Outer vesicle of bifidobacterium longum and application thereof

Abstract

The invention discloses an outer vesicle of bifidobacterium longum. The outer vesicle is obtained by carrying out anaerobic culture on bifidobacterium longum AY15 through a BS liquid culture medium and then centrifuging. The invention also relates to the effect of the bifidobacterium longum outer vesicle in preparation of drugs for enhancing immune systems and the like. The exovesicles of bifidobacterium longum can change the composition of intestinal flora, influence the metabolism of short-chain fatty acid and activate a short-chain fatty acid receptor, so that the immune system is regulated, the generation of related immune cells is regulated, the balance of the immune cells is promoted, and the immune injury caused by cyclophosphamide is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and provides an extracellular vesicle of Bifidobacterium longum and its application. Background Art

[0002] The immune system consists of immune organs, immune cells, immune active substances, etc., and can prevent the invasion of pathogens through immune responses to maintain the environmental stability in the human body. However, when the function of the immune system is reduced, the phenomenon of immunosuppression will occur, the immune response and protection function will be reduced, and then the body will develop immune system diseases.

[0003] Extracellular vesicles are small molecules that play important roles in cell communication, immune response, etc., and can activate people's innate immunity and adaptive immunity. They naturally contain a series of highly immunostimulatory ligands, called pathogen-associated molecular patterns (PAMPs), which can be recognized by pathogen recognition receptors (PRRs) on epithelial cells and immune cells. The activation of PRRs induces an innate immune response, which can produce pro-inflammatory factors and chemokines to recruit immune cells. In addition, since extracellular vesicles can carry PAMPs distally throughout the body as carriers, they are inherently effective in activating the systemic innate immune response of the host.

[0004] Bifidobacterium is a common probiotic and is considered an important component of the human gut microbiota and the most abundant species in the infant gut. Breast milk contains a large number of bacterial species and a unique microbiome, including Bifidobacterium, which can help promote the maturation of infant intestinal cells and improve the intestinal immune system. Since Bifidobacterium has multiple functions such as regulating immunity, anti-tumor, and anti-inflammatory effects, it is considered an immune regulator or biomarker for human diseases and is applied to the prevention and adjuvant treatment of various diseases.

[0005] Currently, the method of directly using live probiotic preparations to regulate the immune system has certain limitations. The survival rate of the strains may decrease after digestion in the gastrointestinal tract, and they cannot exert their due regulatory effects. Moreover, other substances in the strains may have certain effects on immunodeficient populations. Therefore, there is an urgent need to develop a natural immune regulator with stability and safety. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides an extracellular vesicle of Bifidobacterium longum, which is the extracellular vesicle of Bifidobacterium longum AY15, can change the composition of the intestinal flora, affect short-chain fatty acid metabolism, activate short-chain fatty acid receptors, thereby regulating the immune system, regulating the production of related immune cells, promoting the balance of immune cells, and improving the immune damage caused by cyclophosphamide.

[0007] To achieve the above object, the technical solution provided by the present invention is as follows:

[0008] An outer vesicle of Bifidobacterium longum is obtained by anaerobic culture of Bifidobacterium longum AY15 in BS liquid medium followed by centrifugation. The preparation method of the outer vesicle of Bifidobacterium longum comprises the following operation steps:

[0009] (1) The activated Bifidobacterium longum AY15 bacterial liquid is inoculated into 200 mL of BS liquid medium at an inoculation amount of 5% (v / v) and anaerobically cultured at 37 °C for 48 hours. The cultured bacterial liquid is aliquoted into 50 mL EP tubes and centrifuged at 10,000×g for 30 minutes to remove the precipitate, and the supernatant is retained.

[0010] (2) The outer vesicles are obtained by sucrose density ultracentrifugation. Centrifuge at 120,000×g for 90 minutes at 4 °C, take out the fraction with a sucrose concentration of 30% (w / v) - 60% (w / v), dilute this fraction with PBS, and further centrifuge (4 °C, 120,000×g, 90 minutes), discard the supernatant, and resuspend the precipitate to obtain the outer vesicles of Bifidobacterium longum AY15.

[0011] Preferably, the Bifidobacterium longum AY15 bacterial liquid in step (1) is the bacterial liquid of Bifidobacterium longum AY15 strain, and the preservation number of the Bifidobacterium longum AY15 strain is GDMCC NO: 65457.

[0012] Preferably, the activated Bifidobacterium longum AY15 bacterial liquid in step (1) is obtained by taking the cryopreserved Bifidobacterium longum AY15 bacteria, inoculating them onto the BS solid medium with a sterile inoculation loop, anaerobically culturing at 37 °C for 72 hours, then picking a single colony on the BS solid medium and inoculating it into the BS liquid medium, anaerobically culturing at 37 °C for 48 hours, inoculating the cultured bacterial liquid into the BS liquid medium at an inoculation amount of 5% (v / v), anaerobically culturing at 37 °C for 48 hours, and repeating the anaerobic culture in the BS liquid medium once, that is, the anaerobic culture in the BS liquid medium is carried out 2 times in total, which is the activated Bifidobacterium longum AY15 bacterial liquid.

[0013] Preferably, the BS liquid medium is obtained by weighing 48.92 g of Bifidobacterium BS medium, adding 1 mL of Tween 80, heating and dissolving it in 1000 mL of distilled water, and autoclaving at 116 °C for 30 minutes; the BS solid medium is obtained by weighing 48.92 g of Bifidobacterium BS medium, adding 1 mL of Tween 80 and 20 g of agar powder, heating and dissolving it in 1000 mL of distilled water, autoclaving at 116 °C for 30 minutes, and pouring the plate after cooling to an appropriate temperature, which is the BS solid medium.

[0014] The role of the outer vesicle of Bifidobacterium longum as described above in the preparation of drugs for enhancing the immune system.

[0015] Furthermore, the immune system enhancement system is to increase immunoglobulins, or enhance the expression of transcription factors Foxp3, T-bet, and GATA3, induce Th cell differentiation, and regulate the Th1 / Th2 balance, thereby regulating the immune system.

[0016] Use of the outer vesicles of Bifidobacterium longum as described above in the preparation of a drug for promoting the secretion of short-chain fatty acids by the intestinal flora. The outer vesicles of Bifidobacterium longum can regulate the intestinal flora, increase the abundance of short-chain fatty acid-producing flora, increase the content of short-chain fatty acids, and enhance the immune system.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) The outer vesicles extracted from Bifidobacterium longum AY15 of the present invention can alleviate the immune damage caused by cyclophosphamide to the spleen and thymus, and can restore the decrease in the content of immunoglobulins caused by cyclophosphamide, and promote the secretion of IgA, IgG, and IgM. It can also increase the mRNA expression levels of Foxp3, T-bet, and GATA3 in the spleen, alleviate the immunosuppression caused by cyclophosphamide, and increase the abundance of beneficial bacteria such as g__Alistipes and g__Bacteroides, promote the secretion of short-chain fatty acids in the intestine, activate the short-chain fatty acid receptors GPR41 and GPR43, and regulate the immune system. The outer vesicles of Bifidobacterium longum AY15 of the present invention have the effects of regulating the immune system and enhancing immunity, and can be used to improve immune function, assist in the treatment of immune system diseases, and improve the immunosuppression situation.

[0019] (2) After being digested by simulated gastric juice and simulated intestinal juice, about 50% of the outer vesicles of Bifidobacterium longum AY15 of the present invention still maintain a size between 100-200 nm, showing stability, ensuring that they can play a role in regulating immunity after entering the intestine.

[0020] Description of Preservation Information

[0021] Bifidobacterium longum AY15 was deposited at the Guangdong Provincial Microbial Culture Collection Center (abbreviation: GDMCC) on November 11, 2024, with the deposit number GDMCC NO: 65457. Description of the Drawings

[0022] Figure 1 It is the structural characterization of the outer vesicles of Bifidobacterium longum AY15 of the present invention: (A) Transmission electron microscopy results; among them, the red arrow: outer vesicles, (B) Nanoparticle tracking analysis results, (C) Protein distribution.

[0023] Figure 2 It is the stability analysis of the outer vesicles of Bifidobacterium longum AY15 of the present invention.

[0024] Figure 3 It is the effect of the outer vesicles of Bifidobacterium longum AY15 of the present invention on the organ indices of mice: (A) spleen index and (B) thymus index.

[0025] Figure 4 It is the H&E staining of spleen tissue; among them, the red arrow: red pulp, and the white arrow: white pulp.

[0026] Figure 5 It is the content of three immunoglobulins in serum.

[0027] Figure 6 It is the mRNA expression level of genes related to immune regulation in the spleen.

[0028] Figure 7 It is the differential microbiota based on LDA scores.

[0029] Figure 8 It is the effect of EVs on short-chain fatty acids and GPR41 / 43 signaling pathways in mice: (A) the content of short-chain fatty acids in mouse feces, (B) the mRNA expression level of GPR41 / 43 in colon tissue. Detailed implementation manners

[0030] The following is a detailed description in combination with the specific implementation manners in the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. The raw materials and reagents used in the examples are all commercially available unless otherwise specified. The Bifidobacterium BS medium is purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0031] Example 1

[0032] Preparation of outer vesicles of Bifidobacterium longum AY15

[0033] (1) Strain information:

[0034] The obtained Bifidobacterium longum AY15 after screening, separation, and purification was inoculated into BS liquid medium at an inoculation amount of 5% (v / v) and anaerobically cultured at 37°C for 48 hours. After the culture, the bacterial solution was mixed with 50% glycerol at a ratio of 1:1 and stored at -80°C. Bifidobacterium longum AY15 was deposited at the Guangdong Microbial Culture Collection Center on November 11, 2024, with the deposit number GDMCC NO: 65457. Amplification was carried out according to the bifidobacterium-specific primers lm3 - 5’-CGGGTGCTYCCCACTTTCATG-3’ and lm26 - 5’-GATTCTGGCTCAGGATGAACG-3’, and the obtained 16S rDNA gene sequence is shown as SEQ ID NO.1.

[0035] (2) Cultivation of Bifidobacterium longum AY15

[0036] BS liquid medium: Weigh 48.92 g of bifidobacterium BS medium, add 1 mL of Tween 80, heat and dissolve it in 1000 mL of distilled water, and autoclave at 116°C for 30 minutes.

[0037] BS solid medium: Weigh 48.92 g of bifidobacterium BS medium, add 1 mL of Tween 80 and 20 g of agar powder, heat and dissolve it in 1000 mL of distilled water, autoclave at 116°C for 30 minutes, and pour plates after cooling to an appropriate temperature.

[0038] Strain resuscitation and activation: Take the Bifidobacterium longum AY15 bacteria frozen at -80°C with glycerol as described above, inoculate them onto BS solid medium with a sterile inoculation loop, and anaerobically culture at 37°C for 72 hours. Pick a single colony on the BS solid medium and inoculate it into BS liquid medium, anaerobically culture at 37°C for 48 hours. The cultured bacterial solution was inoculated into BS liquid medium at an inoculation amount of 5% (v / v) and anaerobically cultured at 37°C for 48 hours. Repeat the anaerobic culture in BS liquid medium once, that is, the anaerobic culture in BS liquid medium was carried out a total of 2 times, which is the activated Bifidobacterium longum AY15 bacterial solution.

[0039] (3) Extraction of outer vesicles of Bifidobacterium longum AY15

[0040] The activated Bifidobacterium longum AY15 bacterial solution was inoculated into 200 mL of BS liquid medium at an inoculation amount of 5% (v / v) and anaerobically cultured at 37 °C for 48 hours. The cultured bacterial solution was aliquoted into 50 mL EP tubes and centrifuged at 10,000×g for 30 minutes to remove the precipitate, and the supernatant was retained. Sucrose solutions of 8% (w / v), 30% (w / v), and 60% (w / v) were respectively prepared using 0.01 mol / L PBS solution, and extracellular vesicles were obtained by sucrose gradient ultracentrifugation. Centrifugation was performed at 120,000×g for 90 minutes at 4 °C, and the fraction with a sucrose concentration of 30% (w / v) - 60% (w / v) was taken out. This fraction was diluted with PBS and further centrifuged (4 °C, 120,000×g, 90 minutes), the supernatant was discarded, and the precipitate was resuspended, which was the extracellular vesicles of Bifidobacterium longum AY15.

[0041] Example 2

[0042] Morphological and Structural Characterization of Extracellular Vesicles of Bifidobacterium longum AY15

[0043] Transmission Electron Microscopy Detection: Take the extracellular vesicles of Bifidobacterium longum AY15 obtained in Example 1(3), adjust to 1×10 10 particles / mL, gently drop it onto a copper mesh with a common carbon film, after 5 min, use absorbent paper to remove the liquid on the copper mesh, and use 2% phosphotungstic acid for negative staining for 5 min and then detect it on the machine.

[0044] Nanoparticle Tracking Analysis: Take the extracellular vesicles of Bifidobacterium longum AY15, adjust to 1×10 10 particles / mL, and then inject it into a nanoparticle analyzer for detection on the machine.

[0045] Detection of Protein Distribution: Take 16 μL of extracellular vesicles of Bifidobacterium longum AY15 and add 4 μL of SDS-PAGE protein loading buffer (5×), mix well and put it into a metal bath, heat at 95 °C for 10 min. Prepare the stacking gel and separating gel respectively, inject them into the gel plate and wait for solidification to obtain the denatured protein sample. After adding the denatured protein sample and protein Marker into the gel wells, perform electrophoresis at 80 V for 20 minutes, and then perform electrophoresis at 150 V until the smallest band reaches the bottom and then end the electrophoresis. Take out the gel after electrophoresis, put it into Coomassie Brilliant Blue staining solution, and stain it on a shaker for 1 hour. Put the stained gel into the decolorizing solution and decolorize it on a shaker for 12 hours.

[0046] The results are as Figure 1 (A) shows that the morphology of the extracellular vesicles of Bifidobacterium longum AY15 can be visually seen through transmission electron microscopy. The extracellular vesicles are round or oval particles, with a size between 100 - 150 nm, and have a phospholipid bilayer structure. Figure 1(B) The nanoparticle tracking data shows that there is only one peak in the size distribution graph of the extracellular vesicles, and its highest peak is 152 nm, indicating that the particle distribution is uniform, and the average particle size is 164.2 ± 0.7 nm. Figure 1 (C) shows the protein distribution of the extracellular vesicles. The molecular weights of the proteins contained in the extracellular vesicles are mainly distributed around 40 kDa and 70 kDa.

[0047] Example 3

[0048] Stability of extracellular vesicles of Bifidobacterium longum AY15

[0049] Prepare simulated gastric juice (2000 U / mL): Adjust the pH value of PBS to 3 with 6 mol / L HCl, add pepsin, dissolve it thoroughly, store it at 4 °C for later use, and filter and sterilize it with a sterile water-based filter membrane before use.

[0050] Prepare simulated intestinal juice (100 U / mL): Adjust the pH value of PBS to 8 with 0.1 mol / L NaOH, add trypsin and bile salts, dissolve it thoroughly, store it at 4 °C for later use, and filter and sterilize it with a sterile water-based filter membrane before use.

[0051] Dilute the extracellular vesicles of Bifidobacterium longum AY15 with PBS to 1×10 9 particles / mL, and then add them to the simulated gastric juice at a ratio of 10% (v / v) and digest at 37 °C for 3 hours. Subsequently, add the mixture digested by the simulated gastric juice to the simulated intestinal juice at a ratio of 10% (v / v) and continue to digest at 37 °C for 3 hours. After completion, take samples, dilute the samples of undigested extracellular vesicles, the mixture digested by the simulated gastric juice, and the mixture digested by the simulated intestinal juice to the same multiple, and use a Malvern particle size analyzer to compare the particle sizes of the extracellular vesicles before and after simulated digestion.

[0052] The results are as Figure 2 shown. The highest peak of the undigested extracellular vesicles in vitro is 122 nm, and most of the particles are between 100 - 200 nm. After the extracellular vesicles are digested by gastric juice, the particles generally become larger, and the highest peak is 166 nm. Some extracellular vesicles deform, but the sizes of most extracellular vesicles still remain between 100 - 200 nm. The changes of the extracellular vesicles digested by gastric juice and intestinal juice are more obvious, and the highest peak increases significantly, about 200 nm. It is thus found that most of the extracellular vesicles show obvious morphological changes, but about 50% of the extracellular vesicles still have a state between 100 - 200 nm. Therefore, most of the extracellular vesicles of Bifidobacterium longum AY15 can pass through the digestion of simulated gastric juice, while the digestion of simulated intestinal juice will cause obvious changes in some extracellular vesicles. Although the extracellular vesicles have low tolerance to simulated intestinal juice, about 50% of the extracellular vesicles still do not show obvious deformation, indicating that they can still enter the intestine and play their functions.

[0053] Example 4

[0054] Effect of Exosomes of Bifidobacterium longum subsp. longum AY15 on Regulating the Immune System

[0055] (1) Animal Model

[0056] Thirty 6-week-old specific pathogen-free (SPF) C57BL / 6J mice were purchased from Beijing Speywood Biotechnology Co., Ltd. and acclimated for 1 week. All animals were housed in an experimental animal room with a 12-hour light-dark cycle, at a room temperature of 20 - 25°C and a relative humidity of 30 - 70%, with free access to water and food.

[0057] After the acclimation period, the 30 C57BL / 6J mice were randomly divided into three groups of 10 mice each. The mice were subjected to intraperitoneal injection for three days to establish a model. Among them, the blank group (NC) was injected with normal saline, and the model group (MC) and the exosome intervention group of Bifidobacterium longum subsp. longum AY15 (EV) were injected with cyclophosphamide (CTX, 100 mg / kg) solution. After the modeling, the blank group and the model group were continuously gavaged with sterile normal saline for seven days, while the exosome intervention group of Bifidobacterium longum subsp. longum AY15 was continuously gavaged with exosomes of Bifidobacterium longum subsp. longum AY15 at a concentration of 2×10 9 per mouse.

[0058] (2) Exosomes of Bifidobacterium longum subsp. longum AY15 Alleviate Damage in Immune Organs

[0059] After the experiment, the spleen and thymus tissues of the mice were removed, weighed and recorded. HE staining was performed on the spleen tissue samples to observe the changes in the spleen tissue.

[0060] Spleen index and thymus index are often used to reflect the degree of lymphocyte proliferation in immune organs and are also indicators for judging the strength of immune function. The results are as Figure 3 shown. It was found that the spleen index of the MC group increased significantly, while after exosome intervention, this situation could be alleviated to a certain extent. The thymus index of the MC group decreased significantly, indicating that its immune function was inhibited, while after exosome intervention, the thymus index increased significantly. The above results show that exosomes of Bifidobacterium longum subsp. longum AY15 can alleviate the immune function inhibition caused by CTX to a certain extent.

[0061] Figure 4 are the H&E staining results of the spleen. It can be seen that the white pulp lymphocytes in the spleen tissue of the NC group are distributed closely, and the boundary with the red pulp is obvious. In the MC group, the number of white pulp lymphocytes decreased, the structure was scattered, and the boundary with the red pulp was unclear. In the EV group, although there was also a decrease in white pulp, compared with the MC group, the boundary between the red pulp and white pulp was clear, and the structure was not as scattered and the boundary was not as disordered as in the MC group. Therefore, exosomes of Bifidobacterium longum subsp. longum AY15 can alleviate the spleen damage caused by CTX to a certain extent.

[0062] (3) Outer vesicles of Bifidobacterium longum AY15 regulate immunoglobulins

[0063] Immunoglobulins in serum were detected using an enzyme-linked immunosorbent assay kit purchased from Nanjing Jiancheng Bioengineering Institute.

[0064] Immunoglobulins are one of the main components of the immune system and can be used to understand the immune function of the body, such as the resistance to various viruses and bacteria and the body's recognition ability of various antigen invasions. Figure 5 The contents of immunoglobulins in the sera of mice in different groups are shown. The results indicate that the contents of the three immunoglobulins (IgA, IgG, IgM) in the MC group after CTX intervention were significantly decreased, indicating that the immune system was significantly inhibited after CTX intervention. However, the intervention with outer vesicles of Bifidobacterium longum AY15 could alleviate this phenomenon, and the recovery of immunoglobulin IgG was the most significant. It can be seen that the intervention with outer vesicles of Bifidobacterium longum AY15 can alleviate the phenomenon of decreased immunoglobulin content caused by CTX.

[0065] (4) Outer vesicles of Bifidobacterium longum AY15 regulate the expression of immune-related genes

[0066] Weigh 50 - 100 mg of the mouse spleen tissue in Example 4(2), and extract total RNA using the Trizol method. React and synthesize cDNA according to the instructions of the BeyoRT TM Q First Strand cDNA Synthesis Kit. Perform RT-qPCR reactions using SYBR Green on a CFX96 (Bio-Rad) instrument. Each sample was normalized with the internal reference GAPDH, and the relative gene expression levels were calculated by the 2 -ΔΔCT -ΔΔCt method.

[0067] Foxp3 is one of the important markers of regulatory T cells (Tregs), a transcription factor of Treg cells, and mainly plays an immune regulatory role by mediating the balance of Treg / Th17. T-bet is a transcription factor of the T-box family and mainly drives the differentiation of Th1 cells by regulating cytokines such as IFN-γ. GATA3 is mutually exclusive with T-bet and plays a corresponding antagonistic role, and its main function is to drive the differentiation of Th2 cells. The results are as Figure 6As shown, compared with the NC group, the mRNA levels of Foxp3, T-bet, and GATA3 in the MC group were all significantly decreased. Compared with the MC group, the mRNA levels of Foxp3, T-bet, and GATA3 in the EV group were all significantly increased. It was thus found that the intervention of the outer vesicles of Bifidobacterium longum AY15 could relieve the inhibition of related immune factors caused by CTX, indicating that the outer vesicles relieve the immunosuppression caused by CTX by promoting the differentiation of Th1 cells and Th2 cells and regulating the Th1 / Th2 balance.

[0068] Example 5

[0069] Effect of Outer Vesicles of Bifidobacterium longum AY15 on Intestinal Flora

[0070] Mouse feces were collected one day before the end of the animal experiment, and the fecal samples were subjected to 16S rDNA determination.

[0071] As Figure 7 shown, by analyzing the differential flora between the MC group and the EV group through linear discriminant analysis effect size (LEfSe) (LDA score > 2 and p < 0.05), it was found that after the intervention of the outer vesicles of Bifidobacterium longum AY15, the abundances of g__Alistipes and g__Bacteroides in the mouse intestine increased, while the abundance of g__Enterorhabdus decreased. g__Bacteroides and g__Alistipes are producers of short-chain fatty acids in the human intestine, and short-chain fatty acids have anti-inflammatory effects and contribute to maintaining the stability of the immune system. Therefore, the outer vesicles of Bifidobacterium longum AY15 can regulate the intestinal flora disorder caused by CTX, possibly by promoting the growth of flora that produce short-chain fatty acids and increasing the content of short-chain fatty acids in the intestine, thereby regulating the immune system.

[0072] Example 6

[0073] Effect of Outer Vesicles of Bifidobacterium longum AY15 on Short-chain Fatty Acid Metabolism

[0074] Short-chain fatty acid detection: Add the fecal samples of mice collected from animal experiments to 1% formic acid solution and mix well. Store at -20°C for 2 hours, thaw and centrifuge to obtain the supernatant. Add an equal volume of ethyl acetate to the supernatant for extraction, take the ethyl acetate phase and add 4-methylvaleric acid as the internal standard. Mix well, dry and filter through a 0.22 μm organic filter membrane, and store at -20°C for detection. Detection is carried out using GC-MS equipped with a TR-Wax chromatographic column. The detection conditions are as follows: ion source temperature 250°C, injection port temperature 250°C; initial temperature 90°C, rise to 150°C at a rate of 15°C / min, then rise to 170°C at a rate of 5°C / min, and finally rise to 250°C at a rate of 20°C / min and hold for 2 min; the carrier gas is helium, with a flow rate of 1 mL / min. The split ratio is 1:1, and the injection volume is 1 μL. The detector is set to electron ionization mode, with an electron energy of 70 eV, a solvent retention time of 3.5 min, and a scanning range of 10 - 350 m / z.

[0075] To determine the effect of the outer vesicles of Bifidobacterium longum AY15 on short-chain fatty acid metabolism, the levels of short-chain fatty acids in feces after outer vesicle intervention were detected. The results are as Figure 8 (A) shown. Compared with the NC group, the contents of all short-chain fatty acids in the MC group were significantly lower than those in the NC group. And compared with the MC group, after the intervention of outer vesicles, the contents of n-valeric acid, acetic acid, isobutyric acid, propionic acid and n-butyric acid were all significantly higher than those in the MC group, while the change in the content of isovaleric acid was not significant accordingly, but the overall trend was that the short-chain fatty acids in the EVs treatment group were higher than those in the MC group.

[0076] Short-chain fatty acid receptors (GPR41 / 43) belong to G protein-coupled receptors. GPR43 is also known as free fatty acid receptor 2 (FFAR2) and is expressed on a variety of immune cells. GPR41 is also called free fatty acid receptor 3 (FFAR3) and is mainly expressed by a part of enteroendocrine cells in the intestinal epithelium. GPR41 and GPR43 can bind to short-chain fatty acids and participate in the regulation of inflammatory responses. Therefore, we detected the expression of short-chain fatty acid receptors (GPR41 / 43), and the results are as Figure 8 (B) shown. Compared with the NC group, the expression of GPR41 / 43 in the MC group was significantly down-regulated, while the EV group significantly up-regulated the expression of GPR41 / 43. The results indicate that the outer vesicles of Bifidobacterium longum AY15 can regulate the metabolism of short-chain fatty acids, increase the content of short-chain fatty acids, and then activate short-chain fatty acid receptors (GPR41 / 43) and play corresponding immunomodulatory roles.

[0077] The foregoing description of the specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the present invention, as well as various different selections and modifications. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. An exosome of Bifidobacterium longum, characterized in that: The exosomes of Bifidobacterium longum AY15 are obtained by centrifugation after anaerobic culture of BS liquid medium. The preparation method of the exosomes of Bifidobacterium longum comprises the following steps: (1) The activated Bifidobacterium longum AY15 bacterial suspension was inoculated into BS liquid culture medium for anaerobic culture for 1 h, and the obtained bacterial suspension was centrifuged to retain the supernatant; (2) The exosomes were obtained by sucrose gradient ultracentrifugation, and the fraction with a sucrose concentration of 30% to 60% was taken out by centrifugation. The fraction was diluted with PBS, further centrifuged, the supernatant was discarded, and the precipitate was resuspended to obtain the exosomes of Bifidobacterium longum AY15.

2. The exosome of Bifidobacterium longum according to claim 1, characterized in that: The bacterial solution of Bifidobacterium longum AY15 described in step (1) is the bacterial solution of Bifidobacterium longum AY15 strain, and the preservation number of the Bifidobacterium longum AY15 strain is GDMCCNO:65457.

3. The exosome of Bifidobacterium longum according to claim 1, characterized in that: In step (1), the activated Bifidobacterium longum AY15 bacterial liquid is inoculated into BS liquid culture medium at a 5% inoculation rate and anaerobically cultured at 37° C. for 48 hours.

4. The exosome of Bifidobacterium longum according to claim 1, characterized in that: The activated Bifidobacterium longum AY15 bacterial liquid described in step (1) is obtained by taking Bifidobacterium longum AY15 bacteria, inoculating it on BS solid culture medium, and culturing it anaerobically at 37°C for 72 hours, then picking a single colony on the BS solid culture medium and inoculating it into BS liquid culture medium, and culturing it anaerobically at 37°C for 48 hours, inoculating the cultured bacterial liquid into BS liquid culture medium at an inoculum amount of 5%, and culturing it anaerobically at 37°C for 48 hours. Repeating the anaerobic culture of BS liquid culture medium once, the activated Bifidobacterium longum AY15 bacterial liquid is obtained.

5. The exosome of Bifidobacterium longum according to claim 1 or 4, characterized in that: The BS liquid culture medium is obtained by weighing 48.92 g of Bifidobacterium BS culture medium, adding 1 mL of Tween 80, heating and dissolving in 1000 mL of distilled water, and sterilizing at 116° C. for 30 minutes; the BS solid culture medium is obtained by weighing 48.92 g of Bifidobacterium BS culture medium, adding 1 mL of Tween 80 and 20 g of agar powder, heating and dissolving in 1000 mL of distilled water, sterilizing at 116° C. for 30 minutes, and pouring onto a plate after cooling to an appropriate temperature, thereby obtaining the BS solid culture medium.

6. The role of the exosomes of Bifidobacterium longum as claimed in any one of claims 1 to 5 in the preparation of drugs for enhancing the immune system.

7. The effect of the exosomes of Bifidobacterium longum in the preparation of a drug for enhancing the immune system according to claim 6, characterized in that: The immunity enhancement system increases immunoglobulins, or enhances the expression of Foxp3, T-bet and GATA3 transcription factors, induces Th cell differentiation, and regulates the Th1 / Th2 balance, thereby regulating the immune system.

8. Use of the exosomes of Bifidobacterium longum as claimed in any one of claims 1 to 5 in the preparation of a drug for promoting the secretion of short-chain fatty acids by intestinal flora.

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