Phytobacterium plantarum 24 and application thereof

Abstract

The invention provides a lactobacillus plantarum 24 which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and the preservation number of the lactobacillus plantarum 24 is CGMCC No.31278. The lactobacillus plantarum 24 has the advantages that the lactobacillus plantarum 24 is prepared from lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum and lactobacillus plantarum; the plant lactobacillus 24 disclosed by the invention can secrete sphingomyelinase and promote HaCaT cells to generate ceramide; the plant lactobacillus 24 or the metabolite or lysate thereof can up-regulate expression of key enzymes in a synthetic route of skin cell sphingomyelinase and / or ceramide, and repair a skin barrier; expression of proinflammatory cytokines is lowered, autophagy is inhibited, and a good anti-inflammatory effect is achieved; meanwhile, the anti-oxidation and whitening effects are relatively good. The plant lactobacillus 24 or the metabolite or the cell lysate of the plant lactobacillus 24 can be applied to preparation of products with the effects or the effects of the plant lactobacillus 24 and the metabolite or the cell lysate of the plant lactobacillus 24.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a plant lactobacillus strain and application thereof. Background Art

[0002] As the largest organ in the human body, the skin has a barrier function, which is composed of microbial barriers, physical barriers, chemical barriers and immune barriers. The 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, which can easily cause inflammatory diseases such as sensitivity, atopic dermatitis, and acne. Ceramide is a key lipid in maintaining the skin barrier. Its synthesis and metabolism involve a variety of enzymes, such as sphingomyelinase, including acid sphingomyelinase (A.SMase, encoded by SMPD1 gene), neutral sphingomyelinase (N.SMase, encoded by SMPD2, SMPD3 and SMPD4 genes), serine palmitoyltransferase (SPT, encoded by genes such as SPTLC1, SPTLC2 and SPTLC3) and ceramide synthase (CerS) family. These enzymes and their corresponding genes work together to finely regulate the synthesis process of ceramide. Abnormalities of these enzymes may cause skin problems.

[0003] Skin barrier damage is also associated with inflammatory responses, such as the secretion of cytokines such as thymic stromal lymphopoietin (TSLP), interleukin 25 (IL-25), and interleukin 33 (IL-33), as well as abnormal expression of keratin 10 (K10) and desmoglein 1 (DSG1). In addition, excessive cell autophagy can 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 quickly produce ceramides by secreting sphingomyelinase to strengthen the skin barrier. In recent years, probiotic skin care has become a research hotspot, which improves skin barrier function and alleviates skin problems by regulating the balance of skin flora and enhancing skin immune function. Studies have found that specific probiotic strains and their metabolites can promote skin cells to secrete ceramides and repair the skin barrier.

[0005] Damage to the skin barrier can lead to a variety of skin diseases, so it is of great significance to develop effective repair methods. The application of probiotics in cosmetics can balance the skin flora, repair the barrier, and prevent and treat inflammation by regulating immune function. There are related invention patent applications in my country. 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 function of repairing the barrier, treating skin inflammation and acne; for example, CN114672442A discloses Lactobacillus plantarum ProfMIC 211, which is used to maintain and repair the skin barrier and treat acne; for example, CN116211764A discloses a composition of Saccharomyces cerevisiae and Lactobacillus, which has oil-inhibiting, anti-inflammatory and antioxidant capabilities; for example, CN114081901B discloses a probiotic composition, including Lactobacillus plantarum and Lactobacillus fermentum, which can regulate the balance of flora, promote skin repair and relieve inflammation.

[0006] In summary, although there are some probiotics and their applications in repairing skin barriers in the existing technology, the efficacy they exhibit is mostly relatively single, and the depth of research on their efficacy mechanisms in the existing technology is also very limited. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a probiotic having multiple functions of repairing skin barrier, anti-inflammation, anti-acne, anti-allergy, anti-oxidation and whitening and its application.

[0008] The technical solution of the present invention is summarized as follows: The present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum )24, the strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 31278, deposit date: July 12, 2024, classification name: Lactobacillus plantarum Lactiplantibacillus plantarum .

[0009] The plant lactobacillus 24 of the present invention is derived from Sichuan pickles, and has an opaque round, milky white colony on a solid plate, a bright and smooth surface, a moist texture, a neat and raised edge, and a colony diameter of 2 to 3 mm.

[0010] The plant lactobacillus 24 of the present invention is non-hemolytic, sensitive to common clinical antibiotics such as erythromycin, tetracycline, ampicillin and chloramphenicol, and has good safety.

[0011] The Lactobacillus plantarum 24 or its metabolites or its cell lysates described in the present invention have the ability to repair skin barrier, resist inflammation, resist acne and / or resist allergy.

[0012] The plant lactobacillus 24 of the present invention can secrete sphingomyelinase, promote skin cells to produce sphingomyelinase, and / or promote skin cells to produce ceramide.

[0013] The Lactobacillus plantarum 24 or its metabolites or its cell lysates described in the present invention can significantly upregulate the expression levels of genes encoding key enzymes SPTLC1, SPTLC2, SPTLC3, CerS3, SMPD1 and / or SMPD3 in the ceramide synthesis pathway.

[0014] The Lactobacillus plantarum 24 or its metabolites or its cell lysates described in the present invention can inhibit starvation-induced HeLa cell autophagy.

[0015] The Lactobacillus plantarum 24 or its metabolites or its cell lysates described in the present invention can significantly down-regulate the expression levels of genes encoding thymic stromal lymphopoietin TSLP, interleukin-25 and / or interleukin-33.

[0016] The Lactobacillus plantarum 24 or its metabolites or its cell lysates described in the present invention can significantly upregulate the expression levels of genes encoding keratin 10 (K10) and / or desmoglein 1 (DSG1).

[0017] The Lactobacillus plantarum 24 or its metabolites or its cell lysates described in the present invention can significantly downregulate the expression levels of genes encoding proinflammatory cytokines (TNF-α, IL-1β and / or IL-6) in a mouse atopic dermatitis model.

[0018] The Lactobacillus plantarum 24 or its metabolites or its cell lysates described in the present invention can scavenge hydroxyl free radicals and / or ABTS free radicals and have an antioxidant effect.

[0019] The Lactobacillus plantarum 24 or its metabolites or its cell lysates described in the present invention can inhibit tyrosinase activity and have whitening effect.

[0020] The present invention also provides the use of the plant lactobacillus in preparing products for repairing skin barrier, anti-inflammatory, anti-acne, anti-allergic, whitening and / or anti-oxidation.

[0021] The present invention also provides the use of the plant lactobacillus in preparing sphingomyelinase and / or products for promoting skin cells to produce sphingomyelinase and / or products for promoting skin cells to produce ceramide.

[0022] The present invention also provides the use of the plant lactobacillus in preparing a product for upregulating the expression of key enzymes in the ceramide synthesis pathway, downregulating the expression of pro-inflammatory cytokines, inhibiting cell autophagy, and upregulating the expression of skin barrier repair proteins.

[0023] The present invention also provides the use of the plant lactobacillus in preparing a product for up-regulating the expression level of genes encoding key enzymes SPTLC1, SPTLC2, SPTLC3, CerS3, SMPD1 and / or SMPD3 in the ceramide synthesis pathway.

[0024] The present invention also provides the use of the Lactobacillus plantarum in preparing a product for up-regulating the expression level of a gene encoding keratin 10 (K10) and / or desmoglein 1 (DSG1).

[0025] The present invention also provides the use of the plant lactobacillus in preparing a product for down-regulating the expression level of genes encoding thymic stromal lymphopoietin TSLP, interleukin-25 and / or interleukin-33.

[0026] The present invention also provides the use of the Lactobacillus plantarum in preparing a product for down-regulating the expression level of genes encoding pro-inflammatory cytokines TNF-α, IL-1β and / or IL-6.

[0027] The present invention also provides application of the Lactobacillus plantarum in preparing a product for inhibiting HeLa cell autophagy.

[0028] In the above applications, the products include microbial preparations, medicines, cosmetics or skin care products.

[0029] In the above application, the Lactobacillus plantarum may include live bacteria, inactivated bacteria, metabolites and / or cell lysates according to the needs of the product, such as the fermentation supernatant used in some embodiments of the present invention.

[0030] The present invention also provides a microbial preparation, which comprises the Lactobacillus plantarum 24 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or auxiliary material.

[0031] The present invention also provides a medicine, which comprises the Lactobacillus plantarum 24 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or auxiliary material.

[0032] The present invention also provides a cosmetic comprising the Lactobacillus plantarum 24 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or auxiliary material.

[0033] The present invention also provides a skin care product, which comprises the Lactobacillus plantarum 24 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or auxiliary materials.

[0034] Beneficial effects: Plant lactobacillus 24 of the present invention can secrete sphingomyelinase and promote HaCaT cells to produce ceramide; plant lactobacillus 24 or its metabolites or its cell lysates can upregulate the expression of key enzymes of skin cell sphingomyelinase and / or ceramide synthesis pathway, and repair skin barrier; downregulate the expression of proinflammatory cytokines, inhibit cell autophagy, and have good anti-inflammatory effect; also have good antioxidant and whitening effects. In summary, plant lactobacillus 24 is a microecologically friendly multi-functional skin probiotic, which can simultaneously play a multi-functional role such as repairing skin barrier, anti-inflammatory, anti-acne, anti-sensitivity, antioxidant and / or whitening. Plant lactobacillus 24 of the present invention or its metabolites or its cell lysates can be used in the preparation of products with the above-mentioned effects or effects.

[0035] The present invention relates to the deposit information of biological materials: Biological materials: Lactobacillus plantarum 24; Deposit date: July 12, 2024; Depository: China General Microbiology Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No. 31278; Classification name: Lactobacillus plantarum Lactiplantibacillus plantarum . BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Colony morphology of strain 24 in Example 1.

[0037] Figure 2 : Hemolytic activity experiment of strain 24 in Example 2.

[0038] Figure 3 : Growth curve of strain 24 in Example 3.

[0039] Figure 4 : The strain 24 in Example 4 produces sphingomyelinase activity.

[0040] Figure 5 : Strain 24 in Example 5 promotes the production of ceramide in HaCaT cells.

[0041] Figure 6 : The effect of strain 24 in Example 6 on the transcription levels of key genes in the ceramide biosynthesis pathway.

[0042] Figure 7 : Strain 24 in Example 7 inhibits HeLa cell autophagy.

[0043] Figure 8 : The effect of strain 24 in Example 8 on the expression level of pro-inflammatory cytokines in HaCaT cells.

[0044] Fig. 9 : The effect of strain 24 in Example 9 on the expression level of skin barrier repair genes in HaCaT cells.

[0045] Fig.10 : The effect of strain 24 in Example 10 on the appearance of skin lesion tissue in the mouse AD model.

[0046] Fig.11 : The effect of strain 24 in Example 10 on the mRNA expression level of pro-inflammatory cytokines in the mouse AD model.

[0047] Fig.12 : The effect of strain 24 in Example 10 on the protein levels of pro-inflammatory cytokines in the mouse AD model. DETAILED DESCRIPTION

[0048] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0049] Example 1: Identification and preservation of strains Strain 24 was isolated and purified from Sichuan pickled vegetables, and identified and preserved by colony morphology, 16S rRNA gene and phes gene sequencing analysis, cell morphology and physicochemical tests. The identification result was Lactobacillus plantarum ( Lactiplantibacillus plantarum ), the deposit number is CGMCC No. 31278.

[0050] Colony morphology of strain 24 ( Figure 1 ): On the solid plate, the colonies are opaque, round, milky white, with a bright and smooth surface, moist texture, neat edges and raised shapes. The diameter of the colonies is 2 to 3 mm.

[0051] The 16S rRNA gene sequence of strain 24 is shown in SEQ ID NO: 1:

[0052] The phes gene sequence of strain 24 is shown in SEQ ID NO: 2:

[0053] The cell morphology and physicochemical test results of strain 24 are shown in Table 1.

[0054] Table 1 Cell morphology and physicochemical test results of strain 24

[0055] Note: "+" represents a positive result, and "-" represents a negative result.

[0056] Example 2: Safety evaluation of strains The safety of the strain was evaluated by hemolytic assay and antibiotic sensitivity assay.

[0057] (1) Hemolytic test Take 10 μL of strain 24 bacterial solution and drop it on Columbia blood agar plate containing 5% sheep blood, set Staphylococcus aureus (ATCC 6538) as positive control, Lactobacillus plantarum (D8) as negative control, LB liquid medium as blank control, culture at 37℃ for 48 h, and observe the hemolysis of the strain on the plate. Figure 2 As shown, strain 24 is not hemolytic.

[0058] (2) Antibiotic sensitivity analysis The disk diffusion method was used to evaluate the antibiotic sensitivity of strain 24. The results are shown in Table 2, and strain 24 was sensitive to all four commonly used clinical antibiotics.

[0059] Table 2 Antibiotic susceptibility test

[0060] Note: S stands for sensitive.

[0061] Example 3: Preparation of strain fermentation supernatant A single colony of strain 24 with good growth status was selected and inoculated into MRS liquid medium for activation. Then, it was transferred to MRS liquid medium at a 1% inoculation volume and cultured statically for 24 h. Every 2 hours, 200 μL of solution was taken to measure the OD value of bacteria at a wavelength of 600 nm. The growth curve of strain 24 was recorded and plotted. Figure 3 The fermentation broth was collected at the late logarithmic phase of the growth curve, and the supernatant was collected by centrifugation at 8000 rcf / min for 10 min, and filtered through a 0.22 μm filter membrane to obtain the fermentation supernatant of strain 24 for later use.

[0062] Example 4: Experiment on secretion of sphingomyelinase by strains The supernatant of Bacillus cereus (positive group) and the fermentation supernatant of strain 24 were prepared, and the negative group (MRS) was set up. The determination was carried out according to the instructions of the sphingomyelinase assay kit (Abcam).

[0063] 1. Operation steps: (1) Dilution of standard: dilute the sample according to the instructions.

[0064] (2) Sample addition: Set up blank wells (blank control wells do not contain samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50 μL of the standard sample to the enzyme-labeled plate, add 40 μL of sample diluent to the test sample wells, and then add 10 μL of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0065] (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min.

[0066] (4) Liquid preparation: Dilute the 30-fold concentrated washing solution with 30-fold distilled water and set aside.

[0067] (5) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard. Repeat this process 5 times and pat dry.

[0068] (6) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0069] (7) Incubation: Same as step 3.

[0070] (8) Washing: Same operation as in step 5.

[0071] (9) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and incubate at 37°C in the dark for 15 min.

[0072] (10) Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0073] (12) Determination: Use blank as zero and measure the absorbance (OD value) of each well at 450 nm. The determination should be performed within 15 min after adding the stop solution.

[0074] 2. Result judgment: Draw a standard curve on the coordinate paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration from the standard curve based on the OD value of the sample; then multiply it by the dilution factor to get the actual concentration of the sample.

[0075] 3. Calculation of sphingomyelinase activity The standard of sphingomyelinase was prepared into a standard solution and diluted step by step into a series of solutions with known concentrations. The OD value (OD 450) at 450 nm was measured by the above ELISA method. The regression equation of the standard curve was fitted and the OD 450 value of each test sample was substituted into the equation to calculate the ceramide content in the test sample. Three replicate wells were tested in parallel in each group, and the mean value and standard deviation (SD) were calculated. SPSS statistical software was used to perform t-test analysis on the sphingomyelinase activity of each sample group measured in the above test, and p < 0.05 was considered statistically significant.

[0076] The results are as follows Figure 4 As 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 in the positive group, upregulated by 28%~45%.

[0077] Example 5: The strain promotes the production of ceramide in HaCaT cells HaCaT cells were added to DMEM high-glucose culture medium containing 10% fetal bovine serum and placed in an incubator (37°C, 5% CO2) for routine culture. When the cell confluence reached 80-90%, the adherent cells were digested with trypsin and the cell density was adjusted to 2×10 5 Cell suspension of 1000 μL / mL was added to a 24-well plate, and 1000 μL of cell suspension was added to each well. The plate was then placed in an incubator (37°C, 5% CO2) for 12 h. When the cell plating rate in the well plate met the experimental requirements, the old culture medium was discarded, and complete culture medium was added. 50 μL of strain 24 fermentation supernatant was added to each well of the experimental group, and the positive group (Bacillus cereus fermentation supernatant) and the negative group (MRS) were cultured for 24 h at the same volume. Three replicate wells were set up in each group as parallel groups. The adherent cells were scraped up with a cell scraper, and the cells were collected by centrifugation at 1000 g for 3 min, and then tested by ELISA.

[0078] 1. Preparation before testing: (1) Take the kit out of the refrigerator 20 min in advance to equilibrate to room temperature. (2) Dilute the 30-fold concentrated washing solution with 30-fold distilled water and set aside.

[0079] 2. Operation steps: (1) Take out the panels required for testing from the sealed bag that has been equilibrated to room temperature.

[0080] (2) Set up standard wells, blank wells, and sample wells. Add 50 μL of standard of different concentrations to each standard well.

[0081] (3) Add 50 μL of the sample to be tested to the sample well; do not add anything to the blank well.

[0082] (4) Set up blank wells (blank control wells without adding samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40 μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10 μL of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix. Seal the reaction wells with a sealing film and incubate at 37°C for 30 min.

[0083] (5) Discard the liquid, pat dry with absorbent paper, and fill each well with washing solution (350 μL); let it stand for 30 seconds, shake off the washing solution, pat dry with absorbent paper, and repeat this process 5 times.

[0084] (6) Add 50 μL of substrate enzyme-labeled reagent to each well, except for the blank well. Incubate at 37°C in the dark for 15 min. (7) Washing: Same operation as in step 5.

[0085] (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, and incubate at 37°C in the dark for 15 min.

[0086] (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 min.

[0087] 3. Result judgment: The OD value of the blank well should be subtracted from the OD value of each standard and sample. The software draws and selects the best fitting curve with the standard concentration as the horizontal axis and the OD value as the vertical axis. The concentration of the sample can be found on the standard curve through the OD value of the sample.

[0088] 4. Calculation of ceramide content The ceramide standard was prepared into a standard solution and diluted step by step into a series of solutions with known concentrations according to the proportion. The OD value (OD 450) at 450 nm was measured by the above ELISA method. The regression equation of the standard curve was fitted and the OD 450 value of each test sample was substituted into the equation to calculate the ceramide content in the test sample. Three replicate wells were tested in parallel in each group, and the mean value and standard deviation (SD) were calculated. SPSS statistical software was used to perform t-test analysis on the ceramide content of each sample group measured in the above test and the blank control group, and p<0.05 was considered statistically significant.

[0089] The results are as follows Figure 5As shown (** p < 0.01, compared with the negative group), the ceramide level in the fermentation supernatant of strain 24 was significantly higher than that in the negative group, upregulated by 52%~55%.

[0090] Example 6: Strains upregulate the expression of key genes in the ceramide synthesis pathway of HaCaT cells See Example 5 for cell culture conditions and operation methods. When the cell plating rate in the well plate meets the test requirements, the old culture medium in the 24-well plate is discarded, 50 μL of the fermentation supernatant of strain 24 is added to each well of 24 (experimental group), and equal volumes of DMEM high-glucose culture medium and MRS culture medium containing 10% fetal bovine serum are added to the blank group and the negative group, respectively. At the same time, Lactobacillus plantarum ( Lactobacillus plantarum ) L16 (CN202410351722.5, deposit number: CGMCCNo.28163) fermentation supernatant was used as the experimental control group. The culture was continued for 24 h. Three replicate wells were set up in each group as parallel groups. The adherent cells were scraped up with a cell scraper and centrifuged at 1000 g for 3 min to collect the cells.

[0091] The above collected cells were used to extract total RNA from HaCaT cells using the TRIZOL kit. Total RNA was reverse transcribed into cDNA using a reverse transcription kit, and the target mRNA expression level 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 of the target gene mRNA was calculated based on 2 －ΔΔCt calculate.

[0092] The results are as follows Figure 6 As 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), which was upregulated by 20%~62%, and was better than L16.

[0093] Example 7: Strain inhibits cell autophagy HeLa cells stably expressing GFP-LC3 were seeded in 96-well plates containing DMEM medium (containing 10% fetal bovine serum), with 7000 cells per well, and incubated overnight at 37°C, 5% CO2. The control group was treated with 200 μL EBSS for 2 h, and the treated 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 an inverted fluorescence microscope.

[0094] The results are as follows Figure 7As shown, the fermentation supernatant of strain 24 could significantly inhibit starvation-induced autophagosome formation, indicating that it has certain anti-inflammatory and anti-tumor effects.

[0095] Example 8: Strains downregulate the expression of inflammation-related genes in HaCaT cells See Example 5 for cell culture conditions and operation methods. When the cell plating rate in the well plate meets the requirements, the old culture medium in the 24-well plate is discarded, and 50 ng / mL IL-4 and IL-13 are added to the experimental group (24) and the model group for 24 hours to induce the HaCaT cell inflammation model. No IL-4 and IL-13 are added to the blank group. Subsequently, 50 μL of strain 24 fermentation supernatant was added to each well of the experimental group, and the blank group and the model group were cultured at the same volume for 24 hours. Three replicate wells were set up in each group as parallel groups. The adherent cells were scraped up with a cell scraper and the cells were collected by centrifugation at 1000 g for 3 minutes.

[0096] The above collected cells were used to extract total RNA from HaCaT cells using the TRIZOL kit. Total RNA was reverse transcribed into cDNA using a reverse transcription kit, and the target mRNA expression level 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 of the target gene mRNA was calculated based on 2 －ΔΔCt calculate.

[0097] The results are as follows Figure 8 As shown (compared with the blank group, * p<0.05, *** p<0.001; compared with the model group, # p<0.05, ## p<0.01, ### p<0.001), the fermentation supernatant of strain 24 can inhibit the expression of thymic stromal lymphopoietin TSLP, interleukin-25 and interleukin-33, down-regulating by 12%~61%, that is, it can inhibit skin inflammation.

[0098] Example 9: The strain promotes the expression of genes related to skin barrier repair The cell culture conditions and experimental operation methods are the same as Example 8.

[0099] The results are as follows Fig. 9 As shown (compared with the blank group, *** p<0.001; compared with the model group, # p<0.05, ## p<0.01), the fermentation supernatant of strain 24 can upregulate the expression of genes encoding keratin 10 and desmoglein 1, which is 33%~44% higher than that of the model group, which is beneficial to skin barrier repair.

[0100] Example 10: Effect of strains on mouse dermatitis model A mouse atopic dermatitis model was established, and the fermentation supernatant of strain 24 was used for intervention. The number of scratching times and skin lesion scores of mice were calculated, and the gene and protein levels of proinflammatory cytokines in skin tissues were measured to evaluate the therapeutic effect of strain 24 on dermatitis.

[0101] 1. Establishment and intervention of mouse atopic dermatitis model The mice were first adaptively raised for 1 week. Female Balb / c mice, 20±2 g, were used. The hair on the abdomen and back of the mice was removed with electric clippers (the hair removal area was 2 cm×3 cm). On the first day, 25 μL 0.4% DNFB was applied to the abdomen of the mice for sensitization. On the fifth day, 20 μL 0.05% DNFB was applied to the back of the mice. After that, DNFB was used for modeling at 9:00 every day, and the application continued for 21 days. The experiment was divided into negative group, model group, positive group, and 24 group, with 6 mice in each group. The intervention drug in the positive group was mometasone furoate cream, and the negative group was intervened with normal saline. The intervention began on the 7th day of the experiment. The negative group, positive group, and 24 group were evenly applied with 200 μL normal saline, mometasone furoate cream, and fermentation supernatant of strain 24 once on the left ear at 15:00 every day, and the application continued for 21 days.

[0102] 2. Number of mouse scratches On the 10th, 15th, and 20th days of drug administration, the filming equipment was set up without interruption, and the filming was performed for 10 minutes to record the number of times the mice scratched.

[0103] Table 3 Number of scratching (times / 10 min)

[0104] Note: Compared with the negative group, ### p<0.001; compared with the model group, *** p<0.001 The results are shown in Table 3. Compared with the negative group, the scratching frequency in the model group increased significantly (p<0.01). Compared with the model group, the scratching frequency decreased significantly after intervention with the fermentation supernatant of strain 24 (p<0.01), and was significantly lower than that in the positive group on the 20th day.

[0105] 3. Epigenetic changes of local skin lesions in mice Before the intervention and on the last day of the intervention, the apparent changes of the back skin of each group of mice were recorded using a camera ( Fig.10 ). Obvious scabs were observed on the back skin of mice in the model group, and the skin was dry and scaly. Compared with the model group, the skin condition of mice in the positive drug group was better, and the scabs on the back skin were smaller. The back skin condition of mice in group 24 was good, and the dry skin and scaly were effectively alleviated, which was better than that of the positive group.

[0106] 4. Scoring of the severity of skin lesions in mice Before and on the last day of intervention, the severity of skin lesions on the back of mice was scored, with the scoring index being 0 (no symptoms), 1 (mild symptoms), 2 (moderate symptoms), and 3 (severe symptoms) (Table 4).

[0107] Table 4 Atopic dermatitis score

[0108] The scoring results showed (Table 5) that the skin lesion score of the model group increased; compared with the model group, the positive group, and the strain 24 group, the score values ​​were all reduced, and there were significant differences (p<0.05); the skin lesion score of the strain 24 group was the lowest, which was better than the positive group.

[0109] Table 5 Atopic dermatitis scores after intervention

[0110] 5. Effects on the mRNA expression of pro-inflammatory cytokines in the back tissue of mice The dorsal tissues of mice in each group were homogenized, and total RNA was extracted and reverse transcribed into cDNA. qRT-PCR was used to detect the mRNA expression of atopic dermatitis-related proinflammatory cytokines TNF-α, IL-1β, and IL-6. GAPDH was used as an internal reference. -△△Ct The relative expression level of each gene was calculated by the method.

[0111] The results are as follows Fig.11 As shown (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; compared with the negative group, positive group, group 24 and the model group), the fermentation supernatant of strain 24 significantly downregulated the mRNA levels of TNF-α, IL-1β, and IL-6 encoding genes (p<0.05), which were downregulated by 23% to 66% compared with the model group.

[0112] 6. Effect of pro-inflammatory cytokine protein levels in mouse back tissue (1) ELISA test a. Preparation before testing: Taking TNF-α kit as an example 1) Take out the kit from the refrigerator 20 min in advance to equilibrate to room temperature. 2) Dilute the 20× concentrated washing solution to 1× working solution with double distilled water.

[0113] b. Operation steps: 1) Take out the required strips 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 return it to 4℃.

[0114] 2) Set up standard wells, blank wells and sample wells. Add 50 μL of standard of different concentrations to each standard well.

[0115] 3) Add 50 μL of the sample to be tested to the sample well; do not add anything to the blank well.

[0116] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the 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.

[0117] 5) Discard the liquid, pat dry with absorbent paper, and fill each well with washing solution (350 μL); let it stand for 1 min, shake off the washing solution, pat dry with absorbent paper, and repeat this process 5 times.

[0118] 6) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min. 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). c. Result judgment: The OD value of the blank well should be subtracted from the OD value of each standard and sample. The software draws and selects the best fitting curve with the standard concentration as the horizontal axis and the OD value as the vertical axis. The concentration of the sample can be found on the standard curve through the OD value of the sample.

[0119] (2) Calculation of TNF-α, IL-1β and IL-6 content The standard samples of TNF-α, IL-1β and IL-6 were prepared into standard solutions, and then diluted step by step into a series of solutions with known concentrations. The OD values ​​at 450 nm were measured by the above ELISA method. The regression equation of the standard curve was fitted, and the OD450 values ​​of each test sample were substituted into the equation to calculate the TNF-α, IL-1β and IL-6 content in the test sample. Three replicate wells were tested in parallel in each group, and the mean value and standard deviation (SD) were calculated. SPSS statistical software was used to perform t-test analysis on the TNF-α, IL-1β and IL-6 contents of each sample group measured in the above test and the blank control group, and p<0.05 was considered statistically significant.

[0120] The results are as follows Fig.12 As shown (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; compared with the negative group, positive group, group 24 and the model group), the fermentation supernatant of strain 24 significantly downregulated the protein levels of atopic dermatitis-related proinflammatory cytokines TNF-α, IL-1β and / or IL-6 (p<0.05), which were downregulated by 15% to 29% compared with the model group.

[0121] Example 11: Antioxidant capacity detection of strains The antioxidant capacity of strain 24 was evaluated by measuring the hydroxyl radical scavenging ability and ABTS radical scavenging ability of the fermentation supernatant of strain 24.

[0122] 1. Hydroxyl free radical scavenging ability test The hydroxyl radical scavenging ability test kit (Beijing Box Biotechnology Co., Ltd.) was used for determination. The hydroxyl radical scavenging rate was calculated as follows: Hydroxyl free radical scavenging rate D% = [(A determination - A control) ÷ (A blank - A control)] × 100%; The calculation results showed that the hydroxyl radical scavenging ability of strain 24 was 32.95%.

[0123] 2. ABTS free radical scavenging ability test Refer to the instructions of the ABTS free radical scavenging ability test kit (Beijing Box Biotechnology Co., Ltd.). The scavenging rate calculation formula is as follows: ABTS free radical scavenging rate D% = [A blank - (A determination - A control)] ÷ A blank × 100%; The calculation results showed that the ABTS free radical scavenging ability of strain 24 was 30.33%.

[0124] The above experimental results show that strain 24 has good antioxidant capacity. Strain 24 can be used to prepare antioxidant products, such as medicines, skin care products or cosmetics with antioxidant and free radical scavenging functions.

[0125] Example 12: Detection of tyrosinase activity inhibition by strains PBS phosphate buffer (pH 6.8), 40 μL of 10% strain 24 fermentation supernatant and L-DOPA / tyrosine solution were added to the 96-well plate in sequence, mixed and incubated at 37°C for 20 min. Then 40 μL of tyrosinase solution was added, incubated at 37°C for 15 min, and the absorbance of the sample group was measured at a wavelength of 475 nm in an ELISA reader. Vc aqueous solution was used as the positive control, and the tyrosinase inhibition rate was calculated according to the following formula: A: 120 μL PBS + 40 μL tyrosinase + 40 μL L-dopa / tyrosine; B: 160 μL PBS + 40 μL L-dopa / tyrosine; C: 80 μL PBS + 40 μL strain 24 fermentation supernatant + 40 μL tyrosinase + 40 μL L-dopa / tyrosine; D: 120 μL PBS + 40 μL strain 24 fermentation supernatant + 40 μL L-dopa / tyrosine; Tyrosinase inhibition rate (%) = [(AB)-(CD)] / (AB)×100%.

[0126] The results are shown in Table 6. The fermentation supernatant of strain 24 has a good inhibitory effect on tyrosinase activity, indicating that it has a whitening effect. Strain 24 can be used to prepare whitening products, such as medicines, skin care products or cosmetics with whitening function.

[0127] Table 6 Inhibition rate of strain 24 on tyrosinase activity

[0128] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles 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 described in claim 1 in the preparation of skin barrier repair, anti-inflammatory, anti-acne, anti-allergic, whitening and / or antioxidant products.

3. Use of the plant lactobacillus according to claim 1 in the preparation of sphingomyelinase and / or products that promote skin cells to produce sphingomyelinase and / or promote skin cells to produce ceramide.

4. Use of the plant lactobacillus described in claim 1 in preparing a product for upregulating the expression level of genes encoding key enzymes SPTLC1, SPTLC2, SPTLC3, CerS3, SMPD1 and / or SMPD3 in the ceramide synthesis pathway, and / or upregulating the expression level of genes encoding keratin 10 and / or desmoglein 1, and / or downregulating the expression level of genes encoding thymic stromal lymphopoietin TSLP, interleukin-25 and / or interleukin-33, and / or downregulating the expression level of genes encoding proinflammatory cytokines TNF-α, IL-1β and / or IL-6.

5. Use of the plant lactobacillus according to claim 1 in preparing a product for inhibiting HeLa cell autophagy.

6. The use according to any one of claims 2 to 5, characterized in that: The products include microbial preparations, medicines, cosmetics or skin care products.

7. A microbial preparation comprising the Lactobacillus plantarum according to claim 1, and an acceptable carrier and / or auxiliary material.

8. A medicine comprising the Lactobacillus plantarum and / or its metabolites and / or its cell lysates as claimed in claim 1, and an acceptable carrier and / or excipient.

9. A cosmetic comprising the Lactobacillus plantarum and / or its metabolites and / or its cell lysates as claimed in claim 1, and an acceptable carrier and / or excipient.

10. A skin care product comprising the Lactobacillus plantarum and / or its metabolites and / or its cell lysates as claimed in claim 1, and an acceptable carrier and / or excipient.

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