Exosome extraction method and application thereof

Abstract

The present application relates to the technical field of exosome, and especially relates to an extraction method of exosome and application thereof. The present application provides an extraction method of exosome. The method adds a protective agent with a specific composition in the steps such as centrifugal separation in the extraction of exosome. Different protective agents work together to achieve efficient and specific protection of exosome, better guarantee the structural integrity and activity of exosome, provide more comprehensive protection for the extraction process of exosome, significantly improve the recovery rate and purity of exosome, and improve the storage stability of exosome. The method has a good application prospect in the extraction of exosome from raw materials such as cow milk.

Description

Technical Field

[0001] This invention relates to the field of exosome technology, and in particular to a method for extracting exosomes and its application. Background Technology

[0002] Exosomes, as key carriers of intercellular communication, have broad application prospects in disease diagnosis, drug delivery, and functional food development. Animal-derived body fluids can serve as sources for exosome extraction. Bovine milk, especially colostrum, is an ideal raw material for large-scale exosome production due to its abundant source and high exosome content. However, isolating exosomes from bovine milk faces significant challenges: the exosome membrane structure is fragile, and the chemical, mechanical, and osmotic stresses generated during separation, such as acid precipitation, high-speed centrifugation, ultrasonic treatment, and freeze-thaw cycles, can easily lead to exosome membrane rupture, leakage of contents, and loss of biological activity. Ruptured exosomes cannot be effectively recovered. Proteases and RNases present in bovine milk may degrade the protein and nucleic acid contents of exosomes, affecting their function and application value.

[0003] Currently, existing exosome isolation technologies mainly focus on optimizing the isolation and purification steps themselves, but lack effective means to protect exosomes during the isolation process. Therefore, there is an urgent need for a solution that can actively protect the integrity of exosomes during the isolation and purification process, thereby comprehensively improving recovery rate, purity, and activity. Summary of the Invention

[0004] This invention provides a method for extracting exosomes and its application.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a method for extracting exosomes, the method comprising: first mixing exosome extraction raw material or pretreated raw material with a first protective agent, and then performing gradient centrifugation purification;

[0007] The gradient centrifugation purification includes low-speed ultracentrifugation and high-speed ultracentrifugation; after the low-speed ultracentrifugation, the recovered material is mixed with a second protective agent and then subjected to high-speed ultracentrifugation.

[0008] The high-speed ultracentrifugation process includes at least a first high-speed ultracentrifugation process and a second high-speed ultracentrifugation process; after the first high-speed ultracentrifugation process, the exosome precipitate is collected, mixed with a third protective agent, and then subjected to a second high-speed ultracentrifugation process.

[0009] The first protective agent comprises sodium citrate and Tween 80 in a mass ratio of 20:1 to 120:1; the mixing of the exosome extract or pretreated raw material with the first protective agent comprises mixing the exosome extract or pretreated raw material with a mixture of sodium citrate and Tween 80, or mixing the exosome extract or pretreated raw material first with sodium citrate and then with Tween 80; the amount of the first protective agent added is calculated as a mass percentage to a final concentration of 0.5% to 1.1%;

[0010] The second protective agent includes sucrose and / or trehalose;

[0011] The third protective agent comprises phosphatidylcholine and cholesterol alcohol in a mass ratio of 3:1 to 6:1; the amount of the third protective agent added is calculated as a mass percentage to a final concentration of 0.3% to 1.5%.

[0012] The exosome extraction method provided by this invention adds specific protective agents in core steps such as centrifugation and washing to provide targeted protection and maximize the maintenance of exosome structural integrity and activity. Before centrifugation purification, adding the aforementioned specific ratio of sodium citrate and Tween 80 can reduce the impact of shear force on the exosome membrane during low-speed ultracentrifugation, while better preventing degradation of exosome contents by nucleases, proteases, etc. Before high-speed ultracentrifugation, adding sucrose and / or trehalose can further reduce exosome membrane denaturation and rupture caused by the shear force of ultracentrifugation, ensuring its structural integrity. Based on the above two protective steps, adding the aforementioned specific ratio of phosphatidylcholine and cholesterol in the resuspension and washing step can effectively resist the mechanical damage of this step and provide protection in post-processing of exosomes (e.g., ultrafiltration concentration, freeze-drying, etc.). In the method of this invention, the three protective agents work synergistically in terms of their composition. For example, the first protective agent, containing sodium citrate and Tween 80, works synergistically with the second protective agent, containing phosphatidylcholine and cholesterol. This not only solves the problem of interference from milk fat / casein in exosome extraction but also simultaneously repairs membrane damage during purification, significantly improving the exosome recovery rate. Furthermore, the three protective agents need to be added in specific steps mentioned above to achieve a good synergistic effect, preventing exosomes from being exposed to an unprotected environment during purification and significantly enhancing the protection of exosome structure and activity. This provides more comprehensive and effective protection for the exosome extraction process. The first protective agent also needs to be added in a specific manner; changing the composition or addition step of the protective agent will destroy its synergistic effect and reduce the protective efficacy.

[0013] Preferably, in the first protective agent, the mass ratio of sodium citrate to Tween 80 is 20:1 to 50:1.

[0014] In the above method, the amount of the second protective agent added is 5% to 10% to the final concentration.

[0015] In this invention, the final concentration of the protective agent is the final concentration in the corresponding treatment system.

[0016] Preferably, the first protective agent is added at a final concentration of 0.9% to 1.1%. The second protective agent is added at a final concentration of 6% to 8%. The third protective agent is added at a final concentration of 0.5% to 0.9%.

[0017] In the above method, the second protective agent can be selected from one or a combination of sucrose and trehalose. A combination of sucrose and trehalose is preferred, as their combined effect provides better protection for exosomes.

[0018] Preferably, the second protective agent comprises sucrose and trehalose in a mass ratio of 1:1 to 5:1.

[0019] Preferably, in the second protective agent, the mass ratio of sucrose to trehalose is 1:1 to 3:1.

[0020] Phosphatidylcholine (PC) typically includes phosphatidylcholine with two saturated fatty acid chains and phosphatidylcholine with one saturated fatty acid chain and the other an unsaturated fatty acid chain.

[0021] Preferably, the phosphatidylcholine is a phosphatidylcholine containing two saturated fatty acid chains. More preferably, it is dipalmitoylphosphatidylcholine (DPPC).

[0022] In this invention, phosphatidylcholine with saturated fatty acid chains is preferred, with dipalmitoylphosphatidylcholine (DPPC) being the most preferred. Phosphatidylcholine containing unsaturated fatty acid chains, such as 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), has a weaker protective effect on exosomes than phosphatidylcholine containing two saturated fatty acid chains.

[0023] For the centrifugation speed in each step, preferably, the low-speed ultracentrifugation treatment is performed at a speed of 8000~20000g, and the high-speed ultracentrifugation treatment is performed at a speed of 60000~150000g.

[0024] More preferably, the low-speed ultracentrifugation treatment has a rotation speed of 8000~12000g. The high-speed ultracentrifugation treatment has a rotation speed of 80000~120000g.

[0025] Preferably, the low-speed ultracentrifugation time is 10-50 min, and the high-speed ultracentrifugation time is 40-120 min for each step.

[0026] More preferably, the low-speed ultracentrifugation treatment time is 20-40 min. The first high-speed ultracentrifugation treatment time is 80-100 min. The second high-speed ultracentrifugation treatment time is 50-70 min.

[0027] In the above method, after the first high-speed ultracentrifugation treatment, the exosome precipitate is collected, the exosome precipitate is resuspended to obtain a resuspension, and the resuspension is mixed with a third protective agent.

[0028] Preferably, the resuspension is performed using a buffer solution. The buffer solution includes, but is not limited to, PBS. The pH of the buffer solution is preferably pH 7.2-7.4.

[0029] In this invention, the raw materials for extracting exosomes include animal body fluids, preferably animal milk. The animal milk includes cow's milk, sheep's milk, human milk, etc.

[0030] Preferably, the raw material is skimmed animal milk.

[0031] In some embodiments of the present invention, the raw material for extracting the exosomes is defatted milk. Centrifugation is preferably used to remove fat. The centrifugation speed is preferably 3000-7000g, and the centrifugation time is preferably 20-40 minutes.

[0032] In the above method, it is preferable to first mix phosphatidylcholine and cholesterol under dissolution conditions, and then mix them with the resuspension. Phosphatidylcholine added alone to the resuspension will rapidly aggregate due to hydrophobic interactions, while cholesterol added alone to the resuspension will precipitate directly due to its extremely low solubility. Therefore, it is necessary to premix and dissolve the two first.

[0033] Preferably, phosphatidylcholine and cholesterol are mixed and then dissolved in ethanol (e.g., anhydrous ethanol), or phosphatidylcholine and cholesterol are dissolved separately in ethanol (e.g., anhydrous ethanol), and then 90%-95% of the ethanol is removed by evaporation (e.g., rotary evaporation) to obtain a lipid-phase residue, which is then mixed with the resuspension. During the ethanol removal process, if the ethanol residue is low, the phosphatidylcholine-cholesterol will lose solvent support and change from a viscous liquid to solid crystals, directly destroying the lipid-phase function; if the ethanol residue is high, the high concentration of ethanol will directly damage exosomes and disrupt the stability of the aqueous system.

[0034] After gradient centrifugation purification, the above method also includes the steps of ultrafiltration concentration and freeze-drying of the exosomes obtained by gradient centrifugation purification.

[0035] The ultrafiltration concentration can be achieved using an ultrafiltration membrane of a suitable size for exosomes, such as 100 kDa. The freeze-drying can be vacuum freeze-drying.

[0036] In some embodiments of the present invention, a method for extracting exosomes is provided, comprising the following steps:

[0037] (1) Remove the milk fat by centrifuging the animal milk at 2-5℃ and 3000-7000g for 20-40 minutes to obtain skimmed animal milk;

[0038] (2) Add the first protective agent to the skimmed animal milk and stir to ensure that the protective agent fully binds to the exosomes;

[0039] (3) Gradient centrifugation purification

[0040] First stage: Centrifuge at 2-5℃ and 8000~12000g for 20-40 minutes to remove casein precipitate, and add a second protective agent to the remaining solution;

[0041] Second stage: Centrifuge at 2-5℃ and 80,000~120,000g for 80-100 minutes, and collect the exosome precipitate;

[0042] Third stage: Resuspend the exosome precipitate with buffer, add the third protective agent, and centrifuge at 2-5℃ and 80,000~120,000g for 50-70 min to obtain purified protective agent-modified exosomes;

[0043] (4) Obtaining the final product: The exosomes collected in step (3) are concentrated by ultrafiltration and freeze-dried. The freeze-dried product obtained in step (4) can be stored at 4°C or -20°C.

[0044] In a second aspect, the present invention provides an exosome protective composition, the composition comprising:

[0045] The first protective agent consists of sodium citrate and Tween 80 in a mass ratio of 20:1 to 120:1;

[0046] Secondary protectants include sucrose and / or trehalose;

[0047] And a third protective agent, including phosphatidylcholine and cholesterol in a mass ratio of 3:1 to 6:1.

[0048] In the protective agent composition provided by the present invention, each component of the three protective agents can be packaged separately, or the first protective agent can be mixed and packaged separately, the second protective agent can be mixed and packaged separately, and the third protective agent can be mixed and packaged separately.

[0049] The second protective agent can be selected from one or a combination of sucrose and trehalose. A combination of sucrose and trehalose is preferred, as their combined effect provides better protection for exosomes.

[0050] Preferably, the second protective agent comprises sucrose and trehalose in a mass ratio of 1:1 to 5:1.

[0051] Preferably, the phosphatidylcholine is a phosphatidylcholine containing two saturated fatty acid chains. More preferably, it is dipalmitoylphosphatidylcholine (DPPC).

[0052] The above-mentioned protective agent composition is used for exosome extraction. The first, second, and third protective agents are added sequentially before gradient centrifugation purification, after low-speed ultracentrifugation of gradient centrifugation purification, and in the resuspension of exosome precipitates collected after high-speed ultracentrifugation of gradient centrifugation purification.

[0053] The first protective agent is added at a final concentration of 0.5-1.1%. The second protective agent is added at a final concentration of 5%-10%. The third protective agent is added at a final concentration of 0.3-1.5%.

[0054] Wherein, the final concentration is the final concentration in the corresponding treatment system.

[0055] Preferably, the first protective agent is added at a final concentration of 0.9% to 1.1%. The second protective agent is added at a final concentration of 6% to 8%. The third protective agent is added at a final concentration of 0.5% to 0.9%.

[0056] The combinations involved in the above-mentioned protective agent compositions of the present invention are all food-grade safe formulations, comply with GB 2760 food additive standards, and can be used in food / cosmetic scenarios.

[0057] Thirdly, the present invention provides the above-described method for extracting exosomes or the application of the exosome protective agent composition in the preparation of exosomes.

[0058] Fourthly, the present invention provides the application of the above-described method for extracting exosomes or the exosome protective agent composition in improving the integrity, recovery rate and / or purity of exosomes.

[0059] The beneficial effects of this invention include at least the following: The exosome extraction method provided by this invention adds a protective agent of a specific composition to the centrifugation and other steps of exosome extraction. Different protective agents work synergistically in terms of composition to achieve efficient and specific protection of exosomes, better ensuring the structural integrity and activity of exosomes, providing more comprehensive protection for the exosome extraction process, significantly improving the recovery rate and purity of exosomes, and improving the storage stability of exosomes. It has good application prospects in the extraction of exosomes from raw materials such as milk. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0061] In the following examples, the preparation method of the mixed solution of phosphatidylcholine and cholesterol is as follows: phosphatidylcholine and cholesterol are mixed in a mass ratio (calculated based on the final concentration of their added amounts), dissolved in anhydrous ethanol, or phosphatidylcholine and cholesterol are dissolved separately in anhydrous ethanol and then mixed in a mass ratio; then 90%-95% of the ethanol is removed by rotary evaporation to obtain the lipid phase residue, which is the mixed solution of phosphatidylcholine and cholesterol.

[0062] The phosphatidylcholine used in the following examples and comparative examples is dipalmitoylphosphatidylcholine (DPPC).

[0063] Example 1

[0064] This embodiment provides a method for extracting exosomes, which includes the following steps:

[0065] (1) Pretreatment: Take fresh milk, centrifuge at 4℃ and 5000g for 30min to remove milk fat, and obtain skim milk;

[0066] (2) Add sodium citrate to skim milk first, then add Tween 80. The amount of sodium citrate added is 0.88% to the final concentration, and the amount of Tween 80 added is 0.02% to the final concentration. Stir gently at 37°C for 10 minutes to allow the protectant to fully combine with the exosomes.

[0067] (3) Gradient centrifugation purification

[0068] First stage: Centrifuge the mixture obtained in step (2) at 4℃ and 8000g for 20min to remove casein precipitate. Add sucrose and trehalose to the remaining solution (sucrose and trehalose can be added together or separately, and the order of addition does not affect the extraction effect). The amount of sucrose added is 3% to the final concentration, and the amount of trehalose added is 3% to the final concentration.

[0069] Second stage: Centrifuge at 4℃ and 80000g for 80 min and collect the exosome precipitate;

[0070] Third stage: Resuspend the exosome precipitate with PBS, and then add a mixed solution of phosphatidylcholine and cholesterol, wherein the amount of phosphatidylcholine added is 0.4% to the final concentration and the amount of cholesterol added is 0.1% to the final concentration; centrifuge at 80,000g for 50 min at 4℃ to obtain purified protective agent modified exosomes.

[0071] (4) Obtaining the final product: The purified protective agent-modified exosomes collected in step (3) were further concentrated using a 100kDa ultrafiltration centrifuge tube, and then freeze-dried under vacuum. The freeze-dried product was stored at -20°C.

[0072] Example 2

[0073] This embodiment provides a method for extracting exosomes, which includes the following steps:

[0074] (1) Take fresh milk and centrifuge at 4℃ and 5000g for 30 minutes to remove milk fat and obtain skim milk;

[0075] (2) Add a mixture of sodium citrate and Tween 80 to skim milk. The amount of sodium citrate added is 1.06% and the amount of Tween 80 added is 0.04%. Stir gently at 37°C for 10 minutes to allow the protectant to fully combine with the exosomes.

[0076] (3) Gradient centrifugation purification

[0077] First stage: Centrifuge the mixture obtained in step (2) at 4℃ and 12000g for 40min to remove casein precipitate. Add sucrose and trehalose to the remaining solution (sucrose and trehalose can be added together or separately, and the order of addition does not affect the extraction effect). The amount of sucrose added is 6% to the final concentration, and the amount of trehalose added is 2% to the final concentration.

[0078] Second stage: Centrifuge at 4℃ and 120000g for 100 min and collect the exosome precipitate;

[0079] Third stage: Resuspend the exosome precipitate with PBS, and then add a mixed solution of phosphatidylcholine and cholesterol, wherein the amount of phosphatidylcholine added is 0.6% to a final concentration and the amount of cholesterol added is 0.2% to a final concentration; centrifuge at 120,000g for 70 min at 4℃ to obtain purified protective agent modified exosomes.

[0080] (4) Obtaining the final product: The purified protective agent-modified exosomes collected in step (3) were further concentrated using a 100kDa ultrafiltration centrifuge tube, and then freeze-dried under vacuum. The freeze-dried product was stored at -20°C.

[0081] Example 3

[0082] This embodiment provides a method for extracting exosomes, which includes the following steps:

[0083] (1) Take fresh milk and centrifuge at 4℃ and 5000g for 30 minutes to remove milk fat and obtain skim milk;

[0084] (2) Add sodium citrate to skim milk first, then add Tween 80. The amount of sodium citrate added is 1.07% to the final concentration, and the amount of Tween 80 added is 0.03% to the final concentration. Stir gently at 37°C for 10 minutes to allow the protectant to fully combine with the exosomes.

[0085] (3) Gradient centrifugation purification

[0086] First stage: Centrifuge the mixture obtained in step (2) at 4℃ and 10000g for 30min to remove casein precipitate. Add sucrose and trehalose to the remaining solution (sucrose and trehalose can be added together or separately, and the order of addition does not affect the extraction effect). The amount of sucrose added is 4% to the final concentration, and the amount of trehalose added is 4% to the final concentration.

[0087] Second stage: Centrifuge at 4℃ and 100,000g for 90 minutes to collect the exosome precipitate;

[0088] Third stage: Resuspend the exosome precipitate with PBS, and then add a mixed solution of phosphatidylcholine and cholesterol, wherein the amount of phosphatidylcholine added is 0.7% to the final concentration and the amount of cholesterol added is 0.12% to the final concentration; centrifuge at 4℃ and 100,000g for 60 min to obtain purified protective agent modified exosomes.

[0089] (4) Obtaining the final product: The purified protective agent-modified exosomes collected in step (3) were further concentrated using a 100kDa ultrafiltration centrifuge tube, and then freeze-dried under vacuum. The freeze-dried product was stored at -20°C.

[0090] Comparative Example 1

[0091] This comparative example provides a method for extracting exosomes, which differs from the method for extracting exosomes in Example 1 only in that the order of adding sodium citrate and Tween 80 in step (2) is changed, i.e., Tween 80 is added first, and then sodium citrate is added.

[0092] Comparative Example 2

[0093] This comparative example provides a method for extracting exosomes, which differs from the method in Example 1 only in that the steps for adding sucrose and trehalose are changed. Sucrose and trehalose are added before adding sodium citrate and Tween 80. That is, step (2) is as follows: sucrose and trehalose are added to skim milk, wherein the amount of sucrose added is 3% to the final concentration and the amount of trehalose added is 3% to the final concentration; then sodium citrate is added, followed by Tween 80, wherein the amount of sodium citrate added is 0.88% to the final concentration and the amount of Tween 80 added is 0.02% to the final concentration. The mixture is then gently stirred at 37°C for 10 minutes to allow the protective agent to fully bind with the exosomes. Sucrose and trehalose are no longer added in step (3).

[0094] Comparative Example 3

[0095] This comparative example provides a method for extracting exosomes, which differs from the method in Example 1 only in that the steps for adding phosphatidylcholine and cholesterol are changed. Phosphatidylcholine and cholesterol are added before sodium citrate and Tween 80 are added. Specifically, step (2) involves adding a mixed solution of phosphatidylcholine and cholesterol to skim milk, wherein the amount of phosphatidylcholine added is 0.4% to a final concentration and the amount of cholesterol added is 0.1% to a final concentration; then sodium citrate is added, followed by Tween 80, wherein the amount of sodium citrate added is 0.88% to a final concentration and the amount of Tween 80 added is 0.02% to a final concentration. The mixture is then gently stirred at 37°C for 10 minutes to allow the protective agent to fully bind with the exosomes. In step (3), the mixed solution of phosphatidylcholine and cholesterol is no longer added.

[0096] Comparative Example 4

[0097] This comparative example provides a method for extracting exosomes, which differs from the method for extracting exosomes in Example 1 only in that the total amount of sodium citrate and Tween 80 added is 1.3% to a final concentration, and the mass ratio of the two remains unchanged.

[0098] Comparative Example 5

[0099] This comparative example provides a method for extracting exosomes, which differs from the method for extracting exosomes in Example 1 only in that Tween 80 is replaced with Tween 20.

[0100] Comparative Example 6

[0101] This comparative example provides a method for extracting exosomes, which differs from the method for extracting exosomes in Example 1 only in that cholesterol is replaced with ergosterol.

[0102] Comparative Example 7

[0103] This comparative example provides a method for extracting exosomes, which differs from the method for extracting exosomes in Example 1 only in that the total amount of phosphatidylcholine and cholesterol added is 0.1% to a final concentration, and the mass ratio of the two remains unchanged.

[0104] Experimental Example

[0105] The exosomes extracted using the extraction methods of the above embodiments and comparative examples were subjected to recovery rate and purity tests and electron microscopy observations. The particle size changes of the exosomes were also detected after being stored at -20°C for 6 months.

[0106] The recovery rate was calculated as follows: Recovery rate (%) = Total number of exosome particles after purification (N1×V1) / Total number of exosome particles before purification (N2×V2) × 100%; where, particle concentration before purification (N1): exosome particle concentration after milk pretreatment and before gradient centrifugation; particle concentration after purification (N2): exosome particle concentration after the third stage of ultracentrifugation; V1: total volume of the system before purification: milk supernatant + sodium citrate and Tween; V2: volume of the resuspension after purification. The exosome particle concentration (total concentration of particles with a diameter between 30 and 150 nm) was detected using the NTA method. Particles with a diameter between 30 and 150 nm were summed to obtain A2 (particles / mL); the total particle concentration was summed to obtain A3 (particles / mL). Exosome purity (%) = A2 / A3 × 100%.

[0107] Particle size was determined using the NTA method. The particle size change rate (%) after 6 months of storage at -20℃ was calculated as follows: (average particle size after storage D2 - initial average particle size D1) / initial average particle size D1 × 100%; where, initial average particle size (D1): the average particle size of the purified exosome suspension, and average particle size after storage (D2): the average particle size of the exosome suspension after 6 months of storage at -20℃.

[0108] The test results are shown in Table 1.

[0109] Table 1

[0110]

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting exosomes, characterized in that, The method includes: first mixing the exosome extract or pretreated raw material with a first protective agent, and then performing gradient centrifugation purification; The gradient centrifugation purification includes low-speed ultracentrifugation and high-speed ultracentrifugation; after the low-speed ultracentrifugation, the recovered material is mixed with a second protective agent and then subjected to high-speed ultracentrifugation. The high-speed ultracentrifugation process includes at least a first high-speed ultracentrifugation process and a second high-speed ultracentrifugation process; after the first high-speed ultracentrifugation process, the exosome precipitate is collected, mixed with a third protective agent, and then subjected to a second high-speed ultracentrifugation process. The first protective agent comprises sodium citrate and Tween 80 in a mass ratio of 20:1 to 120:1; the mixing of the exosome extract or pretreated raw material with the first protective agent comprises mixing the exosome extract or pretreated raw material with a mixture of sodium citrate and Tween 80, or mixing the exosome extract or pretreated raw material first with sodium citrate and then with Tween 80; the amount of the first protective agent added is calculated as a mass percentage to a final concentration of 0.5% to 1.1%; The second protective agent includes sucrose and trehalose; the amount of the second protective agent added is calculated as a final concentration of 5% to 10% by mass percentage. The third protective agent comprises phosphatidylcholine and cholesterol in a mass ratio of 3:1 to 6:1; the amount of the third protective agent added is calculated as a mass percentage to a final concentration of 0.3% to 1.5%. The low-speed ultracentrifugation treatment time is 10-50 min; The time for the first high-speed ultracentrifugation treatment and the second high-speed ultracentrifugation treatment is 40-120 min; The raw material is skimmed animal milk.

2. The method for extracting exosomes according to claim 1, characterized in that, The second protective agent comprises sucrose and trehalose in a mass ratio of 1:1 to 5:

1.

3. The method for extracting exosomes according to claim 1 or 2, characterized in that, The phosphatidylcholine is a phosphatidylcholine containing two saturated fatty acid chains.

4. The method for extracting exosomes according to claim 1 or 2, characterized in that, The speed of the low-speed ultracentrifugation process is 8000~20000g.

5. The method for extracting exosomes according to claim 1 or 2, characterized in that, The high-speed ultracentrifugation process involves centrifugation at speeds of 60,000 to 150,000 g.

6. The method for extracting exosomes according to claim 1 or 2, characterized in that, The method further includes the steps of ultrafiltration concentration and freeze-drying of the exosomes obtained by gradient centrifugation purification.

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