Lactobacillus reuteri strain as well as exovesicles and application thereof

Abstract

The invention discloses a lactobacillus reuteri strain as well as an outer vesicle and application thereof. The lactobacillus reuteri strain is named as C186 and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the microbial preservation number of the lactobacillus reuteri strain is CGMCC No.30337, and the preservation date is April 15, 2024. Researches show that the strain has excellent immunoregulation characteristics, acid resistance, cholate resistance and the like, the strain can generate extracellular vesicles with immunological enhancement activity, and the extracellular vesicles can directly activate chicken Hastelloid lymphocytes and tracheal endothelium lymphocytes only by dripping nasal drops to eyes, so that mucosal immune response is remarkably enhanced. When the lactobacillus reuteri strain C186 or the exovesicles thereof are added into feed or medicines, the effects of the feed or the medicines on enhancing the immune response and health care of the body can be improved, and the lactobacillus reuteri strain C186 or the exovesicles thereof is expected to be used as a natural delivery carrier for researching and developing novel oral vaccines and mucous membrane delivery vaccines.

Description

Technical Field

[0001] The present invention relates to a new strain of Lactobacillus reuteri and its extracellular vesicles, and also relates to the use of the new strain of Lactobacillus reuteri and its extracellular vesicles in preparing immunopotentiators, vaccine immune adjuvants, and preparations for enhancing the body's immune regulation activity; and also relates to the use of the new strain of Lactobacillus reuteri in preparing mucosal immune vaccines or oral immune vaccines. The present invention belongs to the field of biotechnology. Background Art

[0002] Lactobacillus reuteri ( Lactobacillus reuteri ) is one of the important members of the genus Lactobacillus. It has been reported that lactic acid bacteria exist naturally in the intestines of almost all vertebrates and mammals. It is the most common type of probiotics, including many genera and species, such as Lactobacillus acidophilus , Lactobacillus rhamnosus , Lactobacillus casei etc., are widely found in fermented dairy products, fermented foods and the human intestine. Studies have shown that the composition of the intestinal flora is closely related to human health, involving growth and development, metabolism and immune response. Studies on germ-free animals have further confirmed the importance of intestinal microorganisms and probiotics in intestinal digestive enzyme activity, intestinal wall thickness, cytokine production and serum immunoglobulin levels. Therefore, in view of the key role of intestinal microbiota and probiotics, probiotics and probiotics are widely used to repair ecological imbalances and regulate the host's immune response. Probiotics, these tiny active organisms, not only play a key role in maintaining the microbial ecological balance of human health, but also show great potential in the prevention and treatment of a variety of diseases.

[0003] Lactobacillus reuteri is one of the lactobacilli naturally found in the gastrointestinal tract of humans and animals. It not only has the main beneficial effects of lactic acid bacteria, but also has the special effect of producing broad-spectrum antibacterial substances. It can effectively inhibit the growth of Helicobacter pylori, Escherichia coli, Salmonella, Shigella, Listeria, Vibrio, Clostridium, and Staphylococcus. Lactobacillus reuteri got its name from German microbiologist Gerhard Reuter, who discovered it in human intestinal and fecal samples in the 1960s. When it was discovered in the 1960s, Lactobacillus reuteri naturally existed in 30-40% of the human body, but now it has dropped to about 10-20%. Scientific researchers have linked this decline to changes in lifestyle. Instead of eating fermented foods like sauerkraut as we used to, we use preservatives, which kill bacteria in food and in the body.

[0004] Lactobacillus reuteri is a lactic acid bacterium that has been reported to exist naturally in the intestines of almost all vertebrates and mammals. Lactobacillus reuteri has a strong adhesion ability to the intestinal mucosa, which can improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, and avoid intestinal diseases. Lactobacillus reuteri can produce a non-protein broad-spectrum antibacterial substance called "Reuterin", which can widely inhibit the growth of Gram-positive bacteria, Gram-negative bacteria, yeast, fungi and pathogens. Lactobacillus reuteri probiotic preparations can improve human functions, enhance immunity and promote human health. In 2003, my country approved Lactobacillus reuteri as a microbial strain for human health products, and the bacteria has been internationally recognized as a new type of probiotic lactic acid bacteria with high theoretical research and production application value. Research on Lactobacillus reuteri began as early as the 1980s, and research on its various probiotic functions is still ongoing, with new research results emerging in an endless stream, giving people a more comprehensive understanding of the probiotic functions of Lactobacillus reuteri.

[0005] Lactobacillus reuteri is able to produce glucans and fructans during fermentation. One of the glucans, alpha-1,4 / 1,6 glucan, has a molecular weight of 40 MDa and a branching degree of about 16%, and is a satiety inducer with favorable effects on insulin and blood glucose levels. The glucan is not degraded in the stomach and jejunum, but is completely degraded in the colon. Due to its slow starch properties, this glucan may be a health-promoting ingredient in baking applications.

[0006] Lactobacillus reuteri is resistant to low pH and bile salts and is able to adhere to mucin and intestinal epithelial cells, with some strains able to adhere to intestinal epithelial cells of a range of vertebrate hosts. One possible mechanism of adhesion is the binding of bacterial surface molecules to the mucus layer. Mucus binding proteins (MUB) and MUB-like proteins, encoded by specific orthologous protein-encoding gene clusters of lactobacilli, serve as adhesion mediators or so-called adhesins. The strain-specific role of MUB in recognizing mucus components and / or their ability to promote aggregation may explain the contribution of MUB to Lactobacillus reuteri adhesion. Probiotics can improve the balance of host microecology, inhibit the growth of pathogens, and enhance host immunity and health.

[0007] Lactobacillus reuteri is a lactic acid bacterium that has been reported to exist naturally in the intestines of almost all vertebrates and mammals. Lactobacillus reuteri has a strong ability to adhere to the intestinal mucosa, which can improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, and avoid intestinal diseases. Summary of the invention

[0008] One of the purposes of the present invention is to provide a new strain of Lactobacillus reuteri and the use of the strain.

[0009] The second object of the present invention is to provide the exovesicles of the new strain of Lactobacillus reuteri and the use of the exovesicles.

[0010] In order to achieve the above object, the present invention adopts the following technical means: The present invention uses 10 probiotic strains with good immunomodulatory properties as starting strains, uses protoplast fusion breeding technology to construct fusion bacteria, and through interaction with J774 Dual™ (a macrophage cell line with stably integrated reporter genes, which carries two reporter genes (alkaline phosphatase reporting NF-κB and luciferase reporting IRF)), a fusion strain derived from Lactobacillus reuteri and Bifidobacterium was screened out from 18 fusion strains, and it was identified as belonging to Lactobacillus reuteri ( Lactobacillus reuteri ), named C186, and classified as Lactobacillus reuteri ( Lactobacillus reuteri ), 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. 30337 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 to large, regular square rods; 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 surface, bulges, opaque medium-sized colonies, and a sour and fragrant taste.

[0011] Based on the above research, the present invention first proposes a Lactobacillus reuteri strain, named C186, which 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 preservation number is CGMCC No. 30337, and the preservation date is April 15, 2024.

[0012] Furthermore, the present invention also proposes the use of the Lactobacillus reuteri strain in the following aspects: (1) Preparation of immune adjuvants or preparations for enhancing immunity; (2) Preparation of anti-inflammatory and anti-infective drug preparations or preparation of drugs for regulating animal intestinal flora or promoting animal growth.

[0013] Among them, preferably, the immune adjuvant or immune enhancement preparation has the function of enhancing mucosal immune response and promoting mucosal secretion of specific antibodies.

[0014] Secondly, the present invention also proposes a Lactobacillus reuteri exovesicle, which is extracted from the culture solution of the Lactobacillus reuteri strain C186.

[0015] Among them, preferably, the exosomes are prepared by the following method: the bacterial liquid of the Lactobacillus reuteri strain C186 is inoculated into a liquid MRS medium at a ratio of 1:100, and after culturing for 48 hours, centrifuged at 3000-3500 g, 4°C for 10 min, repeated 1-2 times, and the supernatant is retained; the supernatant is passed through a 0.45 μm hollow fiber sieve, the filtered liquid is collected, centrifuged at 35000 g, 4°C for 1 hour, and the supernatant is retained; centrifuged at 120000 g, 4°C for 1 hour, the supernatant is removed, the precipitate is resuspended with an exosome preservation solution, temporarily stored at 4°C, and stored at -80°C.

[0016] Preferably, the method further comprises the step of purifying the extracted exosomes by glucose density gradient centrifugation.

[0017] Among them, preferably, the purity of glucose used in the glucose density gradient centrifugation method is, by mass percentage, 5 ml 60% glucose solution, 10 ml 40% glucose solution, 25 ml 30% glucose solution, 10 ml 20% glucose solution, 5 ml 5% glucose solution + 5 ml extracellular vesicle solution from bottom to top, centrifuged at 100,000 g for 18 h, and the extracellular vesicle solutions of different layers were taken and supplemented with extracellular vesicle preservation solution to 60 ml, centrifuged at 120,000 g-130,000 g for 1.5 h, the supernatant was removed, and the precipitate was resuspended to 300-500 μL with PBS at pH 8.0.

[0018] Furthermore, the present invention also proposes the use of the Lactobacillus reuteri exovesicles in the following aspects: (1) Preparation of immune adjuvants or mucosal delivery vectors; (2) Preparation of mucosal immune preparations; (3) Preparation of immunomodulatory preparations; (4) Preparation of anti-inflammatory and anti-infective drug preparations or preparation of drugs for regulating animal intestinal flora or promoting animal growth.

[0019] Among them, preferably, the mucosal delivery vector is used to deliver all components or subunit components of antigens from inactivated bacteria or inactivated viruses, and the mucosal delivery vector can be used for intestinal immunization or oral immunization.

[0020] Among them, preferably, the mucosal immune preparation is a mucosal immune vaccine.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses 10 probiotic strains with good immunomodulatory properties as starting strains, uses protoplast fusion breeding technology to construct fusion bacteria, and screens a fusion strain derived from Lactobacillus reuteri and Bifidobacterium from 18 fusion strains after interacting with J774 Dual™, and it is identified as belonging to Lactobacillus reuteri and named C186. Studies have shown that the strain has excellent immunomodulatory properties with strong immunoenhancing activity, and also has properties such as acid resistance and bile resistance. Moreover, the strain can produce extracellular vesicles with strong immunoadjuvant activity and immunoenhancing activity. Only nasal drops and eye drops of the strain's extracellular vesicles 186-BEVs can directly activate chicken Harderian gland lymphocytes and tracheal epithelial lymphocytes, significantly enhancing mucosal immune response. Adding the Lactobacillus reuteri strain C186 or its extracellular vesicles of the present invention as additives to feed and medicines can improve the effects of feed and medicines in enhancing the body's immune response and health care. The natural probiotic C186 strain with both anti-inflammatory and anti-infective effects is expected to be used as an auxiliary therapeutic preparation for various acute inflammations. 186-BEVs is also expected to be used as a natural delivery carrier for the development of new oral vaccines and mucosal delivery vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The aerobic growth of Lactobacillus reuteri strain C186 on solid media MRS-X-Gal, MC and MRS; Figure 2 The anaerobic growth of Lactobacillus reuteri strain C186 on solid media MRS-X-Gal, MC and MRS; Figure 3 The result is that Lactobacillus reuteri C186 activates the NF-κB signaling pathway and the IRF signaling pathway, and has strong immune enhancement activity characteristics; Among them, A is the result of activating the NF-κB signaling pathway; B is the result of activating the IRF signaling pathway; Figure 4 The results of extraction and purification of extracellular vesicles of Lactobacillus reuteri C186 strain; Figure 5A The particle size test results of Lactobacillus reuteri C186 strain extracellular vesicles 186-BEVs; Figure 5B Zeta potential test results of extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 strain; Figure 6 The results of small RNA detection of Lactobacillus reuteri C186-BEVs; Figure 7The results showed that extracellular vesicles (186-BEVs) of Lactobacillus reuteri C186 strain could continuously activate the IRF and NF-κB signaling pathways in macrophages; Among them, A is the result of activating the NF-κB signaling pathway; B is the result of activating the IRF signaling pathway; Figure 8 The results are for the in vitro immune activation ability of Lactobacillus reuteri C186 extracellular vesicles 186-BEVs at 24 hours; Among them, A is the result of activating the NF-κB signaling pathway; B is the result of activating the IRF signaling pathway; Fig. 9 The results of cytokine expression in Harderian gland lymphocytes (HGLs) of SPF chickens after intranasal and ocular administration of extracellular vesicles (186-BEVs) of Lactobacillus reuteri strain C186. Among them, AG are the detection results of IL-1β, IL-17A, IL-6, IL-23, IL-4, IL-10, and TGF-β; Fig.10 The results of the detection of Th1 and Th2 CK in Harderian gland lymphocytes (HGLs) of SPF chickens after intranasal and ocular administration of extracellular vesicles (186-BEVs) of Lactobacillus reuteri C186 strain. Among them, AH are the detection results of IFN-β, IL-28B, IL-2, IL-12p70, IFN-γ, IFN-γ / IL-4 ratio, IL-10, and IL-12 / IL-10 ratio; Fig.11 The results of the detection of interferon regulatory factors that regulate the antiviral innate immune signaling pathway and lymphocyte homing chemokine receptors in Harderian gland lymphocytes (HGLs) of SPF chickens after nasal and eye drops of extracellular vesicles 186-BEVs of Lactobacillus reuteri strain C186 were obtained. Among them, AE are the detection results of IRF1, IRF7, TBK1, CCR5, and CXCR5; Fig.12 The results of the detection of co-stimulatory molecules highly expressed by DC or APC cells and T cell co-stimulatory molecules in Harderian gland lymphocytes HGLs of SPF chickens after intranasal and ocular administration of extracellular vesicles 186-BEVs of Lactobacillus reuteri strain C186; Among them, AE are the detection results of CD40, CD80, TLR4, CD40 / CD40L ratio, and CD40L; Fig.13The fluorescence quantitative detection results of signaling molecules of the JAK / STAT signaling pathway (second signaling pathway) that regulate immune cells in Harderian gland lymphocytes HGLs of SPF chickens after nasal and eye drops of extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 strain; Among them, AF are STAT1 (Th1, M1 cells), STAT3 (Th17 cells), STAT5 (Tregs cells), STAT1 / STAT3 ratio (Th1 / Th17 ratio), STAT1 / STAT5 ratio (Th1 / Tregs ratio), and STAT1 / STAT6 ratio; Fig.14 The results of the detection of cytokines and related immunoregulatory molecules in the intraepithelial lymphocytes of the hind trachea of ​​SPF chickens by nasal and eye drops of extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 strain; Among them, AN are IL-1β, IL-17A, IL-6, IL-12P70, IL-2, IL-23, IL-4, IL-10, TGF-β, CCR7, CXCR5, CCR5, IRF-1, and IRF-7; Fig.15 The results of the detection of regulatory signal molecules related to the JAK / STAT signaling pathway (second signaling pathway) of immune cells in the hind tracheal intraepithelial lymphocytes (IELs) of SPF chickens by nasal and eye drops of extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 strain; Among them, AE are the detection results of CD40, CD80, TLR4, CD40L, and CD40 / CD40L ratio; Fig.16 The results of the detection of mucosal secretory specific neutralizing antibodies after the Lactobacillus reuteri C186 strain extracellular vesicles 186-BEVs were used as mucosal delivery carriers (nasal drops and eye drops) to deliver inactivated avian influenza virus to efficiently activate chicken respiratory mucosal immunity; Fig.17 The results of the detection of humoral specific circulating antibodies after the extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 strain were used as mucosal delivery vectors (nasal drops and eye drops) to deliver inactivated avian influenza virus to efficiently activate chicken respiratory mucosal immunity. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1 Isolation and identification of Lactobacillus reuteri C186 strain 1. Isolation and identification of strains Colony characteristics of strains Figure 1 As shown in the left picture.

[0025] The present invention uses 10 probiotic strains with good immunomodulatory properties isolated from the intestinal mucosa of healthy wild boars in the Fuyuan primeval forest of Mudanjiang, Heilongjiang Province, China as starting strains, uses protoplast fusion breeding technology to construct fusion bacteria, and after interacting with J774 Dual™ (a macrophage cell line with stably integrated reporter genes, which carries two reporter genes (alkaline phosphatase reporting NF-κB and luciferase reporting IRF)), a fusion strain derived from Lactobacillus reuteri and Bifidobacterium was screened out from 18 fusion strains, and it was identified as Lactobacillus reuteri, named C186, and classified as Lactobacillus reuteri ( Lactobacillus reuteri ), 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. 30337 and the deposit date is April 15, 2024.

[0026] Lactobacillus reuteri C186 strain grows well on MRS medium, and its microbiological characteristics are: typical Gram staining positive, the bacteria are medium to large, regular in morphology, aerobic or facultative anaerobic bacteria, grow well on solid and liquid MRS medium, the optimal culture temperature for this bacterium is 37°C, the colonies on MRS medium are milky white, with neat edges, smooth and moist surface, raised, opaque medium-sized colonies, and a sour and fragrant taste.

[0027] 2. Acid resistance test of strains: The isolated strain Lactobacillus reuteri C186 was inoculated into MRS liquid culture medium with pH values ​​of 3.0, 4.0, 5.0, and 6.5 at an inoculum volume of 10% of the volume of the MRS liquid culture medium. Three parallel controls were set for each acidity gradient and cultured at 37°C. The bacterial liquid samples were taken at 30 min, 60 min, 90 min, and 120 min of culture to determine the OD 600 The experiment showed that Lactobacillus reuteri had strong acid resistance and could grow on MRS at pH 4.0.

[0028] 3. Chicken bile resistance test of strains: The isolated strain Lactobacillus reuteri C186 was inoculated into MRS medium containing fresh chicken bile at 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% of the volume of MRS liquid culture medium at an inoculum volume of 10%. Three parallel experiments were set for each concentration and cultured at 37°C. The bacterial liquid samples were taken after 30 min, 60 min, 90 min, and 120 min of culture to determine the OD 600 nm value. The results showed that Lactobacillus reuteri C186 strain could grow well in the culture medium containing 30% chicken bile and had a strong resistance to chicken bile.

[0029] Example 2 Cultivation characteristics of Lactobacillus reuteri C186 strain on solid culture medium 1. Colony characteristics of Lactobacillus reuteri C186 strain on ordinary solid culture medium: Experimental method: Lactobacillus reuteri C186 was streaked on MRS-X-Gal, MC and MRS solid culture media for aerobic culture, and the colony morphology of C186 was observed.

[0030] Experimental results: Figure 1 The colony morphology of Lactobacillus reuteri C186 is a medium-sized colony with neat edges, smooth and moist surface, ridges, opaque, and a sour and fragrant smell. When aerobically cultured on MRS-X-Gal, the colony morphology is similar to that on MRS medium, and the color is light blue (indicating that C186 strain can produce β-glucosidase, decompose X-gal substrate, and make the colony blue); after aerobic culture of C186 strain on MC medium, transparent rings appear around the colony, indicating that C186 strain produces acid metabolites and dissolves the transparent ring formed by calcium carbonate.

[0031] 2. Anaerobic culture characteristics of Lactobacillus reuteri C186 strain on MRS-X-Gal medium, MC medium and MRS solid medium: Experimental method: Lactobacillus reuteri C186 strain was streaked on MRS-X-Gal medium, MC and MRS solid culture medium, cultured anaerobically at 37℃ for 48 h, and the colony characteristics were observed.

[0032] Experimental results: Figure 2 When Lactobacillus reuteri C186 was cultured anaerobically on MRS-X-Gal, the colony morphology was similar to that on MRS-X-Gal aerobic medium and aerobic MRS medium. After strict anaerobic culture on MC medium, transparent rings appeared around the colonies of C186, indicating that Lactobacillus reuteri C186 produced acid metabolites and dissolved calcium carbonate to form transparent rings.

[0033] Example 3 Lactobacillus reuteri C186 strain can activate NF-κB signaling pathway and IRF signaling pathway and has strong immune enhancement activity Experimental method: Cultivate macrophages: J774-Dual™ macrophages were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase.

[0034] Stimulate or treat cells: Use MRS medium to culture Lactobacillus reuteri C186, 538 or 606 to the logarithmic phase, measure OD600 of 2.0 and stimulate with 50 μL of bacterial solution. Before stimulation, accurately adjust the cell suspension concentration to 1×10 7 To ensure that the number of cells in each sample is consistent.

[0035] Use specific stimuli, including: 1. Lipopolysaccharide (LPS): dilute LPS to a working concentration of 25 μg / ml, add 40 μL to each sample to reach a final concentration of 1 ng / ml; 2. 2',3'-cGAMP (2'3'-Cyclic GMP-AMP): dilute to a working concentration of 0.1 mg / ml, add 10 μL to each sample to reach a final concentration of 1 μg / ml; 3. Pam3csk4: dilute to a working concentration of 1 μg / ml, add 50 μL to each sample to reach a final concentration of 50 ng / ml as a control. Ensure that all samples are added with stimuli at the same time and cultured under the same conditions for 24 h. After 24 h of culture, centrifuge at 300 g, 5 min, 4°C to obtain the supernatant.

[0036] Before performing the spectrophotometric test, calibrate the instrument to ensure the accuracy of the reading. Use a spectrophotometer to measure the OD450 value to observe the activation status.

[0037] Statistical analysis: Use appropriate statistical methods to compare differences between treatment groups to ensure the significance of the results. Consider using ANOVA or t-test, depending on the experimental design.

[0038] Experimental results: The J774-Dual™ macrophage dual signal reporter cell line (mouse macrophage reporter cell line) was used to verify the activation of the C186 strain on the macrophage NF-κB signaling pathway and the IRF signaling pathway. Figure 3 As shown in A, by detecting the expression level of SEAP in the supernatant (OD630 nm), it was shown that strain C186 can activate the NF-κB signaling pathway (compared with two standard control agonists LPS and Pam3CSK4, it was extremely significantly higher than the standard agonists (p<0.0001), indicating that strain C186 can extremely significantly activate the immune response); and as Figure 3 As shown in B, by detecting the expression level of luciferase, it was shown that strain C186 can activate the IRF signaling pathway (interferon signaling pathway, compared with the two standard agonists LPS and 2'3'-cGAMP, it was extremely significantly higher than the standard agonist (p<0.0001), indicating that strain C186 can extremely significantly activate the interferon signaling pathway and can activate anti-infection immune response).

[0039] Example 4 Extraction and purification of extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 strain Experimental methods: Lactobacillus reuteri C186 strain was inoculated into liquid MRS medium at a ratio of 1:100. After 48 h of culture, centrifuged at 3000-3500 g, 4°C for 10 min, repeated 1-2 times, and the supernatant was retained. The supernatant was passed through a 0.45 μm hollow fiber sieve, the filtered liquid was collected, centrifuged at 35000 g, 4°C for 1 h, and the supernatant was retained. Centrifuged at 120000 g, 4°C for 1 h, the supernatant was removed, and the precipitate was resuspended in pH8.0 PBS. Temporarily stored at 4°C, stored at -80°C.

[0040] Purification: Density gradient centrifugation, the purity from bottom to top is 5 ml 60% glucose solution; 10 ml 40% glucose solution; 25 ml 30% glucose solution; 10 ml 20% glucose solution; 5 ml 5% glucose solution + 5 ml 186-BEVs. Centrifuge at 100000 g for 18 h. Take the extracellular vesicle solution of different layers and fill it with extracellular vesicle preservation solution to 60 ml, centrifuge at 120000g-130000g for 1.5 h, remove the supernatant, and resuspend the precipitate to 300-500μL with pH8.0 PBS.

[0041] Experimental results: Figure 4 Shown is an electron micrograph of the extracellular vesicles of Lactobacillus reuteri C186.

[0042] The concentration of extracellular vesicles of the extracted 186-BEVs was measured by ultraviolet absorption method (UV method), and the concentration was 9.39 mg / ml; while the concentration of the purified 186-BEVs liquid protein was 12.36 mg / ml. The yield of the extracted extracellular vesicles was 2 ml of extracellular vesicles per 1 L of stock solution.

[0043] Example 5 Detection of the particle size and zeta potential of extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 strain 5.1 Detection of the particle size of Lactobacillus reuteri C186 extracellular vesicles 186-BEVs Turn on the power on the back of the instrument, preheat for about half an hour, press the large round button on the instrument to open the instrument sample slot, inject the prepared Lactobacillus reuteri C186 extracellular vesicles 186-BEVs into the corresponding sample pool and insert it into the slot (insert to the bottom), close the sample slot cover, and let the sample temperature balance. Open the Zetasizer Software software, and the large round button on the instrument changes from red to stable green, indicating that the instrument is in normal standby. During the test, the button is flashing green, so do not open the sample slot cover at this time.

[0044] Measurement preparation: Create or open a test file with the file extension of .dts. Inject the sample into the particle size sample pool at a height of 1~1.5cm to avoid bubbles. Do not touch the bottom 1cm part of the sample pool with your hands to avoid contaminating the light path. The NanoZS90 / S90 instrument must use a four-sided transparent sample pool. Select Measure-Manual to open the manual measurement setting window.

[0045] Left-click on Measurement type and select Size to enter the sample name and remarks. The optical parameters of the sample have no effect on the average particle size, PDI and light intensity distribution. Select the correct dispersion medium parameters. Balance time: The sample temperature must reach the set temperature. Select the sample pool used - the instrument will determine the light path based on the selected sample pool. Test the number of cycles for more than 3 times and check the stability of the results. The rest of the settings are set by default. The instrument will automatically optimize the test conditions based on the sample signal. After the settings are completed, click OK to close the settings dialog box. The measurement window automatically opens. Click Star to start the measurement.

[0046] Multi-window display: real-time correlation equations, light intensity fluctuations and particle size distribution, record instrument working status and optimization process, automatically save results, select Size worksheet; select Intensity PSD report to view.

[0047] 5.2 Detection of Zeta Potential of C186-BEVs Turn on the power on the back of the instrument and preheat for about half an hour. Press the large round button on the instrument to open the sample slot. Inject the prepared sample into the corresponding sample cell and make sure the sample cell is inserted into the bottom of the sample cell. Close the sample cell cover and wait for the sample temperature to equilibrate. Open the Zetasizer Software. Confirm that the large round button on the instrument changes from red to green, indicating that the instrument is in normal standby. During the measurement, the button will flash green; at this time, do not open the sample cell cover. Create or open a test file (the file suffix should be .dts). Inject the sample into the particle size sample cell, be careful to avoid bubbles, and the height should be 1~1.5 cm. Select "Measure-Manual" to enter the manual measurement settings. Select "Size" in Measurement type for particle size measurement. Enter the sample name and remarks. Select the correct dispersion medium parameters, which have a great impact on the results. Make sure the sample temperature reaches the set equilibrium time temperature. Select the sample cell you are using, and the instrument will determine the optical path based on this selection. Set the number of test cycles to more than 3 times to check the stability of the results. Keep the rest of the settings as default, and the instrument will optimize the test conditions based on the sample signal. After the settings are completed, click "OK" to close the settings dialog box. The measurement window opens automatically, click "Start" to start the measurement. The instrument automatically determines the number of sub-tests, and the displayed results are not the final results. Real-time display of multi-window correlation equations, light intensity fluctuations and particle size distribution. The work log records the instrument working status and optimization process. The results are automatically saved. Select the Size worksheet and Intensity PSD report to view the final results. This process needs to be carried out strictly in accordance with the operating instructions to ensure accurate measurement of the particle size of the sample.

[0048] Experimental results: Figure 5A As shown in Figure 5B, the particle size and Zeta potential of the extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 strain are detected. The particle size distribution of 186-BEVs measured by the dynamic light scattering (DLS) technology is shown after analysis by the Malvern particle size Z potential instrument, which is used to analyze the size of 186-BEVs particles. Z-Average: represents the average dynamic diameter of the measured particles, and the result is 104.8 nm. Intercept: is 0.929, which is usually related to the quality of the scattering data. A value close to 1 indicates good data quality. Peak 1, 2, 3: represents the size of the three different sizes of particle groups detected for 186-BEVs, which are 144.7 nm, 34.40 nm and 2629 nm, respectively, and the main peak is approximately at 144.7 nm. The Zeta potential of 186-BEVs is -14.6 mV. This indicates that 186-BEVs carry negative charges and can stably exist in alkaline solutions (stable dispersion system).

[0049] The average particle size of 186-BEVs is 104.8nm, and the main peak is 144.7nm (accounting for 88.7%).

[0050] The Zeta potential of 186-BEVs is -14.6 mV, indicating that 186-BEVs carry negative charges and can stably exist in alkaline solutions (stable dispersion system).

[0051] Example 6 Small RNA carried by extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 Experimental method: Before isolating exosomes, no RNA protectants (such as Trizol) should be added to the sample.

[0052] If the separated exosomes need to be observed by electron microscopy, they should be stored at 4°C to avoid long-term storage. If long-term storage is necessary, a large amount of buffer (10 to 100 times the normal volume) should be added when resuspending the exosomes to reduce the exosome concentration. After sufficient resuspension, they can be frozen at -80°C. The prepared samples were sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for testing.

[0053] Experimental results: The results of small RNA identification and analysis of 186-BEVs showed that 186-BEVs mainly carried 14 small RNAs, with the lengths and characteristics as follows Figure 6 .

[0054] Example 7 Lactobacillus reuteri C186 extracellular vesicles 186-BEVs continuously activate macrophage IRF and NF-κB signaling pathways Cultivate macrophages: J774-Dual™ macrophages were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase.

[0055] Stimulate or treat cells: adjust the extracellular vesicle concentration to 12 mg / ml, add 5 μL, 10 μL, or 15 μL to each well before stimulation, and accurately adjust the cell suspension concentration to 1×10 7 To ensure that the number of cells in each sample is consistent.

[0056] Use specific stimuli: 1. Lipopolysaccharide (LPS): dilute LPS to a working concentration of 25 μg / ml, add 40 μL to each sample to reach a final concentration of 1 ng / ml; 2. 2'3'-cGAMP (2'3'-Cyclic GMP-AMP): dilute to a working concentration of 0.1 mg / ml, add 10 μL to each sample to reach a final concentration of 1 μg / ml; 3. Pam3csk4: dilute to a working concentration of 1 μg / ml, add 50 μL to each sample to reach a final concentration of 50 ng / ml as a control, ensure that all samples are added with stimuli at the same time, and cultured under the same conditions for 24 h.

[0057] After 24 h of incubation, the supernatant was collected by centrifugation at 300 g for 5 min at 4°C; Before performing the spectrophotometric test, calibrate the instrument to ensure the accuracy of the reading. Use a spectrophotometer to measure the OD450 value to observe the activation status.

[0058] Statistical analysis: Use appropriate statistical methods to compare differences between treatment groups to ensure the significance of the results. Consider using ANOVA or t-test, depending on the experimental design.

[0059] Experimental results: Figure 7 Shown are the results of testing the in vitro immunomodulatory properties of Lactobacillus reuteri C186 extracellular vesicles 186-BEVs.

[0060] Activation results of NF-κB signaling pathway of 186-BEVs: The activation effect of NF-κB signaling pathway of 186-BEVs was higher than that of the parent strain C186 ( Figure 7 A) Its activation effect is comparable to that of the standard agonist Pam3csk4 in activating the NF-κB signaling pathway.

[0061] Activation results of the IRF signaling pathway of 186-BEVs: The activation effect of the IRF signaling pathway of 186-BEVs was significantly higher than that of the parent strain C186 ( Figure 7 B), and significantly higher than the standard activators of the IRF signaling pathway.

[0062] The above results show that 186-BEVs have the ability to continuously activate macrophage IRF and NF-κB, which is significantly higher than its parental strain and standard agonists, suggesting that 186-BEVs have strong immune adjuvant and immune enhancement functions.

[0063] Example 8 In vitro immune activation ability of Lactobacillus reuteri C186 extracellular vesicles 186-BEVs Experimental methods: After different doses of 186-BEVs interacted with macrophage J774-Dual™ reporter cells for 4 h / 8 h / 12 ​​h / 16 h / 24 h, the activation of NF-κB signaling pathway was detected.

[0064] After different doses of 186-BEVs interacted with macrophage J774-Dual™ reporter cells for 4 h / 8 h / 12 ​​h / 16 h / 24 h, the activation of interferon regulatory factor (IRF) signaling pathway was detected.

[0065] Use specific stimuli: 1. Lipopolysaccharide (LPS): dilute LPS to a working concentration of 25 μg / ml, add 40 μL to each sample to reach a final concentration of 1 ng / ml; 2. 2'3'-cGAMP (2'3'-Cyclic GMP-AMP): dilute to a working concentration of 0.1 mg / ml, add 10 μL to each sample to reach a final concentration of 1 μg / ml; 3. Pam3csk4: dilute to a working concentration of 1 μg / ml, add 50 μL to each sample to reach a final concentration of 50 ng / ml As a control, ensure that all samples are added with stimuli at the same time and cultured under the same conditions for 4 h / 8 h / 12 ​​h / 16 h / 24 h.

[0066] Statistical analysis: Use appropriate statistical methods to compare differences between treatment groups to ensure the significance of the results. Consider using ANOVA or t-test, depending on the experimental design.

[0067] Experimental results: Figure 8 Shown are the results of the dose-dependent test of the in vitro immune activation properties of Lactobacillus reuteri C186 extracellular vesicles 186-BEVs.

[0068] The results of the 186-BEVs immune activation dose-dependent test showed that different doses of 186-BEVs continuously activated macrophages within 24 hours, and the activation ability of only 10 μL of 186-BEVs at 24 hours was significantly higher than that of the standard agonist ( Figure 8 A, Figure 8 B).

[0069] Example 9: Direct activation of chicken Harderian gland lymphocytes by nasal and eye drops of extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 Experimental methods: The experimental animals were 12 SPF white Leghorn chickens aged 21 days, divided into two groups: the experimental group (6 chickens) orally administered 200 μL of C186 extracellular vesicles 186-BEVs solution with a concentration of 12.36 mg / ml; and the control group (6 chickens) orally administered 200 μL of PBS solution.

[0070] Experimental time points and sampling, blood sampling: 5 ml of blood was collected at 24 h, 48 h, and 72 h after administration for the isolation of peripheral blood mononuclear cells (PBMCs). End point treatment: At the 72nd hour of the experiment, all experimental chickens were humanely killed, and then Harderian gland lymphocytes (HGLs) were isolated for further analysis. First, samples were obtained from the lower part of the canthus of the chicken. The collected tonsil tissue was finely cut under sterile conditions and then treated with an appropriate buffer to release lymphocytes. Lymphocytes were isolated using density gradient centrifugation. This step can help isolate lymphocytes and remove dead cells and other tissue debris. The separated cells need to be washed with sterile saline or buffer to remove residual density gradient medium. The number of cells was counted using a hemocytometer or an automated cell counter, and dead cells were excluded by staining to assess cell activity and purity.

[0071] Experimental results: After 186-BEVs was dripped into the nose and eyes of SPF chickens, the Harderian gland lymphocytes (HGLs) in the chicken eyes were extracted and the expression of immune cell CK in HGLs was detected by fluorescence quantitative analysis. The expression of inflammatory cytokines IL-1β, IL-17A, IL-6, and IL-23 in HGLs of the immunized group was significantly higher than that in the control group ( Fig. 9 , AD); while the immune homeostasis and immune negative regulatory cytokines IL-10, IL-4, TGF-β were also significantly higher than those in the control group ( Fig. 9 , EG). The results showed that 186-BEVs nasal drops and eye drops directly activated the immune response of chicken Harderian gland lymphocytes HGLs, while also activating immune regulatory cells, keeping immune activation in a proper steady state.

[0072] The results of the detection of Th1 and Th2 CK in Harderian gland lymphocytes HGLs showed that the expression of IFN-β, IL-28B, IL-2, IL-12p70, IFN-γ, and IL-10 was significantly higher than that in the control group ( Fig.10 , AE, G); IFN-γ / IL-4 ratio; IL-12 / IL-10 ratio in the experimental group were significantly higher than those in the control group ( Fig.10 , F, H). The results showed that 186-BEVs could significantly activate the cellular immune response and humoral immune response of Harderian gland lymphocytes (HGLs); in particular, the net effect of immune regulation tended to be cellular immune response.

[0073] The results of the detection of interferon regulatory factors IRF1 / 7, TBK1 molecules, and lymphocyte homing chemokine receptors CCR5 and CXCR5 in Harderian gland lymphocytes HGLs that regulate the antiviral innate immune signaling pathway showed that the expression of these regulatory signaling molecules was extremely significantly higher than that in the control group ( Fig.11, AE). The results showed that 186-BEVs nasal drops and eye drops directly activated the Th1 immune cells of chicken Harderian gland lymphocytes HGLs.

[0074] The costimulatory molecules CD40, TLR4 and T cell costimulatory molecule CD40L highly expressed in DC or APC cells in Harderian gland lymphocytes HGLs were detected, and the CD40 / CD40L ratio was also significantly higher than that in the control group ( Fig.12 , AE). The results showed that 186-BEVs significantly activated antigen-presenting cells and T cells of HGLs.

[0075] The results of fluorescence quantitative detection of signaling molecules in the JAK / STAT signaling pathway (second signaling pathway) that regulates immune cells in Harderian gland lymphocytes HGLs showed that the related signaling molecules STAT1 (Th1, M1 cells), STAT5 (Tregs cells), and STAT3 (Th17 cells) that activate immune cells were significantly higher than those in the control group ( Fig.13 , AC); STAT1 / STAT3 ratio (Th1 / Th17 ratio) and STAT1 / STAT5 ratio (Th1 / Tregs ratio) were significantly higher than those in the control group ( Fig.13 , DE); STAT1 / STAT6 ratio was significantly higher than that of the control group ( Fig.13 , F). The above results show that 186-BEVs significantly activated HGLs lymphocytes comprehensively and significantly activated anti-infection cellular immune responses.

[0076] Example 10: Direct activation of chicken tracheal intraepithelial lymphocytes (IELs) by nasal and eye drops of extracellular vesicles 186-BEVs of Lactobacillus reuteri C186 Experimental methods: The experimental animals were 12 SPF white Leghorn chickens aged 21 days, divided into two groups: the experimental group (6 chickens) orally administered 200 μL of C186 extracellular vesicles 186-BEVs solution with a concentration of 12.36 mg / ml; and the control group (6 chickens) orally administered 200 μL of PBS solution.

[0077] Experimental time points and sampling, blood sampling: 5 ml of blood was collected at 24 h, 48 h, and 72 h after administration for the isolation of peripheral blood mononuclear cells (PBMCs). End point treatment: At the 72nd hour of the experiment, all experimental chickens were humanely killed, and then tracheal intraepithelial lymphocytes (IELs) were isolated for further analysis.

[0078] Isolation of intraepithelial lymphocytes (IELs) is used to extract lymphocytes from tracheal epithelial tissue.

[0079] Tissue collection: Collect samples from experimental animals or human tracheal epithelial tissue. Ensure that the operation is carried out under sterile conditions to prevent bacterial contamination.

[0080] Tissue processing: The collected tissues were rinsed with pre-cooled PBS (phosphate-buffered saline) under sterile conditions to remove blood and impurities.

[0081] Enzymatic hydrolysis: Treating tissue with appropriate enzymes, such as collagenase and DNase. These enzymes help break down the extracellular matrix and release cells. Usually, tissue fragments are mixed with enzymes and incubated at 37°C for a certain period of time.

[0082] Mechanical separation: After enzyme treatment, mechanical methods are used to separate IELs using the 37-m-LPDK-1 procedure; 37 degrees-Multi-B, 37 degrees-Multi-E to separate LPLs to separate cells from tissues.

[0083] Experimental results: After 186-BEVs were dripped into the nose and eyes of SPF chickens, tracheal intraepithelial lymphocytes were extracted, and the results of fluorescence quantitative detection of cytokines and related immunoregulatory molecules showed that the expression levels of inflammatory cytokines IL-1β, IL-17A, IL-6, and IL-23 in the 186-BEVs dripped nose and eyes experimental groups were significantly higher than those in the control group ( Fig.14 , A, B, C, F); Th1 cytokines IL-2 and IL-12 were also significantly higher than those in the control group ( Fig.14 , D, E); immune homeostasis and immune negative regulatory cytokines IL-10 and TGF-β were significantly increased ( Fig.14 , H, I); lymphocyte homing chemokine receptors CCR5, CCR7 and CXCR5 were significantly higher than those in the control group ( Fig.14 , J, K, L); the anti-infection Th1 interferon regulatory factor IRF1 / 7 was also significantly higher than that in the control group ( Fig.14 , M, N). The results showed that 186-BEVs could significantly activate the anti-infection immune response of tracheal intraepithelial lymphocytes (IELs), especially the anti-infection cellular immune response, by nasal and eye drops.

[0084] After 186-BEVs were dripped into the nose and eyes of SPF chickens, tracheal intraepithelial lymphocytes (IELs) were extracted, and the results of fluorescence quantitative detection of regulatory signal molecules related to the JAK / STAT signaling pathway (second signaling pathway) of immune cells showed that the related signal molecules STAT1 (Th1, M1 cells), STAT5 (Tregs cells), STAT3 (Th17 cells), and STAT6 (Th2 cells) of activated immune cells were significantly higher than those in the control group; STAT1 / STAT3 ratio (Th1 / Th17 ratio) and STAT1 / STAT5 ratio (Th1 / Tregs ratio) were significantly higher than those in the control group; STAT3 / STAT5 ratio (Th17 / Tregs ratio) was significantly higher than that in the control group; STAT1 / STAT6 ratio (Th1 / Th2 ratio) had no significant difference between the immunization group and the control group. The above results show that 186-BEVs fully and significantly activated IELs lymphocytes, and at the same time significantly activated anti-infection cellular immune responses and humoral immune responses.

[0085] The results of detecting the costimulatory molecules CD40, TLR4 and T cell costimulatory molecule CD40L highly expressed by DC or APC cells showed that the expression of costimulatory molecule CD40 highly expressed by DC or APC cells in the immunization group was significantly higher than that in the control group ( Fig.15 , AC); the expression of T cell activation co-stimulatory molecule CD40L and CD40 / CD40L ratio in the immunization group were also significantly higher than those in the control group ( Fig.15 , D, E). The results showed that 186-BEVs significantly activated antigen-presenting cells and T cells of IELs.

[0086] Example 11 Lactobacillus reuteri C186 extracellular vesicles 186-BEVs are used as delivery vectors to deliver inactivated avian influenza virus by nasal drops or eye drops to directly activate mucosal immune responses and produce specific secretory neutralizing antibodies Experimental subjects: 12 7-day-old SPF (Specific Pathogen Free) white broiler chickens were selected. The chickens were divided into four groups, 3 in each group. Experimental group setting: 1 experimental group and 3 control groups were set up.

[0087] Experimental group: Oral administration of 186-H5-BEVs (extravesicle 186-BEVs mixed with H5 subtype influenza virus inactivated virions, 37°C for 30 minutes, the formed H5 virus particles adsorbed extravesicle particles, named C186-H5-BEVs) 200 μL. Control group A: Oral administration of H5-R14 inactivated antigen (H5 subtype influenza virus inactivated virions, named H5-R14) 200 μL. Control group B: Oral administration of C186 extravesicle 186-BEVs solution with a concentration of 12.36 mg / ml 200 μL. Control group C: Oral administration of PBS (phosphate buffered saline) 200 μL.

[0088] Experimental procedures: Blood was collected from all chickens on days 7, 14, and 21 of the experiment for antibody detection. Peripheral blood mononuclear cells (PBMCs) were also isolated for further analysis.

[0089] Specimen collection: In addition to blood samples, oropharyngeal swabs are collected at the same time points to test the level of secretory immunoglobulin A (sIgA).

[0090] Data Recording and Analysis: All collected samples should be properly labeled and stored. Samples should be analyzed using appropriate immunological and molecular biological techniques, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), etc. All operation steps, observations, and any abnormalities should be recorded to facilitate subsequent data analysis and the writing of laboratory reports.

[0091] Experimental results: Fig.16 As shown in the animal experiment, 186-BEVs-H5 was immunized once (nasal drops, eye drops), and sIgA antibodies could be detected in some chickens one week after immunization. After two weeks, the slgA antibody titer in the oral cavity and pharynx of all chickens exceeded the immune critical value by 4log2; at three weeks, the slgA-specific anti-H5 slgA antibody reached 4 to 7log2. The results show that a single immunization (nasal drops, eye drops) with 186-BEVs-H5 successfully induced a protective mucosal immune response.

[0092] Example 12: Lactobacillus reuteri C186 extracellular vesicles 186-BEVs are used as delivery vectors to deliver inactivated avian influenza virus by nasal drops or eye drops to directly activate mucosal immune responses while activating systemic immune responses to produce specific circulating antibodies Experimental method: Experimental subjects: 12 7-day-old SPF (Specific Pathogen Free) white-feathered broilers were selected. The chickens were divided into four groups, 3 in each group. Experimental group setting: 1 experimental group and 3 control groups were set up.

[0093] Experimental group: Oral administration of 200 μL of C186-H5-BEVs. Control group A: Oral administration of 200 μL of H5-R14 inactivated antigen. Control group B: Oral administration of 200 μL of C186 extracellular vesicle 186-BEVs solution with a concentration of 12.36 mg / ml. Control group C: Oral administration of 200 μL of PBS (phosphate buffered saline).

[0094] Experimental procedures: Blood was collected from all chickens on days 7, 14, and 21 of the experiment for antibody detection. Peripheral blood mononuclear cells (PBMCs) were also isolated for further analysis.

[0095] Specimen collection: In addition to blood samples, oropharyngeal swabs are collected at the same time points to test the level of secretory immunoglobulin A (sIgA).

[0096] Data Recording and Analysis: All collected samples should be properly labeled and stored. Samples should be analyzed using appropriate immunological and molecular biological techniques, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), etc. All operation steps, observations, and any abnormalities should be recorded to facilitate subsequent data analysis and the writing of laboratory reports.

[0097] Experimental results: Fig.17 The results of serum test of 186-BEVs-H5 in animal experiments after one immunization (nasal drops, eye drops and oral administration) were shown. Two weeks after one immunization (nasal drops, eye drops and oral administration) of 186-BEVs-H5, the serum specific HI antibody titer reached or exceeded the immune critical value of 4log2; three weeks later, the serum specific anti-H5 antibody titer reached 6 to 8log2. The results showed that one immunization (nasal drops, eye drops) of 186-BEVs-H5 successfully induced a systemic immune response and produced specific protective serum antibodies.

Claims

1. A strain of Lactobacillus reuteri ( Lactobacillus reuteri ) strain, named C186, 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. 30337, and the deposit date is April 15, 2024.

2. Use of the Lactobacillus reuteri strain according to claim 1 in the following aspects: (1) Preparation of immune adjuvants or preparations for enhancing immunity; (2) Preparation of anti-inflammatory and anti-infective drug preparations or preparation of drugs for regulating animal intestinal flora or promoting animal growth.

3. The use according to claim 2, characterized in that The immune adjuvant or immune enhancement preparation has the function of improving mucosal immune response and promoting mucosal secretion of specific antibodies.

4. A Lactobacillus reuteri exovesicle, characterized in that The Lactobacillus reuteri is extracted from the culture solution of the Lactobacillus reuteri strain C186 described in claim 1.

5. The exosome according to claim 4, characterized in that The exosomes are prepared by the following method: the bacterial liquid of the Lactobacillus reuteri strain C186 is inoculated into a liquid MRS medium at a ratio of 1:100, and after culturing for 48 hours, the liquid is centrifuged at 3000-3500 g and 4°C for 10 min, and the process is repeated 1-2 times to retain the supernatant; the supernatant is passed through a 0.45 μm hollow fiber sieve, the filtered liquid is collected, and the liquid is centrifuged at 35000 g and 4°C for 1 hour to retain the supernatant; the supernatant is centrifuged at 120000 g and 4°C for 1 hour to remove the supernatant, and the precipitate is resuspended with PBS at pH 8.0, and the precipitate is temporarily stored at 4°C and stored at -80°C.

6. The exosome according to claim 5, characterized in that The method also includes the step of purifying the extracted exosomes by glucose density gradient centrifugation.

7. The exosome according to claim 5, characterized in that The purity of the glucose used in the glucose density gradient centrifugation method is, by mass percentage, 5 ml of 60% glucose solution, 10 ml of 40% glucose solution, 25 ml of 30% glucose solution, 10 ml of 20% glucose solution, 5 ml of 5% glucose solution + 5 ml of extracellular vesicle solution, centrifuged at 100,000 g for 18 h, and the extracellular vesicle solutions of different layers were taken and supplemented with extracellular vesicle preservation solution to 60 ml, centrifuged at 120,000 g-130,000 g for 1.5 h, the supernatant was removed, and the precipitate was resuspended to 300-500 μL with PBS at pH 8.

0.

8. Use of the Lactobacillus reuteri exovesicles according to any one of claims 4 to 7 in the following aspects: (1) Preparation of immune adjuvants or mucosal delivery vectors; (2) Preparation of mucosal immune preparations; (3) Preparation of immunomodulatory preparations; (4) Preparation of anti-inflammatory and anti-infective drug preparations or preparation of drugs for regulating animal intestinal flora or promoting animal growth.

9. The use according to claim 8, characterized in that The mucosal delivery carrier is used to deliver all components or subunit components of antigens from inactivated bacteria or inactivated viruses, and the mucosal delivery carrier can be used for intestinal immunization or oral immunization.

10. The use according to claim 8, characterized in that The mucosal immune preparation is a mucosal immune vaccine.

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