Extracellular vesicles of bifi dobacterium longum and uses thereof
By preparing the outer vesicles of Bifidobacterium longum AY15, the problem of low survival rate of probiotics after digestion in the gastrointestinal tract was solved, achieving stable and safe immunomodulation, alleviating immune damage, restoring immune function, regulating intestinal flora and short-chain fatty acid metabolism, and promoting immune system balance.
Patent Information
- Application Number
- CN202510370064.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2026-05-15
- Estimated Expiration
- 2045-03-27
AI Technical Summary
Existing technologies that directly use probiotics to regulate the immune system have problems such as low survival rate of strains after digestion in the gastrointestinal tract and potential impact on immunocompromised individuals. There is an urgent need to develop stable and safe natural immunomodulators.
The outer vesicles of Bifidobacterium longum AY15 were extracted and prepared through anaerobic culture, centrifugation and other steps. The preparation method included sucrose gradient ultracentrifugation. The obtained outer vesicles were used to regulate the immune system, activate short-chain fatty acid receptors and regulate the production of related immune cells.
Bifidobacterium longum AY15 exovesicles can alleviate immune damage caused by cyclophosphamide, restore immunoglobulin levels, increase the expression of Foxp3, T-bet and GATA3 in the spleen, increase the abundance of beneficial bacteria, promote the secretion of short-chain fatty acids, regulate the immune system, and have stability, ensuring that they play a regulatory role in the gut.
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Figure CN120192883B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to an external vesicle of Bifidobacterium longum and its applications. Background Technology
[0002] The immune system, composed of immune organs, immune cells, and immune-active substances, prevents the invasion of pathogens and maintains homeostasis within the body through immune responses. However, when the function of the immune system is reduced, immunosuppression occurs, decreasing the immune response and protective function, which can lead to immune system diseases.
[0003] Extracellular vesicles are small molecules that play important roles in cell communication and immune responses, activating both innate and adaptive immunity. They naturally contain a series of highly immunostimulatory ligands known as pathogen-associated molecular patterns (PAMPs), which can be recognized by pathogen recognition receptors (PRRs) on epithelial and immune cells. Activation of PRRs induces an innate immune response, producing pro-inflammatory and chemokine-producing factors and recruiting immune cells. Furthermore, because extracellular vesicles act as carriers, they can transport PAMPs to various parts of the body, thus possessing inherent effectiveness in activating the host's systemic innate immune response.
[0004] Bifidobacteria are common probiotics, considered an important component of the human gut microbiota, and the most abundant species in the infant's gut. Breast milk contains a large number of bacterial species and a unique microbiome, including Bifidobacteria, which help promote the maturation of infant intestinal cells and develop a robust intestinal immune system. Because Bifidobacteria have multiple functions, including immune regulation, anti-tumor activity, and anti-inflammation, they are considered immunomodulators or biomarkers for human diseases and are used in 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, failing to exert their intended regulatory effects. Furthermore, other substances in the strains may have certain effects on immunocompromised individuals. Therefore, there is an urgent need to develop a stable and safe natural immunomodulator. Summary of the Invention
[0006] To address the aforementioned technical problems, this invention provides an outer vesicle of Bifidobacterium longum AY15, which can alter 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 immune damage caused by cyclophosphamide.
[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows:
[0008] An external vesicle of *Bifidobacterium longum* is obtained by centrifugation after anaerobic culture of *Bifidobacterium longum* AY15 in BS liquid medium. The preparation method of the external vesicle of *Bifidobacterium longum* includes the following steps:
[0009] (1) The activated Bifidobacterium longum AY15 bacterial culture was inoculated into 200mL BS liquid medium at an inoculation rate of 5% (v / v) and anaerobic cultured at 37℃ for 48 hours. The bacterial culture obtained after culture was dispensed into 50mL EP tubes, centrifuged at 10000×g for 30 minutes, the precipitate was removed, and the supernatant was retained.
[0010] (2) The outer vesicles were obtained by sucrose gradient ultracentrifugation. The fraction with sucrose concentration of 30% (w / v)-60% (w / v) was taken out at 4℃ and centrifuged at 120000×g for 90 minutes. The fraction was diluted with PBS and centrifuged again (4℃, 120000×g, 90 minutes). The supernatant was discarded and the precipitate was resuspended to obtain the outer vesicles of Bifidobacterium longum AY15.
[0011] Preferably, the Bifidobacterium longum AY15 bacterial solution mentioned in step (1) is a bacterial solution 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 culture in step (1) is obtained by taking frozen Bifidobacterium longum AY15 bacteria, inoculating it onto BS solid medium with a sterile inoculation loop, and anaerobically culturing it at 37°C for 72 hours. Then, a single colony on the BS solid medium is picked and inoculated into BS liquid medium, and anaerobically cultured at 37°C for 48 hours. The resulting bacterial culture is then inoculated into BS liquid medium at an inoculation rate of 5% (v / v), and anaerobically cultured at 37°C for 48 hours. The anaerobic culture on BS liquid medium is repeated once, that is, the anaerobic culture on BS liquid medium is repeated twice, which is the activated Bifidobacterium longum AY15 bacterial culture.
[0013] Preferably, the BS liquid culture medium is obtained by weighing 48.92g of Bifidobacterium BS culture medium, adding 1mL of Tween 80, heating and dissolving in 1000mL of distilled water, and then autoclaving at 116℃ for 30 minutes; the BS solid culture medium is obtained by weighing 48.92g of Bifidobacterium BS culture medium, adding 1mL of Tween 80 and 20g of agar powder, heating and dissolving in 1000mL of distilled water, autoclaving at 116℃ for 30 minutes, and then pouring the mixture into plates after cooling to a suitable temperature.
[0014] The role of the exovesicles of Bifidobacterium longum in the preparation of drugs that enhance the immune system, as described above.
[0015] Furthermore, the immune-enhancing 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 modulating the immune system.
[0016] As described above, the outer vesicles of *Bifidobacterium longum* are used in the preparation of drugs that promote the secretion of short-chain fatty acids by intestinal flora. The outer vesicles of *Bifidobacterium longum* can regulate intestinal flora, increase the abundance of short-chain fatty acid-producing bacteria, 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 extravesicles extracted from *Bifidobacterium longum* AY15 in this invention can alleviate the immune damage to the spleen and thymus caused by cyclophosphamide, and can restore the reduction in immunoglobulin content caused by cyclophosphamide, promoting 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, promoting the secretion of short-chain fatty acids in the intestine, activating short-chain fatty acid receptors GPR41 and GPR43, and regulating the immune system. The extravesicles of *Bifidobacterium longum* AY15 in this invention have the effect 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 immunosuppression.
[0019] (2) After being digested by simulated gastric juice and simulated intestinal juice, about 50% of the outer vesicles of the present invention, Bifidobacterium longum AY15, still maintain a size between 100-200nm, which is stable and ensures that they can play a role in regulating immunity after entering the intestine.
[0020] Preservation Information
[0021] Bifidobacterium longum AY15 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on November 11, 2024, with accession number GDMCC NO: 65457. Attached Figure Description
[0022] Figure 1 The structural characterization of the outer vesicles of Bifidobacterium longum AY15 of this invention is as follows: (A) Transmission electron microscopy results; where red arrows indicate outer vesicles, (B) Nanoparticle tracking analysis results, and (C) Protein distribution.
[0023] Figure 2 This invention relates to the stability analysis of the extravesicles of Bifidobacterium longum AY15.
[0024] Figure 3 The effects of the extravesicles of Bifidobacterium longum AY15 on organ indices in mice are as follows: (A) spleen index and (B) thymus index.
[0025] Figure 4 This is H&E staining of spleen tissue; red arrows indicate red pulp, and white arrows indicate white pulp.
[0026] Figure 5 It refers to the content of three immunoglobulins in the serum.
[0027] Figure 6 It refers to the mRNA expression level of genes related to immune regulation in the spleen.
[0028] Figure 7 It is a differential microbiome based on LDA scores.
[0029] Figure 8 The effects of EVs on short-chain fatty acids and the GPR41 / 43 signaling pathway in mice were investigated: (A) the content of short-chain fatty acids in mouse feces, and (B) the mRNA expression level of GPR41 / 43 in colon tissue. Detailed Implementation
[0030] The following detailed description, in conjunction with the accompanying drawings, outlines specific embodiments. However, it should be understood that the scope of protection of this invention is not limited to these specific embodiments. Unless otherwise specified, all raw materials and reagents used in the examples are commercially available. Bifidobacterium BS culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.
[0031] Example 1
[0032] Preparation of Bifidobacterium longum AY15 exovesicles
[0033] (1) Strain information:
[0034] The screened, isolated, and purified *Bifidobacterium longum* AY15 was inoculated into BS liquid medium at a 5% (v / v) inoculum and anaerobically cultured at 37°C for 48 hours. After culture, the bacterial culture was mixed with 50% glycerol at a 1:1 ratio and stored at -80°C. *Bifidobacterium longum* AY15 was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 11, 2024, with accession number GDMCC NO: 65457. The 16S rDNA gene sequence was amplified using the *Bifidobacterium*-specific primers lm3-5'-CGGGTGCTYCCCACTTTCATG-3' and lm26-5'-GATTCTGGCTCAGGATGAACG-3', and is shown in SEQ ID NO. 1.
[0035] (2) Culture of Bifidobacterium longum AY15
[0036] BS liquid culture medium: Weigh 48.92g of Bifidobacterium BS culture medium, add 1mL of Tween 80, heat to dissolve in 1000mL of distilled water, and autoclave at 116℃ for 30 minutes.
[0037] BS solid medium: Weigh 48.92g of Bifidobacterium BS medium, add 1mL of Tween 80 and 20g of agar powder, heat to dissolve in 1000mL of distilled water, autoclave at 116℃ for 30 minutes, and pour into plates after cooling to the appropriate temperature.
[0038] Bacterial resuscitation and activation: The *Bifidobacterium longum* AY15 strain, frozen with glycerol at -80℃, was inoculated onto BS solid medium using a sterile inoculation loop and anaerobically cultured at 37℃ for 72 hours. Single colonies from the BS solid medium were then inoculated into BS liquid medium and anaerobically cultured at 37℃ for 48 hours. The cultured bacterial suspension was then inoculated into BS liquid medium at a 5% (v / v) inoculation rate and anaerobically cultured at 37℃ for 48 hours. This process was repeated once more, resulting in a total of two anaerobically cultured BS liquid medium cultures, which constitutes the activated *Bifidobacterium longum* AY15 bacterial suspension.
[0039] (3) Extraction of Bifidobacterium longum AY15 exovesicles
[0040] The activated *Bifidobacterium longum* AY15 bacterial culture was inoculated into 200 mL of BS liquid medium at a 5% (v / v) inoculum and anaerobically cultured at 37°C for 48 hours. The cultured bacterial culture was aliquoted into 50 mL EP tubes, centrifuged at 10000×g for 30 minutes, the precipitate was removed, and the supernatant was retained. Sucrose solutions of 8% (w / v), 30% (w / v), and 60% (w / v) were prepared using 0.01 mol / L PBS. The outer vesicles were obtained by sucrose gradient ultracentrifugation, centrifuged at 120000×g for 90 minutes at 4°C, and fractions with sucrose concentrations of 30% (w / v)–60% (w / v) were collected. These fractions were diluted with PBS, centrifuged again (4°C, 120000×g, 90 minutes), the supernatant was discarded, and the precipitate was resuspended; these were the *Bifidobacterium longum* AY15 outer vesicles.
[0041] Example 2
[0042] Morphological and structural characterization of Bifidobacterium longum AY15 extravesicles
[0043] Transmission electron microscopy: Take the Bifidobacterium longum AY15 outer vesicles obtained in Example 1(3) and adjust to 1×10 10 Particles / mL were gently dropped onto a copper grid containing a common carbon film. After 5 minutes, the liquid on the copper grid was removed with absorbent paper. The sample was then negatively stained with 2% phosphotungstic acid for 5 minutes before being tested on the instrument.
[0044] Nanoparticle tracking analysis: Exovesicles of *Bifidobacterium longum* AY15 were taken and adjusted to 1×10⁻⁶. 10 After the particles / mL is measured, it is injected into the nanoparticle analyzer for detection.
[0045] Protein distribution detection: 16 μL of Bifidobacterium longum AY15 exovesicles were added to 4 μL of DS-PAGE protein loading buffer (5×), mixed thoroughly, and then placed in a metal bath and heated at 95℃ for 10 min. Stacking and separating gels were prepared separately, poured into gel plates, and allowed to solidify to obtain denatured protein samples. The denatured protein samples and protein markers were added to the gel wells, and electrophoresis was performed at 80V for 20 minutes, followed by electrophoresis at 150V until the smallest band reached the bottom, at which point the electrophoresis was stopped. The gel was then removed and stained in Coomassie Brilliant Blue staining solution for 1 hour on a shaker. The stained gel was then placed in destaining solution and destained for 12 hours on a shaker.
[0046] The results are as follows Figure 1 As shown in (A), the morphology of the outer vesicles of Bifidobacterium longum AY15 can be directly observed by transmission electron microscopy. The outer vesicles are round or elliptical 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 outer vesicle particle size diagram, with the highest peak being 152 nm, indicating that the particles are uniformly distributed and the average particle size is 164.2 ± 0.7 nm. Figure 1 (C) shows the protein distribution of the outer vesicles. The molecular weight of the proteins contained in the outer vesicles is mainly distributed at around 40kDa and 70kDa.
[0047] Example 3
[0048] Stability of Bifidobacterium longum AY15 exovesicles
[0049] Prepare simulated gastric fluid (2000 U / mL): Adjust the pH of PBS to 3 with 6 mol / L HCl, add pepsin, dissolve thoroughly, store at 4℃ for later use, and filter with a sterile aqueous filter membrane before use.
[0050] Prepare simulated intestinal fluid (100 U / mL): Adjust the pH of PBS to 8 with 0.1 mol / L NaOH, add pancreatin and bile salts, dissolve thoroughly, store at 4°C for later use, and filter with a sterile aqueous filter membrane before use.
[0051] Bifidobacterium longum AY15 exovesicles were diluted with PBS to 1×10⁻⁶. 9 After determining the particle size at 10% (v / v), the mixture was added to simulated gastric juice at 10% (v / v) and digested at 37°C for 3 hours. Subsequently, the mixture digested with simulated gastric juice was added to simulated intestinal juice at 10% (v / v) and digested again at 37°C for 3 hours. Afterward, samples were taken, and the undigested vesicles, the mixture digested with simulated gastric juice, and the mixture digested with simulated intestinal juice were diluted to the same concentration. The particle size of the vesicles before and after simulated digestion was compared using a Malvern particle size analyzer.
[0052] The results are as follows Figure 2 As shown, the highest peak size of the undigested exovesicles was 122 nm, with most particles falling between 100-200 nm. After digestion by gastric juice, the exovesicles generally enlarged, with the highest peak reaching 166 nm. Some exoves underwent deformation, but most remained within the 100-200 nm range. The changes in exoves digested by both gastric and intestinal juices were more pronounced, with the highest peak significantly increasing to approximately 200 nm. This indicates that while most exoves showed significant morphological changes, about 50% remained within the 100-200 nm range. Therefore, most *Bifidobacterium longum* AY15 exoves can be digested by simulated gastric juice, while simulated intestinal juice digestion causes significant changes in some exoves. Although the exoves showed poor tolerance to simulated intestinal juice, approximately 50% remained unchanged, indicating they can still enter the intestines and exert their effects.
[0053] Example 4
[0054] The role of Bifidobacterium longum AY15 exovesicles in regulating the immune system
[0055] (1) Animal model
[0056] Thirty 6-week-old specific pathogen-free (SPF) C57BL / 6J mice were purchased from Beijing Spefol Biotechnology Co., Ltd. and acclimatized for one week. All animals were housed in the laboratory animal room under a 12-hour diurnal cycle, with a room temperature of 20-25℃ and a relative humidity of 30-70%, and free access to water and food.
[0057] After the adaptation period, 30 C57BL / 6J mice were randomly divided into three groups of 10 each. Mice underwent intraperitoneal injection to establish the model for seven consecutive days. The control group (NC) received saline, while the model group (MC) and the *Bifidobacterium longum* AY15 vesicle intervention group (EV) received cyclophosphamide (CTX, 100 mg / kg) solution. After modeling, the control and model groups were administered sterile saline by gavage for seven consecutive days, while the *Bifidobacterium longum* AY15 vesicle intervention group was administered *Bifidobacterium longum* AY15 vesicles by gavage for seven consecutive days at a concentration of 2 × 10⁻⁶. 9 Each / each.
[0058] (2) Bifidobacterium longum AY15 exovesicles alleviate damage in immune organs
[0059] After the experiment, the spleen and thymus tissue of the mice were removed, weighed, and recorded. The spleen tissue samples were stained with hematoxylin and eosin (HE) to observe changes in the spleen tissue.
[0060] Spleen index and thymus index are commonly used to reflect the degree of lymphocyte proliferation in immune organs and are also indicators of immune function strength. Results are as follows... Figure 3 As shown, the spleen index in the MC group was significantly increased, and this condition could be alleviated to some extent after intervention with external vesicles. The thymus index in the MC group was significantly decreased, indicating that its immune function was suppressed, but the thymus index rebounded significantly after intervention with external vesicles. These results indicate that intervention with Bifidobacterium longum AY15 external vesicles can alleviate the immunosuppression caused by CTX to some extent.
[0061] Figure 4 The results of H&E staining of the spleen show that in the NC group, the white pulp lymphocytes are densely distributed with a clear boundary from the red pulp. In the MC group, however, the number of white pulp lymphocytes is reduced, their structure is dispersed, and the boundary with the red pulp is unclear. In the EV group, although a reduction in white pulp was also observed, the boundary between the red and white pulp was clearer compared to the MC group, unlike the dispersed structure and disordered boundary in the MC group. Therefore, Bifidobacterium longum AY15 external vesicles can alleviate spleen damage caused by CTX to a certain extent.
[0062] (3) Bifidobacterium longum AY15 exovesicles regulate immunoglobulins
[0063] Immunoglobulins in serum were detected using an enzyme-linked immunosorbent assay (ELISA) kit, which was purchased from Nanjing Jiancheng Biotechnology Institute.
[0064] Immunoglobulins are one of the main components of the immune system and can be used to understand the body's immune function, such as resistance to various viruses and bacteria, and the body's ability to recognize various invading antigens. Figure 5 The levels of serum immunoglobulins in different groups of mice were displayed. The results showed that the levels of all three immunoglobulins (IgA, IgG, and IgM) in the MC group after CTX intervention were significantly reduced, indicating that the immune system was significantly suppressed after CTX intervention. Intervention with *Bifidobacterium longum* AY15 outer vesicles could alleviate this phenomenon, with the recovery of immunoglobulin IgG being the most significant. This demonstrates that intervention with *Bifidobacterium longum* AY15 outer vesicles can alleviate the reduction in immunoglobulin levels caused by CTX.
[0065] (4) Bifidobacterium longum AY15 extravesicles regulate the expression of immune-related genes.
[0066] Weigh 50-100 mg of mouse spleen tissue from Example 4(2) and extract total RNA using the Trizol method. Follow the BeyoRT... TM The Q First Strand cDNA Synthesis Kit was followed according to the instructions to synthesize cDNA. RT-qPCR was performed using SYBR Green on a CFX96 (Bio-Rad) instrument. Each sample was normalized with the internal control GAPDH and analyzed using 2... -ΔΔCT The method calculates the relative expression level of genes.
[0067] Foxp3 is an important marker of regulatory T cells (Tregs) and a transcription factor for Tregs, primarily exerting its immunomodulatory role by mediating the Treg / Th17 balance. T-bet is a transcription factor of the T-box family, mainly driving Th1 cell differentiation by regulating cytokines such as IFN-γ. GATA3, however, is mutually exclusive with T-bet, exerting a corresponding antagonistic effect, and its main function is to drive Th2 cell differentiation. Results are as follows... Figure 6As shown, compared with the NC group, the mRNA levels of Foxp3, T-bet, and GATA3 in the MC group were significantly decreased. Conversely, compared with the MC group, the mRNA levels of Foxp3, T-bet, and GATA3 in the EV group were significantly increased. This indicates that intervention with Bifidobacterium longum AY15 external vesicles can alleviate the suppression of CTX-related immune factors, suggesting that external vesicles alleviate CTX-induced immunosuppression by promoting Th1 and Th2 cell differentiation and regulating the Th1 / Th2 balance.
[0068] Example 5
[0069] The effects of Bifidobacterium longum AY15 outer vesicles on gut microbiota
[0070] Mouse feces were collected one day before the end of the animal experiment, and 16S rDNA was measured from the fecal samples.
[0071] like Figure 7 As shown, linear discriminant analysis (LEfSe) was used to analyze the differential flora between the MC and EV groups (LDA score > 2 and p < 0.05). The results showed that intervention with *Bifidobacterium longum* AY15 outer vesicles increased the abundance of *g__Alistipes* and *g__Bacteroides* in the mouse gut, while decreasing the abundance of *g__Enterorhabdus*. *g__Bacteroides* and *g__Alistipes* are producers of short-chain fatty acids in the human gut, and short-chain fatty acids have anti-inflammatory effects, helping to maintain the stability of the immune system. Therefore, *Bifidobacterium longum* AY15 outer vesicles can regulate the gut microbiota dysbiosis caused by CTX, possibly by promoting the growth of bacteria that produce short-chain fatty acids, thereby increasing the content of short-chain fatty acids in the gut and thus regulating the immune system.
[0072] Example 6
[0073] Effects of Bifidobacterium longum AY15 exovesicles on short-chain fatty acid metabolism
[0074] Detection of short-chain fatty acids: Mouse fecal samples collected from animal experiments were added to 1% formic acid solution and mixed thoroughly. The mixture was stored at -20℃ for 2 hours, thawed, and centrifuged to collect the supernatant. An equal volume of ethyl acetate was added to the supernatant for extraction. The ethyl acetate phase was collected, and 4-methylvaleric acid was added as an internal standard. After mixing thoroughly, the mixture was dried and filtered through a 0.22 μm organic filter membrane. The mixture was stored at -20℃ for later detection. GC-MS with a TR-Wax column was used for detection. The detection conditions were as follows: ion source temperature 250℃, injection port temperature 250℃; initial temperature 90℃, increased to 150℃ at a rate of 15℃ / min, then increased to 170℃ at a rate of 5℃ / min, and finally increased to 250℃ at a rate of 20℃ / min and held for 2 min; carrier gas was helium, flow rate 1 mL / min; split ratio 1:1, injection volume 1 μL. The detector was set to electron ionization mode with an electron energy of 70 eV, a solvent residence time of 3.5 min, and a scan range of 10-350 m / z.
[0075] To determine the effect of Bifidobacterium longum AY15 outer vesicles on short-chain fatty acid metabolism, the levels of short-chain fatty acids in feces were measured after outer vesicle intervention. Results are as follows: Figure 8 As shown in (A), the content of all short-chain fatty acids in the MC group was significantly lower than that in the NC group. Compared with the MC group, after intervention with external vesicles, the contents of valeric acid, acetic acid, isobutyric acid, propionic acid and valeric acid were significantly higher in the MC group, while the content of isovaleric acid did not change significantly. However, the overall trend was that the short-chain fatty acid content in the EVs treatment group was higher than that in the MC group.
[0076] Short-chain fatty acid receptors (GPR41 / 43) belong to the G protein-coupled receptor family. GPR43, also known as free fatty acid receptor 2 (FFAR2), is expressed on various immune cells, while GPR41, also known as free fatty acid receptor 3 (FFAR3), is mainly expressed by a subset of enteroendocrine cells in the intestinal epithelium. Both GPR41 and GPR43 can bind to short-chain fatty acids and participate in the regulation of inflammatory responses. Therefore, we examined the expression of short-chain fatty acid receptors (GPR41 / 43), and the results are as follows: Figure 8 (B) shows that, compared with the NC group, GPR41 / 43 expression was significantly downregulated in the MC group, while it was significantly upregulated in the EV group. The results indicate that the extravesicles of *Bifidobacterium longum* AY15 can regulate the metabolism of short-chain fatty acids, increase their content, and thereby activate short-chain fatty acid receptors (GPR41 / 43), exerting a corresponding immunomodulatory effect.
[0077] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.
Claims
1. An exovesicle of Bifidobacterium longum, characterized in that: The extravesicles of *Bifidobacterium longum* AY15 were obtained by centrifugation after anaerobic culture in BS liquid medium. The preparation method of the extravesicles of *Bifidobacterium longum* includes the following steps: (1) The activated Bifidobacterium longum AY15 bacterial culture was inoculated into BS liquid medium for anaerobic culture. The cultured bacterial culture was centrifuged at 10000×g for 30 minutes to remove the precipitate and retain the supernatant. The preservation number of Bifidobacterium longum AY15 bacterial culture is GDMCC NO: 65457. (2) The outer vesicles were obtained by sucrose gradient ultracentrifugation. The fraction with a sucrose concentration of 30%-60% was taken out at 4°C and diluted with PBS. The fraction was then centrifuged at 120,000×g at 4°C for 90 minutes. The supernatant was discarded and the precipitate was resuspended to obtain the outer vesicles of Bifidobacterium longum AY15.
2. The exovesicles of *Bifidobacterium longum* according to claim 1, characterized in that: In step (1), the activated Bifidobacterium longum AY15 bacterial suspension is inoculated into BS liquid culture medium at an inoculation rate of 5% and cultured anaerobically at 37°C for 48 hours.
3. The exovesicles of *Bifidobacterium longum* according to claim 1, characterized in that: The activated Bifidobacterium longum AY15 bacterial solution mentioned in step (1) is obtained by taking the Bifidobacterium longum AY15 bacteria, inoculating it onto BS solid medium, anaerobically culturing it at 37°C for 72 hours, then picking a single colony from the BS solid medium and inoculating it into BS liquid medium, anaerobically culturing it at 37°C for 48 hours, and then inoculating the resulting bacterial solution into BS liquid medium at a 5% inoculation rate, anaerobically culturing it at 37°C for 48 hours, and repeating the anaerobic culture on BS liquid medium once, which is the activated Bifidobacterium longum AY15 bacterial solution.
4. The exovesicles of *Bifidobacterium longum* according to claim 3, characterized in that: The BS liquid culture medium is obtained by weighing 48.92g of Bifidobacterium BS culture medium, adding 1mL of Tween 80, heating and dissolving in 1000mL of distilled water, and then autoclaving at 116℃ for 30 minutes; the BS solid culture medium is obtained by weighing 48.92g of Bifidobacterium BS culture medium, adding 1mL of Tween 80 and 20g of agar powder, heating and dissolving in 1000mL of distilled water, autoclaving at 116℃ for 30 minutes, and then pouring the mixture into plates after cooling to a suitable temperature.
5. The role of the exovesicles of *Bifidobacterium longum* as described in any one of claims 1-4 in the preparation of drugs that enhance the immune system, characterized in that: The enhancement of the immune system is achieved by increasing the expression of immunoglobulins IgA, IgG, or IgM, or by enhancing the expression of Foxp3, T-bet, and GATA3 transcription factors, inducing Th cell differentiation, and regulating the Th1 / Th2 balance, thereby regulating the immune system.
6. The use of the outer vesicles of Bifidobacterium longum as described in any one of claims 1-4 in the preparation of a drug that promotes the secretion of short-chain fatty acids by intestinal flora.