A Lactobacillus fermentum strain, its extracellular vesicles and uses

Abstract

The present invention discloses a Lactobacillus fermentum strain, its extracellular vesicles and uses thereof. The Lactobacillus fermentum strain ( Lactobacillus fermentum ), named C283, is deposited in the China General Microbiological Culture Collection Center, and its microbial deposit number is CGMCC No. 30338. Research shows that this strain has unique dual anti-inflammatory and anti-infective properties, and also has properties such as acid tolerance and bile salt tolerance; in particular, the C283 strain can produce extracellular vesicles with strong immune adjuvant activity and immune enhancement activity. Only by intranasal and ocular instillation of the extracellular vesicles of the C283 strain can chicken mucosal intraepithelial lymphocytes be directly activated, significantly enhancing the mucosal immune response; in particular, the extracellular vesicles of this strain can directly serve as an efficient mucosal delivery carrier for inactivated viruses or antigens, nucleic acid vaccines or mRNA vaccines, and can achieve efficient delivery of inactivated vaccines or antigens to the mucosal immune system through mucosal immunization. The present invention provides a new technical means for the preparation of novel oral vaccines and mucosal delivery vaccines.

Description

Technical Field

[0001] The invention relates to a new strain of Lactobacillus fermentum, and also to the new strain of Lactobacillus fermentum and its exovesicles, as well as uses of the new strain of Lactobacillus fermentum and its exovesicles, and the use of the two in preparing anti-inflammatory and anti-infective preparations and preparing mucosal vaccines or mucosal delivery carriers. The invention belongs to the field of biotechnology. Background Art

[0002] As part of the intestinal flora, probiotics play many positive roles. First, probiotics regulate the pH of the intestinal environment by producing beneficial metabolites such as lactic acid and short-chain fatty acids, thereby inhibiting the growth of harmful bacteria. These metabolites help create an environment that is not conducive to the survival and reproduction of harmful bacteria. Secondly, probiotics can also strengthen the function of the intestinal mucosal barrier, forming a protective barrier to prevent harmful bacteria and toxins from entering the blood circulation and protecting the health of intestinal tissues. Probiotics can promote the repair and growth of mucosal cells, enhance the integrity and barrier function of the mucosal barrier, and thus reduce the absorption of harmful substances. In addition, probiotics enhance the immunity of the host body by regulating the function of the intestinal immune system. Studies have shown that probiotics can stimulate immune cells to increase the production of plasma cells and antibodies, thereby enhancing the activity of immune cells and improving the body's defense against pathogens. This regulatory effect helps to enhance the function of the overall immune system and make the body more resistant and immune. In general, there is a close relationship between probiotics and intestinal flora. By optimizing the structure of the intestinal flora and performing its functions, probiotics can have a positive impact on the health of the host.

[0003] Probiotics have the ability to regulate the body's immune system. Probiotics can directly interact with lymphocytes in the mucosa, thereby regulating and enhancing the activity of mucosal immune cells, promoting their differentiation and function, and improving the immune cells' ability to eliminate pathogens and harmful substances. This effect helps to strengthen the immune system's effective response and enhance the body's defense against external threats.

[0004] In summary, probiotics regulate the immune system and maintain the health of the host through various mechanisms, including regulating the activity of immune cells, regulating the production of cytokines, and maintaining the intestinal barrier.

[0005] Lactobacillus fermentum ( Lactobacillus fermentum ), belongs to the Lactobacillaceae family, Lactobacillus genus. Gram-positive, facultative anaerobic, widely distributed in the gastrointestinal tract of humans and animals, and is a normal flora of the intestines, oral cavity and vagina. Lactobacillus fermentum is a heterotypic fermentative lactobacillus that can metabolize lactose, galactose and other sugars to produce metabolites such as lactic acid, acetic acid, succinic acid, ethanol, etc. Current research has confirmed that Lactobacillus fermentum is a dominant microorganism in traditional fermented dairy products, meat products, soy products, vegetable products and other foods, and plays a unique role in the production and efficacy of fermented foods.

[0006] In recent years, with the continuous development of molecular biology techniques, various physiological and biochemical methods and molecular identification techniques have been used to classify and identify lactic acid bacteria in traditional fermented foods. Scholars have isolated and identified lactic acid bacteria in fermented dairy products collected from different regions such as Tibet, Xinjiang, Yunnan, Gansu, and Sichuan in China. The results show that fermented lactic acid bacteria are the dominant Lactobacilli among them. In the case of fermented vegetables, Lactobacillus fermentum accounts for a large proportion among the Lactobacilli isolated from Sichuan pickles and sauerkraut in China, and traditional fermented vegetables in Iran and Nepal.

[0007] The main functions of Lactobacillus fermentum include: (1) Lactobacillus fermentum has antibacterial activity, can protect the intestine from infection, inhibit the growth of pathogenic bacteria, it can survive in the gastrointestinal environment, and has a strong intestinal cell adhesion ability, which can effectively block the adhesion of pathogenic bacteria (such as Salmonella, Escherichia coli, Staphylococcus aureus, Listeria, etc.) in the intestine. (2) Immunomodulatory activity, activating the immune response, effectively improving the body's immunity and disease resistance. Some studies have pointed out that one of the reasons for the immunomodulatory effect of Lactobacillus is from its cell wall components (such as teichoic acid and peptidoglycan); it has the effect of alleviating allergic reactions, blocking the secretion of IgE and balancing the number of T lymphocyte subsets. (3) Maintaining the balance of the intestinal microecological flora, it can regulate the balance of the microbial flora in the host body, can reach the intestine through oral administration, adsorb well on the epithelial cells of the small intestine, and produce surface active components to prevent the adhesion of harmful bacteria to the intestine, thereby improving the internal system environment of the host, promoting the health of the host, promoting the health of the gastrointestinal digestive system, and reducing the incidence of infectious diseases, especially gastrointestinal infections. (4) Antioxidant effect, Lactobacillus fermentum strains have antibacterial activity against intestinal pathogenic bacteria, high antioxidant activity and antioxidant status in the overall intact cell state. It is mainly manifested in the ability to eliminate Salmonella in the small intestine and liver and the ability to degrade typhoid nodules in the liver; Lactobacillus fermentum can reduce oxidative stress and intestinal inflammation in thrombotic mice.

[0008] As a normal flora in the animal intestine, Lactobacillus fermentum can promote the development of the immune system, regulate the body's immunity, and enhance the immune tolerance of animals. However, due to the lack of in-depth research on the role of Lactobacillus fermentum in fermented foods and its probiotic effects, Lactobacillus fermentum is rarely used as a starter in industrial fermented foods at present. Therefore, as a potential probiotic widely present in traditional foods, Lactobacillus fermentum will have great application prospects. Summary of the Invention

[0009] The object of the present invention is to provide a new strain of Lactobacillus fermentum, extracellular vesicles (EVs) produced by the new strain, and the uses of both in the preparation of anti-inflammatory and anti-infective preparations and in the preparation of mucosal vaccines or mucosal delivery carriers.

[0010] To achieve the above object, the present invention adopts the following technical means:

[0011] The strain of the present invention was isolated from the intestinal mucosa of wild healthy red mountain pigs in the virgin forest of Fuyuan, Jiamusi City, Heilongjiang Province, China. It is a lactic acid strain obtained by inoculating and culturing on MRS, separating and purifying. After identification, it belongs to Lactobacillus fermentum and is named C283. The taxonomic name is Lactobacillus fermentum ( Lactobacillus fermentum ), and it is preserved in the China General Microbiological Culture Collection Center, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing. Its microbial preservation number is CGMCC No. 30338, and the preservation date is April 15, 2024. The microbiological characteristics of this strain are: ①Typical Gram-positive staining, medium to large-sized cells, regular-shaped coccobacilli; aerobic or facultative anaerobic bacteria, growing well on solid and liquid MRS media; ②Having acid tolerance, growing well on MRS at pH 4.0; ③Having bile salt tolerance, surviving and growing in a culture medium containing 50% chicken bile MRS; ④Having unique anti-inflammatory and anti-infective characteristics; ⑤The optimal temperature for culturing this bacterium is 37 °C. The colonies on the MRS medium are milky white, with neat edges, smooth and moist surfaces, raised, opaque medium-sized colonies, and have a sour and fragrant smell.

[0012] Furthermore, the present invention also proposes the uses of the above-mentioned Lactobacillus fermentum strain in the following aspects:

[0013] (1) Use in the preparation of anti-inflammatory and anti-infective microbial preparations;

[0014] (2) Use in the preparation of mucosal immune vaccine preparations;

[0015] (3) Use in the preparation of feeds or drugs for regulating the intestinal flora of animals or promoting animal growth;

[0016] (4) Use in the preparation of highly efficient natural immune adjuvants or preparations for enhancing immunity.

[0017] Among them, preferably, the above-mentioned Lactobacillus fermentum strain has the effects of enhancing mucosal immune response and promoting mucosal secretion of specific antibodies.

[0018] Furthermore, the present invention also proposes an extracellular vesicle of a Lactobacillus fermentum strain, and the extracellular vesicle is extracted from the fermentation broth of the Lactobacillus fermentum.

[0019] Among them, preferably, the outer vesicles are prepared by the following method:

[0020] Inoculate the Lactobacillus fermentum C283 into MRS medium and culture at 37 °C for 48 h. Centrifuge the cultured bacterial solution at 4 °C and 5000 g for 20 min, collect the supernatant, filter it through a 0.45 μm filter membrane, then centrifuge the filtrate at 4 °C and 35000 g for 1 h, and then centrifuge the supernatant at 120000 g for 1 h. Resuspend the precipitate with PBS at pH 8.5, and finally filter it through a 0.45 μm filter. Store the filtrate at -80 °C for later use, which is the outer vesicles of Lactobacillus fermentum C283 strain, named C283-BEVs.

[0021] Among them, preferably, the method further includes a step of purifying the extracted outer vesicles, and the purification is carried out by sucrose density gradient ultracentrifugation.

[0022] Among them, preferably, the specific operation of the purification is as follows:

[0023] (1) Slowly add sucrose solutions with mass concentrations of 60%, 50%, 40%, 30%, and 20% into the centrifuge tube in sequence;

[0024] (2) Mix a 5% sucrose solution with C-283-BEV at a volume ratio of 1:1, and carefully add it along the centrifuge tube wall on the prepared density gradient;

[0025] (3) Centrifuge at 4 °C and 120000 g for 18 h with an ultracentrifuge;

[0026] (4) After ultracentrifugation, sequentially take out the bands formed in the middle of the density gradient solution and put them into centrifuge tubes, dissolve them with PBS respectively, centrifuge again at 4 °C and 120000 g for 2 h, resuspend the precipitate with PBS, and finally filter it through a 0.45 μm filter and store it at -80 °C for later use.

[0027] Furthermore, the present invention also proposes the uses of the outer vesicles of the Lactobacillus fermentum strain in the following aspects:

[0028] (1) Use in the preparation of anti-inflammatory and anti-infective microbial preparations;

[0029] (2) Use in the preparation of mucosal immune vaccine preparations;

[0030] (3) Use in the preparation of feeds or drugs for regulating the intestinal flora of animals or promoting animal growth;

[0031] (4) Use in the preparation of highly efficient natural immune adjuvants or preparations for enhancing immunity;

[0032] (5) Use in the preparation of a mucosal delivery vehicle.

[0033] Among them, preferably, the mucosal delivery vehicle is used to deliver all components or subunit components of antigens from inactivated bacteria or inactivated viruses, nucleic acids or mRNAs.

[0034] Among them, preferably, the outer vesicles of the Lactobacillus fermentum strain have the effects of enhancing mucosal immune responses and promoting mucosal secretion of specific antibodies.

[0035] Furthermore, the present invention also provides a mucosal immune complex, which is obtained by loading the VP2 structural protein of chicken bursal disease virus expressed and purified by Escherichia coli onto the outer vesicles of the Lactobacillus fermentum strain through a Polyjet transfection reagent. The specific loading method includes: (1) fully mixing 20 μL of Polyjet transfection reagent, 80 μL of the outer vesicles of the Lactobacillus fermentum strain, and 500 μL of opti-DMEM medium as solution A; (2) fully mixing 50 μL of VP2 structural protein solution and 500 μL of opti-DMEM medium as solution B; (3) adding solution A to solution B and fully mixing to obtain a successfully loaded mixture, and the obtained mucosal immune complex is named C283-BEVs-VP2.

[0036] Furthermore, the present invention also provides the use of the mucosal immune complex in the following aspects:

[0037] (1) Use in the preparation of anti-inflammatory and anti-infective microbial agents;

[0038] (2) Use in the preparation of mucosal immune vaccine agents;

[0039] (3) Use in the preparation of feeds or drugs for regulating animal intestinal flora or promoting animal growth;

[0040] (4) Use in the preparation of natural immune adjuvants or agents for enhancing immunity.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] The present invention discloses a new strain of Lactobacillus fermentum and its uses. The Lactobacillus fermentum strain is named C283. Research shows that this strain has unique dual anti-inflammatory and anti-infective properties, and also has properties such as acid resistance and bile salt resistance; in particular, the C283 strain can produce extracellular vesicles with strong immune adjuvant activity and immune enhancement activity. Only by intranasal and eye-drop administration of the C283 strain extracellular vesicles C283-BEVs can directly activate chicken mucosal intraepithelial lymphocytes and significantly enhance mucosal immune responses; in particular, the extracellular vesicles C283-BEVs of this strain can directly serve as an efficient mucosal delivery carrier for inactivated viruses or antigens, nucleic acid vaccines or mRNA vaccines, and can achieve efficient delivery of inactivated vaccines or antigens to the mucosal immune system through mucosal immunity. Using the extracellular vesicles C283-BEVs of this strain, mucosal vaccines of novel inactivated vaccines or subunit antigens can be obtained. The Lactobacillus fermentum strain C283 disclosed in the present invention can be used as a probiotic strain for additives in feed and drugs, and adding it to feed or drugs can achieve its functions of enhancing the body's immune response and health care effects, etc. The natural probiotic C283 strain with strong immune enhancement activity and its extracellular vesicles are expected to provide an important tool for developing novel oral vaccines and mucosal delivery vaccines as natural delivery carriers. In particular, Lactobacillus fermentum C283-BEVs are expected to be an efficient mucosal delivery carrier for subunit antigens or nucleic acid vaccines or mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 shows the aerobic culture growth of Lactobacillus fermentum C283 strain on solid media X-gal-MRS, MC and MRS;

[0044] Figure 2 shows the anaerobic culture growth of Lactobacillus fermentum C283 strain on solid media X-gal-MRS, MC and MRS;

[0045] Figure 3 shows the activation effect of Lactobacillus fermentum C283 strain on the immune signaling pathway of J774-Dual™ reporter cell line;

[0046] Among them, A shows the activation of the NF-κB signaling pathway in macrophages by the C283 strain; B shows the activation of the IRF signaling pathway in macrophages by the C283 strain;

[0047] Figure 4 is the electron micrograph showing the morphological characteristics of C283-BEVs observed by transmission electron microscope;

[0048] Among them, the scale bar of Figure A is 500nm; the scale bar of Figure B is 200nm;

[0049] Figure 5Measurement results of the particle size (A) and Zeta potential (B) of C283-BEVs;

[0050] Figure 6 Results of the activation of the NF-κB (A) and IRF signaling pathways (B) of J774-Dual™ macrophages by C283-BEVs;

[0051] Figure 7 Results of the detection of the proliferative activity of PBMCs activated by C283-BEVs;

[0052] Figure 8 Results of the flow cytometry detection of PBMCs activated by C283-BEVs;

[0053] Among them, A shows the difference in the content of CD4+ and CD8+ in peripheral blood mononuclear cells between the C283-BEVs group and the blank control group after immunization; B is the statistical bar chart of the CD8+ / CD4+ T cell ratio between the C283-BEVs group and the blank group after immunization; C is the statistical bar chart of CD4+ and CD8+ in PBMCs between the C283-BEVs group and the blank control group after immunization;

[0054] Figure 9 Results of the detection of the transcriptional level of cytokine mRNA in tracheal IELs activated by C283-BEVs;

[0055] Among them, A-I are IL-1β, IFN-β, IRF 1, CCR 5, IL-10, IL-12p70, IL-12p70 / IL-10 ratio, CD40, and CD80 respectively;

[0056] Figure 10 Transcriptional levels of cytokine mRNA in LPLs and PBMC lymphocyte populations activated by IELs activated by C283-BEVs;

[0057] Among them, A-F are IL-1β, TGF-β, IRF 1, CCR 7, STAT 1 / STAT 5 ratio, and CD80 respectively;

[0058] Figure 11 Transcriptional levels of cytokine mRNA in LPLs and PBMC lymphocyte populations activated by LPLs activated by C283-BEVs;

[0059] Among them, A-E are IL-1β, IRF 1, TBK 1, CD80, and CD40L respectively;

[0060] Figure 12Electron micrograph of C283 - BEVs loaded with subunit VP2 complex observed by transmission electron microscope;

[0061] Figure 13 Schematic diagram of the immunization experiment of C283 - BEVs - VP2 complex by intranasal and ocular instillation in animals;

[0062] Figure 14 ELISA serum antibody titer detection results of sera from animals immunized with C283 - BEVs - VP2;

[0063] Figure 15 Flow cytometry analysis diagram of peripheral blood mononuclear cells (PBMCs) from animals immunized with C283 - BEVs - VP2;

[0064] Among them, A is the flow cytometry detection results of CD4+ and CD8+ in peripheral blood mononuclear cells of the experimental group and the blank control group; B is the statistical histogram of the CD8+ / CD4+ T cell ratio in the C283 - BEVs group and the blank group after immunization; C is the statistical histogram of CD4+ and CD8+ in PBMCs of the C283 - BEVs group and the blank control group after immunization. Detailed implementation manners

[0065] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, the embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and such modifications and substitutions all fall within the protection scope of the present invention.

[0066] Example 1 Isolation and identification of Lactobacillus fermentum strain C283

[0067] 1. Isolation and identification of Lactobacillus fermentum strain C283

[0068] This strain was isolated from the intestinal mucosa of a wild and healthy red mountain wild boar in the virgin forest of Fuyuan, Jiamusi City, Heilongjiang Province, China. A lactic acid strain was obtained after inoculation and cultivation on MRS medium and separation and purification. It was identified as Lactobacillus fermentum, named C283, and taxonomically named Lactobacillus fermentum ( Lactobacillus fermentum) , deposited in the China General Microbiological Culture Collection Center, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing. Its microbial deposit number is CGMCC No. 30338, and the deposit date is April 15, 2024.

[0069] The microbiological characteristics of this strain are as follows: ①Typical Gram-positive, with medium to large-sized, regular-shaped coccobacilli; aerobic or facultative anaerobic bacteria, growing well on solid and liquid MRS media; ②Having acid tolerance and being able to grow well on MRS at pH 4.0; ③Having bile salt tolerance and being able to survive and grow in a culture medium containing 50% chicken bile MRS; ④Having unique anti-inflammatory and anti-infective characteristics; ⑤The optimal temperature for culturing this bacterium is 37°C. The colonies on MRS medium are milky white, with neat edges, smooth and moist surfaces, raised, opaque, medium-sized colonies, and have an acid fragrance.

[0070] Example 2: Culture characteristics of Lactobacillus fermentum C283 strain on solid medium

[0071] 1. Colony characteristics of Lactobacillus fermentum C283 strain on common MRS, X-gal-MRS, and MC media:

[0072] Experimental method: Streak Lactobacillus fermentum C283 on MRS, X-gal-MRS, and MC solid media respectively, and conduct aerobic and anaerobic cultures, and observe the colony morphology of C283.

[0073] Experimental results: As shown in Figure 1 and Figure 2 , Lactobacillus fermentum C283 can grow under both aerobic and anaerobic culture conditions on MRS medium. The colony morphology is that of medium-sized colonies with neat edges, smooth and moist surfaces, raised, opaque, milky white, and having an acid fragrance. When Lactobacillus fermentum C283 strain is anaerobically cultured on X-gal-MRS, the colony morphology is similar to that on MRS medium; when aerobically cultured on X-gal-MRS, the colony morphology is similar to that on MRS medium, and the color is light blue (indicating that Lactobacillus fermentum C283 strain can produce β-glucosidase to decompose the X-gal substrate, making the colonies light blue); when Lactobacillus fermentum C283 strain is aerobically cultured on MC medium, a transparent ring appears around the colonies, indicating that Lactobacillus fermentum C283 strain produces acid metabolites that dissolve calcium carbonate.

[0074] Example 3: Detection of immunomodulatory characteristics of Lactobacillus fermentum C283 strain

[0075] Experimental method: Take out the mouse macrophage reporter cell line stored in the liquid nitrogen tank, immediately place it in a 37 °C water bath to thaw until it becomes semi-solid. Break the bottle mouth, take out the cell solution and put it into a 15 mL centrifuge tube containing growth medium. Centrifuge at 300 g for 5 min, discard the supernatant, resuspend with growth medium and transfer it into a 60 mm culture dish. Then add 5 mL of growth medium and observe the culture in a CO2 incubator at 37 °C. After a period of time, passage the cells. After passage three times or more, perform pressure selection with growth medium containing 5 μg / mL Blasticdin and 100 μg / L Zeocin. After passage culture for three generations or more, until 80%-90% of the cells in the cell plate adhere to the wall, and set aside for use.

[0076] Carry out the interaction between Lactobacillus fermentum strain C283 and the macrophage reporter cell line according to the J774-Dual™ Cells product manual. The specific steps are as follows: (1) Add 500 μL of J774-Dual™ cells (2.8×10 5 -1×10 6 cells / mL) into a 24-well plate. (2) Add 1 μg / mL Pam3CSK4, 1 μg / mL 2',3'-cGAMP and 1 μg / mL LPS into the corresponding wells as positive controls. Then add Lactobacillus fermentum strain C283 (1×10 9 CFU / mL) into the remaining 24-well plates containing cells at three gradients of 5 μL, 10 μL, and 15 μL. Set 3 replicates for the test samples and culture them in a 37 °C, 5% CO2 incubator. After stimulation for 24 h, collect the supernatant.

[0077] Detect the NF-κB signaling pathway and the IRF signaling pathway according to the J774-Dual™ Cells product manual. The specific steps are as follows: (1) According to the Quanti-BlueTM (InvivoGen, France) product manual, add 170 μL of Quanti-BlueTM and 30 μL of the supernatant into a 96-well plate, incubate in a 37 °C, 5% CO2 incubator for 4 - 8 h in the dark, and measure the absorbance at 630 nm using an absorbance microplate reader to detect the expression level of SEAP induced by NF-κB. (2) According to the QUANTI-Luc™ 4 Reagent manual, add 20 μL of the supernatant into an opaque 96-well plate, and put it into a microplate chemiluminescence detector (LB 960) to evaluate the activation level of the interferon regulatory factor (IRF) pathway by monitoring the activity of luciferase.

[0078] Experimental results: The activation of the NF-κB signaling pathway and the IRF signaling pathway in macrophages by strain C283 was verified using the J774-Dual™ macrophage dual-signal reporter cell line (mouse macrophage reporter cell line). The results are as Figure 3 shown in A. By detecting the expression level of SEAP in the supernatant (OD630 nm), it was shown that strain C283 could activate the NF-κB signaling pathway (the red column was extremely significantly higher than the standard agonists (p < 0.0001) compared with the two standard control agonists LPS and Pam3CSK4, indicating that strain C283 could extremely significantly activate the immune response); and as Figure 3 shown in B. By detecting the expression level of luciferase, it was shown that strain C283 could activate the IRF signaling pathway (interferon signaling pathway, the red column was extremely significantly higher than the standard agonists (p < 0.0001) compared with the two standard agonists LPS and 2'3'-cGAMP, indicating that strain C283 could extremely significantly activate the interferon signaling pathway and could activate the anti-infection immune response).

[0079] Example 4 Extraction of exosomes C283-BEVs from Lactobacillus fermentum strain C283

[0080] Experimental method: Lactobacillus fermentum strain C283 was inoculated into MRS medium at 1% (v / v) and cultured at 37 °C for 48 h. The cultured bacterial solution was centrifuged at 5000 g for 20 min at 4 °C, and the supernatant was collected. After filtering with a 0.45 μm filter membrane, the filtrate was centrifuged at 35000 g for 1 h at 4 °C, and then the supernatant was centrifuged at 120000 g for 1 h. The precipitate was resuspended with PBS (pH 8.5) and finally filtered through a 0.45 μm filter. The filtrate was stored at -80 °C for later use, which was the exosomes of Lactobacillus fermentum strain C283, named C283-BEVs.

[0081] The above-extracted C283-BEVs were separated and purified by sucrose density gradient ultracentrifugation. The specific operations are as follows: (1) Sequentially and slowly add sucrose solutions with mass concentrations of 60%, 50%, 40%, 30%, and 20% into a Polyallomer centrifuge tube. (2) Mix a sucrose solution with a mass concentration of 5% and C283-BEVs at a ratio of 1:1, and then carefully add it along the centrifuge tube wall onto the prepared density gradient. (3) Centrifuge at 4 °C and 120,000 g for 18 h using an ultracentrifuge. (4) After ultracentrifugation, sequentially take out the bands formed in the middle of the density gradient solution and place them into Polyallomer centrifuge tubes. Dissolve them with PBS respectively, centrifuge again at 4 °C and 120,000 g for 2 h, resuspend the precipitate with PBS, and finally filter it through a 0.45 μm filter and store it at -80 °C for standby.

[0082] Results: As Figure 4 shown, it is the electron micrograph of C283-BEVs, mostly spherical or oval particles with typical bilayer membrane structures in various forms.

[0083] Example 5 Measurement of the particle size and Zeta potential of exosomes C283-BEVs of Lactobacillus fermentum strain C283

[0084] Experimental method: The exosomes of Lactobacillus fermentum strain C283 (C283-BEVs) obtained in Example 4 were measured for their nanoparticle size using a ZETASIZER Nano ZS / ZS90 (Malvern) instrument, that is, the particle size distribution was evaluated. The zeta potential was measured to optimize the sample stability and shelf life by changing the pH value, ion concentration, ion type, and using additives such as surfactants and polyelectrolytes. For the detailed operations, refer to the operating procedures of Nano ZS / ZS90:

[0085] Particle size measurement: (1) Inject the sample into the particle size sample cell (DTS0012 square sample cell) with a height of 1 cm, avoiding air bubbles. (2) Pretreatment: Dilute the sample 200 times with PBS, and then put it into the instrument for measurement. (3) Set the test conditions of the particle size measuring instrument, start the measurement, and export the data after the measurement is completed.

[0086] Zeta potential measurement: (1) Inject the sample into the potential sample cell (DTS1060C U-shaped capillary sample cell), avoiding air bubbles. (2) Pretreatment: Dilute the sample 200 times with PBS (pH 8.5), and then put it into the instrument for measurement. (3) Set the test conditions of the particle size measuring instrument, start the measurement, and export the data after the measurement is completed.

[0087] Experimental results: The results are as Figure 5As shown, the average particle size Z-Average (r.nm) of C283-BEVs was 116.8 nm, and the main peak was at 143.3 nm (accounting for 50.9%). The particle size distribution range of BEVs was relatively large. The Zeta potential was -15.8 mV. This indicates that C283-BEVs carry negative charges, that is, they can stably exist in the alkaline extracellular vesicle storage solution.

[0088] Example 6 Detection of the immunomodulatory properties of Lactobacillus fermentum C283 extracellular vesicles C283-BEVs

[0089] Experimental method: Take out the cells stored in the liquid nitrogen tank, immediately put them into a 37°C water bath to thaw until semi-solid, break the bottle mouth, take out the cell liquid and put it into a 15 mL centrifuge tube containing growth medium, centrifuge at 300 g for 5 min, discard the supernatant, resuspend with growth medium and put it into a 60 mm Dish, then supplement 5 mL of growth medium, and observe the culture in a CO2 incubator at 37°C. After a period of time, passage the cells. After passage three times or more, use a growth medium containing 5 μg / mL Blasticdin and 100 μg / L Zeocin for pressure selection. After passage culture three times or more, until 80%-90% of the cells in the cell plate adhere to the wall, and set aside for use.

[0090] According to the J774-Dual™ Cells product manual, the interaction between Lactobacillus fermentum C283 extracellular vesicles C283-BEVs (prepared in Example 4) and the macrophage reporter cell line was carried out. The specific steps are as follows: (1) Add 500 μL of J774-Dual™ cells (2.8×10 5 -1×10 6 cells / mL) to a 24-well plate. (2) Add 1 μg / mL Pam3CSK4, 1 μg / mL 2',3'-cGAMP, and 1 μg / mL LPS to the corresponding wells as positive controls. Then add Lactobacillus fermentum C283 extracellular vesicles C283-BEVs to the remaining 24-well plates containing cells at three gradients of 5 μL, 10 μL, and 15 μL. Set 3 replicates for the test samples, and culture them in a 37°C, 5% CO2 incubator. After stimulation for 24 h, collect the supernatant.

[0091] Detect the NF-κB signaling pathway and IRF signaling pathway according to the product manual of J774-Dual™ Cells. The specific steps are as follows: (1) Add 170 μL of Quanti-BlueTM and 30 μL of Lactobacillus fermentum C283-derived extracellular vesicles C283-BEVs to a 96-well plate according to the product manual of Quanti-BlueTM (InvivoGen, France). Incubate in an incubator at 37°C and 5% CO2 for 4-8 h in the dark. Measure the absorbance at 650 nm using an absorbance microplate reader to detect the expression level of NF-κB-induced SEAP. (2) Add 20 μL of Lactobacillus fermentum C283-derived extracellular vesicles C283-BEVs to an opaque 96-well plate according to the QUANTI-Luc™ 4 Reagent manual. Place it in a microplate chemiluminescence detector (LB960) to evaluate the activation level of the interferon regulatory factor (IRF) pathway by monitoring the activity of luciferase.

[0092] Experimental results: As Figure 6 shown, at 4 h, C283-BEVs had already activated the NF-κB signaling pathway compared with the blank control. As the reaction time increased, C283-BEVs continuously activated the NF-κB signaling pathway in macrophages. When the reaction time was 24 h, the activation ability of C283-BEVs was comparable to that of the standard agonists LPS and Pam3CSK (Figure 6A); at 4 h, C283-BEVs significantly activated the IRF signaling pathway compared with the blank control (p<0.0001; Figure 6B), and continuously activated the IRF signaling pathway in macrophages. When the reaction time was 24 h, the activation ability was extremely significantly higher than that of the standard agonist 2',3'-cGAMP (p<0.0001; Figure 6B). In vitro studies have shown that C283-BEVs have strong immune-enhancing activity.

[0093] Example 7 Detect the mucosal immune activation characteristics of C283-BEVs through animal experiments

[0094] Experimental method: Randomly divide 7-day-old SPF chickens into 2 groups, the blank control group and the C283-BEVs group, with 9 chickens in each group. Perform intranasal and ocular mucosal immunization at 7 days old. Each chicken in the experimental group was instilled with 200 μL of C283-BEVs intranasally and ocularly, and each chicken in the blank control group was instilled with 200 μL of PBS intranasally and ocularly. Collect anticoagulated blood at 48 h and isolate and obtain tracheal and Harderian gland tissues.

[0095] 1. Detect the proliferative activity of C283-BEVs in activating peripheral blood mononuclear cells (PBMCs)

[0096] Experimental method: PBMCs isolated from the C283-BEVs group and the PBS blank control group after mucosal immunization of SPF chickens for 48 h were used as the detection objects for the proliferation assay. The Cell Counting Kit-8 (CCK-8) for detecting proliferation activity was used to detect the cell proliferation activity and the activation of PBMCs by the extracted C283-BEVs. The operation was as follows according to the instructions: (1) Add 100 μL of PBMCs cell suspension (usually at a density of 5×10 5 cells / mL) to a 96-well culture plate. (2) Add 5 μL, 10 μL, and 15 μL of three gradients of C283-BEVs to the culture plate respectively, and perform 3 experimental replicates. (3) Then place it in an incubator at 37°C with 5% CO2 for 36 h. (4) After incubation, add 10 μL of CCK-8 detection solution to each well, and then place the culture plate in an incubator at 37°C with 5% CO2 for 4 h. Measure the absorbance at 450 nm using an absorption spectrophotometer.

[0097] Experimental results: As Figure 7 shown, after mucosal immunization for 48 h, the proliferation activity of PBMCs in the C283-BEVs group was extremely significantly higher than that in the blank control group (P < 0.0001). This indicates that immunization with C283-BEVs can significantly improve the proliferation activity of T lymphocytes in PBMCs of SPF chickens, which is beneficial to the body's immune response and anti-infection effect.

[0098] 2. Flow cytometry detection of chicken PBMCs stimulated by C283-BEVs

[0099] Experimental method: After SPF chickens were instilled with C283-BEVs through nasal drops and eye drops, anticoagulated blood was aseptically collected at 48 h, and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium. The specific operation was as follows: (1) Add 2 mL of lymphocyte separation medium and 2 mL of anticoagulated blood in equal proportion to a 15 mL centrifuge tube, and centrifuge at 800 g for 24 min. (2) Aspirate the PBMCs layer and put it into a 15 mL centrifuge tube containing 1640 culture medium, add PBMCs washing solution and then centrifuge at 800 g for 10 min, and wash continuously for 2 times. (3) Resuspend PBMCs with lymphocyte 1640 medium (containing 10% fetal bovine serum FCS), use a cell counter to detect the concentration of PBMCs, and make the total number of cells in the flow cytometry sample 1×10 6cells / mL. (4) Refer to the instruction manual of the purchased flow cytometry CD3+ / CD4+ / CD8+ antibodies to prepare the blank control tube, single-stain control tube and experimental tubes in the dark. (5) After incubating in the dark on a shaker at 4 °C for 30 min, add 1 mL of PBS (containing 2% fetal bovine serum) to wash, centrifuge at 1000 g for 7 min, and repeat the operation 3 times. (6) Finally, to determine the relative content of each T cell subset, use a flow cytometer (A60 Universal, Apogee Flow Systems, UK) to analyze the relative fluorescence intensity of the cells.

[0100] Experimental results: As Figure 8 shown, after immunization, significant changes occurred in the CD4+ and CD8+ contents of peripheral blood mononuclear cells in the experimental group and the blank control group. As Figure 8 shown in A, in the blank control group, the proportion of CD4+ cells was 20.9% (abscissa), and the proportion of CD8+ cells was 14.7% (ordinate); while in the C283-BEVs group, the proportion of CD4+ cells was 10.8% (abscissa), and the proportion of CD8+ cells was 24.7% (ordinate). According to the statistical analysis shown in Figure 8 C, the number of CD4+ immune cells in PBMCs of the C283-BEVs group was significantly lower than that of the blank control group, but the number of CD8+ immune cells was significantly higher than that of the blank control group, and the difference was extremely significant (p < 0.0001); and as Figure 8 shown in the statistical analysis of B, the ratio of CD8+ / CD4+ T cells in the C283-BEVs group (2.287) was extremely significantly higher than that of the blank group (0.703), and the difference was extremely significant (p < 0.001). The above results indicate that mucosal immunization with C283-BEVs significantly enhances the immune response of animals, especially the cellular immune response of chickens.

[0101] 3. Detection of activated mucosal immune response after mucosal immunization with C283-BEVs

[0102] Experimental method: After mucosal immunization, mucosal lymphocyte populations (IELs, LPLs, HGLs) were prepared using a GentleMACS automated tissue processor and the accompanying MACS multi-tissue dissociation kit as the main tools. First, the trachea and Harderian glands of SPF chickens were obtained under aseptic conditions and could be directly separated by pulling with forceps. The separated tissues were placed in 1640 medium (containing 10% fetal bovine serum), and then cut into small pieces using surgical scissors. Referring to the multi-tissue dissociation kit instructions, the fragmented tissues were placed in a tissue dissociation tube, relevant reagents were added and mixed, and then the tissue dissociation tube was inserted upside down on the tissue processor. The corresponding tissue dissociation program was selected and the run button was clicked. The mucosal tissues and Harderian glands were dissociated in a standardized manner, and finally the mucosal tissue lymphocyte populations (IELs, LPLs, HGLs) were obtained, with the cell density maintained at ≥1×10 5 cells / mL. Finally, the cells were cryopreserved using a cell cryopreservation solution for future use.

[0103] Taking the above-separated IELs as the research object, first, the separated IELs were resuspended in 1640 medium (containing 10% fetal bovine serum), and the cell density was adjusted to 2×10 5 cells / mL, and (3) cultured in an incubator at 37°C and 5% CO2 for 24 h. The IELs cell pellet was obtained for the following fluorescence quantitative detection.

[0104] IELs (or HGLs) were used to perform migration experiments using the Tanswell chamber system to stimulate the LPLs and PBMCs lymphocyte populations. First, IELs, LPLs, HGLs, and PBMCs were resuspended in 1640 medium (containing 10% fetal bovine serum), and the cell density was adjusted to 2×10 5 cells / mL, and the operation was carried out according to the Corning® Transwell chamber product instructions: (1) Add 1.5 mL of the LPLs and PBMCs mixed cell suspension to each well in the lower chamber of a 12-well plate. (2) Place the chamber in the 12-well plate, and add 500 μL of the IELs (or HGLs) cell suspension to the Transwell chamber. (3) Culture in an incubator at 37°C and 5% CO2 for 24 h. Keep for future use.

[0105] Using the cells collected indoors as the detection object, the relative expression levels of cytokines produced by activated lymphocyte populations in SPF chickens after mucosal immunization with Fusant-283-BEVs were detected by fluorescence quantitative method, and Table 1 was used as primers respectively. The specific operations are as follows: (1) Operate according to the product instruction manual of the Baypure universal magnetic bead method virus DNA / RNA rapid extraction kit to obtain the RNA of the collected cells. (2) Use the UltraSYBR One Step RT-qPCR Kit to determine the relative fold change of cytokine levels and analyze the results according to the CT value. (The detailed PCR reaction system is shown in Table 2, and the detailed RT-qPCR reaction conditions are shown in Table 3).

[0106]

[0107]

[0108]

[0109] Experimental results: As Figure 9 shown, C283-BEVs can effectively stimulate chicken IELs to produce cytokines IL-1β, IL-10, IL-12p70, IFN-β, IRF 1, and CCR 5. Among them, the levels of IL-1β, IFN-β, IRF 1, and CCR 5 in the C283-BEVs group were extremely significantly higher than those in the blank control group, P < 0.0001 (see Figure 9 A, B, C, D); the level of IL-10 in the C283-BEVs group was extremely significantly lower than that in the blank control group, P < 0.0001 (see Figure 9 E); the level of IL-12p70 in the Fusant-283-BEVs group was extremely significantly higher than that in the blank control group, P < 0.0001 (see Figure 9 F), and the IL-12p70 / IL-10 ratio in the C283-BEVs group was extremely significantly higher than that in the blank control group, P < 0.0001 (see Figure 9 G). C283-BEVs activated the CD80 and CD40 molecules of the lymphocyte population. The results showed that: compared with the blank control group, C283-BEVs extremely significantly increased the expression of CD80 and CD40 in chicken IELs, P < 0.0001 ( Figure 9 H, I); indicating that C283-BEVs extremely significantly activated antigen-presenting cells and cellular immune responses.

[0110] As Figure 10As shown, the IELs activated by C283-BEVs can effectively stimulate the cytokine production of IL-1β, IRF 1, CCR 7, TGF-β, STAT1, and STAT5 in chicken LPLs and PBMC lymphocyte populations (see Figure 10 ). Among them, the levels of IL-1β, IRF 1, CCR7, and TGF-β in the C283-BEVs group were extremely significantly higher than those in the blank control group, with P < 0.0001 (see Figure 10 A, B, C, D), and the STAT 1 / STAT 5 ratio in the C283-BEVs group was extremely significantly higher than that in the blank control group, with P < 0.0001 (see Figure 10 E). Compared with the blank control group, C283-BEVs extremely significantly increased the expression of CD80 in chicken LPLs and PBMC lymphocyte populations, with P < 0.0001 ( Figure 10 F); indicating that C283-BEVs extremely significantly activated antigen-presenting cells and cellular immune responses.

[0111] As Figure 11 shown, the LPLs activated by C283-BEVs can effectively stimulate the cytokine production of IL-1β, IRF 1, and TBK 1 in chicken LPLs and PBMC lymphocyte populations ( Figure 11 ), and the levels in the C283-BEVs group were extremely significantly higher than those in the blank control group, with P < 0.0001 (see Figure 11 A, B, C). Compared with the blank control group, C283-BEVs extremely significantly increased the expression of CD80 and CD40L in chicken HGLs and PBMC lymphocyte populations, with P < 0.0001 ( Figure 11 D, E), indicating that C283-BEVs extremely significantly activated antigen-presenting cells and cellular immune responses.

[0112] In summary, C283-BEVs can effectively activate IELs, LPLs, HGLs, and PBMC lymphocyte populations, increasing the production of the acute inflammatory cytokine IL-1 β, reducing the anti-inflammatory downregulated cytokine IL-10 and the upregulated cytokine TGF-β, upregulating the expression of IL-12p70, IFN-β, IRF 1, TBK 1, and chemokine receptors CCR 5 and CCR 7 for cellular immune responses, and activating the CD40 and CD80 molecules of lymphocyte populations, thereby maintaining immune homeostasis.

[0113] Example 8 C283-BEVs can load subunit VP2 protein to form complex particles

[0114] Experimental method:

[0115] Ultrasonic oscillation loading: Mix C283 - BEVs (diluted with PBS, protein concentration 480 μg / ml) and the VP2 structural protein of chicken bursal disease virus expressed and purified by Escherichia coli (diluted with PBS, protein concentration 680 μg / mL) in a volume ratio of 4:1, and then use an ultrasonic crusher for oscillation (time 1 min 12 s, ultrasound for 3 s, cooling for 3 s, Aim 20%). Repeat the operation 2 times to obtain a mixed solution suspected of successful loading.

[0116] Incubation loading: Mix C283 - BEVs (diluted with PBS, protein concentration 480 μg / ml) and VP2 (diluted with PBS, protein concentration 680 μg / mL) in a volume ratio of 4:1. Pipette several times, and then place it in a 37°C constant temperature oscillator for incubation for 3 h to obtain a mixed solution suspected of successful loading.

[0117] Transfection reagent loading: Refer to the instruction manual of Polyjet transfection reagent: (1) Mix 20 μL of Polyjet transfection reagent, 80 μL of C283 - BEVs (diluted with PBS, protein concentration 480 μg / ml), and 500 μL of opti - DMEM medium thoroughly as solution A. (2) Mix 50 μL of VP2 (diluted with PBS, protein concentration 680 μg / mL) and 500 μL of opti - DMEM medium thoroughly as solution B. (3) Add solution A to solution B and mix thoroughly to obtain a mixed solution suspected of successful loading.

[0118] Send the above - loaded mixed solution to the electron microscopy room of Harbin Veterinary Research Institute for further sample processing, and use an H - 7650 transmission electron microscope to determine whether the loading is successful.

[0119] Experimental results: Loading the VP2 subunit by ultrasonic and incubation methods in C283 - BEVs will cause a large number of BEVs to break and disappear ( Figure 12 A, B) Only after loading the VP2 subunit protein antigen by Polyjet transfection reagent, some C283 - BEVs loaded the VP2 protein, that is, the C283 - BEVs - VP2 complex was obtained ( Figure 12 C).

[0120] Example 9 Evaluation of the immunization effect of intranasal and ocular instillation of C283 - BEVs - VP2 complex

[0121] Experimental method: As Figure 13As shown in the figure, 21-day-old SPF chickens were randomly divided into 4 groups: blank control, VP2 control, VP2-Polyjet (prepared by thoroughly mixing 20 μL of Polyjet transfection reagent, 50 μL of VP2, and 500 μL of opti-DMEM medium) control, and C283-BEVs-VP2 (prepared in Example 11), with 8 chickens in each group. Nasal and ocular immunization was performed at 21 days of age. Each chicken in the experimental group was immunized with 200 μL of VP2 protein, VP2-Polyjet, or C283-BEVs-VP2 by nasal and ocular drops, and each chicken in the blank control group was immunized with 200 μL of PBS by nasal and ocular drops. Serum samples were collected at 1 week and 2 weeks after immunization for IBDV antibody detection. At 2 weeks after immunization, the chickens were sacrificed, anticoagulated blood was collected, and visceral tissues were isolated.

[0122] Experimental results:

[0123] Detection of serum IBDV-ELISA antibody titer:

[0124] Experimental method: The IBDV antibody titer in chicken serum was detected according to the instructions of the ID Screen® Infectious Bursal Disease Virus Indirect ELISA Antibody Detection Kit. The specific steps are as follows: (1) First, on the dilution plate, dilute the sample to be tested 500-fold with Diluent 14 and mix well. (2) Then, add 100 μL of negative control to wells A1 and B1, 100 μL of positive control to wells C1 and D1 on the ELISA reaction plate, and add 100 μL of the diluted sample to be tested to the remaining wells. Cover the ELISA reaction plate and incubate at (21 ± 5°C) for 30 ± 3 min. (3) Discard the liquid in the ELISA plate wells, add 300 μL of Wash Solution (1×) to wash the plate 3 times, then add 100 μL of Enzyme Conjugate (1×) to each well. Cover the ELISA reaction plate and incubate at (21 ± 5°C) for 30 ± 3 min. (4) Discard the liquid in the ELISA plate wells, add 300 μL of Wash Solution (1×) to wash the plate 3 times, then add 100 μL of Substrate Solution (TMB) to each well. Incubate at (21 ± 5°C) for 15 ± 2 min, then add 100 μL of Stop Solution to each well to terminate the reaction, and then read and record the absorbance value at a wavelength of 450 nm. (5) Use the IDSoftTM data analysis software provided by IDvet to calculate the S / P value and titer value and determine the immune status.

[0125] Experimental results: As Figure 14As shown in the figure, the specific IgG of IBDV in the sera of each group was detected 1 week and 2 weeks after immunization according to the instructions of the kit. The results showed that 1 week after intranasal and ocular mucosal immunization, the serum antibodies of the experimental groups were all negative. 2 weeks after immunization, the serum ELISA antibodies of the C283-BEVs, VP2 control, and blank control groups were negative, while the serum antibody titers of 3 / 8 chickens in the C283-BEVs-VP2 immunization group were positive, and the ELISA antibody titer was about 2000.

[0126] Flow cytometry detection of PBMCs activated by C283-BEV-VP2 mucosal immunization:

[0127] Experimental method: After SPF chickens were intranasally and ocularly immunized with C283-BEVs-VP2, anticoagulated blood was aseptically collected 48 h later, and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium. The specific operations were as follows: (1) Add 2 mL of lymphocyte separation medium and 2 mL of anticoagulated blood in equal proportion to a 15 mL centrifuge tube, and centrifuge at 800 g for 24 min. (2) Aspirate the PBMCs layer and put it into a 15 mL centrifuge tube containing 1640 culture medium. After adding PBMCs washing solution, centrifuge at 800 g for 10 min, and wash continuously for 2 times. (3) Resuspend PBMCs with lymphocyte 1640 medium (containing 10% fetal bovine serum FCS), and use a cell counter to detect the concentration of PBMCs to make the total number of cells in the flow cytometry sample 1×10 6 cells / mL. (4) Refer to the instruction manual of the purchased flow cytometry CD3+ / CD4+ / CD8+ antibody to prepare the blank control tube, single stain control tube, and experimental tube in the dark. (5) After incubating in the dark on a shaker at 4°C for 30 min, add 1 mL of PBS (containing 2% fetal bovine FBS serum) to wash, centrifuge at 1000 g for 7 min, and repeat the operation 3 times. (6) Finally, in order to determine the relative content of each T cell subset, a flow cytometer (A60Universal, Apogee Flow Systems, UK) was used to analyze the relative fluorescence intensity of the cells.

[0128] Experimental results: The flow cytometry detection results showed that 2 weeks after immunization, there were differences in the contents of CD4+ and CD8+ in peripheral blood mononuclear cells between the experimental group and the blank control group (Figure 15A). According to statistical analysis, it was found that the number of CD4+ and CD8+ immune cells in PBMCs of the C283-BEVs-VP2 group was higher than that of the blank control group, and the difference was extremely significant (p<0.0001, Figure 15C), the ratio of CD4+ and CD8+ T cells in the mucosal immune C283-BEVs group (0.422) was significantly different from that in the blank group (0.523) (p<0.01, Figure 15 B). It indicates that the mucosal immune C283-BEVs-VP2 complex can significantly activate the systemic immune response.

Claims

1. A strain of Lactobacillus fermentum ( Lactobacillus fermentum ) strain, named C283, is deposited in the General Microbiology Center of China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its microbial accession number is CGMCC No.30338, and the deposit date is April 15, 2024.

2. The use of the fermented lactobacillus strain according to claim 1 in the following aspects: (1) Use in the preparation of anti-inflammatory and anti-infective microbial preparations; (2) Use in the preparation of highly effective natural immune adjuvants.

3. The use according to claim 2, characterized in that The fermented lactobacillus strain has the effect of improving mucosal immune response.

4. A fermented lactobacillus strain extracellular vesicle, characterized in that The exosomes are extracted from the fermentation broth of Lactobacillus fermentum. The Lactobacillus fermentum is named C283, its microbial preservation number is CGMCC No.30338, and the preservation date is April 15, 2024.

5. A method for preparing the extracellular vesicles of the fermented Lactobacillus strain according to claim 4, characterized in that: The method comprises the following steps: The fermented Lactobacillus C283 was inoculated into MRS medium and cultured at 37°C for 48 hours. The culture solution was centrifuged at 5000 g for 20 minutes at 4°C, the supernatant was collected, and then filtered through a 0.45 μm filter membrane. The filtrate was centrifuged at 35000 g for 1 hour at 4°C, and then the supernatant was centrifuged at 120000 g for 1 hour. The precipitate was resuspended with pH 8.5 PBS and finally filtered through a 0.45 μm filter. The filtrate was stored at -80°C for later use, which is the extracellular vesicles of Lactobacillus fermented Lactobacillus C283 strain, named C283-BEVs.

6. The method according to claim 5, characterized in that The method further comprises the step of purifying the extracted exosomes, wherein the purification is performed by sucrose density gradient ultracentrifugation, and the specific operation steps are as follows: (1) Slowly add sucrose solutions with mass concentrations of 60%, 50%, 40%, 30%, and 20% into the centrifuge tube in sequence; (2) Mix 5% sucrose solution and C283-BEVs in a volume ratio of 1:1, and then carefully add them along the wall of the centrifuge tube on the prepared density gradient; (3) Centrifuge at 120,000 g for 18 h at 4°C in an ultracentrifuge. (4) After ultracentrifugation, take out the bands formed in the middle of the density gradient solution and place them in centrifuge tubes one by one. Dissolve them separately with PBS and centrifuge them again at 120,000 g at 4°C for 2 h. Resuspend the precipitate with PBS and finally filter it with a 0.45 μm filter and store it at -80°C for later use.

7. Use of the extracellular vesicles of the fermented Lactobacillus strain according to claim 4 in the following aspects: (1) Use in the preparation of anti-inflammatory and anti-infective drugs; (2) Use in the preparation of natural immune adjuvants; (3) Use in the preparation of a mucosal delivery vector, wherein the mucosal delivery vector is used to deliver the VP2 structural protein of chicken bursal disease virus.

8. The use according to claim 7, characterized in that The extracellular vesicles of the fermented lactobacillus strain have the effect of improving mucosal immune response.

9. A mucosal immune complex, characterized in that The mucosal immune complex is obtained by loading the VP2 structural protein of chicken bursal disease virus expressed and purified by Escherichia coli onto the extracellular vesicle of the fermentation lactobacillus strain according to claim 4 through a Polyjet transfection reagent.

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