Improved method for purifying environment and pure bacteria-derived extracellular vesicles

Abstract

The invention relates to an improved purification environment and pure bacterium source extracellular vesicle method, and relates to the technical field of biology, the method comprises the following steps: S1, obtaining a first crude extraction extracellular vesicle sample, carrying out exploratory multi-layer density gradient ultracentrifugation on the first sample, and carrying out characterization analysis on each centrifuged component, to determine an enrichment zone of extracellular vesicles; and S2, obtaining an extracellular vesicle sample of a second crude extraction homologous with the extracellular vesicle sample of the first crude extraction, constructing an optimized simplified density gradient according to the enrichment regions determined in the step S1, and carrying out optimized simplified density gradient ultracentrifugation on the second sample to collect and purify the extracellular vesicles. According to the method disclosed by the invention, a standardized scheme is established for a specific sample through two steps of exploratory purification and purification after optimization, and enrichment regions of the extracellular vesicles are determined by adopting an identification method combining a transmission electron microscope and nanoflow cytometry, so that the repeatability of a purification result is improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to an improved purification environment and method for pure bacterial-derived extracellular vesicles. Background Technology

[0002] Extracellular vesicles (EVs) are nanoscale, spherical substances with a phospholipid bilayer structure produced by all living organisms, playing a crucial role in material transport and information transmission. The isolation and purification of EVs from complex environmental samples such as water and soil, as well as pure cultures, is of great significance for research in fields such as microbial ecology. Density gradient ultracentrifugation is a commonly used technique for purifying EVs, but existing techniques still have some inherent technical challenges when applied to environmental samples.

[0003] Current density gradient centrifugation techniques typically employ a fixed set of non-customized centrifugation parameters and gradient protocols for samples from different sources. However, environmental samples exhibit high heterogeneity in their physicochemical properties (such as salinity and pH) and biological components (such as microbial community structure), leading to variations in the physical density distribution of extracellular vesicles within samples from different sources. Using a uniform purification protocol for these samples with varying densities results in inconsistent purification outcomes across different batches, making it difficult to obtain reproducible experimental results.

[0004] After centrifugation, existing techniques also suffer from insufficient accuracy in determining the enrichment region of extracellular vesicles. Some methods rely on a single physical or biochemical indicator, such as visually observing turbidity bands in a gradient or analyzing the protein content of each component by gel electrophoresis. Turbidity may be caused by non-vesicle protein aggregates or lipoproteins, and high abundance of soluble protein contaminants may also interfere with the identification of the target protein. This localization method based on single-dimensional information is not accurate enough to effectively distinguish extracellular vesicles from other impurity particles of similar density, thus affecting the purity of the final product.

[0005] Furthermore, in pursuit of separation precision, some techniques construct complex density gradients containing multiple fine layers. While multi-layer gradients can theoretically provide high separation resolution, in practice, collecting multiple dispersed, fine bands increases the complexity and technical difficulty of the operation. Simultaneously, collecting multiple fractions for subsequent processing increases the overall sample volume, complicating subsequent washing and concentration steps, thus increasing the overall complexity of the purification process. Summary of the Invention

[0006] The purpose of this application is to provide an improved purification environment and method for pure bacterial extracellular vesicles, which solves the problems of poor reproducibility and low purity in the existing technology when purifying extracellular vesicles from diverse sources and heterogeneous densities in environments and from pure bacteria.

[0007] This application provides an improved purification environment and a method for pure bacterial-derived extracellular vesicles, the method comprising the following steps:

[0008] S1. Exploratory purification step: Obtain the first crude extracellular vesicle sample, perform exploratory multilayer density gradient ultracentrifugation on the first sample, and characterize and analyze each component obtained after centrifugation to determine the enrichment range of extracellular vesicles.

[0009] S2. Optimized purification step: Obtain a second crude extracellular vesicle sample homologous to the first crude extracellular vesicle sample. Based on the enrichment interval determined in step S1, construct an optimized simplified density gradient and perform ultracentrifugation on the second sample using the optimized simplified density gradient to collect and purify the extracellular vesicles.

[0010] In one specific embodiment, the characterization analysis in step S1 is performed by combining transmission electron microscopy and nanoflow cytometry to analyze each component. Transmission electron microscopy is used to observe the presence of vesicle-like particles in each component and to identify their purity, while nanoflow cytometry is used to determine the particle size distribution and particle concentration in each component.

[0011] In another optional specific embodiment, the characterization analysis further includes protein analysis of each component using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain protein band distribution information of each component.

[0012] In one specific implementation, the exploratory multilayer density gradient ultracentrifugation in step S1 and the optimized simplified density gradient ultracentrifugation in step S2 are set with the following centrifugation conditions: relative centrifugal force of 100,000 × g, centrifugation temperature of 4 °C, and centrifugation time of 16 hours.

[0013] In one specific embodiment, the exploratory multilayer density gradient in step S1 is formed by laying 10-16 working solutions of iodixanol with different concentrations.

[0014] In one specific embodiment, the optimized simplified density gradient in step S2 is formed by laying 4-6 different concentrations of iodixanol working solutions, and the concentration range of the 4-6 different concentrations of iodixanol working solutions covers the enrichment interval determined in step S1.

[0015] In one specific embodiment, the iodixanol working solution is prepared from a 60% (w / v) iodixanol stock solution and a dilution buffer. The dilution buffer consists of 8.5 g of NaCl and 10 mM HEPES per liter of aqueous solution, with a pH of 7.4. The salinity and pH of the dilution should be consistent with the sample source environment.

[0016] In one specific embodiment, the first and second crude extracts of extracellular vesicle samples are prepared by differential centrifugation combined with ultracentrifugation of natural environmental samples such as water, soil, dust or feces or laboratory pure cultures.

[0017] In one specific embodiment, the method further includes a follow-up step: after step S2, the collected purified extracellular vesicles are diluted with buffer and then residual density gradient medium is removed by washing and centrifugation.

[0018] In summary, this application includes at least one of the following beneficial technical effects:

[0019] 1. This invention establishes a set of density gradient parameters and collection protocols for extracellular vesicle samples from specific sources by setting up exploratory purification steps and optimized purification steps. When processing samples homologous to the first sample, the optimized simplified density gradient can be directly used. This standardizes the purification process for specific sample sources, thereby improving the reproducibility of purification results across different batches;

[0020] 2. In the exploratory purification step of this invention, transmission electron microscopy and nanoflow cytometry are used to jointly characterize and analyze all components after centrifugation. This method uses multiple physical parameters, such as morphology (vesicle structure, purity), particle size distribution, and particle concentration, to define the enrichment zone of extracellular vesicles. Compared with methods that rely on a single dimension (such as protein distribution or morphological observation), it provides more precise localization of the enrichment zone.

[0021] 3. In the optimized purification step, this invention employs a simplified density gradient with fewer layers. This design enriches the target extracellular vesicles in one or two wider, more concentrated bands. This simplifies the physical collection of the target component and reduces operational errors or sample loss that may be introduced by collecting multiple dispersed, small bands, thus lowering the complexity of routine purification procedures. Detailed Implementation

[0022] This application will be further described in detail below.

[0023] Example 1: An improved method for purifying extracellular vesicles from a pure bacterial environment, comprising the following steps:

[0024] 1. Exploratory purification steps

[0025] Obtain a crude extracellular vesicle sample prepared by differential centrifugation combined with ultracentrifugation. Twelve working solutions of iodixanol with volume percentages of 40%, 37%, 34%, 31%, 28%, 25%, 22%, 19%, 16%, 13%, 10%, and 7% were prepared using 60% iodixanol stock solution and dilution buffer (DB). In a 14x89 mm ultracentrifuge tube, 1 mL of each of the 40%, 37%, 34%, 31%, 28%, 25%, 22%, 19%, 16%, 13%, 10%, and 7% working solutions was slowly added sequentially from bottom to top, creating a 12-layer fine density gradient. A 1.0 mL resuspended crude extracellular vesicle sample was stacked on top of the gradient. After balancing, the sample was ultracentrifuged at 100,000 × g for 16 hours at 4°C. After centrifugation, each layer was collected sequentially from the top, with each 1.0 mL fraction constituting one sample.

[0026] The collected fractions were analyzed by transmission electron microscopy and nanoflow cytometry. The results showed that the fractions corresponding to the original 22% and 31% iodixanol layers contained a large number of intact vesicle structures with diameters concentrated between 50 and 150 nm, and few impurity particles. Based on this, the extracellular vesicle enrichment range of this sample was determined to be the 22%–31% iodixanol concentration range.

[0027] Obtain another crude extracellular vesicle sample homologous to the aforementioned sample. Construct a simplified, optimized density gradient consisting of four layers, from bottom to top: 3.0 mL of 40% iodixanol, 3.0 mL of 30% iodixanol, 3.0 mL of 20% iodixanol, and 3.0 mL of 10% iodixanol. Add 1.0 mL of the resuspended sample to the top of the gradient and centrifuge under the same conditions (4°C, 100,000 × g, 16 hours).

[0028] After centrifugation, a distinct band was observed at the 20% / 30% iodixanol interface. This band was collected. The collected fraction was diluted with at least 5 volumes of sterile PBS and washed by centrifugation at 100,000 × g for 2 hours at 4°C. The supernatant was discarded, and the precipitate was resuspended in 200 µL of PBS to obtain purified extracellular vesicles.

[0029] Example 2: An improved method for purifying the environment and pure bacterial-derived extracellular vesicles, comprising the following steps:

[0030] 1. Exploratory purification steps

[0031] Obtain a crude sample of extracellular vesicles. Prepare six working solutions of iodixanol with volume percentages of 35%, 30%, 25%, 20%, 15%, and 10% using 60% iodixanol stock solution and dilution buffer (DB). Construct a six-layer fine density gradient in an ultracentrifuge tube, with each layer having a volume of 2.0 mL. Add 1.0 mL of resuspended sample to the top of the gradient. After balancing, ultracentrifuge at 100,000 × g for 16 hours at 4 °C. After centrifugation, collect the fractions from each layer.

[0032] Characterization analysis of each component showed that the component corresponding to the original 20%-30% iodixanol layer had the highest concentration of vesicle particles and the most uniform morphology. Based on this, the extracellular vesicle enrichment range of this sample was determined to be around the 20%-30% iodixanol concentration.

[0033] 2. Optimized purification steps

[0034] Obtain another homologous crude extracellular vesicle sample. Construct an optimized density gradient consisting of three layers, from bottom to top: 2.0 mL of 30% iodixanol, 4.0 mL of 20% iodixanol, and 4.0 mL of 10% iodixanol. Add 1.5 mL of the resuspended sample to the top of the gradient and centrifuge under the same conditions (4°C, 100,000 × g, 16 h).

[0035] After centrifugation, the target band was collected at the 10% / 20% iodixanol interface. The collected fraction was diluted with PBS and washed by centrifugation at 100,000×g for 2 hours. The supernatant was discarded, and the precipitate was resuspended in 200µL PBS to obtain purified extracellular vesicles.

[0036] Example 3: An improved method for purifying the environment and pure bacterial-derived extracellular vesicles, comprising the following steps:

[0037] 1. Exploratory purification steps

[0038] Obtain a crude sample of extracellular vesicles. Prepare five working solutions of iodixanol with volume percentages of 45%, 35%, 25%, 15%, and 5% using 60% iodixanol stock solution and dilution buffer (DB). Construct a fine density gradient of five layers, each with a volume of 2 mL, in an ultracentrifuge tube. Add 2.0 mL of the resuspended sample to the top of the gradient. After balancing, ultracentrifuge at 100,000 × g for 16 hours at 4°C. After centrifugation, collect the fractions from each layer.

[0039] Characterization analysis of each component showed that the components corresponding to the original 25% and 35% iodixanol layers had the highest enrichment of vesicle particles. Based on this, the extracellular vesicle enrichment range of this sample was determined to be the 25%-35% iodixanol concentration range.

[0040] 2. Optimized purification steps

[0041] Obtain another homologous crude extracellular vesicle sample. Construct an optimized density gradient consisting of two layers, with 6.0 mL of 40% iodixanol and 6.0 mL of 20% iodixanol added sequentially from bottom to top. Stack 1.0 mL of the resuspended sample on top of the gradient and centrifuge under the same conditions (4°C, 100,000 × g, 16 h).

[0042] After centrifugation, the target band was collected at the 20% / 40% iodixanol interface. The collected fraction was diluted with PBS and washed by centrifugation at 100,000×g for 2 hours. The supernatant was discarded, and the precipitate was resuspended in 200 µL PBS to obtain purified extracellular vesicles.

[0043] Comparative Example

[0044] Comparative Example 1

[0045] Compared to Example 1, the difference is that, instead of performing an exploratory purification step, a pre-defined density gradient constructed from four iodixanol working solutions of 40%, 30%, 20%, and 10% was used to purify the crudely extracted extracellular vesicle sample, and the band at the 20% / 30% interface was collected. All other steps were the same.

[0046] Comparative Example 2

[0047] Compared to Example 1, the difference lies in that, in the exploratory purification step, instead of using a combination of transmission electron microscopy and nanoflow cytometry, the enrichment zone was determined solely by visually observing the turbidity of each layer in the centrifuge tube, with the most turbid component layer (assumed to be the original 15% iodixanol layer) designated as the enrichment zone for extracellular vesicles. Subsequent optimized purification steps were performed based on this zone. Everything else remained the same.

[0048] Comparative Example 3

[0049] Compared to Example 2, the difference is that, instead of performing an exploratory purification step, a pre-defined density gradient constructed from 30%, 20%, and 10% iodixanol working solutions was used to purify the crudely extracted extracellular vesicle sample, and the band at the 10% / 20% interface was collected. All other steps were the same.

[0050] Comparative Example 4

[0051] Compared to Example 2, the difference lies in that, in the exploratory purification step, instead of using a combination of transmission electron microscopy and nanoflow cytometry, only sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the total protein content of each fraction, and the fraction layer with the richest protein bands (assumed to be the original 20% iodixanol layer) was used as the enrichment region for extracellular vesicles. Subsequent optimized purification steps were performed based on this region. Everything else was the same.

[0052] Comparative Example 5

[0053] Compared to Example 3, the difference is that, instead of performing an exploratory purification step, a pre-defined density gradient constructed from 40%, 20%, and 10% iodixanol working solutions was used to purify the crudely extracted extracellular vesicle sample, and the band at the 20% / 40% interface was collected. All other steps were the same.

[0054] Comparative Example 6

[0055] Compared to Example 3, the difference lies in that, in the exploratory purification step, only transmission electron microscopy was used for observation, without quantitative analysis using nanoflow cytometry, and the enrichment zone was determined based on the component layer with the clearest observed vesicle morphology (assumed to be the original 20% iodixanol layer). Subsequent optimized purification steps were performed based on this zone. Everything else was the same.

[0056] Test Example 1: Comparison Test of Final Product Purity and Recovery Rate

[0057] 1. Experimental Procedure

[0058] The final purified products prepared according to Examples 1, 2, and 3 (denoted as Experimental Group A, Experimental Group B, and Experimental Group C, respectively) and the final products prepared according to Comparative Examples 1, 3, and 5 (denoted as Control Group A, Control Group B, and Control Group C, respectively) were obtained. The following tests were performed on these six groups of samples:

[0059] Step 1.1: Determination of total particle count

[0060] The assay was performed using a nanoflow cytometer. 10 µL of the final product suspension from each group was diluted with 190 µL of PBS filtered through a 0.1 µm filter. The instrument was calibrated for particle size and concentration using silica nanoparticle standards provided by the instrument manufacturer. The diluted samples were analyzed, and particle concentration data (unit: particles / mL) were recorded. The total number of particles in each group was calculated based on the initial resuspending volume.

[0061] Step 1.2: Total Protein Measurement

[0062] Protein quantification was performed using the BCA method protein quantification kit. Take 2 µL of the final product suspension from each group and follow the kit instructions. A standard curve was constructed using bovine serum albumin (BSA) standards. After mixing the samples with the BCA working reagent, incubate at 37°C for 30 minutes. Measure the absorbance at 562 nm using a micro-spectrophotometer. Calculate the protein concentration (μg / mL) for each group based on the standard curve. Calculate the total protein content for each group based on the initial resuspending volume.

[0063] Step 1.3: Data Calculation

[0064] Based on the data obtained in steps 1.1 and 1.2, calculate the ratio of particle number to protein mass for each group of samples. The calculation formula is: Ratio = Total number of particles / Total protein mass (μg).

[0065] The experimental data are shown in Table 1:

[0066] Table 1. Comparison of product purity and recovery rate test data

[0067] Group <![CDATA[Total number of particles (×10 9 )]]> Total protein content (μg) <![CDATA[Particle number / protein mass ratio (×10 7 particles / μg)]]> Experimental group (Example 1) 8.7 12.5 6.96 Control group A (Comparative example 1) 4.2 18.1 2.32 Experimental Group B (Example 2) 9.1 13.2 6.89 Control group B (Comparative example 2) 3.8 17.5 2.17 Experimental Group C (Example 3) 7.9 11.8 6.69 Control group C (Comparative example 5) 4.5 19.3 2.33

[0068] Experimental conclusion:

[0069] Experimental data show that, compared to their corresponding control groups A, B, and C, experimental groups A, B, and C produced a final product with a higher total particle count and a lower total protein content. The calculated particle count to protein mass ratio was significantly higher in the experimental groups than in the control groups. This data indicates that, compared to the method used in the control groups, the method of this invention produces a final product containing a higher number of target particles and a relatively lower content of protein impurities.

[0070] The control group used a single centrifugation purification method with a fixed density gradient and a small number of layers, without pre-determining the sample density distribution. Because the physical density of environmentally derived extracellular vesicles is unknown and heterogeneous, a pre-defined gradient interface may not precisely match the actual isodense points of extracellular vesicles in a specific sample. This mismatch results in some target vesicles failing to accumulate in the collection area, thus reducing the total number of recovered particles. Simultaneously, it also causes protein contaminants with similar densities to the target vesicles or those non-specifically aggregated in this region to be collected along with them, leading to a relatively high protein content in the final product.

[0071] The method of this invention comprises an exploratory purification step and an optimized post-purification step. The exploratory purification step utilizes multiple fine density gradients to determine the actual enrichment range of extracellular vesicles for a sample from a specific source. The subsequent optimized post-purification step then constructs a simplified density gradient to match this range. This two-step framework ensures that the final collection operation is performed on a known and precise target density range, thereby guaranteeing the enrichment efficiency of the target extracellular vesicles and their effective separation from major protein contaminants. This is consistent with the results in the test data showing a higher total particle count and a higher particle number to protein mass ratio.

[0072] Test Example 2: Comparative Test of the Accuracy of Enrichment Interval Determination Methods

[0073] 1. Experimental Procedure

[0074] The crude extracellular vesicle samples from the same batch were divided into four equal parts, and the exploratory multilayer density gradient ultracentrifugation step described in Example 1 was performed on each sample. After centrifugation, each component was analyzed using the following different methods to determine the enrichment range, and subsequent optimized purification steps were performed based on this range.

[0075] Example 1: Each collected component was analyzed by transmission electron microscopy (TEM) to confirm vesicle morphology, and nanoflow cytometry (NanoFCM) was performed to determine particle concentration and size distribution. The continuous component layer with the correct vesicle morphology and the highest particle concentration was identified as the enrichment zone.

[0076] Comparative Example 2: By visually observing the turbidity of each component layer, the component layer with the highest turbidity was determined as the enrichment zone.

[0077] Comparative Example 4: SDS-PAGE analysis was performed on each component, and the layer with the richest total protein band was identified as the enrichment region.

[0078] Comparative Example 6: Only transmission electron microscopy (TEM) analysis was performed on each component, and the component layer with the clearest and most uniform vesicle morphology was identified as the enrichment region.

[0079] Based on the determined enrichment regions for each group, corresponding optimized simplified density gradients were constructed, and subsequent purification and washing steps were completed to obtain four final product groups. The following tests were performed on these four product groups:

[0080] Step 1.1: Product Purity Determination. The total protein content of the final product in each group was determined using the BCA method. The total number of particles in the final product of each group was determined using nanoflow cytometry (NanoFCM). The ratio of particle number to protein mass was calculated.

[0081] Step 1.2: Determination of particle size distribution uniformity. Using the analytical data from nanoflow cytometry (NanoFCM), the particle size distribution of the final products of each group was statistically analyzed, and the standard deviation (SD) of the particle size distribution was calculated as an indicator of sample uniformity.

[0082] The experimental data are shown in Table 2:

[0083] Table 2. Comparative test data of products obtained by different enrichment interval determination methods

[0084] Group <![CDATA[Total number of particles (×10 9 )]]> <![CDATA[Particle number / protein mass ratio (×10 7 particles / μg)]]> Particle size distribution standard deviation (nm) Example 1 8.5 6.81 65.1 Comparative Example 2 3.1 1.53 85.8 Comparative Example 4 6.9 3.24 78.4 Comparative Example 6 7.5 5.12 70.2

[0085] Experimental conclusion:

[0086] Experimental data showed that the experimental group, analyzed using a combination of transmission electron microscopy and nanoflow cytometry, produced the highest ratio of particle number to protein mass and the smallest standard deviation of particle size distribution. In contrast, the products from controls 2, 4, and 6 showed inferior results in both of these metrics compared to Example 1. This data indicates that the enrichment interval determination method used in the experimental group yields a final product with higher purity and more uniform particle size.

[0087] The single-dimensional analysis method used in the control group has limitations in its physical basis. The turbidity based on Comparative Example 2 may originate from large protein aggregates or other non-vesicle particles, not perfectly corresponding to the actual distribution of extracellular vesicles. The total protein concentration based on Comparative Example 4 cannot distinguish between membrane / luminal proteins of the target vesicle and co-precipitated, high-abundance protein contaminants from non-vesicle sources, potentially leading to misclassification of contaminant-enriched layers as the target layer. While vesicle morphology observation based on Comparative Example 6 can identify particles, it cannot provide concentration information, potentially missing the core enriched layer with the highest particle concentration. These limitations explain the lower purity and uniformity of the final product.

[0088] The method of this invention specifies multi-dimensional characterization analysis of each gradient component in its exploratory purification step. Transmission electron microscopy provides qualitative information at the morphological level to confirm whether the particles are vesicles with a bilayer membrane structure. Nanoflow cytometry provides quantitative information at the physical level, including the precise concentration and particle size distribution of particles in each component. By combining morphological confirmation with quantitative concentration data, this method can accurately identify the core enrichment region with the highest concentration from all components containing the target vesicles, thereby providing more accurate target parameters for subsequent optimized purification steps.

[0089] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made to the structure, shape, and principle of this application should be covered within the scope of protection of this application.

Claims

1. An improved method for purifying extracellular vesicles from a pure bacterial environment, characterized in that, Includes the following steps: S1. Exploratory purification step: Obtain the first crude extracellular vesicle sample, perform exploratory multilayer density gradient ultracentrifugation on the first sample, and characterize and analyze each component obtained after centrifugation to determine the enrichment range of extracellular vesicles. S2. Optimized purification step: Obtain a second crude extracellular vesicle sample homologous to the first crude extracellular vesicle sample. Based on the enrichment interval determined in step S1, construct an optimized simplified density gradient and perform ultracentrifugation on the second sample using the optimized simplified density gradient to determine the collected density layer, so as to collect and purify the extracellular vesicles.

2. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 1, characterized in that, The characterization analysis in step S1 includes: The components were analyzed by a combination of transmission electron microscopy and nanoflow cytometry to determine the enrichment regions of the extracellular vesicles.

3. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 2, characterized in that, The characterization analysis may also include: Protein analysis of each component was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This step is optional.

4. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 1, characterized in that, The centrifugation conditions for the exploratory multilayer density gradient ultracentrifugation in step S1 and the optimized simplified density gradient ultracentrifugation in step S2 are as follows: The relative centrifugal force was 100,000 × g, the temperature was 4 °C, and the time was 16 hours.

5. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 1, characterized in that, The exploratory multilayer density gradient in step S1 is formed by laying 10-16 working solutions of iodixanol at different concentrations. The number of density gradient layers can be set according to the complexity of the extracellular vesicle sample source.

6. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 1, characterized in that, The optimized simplified density gradient in step S2 is formed by laying 4-6 different concentrations of iodixanol working solution, and the concentration range of the 4-6 different concentrations of iodixanol working solution covers the enrichment interval. After the enrichment interval is determined, subsequent samples from the same source can be directly purified using the enrichment interval. However, when the sample source is changed, the exploratory multilayer density gradient in step S1 needs to be repeated to redefine the enrichment interval of extracellular vesicles from the new sample source.

7. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 1, characterized in that, The iodixanol working solutions used in the exploratory multilayer density gradient in step S1 and the optimized simplified density gradient in step S2 are both prepared from 60% (w / v) iodixanol stock solution and dilution buffer.

8. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 1, characterized in that, The formulation of the dilution buffer is as follows: It contains 8.5 g / L NaCl and 10 mM HEPES, and the pH is adjusted to 7.

4. The addition of NaCl and the adjustment of pH must be consistent with the salinity and pH of the sample source environment.

9. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 1, characterized in that, The first and second crude extracts of extracellular vesicles can be derived from natural environmental samples such as water, soil, dust or feces, as well as liquid cultures of pure bacteria in the laboratory.

10. The method for improving the purification environment and pure bacterial-derived extracellular vesicles according to claim 1, characterized in that, Also includes: After step S2, the purified extracellular vesicles are collected and diluted, and the density gradient medium is removed by washing and centrifugation. During the washing process, PBS with a volume at least 5 times larger than the collected enriched layer is added.