Bifidobacterium breve extracellular vesicle as well as preparation method and application thereof

Abstract

The invention discloses a bifidobacterium breve extracellular vesicle as well as a preparation method and application thereof, and belongs to the field of biological medicines. The extracellular vesicles are obtained by the following steps: carrying out anaerobic culture on Bifidobacterium breve FB-342 in a culture medium containing L-cysteine, and then carrying out differential centrifugation, filtration and ultracentrifugation on the cultured Bifidobacterium breve FB-342. According to the invention, the extracellular vesicles with definite physicochemical characteristics (the average particle size is about 143.1 nm and the purity is about 1.76 * 10 < 11 > parts / g protein) are successfully prepared from the bifidobacterium breve FB-342 for the first time, and experimental results show that the prepared extracellular vesicles of the bifidobacterium breve FB-342 have obvious potential for treating ulcerative colitis in cell and animal models, and can be used for preparing the extracellular vesicles of the bifidobacterium breve FB-342 for treating ulcerative colitis. And a new strategy based on the probiotic vesicles is provided for treatment of ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an extracellular vesicle of Bifidobacterium brevesium, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic inflammatory gastrointestinal diseases, mainly including ulcerative colitis and Crohn's disease, with a continuously rising global incidence. Currently, first-line drugs such as mesalazine, thiopurines, and biologics are widely used in clinical treatment. Meanwhile, probiotic intervention has become a research hotspot. Studies have shown that specific probiotic strains, such as *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Escherichia coli* Nissle 1917, can reduce inflammation by regulating intestinal barrier function or immune responses, providing new strategies for IBD treatment. In recent years, bacterial extracellular vesicles (EVs) have received widespread attention due to their crucial roles in intercellular communication and immune regulation. Especially in the field of probiotics, although the traditional view is that most probiotics (such as Gram-positive bacteria) secrete few extracellular vesicles, studies have confirmed that some probiotic-derived extracellular vesicles can play a protective role in intestinal inflammation models, such as improving intestinal barrier integrity and downregulating pro-inflammatory factors, showing good therapeutic potential.

[0003] However, existing technologies do not address the presence of Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium breve Research on extracellular vesicles remains significantly insufficient. Although *Bifidobacterium brevesii* itself has been shown to possess various physiological functions, such as regulating intestinal metabolism, enhancing immunity, and alleviating Crohn's disease, and extracellular vesicles of related strains, such as *Bifidobacterium longum* and *Lactobacillus plantarum*, have been reported to improve ulcerative colitis, the specific role of *Bifidobacterium brevesii* extracellular vesicles in the treatment of ulcerative colitis has not been systematically explored. In particular, as a probiotic, the secretion mechanism, content composition, and correlation with the bacterial cell's function of its extracellular vesicles remain unclear, leading to uncertainty as to whether these extracellular vesicles possess anti-inflammatory and intestinal protective functions similar to their bacterial cells. Currently, only a limited number of cellular-level studies focus on its immunomodulatory effects, lacking functional validation in specific disease models such as ulcerative colitis, which limits the expansion of this natural biomaterial's application in clinical treatment.

[0004] The main reason for the above problems lies in the complexity of the secretion and packaging mechanisms of bacterial extracellular vesicles, and the relatively lagging research technology on vesicles of Gram-positive bacteria such as *Bifidobacterium breve*, making isolation, identification, and functional evaluation extremely difficult. Furthermore, existing technologies mostly focus on common probiotic strains, lacking in-depth exploration of the unique functions of *Bifidobacterium breve* and the specific effects of its extracellular vesicles, resulting in its potential application in the treatment of ulcerative colitis not being fully explored. Therefore, it is urgent to clarify whether *Bifidobacterium breve* extracellular vesicles have therapeutic effects such as reducing intestinal inflammation and repairing the epithelial barrier, to fill the technological gap in this field and provide a basis for developing novel IBD treatment strategies based on extracellular vesicles. Summary of the Invention

[0005] The purpose of this invention is to provide extracellular vesicles of *Bifidobacterium brevesicum*, their preparation method, and applications, thereby addressing the problems existing in the prior art. This invention successfully prepares extracellular vesicles from *Bifidobacterium brevesicum* FB-342, and for the first time systematically demonstrates that these vesicles can effectively alleviate ulcerative colitis through multi-target synergistic effects (inhibiting inflammation, repairing the barrier, and regulating the microbiota), providing a new strategy for the treatment of this disease based on probiotic vesicles.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an extracellular vesicle of bacteria, said extracellular vesicle being composed of Bifidobacterium brevesium (Bifidobacterium brevesium). Bifidobacterium breve FB-342 was obtained by anaerobic culture in a medium containing L-cysteine, followed by differential centrifugation, filtration, and ultracentrifugation. The Bifidobacterium breve FB-342 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 25, 2025, with accession number GDMCC No. 67352.

[0007] Optionally, the differential centrifugation is performed at 4°C for 10 min at 2000 g, 20 min at 5000 g, and 30 min at 10000 g.

[0008] Optionally, the filtration is performed using a filter membrane with a pore size of 0.45 μm.

[0009] Optionally, the ultracentrifugation is centrifugation at 150,000 g for 120 min at 4°C.

[0010] The present invention also provides the use of the extracellular vesicles described above in the preparation of a medicament for treating ulcerative colitis.

[0011] Optionally, the extracellular vesicles treat ulcerative colitis by inhibiting the expression of pro-inflammatory factors, enhancing the expression of intestinal epithelial tight junction proteins, inhibiting the growth of intestinal pathogens, and regulating intestinal flora homeostasis.

[0012] The present invention also provides a medicament for treating ulcerative colitis, wherein the active ingredient comprises the aforementioned extracellular vesicles.

[0013] Optionally, the drug may also include pharmaceutically acceptable carriers and excipients.

[0014] Optionally, the drug may be in the form of an oral formulation.

[0015] Optionally, the oral formulation includes capsules, granules, tablets, and oral liquids.

[0016] The present invention discloses the following technical effects: This invention, through specific cultivation and purification processes, successfully prepared for the first time from Bifidobacterium breve FB-342 a strain with distinct physicochemical characteristics (average particle size of approximately 143.1 nm and purity of approximately 1.76 × 10⁻⁶). 11 This method produces extracellular vesicles (particles / µg protein), thus solving the technical problems of lack of systematic research and difficulty in isolation and identification of extracellular vesicles derived from Bifidobacterium breve in existing technologies. The preparation method has good reproducibility, providing a material basis for in-depth research on this bioactive material.

[0017] Experimental results show that the extracellular vesicles of *Bifidobacterium brevesii* FB-342 prepared in this invention exhibit significant therapeutic potential for ulcerative colitis in cell and animal models. They not only effectively inhibit the expression of pro-inflammatory factors and enhance intestinal epithelial tight junction proteins, but also inhibit the growth of pathogenic bacteria and regulate the transformation of the intestinal flora towards a beneficial direction. These multifaceted synergistic mechanisms collectively achieve effective relief of ulcerative colitis, providing a safe and effective novel intervention strategy based on probiotic extracellular vesicles for the treatment of this type of disease. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a particle size distribution diagram of extracellular vesicles from Bifidobacterium breve FB-342. Figure 2 This is a comparison of extracellular vesicles and bacterial omics results. The horizontal axis represents the logarithmic transformation of FC, and the vertical axis represents the negative logarithmic transformation of p-value. Each point represents a protein. Red points are proteins with significantly high expression; blue points are proteins with significantly low expression; and gray points are proteins with no differential expression. Figure 3Figure 1 shows the effect of different concentrations of Bifidobacterium breve FB-342 extracellular vesicles on the proliferation of RAW264.7 and Caco-2 cells. Figure 4 The figure shows the statistical results of mRNA expression levels of inflammatory factors TNF-α (A) and IL-6 (B) in different treatment groups; where ns P > 0.05, P < 0.05 P < 0.01, P < 0.0001; Figure 5 The graph shows the expression results of intestinal tight junction proteins in different treatment groups; where A is the Western Blot detection result of intestinal tight junction proteins ZO-1 and Occludin in different treatment groups; B and C are the quantitative results of the expression levels of intestinal tight junction proteins ZO-1 and Occludin, respectively. P < 0.05 P < 0.01, P < 0.001; Figure 6 The growth curves of Escherichia coli LF82 (A) and Escherichia coli O157 (B) in media containing and without Bifidobacterium breve FB-342 extracellular vesicles are shown. Figure 7 Phenotypic diagram of the colon (A) and colon length statistics (B) of mice in different treatment groups; Figure 8 This graph shows the changes in the Disease Activity Index (DAI) scores of different groups of mice during the experiment. Figure 9 This is a graph showing the changes in body weight of mice in different groups during the experiment; Figure 10 The image shows the H&E staining results of mouse colon, where A represents the NC group; B represents the DSS group; C represents the 5-ASA group; and D represents the FB-342 group. Figure 11 The images show the results of PAS staining in the colon of mice. In the images, A represents the NC group; B represents the DSS group; C represents the 5-ASA group; D represents the FB-342 group; and E represents the colonic goblet cell count results for each group of mice. P < 0.001; Figure 12 The image shows the ELISA results of detecting the protein levels of inflammatory factors TNF-α (A), IL-6 (B), and IL-1β (C) in the intestinal tissue of mice in different treatment groups. P < 0.05 P < 0.01, P < 0.001; Figure 13The results show the detection of tight junction proteins in the intestinal tissue of mice in different treatment groups; where A is a gel electrophoresis image detected by Western Blot; B is a grayscale analysis image of tight junction protein ZO-1; C is a grayscale analysis image of tight junction protein Occludin; and D is a grayscale analysis image of tight junction protein Claudin-1. Figure 14 Alpha diversity of gut microbiota in mice under different treatment groups; where A is the Shannon index; B is the Chao1 index; and C is the Simpson index. Figure 15 The relative abundance of gut microbiota at the phylum level in mice under different treatment groups; Figure 16 The relative abundance of gut microbiota species at the genera level in mice under different treatment groups; Figure 17 Bar chart showing the distribution of LDA values ​​of gut microbiota in mice under different treatment groups. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: Isolation and Identification of Bifidobacterium breve FB-342 In this embodiment, a novel strain of Bifidobacterium breve was isolated, purified, and identified from an infant fecal sample, and named Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve FB-342, details are as follows: 1. Isolation, screening, and purification of bacterial strains Sample collection and processing: Fresh infant fecal samples were collected. In an anaerobic workstation, sterile anaerobic PBS (containing 0.05 g / 100 mL L-cysteine) was added to the samples at a ratio of 1:9 (g / mL). Several sterile glass beads were added, and the mixture was vortexed until no obvious solids were found. The mixture was centrifuged at 200×g and 4 ℃ for 2 min to remove impurities. The supernatant was collected in a new centrifuge tube, shaken evenly, and a sample suspension was prepared.

[0026] Dilution and Coating: Take 1 mL of sample suspension, add 9 mL of sterile, oxygen-free PBS, mix thoroughly, and prepare 10 mL of the solution. -1 Diluent; take 1 mL of diluent and perform serial dilution to prepare 10... -2 10 -3 10 -4 10 -5 10 -6 A series of gradient dilutions were selected, with 10... -4 10 -5 10 -6 For each dilution, 0.1 mL of the diluted solution was spread onto MRS agar plates (Solepro, MRS agar) containing 5 mg / 100 mL mupirocin lithium and 0.05 g / 100 mL L-cysteine, and incubated anaerobicly at 37 ℃ for 48-72 h. After incubation, the colony morphology on the plates was observed, and colonies conforming to the typical characteristics of Bifidobacterium were selected (colonies are round, raised, with neat edges, smooth surface, milky white or milky yellow in color, 1-3 mm in diameter, and soft in texture), to preliminarily exclude contaminating colonies.

[0027] Purification culture: The suspected Bifidobacterium colonies obtained from the initial screening were inoculated onto fresh MRS agar plates containing 0.05 g / 100 mL L-cysteine ​​using the streak plating method and incubated at 37 ℃ for 48 h under the same anaerobic conditions as described above. The streak purification process was repeated 1-2 times until single colonies were obtained on the plates, and all single colonies were identical in morphology, color, and size. Microscopic examination confirmed the absence of contamination, thus obtaining the purified strain FB-342.

[0028] 2. Identification of strains Morphological identification: Purified single colonies were picked and Gram-stained. The staining procedure was strictly performed according to the instructions of the Gram staining kit (Cyclocarya): After fixation, crystal violet was added for staining for 1 min, followed by rinsing with tap water; iodine solution was added for mordanting for 1 min, followed by rinsing; 95% ethanol was added for decolorization for 30 s, followed by rinsing; safranin was added for counterstaining for 1 min, followed by rinsing and air drying. The strain morphology was then observed under an optical microscope (100× oil immersion). The morphological characteristics of *Bifidobacterium breve* are Gram-positive bacteria, non-spore-forming, non-flagellated, and non-motile. The cell morphology is short rod-shaped, dumbbell-shaped, or V-shaped, and they are often arranged in pairs, chains, or clusters, which is consistent with the typical morphological characteristics of *Bifidobacterium breve*.

[0029] Liquid culture: A single colony of Bifidobacterium was picked and placed in 5 mL of MRS broth medium (Solepro, MRS broth) containing 0.05 g / 100 mL L-cysteine. The culture was placed in an anaerobic container, and an anaerobic bag (Mitsubishi, 2.5 L anaerobic gas-generating bag) and an anaerobic indicator (Mitsubishi, oxygen indicator) were added. The culture was then incubated at 37 °C for 48 h.

[0030] Genomic DNA extraction: The bacterial genomic DNA was extracted and purified using a bacterial genomic DNA extraction kit (Tiangen) according to the kit instructions.

[0031] PCR amplification of the 16S rRNA gene: PCR amplification was performed using universal primers for the bacterial 16S rRNA gene. The upstream primer was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', and the downstream primer was 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction mixture (25 μL): 2:1 (TTACCTTGTTACGAC (containing Taq enzyme, dNTPs, Mg) 2+ 12.5 μL of reagent, 1 μL of upstream primer (10 μg primer / L), 1 μL of downstream primer (10 μg primer / L), 2 μL of genomic DNA template, and 8.5 μL of sterile double-distilled water. PCR reaction program: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 1.5 min, for a total of 35 cycles; final extension at 72 ℃ for 10 min, and storage at 4 ℃. The amplified product was detected by 1.5% agarose gel electrophoresis to confirm the target band (approximately 1500 bp in length).

[0032] Sequencing: The PCR stock solution was sent to a sequencing company for bidirectional sequencing to obtain the 16S rRNA gene sequence. The sequenced sequence was submitted to the NCBI database, and BLAST was used to perform homology comparison with the bacterial 16S rRNA gene sequences already registered in the database. The 16S rRNA gene sequence of this strain is similar to that of *Bifidobacterium breve* (…). Bifidobacterium brevumThe homology of the type strain is ≥99%, and combined with the morphological results, the strain can be identified as Bifidobacterium breve.

[0033] 3. Preservation of bacterial strains The taxonomic name of strain FB-342 is Bifidobacterium breve It was deposited on November 25, 2025 at the Guangdong Provincial Center for the Preservation of Microbial Cultures, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, with accession number GDMCC No. 67352.

[0034] Example 2: Extraction and characterization of extracellular vesicles from Bifidobacterium brevesicans FB-342 1. Extraction of extracellular vesicles Cell culture: Take an appropriate amount of *Bifidobacterium breve* FB-342 bacterial suspension stored in glycerol at -80℃, streak it onto an MRS agar plate (Solepro, MRS agar), and incubate at 37℃ for 48-72 h. Once colonies are clear, pick a single colony and add it to 5 mL of MRS broth (Solepro, MRS broth) containing 0.05 g / 100 mL L-cysteine. Place the plate in an anaerobic chamber, add an anaerobic bag (Mitsubishi, 2.5 L anaerobic gas generator) and an anaerobic indicator (Mitsubishi, oxygen indicator), and incubate at 37℃ for 48 h. Then, take 500 μL and inoculate it into 50 mL of MRS broth containing 0.05 g / 100 mL cysteine, place it in an anaerobic chamber, add an anaerobic bag and an anaerobic indicator, and incubate at 37℃ for 48 h to complete the culture.

[0035] Supernatant treatment: Centrifuge the bacterial culture at 4°C and 2000 g for 10 min to remove bacterial cells. The collected supernatant is then centrifuged sequentially at 4°C and 5000 g for 20 min and at 10000 g for 30 min to thoroughly remove residual bacterial cells and cell debris.

[0036] Filtration and ultracentrifugation: The supernatant was filtered using a 0.45 μm pore size filter membrane (Merck-Millipore). The filtrate was placed in an ultracentrifuge tube and centrifuged at 150,000 g for 120 min at 4 °C using an ultracentrifuge (Hitachi, CP70 ME). The supernatant was gently discarded, and the precipitate (i.e., crude vesicle extraction) was collected.

[0037] Washing: Gently resuspend the precipitate in pre-cooled sterile PBS (pH 7.4), centrifuge again at 4°C and 150,000 g for 120 min, and finally resuspend the precipitate with an appropriate amount of PBS to obtain a purified extracellular vesicle suspension. After aliquoting, store at -80°C for later use.

[0038] 2. Characterization of extracellular vesicles (1) Particle size determination The particle size distribution and number of extracellular vesicle samples were determined using the NanoSight NS 300 system (Malvern Panalytical), and data analysis was performed using the accompanying NanoSight NTA version 3.4 analysis software. The specific operating procedure is as follows: Install the dedicated plastic sample carrier, place the detection module in the detection chamber, and turn on the instrument power, computer system, and analysis software in sequence; start the camera and perform focus calibration to stabilize the camera level parameter in the range of 13-14; rinse the system with PBS until the background cleanliness meets the requirements (particle count <5 per frame), at which point the sample is ready for detection.

[0039] Before testing, the stock solution needs to be serially diluted. After injecting the sample at a uniform rate, the real-time imaging screen is monitored simultaneously. The display interface is switched to the bottom control panel, and the optical parameters and mechanical focal length are adjusted collaboratively to ensure that the visualized particles achieve the best resolution. The single acquisition time is set to 60 seconds. After the first detection is completed, the position of the sample injector is moved to switch the observation field of view. Each sample needs to be repeatedly detected in 3 independent microscopic fields of view.

[0040] After individual sample testing is completed, immediately verify whether the measured concentration value is within the instrument's standard detection range (1×10⁻⁶). 8 -1×10 9 (particles / mL). If the range is exceeded, the dilution ratio needs to be optimized again, and the gradient dilution and detection steps need to be repeated. Analyze the test report, and the final formula for calculating the original sample concentration is: instrument detection value × sample dilution factor.

[0041] The results are shown in Table 1. The average particle size of the exovesicles extracted by differential centrifugation was 143.1 nm, and the particle concentration was 1.84 × 10⁻⁶. 11 particles / mL, specific particle size distribution as follows Figure 1 As shown.

[0042] (2) Protein concentration determination (BCA method) Protein concentrations were determined using the Pierce™ BCA protein assay kit (Thermo Fisher). Bovine serum albumin (BSA) standards (2 mg / mL) were serially diluted using PBS (pH 7.4) buffer; the prepared extracellular vesicle solutions were also moderately diluted using PBS (pH 7.4) buffer. First, 10 μL of different concentrations of standard (AI) were added to each well of a 96-well plate, with three replicates per sample. Then, 10 μL of sample diluent was added to each well, also with three replicates. The chromogenic solution was prepared with a 50:1 ratio of solution A to solution B, thoroughly mixed by pipetting, and used immediately. 100 μL of the chromogenic solution was added to each well of the 96-well plate containing the standard and sample. The 96-well plate was incubated at 37°C in the dark for 30 min. The absorbance at 562 nm was measured using a microplate reader, and a standard curve was plotted based on the standard concentrations and the measured OD values. The protein concentration of the sample is calculated by substituting the absorbance value of the sample into the curve formula of the standard.

[0043] The results are shown in Table 1. The concentration of extravesicular protein extracted by differential centrifugation was 1.045 μg / mL, and its purity was 1.76 × 10⁻⁶. 11 particles / μg.

[0044] Table 1. Characterization results of extracellular vesicles of Bifidobacterium brevesicans FB-342 3. Comparative analysis of vesicle and bacterial cell proteomics Total protein from *Bifidobacterium breve* FB-342 cells and its extracellular vesicles was extracted and subjected to mass spectrometry-based proteomics analysis. The results are as follows: Figure 2 As shown, comparative analysis revealed differences in the proteins contained within bacterial cells and their extracellular vesicles. Compared to bacterial cells, most proteins in extracellular vesicles were downregulated; these proteins are mostly related to DNA replication, protein synthesis, basal metabolism, and substance transport. However, a very small number of proteins were enriched in extracellular vesicles (OMVs), and these proteins are mostly related to carbohydrate metabolism, energy metabolism, lipid metabolism, and DNA-related functions. This indicates that bacteria selectively package specific proteins into vesicles, suggesting that the function of vesicles may differ from that of bacteria.

[0045] Example 3: In vitro functional verification of extracellular vesicles of Bifidobacterium brevesicans FB-342 1. Effects on cell proliferation Cell status was observed using a microscope. Cells were subjected to either digestion (Caco-2) or direct blow-off (RAW264.7), and cell counting and viability were performed. When cell viability reached 90% or higher, cells were further analyzed using a 1.5 × 10⁻⁶ micrometer scale. 4 Cells were seeded at a density of 100 cells / well in 96-well plates, with 100 μL of culture medium per well. Five replicates were performed for each sample. The outermost wells of the 96-well plate were left unfilled with cells and culture medium, but instead contained 100 μL of PBS buffer to minimize evaporation from the inner wells. Cells were cultured overnight to allow adhesion. After removing the culture medium, fetal bovine serum (FBS)-free medium was added for 2 hours of starvation culture. The FBS-free medium was then removed, and different cell culture media were added. The control group used FBS-free medium, while the experimental groups were prepared with five concentration gradients of FBS-free medium containing FB-342 extracellular vesicles at concentrations of 0 ng / mL, 256 ng / mL, 640 ng / mL, 1600 ng / mL, and 4000 ng / mL. The blank control group received cell-free FBS-free medium. Cells were cultured for another 24 hours. Add 10 μL of CCK8 reagent (Beyotime) to each sample well and incubate at 37℃ for 3 h. Then, use an ELISA reader to measure the absorbance (OD value) at 450 nm and 630 nm. Calculate the cell viability (%) according to the following formula: Cell viability (%) = [(A experimental group - A blank group) / (A control group - A blank group)] × 100%.

[0046] The measurement results are as follows Figure 3 As shown, FB-342 extracellular vesicles at a concentration of 4000 ng / mL promoted the proliferation of RAW264.7 cells but not Caco-2 cells. This suggests that it has a specific growth-promoting or activating effect on immune cells.

[0047] 2. Effects on the expression of macrophage inflammatory factors RAW264.7 cells were observed under a microscope. Cells were detached using PBS, and cell counting and viability were performed. When cell viability reached over 90%, cells were counted at 5 × 10⁻⁶ cells / day. 6Cells were seeded at a density of cells / well in 6-well plates with 2 mL of culture medium per well. Each sample was tested in triplicate and cultured overnight. Dexamethasone (DEX) was used as a positive control. A stock solution of 2 mg / mL DEX was prepared in anhydrous ethanol and added to the culture medium at a 1:100 ratio, resulting in a final concentration of 20 μg / mL. After discarding the culture medium, medium containing F-342 extracellular vesicles (4000 ng / mL) was added. The NC group received standard culture medium. Cells were cultured for 4 h, followed by the addition of LPS and another 24 h. The supernatant was discarded, and the cells were slowly washed twice with PBS. Trypsin was added, and the cells were digested at 37°C for an appropriate time. Digestion was then stopped by adding complete culture medium. All cells were slowly detached and collected into centrifuge tubes. The cell suspension was centrifuged at 200 g for 5 min, and the supernatant was discarded. The following kits were used: Rapid RNA Extraction Kit (TransGen Biotech), HiFiScript All-in-one RT Master Mix for qPCR (Kangwei Century), and dye-based quantitative PCR premix (TransGen Biotech). RNA extraction, reverse transcription, and qRT-PCR detection were performed strictly according to their instructions. RNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed.

[0048] The results are as follows Figure 4 As shown, F-342 extracellular vesicles can alleviate the LPS-induced increase in TNF-α inflammatory factor levels. Compared with the NC group, the LPS group had significantly higher levels of IL-6 inflammatory factor (P < 0.05), however, there was no statistically significant difference between the F-342 extracellular vesicle group and the LPS group.

[0049] 3. Effects on intestinal epithelial barrier function Cell status was observed using a microscope, followed by cell digestion, counting, and viability testing. When cell viability reached 90% or higher, cells were analyzed at a concentration of 5 × 10⁻⁶ cells / mL. 6Cells were seeded at a density of 2 cells / well into 6-well plates, with 2 mL of culture medium per well. Each sample was tested in triplicate and cultured overnight. Dexamethasone (DEX) was used as a positive control. DEX was dissolved in anhydrous ethanol to prepare a stock solution at a concentration of 2 mg / mL, which was then added to the culture medium at a ratio of 1:100, resulting in a final concentration of 20 μg / mL. After discarding the culture medium, culture medium containing F-342 extracellular vesicles (4000 ng / mL) was added. For the NC group, culture medium was added only. After culturing for 4 h, LPS was added, and the cells were cultured for another 24 h. The supernatant was discarded, and the cells were slowly washed twice with PBS. RIPA lysis buffer containing protease inhibitors was added, and the cells were lysed on ice for 30 min. Cells were scraped off using a cell scraper. The lysate was collected and centrifuged at 12000 g at 4°C for 30 min. The supernatant was collected as the total protein solution from cell lysis and stored at -80°C for later use.

[0050] After sample preparation and SDS-PAGE gel electrophoresis, the gel was cut according to the location of the target protein. The sponge, transfer filter paper, and nitrocellulose membrane were moistened with pre-cooled transfer buffer. The transfer clamp was assembled in the order of "sponge - transfer filter paper - gel - nitrocellulose membrane - transfer filter paper - sponge," and placed in the transfer tank with the positive and negative electrodes aligned. A constant current of 300 mA was set, and the transfer time was adjusted according to the size of the target protein (30-180 min). After transfer, the nitrocellulose membrane was removed and placed face up in the incubation chamber. An appropriate amount of TBST was added for washing. The TBST was discarded, and freshly prepared blocking buffer was added. The membrane was blocked on a shaker at room temperature for 1 h. The TBST blocking buffer was discarded, and an appropriate amount of TBST was added again for three washes, each lasting 7 min. Add the primary antibody (Anti-GAPDH, Proteintech; Anti-ZO-1, Proteintech; Anti-occludin, Proteintech) diluted with antibody diluent and incubate overnight on a shaker at 4°C. Recover the primary antibody, add an appropriate amount of TBST, and wash the membrane three times for 7 minutes each time. Add the secondary antibody (Anti-mouse IgG-HRP, Proteintech; Anti-rabbit IgG-HRP, Proteintech) diluted with blocking buffer and incubate on a shaker at room temperature for 1 hour. Discard the secondary antibody, add an appropriate amount of TBST, and wash the membrane three times for 7 minutes each time. Prepare ECL ultrasensitive luminescent developing solution (NewCeMed) at a ratio of A:B = 1:1, preparing it fresh each time and storing it away from light. Evenly drop an appropriate amount of developing solution onto the nitrocellulose membrane transferred with the protein, develop and photograph using a gel imaging system.

[0051] The results are as follows Figure 5 As shown, the levels of ZO-1 and Occludin tight junction proteins in the LPS group showed a significant decreasing trend compared to the NC group, while those in the F-342 group showed a significant increasing trend compared to the LPS group. This indicates that F-342 extracellular vesicle treatment can significantly restore the expression levels of these two proteins, with an effect comparable to that of the DEX group, suggesting that it has the function of enhancing the integrity of the intestinal epithelial barrier.

[0052] 4. Inhibitory effect on the growth of pathogenic bacteria in the intestines Escherichia coli LF82 ( Escherichia coli LF82, an enteropathogenic strain associated with Crohn's disease, induces intestinal inflammatory responses and may cause tissue damage through mechanisms such as adhesion and invasion, induction of inflammation, destruction of the intestinal mucosa, and immune escape. Escherichia coli O157 ( Escherichia coli O157:H7 is a foodborne pathogen that mainly causes serious intestinal infections and related diseases by producing toxins, adhering to and invading host cells, and genetically facilitating flight, posing a significant threat to public health.

[0053] Escherichia coli LF82 and O157, stored at -80℃, were inoculated into 10 mL of LB liquid medium at a 2% inoculum and cultured overnight at 37℃ and 220 rpm with shaking until OD (Oxygen Demand). 600 Reach 0.4-0.6. Take 100 μL of bacterial culture (OD... 600 = 0.4 - 0.6) Inoculate into 5 mL LB liquid medium.

[0054] Take a 1.5 mL EP tube, add F-342 extracellular vesicle solution to the experimental group to make the final protein concentration reach 100 μg / mL; add an equal volume of PBS solution to the control group, and then add the diluted bacterial solution to make the final volume of each reaction system 1 mL, and gently shake to mix.

[0055] A 96-well cell culture plate was used. The reaction solution was inoculated into each well at a rate of 200 μL, with three replicates per group. The 96-well plate was placed in a microplate reader, the monitoring wavelength was set to 600 nm, the shaking speed was set to low, and the monitoring time was 24 h. The absorbance was measured every 30 min, and a bacterial growth curve was plotted after the monitoring was completed.

[0056] The results showed that, compared with the PBS control group, the extracellular vesicles of F-342 had a certain degree of inhibitory effect on both pathogens. Figure 6 ).

[0057] Example 4: The therapeutic effect of extracellular vesicles of Bifidobacterium brevesicans FB-342 on mice with ulcerative colitis. 1. Establishment and treatment of animal models Grouping and Model Establishment: Twenty-four 8-week-old SPF-grade male C57BL / 6 mice were randomly divided into four groups (n=6) after one week of acclimatization: normal control group (NC), sodium dextran sulfate model control group (DSS), positive drug mesalazine control group (5-ASA), and Bifidobacterium breve FB-342 extracellular vesicle treatment group (FB-342). Except for the NC group, the other three groups of mice were allowed free access to drinking water containing 3% (w / v) sodium dextran sulfate (DSS) for 7 consecutive days (referred to as days 1 to 7 of the experiment) to induce an acute ulcerative colitis model; the NC group had free access to ordinary tap water throughout the experiment.

[0058] Starting from day 4 of the experiment, medication was administered via gavage once daily for 7 consecutive days (until day 10 of the experiment), while the model was being established. Details are as follows: FB-342 group: Bifidobacterium breve FB-342 extracellular vesicles were administered via gavage at a dose of 1 mg / kg (based on vesicle protein content) and a gavage volume of 200 μL.

[0059] 5-ASA group: Mesalazine (5-ASA) was administered via gavage in a saline solution at a dose of 75 mg / kg and a volume of 200 μL.

[0060] NC group and DSS group: The same volume (200 μL) of physiological saline was administered by gavage.

[0061] On day 7 of the experiment, all DSS-inducing groups were stopped receiving DSS water and replaced with ordinary tap water. On day 11 of the experiment, all mice were anesthetized and sacrificed for sample collection.

[0062] 2. Assessment of disease activity and macroscopic indicators Disease Activity Index (DAI) score: Daily records of mouse weight, fecal characteristics, and fecal blood were used to calculate the DAI score.

[0063] Colon length measurement: After euthanizing the mice, the colon was completely dissected (from the end of the cecum to the anus), placed on ice, and gently flattened before measuring its length.

[0064] The results are as follows Figures 7-9 As shown, compared with the NC group, the DSS model group mice had significantly higher DAI scores, significantly lower body weight, and significantly shorter colon length. Compared with the DSS group, FB-342 extracellular vesicle treatment significantly reduced DAI scores, improved body weight loss, and increased colon length, with effects comparable to the positive control drug 5-ASA.

[0065] 3. Histopathological analysis of colon tissue The colon of mice was harvested from the distal 1 cm and then embedded and stained by Xavier Biosciences.

[0066] Embedding and sectioning: In a fume hood, the colonic segment was trimmed and leveled using a scalpel and forceps. The trimmed colonic segment and its corresponding label were then carefully placed in the embedding frame. After placing the sample in the dehydration box, it was placed in a dehydrator for sequential dehydration with a gradient of alcohols, followed by clearing and paraffin impregnation (75% ethanol for 4 h, 85% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, anhydrous ethanol I for 0.5 h, anhydrous ethanol II for 0.5 h, benzene for 5-10 min, xylene I for 5-10 min, xylene II for 5-10 min, 65℃ molten paraffin I for 1 h, 65℃ molten paraffin II for 1 h, 65℃ molten paraffin III for 1 h). h); First, carefully place the melted paraffin into the embedding frame. Before the wax solidifies, carefully remove the colon from the dehydration box with forceps and place it into the embedding frame according to the embedding surface requirements, and attach the corresponding label. Cool at -20℃. After the paraffin solidifies, remove the wax block from the embedding frame and trim the paraffin block to the ideal state. Place the trimmed paraffin block into a paraffin microtome and slice it to a thickness of 4 μm. Spread the slices in a 40℃ deionized water bath for 30 seconds. Attach cationic coated glass slides and dry and solidify them in a 60℃ oven for 2 hours. After the paraffin is dried by baking and melted, remove the slices and store them at room temperature for later use. Dewax and rehydrate the paraffin sections (environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% ethanol for 5 min, deionized water for 5 min). Immerse the sections in histopathological grade high-resolution constant staining pretreatment solution and incubate at room temperature for 1 min to enhance the penetration of subsequent stains.

[0067] Staining: Immerse the sections in hematoxylin staining solution for 5 min, rinse slowly with tap water, add differentiation solution for differentiation, rinse slowly with tap water, add blueing solution for blueing, rinse slowly with tap water; add 95% ethanol for dehydration for 1 min, immerse in eosin staining solution for 15 s; dehydrate (anhydrous ethanol I 2 min, anhydrous ethanol II 2 min, anhydrous ethanol III 2 min, n-butanol I 2 min, n-butanol II 2 min, xylene I 2 min, xylene II 2 min); add high refractive index neutral resin to a glass slide, cover with a coverslip and mount. The resulting section is a HE-stained section of colon tissue.

[0068] Perform dewaxing and rehydration treatment as described above; slowly immerse the dewaxed and rehydrated sections in PAS staining solution B for 10-15 min, then rinse once with tap water and twice with ultrapure water; immerse the sections in PAS staining solution A in the dark for 30 min, then rinse slowly with ultrapure water for 5 min; immerse the sections in PAS staining solution C for 30 sec, then rinse slowly with tap water; add hydrochloric acid solution for differentiation, then rinse slowly with tap water, then rinse with ammonia solution for blue reversion, and finally rinse slowly with ultrapure water; dehydrate and mount with resin as described above; the resulting section is the PAS-stained section of colon tissue.

[0069] The results of hematoxylin-eosin (H&E) staining are as follows: Figure 10 As shown, extensive ulcers were observed in the colon of mice in the DSS group, with necrosis and sloughing of epithelial mucosal cells. The crypt structures in the lamina propria disappeared, and there was extensive inflammatory cell infiltration. The muscular layer was significantly thinned and showed partial inflammatory cell infiltration. In mice in the FB-342 group, partial ulceration was observed in the colon tissue, but crypt structures were still visible in the lamina propria. However, the base thickness of these crypt structures was increased, and partial inflammatory cell infiltration was observed. Partial inflammatory cell infiltration was also observed in the muscular layer.

[0070] PAS staining results are as follows Figure 11 As shown, the colon of the NC group contained a large number of goblet cells, while almost no goblet cells were observed in the DSS group. After treatment with FB-342 extracellular vesicles, the number of goblet cells in the colonic tissue of mice increased significantly, indicating that FB-342 extracellular vesicles can alleviate the reduction of goblet cells in colonic tissue caused by DSS.

[0071] 4. Detection of inflammatory factor levels in colon tissue Sample preparation: Mouse colon tissue was collected and placed in 1.5 mL centrifuge tubes; an appropriate amount of pre-chilled RIPA lysis buffer (Beyotime) with added protease inhibitor (Beyotime) was added, with an addition ratio of 250 μL of RIPA lysis buffer for every 20 mg of colon tissue; two steel grinding beads were added to each centrifuge tube, leveled, and placed on a pre-chilled grinding rack, which was then placed in a tissue homogenizer (Shanghai Jingxin). The program was set to grind for 30 seconds, stop for 30 seconds, and repeated 15 times for a total duration of 15 minutes. If the grinding effect was not as expected, the operation was repeated once; the lysis buffer was collected and centrifuged at 12000 g and 4℃ for 30 minutes, and the supernatant was collected. This supernatant is the total protein solution of colon tissue lysis.

[0072] ELISA Assay: The ELISA kit (Jianglai Biotechnology) was placed at room temperature until all reagents in the kit reached room temperature. The strips used for ELISA assay were then removed. The standards were serially diluted, with 100 μL of each diluted standard added to the corresponding well. Simultaneously, 100 μL of the test sample was added to the corresponding well. The plate was then sealed with sealing film and incubated at 37°C for 60 min. After incubation, the plate was removed, the liquid in the wells was discarded, and 100 μL of biotinylated antibody working solution was added to each well. The plate was sealed again and incubated at 37°C for 60 min. The plate was then removed, the liquid was discarded, and 300 μL of washing buffer was added to each well. The plate was allowed to stand for 1 min, the washing buffer was discarded, and the plate was patted dry on absorbent paper. This washing procedure was repeated three times. Finally, 100 μL of enzyme conjugate working solution was added to each well, the plate was sealed with sealing film, and incubated at 37°C for 30 min. After incubation, discard the liquid and wash the microplate five times using the washing method described above. Then, add 90 μL of substrate (TMB) to each well, seal with sealing film, and incubate at 37°C for 15 min in the dark. Finally, remove the microplate, add 50 μL of stop solution directly to each well, and measure the absorbance (OD value) at 450 nm.

[0073] Result: As Figure 12 As shown, compared with the NC group, the DSS group showed an increase in the levels of three inflammatory factors, TNF-α, IL-6, and IL-1β, in the colon tissue, while FB-342 extracellular vesicles could simultaneously reduce the upregulation of TNF-α, IL-6, and IL-1β induced by DSS.

[0074] 5. Detection of tight junction protein expression in colon tissue Sample preparation: Same as the ELISA sample preparation method described above.

[0075] Western Blot: After sample preparation and SDS-PAGE gel electrophoresis, cut the gel according to the location of the target protein. Soak the sponge, transfer paper, and nitrocellulose membrane in pre-cooled transfer buffer. Assemble the transfer clamps in the order of "sponge-transfer paper-gel-nitrocellulose membrane-transfer paper-sponge," placing them in the transfer tank with the positive and negative electrodes aligned. Set a constant current of 300 mA and adjust the time according to the size of the target protein (30-180 min). After transfer, remove the nitrocellulose membrane, place it face up in the incubation box, and wash with an appropriate amount of TBST. Discard the TBST, add freshly prepared blocking buffer, and block on a shaker at room temperature for 1 hour. Discard the TBST blocking buffer, add an appropriate amount of TBST, and wash the membrane three times, 7 ml each time. Add the primary antibody (Anti-GAPDH, Proteintech; Anti-βacting, Proteintech; Anti-ZO-1, Proteintech; Anti-occludin, Proteintech; Anti-claudin-1, Proteintech) diluted with antibody diluent, and incubate overnight at 4°C on a shaker; recover the primary antibody, add an appropriate amount of TBST, wash the membrane three times for 7 min each time; add the secondary antibody (Anti-mouse IgG-HRP, Proteintech; Anti-rabbit IgG-HRP, Proteintech) diluted with blocking buffer, and incubate at room temperature on a shaker for 1 h; discard the secondary antibody, add an appropriate amount of TBST, wash the membrane three times for 7 min each time; prepare ECL ultrasensitive luminescent developing solution (New Semiconductor) according to the ratio of solution A to solution B = 1:1, prepare fresh and use immediately, and store in the dark; take an appropriate amount of developing solution and evenly drop it onto the nitrocellulose membrane transferred with protein, develop and photograph it using a gel imaging system.

[0076] Result: As Figure 13 As shown, compared to the NC group, the expression levels of the three tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon tissue of the DSS group were significantly downregulated. Furthermore, FB-342 extracellular vesicles significantly reversed the DSS-induced inhibition of ZO-1, Occludin, and Claudin-1 expression.

[0077] 6. Intestinal flora analysis After isolating the intestinal contents, 16S rRNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. to analyze the gut microbiota. The specific procedure is as follows: First, genomic DNA was extracted. DNA concentration was measured using a Qubit® 4.0 fluorometer to ensure sufficient and high-quality genomic DNA. PCR amplification was performed immediately after DNA extraction, targeting the hypervariable region of the bacterial 16S rRNA gene V3-V4. Free primers and dimers in the amplification products were purified using Hieff NGS™ magnetic beads. The purified samples were used to construct Illumina standard libraries. Before sequencing, DNA quantification was performed using a Qubit® 4.0 fluorometer, and quality control was conducted using an Agilent 2100 bioanalyzer. After mixing samples proportionally according to sequencing coverage requirements, paired-end sequencing was performed using the Illumina MiSeq sequencing platform.

[0078] Alpha diversity analysis of gut microbiota is a method used in microbiome research to assess microbial diversity within a single sample, encompassing both species richness and evenness. The Chao1 index focuses on richness based on species rarity; the Shannon index reflects both species richness and evenness of the sample; while the Simpson index places more emphasis on evenness (sensitive to dominant species).

[0079] Alpha diversity analysis: Results of alpha diversity analysis of gut microbiota are as follows Figure 14 As shown, the Shannon and Chao1 indices in the DSS group were significantly lower than those in the NC group, and F-342 extracellular vesicle treatment showed a trend of restoring these two indices.

[0080] Species composition analysis: (1) Analysis of relative abundance of species at the phylum level The results of the relative abundance analysis of species at the phylum level are as follows: Figure 15 As shown, Firmicutes and Bacteroidetes had relatively high abundances in the gut microbiota of the four groups of mice. The content of Proteobacteria in the gut microbiota of the DSS group was higher than that of the NC group, while the content of Bacillus was lower in the DSS group than in the NC group. After treatment with F-342 extracellular vesicles, the relative abundance of Proteobacteria decreased compared to the DSS group, while the relative abundance of Firmicutes increased. The F-342 group had a higher relative abundance of Verrucous Microbes compared to the other three groups.

[0081] (2) Species relative abundance analysis at the genus level The results of the relative abundance analysis of species at the genus level are as follows: Figure 16As shown, at the genus level, the relative abundance of *Escherichia coli*-*Shigella* and *Streptococcus* was significantly higher in the DSS group than in the NC group. *Escherichia coli*-*Shigella* may invade colonic epithelial cells, causing inflammation and ulcers, with symptoms including bloody diarrhea, fever, and abdominal pain. In contrast, the relative abundance of these two genera was lower in the F-342 group than in the DSS group, mitigating the adverse effects of *Escherichia coli*-*Shigella*.

[0082] Marker species analysis: In the F-342 extracellular vesicle treatment group, the relative abundance of *AKK* bacteria was significantly higher than in the other three groups. LEfSe analysis results of the gut microbiota are as follows: Figure 17 As shown, the dominant bacteria in the gut microbiota of the DSS group include Proteobacteria, while the dominant bacteria in the F-342 group are Bacillus zurichae and Clostridium.

[0083] This embodiment demonstrates that oral administration of extracellular vesicles of Bifidobacterium brevesicans FB-342 can effectively improve the clinical symptoms of DSS-induced ulcerative colitis in mice and reduce the pathological damage to colon tissue. Its mechanism of action is closely related to inhibiting the release of pro-inflammatory factors, enhancing the integrity of the intestinal epithelial barrier, and regulating intestinal flora homeostasis (inhibiting harmful bacteria and promoting beneficial bacteria).

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An extracellular vesicle of bacteria, characterized in that, The extracellular vesicles are composed of Bifidobacterium brevesin ( Bifidobacterium breve FB-342 was obtained by anaerobic culture in a medium containing L-cysteine, followed by differential centrifugation, filtration, and ultracentrifugation. The Bifidobacterium breve FB-342 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 25, 2025, with accession number GDMCC No. 67352.

2. The extracellular vesicle according to claim 1, characterized in that, The differential centrifugation was performed at 4°C for 10 minutes at 2000 g, 20 minutes at 5000 g, and 30 minutes at 10000 g.

3. The extracellular vesicle according to claim 1, characterized in that, The filtration is performed using a filter membrane with a pore size of 0.45 μm.

4. The extracellular vesicle according to claim 1, characterized in that, The ultracentrifugation was performed at 150,000 g for 120 min at 4°C.

5. The use of an extracellular vesicle as described in any one of claims 1-4 in the preparation of a medicament for treating ulcerative colitis.

6. The application according to claim 5, characterized in that, The extracellular vesicles treat ulcerative colitis by inhibiting the expression of pro-inflammatory factors, enhancing the expression of tight junction proteins in intestinal epithelium, inhibiting the growth of intestinal pathogens, and regulating intestinal flora homeostasis.

7. A drug for treating ulcerative colitis, characterized in that, The active ingredient includes the extracellular vesicles as described in any one of claims 1-4.

8. The medicament according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable carriers and excipients.

9. The drug according to claim 7, characterized in that, The drug is an oral formulation.

10. The medicament according to claim 9, characterized in that, The oral preparations include capsules, granules, tablets, and oral liquids.

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