Lactobacillus paracasei extracellular vesicle for relieving ulcerative colitis and application of lactobacillus paracasei extracellular vesicle

Abstract

The invention discloses a lactobacillus paracasei extracellular vesicle for relieving ulcerative colitis and application of the lactobacillus paracasei extracellular vesicle, and belongs to the technical field of microbial extracellular vesicles. The invention provides a strain of Lactobacillus paracasei Li-1, and the preservation number of the Lactobacillus paracasei Li-1 is CCTCC (China Center For Type Culture Collection) NO: M 2025435. The invention further provides the extracellular vesicles of the lactobacillus paracasei Li-1, and the extracellular vesicles are extracted from fermentation liquor of the lactobacillus paracasei Li-1. The application of the Lactobacillus paracasei extracellular vesicles in relieving and / or treating ulcerative colitis shows that the Lactobacillus paracasei extracellular vesicles significantly improve the weight change rate, DAI index and colon length of mice, and intervene the progress of ulcerative colitis. The lactobacillus paracasei extracellular vesicles can relieve ulcerative colitis, and a theoretical basis is provided for developing the lactobacillus paracasei extracellular vesicles into microecologics to be applied to the fields of medicines and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial extracellular vesicles, and particularly to an extracellular vesicle of Lactobacillus paracasei for alleviating ulcerative colitis and its application. Background Art

[0002] Ulcerative colitis is a lifelong inflammatory disease that affects the rectum and colon to varying degrees. In 2023, the global prevalence of ulcerative colitis was estimated to be 5 million cases, and the global incidence is on the rise. Patients usually present with symptoms such as bloody diarrhea, intestinal epithelial barrier defects, and microbiota dysbiosis. Currently, clinical treatment drugs include 5-aminosalicylic acid drugs, thiopurine drugs, biological agents (e.g., anti-cytokines and anti-integrins), and small molecule drugs (Janus kinase inhibitors and sphingosine-1-phosphate receptor modulators). Although the means of clinical drug treatment are increasing year by year, drug treatment will ultimately lead to drug resistance in the host. In recent years, treatment regimens for alleviating ulcerative colitis through probiotic intervention have been increasing year by year. However, probiotics also have their limitations: firstly, probiotics may carry resistance genes and transfer these genes to pathogenic bacteria through horizontal gene transfer; secondly, since probiotics need to function under live bacteria conditions, there are high requirements for their shelf life, transportation methods, and preparation processes. In contrast, extracellular vesicles derived from probiotics are more stable, have a longer shelf life, better environmental tolerance, and are more suitable for the food and drug fields.

[0003] Bacterial extracellular vesicles are spherical lipid bilayer nanostructures with sizes ranging from 20 to 300 nm. Both Gram-negative and Gram-positive bacteria can produce them. They are natural carriers of bacterial molecules, including biomacromolecules such as peptidoglycan, lipids, proteins, and nucleic acids. As a secretion mechanism, they allow for the long-distance delivery of bacterial active compounds in a protected environment, avoiding direct cell-to-cell contact. The composition and content of extracellular vesicles are derived from the producer strain and vary according to the growth stage and conditions. Probiotic extracellular vesicles have been proven to be able to alleviate intestinal inflammation, but their alleviation efficiency varies greatly. Lingjun Tong et al. found that extracellular vesicles of Lactobacillus rhamnosus can relieve colon tissue damage, reduce intestinal inflammatory responses by inhibiting the activation of the TLR4-NF-κB-NLRP3 axis, and pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-2 were effectively inhibited after treatment, but the weight change rate of the mice in the treatment group did not recover. Morishita et al. showed that extracellular vesicles of Lactiplantibacillus plantarum WCFS1 were taken up by Raw264.7 and DC2.4 cells through clathrin-mediated endocytosis and phagocytosis, and these extracellular vesicles produced inflammatory cytokines TNF-α and IL-6 by immune cells, but in vivo animal experiments were not conducted, and its efficiency in vivo could not be determined. Summary of the Invention

[0004] The object of the present invention is to provide an extracellular vesicle of Lacticaseibacillus paracasei for alleviating ulcerative colitis and its application, so as to solve the problems existing in the above-mentioned prior art. The extracellular vesicle of Lacticaseibacillus paracasei of the present invention can alleviate ulcerative colitis, providing a theoretical basis for its development as a probiotic preparation for application in the fields of medicine and the like.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a strain of Lacticaseibacillus paracasei Li-1, and the preservation number of the Lacticaseibacillus paracasei Li-1 is CCTCC NO: M 2025435, the preservation date is March 10, 2025, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

[0007] The present invention also provides an extracellular vesicle of Lacticaseibacillus paracasei Li-1, and the extracellular vesicle is extracted from the fermentation broth of Lacticaseibacillus paracasei Li-1, and the preservation number of the Lacticaseibacillus paracasei Li-1 is CCTCC M2025435.

[0008] The present invention also provides a method for preparing the extracellular vesicles, comprising the following steps:

[0009] Centrifuge the fermentation broth of Lactobacillus paracasei Li-1 for the first time, collect the supernatant, and filter and sterilize it to obtain the sterilized supernatant;

[0010] Add the sterilized supernatant to a 100KD ultrafiltration tube, centrifuge it for the second time, and collect the concentrate;

[0011] Centrifuge the concentrate for the third time, discard the supernatant, resuspend it, and then centrifuge it for the fourth time, discard the supernatant, and thus obtain the extracellular vesicles.

[0012] Optionally, the first centrifugation is carried out at 5000-9000g for 5-15 min.

[0013] Optionally, the second centrifugation is carried out at 1500-3000g for 5-15 min.

[0014] Optionally, both the third centrifugation and the fourth centrifugation are carried out at 100000-130000g at 4°C for 1-2 h.

[0015] Optionally, the filtration and sterilization is carried out by filtering with a 0.22μm filter membrane.

[0016] The present invention also provides the application of the extracellular vesicles of Lactobacillus paracasei Li-1 in the preparation of a drug for relieving ulcerative colitis.

[0017] The present invention also provides a drug for relieving ulcerative colitis, and the drug comprises the extracellular vesicles of Lactobacillus paracasei Li-1.

[0018] Optionally, the drug further comprises a pharmaceutically acceptable excipient.

[0019] The present invention discloses the following technical effects:

[0020] The present invention isolates and screens a strain of Lactobacillus paracasei Li-1 capable of producing extracellular vesicles from the intestinal contents of healthy infants, which was deposited at the China Center for Type Culture Collection on March 10, 2025, with the deposit number CCTCC NO: M2025435. By optimizing the extraction method, the number of extracellular vesicle particles extracted reaches 7.2×10 8 particles / mL (7.2×10 8 extracellular vesicles are contained in every 1 mL of the fermentation broth supernatant).

[0021] The present invention applies the extracellular vesicles of Lactobacillus paracasei to relieve and / or treat ulcerative colitis, and finds that it significantly improves the weight change rate, DAI index and colon length of mice, and intervenes in the process of ulcerative colitis.

[0022] The extracellular vesicles of Lactobacillus paracasei were applied to LPS-induced MODE-K cells, significantly increasing the proliferation rate of MODE-K cells.

[0023] In summary, the extracellular vesicles of Lactobacillus paracasei of the present invention can alleviate ulcerative colitis, providing a theoretical basis for its development as a probiotic preparation for application in the fields of medicine and others. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is a morphological diagram of Lactobacillus paracasei colonies;

[0026] Figure 2 It is a Gram staining diagram of Lactobacillus paracasei;

[0027] Figure 3 It is a liquid chromatography diagram of lactic acid content;

[0028] Figure 4 It is a liquid chromatography diagram of γ-aminobutyric acid content;

[0029] Figure 5 It is a scanning electron microscopy diagram of Lactobacillus paracasei; the scale bar is 2.00 μm;

[0030] Figure 6 It is a scanning electron microscopy diagram of Lactobacillus paracasei; the scale bar is 500 nm;

[0031] Figure 7 It is a transmission electron microscopy diagram of the extracellular vesicles of Lactobacillus paracasei; the scale bar is 200 nm;

[0032] Figure 8 It is a transmission electron microscopy diagram of Lactobacillus paracasei and its extracellular vesicles; the scale bar is 200 nm;

[0033] Figure 9 It is an NTA particle size analysis diagram of the extracellular vesicles of Lactobacillus paracasei;

[0034] Figure 10 It is a diagram of the DNA, RNA, and protein contents of the extracellular vesicles of Lactobacillus paracasei;

[0035] Figure 11 It is a diagram of the body weight change rate (A) and DAI index (B) of the extracellular vesicles of Lactobacillus paracasei on DSS-induced murine colitis;

[0036] Figure 12 The intestinal tissue length diagram of the extracellular vesicles of Lactobacillus paracasei against DSS-induced colitis in mice;

[0037] Figure 13 The attack result diagram of LPS on MODE-K;

[0038] Figure 14 The diagram of the effect of the extracellular vesicles of Lactobacillus paracasei on the proliferation rate of LPS-induced MODE-K. Detailed implementation mode

[0039] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing particular implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0042] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0043] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0044] Example 1 Screening and identification of Lactobacillus paracasei

[0045] 1. Screening of strains

[0046] First, take 1 g of intestinal contents from healthy infants, and select 10 -6 dilution factors for plate coating according to the dilution and spread plate method. The coating volume is 100 μL, and the culture is carried out at 37 °C for 48 h. Select the strains with larger diameters of yellow transparent zones for rescreening by streaking on the plate, and finally select single colonies and inoculate them into MRS liquid medium for enrichment culture at 37 °C and 180 rpm / min.

[0047] 2. Identification of strains

[0048] 2.1 Morphological identification

[0049] Streak the strain on the MRS plate. As Figure 1 shown, the colonies of this strain are milky white, the single colonies are small, the surface is smooth and moist, round, and the colony diameter is about 1 mm.

[0050] Perform Gram staining on the strain. The result is as Figure 2 shown. By observing with an oil immersion lens of an optical microscope, this strain is a Gram-positive bacterium.

[0051] 2.2 Identification of the ability to produce organic acids

[0052] 2.2.1 Determination of lactic acid content

[0053] (1) Mobile phase: 5 mM H2SO4.

[0054] (2) Plot the standard curve

[0055] Dilute the lactic acid standard sample of 1.2 g / L to 0.6 g / L, 0.2 g / L, 0.1 g / L, and 0.05 g / L in turn, and perform sample injection for detection.

[0056] Chromatographic column: Bio-rad HPX-87H ion-exclusion column (300×7.8 mm); HPLC conditions are: isocratic elution, flow rate is 0.6 mL / min; detector: differential refractive index detector; detection wavelength: 210 nm; injection volume: 20 μL; column temperature 40 °C, detector temperature 35 °C.

[0057] According to Figure 3 it can be known that organic acids such as lactic acid, acetic acid, and propionic acid are produced in the bacterial liquid. Among them, the peak time of lactic acid is 13 min, the peak time of acetic acid is 15 min, and the peak time of propionic acid is 18 min. Among them, the peak at 13.399 is lactic acid, the peak at 15.690 is acetic acid, and the peak at 18.512 is propionic acid. According to the lactic acid standard curve y = 253019x - 5161 (where y is the peak area and x is the lactic acid content), the lactic acid content is calculated to be 1.016 g / L.

[0058] 2.2.2 Determination of γ-aminobutyric acid content

[0059] (1) Reagent preparation

[0060] Derivatization reagent: Dissolve 100 mg of OPA and 100 μL of β-mercaptoethanol in 25 mL of acetonitrile;

[0061] 0.4 mol / L boric acid buffer (pH 10.2): Dissolve 2.47 g of boric acid in 100 mL of ultrapure water and adjust the pH to 10.2.

[0062] Mobile phase: Phase A was 20 mM sodium acetate (pH 7.5); Phase B was acetonitrile.

[0063] (2) Sample derivation

[0064] Pipette 400 μL of borate buffer, 80 μL of sample solution and 80 μL of OPA derivatization reagent, mix and allow to react fully at room temperature.

[0065] (3) Draw a standard curve

[0066] 1g / L γ-aminobutyric acid standard was diluted to 0.8g / L, 0.6g / L, 0.4g / L, and 0.2g / L in sequence, and 20μL was injected after derivatization. HPLC conditions were: gradient elution, chromatographic column was Zorbax Eclipse-AAA column (4.6mm×150mm, 3.5μm), column temperature was 40℃, detector was UV detector, detection wavelength was 338nm, flow rate was 1mL / min, elution time was 26min, injection volume was 20μL. The standard curve was drawn with the concentration of γ-aminobutyric acid as the horizontal axis and the peak area as the vertical axis.

[0067] like Figure 4 As shown, 15.1 min is the γ-aminobutyric acid peak time. According to the γ-aminobutyric acid standard curve y=6911026x-74008 (where y is the peak area and x is the γ-aminobutyric acid content), the γ-aminobutyric acid content is calculated to be about 0.14 g / L.

[0068] 2.3 Molecular Biological Identification

[0069] The DNA of the strain was extracted and amplified using bacterial universal primers, and then 16S rDNA sequencing analysis was performed to obtain a partial sequence of the 16S rDNA gene as shown in SEQ ID NO.1.

[0070] SEQ ID NO.1:

[0071]

[0072] Through NCBI sequence alignment, the results showed that the highest similarity was with Lactobacillus paracasei, reaching 99.86%. This strain was identified as Lactobacillus paracasei and named Lactobacillus paracasei Li-1. This strain was deposited in the China Center for Type Culture Collection on March 10, 2025. The taxonomic name is Lacticaseibacillus paracasei, and the deposit number is CCTCC NO: M 2025435. The deposit address is Wuhan University, Wuhan, China.

[0073] Example 2 Optimization of the extraction method and identification and characterization of extracellular vesicles of Lactobacillus paracasei

[0074] 2.1 Collection and purification of extracellular vesicles

[0075] (1) Collection: Collect the fermentation broth (MRS liquid medium) into a 50 mL centrifuge tube and centrifuge at 5000 - 9000 g for 5 - 15 min (in this example, 5000 g for 10 min). Collect the supernatant and filter it through a 0.22 μm filter membrane to remove bacteria.

[0076] (2) Concentration: Add the supernatant to a 100 KD ultrafiltration tube and centrifuge at 1500 - 3000 g for 5 - 15 min (in this example, 1500 g for 10 min). Collect the concentrated solution.

[0077] (3) Ultracentrifugation: Transfer the concentrated supernatant to an ultracentrifuge tube, centrifuge at 100000 - 130000 g at 4°C for 1 - 2 h (in this example, 100000 g for 1 h). Then discard the supernatant, add an equal volume of 1×PBS to resuspend, centrifuge at 100000 - 130000 g at 4°C for 1 - 2 h (in this example, 100000 g for 1 h), discard the supernatant, add 1 mL of 1×PBS to resuspend to obtain purified extracellular vesicles, and store them in a -80°C refrigerator.

[0078] 2.2 Characterization of extracellular vesicles

[0079] 2.2.1 Observation of cell morphology by scanning electron microscopy

[0080] (1) Bacterial cell collection: For liquid fermentation of bacteria, after culturing to the logarithmic growth phase, take an appropriate amount of the bacterial solution and centrifuge at 2000 - 3000 rpm / min for 5 - 10 min (in this example, 2000 rpm / min for 5 min).

[0081] (2) Bacterial cell washing: Discard the supernatant, wash the precipitate with PBS, gently pipette and invert several times, and then centrifuge at 2000 - 3000 rpm / min for 5 - 10 min (in this example, 2000 rpm / min for 5 min). Repeat this operation 3 - 5 times.

[0082] (3) Bacterial cell fixation: Discard the PBS, add the fixative (2.5% glutaraldehyde), and incubate in the dark at 4 °C for more than 4 h.

[0083] (4) Bacterial cell washing: Discard the supernatant, wash the precipitate with PBS, gently pipette up and down, invert several times, and then centrifuge at 2000 - 3000 rpm for 5 - 10 min (in this example, centrifuge at 2000 rpm for 5 min). Repeat this operation 3 - 5 times.

[0084] (5) Ethanol dehydration: Prepare ethanol with gradient concentrations of 30%, 50%, 70%, 80%, 90%, and 100%, and perform gradient dehydration. Add an appropriate amount of ethanol solution, invert and mix well for 5 - 10 min, let stand for 5 - 10 min, and then centrifuge at 2000 - 3000 rpm for 5 - 10 min (in this example, centrifuge at 2000 rpm for 5 min).

[0085] The results are as Figures 5 - 6 shown. It can be seen from Figure 5 that there are raised vesicles on the surface of the bacterial cells, Figure 6 and it can be seen that it is the budding morphology of extracellular vesicles of Lactobacillus paracasei Li - 1.

[0086] 2.2.2 Observation of EVs morphology by transmission electron microscopy

[0087] Prepare the transmission electron microscopy sample of EVs by the hanging drop method. The steps are as follows:

[0088] (1) Adjust the EVs concentration to about 10 8 -10 9 particles / mL, pipette 5 - 20 μL of the sample and drop it onto the copper grid; let stand at room temperature for 3 - 5 min;

[0089] (2) Use filter paper to absorb the excess liquid. When it is about to dry, use a dropper to suck up a drop of 2% (w / v) sodium phosphotungstate staining solution (pH 7.0) and stain for 3 - 5 min;

[0090] (3) Use filter paper to suck away the excess dye, let stand, and observe under the microscope after drying in the light;

[0091] (4) Observe and photograph the sample using the HT7800 transmission electron microscope. The accelerating voltage of the transmission electron microscope is set to 90 - 120 kV, and other parameters are set to default. It can be seen from Figures 7 - 8 that the extracellular vesicles of Lactobacillus paracasei Li - 1 have an obvious lipid bilayer structure.

[0092] The DNA of EVs was measured using the Thermo Fisher Qubit dsDNA Quantification Kit, the RNA of EVs was measured using the Thermo Fisher Qubit RNA Quantification Kit, and the protein content of EVs was measured using the Novizan BCA Protein Quantification Detection Kit. The size of extracellular vesicles was measured using a nanoparticle tracking analyzer.

[0093] As Figures 9 - 10 shown, the average size of EVs was 174.6 nm. The number of particles measured after diluting the extracellular vesicles extracted from 1 L of fermentation broth was 1.8×10 8 particles / mL, and the dilution factor was 4000 times. So the final number of particles was 7.2×10 11 particles / mL, that is, 1 L of fermentation broth contained 7.2×10 11 extracellular vesicles; the DNA content was 1.44 μg / 10 9 particles; the RNA content was 4.13 μg / 10 9 particles; the protein content was 97.67 μg / 10 9 particles.

[0094] Example 3: Remission effect of extracellular vesicles of Lactobacillus paracasei on DSS-induced colitis in mice

[0095] Male C57BL / 6 mice aged 6 - 8 weeks with a body weight of 18 - 20 g were selected for the experiment of intervening in ulcerative colitis mice with extracellular vesicles derived from Lactobacillus paracasei. They were randomly divided into 3 groups (n = 8): NC group (control), MC group (model), and Li-1-EVs group (treatment with extracellular vesicles prepared in Example 2). The model mice were allowed to freely drink 2% DSS, and during the recovery period, the DSS was replaced with sterile water. The mice in the NC group and the MC group were gavaged with 200 μL of PBS every day, and the mice in the extracellular vesicle treatment group were gavaged with 200 μL of Li-1-EVs (0.3 - 0.8 mg / mL, preferably 0.25 mg / mL) every day. Finally, the mice were euthanized using isoflurane, and then the intestinal tissues of the mice were collected.

[0096] As Figure 11 shown in A, the body weight of the NC group increased steadily during the whole experiment, while the body weight of the MC group of mice induced by DSS decreased significantly. Compared with the MC group, the Li-1-EVs group had less weight loss during the modeling period. Further, the DAI was measured based on body weight changes, diarrhea conditions, and fecal occult blood to evaluate the development of DSS-induced colitis symptoms and the remission effect of the vesicles. As Figure 11As shown in Figure B, the DAI index of the MC group began to increase rapidly on the third day and slowly decreased from the ninth day. The DAI index of the Li-1-EVs group began to increase rapidly on the fourth day (except for NC) and gradually decreased from the eighth day. On the 12th day, the DAI score of the MC group was significantly higher (p < 0.01) than that of the Li-1-EVs group.

[0097] The mice were sacrificed by euthanasia on the 12th day and dissected, and the colon part was taken to measure the length. As Figure 12 shown, the colon length of the Li-1-EVs group was significantly higher than that of the MC group.

[0098] Example 4 Protective effect of extracellular vesicles of Lactobacillus paracasei on LPS-induced MODE-K mouse intestinal epithelial cells

[0099] 4.1 LPS challenge of MODE-K cells

[0100] (1) The resuscitated MODE-K cells were seeded into 96-well plates and cultured for 24 h;

[0101] (2) Different concentrations of LPS (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL) were added to the 96-well plates and co-incubated for 24 h;

[0102] (3) The CCK-8 detection kit was used to observe the effect of LPS on the proliferation rate of MODE-K.

[0103] 4.2 Protective effect of EVs on LPS-induced MODE-K

[0104] (1) The resuscitated MODE-K cells were seeded into 96-well plates and cultured for 24 h;

[0105] (2) Different concentrations of EVs prepared in Example 2 (0 μg / mL, 10 μg / mL, 30 μg / mL, 60 μg / mL, 90 μg / mL, 120 μg / mL) were added to the 96-well plates and co-incubated for 24 h; at the same time, a control NC group (without adding LPS and EVs) was set;

[0106] (3) 80 μg / mL LPS was added to the 96-well plates and co-incubated for 24 h;

[0107] (4) The CCK-8 detection kit was used to observe the effect of EVs on the proliferation rate of LPS-induced MODE-K.

[0108] As Figure 13 shown, under the action of 80 μg / mL LPS, the proliferation rate of MODE-K was the lowest, showing a dose-dependent trend; from Figure 14It can be seen that EVs at a concentration of 60-120 μg / mL significantly increased the proliferation rate of MODE-K. At 120 μg / mL, the proliferation rate was 84.11%.

[0109] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Lacticaseibacillus paracasei Li-1, characterized in that, The deposit number of the Lactobacillus paracasei Li-1 is CCTCC NO: M 2025435.

2. An extracellular vesicle of Lactobacillus paracasei Li-1, characterized in that, The extracellular vesicles are obtained by extracting from the fermentation broth of Lactobacillus paracasei Li-1, and the deposit number of the Lactobacillus paracasei Li-1 is CCTCC M2025435.

3. The method for preparing extracellular vesicles according to claim 2, wherein It includes the following steps: Centrifuge the fermentation broth of Lactobacillus paracasei Li-1 for the first time, collect the supernatant, and filter and sterilize it to obtain the sterilized supernatant. Add the sterilized supernatant to a 100KD ultrafiltration tube, centrifuge it for the second time, and collect the concentrate. Centrifuge the concentrate for the third time, discard the supernatant, resuspend it, and then centrifuge it for the fourth time, discard the supernatant, and that's it.

4. The preparation method according to claim 3, characterized in that, The first centrifugation is at 5000 - 9000g for 5 - 15 minutes.

5. The preparation method according to claim 3, characterized in that, The second centrifugation is at 1500 - 3000g for 5 - 15 minutes.

6. The preparation method according to claim 3, characterized in that, Both the third centrifugation and the fourth centrifugation are at 100000 - 130000g, and centrifuge at 4°C for 1 - 2 hours.

7. The preparation method according to claim 3, characterized in that, The filtration and sterilization is to filter and sterilize with a 0.22μm filter membrane.

8. Use of the extracellular vesicles of Lactobacillus paracasei Li-1 according to claim 2 in the preparation of a drug for relieving ulcerative colitis.

9. A drug for relieving ulcerative colitis, characterized in that, The drug contains the extracellular vesicles of Lactobacillus paracasei Li-1 according to claim 2.

10. The drug according to claim 9, characterized in that, The drug also contains pharmaceutically acceptable excipients.

Images