skin tissue pathology sections; "*" 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

[0033] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0034] The BALB / c mice used in the following examples were purchased from Vital Rivers.

[0035] The *Lactobacillus paracasei* M207F01L421, *Lactobacillus paracasei* FZJHZ22L2, *Lactobacillus paracasei* FJSWXBB6L2, *Lactobacillus paracasei* C57BLB52, and *Lactobacillus paracasei* CCFM1503 involved in the following examples were self-screened strains from the Food Biotechnology Center of Jiangnan University.

[0036] 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.

[0037] 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.

[0038] 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).

[0039] 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.

[0040] Example 1: Screening of Lactobacillus paracasei 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 paracasei* 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 (by Suzhou Genewiz Biotechnology Co., Ltd.), and the results, confirmed by NCBI sequence alignment, identified them as *Lactobacillus paracasei*, and named *Lactobacillus paracasei* M207F01L421, *Lactobacillus paracasei* FZJHZ22L2, *Lactobacillus paracasei* FJSWXBB6L2, *Lactobacillus paracasei* C57BLB52, and *Lactobacillus paracasei* CCFM1503, respectively.

[0041] Example 2: Preparation of Lactobacillus paracasei CCFM1503 and its postbiotics (1) Preparation of bacterial suspension: The frozen Lactobacillus paracasei CCFM1503 was streaked on modified MRS solid medium for recovery and placed in a 37°C water-jacketed constant temperature incubator for 36-48 h. Single colonies were picked and added to 5 mL of modified MRS liquid medium and cultured at 37°C for 16-22 h. Then, 2% (v / v) inoculum was added to a new 5 mL of modified MRS liquid medium and cultured at 37°C for 18 h. Then, 2% (v / v) inoculum was added 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. (2) Preparation of bacterial supernatant: Centrifuge the 200 mL bacterial suspension prepared in step (1) at 8000 g for 25 min, and collect the supernatant and bacterial sludge respectively. The obtained supernatant needs to be adjusted for pH and filtered through a membrane (0.22 µm aqueous filter membrane) for sterilization. Then freeze-dry at -50℃ for 36 h to obtain freeze-dried powder of fermentation supernatant of Lactobacillus paracasei CCFM1503 (denoted as CCFM1503-S) for later use.

[0042] (3) Preparation of bacterial lysate: The bacterial sludge prepared in step (2) was added to sterile water at a wet weight ratio of 1:50 and resuspended. The resulting bacterial concentration of 20 g / L was subjected to high pressure homogenization (1200 MPa, 12 times). After homogenization, the supernatant was collected by centrifugation at 8000 g / min for 25 min and then heat-treated (85℃, 15 min) to obtain bacterial lysate (denoted as CCFM1503-J).

[0043] (4) The preparation method of live bacteria of Lactobacillus paracasei CCFM1503 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 and 25 min is resuspended in a ratio of 1 g: 2 mL of glycerol to obtain live bacteria of Lactobacillus paracasei CCFM1503 glycerol tube, which is denoted as CCFM1503-H.

[0044] The preparation methods of Lactobacillus paracasei M207F01L421, Lactobacillus paracasei FZJHZ22L2, Lactobacillus paracasei FJSWXBB6L2, and Lactobacillus paracasei C57BLB52 postbiotics are as follows (1) to (4).

[0045] Example 3: 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.

[0046] (2) After the cells adhered to the wall, a control group and an experimental group were set up for 12 h of incubation.

[0047] 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 Lactobacillus paracasei M207F01L421, Lactobacillus paracasei FZJHZ22L2, Lactobacillus paracasei FJSWXBB6L2, Lactobacillus paracasei C57BLB52 and Lactobacillus paracasei CCFM1503, respectively).

[0048] 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). 7The amount of metabiotics prepared from bacterial cultures of CFU / mL is equivalent. 2 mL of metabiotics prepared from *Lactobacillus paracasei* M207F01L421 (M207F01L421-S and M207F01L421-J), *Lactobacillus paracasei* FZJHZ22L2 (FZJHZ22L2-S and FZJHZ22L2-J), *Lactobacillus paracasei* FJSWXBB6L2 (FJSWXBB6L2-S and FJSWXBB6L2-J), *Lactobacillus paracasei* C57BLB52 (C57BLB52-S and C57BLB52-J), and *Lactobacillus paracasei* CCFM1503 (CCFM1503-S and CCFM1503-J) were added respectively.

[0049] (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.

[0050] Normalization of the data revealed that, compared with the control group (100%), the post-biotics prepared by *Lactobacillus paracasei* M207F01L421 (M207F01L421-J), *Lactobacillus paracasei* FZJHZ22L2 (FZJHZ22L2-J), *Lactobacillus paracasei* FJSWXBB6L2 (FJSWXBB6L2-J), and *Lactobacillus paracasei* CCFM1503 (CCFM1503-S and CCFM1503-J) all significantly increased the ceramide content in HaCaT, increasing it to 124.13%, 124.70%, 131.44%, 138.50%, and 163.86%, respectively. Among them, the post-biotic prepared by *Lactobacillus paracasei* CCFM1503 showed the best effect, while the remaining experimental groups did not show a significant improvement in promoting ceramide secretion in HaCaT.

[0051] Example 4: Effect of post-biotic prepared from Lactobacillus paracasei CCFM1503 on HaCaT cell activity (1) HaCaT cells in the logarithmic growth phase were taken at 1.0 × 10⁻⁶. 4 Cells were seeded at a concentration of 100 μL / well in 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 control group, control group and post-genetic 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 post-genetic treatment group contained cell culture medium and HaCaT cells, as well as post-genetic agents.

[0052] 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 metabiotic prepared from bacterial culture of CFU / mL is equivalent to that prepared from Lactobacillus paracasei CCFM1503. 100 μL of metabiotic prepared from Lactobacillus paracasei CCFM1503 was added.

[0053] (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.

[0054] 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%. The results are as follows: Figure 2 As shown.

[0055] Keratinocytes are core functional cells that secrete ceramides and regulate skin inflammatory factors. Ceramide metabolism occurs entirely within keratinocytes; simultaneously, keratinocytes are also inflammatory signal receptors and effectors that execute inflammatory responses. Therefore, keratinocyte viability is crucial for ceramide synthesis and the intensity of skin inflammatory responses. The effects of *Lactobacillus paracasei* CCFM1503 on cell viability are as follows... Figure 2 As shown, compared with the control group (defined as having a cell proliferation rate of 100%), the addition of postbiotics (CCFM1503-S and CCFM1503-J) prepared from Lactobacillus paracasei CCFM1503 at an inactivated cell concentration of 5.0 × 10⁻⁶ cells significantly improved cell proliferation. 7 The cell proliferation rates at CFU / mL were 112.238% and 117.81%, respectively. The results showed that the postbiotics (CCFM1503-S and CCFM1503-J) prepared from Lactobacillus paracasei CCFM1503 could both enhance the activity of HaCaT, but the effect of CCFM1503-J group was significant and statistically significant.

[0056] Example 5: Effects of Lactobacillus paracasei CCFM1503 and its postbiotics on increasing the water content of the stratum corneum in mouse skin. Based on Examples 3 and 4, *Lactobacillus paracasei* CCFM1503 was determined to have the greatest potential to increase skin ceramide content, especially its post-biotic CCFM1503-J. Therefore, *Bifidobacterium breve* CCFM1505 and its prepared post-biotic (inactivated bacterial cell group CCFM1503-J) were selected for in vivo experiments.

[0057] In the implementation plan, the preparation method of live Lactobacillus paracasei CCFM1503 and its post-biotic (CCFM1503-J) is the same as in Example 2. The difference is that in the preparation method of the post-biotic cells (CCFM1503-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.

[0058] Twenty-four healthy male BALB / c mice aged 7 weeks from Vitallii were purchased and randomly divided into four cages of six mice each. The four cages consisted of one model group, one control group, and two experimental groups (one metatrophic bacterial cell group (CCFM1503-J) and one live bacteria group (CCFM1503-H)). 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 were treated with 3M tape for 3 times and MC903 was applied.

[0059] CCFM1503-H group: The gavage sample was live Lactobacillus paracasei CCFM1503, at a dose of 1×10⁻⁶. 9 CFU / Rat / Day; CCFM1503-J group: The gavage sample was postbiotic (inactivated bacteria), and the dosage was: 5 × 10⁶ live bacteria count. 9 CFU-prepared postbiotic inactivated bacterial cells; 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, with the shaved area measuring 3 cm long × 2 cm wide. Modeling began on day 8, and mice were administered the model via gavage daily from day 8 to day 28. A mouse skin barrier damage model was constructed using a combination of 3M tape and MC903 application. 3M tape was applied to the backs of the mice and peeled off three times, followed by application of 10 μM MC903, 200 μL per mouse per application, twice a week. The control group received an equal volume of saline solution. Specific animal experimental methods are as follows: Figure 3 As shown.

[0060] After the experiment, mice were briefly anesthetized via isoflurane inhalation, and the stratum corneum water content of the mouse skin was measured using a Cutometer DUAL MPA580 skin analyzer equipped with a CM825 probe for stratum corneum water content. The results are as follows: Figure 4The 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 Lactobacillus paracasei CCFM1503-J and CCFM1503-H, the stratum corneum water content of mouse skin recovered to 61.71% and 59.26%, respectively. Lactobacillus paracasei CCFM1503 and its post-biotic (CCFM1503-J) both significantly improved the reduced stratum corneum water content in mouse skin.

[0061] Example 6: Effects of Lactobacillus paracasei CCFM1503 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 5. 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 caused by decreased ceramide content and can represent skin health and inflammation. The experimental results are as follows: Figure 5 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 paracasei* CCFM1503-J and CCFM1503-H via gavage, respectively, transepidermal water loss was significantly reduced to 17.72 gm compared to the model group. -2 h -1 14.24 gm -2 h -1 Both CCFM1503-J and CCFM1503-H groups effectively reduced transepidermal water loss (TWEL) on the skin surface of mice, indicating that Lactobacillus paracasei CCFM1503 can improve skin barrier damage caused by decreased skin ceramide content.

[0062] Example 7: Effects of Lactobacillus paracasei CCFM1503 and postbiotics on the expression of ceramide degradation-related enzymes in mouse skin tissue The animal experiment design and experimental grouping were the same as in Example 5. After completing the determination of the skin appearance index of mice, the mice were sacrificed, the skin tissue on the back was cut off, an appropriate weight of skin tissue was weighed, pre-cooled PBS (1:10, tissue:PBS) was added, the tissue was ground into a homogenate, centrifuged at 3000 r / min for 20 min at 4℃, and the supernatant was taken for use.

[0063] Sphingomyelin synthase (SMS) is a key regulatory enzyme in the ceramide metabolic pathway, primarily responsible for converting ceramides into sphingomyelin and conversely regulating ceramide levels. It is crucial for maintaining skin barrier function, cell signaling, and lipid homeostasis. Excessive SMS levels in skin tissue lead to abnormal ceramide breakdown, significantly reducing ceramide levels and causing skin damage and inflammation. Therefore, reducing SMS overexpression can effectively prevent ceramide breakdown, maintain the balance between ceramides and sphingomyelin, and thus influence the health of the skin barrier.

[0064] The content of SMS in mouse skin tissue was determined using ELISA, and the results are as follows: Figure 6 Data showed that the SMS content in the skin tissue of the control group was 193.15 ng / mg, while in the model group, the SMS content increased to 225.73 ng / mg. After gavage administration of Lactobacillus paracasei CCFM1503 and its post-biotic (CCFM1503-J), the SMS content in the skin of the CCFM1503-J group was 156.19 ng / mg, and in the CCFM1503-H group, the SMS content in the skin was 157.78 ng / mg. Compared with the model group, the CCFM1503-J group and the CCFM1503-H group could significantly reduce the expression of SMS in the skin tissue and inhibit the decomposition of skin ceramides.

[0065] The core function of acidic ceramide enzyme (ASAH1) is to hydrolyze the amide bond of ceramide within lysosomes (or secreted into the extracellular matrix) to generate sphingosine and free fatty acids. This is a crucial step in the catabolism of ceramide, irreversibly degrading it and directly determining its lifespan and biological activity. Therefore, reducing the expression of ASAH1 can effectively inhibit the degradation of ceramide.

[0066] 30 mg of the obtained mouse skin tissue was weighed and added to the lysis buffer (the lysis buffer from the RNA extraction kit). The tissue was homogenized, and RNA was extracted from the skin tissue according to the kit instructions. cDNA was obtained by reverse transcription. The expression level of the acid ceramide 1 (ASAH1) gene in the mouse skin tissue was determined using RT-qPCR. The results are as follows: Figure 6 The primer sequences used for the ASAH1 gene are shown in Table 1.

[0067] Table 1 Primer Sequences

[0068] The relative expression levels of acid ceramidinase (ASAH1) mRNA were determined by oral administration of CCFM1503-J and CCFM1503-H. The relative expression levels of acid ceramidinase (ASAH1) mRNA were 1.07 and 0.66, respectively, which significantly downregulated the gene expression level of ASAH1 compared with the model group (2.29).

[0069] In summary, the results indicate that *Lactobacillus paracasei* CCFM1503 and its postbiotic (CCFM1503-J) can inhibit the abnormal decomposition of ceramides by reducing the expression of key enzymes in the process of ceramide degradation, thereby preventing the failure of the skin barrier function and the initiation of skin inflammatory responses caused by the rapid decomposition of ceramides in the skin.

[0070] Example 8: Effects of Lactobacillus paracasei CCFM1503 and its postbiotics on inflammatory responses in mouse skin tissue; The animal experimental design and experimental grouping involved in the following examples are the same as in Example 5, and the sample processing and preparation methods are the same as in Example 7.

[0071] (1) Effects of Lactobacillus paracasei CCFM1503 and post-biotics on the content of pro-inflammatory factor IL-1 in mouse skin tissue Interleukin-1 (IL-1) is the first signaling molecule to initiate inflammation in keratinocytes and is one of the first cytokines released. When the skin barrier is damaged, keratinocytes produce large amounts of IL-1, which can rapidly cause vasodilation, increased vascular permeability, and attract immune cells such as neutrophils to the damaged site, triggering a more severe inflammatory response in the skin.

[0072] The pro-inflammatory factor IL-1 in mouse skin tissue was measured using ELISA, and the results are as follows: Figure 7 As shown in the data, the IL-1 content in the model group was 15.14 pg / mg, which was significantly higher than that in the control group (11.75 pg / mg). Oral administration of *Lactobacillus paracasei* CCFM1503 and its post-biotics (CCFM1503-J and CCFM1503-H) reduced IL-1 levels to 11.43 pg / mg and 11.32 pg / mg, respectively, significantly reducing IL-1 expression, alleviating skin inflammation, and repairing tissue damage.

[0073] (2) Effects of Lactobacillus paracasei CCFM1503 and post-biotics on the content of pro-inflammatory factor IL-4 in mouse skin tissue Interleukin-4 (IL-4) is a key cytokine that induces naive T cells to differentiate into Th2 cells. The formation of Th2 cells produces large amounts of IL-4, amplifying the type 2 immune response, which can directly lead to skin itching, barrier dysfunction, and chronic inflammation.

[0074] The inflammatory factor IL-4 in mouse skin tissue was measured using ELISA, and the results are as follows: Figure 7 As shown in the figure. The results showed that after gavage administration of *Lactobacillus paracasei* CCFM1503 and its postbiotic, the IL-4 levels in mouse skin tissue were 2.42 pg / mg and 2.47 pg / mg, respectively, which were significantly lower than those in the model group (3.65 pg / mg). Therefore, the CCFM1503-J and CCFM1503-H groups can regulate IL-4 levels, reduce skin inflammation, maintain the skin barrier, and reduce the occurrence of type 2 immune responses in mouse skin tissue inflammation.

[0075] (3) Effects of Lactobacillus paracasei CCFM1503 and post-biotics on the content of pro-inflammatory factor TSLP in mouse skin tissue Thymic stromal lymphopoietin (TSLP) links skin barrier damage with skin inflammation. When the skin barrier is damaged, keratinocytes produce large amounts of TSLP, which in turn induces a type 2 immune response, directly activating sensory nerve fibers in the skin and causing intense itching.

[0076] The inflammatory factor TSLP in mouse skin tissue was measured using ELISA, and the results are as follows: Figure 7 As shown in the figure. The results showed that compared with the control group (2.42 pg / mg), the expression level of TSLP in the skin tissue of mice in the model group was significantly increased, with a content of 2.93 pg / mg. After gavage administration of Lactobacillus paracasei CCFM1503 and its postbiotic, the TSLP content was significantly reduced to 2.24 pg / mg and 2.22 pg / mg, respectively. Therefore, the CCFM1503-J and CCFM1503-H groups were effective in reducing the pro-inflammatory factor TSLP, which could not only alleviate the skin inflammatory response, but also reduce itching caused by inflammation and maintain skin health.

[0077] Example 9: Visual manifestation of Lactobacillus paracasei CCFM1503 and its postbiotics in alleviating skin inflammation in mice in skin tissue pathological sections; The animal experimental design and grouping involved in the following examples are the same as in Example 4. After completing the skin index measurement, the mice were sacrificed, and skin tissue from the back was excised. A 1×1 cm square tissue sample was taken and placed in a centrifuge tube containing tissue fixative for hematoxylin-eosin staining (HE staining). HE staining can directly assess the inflammatory status of mouse skin tissue. The experimental results are as follows: Figure 8The results showed that, compared with the control group, the model group had a significantly increased epidermal thickness and obvious inflammatory cell infiltration. After gavage administration of Lactobacillus paracasei CCFM1503 and its postbiotic (CCFM1503-J), the inflammatory infiltration was significantly reduced and the epidermal thickness showed a recovery trend.

[0078] In summary, in Examples 8 and 9, Lactobacillus paracasei CCFM1503 and its post-biotic (CCFM1503-J) can effectively inhibit the production of pro-inflammatory factors, alleviate skin inflammation, and to a certain extent, effectively maintain skin health and repair skin damage.

[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. Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1503 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66741.

2. The metabiotic prepared using Lactobacillus paracasei CCFM1503 as described in claim 1.

3. The epigenetic agent according to claim 2, characterized in that, The metabiotic includes bacterial lysate, inactivated or dead cells, fermentation supernatant, or any of the above-mentioned powders prepared by drying.

4. The epigenetic agent according to claim 3, characterized in that, The inactivated or dead cells are prepared as follows: Lactobacillus paracasei CCFM1503 is cultured in a culture medium for a period of time, bacterial cells in the bacterial culture medium are collected, and inactivated bacterial cells are obtained after heat treatment.

5. A composition containing *Lactobacillus paracasei* CCFM1503 as described in claim 1 and / or the metagener as described in any one of claims 2 to 4.

6. The composition according to claim 5, characterized in that, The content of *Lactobacillus paracasei* CCFM1503 is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

7. The composition according to claim 5 or 6, characterized in that, The composition includes, but is not limited to, pharmaceuticals or daily chemical products.

8. The composition according to claim 7, characterized in that, The daily chemical products include skin care products, cosmetics, or toiletries; the medicines include topical medicines or oral medicines.

9. The use of Lactobacillus paracasei CCFM1503 and / or its metabiotics as described in claim 1 in the preparation of pharmaceuticals or daily chemical products that increase the ceramide content in the skin.

10. The use of *Lactobacillus paracasei* CCFM1503 and / or its metabiotics as described in claim 1 in the preparation of a medicament for repairing the skin barrier or skin damage, characterized in that... The drug has at least one of the following effects: (1) Promotes the secretion of ceramides by keratinocytes; (2) Promotes the vitality of keratinocytes; (3) Increase the moisture content of the individual's stratum corneum; (4) Relieves transepidermal water loss in individual skin; (5) Inhibits the expression of ceramide degradation-related enzymes in individual skin tissue; (6) Relieve the inflammatory response of individual skin tissues.

Images