Extracellular vesicles of irpex lacteus, preparation method and application thereof

Abstract

This invention relates to the fields of biotechnology and pharmaceutical technology, and discloses a species of white burr fungus ( ). Irpex lacteus Extracellular vesicles, their preparation method, and applications. The preparation method includes: inoculating *Alternaria alternata* strain onto a solid culture medium lined with cellophane and culturing, collecting the mycelium on the cellophane surface; immersing the mycelium in an enzymatic hydrolysate containing an osmotic stabilizer and fungal cell wall hydrolytic enzymes for incubation, and collecting the supernatant after filtration; subjecting the supernatant to multiple differential centrifugations to remove the precipitate, obtaining the initial separation solution of extracellular vesicles; subjecting the initial separation solution of extracellular vesicles to multiple ultracentrifugations, collecting the precipitate, and resuspending the precipitate in a buffer solution to obtain *Alternaria alternata* extracellular vesicles. This invention significantly increases vesicle yield by inducing secretion through protoplastization, resulting in vesicles with high purity and strong activity.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a type of *Bacillus thuringiensis* (…). Irpex lacteus Extracellular vesicles, their preparation methods and applications. Background Technology

[0002] Extracellular vesicles are nanoscale particles secreted by almost all life forms and enclosed in a lipid bilayer, playing a crucial role in life processes such as intercellular communication, substance transport, and immune regulation. In fungi, extracellular vesicles are considered important carriers of bioactive molecules (such as proteins, RNA, lipids, and metabolites), participating in processes such as interspecies competition, host interaction, and environmental adaptation. *Alternaria alternata*, a filamentous fungus with potential for biocontrol, has shown potential application value in antagonizing plant pathogens (such as *Fusarium oxysporum*) through its secreted extracellular vesicles, providing new ideas for developing novel, green fungal-derived biopesticides or plant immune inducers.

[0003] However, the efficient preparation and functional analysis of extracellular vesicles from *Alternaria alternata* and other filamentous fungi still face a series of technical bottlenecks. Firstly, in terms of preparation, traditional fungal vesicle extraction methods mainly rely on collecting the supernatant after submerged liquid fermentation. This method often results in low vesicle release efficiency and unstable yield because filamentous fungi easily entangle and form dense spheres in the liquid. Furthermore, the supernatant contains a large amount of soluble proteins, metabolites, and cell debris secreted by the fungi, making subsequent purification steps complex and difficult to obtain high-purity vesicle products. Secondly, in terms of mechanism research, existing technologies have limited understanding of the key functional molecules (such as specific proteins and microRNAs) carried by *Alternaria alternata* extracellular vesicles during their antagonistic activity against pathogenic bacteria. The lack of systematic omics analysis data severely restricts a deeper understanding of their mechanism of action and the development and application based on this understanding. Furthermore, in terms of extraction processes, conventional methods such as differential centrifugation or ultrafiltration are prone to causing vesicle loss, aggregation, or membrane structure damage. They also have low extraction efficiency for small RNA molecules (such as miRNA) within vesicles, making it difficult to obtain sufficient quantities of high-quality RNA from trace samples for subsequent high-throughput sequencing analysis.

[0004] Therefore, there is an urgent need in this field for a method to prepare extracellular vesicles of *Bacillus albus* with high efficiency and purity, and to establish a supporting omics analysis platform to analyze its molecular composition and function in biological antagonism, thereby laying a solid technical and theoretical foundation for its industrial application in the fields of biological control of agricultural diseases and drug delivery. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides extracellular vesicles of *Alternaria alternata*, their preparation method, and their applications.

[0006] In a first aspect, embodiments of the present invention provide a method for preparing extracellular vesicles of *Pterygodium japonicum*, comprising the following steps: S10: Inoculate the white spores of *Bacillus thunbergii* onto a solid culture medium lined with cellophane and culture them, then collect the mycelium on the cellophane surface. S20: The mycelium is immersed in an enzymatic hydrolysate containing an osmotic stabilizer and fungal cell wall hydrolytic enzymes for incubation, and the supernatant is collected after filtration; S30: Perform differential centrifugation on the supernatant multiple times to remove the precipitate and obtain the initial separation solution of extracellular vesicles; S40: Perform multiple ultracentrifugations on the initial separation solution of extracellular vesicles, collect the precipitate, and resuspend the precipitate in buffer solution to obtain extracellular vesicles of *Alternaria alternata*.

[0007] Furthermore, the solid culture medium in step S10 is potato dextrose agar medium; The cellophane is autoclaved and pre-soaked in deionized water before use. The cultivation conditions include: incubation at 25-27℃ in the dark for 72-96 hours.

[0008] Furthermore, the fungal cell wall hydrolytic enzyme in step S20 includes a complex enzyme system composed of chitinase and glucanase; Incubation conditions include: shaking incubation at 25-28℃ and 100-120 rpm for 3-5 hours.

[0009] Furthermore, the process of step S30 includes: The supernatant was centrifuged at low temperature at 3000-4000g and 7000-8000g respectively to remove the precipitate; The obtained supernatant was centrifuged at 10,000-12,000 g under low temperature conditions, and filtered to obtain the initial separation solution of extracellular vesicles.

[0010] Further, step S40 includes the following process: The initial separation solution of the extracellular vesicles was subjected to ultracentrifugation at 100,000-120,000 g under low temperature conditions, and the precipitate was collected. After resuspending the precipitate in physiological buffer, it was ultracentrifuged again at 100,000-120,000 g under low temperature conditions, and the precipitate was collected. The precipitate was resuspended in physiological buffer to obtain extracellular vesicles of *Alternaria alternata*.

[0011] Secondly, the present invention also provides extracellular vesicles of *Alternaria alternata*, prepared by the above method; the extracellular vesicles of *Alternaria alternata* have an average particle size of 120-220 nm, a Zeta potential of -15 to -25 mV, and a protein concentration of 0.8-1.5 mg / mL.

[0012] Thirdly, the present invention also provides the application of the extracellular vesicles of *Echinococcus solani* prepared by the above method in the preparation of antifungal agents, biopesticides or plant immune inducers, wherein the pathogenic bacterium is *Fusarium oxysporum*.

[0013] Fourthly, the present invention also provides a method for extracting miRNA from extracellular vesicles of *Pterygodium japonicum*, comprising the following steps: S101: Add RNase solution to the extracellular vesicle sample of *Bacillus thuringiensis* and incubate under ice bath conditions to remove free RNA outside the vesicles; S102: Add RNA lysis reagent to the incubated extracellular vesicle sample of *Bacillus thuringiensis*, and add small RNA precipitation agent and alcohol precipitation agent for separation and extraction at low temperature to obtain RNA; S103: Construct sequencing libraries based on extracted RNA, sequence cDNA fragments of 10-40 nt in length, and predict miRNA sequences using a fungal miRNA database.

[0014] Furthermore, in step S101, the concentration of the extracellular vesicle sample of *Bacillus thuringiensis* after adding RNase solution is 0.4-0.6 mg / mL; In step S101, the volume ratio of the incubated *Bacillus thuringiensis* extracellular vesicle sample to the RNA lysis reagent is 1:3.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes cellophane solid-state culture of mycelium, combined with protoplast enzymatic hydrolysis, to prepare protoplasts using brewing enzymes and induce vesicle release. The vesicles are then enriched and purified using a combination of differential centrifugation, ultracentrifugation, and molecular sieve chromatography. Furthermore, the proteins and miRNAs within the vesicles involved in the antagonistic process against pathogenic bacteria are analyzed using omics. This application significantly increases vesicle yield through protoplastization-induced secretion, resulting in high-purity, highly bioactive extracellular vesicles of *Amanita muscaria*. This application elucidates the molecular mechanism by which *Amanita muscaria* antagonizes pathogenic bacteria, providing an efficient preparation platform and theoretical support for the development of novel fungal-derived biopesticides, plant immune inducers, and drug delivery systems, and possesses significant industrialization value. Attached Figure Description

[0016] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention.

[0017] Figure 1 The results of NTA detection in the solid enzymatic digestion group of *E. leucocephala* EVs in this invention; Figure 1 In the image, A represents a transmission electron microscope (TEM) image; Figure 1 In this figure, B represents the particle size distribution curve; Figure 1 In this context, C represents the statistical data on particle size and concentration. Figure 2 The results of NTA detection in the liquid enzymatic digestion group of *E. leucocephala* EVs in this invention; Figure 2 In the image, A represents a transmission electron microscope (TEM) image; Figure 2 In this figure, B represents the particle size distribution curve; Figure 2 In this context, C represents the statistical data on particle size and concentration. Detailed Implementation

[0018] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0019] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0020] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0021] As described above, this invention provides extracellular vesicles of *Gnaphalium affine*, their preparation method, and applications. *Gnaphalium affine*, an important filamentous fungus, plays a crucial role in interspecies competition and biocontrol through its secreted extracellular vesicles. Traditional fungal vesicle extraction often relies on recovery from liquid culture media; however, because filamentous fungi easily form spheroids during deep liquid culture, vesicle release efficiency is limited and contains a high amount of impurity proteins.

[0022] Based on this, the present invention uses a specific solid culture combined with protoplast enzymatic hydrolysis technology, and an optimized process of ultracentrifugation and molecular sieve chromatography to prepare extracellular vesicles of *Bacillus thuringiensis* with high purity and high biological activity.

[0023] In a first aspect, the present invention provides a method for preparing extracellular vesicles of *Pteris vittata*, comprising the following steps: S10, strain revival and mycelial culture: The *Alternaria alternata* strain was inoculated onto a solid culture medium lined with cellophane and cultured. The mycelium on the cellophane surface was collected.

[0024] Specifically, step S10 includes: inoculating the white spores of *Bacillus thunbergii* into the center of a solid culture medium covered with cellophane, culturing it at 25-27°C in the dark for 72-96 h, and collecting the mycelium on the surface of the cellophane.

[0025] Furthermore, in this step, the use of cellophane prevents the hyphae from penetrating the agar medium, thus ensuring complete collection of the hyphae and avoiding interference from the medium components in subsequent vesicle purification. Simultaneously, the cellophane undergoes autoclaving before use and is pre-soaked in deionized water to remove industrial additives, preventing chemical interference from impurities on hyphal growth. By controlling the hyphal culture time to 72-96 hours, it is ensured that the hyphae are in the logarithmic growth phase, at which point cell metabolism is vigorous, and the secreted vesicles contain a higher proportion of growth regulators and stress-related proteins.

[0026] In some embodiments, the solid culture medium is potato dextrose agar (PDA) medium, which has the following formula: 200 g of potato, 20 g of glucose, and 15-20 g of agar per 1000 mL of deionized water.

[0027] S20, Protoplast preparation and enzymatic hydrolysate collection: The mycelium is immersed in an enzymatic hydrolysate containing an osmotic stabilizer and fungal cell wall hydrolytic enzymes for incubation, and the supernatant is collected after filtration.

[0028] Specifically, step S20 includes: immersing the mycelium in an enzymatic hydrolysate containing an osmotic stabilizer and fungal cell wall hydrolytic enzymes, and incubating with shaking at 25-28°C and 100-120 rpm for 3-5 hours. After the reaction, the solution is filtered through sterile gauze, and the filtrate is collected to obtain a supernatant containing protoplasts.

[0029] In this example, the enzymatic hydrolysate contains 0.6-0.8 M NaCl as an osmotic stabilizer, 8-12 mM phosphate buffer, and 80-120 mg / mL of a yeast enzyme as a fungal cell wall hydrolase, with a pH of 5.2-5.8. The fungal cell wall hydrolase comprises a complex enzyme system composed of chitinase and glucanase. This enzyme effectively degrades the chitin and glucan cross-linked cell wall structure of *Amanita muscaria*, inducing the release of vesicles from the cell membrane via microvesicle formation. Simultaneously, by controlling the enzymatic hydrolysis temperature at 25-28°C, the fluidity of the protoplast membrane is maintained, which is beneficial for the continuous secretion of vesicles.

[0030] The NaCl concentration is preferably 0.7 M. The osmotic pressure stabilizer is used to balance the solute concentration inside and outside the protoplast, preventing osmotic lysis of the protoplast during enzymatic hydrolysis, thereby ensuring that the released vesicles mainly come from active secretion rather than cell lysis fragments.

[0031] In some embodiments, the pH of the enzymatic hydrolysate is adjusted using a 0.1 M citric acid or sodium dihydrogen phosphate solution, precisely controlling the pH at 5.5. This acidic environment is optimal for the activity of chitinase and glucanase in the fermentation enzymes, while inhibiting the activity of the *Alternaria alternata* proteases, thus protecting the integrity of the vesicle membrane proteins.

[0032] In some implementations, the shaking speed is preferably controlled at 100-110 rpm. This shear force intensity promotes contact between enzyme molecules and the hyphal surface without causing large-scale damage to the protoplasts due to excessive mechanical force. The shaking incubation time is controlled at 3 hours, which is the optimal point at which the protoplast release and vesicle secretion rates reach equilibrium. Exceeding 5 hours will lead to a decrease in protoplast viability and an increase in cell debris.

[0033] In some embodiments, in step S20, a mixture of protease inhibitors is added to the collected filtrate before it is stored at -20°C for later use, further preventing protein degradation before subsequent centrifugation.

[0034] In some embodiments, step S20 further includes: detecting protoplast activity by Evans blue staining, specifically: taking 100 μL of protoplast resuspension and mixing it with an equal volume of 0.5% w / v Evans blue staining solution, incubating at room temperature in the dark for 5 min, and counting the protoplasts using a hemocytometer to control the protoplast survival rate to be above 90%.

[0035] S30, Preliminary separation of extracellular vesicles: The supernatant is centrifuged multiple times at different speeds to remove the precipitate and obtain the initial separation solution of extracellular vesicles.

[0036] Specifically, step S30 includes: centrifuging the supernatant sequentially at 3000-4000 g for 15-25 min and 7000-8000 g for 15-25 min at 4°C to remove precipitate. Then, centrifuging the supernatant at 10000-12000 g for 50-70 min at 4°C, and filtering the resulting supernatant through a 0.22 µm sterile syringe filter to obtain the initial separation solution of extracellular vesicles.

[0037] Furthermore, in this example, the preferred differential centrifugation gradient is set as follows: first, centrifugation at 3500 g for 20 min at 4°C, followed by centrifugation at 7500 g for 20 min, and finally centrifugation at 11000 g for 60 min. This three-stage gradient centrifugation process can remove bacterial residues, large organelle fragments, and micron-sized membrane vesicles step by step, providing a high-purity pretreatment solution for subsequent ultracentrifugation. The washing step after the second ultracentrifugation uses sterile PBS filtered through a 0.22 µm filter membrane, removing any fine salt particles or impurities in the PBS solution that might affect NTA detection. The 11000 g centrifugation step for 60 min effectively removes residual small agar particles and insoluble metabolites from the culture medium, reducing the load on the 0.22 µm filtration step and preventing filter clogging.

[0038] Furthermore, the 0.22 µm sterile needle filter is preferably made of polyethersulfone (PES), which has low protein binding properties, reducing the loss of vesicle particles during filtration.

[0039] S40, Enrichment and purification of extracellular vesicles: The initial separation solution of extracellular vesicles was subjected to multiple ultracentrifugations, the precipitate was collected, and the precipitate was resuspended in buffer solution to obtain extracellular vesicles of *Alternaria alternata*.

[0040] Specifically, step S40 includes: centrifuging the initial separation solution at 4°C and 100,000-120,000 g for 60-80 min, and collecting the precipitate. Resuspending the precipitate in sterile PBS and making up to 20-30 mL, centrifuging again under the same ultracentrifugation conditions for 60-80 min, discarding the supernatant, and resuspending the final precipitate in 150-250 µL PBS to obtain concentrated extracellular vesicles of *Alternaria alternata*.

[0041] Furthermore, the ultracentrifugation parameters used in this invention have been optimized. A force of 100,000 g combined with a centrifugation time of 60-80 min ensures efficient vesicle sedimentation while avoiding vesicle membrane rupture or excessive aggregation caused by excessive centrifugation force. Simultaneously, the PBS volume is increased to 25 mL during the washing step to dilute soluble proteins and remove them with the supernatant. The final product is resuspended in 200 µL of PBS, achieving approximately 125-fold volume concentration.

[0042] In some implementations, the pH of the PBS resuspension is maintained at 7.4, which utilizes a physiological buffering environment to maintain the electrostatic stability of the vesicle lipid bilayer membrane and prevent non-specific aggregation between particles.

[0043] In some implementations, the rotor used in ultracentrifugation is a horizontal rotor. Compared to a fixed-angle rotor, a horizontal rotor causes the vesicles to settle at the center of the tube bottom during centrifugation, which facilitates subsequent resuspension and recovery.

[0044] Furthermore, step S40 also includes wiping the inner wall of the ultracentrifuge tube with lint-free paper to remove non-vesicular lipid impurities and residual protein membranes adsorbed on the tube wall, thereby improving the purity of the final resuspension.

[0045] In some embodiments, after step S40, a step of characterizing the extracellular vesicles is further included, including: The particle size distribution and concentration of vesicles were measured using nanoparticle tracking analysis technology, with the dilution factor controlled at 800-1200 times and the number of particles per frame at 30-200. Vesicle morphology was observed using transmission electron microscopy. 10 μL of sample was loaded onto a 200-mesh copper grid and negatively stained with 1% w / v uranyl acetate for 1 min. The protein content of vesicles was determined using a BCA kit.

[0046] It should be noted that, according to the present invention, the cellophane solid culture method combined with a high-concentration fermentation enzyme hydrolysis process directly acts on the mycelial cell wall of *Aureobasidium alatum*, causing partial degradation of the cell wall and release of protoplasts. During this process, the cell membrane releases a large number of extracellular vesicles through budding or microvesicle formation. Compared with the traditional method of directly recovering the supernatant from liquid culture, the present invention increases the vesicle yield per unit mass of mycelium by inducing vesicle release through protoplastization. Simultaneously, a purification gradient combining differential centrifugation and ultracentrifugation is used to remove mycelial debris, apoptotic bodies, and soluble proteins, retaining nanoparticles with intact lipid bilayer structures in the particle size range of 50-200 nm.

[0047] Meanwhile, this invention, through a combination of solid-state culture and enzymatic hydrolysis, increases the number of vesicles per unit mycelial biomass by 2 to 3 times compared to liquid culture. This improvement stems from the fact that solid-state culture simulates the growth state of fungi in their natural environment, and the introduction of cellophane facilitates mycelial collection while avoiding interference from agar medium components on vesicle purification. Combined with precise ultracentrifugation parameters, this invention achieves efficient and high-purity acquisition of *Alternaria alternata* extracellular vesicles, laying a solid foundation for their application in the biocontrol of agricultural pathogens and pharmaceutical development.

[0048] Secondly, this invention provides the application of the above-mentioned *Alternaria alternata* extracellular vesicles in antagonizing pathogenic bacteria, wherein the pathogenic bacteria is *Fusarium oxysporum*. When subjected to *Fusarium oxysporum* stress, the composition of proteins and miRNAs in the extracellular vesicles secreted by *Alternaria alternata* undergoes specific changes.

[0049] In some embodiments, the application involves analyzing the physiological response of *Bacillus thuringiensis* to pathogenic antagonism. This analysis, conducted under *Fusarium oxysporum* stress, elucidates the proteomics and miRNA composition within its vesicles. Specific steps include: Fusarium oxysporum was inoculated into PDB medium and cultured at 25-27℃ and 180-200 rpm for 7-9 days. The culture product was obtained by centrifugation and filtration through a 0.22 μm filter membrane. The aseptic fermentation product was added to PDA medium at a volume ratio of 15%-25% to prepare an antagonistic medium. Bacteroides leucocephala was cultured on antagonistic medium, and extracellular vesicles in the antagonistic state were collected and extracted (Fo group), with vesicles in normal culture as the control (CK group).

[0050] In some embodiments, the application further includes proteomic analysis of extracellular vesicles, specifically including the following steps: Vesicle proteins were extracted using RIPA strong lysis buffer containing PMSF at a final concentration of 1 mM, and incubated on ice for 20-30 min. Protein distribution was detected by SDS-PAGE electrophoresis and silver staining, with the loading amount controlled at 8-12 µg per lane. Mass spectrometry identification was performed using a data-independent acquisition DIA strategy, and peptide separation was performed using a nanoliter liquid chromatography system with a high performance liquid chromatography column, with the column temperature maintained at 50-60℃.

[0051] Furthermore, a data-independent acquisition (DIA) strategy was employed for mass spectrometry identification. Mobile phase A for mass spectrometry identification consisted of 0.1% formic acid, 2% acetonitrile, and 97.9% ultrapure water, while mobile phase B consisted of 0.1% formic acid, 80% acetonitrile, and 19.9% ​​ultrapure water. The gradient elution program was set as follows: 4% to 12% mobile phase B for 0.2 min, 12% to 25% mobile phase B for 3 min, 25% to 50% mobile phase B for 2.6 min, and 50% to 99% mobile phase B for 0.4 min.

[0052] Furthermore, in the proteomics analysis, the criteria for screening differentially expressed proteins are: a P-value less than 0.05 and a fold change greater than 0.38.

[0053] Thirdly, the present invention provides the application of the above-mentioned extracellular vesicles of *Gnaphalium affine* in the preparation of biological pesticides or plant immune inducers.

[0054] Fourthly, the present invention provides a method for extracting miRNA from extracellular vesicles of *Bacillus thuringiensis*, comprising the following steps: S101: Add RNase solution to the extracellular vesicle sample of *Bacillus thuringiensis* and incubate under ice bath conditions to remove free RNA outside the vesicles.

[0055] Specifically, RNase solution was added to 300 µL of freshly extracted extracellular vesicle sample to a final concentration of 0.4-0.6 mg / mL, and the sample was incubated on ice for 5-10 min.

[0056] S102: Trizol reagent was added to the incubated *Echinococcus faecium* extracellular vesicle sample, and MgCl2 solution was added to promote small RNA precipitation. Isopropanol was then added for precipitation extraction at low temperature to extract RNA.

[0057] Specifically, Trizol reagent was added to the sample at a volume ratio of 1:3, 10 mM MgCl2 solution was added to promote small RNA precipitation, and an equal volume of isopropanol was added to precipitate at -20℃ for 12-16 h.

[0058] S103: Construct sequencing libraries based on extracted RNA, sequence cDNA fragments of 10-40 nt in length, and predict miRNA sequences using a fungal miRNA database.

[0059] Specifically, sequencing libraries were constructed, and high-throughput sequencing of cDNA fragments with lengths of 10-40 nt was performed using the Illu mina platform. miRNA sequence prediction was performed using miRDeep software in conjunction with a fungal miRNA database.

[0060] The following describes embodiments of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or in the product manual.

[0061] Example 1 This embodiment provides a method for preparing extracellular vesicles of *Bacillus thuringiensis*, the specific steps of which are as follows: (1) Mycelial culture: Prepare PDA medium (containing 200 g potato, 20 g glucose, and 18 g agar per 1000 mL), sterilize and pour into plates. Lay pre-moistened cellophane that has been autoclaved on the surface of the plates. Inoculate the white buds fungus in the center of the cellophane and incubate at 26°C in the dark for 72 h.

[0062] (2) Protoplastization induction: Mycelia were collected from the surface of cellophane, weighed, and mixed at a ratio of 1 gram of mycelia to 10 mL of enzymatic hydrolysate. The enzymatic hydrolysate consisted of: 0.7 M NaCl, 10 mM phosphate buffer, 100 mg / mL brewer's enzyme, and pH adjusted to 5.5 with 0.1 M citric acid. The mixture was incubated at 26°C and 110 rpm for 3 h with shaking. The filtrate was collected after filtration through four layers of sterile gauze.

[0063] (3) Differential centrifugation pretreatment: Centrifuge the filtrate at 3500 g for 20 min at 4℃ and discard the precipitate; centrifuge the supernatant at 7500 g for 20 min and discard the precipitate; centrifuge the supernatant again at 11000 g for 60 min and collect the supernatant. Filter the supernatant using a 0.22 µm PES filter membrane.

[0064] (4) Ultracentrifugation purification: Transfer the filtrate to an ultracentrifuge tube and centrifuge at 110,000 g for 70 min at 4 °C. Discard the supernatant and resuspend the precipitate in 25 mL of sterile PBS (pH 7.4), and centrifuge again at 110,000 g for 70 min. After discarding the supernatant, resuspend the precipitate in 200 µL of PBS.

[0065] (5) Characterization: The average particle size of the vesicles was 138 nm by NTA analysis, and the concentration was 1.2 x 10^11 particles per milliliter. The protein concentration was 1.15 mg / mL by BCA analysis. The intact lipid bilayer cup structure was observed by TEM.

[0066] Figure 1 The extracellular vesicles of *Pteris vittata* prepared by solid-state enzymatic hydrolysis were comprehensively and intuitively characterized from three dimensions: morphology, particle size distribution, and extraction concentration, using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Figure 1Image A in the figure is a transmission electron microscope (TEM) image, showing a large number of uniformly distributed nanoscale particles. As indicated by the red arrow in the figure, the vesicles exhibit a well-defined teacup-shaped or spherical concave morphology, a typical characteristic of an intact lipid bilayer membrane resulting from vesicle dehydration and shrinkage during negative staining preparation for electron microscopy. The background of the entire TEM field is relatively clean, with no obvious large fragments of hyphae or severe nonspecific protein aggregates. This directly confirms that the *E. leucocephala* EVs extracted by solid-state enzymatic digestion are morphologically intact, and that the extraction process effectively removed background impurities. Figure 1 In the graph, B represents a scatter plot of NTA particle size versus light scattering intensity. This scatter plot shows the particle size (Size) of a single particle in the test sample and its corresponding light scattering signal intensity (Intensity). Different colored dots in the graph represent multiple independent data collections. Figure 1 As can be clearly observed in B, the vast majority of particles are distributed in the core range of 50 nm to 300 nm, and there are very few large impurity particles larger than 400 nm, which is consistent with the typical physiological particle size range of fungal extracellular vesicles. Figure 1 In the figure, C represents the NTA particle size and concentration distribution curve. The solid green line in the figure represents the average distribution curve of particle concentration, with an average particle size of 188.6 ± 3.6 nm and a peak particle size of 171.6 ± 3.6 nm. NTA detection shows a particle concentration of 3.48 × 10⁻⁶. 8 particles / mL (converted to 3.48×10⁻⁶) 11 Based on the final yield conversion index (particles / mL), this method produced 1.70 × 10⁻⁶ vesicle unit protein particles. 8 The number of particles per μg was as high as 1.61 × 10⁻⁶. 12 pcs / g. Therefore Figure 1 This demonstrates that solid culture combined with enzymatic hydrolysis has extremely high vesicle release induction ability and extraction efficiency.

[0067] Comparative Example 1 Comparative Example 1 provides a process for preparing extracellular vesicles of *Bacillus thuringiensis* using a liquid enzymatic hydrolysis method, specifically including the following steps: The mycelium of *Gnaphalium affine* was inoculated into 400 mL of PDB liquid medium and cultured at 25°C and 120 rpm in the dark for 72 h with shaking. The mycelium was collected and incubated for 3 h at 25°C and 100 rpm with shaking using the same enzymatic hydrolysis solution as in Example 1 (0.7 M NaCl, 10 mM phosphate buffer, 100 mg / mL brewer's enzyme, pH 5.5). Subsequent differential centrifugation and ultracentrifugation procedures were exactly the same as in Example 1. The resulting vesicles were designated as the liquid enzymatic hydrolysis group.

[0068] Figure 2The morphological characteristics and physical parameters of extracellular vesicles of *Pteris vittata* prepared by liquid culture combined with enzymatic hydrolysis were demonstrated by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), which demonstrates the advantages of the solid enzymatic hydrolysis process of this invention from the opposite perspective. Figure 2 In the image, A represents transmission electron microscopy (TEM). Although some cup-shaped or spherical vesicles are still visible in the field of view, the overall electron microscopic background appears quite cluttered. Compared to the solid-state enzymatic digest, the image shows numerous irregular, dark, electron-dense clumps (such as large protein polymers or cell debris), and the vesicles are adhered to some extent with the impurities. Figure 2 The area highlighted by the red arrow in section A shows clearly visible flocculent or filamentous material (possibly nucleic acid or denatured fibrin released from bacterial cell rupture). This indicates that the liquid submerged culture system is prone to causing impurities to co-precipitate with the supernatant, and the purity of the vesicles is far lower than that of the solid enzymatic digestion group. Figure 2 B in the graph represents the scatter plot of NTA particle size versus light scattering intensity. The scatter plot shows that although particles are enriched in specific regions, the entire lattice community is significantly shifted to the right of the horizontal axis (in the direction of larger particle size). Furthermore, in the large particle size range of 400 nm to 800 nm, the number of discrete impurity signal points increases significantly, which corroborates the agglomeration phenomenon observed in the electron microscope field of view. Figure 2 C in the figure represents the NTA particle size distribution curve. The average particle size of EVs in the liquid enzymatic hydrolysis group was 210.2 ± 3.3 nm, with a peak particle size of 187.5 ± 4.0 nm, slightly larger than that in the solid enzymatic hydrolysis group; the particle concentration was 2.06 × 10⁻⁶. 8 particles / mL (converted to 2.06×10⁻⁶) 11 (particles / mL), number of protein particles per unit: 1.32 × 10⁻⁶ 8 Cells / μg, number of particles per unit biomass 4.95×10 11 In terms of both absolute particle concentration and relative biomass conversion rate, the overall yield of the liquid enzymatic hydrolysis group was significantly lower than that of the solid enzymatic hydrolysis group. Combined with the impurity characteristics observed under electron microscopy, this fully demonstrates the technical bottlenecks of the traditional liquid method in terms of vesicle release efficiency and purification.

[0069] Table 1 compares the characteristic parameters of EVs in the solid-state and liquid-state enzymatic hydrolysis groups of this invention. As shown in Table 1, the particle concentration in the solid-state enzymatic hydrolysis group is as high as 3.48 × 10⁻⁶. 11 The concentration of enzymes per mL is significantly higher than that of the liquid enzymatic digestion group (2.06 × 10⁻⁶). 11 The number of particles per unit biomass in the solid enzymatic digestion group reached 1.61 × 10⁻⁶ per mL. 12 The concentration of ions / g is in the liquid group (4.95 × 10⁻⁶). 11The yield of vesicles (cells / g) was 3.2 times higher. This clearly demonstrates that solid-film culture effectively avoids the problem of bacterial entanglement in liquid fermentation, allowing for more complete enzymatic hydrolysis and protoplast release, thus greatly increasing vesicle yield. The contamination protein concentration in the solid-film enzymatic hydrolysis group was 2.053 mg / mL, while that in the liquid enzymatic hydrolysis group was 1.559 mg / mL, indicating a significantly lower background protein concentration in liquid enzymatic hydrolysis. Although the solid-film group brought in slightly more background protein due to the extremely high total extraction yield, the effective proportion of vesicles in the final product was higher, and there were fewer impurity agglomerates, resulting in significantly higher overall product purity compared to the liquid method.

[0070] Table 1: Comparison of characteristic parameters of EVs from solid-state and liquid-state enzyme digestion groups Example 2 This embodiment, based on Example 1, further investigated vesicle extraction under Fusarium oxysporum antagonistic conditions (Fo group): (1) Construction of antagonistic environment: Fusarium oxysporum was inoculated into PDB medium and cultured at 26°C and 190 rpm for 8 days. The sterile fermentation broth was obtained by filtration. The fermentation broth was added to PDB medium at a volume ratio of 20%.

[0071] (2) Vesicle extraction: The white vesicles were cultured on the culture medium containing Fusarium oxysporum metabolites and extracellular vesicles were extracted according to the method in Example 1.

[0072] (3) Proteomics analysis: Vesicle proteins were extracted from the Fo group and the normal culture control group (CK group) using RIPA lysis buffer. Mass spectrometry identification was performed using DIA technology. The results showed that the expression levels of β1,3-glucanase and chitinase were significantly upregulated in the Fo group, with log2FC greater than 1.5.

[0073] (4) miRNA extraction: 0.5 mg / mL RNase A was added to 300 µL of vesicle sample and incubated on ice for 10 min. 900 µL of Trizol and 10 mM MgCl2 were added and precipitated with isopropanol at -20℃ for 14 h. Sequencing analysis revealed 12 miRNAs that were specifically highly expressed under antagonistic pressure.

[0074] Example 3 This embodiment investigated the effect of the pH value of the enzymatic hydrolysate on vesicle yield. Except for setting the pH value to 4.5, 5.5 (same as in Example 1), and 6.5, the other steps were the same as in Example 1.

[0075] Experimental results showed that when the pH was 4.5, enzyme activity was limited, protoplast release rate was less than 60%, and vesicle protein production was only 0.42 mg / mL; when the pH was 5.5, protoplast release rate reached 92%, and vesicle protein production was 1.15 mg / mL; when the pH was 6.5, although protoplast release was faster, the endogenous protease activity of *Tetranychus leucocephala* increased under alkaline conditions, leading to degradation of vesicle membrane proteins. NTA detection showed an increase in small-diameter fragments and uneven particle size distribution.

[0076] Example 4 This embodiment investigates the effect of ultracentrifugation parameters on vesicle integrity. Centrifugation forces were set to 80,000 g, 110,000 g (same as in Example 1), and 150,000 g, with centrifugation times of 70 min for all three.

[0077] Experimental results showed that at 80,000 g, a large number of nanoparticles remained in the supernatant, with a recovery rate of only 45%; at 110,000 g, the recovery rate reached 88%, and the vesicle structure remained intact under TEM; at 150,000 g, although the recovery rate improved slightly, NTA detection revealed that the particles were significantly aggregated, and some vesicles showed membrane rupture.

[0078] Example 5 This example verifies the role of MgCl2 in miRNA extraction. The control group did not include MgCl2; the remaining steps were the same as in Example 2.

[0079] The results showed that the RNA concentration in the group with 10 mM MgCl2 was 45 ng / µL, and Agilent 2100 assay showed a significant enrichment peak in the 10-40 nt region; the RNA concentration in the control group without MgCl2 was only 8 ng / µL, which could not meet the library preparation requirements for high-throughput sequencing.

[0080] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing extracellular vesicles of *Pterygodium japonicum*, characterized in that, Includes the following steps: S10: Inoculate the white spores of *Bacillus thunbergii* onto a solid culture medium lined with cellophane and culture them, then collect the mycelium on the cellophane surface. S20: The mycelium is immersed in an enzymatic hydrolysate containing an osmotic stabilizer and fungal cell wall hydrolytic enzymes for incubation, and the supernatant is collected after filtration; S30: Perform differential centrifugation on the supernatant multiple times to remove the precipitate and obtain the initial separation solution of extracellular vesicles; S40: Perform multiple ultracentrifugations on the initial separation solution of extracellular vesicles, collect the precipitate, and resuspend the precipitate in buffer solution to obtain extracellular vesicles of *Alternaria alternata*.

2. The method for preparing extracellular vesicles of *Pteris vittata* according to claim 1, characterized in that, The solid culture medium used in step S10 is potato dextrose agar medium. The cellophane is autoclaved and pre-soaked in deionized water before use. The cultivation conditions include: incubation at 25-27℃ in the dark for 72-96 hours.

3. The method for preparing extracellular vesicles of *Pteris vittata* according to claim 1, characterized in that, The fungal cell wall hydrolytic enzyme in step S20 includes a complex enzyme system composed of chitinase and glucanase. Incubation conditions include: shaking incubation at 25-28℃ and 100-120 rpm for 3-5 hours.

4. The method for preparing extracellular vesicles of *Leymus chinensis* according to claim 1, characterized in that, The process of step S30 includes: The supernatant was centrifuged at low temperature at 3000-4000g and 7000-8000g respectively to remove the precipitate; The obtained supernatant was centrifuged at 10,000-12,000 g under low temperature conditions, and filtered to obtain the initial separation solution of extracellular vesicles.

5. The method for preparing extracellular vesicles of *Pteris vittata* according to claim 1, characterized in that, The process of step S40 includes: The initial separation solution of the extracellular vesicles was subjected to ultracentrifugation at 100,000-120,000 g under low temperature conditions, and the precipitate was collected. After resuspending the precipitate in physiological buffer, it was ultracentrifuged again at 100,000-120,000 g under low temperature conditions, and the precipitate was collected. The precipitate was resuspended in physiological buffer to obtain extracellular vesicles of *Alternaria alternata*.

6. An extracellular vesicle of *Alternaria solani*, characterized in that, Prepared by the method according to any one of claims 1-5.

7. The use of the extracellular vesicles of *Pteris vittata* as described in claim 6 or the extracellular vesicles of *Pteris vittata* prepared by the method described in any one of claims 1-5 in the preparation of antifungal agents, biopesticides, or plant immune inducers.

8. The application according to claim 7, characterized in that, The pathogenic fungi include Fusarium oxysporum.

9. A method for extracting miRNA from extracellular vesicles of *Pteris vittata* as described in claim 6, characterized in that, Includes the following steps: S101: Add RNase solution to the extracellular vesicle sample of *Bacillus thuringiensis* and incubate under ice bath conditions to remove free RNA outside the vesicles; S102: Add RNA lysis reagent to the incubated extracellular vesicle sample of *Bacillus thuringiensis*, and add small RNA precipitation agent and alcohol precipitation agent for separation and extraction at low temperature to obtain RNA; S103: Construct sequencing libraries based on extracted RNA, sequence cDNA fragments of 10-40 nt in length, and predict miRNA sequences using a fungal miRNA database.

10. The method for extracting miRNA from extracellular vesicles of *Bacillus thuringiensis* according to claim 9, characterized in that, The concentration of the extracellular vesicle sample of *Bacillus thunbergii* after adding RNase solution in step S101 is 0.4-0.6 mg / mL. In step S101, the volume ratio of the incubated *Bacillus thuringiensis* extracellular vesicle sample to the RNA lysis reagent is 1:3.

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