Preparation method of plant exosome

Abstract

The invention relates to a preparation method of a plant exosome, which comprises the following steps: 1) crushing and sieving a dry plant, stirring and filtering plant raw material powder and a buffer solution according to a material-liquid ratio of 1g: (5-15) mL; the method comprises the following steps: co-grinding a fresh plant and a buffer solution, and filtering according to a material-liquid ratio of 1: (1-5) mL; 2) centrifuging the filtrate, and collecting supernate; 3) performing filtration concentration and impurity removal on the supernate through a tangential flow filtration system to obtain a concentrated solution; 4) filtering the concentrated solution, adding polyglutamic acid and glucan, and carrying out polymerization precipitation; after polymerization is finished, centrifuging and collecting precipitates; 5, the precipitate is resuspended with pure water and centrifuged, supernate is taken, and the exosomes are obtained.By means of the method, the extraction efficiency is remarkably improved, the method has the advantage of being easy and convenient to operate, and a solid foundation is laid for wide application of the plant exosomes in industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant exosomes, and particularly relates to a method for preparing plant exosomes. Background Art

[0002] In nature, plants communicate information between cells through complex signal transduction mechanisms. Exosomes, as an emerging form of intercellular communication, have attracted widespread attention and research in recent years. Plant exosomes are tiny vesicles released by plant cells that carry a variety of bioactive molecules, such as proteins, nucleic acids, and lipids. These molecules play a key role in plant growth, development, defense response, and interaction with other organisms.

[0003] To overcome these challenges, researchers are constantly exploring new methods for preparing plant exosomes. Some studies have attempted to destroy plant cell walls through physical or chemical methods to release more exosomes. However, these methods often damage the structure and function of exosomes and reduce their biological activity. It is particularly important to develop a preparation method for different morphological plants that can not only efficiently release exosomes but also maintain their integrity and biological activity.

[0004] Based on this, this application was developed. Summary of the invention

[0005] The present invention aims to overcome the defects of the prior art and provide a method for preparing plant exosomes. This method not only significantly improves the extraction efficiency, but also has the advantage of simple operation, laying a solid foundation for the wide application of plant exosomes in industrial production.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for preparing plant exosomes, comprising the following steps: 1) Pretreatment: For dry plants, the plants are crushed and sieved, and the plant raw material powder is stirred and filtered with the buffer solution, and the solid-liquid ratio is 1g:5~15mL; for fresh plants, the plants and the buffer solution are co-grinded and filtered, and the solid-liquid ratio is 1g:1~5mL; for the buffer solution, when it is for plants with alkaline secondary metabolites, MES buffer with pH 5-6 is used; when it is for plants with acidic secondary metabolites, TBS buffer with pH 7.5-9 is selected; 2) Centrifuge the filtrate and collect the supernatant; 3) filtering, concentrating and removing impurities from the supernatant through a tangential flow filtration system to obtain a concentrate; the setting conditions of the tangential flow filtration are: a rotation speed of 200-600 rpm (preferably 400 rpm); and an eluent of TBS buffer (pH 7.0-7.5); 4) The concentrated solution is filtered, and polyglutamic acid and dextran are added for polymerization precipitation; after the polymerization is completed, centrifuge and collect the precipitate; 5) The precipitate is resuspended in pure water, centrifuged, and the supernatant is taken, which is the exosome. The average particle size of the plant-derived exosomes is 60-120 nm, and the concentration is 2.4E+11-3.1E+11 Particles / mL.

[0007] Specifically, the plants in step 1) include but are not limited to one or more selected from grapefruit, sweet orange, sour orange, lemon, grape, water chestnut, strawberry, dried ginger, dried peony leaves, dried Arabidopsis, soybean, etc.

[0008] Specifically, the buffer described in step 1) is selected from different buffers for plants with different secondary metabolites: for plants with alkaline secondary metabolites, use MES buffer with pH 5-6; for plants with acidic secondary metabolites, select TBS buffer with pH 7.5-9.0. The composition of MES buffer with pH 5-6 is: 20 mM MES, 100 mM NaCl, 10 mM EDTA, 10 M NaOH, and the pH is adjusted to 5-6; the composition of TBS buffer with pH 7.5-9.0 is: 100 mMTris, 100 mM NaCl, 10 mM EDTA, 10% HCl, and the pH is adjusted to 7.5-9.0. Preferably, in step 1), the solid-liquid ratio of the dried plant raw material powder to the buffer is 1g:10 mL; the solid-liquid ratio of the fresh plant to the buffer is 1g:3 mL.

[0009] Specifically, in step 4), the mass ratio of polyglutamic acid to dextran can be 1:0.5-1.5. The mass ratio of the sum of the mass of polyglutamic acid and dextran to the concentrated solution can be 1:5-20, preferably 1:15.

[0010] Furthermore, in step 4), the polymerization precipitation is carried out at a temperature of 2-6°C for 6-10 hours.

[0011] Further, in step 2), the filtrate is centrifuged at 8000-15000 rpm for 40-70 min, and the centrifugation conditions in step 2) are preferably: 10000 rpm, 50 min. In step 4), the filtrate is centrifuged at 6000-20000 rpm for 30-60 min, and the precipitate is collected, and the preferred centrifugation conditions are: 13000 rpm, 40 min.

[0012] Furthermore, in step 5), the precipitate is resuspended in TBS buffer and centrifuged at 8000-12000 rpm for 15-30 min, preferably at 10000 rpm for 20 min; the mixed solution is centrifuged at 4000-10000 rpm for 20-50 min, preferably at 8000 rpm for 25 min.

[0013] The present invention provides plant exosomes prepared by the above preparation method.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The core goal of the present invention is to innovate a method for preparing plant exosomes, aiming to effectively extract plant exosomes through the preparation process described in detail in the present invention. The method of the present invention not only significantly improves the extraction efficiency, but also has the advantage of simple operation, laying a solid foundation for the wide application of plant exosomes in industrial production.

[0015] The preparation method disclosed in the present invention can prepare exosomes for plant materials of different forms, and perform different treatments for plants with different secondary metabolites. In addition, it has the advantages of improved extraction efficiency and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The transmission electron microscopy (TEM) characterization results of lemon exosomes prepared in Example 1; Figure 2 NanoFCM characterization results of lemon exosomes prepared in Example 1; Figure 3 The transmission electron microscopy (TEM) characterization results of soybean exosomes prepared in Example 2; Figure 4 NanoFCM characterization results of soybean exosomes prepared in Example 2; Figure 5 This is the transmission electron microscopy (TEM) characterization result of lemon exosomes prepared in Comparative Example 1; Figure 6 NanoFCM characterization results of lemon exosomes prepared in Comparative Example 1; Figure 7 This is the transmission electron microscopy (TEM) characterization result of soybean exosomes prepared in Comparative Example 2; Figure 8 This is the NanoFCM characterization result of soybean exosomes prepared in Comparative Example 2. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0018] In the following examples, unless otherwise specified, the raw materials used are all common commercially available products that can be directly purchased, and the processes not described in detail can be based on conventional techniques in the art.

[0019] Embodiment 1: A method for preparing plant exosomes, which specifically comprises the following steps: 1) Pretreatment: For lemon, grind lemon with pH 8.0 TBS buffer and filter, the solid-liquid ratio is 1g:3 mL; the composition of pH 8.0 TBS buffer is: 100 mM Tris, 100 mM NaCl, 10 mM EDTA, 10% HCl, and the pH is adjusted to 8.0; 2) The filtrate was centrifuged (10000 rpm, 50 min) and the supernatant was harvested; 3) The supernatant is filtered, concentrated and impurity-removed through a tangential flow filtration system (the tangential flow filtration system can be conventionally used in the art, and its structure is not the innovation of the present application, so it will not be described in detail) to obtain a concentrate. The setting conditions of the tangential flow filtration are: the rotation speed is 400 rpm; the eluent is: TBS buffer (pH 7.2); 4) The concentrate was filtered, and polyglutamic acid and dextran (mass ratio 1:1) were added for polymerization precipitation (standing at 4°C for 8h), and the mass ratio of the sum of the mass of polyglutamic acid and dextran to the concentrate was 1:15; after the polymerization was completed, centrifuged (13000 rpm, 40min), and the precipitate was harvested; 5) The precipitate was resuspended in pure water, centrifuged (10,000 rpm, 20 min), and the supernatant was taken, which was the lemon exosomes.

[0020] Figure 1 The transmission electron microscopy (TEM) characterization results of lemon exosomes prepared in Example 1 are given. Figure 1 It can be seen that the lemon exosomes prepared in this example have a typical hemispherical or saucer-like structure and an ideal particle size.

[0021] Figure 2 The NanoFCM characterization results of lemon exosomes prepared in Example 1 are given. Figure 2 It can be seen that the lemon exosomes prepared in this example have a uniform particle size distribution, with an average particle size of 80.4 nm.

[0022] Embodiment 2: A method for preparing plant exosomes, which specifically comprises the following steps: 1) Pretreatment: For dried soybeans, crush and sieve them, stir and filter the plant raw material powder with pH 5.5 MES buffer, and the solid-liquid ratio is 1g:10 mL; the composition of pH 5.5 MES buffer is: 20 mM MES, 100 mM NaCl, 10 mM EDTA, 10 M NaOH, and the pH is adjusted to 5.5; 2) The filtrate was centrifuged (10000 rpm, 50 min) and the supernatant was harvested; 3) The supernatant is filtered, concentrated and impurity-removed through a tangential flow filtration system to obtain a concentrate. The setting conditions of the tangential flow filtration are: a rotation speed of 400 rpm; an eluent: TBS buffer (pH 7.2); 4) The concentrate was filtered, and polyglutamic acid and dextran (mass ratio 1:1) were added for polymerization precipitation (standing at 4°C for 8 hours), and the mass ratio of the sum of the mass of polyglutamic acid and dextran to the concentrate was 1:15; after the polymerization was completed, centrifuged (13000 rpm, 40 min) and the precipitate was harvested; 5) The precipitate was resuspended in pure water, centrifuged (10,000 rpm, 20 min), and the supernatant was taken, which was the soybean exosomes.

[0023] Figure 3 The transmission electron microscopy (TEM) characterization results of soybean exosomes prepared in Example 2 are given. Figure 3 It can be seen that the soybean exosomes prepared in this example have a typical hemispherical or saucer-like structure and an ideal particle size.

[0024] Figure 4 The NanoFCM characterization results of soybean exosomes prepared in Example 2 are given. Figure 4 It can be seen that the soybean exosomes prepared in this example have a uniform particle size distribution, with an average particle size of 77.2 nm.

[0025] Comparative Example 1 A method for preparing plant exosomes, which specifically comprises the following steps: 1) Pretreatment: For lemon, grind lemon with pH 7.4 PBS buffer and filter, with a solid-liquid ratio of 1 g: 3 mL; 2) The filtrate was centrifuged (10000 rpm, 50 min) and the supernatant was harvested; 3) The supernatant is filtered, concentrated and impurity-removed through a tangential flow filtration system to obtain a concentrate. The setting conditions of the tangential flow filtration are: a rotation speed of 400 rpm; an eluent: TBS buffer (pH 7.2); 4) The concentrate was filtered, and polyglutamic acid and dextran (mass ratio 1:1) were added for polymerization precipitation (standing at 4°C for 8 hours), and the mass ratio of the sum of the mass of polyglutamic acid and dextran to the concentrate was 1:15; after the polymerization was completed, centrifuged (13000 rpm, 40 min) and the precipitate was harvested; 5) The precipitate was resuspended in pure water, centrifuged (10,000 rpm, 20 min), and the supernatant was taken, which was the lemon exosomes.

[0026] Figure 5 The transmission electron microscopy (TEM) characterization results of lemon exosomes prepared in Comparative Example 1 are given. Figure 5 It can be seen that the particle size of the lemon exosomes prepared in this comparative example does not have a typical hemispherical or saucer-like structure and an ideal particle size.

[0027] Figure 6 The NanoFCM characterization results of lemon exosomes prepared in Comparative Example 1 are given. Figure 6 It can be seen that the lemon exosomes prepared in this comparative example have a uniform particle size distribution, and the average particle size is 77.7 nm.

[0028] Comparative Example 2 A method for preparing plant exosomes, which specifically comprises the following steps: 1) Pretreatment: For dried soybeans, crush and sieve them, stir and filter the plant raw material powder with pH 7.4 PBS buffer, and the solid-liquid ratio is 1g:10 mL; 2) The filtrate was centrifuged (10000 rpm, 50 min) and the supernatant was harvested; 3) The supernatant is filtered, concentrated and impurity-removed through a tangential flow filtration system to obtain a concentrate. The setting conditions of the tangential flow filtration are: a rotation speed of 400 rpm; an eluent: TBS buffer (pH 7.2); 4) The concentrate was filtered, and polyglutamic acid and dextran (mass ratio 1:1) were added for polymerization precipitation (standing at 4°C for 8 hours), and the mass ratio of the sum of the mass of polyglutamic acid and dextran to the concentrate was 1:15; after the polymerization was completed, centrifuged (13000 rpm, 40 min) and the precipitate was harvested; 5) The precipitate was resuspended in pure water, centrifuged (10,000 rpm, 20 min), and the supernatant was taken, which was the soybean exosomes.

[0029] Figure 7 The transmission electron microscopy (TEM) characterization results of soybean exosomes prepared in Comparative Example 2 are given. Figure 5 It can be seen that the particle size of the lemon exosomes prepared in this comparative example does not have a typical hemispherical or saucer-like structure and an ideal particle size.

[0030] Figure 8The NanoFCM characterization results of soybean exosomes prepared in Comparative Example 2 are given. Figure 6 It can be seen that the lemon exosomes prepared in this comparative example have uniform particle size distribution, and the average particle size is 81.8 nm.

[0031] Table 1 shows the concentrations of plant exosomes prepared in Examples 1 and 2 and Comparative Examples 1 and 2.

[0032] Table 1 Concentration of plant exosomes prepared in different examples As shown in Table 1, the method of the present invention can be used to prepare exosomes from plant materials of different forms, and the concentration of the exosomes prepared by performing different treatments on plants with different secondary metabolites is significantly improved, thereby greatly improving the extraction efficiency.

Claims

1. A method for preparing plant exosomes, characterized in that: The steps include: 1) Pretreatment: For dry plants, the plants are crushed and sieved, and the plant raw material powder is stirred and filtered with the buffer solution, with a solid-liquid ratio of 1g:5~15mL; for fresh plants, the plants and the buffer solution are co-grinded and then filtered, with a solid-liquid ratio of 1g:1~5mL; for plants with alkaline secondary metabolites, the buffer solution is MES buffer with a pH of 5-6; For plants producing acidic secondary metabolites, choose TBS buffer with a pH of 7.5-9.0; 2) Centrifuge the filtrate and collect the supernatant; 3) filtering, concentrating and removing impurities from the supernatant through a tangential flow filtration system to obtain a concentrate; the setting conditions of the tangential flow filtration are: a rotation speed of 200-600 rpm; and an eluent of TBS buffer; 4) The concentrated solution is filtered, and polyglutamic acid and dextran are added for polymerization precipitation; after the polymerization is completed, centrifuge and collect the precipitate; 5) Resuspend the precipitate with pure water, centrifuge, and take the supernatant, which is the exosomes.

2. The method for preparing plant exosomes according to claim 1, characterized in that: The plant in step 1) is selected from one or more of grapefruit, sweet orange, sour orange, lemon, grape, water chestnut, strawberry, dried ginger, dried peony leaves, dried Arabidopsis, and soybean.

3. The method for preparing plant exosomes according to claim 1, characterized in that: The composition of MES buffer at pH 5-6 is: 20 mM MES, 100 mM NaCl, 10 mM EDTA, 10 M NaOH, and the pH is adjusted to 5-6; the composition of TBS buffer at pH 7.5-9.0 is: 100 mM Tris, 100 mM NaCl, 10 mM EDTA, 10% HCl, and the pH is adjusted to 7.5-9.

0.

4. The method for preparing plant exosomes according to claim 1, characterized in that: In step 4), the mass ratio of polyglutamic acid to dextran is 1:0.5-1.

5.

5. The method for preparing plant exosomes according to claim 1, characterized in that: In step 4), the mass ratio of the sum of the mass of polyglutamic acid and dextran to the mass of the concentrated solution is 1:5-20.

6. The method for preparing plant exosomes according to claim 1, characterized in that: In step 4), the polymerization precipitation is carried out at a temperature of 2-6°C for 6-10 hours.

7. The method for preparing plant exosomes according to claim 1, characterized in that: In step 2), the filtrate is centrifuged at 8000-15000 rpm for 40-70 min; in step 4), the filtrate is centrifuged at 6000-20000 rpm for 30-60 min, and the precipitate is collected.

8. The method for preparing plant exosomes according to claim 1, characterized in that: In step 5), the precipitate is resuspended in TBS buffer and centrifuged at 8000-12000 rpm for 15-30 min; the mixture is centrifuged at 4000-10000 rpm for 20-50 min.

9. Plant exosomes prepared by the preparation method according to any one of claims 1 to 8.

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