CN-121203938-B - Plant extracellular vesicle extraction method based on bionic low-permeability vibrating membrane separation technology

Abstract

The invention discloses a plant extracellular vesicle extraction method based on a bionic low-permeability vibrating membrane separation technology. According to the invention, the vesicle release liquid is obtained by mixing and incubating the plant sample with the specific hypotonic induction liquid (phytohormone, osmotic regulator and activity protective agent) through a specific pretreatment method, so that the vesicle yield can be remarkably improved. The process simulates the natural vesicle release process of plant cells after being stimulated by the outside temperature, and activates the vesicle release mechanism of the cells, so that the vesicle release efficiency is far higher than that of the traditional method. On the basis, the method for extracting the plant extracellular vesicles based on the bionic low-permeability vibrating membrane separation technology can remarkably improve the yield and purity of vesicles after the pre-treated vesicle release liquid is subjected to specific high-frequency micro-vibrating membrane separation and concentration steps and photoacoustic flow control separation and purification treatment, overcomes the defects of the traditional method, and provides a plant extracellular vesicle extraction scheme which can be used for achieving high yield, high purity, high activity, standardization and amplification.

Inventors

• LI SENPENG
• LIU YU
• ZHAO KAI
• SUN MEIYU
• HUANG FUSHAN
• TIAN XIAOTING
• LIU SHUANG
• LI QIAN

Assignees

• 山东杰凯生物科技有限公司

Dates

Publication Date

20260505

Application Date

20251126

Claims (4)

1. 1. The method for extracting the plant extracellular vesicles based on the bionic low-permeability vibrating membrane separation technology is characterized by comprising the following steps of: s1, treating by adopting a pretreatment method to obtain a release liquid containing plant extracellular vesicles; the pretreatment method comprises the steps of taking a plant sample, cleaning, draining, cutting into pieces, mixing with hypotonic induction liquid, and incubating for 1-2 hours at 30-50rpm and 25-30 ℃ to obtain a release liquid containing plant extracellular vesicles; The ratio of the plant sample to the hypotonic induction liquid is 1 (2-8) g/mL; the hypotonic induction liquid is prepared by mixing 1nM-1 mu M plant hormone, 5mM-50mM osmotic regulator and 0.1mM-5mM active protective agent; the plant hormone is one or more of methyl jasmonate, salicylic acid, gibberellin, brassinolide, jasmonate, abscisic acid and ethephon; The osmotic regulator is one or more of trehalose, sucrose, fructose, mannitol, glucose, sorbitol and inositol; The active protective agent is one or more of ascorbic acid, glutathione, citric acid, sodium thiosulfate, cysteine, EGCG and vitamin E; S2, separating and concentrating by a high-frequency micro-vibration membrane, namely removing impurities from a release liquid containing plant extracellular vesicles, collecting filtrate, and filtering the filtrate by a membrane component under the conditions of 80-200Hz high frequency and low amplitude vibration of <10 mu m to obtain concentrated plant extracellular vesicle suspension; S3, optoacoustic flow control sorting and purifying, namely irradiating the vesicle suspension concentrated by the membrane under nanosecond pulse laser with the wavelength of 400-1200 nm and the energy density of less than 20mJ/cm 2 , sorting in 1-10MHz ultrasonic and 0.1-1MPa sound pressure, and removing impurities to obtain the high-purity plant extracellular vesicles.
2. 2. The method of claim 1, wherein the membrane module employed in S2 is a ceramic membrane or PES membrane.
3. 3. The method of claim 1, wherein the S3 treatment is followed by further concentration by tangential flow filtration or ultrafiltration centrifugation and lyophilization to produce the plant extracellular vesicle lyophilized powder.
4. 4. Use of the method according to any one of claims 1 to 3 for increasing yield and purity of plant extracellular vesicles.

Description

Plant extracellular vesicle extraction method based on bionic low-permeability vibrating membrane separation technology Technical Field The invention relates to the technical field of medicines and cosmetics, in particular to a method for extracting plant extracellular vesicles based on a bionic low-permeability vibrating membrane separation technology. Background Plant extracellular vesicles (Plant-derived extracellular vesicles, PDEVs) are nanoscale lipid bilayer vesicles secreted naturally by Plant cells, and can accurately deliver bioactive substances such as proteins, nucleic acids, metabolites and the like to target cells, so that inter-species intercellular communication is realized. Because of its low immunogenicity, good biocompatibility and diverse biological activities, it has great potential in the fields of drug delivery, disease treatment, cosmetics, functional foods, etc. Despite its broad prospects, PDEVs, how to obtain high yields, high purity, high activity, high efficiency, remains a central challenge in current scientific research and industry. Particularly for vesicle extraction with complex composition, high starch content and dry plant-based materials. PDEVs is far less standardized than animal-derived EVs, and the yield is affected by various factors such as plant sources, tissue sites, growth conditions, extraction methods and the like. The prior studies disclose PDEVs pretreatment/release methods aimed at facilitating the release of vesicles from the interstitial space, apoplast or specific organelles into the liquid phase for subsequent separation without thoroughly destroying the plant cell structure. The conventional method is (1) a grinding method, namely, directly mechanically grinding fresh or frozen plant tissues (such as leaves and fruits) in a buffer solution, thoroughly breaking cell walls and cell membranes by physical force, releasing all cell contents including organelles, cytoplasmic proteins and EVs, and has the defects of extremely high impurity content (cell fragments, protein polymers and various organelles), high subsequent purification difficulty and possibility of mixing unnatural intracellular vesicles. (2) Soaking/diafiltration the whole plant tissue or purified cell wall is soaked in isotonic buffer, allowed to stand or gently shaken at a temperature (e.g. 4 ℃ or 25 ℃) for several hours to tens of hours. The apoplast transport in plants is simulated, allowing vesicles pre-existing in the cell gap or naturally secreted from the cells to diffuse into the buffer. The disadvantage is that the yields are generally very low and take a long time. (3) Enzymolysis method comprises separating plant tissue with mixed enzyme such as cellulase, pectase, and hemicellulase. Specifically degrading the major components of the cell wall, releasing the protoplasts and "freeing" the vesicles in the cell space. The disadvantage is the high cost of the enzyme and the need to optimise the enzymatic conditions (pH, temperature, time) and the residual enzyme may affect downstream analysis or application. (4) Extrusion is a process in which a lightly ground plant tissue homogenate or mass is extruded through a screen or membrane module of a specified pore size (e.g., 10-40 μm). The vesicles in the tissue are "squeezed" out by mechanical pressure. The disadvantage is the possible introduction of subcellular fragments. (5) Vacuum infiltration, in which plant tissue is immersed in a buffer solution and released rapidly after application of vacuum. This process causes the gas inside the tissue to be evacuated and when the vacuum is released, the buffer rapidly penetrates into the interstitial space, and the pressure differential is used to "flush" the vesicles out of the interstitial space. The disadvantage is that specific equipment is required and that misoptimization may cause damage to certain delicate tissues. In summary, the vesicle yields of the prior PDEVs pretreatment/release methods are generally not high, and compared with the prior art, the vesicle yields are generally low (from high to low) by a grinding method, an extrusion method, an enzymolysis method, and a vacuum infiltration/soaking method. At present, "grinding/extrusion+differential centrifugation" is the most mainstream P-EVs release and primary separation strategy due to its high yield, but its yield is also around 10 8~109 (particle count/gram fresh weight) and still needs to be improved. Regardless of the pretreatment method, the crude extract obtained requires a series of downstream steps to purify the vesicles. However, in performing bulk extraction preparations, a tradeoff must be made between yield and purity. Because, few methods exist at present, the method can achieve the combination of high yield and high purity, and particularly the vesicle extraction of the plant materials with complex components, high starch content and dryness is aimed at. Therefore, in order to overcome the defects of low efficiency,