A plant lactobacillus P-8 extracellular vesicle and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a plant lactobacillus P-8 extracellular vesicle and a preparation method and application thereof. Lactiplantibacillus plantarum The extracellular vesicle is extracted from a plant lactobacillus (Lactobacillus plantarum) P-8 strain culture suspension through purification and ultracentrifugation. The application provides a technical scheme for the extraction method, basic characterization and optimized preservation of the extracellular vesicle. A specific freeze-drying protective agent and freeze-drying method for long-term preservation of the plant lactobacillus P-8 extracellular vesicle are also provided. The application also provides an application of the plant lactobacillus P-8 extracellular vesicle in preparation of a drug for relieving intestinal inflammation and / or depression-related diseases. Cell and animal experiments prove that the extracellular vesicle can effectively relieve intestinal inflammation and improve depression-like behavior by regulating the “gut-brain axis”. Therefore, the extracellular vesicle can be used for preparing functional food or related products for assisting in relieving intestinal inflammation and / or depression.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an extracellular vesicle of Bacillus plantarum P-8, its preparation method and application. Background Technology

[0002] Probiotics play a vital role in maintaining bodily health, with functions including regulating gut microbiota balance, strengthening the intestinal barrier, and immune regulation. *Lactobacillus plantarum* (…) Lactiplantibacillus plantarum As an important class of probiotics, probiotics have been extensively studied for their effects in improving inflammatory bowel disease, enhancing intestinal immunity, and alleviating depression. Their benefits are mainly attributed to the direct effects of live bacteria or their metabolites in the intestine.

[0003] Bacterial extracellular vesicles (EVs) are nanoscale lipid bilayer structures released by bacteria, carrying bioactive components such as proteins, nucleic acids, and metabolites, and are key carriers mediating bacterial-host interactions. Compared with live bacteria, their nanoscale size allows them to easily penetrate the intestinal mucus layer and target host cells, achieving efficient, "colonization-independent" functional regulation. Studies have shown that EVs are significantly more stable than live bacteria in the gastrointestinal environment, demonstrating their potential as a stable and precise delivery system. However, EVs are susceptible to temperature and freeze-thaw cycles during storage and transportation, resulting in structural damage and decreased activity. Current low-temperature preservation methods are costly, and freeze-drying processes easily damage membrane integrity, hindering their industrial application. Currently, research on the efficient extraction, stable preservation, and mechanism elucidation of probiotic-derived EVs is still in its developmental stage. Developing efficient, stable, and low-cost large-scale preparation and preservation strategies has become a key challenge in this field.

[0004] Intestinal inflammation can affect central nervous system function through multiple pathways, including neural, immune, and endocrine pathways, and is closely related to the occurrence and development of depression. Regulating the gut environment has become a novel strategy for intervening in depression. Current medications for enteritis suffer from numerous adverse reactions with long-term use and difficulty in repairing the intestinal barrier. Probiotic extracellular vesicles, due to their ability to target and deliver anti-inflammatory components, regulate immune pathways, and enhance barrier function, can act as key messengers between the gut and brain. They can simultaneously combat intestinal inflammation and deliver neuromodulatory substances to the central nervous system. However, the specific mechanisms by which vesicles from specific strains improve depression through this pathway remain unclear.

[0005] Studies on Lactobacillus plantarum strain P-8 have been reported, but the specific preparation process, physicochemical characterization, stability maintenance methods, and dual efficacy and mechanism in alleviating intestinal inflammation and improving depressive-like behavior through the gut-brain axis have not yet been studied or disclosed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes an extracellular vesicle of *Lactobacillus plantarum* P-8, its preparation method, and its application. An extraction method for this extracellular vesicle was constructed, and a specific freeze-drying protection system capable of effectively maintaining the structural integrity and biological activity of the vesicle was developed. This system demonstrates potential application value in alleviating intestinal inflammation and depression.

[0007] To achieve the above objectives, the present invention provides a method for preparing extracellular vesicles of *Lactobacillus plantarum* P-8, comprising the following steps: S1. Inoculate Lactobacillus plantarum P-8 strain into liquid culture medium and culture. Collect the culture supernatant, centrifuge, and collect the supernatant. S2. Filter the supernatant to remove bacteria and collect the filtrate; S3. Centrifuge the filtrate, discard the supernatant, resuspend the precipitate, and obtain extracellular vesicles of *Lactobacillus plantarum* P-8.

[0008] Preferably, in S1, the culture time is 6 hours and the centrifugation is performed at 4°C and 10000×g for 30 minutes.

[0009] Preferably, in S2, the filtration is performed using a 0.22μm filter membrane.

[0010] Preferably, in S3, the filtrate is centrifuged at 4°C and 120,000 × g for 90 min; the precipitate is resuspended in 100 μL of pre-cooled 1×PBS buffer.

[0011] A method for long-term preservation of *Lactobacillus plantarum* P-8 extracellular vesicles by freeze-drying is also provided, comprising the following steps: mixing the *Lactobacillus plantarum* P-8 extracellular vesicles with a freeze-drying protectant to obtain a vesicle protection solution; placing the vesicle protection solution at -80°C for overnight slow freezing to complete the pre-freezing treatment; and freeze-drying the pre-frozen sample to obtain a freeze-dried extracellular vesicle product.

[0012] Preferably, the lyophilization protectant consists of lysine, albumin, and PBS buffer; the final concentration of lysine is 5% (w / v); the final concentration of albumin is 1% (w / v); and the pH of the PBS buffer is 7.4.

[0013] The use of the aforementioned *Lactobacillus plantarum* P-8 extracellular vesicles in the preparation of drugs or functional foods for relieving or treating enteritis is also provided.

[0014] Preferably, the extracellular vesicles of *Lactobacillus plantarum* P-8 can repair the intestinal epithelial barrier function and alleviate intestinal inflammation by inhibiting the secretion of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α, and upregulating the expression of ZO-1 and Occludin tight junction proteins.

[0015] The use of the aforementioned *Lactobacillus plantarum* P-8 extracellular vesicles in the preparation of drugs or functional foods for relieving or treating depression is also provided.

[0016] Preferred, extracellular vesicles of *Lactobacillus plantarum* P-8 regulate the 5-HT1AR / cAMP / PKA / CREB signaling pathway and improve depressive-like behavior.

[0017] Preferably, the extracellular vesicles of *Lactobacillus plantarum* P-8 carry miR-92b-3p, miR-92a sRNA, key enzymes for short-chain fatty acid synthesis, and functional proteins of the type II fatty acid synthase system. They exert neuroprotective effects by catalyzing the production of short-chain fatty acids such as acetate and butyrate to activate the cAMP / PKA / CREB pathway.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The vesicles obtained by the ultracentrifugation method of this invention have higher particle concentration, smaller average particle size and better sample purity. The vesicles have typical morphology and uniform distribution, providing a high-quality and highly consistent experimental material basis for subsequent functional research and application of vesicles.

[0019] (2) This invention optimizes the extraction, freeze-drying, and preservation of *Lactobacillus plantarum* P-8 extracellular vesicles: the optimal extraction time was determined, an optimal freeze-drying protection system consisting of lysine, albumin, and PBS buffer was established, and a slow-freezing pre-freezing process was employed. The freeze-dried product obtained by this method maintains a typical bilayer membrane structure, key physicochemical indicators remain stable before and after freeze-drying, surface negative charge increases, and it exhibits good colloidal and short-term storage stability as well as simulated gastrointestinal tolerance, providing technical support for the preparation of high-quality, highly stable *Lactobacillus plantarum* extracellular vesicle products.

[0020] (3) This invention systematically analyzed the molecular composition of extracellular vesicles of P-8 *Lactobacillus plantarum* through sRNA sequencing and proteomics analysis: 54 sRNAs (including 21 novel sRNAs) and 411 proteins were identified, and core sRNAs (miR-92b-3p and miR-92a) and 7 key functional proteins related to depression were screened out. The molecular mechanism by which they exert their effects through the regulation of the cAMP / PKA / CREB pathway was clarified, providing omics basis for elucidating the regulatory mechanism of the gut-brain axis.

[0021] (4) The extracellular vesicles of *Lactobacillus plantarum* P-8 of the present invention can be effectively taken up by intestinal epithelial cells, significantly improving the cell viability of LPS-damaged cells and inhibiting cell apoptosis; at the same time, by inhibiting the secretion of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α, and upregulating the expression of tight junction proteins such as ZO-1 and Occludin, the intestinal epithelial barrier function is systematically repaired.

[0022] (5) The extracellular vesicles of *Lactobacillus plantarum* P-8 of the present invention can be effectively taken up by nerve cells, significantly enhance the vitality of CORT-induced SH-SY5Y cells and inhibit cell apoptosis, while effectively reducing the level of intracellular reactive oxygen species (ROS) and alleviating oxidative stress damage. It has good neuroprotective potential and provides a new candidate preparation for the intervention of depression-related diseases.

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] Figure 1 Characterization results of P-8 extracellular vesicles prepared by different extraction methods. In the figure, A is the NTA detection result of P-8 extracellular vesicles extracted by the kit, B is the transmission electron micrograph of P-8 extracellular vesicles extracted by the kit, C is the NTA detection result of P-8 extracellular vesicles extracted by ultracentrifugation, and D is the transmission electron micrograph of P-8 extracellular vesicles extracted by ultracentrifugation. The scale bar is 100 nm. Figure 2 The results of the freeze-drying process parameter optimization are shown in the figure. A shows the effect of different extraction times on the concentration of P-8 extracellular vesicles, B shows the effect of different protective agents on the concentration and particle size of P-8 extracellular vesicles, C shows the effect of different concentrations of protective agents on the concentration and particle size of P-8 extracellular vesicles, D shows the effect of different concentrations of auxiliary protective agents on the concentration and particle size of P-8 extracellular vesicles, E shows the effect of different pH buffers on the concentration and particle size of P-8 extracellular vesicles, and F shows the effect of different pre-freezing methods on the zeta potential of P-8 extracellular vesicles. Figure 3 To illustrate the effect of lyophilization on the physicochemical properties of P-8 extracellular vesicles, Figure A shows the microstructure of fresh vesicles (scale bar: 100 nm), B shows the microstructure of lyophilized and reconstituted vesicles (scale bar: 100 nm), C shows the effect of lyophilization on the concentration, particle size, and protein concentration of extracellular vesicles, D shows the effect of different pre-freezing methods on the zeta potential of P-8 extracellular vesicles, and E shows the effect of different pre-freezing methods on the zeta potential of P-8 extracellular vesicles. P-8 represents fresh vesicles, and P-8-D represents lyophilized and reconstituted vesicles. Uppercase and lowercase letters indicate significant differences. Figure 4This study analyzed the gastrointestinal tolerance of fresh and lyophilized P-8 extracellular vesicles. In the figure, A represents the particle concentration analysis in gastric juice tolerance of fresh and lyophilized extracellular vesicles, B represents the particle size analysis in gastric juice tolerance of fresh and lyophilized extracellular vesicles, C represents the particle concentration analysis in intestinal juice tolerance of fresh and lyophilized extracellular vesicles, D represents the particle size analysis in intestinal juice tolerance of fresh and lyophilized extracellular vesicles, E represents the particle concentration analysis in simulated continuous gastrointestinal digestion tolerance of fresh and lyophilized extracellular vesicles, and F represents the particle size analysis in simulated continuous gastrointestinal digestion tolerance of fresh and lyophilized extracellular vesicles. D-P8 represents lyophilized P-8 extracellular vesicles, and P8 represents fresh P-8 extracellular vesicles. Uppercase and lowercase letters indicate significant differences. Figure 5 To illustrate the effect of lyophilization on the composition of P-8 extracellular vesicle metabolites, Figure A shows the PCA diagram of metabolites from fresh vesicles and lyophilized vesicles treated with a protectant; Figure B shows the PLS-DA diagram of metabolites from fresh vesicles and lyophilized vesicles treated with a protectant; Figure C shows the volcano diagram of differential metabolites from fresh vesicles and lyophilized vesicles treated with a protectant; Figure D shows the scatter plot of differential metabolites; Figure E shows the radar diagram of the top 10 substances with the most significant changes in differential metabolites; and Figure F shows the KEGG pathway enrichment map of differential metabolites. Figure 6 To illustrate the effect of lyophilization on the lipid composition of P-8 extracellular vesicle membranes, Figure A shows the PCA diagram of lipids from fresh vesicles and lyophilized vesicles treated with a protectant; Figure B shows the PLS-DA diagram of lipids from fresh vesicles and lyophilized vesicles treated with a protectant; Figure C shows the volcano diagram of differential lipids from fresh vesicles and lyophilized vesicles treated with a protectant; Figure D shows the scatter plot of differential lipids; Figure E shows the radar diagram of the top 10 substances with the most significant changes in differential lipids; and Figure F shows the enrichment of differential lipids in the KEGG pathway. Figure 7 For the storage stability analysis of lyophilized extracellular vesicles, Figure A shows the microscopic morphological changes of extracellular vesicles after 0 days of storage, B shows the microscopic morphological changes of extracellular vesicles after 15 days of storage, C shows the microscopic morphological changes of extracellular vesicles after 30 days of storage, D shows the microscopic morphological changes of extracellular vesicles after 90 days of storage, E shows the changes in concentration and particle size of extracellular vesicles during storage, F shows the changes in zeta potential of extracellular vesicles during storage, and G shows the changes in protein concentration of extracellular vesicles during storage. Uppercase and lowercase letters indicate significant differences. Figure 8 A statistical distribution of the total length of P-8 extracellular vesicle sRNA; Figure 9 The results of P-8 extracellular vesicle sRNA identification and expression characteristics are shown in the figure. A represents the number of sRNAs identified in P-8 exosomes of Lactobacillus plantarum, and B represents the top 20 sRNAs with the highest expression levels in P-8 exosomes of Lactobacillus plantarum. Figure 10For the functional enrichment analysis of P-8 extracellular vesicle sRNA target genes, A in the figure is the GO analysis of P-8 exosome sRNA target genes, B is the KEGG analysis of exosome sRNA target genes, C is the GO functional analysis of TOP20 expression level sRNA, and D is the KEGG functional analysis of TOP20 expression level sRNA of exosomes. Figure 11 A schematic diagram of the target genes and sRNAs regulated by the 5-HT1AR / cAMP / PKA / CREB pathway; Figure 12 The figure shows the protein interaction network and core gene screening for cAMP pathway-related target genes. A represents the interaction network of genes predicted to be in the 5-HT1AR / cAMP / PKA / CREB pathway, B represents the top 10 core genes analyzed by cytoHubba, C represents the key sub-network analyzed by MCODE, and D represents the top 10 core genes analyzed by cytoNCA. Figure 13 The results are qPCR validations of the key sRNA. Figure 14 The figure shows the proteomic identification results of P-8 extracellular vesicles and ultracentrifuged supernatant. In the figure, A is the scatter plot of PCA, B is the Venn diagram of protein identification results, C is the volcano plot of differentially expressed proteins, and D is the statistical plot of the number of differentially expressed proteins. P8-Con represents P-8 extracellular vesicles, and P8-Exo represents the ultracentrifuged supernatant of P-8 extracellular vesicles. Figure 15 For the functional enrichment analysis of differentially expressed proteins, A in the figure is a GO enrichment triple bar chart, and B is a KEGG differential protein pathway analysis. Figure 16 This is a heatmap of clusters of depression-related functional proteins. In the figure, P8-Con represents P-8 extracellular vesicles, and P8-Exo represents the supernatant of P-8 extracellular vesicles after ultracentrifugation. Each group has 3 replicates. Figure 17 The results of the HIEC-6 cell uptake experiment on P-8 extracellular vesicles are shown. In the figure, LPS (50 μm / ml) represents the LPS-damaged group, and LPS (50 μm / ml) + p8-Exo represents the P-8 extracellular vesicle intervention group. The scale bar is 25 μm. Figure 18 The effect of P-8 extracellular vesicles on LPS-induced HIEC-6 cell viability and apoptosis is shown in the figure. A represents the cell viability results detected by CCK-8 assay, B represents the cell apoptosis results detected by flow cytometry, HICE-6 represents the control group, HICE-6+LPS represents the LPS-damaged group, and HICE-6+LPS+P8-Exo represents the P-8 extracellular vesicle intervention group. ** represents P<0.01, and *** represents P<0.001. Figure 19The effect of P-8 extracellular vesicles on LPS-induced secretion of inflammatory factors in intestinal epithelial cells is shown in the figure. A represents inflammatory factor IL-6, B represents inflammatory factor IL-1β, C represents inflammatory factor TNF-α, D represents inflammatory factor IL-10, HICE-6 represents the control group, HICE-6+LPS (50 μg / ml) represents the LPS-damaged group, and HICE-6+LPS (50 μg / ml)+P8-Exo represents the P-8 extracellular vesicle intervention group. * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. Figure 20 The effect of P-8 extracellular vesicles on LPS-induced tight junction protein expression in HIEC-6 cells is shown in the figure. HICE-6 represents the control group, HICE-6+LPS represents the LPS-damaged group, and HICE-6+LPS+P8-Exo represents the P-8 extracellular vesicle intervention group. The scale bar is 10 μm. Figure 21 The effect of different concentrations of corticosterone (CORT) on the viability of SH-SY5Y cells is shown in the figure. SH-SY5Y represents the control group, and SH-SY5Y+CORT represents the experimental groups with different concentrations of CORT (10μm, 25μm, 50μm, 100μm, 150μm). ns represents P>0.05, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. Figure 22 The figure shows the results of the uptake experiment of P-8 extracellular vesicles by SH-SY5Y cells. CORT represents the model group and CORT+Exo represents the vesicle group. Figure 23 The effect of P-8 extracellular vesicles on CORT-induced viability and apoptosis of SH-SY5Y cells is shown in the figure. A represents the cell viability results detected by CCK-8 assay, and B represents the cell apoptosis results detected by flow cytometry. SH-SY5Y represents the control group, SH-SY5Y+CORT represents the model group, SH-SY5Y+CORT+Exo represents the vesicle group, and SH-SY5Y+CORT+fluoxetine represents the fluoxetine treatment group. * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. Figure 24 The effect of P-8 extracellular vesicles on CORT-induced reactive oxygen species (ROS) levels in SH-SY5Y cells is shown in the figure. Figure A is a bar chart of intracellular ROS levels, and Figure B is a flow cytometry fluorescence intensity distribution of intracellular ROS levels. SH-SY5Y represents the control group, SH-SY5Y+CORT represents the model group, SH-SY5Y+CORT+Exo represents the vesicle group, and SH-SY5Y+CORT+fluoxetine represents the fluoxetine treatment group. ** represents P<0.01, and *** represents P<0.001. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0027] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0028] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0029] This invention relates to *Lactobacillus plantarum* P-8, which is classified and named *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The *Lactobacillus plantarum* P-8 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 6312. It has been disclosed in a prior patent application (Invention title: A metabiotic composition for preservation and antibacterial purposes in food and its preparation method and application; Application No.: 202510089302.9; Publication No.: CN119522963A; Authorization Announcement No.: CN119522963B).

[0030] Example 1 Extraction of P-8 extracellular vesicles (P-8-EVs) from Lactobacillus plantarum.

[0031] 1.1 Ultracentrifugation: Take the supernatant of *Lactobacillus plantarum* P-8 cells, centrifuge at 10000×g for 30 min at 4℃ to remove impurities, and transfer the supernatant to a new centrifuge tube (if the sample volume is large, it needs to be concentrated; the concentration method is to centrifuge at 3500×g for 15 min in a 100KD ultrafiltration tube); then filter through a 0.22μm filter membrane and collect the filtrate; transfer the filtrate to an ultracentrifuge tube and centrifuge at 120000×g for 90 min at 4℃; remove the supernatant, and resuspend the precipitate with 100μL of pre-cooled 1×PBS buffer to obtain *Lactobacillus plantarum* P-8 extracellular vesicles (P-8 extracellular vesicles).

[0032] 1.2 Kit Method (Control Group): Take 8 mL of *Lactobacillus plantarum* P-8 cell supernatant sample, add an equal volume of buffer XBP, gently invert and mix 5 times, and allow the mixture to warm to room temperature. Add 16 mL of sample / XBP mixture to an exoEasy centrifuge column and centrifuge at 500×g for 1 min. Discard the eluent and return the column to the same collection tube (if the sample volume is greater than 8 mL, repeat this step until the entire volume passes through the column). Then centrifuge at 5000×g for 1 min to remove residual liquid from the membrane; add 10 mL of buffer XWP, centrifuge at 5000×g for 5 min to remove residual buffer from the column, and discard the eluent and collection tube. Transfer the centrifuge column to a new collection tube, add 250 µL of buffer XE to the membrane, and incubate for 1 min. Centrifuge at 500×g for 5 min, collect the eluent, and obtain the extracellular vesicles extracted using the kit method.

[0033] The EVs prepared by ultracentrifugation and the kit method were stored at -80℃ for later use.

[0034] Example 2 The effects of different extraction methods on the biological characteristics of extracellular vesicles (EVs).

[0035] 2.1 Characterization methods.

[0036] (1) Transmission electron microscopy (TEM) characterization: Take the EVs prepared by ultracentrifugation and the kit method, use a pipette to draw 10 μL of suspension and drop it onto the ParafiLm sealing film (the back of the sealing film is attached to the table), place the copper mesh of the carrier film face down, and let it naturally absorb the suspension drop for 10-15 min. Then use filter paper strips to remove excess droplets and let it dry slightly. Use a pipette to draw 10 μL of 2% phosphotungstic acid solution and drop it onto the sealing film. Place the copper mesh with the front side facing the staining solution, invert it and let it stand for 3-5 min. Use filter paper strips to remove excess droplets and let it dry under an incandescent lamp. Observe and photograph under a transmission electron microscope.

[0037] (2) Nanoparticle tracking analysis (NTA): EVs frozen at -80℃ were removed, thawed in a 25℃ water bath, and placed on ice. They were diluted 8000 times with PBS and analyzed using a particle size analyzer (ZetaviewS / PMX-120) to directly image and observe specific exosomes and vesicles with diameters of 50-1000 nm in the suspension in real time. The exosome particle size distribution was calculated using Zetaview 8.04.02 software. The analysis was repeated three times using completely independent samples.

[0038] 2.2 Results: The results are as follows Figure 1 As shown, the NTA detection results indicate that the concentration of EV particles obtained by ultracentrifugation was 1.3 × 10⁻⁶. 11Particles / mL, particle size exhibiting a unimodal normal distribution with a peak value of 125.4 nm; the EV particle concentration obtained by the kit method was 1.6 × 10⁻⁶. 10 The particle size distribution was 140.6 nm. It is evident that the extracellular vesicles extracted by both methods conformed to the normal particle size of extracellular vesicles. However, the vesicles extracted by ultracentrifugation had smaller particle sizes and a significantly higher particle concentration than those extracted by the kit method, demonstrating a significant advantage in both yield and particle size uniformity.

[0039] TEM observations showed that both methods yielded EVs with a typical saucer-like bilayer structure, uniform in size, ranging from 50 to 150 nm in diameter, exhibiting either a single distribution or clustered formations, with visible low-electron-density material inside. However, the EVs extracted using the kit method had more background impurities, while the EVs extracted using the ultracentrifugation method had a clearer background, fewer impurities, and superior EV purity.

[0040] In summary, the *Lactobacillus plantarum* P-8 EVs prepared by ultracentrifugation exhibit high particle concentration, uniform particle size, and good purity.

[0041] Example 3 Optimization of the freeze-drying process of P-8 extracellular vesicles of Lactobacillus plantarum.

[0042] 3.1 Determination of optimal extraction time: The growth curve of *Lactobacillus plantarum* P-8 and the concentration changes of EVs at different culture times were monitored. Results are as follows: Figure 2 As shown in Figure A, the lag phase of the strain is 0-3 hours, and the logarithmic growth phase is 4-10 hours. The extracellular vesicle concentration of P-8 shows a trend of first increasing and then decreasing, reaching a peak of 6.60 × 10⁻⁶ at 6 hours of the logarithmic growth phase. 9 The particle concentration was significantly higher than at 3h and 9h (P < 0.05). After entering the stationary phase, the vesicle concentration decreased significantly and remained stable until 24h without a significant increase, indicating that the release of extracellular vesicles from P-8 cells may be more dependent on the specific physiological state during the logarithmic phase, and the environmental pressure during the stationary phase did not significantly stimulate its yield increase. Therefore, 6h was determined to be the optimal extraction time.

[0043] 3.2 Screening of the best protective agent.

[0044] Fresh vesicles were used as the blank group, and freeze-thawed samples without protective agents were used as the control group.

[0045] The concentration and particle size changes of vesicles after freeze-thaw cycles were compared after treatment with different cryoprotectants, including trehalose, mannitol, glycine, sucrose, dextran, and lysine, to evaluate the protective effect. Results are as follows: Figure 2As shown in Figure B, the vesicle concentration in the lysine-treated group was not significantly different from that in the control group, the particle size deviation was the smallest (difference from the control group was 6.8 nm), the structural integrity was best maintained, and the protective effect was significantly better than other protective agents. Therefore, lysine was selected as the main protective agent.

[0046] The protective effects of different concentrations of lysine (2%, 5%, and 10%) were further investigated, and the results are as follows: Figure 2 As shown in Figure C, the 2% lysine group had the highest vesicle concentration (8.80 × 10⁻⁶). 9 The EV concentration in the 5% concentration group was significantly higher than all other groups (P < 0.05), but the particle size (148.4 nm) was significantly lower than the control group (P < 0.05). This may be due to the vesicles breaking down into microvesicles, leading to a significant increase in concentration. The EV concentration in the 5% concentration group was 6.70 × 10⁻⁶. 9 The particle count / mL was not significantly different from the control group, and the particle size (154.3 nm) was also quite similar, indicating a relatively balanced overall protective effect. The EV concentration in the 10% concentration group was 7.90 × 10⁻⁶. 9 Although the particle count / mL was still significantly higher than the control group, the particle size increased significantly to 175.3 nm (P < 0.05), which may indicate the presence of large vesicle aggregation or protective agent molecule attachment. In summary, 5% lysine achieved the best balance in maintaining the number and structural integrity of EVs; therefore, 5% was determined to be the optimal concentration of lysine.

[0047] 3.3 Screening of the optimal co-protective agent: Albumin was selected as the co-protective agent and combined with 5% lysine. The typical concentration range of albumin in lyophilized formulations is 1%~5%, and its protective effect usually shows a concentration-dependent effect. Too low a concentration is insufficient to effectively maintain the stability of EVs, while too high a concentration may have adverse effects due to osmotic pressure. The protective effect of different concentrations of albumin was tested. The results are as follows: Figure 2 As shown in Figure D, the EV concentration in the 1% albumin group was closest to that in the control group, and the particle size was not significantly different from that of the control group, indicating the best protective effect. Therefore, 1% albumin was determined to be the optimal concentration of auxiliary protective agent.

[0048] 3.4 Determination of the optimal buffer system: Under the protective agent system of 5% lysine + 1% albumin, protective solutions were prepared using three buffers: MES (pH 6.0), PBS (pH 7.4), and Tris-HCl (pH 8.8), respectively. The stability of EVs was tested after repeated freeze-thaw cycles.

[0049] The results are as follows Figure 2 As shown in Figure E, the concentration of EVs in the PBS (pH 7.4) group was not significantly different from that in the control group, and the particle size was closest to that of the control group, indicating the best effect in maintaining EV stability. Therefore, the PBS buffer at pH 7.4 was determined as the standardized buffer system.

[0050] 3.5 Determination of the optimal pre-freezing method: Based on the optimized cryoprotectant system described above, EVs were freeze-dried using two pre-freezing methods: rapid freezing with liquid nitrogen and slow freezing overnight at -80℃, to screen for the most suitable pre-freezing conditions for maintaining vesicle stability. The results are as follows: Figure 2 As shown in Figure F, the vesicle concentration in the liquid nitrogen quick-freezing group was significantly higher than that in the control group. This indicates that the rapid freezing rate of liquid nitrogen may cause vesicles under different cryoprotectant systems to experience varying degrees of permeation and mechanical stress, resulting in some degree of breakage and abnormal shrinkage or aggregation of particle size, affecting the uniformity and stability of their structure. The vesicle concentration in the -80℃ overnight slow-freezing group was not significantly different from that in the control group, indicating that the slow freezing process is conducive to the slow precipitation of water, reducing the physical damage of ice crystals to the vesicle membrane and the excessive aggregation caused by drastic changes in local solute concentration, thus better maintaining the original structural integrity of the vesicles.

[0051] like Figure 2 As shown in Figure F, the extracellular vesicle potential of P-8 cells treated with slow freezing at -80℃ was -23.06 mV, which is higher than the -20.43 mV of the liquid nitrogen quick-freezing group. This indicates that the charge is more stable during slow freezing, which is more conducive to maintaining the long-term stability of the dispersion system. Therefore, overnight slow freezing at -80℃ was determined to be the optimal pre-freezing method.

[0052] 3.6 Optimal freeze-drying process: Based on the above optimization results, the optimal freeze-drying preservation process for Lactobacillus plantarum P-8 extracellular vesicles is as follows: Prepare a freeze-drying protection solution containing 5% (w / v) lysine and 1% (w / v) albumin in pH 7.4 PBS buffer, mix it with the purified extracellular vesicles, and then pre-freeze it overnight at -80℃, followed by freeze-drying.

[0053] Example 4 Effects of freeze-drying on the physicochemical and functional properties of P-8 extracellular vesicles of Lactobacillus plantarum (P-8 extracellular vesicles).

[0054] In optimizing the freeze-drying system, the effects of the freeze-thaw process on vesicle concentration and particle size were primarily investigated. However, the drying process also affects vesicle membrane structure, protein concentration, and particle size. Therefore, the differences between freeze-dried vesicles using the optimized freezing scheme and fresh vesicles were compared to evaluate the overall effect of freeze-drying on vesicle quality characteristics.

[0055] 4.1 Effect on microstructure: TEM was used to observe the morphology of vesicles after lyophilization and rehydration. The results are as follows: Figure 3 China A Figure 3As shown in Figure B, the P-8 extracellular vesicles, after freeze-drying using the optimized process, still maintain their typical bilayered cup / spherical structure. The membrane structure is continuous and intact, with no obvious rupture or leakage of contents, good dispersibility, and no large-scale aggregation. This indicates that the established freeze-drying protection system effectively reduces the damage to P-8 extracellular vesicles caused by ice crystal formation and drastic changes in osmotic pressure during freeze-drying, and morphologically confirms the effectiveness of the process in protecting P-8 extracellular vesicles.

[0056] 4.2 Effects on concentration, particle size and protein concentration: The particle concentration, average particle size and protein concentration of vesicles before and after freeze-drying were measured.

[0057] The particle concentration and average particle size results are as follows: Figure 3 As shown in Figure C, there was no significant difference in particle concentration of P-8 extracellular vesicles before and after freeze-drying, and the average particle size also did not change significantly. This indicates that the protective agent combination under optimized conditions can effectively maintain the structural integrity and counting consistency of vesicles, resulting in better concentration stability of P-8 extracellular vesicles after freeze-drying; protein concentration results are shown in Figure C. Figure 3 As shown in Figure D, there was no significant difference in protein concentration in P-8 extracellular vesicles before and after lyophilization. This indicates that the combination of protective agents used in P-8 can effectively maintain the stability of proteins within its vesicles.

[0058] 4.3 Effect on zeta potential: Detection results are as follows Figure 3 As shown in Figure E, the absolute value of the Zeta potential of P-8 extracellular vesicles significantly increased after lyophilization (P<0.05), and the surface negative charge density increased, which enhanced the electrostatic repulsion between vesicles, improved the long-term stability of the colloidal dispersion system, and reduced secondary aggregation. Therefore, the optimized lyophilization method can effectively improve the surface charge stability of its vesicles.

[0059] 4.4 Effects on gastrointestinal digestive tolerance: To assess the stability of Lactobacillus plantarum P-8 extracellular vesicles (P-8 extracellular vesicles) and their lyophilized extracellular vesicles (D-P8) in the gastrointestinal environment, the dynamic changes of P-8 extracellular vesicles in simulated gastric juice (SGF), simulated intestinal juice (SIF), and continuous gastrointestinal digestion were monitored to reveal their gastrointestinal tolerance and evaluate the gastrointestinal stability of fresh and lyophilized vesicles.

[0060] (1) Analysis of gastric juice digestive stability: such as Figure 4 China A Figure 4As shown in Figure B, after 3 hours of digestion of fresh vesicles, the particle concentration increased significantly while the particle size decreased significantly, indicating that the acidic environment may induce vesicle membrane dehydration and shrinkage. At the same time, the membrane structure disturbance caused some vesicles to rupture, generating microvesicles, which resulted in a decrease in particle size and an increase in particle count. After 6 hours, the concentration continued to increase significantly while the particle size tended to stabilize, indicating that the continuous action of pepsin and strong acid caused the vesicles to further disintegrate into membrane fragments of similar size. The system was characterized by a continuous increase in the number of particles, and the membrane integrity was basically lost.

[0061] After 3 hours of digestion of lyophilized vesicles, the concentration increased significantly while the particle size remained unchanged. This indicates that the hydration and dissolution of the lyophilization protectant matrix caused previously embedded or aggregated intact vesicles to be released into the solution, increasing the number of particles, but the vesicles themselves did not undergo significant morphological changes. After 6 hours, the concentration decreased significantly and the particle size increased, indicating that after the protectant was exhausted, the exposed vesicle membranes were subjected to acid hydrolysis and enzymatic attack, leading to significant aggregation or fusion, resulting in a decrease in countable particles and an increase in average size. In summary, lyophilization provided a short-term protective window for P-8 extracellular vesicles, but failed to withstand continuous digestion pressure; the later structural features were mainly aggregated and fused.

[0062] (2) Analysis of the digestive stability of intestinal fluid: such as Figure 4 C, Figure 4 As shown in Figure D, there was a significant difference in tolerance between fresh and lyophilized vesicles. After 4 hours of digestion of fresh P-8 extracellular vesicles, the particle concentration decreased significantly while the particle size remained unchanged; after 8 hours, both concentration and particle size remained stable. This result exhibits a "selective degradation-residual homeostasis" pattern, meaning that bile salts and pancreatic enzymes in the intestinal fluid preferentially cleared the fragile vesicle subpopulations, while the remaining vesicle membranes maintained good integrity, without further loss or morphological changes.

[0063] The lyophilized P-8 extracellular vesicles exhibited a progressively intensified aggregation trend. At 4 hours of digestion, the concentration decreased significantly while the particle size increased significantly, indicating that after the lyophilization protectant matrix hydrated and dissolved, the exposed vesicle membranes were attacked by bile salts and trypsin, resulting in the destruction and dissolution of some vesicles. Simultaneously, significant aggregation or fusion occurred among the remaining vesicles, leading to a decrease in countable particles and an increase in average size. At 8 hours, both the concentration and particle size continued to increase synchronously. It is speculated that under sustained digestion pressure, further fusion of aggregates or membrane damage induced vesicle expansion, while large fragments from the disintegration of some vesicles were included in the particle count, collectively driving up both the concentration and particle size.

[0064] (3) Continuous gastrointestinal digestive stability analysis: such as Figure 4 E, Figure 4 As shown in Figure F, the overall trend of P-8 extracellular vesicles in the fresh and freeze-dried states during continuous gastrointestinal digestion is similar, both exhibiting a three-stage pattern of "dispersion in the gastric stage - initial damage from intestinal fluid - stabilization in the later stage", but there are subtle differences in the magnitude of change and microscopic mechanisms.

[0065] After 3 hours of digestion by gastric juice, the concentration of fresh vesicles increased significantly while the particle size decreased significantly. This indicates that the acidic gastric juice environment induced dehydration and contraction of the vesicle membrane, leading to its rupture and the generation of numerous, smaller microvesicles, resulting in an increase in particle count and a decrease in average particle size. Upon transfer to intestinal juice (0 hours), i.e., the initial contact stage, both the concentration and particle size decreased significantly to their lowest points. This suggests that the sudden change in pH, combined with the synergistic impact of bile salts and pancreatic enzymes, caused the rapid disintegration and clearance of a large number of vesicles and fragments. The remaining particles also further contracted, indicating that the system entered its most severely damaged phase. From 3 to 6 hours in the intestinal juice, the concentration decreased slightly and then stabilized, while the particle size remained stable or continued to decrease slightly. This indicates that the remaining particles no longer suffered large-scale loss under continuous digestive pressure, and their structure tended towards a steady state.

[0066] After 3 hours in gastric fluid, the concentration of lyophilized vesicles increased significantly while the particle size remained unchanged. This indicates that the hydration and dissolution of the lyophilization protectant matrix released intact vesicles, increasing the number of particles, but the vesicles themselves did not undergo significant shrinkage or rupture. Upon transfer to intestinal fluid at 0 hours, both the concentration and particle size plummeted to their lowest points, indicating that after the protectant was exhausted, the exposed vesicles experienced the same severe impact as the fresh group, resulting in the disintegration and removal of a large number of particles, and the dehydration and shrinkage of remaining vesicles. From 3 to 6 hours in intestinal fluid, the concentration initially decreased slightly and then rebounded, while the particle size increased slightly and then stabilized. This suggests that the remaining vesicles underwent slight reversible aggregation or deagglomeration under the scrubbing effect of bile salts, and the system gradually reached equilibrium.

[0067] In summary, critical structural damage to P-8 extracellular vesicles during continuous digestion is concentrated in the initial contact stage with intestinal fluid. Lyophilization can delay gastric disintegration and impart some reversibility to later structural components. The inherent properties of its vesicle membrane components, combined with the compatibility of lyophilization protectants, preserve the basic gastrointestinal tolerance required for oral delivery.

[0068] 4.5 Effects on metabolite composition.

[0069] (1) Comparative analysis of samples: To evaluate the effect of lyophilization on the metabolome of P-8 extracellular vesicles, fresh vesicles were used as a control, and PCA and PLS-DA analyses were performed on lyophilized samples with added preservatives. The PCA results are as follows: Figure 5 As shown in Figure A, the cumulative contribution rate of the first two principal components reached 94.14%. The fresh group and the freeze-dried group showed a clear spatial separation trend in the score plot, and the samples within each group were well clustered. The PLS-DA results are as follows: Figure 5 As shown in Figure B, the model parameters R 2 Y=1, Q 2 =0.999, indicating strong explanatory power and robust prediction of the model. In summary, lyophilization significantly altered the metabolite composition of P-8 extracellular vesicles, providing a reliable basis for subsequent differential metabolite screening.

[0070] (2) Comparative analysis of differential metabolites: The results of the volcano plot are as follows Figure 5As shown in Figure C, a total of 1186 differentially expressed metabolites were screened from P-8 extracellular vesicles before and after freeze-drying (P<0.05, VIP>1, |log2 FC|>1.3), of which 217 were upregulated and 562 were downregulated. These differentially expressed metabolites mainly belong to 16 major categories, including amino acids and their metabolites, glycerophospholipids, nucleotides and their metabolites, and fatty acids.

[0071] radar image Figure 5 As shown in Figure D, Lys-Tyr (an antioxidant dipeptide) and 2,2-diphenylglycine were significantly upregulated after lyophilization, but cGMP, various dipeptides (such as Ile-Arg and Ile-Thr), and organic acids were lost. This indicates that the lyophilization method can retain some functional peptides, but there is a certain loss of nucleotides.

[0072] (3) Differential metabolite pathway enrichment analysis: KEGG pathway enrichment analysis, such as Figure 5 As shown in Figure E, the differential metabolites of P-8 extracellular vesicles before and after freeze-drying were mainly enriched in the D-amino acid metabolism and purine metabolism pathways, with the highest number of compounds contained in these pathways. P-8-derived vesicles may maintain energy balance by regulating D-amino acid metabolism and purine metabolism to cope with oxidative stress induced by freeze-drying.

[0073] 4.6 Effect on membrane lipid composition: (1) Comparative analysis of samples: To evaluate the effect of lyophilization on the lipid composition of P-8 extracellular vesicle membranes, fresh vesicles were used as controls, and PCA and PLS-DA analyses were performed on lyophilized samples with added preservatives. The PCA results are as follows: Figure 6 As shown in Figure A, the total contribution rate was 66.85%, indicating that the model effectively revealed the separation trend between samples, demonstrating that the freeze-drying process significantly altered the lipid composition of the vesicle membrane. PLS-DA analysis is shown below. Figure 6 As shown in Figure B, the model parameters R 2 Y=0.999, Q 2 =0.986, the result is robust and reliable. This indicates that the protectant regimen can protect the physical properties of the vesicles.

[0074] (2) Comparative analysis of differential lipid molecules: volcano diagram as shown Figure 6 As shown in Figure C, a total of 357 differentially expressed metabolites (5 upregulated and 12 downregulated) were generated from the extracellular vesicle membrane components of P-8 cells before and after lyophilization, involving 25 lipid classes (e.g., ...). Figure 6 (As shown in D). Radar image as follows. Figure 6As shown in Figure E, the abundance of sphingomyelin SM (d18:1 / 18:1) increased after lyophilization, while the abundance of various lipids such as prostaglandin D2, thromboxane B3, MGDG, TG, LPG, PG, LPC, and LPA decreased. This indicates that optimizing the lyophilization process can inhibit membrane lipid peroxidation and enhance membrane structural stability, but the reduction in MGDG and TG suggests that neutral lipid reserves have been depleted.

[0075] KEGG annotation was used to perform pathway mining and enrichment analysis on differential lipids between the lyophilized and fresh groups with protective agents. Results are as follows: Figure 6 As shown in Figure F, before and after the lyophilization treatment, the pathway with the most significant enrichment and the largest number of compounds in the P-8 extracellular vesicle membrane components was sphingolipid metabolism. This indicates that sphingolipid metabolism is the core regulatory hub for P-8 extracellular vesicles to cope with lyophilization stress. The optimized treatment significantly promoted the synthesis and remodeling of sphingolipids, thereby enhancing the lyophilization tolerance of the membrane by strengthening the membrane microdomain structure and signal transduction capabilities.

[0076] 4.7 Storage Stability Analysis: The lyophilized vesicles were stored at 37℃ under accelerated storage conditions for 90 days, and changes in their physicochemical properties were monitored. The results are as follows: Figure 7 As shown, the vesicle membrane structure remained intact during short-term storage (0-15 days); at 30 days, the vesicles began to shrink, with a sharp increase in concentration and a decrease in particle size, accompanied by leakage of contents; at 90 days, vesicle fragments aggregated again, and the protein content decreased by 15.9% compared to the initial value. This lyophilized formulation exhibits certain short-term storage stability under accelerated storage conditions.

[0077] Example 5 sRNA omics analysis of extracellular vesicles of Lactobacillus plantarum P-8.

[0078] 5.1 sRNA Sequencing and Identification: 3' and 5' adapters were sequentially ligated using T4 RNA ligase. After reverse transcription and PCR amplification, target fragments were excised by PAGE electrophoresis and quality control was performed. The raw data was filtered using ACGT101-miR to screen for 18-26 nt sequences. These sequences were then aligned to a reference genome and non-coding RNA databases to remove rRNAs and tRNAs. The remaining sequences were aligned to miRBase to identify known sRNAs. Unmatched sequences were aligned to the genome for flanking extensions, and hairpin structures were predicted to identify novel sRNAs. sRNAs were normalized and counted, differentially expressed sRNAs were screened, target genes were predicted, and GO and KEGG enrichment analyses were performed.

[0079] (1) Quality control of exosome sRNA sequencing data of Lactobacillus plantarum P-8.

[0080] As shown in Table 1, based on the sequencing data quality control results, the original read counts for the three parallel samples of *Lactobacillus plantarum* P-8 (P8_Exo_1, P8_Exo_2, and P8_Exo_3) were 8,755,509, 1,453,643, and 8,611,118, respectively. After removing adapters, length filtering (18-26 nt), and filtering for low-complexity sequences, 4,344,486, 7,938,559, and 4,365,226 clean reads were obtained, respectively. Further alignment with Rfam, mRNA, and repetitive sequence databases, and after removing various non-target RNAs and repetitive sequences, the effective reads that could be used for sRNA alignment and identification were finally obtained, numbering 2,682,805, 4,783,253, and 2,658,632, respectively, providing a reliable data foundation for subsequent analysis.

[0081] Table 1. Sequencing data alignment results

[0082] Length distribution analysis was performed on the total effective sequences obtained from sequencing, such as... Figure 8 As shown, the sequence lengths of the three parallel samples were concentrated between 18 and 26 nucleotides, and the distribution patterns were highly consistent. Specifically, P8_Exo_2 and P8_Exo_3 exhibited significant abundance peaks at 22 nt (accounting for 13.40% and 13.70%, respectively), while P8_Exo_1 had the highest proportion at 18 nt (18.17%). This difference may reflect the heterogeneity of sRNA expression among the samples. The overall distribution characteristics are consistent with the typical length range of mature animal sRNAs, indicating that the sequencing data successfully enriched high-quality sRNAs, laying a reliable foundation for subsequent sRNA identification and expression analysis.

[0083] (2) Identification results and expression characteristics of exosome sRNA of Lactobacillus plantarum P-8.

[0084] like Figure 9 As shown in Figure A, 32, 32, and 35 sRNAs were identified in P8_Exo_1, P8_Exo_2, and P8_Exo_3, respectively, with a total of 15 sRNAs expressed in the *Lactobacillus plantarum* P-8 samples. In total, 54 sRNAs were identified from *Lactobacillus plantarum* P-8 exosomes. As shown in Table 2, based on the alignment results with the miRBase database, these sRNAs can be divided into three categories: 13 are known sRNAs with completely identical sequences; 20 are sRNAs that are highly similar to known sequences but have differences; and the remaining 21 are new predicted candidate sRNAs for which no matching was found in the database.

[0085] Table 2 Classification of sRNA alignment results

[0086] The top 20 sRNAs expressed in P-8 exosomes of Lactobacillus plantarum (e.g.) Figure 9 (As shown in B). Based on the expression levels determined by the normalized expression level (norm value), high abundance expression includes let-7, miR-125, miR-10, miR-10-5p, miR-100a-5p, miR-92b-3p, and miR-92a; medium abundance expression includes miR-1, PC-5p-53448, miR-31b-5p, PC-3p-89874, PC-5p-50828, miR-9-5p, miR-7-5p, miR-7, and miR-29b; and low abundance expression includes miR-3505-p3, PC-3p-176478, miR-263, and miR-263b.

[0087] 5.2 Target gene functional enrichment analysis.

[0088] (1) Based on gene ontology (GO), the target genes regulated by P-8 exosome sRNA of Lactobacillus plantarum were analyzed from three dimensions: biological process (BP), molecular function (MF), and cellular component (CC). The target genes of sRNA involve 3168 biological processes, regulate 895 molecular functions, and constitute 567 cellular components.

[0089] Analysis based on GO annotation results shows (e.g.) Figure 10 As shown in Figure A), the target genes of *Lactobacillus plantarum* P-8 exosome sRNA exhibit different enrichment characteristics across the three functional categories.

[0090] Within the biological processes (BP) category, there are numerous entries, but the number of enriched genes in each specific entry is relatively limited, indicating that the target genes are involved in a wide range of biological processes, albeit dispersed. They are significantly enriched in transcriptional regulation-related processes, such as RNA polymerase II-mediated transcriptional regulation and DNA-templated transcriptional regulation, and also involve post-translational modifications such as phosphorylation. sRNAs may broadly influence host cell function by regulating multiple aspects of gene expression and signal transduction.

[0091] Within the cellular component (CC) category, entries are concentrated, and the number of enriched genes is moderate and stable, indicating consistent localization of target genes in specific cellular compartments or structures. They are primarily enriched in key subcellular structures such as the cytoplasm, nucleus, endoplasmic reticulum, and Golgi apparatus, and show significant enrichment in specialized regions such as synapses, suggesting that their functions are closely related to the basic cellular structure, endomembrane system, and intercellular communication.

[0092] Within the molecular function (MF) category, the distribution pattern is most prominent. One entry shows an extremely high enrichment of genes (approaching 10,000), significantly exceeding all other functional entries, while the remaining MF entries generally show a lower enrichment of genes. This indicates that the target genes are highly concentrated in a specific type of activity, such as protein binding; simultaneously, they are also distributed in metal ion binding, nucleotide binding, DNA binding, as well as transferase and hydrolase activities. This reflects the existence of a dominant core mechanism of action within their function, while simultaneously possessing the potential to participate in multiple biochemical reactions.

[0093] (2) KEGG analysis of target genes.

[0094] After KEGG annotation analysis (such as...) Figure 10As shown in Figure B, the target genes are involved in 181 KEGG pathways. The top 20 most significantly enriched pathways are widely distributed across signal transduction, cancer, cytoskeleton regulation, nervous system, and cardiovascular system pathways. In signal transduction-related pathways, the target genes are significantly enriched in the Ras signaling pathway, Rap1 signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and calcium signaling pathway. In cancer-related pathways, the target genes are enriched in cancer pathways, proteoglycans in cancer, and pancreatic cancer. In cytoskeleton and adhesion-related pathways, the target genes are enriched in the regulation of actin cytoskeleton and focal adhesion. Furthermore, the axon guidance pathway is also enriched.

[0095] These significantly enriched pathways suggest that *Lactobacillus plantarum* P-8 exosomal sRNAs may play a broad and synergistic regulatory role in host cell proliferation, differentiation, migration, communication, and specific physiological and pathological processes by regulating multiple key signaling networks, cell structures, growth cycles, and tissue-specific functional pathways.

[0096] (3) Functional analysis of exosome-highly expressed sRNA GO.

[0097] Analysis based on GO annotation results shows (e.g.) Figure 10 As shown in Figure C), the top 20 most expressed sRNAs in the P-8 exosomes of Lactobacillus plantarum have significantly enriched their target gene functions in two major categories: molecular function (MF) and biological process (BP), and exhibit different enrichment characteristics.

[0098] In the molecular function (MF) category, the enriched entries were highly concentrated on enzymatic catalytic activity and specific binding activities. The proteins encoded by the target genes primarily possessed the activities of multiple key metabolic enzymes, such as tRNA-guanosine (34) queuine transglycosylase activity, asparagine synthase (glutamine hydrolysis) activity, carbamoyl phosphate synthase activity, and methylenetetrahydrofolate dehydrogenase activity. Simultaneously, binding functions such as carboxylic acid binding and NADH / NADPH binding were also enriched. This indicates that the regulatory functions of these sRNAs are highly biochemically specific and directly related to the basal metabolic catalysis of bacteria.

[0099] Within the biological processes (BP) category, enriched entries closely revolve around a series of core synthetic and metabolic processes. Target genes are significantly involved in the metabolism of amino acids and their derivatives, such as asparagine biosynthesis and glutamine metabolism; in nucleoside modification, such as quinoline biosynthesis; and in nitrogen metabolism and one-carbon unit metabolism, such as the urea cycle and one-carbon unit metabolism. Furthermore, lipopolysaccharide biosynthesis and DNA recombination are also enriched. This suggests that these highly expressed sRNAs may synergistically regulate multiple fundamental life processes in bacteria, from nutrient metabolism and the synthesis of important cofactors to cell wall formation and maintenance of genetic stability.

[0100] In summary, the functional network of target genes with highly expressed sRNAs points to the fine regulation of bacterial basal metabolism (amino acids, one-carbon units, nitrogen metabolism), the synthesis of key biomolecules (tRNA, lipopolysaccharide), and the catalytic functions of related enzymes. This suggests that *Lactobacillus plantarum* P-8 may deliver these sRNAs via exosomes, remotely influencing the core physiological processes of itself and its symbiotic bacteria, and exerting potential probiotic functions.

[0101] (4) Analysis of the KEGG pathway with high expression of sRNA in exosomes.

[0102] Based on KEGG pathway enrichment analysis (e.g.) Figure 10As shown in Figure D, the top 20 most expressed sRNAs in *Lactobacillus plantarum* P-8 exosomes significantly enriched their target genes in five core metabolic pathways, ranked by enrichment significance: alanine, aspartate, and glutamate metabolism; glyoxylate and dicarboxylate metabolism; one carbon pool by folate; glycine, serine, and threonine metabolism; and pyrimidine metabolism. These pathways collectively constitute the core network of bacterial basal metabolism, mainly involving amino acid tautomerism and degradation, one-carbon unit generation and transfer, and the supply of nucleotide synthesis precursors. These results suggest that these highly abundant sRNAs may play a crucial role in adapting to the environment and maintaining physiological functions by synergistically regulating fundamental life processes such as carbon and nitrogen metabolic flux, energy metabolism, and genetic material synthesis within bacteria.

[0103] 5.3 Screening of target genes and sRNAs in depression-related pathways.

[0104] Based on three depression-related pathways (5-HT1AR / cAMP / PKA / CREB, GABA_B receptor / CaMKIIa, BDNF / TrkB), GO enrichment analysis was performed on the target genes of P-8 exosome sRNA in *Lactobacillus plantarum*. As shown in Table 3, a total of 37 GO entries were matched, of which 19 were significantly enriched (P<0.05), and all belonged to the 5-HT1AR / cAMP / PKA / CREB pathway. Entries related to the other two pathways did not reach significance (P>0.05), so this pathway was selected. Further, the top 10 entries were screened based on P-value, and entries 1 (cAMP-mediated signaling) and 2 (cellular response to cAMP) were selected based on significance. To elucidate the mechanism of cAMP signal downregulation mediated by the 5-HT1AR pathway, entries 6 (negative regulation of cAMP-mediated signaling) and 7 (cAMP catabolic process) were added to characterize the pathway's inhibition of cAMP transmission and the potential parallel mechanism of reducing cAMP levels through accelerated degradation, respectively. Ultimately, 66 target genes were enriched from GO entries related to this pathway, and 19 sRNAs (such as...) were identified. Figure 11 (As shown).

[0105] Table 3. Enrichment analysis of pathway-related GO items

[0106] A protein-protein interaction network for the target gene was constructed based on the STRING database. Co-expression correlation scores were used as the connection criterion, and the network was visualized using color intensity. Darker colored nodes indicated stronger interactions, suggesting potentially more important biological functions within the network. Figure 12 As shown in Figure A, the network contains 111 nodes and 604 interaction relationships. Among them, CREB1, PDE4D, RAPGEF3, RAPGEF4, and PDE4A occupy core positions in the network topology and may be key functional genes.

[0107] Three plugins were installed in Cytoscape software, and three methods were used to screen for target genes.

[0108] (1) The nodes were sorted using the MCC algorithm of the Cytohubba plugin, and the top 10 key genes (such as...) were selected. Figure 12 As shown in Figure B), the genes are PDE4D, PDE4A, ADCY1, PDE7A, PDE7B, PDE4C-2, PDE2A, PDE11A, PDE10A, and PDE8A, in that order. These genes have high topological importance in the network and may serve as core hubs in the regulatory network, playing a key role in related biological processes.

[0109] (2) Modular analysis of the interaction network was performed using the MCODE plugin, and three highly cohesive subnetworks were identified (e.g., Figure 12 (C). The highest-scoring subnetwork (scoring 5 points) contains key target genes such as PDE4D, PDE10A, PDE4A, PDE11A, PDE7B, PDE4C-2, ADCY1, PDE7A, PDE2A, and PDE8A, with PDE4A being the seed gene of this network.

[0110] (3) Using the cytoNCA plugin algorithm, these genes are filtered according to six dimensions (Degree, Eigenvector, LAC, Betweenness, Closeness, Network) in the interaction network to obtain the top 10 key genes (e.g. Figure 12 As shown in D), they are CREB1, RAPGEF4, RAPGEF3, PDE4D, PDE4A, ADCY1, RAP1A, PDE11A, PDE4C-2 and AKAP9 in sequence.

[0111] Finally, the intersection of the key target genes obtained by the three algorithms cytoHubba-MCC, MCODE, and cytoNCA was taken, resulting in four intersection target genes: PDE4D, PDE4A, ADCY1, PDE4C-2, and PDE11A. Based on a review of relevant literature, PDE4D was determined to be the target gene. The sRNAs corresponding to PDE4D include miR-92b-3p, miR-92a, and miR-7-5p.

[0112] 5.4 qPCR Validation: qPCR was used to validate the key target genes and corresponding 8 sRNAs (miR-92b-3p, miR-10-5p, miR-92a, miR-31b-5p, miR-7-5p, miR-263b, miR-3505, miR-263) screened from P-8 extracellular vesicles. Figure 13 As shown, miR-3505 and miR-263 showed no effective amplification (CT > 35), indicating they were either not expressed or had extremely low abundance in the sample; all other sRNAs were stably detected. Among them, miR-7-5p and miR-263b had low expression levels (CT > 30), while miR-92b-3p, miR-92a, miR-10-5p, and miR-31b-5p showed high expression abundance, providing candidate molecules for subsequent mechanistic studies.

[0113] Example 6 Proteomic analysis of P-8 extracellular vesicles from Lactobacillus plantarum. Proteomic analysis was performed on the supernatant of P-8 extracellular vesicles (P-8-EVs) and P-8 extracellular vesicles after ultracentrifugation (P-8-Exo).

[0114] Proteomics detection method: The DIA quantitative proteomics method was adopted. Proteins were identified and quantified through the directDIA workflow. Sample preparation was performed first, including protein extraction with RIPA lysis buffer and acetone precipitation, BCA quantification (concentration calculated based on absorbance to ensure sample usability), reductive alkylation, and trypsin digestion to purify peptides. Mass spectrometry detection was performed using a nanoLC-MS / MS system in DIA-PASEF mode. Sensitivity was improved by chromatographic gradient separation and mass spectrometry parameter optimization. Finally, Spectronaut software was used for data retrieval and qualitative analysis in combination with specific databases and modification parameters, and quality control measures such as iRT correction retention time were integrated.

[0115] 6.1 Protein identification and differential analysis.

[0116] PCA analysis showed that the two groups of samples were clearly separated and that there were significant differences in their composition. Figure 14(As shown in Figure A). A total of 411 proteins were identified, of which 295 were specific to the P-8 extracellular vesicle group. Compared with the P-8 extracellular vesicle group, the P-8 extracellular vesicle ultracentrifugation supernatant group had 187 differentially expressed proteins, of which 29 were upregulated and 158 were downregulated, indicating that vesicle formation is a highly selective process (e.g., ...). Figure 14 (As shown in BD).

[0117] 6.2 Functional enrichment analysis.

[0118] GO enrichment analysis showed that the differentially expressed proteins are mainly involved in biological processes such as cell wall organization, fatty acid biosynthesis, and glycolysis, and are located in the cytoplasm, cell membrane, and cytosol. Their molecular functions are mainly ATP binding, transferase activity, and ATP hydrolysis activity (e.g., Figure 15 (As shown in A).

[0119] KEGG pathway analysis revealed that differentially expressed proteins were involved in 57 pathways, with significant enrichment in pathways such as methane metabolism, fatty acid biosynthesis, tyrosine metabolism, and butyrate metabolism (e.g., Figure 15 (As shown in Figure B). Among them, tyrosine metabolism and butyrate metabolism are closely related to neural regulation and antidepressant function.

[0120] 6.3 Screening of depression-related functional proteins.

[0121] Seven functional proteins associated with depression were screened from 411 identified proteins (such as...). Figure 16 As shown, the enzymes include phosphoacetyltransferase, acetate kinase (catalyzing the production of acetic acid), and the complete enzyme system for type II fatty acid synthesis (FAS-II) (enoyl-ACP reductase, β-ketoacyl-ACP reductase, β-ketoacyl-ACP synthase II, 3-hydroxyacyl-ACP dehydratase, acyl carrier protein, and acyl-ACP thioesterase), the latter of which mediates butyrate synthesis. These functional proteins catalyze the production of short-chain fatty acids such as acetic acid and butyrate, thereby activating the cAMP / PKA / CREB signaling pathway, exerting antidepressant effects, and serving as an important material basis for the regulation of central nervous system function via the gut-brain axis.

[0122] Example 7 Extracellular vesicles of Lactobacillus plantarum P-8 alleviate LPS-induced inflammatory damage to intestinal epithelial cells.

[0123] 7.1 PKH26 cell uptake experiment.

[0124] Prepare a staining working solution by mixing PKH26 linker and Diluent C at a ratio of 1:9. Add P-8 extracellular vesicles and incubate at room temperature in the dark for 10 min. Centrifuge at 120,000g for 90 min to remove free dye. Collect logarithmic growth phase cells, seed them in 24-well plates, and incubate with P-8 extracellular vesicles for 12 h. Discard the supernatant, wash three times with PBS, fix with 4% paraformaldehyde for 15 min, permeate with 0.2% Triton X-100 for 10 min, wash with PBS, and incubate with phalloidin (2 drops / mL PBS) at 37°C in the dark for 30 min. Wash 3-5 times with PBS, stain the nuclei with Hoechst 33342, mount the slides, and photograph using a laser confocal microscope.

[0125] The results are as follows Figure 17 As shown, a distinct red fluorescent signal is visible around the cell nucleus, indicating that P-8 extracellular vesicles can be effectively taken up by HIEC-6 cells.

[0126] 7.2 Effects of P-8 extracellular vesicles on cell viability and apoptosis.

[0127] (1) Detection of CCK8 cell proliferation: HICE-6 cells were digested with trypsin and counted. 1000 cells / well were seeded in 96-well plates and cultured at 37℃ in a 5% CO2 incubator. After cell attachment, the cells were divided into groups: ① Control group: only an equal volume of culture medium was added, without any drug intervention; ② LPS damage group: 50ug / ml LPS was added to establish a cell damage model; ③ P-8 extracellular vesicle intervention group: in addition to LPS stimulation, 1×10 10 P-8 extracellular vesicles (pp / ml) were co-incubated for 24 h. After treatment, 10 μL of CCK-8 solution was added to each well for further incubation for 1-2 h. The absorbance at 450 nm was measured using a microplate reader to assess cell proliferation capacity.

[0128] The results are as follows Figure 18 As shown in Figure A, compared with the control group, the OD of HICE-6 cells stimulated with 50 μg / mL LPS for 24 h was significantly higher. 450 The value decreased significantly (P<0.001), indicating that LPS successfully constructed an intestinal epithelial cell injury model. Furthermore, after intervention with P-8 extracellular vesicles, OD... 450 The value significantly recovered to 0.70, which was significantly different from the LPS-damaged group (P<0.01), demonstrating that P-8 extracellular vesicles can effectively protect against LPS-induced decline in intestinal epithelial cell viability.

[0129] (2) Flow cytometry detection of apoptosis: Cells from the control group, LPS-damaged group and P-8 extracellular vesicle intervention group were collected, washed twice with PBS, resuspended with 100 μL 1×binding buffer, and 10 μL Annexin V-FITC and 5 μL PI were added respectively. After mixing, the cells were incubated at room temperature for 15 min, and 400 μL 1×binding buffer was added before immediate flow cytometry detection.

[0130] The results are as follows Figure 18 As shown in Figure B, LPS stimulation increased the apoptosis rate of HICE-6 cells from 7.04% in the control group to 21.88%, indicating that LPS successfully induced apoptosis of intestinal epithelial cells. However, after intervention with P-8 extracellular vesicles, the apoptosis rate significantly decreased to 15.42%, which was significantly different from the LPS-damaged group (P<0.001), indicating that P-8 extracellular vesicles can significantly inhibit LPS-induced apoptosis of intestinal epithelial cells.

[0131] 7.3 Effects on the secretion of inflammatory factors.

[0132] Immunofluorescence staining (IF): Cells were fixed with 4% paraformaldehyde for 10-15 min, washed 3 times with PBS, permeabilized with 0.2% Triton X-100 for 10 min, washed 3 times with PBS, and blocked with 10% goat serum at 37°C for 30 min. Primary antibody (diluted with 5% goat serum) was incubated overnight at 4°C, washed 3-5 times with PBS, secondary antibody (diluted with 5% goat serum) was incubated at 37°C in the dark for 1 h, washed 3-5 times with PBS in the dark, nuclei were stained with DAPI, and the slides were mounted and photographed using a laser confocal microscope.

[0133] The results are as follows Figure 19 As shown, the levels of IL-1β, IL-6, and TNF-α were significantly increased in the LPS-damaged group, while the level of the anti-inflammatory factor IL-10 was significantly decreased (P<0.01). After intervention with P-8 extracellular vesicles, the levels of pro-inflammatory factors decreased significantly, while the level of IL-10 increased significantly (P<0.05), indicating that P-8 extracellular vesicles can regulate the balance of inflammatory factors and exert an anti-inflammatory effect.

[0134] 7.4 Effects on tight junction proteins.

[0135] qPCR: Total RNA was extracted using the RNAfast200 kit, and its concentration and purity were determined using a nucleic acid and protein analyzer. Reverse transcription was performed using the ReverTra Ace qPCR RT Kit (10 μL system, containing 1 μg RNA, 37℃ for 15 min, 98℃ for 5 min). The cDNA was diluted and stored at -20℃. qPCR was performed using SYBR High-Sensitivity reagent (20 μL system, 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 40 cycles), with GAPDH as an internal control. Triple replicates were used. -ΔΔCt The method analyzes the relative expression levels of genes.

[0136] Western blot (WB): Lyse cell or tissue samples using RIPA lysis buffer, release proteins by shaking or freeze-thaw, and determine protein concentration using the BCA method. Prepare separating and stacking gels according to a 10% PAGE gel kit, load equal volumes of samples, and perform electrophoresis at a constant voltage of 80V until bromophenol blue enters the separating gel, then switch to 120V until the indicator reaches the bottom. Activate the PVDF membrane with methanol for 5 min, and transfer it under a constant current of 300mA. After transfer, block with 5% skim milk at room temperature for 1 h, incubate with primary antibody at 4°C overnight, wash with TBST, add HRP-labeled secondary antibody, and incubate at 37°C for 30 min. After washing, develop with ECL chemiluminescence buffer, and quantify the relative expression level of the target protein using image analysis software.

[0137] The results are as follows Figure 20 As shown, LPS stimulation significantly downregulated the mRNA and protein expression levels of tight junction proteins such as ZO-1, Occludin, and Collagen IV, and weakened the fluorescence signal at the cell boundary. After intervention with P-8 extracellular vesicles, the expression and localization of tight junction proteins were significantly restored. This indicates that P-8 extracellular vesicles can upregulate the expression of tight junction proteins and repair the intestinal epithelial barrier structure.

[0138] In summary, the extracellular vesicles of Lactobacillus plantarum P-8 can be effectively taken up by intestinal epithelial cells and exert a protective effect. They can effectively alleviate intestinal inflammatory damage through anti-inflammatory, anti-apoptotic, and intestinal barrier repair pathways.

[0139] Example 8 Extracellular vesicles of Lactobacillus plantarum P-8 alleviate CORT-induced depression-like damage to nerve cells.

[0140] The cell uptake assay, CCK8 cell proliferation assay, flow cytometry assay for apoptosis, qPCR and WB assay were performed using the same methods as in Example 7 above.

[0141] ROS reactive oxygen species level detection: Dilute DCFH-DA to a final working concentration of 10 μmol / L using serum-free culture medium at a ratio of 1:1000. After removing the cell culture medium, add at least 1 mL of working solution to each well of a six-well plate, ensuring complete coverage of the cells. Incubate the cells at 37°C for 20 minutes, then wash the cells three times with serum-free culture medium to thoroughly remove any probes that have not entered the cells. Finally, collect the cells and perform fluorescence detection using flow cytometry.

[0142] 8.1 Determination of damage model concentration: SH-SY5Y nerve cells were treated with different concentrations of corticosterone (CORT) to screen the model concentration.

[0143] The results are as follows Figure 21 As shown, SH-SY5Y cells were damaged by different concentrations of CORT (10 μM, 25 μM, 50 μM, 100 μM, and 150 μM). The cell survival rate decreased with increasing CORT concentration, and the cell damage rate remained stable at around 50% with 100 μM CORT (P < 0.001). Therefore, a CORT concentration of 100 μM was used as the damage model in subsequent experiments.

[0144] 8.2 SH-SYSY cell uptake experiment.

[0145] To verify the uptake capacity of SH-SY5Y cells by P-8 extracellular vesicles, DiI (red)-labeled P-8-EVs were co-cultured with cells for 24 hours, and then observed using a confocal microscope. The results are as follows: Figure 22 As shown, obvious red fluorescent signals are visible around the cell nucleus marked with blue fluorescent markers and in the cytoskeleton region marked with green fluorescent markers, indicating that P-8 extracellular vesicles can be effectively internalized by SH-SY5Y cells.

[0146] 8.3 Effects of P-8 extracellular vesicles on CORT-induced viability and apoptosis in SH-SYSY cells.

[0147] The grouping and treatment were as follows: ① Control group (SH-SY5Y-6) received only an equal volume of culture medium or solvent, without CORT, extracellular vesicles, or drug intervention; ② Model group (SH-SY5Y+CORT) received 100 μM CORT to establish a cell damage model; ③ Vesicle group (SH-SY5Y+CORT) received 1×10⁻⁶ CORT in addition to 100 μM CORT stimulation. 10 particle / ml extracellular vesicles; ④ Fluoxetine treatment group (SH-SY5Y+CORT), 10μM fluoxetine was added to 100μM CORT stimulation. All groups were cultured in a 37℃, 5% CO2 incubator for 24h, followed by subsequent detection. The results of CCK8 cell proliferation detection are as follows: Figure 23As shown in Figure A, compared with the control group, the survival rate of SH-SYSY cells significantly decreased to 49% after 24 hours of stimulation with 100 μM CORT (P<0.001), indicating that CORT successfully established the SH-SYSY cell depression model. However, after intervention with P-8 extracellular vesicles, the survival rate of SH-SYSY cells increased to 65%, a significant difference compared with the model group (P<0.05), demonstrating that P-8 extracellular vesicles can effectively protect against the CORT-induced decrease in neuronal viability.

[0148] Flow cytometry results as follows Figure 23 As shown in Figure B, CORT stimulation significantly induced apoptosis in SH-SY5Y cells, increasing the apoptosis rate from 4.41% in the control group to 26.27%. However, after intervention with P-8 extracellular vesicles, the apoptosis rate significantly decreased to 10.52%, showing a highly significant difference compared to the model group (P<0.01). This indicates that P-8 extracellular vesicles can effectively inhibit CORT-induced neuronal apoptosis.

[0149] 8.4 Effect of P-8 extracellular vesicles on CORT-induced reactive oxygen species levels in SH-SYSY

[0150] Intracellular reactive oxygen species (ROS) levels were measured to assess oxidative stress status. Results Figure 24 As shown, the ROS level in the control group was 5.84%, while it significantly increased to 29.8% in the model group, indicating that the modeling successfully induced strong oxidative stress. After intervention with P-8 extracellular vesicles, the ROS level decreased to 21.02%, and in the fluoxetine treatment group it decreased to 13.55%. Compared with the model group, the ROS levels in both the vesicle group and the fluoxetine treatment group showed a significant decrease, indicating that P-8 extracellular vesicles can effectively alleviate oxidative stress damage and reduce the toxic effects of excessive free radicals on nerve cells, thus demonstrating potential neuroprotective and repair functions.

[0151] In summary, P-8 extracellular vesicles can be effectively internalized by nerve cells. Based on the successful establishment of a CORT injury model (100 μM), it was found that P-8 extracellular vesicles can significantly enhance cell viability, inhibit apoptosis, and reduce intracellular reactive oxygen species (ROS) levels induced by CORT, demonstrating their neuroprotective potential in alleviating oxidative stress and inhibiting apoptosis.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing extracellular vesicles of *Lactobacillus plantarum* P-8, characterized in that, Includes the following steps: S1. Inoculate Lactobacillus plantarum P-8 strain into liquid culture medium and culture. Collect the culture supernatant, centrifuge, and collect the supernatant. S2. Filter the supernatant to remove bacteria and collect the filtrate; S3. Centrifuge the filtrate, discard the supernatant, resuspend the precipitate, and obtain extracellular vesicles of *Lactobacillus plantarum* P-8.

2. The preparation method according to claim 1, characterized in that, In S1, the culture time was 6 hours, and the centrifugation was carried out at 4℃ and 10000×g for 30 minutes.

3. The preparation method according to claim 1, characterized in that, In S2, filtration is performed using a 0.22μm filter membrane.

4. The preparation method according to claim 1, characterized in that, In S3, the filtrate was centrifuged at 4°C and 120,000 × g for 90 min; the precipitate was resuspended in 100 μL of pre-cooled 1×PBS buffer.

5. A method for freeze-drying and preserving the *Lactobacillus plantarum* P-8 extracellular vesicles prepared by the preparation method according to any one of claims 1-4, characterized in that, Includes the following steps: The extracellular vesicles of Lactobacillus plantarum P-8 were mixed evenly with the freeze-drying protectant and then placed at -80℃ overnight for slow freezing to complete the pre-freezing treatment; The pre-frozen sample was freeze-dried to obtain freeze-dried extracellular vesicle products.

6. The freeze-drying preservation method according to claim 5, characterized in that, The lyophilization protectant consists of lysine, albumin, and PBS buffer; the final concentration of lysine is 5% (w / v); the final concentration of albumin is 1% (w / v); and the pH of the PBS buffer is 7.

4.

7. The use of *Lactobacillus plantarum* P-8 extracellular vesicles prepared by the preparation method according to any one of claims 1-4 in the preparation of drugs or functional foods for relieving or treating enteritis.

8. The use of *Lactobacillus plantarum* P-8 extracellular vesicles prepared by the preparation method according to any one of claims 1-4 in the preparation of drugs or functional foods for relieving or treating depression.

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