Phytobacterium plantarum 18 and application thereof

Abstract

The invention provides a lactobacillus plantarum 18 which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No.31277. The lactobacillus plantarum 18 has the advantages that the lactobacillus plantarum 18 is prepared from lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum, lactobacillus plantarum and lactobacillus plantarum; the plant lactobacillus 18 can inhibit growth of propionibacterium acnes, inhibit expression of virulence factors (biological membranes, lipase and the like) of the propionibacterium acnes, relieve inflammation, inhibit autophagy of cells, scavenge free radicals and inhibit activity of tyrosinase, so that the plant lactobacillus 18 has the effects of resisting acnes, dermatitis and oxidation and whitening skin. The plant lactobacillus 18 or the metabolite of the plant lactobacillus 18 can be used for preparing products with the effects or effects.

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] Microbial infection, damaged skin barrier, inflammation, and pigmentation caused by inflammation are common problems for most skin diseases. There are a variety of microorganisms on the surface of the skin, including bacteria, fungi, viruses, protozoa, etc., which together constitute the skin microbial community. Under normal circumstances, these microorganisms maintain a dynamic balance. However, when skin problems occur, they are often accompanied by skin microecological imbalance. Probiotics can fight against harmful bacterial infections, enhance the skin barrier, and reduce skin inflammation by antagonism, secreting antimicrobial peptides, metabolizing skin lipids to form an organic acid "acid mask", and mobilizing immunity. 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 more and more attention because of its good effect and few side effects.

[0003] According to my country's "Technical Specifications for Safety of Cosmetics" (2015 edition), live bacteria cannot be added to cosmetics. "Probiotics" or "probiotic ingredients" in skin care products usually refer to inactive 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 most common skin probiotic ingredients in cosmetics come from Lactobacillus plantarum ( Lactobacillus plantarum )、Lactobacillus rhamnosus( L. rhamnosus )、Lactobacillus reuteri( L. reuteri )、Lactobacillus acidophilus( L. acidophilus ) and Bifidobacterium ( Bifidobacterium bifidum )wait.

[0004] With the deepening of research on skin microecology, the application of skin probiotics has gradually become a new way to solve skin problems. Probiotics are mainly used in skin care in the form of dead bacteria, cell lysates or fermentation products, namely postbiotics. Existing reports show that their effects include the following aspects. First, antibacterial effect. Antimicrobial peptides and other ingredients in postbiotics can antagonize and kill skin pathogens; second, anti-inflammatory effect. Postbiotics help reduce the expression of various inflammatory factors and reduce skin inflammation. Third, inhibit melanin synthesis. Postbiotics can directly inhibit the synthesis of melanin by inhibiting the activity of tyrosinase, inhibit the secretion of melanin in cells, and achieve a whitening effect. In addition, there are reports that probiotics can enhance the skin barrier and play an anti-aging role.

[0005] The existing technical solutions for the application of skin probiotics cover multiple application aspects of probiotics in skin care. However, there are still some problems in practical applications. First of all, compared with antibacterial drugs, the advantage of probiotic treatment is to solve skin problems and treat skin diseases by regulating the skin microecology. However, existing studies are mostly carried out around the antibacterial or bactericidal effects of probiotics on pathogenic bacteria. In fact, inhibiting the expression of virulence factors (biofilm, lipase, etc.) of harmful bacteria can control harmful bacteria without destroying the flora, which is more in line with the concept of microecological regulation. Secondly, 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 its promotion and application. Thirdly, common skin problems (acne, atopic dermatitis, sensitivity, etc.) often have commonalities, namely microbial infection, inflammation, pigmentation, etc., and multifunctional plant lactobacillus that can improve these problems at the same time has not been reported.

[0006] At present, my country has some invention patents and patent applications on probiotics to improve skin conditions. For example, CN116555128A discloses a plant lactobacillus that can improve skin aging and its application. Lactobacillus plantarum ) is deposited as CGMCC NO.23434, which has the effect of inhibiting the degradation of skin collagen. For example, CN116042426A discloses a plant lactobacillus WSH048, characterized in that the plant lactobacillus WSH048 has the effects of repairing ultraviolet damage, relieving inflammation, and preventing skin photoaging, and the deposit number is: CGMCC No.23159. For example, CN112920983B discloses a plant lactobacillus ( Lactobacillus plantarum )CCFM1158. For example, CN118667687A discloses a strain of Lactobacillus plantarum with anti-ultraviolet activity and its application, which relates to the field of skin care product raw materials, and the deposit number is: CCTCC NO:M20231222. For example, CN114854649A discloses a strain of Lactobacillus salivarius, which regulates the microbial flora of human skin, promotes the proliferation and repair of epidermal cells, and has anti-inflammatory and antioxidant effects. For example, CN114574408B discloses that Lactobacillus plantarum SEUNEU-107 has the function of treating acne and improving sensitive skin.

[0007] In summary, although there are some probiotics in the existing technology for improving skin microecology, the effects they exhibit are mostly relatively simple, and the depth of research on their efficacy mechanisms in the existing technology is also very limited. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention aims to provide a multifunctional probiotic for improving skin microecology and its application.

[0009] The technical solution of the present invention is summarized as follows: The present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum )18, 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. 31277, deposit date: July 12, 2024, classification name: Lactobacillus plantarum Lactiplantibacillus plantarum .

[0010] The plant lactobacillus 18 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.

[0011] The Lactobacillus plantarum 18 of the present invention is non-hemolytic, sensitive to common clinical antibiotics such as erythromycin, tetracycline, ampicillin and chloramphenicol, and has good safety.

[0012] The Lactobacillus plantarum 18 or its metabolites or its cell lysates described in the present invention have the ability to antagonize Propionibacterium acnes and significantly inhibit the growth of Propionibacterium acnes. The diameter of the inhibition zone of the double-layer plate Oxford cup method is 22±0.1 mm.

[0013] The Lactobacillus plantarum 18 or its metabolites or its cell lysates described in the present invention can inhibit the biofilm formation of Propionibacterium acnes, and the inhibition rate measured by bacterial biomass is 80%.

[0014] The Lactobacillus plantarum 18 or its metabolites or its cell lysates described in the present invention can destroy the biofilm structure of Propionibacterium acnes and shorten the long axis of the rod-shaped cells of Propionibacterium acnes.

[0015] The Lactobacillus plantarum 18 or its metabolites or its cell lysates described in the present invention can significantly reduce the activity of Propionibacterium acnes lipase.

[0016] The Lactobacillus plantarum 18 or its metabolites or cell lysates of the present invention can significantly downregulate the virulence genes of Propionibacterium acnes, including: biofilm formation encoding genes ( cbiL, roxP, btuR ) and / or lipase encoding genes ( PPA2105 , PPA1761 , PPA1796 ) expression level.

[0017] The Lactobacillus plantarum 18 or its metabolites or its cell lysates described in the present invention can significantly reduce 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).

[0018] The Lactobacillus plantarum 18 or its metabolites or its cell lysates described in the present invention can significantly downregulate the expression levels of genes encoding proinflammatory cytokines (IL-6, IL-8 and / or IL-17A) in HaCaT cells.

[0019] The Lactobacillus plantarum 18 or its metabolites described in the present invention can significantly inhibit starvation-induced HeLa cell autophagy.

[0020] The Lactobacillus plantarum 18 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.

[0021] The Lactobacillus plantarum 18 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.

[0022] The Lactobacillus plantarum 18 or its metabolites or its cell lysates described in the present invention can inhibit tyrosinase activity and have whitening effect.

[0023] The present invention also provides application of the plant lactobacillus in preparing anti-acne, anti-dermatitis, anti-oxidation and / or whitening products.

[0024] The present invention also provides application of the Lactobacillus plantarum in preparing a product for inhibiting Propionibacterium acnes.

[0025] The present invention also provides the plant lactobacillus in the preparation of down-regulating toxicity genes of Propionibacterium acnes cbiL、roxP、 btuR, PPA2105 , PPA1761 and / or PPA1796 Application of products with expression levels.

[0026] The present invention also provides the use of the Lactobacillus plantarum in preparing a product for down-regulating the gene expression level of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and / or IL-17A.

[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 18 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 18 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or excipient.

[0032] The present invention also provides a cosmetic comprising the Lactobacillus plantarum 18 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 18 and / or its metabolites and / or its cell lysates, and an acceptable carrier and / or auxiliary materials.

[0034] Beneficial effects: The plant lactobacillus 18 of the present invention has good safety, has the effects of treating acne, dermatitis, anti-inflammatory, anti-oxidation and whitening, and is a microecologically friendly multi-functional skin probiotic.

[0035] The plant lactobacillus 18 or its metabolites or cell lysates of the present invention can down-regulate the toxicity gene 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 aimed at killing Propionibacterium acnes often lead to microecological imbalance. The treatment idea of ​​antagonizing the virulence factors of Propionibacterium acnes (such as biofilm, lipase, etc.) is not only effective, but also safer and has fewer side effects.

[0036] The present invention relates to the deposit information of biological materials: Biological materials: Lactobacillus plantarum 18; Deposit date: July 12, 2024; Depository: China General Microbiology Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No. 31277; Classification name: Lactobacillus plantarum Lactiplantibacillus plantarum . BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Colony morphology of strain 18 in Example 1.

[0038] Figure 2: Hemolytic activity experiment of strain 18 in Example 2.

[0039] Figure 3 : Growth curve of strain 18 in Example 3.

[0040] Figure 4 : The inhibition zone of strain 18 in Example 4 inhibits Propionibacterium acnes.

[0041] Figure 5 : The strain 18 in Example 5 inhibited the biofilm of Propionibacterium acnes (Scanning electron micrograph, magnification: 5000×).

[0042] Figure 6 : Strain 18 in Example 5 inhibits Propionibacterium acnes biofilm (confocal image, magnification 40×).

[0043] Figure 7 : Strain 18 in Example 6 inhibited the lipase activity of Propionibacterium acnes.

[0044] Figure 8 : The effect of strain 18 in Example 7 on the expression level of virulence genes of Propionibacterium acnes.

[0045] Fig. 9 : The effect of strain 18 in Example 8 on the transcription level of pro-inflammatory cytokines in the rat auricular acne model.

[0046] Fig.10 : The effect of strain 18 in Example 8 on the protein levels of proinflammatory cytokines in the rat auricular acne model.

[0047] Fig.11 : The effect of strain 18 in Example 9 on the expression level of pro-inflammatory cytokine genes in HaCaT cells.

[0048] Fig.12 : Strain 18 in Example 10 inhibits HeLa cell autophagy.

[0049] Fig.13 : The effect of strain 18 in Example 11 on the transcription level of pro-inflammatory cytokines in the mouse AD model.

[0050] Fig.14 : The effect of strain 18 in Example 11 on the protein levels of pro-inflammatory cytokines in the mouse AD model. DETAILED DESCRIPTION

[0051] 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.

[0052] Example 1: Identification and preservation of strains Strain 18 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. 31277.

[0053] Colony morphology of strain 18 ( 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.

[0054] The 16S rRNA gene sequence of strain 18 is shown in SEQ ID NO: 1:

[0055] The phes gene sequence of strain 18 is shown in SEQ ID NO: 2:

[0056] The cell morphology and physicochemical test results of strain 18 are shown in Table 1.

[0057] Table 1 Cell morphology and physicochemical test results of strain 18

[0058] Note: "+" represents a positive result, and "-" represents a negative result.

[0059] Example 2: Safety evaluation of strains The safety of the strain was evaluated by hemolytic assay and antibiotic sensitivity assay.

[0060] (1) Hemolytic test Take 10 μL of the bacterial solution of strain 18 and drop it on Columbia blood agar plate containing 5% sheep blood, set Staphylococcus aureus (ATCC 6538) as the positive control, Lactobacillus plantarum (D8) as the negative control, LB liquid medium as the blank control, and culture at 37℃ for 48 h to observe the hemolysis of the strain on the plate. Figure 2 As shown, strain 18 is not hemolytic.

[0061] (2) Antibiotic sensitivity analysis The disk diffusion method was used to evaluate the antibiotic sensitivity of strain 18. The results are shown in Table 2, and strain 18 was sensitive to all four commonly used clinical antibiotics.

[0062] Table 2 Antibiotic susceptibility test

[0063] Note: S stands for sensitive.

[0064] Example 3: Preparation of strain fermentation supernatant A single colony of strain 18 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 aspirated to measure the OD value of bacteria at a wavelength of 600 nm. The growth curve of strain 18 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 18 for later use.

[0065] Example 4: Experiment on the inhibition of growth of Propionibacterium acnes by strains The double-layer plate method was used to evaluate the effect of the fermentation supernatant of strain 18 on the growth of Propionibacterium acnes.

[0066] Propionibacterium acnes (ATCC 6919) was selected as the test strain. This strain is type IA, which is the main type that causes acne.

[0067] A reinforced Clostridium difficile (RCM) solid culture medium containing 2% agar was used as the bottom layer of the double-layer plate. A sterilized Oxford cup was placed on it after solidification. A 5% Propionibacterium acnes liquid was added to the RCM culture medium which had not yet solidified at about 50°C and poured on the bottom layer. After solidification, the Oxford cup was clipped out to form the top layer of the double-layer plate with Oxford cup holes.

[0068] 0.2 mL of the fermentation supernatant of strain 18 was injected into the well of the Oxford cup, 0.5% triclosan (CHX) solution was set as the positive control, and PBS buffer solution was set as the negative control, and the culture was kept at 37°C for 48 h. Figure 4 As shown, strain 18 significantly inhibited the growth of Propionibacterium acnes, and the diameter of the inhibition zone was about 22±0.1 mm.

[0069] Example 5: Experiment on the inhibition of Propionibacterium acnes biofilm formation by strains Propionibacterium acnes was inoculated into RCM liquid medium at a 2% inoculum, and 10%-30% (v / v) fermentation supernatant of strain 18 was added. The culture was anaerobically cultured at 37℃ for 48 h in 96-well or 24-well plates to form a P. acnes biofilm at the bottom of the well or on the cell slide. 0.5% triclosan (CHX) solution was set as a positive control, and MRS medium was set as a negative control. Crystal violet staining, scanning electron microscopy and confocal microscopy were used to evaluate the inhibitory effect of the fermentation supernatant of strain 18 on the P. acnes biofilm.

[0070] 1. Crystal violet staining The biomass of P. acnes biofilm was determined by crystal violet staining. After the biofilm was rinsed with PBS three times, it was stained with 0.1% crystal violet at room temperature for 20 min, and the excess unbound dye was washed away with PBS. Then, the biofilm was decolorized with 95% ethanol for 30 min, and gently shaken until no crystal violet was released from the biofilm. The OD value at 570 nm was measured using an enzyme reader. The results are shown in Table 3. The inhibition rate of the fermentation supernatant of strain 18 on P. acnes biofilm = (0.995-0.199) / 0.995×100%=80%, which was better than the positive group.

[0071] Table 3 Crystal violet assay for Propionibacterium acnes biofilm

[0072] 2. Scanning electron microscopy observation The morphology of Propionibacterium acnes cells in the biofilm was observed by scanning electron microscopy. The biofilm was rinsed with PBS three times, 1.5 mL of 2.5% glutaraldehyde was added, and it was stored at 4°C overnight. It was dehydrated with gradient ethanol (50%, 60%, 70%, 80%, 90%, 95%, 100%), dried at room temperature, and fixed by spraying gold for 30 s. The morphological changes of Propionibacterium acnes were observed by scanning electron microscopy at 10 kV. The results are shown in Figure 2. Figure 5 As shown, the fermentation supernatant of strain 18 inhibited the growth of P. acnes in the biofilm and shortened the long axis of the rod-shaped cells.

[0073] 3. Laser Confocal Microscopy Observation The working solution was prepared using the LIVE / DEAD™ Cell Viability Kit and Dextran, Alexa Fluor™ 647 instructions, and SYTO 9, propidium iodide (PI) and Dextran, Alexa Fluor™ 647 were added to the well plate. 500 μL of the mixed working solution was stained and incubated for 15 min in a light-proof environment. The biofilm slide was then placed on a dedicated slide for a laser confocal microscope to observe the distribution of bacteria in the dark. The results are shown in Figure 2. Figure 6As shown, PI dye stains dead bacteria in red, and SYTO-9 dye stains live bacteria in green. The fermentation supernatant of strain 18 inhibited the formation of P. acnes biofilm.

[0074] Example 6: Experiment on the inhibition of lipase activity of Propionibacterium acnes by strains Propionibacterium acnes (1×10 8 CFU / mL) and the fermentation supernatant of strain 18 were co-cultured at 37°C under anaerobic conditions for 48 h. 0.5% triclosan (CHX) solution was set as a positive control and MRS medium was set as a negative control. The supernatant was centrifuged and the lipase activity was detected using a lipase assay kit. The results are shown in Figure 7 As shown (* p<0.05, ** p<0.01, compared with the negative group), strain 18 significantly inhibited the activity of Propionibacterium acnes lipase (p<0.05).

[0075] Example 7: Experiment on the inhibition of virulence genes of Propionibacterium acnes by strains Propionibacterium acnes (1×10 8 CFU / mL) and the fermentation supernatant of strain 18 were co-cultured at 37°C under anaerobic conditions for 48 h. 0.5% triclosan (CHX) solution was set as a positive control, MRS medium was set as a negative control, and Lactobacillus plantarum ( Lactobacillus plantarum )L16 (CN202410351722.5, collection number: CGMCC No.28163) fermentation supernatant was used as the experimental control group.

[0076] After the culture was completed, the supernatant was centrifuged and the total RNA was extracted using the TIANGEN kit and reverse transcribed into cDNA. Real-time quantitative PCR was used to detect the virulence genes of Propionibacterium acnes: biofilm formation encoding genes ( cbiL, roxP, btuR ), lipase encoding genes ( PPA2105, PPA1761 , PPA1796 ). GAPDH was used as the internal reference and 2 -△△Ct The relative expression level of each gene was calculated by the method.

[0077] The results are as follows Figure 8 As shown in Figure 2 (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared with the negative group), the fermentation supernatant of strain 18 can significantly down-regulate the virulence genes of Propionibacterium acnes cbiL, roxP, btuR, PPA2105 , PPA1761 and PPA1796 The expression level of virulence genes was significantly downregulated by 43% to 74% (p < 0.05). roxP and PPA1796(p<0.05), but for virulence genes LqCy , btuR, PPA2105 and PPA1761 No significant inhibition was observed, indicating that strain 18 has a wider range of inhibition on the virulence genes of Propionibacterium acnes.

[0078] Example 8: Experiment on the effect of strains on rat acne model A rat auricle acne model was established, and the fermentation supernatant of strain 18 was used for intervention. The therapeutic effect of strain 18 on acne was evaluated by measuring the auricle thickness, the mRNA level of pro-inflammatory cytokines in auricle tissue, and the protein level of pro-inflammatory cytokines in auricle tissue.

[0079] 1. Establishment and intervention of rat auricle acne model The rats were first adapted for 1 week. Male SD rats, 200±20 g, were used. On the first day of modeling, 100% oleic acid solution was applied once to the opening of the ear tube on the inner side of the left ear of the rat, 0.1 mL each time, and 50 μL of inactivated Propionibacterium acnes solution (1×10 8 CFU / mL), modeling for 8 days.

[0080] The experiment was divided into model group, negative group, positive group, and strain 18 group (18), with 6 rats in each group. The intervention drug in the positive group was adapalene gel, and the negative control group was intervened with normal saline. After the model was successfully established, the intervention began on the 9th day of the experiment. The negative control group, positive group, and strain 18 group were evenly applied with 200 μL of normal saline, adapalene gel, and fermentation supernatant of strain 18 once a day on the left ear for 8 consecutive days.

[0081] 2. Rat ear thickness During the measurement, the rats were anesthetized using an animal anesthesia machine, and the thickness of the rats' left ears was measured using a vernier caliper before modeling, after modeling, 4 days after drug administration, and 8 days after drug administration. The measurements were taken three times at different locations. The measurement results are shown in Table 4. After intervention with the fermentation supernatant of strain 18, the auricular cysts in rats significantly subsided, and the subsidence rate was better than that of the positive group drug.

[0082] Table 4 Changes in the thickness of the auricle in rats

[0083] 3. Gene expression of pro-inflammatory cytokines in rat auricle tissue The rat auricle tissue was homogenized, total RNA was extracted, and reverse transcribed into cDNA. qRT-PCR was used to detect the mRNA expression levels of acne-related proinflammatory cytokines TNF-α, IL-1β, and IL-6. GAPDH was used as an internal reference. -△△CtThe relative expression level of each gene was calculated by the method.

[0084] The results are as follows Fig. 9 As shown (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, compared with the model group), strain 18 significantly downregulated the gene expression levels of acne-related proinflammatory cytokines TNF-α, IL-1β and / or IL-6 (p<0.05), down-regulated by 63%~71%.

[0085] ELISA detection of pro-inflammatory cytokine protein levels in rat auricle tissue (1) ELISA detection a. Preparation before testing: Take the TNF-α kit as an example.

[0086] 1) Take the kit out of the refrigerator 20 minutes in advance to equilibrate to room temperature. 2) Dilute the 20× concentrated washing solution to 1× working solution with double distilled water.

[0087] 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℃.

[0088] 2) Set up standard wells, blank wells and sample wells. Add 50 μL of standard of different concentrations to each standard well.

[0089] 3) Add 50 μL of the sample to be tested to the sample well; do not add anything to the blank well.

[0090] 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.

[0091] 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.

[0092] 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).

[0093] 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.

[0094] (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 value (OD450) at 450 nm was measured by the above ELISA method. The regression equation of the standard curve was fitted, and the OD450 value of each test sample was substituted into the equation to calculate the content of TNF-α, IL-1β and IL-6 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 content of each sample group measured in the above test and the blank control group, and p<0.05 was considered statistically significant.

[0095] The results are as follows Fig.10 As 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 downregulated the protein levels of acne-related proinflammatory cytokines TNF-α, IL-1β and / or IL-6 (p<0.05), with a decrease of 13%~32%.

[0096] Example 9: Experiment on the effect of strains on the expression level of pro-inflammatory cytokine genes in HaCaT cells 1. Test Article Preparation Stimulant: Accurately weigh 5 mg LPS, fully dissolve it in 5 mL PBS, and prepare a LPS stock solution with a mass concentration of 1 mg / mL. Take 100 μL and dispense it into a 0.5 mL EP tube and store it in a -80℃ refrigerator. Dilute it with cell culture medium to a mass concentration of 500 μg / mL as a stimulant before use.

[0097] Experimental group (18): fermentation supernatant of strain 18 prepared according to Example 3.

[0098] 2. Cell Culture 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%, adherent cells were digested and passaged with trypsin, and the culture medium was changed 2-3 times a week.

[0099] 3. Detection of pro-inflammatory cytokine gene expression levels in HaCaT cells When the cell confluence reached 80% to 90%, the cell suspension was obtained using the above cell passage method and the cell density was adjusted to 2 × 10 5 / mL, added to a 24-well plate, 1000 μL of cell suspension per well, and continued to be placed in an incubator (37°C, 5% CO2 by volume) for 12 h. When the cell plating rate in the well plate meets the experimental requirements, the old culture medium in the 24-well plate is first aspirated. Except for the blank group, LPS (500 μg / mL) is added to the complete culture medium in the experimental group and the model group to a final concentration of 20 μg / mL, and the incubation is continued for 12 h. Subsequently, 50 μL of strain 18 fermentation supernatant was added to each well of the experimental group, and the blank group and the model group were cultured for 12 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 1000g for 3 min.

[0100] 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.

[0101] The results are as follows Fig.11 As 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 downregulated the expression levels of proinflammatory cytokines IL-6, IL-8 and / or IL-17A genes in HaCaT cells (p<0.05), down by 29%~63%.

[0102] Example 10: Experiment on the inhibition of cell autophagy by bacterial strains 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 18) for 2 h. The punctate distribution of GFP-LC3 (autophagosome formation) was observed using an inverted fluorescence microscope.

[0103] The results are as follows Fig.12 As shown, the fermentation supernatant of strain 18 could significantly inhibit starvation-induced autophagosome formation, indicating that it has certain anti-inflammatory and anti-tumor effects.

[0104] Example 11: Experiment on the effect of strains on mouse dermatitis model A mouse atopic dermatitis model was established, and the fermentation supernatant of strain 18 was used for intervention. The gene and protein levels of pro-inflammatory cytokines in skin tissue were measured to evaluate the therapeutic effect of strain 18 on dermatitis.

[0105] 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.

[0106] The experiment was divided into model group, negative group, positive group, and strain 18 group (18), with 6 mice in each group. The intervention drug in the positive group was mometasone furoate cream, and the intervention drug in the negative group was normal saline. The intervention began on the 7th day of the experiment. The negative group, positive group, and strain 18 group were evenly applied with 200 μL normal saline, mometasone furoate cream, and fermentation supernatant of strain 18 once on the left ear at 15:00 every day, and the application continued for 21 days.

[0107] 2. qRT-PCR detection of mRNA expression levels of TNF-α, IL-1β, and IL-6 in mouse back tissue The dorsal tissue of mice was homogenized, total RNA was extracted, and reverse transcribed into cDNA. qRT-PCR was used to detect the mRNA expression levels of acne-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.

[0108] The results are as follows Fig.13 As 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 downregulated the expression levels of atopic dermatitis-related proinflammatory cytokines TNF-α, IL-1β and / or IL-6 genes (p<0.05), down by 41%~63%.

[0109] 3. ELISA detection of TNF-α, IL-1β, and IL-6 protein levels in mouse back tissue Refer to the ELISA detection and calculation method of Example 8. The results are as follows Fig.14As 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 downregulated the protein levels of atopic dermatitis-related proinflammatory cytokines TNF-α, IL-1β and / or IL-6 (p<0.05), with a decrease of 11%~26%.

[0110] Example 12: Antioxidant capacity test of bacterial strains 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.

[0111] 1. Hydroxyl free radical scavenging ability test The ABTS free radical scavenging ability test kit (Beijing Box Biotechnology Co., Ltd.) was used for determination. The hydroxyl free radical scavenging rate was calculated as follows: Hydroxyl free radical scavenging rate D% = [(A measurement - A control) ÷ (A blank - A control)] × 100%.

[0112] The calculation results showed that the hydroxyl radical scavenging ability of strain 18 was 78.63%.

[0113] 2. ABTS free radical scavenging ability test Refer to the instructions of the Hydroxyl 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 measurement - A control)] ÷ A blank × 100%.

[0114] The calculation results showed that the ABTS free radical scavenging ability of strain 18 was 25.48%.

[0115] The above experimental results show that strain 18 has good antioxidant capacity. Strain 18 can be used to prepare antioxidant products, such as medicines, skin care products or cosmetics with antioxidant and free radical scavenging functions.

[0116] Example 13: Experimental study on the inhibition of tyrosinase activity by strains PBS phosphate buffer (pH 6.8), 40 μL of 10% strain 18 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 18 fermentation supernatant + 40 μL tyrosinase + 40 μL L-dopa / tyrosine; D: 120 μL PBS + 40 μL strain 18 fermentation supernatant + 40 μL L-dopa / tyrosine; Tyrosinase inhibition rate (%) = [(AB)-(CD)] / (AB)×100%.

[0117] The results are shown in Table 5. The fermentation supernatant of strain 18 has a good inhibitory effect on tyrosinase activity, indicating that it has a whitening effect. Strain 18 can be used to prepare whitening products, such as medicines, skin care products or cosmetics with whitening function.

[0118] Table 5 Inhibition rate of strain 18 on tyrosinase activity

[0119] In summary, the plant lactobacillus 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, so it has anti-acne, anti-dermatitis, anti-oxidation, and whitening effects. The plant lactobacillus 18 of the present invention or its metabolites can be used to prepare products with the above-mentioned effects or functions.

[0120] 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. 31277.

2. Use of the plant lactobacillus described in claim 1 in the preparation of anti-acne, anti-dermatitis, antioxidant and / or whitening products.

3. Use of the plant lactobacillus according to claim 1 in preparing a product for inhibiting Propionibacterium acnes.

4. The plant lactobacillus of claim 1 in the preparation of a method for down-regulating the virulence gene of Propionibacterium acnes cbiL, roxP, btuR, PPA2105 , PPA1761 and / or PPA1796 The invention relates to a product for reducing the expression level of genes encoding proinflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and / or IL-17A.

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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