Cascade fermentation product related to lactobacillus plantarum VB173 and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a cascade fermentation product related to lactobacillus plantarum VB173 and application of the cascade fermentation product. The invention provides lactobacillus plantarum, a fermentation product obtained by cascade fermentation of the lactobacillus plantarum and cordyceps sinensis fermentation supernatant and application of the fermentation product. The inventor obtains a lactobacillus plantarum strain through self-screening, and the lactobacillus plantarum strain is named as lactobacillus plantarum VB173. Lactobacillus plantarum VB173 and cordyceps sinensis fermentation supernatant are subjected to cascade fermentation, cascade fermentation supernatant and / or cascade fermentation lysate are / is obtained through multi-step fermentation culture and post-treatment, the cascade fermentation product remarkably reduces the content of endotoxin in cordyceps sinensis fermentation filtrate, has an inhibition effect on inflammatory factors caused by lipopolysaccharide (LPS), and can be used for preparing the cordyceps sinensis polysaccharide. Meanwhile, the cascade fermentation product has an obvious inhibition effect on a skin resident strain corynebacterium. The cascade fermentation product obtained by the invention has wide application prospects in the fields of cosmetics, medicines and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a cascade fermentation product related to Lactobacillus plantarum VB173 and an application thereof, and further to a fermentation product obtained by cascade fermentation of Lactobacillus plantarum VB173 and a Cordyceps sinensis fermentation supernatant and an application thereof. Background Art

[0002] Lactobacillus plantarum ( Lactobacillus plantarum ) is a type of lactic acid bacteria with an optimal growth temperature of 30-35°C. It grows in anaerobic or facultative anaerobic environments and is a homofermentative lactic acid bacterium. Lactobacillus plantarum is widely distributed in nature, primarily found in fermented vegetables, fruits, and traditional fermented foods. It is also common in the gastrointestinal tracts of humans and animals. Lactobacillus plantarum is widely used in food production, including kimchi, winemaking, and probiotic preparations. Lactobacillus plantarum can produce the biopreservative lactobacillus, and is therefore also used in aquaculture, animal husbandry, and other industries. As a beneficial intestinal microorganism, Lactobacillus plantarum can occupy and colonize the gastrointestinal tract through competitive inhibition, inhibiting pathogenic bacteria from invading the gastrointestinal tract, regulating the balance of intestinal flora, and also exhibiting immunomodulatory functions. Lactobacillus plantarum has potential anti-inflammatory effects on the skin, which is mainly based on its anti-inflammatory properties in the intestine and some research results specifically on the skin. Based on the anti-inflammatory, immune-modulating, and microbial community-improving properties of Lactobacillus plantarum, it shows potential for application in the prevention and treatment of certain skin diseases.

[0003] Cordyceps sinensis ( Cordyceps sinensis(Berk)Sacc. ), is an entomogenous fungus belonging to the order Clavicipitales, family Clavicipitaceae, genus Cordyceps. Natural Cordyceps sinensis has a variety of pharmacological functions and anti-cancer active substances, and is listed as one of the three major tonics along with ginseng and deer antler. Due to the strict restrictions on growth conditions, Cordyceps sinensis is listed in the "China Biodiversity Red List - Large Fungi Volume" and its protection level is vulnerable. Natural Cordyceps sinensis is expensive and has limited production, which is far from meeting market demand. Artificial Cordyceps powder prepared by fermentation technology has similar chemical composition and pharmacological effects to wild Cordyceps, making up for the serious shortage of wild Cordyceps production. With the rapid development of biotechnology, fermented Cordyceps sinensis powder can be produced on a large scale, effectively reducing the edible cost of Cordyceps.

[0004] However, there are few reports on the fermentation culture of Lactobacillus plantarum using the fermentation products of Cordyceps sinensis and the use of the obtained fermentation products. Summary of the Invention

[0005] The present invention aims to at least partially address one of the technical problems in the related art. To this end, one object of the present invention is to provide a strain of Lactobacillus plantarum, as well as a fermentation product obtained by cascade fermentation of this strain with the fermentation supernatant of Cordyceps sinensis, and its applications. The inventors of this application independently screened and obtained a strain of Lactobacillus plantarum, named Lactobacillus plantarum VB173, which was deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection on September 28, 2022, under the deposit number CGMCC No. 25840. Cascade fermentation of Lactobacillus plantarum VB173 with the fermentation supernatant of Cordyceps sinensis is performed. After multiple fermentation steps and post-processing, a cascade fermentation supernatant and / or cascade fermentation lysate are obtained. This cascade fermentation product significantly reduces the endotoxin content in the Cordyceps sinensis fermentation filtrate and inhibits inflammatory factors induced by lipopolysaccharide (LPS). Furthermore, this cascade fermentation product has a significant inhibitory effect on Corynebacterium, a skin-resident strain (Corynebacterium is one of the skin bacteria that contributes to increased body odor). The cascade fermentation product obtained by the present invention has broad application prospects in cosmetics, pharmaceuticals, and other fields.

[0006] To this end, the first aspect of the present invention provides a plant lactobacillus strain. According to an embodiment of the present invention, the plant lactobacillus strain is plant lactobacillus Lactobacillus plantarum VB173 was deposited in the General Microbiology Center of China Culture Collection Administration on September 28, 2022, with the deposit number CGMCC No.25840.

[0007] The inventors screened out a strain of Lactobacillus plantarum VB173, and used this Lactobacillus plantarum to carry out cascade fermentation with the supernatant of Cordyceps sinensis fermentation to obtain cascade fermentation supernatant and / or cascade fermentation lysate. The cascade fermentation product significantly reduced the endotoxin content in the Cordyceps sinensis fermentation filtrate and had an inhibitory effect on inflammatory factors caused by lipopolysaccharide (LPS). At the same time, the cascade fermentation product had a significant inhibitory effect on the skin-resident strain Corynebacterium.

[0008] A second aspect of the present invention provides a fermentation supernatant. According to an embodiment of the present invention, the fermentation supernatant comprises metabolites of the Lactobacillus plantarum strain described in the first aspect.

[0009] A third aspect of the present invention provides a bacterial suspension. According to an embodiment of the present invention, the bacterial suspension comprises the Lactobacillus plantarum strain described in the first aspect.

[0010] A fourth aspect of the present invention provides a fermentation product. According to an embodiment of the present invention, the fermentation product is obtained by cascade fermentation of the Lactobacillus plantarum strain described in the first aspect and / or the bacterial suspension described in the third aspect with Cordyceps sinensis fermentation supernatant.

[0011] The fermentation product provided by the present invention is obtained by cascade fermentation of Lactobacillus plantarum strain VB173 in Cordyceps sinensis fermentation supernatant. The cascade fermentation product has antibacterial and anti-inflammatory activities, significantly reduces the endotoxin content in the Cordyceps sinensis fermentation filtrate, has an inhibitory effect on inflammatory factors caused by lipopolysaccharide (LPS), and has a significant inhibitory effect on skin-resident strains of Corynebacterium.

[0012] According to an embodiment of the present invention, the fermentation product includes a fermentation supernatant and / or a lysate obtained by cascade fermentation.

[0013] A fifth aspect of the present invention provides a method for reducing the endotoxin content in a Cordyceps sinensis fermentation filtrate. According to an embodiment of the present invention, the method comprises: The fermentation product of the fourth aspect is contacted with the Cordyceps sinensis fermentation supernatant.

[0014] The sixth aspect of the present invention provides use of the Lactobacillus plantarum strain described in the first aspect, the bacterial suspension described in the third aspect, and the fermentation product described in the fourth aspect in reducing the endotoxin content in Cordyceps sinensis fermentation filtrate.

[0015] The seventh aspect of the present invention provides use of the Lactobacillus plantarum strain described in the first aspect, the bacterial suspension described in the third aspect, and the fermentation product described in the fourth aspect in preparing a drug for inhibiting inflammatory factors.

[0016] According to an embodiment of the present invention, the inflammatory factors are released by lipopolysaccharide.

[0017] According to an embodiment of the present invention, the inflammatory factors include IL-6, IL-1β, TNF-α, and IL-8.

[0018] In an eighth aspect, the present invention provides a drug for inhibiting inflammatory factors. According to an embodiment of the present invention, the drug comprises the fermentation product described in the fourth aspect.

[0019] According to an embodiment of the present invention, the inflammatory factors are released by lipopolysaccharide.

[0020] According to an embodiment of the present invention, the inflammatory factors include IL-6, IL-1β, TNF-α, and IL-8.

[0021] The ninth aspect of the present invention provides use of the Lactobacillus plantarum strain of the first aspect, the bacterial suspension of the third aspect, and the fermentation product of the fourth aspect in the preparation of medicines, skin care products, or cosmetics that inhibit Corynebacterium.

[0022] The tenth aspect of the present invention provides a medicine, skin care product or cosmetic for inhibiting Corynebacterium. According to an embodiment of the present invention, the medicine, skin care product or cosmetic comprises the fermentation product of the fourth aspect.

[0023] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention.

[0024] Collection information: Strain name: VB173 Date of deposit: September 28, 2022 Depository: China General Microbiology Center (CGMCC) Deposit number: CGMCC No.25840. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which: Figure 1 An electron micrograph of Lactobacillus plantarum VB173 obtained by screening in Example 1 of the present invention is shown; Figure 2 The results of endotoxin content determination of the Cordyceps sinensis fermentation supernatant group, cascade fermentation supernatant group, and cascade fermentation lysis group in Example 3 of the present invention are shown; Figure 3-Figure 5 The effects of different samples on the production of inflammatory factors IL-6, IL-1β and TNF-α by mouse macrophages are shown respectively. DETAILED DESCRIPTION

[0026] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0027] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0028] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0029] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0030] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0031] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0032] According to a specific embodiment of the present invention, the present invention provides a Lactobacillus plantarum strain, which is Lactobacillus plantarum Lactobacillus plantarum VB173 was deposited in the General Microbiology Center of China Culture Collection Administration on September 28, 2022, with the deposit number CGMCC No.25840.

[0033] According to a specific embodiment of the present invention, the present invention further provides a fermentation supernatant, which includes the metabolites of the aforementioned Lactobacillus plantarum strain.

[0034] It should be noted that the fermentation supernatant mentioned in the present invention refers to the liquid portion obtained by fermenting and culturing the Lactobacillus plantarum strain, then centrifuging and removing solid components such as cells. The fermentation supernatant generally contains organic acids, fatty acids, amino acids, and small molecule peptides.

[0035] According to a specific embodiment of the present invention, the present invention further provides a bacterial suspension, which includes the aforementioned Lactobacillus plantarum strain.

[0036] It should be noted that the bacterial suspension mentioned in the present invention refers to the uniform suspension formed by microbial dispersion in a liquid medium. Bacterial suspension described in the present invention refers in particular to a bacterial suspension containing plant lactobacillus strain VB173. There are no particular restrictions about the liquid medium, and any liquid medium known in the art that can stably survive plant lactobacillus strain VB173 is encompassed within the scope of protection of the present invention.

[0037] According to a specific embodiment of the present invention, the present invention further provides a fermentation product. According to an embodiment of the present invention, the fermentation product is obtained by cascade fermentation of the Lactobacillus plantarum strain described in the first aspect and / or the bacterial suspension described in the third aspect with Cordyceps sinensis fermentation supernatant.

[0038] As used herein, fermentation products refer to various metabolites produced by microorganisms during fermentation. Specifically, the present invention refers to fermentation products obtained by cascade fermentation of the supernatant of Lactobacillus plantarum strain VB173 and Cordyceps sinensis. Fermentation products have a wide range of applications in various fields, including food, pharmaceuticals, and chemicals.

[0039] According to a specific embodiment of the present invention, the fermentation product includes fermentation supernatant and / or lysate obtained by cascade fermentation.

[0040] It should be noted that the lysate mentioned in the present invention refers to the cell contents released by rupturing the cells of Lactobacillus plantarum VB173 after fermentation by a specific process, as well as the metabolites produced during the fermentation process.

[0041] According to a specific embodiment of the present invention, the present invention also provides a method for reducing the endotoxin content in Cordyceps sinensis fermentation filtrate, the method comprising: The fermentation product mentioned above is contacted with the Cordyceps sinensis fermentation supernatant.

[0042] The inventors have found that the fermentation product provided by the present invention can significantly reduce the endotoxin content in the Cordyceps sinensis fermentation filtrate.

[0043] According to a specific embodiment of the present invention, the present invention also provides the use of the aforementioned Lactobacillus plantarum strain, the aforementioned bacterial suspension, and the aforementioned fermentation product in reducing the endotoxin content in Cordyceps sinensis fermentation filtrate.

[0044] According to a specific embodiment of the present invention, the present invention also provides use of the aforementioned Lactobacillus plantarum strain, the aforementioned bacterial suspension, and the aforementioned fermentation product in the preparation of a drug for inhibiting inflammatory factors.

[0045] The inventors have discovered that the fermentation product provided by the present invention can inhibit the release of inflammatory factors.

[0046] According to a specific embodiment of the present invention, the inflammatory factors may be released due to various reasons, including but not limited to being released due to lipopolysaccharide.

[0047] According to a specific embodiment of the present invention, the inflammatory factors include but are not limited to IL-6, IL-1β, TNF-α, IL-8 and the like.

[0048] According to a specific embodiment of the present invention, the present invention also provides a drug for inhibiting inflammatory factors, wherein the drug comprises the aforementioned fermentation product.

[0049] According to a specific embodiment of the present invention, the inflammatory factors may be released due to various reasons, including but not limited to being released due to lipopolysaccharide.

[0050] According to a specific embodiment of the present invention, the inflammatory factors include but are not limited to IL-6, IL-1β, TNF-α, IL-8 and the like.

[0051] According to a specific embodiment of the present invention, the present invention also provides the use of the aforementioned Lactobacillus plantarum strain, the aforementioned bacterial suspension, and the aforementioned fermentation product in the preparation of medicines, skin care products, or cosmetics that inhibit Corynebacterium.

[0052] According to a specific embodiment of the present invention, the present invention also provides a medicine, skin care product or cosmetic for inhibiting Corynebacterium, wherein the medicine, skin care product or cosmetic comprises the aforementioned fermentation product.

[0053] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0054] Example 1 Isolation and Identification of Lactobacillus plantarum 1. Isolation and Purification of Lactobacillus plantarum VB173 (1) Take 0.1 g of healthy human feces, place it in a sterile test tube, add 0.9 ml of sterile water, and shake thoroughly; (2) Dilute the bacterial solution by the doubling dilution method, with a total of 7 gradients: 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ; (3) Take 100 μl of bacterial solution from each gradient and spread it on a sterilized MRS solid plate, then place it in a 37°C incubator for anaerobically incubating for 48 h; (4) Pick a single colony for subculture, streak it onto an MRS solid plate, place it in a 37°C incubator for anaerobically incubating for 48 h, and subculture it five times continuously until the colony morphology is uniform; (5) The obtained single colonies were identified by 16S rRNA. The strains identified as Lactobacillus plantarum were inoculated into MRS liquid culture medium and cultured anaerobically at 37°C for 48 h. (6) Take 100 μl of culture medium and dilute to 10 -3 Then, the cells were spread on MRS plates and cultured anaerobically at 37 °C for 24 h. (7) Pick a single colony and culture it in MRS liquid medium at 37℃ for 24 hours to obtain the target strain. Then store the bacteria in glycerol with a final concentration of 20% and store it at -80℃ for future use.

[0055] 2. Identification of Lactobacillus plantarum VB173 The experiment was conducted according to the Molecular Cloning Guide. Purified bacteria were cultured in MRS medium for 24 hours. One ml of the culture was centrifuged at 12,000 rpm for 3 minutes, and the supernatant was discarded. DNA was extracted using the MAGEN Bacterial Genomic DNA Extraction Kit. The extracted DNA served as a template for 16S rDNA amplification using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, as shown in SEQ ID NO: 1) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′, as shown in SEQ ID NO: 2). The PCR reaction system and procedure were performed according to the instructions for Phusion High-Fidelity DNA Polymerase. PCR products were sequenced by Hangzhou Qingke Biotechnology Co., Ltd.

[0056] The 16S rDNA sequence was compared with the strains in GenBank, and the strain of the present invention was homologous to Lactobacillus plantarum. Lactobacillus plantarum The highest homology was 99%, and it was identified as Lactobacillus plantarum and named Lactobacillus plantarum VB173. It was deposited in the General Microbiology Center of China Culture Collection Administration on September 28, 2022. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25840. The strain morphology is as follows Figure 1 .

[0057] The 16S rDNA sequence of the strain is as follows:

[0058] Example 2 Preparation of cascade fermentation products (1) Preparation of solid culture medium 1% tryptone, 0.5% yeast extract powder, 1% sodium chloride, 2% agar, adjust pH to 6.5, sterilize at 121°C for 30 minutes; (2) Single colony culture Take the Lactobacillus plantarum solution (prepared in Example 1) in the glycerol tube and dilute it and apply it to the solid culture medium of step (1). The coating gradient is 6 times that of the original solution. Incubate anaerobically at 37°C for 24 hours to obtain a single colony on the plate. (3) Seed preparation The seed culture medium is 2% sucrose and 0.5% yeast extract powder, the pH is adjusted to 6.5, and sterilized at 121°C for 30 min. A single colony of Lactobacillus plantarum from step (2) is picked and inoculated into a 500 ml seed bottle, and anaerobically cultured at 37°C for 18 h. (4) Scaled-up fermentation The mature seed liquid from step (3) was introduced into the tank, with a transplanting volume of 2%-4%. The initial tank pressure was controlled at 0.05 MPa, and after bubbling began, the tank pressure was raised to 0.08 MPa. The air ratio was 1:0.8, dissolved oxygen was not stirred, the temperature was 37 °C, and the fermentation time was 24 h. The fermentation tank system was: 2% glycerol, 1.5% yeast extract powder, and the balance was the supernatant of Cordyceps sinensis fermentation. The pH was adjusted to 6.5, and sterilized at 121 °C for 30 min. 600 A value of 15 indicates that the residual glycerol content is less than 10 mg / ml; (5) Preparation of fermentation products Preparation of cascade fermentation supernatant: take the fermentation broth from step (4) above and centrifuge it at 14,000 × g for 10 min, take the supernatant and heat it at 100°C for 20 min, filter it through 0.22 μm for sterilization, collect the filtrate and seal it to obtain the cascade fermentation supernatant; Preparation of cascade fermentation lysate: The bacterial pellet was resuspended in a bacterial cell to water ratio of 1:10 and lysed three times in a homogenizer at 1000 bar and 50 Hz. The lysed fluid was centrifuged at 14,000 × g for 10 min, and the supernatant was collected. 3% mannitol was added, the mixture was stirred evenly, and the supernatant was transferred to a freeze dryer for lyophilization to obtain the cascade fermentation lysate.

[0059] Example 3 Endotoxin test of cascade fermentation products Test sample preparation: The Cordyceps sinensis fermentation supernatant, the cascade fermentation supernatant prepared in Example 2, and the cascade fermentation lysate were prepared into a 0.5 mg / ml sample solution using water for bacterial endotoxin testing.

[0060] Bacterial endotoxin working standard was prepared as a 30 EU / mL stock solution, vortexed on a vortex shaker for 15 minutes, and then serially diluted to create endotoxin standard solutions of varying concentrations. The dilution process is shown in Table 1. Each dilution step required vortexing on a vortex shaker for at least 1 minute.

[0061] Table 1 Endotoxin standard solution preparation system

[0062] Place 24 black microplate strips in a microplate rack. Add 100 μl each of bacterial endotoxin test water, endotoxin standard solution, and the three test samples listed above to the corresponding wells, setting up three replicates. Place the microplate in a preheated fluorescence microplate reader and incubate at 37°C for 10 min. Dissolve the indicated amount of recombinant Factor C and shake well for later use. After the 10-min incubation, remove the microplate and add 50 μl of recombinant Factor C to each well. Place the microplate in a fluorescence microplate reader at 37°C, set the excitation wavelength to 380 nm and the emission wavelength to 440 nm, and read the relative fluorescence units (RFUs) at the start of the reaction (0 min) and at T minutes into the reaction. Shake the plate at medium speed for 15 seconds before each reading.

[0063] To calculate endotoxin concentration, record the relative fluorescence units (RFUs) for each well at the start of the reaction (0 minutes) and at T minutes. Subtract the RFU value at the start of the reaction (0 minutes) from the RFU value at T to obtain the RFU increment (ΔRFU). Subtract the ΔRFU of the negative control well from the RFU value to obtain the net ΔRFU. Construct a standard curve using the formula: lg(Y) = A * lg X + B, where Y is the net ΔRFU, X is the endotoxin concentration, A is the slope, and B is the Y-intercept.

[0064] Endotoxin content in the Cordyceps sinensis fermentation supernatant group, cascade fermentation supernatant group, and cascade fermentation lysis group Figure 2 As shown in the figure, it can be seen from the data that compared with the Cordyceps sinensis fermentation supernatant, the cascade fermentation supernatant and cascade fermentation lysate provided by the present invention have lower endotoxins.

[0065] Example 4 Detection of Corynebacterium Inhibition by Cascade Fermentation Products Determination of antibacterial rate: The samples to be tested were cascade fermentation product supernatant, cascade fermentation lysate, Lactobacillus plantarum fermentation supernatant, Lactobacillus plantarum fermentation lysate, and Cordyceps sinensis fermentation filtrate. The positive control was culture medium without any test sample or drug. The negative control was supplemented with 50 μg / ml ciprofloxacin.

[0066] 1) Sample preparation: Prepare the sample to be tested into a 1 mg / ml sample solution; 2) Culture medium preparation: Mueller-Hinton broth, purchased from Qingdao Hibo Biotechnology, catalog number HB6231; 3) Preparation of colony suspension: Prepare a 24-hour culture of Corynebacterium spp. (Beina Biotech BNCC353961) with a 0.5 McFarland turbidimetric standard. Dilute the suspension 1:100 with MH broth and set aside. 4) Dilution of the test sample and bacterial inoculation: Take 7 sterile test tubes, add 1.6 ml of MH broth to the first tube, and add 1 ml of MH broth to each of the remaining tubes. Add 0.4 ml of the test sample to the first tube and mix well. Then pipette 1 ml into the second tube, mix well, and then pipette 1 ml from the second tube into the third tube. Continuously dilute the sample to the fifth tube, and discard 1 ml from the fifth tube. The sixth tube is a growth control without the test sample. The seventh tube is added with 50 μg / ml ciprofloxacin. At this time, the drug concentrations in each tube are 200, 100, 50, 25, and 12.5 μg / ml, respectively. Then, add 1 ml of the bacterial suspension prepared in step 3) to each tube. The final bacterial concentration in each tube is approximately 5×10 5 CFU / ml; 5) Incubation: Stopper the inoculated dilution tube and incubate in a 37°C incubator for 20 hours. 6) Calculation of inhibition rate: Inhibition rate (%) = (OD value of positive control - OD value of test group) / (OD value of positive control - OD value of negative control) × 100%.

[0067] Table 2 below shows the antibacterial rate of each group of fermentation products against Corynebacterium.

[0068] Table 2 Antibacterial rate of fermentation products

[0069] As shown in Table 2, the Cordyceps filtrate has almost no antibacterial effect, and the cascade fermentation product has a higher antibacterial rate than the single Lactobacillus plantarum fermentation product. The cascade fermentation product has an increased antibacterial rate compared to the Lactobacillus plantarum fermentation product. The cascade fermentation product supernatant and cascade lysis provided by the present invention can inhibit the growth of Corynebacterium.

[0070] Example 5 Anti-inflammatory Experiment of Cascade Fermentation Product Supernatant A mouse macrophage model was established, and lipopolysaccharide (LPS) was used to stimulate mouse macrophage RAW264.7 to produce inflammatory factors to investigate whether the sample could inhibit the secretion of inflammatory factors. LPS was prepared into a stock solution with a concentration of 1 mg / ml using PBS buffer solution, sterilized by filtration with a 0.22 µm filter membrane, and stored in a -20 ℃ refrigerator until use. The working concentration was 10 μg / ml. When RAW264.7 cells grew to a confluence of approximately 90%, the culture supernatant was discarded and the cells were washed three times with PBS buffer. The cells were scraped off with a cell scraper, centrifuged for 5 minutes, and resuspended in 4 ml RPMI1640 medium and counted. RAW264.7 cells in the logarithmic growth phase were taken at 5×10 5 Cells were inoculated into 24-well plates at 100 μg / ml and cultured at 37 °C and 5% CO2 for 24 h. 1 ml of the test sample was added to each well. The model group was added with LPS solution without the test sample, and the negative control group was added with RPMI1640 culture medium without LPS. After 24 h of culture, the content of inflammatory factors in the culture supernatant was determined according to the instructions of the mouse ELISA detection kit for IL-6, TNF-α, and IL-1β (e.g. Figure 3 、 Figure 4 、 Figure 5 Calculate the inhibition rate of each test sample on inflammatory factors: Inhibition rate (%) = 100% - (inflammatory factor content in sample group - inflammatory factor content in negative control group) / (inflammatory factor content in model group - inflammatory factor content in negative control group) × 100%. The results of the fermentation product's inhibition rate on inflammatory factors are shown in Table 3.

[0071] Table 3 Inhibition rate of fermentation products on inflammatory factors

[0072] From the results in Table 3, it can be seen that the supernatant of the cascade fermentation product has an inhibitory effect on inflammatory factors, and the ability of the cascade fermentation product to inhibit inflammation is significantly better than that of the single supernatant of Lactobacillus plantarum fermentation product and Cordyceps sinensis fermentation filtrate.

[0073] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0074] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A Lactobacillus plantarum strain, characterized in that The plant lactobacillus strain is plant lactobacillus Lactobacillus plantarum VB173 was deposited in the General Microbiology Center of China Culture Collection Administration on September 28, 2022, with the deposit number CGMCC No.25840.

2. A fermentation supernatant, characterized in that: The invention relates to a method for preparing a lactobacillus plantarum product.

3. A bacterial suspension, characterized in that The invention comprises the plant lactobacillus strain according to claim 1.

4. A fermentation product, characterized in that The fermentation product is obtained by cascade fermentation of the plant lactobacillus strain according to claim 1 and / or the bacterial suspension according to claim 3 with the Cordyceps sinensis fermentation supernatant.

5. The fermentation product according to claim 4, characterized in that The fermentation product includes a fermentation supernatant and / or a lysate obtained by cascade fermentation.

6. A method for reducing endotoxin content in Cordyceps sinensis fermentation filtrate, characterized in that: The method comprises contacting the fermentation product according to claim 4 or 5 with the Cordyceps sinensis fermentation supernatant.

7. Use of the Lactobacillus plantarum strain according to claim 1, the bacterial suspension according to claim 3, or the fermentation product according to claim 4 or 5 for reducing the endotoxin content in Cordyceps sinensis fermentation filtrate.

8. Use of the Lactobacillus plantarum strain according to claim 1, the bacterial suspension according to claim 3, or the fermentation product according to claim 4 or 5 in the preparation of a medicament for inhibiting inflammatory factors.

9. The use according to claim 8, characterized in that The inflammatory factors are released by lipopolysaccharide.

10. The use according to claim 8, characterized in that The inflammatory factors include IL-6, IL-1β, TNF-α, and IL-8.

11. A drug for inhibiting inflammatory factors, characterized in that: The method comprises the fermentation product according to claim 4 or 5.

12. The drug according to claim 11, characterized in that The inflammatory factors are released by lipopolysaccharide.

13. The drug according to claim 11, characterized in that The inflammatory factors include IL-6, IL-1β, TNF-α, and IL-8.

14. Use of the Lactobacillus plantarum strain according to claim 1, the bacterial suspension according to claim 3, or the fermentation product according to claim 4 or 5 in the preparation of medicines, skin care products, or cosmetics for inhibiting Corynebacterium.

15. A medicine, skin care product or cosmetic for inhibiting Corynebacterium, characterized in that: The method comprises the fermentation product according to claim 4 or 5.

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