A preparation method and application of extracellular vesicles of Lactobacillus amylovora

Abstract

The invention discloses a method for preparing extracellular vesicles of Lactobacillus amylovora, which comprises the steps of: inoculating Lactobacillus amylovora for cultivation, centrifuging the fermented bacterial liquid, removing bacterial precipitates, filtering the supernatant, centrifuging the obtained filtrate using a high-grade centrifuge tube, ultracentrifuging the ultrafiltrate, resuspending the obtained precipitate, filtering, and obtaining the extracellular vesicles of Lactobacillus amylovora. The above-mentioned preparation method or the extracellular vesicles of Lactobacillus amylovora prepared therefrom can be used to prepare preparations for preventing or treating cholestatic liver disease, prepare extracellular vesicle drug delivery systems, etc. The present invention takes the growth curve of Lactobacillus amylovora as the starting point, expands the bacterial yield with a specific culture time, combines ultrafiltration to filter impurities such as small molecule proteins, and then separates the extracellular vesicles with ultracentrifugation, thereby obtaining high-concentration bacterial-derived extracellular vesicles, providing an efficient, high-purity, low-cost method for preparing extracellular vesicles without destroying the vesicle structure.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial-derived extracellular vesicle extraction, and in particular to a preparation method and application of extracellular vesicles of Lactobacillus amylovora. Background Art

[0002] As an important strain of microecological preparations, lactic acid bacteria plays an important role in maintaining the intestinal health of humans and animals and promoting the balance of microecology in the body. A large number of studies have shown that lactic acid bacteria can regulate the normal gastrointestinal flora of the body, maintain the balance of microecology, improve food digestibility and biological value, reduce serum cholesterol, control endotoxins, inhibit the growth and reproduction of putrefactive bacteria in the intestine and the production of putrefactive products, produce nutrients, stimulate tissue development, and thus have effects on the body's nutritional status, physiological functions, cell infection, drug effects, toxic reactions, immune reactions, tumorigenesis, aging processes and sudden emergency reactions. Lactobacillus amylovorus (La) is a type of lactic acid bacteria that can hydrolyze starch. Nakamura originally discovered and isolated it from the mixed fermentation of corn and waste in cattle farms, and named it Lactobacillus amylovorus. Later, researchers also successfully isolated Lactobacillus amylovorus from the intestines of animals. The bacteria are Gram-positive bacteria, convex, round, white, smooth colonies, and neat and opaque edges. Under the microscope, they are chain-shaped or single, without spores, a few have peritrichous, and most are non-motile. The optimum pH is 5.5-6.0, but they can survive at pH 3.0-4.5. Lactobacillus amylovora reaches the intestine from the mouth, forms a stable flora through adhesion, colonization, and reproduction, and maintains the stability and health of the intestinal environment. Lactobacillus amylovora can produce antibiotic substances, including hydrogen peroxide, organic acids, peptides, and bacteriocins.

[0003] Bacteria can actively release nano-sized membrane vesicles into the extracellular environment. This spherical structure of 20 to 400 nm is called extracellular vesicles (EVs). EVs can carry a variety of biological genetic information, such as nucleic acids, enzymes, effector proteins and virulence factors. Its structure enables EVs to play an important role in bacterial competition, survival, material exchange, host immune escape, regulation, infection and invasion. Based on the many health-promoting effects of probiotics, probiotic-derived EVs show beneficial effects such as anti-inflammatory and anti-tumor, which have been clearly demonstrated in many studies. Probiotic EVs can carry the bioactive components of bacteria, migrate directly to other tissues through the mucus layer or interact with different cells of the host immune system to exert their biological functions. EVs can be regarded as a safe and effective communication medium for probiotics to exert their beneficial effects. Therefore, probiotic-derived EVs are expected to become efficient, safe and clinically applicable biological preparations. Extracellular vesicles play an important role in participating in intercellular communication, disease diagnosis, and disease treatment due to their advantages such as size and bimolecular membrane. In recent years, the use of EVs as therapeutic agents has become a promising area of ​​research. The main advantage of nanoparticles, such as EVs, over traditional therapeutic drugs, such as small molecules and antibodies, is that the large size of the nanoparticles enables the incorporation of multiple ingredients. For example, several drugs can be loaded into the same nanoparticle in an optimal ratio to achieve improved efficacy.

[0004] There are several main methods for isolating extracellular vesicles: The first method is to use different ultracentrifugation speeds or density gradient centrifugation: This method is currently considered the gold standard for extracellular vesicle isolation. The purity of the extracellular vesicles separated by this method is high, but the RNA content of the extracellular vesicles extracted by this method is very low, and the ultracentrifuge is expensive and cannot be popularized, and the structure of the extracellular vesicles extracted by this method may be destroyed; The second method is ultrafiltration: Although this method is simple to operate, the purity of the extracellular vesicles obtained is not high; The third method is a commercial kit, which can be divided into two categories: One is to add the extraction reagent to the sample and centrifuge to obtain the precipitate of the extracellular vesicles; the other method is to add the sample to the column. In the process of passing through the column, impurities such as proteins in the sample will enter the column, and extracellular vesicles with larger particle sizes will flow out of the column first, so that extracellular vesicles with higher purity can be obtained more quickly. Comparing the advantages and disadvantages of these three separation and preparation methods, it can be found that although EVs prepared by density gradient centrifugation have high purity, they are usually not used for routine research due to their low yield. Although probiotic EVs can be extracted using a kit method under mild conditions without the use of an ultracentrifuge, only a small amount of bacterial liquid can be extracted at a time, and the steps involved are cumbersome. Ultracentrifugation is the most widely used method in the field of separation and preparation. In the early stage, the purity of EVs can be improved by removing bacteria and their fragments, flagella, etc. through differential centrifugation, filtration and other steps.

[0005] At present, the research and development of extracellular vesicles of Lactobacillus amylovora and their extraction methods are still in a blank stage. How to obtain an efficient, high-purity, low-cost method for preparing extracellular vesicles of Lactobacillus amylovora without destroying the vesicle structure during the preparation process is currently an urgent problem to be solved. Summary of the invention

[0006] In order to overcome at least one problem existing in the prior art, the present invention is comprehensively optimized from the aspects of low cost, high efficiency, high purity, etc., taking the growth curve of Lactobacillus amylovora as the starting point, expanding the bacterial yield with a specific culture time, combining ultrafiltration to filter impurities such as small molecule proteins, and then separating extracellular vesicles by ultracentrifugation, thereby obtaining high-concentration bacterial extracellular vesicles. The present invention provides a method for quickly extracting Lactobacillus amylovora extracellular vesicles (LaEVs) with high efficiency, high purity and low cost without destroying the vesicle structure, and proves that the extracellular vesicles secreted by Lactobacillus amylovora have an effective concentration and the morphological structure is intact.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention is to provide a method for preparing extracellular vesicles of Lactobacillus amylovora, which comprises the steps of:

[0009] A) inoculating Lactobacillus amylovora for culturing to obtain a fermentation broth containing extracellular vesicles;

[0010] B) centrifuging the fermented bacterial liquid obtained in step A) to remove bacterial precipitates, filtering the collected supernatant, and collecting the filtrate;

[0011] C) centrifuging the filtrate obtained in step B) using a high-grade centrifuge tube to collect the ultrafiltrate;

[0012] D) ultracentrifuging the ultrafiltrate obtained in step C), resuspending and filtering the obtained precipitate to obtain the extracellular vesicles of Lactobacillus amylovora.

[0013] Further, in step A), after the strain is revived, the strain is picked in the culture medium, placed in a constant temperature incubator at 37°C for 4 to 8 hours, and then inoculated in 1 to 3L culture medium at 37°C for 12 to 24 hours with an inoculum of 1 to 4wt%. Preferably, after the strain is revived, the strain is picked in MRS broth, placed in a constant temperature incubator at 37°C for 6 hours, and then inoculated in 2L MRS broth at 37°C for 18 hours with an inoculum of 2wt%. When other conditions remain the same, if the volume of the fermentation liquid is reduced, the yield of extracellular vesicles will decrease. In the above steps, the inoculum size and culture time are combined with the growth curve of Lactobacillus amylovora to determine that: 2wt% is the optimal inoculum size, 6h is the early logarithmic stage of the growth period, and 18h ​​is the late logarithmic stage of the growth period. According to this step and conditional culture, the bacterial activity and concentration of Lactobacillus amylovora are high, which can maximize the yield of extracellular vesicles secreted by it.

[0014] Furthermore, the classification name of the amylovorus Lactobacillus is Lactobacillus amylovorus, its preservation number is CCTCC NO: M2023427, the preservation date is March 28, 2023, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation unit address is Wuhan University, Wuhan, China.

[0015] Further, the step B) specifically includes: centrifuging the fermented bacterial liquid at 4000-6000g and 4°C for 10-20 minutes, discarding the bacterial precipitate, filtering the collected supernatant with a 0.2-0.3 μm filter, and collecting the filtrate. Preferably, the fermented bacterial liquid is centrifuged at 5000g and 4°C for 15 minutes, discarding the bacterial precipitate, at which time the obtained supernatant volume is high, ensuring a high yield of extracellular vesicles; filtering the collected supernatant with a 0.22 μm filter, collecting the filtrate, this filtration step further removes the bacterial precipitate and substances of similar mass, which is beneficial to improve the ultrafiltration efficiency and reduce the loss caused by filter membrane clogging.

[0016] Furthermore, the step C) specifically comprises: centrifuging the filtrate using an ultrafiltration tube at 4000-6000g and 4°C for 15-60 minutes, and collecting the ultrafiltrate. Preferably, the filtrate is centrifuged using an ultrafiltration tube at 5000g and 4°C for 30 minutes.

[0017] Furthermore, the ultrafiltration tube contains an ultrafiltration membrane that can retain vesicles larger than 100 kDa. At this time, the ultrafiltration membrane can retain vesicles larger than 100 kDa in the ultrafiltrate, and substances smaller than 100 kDa, such as small peptides, oligonucleotides, nucleic acids, enzymes and other similar macromolecules, will be removed. After the ultrafiltration step, the purity of the preparation can be effectively improved.

[0018] Furthermore, the ultrafiltration tube comprises a filtrate receiver, the filtrate receiver has a built-in sample pool, a packaging membrane (ultrafiltration membrane) is fixed in the sample pool, and the top of the filtrate receiver is detachably connected to the sample pool cover.

[0019] Furthermore, the ultrafiltration tube is Advanced centrifuge tube. It is understandable that the model of the ultrafiltration tube and the volume of its filter can be selected according to specific needs. In a specific embodiment, the volume of the filter of the ultrafiltration tube is 15mL. The centrifugal force for concentrating the extracellular vesicles in the liquid containing extracellular vesicles using the ultrafiltration tube can be 5000g. The centrifugation time for concentrating the extracellular vesicles in the liquid containing extracellular vesicles using the ultrafiltration tube can be 15-60 minutes. Concentrating the extracellular vesicles in the liquid containing extracellular vesicles using the ultrafiltration tube is carried out at 4°C.

[0020] Further, the step D) specifically includes: ultracentrifuging the ultrafiltrate at 11000-160000g and 4°C for 1-3 hours; resuspending the obtained precipitate with PBS, filtering with a 0.2-0.3 μm filter, and obtaining the extracellular vesicles of Lactobacillus amylovora. Preferably, the ultrafiltrate is ultracentrifuged at 160000g and 4°C for 2 hours, at which time the relatively heavy extracellular vesicles will be precipitated, and other conditions remain the same. Reducing the ultracentrifugation speed can lead to a decrease in the yield of extracellular vesicles; resuspending the obtained precipitate with PBS, filtering with a 0.22 μm filter, removing possible bacterial contamination in the above process, and improving the success rate of the separation and preparation test.

[0021] The second aspect of the present invention is to provide an extracellular vesicle of Lactobacillus amylovora prepared by any of the above-mentioned preparation methods, wherein the extracellular vesicle of Lactobacillus amylovora contains an effective amount of extracellular vesicles with intact structure.

[0022] Furthermore, in a specific embodiment, the particle size of the extracellular vesicles of Lactobacillus amylovora is between 100 and 313 nm, the vesicles with a particle size of 170.4 nm are the largest, accounting for about 59.3%, and the particle size concentration is 2.8×10 10particles / mL.

[0023] Furthermore, in a specific embodiment, the concentration of the extracellular vesicles of Lactobacillus amylovora is 3.60-9.16 μg / μL.

[0024] The third aspect of the present invention is to provide any of the above-mentioned preparation methods or any of the above-mentioned applications of the extracellular vesicles of Lactobacillus amylovora, which is selected from at least one of the following applications: application in the preparation of a preparation for preventing or treating cholestatic liver disease, application in the preparation of an extracellular vesicle drug delivery system, and application in transcriptomics / proteomics / lipidomics association analysis.

[0025] Furthermore, in the use of preparing a preparation for preventing or treating cholestatic liver disease, the preparation includes a preparation for improving liver function damage, inflammatory lesions and / or liver fibrosis caused by cholestatic liver disease.

[0026] Furthermore, in the application of preparing extracellular vesicle drug delivery systems, it can be used as an effective transporter to deliver therapeutic drug components to the central nervous system region.

[0027] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0028] The present invention takes the growth curve of Lactobacillus amylovora as the starting point, expands the bacterial yield with a specific culture time, combines ultrafiltration to filter out impurities such as small molecule proteins, and then separates extracellular vesicles by ultracentrifugation, thereby obtaining high-concentration bacterial-derived extracellular vesicles, providing an efficient, high-purity, low-cost method for preparing extracellular vesicles without destroying the vesicle structure. The extracellular vesicles of Lactobacillus amylovora prepared by the present invention have potential application value in the fields of disease treatment, antibacterial drug targets and drug development, industrial fermentation lactic acid bacteria regulation and animal health care. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0030] Figure 1 is a schematic diagram of a high-grade centrifuge tube used in one embodiment of the present invention;

[0031] Figure 2 This is a schematic diagram of Lactobacillus amylovora extracellular vesicles (LaEVs) under a high-resolution transmission electron microscope in one embodiment of the present invention (the scale bar is 200 nm);

[0032] Figure 3This is a particle size diagram of Lactobacillus amylovora extracellular vesicles (LaEVs) measured by a particle size analyzer in one embodiment of the present invention (the main particle size is 170.1 nm, followed by 135.4 nm);

[0033] Figure 4 The results of the effect of Lactobacillus amylovora extracellular vesicles (LaEVs) on ALT, AST, ALP and liver total bile acid levels in mice with DDC-induced cholestatic liver injury and fibrosis are shown in one embodiment of the present invention; specifically: serum ALT activity, serum AST activity, serum ALP activity, liver TBA level;

[0034] Figure 5 This is a result diagram of the effect of extracellular vesicles of Lactobacillus amylovora (LaEVs) on the pathological changes of liver tissues in mice with DDC-induced cholestatic liver injury and fibrosis in one embodiment of the present invention; wherein, H&E and Sirius red staining (100X);

[0035] Figure 6 This is a result diagram showing the effect of Lactobacillus amylovora extracellular vesicles (LaEVs) on the gene expression levels of IL-1β, IL-6, and TNF-α in the liver of mice with DDC-induced cholestatic liver injury and fibrosis in one embodiment of the present invention;

[0036] Figure 7 This is a graph showing the effect of Lactobacillus amylovora extracellular vesicles (LaEVs) on the gene expression levels of α-SMA and Vimentin in the liver of mice with DDC-induced cholestatic liver injury and fibrosis in one embodiment of the present invention.

[0037] All test results in the above figures are expressed as means±SEM (n=5), and different letters between data groups indicate significant differences between them (P<0.05). DETAILED DESCRIPTION

[0038] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0039] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0040] In the following examples, the composition of MRS broth: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 5.0 g, dipotassium hydrogen phosphate 2.0 g, glucose 20.0 g, Tween 80 1 mL, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2, manganese sulfate 0.04, distilled water 1000 mL, 118 ° C high pressure sterilization for 15 minutes before use.

[0041] In a preferred embodiment, a method for preparing extracellular vesicles of Lactobacillus amylovora comprises the steps of: after resuscitating the Lactobacillus amylovora strain, picking the strain into a culture medium, placing it in a constant temperature incubator at 37°C and culturing it for 4 to 8 hours, and then inoculating it in a 1 to 3 L culture medium at an inoculum size of 1 to 4 wt% and culturing it at 37°C for 12 to 24 hours; centrifuging the fermented bacterial liquid at 4000 to 6000 g and 4°C for 10 to 20 minutes, discarding the bacterial precipitate, filtering the collected supernatant using a 0.2 to 0.3 μm filter, and collecting the filtrate; centrifuging the filtrate using an ultrafiltration tube at 4000 to 6000 g and 4°C for 15 to 60 minutes, and collecting the ultrafiltrate; ultracentrifuging the ultrafiltrate at 120,000 to 160,000 g and 4°C for 1 to 3 hours; resuspending the obtained precipitate with PBS, filtering it using a 0.2 to 0.3 μm filter, and obtaining the extracellular vesicles of Lactobacillus amylovora.

[0042] Example 1 - Isolation, screening and identification of starch-eating lactic acid bacteria (Lactobacillus amylovorus)

[0043] In this embodiment, the following method is used to obtain starch-eating lactic acid bacteria:

[0044] (1) Isolation and screening of starch-eating lactic acid bacteria

[0045] Weigh 0.1 g of feces from piglets, sows, etc. and add them to 0.9 mL of sterile saline. Mix by pipetting and dilute with saline by 10 times. -5 -10 -7 times, take 100μL of each dilution multiple and spread it on MRS agar plate, invert it in a constant temperature incubator at 37℃ and culture it for 48h; then use a sterile inoculation loop to pick colonies with lactic acid bacteria morphology, inoculate them in MRS broth, and culture them at 37℃ for 48h; then take the liquid bacterial liquid to streak the MRS agar plate, invert it in a constant temperature incubator at 37℃ and culture it for 48h; pick a single colony and inoculate it in MRS broth again, repeat the above steps 3 times to obtain the purified primary screening bacteria. After further screening by Gram staining, it was identified as amylovorax lactobacillus.

[0046] Main biological characteristics: Under the microscope, it is blue-purple, long rod-shaped, mostly single, not chain-shaped; after culturing at 37°C on an MRS plate for 24 hours, it appears as yellow colonies with a smooth surface, slightly raised, powdery and opaque, uniform texture, neat edges, and a diameter of about 2-3 mm.

[0047] (2) Identification of starch-eating lactic acid bacteria

[0048] The selected strains were activated by MRS agar plates and MRS broth culture, and then sent to Sangon Biotech Co., Ltd. for strain sequencing. The sequencing results were compared with the GenBank database of the National Center for Biotechnology Information (NCBI) of the United States, and strains with more than 99% homology were found for comparison to determine the type of lactic acid bacteria to be measured. The sequence (5'-3') measured by the strain is shown below, and the homology with the base sequence of Lactobacillus amylovorus currently available on NCBI reached 100%.

[0049]

[0050] The above-mentioned Lactobacillus amylovorus has been preserved, and its classification name is Lactobacillus amylovorus. Its preservation number is CCTCC NO: M2023427, the preservation date is March 28, 2023, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation unit address is Wuhan University, Wuhan, China.

[0051] Example 2 - Preparation and characterization of extracellular vesicles (LaEVs) of lactic acid bacteria

[0052] This example uses a preferred method to prepare extracellular vesicles of lactic acid bacteria and characterize and analyze them, including the following steps:

[0053] (1) Obtaining extracellular vesicle-containing fermentation broth

[0054] The amylovoric Lactobacillus used in this example is obtained by screening lactic acid bacteria from pig manure, and its deposit number is CCTCCNO: M2023427. After the strain is revived, the strain is selected and placed in MRS broth, placed in a constant temperature incubator at 37°C for 6 hours, and inoculated with 2% inoculum in 2L of MRS broth at 37°C for 18 hours.

[0055] (2) Pretreatment of extracellular vesicle-containing fermentation broth

[0056] The bacterial solution was dispensed into 50mL EP tubes and centrifuged at 5000g speed and 4℃ for 15 minutes to remove the bacterial precipitate. The inoculation amount and culture time comprehensively considered the growth curve of Lactobacillus amylovora, 2% was the optimal inoculation amount, 6h was the early logarithmic growth period, and 18h ​​was the late logarithmic growth period. According to this step and condition, the activity and concentration of Lactobacillus amylovora were high, which could maximize the production of extracellular vesicles secreted by it. The supernatant was filtered with a 0.22μm filter to completely remove macromolecular substances such as the bacteria and its fragments. This process can improve the ultrafiltration efficiency and reduce the loss caused by membrane clogging.

[0057] (3) Concentration of fermentation broth

[0058] After filtration, centrifuge at 5000g for 30 minutes using a high-grade centrifuge tube, concentrate by ultrafiltration to 320 mL, and collect the ultrafiltrate. In the above method, the extracellular vesicles in the liquid containing extracellular vesicles are concentrated using a filter membrane with a molecular weight cutoff of 100 kDa, which can be performed by using an ultrafiltration tube containing the filter membrane. At this time, the ultrafiltration membrane can retain vesicles greater than 100 kDa in the ultrafiltrate, and substances less than 100 kDa such as small peptides, oligonucleotides, nucleic acids, enzymes and other similar macromolecules will be removed. After the ultrafiltration step, the purity of the preparation can be effectively improved.

[0059] The ultrafiltration tube is specifically Advanced centrifuge tube (Pall, USA), its structure is: the ultrafiltration tube includes a filtrate receiver with a built-in sample pool, a packaging membrane (ultrafiltration membrane) is fixed in the sample pool, and the top of the filtrate receiver is detachably connected to the sample pool cover. The volume of the filter of the ultrafiltration tube can be selected according to specific needs. In this embodiment, the volume of the filter of the ultrafiltration tube is 15mL. The centrifugal force for concentrating the extracellular vesicles in the liquid containing extracellular vesicles using the ultrafiltration tube can be 5000g. The centrifugation time for concentrating the extracellular vesicles in the liquid containing extracellular vesicles using the ultrafiltration tube can be 15-60 minutes. Concentrating the extracellular vesicles in the liquid containing extracellular vesicles using the ultrafiltration tube is carried out at 4°C.

[0060] (4) Isolation of extracellular vesicles

[0061] The obtained ultrafiltrate was ultracentrifuged at 160000g and 4°C for 2 hours, at which time the relatively heavy extracellular vesicles were precipitated. The obtained precipitate was resuspended with a small amount of PBS, and the multiple tube suspensions were mixed and filtered using a 0.22 μm filter to remove possible bacterial contamination in the above process, improve the success rate of the separation and preparation test, and finally obtain the extracellular vesicles of Lactobacillus amylovora.

[0062] Example 3 - Characterization and analysis of extracellular vesicles (LaEVs) of Lactobacillus amylovora

[0063] This example characterizes and analyzes the extracellular vesicles of lactic acid bacteria prepared in Example 2, including the following steps:

[0064] First, the total protein content of the collected extracellular vesicles was determined using a BSA kit, and then their morphology was observed using a high-resolution transmission electron microscope, and their particle size was determined using a particle size analyzer.

[0065] The steps of observing the morphology of extracellular vesicles by high-resolution transmission electron microscopy are as follows: take 15 μL of the collected extracellular vesicles, add 15 μL PBS to dilute, then drop the solution onto the copper mesh, let it stand for 2 minutes, and remove the excess solution on the copper mesh with filter paper. Use 15 μL of 2% phosphotungstic acid staining solution for negative staining for 2-3 minutes, remove the excess staining solution with filter paper, dry for 20-30 minutes, and perform transmission electron microscopy on the machine to observe its morphology.

[0066] The steps for measuring the particle size of extracellular vesicles by a particle size analyzer are as follows: take the collected extracellular vesicles, use ParticleMetrix'ZetaView nanoparticle tracking analyzer, calibrate the instrument, and dilute the extracellular vesicle sample to an appropriate concentration, so that the number of particles displayed on the instrument detection interface of the extracellular vesicle sample is between 50-400, preferably around 200; enter the dilution factor of the sample in the software interface, and observe that the number of particles displayed at the detection position is between 50-400. And use Zeta View software to analyze the acquired data. The instrument detection parameters are set as follows: temperature is 21.25℃, pH is 7.0, laser wavelength: 520nm, filter wavelength: scattering, and other parameters use default parameters. Among them, the formula for calculating the concentration of extracellular vesicle particles is:

[0067] EVs particle concentration = detected concentration × sample dilution factor

[0068] The total protein concentration of the extracted extracellular vesicles was measured after dilution and was found to be 9.16 μg / μl. The morphology of the obtained extracellular vesicles was observed using a high-resolution transmission electron microscope ( Figure 2 ), and the molecular size was measured using a particle size analyzer ( Figure 3 ). Characterization and analysis showed that the prepared solution contained extracellular vesicles of Lactobacillus amylovora. Figure 2 These vesicles present a typical vesicle structure containing a phospholipid bilayer, are spherical particles, and have a diameter of about 135nm, which is consistent with the results of the nanoparticle size analyzer (Zeta ViewS / N22-779). Figure 3 As shown in the figure, it can be seen that the LaEVs particles are evenly distributed and the peaks are relatively neat and uniform, which indicates that the concentration of LaEVs prepared in this experiment is high and contains fewer impurities, which also ensures the reliability of our subsequent experiments. Finally, the particle size distribution and concentration of LaEVs were obtained by NTA software analysis. The results showed that the particle size of LaEVs was between 100 and 313 nm, and the vesicles with a particle size of 170.4 nm were the largest, accounting for about 59.3%, and the particle size concentration was 2.8×10 10 This is consistent with the results observed by electron microscopy and the particle size distribution range of bacterial EVs reported in other literature.

[0069] Example 4 - Screening of the best solution for preparing high-concentration extracellular vesicles

[0070] In this embodiment, the volume of the fermentation broth in the preparation method described in Example 2 is changed to 1L, or the speed of the ultracentrifuge is reduced to 120000g, and other conditions remain the same. The total protein concentration of the obtained extracellular vesicle solution is determined by BCA, and the extraction efficiency is compared. The results are shown in Table 1 below. When the volume of the fermentation broth is 1L and the speed of the ultracentrifuge is 160000g, the protein concentration is 3.60μg / μL, and when the volume of the fermentation broth is 2L and the speed of the ultracentrifuge is 120000g, the protein concentration is 3.94μg / μL. And as the centrifugal speed increases moderately, the extracellular vesicle yield can increase, but blindly increasing it may be difficult to ensure the integrity and function of the extracellular vesicles.

[0071] Table 1 - Total protein concentration obtained by different protocols

[0072]

[0073] Example 5 - Effect of Lactobacillus amylovora extracellular vesicles (LaEVs) on the alleviation of cholestatic liver injury

[0074] This example verifies the effect of extracellular vesicles of lactic acid bacteria amylovora prepared according to the steps of Example 2 (wherein: the volume of the fermentation broth is 2 L, and the speed of the ultracentrifuge is 120000 g) in alleviating cholestatic liver injury, which includes the steps of:

[0075] (1) Materials and methods

[0076] 1.1- Chemical reagents;

[0077] Main reagents: alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bile acid detection kit (Nanjing Jiancheng Bioengineering Institute), RNAtrizol (Nanjing Novozyme Biotechnology Co., Ltd.), RT-PCR kit (Eric Biotech).

[0078] 1.2-Animal testing methods;

[0079] Eighteen 7-week-old specific-pathogen-free (SPF) male C57BL / 6 mice purchased from Jicui Yaokang were used in this experiment (weight 22.27±0.40g). The mice were randomly divided into 3 groups, 6 in each group: control group; DDC group; DDC+LaEVs group. The mice were kept in an SPF environment (12h light and 12h dark alternately, free access to food and water, constant temperature and humidity suitable for automatic control) in 1 cage per 6 mice. The experiment began after 1 week of adaptive feeding. The control group was fed with normal mouse diet for 14 consecutive days. On the first day, DDC purified diet and normal mouse diet were given to the DDC group and DDC+LaEVs group at a ratio of 1:2; on the second day, DDC purified diet and normal mouse diet were given to the DDC group and DDC+LaEVs group at a ratio of 2:1; on the 3rd to 14th day, the DDC group and DDC+LaEVs group were only given DDC purified diet. The mice in the DDC+LaEVs group were gavaged with 1.2 μg / kg extracellular vesicles of Lactobacillus amylovora for 14 consecutive days at the same time every day. The experiment lasted for 2 weeks, and the mice had free access to food and water throughout the experiment.

[0080] 1.3-Sample collection

[0081] After the experiment, the mice were fasted for 12 h before euthanasia, and their blood was collected by enucleation of the eyeballs, centrifuged at 3500 rpm × 25 min, and the serum was separated and stored at -80 ° C. After the death of the mice, the abdominal cavity was opened and liver tissue samples were collected. One part of the tissue was fixed with 4% paraformaldehyde solution for subsequent tissue morphology analysis, and the other part was quickly frozen with liquid nitrogen and stored at -80 ° C for subsequent molecular biology analysis.

[0082] 1.4- Serum biochemical indexes and liver total bile acid determination

[0083] Alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bile acid in the liver were detected using kits.

[0084] 1.5-H&E and Sirius Red Staining of Liver Tissue

[0085] The liver tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. A piece of tissue (1.5 cm × 1 cm × 0.5 cm) was cut and placed in an embedding box. After dehydration, wax immersion, embedding, freezing and other operations, the liver tissue was cut into 3 μm thick slices using a tissue slicer. The prepared tissue slices were stained with HE and Sirius red, and then dehydrated, transparentized, and sealed for observation under a microscope. The slices were observed and photographed using an upright optical microscope.

[0086] 1.6-Real-Time PCR detection of relative expression levels of IL-1β, IL-6, TNF-α, α-SMA, and Vimentin in liver tissue

[0087] According to the sequences of GAPDH, IL-1β, IL-6, TNF-α, α-SMA, and Vimentin of C67BL / 6 mice in GenBank, the primers were designed using Premier 5.0 in accordance with the principles of quantitative PCR primer design. Each primer was synthesized by Sangon Biotech Co., Ltd. The specific sequences are shown in Table 2. The total RNA was extracted using the TRIZOL kit according to the instructions, and the concentration of the extracted RNA was finally detected by the Nanodrop 2000 (Thermoscientific, USA) analyzer. The real-time PCR reaction system and PCR amplification conditions were referred to the instructions of the PrimeScript RT-PCR Kit of Myrica. Real-time PCR amplification was performed on the 7300 Real-time PCR System produced by Applied Biosystems (ABI).

[0088] Table 2 - Primer sequences

[0089]

[0090] (2) Results

[0091] 2.1-Effects of extracellular vesicles of Lactobacillus amylovora on serum ALP, AST, ALT, and liver TBA;

[0092] Changes in serum liver enzyme indicators such as Figure 4 As shown (C is serum ALT activity, D is serum AST activity, E is serum ALP activity, F is liver TBA level): compared with the control group, serum ALP, AST, and ALT in the DDC group increased significantly (P < 0.05), and compared with the DDC group, ALP, AST, and ALT in the DDC+LaEVs group decreased significantly (P < 0.05). Under the influence of LaEVs, the level of total bile acid in the liver decreased significantly (P < 0.05).

[0093] 2.2-Effects of extracellular vesicles of Lactobacillus amylovora on pathological changes in liver tissues;

[0094] H&E staining showed that ( Figure 5, H&E, Sirius red staining (100×): In the control group, liver sections showed hepatic lobules composed of normal central veins and cell structures. In the DDC group, obvious punctate hemorrhage necrosis was observed in the liver, with a large number of inflammatory granulocytes and lymphocytes infiltrating the necrosis. Most of the hepatic lobules had disordered structures, the morphology of hepatocyte cords disappeared, the cells in the hepatic lobules were swollen, the cell cytoplasm was lightly stained and loose, and showed balloon-like changes. LaEVs can improve DDC-induced liver lesions.

[0095] Sirius red staining was used to detect collagen deposition in the liver ( Figure 5 ), in the DDC group, there was a large amount of collagen deposition in the portal vein and portal duct areas. LaEVs could significantly reduce the collagen deposition induced by DDC.

[0096] 2.3-Effect of extracellular vesicles of Lactobacillus amylovora on the gene expression levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in the liver;

[0097] like Figure 6 As shown, compared with the control group, the gene expression levels of IL-6, IL-1β, and TNF-α in the DDC group were significantly increased (P<0.05). Compared with the DDC group, the gene expression levels of IL-6, IL-1β, and TNF-α in the LaEVs group were significantly decreased (P<0.05).

[0098] 2.4 Effects of extracellular vesicles of Lactobacillus amylovora on the expression levels of α-SMA and Vimentin genes in the liver

[0099] like Figure 7 As shown, compared with the control group, the gene expression levels of α-SMA and Vimentin in the DDC group were significantly increased (P<0.05). Compared with the DDC group, the gene expression levels of α-SMA and Vimentin in the LaEVs group were significantly decreased (P<0.05).

[0100] It can be seen from the above embodiments that the present invention uses the growth curve of isolated amylovorax as the starting point, expands the bacterial yield with a specific culture time, combines ultrafiltration to filter out impurities such as small molecule proteins, and then separates extracellular vesicles by ultracentrifugation, thereby obtaining high-concentration bacterial extracellular vesicles, providing an efficient, high-purity, low-cost method for preparing extracellular vesicles without destroying the vesicle structure. The above preparation can improve cholestatic liver disease and can also be used to load other drug components, and has good application prospects.

[0101] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing extracellular vesicles of Lactobacillus amylovora, characterized in that: Includes steps: A) Inoculation of Lactobacillus amylovora Lactobacillus amylovorus ) to culture and obtain a fermentation broth containing extracellular vesicles; B) centrifuging the fermented bacterial liquid obtained in step A) to remove bacterial precipitate, filtering the collected supernatant, and collecting the filtrate; C) centrifuging the filtrate obtained in step B) using a high-grade centrifuge tube to collect the ultrafiltrate; D) ultracentrifuging the ultrafiltrate obtained in step C), resuspending and filtering the obtained precipitate to obtain the extracellular vesicles of Lactobacillus amylovora; Wherein, the preservation number of the amylovora bacillus is CCTCC NO: M 2023427.

2. The preparation method according to claim 1, characterized in that The step A) comprises: after the bacterial strain is revived, the bacterial strain is picked out in a culture medium, placed in a constant temperature incubator and cultured at 37° C. for 4 to 8 hours, and then inoculated in 1 to 3 L of culture medium at an inoculum amount of 1 to 4 wt % and cultured at 37° C. for 12 to 24 hours.

3. The preparation method according to claim 1, characterized in that The step B) comprises: centrifuging the fermented bacterial liquid at 4000-6000g and 4°C for 10-20 minutes, discarding the bacterial precipitate, filtering the collected supernatant using a 0.2-0.3 μm filter, and collecting the filtrate.

4. The preparation method according to claim 1, characterized in that: The step C) comprises: centrifuging the filtrate at 4000-6000g and 4°C for 15-60 minutes using an ultrafiltration tube to collect the ultrafiltrate.

5. The preparation method according to claim 4, characterized in that The ultrafiltration tube contains an ultrafiltration membrane with a molecular weight cutoff of 100 kDa.

6. The preparation method according to claim 1, characterized in that: The step D) comprises: ultracentrifuging the ultrafiltrate at 11000-160000g and 4°C for 1-3 hours; resuspending the obtained precipitate with PBS, and filtering with a 0.2-0.3 μm filter to obtain the extracellular vesicles of Lactobacillus amylovora.

7. Use of the extracellular vesicles of Lactobacillus amylovora prepared by the preparation method according to any one of claims 1 to 6 in the preparation of a medicament for treating cholestatic liver disease.

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