Lactiplantibacillus plantarum 18 and its applications

Abstract

The present invention provides a Lactiplantibacillus plantarum ( Lactiplant ibacillus plantarum ), strain 18, which is deposited in the China General Microbiological Culture Collection Center with the deposit number: CGMCC No. 31277. The Lactiplantibacillus plantarum 18 of the present invention can inhibit the growth of Propionibacterium acnes, inhibit the expression of virulence factors (biofilm, lipase, etc.) of Propionibacterium acnes, reduce inflammation, inhibit autophagy, scavenge free radicals, and inhibit tyrosinase activity. Therefore, it has the effects of anti-acne, anti-dermatitis, antioxidant, and whitening. The Lactiplantibacillus plantarum 18 of the present invention or its metabolites can be used to prepare products with 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 and its application. Background Art

[0002] Microbial infection, impaired barrier, inflammation, and pigment deposition caused by inflammation are common problems in most skin diseases. There are various microorganisms on the skin surface, including bacteria, fungi, viruses, protozoa, etc., which together constitute the skin microbial community. Under normal circumstances, these microorganisms maintain a dynamic balance. However, when the skin has problems, skin microecological disorders often occur. Probiotics can antagonize, secrete antibacterial peptides, metabolize skin lipids to form an organic acid "acid shield", and mobilize the immune system, etc., to combat harmful bacterial infections, enhance the skin barrier, and reduce skin inflammation. Therefore, regulating the skin microecology through probiotics has become one of the effective means to improve skin problems and treat skin diseases, and it has received increasing attention due to its good effect and small side effects.

[0003] According to the "Technical Specifications for Cosmetics Safety" (2015 Edition) of our country, live bacteria cannot be added to cosmetics. The "probiotics" or "probiotic ingredients" in skin care products usually refer to non-living bacteria, bacterial fermentation products or cell lysates, which are actually closer to the concept of "postbiotics" defined by the International Scientific Association for Probiotics and Prebiotics (ISAPP) in 2021. Currently, the common skin probiotic ingredients in cosmetics come from Lactiplantibacillus plantarum ( Lactobacillus plantarum ), Lactobacillus rhamnosus ( L. rhamnosus ), Lactobacillus reuteri ( L. reuteri ), Lactobacillus acidophilus ( L. acidophilus ), and Bifidobacterium ( Bifidobacterium bifidum ), etc.

[0004] With the in-depth study of skin microecology, the application of skin probiotics has gradually become a new way to solve skin problems. Probiotics are mainly applied in skin care with their dead bacteria, cell lysates or fermentation products, that is, postbiotics. The reported ways for them to play roles include the following aspects. First, antibacterial effect. Components such as antibacterial peptides in postbiotics can play the role of antagonizing and killing skin pathogenic bacteria. Second, anti-inflammatory effect. Postbiotics help reduce the expression of various inflammatory factors and alleviate skin inflammation. Third, inhibiting melanin synthesis. Postbiotics can directly inhibit melanin synthesis by inhibiting the activity of tyrosinase and inhibit the secretion of melanin in cells to achieve the effect of whitening. In addition, there are also reports that probiotics can enhance the skin barrier and play an anti-aging role.

[0005] Existing technical solutions for skin probiotic applications cover multiple aspects of probiotic applications in skin care. However, there are still certain problems in actual applications. First, compared with antibacterial drugs, the advantage of probiotic treatment is to achieve the purpose of solving skin problems and treating skin diseases by regulating the skin microecology. However, existing research mainly focuses on the antibacterial or bactericidal effects of probiotics on pathogenic bacteria. In fact, inhibiting the expression of virulence factors (biofilms, lipases, etc.) of harmful bacteria can control harmful bacteria without destroying the flora, which is more in line with the concept of microecological regulation. Second, there is currently a lack of in-depth understanding of the molecular mechanism of the efficacy of skin probiotics (postbiotics), which is not conducive to their popularization and application. Third, common skin problems (acne, atopic dermatitis, sensitivity, etc.) often have commonalities, namely microbial infections, inflammation, pigment deposition, etc., and multi-functional Lactobacillus plantarum that can simultaneously improve these problems has not been reported.

[0006] Currently, there are already some invention patents and patent applications in China regarding the improvement of skin conditions by probiotics. For example, CN116555128A discloses a Lactobacillus plantarum that can improve skin aging and its application. This Lactobacillus plantarum ( Lactobacillus plantarum ) has a preservation number of CGMCC NO.23434 and has the effect of inhibiting the degradation of skin collagen. For example, CN116042426A discloses a Lactobacillus plantarum WSH048, which is characterized in that the Lactobacillus plantarum WSH048 has the effects of repairing ultraviolet damage, alleviating inflammation, and preventing skin photoaging, and its preservation number is: CGMCC No.23159. For example, CN112920983B discloses a Lactobacillus plantarum ( Lactobacillus plantarum ) CCFM1158 that has the effect of improving facial sensitive skin and repairing the skin barrier. For example, CN118667687A discloses a Lactobacillus plantarum with anti-ultraviolet activity and its application. It relates to the field of raw materials for skin care products, and its preservation number is: CCTCC NO:M20231222. For example, CN114854649A discloses a Lactobacillus salivarius that regulates the human skin microflora, promotes the proliferation and repair of epidermal cells, and simultaneously has anti-inflammatory and antioxidant effects. For example, CN114574408B discloses that Lactobacillus plantarum SEUNEU-107 has the functions of treating acne and improving sensitive skin.

[0007] In summary, although there are already some probiotics for improving skin microecology in the prior art, their exhibited effects are mostly relatively single, and the depth of research on their efficacy mechanisms in the prior art is also very limited. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a multi-functional probiotic for improving skin microecology and its application.

[0009] The technical solution of the present invention is outlined as follows:

[0010] The present invention provides a Lactiplantibacillus plantarum Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) 18, which is deposited in the China General Microbiological Culture Collection Center (abbreviated as CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 31277, deposit date: July 12, 2024, taxonomic name: Lactiplantibacillus plantarum

[0011] The Lactiplantibacillus plantarum 18 of the present invention is derived from Sichuan pickled vegetables. On a solid plate, the colony is opaque, round, milky white, with a bright and smooth surface, moist texture, neat edges, and is raised. The colony diameter is 2 - 3 mm.

[0012] The Lactiplantibacillus plantarum 18 of the present invention is non-hemolytic, sensitive to clinically common antibiotics such as erythromycin, tetracycline, ampicillin, and chloramphenicol, and has good safety.

[0013] The Lactiplantibacillus plantarum 18 of the present invention, or its metabolite, or its cell lysate has the ability to antagonize Propionibacterium acnes, significantly inhibits the growth of Propionibacterium acnes, and the diameter of the inhibition zone by the double-layer plate Oxford cup method is 22 ± 0.1 mm.

[0014] The Lactiplantibacillus plantarum 18 of the present invention, or its metabolite, or its cell lysate can inhibit the formation of Propionibacterium acnes biofilm, and the inhibition rate measured according to the bacterial biomass is 80%.

[0015] The Lactiplantibacillus plantarum 18 of the present invention, or its metabolite, or its cell lysate can destroy the biofilm structure of Propionibacterium acnes, making the long axis of the rod-shaped cells of Propionibacterium acnes shorter.

[0016] The Lactiplantibacillus plantarum 18 of the present invention, or its metabolite, or its cell lysate can significantly reduce the activity of Propionibacterium acnes lipase.

[0017] The Lactiplantibacillus plantarum 18 of the present invention, or its metabolite, or its cell lysate can significantly down-regulate the virulence genes of Propionibacterium acnes, including: biofilm formation encoding genes ( cbiL, roxP, btuR ) and / or lipase encoding genes ( PPA2105 , PPA1761 , PPA1796 ) expression levels.

[0018] The Lactiplantibacillus plantarum 18 of the present invention, or its metabolite, or its cell lysate can significantly regress rat acne cysts and down-regulate the expression levels of genes encoding acne-related pro-inflammatory cytokines (TNF-α, IL-1β, and / or IL-6).

[0019] The Lactiplantibacillus plantarum 18 or its metabolite or its cell lysate according to the present invention can significantly down-regulate the expression levels of genes encoding pro-inflammatory cytokines (IL-6, IL-8, and / or IL-17A) in HaCaT cells.

[0020] The Lactiplantibacillus plantarum 18 or its metabolite according to the present invention can significantly inhibit starvation-induced autophagy in HeLa cells.

[0021] The Lactiplantibacillus plantarum 18 or its metabolite or its cell lysate according to the present invention can significantly down-regulate the expression levels of genes encoding pro-inflammatory cytokines (TNF-α, IL-1β, and / or IL-6) in a murine atopic dermatitis model.

[0022] The Lactiplantibacillus plantarum 18 or its metabolite or its cell lysate according to the present invention can scavenge hydroxyl radicals and / or ABTS radicals and has antioxidant efficacy.

[0023] The Lactiplantibacillus plantarum 18 or its metabolite or its cell lysate according to the present invention can inhibit tyrosinase activity and has whitening efficacy.

[0024] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of anti-acne, anti-dermatitis, antioxidant, and / or whitening products.

[0025] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of products for inhibiting Propionibacterium acnes.

[0026] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of products for down-regulating the virulence genes of Propionibacterium acnes cbiL, roxP, btuR, PPA2105 , PPA1761 and / or PPA1796 the expression levels of which.

[0027] The present invention also provides the use of the Lactiplantibacillus plantarum in the preparation of products for down-regulating the gene expression levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and / or IL-17A.

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

[0029] In the above applications, the products include microbial agents, drugs, cosmetics, or skin care products.

[0030] In the above applications, the Lactiplantibacillus plantarum may include its live bacteria, inactivated bacteria, metabolites, and / or cell lysates and the like in forms according to the needs of the products, 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 18 and / or its metabolites and / or its cell lysates, as well as an acceptable carrier and / or excipient.

[0032] The present invention also provides a medicine, which comprises the Lactiplantibacillus plantarum 18 and / or its metabolites and / or its cell lysates, as well as an acceptable carrier and / or excipient.

[0033] The present invention also provides a cosmetic, which comprises the Lactiplantibacillus plantarum 18 and / or its metabolites and / or its cell lysates, as well as an acceptable carrier and / or excipient.

[0034] The present invention also provides a skin care product, which comprises the Lactiplantibacillus plantarum 18 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 18 of the present invention has good safety and has the effects of treating acne, dermatitis, anti-inflammation, antioxidant and whitening, etc., and is a multi-functional skin probiotic friendly to the microecology.

[0037] The Lactiplantibacillus plantarum 18 or its metabolites or its cell lysates of the present invention can down-regulate the expression levels of the virulence genes of Propionibacterium acnes cbiL, roxP, btuR, PPA2105 , PPA1761 and / or PPA1796 . Propionibacterium acnes is an important pathogenic bacterium of acne, but it is also a resident bacterium on the skin of healthy people. Treatments targeting the killing of Propionibacterium acnes often lead to microecological disorders. The treatment idea of acting by antagonizing the virulence factors of Propionibacterium acnes (such as biofilm, lipase, etc.) not only has a definite effect, but also is safer and has fewer side effects.

[0038] The present invention relates to the preservation information of biological materials:

[0039] Biological material: Lactiplantibacillus plantarum 18;

[0040] Preservation date: July 12, 2024;

[0041] Preservation unit: China General Microbiological Culture Collection Center (abbreviated as CGMCC), Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0042] Preservation number: CGMCC No. 31277;

[0043] Taxonomic name: Lactiplantibacillus plantarum Lactiplantibacillus plantarum . Description of the drawings

[0044] Figure 1 : Colony morphology of strain 18 in Example 1.

[0045] Figure 2 : Hemolytic experiment of strain 18 in Example 2.

[0046] Figure 3 : Growth curve of strain 18 in Example 3.

[0047] Figure 4 : Inhibition zone of strain 18 against Propionibacterium acnes in Example 4.

[0048] Figure 5 : Inhibition of Propionibacterium acnes biofilm by strain 18 in Example 5 (scanning electron micrograph, magnification: 5000×).

[0049] Figure 6 : Inhibition of Propionibacterium acnes biofilm by strain 18 in Example 5 (confocal micrograph, magnification 40×).

[0050] Figure 7 : Inhibition of lipase activity of Propionibacterium acnes by strain 18 in Example 6.

[0051] Figure 8 : Effect of strain 18 on the expression level of virulence genes of Propionibacterium acnes in Example 7.

[0052] Figure 9 : Effect of strain 18 on the transcriptional level of pro-inflammatory cytokines in a rat auricular acne model in Example 8.

[0053] Figure 10 : Effect of strain 18 on the protein level of pro-inflammatory cytokines in a rat auricular acne model in Example 8.

[0054] Figure 11 : Effect of strain 18 on the gene expression level of pro-inflammatory cytokines in HaCaT cells in Example 9.

[0055] Figure 12 : Inhibition of autophagy in HeLa cells by strain 18 in Example 10.

[0056] Figure 13 : Effect of strain 18 on the transcriptional level of pro-inflammatory cytokines in a mouse AD model in Example 11.

[0057] Figure 14 : Effect of strain 18 on the protein level of pro-inflammatory cytokines in a mouse AD model in Example 11. Detailed implementation manners

[0058] 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 fall within the protection scope of the present invention.

[0059] Example 1: Identification and Preservation of Strains

[0060] Strain 18 was isolated and purified from Sichuan pickles, and the strain was identified and preserved through colony morphology, 16S rRNA gene and phes gene sequencing analysis, cell morphology, and physical and chemical tests. The identification result was Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), and the preservation number was CGMCC No. 31277.

[0061] Colony morphological characteristics of strain 18 ( 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.

[0062] The 16S rRNA gene sequence of strain 18 is shown in SEQ ID NO:1:

[0063]

[0064] The phes gene sequence of strain 18 is shown in SEQ ID NO:2:

[0065]

[0066] The cell morphology and physical and chemical test results of strain 18 are shown in Table 1.

[0067] Table 1 Cell Morphology and Physical and Chemical Test Results of Strain 18

[0068]

[0069] Note: “+” represents a positive result, and “-” represents a negative result.

[0070] Example 2: Safety Evaluation of Strains

[0071] The safety of the strain was evaluated through hemolytic experiments and antibiotic sensitivity experiments.

[0072] (1) Hemolytic experiment

[0073] Add 10 μL of the bacterial liquid of strain 18 dropwise onto a Columbia blood agar plate containing 5% sheep blood. Set Staphylococcus aureus (ATCC 6538) as the positive control, Lactobacillus plantarum (D8) as the negative control, and LB liquid medium as the blank control. Incubate at 37 °C for 48 h and observe the hemolysis of the strain on the plate. The results are as Figure 2 shown, and strain 18 does not hemolyze.

[0074] (2) Antibiotic sensitivity analysis

[0075] The Kirby-Bauer disk diffusion method was used to evaluate the antibiotic sensitivity of strain 18. The results are shown in Table 2, and strain 18 is sensitive to 4 commonly used clinical antibiotics.

[0076] Table 2 Antibiotic susceptibility test

[0077]

[0078] Note: S represents sensitive.

[0079] Example 3: Preparation of the fermentation supernatant of the strain

[0080] Pick a single colony of strain 18 with good growth state, inoculate it into MRS liquid medium for activation, and then transfer it to MRS liquid medium at an inoculation amount of 1%. Incubate statically for 24 h. Every 2 hours, take 200 μL of the solution to measure the OD value of the bacteria at a wavelength of 600 nm, record and plot the growth curve of strain 18, 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 obtain the fermentation supernatant of strain 18 for standby.

[0081] Example 4: Experiment on the inhibition of the growth of Propionibacterium acnes by the strain

[0082] The double-layer plate method was used to evaluate the effect of the fermentation supernatant of strain 18 on the growth of Propionibacterium acnes.

[0083] Propionibacterium acnes (ATCC6919) was selected as the test strain, and this strain is type IA, which is the main type causing acne.

[0084] Reinforced Clostridium medium (RCM) solid medium containing 2% agar was used as the bottom layer of the double-layer plate. After solidification, place a sterilized Oxford cup. Add 5% Propionibacterium acnes bacterial liquid to the RCM medium that has not solidified at about 50 °C and pour it on the bottom layer. After solidification, clamp out the Oxford cup to form the top layer of the double-layer plate with Oxford cup holes.

[0085] Inject 0.2 mL of the fermentation supernatant of strain 18 into the Oxford cup wells. Set the 0.5% triclosan (CHX) solution as the positive control and the PBS buffer solution as the negative control. Incubate anaerobically at 37 °C for 48 h. The results are as Figure 4 shown. Strain 18 significantly inhibited the growth of Propionibacterium acnes, and the diameter of the inhibition zone was approximately 22 ± 0.1 mm.

[0086] Example 5: Experiment on the inhibition of Propionibacterium acnes biofilm formation by the strain

[0087] Inoculate Propionibacterium acnes into the RCM liquid medium at an inoculum size of 2%. At the same time, add 10% - 30% (v / v) of the fermentation supernatant of strain 18. Incubate anaerobically at 37 °C for 48 h in a 96-well plate or a 24-well plate to form a Propionibacterium acnes biofilm on the bottom of the wells or on the cell slides. Set the 0.5% triclosan (CHX) solution as the positive control and the MRS medium as the negative control. The inhibitory effect of the fermentation supernatant of strain 18 on the Propionibacterium acnes biofilm was evaluated by crystal violet staining, scanning electron microscopy, and confocal microscopy.

[0088] 1. Crystal violet staining method

[0089] The biomass of the Propionibacterium acnes biofilm was determined by crystal violet staining. After washing the biofilm 3 times with PBS, stain it with 0.1% crystal violet at room temperature for 20 min, and wash away the excess unbound dye with PBS. Then, decolorize the biofilm with 95% ethanol for 30 min, gently shake until no crystal violet is released from the biofilm, and measure the OD value at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Table 3. The inhibition rate of the fermentation supernatant of strain 18 on the Propionibacterium acnes biofilm = (0.995 - 0.199) / 0.995 × 100% = 80%, and the result is better than that of the positive group.

[0090] Table 3 Crystal violet determination of Propionibacterium acnes biofilm

[0091]

[0092] 2. Scanning electron microscopy observation

[0093] The cell morphology of Propionibacterium acnes in the biofilm was observed by scanning electron microscopy. Wash the biofilm 3 times with PBS, add 1.5 mL of 2.5% glutaraldehyde, and store it overnight at 4 °C. Dehydrate it with gradient ethanol (50%, 60%, 70%, 80%, 90%, 95%, 100%), dry it at room temperature, fix it by spraying gold for 30 s, and observe the morphological changes of Propionibacterium acnes under a scanning electron microscope at a voltage of 10 kV. The results are as Figure 5 shown. The fermentation supernatant of strain 18 inhibited the growth of Propionibacterium acnes in the biofilm and shortened the long axis of the rod-shaped cells.

[0094] 3. Observation with a laser confocal microscope

[0095] Prepare the working solution according to the instructions of the LIVE / DEAD™ Cell Viability Kit and Dextran, Alexa Fluor™ 647. Add SYTO 9, Propidium-Iodide (PI), and Dextran, Alexa Fluor™ 647 to the well plate. Stain with 500 μL of the mixed working solution and incubate for 15 min in the dark. Then, place the biofilm coverslip on a special glass slide for the laser confocal microscope and observe the bacterial distribution in the dark. The results are as Figure 6 shown. The PI dye stains dead bacteria red, and the SYTO-9 dye stains live bacteria green. The fermentation supernatant of strain 18 inhibited the formation of Propionibacterium acnes biofilm.

[0096] Example 6: Experiment on the inhibition of Propionibacterium acnes lipase activity by the strain

[0097] Mix Propionibacterium acnes (1×10 8 CFU / mL) with the fermentation supernatant of strain 18 under anaerobic conditions at 37°C for 48 h. Set 0.5% chlorhexidine (CHX) solution as the positive control and MRS medium as the negative control. Centrifuge to obtain the supernatant and detect the change in lipase activity using a Lipase assay kit. The results are as Figure 7 shown (* p<0.05, ** p<0.01, compared with the negative group). Strain 18 significantly inhibited the lipase activity of Propionibacterium acnes (p<0.05).

[0098] Example 7: Experiment on the inhibition of Propionibacterium acnes virulence genes by the strain

[0099] Mix Propionibacterium acnes (1×10 8 CFU / mL) with the fermentation supernatant of strain 18 under anaerobic conditions at 37°C for 48 h. Set 0.5% chlorhexidine (CHX) solution as the positive control, MRS medium as the negative control, and at the same time set the fermentation supernatant of Lactobacillus plantarum ( Lactobacillus plantarum ) L16 (CN202410351722.5, deposit number: CGMCC No.28163) as the experimental control group.

[0100] After incubation, centrifuge to obtain the supernatant, extract total RNA using the TIANGEN kit, and reverse transcribe it into cDNA. Detect the virulence genes of Propionibacterium acnes by real-time quantitative PCR: the gene encoding biofilm formation ( cbiL, roxP, btuR ), the gene encoding lipase ( PPA2105, PPA1761 , PPA1796 ). Using GAPDH as an internal reference, adopt 2-△△Ct The relative expression levels of each gene were calculated by the method.

[0101] The results are shown as Figure 8 (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared with the negative group). The fermentation supernatant of strain 18 could significantly down-regulate the expression levels of the virulence genes of Propionibacterium acnes cbiL, roxP, btuR, PPA2105 , PPA1761 and PPA1796 (p<0.05), with a down-regulation of 43% - 74%. The fermentation supernatant of strain L16 could significantly down-regulate the expression levels of the virulence genes roxP and PPA1796 (p<0.05), but there was no significant inhibition on the virulence genes cbiL , btuR, PPA2105 and PPA1761 . It can be seen that strain 18 has a wider inhibitory range on the virulence genes of Propionibacterium acnes.

[0102] Example 8: Experiment on the effect of the strain on the rat acne model

[0103] A rat auricular acne model was established and intervened with the fermentation supernatant of strain 18. By measuring the auricular thickness, the mRNA levels of pro-inflammatory cytokines in auricular tissues, and the protein levels of pro-inflammatory cytokines in auricular tissues, the therapeutic effect of strain 18 on acne was evaluated.

[0104] 1. Establishment and intervention of the rat auricular acne model

[0105] The rats were adaptively fed for 1 week first. Sprague-Dawley (SD) rats, male, weighing 200 ± 20 g were used. On the first day of modeling, 100% oleic acid solution (0.1 mL each time) was applied once to the inner side of the left ear at the opening of the ear duct of the rats. On the same day, 50 μL of inactivated Propionibacterium acnes bacterial solution (1×10 8 CFU / mL) was injected intradermally into the left auricle of the rats every day in an alternating manner for 8 days.

[0106] The experiment was divided into a model group, a negative group, a positive group, and a strain 18 group (18), with 6 rats in each group. The intervention drug for the positive group was adapalene gel, and the negative control group was intervened with normal saline. After successful modeling, that is, starting from the 9th day of the experiment, the negative control group, the positive group, and the strain 18 group were respectively applied with 200 μL of normal saline, adapalene gel, and the fermentation supernatant of strain 18 evenly on the left ear once a day for 8 consecutive days.

[0107] 2. Rat auricular thickness

[0108] During the measurement, the rats were anesthetized using an animal anesthesia machine. Before modeling, after modeling, 4 days after drug administration, and 8 days after drug administration, the thickness of the left ear of the rats was measured using a vernier caliper, and three measurements were taken at different positions.

[0109] The measurement results are shown in Table 4. After the intervention with the fermentation supernatant of strain 18, the auricular cysts of the rats significantly regressed, and the regression rate was better than that of the positive group drug.

[0110] Table 4 Changes in the thickness of the auricles of rats

[0111]

[0112] 3. Gene expression of pro-inflammatory cytokines in rat auricular tissues

[0113] The rat auricular tissues were homogenized, total RNA was extracted, reverse transcribed into cDNA, and qRT-PCR was used to detect the mRNA expression levels of acne-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.

[0114] The results are as Figure 9 shown (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared with the model group). Strain 18 significantly down-regulated the gene expression levels of acne-related pro-inflammatory cytokines TNF-α, IL-1β, and / or IL-6 (p<0.05), with a down-regulation of 63% - 71%.

[0115] ELISA detection of the protein levels of pro-inflammatory cytokines in rat auricular tissues (1) ELISA detection

[0116] a. Preparation before detection: Taking the TNF-α kit as an example.

[0117] 1) Take out the kit from the refrigerator 20 minutes in advance to balance to room temperature.

[0118] 2) Dilute the 20× concentrated washing solution with double-distilled water into 1× working solution.

[0119] b. Operating steps:

[0120] 1) Take out the strip required for the test from the sealed bag that has been balanced 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℃.

[0121] 2) Set up the standard wells, blank wells, and sample wells. Add 50 μL of standards with different concentrations to each standard well.

[0122] 3) Add 50 μL of the sample to be tested into the sample wells; do not add anything to the blank wells.

[0123] 4) Except for the blank wells, add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C incubator or water bath for 60 min.

[0124] 5) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL); let stand for 1 min, discard the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times.

[0125] 6) Add 50 μL of each of Substrate A and B to each well, and incubate at 37°C in the dark for 15 min.

[0126] 7) Add 50 μL of the 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).

[0127] c. Result judgment:

[0128] 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.

[0129] (2) Calculation of TNF-α, IL-1β and IL-6 contents

[0130] 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 ratio. Measure their OD values at 450 nm (OD450) using the above ELISA method, fit the regression equation of the standard curve, substitute the OD450 values of each test sample into the equation, and 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 and the blank control group. A p < 0.05 is considered statistically significant.

[0131] The results are as Figure 10 shown (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the model group). The fermentation supernatant of strain 18 significantly down-regulated the protein levels of acne-related pro-inflammatory cytokines TNF-α, IL-1β and / or IL-6 (p < 0.05), with a down-regulation of 13% - 32%.

[0132] Example 9: Experiment on the Effect of the Strain on the Gene Expression Level of Pro-inflammatory Cytokines in HaCaT Cells

[0133] 1. Preparation of Test Samples

[0134] Stimulus: Accurately weigh 5 mg of LPS, dissolve it thoroughly with 5 mL of PBS to prepare an LPS stock solution with a mass concentration of 1 mg / mL. Take 100 μL and dispense it into 0.5 mL EP tubes, and store them in a -80°C refrigerator. Dilute it with cell culture medium to a mass concentration of 500 μg / mL before use as the stimulus.

[0135] Experimental group (18): The fermentation supernatant of strain 18 prepared according to Example 3.

[0136] 2. Cell Culture

[0137] 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% CO2 by volume) for routine culture. When the cell confluence reaches 80 - 90%, digest and passage the adherent cells with trypsin, and passage or change the medium 2 - 3 times a week.

[0138] 3. Detection of the Gene Expression Level of Pro-inflammatory Cytokines in HaCaT Cells

[0139] When the cell confluence reaches 80% - 90%, obtain a cell suspension by the method of cell passage described above, adjust the cell density to 2×10 5 cells / mL, add it to a 24-well plate, 1000 μL of cell suspension per well, and continue to culture it in an incubator (37°C, 5% CO2 by volume) for 12 h. When the cell plating rate in the well plate meets the test requirements, first aspirate the old culture medium in the 24-well plate. Except for the blank group, add LPS (500 μg / mL) to the experimental group and the model group in the complete culture medium to a final concentration of 20 μg / mL, and continue to incubate for 12 h. Subsequently, add 50 μL of the fermentation supernatant of strain 18 to each well of the experimental group, and keep the same volume in the blank group and the model group and continue to culture for 12 h. Set 3 replicate wells for each group as parallel groups. Use a cell scraper to scrape up the adherent cells, and centrifuge at 1000g for 3 min to collect the cells.

[0140] Extract the total RNA of HaCaT cells from the collected cells above using the TRIZOL kit. Reverse transcribe the total RNA into cDNA using a reverse transcription kit, and detect the expression level of the target mRNA using the SYBR Green kit on a Thermo Fisher Q1 PLUS real-time quantitative PCR instrument. Using GAPDH as an internal reference, the relative expression level of the mRNA of the target gene is calculated according to 2 －ΔΔCt Calculate.

[0141] The results were as follows Figure 11 ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the model group). The fermentation supernatant of strain 18 significantly down-regulated the expression levels of pro-inflammatory cytokine genes IL-6, IL-8 and / or IL-17A in HaCaT cells (p < 0.05), with a down-regulation of 29% - 63%.

[0142] Example 10: Experiment on the inhibition of cell autophagy by the strain

[0143] HeLa cells stably expressing GFP-LC3 were seeded into 96-well plates containing DMEM medium (containing 10% fetal bovine serum), with 7000 cells per well, and incubated overnight at 37 °C in 5% CO2. 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 18) for 2 h. The punctate distribution of GFP-LC3 (autophagosome formation) was observed using a fluorescence inverted microscope.

[0144] The results were as follows Figure 12 shown. The fermentation supernatant of strain 18 could significantly inhibit the autophagosome formation induced by starvation, indicating its certain anti-inflammatory and anti-tumor effects.

[0145] Example 11: Experiment on the effect of the strain on a mouse dermatitis model

[0146] An atopic dermatitis model in mice was established and intervened with the fermentation supernatant of strain 18. The therapeutic effect of strain 18 on dermatitis was evaluated by measuring the gene levels and protein levels of pro-inflammatory cytokines in skin tissues.

[0147] 1. Establishment and intervention of a mouse specific dermatitis model

[0148] The mice were first adaptively fed for 1 week. Balb / c mice, female, 20 ± 2 g, had their abdominal and dorsal hairs removed using an electric 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, and on the fifth day, 20 μL of 0.05% DNFB was applied to the back of the mice. After that, DNFB was used for modeling every day at 9:00, and the application was continued for 21 days.

[0149] The experiment was divided into a model group, a negative group, a positive group, and a strain 18 group (18), 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. The negative group, the positive group, and the strain 18 group were evenly applied with 200 μL of normal saline, mometasone furoate cream, and the fermentation supernatant of strain 18 once at 15:00 every day on the left ear, and the application was continued for 21 days.

[0150] 2. Detection of mRNA expression levels of TNF-α, IL-1β, and IL-6 in the back tissues of mice by qRT-PCR

[0151] The back tissues of the mice were homogenized, total RNA was extracted, reverse transcribed into cDNA, and the mRNA expression levels of acne-related pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were detected by qRT-PCR. GAPDH was used as an internal reference, and the 2 -△△Ct method was used to calculate the relative expression levels of each gene.

[0152] The results were as Figure 13 shown (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared with the model group). The fermentation supernatant of strain 18 significantly down-regulated the expression levels of atopic dermatitis-related pro-inflammatory cytokines TNF-α, IL-1β, and / or IL-6 genes (p<0.05), with a down-regulation of 41% - 63%.

[0153] 3. Detection of protein levels of TNF-α, IL-1β, and IL-6 in the back tissues of mice by ELISA

[0154] Referring to the ELISA detection and calculation method in Example 8. The results were as Figure 14 shown (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared with the model group). The fermentation supernatant of strain 18 significantly down-regulated the protein levels of atopic dermatitis-related pro-inflammatory cytokines TNF-α, IL-1β, and / or IL-6 (p<0.05), with a down-regulation of 11% - 26%.

[0155] Example 12: Experiment on the antioxidant capacity of the strain

[0156] The antioxidant capacity of strain 18 was evaluated by measuring the hydroxyl radical scavenging ability and ABTS radical scavenging ability of the fermentation supernatant of strain 18.

[0157] 1. Detection of hydroxyl radical scavenging ability

[0158] The determination was carried out with reference to the instruction manual of the ABTS free radical scavenging ability detection kit (Beijing Box Bioscience and Technology Co., Ltd.). The calculation formula for the hydroxyl radical scavenging rate is as follows:

[0159] Hydroxyl radical scavenging rate D% = [(A determination - A control) ÷ (A blank - A control)] × 100%.

[0160] The calculation results showed that the hydroxyl radical scavenging ability of strain 18 was 78.63%.

[0161] 2. Detection of ABTS free radical scavenging ability

[0162] The detection was carried out with reference to the instruction manual of the hydroxyl radical scavenging ability detection kit (Beijing Box Bioscience and Technology Co., Ltd.). The calculation formula for the scavenging rate is as follows:

[0163] ABTS free radical scavenging rate D% = [A blank - (A determination - A control)] ÷ A blank × 100%.

[0164] The calculation results showed that the ABTS free radical scavenging ability of strain 18 was 25.48%.

[0165] The above experimental results indicated that strain 18 had good antioxidant ability. Strain 18 could be used to prepare antioxidant products, such as drugs, skin care products or cosmetics with antioxidant and free radical scavenging functions.

[0166] Example 13: Experiment on the detection of the inhibitory activity of the strain on tyrosinase

[0167] PBS phosphate buffer (pH 6.8), 40 μL of 10% fermentation supernatant of strain 18 and L-dopa / tyrosine solution were successively added to a 96-well plate. After mixing, it was incubated at a constant temperature of 37 °C for 20 min. Then 40 μL of tyrosinase solution was added, and it was incubated at a constant temperature of 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:

[0168] A: 120 μL PBS + 40 μL tyrosinase + 40 μL L-dopa / tyrosine;

[0169] B: 160 μL PBS + 40 μL L-dopa / tyrosine;

[0170] C: 80 μL PBS + 40 μL fermentation supernatant of strain 18 + 40 μL tyrosinase + 40 μL L-dopa / tyrosine;

[0171] D: 120 μL PBS + 40 μL fermentation supernatant of strain 18 + 40 μL L-dopa / tyrosine;

[0172] Inhibitory rate of tyrosinase (%) = [(A - B) - (C - D)] / (A - B) × 100%.

[0173] As shown in Table 5, the fermentation supernatant of strain 18 has a good effect on inhibiting tyrosinase activity, indicating its whitening effect. Strain 18 can be used to prepare whitening products, such as drugs, skin care products or cosmetics with whitening functions.

[0174] Table 5 Inhibitory rate of strain 18 on tyrosinase activity

[0175]

[0176] In summary, Lactiplantibacillus plantarum 18 of the present invention can inhibit the growth of Propionibacterium acnes, inhibit the expression of virulence factors (biofilm, lipase, etc.) of Propionibacterium acnes, reduce inflammation, scavenge free radicals, and inhibit tyrosinase activity. Therefore, it has the effects of anti-acne, anti-dermatitis, antioxidant and whitening. Lactiplantibacillus plantarum 18 of the present invention or its metabolites can be used in the preparation of products with the above effects or functions.

[0177] 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 preservation number of the Lactiplantibacillus plantarum is CGMCC No. 31277.

2. Use of the Lactiplantibacillus plantarum according to claim 1 in the preparation of anti-acne, anti-dermatitis, antioxidant and / or skin-whitening products; the products are microbial agents, drugs, cosmetics or skin care products; The Lactiplantibacillus plantarum in the cosmetics or skin care products is inactivated cells and / or the fermentation supernatant of its strains.

3. Use of the Lactiplantibacillus plantarum according to claim 1 in the preparation of products for inhibiting Propionibacterium acnes; the products are microbial agents, drugs, cosmetics or skin care products; The Lactiplantibacillus plantarum in the cosmetics or skin care products is inactivated cells and / or the fermentation supernatant of its strains.

4. A microbial agent, which comprises the Lactiplantibacillus plantarum according to claim 1 and acceptable excipients.

5. A drug, which comprises the Lactiplantibacillus plantarum according to claim 1 and / or the fermentation supernatant of its strains, and acceptable excipients.

6. A cosmetic, which comprises inactivated cells of the Lactiplantibacillus plantarum according to claim 1 and / or the fermentation supernatant of its strains, and acceptable excipients.

7. A skin care product, which comprises inactivated cells of the Lactiplantibacillus plantarum according to claim 1 and / or the fermentation supernatant of its strains, and acceptable excipients.

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