CN114672442A - ProfMIC-211 of probiotics and application - Google Patents
ProfMIC-211 of probiotics and application Download PDFInfo
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- CN114672442A CN114672442A CN202210485444.3A CN202210485444A CN114672442A CN 114672442 A CN114672442 A CN 114672442A CN 202210485444 A CN202210485444 A CN 202210485444A CN 114672442 A CN114672442 A CN 114672442A
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- lactobacillus plantarum
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention relates to the technical field of microorganisms, in particular to probiotic ProfMIC-211 and application thereof. The invention provides a Lactobacillus plantarum ProfMIC-211 and application thereof, and the preservation number is CCTCC NO: M20211568. The strain ProfMIC-211 has functions of maintaining and repairing skin barrier, and treating acne and sinusitis, and can be used in food, medicine, cosmetic, etc.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to probiotic ProfMIC-211 and application thereof.
Background
The skin is the largest organ in the human body, the total weight accounts for about 16% of the body weight of an individual, and the skin is the first defense line for maintaining the stability of the body and resisting the invasion of external adverse factors. Studies have shown that skin diseases are induced if the external environment causes abnormalities in the relevant genes in the skin barrier.
The skin barrier is a structural barrier formed by the epidermal keratinocytes of the stratum corneum and the lipids between the cutin. The skin barrier prevents the release of excess water from the human body and prevents harmful substances such as chemicals or microorganisms from entering our body. The corneocyte cortex, which constitutes the surface of dead keratinocytes, plays an important role in the stability of intercellular lipids. Skin barrier damage can cause skin dryness, skin aging, atopic dermatitis, eczema, psoriasis, ichthyosis, skin sensitivity such as solar optical dermatitis, irritant dermatitis, and hormone dependent dermatitis, and seborrheic diseases such as acne, rosacea, and seborrheic dermatitis.
The probiotic is used in cosmetics, and can balance skin epidermal flora and repair skin barrier. As reported in the literature, the topical application of probiotics can treat atopic dermatitis, promoting the repair of the skin barrier. Meanwhile, researches show that the probiotics can prevent and treat various inflammatory diseases by regulating the immune function and phagocytic capacity of macrophages, can effectively increase the absorption of skin on nutrient substances, resist free radicals and enhance the immunity of the skin.
Acne is a common chronic inflammatory skin disease, and is closely related to factors such as excessive sebum secretion, blockage of pilosebaceous ducts, bacterial infection and inflammatory reaction. Research has shown that Propionibacterium acnes (Propionibacterium acnes), considered as the main pathogenic bacterium causing acne, can induce and activate the initial loop of acne inflammation and convert glycerol into fatty acids, leading to an inflammatory response; simultaneously, protease, hyaluronidase and chemotactic factor are produced, so that the hair follicle is hyperkeratotic and acne is formed. On the other hand, Staphylococcus epidermidis (Staphylococcus epidermidis) which is resident on the skin and Propionibacterium acnes can compete with each other in an antagonistic manner, and the increase in the number of Staphylococcus epidermidis can inhibit the proliferation of Propionibacterium acnes. Therefore, reducing the flora ratio of propionibacterium acnes/staphylococcus epidermidis can effectively relieve acne inflammation, thereby maintaining skin health by regulating the skin micro-ecological balance.
Nasosinusitis is a common disease in otorhinolaryngology, and is mainly caused by respiratory tract allergy, nasal-paranasal sinus mucosa cleaning dysfunction, respiratory tract microbial infection, abnormal local anatomical structure of nose and the like. Researches show that Pseudomonas aeruginosa (Pseudomonas aeruginosa) is an important pathogenic bacterium of nasosinusitis, and Lactobacillus sakei (Lactobacillus sakei) has a positive effect on maintaining normal functions of nasal cavities. Therefore, the ratio of the pseudomonas aeruginosa to the lactobacillus saka flora is reduced, and the treatment of the nasosinusitis is facilitated.
Therefore, it is of great practical significance to provide probiotics that can prevent and treat various inflammatory diseases.
Disclosure of Invention
In view of the above, the invention provides the probiotic ProfMIC-211 and the application thereof, which can promote the repair of skin barriers, resist inflammation and treat acne and nasosinusitis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, an extract and/or a metabolite thereof, and the preservation number is CCTCC NO: M20211568.
In some embodiments of the invention, the Lactobacillus plantarum (Lactobacillus plantarum) described above is live or dead or is tyndallized, or is in the form of a lysate and/or extract, or is in the form of a bacterial product or is in the form of a supernatant or in the form of a derivative, including: metabolites, metabolic biologicals, prebiotics, cell walls and components thereof, exopolysaccharides, and compounds containing immunogenic components including: supernatant and inactivated bacteria.
The invention also provides application of the Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, an extract and/or a metabolite thereof in preparation of a product for adjusting flora, inflammation and/or allergy.
The invention also provides application of the Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, an extract and/or a metabolite thereof in preparation of products for repairing skin barriers, improving skin conditions, treating acne and/or treating nasosinusitis.
The invention also provides application of the Lactobacillus plantarum or a lysate, an extract and/or a metabolite thereof in preparing products for up-regulating skin barrier repair related factor expression, down-regulating cell inflammatory factor expression, reducing inflammatory factor release, inhibiting pathogenic bacteria growth, promoting probiotic proliferation and/or changing flora ratio.
The invention also provides application of the Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, an extract and/or a metabolite thereof in preparation of products for up-regulating the expression of FLG, OVOL1 and/or LOR, down-regulating the expression of IL-8, COX-2, IL-8, TNF-alpha, IFN-gamma and/or IL-4, reducing the amount of nitric oxide produced by cells, inhibiting the growth of Staphylococcus aureus, inhibiting the growth of Staphylococcus hominis, inhibiting the growth of hemolyticus, inhibiting the growth of Corynebacterium xerosis, inhibiting the growth of Propionibacterium acnes, promoting the proliferation of Staphylococcus epidermidis, inhibiting the growth of Pseudomonas aeruginosa and/or promoting the growth of Lactobacillus sake.
In some embodiments of the invention, the Lactobacillus plantarum described above, or a lysate, extract and/or metabolite thereof, reduces the propionibacterium acnes/staphylococcus epidermidis ratio, reduces the pseudomonas aeruginosa/Lactobacillus sake ratio.
In some embodiments of the invention, the product for the above-mentioned application comprises a microbial preparation, a food, a pharmaceutical or a cosmetic.
The invention also provides a microbial preparation which comprises the Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, an extract and/or a metabolite thereof as well as acceptable auxiliary materials and/or auxiliary agents.
In some embodiments of the present invention, the microbial preparation further comprises yeast, probiotic bacillus, clostridium butyricum, lactobacillus, bifidobacterium, actinomycetes, and/or the like.
The invention also provides food, which comprises the Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, an extract and/or a metabolite thereof, and/or the microbial preparation and acceptable auxiliary materials and/or auxiliary agents.
The invention also provides a medicament, which comprises the Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, an extract and/or a metabolite thereof, and/or the microbial preparation and acceptable auxiliary materials and/or auxiliary agents.
The invention also provides cosmetics which comprise the Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, an extract and/or a metabolite thereof, and/or the microbial preparation and acceptable auxiliary materials and/or auxiliary agents.
In some embodiments of the present invention, the above-mentioned adjuvants or adjuvants include a nutrition enhancer, a sweetness modifier, an acidity modifier, an isotonicity modifier, a filler, a binder, a disintegrant, a solubilizer, a cosolvent, a preservative, a flavoring agent, a coloring agent, a suspending agent, a wetting agent, an emulsifier, and/or a surfactant.
The strain of the invention has the following effects:
1. ProfMIC-211 of the present invention was identified as Lactobacillus plantarum (Lactobacillus plantarum) by 16S rRNA.
2. In vitro cell experiments show that the lactobacillus plantarum ProfMIC-211 has the effect of up-regulating the expression of a skin barrier repair related factor silk fibroin gene FLG, a OVO-like transcription factor 1 gene OVOL1 and an lorir of an loricrin gene, and the gene expression level is up-regulated by 1.34-1.48 times.
3. In vitro cell experiments show that the lactobacillus plantarum ProfMIC-211 has the effect of inhibiting the release of inflammatory factors and can reduce the generation amount of Nitric Oxide (NO) of mouse macrophage Raw264.7 induced by Lipopolysaccharide (LPS) by 24.76-27.45%.
4. In vitro cell experiments show that the lactobacillus plantarum ProfMIC-211 has the function of reducing the expression of HaCaT cell inflammation related factor IL-8 and cyclooxygenase-2 gene COX-2 gene induced by staphylococcus aureus, and the gene expression level is reduced by 49.00-68.00%.
5. In vitro experiments show that the lactobacillus plantarum ProfMIC-211 has the function of reducing the ratio of propionibacterium acnes to staphylococcus epidermidis, thereby treating acne.
6. In vitro experiments show that the lactobacillus plantarum ProfMIC-211 has the function of reducing the ratio of pseudomonas aeruginosa to lactobacillus sake, thereby treating nasosinusitis.
7. In vitro experiments show that the lactobacillus plantarum ProfMIC-211 has the function of inhibiting skin pathogenic bacteria, and has the bacteriostatic rate of 37.25-60.52% for staphylococcus aureus, staphylococcus hominis, staphylococcus haemolyticus, corynebacterium xerosis and pseudomonas aeruginosa.
Biological preservation Instructions
Biological material: ProfMIC-211, taxonomic nomenclature: lactobacillus plantarum ProfMIC-211 (Lactobacillus plantarum ProfMIC-211), which was deposited in the China center for type culture Collection at 12 months and 08 days 2021, with the collection addresses: wuhan, Wuhan university; the preservation number is CCTCC NO: M20211568.
The ProfMIC-211 is the strain with the preservation number of CCTCC NO: M20211568.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows that ProfMIC-211 promotes HaCaT cell repair, and supernatant upregulation of repair gene expression;
FIG. 2 shows ProfMIC-211 pathogen inhibition;
FIG. 3 shows that ProfMIC-211 down-regulates the expression of HNEC inflammatory-related factors in nasal mucosal cells.
Detailed Description
The invention discloses probiotic ProfMIC-211 and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the probiotic ProfMIC-211 as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Probiotic related products developed by using micro-ecological technology can effectively solve the problems as described above. The lactobacillus plantarum ProfMIC-211 separated and identified from the perilla delavayi leaves has the effects of repairing skin barriers, resisting inflammation and treating acne and nasosinusitis, and has great market potential in the fields of food, medicines, cosmetics and the like.
The strain is gram-positive and rod-shaped under a microscope; the bacterial colony grows on an MRS plate, can form a round bacterial colony with a smooth and opaque surface, is white and has a regular edge; the strain grows uniformly and turbulently in MRS liquid culture medium, and the strain is white precipitate after long-term storage, and the optimal growth temperature is 37 ℃.
The invention further provides the presence of lactobacillus plantarum ProfMIC mic-211, either live or dead or tyndallized, or in the form of a lysate and/or extract, or in the form of a bacterial product or in the form of a supernatant or in the form of a derivative, preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and components thereof, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: supernatant and inactivated bacteria.
The raw materials and reagents used in the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1: isolation of ProfMIC-211
Sampling in the characteristic perilla leaves of the Korean along with the growing edges. Properly processing a sample, uniformly mixing the sample in normal saline by oscillation, taking a supernatant, streaking the supernatant on an MRS solid plate, culturing the MRS solid plate at a constant temperature of 37 ℃ for 24-48 hours, and then selecting a white colony to repeatedly inoculate and screen until a uniform single colony is obtained, wherein the colony is named as ProfMIC-211.
Gram staining microscopy: the strain ProfMIC-211 is gram-positive and rod-shaped under a microscope; growing on an MRS plate to form white round microcolonies with smooth, mellow and opaque surfaces and regular edges; the strain grows in MRS liquid culture medium in a uniform turbid way, and the strain is white and precipitated after being placed for a long time.
Example 2: nucleic acid identification of ProfMIC-211
1. 16s rRNA Gene sequence analysis:
picking single colony in MRS liquid culture medium, culturing overnight at 37 ℃, centrifuging and collecting thallus, and operating according to the steps of DNA extraction kit. The primers are bacterial universal primers 27F and 1492R, the PCR amplification system is a 50 mu L system, and the pre-denaturation is carried out for 5min at 95 ℃; 15s at 94 ℃, 15s at 57 ℃, 40s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min.
2. Results
The homology comparison (BLASTN) of the PCR product sequencing result with the published standard sequence in GenBank indicates that the strain ProfMIC-211 is Lactobacillus plantarum (Lactobacillus plantarum).
Example 3: ProfMIC-211 gene expression experiment for promoting HaCaT barrier repair
Inoculation of human immortalized keratinocytes HaCaT (5X 10)5cells/well) to 6-well plates and cultured overnight until cells adhere. Adding 5% of strain supernatant and 10% of inactivated bacteria, respectively culturing for 24h, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, adjusting all samples to 1 mu g, carrying out reverse transcription to cDNA, and carrying out qPCR (quantitative polymerase chain reaction) to detect the expression of FLG (Ribose nucleic acid), OVOL1 and LOR (LOR) genes. And (3) taking a treatment group without adding the strain supernatant and the inactivated bacteria as a control, wherein the gene expression amount is 1, and calculating the expression change multiple according to a formula.
The formula: f is 2-ΔΔCT
The results are shown in fig. 1 and table 1:
TABLE 1 expression of the repair genes regulated in supernatant of ProfMIC-211
The results show that FLG, OVOL1 and LOR gene expression are all up-regulated. The addition of ProfMIC-211 was shown to have the effect of promoting skin barrier repair. Results of ProfMIC-211 in promoting skin barrier repair are shown in FIG. 1.
Example 4: bacterial liquid concentration change in bacterial experiments for inhibiting pathogenic bacteria such as staphylococcus aureus by ProfMIC-211
1. Preparing a bacterial liquid of lactobacillus plantarum ProfMIC-211:
culturing the activated lactobacillus plantarum ProfMIC-211 bacterial liquid in an MRS liquid culture medium in an incubator at 37 ℃ for standing culture for 16-18 h, detecting and adjusting OD600Inactivating at 121 deg.C for 30min, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain sample to be tested.
2. Preparing a pathogenic bacterium liquid:
4 pathogenic bacteria: respectively culturing Staphylococcus aureus CGMCC 1.8721, human Staphylococcus aureus CGMCC 1.493, Staphylococcus haemolyticus CGMCC 1.540 and Corynebacterium siccus CGMCC 1.1919 in BHI culture medium at 37 deg.C for 18h, detecting and adjusting OD600＝0.2。
3. Experiment for inhibiting pathogenic bacteria
Adding the inactivated supernatant into pathogenic bacteria at a ratio of 10%, culturing at 37 deg.C for 2 hr to obtain bacterial liquid concentration (OD)600) The percentage reduction was evaluated for the effect on the growth of pathogenic bacteria.
4. The results are shown in fig. 2 and table 2:
TABLE 2 inhibition of ProfMIC-211 against skin pathogens
The result shows that the ProfMIC-211 has an inhibiting effect on staphylococcus aureus, human staphylococcus, staphylococcus haemolyticus and corynebacterium siccatum. The results of the inhibition rate of ProfMIC-211 on pathogenic bacteria such as staphylococcus aureus are shown in figure 2.
Example 5: ProfMIC-211 reduces NO production of Raw264.7 cells
1. ProfMIC-211 bacterial liquid preparation
ProfMIC-211 was cultured overnight in MRS medium and OD was detected600Adjusting the bacterial liquid concentration to OD600After centrifugation, the cells were autoclaved at 121 ℃ for 30min to obtain cells, and the centrifuged supernatant was filtered through a 0.22 μm filter to obtain a supernatant.
2. Raw264.7 cell preparation
Raw264.7 cells were digested and then digested at 2X 105Inoculating to 24-well plate, culturing at 37 deg.C in 5% carbon dioxide incubatorAnd (5) culturing overnight.
3. ProfMIC-211 addition and LPS stimulation
Adding 5% of the supernatant and 10% of the inactivated thallus into Raw264.7 cells which are cultured overnight in different groups, adding 0.5 mu L of LPS solution with the concentration of 0.2 mu g/mu L after 2h to induce Raw264.7 cells to be inflamed, taking the cell culture supernatant after 20h, drawing a standard curve according to the method of the Biyunyan NO detection kit, and calculating the concentration and the inhibition rate of NO in the sample.
4. The results are shown in Table 3:
TABLE 3ProfMIC-211 reduction of NO production by Raw264.7 cells
The results show that ProfMIC-211 has anti-inflammatory effect, can inhibit the NO production of Raw264.7 cells induced by LPS, and compared with an LPS control group, the supernatant is reduced by 27.45 percent, and the inactivated thallus is reduced by 24.76 percent.
Example 6: ProfMIC-211 downregulating expression of HaCaT cell inflammatory factor
1. ProfMIC-211 sample preparation
ProfMIC-211 was cultured overnight in MRS and OD was detected600Adjusting the concentration of the bacterial liquid to OD600After centrifugation, the cells were autoclaved at 121 ℃ for 30min to obtain cells, and the centrifuged supernatant was filtered through a 0.22 μm filter to obtain a supernatant.
2. HaCaT cell preparation
Human immortalized keratinocyte HaCaT cells were digested and then treated at 2X 105cells/well were inoculated into 24-well plates and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. Preparation and addition of staphylococcus aureus
Inoculating Staphylococcus aureus into nutrient broth culture medium, shake culturing at 37 deg.C overnight, and adjusting bacterial liquid concentration to OD with MEM serum-free culture medium600Adding 100 mu L of the suspension into HaCaT cells cultured overnight, stimulating the production of inflammatory factors, removing cell culture medium after 3h, washing with PBS 5 times, and adding 1 mu L of MEM into each wellAnd (5) nutrient base.
4. ProfMIC-211 sample addition
The ProfMIC-211 supernatant was added at 5% to Staphylococcus aureus-stimulated HaCaT cells in 3 duplicate wells per group and incubated overnight.
5. qPCR method for detecting relative expression multiple of cell inflammatory factor mRNA
After the culture medium of the cells is removed, RNA is extracted by using an RNA extraction kit, the concentration and purity of the RNA are detected, all samples are adjusted to 1 mu g, RT-PCR and qPCR are carried out by using a reverse transcription kit and a SYBR Green qPCR kit, a treatment group without adding strain supernatant is used as a control, the gene expression quantity is 1, and the relative expression multiple F of the inflammatory factor gene is calculated.
The formula: f is 2-ΔΔCT
6. The results are shown in Table 4:
TABLE 4ProfMIC-211 downregulating expression of HaCaT cell inflammation-associated factor
The result shows that ProfMIC-211 can down-regulate the expression of HaCaT inflammatory factors IL-8 and COX-2 genes induced by staphylococcus aureus, the expression level of IL-8 is reduced by 68.00%, and the expression level of COX-2 is reduced by 49.00%. Thus, ProfMIC-211 has an anti-inflammatory effect.
Example 7: ProfMIC-211 for treating acne by changing flora ratio
1. Preparing a lactobacillus plantarum ProfMIC-211 bacterial liquid:
culturing the activated lactobacillus plantarum ProfMIC-211 bacterial liquid in an MRS liquid culture medium in an incubator at 37 ℃ for standing culture for 16-18 h, detecting and adjusting OD600Inactivating at 121 deg.C for 30min, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain sample to be tested.
2. Preparing a skin flora bacterial liquid:
culturing Propionibacterium acnes CGMCC 1.5003 and Staphylococcus epidermidis CGMCC 1.4260 in BHI culture medium at 37 deg.C for 18 hr, detecting and adjusting OD600＝0.2。
3. Experiment for influencing growth of skin flora by adding supernatant
Adding the inactivated supernatant into two bacterial solutions at a ratio of 10%, culturing to logarithmic phase, and measuring the concentrations (OD) of the two bacterial solutions600) And calculating the relative concentration of the two bacterial liquids by taking the bacterial liquid without the added supernatant as a control, and evaluating the influence on the growth of the acne-related flora by using the relative concentration ratio of the propionibacterium acnes to the staphylococcus epidermidis.
4. The results are shown in table 5:
TABLE 5ProfMIC-211 Effect on growth of acne-related flora
The results show that ProfMIC-211 has a significant inhibitory effect on Propionibacterium acnes, while the inhibitory effect on Staphylococcus epidermidis is relatively low. ProfMIC-211 can obviously change the proportion of flora related to acne, thereby achieving the purpose of treating acne.
Example 8: ProfMIC-211 for treating nasosinusitis by changing flora ratio
1. Preparing a lactobacillus plantarum ProfMIC-211 bacterial liquid:
culturing the activated lactobacillus plantarum ProfMIC-211 bacterial liquid in an MRS liquid culture medium in an incubator at 37 ℃ for standing culture for 16-18 h, detecting and adjusting OD600Inactivating at 121 deg.C for 30min, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain sample to be tested.
2. Preparing nasal cavity flora bacterial liquid:
culturing Pseudomonas aeruginosa CGMCC 1.1785 in BHI culture medium and Lactobacillus sake in MRS culture medium at 37 deg.C for 18 hr, detecting and adjusting OD600＝0.2。
3. Experiment for influencing growth of nasal flora by adding supernatant
Adding the inactivated supernatant into two bacterial solutions at a ratio of 10%, culturing to logarithmic phase, and measuring the concentrations (OD) of the two bacterial solutions600) The relative concentration of the two types of bacterial suspension was calculated (control: 100%) using the bacterial suspension without the supernatant as a control, and the ratio was calculatedThe relative concentration ratio of pseudomonas aeruginosa/lactobacillus sake is evaluated to influence the growth of the nasal flora.
4. The results are shown in Table 6:
TABLE 6 influence of ProfMIC-211 on the bacterial flora associated with sinusitis
The result shows that the ProfMIC-211 has obvious inhibition effect on pseudomonas aeruginosa and certain proliferation promoting effect on lactobacillus sake. ProfMIC-211 can obviously change the proportion of the flora related to the nasosinusitis, thereby effectively treating the nasosinusitis.
Example 9: ProfMIC-211 down-regulates the expression of HNEC inflammatory factors in nasal mucosa
1. ProfMIC-211 sample preparation
ProfMIC-211 was cultured overnight in MRS and OD was detected600Adjusting the concentration of the bacterial liquid to OD600Centrifuging at 121 deg.C for 30min, inactivating, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain sample to be tested.
2. Preparation of HNEC nasal mucosa cells
The HNEC nasal mucosa cells are digested and then treated with 1 × 106cells/well were plated onto 6-well plates and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. LPS preparation and addition
Culturing HNEC nasal mucosa cells overnight, then replacing a serum-free culture medium, adjusting the concentration of LPS to 100 ng/mu L by using a DMEM serum-free culture medium, adding 500 mu L of LPS into the HNEC nasal mucosa cells per hole, stimulating the cells to generate inflammatory factors, removing the cell culture medium after 6h, and adding 2.5 mu L of DMEM serum-free culture medium again per hole to continue culturing overnight.
4. ProfMIC-211 sample addition
The ProfMIC-211 supernatant was added at 10% to LPS-stimulated HNEC nasal mucosal cells in 3 duplicate wells per group and incubated overnight.
5. qPCR method for detecting relative expression multiple of cell inflammatory factor mRNA
After the culture medium of the cells is removed, RNA is extracted by using an RNA extraction kit, the concentration and purity of the RNA are detected, all samples are adjusted to 1 mu g, RT-PCR and qPCR are carried out by using a reverse transcription kit and a SYBR Green qPCR kit, a treatment group without adding strain supernatant is used as a control, the gene expression quantity is 1, and the relative expression multiple F of the inflammatory factor gene is calculated.
The formula: f is 2-ΔΔCT
6. The results are shown in fig. 3 and table 7:
the results show that ProfMIC-211 can down-regulate the expression level of HNEC nasal mucosa cell inflammatory factors TNF-alpha, IFN-gamma and IL-4 genes induced by LPS (figure 3). Thus, ProfMIC-211 had an anti-inflammatory effect on nasal mucosal cells (table 7).
TABLE 7ProfMIC-211 downregulation of expression of HNEC inflammation-associated factors
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, extract and/or metabolite thereof, characterized in that it has the deposit number CCTCC NO: M20211568.
2. Use of Lactobacillus plantarum (Lactobacillus plantarum) according to claim 1, or a lysate, extract and/or metabolite thereof, for the preparation of a flora-regulating, anti-inflammatory and/or anti-allergic product.
3. Use of Lactobacillus plantarum (Lactobacillus plantarum) or a lysate, extract and/or metabolite thereof according to claim 1 for the preparation of a product for repairing skin barriers, improving skin condition, treating acne and/or treating sinusitis.
4. Use of Lactobacillus plantarum (Lactobacillus plantarum) according to claim 1, or a lysate, extract and/or metabolite thereof, for the preparation of a product for up-regulating skin barrier repair-related factor expression, down-regulating cellular inflammatory factor expression, reducing inflammatory factor release, inhibiting pathogenic growth, promoting probiotic proliferation and/or modifying flora ratio.
5. Use of Lactobacillus plantarum (Lactobacillus plantarum) according to claim 1, or a lysate, extract and/or metabolite thereof, for the preparation of a product for upregulating the expression of cellular FLG, OVOL1 and/or LOR, downregulating the expression of cellular IL-8, COX-2, IL-8, TNF- α, IFN- γ and/or IL-4, decreasing cellular nitric oxide production, inhibiting the growth of Staphylococcus aureus, inhibiting the growth of human Staphylococcus aureus, inhibiting the growth of hemolytic Staphylococcus aureus, inhibiting the growth of Corynebacterium xerosis, inhibiting the growth of Propionibacterium acnes, promoting the proliferation of Staphylococcus epidermidis, inhibiting the growth of Pseudomonas aeruginosa and/or promoting the growth of Lactobacillus sake.
6. Use according to claims 2 to 5, wherein the product comprises a microbial preparation, a food product, a pharmaceutical or a cosmetic product.
7. Microbial preparation, characterized by the presence of Lactobacillus plantarum (Lactobacillus plantarum) according to claim 1, or a lysate, extract and/or metabolite thereof, together with acceptable adjuvants and/or adjuvants.
8. Food product, characterized by having a Lactobacillus plantarum (Lactobacillus plantarum) according to claim 1 or a lysate, extract and/or metabolite thereof, and/or a microbial preparation according to claim 7, together with acceptable adjuvants and/or adjuvants.
9. Medicament, characterized by having a Lactobacillus plantarum (Lactobacillus plantarum) according to claim 1 or a lysate, extract and/or metabolite thereof, and/or a microbial preparation according to claim 7, together with acceptable adjuvants and/or adjuvants.
10. Cosmetic product, characterized by having a Lactobacillus plantarum (Lactobacillus plantarum) or lysate, extract and/or metabolite thereof according to claim 1, and/or a microbial preparation according to claim 7, together with acceptable adjuvants and/or adjuvants.
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