A method for promoting the secretion of extracellular vesicles from lactobacillus plantarum

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to a method for promoting the secretion of extracellular vesicles from *Lactobacillus plantarum*, comprising: S1. Adding H2O2 to the culture system during cultivation, inoculating *Lactobacillus plantarum* to induce oxidative stimulation, thus subjecting *Lactobacillus plantarum* to an adaptive oxidative stress state; the concentration of H2O2 is 2mM~4mM; S2. After culturing for 12 hours, collecting the supernatant and centrifuging, filtering, and enriching and purifying it to obtain extracellular vesicles derived from *Lactobacillus plantarum*. This method induces enhanced natural vesicle secretion through "sublethal, adaptive" oxidative stress, avoiding the impurities and interpretability issues associated with methods that increase yield at the expense of cell lysis. It increases the secretion of extracellular vesicles from *Lactobacillus plantarum* while maintaining the viable state of the bacteria, thus making it more suitable for obtaining physiologically significant and batch-comparable vesicle preparations.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for promoting the secretion of extracellular vesicles of Lactobacillus plantarum. Background Technology

[0002] Extracellular vesicles derived from lactic acid bacteria have attracted attention due to their small size (nanoscale), membrane structure, and ability to carry various bioactive substances for drug delivery and disease treatment. Existing technologies have disclosed compositions containing extracellular vesicles derived from lactic acid bacteria (including *Lactobacillus plantarum*) as immunomodulatory components, and described technical solutions for separating and preparing extracellular vesicles from lactic acid bacteria cultures using methods such as centrifugation, filtration, and ultracentrifugation. Simultaneously, existing technologies also include methods to improve the yield of microbial-derived vesicles through bacterial lysis or disruption; for example, obtaining lysate through bacterial lysis and then preparing microbial-derived extracellular vesicles through steps such as filtration and ultrafiltration.

[0003] However, the existing methods for preparing extracellular vesicles still have many key technical problems: First, while the preparation route using cell lysis or fragmentation can increase the recovery of particles or vesicle-like structures, it cannot guarantee that the obtained product is a population of extracellular vesicles naturally secreted by the bacteria and retaining physiological functions. It also easily leads to leakage of cell contents, causing co-segregation of impurities, ultimately resulting in poor batch-to-batch consistency of the prepared product and reduced interpretability of subsequent functional evaluations. Second, for Gram-positive probiotics, their cell walls constitute a natural barrier, significantly limiting the release efficiency of extracellular vesicles. The yield of extracellular vesicles is greatly affected by culture conditions and the stress state of the bacterial envelope. Therefore, there is an urgent need to find a method for preparing extracellular vesicles of *Lactobacillus plantarum* that does not rely on cell lysis and focuses on stress-induced enhancement of natural secretion, in order to balance increased yield, batch stability, and subsequent functional development. Summary of the Invention

[0004] Based on this, the present invention provides a method for promoting the secretion of extracellular vesicles of Lactobacillus plantarum, avoiding the impurities and interpretability problems associated with methods that increase yield at the expense of cell lysis. This method increases the secretion of extracellular vesicles of Lactobacillus plantarum while maintaining the viable state of the cells, thus making it more suitable for obtaining physiologically significant and batch-comparable vesicle preparations.

[0005] The purpose of this invention is to provide a method for promoting the secretion of extracellular vesicles of Lactobacillus plantarum, comprising the following steps: S1. During the cultivation process, H2O2 is added to the culture system, and Lactobacillus plantarum is inoculated to stimulate oxidation, so that Lactobacillus plantarum is in an adaptive oxidative stress state. The concentration of H2O2 is 2mM~6mM. S2. After culturing for at least 12 hours, collect the supernatant and centrifuge, filter, and enrich and purify it to obtain extracellular vesicles derived from Lactobacillus plantarum.

[0006] Preferably, the concentration of H2O2 is 4 mM.

[0007] Preferably, the inoculum amount of *Lactobacillus plantarum* is 1% (v / v) of the culture system volume.

[0008] Compared with existing technologies, the present invention, by adopting the above-mentioned technical solution, has the following effective effects: This invention induces enhanced natural vesicle secretion through sublethal, adaptive oxidative stress, avoiding the impurities and interpretability issues associated with methods that increase yield at the expense of cell lysis. By increasing the secretion of extracellular vesicles in *Lactobacillus plantarum* while maintaining the viable state of the bacteria, it is more suitable for obtaining physiologically significant and batch-comparable vesicle preparations. This invention is simple to operate and has a clear technical route, applicable not only to the efficient preparation of *Lactobacillus plantarum* extracellular vesicles but also to the screening of optimal stimulus concentrations and the study of their yield-enhancing mechanisms.

[0009] During the research process of this invention, it was found that 4 mM H2O2 could induce a significant oxidative stress response in *Lactobacillus plantarum*, resulting in significant membrane damage and morphological remodeling. 4 mM H2O2 caused significant shrinkage, membrane-wall separation, and morphological heterogeneity in some cells, but the hydrodynamic mean particle size at the population level did not change significantly. Treatment with 4 mM H2O2 led to significant but recoverable growth inhibition, accompanied by cell surface roughness, shrinkage, widening of the membrane-wall gap, and a decrease in zeta potential, while the mean particle size did not change significantly. 4 mM H2O2 could significantly increase the output of extracellular vesicles per unit of viable bacteria while maintaining the cells in a scalable state. Simultaneously, 4 mM H2O2 treatment induced extensive metabolic reprogramming and protein expression remodeling in *Lactobacillus plantarum*, with both omics studies pointing to synergistic changes in biological processes such as oxidative stress response, cell wall synthesis, membrane lipid metabolism, central carbon metabolism, and the ABC transport system. Attached Figure Description

[0010] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0011] Figure 1 The effects of different concentrations of H2O2 treatment on the growth, cell morphology, surface properties, and extracellular vesicle production of *Lactobacillus plantarum* were investigated. A shows the growth curves of Lactobacillus plantarum under different concentrations of H2O2 treatment; B represents the SDS-PAGE analysis results of total protein from Lactobacillus plantarum in different H2O2 treatment groups; C represents the scanning electron microscope observation results of Lactobacillus plantarum in different H2O2 treatment groups; D represents the transmission electron microscopy observation results of Lactobacillus plantarum in different H2O2 treatment groups; E represents the change in the zeta potential of the bacterial cell; F represents the change in bacterial cell size; G represents the change in pH of the culture medium; H represents the yield of extracellular vesicle protein in Lactobacillus plantarum after normalization of viable cell count; I represents the yield of Lactobacillus plantarum extracellular vesicle particles after normalization of viable cell count.

[0012] Figure 2 The effects of different concentrations of H2O2 treatment on the oxidative stress level and membrane physiological state of *Lactobacillus plantarum* were investigated. A shows representative fluorescence peaks of intracellular reactive oxygen species in Lactobacillus plantarum under different treatment groups detected by flow cytometry using the DCFH-DA probe. B represents the quantitative analysis results of intracellular ROS fluorescence intensity; C represents the representative peaks of the fluorescence changes of Lactobacillus plantarum JC-1 in different treatment groups detected by flow cytometry using the JC-1 probe; D represents the JC-1 PE / FITC fluorescence intensity ratio; E represents the representative peaks of membrane lipid-related fluorescence in different treatment groups of Lactobacillus plantarum detected by Nile Red staining combined with flow cytometry; F represents the quantitative analysis result of Nile Red fluorescence intensity.

[0013] Figure 3 The effect of different concentrations of H2O2 treatment on the cell membrane integrity of Lactobacillus plantarum was investigated. AD is a representative scatter plot of Lactobacillus plantarum cell membrane status detected by flow cytometry using SYTO 9 / PI double staining. A, B, C, and D represent the blank group, control group, 2 mM H2O2 treatment group, and 4 mM H2O2 treatment group, respectively. Quadrant Q1 represents PI-positive and SYTO 9-negative cells, quadrant Q2 represents SYTO 9 and PI double-positive cells, quadrant Q3 represents SYTO 9-positive and PI-negative cells, and quadrant Q4 represents SYTO 9 and PI double-negative cells. E represents the quantitative analysis result of SYTO 9 fluorescence intensity; F represents the quantitative analysis result of PI fluorescence intensity; G is a representative image from a SYTO 9 / PI double-stain fluorescence microscope, showing bright field, SYTO 9 green fluorescence, PI red fluorescence, and the combined image in sequence; H represents the fluorescence intensity ratio of PI / SYTO 9.

[0014] Figure 4 The effects of different concentrations of H2O2 treatment on the physicochemical properties and antioxidant activity of extracellular vesicles of *Lactobacillus plantarum* were investigated. A shows the particle size distribution of extracellular vesicles of Lactobacillus plantarum in different treatment groups and the results of transmission electron microscopy. B represents the extracellular vesicle zeta potential; C represents the ABTS free radical scavenging rate of extracellular vesicles; D represents the DPPH free radical scavenging rate of extracellular vesicles.

[0015] Figure 5 The results are from the metabolomics analysis of the supernatant of *Lactobacillus plantarum* culture after treatment with 4 mM H2O2. A represents the statistical analysis of the number of differentially expressed metabolites between the 4 mM H2O2 treatment group and the control group; B shows the Venn diagrams for the two groups of metabolite detections; C is the principal component analysis score graph of metabolites; D is the volcano diagram of the differential metabolites between the 4 mM H2O2 treatment group and the control group; E represents the expression profiles of representative differentially expressed metabolites and VIP values; F represents the heatmap of differential metabolite clustering; G represents the chemical classification and composition analysis of differentially metabolites; H represents the enrichment analysis of the differential metabolite KEGG pathway.

[0016] Figure 6 The results are the proteomic analysis of *Lactobacillus plantarum* after treatment with 4 mM H2O2. A represents the statistical analysis of the number of differentially expressed proteins between the 4 mM H2O2 treatment group and the control group; B is the Venn diagram of the two groups of detected proteins; C represents the principal component analysis results of the protein expression profile; D is a volcano plot of differentially expressed proteins between the 4 mM H2O2 treatment group and the control group; E represents the functional enrichment analysis of the differentially expressed protein GO; F represents the KEGG pathway enrichment analysis of differentially expressed proteins; G represents the upregulated protein and its fold change in expression; H represents a protein-protein interaction network constructed based on differentially expressed proteins.

[0017] Figure 7 The results are a combined analysis of the proteome and metabolome of *Lactobacillus plantarum* after treatment with 4 mM H2O2. A represents the results of DIABLO analysis of the proteome and metabolome; B represents a protein-metabolite association network constructed based on important proteins and metabolites screened using DIABLO; C represents the protein-metabolite-pathway network constructed from proteins, metabolites, and related pathways; D represents a comparative analysis of KEGG pathway enrichment between the proteome and metabolome; E represents the KEGG pathway enrichment analysis using both proteomics and metabolomics. F is a heatmap showing the correlation between representative differentially metabolites and differentially expressed proteins; G is a heatmap of KEGG secondary functional classification clusters for proteomics and metabolomics. Detailed Implementation

[0018] 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.

[0019] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0020] 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.

[0021] 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 obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0022] 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.

[0023] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0025] Lactobacillus plantarum Lactiplantibacillus plantarumATCC8014 comes from the Shanghai Center for Preservation of Biotechnology (SHBCC).

[0026] MRS agar medium consisted of: 5.0 g peptone, 5.0 g beef extract, 10.0 g tryptone, 5.0 g yeast extract, 10.0 g glucose, 1.0 g Tween 80, 2.0 g K₂HPO₄, 5.0 g sodium acetate, 2.0 g diammonium citrate, 0.25 g ZnSO₄·7H₂O, 0.1 g MgSO₄·7H₂O, 20.0 g calcium carbonate, 15 g agar powder, and distilled water to a final volume of 1 L. The pH was 6.2–6.4. The culture temperature was 37℃, the shaking speed was 180 rpm, the inoculum size was 1%, and the culture was conducted under anaerobic conditions. SYTO 9 live cell imaging nucleic acid staining agent, propidium iodide (PI) staining agent, and ROS assay kit were provided by Jiangsu Kaiji Biotechnology Co., Ltd. and Yongtai Biotechnology Research Institute, respectively. The mitochondrial membrane potential assay kit (JC-1) was provided by Beijing Solarbio Technology Co., Ltd. All other chemicals were of analytical grade (e.g., H2O2 used in this experiment). Example 1 I. Cell Characterization (a) Bacterial activation culture and counting Take 300 μL of *Lactobacillus plantarum* preserved in a glycerol tube in a refrigerator (containing 150 μL of a 1:1 mixture of glycerol and water and 150 μL of bacterial culture) and aseptically inoculate it into 30 mL of MRS liquid medium at an inoculation ratio of 1%. Incubate anaerobically at 37°C on a shaker for 12 h. Prepare 75 mL of sterilized MRS medium with H2O2 concentrations of 0 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM using 30% H2O2. Inoculate the first generation of bacteria, which have been cultured for 12 h, into MRS liquid medium containing different concentrations of H2O2. After anaerobic incubation at 37°C on a shaker for 12 h, take the corresponding bacterial culture, serially dilute it to the appropriate multiple, spread it on solid plates, and incubate anaerobically at 37°C until single bacteria grow on the plates before counting. Lactobacillus plantarum is a Gram-positive facultative anaerobic bacterium that thrives at 30-35°C and a pH of 6.5. Its colonies are round with a smooth surface and are white, light yellow, or dark yellow in color. It produces metabolites such as organic acids, bacteriocins, and H2O2.

[0027] (II) Determination of growth curve After activating the bacterial strain, 40 mL of MRS medium was used to prepare media with final H2O2 concentrations of 0, 2, 4, 6, 8, and 10 mM, respectively, by adding 0 μL, 8.17 μL, 16.34 μL, 24.51 μL, 32.68 μL, and 40.68 μL of 30% H2O2. After the bacterial culture reached the logarithmic growth phase, it was inoculated into media containing different concentrations of H2O2. 200 μL of the corresponding medium was added to each well, and four blank wells were prepared for zeroing (two plates were prepared: one for the growth curve analyzer and one for the microplate reader). Take 1 mL centrifuge tubes and add 900 μL of the corresponding culture medium to each tube to prepare gradient dilutions (1%, 0.1%, 0.01%), for a total of 5 dilution gradients. Select bacterial cultures at three dilutions (1%, 0.1%, 0.01%) and measure the growth curve using a microplate reader, taking readings every 4 hours for 24 hours. Subtract the readings from the blank culture medium readings and plot the results to obtain the growth curves. Take care to prevent contamination during the operation and incubate at 37℃.

[0028] (iii) pH measurement The initial pH values ​​of the basal culture medium and the H2O2-added culture medium under sterile and sterile conditions were determined. At the same time, the pH values ​​of the culture medium after Lactobacillus plantarum was cultured for 12 h under 0, 2, and 4 mM H2O2 conditions, and after 24 h under 0 mM H2O2 conditions were determined.

[0029] (iv) Bacterial cell SDS-PAGE electrophoresis All frozen samples were removed and transferred to MP lysis tubes; an appropriate amount of protein lysis buffer (8 M urea + 1% SDS, containing protease inhibitors) was added; the samples were homogenized three times using a high-throughput tissue homogenizer, 180 s each time; non-contact cryogenic sonication was performed for 30 min; and the samples were centrifuged at 8℃ and 14000 g for 15 min, and the supernatant was collected. Protein concentration was then determined using the BCA method, strictly following the BCA kit instructions. After quantification, 10 μg of each sample was loaded for SDS-PAGE electrophoresis.

[0030] (v) Colony count After 12 h of culture of activated first-generation bacteria, 40 mL of MRS medium was taken to prepare medium with final H2O2 concentrations of 0, 2, 4 and 6 mM, respectively. After inoculation, the medium was plated after 12 h of culture and cultured for 24 h. After the colony morphology stabilized, the colonies were counted.

[0031] (vi) Scanning electron microscopy Cultures with an OD value of 0.5-0.8 were centrifuged, the culture medium was discarded, and the bacterial precipitate (at least the size of a mung bean) was collected. The precipitate was washed 1-2 times with PBS, and the supernatant was discarded. Pre-cooled 4% glutaraldehyde fixative solution (4℃) was slowly added along the tube wall, and the cells were fixed overnight at 4℃. After fixation, the cells were centrifuged at 8000×g for 5 min at 4℃ and washed 3 times with PBS. The cells were then subjected to a gradient dehydration process with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol (10 min per stage, repeated twice with 100% ethanol), followed by two tert-butanol replacements, and then freeze-dried. The dried cells were adhered to a conductive gel sample stage, sputter-coated with gold, and observed under a scanning electron microscope (5 kV accelerating voltage). Magnifications from 5000× to 50000× were used to observe and photograph the surface morphology and integrity of the cells in different H2O2 treatment groups for comparison.

[0032] (vii) Transmission electron microscopy Transfer bacterial culture with an OD of 0.5-0.8 to a 1.5 mL centrifuge tube, centrifuge at 7000 rpm for 5 min, and collect the bacterial precipitate at the bottom of the tube. The precipitate should be about the size of a mung bean (if the precipitate volume is insufficient for the 4 mM group, multiple tubes can be combined). Discard the supernatant, add 4% glutaraldehyde fixative at room temperature until the tube is full, resuspend the precipitate by pipetting, incubate at room temperature for 30 min, then transfer to a 4°C refrigerator for overnight fixation. Seal the tube with sealing film before shipping. Gram-positive bacteria are fixed with 4% glutaraldehyde (control bacteria are in the logarithmic growth phase), followed by embedding, ultrathin sectioning, staining, and transmission electron microscopy. Take approximately 1 mm... 3 Bacterial samples were immediately placed in 1.5 mL centrifuge tubes containing 2.5% glutaraldehyde and fixed at 4°C for 12–24 h. The fixative was discarded, and the samples were washed three times with PBS buffer for 15 min each time. The samples were then fixed with 1% osmium tetroxide for 1–2 h; the osmium tetroxide waste was discarded, and the samples were washed three times with 0.1M PBS (pH 7.4) for 15 min each time. The samples were then subjected to a gradient dehydration process with 30%, 50%, 70%, and 90% ethanol for 15 min each time; followed by two 20-min dehydration processes with 100% ethanol; and then two 20-min dehydration processes with 100% acetone. Acetone and embedding medium were mixed at a 1:1 ratio and infiltrated at 37°C for 3 h; pure embedding medium was infiltrated overnight at 37°C. The pure embedding medium was injected into embedding plates, and after inserting the samples, the plates were polymerized in an oven at 70°C for 12–48 h. The resin blocks were then removed for later use. The resin block was cut into ultrathin sections of 70-90 nm using an ultramicrotome, and the sections were retrieved using a copper mesh. The sections were stained with uranyl acetate for 8-15 min and lead citrate for 5-10 min, then dried for examination. The bacterial morphology was observed under a transmission electron microscope, and images were acquired for analysis.

[0033] (viii) Bacterial cell characterization Zeta potential: 1 mL of bacterial cells was prepared, and the zeta potential was measured using a Zetasizer 3000HSA (Malvern). In-instruments Ltd, Malvern, United Kingdom, the cell potentials obtained for each sample were measured.

[0034] Particle size: After preparing the bacterial cells, 1 mL of the sample was added and the particle size of the vesicles obtained in each sample was measured (Malvern In-instruments Ltd, Malvern, United Kingdom).

[0035] The above results are as follows Figure 1 As shown, where, as Figure 1 As shown in A, the 0 mM group entered the rapid growth phase after 8 h of culture, OD 600 The levels then continued to rise, reaching approximately 1.45 at 24 hours. The 2 mM group entered the logarithmic growth phase later than the 0 mM group, but its OD... 600 The growth continued in the later stages of culture and approached that of the control group at 24 h. The 4 mM group showed only slight growth in the first 24 h, with OD... 600 Overall, the levels remained low. After extended incubation, the 4 mM group culture medium gradually became significantly turbid and entered a rapid growth phase. In contrast, the 6, 8, and 10 mM groups showed no significant growth within 24 hours, and no visible proliferation was observed in the culture medium after 7 consecutive days of observation. Based on these results, 4 mM H2O2 was determined to be the treatment concentration that could cause significant but recoverable growth inhibition, while ≥6 mM H2O2 led to persistent growth inhibition. SDSPAGE results of whole-cell proteins showed that the 0, 2, and 4 mM groups all exhibited multiple continuously distributed protein bands, with relatively similar overall band patterns. Figure 1 (B) No significant large-scale band loss or severe tailing was observed in any of the treatment groups, but there were some differences in band intensity in certain molecular weight regions. This indicates that under the tested conditions, 4 mM H2O2 treatment did not cause a complete breakdown of the whole bacterial protein composition. However, the differences in band intensity in certain molecular weight regions among the different treatment groups suggest that H2O2 treatment may have altered the relative abundance of some bacterial proteins.

[0036] Scanning electron microscopy revealed that H2O2 treatment caused significant concentration-dependent changes in the surface morphology of *Lactobacillus plantarum* (C in Figure 1). In the untreated group (0 mM), the cells were regular short rods with rounded ends, clear cell outlines, and a relatively smooth and continuous surface. The cells were also relatively uniform in size and shape. After treatment with 2 mM H2O2, most cells maintained their intact rod-like structure, but some cell surfaces began to show slight roughness, with localized shallow wrinkles and slight depressions. The cells were more tightly packed than in the control group. After treatment with 4 mM H2O2, the morphological heterogeneity of the cells increased significantly. Many cells gradually transformed from plump, rounded-end rods into irregularly shaped short rods or even spherical shapes. Some cells shortened in length, and more pronounced wrinkles, bends, depressions, and localized collapses appeared on the surface. Cell surface continuity decreased, and a few cells showed localized damage and fragmented structures. Simultaneously, some cells aggregated, with multiple cells tightly adhering to form larger aggregates, whose surface roughness was significantly higher than that of the 0 mM and 2 mM groups. Overall, with increasing H2O2 concentration, the bacterial cell surface gradually changed from smooth to rough, accompanied by a gradual increase in shrinkage and local structural damage. Transmission electron microscopy further revealed the H2O2-induced ultrastructural changes. Figure 1(D) In ​​the 0 mM group, the bacterial cell walls were intact and clearly defined, the cytoplasmic membrane was tightly attached to the cell wall, the cytoplasmic electron density was uniform, and the bacteria were generally plump, with only a narrow membrane-wall gap visible. After treatment with 2 mM H2O2, some cells began to show slight ultrastructural changes. The distribution of cytoplasmic electron density was slightly uneven compared to the control group, and slight separation between the cytoplasmic membrane and cell wall was observed in some cells. The membrane-wall gap was wider than that in the control group, but the overall cell structure remained intact. After treatment with 4 mM H2O2, the ultrastructural changes were more obvious. The cytoplasm of most bacteria showed varying degrees of shrinkage, electron-dense material accumulated towards the center of the cell, and a wider low-electron-density region was formed between the cytoplasmic membrane and cell wall, significantly widening the membrane-wall gap. At the same time, the cell wall outline of some bacteria became irregular, the cytoplasmic membrane showed local wavy undulations or even slight collapse, and obvious separation of the membrane structure from the cell wall was observed in some areas. Compared to the control group, the bacterial cells gradually changed from their originally plump, nearly circular cross-sections to irregular shapes, and the integrity of the envelope decreased. However, most cells still maintained continuous cell boundaries, and no large-scale cell lysis or complete cytoplasmic leakage was observed. Combined SEM and TEM observations showed that with increasing H2O2 concentration, the morphological changes related to the Lactobacillus plantarum cell envelope gradually worsened, mainly manifested as cell surface roughening, wrinkling, and local collapse, as well as ultrastructural changes such as widening of the membrane-wall gap and cytoplasmic contraction. This is consistent with the characteristics of cell envelope stress and structural remodeling in Gram-positive bacteria under environmental stress. A few cells showed severe deformation or surface damage, but not all cells underwent complete disintegration, indicating that 4 mM treatment mainly induced significant envelope damage and morphological remodeling, rather than causing uniform and complete lysis of the entire bacterial community. The zeta potential results after reconstitution of the bacterial cells in PBS showed that the 0 and 2 mM groups were approximately [value missing]. 14 and At 13 mV, there was no significant difference between the two groups; at 4 mM, the value decreased to approximately [value missing]. 21 mV, significantly lower than the 0 and 2 mM groups ( Figure 1 The change suggests alterations in the composition, exposure, or spatial arrangement of charged groups on the cell wall or cell membrane surface, consistent with microscopic observations of cell membrane remodeling. Culture medium pH results showed that the initial pH of the uninoculated medium was approximately 5.6 (…). Figure 1(F in the text). After 12 h of culture, the pH of the 0 and 2 mM groups decreased to approximately 4.1 and 4.3, respectively, while the 4 mM group remained at approximately 5.1. After 24 h of culture, the pH of the 0 mM group further decreased to approximately 3.8. This indicates that the cells continuously produce acidic metabolites with prolonged culture time. Since there were significant differences in biomass among the groups at the same time point, pH changes mainly reflected the overall growth and fermentation metabolism of the cells. The average particle size of the cells after reconstitution in PBS was approximately 1.3-1.4 μm, with no significant differences among the 0, 2, and 4 mM groups, and the 0 mM group cultured for 24 h. Figure 1 G in the figure. After normalization by endpoint colony forming units (CFU), the extracellular vesicle protein production in the 4 mM H2O2 treatment group was significantly higher than that in the control group and the 2 mM group, while there was no significant difference between the control group and the 2 mM group. Figure 1 The yield of extracellular vesicle particles increased with increasing H2O2 treatment concentration, with the 2 mM group significantly higher than the control group, and the 4 mM group significantly higher than both the 2 mM group and the control group. Figure 1 (I) This result indicates that although 4 mM H2O2 caused significant shrinkage, membrane-wall separation, and morphological heterogeneity in some cells, the hydrodynamic mean particle size at the population level did not change significantly. The lack of significant difference in mean particle size does not negate the local morphological damage observed under the microscope, because population particle size measurement is difficult to reflect changes in a small number of severely deformed cells and fine cell surface structures. Gram staining results showed that after 12 h of culture, cells in the 0 and 2 mM groups were mainly purple; most cells in the 4 mM group were still purple, with only a few cells being red; cells in the 6 mM group were mainly red. After 24 h of culture, cells in the 0 mM group were also mainly red. In summary, H2O2 has a concentration-dependent effect on the growth and cell membrane morphology of *Lactobacillus plantarum*. 2 mM treatment mainly caused growth delay and mild morphological changes, while 4 mM treatment led to significant but recoverable growth inhibition, accompanied by cell surface roughness, shrinkage, widening of membrane-wall gaps, and a decrease in zeta potential, while the mean particle size did not change significantly.

[0037] II. Characterization of bacterial cell membranes SYTO / PI double staining was used to assess membrane integrity, Nile red was used to characterize membrane lipid / hydrophobic structure signals, JC-1 was characterized by red / green fluorescence ratio to represent membrane potential status, and ROS probes were used to characterize intracellular reactive oxygen species levels after oxidative stress. Particle numbers were counted by nanoflow cytometry and normalized to CFU.

[0038] (a) Determination of the effect of cell membrane The experiment was designed based on methods for characterizing bacterial cell surface properties. Logarithmic-phase bacterial cells were diluted to 10⁻⁶ PBS. 8CFU / mL, the bacterial suspension was washed with PBS and resuspended. After H2O2 incubation, PBS was washed again to remove residual treatment solution. After PBS resuspending, 1 mL of bacterial suspension was added to 8 μL SYTO9 and 40 μLPI, and incubated at 37℃ in the dark for 30 min. The fluorescence intensity at Ex490 nm / Em525 nm (SYTO9, live bacteria) and Ex540 nm / Em620 nm (PI, dead bacteria) was measured using a fluorescence spectrophotometer. To eliminate interference from the autofluorescence of *Lactobacillus plantarum*, an unstained blank group was set up to subtract background, and the PBS-treated group served as the experimental control.

[0039] (II) Determination of the influence of bacterial film potential The JC-1 fluorescent probe was used to assess the membrane potential-related changes in *Lactobacillus plantarum* after H2O2 treatment, based on the principle of ratiometric fluorescence detection and flow cytometry analysis of bacterial membrane potential. Bacterial cells were incubated, washed, and resuspended. 1 mL of bacterial suspension was added to 500 μL of JC-1 staining working solution, vortexed, and incubated at 37 °C for 20 min in the dark. After staining, the cells were centrifuged at 6000 g, 4 °C for 5 min, washed twice with JC-1 buffer, and resuspended. Bacterial suspension without H2O2 was used as a negative control, and bacterial suspension treated with 10 mmol / L CCCP for 20 min was used as a positive control. Flow cytometry was used for detection. JC-1 monomers (green) detected the FL1 channel (Ex 488 nm / Em 525 nm), and JC-1 aggregates (red) detected the FL2 channel (Ex 488 nm / Em 590 nm). The red / green fluorescence intensity ratio represented the degree of membrane potential depolarization.

[0040] (III) Biofilm lipid structure Nile Red staining was used to characterize changes in the membrane-associated hydrophobic microenvironment and lipid arrangement of *Lactobacillus plantarum* after H2O2 treatment. 5 μL of Nile Red (final concentration 5 μg / mL) was added to 1 mL of bacterial suspension, and the mixture was reacted in the dark for 30 min. After centrifugation at 6000 g, 4°C, for 5 min, the suspension was washed twice with PBS and resuspended. Flow cytometry was used to detect Nile Red fluorescence, which was collected through the FL2 channel (Ex488 nm / Em620 nm). Changes in fluorescence intensity reflected the degree of damage to membrane lipid polarity and structural integrity.

[0041] (iv) Determination of the effect of bacterial ROS content The DCFH-DA probe was used to assess H2O2-induced intracellular reactive oxygen species (ROS) generation according to literature methods. 1 mL of bacterial suspension was added to 10 μmol / L DCFH-DA (final concentration) and incubated at 37 °C in the dark for 30 min. A positive control group was included (containing the ROS inducer provided in the kit and incubated under the same conditions). After staining, the cells were centrifuged at 6000 g at 4 °C for 5 min, washed twice with PBS, and resuspended. Flow cytometry was used to detect the fluorescence intensity of the FL1 channel (Ex 488 nm / Em 525 nm). SYTO 9 / PI was counterstained to distinguish live and dead bacteria from the background. An unstained blank group was included for all samples to subtract bacterial autofluorescence. Fluorescence values ​​were expressed as relative fluorescence intensity (treatment group / control group) or normalized to the total bacterial count. The ability of different concentrations of H2O2 to induce intracellular ROS levels was systematically evaluated.

[0042] The following figure shows the H2O2-induced oxidative stress and membrane physiological changes in *Lactobacillus plantarum*. Figure 2 As shown in the representative flow cytometry fluorescence peak diagram, with the increase of H2O2 concentration, the fluorescence peaks gradually shift towards the strong fluorescence region, with the shift being most significant in the 4 mM group. Figure 2 (A) Quantitative results showed that, compared with the control group, there were no significant changes in ROS fluorescence intensity in the 0 mM and 2 mM groups, while the intracellular ROS level was significantly increased after 4 mM treatment (P<0.05). Figure 2 The presence of B in the figure indicates that 4 mM H2O2 induced significant oxidative stress. Further evaluation of cell membrane potential changes was conducted using the JC-1 probe. The overall positions of the green fluorescence peaks in each treatment group were similar (…). Figure 2 C in the figure). Quantitative results of PE / FITC fluorescence ratio showed that the 4 mM group showed a decreasing trend compared with the control group, but the differences between the groups did not reach the statistical significance level. Figure 2 (D in the text). Subsequently, changes in cell membrane lipid fluorescence were detected using the Nile Red probe. Representative flow cytometry peaks showed that the fluorescence peak in the 4 mM group shifted significantly to the right, while the changes in the 0 mM and 2 mM groups were relatively small compared to the control group. Figure 2 The quantitative analysis further showed that the Nile Red fluorescence intensity after 4 mM treatment was significantly higher than that of other groups (P<0.05), while there was no significant difference between the 0 mM and 2 mM groups (E). Figure 2 (F in the middle).

[0043] SYTO 9 / PI double staining was used to further evaluate cell membrane integrity (results are shown below). Figure 3 (As shown in AD). With increasing H2O2 concentration, the proportion of PI-positive cells gradually increased, and the number of double-positive cells also increased. The fluorescence intensity of SYTO 9 in the 0, 2, and 4 mM groups was significantly higher than that in the untreated control, with the 4 mM group reaching the highest level (as shown in AD). Figure 3The fluorescence intensity of PI also gradually increased with increasing H2O2 concentration, with the 4 mM group being significantly higher than the 0 mM and 2 mM groups (E). Figure 3 The results observed by fluorescence microscopy were consistent with those by flow cytometry. With increasing H2O2 concentration, the red PI positive signal gradually increased, while the green SYTO 9 signal remained widely present. Figure 3 The PI / SYTO fluorescence intensity ratio calculated using ImageJ further confirmed that cell membrane permeability gradually increased with increasing H2O2 treatment concentration, with the 4 mM group showing significantly higher permeability than other treatment groups. Figure 3 (H in the text).

[0044] III. Extraction and Characterization of Vesicles (a) Extraction and purification of vesicles Take 35 mL of bacterial culture from each group treated with different concentrations of H2O2 for 12 h, centrifuge at 10000×g for 15 min, and collect the supernatant. After filtration through a 0.22 μm microporous membrane, take 12 mL of the filtrate and concentrate it approximately 10 times using a 100 kD ultrafiltration tube. Extract vesicles using the Enzekangtai Cat#ESSO500 exosome purification kit. Mix the concentrated supernatant with solutions A and B in the specified proportions and incubate overnight at 4℃ (at least 12 h). Centrifuge the mixture at 10000×g at 4℃ for 60 min. Resuspend the vesicle precipitate corresponding to every 25 mL of initial supernatant in approximately 0.5 mL of PBS. Centrifuge the resuspended solution at 10000×g at 4℃ for 2 min, and collect the supernatant for purification by centrifugation using the kit's filter column. The purified vesicles are then stored at 4℃ for later use.

[0045] (ii) Concentration determination Nanoparticle concentration: When using a single-particle nanobiocharacterization system to detect the particle concentration of secreted vesicles of Lactobacillus plantarum, the vesicle solution is diluted to a suitable concentration (generally 1000 times) with ultrapure water or buffer. Then, the instrument is calibrated with standard nanoparticles of known size and concentration. After setting parameters such as laser wavelength and detection temperature, the diluted sample is injected into the detection cell. The instrument tracks the Brownian motion trajectory of the vesicles, and the software automatically calculates and outputs the particle concentration. During detection, attention should be paid to sample purification, avoiding air bubbles, and repeating experiments to ensure the accuracy of the results.

[0046] Protein concentration: Protein concentration was determined using a BCA kit, and the vesicle protein yield per CFU was calculated based on colony count. The OD of each well was measured using a microplate reader. 562 Value, based on the concentration of the standard and OD 562 Plot a standard curve and calculate the protein concentration in the sample.

[0047] (III) Vesicle Characterization Potential: Take 1 mL of the prepared vesicle sample and measure the vesicle zeta potential of each sample using a Zetasizer 3000HSA (Malvern Instruments Ltd, Malvern, United Kingdom).

[0048] Particle size: Take 1 mL of the prepared vesicle sample and measure the vesicle particle size of each sample using the same instrument.

[0049] (iv) Vesicle TEM Take 50-100 μL of the prepared vesicle sample (protein concentration ≥0.5 mg / mL) and perform the instrument detection procedure.

[0050] (v) Vesicle antioxidant capacity assay After vesicle extraction, the protein concentration was determined using a BCA kit and adjusted to 0.2 mg / mL before subsequent experiments.

[0051] ABTS: Take an appropriate amount of vesicle sample resuspended in PBS and measure it using the ABTS kit. Prepare the ABTS working solution and the working solutions of each reagent. Add the sample, distilled water, reagent IV working solution, ABTS working solution, and reagent I to a 96-well plate as required. Set up blank tubes, test tubes, and control tubes, with 3 replicates for each. Incubate in the dark for 6 min, using PBS as a blank control, and measure the absorbance at 734 nm. Calculate the scavenging rate of the sample for ABTS free radicals using the formula: ABTS clearance rate (%) = [1 - (A sample - A sample blank) / A control] × 100%, where A control is the absorbance of ABTS working solution mixed with PBS, A sample is the absorbance of sample mixed with ABTS working solution, and A sample blank is the absorbance of sample mixed with PBS.

[0052] DPPH: The vesicle samples resuspended in PBS were diluted to different concentrations with PBS; a 0.1 mM DPPH ethanol solution was prepared (stored protected from light); 100 μL of the sample solution and 100 μL of the DPPH solution were mixed in a 96-well plate, thoroughly mixed by pipetting, and reacted in the dark for 30 min; using ethanol as a blank control, the absorbance was measured at 517 nm. The DPPH radical scavenging rate of the sample was calculated using the following formula: DPPH clearance rate (%) = [1 - (A sample - A sample blank) / A control] × 100%, where A control is the absorbance of the DPPH solution mixed with ethanol, A sample is the absorbance of the sample mixed with DPPH solution, and A sample blank is the absorbance of the sample mixed with ethanol.

[0053] (vi) Vesicle purity The concentration (C1) of the extracted vesicles was determined using NTA. The vesicles were then heated at 37°C until the final concentration was 1%. The Triton-X100 reaction was degraded for 2 hours, and its concentration C2 was obtained using NTA. Purity (%) = (1 - C2 / C1) × 100%.

[0054] (vii) Vesicle SDS-PAGE electrophoresis The frozen samples were removed and transferred to MP lysis tubes; an appropriate amount of protein lysis buffer (8M urea + 1% SDS, containing protease inhibitors) was added; the samples were homogenized three times using a high-throughput tissue homogenizer, 180 s each time; non-contact cryogenic sonication was performed for 30 min; and the samples were centrifuged at 8°C and 14000×g for 15 min, and the supernatant was collected. Protein concentration was then determined using the BCA method, strictly following the BCA kit instructions. After quantification, 10 μg of each sample was loaded for SDS-PAGE electrophoresis.

[0055] The effect of H2O2 on the release of extracellular vesicles (EVs) from Lactobacillus plantarum, such as Figure 4 As shown. Nearly spherical extracellular vesicles with clear membrane boundaries were obtained in all treatment groups. Particle size distribution results showed that the representative particle sizes of the extracellular vesicles in the control group, 2 mM group, and 4 mM group were approximately 69.2, 68.2, and 68.8 nm, respectively, with similar overall particle size distributions across all treatment groups. Figure 4 (A) The extracellular vesicle zeta potentials in each group were approximately [value missing]. 8 mV, no significant difference between groups ( Figure 4 (B in the text). At the same protein concentration, there was no significant difference in the ABTS free radical scavenging rate of extracellular vesicles among different treatment groups ( Figure 4 The DPPH free radical scavenging rate of extracellular vesicles in the 4 mM H2O2 treatment group was significantly lower than that in the control group and the 2 mM group, while there was no significant difference between the control group and the 2 mM group. Figure 4 (D in the middle).

[0056] IV. Omics Analysis (I) Metabolomics Pipettes 100 μL of liquid sample into a 1.5 mL centrifuge tube, adds 400 μL of extraction buffer (acetonitrile:methanol = 1:1) containing four internal standards (L-2-chlorophenylalanine (0.02 mg / mL), etc.), vortexes for 30 s, and then performs low-temperature ultrasonic extraction for 30 min (5℃, 40 kHz). The sample is then allowed to stand at -20℃ for 30 min. After centrifugation at 4℃, the sample is centrifuged at 13000 g for 15 min. The supernatant is collected, dried under nitrogen, and reconstituted with 100 µL of reconstitution solution (acetonitrile:water = 1:1). The sample is then extracted at low-temperature ultrasonic extraction for 5 min (5℃, 40 kHz), and centrifuged at 4℃, 13000 g for 10 min. The supernatant is transferred to a vial with an inner tube for analysis. Equal volumes of metabolites from all samples are mixed to prepare a quality control (QC) sample. During instrumental analysis, one QC sample is inserted every 5–10 samples to assess the repeatability of the entire analytical process.

[0057] Chromatographic conditions: 3 μL of sample was separated using an HSS T3 column (100 mm × 2.1 mm id, 1.8 µm) and then detected by mass spectrometry. Mobile phase A was a 95 / 5, v / v solution of water / acetonitrile containing 0.1% formic acid, and mobile phase B was a 47.5 / 47.5 / 5, v / v / v solution of acetonitrile / isopropanol / water containing 0.1% formic acid. The flow rate was 0.40 mL / min, and the column temperature was 40 °C.

[0058] Mass spectrometry conditions: Sample mass spectrometry signal acquisition adopted positive and negative ion scanning mode, with a mass scanning range of m / z: 70-1050. Ion spray voltage: positive ion voltage 3500V, negative ion voltage -3000V, sheath gas 50 arb, auxiliary heating gas 13 arb, ion source heating temperature 450℃, cyclic collision energy 20-40-60 V.

[0059] (II) Proteomics Enzymatic digestion: Take 100 μg of protein sample, add lysis buffer, and add 100 mM triethylammonium bicarbonate buffer (TEAB); add 10 mM tris(2-carboxyethyl)phosphine (TCEP) and react at 37℃ for 60 min; add 40 mM iodoacetamide and react at room temperature in the dark for 40 min; add pre-cooled acetone (acetone:sample v:v=6:1) to each tube and precipitate at -20℃ for 4 h; centrifuge at 10,000 g for 20 min and collect the precipitate; dissolve the sample thoroughly with 100 µL of 100 mM TEAB; add Trypsin at a mass ratio of 1:50 (enzyme:protein) and digest overnight at 37℃; peptide desalting and quantification: After trypsin digestion, vacuum pump to dry the peptides; reconstitute the digested peptides with 0.1% trifluoroacetic acid (TFA); desalt the peptides using HLB and vacuum concentrate; use NANO... DROP ONE (Thermo Scientific) was used for ultraviolet spectrophotometric peptide quantification. Mass spectrometry detection: Equal volumes of peptides were dissolved in mass spectrometry loading buffer for DIA detection and analysis; peptide separation was performed using a Vanquish Neo (Thermo) chromatograph with a uPAC High Throughput column. The column (75 μm × 5.5 cm, Thermo, USA) was used. Mobile phase A was water (2% acetonitrile + 0.1% formic acid), and mobile phase B was water (80% acetonitrile + 0.1% formic acid). The chromatographic duration was 8 min. Data acquisition software was Thermo Xcalibur 4.7 (Thermo, USA). The samples separated by nano-level high-performance liquid chromatography (HPLC) were analyzed by mass spectrometry using an Orbitrap Astral mass spectrometer (Thermo). The Orbitrap Astral mass spectrometer (Thermo) was operated in DIA mode, with positive ion detection mode and an ion source voltage set to 1.5 kV. The mass spectrometry scan range was 100–1700 m / z.

[0060] Statistical results of differential metabolites showed that ( Figure 5 The number of differentially expressed metabolites was significantly increased in the 4 mM group, with a total of 962 differentially expressed metabolites detected, of which 669 were upregulated and 293 were downregulated. Figure 5 The result (A) suggests that *Lactobacillus plantarum* underwent more extensive metabolic reprogramming with increasing H2O2 concentration. Venn analysis showed a total of 3414 metabolites across different treatment groups, while the 4 mM group had 59 specific metabolites, higher than the 20 in the 2 mM group. Figure 5(B in the original text). PCA analysis further showed that the 4 mM group and the 0 mM group samples were clearly separated along the PC1 direction, while the intra-group repetitive aggregation was good, indicating that oxidative stress significantly altered the overall metabolic profile. Figure 5 (C in the figure). The volcano plot shows that 4 mM treatment induced significant changes in a large number of metabolites, with the number of upregulated metabolites significantly exceeding the number of downregulated metabolites. Figure 5 (D in the text). VIP sequencing combined with expression heatmaps further revealed significant changes in various amino acids, organic acids, lipids, and nucleotide-related metabolites, with different treatment groups exhibiting stable and consistent expression patterns. Figure 5 E in the diagram). Hierarchical cluster analysis further verified that within-group repetitions showed high consistency, while different treatment groups showed significant separation (E). Figure 5 F in the text). Chemical classification analysis showed that the differential metabolites were mainly concentrated in lipids and lipid-like molecules (29.21%), organic acids and their derivatives (28.22%), and organic oxygen-containing compounds (10.40%). Figure 5 KEGG enrichment analysis showed that differentially metabolites were mainly enriched in pathways such as starch and sucrose metabolism, glycolysis / gluconeogenesis, galactose metabolism, cysteine ​​and methionine metabolism, alanine / aspartate / glutamate metabolism, and the ABC transport system. Figure 5 (H in the text).

[0061] To further elucidate the molecular response induced by H2O2 treatment, label-free proteomics analysis was performed on the bacterial cells. The results of differential protein analysis showed a significant increase in the number of differentially expressed proteins after 4 mM treatment, with a total of 771 differentially expressed proteins detected, of which 137 proteins were upregulated and 634 proteins were downregulated. Figure 6 A). Venn analysis showed that a total of 2380 proteins were commonly identified across different treatment groups, with the 4 mM group exhibiting 8 specifically expressed proteins (A). Figure 6 (B in the text). PCA results showed that the two groups of samples were completely separated along the PC1 direction, and the intragroups showed good reproducibility and aggregation, indicating that 4 mM H2O2 significantly altered the overall protein expression profile of the bacteria. Figure 6 (C in the diagram). The volcano plot further shows that differentially regulated proteins are widely distributed in both up- and down-regulated forms, with down-regulated proteins being significantly more numerous than up-regulated proteins. Figure 6 D in the text). GO enrichment analysis showed that the differentially expressed proteins were mainly involved in functions such as redox-related enzyme activity, membrane lipid metabolism, glycolipid biosynthesis, and the organization of membrane and cellular components. Figure 6 E in the text). KEGG analysis further showed that the differentially expressed proteins were mainly enriched in pathways such as glycine / serine / threonine metabolism, lysine biosynthesis, glutathione metabolism, pyruvate metabolism, amino sugar and nucleotide sugar metabolism, citric acid cycle, and cellular metabolism. Figure 6The F in the text. Further results from representative differentially expressed proteins indicate that significant changes occurred in various proteins involved in oxidative stress response, amino acid metabolism, glucose metabolism, and cell wall synthesis. Figure 6 The protein interaction network constructed by STRING showed that multiple differentially expressed proteins formed a highly interconnected network structure, among which MetS, LysS, PurA, ThrS, MurA, Pgi, and GroEL nodes had high connectivity, suggesting that these proteins may play an important role in oxidative stress adaptation. Figure 6 (H in the original text). To further elucidate the synergistic changes between the proteome and metabolome, a DIABLO integration analysis was performed on the two omics data. The DIABLO analysis results showed that, regardless of whether it was the proteome or metabolome, different treatment groups exhibited clear intergroup separation, and the two omics showed consistent trends (H in the original text). Figure 7 (A) The protein-metabolite Circos network further revealed significant correlations between numerous differentially expressed proteins and representative differentially expressed metabolites, forming a complex synergistic regulatory network (A). Figure 7 (B in the text). Protein-metabolism pathway interaction network shows that both omics and protein-metabolism jointly participate in multiple functional modules, including biosynthetic cofactors, ABC transport system, starch and sucrose metabolism, purine metabolism, and pyruvate metabolism. Figure 7 The KEGG comparative analysis further revealed that both the proteome and metabolome were enriched in metabolic pathways, ABC transport systems, nucleotide metabolism, amino acid metabolism, and carbon metabolism, showing a high degree of consistency. Figure 7 (D in the text). Further screening of common enrichment pathways revealed that both omics studies involve important biological processes such as lysine biosynthesis, peptidoglycan biosynthesis, the PTS system, pyruvate metabolism, nicotinic acid and nicotinamide metabolism, glycine / serine / threonine metabolism, and the ABC transport system. Figure 7 E in the figure). The differential metabolite correlation heatmap shows that there are obvious co-expression patterns among different categories of metabolites (E). Figure 7 The joint heatmap constructed based on KEGG secondary classification further shows that the proteome and metabolome exhibit consistent trends in multiple functional modules, including amino acid metabolism, glucose metabolism, cell wall synthesis, and energy metabolism. Figure 7 (G in the middle).

[0062] The combined metabolomics, proteomics and multi-omics analysis results show that 4 mM H2O2 treatment induced extensive metabolic reprogramming and protein expression reconstruction in Lactobacillus plantarum. Both omics studies point to synergistic changes in biological processes such as oxidative stress response, cell wall synthesis, membrane lipid metabolism, central carbon metabolism and ABC transport system.

[0063] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for promoting the secretion of extracellular vesicles of Lactobacillus plantarum, characterized in that, Includes the following steps: S1. During the cultivation process, H2O2 is added to the culture system, and Lactobacillus plantarum is inoculated to stimulate oxidation, so that Lactobacillus plantarum is in an adaptive oxidative stress state. The concentration of H2O2 is 2mM~4mM. S2. After culturing for 12 hours, the supernatant was collected and centrifuged, filtered, and enriched to obtain extracellular vesicles derived from Lactobacillus plantarum.

2. The method according to claim 1, characterized in that, The concentration of H2O2 is 4 mM.

3. The method according to claim 1, characterized in that, The inoculum amount of *Lactobacillus plantarum* is 1% (v / v) of the culture system volume.

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