Lactobacillus fermentum strain as well as extracellular vesicles and application thereof

Abstract

The invention discloses a lactobacillus fermentum strain as well as extracellular vesicles and application thereof. The lactobacillus fermentum strain is named as C283, is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and has the microbial preservation number of CGMCC No.30338. The lactobacillus fermentum strain can be used for preparing the lactobacillus fermentum. Researches show that the strain has unique anti-inflammatory and anti-infection dual characteristics, and also has the characteristics of acid resistance, cholate resistance and the like; particularly, the C283 strain can generate extracellular vesicles with very strong immunologic adjuvant activity and immunological enhancement activity, and the C283 strain outer vesicles can directly activate chicken mucous membrane epithelium lymphocytes only through nasal dripping and eye dripping, so that mucous membrane immune response is remarkably enhanced; particularly, the strain exovesicle can be directly used as an efficient mucosal delivery carrier for inactivated viruses or antigens, nucleic acid vaccines or mRNA vaccines, and efficient mucosal immune system delivery of the inactivated vaccines or antigens can be achieved in a mucosal immune mode. The invention provides a new technical means for preparing novel oral vaccines and mucous membrane 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 technology, people have used various physiological and biochemical methods and molecular identification techniques 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 of my country, such as Tibet, Xinjiang, Yunnan, Gansu, and Sichuan. The results show that fermentative lactic acid bacteria are the dominant lactic acid bacteria. From the perspective of fermented vegetables, fermentative lactobacilli account for a large proportion of lactic acid bacteria isolated from Sichuan pickles and sauerkraut in my country, and traditional fermented vegetables in Iran and Nepal.

[0007] The main effects of fermented lactobacillus include: (1) fermented lactobacillus has antibacterial activity, which can protect the intestine from infection and inhibit the growth of pathogens. 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, activates immune response, and effectively improves the body's immunity and disease resistance. Studies have pointed out that one of the reasons why lactobacillus exerts its immunomodulatory effect comes from its cell wall components (such as teichoic acid and peptidoglycan); relieves allergic reactions, blocks IgE secretion and balances the number of T lymphocyte subsets. (3) Maintains the balance of intestinal microecological flora. It can regulate the balance of microbial flora in the host. It can reach the intestine through oral administration, adsorb well on the epithelial cells of the small intestine, and produce surfactant components to prevent harmful bacteria from adhering to the intestine, thereby improving the system environment in the host body and promoting the health of the host. It promotes the health of the gastrointestinal digestive system and reduces the incidence of infectious diseases, especially gastrointestinal infections. (4) Antioxidant effect: Lactobacillus fermentum strains have antibacterial activity against intestinal pathogens and have high antioxidant activity and antioxidant status of the overall intact cell state. This 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 intestines of animals, 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 fermentation agent in industrial fermented foods. Therefore, as a potential probiotic widely present in traditional foods, Lactobacillus fermentum will have a greater application prospect. Summary of the invention

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

[0010] In order to achieve the above object, the present invention adopts the following technical means: The strain of the present invention is isolated from the intestinal mucosa of wild healthy Hongshan wild boar in Fuyuan primitive forest, Jiamusi City, Heilongjiang Province, China, and is a lactic acid strain isolated after MRS inoculation, culture, separation and purification. It is identified as fermentation lactobacillus, named C283, and classified as fermentation lactobacillus ( Lactobacillus fermentum ), deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with a microbiological deposit number of CGMCC No. 30338 and a deposit date of April 15, 2024. The microbiological characteristics of this strain are: ① Gram staining is typical positive, the bacteria are medium-large, regular-shaped coccobacilli; aerobic or facultative anaerobic bacteria, growing well on solid and liquid MRS medium; ② It has acid resistance and can grow well on MRS with pH 4.0; ③ It has bile resistance and can survive and grow in culture medium containing 50% chicken bile MRS; ④ It has unique anti-inflammatory and anti-infection properties; ⑤ The optimal temperature for the culture of this bacterium is 37°C, and the colonies on the MRS medium are milky white, with neat edges, smooth and moist surfaces, bulges, opaque medium-sized colonies, and a sour and fragrant taste.

[0011] Furthermore, the present invention also proposes the use of the fermented Lactobacillus strain in the following aspects: (1) Use in the preparation of anti-inflammatory and anti-infective microbial preparations; (2) Use in the preparation of mucosal immune vaccine preparations; (3) Use in the preparation of feed or medicine for regulating animal intestinal flora or promoting animal growth; (4) Use in the preparation of highly effective natural immune adjuvants or preparations for enhancing immunity.

[0012] Among them, preferably, the fermented Lactobacillus strain has the effect of enhancing mucosal immune response and promoting mucosal secretion of specific antibodies.

[0013] Furthermore, the present invention also provides an exovesicle of a fermented Lactobacillus strain, wherein the exovesicle is extracted from the fermentation broth of the fermented Lactobacillus strain.

[0014] Preferably, the exosomes are prepared by the following method: The fermented Lactobacillus C283 was inoculated into MRS medium and cultured at 37°C for 48h. The culture solution was centrifuged at 5000g for 20 min at 4°C, the supernatant was collected, and then filtered through a 0.45 μm filter membrane. The filtrate was centrifuged at 35000g for 1 h at 4°C, and then the supernatant was centrifuged at 120000g for 1 h. 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.

[0015] Preferably, the method further comprises the step of purifying the extracted extracellular vesicles, and the purification is performed by sucrose density gradient ultracentrifugation.

[0016] Wherein, preferably, the purification is specifically performed 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 C-283-BEV in a volume ratio of 1:1, and then carefully add it 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, the bands formed in the middle of the density gradient liquid were taken out one by one and placed in centrifuge tubes. They were dissolved with PBS and centrifuged again at 120,000 g for 2 h at 4°C. The precipitate was resuspended with PBS and finally filtered with a 0.45 μm filter and stored at -80°C for later use.

[0017] Furthermore, the present invention also proposes the use of the Lactobacillus fermentum extracellular vesicles in the following aspects: (1) Use in the preparation of anti-inflammatory and anti-infective microbial preparations; (2) Use in the preparation of mucosal immune vaccine preparations; (3) Use in the preparation of feed or medicine for regulating animal intestinal flora or promoting animal growth; (4) Use in the preparation of highly effective natural immune adjuvants or preparations for enhancing immunity; (5) Use in the preparation of mucosal delivery vehicles.

[0018] Among them, preferably, the mucosal delivery vector is used to deliver all components or subunit components, nucleic acids or mRNA of antigens from inactivated bacteria or inactivated viruses.

[0019] Among them, preferably, the extracellular vesicles of the fermented Lactobacillus strain have the effect of enhancing mucosal immune response and promoting mucosal secretion of specific antibodies.

[0020] Furthermore, the present invention also proposes 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 extracellular vesicles of the fermented Lactobacillus strain through a Polyjet transfection reagent. The specific loading method includes: (1) fully mixing 20 μL of Polyjet transfection reagent, 80 μL of the fermented Lactobacillus strain extracellular vesicles and 500 μL of opti-DMEM culture medium to form liquid A; (2) fully mixing 50 μL of VP2 structural protein solution and 500 μL of opti-DMEM culture medium to form liquid B; (3) adding liquid A to liquid B and mixing thoroughly to obtain a successfully loaded mixed solution, and the obtained mucosal immune complex is named C283-BEVs-VP2.

[0021] Furthermore, the present invention also proposes the use of the mucosal immune complex in the following aspects: (1) Use in the preparation of anti-inflammatory and anti-infective microbial preparations; (2) Use in the preparation of mucosal immune vaccine preparations; (3) Use in the preparation of feed or medicine for regulating animal intestinal flora or promoting animal growth; (4) Use in the preparation of natural immune adjuvants or preparations for enhancing immunity.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a new strain of fermented Lactobacillus and its use. The fermented Lactobacillus strain is named C283. Studies have shown that the strain has unique dual characteristics of anti-inflammatory and anti-infection, and also has characteristics such as acid resistance and bile resistance; in particular, the C283 strain can produce extracellular vesicles with strong immune adjuvant activity and immune enhancement activity. Only nasal drops and eye drops of the C283 strain extracellular vesicles C283-BEVs can directly activate chicken mucosal intraepithelial lymphocytes and significantly enhance mucosal immune response; in particular, the strain extracellular vesicles C283-BEVs can be directly used as an efficient mucosal delivery carrier for inactivated viruses or antigens, nucleic acid vaccines or mRNA vaccines, and can achieve efficient mucosal immune system delivery of inactivated vaccines or antigens by mucosal immunization. Using the strain extracellular vesicles C283-BEVs, new inactivated vaccines or subunit antigen mucosal vaccines can be obtained. The fermented Lactobacillus strain C283 disclosed in the present invention can be used as a probiotic strain for feed and drug additives, and added to feed or drugs to achieve its enhanced immune response and health care effects, etc. The natural probiotic C283 strain with strong immune enhancement activity and its extracellular vesicles are expected to be used as natural delivery carriers to provide important tools for the development of new oral vaccines and mucosal delivery vaccines. In particular, fermented Lactobacillus C283-BEVs are expected to be used as a mucosal immune efficient delivery carrier for subunit antigens or nucleic acid vaccines or mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The aerobic growth of Lactobacillus fermentum C283 strain on solid medium X-gal-MRS, MC and MRS; Figure 2 The anaerobic growth of Lactobacillus fermentum C283 strain on solid medium X-gal-MRS, MC and MRS; Figure 3 The activation effect of Lactobacillus fermentum C283 strain on the immune signaling pathway of J774-Dual™ reporter cell line; Among them, A is the activation of the NF-κB signaling pathway of macrophages by the C283 strain; B is the activation of the IRF signaling pathway of macrophages by the C283 strain; Figure 4 This is the transmission electron microscopy image of the morphological characteristics of C283-BEVs; The scale of Figure A is 500nm; the scale of Figure B is 200nm; Figure 5 The particle size (A) and Zeta potential (B) measurement results of C283-BEVs; Figure 6Results of C283-BEVs activating NF-κB (A) and IRF signaling pathways (B) in J774-Dual™ macrophages; Figure 7 The results of the C283-BEVs-activated PBMCs proliferation activity test; Figure 8 Flow cytometry results of C283-BEVs activating PBMCs; Among them, A is 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 graph of the CD8+ / CD4+ T cell ratio between the C283-BEVs group and the blank group after immunization; C is the statistical bar graph of CD4+ and CD8+ in PBMCs between the C283-BEVs group and the blank control group after immunization; Fig. 9 The results are the results of the detection of cytokine mRNA transcription levels in tracheal IELs activated by C283-BEVs; Among them, AIs were IL-1β, IFN-β, IRF 1, CCR 5, IL-10, IL-12p70, IL-12p70 / IL-10ratio, CD40, and CD80; Fig.10 Transcription levels of cytokine mRNA in lymphocyte populations of activated LPLs and PBMCs for IELs activated by C283-BEVs; Among them, AF were IL-1β, TGF-β, IRF 1, CCR 7, STAT 1 / STAT 5 ratio, and CD80; Fig.11 Transcription levels of cytokine mRNA in lymphocyte populations activated by LPLs and PBMCs for C283-BEVs; Among them, AEs were IL-1β, IRF 1, TBK 1, CD80, and CD40L; Fig.12 Transmission electron microscopy observation of C283-BEVs loading subunit VP2 complex; Fig.13 Schematic diagram of animal immunization experiment with C283-BEVs-VP2 complex via nasal and eye drops; Fig.14 The results of ELISA serum antibody titer test of serum from animals immunized with C283-BEVs-VP2; Fig.15This is a flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) from animals immunized with C283-BEVs-VP2; Among them, A is the flow cytometric detection results of CD4+ and CD8+ in peripheral blood mononuclear cells of the experimental group and the blank control group; B is the statistical bar graph of the CD8+ / CD4+ T cell ratio in the C283-BEVs group and the blank group after immunization; C is the statistical bar graph of CD4+ and CD8+ in PBMCs in the C283-BEVs group and the blank control group after immunization. DETAILED DESCRIPTION

[0024] 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 proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention. Example 1 Isolation and identification of Lactobacillus fermentum C283 strain

[0025] 1. Isolation and identification of Lactobacillus fermentum C283 strain The strain was isolated from the intestinal mucosa of a healthy wild boar from Fuyuan primitive forest in Jiamusi City, Heilongjiang Province, China. It was isolated and cultured by MRS and purified. It was identified as a lactic acid strain of Lactobacillus fermentum and named C283. It was classified as Lactobacillus fermentum ( Lactobacillus fermentum It 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 deposit number is CGMCC No.30338, and the deposit date is April 15, 2024.

[0026] The microbiological characteristics of this strain are: ① Gram staining is typically positive, the bacteria are medium to large, and the morphology is regular; aerobic or facultative anaerobic bacteria, growing well on solid and liquid MRS culture media; ② It has acid resistance and can grow well on MRS with a pH of 4.0; ③ It has bile resistance and can survive and grow in culture medium containing 50% chicken bile MRS; ④ It has unique anti-inflammatory and anti-infection properties; ⑤ The optimal temperature for the cultivation of this bacterium is 37°C, and the colonies on the MRS culture medium are milky white, with neat edges, a smooth and moist surface, raised, opaque medium-sized colonies, and a sour and fragrant smell. Example 2 Cultivation characteristics of Lactobacillus fermentum C283 strain on solid culture medium

[0027] 1. Colony characteristics of Lactobacillus fermentum C283 strain on ordinary MRS, X-gal-MRS and MC culture media: Experimental method: Lactobacillus fermentum C283 was streaked on MRS, X-gal-MRS and MC solid culture media, and cultured aerobically and anaerobicly, respectively, and the colony morphology of C283 was observed.

[0028] Experimental results: Figure 1 and Figure 2 , Lactobacillus fermentum C283 can grow on MRS medium under both aerobic and anaerobic culture conditions. The colony morphology is neatly edged, smooth and moist surface, raised, opaque medium-sized colonies, milky white, and has a sour and fragrant taste. When Lactobacillus fermentum C283 is cultured anaerobically on X-gal-MRS, the colony morphology is similar to that on MRS medium; when cultured aerobically 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 can produce β-glucosidase, decompose X-gal substrate, and make the colony light blue); after aerobic culture of Lactobacillus fermentum C283 on MC medium, transparent rings appear around the colony, indicating that Lactobacillus fermentum C283 produces acid metabolites, which dissolve calcium carbonate. Example 3 Detection of the immunomodulatory properties of Lactobacillus fermentum C283 strain

[0029] Experimental method: Take out the mouse macrophage reporter cell line stored in the liquid nitrogen tank, immediately put it into a 37℃ water bath to thaw to a semi-solid state, break the bottle mouth, take out the cell fluid and put it into a 15 mL centrifuge tube containing growth medium, centrifuge at 300 g for 5 min, discard the supernatant, resuspend it with growth medium and put it into a 60 mm culture dish, add 5 mL growth medium, observe and culture in a CO2 incubator at 37℃, subculture after a period of time, after three or more subcultures, use growth medium containing 5 μg / mL Blasticdin and 100 μg / L Zeocin for pressure selection, subculture for three generations or more, until the cells in the cell plate account for 80%-90% of the cells attached to the wall, set aside.

[0030] The interaction between Lactobacillus fermentum C283 strain and macrophage reporter cell line was carried out according to the J774-Dual™ Cells product manual. The specific steps are as follows: (1) Add 500 μL J774-Dual™ cells (2.8×10 5 -1×10 6(2) 1 μg / mL Pam3CSK4, 1 μg / mL 2',3'-cGAMP, and 1 μg / mL LPS were added to the corresponding wells as positive controls, and Lactobacillus fermentum C283 (1×10 9 CFU / mL) was added to the remaining 24-well plate containing cells in three gradients of 5 μL, 10 μL, and 15 μL. The test samples were set up in triplicate and cultured in a 37°C, 5% CO2 incubator. After 24 h of stimulation, the supernatant was collected.

[0031] The NF-κB signaling pathway and IRF signaling pathway were detected according to the J774-Dual™ Cells product manual. The specific steps are as follows: (1) According to the Quanti-BlueTM (InvivoGen, France) product manual, 170 μL Quanti-BlueTM and 30 μL supernatant were added to a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 4-8 h. The plate was protected from light and the absorbance was measured at 630 nm using an absorbance microplate reader to detect the expression level of NF-κB-induced SEAP. (2) According to the QUANTI-Luc™ 4 Reagent manual, 20 μL supernatant was added to an opaque 96-well plate and placed in a microplate chemiluminescence detector (LB 960) to monitor the activity of luciferase to evaluate the activation level of the interferon regulatory factor (IRF) pathway.

[0032] Experimental results: The J774-Dual™ macrophage dual signal reporter cell line (mouse macrophage reporter cell line) was used to verify the activation of the macrophage NF-κB signaling pathway and the IRF signaling pathway by the C283 strain. Figure 3 As shown in A, by detecting the expression level of SEAP in the supernatant (OD630 nm), it was shown that strain C283 can activate the NF-κB signaling pathway (the red column is significantly higher than the two standard control agonists LPS and Pam3CSK4 (p<0.0001), indicating that strain C283 can significantly activate the immune response); and as Figure 3 As shown in B, by detecting the expression level of luciferase, it is shown that strain C283 can activate the IRF signaling pathway (interferon signaling pathway, the red column is significantly higher than the two standard agonists LPS and 2'3'-cGAMP (p<0.0001), indicating that strain C283 can extremely significantly activate the interferon signaling pathway and activate anti-infection immune response). Example 4 Extraction of extracellular vesicles C283-BEVs of Lactobacillus fermentum C283 strain

[0033] Experimental method: Lactobacillus fermentum C283 strain was inoculated into MRS medium at 1% (v / v) and cultured at 37℃ for 48h. The culture solution was centrifuged at 5000g for 20 min at 4℃, the supernatant was collected, and then filtered with a 0.45 μm filter membrane. The filtrate was centrifuged at 35000g for 1 h at 4℃, and then the supernatant was centrifuged at 120000g for 1 h. The precipitate was resuspended in PBS (pH 8.5), and finally filtered with a 0.45 μm filter. The filtrate was stored at -80℃ for later use, which is the extracellular vesicles of Lactobacillus fermentum C283 strain, named C283-BEVs.

[0034] The extracted C283-BEVs were separated and purified by sucrose density gradient ultracentrifugation. The specific steps were as follows: (1) Slowly add sucrose solutions with mass concentrations of 60%, 50%, 40%, 30%, and 20% into a Polyallomer centrifuge tube. (2) Mix 5% sucrose solution with C283-BEVs in a 1:1 ratio and then carefully add it 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 put them into a Polyallomer centrifuge tube, dissolve them with PBS, centrifuge them again at 120,000 g for 2 h at 4°C, resuspend the precipitate with PBS, and finally filter it with a 0.45 μm filter and store it at -80°C for later use.

[0035] Results: Figure 4 Shown are electron micrographs of C283-BEVs, most of which are spherical or elliptical particles of various shapes with typical double-layer membrane structures. Example 5 Particle size and Zeta potential measurement of Lactobacillus fermentum C283 extracellular vesicles C283-BEVs

[0036] Experimental method: The extracellular vesicles (C283-BEVs) of Lactobacillus fermentum C283 obtained in Example 4 were measured for nanoparticle size using a ZETASIZERNano ZS / ZS90 (Malvern) instrument, i.e., 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 polyelectroactive agents. For detailed operations, refer to the Nano ZS / ZS90 operating procedures: Particle size measurement: (1) Inject the sample into the particle size sample pool (DTS0012 square sample pool) at a height of 1 cm to avoid bubbles. (2) Pre-treatment: 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.

[0037] Zeta potential measurement: (1) Inject the sample into the potential sample cell (DTS1060C U-shaped capillary sample cell), avoiding bubbles. (2) Pre-treatment, dilute the sample 200 times with PBS (pH 8.5), then put it into the instrument for measurement. (3) Set the particle size measuring instrument test conditions, start the measurement, and export the data after the measurement is completed.

[0038] Experimental results: The results are as follows Figure 5 As shown, the average particle size Z-Average (r.nm) of C283-BEVs is 116.8nm, and the main peak is at 143.3 nm (accounting for 50.9%). The particle size distribution range of BEVs is relatively large. The Zeta potential is -15.8 mV. This indicates that C283-BEVs carry a negative charge, that is, they can stably exist in an alkaline extracellular vesicle storage solution. Example 6 Detection of the immunomodulatory properties of Lactobacillus fermentum C283 extracellular vesicles C283-BEVs

[0039] Experimental method: Take out the cells stored in the liquid nitrogen tank, immediately put them in a 37℃ water bath to thaw to a semi-solid state, 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 in a 60 mm dish, add 5 mL growth medium, observe and culture in a CO2 incubator at 37℃, subculture after a period of time, after three or more subcultures, use growth medium containing 5 μg / mL Blasticdin and 100μg / L Zeocin for pressure selection, subculture for three generations or more, until the cells in the cell plate are 80%-90% attached to the wall, set aside.

[0040] The interaction between Lactobacillus fermentum C283 extracellular vesicles C283-BEVs (prepared in Example 4) and macrophage reporter cell lines was carried out according to the J774-Dual™ Cells product manual. The specific steps are as follows: (1) 500 μL J774-Dual™ cells (2.8×10 5 -1×10 6cells / mL). (2) 1 μg / mL Pam3CSK4, 1 μg / mL 2',3'-cGAMP and 1 μg / mL LPS were added to the corresponding wells as positive controls, and then Lactobacillus fermentum C283 extracellular vesicles C283-BEVs were added to the remaining 24-well plates containing cells in three gradients of 5 μL, 10 μL and 15 μL. The test samples were set up in triplicate and cultured in a 37°C, 5% CO2 incubator. After 24 h of stimulation, the supernatant was collected.

[0041] The NF-κB signaling pathway and IRF signaling pathway were detected according to the J774-Dual™ Cells product manual. The specific steps are as follows: (1) According to the Quanti-BlueTM (InvivoGen, France) product manual, 170 μL Quanti-BlueTM and 30 μL Lactobacillus fermentum C283 extracellular vesicles C283-BEVs were added to a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 4-8 h. The plate was protected from light and the absorbance was measured at 650 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, 20 μL Lactobacillus fermentum C283 extracellular vesicles C283-BEVs were added to an opaque 96-well plate and placed in a microplate chemiluminescence detector (LB960) to monitor the activity of luciferase to evaluate the activation level of the interferon regulatory factor (IRF) pathway.

[0042] Experimental results: Figure 6 As shown, at 4 h, C283-BEVs activated the NF-κB signaling pathway compared with the blank control. As the reaction time increased, C283-BEVs continued to activate the NF-κB signaling pathway of macrophages. When the reaction lasted for 24 h, C283-BEVs had the same activation ability as 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 6 B), and continued to activate the IRF signaling pathway of macrophages. When the reaction lasted for 24 h, the activation ability was significantly higher than that of the standard agonist 2',3'-cGAMP (p<0.0001; Figure 6 B). In vitro studies have shown that C283-BEVs have strong immune enhancement activity. Example 7 Detection of mucosal immune activation properties of C283-BEVs through animal experiments

[0043] Experimental methods: 7-day-old SPF chickens were randomly divided into 2 groups, a blank control group and a C283-BEVs group, with 9 chickens in each group. Nasal drops and eye drops were performed for mucosal immunization at 7 days of age. Each chicken in the experimental group was given 200 μL C283-BEVs in the nose and eyes, and each chicken in the blank control group was given 200 μL PBS in the nose and eyes. Anticoagulated blood was collected at 48 h, and trachea and Harderian gland tissues were isolated and obtained. 1. Detection of the proliferation activity of peripheral blood mononuclear cells (PBMCs) activated by C283-BEVs

[0044] Experimental method: PBMCs isolated from the C283-BEVs group and PBS blank control group 48 h after SPF chicken mucosal immunization were used as the detection objects of proliferation test. Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation activity and the activation of PBMCs by extracted C283-BEVs was detected. The operation was performed according to the instruction manual as follows: (1) 100 μL of PBMCs cell suspension (generally with a density of 5×10 5 cells / mL). (2) Add 5 μL, 10 μL, and 15 μL of three gradients of C283-BEVs to the culture plate, and repeat the experiment three times. (3) Then place the plate in a 37℃ 5% CO2 incubator and incubate for 36 h. (4) After the incubation time, add 10 μL of CCK-8 detection solution to each well, and then place the plate in a 37℃ 5% CO2 incubator for 4 h. Measure the absorbance at 450 nm using an absorption spectrophotometer.

[0045] Experimental results: Figure 7 As shown in the figure, 48 hours after mucosal immunization, the proliferation activity of PBMCs in the C283-BEVs group was significantly higher than that in the blank control group (P < 0.0001). This indicates that immunization with C283-BEVs can significantly increase the proliferation activity of T lymphocytes in SPF chicken PBMCs, which is beneficial to the body's immune response and anti-infection effect. 2. Flow cytometric detection of chicken PBMCs stimulated by C283-BEVs

[0046] Experimental method: After SPF chickens were given C283-BEVs by nose and eye drops, anticoagulated blood was collected aseptically 48 hours later, and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium. The specific operation was as follows: (1) 2 mL of lymphocyte separation medium and anticoagulated blood were added to a 15 mL centrifuge tube in equal proportions, and the tube was centrifuged at 800 g for 24 min. (2) The PBMCs layer was aspirated and placed in a 15 mL centrifuge tube containing 1640 culture medium. After adding PBMCs washing solution, the tube was centrifuged at 800 g for 10 min, and washed twice in succession. (3) PBMCs were resuspended in lymphocyte 1640 culture medium (containing FCS 10% fetal bovine serum), and the concentration of PBMCs was detected using a cell counter, so that the total cell number of the flow cytometry sample was 1×10 6 cells / mL. (4) Refer to the instructions for use of the purchased flow cytometry CD3+ / CD4+ / CD8+ antibody to prepare blank control tubes, single staining control tubes and experimental tubes in the dark. (5) After incubation in a shaker at 4°C in the dark for 30 min, add 1 mL PBS (containing 2% peptide bovine FBS serum) for washing, 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, the relative fluorescence intensity of the cells was analyzed using a flow cytometer (A60 Universal, Apogee Flow Systems, UK).

[0047] Experimental results: Figure 8 As shown in Figure 2, after immunization, the content of CD4+ and CD8+ in peripheral blood mononuclear cells in the experimental group and the blank control group changed significantly, as shown in Figure 2. Figure 8 As shown in A, the blank control group had a CD4+ cell ratio of 20.9% (horizontal axis) and a CD8+ cell ratio of 14.7% (vertical axis); while the C283-BEVs group had a CD4+ cell ratio of 10.8% (horizontal axis) and a CD8+ cell ratio of 24.7% (vertical axis). Figure 8 Statistical analysis shown in C shows that 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 Figure 8 B Statistical analysis showed that the ratio of CD8+ / CD4+T cells in the C283-BEVs group (2.287) was significantly higher than that in the blank group (0.703), and the difference was extremely significant (p<0.001). The above results show that mucosal immunization with C283-BEVs significantly improves the immune response of animals, especially the cellular immune response of chickens. 3. Detection of mucosal immune response activation after mucosal immunization with C283-BEVs

[0048] Experimental method: After mucosal immunization, mucosal lymphocyte populations (IELs, LPLs, HGLs) were prepared, using the GentleMACS automatic tissue processor and the matching MACS multi-tissue dissociation kit as the main tools. First, the trachea and Harderian glands of SPF chickens were obtained by aseptic operation, which can be directly pulled and separated with forceps. The separated tissue was placed in 1640 (containing 10% fetal bovine serum) culture medium and cut into pieces with surgical scissors. Referring to the instructions of the multi-tissue dissociation kit, the broken tissue was placed in the tissue dissociation tube, and the relevant reagents were added to mix. Then the tissue dissociation tube was inserted upside down on the tissue processor, the corresponding program for tissue dissociation was selected, and clicked to run. Standardized tissue dissociation of mucosal tissues and Harderian glands was performed, and finally the mucosal tissue lymphocyte populations (IELs, LPLs, HGLs) were obtained, and the cell density was maintained at ≥1×10 5 cells / mL. Finally, use cell freezing solution and freeze for later use.

[0049] The isolated IELs were used as the research object. First, the isolated IELs were resuspended in 1640 culture medium (containing 10% fetal bovine serum) and the cell density was adjusted to 2×10 5 cells / mL, (3) incubated at 37°C, 5% CO2 for 24 h. Obtain IELs cell pellet for the following fluorescence quantitative detection.

[0050] IELs (or HGLs) were stimulated to perform migration experiments using the Tanswell chamber system. IELs, LPLs, HGLs, and PBMCs were first resuspended in 1640 medium (containing 10% fetal bovine serum) and the cell density was adjusted to 2×10 5 cells / mL, follow the instructions for use of Corning® Transwell chamber products: (1) Add 1.5 mL of LPLs and PBMCs mixed cell suspension to each well of the lower chamber of a 12-well plate. (2) Place the chamber into a 12-well plate and take 500 μL of IELs (or HGLs) cell suspension and add it to the Transwell chamber. (3) Place in a 37°C, 5% CO2 incubator and culture for 24 h. Set aside.

[0051] The cells collected in the lower chamber were used as the detection object, and the relative expression of cytokines produced by the activated lymphocyte population in SPF chickens after mucosal immunization with Fusant-283-BEVs was detected by fluorescence quantitative method, with primers in Table 1. The specific operation is as follows: (1) According to the product instructions of Baypure Universal Magnetic Bead Viral DNA / RNA Rapid Extraction Kit, RNA from the collected cells was obtained. (2) The relative fold change of cytokine levels was determined using UltraSYBR One Step RT-qPCR Kit, and the results were analyzed according to the CT value. (The PCR reaction system is detailed in Table 2, and the RT-qPCR reaction conditions are detailed in Table 3).

[0052]

[0053]

[0054]

[0055] Experimental results: Fig. 9 As shown in Figure 2, 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, IL-1β, IFN-β, IRF 1, and CCR 5 in the C283-BEVs group were significantly higher than those in the blank control group, P < 0.0001 (see Fig. 9 A, B, C, D); IL-10 in the C283-BEVs group was significantly lower than that in the blank control group, P < 0.0001 (see Fig. 9 E); IL-12p70 in the Fusant-283-BEVs group was 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 significantly higher than that in the blank control group, P < 0.0001 (see Fig. 9 G). C283-BEVs activated CD80 and CD40 molecules of lymphocyte populations. The results showed that C283-BEVs significantly increased the expression of CD80 and CD40 in chicken IELs compared with the blank control group, P < 0.0001 ( Fig. 9 H, I); indicating that C283-BEVs significantly activated antigen-presenting cells and cellular immune responses.

[0056] like Fig.10As shown, IELs activated by C283-BEVs can effectively stimulate chicken LPLs and PBMCs lymphocyte populations to produce cytokines IL-1β, IRF 1, CCR 7, TGF-β, STAT1, and STAT5 (see Fig.10 ). Among them, IL-1β, IRF 1, CCR7, and TGF-β in the C283-BEVs group were significantly higher than those in the blank control group, P < 0.0001 (see Fig.10 A, B, C, D), and the STAT 1 / STAT 5 ratio in the C283-BEVs group was significantly higher than that in the blank control group, P < 0.0001 (see Fig.10 E). C283-BEVs significantly increased the expression of CD80 in chicken LPLs and PBMCs lymphocyte populations compared with the blank control group, P < 0.0001 ( Fig.10 F); This indicates that C283-BEVs significantly activated antigen-presenting cells and cellular immune responses.

[0057] like Fig.11 As shown, C283-BEVs activated LPLs can effectively stimulate chicken LPLs and PBMCs lymphocyte populations to produce cytokines IL-1β, IRF 1, TBK 1 ( Fig.11 ), and the C283-BEVs group was significantly higher than the blank control group, P < 0.0001 (see Fig.11 A, B, C), C283-BEVs significantly increased the expression of CD80 and CD40L in chicken HGLs and PBMCs lymphocyte populations compared with the blank control group, P < 0.0001 ( Fig.11 D, E), indicating that C283-BEVs significantly activated antigen-presenting cells and cellular immune responses.

[0058] In summary, C283-BEVs can effectively activate IELs, LPLs, HGLs, and PBMCs lymphocyte populations, increase the production of acute inflammatory cytokine IL-1β, reduce anti-inflammatory down-regulated cytokine IL-10 and up-regulated cytokine TGF-β, up-regulate the expression of IL-12p70, IFN-β, IRF 1, TBK 1 and chemokine receptors CCR 5 and CCR 7 for cellular immune response, and activate CD40 and CD80 molecules of lymphocyte populations, thereby maintaining immune homeostasis. Example 8 C283-BEVs can be loaded with subunit VP2 protein to form complex particles

[0059] Experimental methods: Ultrasonic oscillation loading: C283-BEVs (diluted with PBS, protein concentration of 480ug / ml) and VP2 structural protein of chicken bursal disease virus expressed and purified by Escherichia coli (diluted with PBS, protein concentration of 680μg / mL) were mixed in a volume ratio of 4:1, and then oscillated using an ultrasonic disruptor (time 1 min 12 s, ultrasound 3 s, cooling 3 s, Aim 20%). Repeat the operation twice to obtain a mixed solution that is suspected to be successfully loaded.

[0060] Incubation and loading: Mix C283-BEVs (diluted with PBS, protein concentration of 480ug / ml) and VP2 (diluted with PBS, protein concentration of 680μg / mL) in a volume ratio of 4:1, blow several times with a gun, and incubate in a 37℃ constant temperature oscillator for 3h to obtain a mixed solution suspected to be successfully loaded.

[0061] Transfection reagent loading: Refer to the Polyjet transfection reagent instruction manual: (1) 20 μL Polyjet transfection reagent, 80 μL C283-BEVs (PBS diluted, protein concentration of 480ug / ml) and 500 μL opti-DMEM medium are mixed thoroughly to form solution A. (2) 50 μL VP2 (PBS diluted, protein concentration of 680μg / mL) and 500 μL opti-DMEM medium are mixed thoroughly to form solution B. (3) Add solution A to solution B and mix thoroughly to obtain a mixture that is suspected to be successfully loaded.

[0062] The mixed solution after loading was sent to the electron microscope room of Harbin Veterinary Research Institute for further sample processing, and an H-7650 transmission electron microscope was used to determine whether the loading was successful.

[0063] Experimental results: The loading of VP2 subunits by ultrasound and incubation of C283-BEVs resulted in the breakage and disappearance of a large number of BEVs ( Fig.12 A, B) Only after the VP2 subunit protein antigen was loaded by Polyjet transfection reagent, some C283-BEVs were loaded with VP2 protein, that is, C283-BEVs-VP2 complex was obtained ( Fig.12 C). Example 9 Evaluation of the immune effect of nasal and eye drops of C283-BEVs-VP2 complex

[0064] Experimental method: Fig.13As shown, 21-day-old SPF chickens were randomly divided into 4 groups, blank control, VP2 control, VP2-Polyjet (20 μL Polyjet transfection reagent, 50 μL VP2 and 500 μL opti-DMEM culture medium were fully mixed) control, C283-BEVs-VP2 (prepared in Example 11), 8 in each group. Nasal drops and eye drops were performed at 21 days of age. Each chicken in the experimental group was nasally dropped with 200 μL VP2 protein, VP2-Polyjet, and C283-BEVs-VP2, and each chicken in the blank control group was nasally dropped with 200 μL PBS. Serum was collected 1 week and 2 weeks after immunization for IBDV antibody detection. The chickens were killed in the second week after immunization, and anticoagulated blood was collected and organ tissues were separated.

[0065] Experimental results: Serum IBDV-ELISA antibody titer detection: Experimental method: The IBDV antibody titer in chicken serum was detected according to the instructions of the ID Screen® Indirect ELISA Antibody Detection Kit. The specific steps are as follows: (1) First, dilute the sample to be tested 500 times with diluent No. 14 on the dilution plate and mix well. (2) Then add 100 μL of negative control to wells A1 and B1 of the ELISA reaction plate, add 100 μL of positive control to wells C1 and D1, and add 100 μL of diluted sample to the remaining wells. Cover the ELISA reaction plate and incubate at (21 ± 5℃) for 30 ± 3 min. (3) Discard the liquid in the ELISA plate wells, add 300 μL of washing solution (1×) and wash the plate three times, then add 100 μL of enzyme conjugate (1×) to each well, cover the ELISA reaction plate, and incubate at (21 ± 5℃) for 30 ± 3 min. (4) Discard the liquid in the ELISA plate wells, add 300 μL of washing solution (1×) and wash the plate three times, then add 100 μL of substrate solution (TMB) to each well, incubate at (21 ± 5°C) for 15 ± 2 min, add 100 μL of stop solution to each well to terminate the reaction, and then read and record the absorbance 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 to determine the immune status.

[0066] Experimental results: Fig.14As shown, according to the instructions of the kit, serum IBDV-specific IgG of each group was detected 1 week and 2 weeks after immunization. The results showed that 1 week after nasal and ocular mucosal immunization, the serum antibodies of the experimental groups were all negative. 2 weeks after immunization, the serum ELISA antibodies of C283-BEVs, VP2 control and blank control groups were negative, while the serum antibody titer of 3 / 8 chickens in the C283-BEVs-VP2 immunization group was positive, and the ELISA antibody titer was about 2000.

[0067] C283-BEV-VP2 mucosal immune activation PBMCs flow cytometry detection: Experimental method: After SPF chickens were nasally and ocularly instilled with C283-BEVs-VP2, anticoagulated blood was collected aseptically 48 hours later, and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium. The specific operation was as follows: (1) 2 mL of lymphocyte separation medium and anticoagulated blood were added to a 15 mL centrifuge tube in equal proportions, and the tube was centrifuged at 800 g for 24 min. (2) The PBMCs layer was aspirated and placed in a 15 mL centrifuge tube containing 1640 culture medium. After adding PBMCs washing solution, the tube was centrifuged at 800 g for 10 min, and washed twice in succession. (3) PBMCs were resuspended in lymphocyte 1640 culture medium (containing FCS 10% fetal bovine serum), and the concentration of PBMCs was detected using a cell counter so that the total cell number of the flow cytometry sample was 1×10 6 cells / mL. (4) Refer to the instructions for use of the purchased flow cytometry CD3+ / CD4+ / CD8+ antibody to prepare blank control tubes, single staining control tubes and experimental tubes in the dark. (5) After incubation in a shaker at 4°C in the dark for 30 min, add 1 mL PBS (containing 2% peptide bovine FBS serum) for washing, 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, the relative fluorescence intensity of the cells was analyzed using a flow cytometer (A60Universal, Apogee Flow Systems, UK).

[0068] Experimental results: Flow cytometry results showed that after 2 weeks of immunization, the content of CD4+ and CD8+ in peripheral blood mononuclear cells in the experimental group and the blank control group was different (Figure 15A). According to statistical analysis, the number of CD4+ and CD8+ immune cells in PBMCs in the C283-BEVs-VP2 group was higher than that in the blank control group, and the difference was extremely significant (p<0.0001, Fig.15C), the ratio of CD4+ and CD8+ T cells in the mucosal immunization C283-BEVs group (0.422) was significantly different from that in the blank group (0.523) (p<0.01, Figure 15 B). This indicates that the mucosal immunization 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 mucosal immune vaccine preparations; (3) Use in the preparation of feed or medicine for regulating animal intestinal flora or promoting animal growth; (4) Use in the preparation of highly effective natural immune adjuvants or preparations for enhancing immunity.

3. The use according to claim 2, characterized in that The fermented lactobacillus strain has the functions of improving mucosal immune response and promoting mucosal secretion of specific antibodies.

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 microbial preparations; (2) Use in the preparation of mucosal immune vaccine preparations; (3) Use in the preparation of feed or medicine for regulating animal intestinal flora or promoting animal growth; (4) Use in the preparation of natural immune adjuvants or immune enhancing preparations; (5) Use in the preparation of mucosal delivery vehicles.

8. The use according to claim 7, characterized in that The mucosal delivery carrier is used to deliver all components or subunit components of antigens, nucleic acids or mRNA from inactivated bacteria or inactivated viruses; the fermented lactobacillus strain extracellular vesicles have the function of improving mucosal immune response and promoting mucosal secretion of specific antibodies.

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.

10. Use of the mucosal immune complex according to claim 9 in the following aspects: (1) Use in the preparation of anti-inflammatory and anti-infective microbial preparations; (2) Use in the preparation of mucosal immune vaccine preparations; (3) Use in the preparation of feed or medicine for regulating animal intestinal flora or promoting animal growth; (4) Use in the preparation of natural immune adjuvants or preparations for enhancing immunity.

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