Lactiplantibacillus plantarum 24 and its applications

Abstract

The present invention provides a Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), strain 24, which is deposited in the China General Microbiological Culture Collection Center, deposit number: CGMCC No. 31278. The Lactiplantibacillus plantarum 24 of the present invention can secrete sphingomyelinase to promote the production of ceramide in HaCaT cells; Lactiplantibacillus plantarum 24 or its metabolite or its lysate can up-regulate the expression of key enzymes in the sphingomyelinase and / or ceramide synthesis pathway of skin cells, repair the skin barrier; down-regulate the expression of pro-inflammatory cytokines, inhibit autophagy, and has a good anti-inflammatory effect; at the same time, it also has good antioxidant and whitening effects. The Lactiplantibacillus plantarum 24 or its metabolite or its cell lysate of the present invention can be used in the preparation of products having the above-mentioned effects or functions.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Lactiplantibacillus plantarum strain and its application. Background Art

[0002] The skin, as the largest organ of the human body, has a barrier function, which is composed of a microbial barrier, a physical barrier, a chemical barrier, and an immune barrier. A normal skin barrier can prevent external stimuli and the loss of water and nutrients in the skin. When the barrier is damaged, the skin's defense ability decreases, and it is prone to inflammatory diseases such as sensitivity, atopic dermatitis, and acne. Ceramide is a key lipid for maintaining the skin barrier, and its synthesis and metabolism involve multiple enzymes, such as sphingomyelinase, including acid sphingomyelinase (A.SMase, encoded by the SMPD1 gene), neutral sphingomyelinase (N.SMase, encoded by the SMPD2, SMPD3, and SMPD4 genes), serine palmitoyltransferase (SPT, encoded by genes such as SPTLC1, SPTLC2, and SPTLC3), and the ceramide synthase (CerS) family. These enzymes and their corresponding genes work together to finely regulate the ceramide synthesis process. Abnormalities in these enzymes may lead to skin problems.

[0003] Skin barrier damage is also related to inflammatory responses, such as the secretion of cytokines such as thymic stromal lymphopoietin (TSLP), interleukin 25 (IL-25), and interleukin 33 (IL-33), and the abnormal expression of keratin 10 (K10) and desmoglein 1 (DSG1). In addition, excessive autophagy of cells will also weaken the skin barrier function.

[0004] Skin microorganisms can maintain skin barrier homeostasis by secreting enzymes (such as sphingomyelinase) and regulating immune factors (such as interleukins). For example, Staphylococcus epidermidis can rapidly generate ceramide by secreting sphingomyelinase, strengthening the skin barrier. In recent years, probiotic skin care has become a research hotspot, which can improve the skin barrier function and relieve skin problems by regulating the skin flora balance and enhancing the skin immune function. Existing studies have found that specific probiotic strains and their metabolites can promote skin cells to secrete ceramide and repair the skin barrier.

[0005] The impairment of the skin barrier can lead to various skin diseases. Therefore, it is of great significance to develop effective repair methods. The application of probiotics in cosmetics can balance the skin microbiota, repair the barrier, and prevent and treat inflammation by regulating immune function. There have been relevant patent applications in China. For example, CN117547559A discloses a composite probiotic postbiotic composition for improving skin allergies, enhancing skin resistance, and repairing the barrier; for example, CN114703106A discloses Lactobacillus plantarum GforU-12, which has the functions of repairing the barrier, treating skin inflammation, and acne; for example, CN114672442A discloses Lactobacillus plantarum ProfMIC 211 for maintaining and repairing the skin barrier and treating acne; for example, CN116211764A discloses a composition of Saccharomyces cerevisiae and Lactobacillus, which has the abilities of oil control, anti-inflammation, and antioxidant; for example, CN114081901B discloses a probiotic composition including Lactobacillus plantarum and Lactobacillus fermentum, which can regulate the balance of the microbiota, promote skin repair, and relieve inflammation.

[0006] In summary, although there are some applications of probiotics and their repair of the skin barrier in the prior art, the effects shown are mostly single, and the depth of research on their efficacy mechanisms in the prior art is also very limited. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a probiotic with multiple functions of repairing the skin barrier, anti-inflammation, anti-acne, anti-allergy, antioxidant, and whitening, and its application.

[0008] The technical solution of the present invention is outlined as follows:

[0009] The present invention provides a Lactiplantibacillus plantarum Lactiplantibacillus plantarum ), strain 24, which is deposited in the China General Microbiological Culture Collection Center (abbreviated as CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 31278, deposit date: July 12, 2024, taxonomic name: Lactiplantibacillus plantarum Lactiplantibacillus plantarum .

[0010] The Lactiplantibacillus plantarum 24 of the present invention is derived from Sichuan pickles. On a solid plate, the colony is opaque, round, milky white, with a bright and smooth surface, moist texture, neat edges, and is raised, and the colony diameter is 2 - 3 mm.

[0011] The Lactiplantibacillus plantarum 24 of the present invention is non-hemolytic and sensitive to clinically common antibiotics such as erythromycin, tetracycline, ampicillin, and chloramphenicol, and has good safety.

[0012] The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention have the ability to repair the skin barrier, reduce inflammation, treat acne and / or relieve allergy.

[0013] The Lactiplantibacillus plantarum 24 of the present invention can secrete sphingomyelinase, promote the production of sphingomyelinase by skin cells, and / or promote the production of ceramides by skin cells.

[0014] The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention can significantly up-regulate the expression levels of the genes encoding the key enzymes SPTLC1, SPTLC2, SPTLC3, CerS3, SMPD1 and / or SMPD3 in the ceramide synthesis pathway.

[0015] The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention can inhibit autophagy of HeLa cells induced by starvation.

[0016] The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention can significantly down-regulate the expression levels of the genes encoding thymic stromal lymphopoietin TSLP, interleukin-25 and / or interleukin-33.

[0017] The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention can significantly up-regulate the expression levels of the genes encoding keratin 10 (K10) and / or desmoglein 1 (DSG1).

[0018] The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention can significantly down-regulate the expression levels of the genes encoding pro-inflammatory cytokines (TNF-α, IL-1β and / or IL-6) in a murine atopic dermatitis model.

[0019] The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention can scavenge hydroxyl radicals and / or ABTS radicals, and has antioxidant efficacy.

[0020] The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention can inhibit tyrosinase activity and has whitening efficacy.

[0021] The present invention also provides the application of the Lactiplantibacillus plantarum in the preparation of products for repairing the skin barrier, reducing inflammation, treating acne, relieving allergy, whitening and / or antioxidant.

[0022] The present invention also provides the application of the Lactiplantibacillus plantarum in the preparation of products for producing sphingomyelinase, and / or promoting the production of sphingomyelinase by skin cells, and / or promoting the production of ceramides by skin cells.

[0023] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of a product for upregulating the expression of key enzymes in the ceramide synthesis pathway, downregulating the expression of pro-inflammatory cytokines, inhibiting autophagy, and upregulating the expression of skin barrier repair proteins.

[0024] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of a product for upregulating the expression levels of the encoding genes of the key enzymes SPTLC1, SPTLC2, SPTLC3, CerS3, SMPD1, and / or SMPD3 in the ceramide synthesis pathway.

[0025] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of a product for upregulating the expression levels of the encoding genes of keratin 10 (K10) and / or desmoglein 1 (DSG1).

[0026] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of a product for downregulating the expression levels of the encoding genes of thymic stromal lymphopoietin TSLP, interleukin-25, and / or interleukin-33.

[0027] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of a product for downregulating the expression levels of the encoding genes of pro-inflammatory cytokines TNF-α, IL-1β, and / or IL-6.

[0028] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of a product for inhibiting autophagy in HeLa cells.

[0029] In the above applications, the product includes a microbial preparation, a drug, a cosmetic, or a skin care product.

[0030] In the above applications, the Lactiplantibacillus plantarum may include its live bacteria, inactivated bacteria, metabolites, and / or cell lysates, etc. according to the needs of the product, such as the fermentation supernatant used in some embodiments of the present invention.

[0031] The present invention also provides a microbial preparation, which comprises the Lactiplantibacillus plantarum 24 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or excipient.

[0032] The present invention also provides a drug, which comprises the Lactiplantibacillus plantarum 24 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or excipient.

[0033] The present invention also provides a cosmetic, which comprises the Lactiplantibacillus plantarum 24 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or excipient.

[0034] The present invention also provides a skin care product, which contains the Lactiplantibacillus plantarum 24 and / or its metabolites and / or its cell lysates, as well as an acceptable carrier and / or excipient.

[0035] Beneficial effects:

[0036] The Lactiplantibacillus plantarum 24 of the present invention can secrete sphingomyelinase to promote the production of ceramide by HaCaT cells; the Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates can up-regulate the expression of key enzymes in the sphingomyelinase and / or ceramide synthesis pathway of skin cells, repair the skin barrier; down-regulate the expression of pro-inflammatory cytokines, inhibit autophagy, and have a good anti-inflammatory effect; at the same time, it also has good antioxidant and whitening effects. In summary, the Lactiplantibacillus plantarum 24 is a microecologically friendly multi-functional skin probiotic that can simultaneously exert multiple functions such as repairing the skin barrier, anti-inflammation, anti-acne, anti-sensitivity, antioxidant and / or whitening. The Lactiplantibacillus plantarum 24 or its metabolites or its cell lysates of the present invention can be used in the preparation of products with the above-mentioned effects or functions.

[0037] The present invention relates to the preservation information of biological materials:

[0038] Biological material: Lactiplantibacillus plantarum 24;

[0039] Preservation date: July 12, 2024;

[0040] Preservation unit: China General Microbiological Culture Collection Center (abbreviated as CGMCC), Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing;

[0041] Preservation number: CGMCC No. 31278;

[0042] Taxonomic name: Lactiplantibacillus plantarum Lactiplantibacillus plantarum . Description of the drawings

[0043] Figure 1 : Colony morphology of strain 24 in Example 1.

[0044] Figure 2 : Hemolytic experiment of strain 24 in Example 2.

[0045] Figure 3 : Growth curve of strain 24 in Example 3.

[0046] Figure 4 : Sphingomyelinase activity produced by strain 24 in Example 4.

[0047] Figure 5 : Promotion of ceramide production by HaCaT cells by strain 24 in Example 5.

[0048] Figure 6 : Influence of Strain 24 on the Transcription Level of Key Genes in the Ceramide Synthesis Pathway in Example 6.

[0049] Figure 7 : Strain 24 in Example 7 Inhibits Autophagy in HeLa Cells.

[0050] Figure 8 : Influence of Strain 24 on the Expression Level of Pro-inflammatory Cytokines in HaCaT Cells in Example 8.

[0051] Figure 9 : Influence of Strain 24 on the Expression Level of Skin Barrier Repair Genes in HaCaT Cells in Example 9.

[0052] Figure 10 : Influence of Strain 24 on the Appearance of Skin Lesion Tissues in a Mouse AD Model in Example 10.

[0053] Figure 11 : Influence of Strain 24 on the mRNA Expression Level of Pro-inflammatory Cytokines in a Mouse AD Model in Example 10.

[0054] Figure 12 : Influence of Strain 24 on the Protein Level of Pro-inflammatory Cytokines in a Mouse AD Model in Example 10. Detailed Implementation Modes

[0055] The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation examples without departing from the essence and scope of the present invention also belong to the protection scope of the present invention.

[0056] Example 1: Identification and Preservation of Strains

[0057] Strain 24 was isolated and purified from Sichuan pickled vegetables, and the strain was identified and preserved through colony morphology, 16S rRNA gene and phes gene sequencing analysis, cell morphology and physicochemical tests. The identification result was Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), and the preservation number was CGMCC No. 31278.

[0058] Colony morphological characteristics of Strain 24 ( Figure 1 ): On the solid plate, the colonies were opaque circles, milky white, with a bright and smooth surface, moist texture, neat edges, and were raised. The colony diameter was 2 - 3 mm.

[0059] The 16S rRNA gene sequence of strain 24 is shown in SEQ ID NO: 1:

[0060]

[0061] The phes gene sequence of strain 24 is shown in SEQ ID NO: 2:

[0062]

[0063] The cell morphology and physicochemical test results of strain 24 are shown in Table 1.

[0064] Table 1 Cell morphology and physicochemical test results of strain 24

[0065]

[0066] Note: “+” represents positive result, and “-” represents negative result.

[0067] Example 2: Safety evaluation of the strain

[0068] The safety of the strain was evaluated by hemolysis experiment and antibiotic sensitivity experiment.

[0069] (1) Hemolysis experiment

[0070] 10 μL of the bacterial solution of strain 24 was dropped onto a Columbia blood agar plate containing 5% sheep blood. Staphylococcus aureus (ATCC 6538) was set as the positive control, Lactobacillus plantarum (D8) as the negative control, and LB liquid medium as the blank control. It was cultured at 37 °C for 48 h, and the hemolysis of the strain on the plate was observed. The results are as Figure 2 shown, and strain 24 did not hemolyze.

[0071] (2) Antibiotic sensitivity analysis

[0072] The antibiotic sensitivity of strain 24 was evaluated by the disk diffusion method. The results are shown in Table 2. Strain 24 was sensitive to 4 commonly used clinical antibiotics.

[0073] Table 2 Antibiotic susceptibility test

[0074]

[0075] Note: S represents sensitive.

[0076] Example 3: Preparation of the fermentation supernatant of the strain

[0077] Pick 24 single colonies of the strain with good growth status, inoculate them into MRS liquid medium for activation, then transfer them to MRS liquid medium at an inoculation amount of 1%, and incubate statically for 24 h. Absorb 200 μL of the solution every 2 hours to measure the OD value of the bacteria at a wavelength of 600 nm, record and plot the growth curve of strain 24, as Figure 3 shown. Collect the fermentation broth in the late logarithmic phase of the growth curve, centrifuge at 8000 rcf / min for 10 min to collect the supernatant, and filter it through a 0.22 μm filter membrane to prepare the fermentation supernatant of strain 24 for standby.

[0078] Example 4: Experiment on the secretion of sphingomyelinase by the strain

[0079] Prepare the supernatant of Bacillus cereus (positive group), the fermentation supernatant of strain 24, set up a negative group (MRS), and measure it with reference to the instructions of the sphingomyelinase assay kit (Abcam).

[0080] 1. Operating steps:

[0081] (1) Dilution of the standard product: Dilute the sample according to the instructions.

[0082] (2) Sample addition: Set blank wells (no sample and enzyme-labeled reagent are added to the blank control wells, and the remaining operations are the same), standard wells, and wells for the samples to be tested. Accurately add 50 μL of the standard product to the standard wells on the enzyme-labeled coated plate. First add 40 μL of the sample dilution solution to the wells for the samples to be tested, and then add 10 μL of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the wells on the enzyme-labeled plate, try not to touch the well walls, and gently shake to mix evenly.

[0083] (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min.

[0084] (4) Solution preparation: Dilute the 30-fold concentrated washing solution 30-fold with distilled water and reserve it for use.

[0085] (5) Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with the washing solution, let it stand for 30 seconds and then discard it. Repeat this 5 times and pat dry.

[0086] (6) Enzyme addition: Add 50 μL of the enzyme-labeled reagent to each well except the blank well.

[0087] (7) Incubation: The operation is the same as in step 3.

[0088] (8) Washing: The operation is the same as in step 5.

[0089] (9) Color development: First add 50 μL of chromogenic reagent A to each well, then add 50 μL of chromogenic reagent B, gently shake to mix evenly, and develop color at 37°C in the dark for 15 min.

[0090] (10) Termination: Add 50 μL of termination solution to each well to terminate the reaction (at this time, the blue color immediately turns yellow).

[0091] (12) Measurement: Zero with the blank well, and sequentially measure the absorbance (OD value) of each well at a wavelength of 450 nm. The measurement should be carried out within 15 minutes after adding the termination solution.

[0092] 2. Result judgment:

[0093] Use the concentration of the standard substance as the abscissa and the OD value as the ordinate to draw a standard curve on the coordinate paper. According to the OD value of the sample, find the corresponding concentration from the standard curve; then multiply by the dilution factor to obtain the actual concentration of the sample.

[0094] 3. Calculation of sphingomyelinase activity

[0095] Prepare a standard solution of sphingomyelinase and serially dilute it step by step into a series of solutions with known concentrations according to a ratio. Use the above ELISA method to measure its OD value (OD 450) at 450 nm, fit and make the regression equation of the standard curve, substitute the OD 450 value of each test sample into the equation, and calculate the result of the ceramide content in the test sample. Each group is tested in 3 parallel replicates, and the mean value and standard deviation (SD) are calculated. Use SPSS statistical software to perform t-test analysis on the sphingomyelinase activities of each sample group measured in the above experiments. A p < 0.05 is considered statistically significant.

[0096] The results are as Figure 4 shown (compared with the negative group, *** p < 0.001; compared with the positive group, ## p < 0.05). The sphingomyelinase activity in the fermentation supernatant of strain 24 was significantly higher than that of the positive group, with an up-regulation of 28% - 45%.

[0097] Example 5: Strain promotes the production of ceramide by HaCaT cells

[0098] Add HaCaT cells to DMEM high-glucose culture medium containing 10% fetal bovine serum by volume, and place them in an incubator (37 °C, 5% CO 2 ) for routine culture. When the cell confluence reaches 80 - 90%, digest the adherent cells with trypsin, adjust and obtain a cell suspension with a cell density of 2×10 5 cells / mL, add it to a 24-well plate, 1000 μL of cell suspension per well, and continue to place it in an incubator (37 °C, 5% CO 2)(12 h) of incubation. When the cell seeding rate in the well plate meets the test requirements, discard the old culture medium, add complete medium, add 50 μL of the fermentation supernatant of strain 24 to each well in the experimental group, and keep the same volume in the positive group (fermentation supernatant of Bacillus cereus) and the negative group (MRS) for continuous culture for 24 h. Set 3 replicate wells in each group as parallel groups. Use a cell scraper to scrape the adherent cells, centrifuge at 1000 g for 3 min to collect the cells, and then perform ELISA detection.

[0099] 1. Preparation before detection:

[0100] (1) Take out the kit from the refrigerator 20 min in advance to equilibrate to room temperature.

[0101] (2) Dilute the 30-fold concentrated washing solution 30-fold with distilled water and set aside.

[0102] 2. Operating steps:

[0103] (1) Take out the strips required for the test from the sealed bag that has been equilibrated to room temperature.

[0104] (2) Set up standard wells, blank wells, and sample wells. Add 50 μL of standards with different concentrations to each standard well.

[0105] (3) Add 50 μL of the sample to be tested to the sample wells; do not add anything to the blank wells.

[0106] (4) Set up blank wells (the blank control wells do not add samples and enzyme-labeled reagents, and the rest of the operations are the same) and sample wells to be tested. First add 40 μL of sample diluent to the sample wells to be tested on the enzyme-coated plate, and then add 10 μL of the sample to be tested (the final dilution of the sample is 5 times). When adding the sample, add it to the bottom of the enzyme plate well, try not to touch the well wall, and gently shake to mix evenly. Seal the reaction wells with a sealing film and incubate at 37 °C for 30 min.

[0107] (5) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL); let it stand for 30 seconds, discard the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times.

[0108] (6) Add 50 μL of substrate enzyme-labeled reagent to each well, except for the blank wells. Incubate at 37 °C in the dark for 15 min.

[0109] (7) Washing: The operation is the same as in step 5.

[0110] (8) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix evenly, and develop color at 37 °C in the dark for 15 min.

[0111] (9) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes (within 15 minutes).

[0112] 3. Result judgment:

[0113] The OD value of each standard and specimen should be subtracted by the OD value of the blank well. Using the standard concentration as the abscissa and the OD value as the ordinate, the software plots and selects the best-fit curve. The concentration of the specimen can be found on the standard curve through its OD value.

[0114] 4. Calculation of ceramide content

[0115] Prepare the ceramide standard into a standard solution and serially dilute it step by step into a series of solutions with known concentrations according to a ratio. Measure its OD value at 450 nm (OD 450) using the above ELISA method, fit and make the regression equation of the standard curve, substitute the OD 450 value of each test sample into the equation, and calculate the ceramide content result in the test sample. Each group is tested in 3 duplicate wells in parallel, and the mean value and standard deviation (SD) are calculated. Apply SPSS statistical software to conduct a t-test analysis on the ceramide content of each sample group measured in the above experiment and the blank control group, with p < 0.05 indicating a statistically significant difference.

[0116] The results are as Figure 5 shown (** p < 0.01, compared with the negative group), the ceramide level in the fermentation supernatant of strain 24 is significantly higher than that of the negative group, with an upregulation of 52% - 55%.

[0117] Example 6: Strains upregulate the expression of key genes in the ceramide synthesis pathway of HaCaT cells

[0118] Refer to Example 5 for the cell culture conditions and operation methods. When the cell seeding rate in the well plate meets the test requirements, first discard the old culture medium in the 24-well plate. Add 50 μL of the fermentation supernatant of strain 24 to each well in the 24 (experimental group). The blank group and the negative group are respectively added with an equal volume of high-glucose DMEM medium containing 10% fetal bovine serum by volume and MRS medium. At the same time, set the fermentation supernatant of Lactobacillus plantarum ( Lactobacillus plantarum ) L16 (CN202410351722.5, preservation number: CGMCC No. 28163) as the experimental control group. Continue to culture for 24 h. Each group is set with 3 duplicate wells as parallel groups. Use a cell scraper to scrape the adherent cells, and centrifuge at 1000 g for 3 min to collect the cells.

[0119] The total RNA of HaCaT cells was extracted from the above - collected cells using a TRIZOL kit. The total RNA was reverse - transcribed into cDNA using a reverse - transcription kit, and the expression level of the target mRNA was detected on a Thermo Fisher Q1 PLUS real - time quantitative PCR instrument using a SYBR Green kit. GAPDH was used as an internal reference, and the relative expression level of the mRNA of the target gene was calculated according to 2 －ΔΔCt Calculation.

[0120] The results were as Figure 6 shown (* p < 0.05, *** p < 0.001, compared with the blank). The fermentation supernatant of strain 24 significantly promoted the gene expression of key enzymes (SPTLC1, SPTLC2, SPTLC3, CerS3, SMPD1, SMPD3) in the ceramide synthesis pathway (p < 0.05), with an up - regulation of 20% - 62%, and was superior to L16.

[0121] Example 7: Inhibition of autophagy by the strain

[0122] HeLa cells stably expressing GFP - LC3 were seeded in a 96 - well plate containing DMEM medium (containing 10% fetal bovine serum) at 7000 cells per well and incubated overnight at 37 °C with 5% CO 2 2. The control group was treated with 200 μL of EBSS for 2 h, and the treatment group was treated with an equal volume of EBSS (containing 5% of the fermentation supernatant of strain 24) for 2 h. The punctate distribution of GFP - LC3 (autophagosome formation) was observed using a fluorescence inverted microscope.

[0123] The results were as Figure 7 shown. The fermentation supernatant of strain 24 could significantly inhibit the formation of autophagosomes induced by starvation, indicating its certain anti - inflammatory and anti - tumor effects.

[0124] Example 8: Down - regulation of the expression of inflammation - related genes in HaCaT cells by the strain

[0125] The cell culture conditions and operation methods refer to Example 5. When the cell seeding rate in the well plate met the requirements, the old culture medium in the 24 - well plate was discarded. The experimental group (24) and the model group were both treated with 50 ng / mL of IL - 4 and IL - 13 for 24 h to induce an inflammation model of HaCaT cells, and the blank group was not added with IL - 4 and IL - 13. Subsequently, 50 μL of the fermentation supernatant of strain 24 was added to each well of the experimental group, and the blank group and the model group continued to be cultured with the same volume for 24 h. Each group was set with 3 replicate wells as parallel groups. The adherent cells were scraped off using a cell scraper, and the cells were collected by centrifugation at 1000 g for 3 min.

[0126] The total RNA of HaCaT cells was extracted from the above - collected cells using a TRIzol kit. The total RNA was reverse - transcribed into cDNA using a reverse - transcription kit, and the expression level of the target mRNA was detected on a Thermo Fisher Q1 PLUS real - time quantitative PCR instrument using a SYBR Green kit. GAPDH was used as an internal reference, and the relative expression level of the mRNA of the target gene was calculated according to 2 －ΔΔCt Calculate.

[0127] The results are as Figure 8 shown (* p < 0.05, *** p < 0.001 compared with the blank group; # p < 0.05, ## p < 0.01, p < 0.001 compared with the model group). The fermentation supernatant of strain 24 can inhibit the expression of thymic stromal lymphopoietin TSLP, interleukin - 25, and interleukin - 33, with a 12% - 61% down - regulation, that is, it can inhibit skin inflammation.

[0128] Example 9: The strain promotes the expression of genes related to skin barrier repair

[0129] The cell culture conditions and experimental operation methods were the same as those in Example 8.

[0130] The results are as Figure 9 shown (* p < 0.001 compared with the blank group; # p < 0.05, ## p < 0.01 compared with the model group). The fermentation supernatant of strain 24 can up - regulate the expression of the genes encoding keratin 10 and desmoglein 1, with a 33% - 44% up - regulation compared with the model group, that is, it is beneficial to skin barrier repair.

[0131] Example 10: The effect of the strain on a mouse dermatitis model

[0132] An atopic dermatitis model of mice was established, and the fermentation supernatant of strain 24 was used for intervention. The scratching times and skin lesion scores of the mice were calculated, and the gene levels and protein levels of pro - inflammatory cytokines in the skin tissue were measured to evaluate the therapeutic effect of strain 24 on dermatitis.

[0133] 1. Establishment and intervention of a mouse specific dermatitis model

[0134] First, the mice were adaptively fed for 1 week. Balb / c mice, female, 20 ± 2 g, had their abdominal and dorsal hairs removed using an electric hair clipper on the back of the mice (the hair - removal area was 2 cm × 3 cm). On the first day, 25 μL of 0.4% DNFB was applied to the abdomen of the mice for sensitization. On the fifth day, 20 μL of 0.05% DNFB was applied to the back of the mice, and then DNFB was used for modeling every day at 9:00, continuously applied for 21 days;

[0135] The experiment was divided into a negative group, a model group, a positive group, and a group 24, with 6 mice in each group. The intervention drug for the positive group was mometasone furoate cream, and the negative group was intervened with normal saline. The intervention started on the 7th day of the experiment. At 15:00 every day, 200 μL of normal saline, mometasone furoate cream, and the fermentation supernatant of strain 24 were evenly applied to the left ear of the mice in the negative group, positive group, and group 24 respectively once, and the application continued for 21 days.

[0136] 2. Scratching times of mice

[0137] On the 10th, 15th, and 20th days of drug administration, in the absence of interference, a filming device was set up to film for 10 minutes to record the scratching times of the mice.

[0138] Table 3 Scratching times (times / 10 min)

[0139]

[0140] Note: Compared with the negative group, p<0.001; compared with the model group, *** p<0.001

[0141] The results are shown in Table 3. Compared with the negative group, the scratching times of the model group increased significantly (p<0.01); compared with the model group, the scratching times decreased significantly after the intervention with the fermentation supernatant of strain 24 (p<0.01), and were significantly lower than those of the positive group on the 20th day.

[0142] 3. Apparent changes in the local skin lesions of mice

[0143] Before the intervention and on the last day of the intervention, a camera was used to record the apparent changes in the back skin of the mice in each group ( Figure 10 ). Obvious scabs could be seen on the back skin of the mice in the model group, and the skin was dry and desquamated; compared with the model group, the skin condition of the mice in the positive drug group was better, and the scabs on the back skin were smaller; the back skin condition of the mice in group 24 was good, and the dryness and desquamation of the skin were effectively alleviated, and the effect was better than that of the positive group.

[0144] 4. Severity score of skin lesions in mice

[0145] Before the intervention and on the last day of the intervention, the severity of the skin lesions on the back of the mice was scored. The scoring criteria were 0 (asymptomatic), 1 (mild symptoms), 2 (moderate symptoms), and 3 (severe symptoms) (Table 4).

[0146] Table 4 Atopic dermatitis score

[0147]

[0148] The scoring results showed (Table 5) that the skin lesion score values in the model group increased; compared with the model group, the score values in the positive group and the strain 24 group decreased, and there were significant differences (p < 0.05); the skin lesion score of the strain 24 group was the lowest, better than that of the positive group.

[0149] Table 5 Atopic dermatitis scores after intervention

[0150]

[0151] 5. Effects on the mRNA expression of pro-inflammatory cytokines in the back tissues of mice

[0152] The back tissues of mice in each group were homogenized, total RNA was extracted, reverse transcribed into cDNA, and qRT-PCR was used to detect the mRNA expression of atopic dermatitis-related pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. GAPDH was used as an internal reference, and the 2 -△△Ct method was used to calculate the relative expression levels of each gene.

[0153] The results were as Figure 11 shown (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; comparison between the negative group, positive group, group 24 and the model group). The fermentation supernatant of strain 24 significantly down-regulated the mRNA levels of the genes encoding TNF-α, IL-1β, and IL-6 (p < 0.05), and was down-regulated by 23% - 66% compared with the model group.

[0154] 6. Effects on the protein levels of pro-inflammatory cytokines in the back tissues of mice

[0155] (1) ELISA detection

[0156] a. Preparation before detection: Taking the TNF-α kit as an example

[0157] 1) Take out the kit from the refrigerator 20 min in advance and equilibrate it to room temperature.

[0158] 2) Dilute the 20× concentrated washing solution with double-distilled water to 1× working solution.

[0159] b. Operating steps:

[0160] 1) Take out the strips required for the test from the sealed bag that has been equilibrated to room temperature. Put the unused strips and desiccant back into the aluminum foil bag, press the self-sealing strip, seal the bag, and put it back at 4°C.

[0161] 2) Set up the standard wells, blank wells, and sample wells. Add 50 μL of standards with different concentrations to each standard well.

[0162] 3) Add 50 μL of the sample to be tested to the sample wells; do not add anything to the blank wells.

[0163] 4) Except for the blank wells, add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each standard well and sample well. Seal the reaction wells with a sealing film and incubate in a 37°C incubator or water bath for 60 min.

[0164] 5) Discard the liquid, pat dry on absorbent paper, and fill each well with washing solution (350 μL); let stand for 1 min, discard the washing solution by centrifugation, and pat dry on absorbent paper. Repeat the washing process 5 times.

[0165] 6) Add 50 μL of each of Substrate A and Substrate B to each well and incubate at 37°C in the dark for 15 min.

[0166] 7) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min (within 3 min).

[0167] c. Result judgment:

[0168] The OD value of each standard and specimen should be subtracted by the OD value of the blank well. Using the standard concentration as the abscissa and the OD value as the ordinate, the software plots and selects the best-fit curve. The concentration of the specimen can be determined from the standard curve according to its OD value.

[0169] (2) Calculation of TNF-α, IL-1β, and IL-6 contents

[0170] Prepare standard solutions of TNF-α, IL-1β, and IL-6 respectively, and serially dilute them step by step into a series of solutions with known concentrations according to a certain ratio. Measure their OD values at 450 nm by the above ELISA method, establish the regression equation of the standard curve by fitting, and substitute the OD450 values of each test sample into the equation to calculate the TNF-α, IL-1β, and IL-6 contents in the test sample. Each group is tested in 3 parallel replicate wells, and the mean and standard deviation (SD) are calculated. Use SPSS statistical software to perform t-test analysis on the TNF-α, IL-1β, and IL-6 contents of each sample group measured in the above experiment compared with the blank control group. A p value < 0.05 is considered statistically significant.

[0171] The results are as Figure 12 shown (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; comparison between the negative group, positive group, group 24 and the model group). The fermentation supernatant of strain 24 significantly down-regulated the protein levels of the atopic dermatitis-related pro-inflammatory cytokines TNF-α, IL-1β, and / or IL-6 (p < 0.05), with a 15% - 29% decrease compared to the model group.

[0172] Example 11: Detection of the antioxidant capacity of the strain

[0173] The antioxidant capacity of strain 24 was evaluated by measuring the hydroxyl radical scavenging capacity and ABTS radical scavenging capacity of the fermentation supernatant of strain 24.

[0174] 1. Detection of hydroxyl radical scavenging capacity

[0175] The measurement was carried out with reference to the instructions of the hydroxyl radical scavenging capacity detection kit (Beijing Box Bioscience & Technology Co., Ltd.). The calculation formula for the hydroxyl radical scavenging rate is as follows:

[0176] Hydroxyl radical scavenging rate D% = [(A measurement - A control) ÷ (A blank - A control)] × 100%;

[0177] The calculation results showed that the hydroxyl radical scavenging capacity of strain 24 was 32.95%.

[0178] 2. Detection of ABTS radical scavenging capacity

[0179] The measurement was carried out with reference to the instructions of the ABTS radical scavenging capacity detection kit (Beijing Box Bioscience & Technology Co., Ltd.). The calculation formula for the scavenging rate is as follows:

[0180] ABTS radical scavenging rate D% = [A blank - (A measurement - A control)] ÷ A blank × 100%;

[0181] The calculation results showed that the ABTS radical scavenging capacity of strain 24 was 30.33%.

[0182] The above experimental results indicated that strain 24 had good antioxidant capacity. Strain 24 could be used to prepare antioxidant products, such as drugs, skin care products or cosmetics with antioxidant and free radical scavenging functions.

[0183] Example 12: Detection of the inhibitory activity of the strain on tyrosinase

[0184] PBS phosphate buffer (pH 6.8), 40 μL of 10% fermentation supernatant of strain 24 and L-dopa / tyrosine solution were successively added to a 96-well plate. After mixing, the mixture was incubated at 37 °C for 20 min. Then 40 μL of tyrosinase solution was added, and the mixture was incubated at 37 °C for 15 min. The absorbance value of the sample group was measured at a wavelength of 475 nm in an enzyme-linked immunosorbent assay reader. An aqueous Vc solution was used as a positive control, and the tyrosinase inhibition rate was calculated according to the following formula:

[0185] A: 120 μL PBS + 40 μL tyrosinase + 40 μL L-dopa / tyrosine;

[0186] B: 160 μL PBS + 40 μL L-dopa / tyrosine;

[0187] C: 80 μL PBS + 40 μL fermentation supernatant of strain 24 + 40 μL tyrosinase + 40 μL L-dopa / tyrosine;

[0188] D: 120 μL PBS + 40 μL fermentation supernatant of strain 24 + 40 μL L-dopa / tyrosine;

[0189] Inhibition rate of tyrosinase (%) = [(A - B) - (C - D)] / (A - B) × 100%.

[0190] The results are shown in Table 6. The fermentation supernatant of strain 24 has a good effect on inhibiting tyrosinase activity, indicating its whitening effect. Strain 24 can be used to prepare whitening products, such as drugs, skin care products or cosmetics with whitening functions.

[0191] Table 6 Inhibition rate of strain 24 on tyrosinase activity

[0192]

[0193] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in the form and details of these embodiments without departing from the spirit and principle of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. Lactobacillus plantarum Lactiplantibacillus plantarum ), characterized in that: The deposit number of the plant lactobacillus is CGMCC No. 31278.

2. Use of the plant lactobacillus according to claim 1 in the preparation of products for repairing skin barrier, anti-inflammatory, anti-acne, anti-allergic, whitening and / or anti-oxidation; the products are microbial preparations, medicines, cosmetics or skin care products; The Lactobacillus plantarum in the cosmetics or skin care products is an inactivated bacterial body and / or a fermentation supernatant of the bacterial strain.

3. A microbial preparation comprising the Lactobacillus plantarum according to claim 1, and acceptable excipients.

4. A medicine comprising the fermentation supernatant of Lactobacillus plantarum and / or its strains as claimed in claim 1, and acceptable excipients.

5. A cosmetic comprising the inactivated bacteria of Lactobacillus plantarum and / or the fermentation supernatant of its strain as claimed in claim 1, and acceptable excipients.

6. A skin care product comprising the inactivated bacteria of Lactobacillus plantarum and / or the fermentation supernatant of its strain as claimed in claim 1, and acceptable excipients.

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