Method for isolating and / or enriching extracellular vesicles

Abstract

The invention provides a method for separating extracellular vesicles from a milk source, which comprises the step of adding a dispersing agent before adding a precipitator into the milk source to precipitate casein so as to reduce the wrapping of protein precipitation on the extracellular vesicles and improve the yield of the extracellular vesicles, thereby obtaining a milk source extract rich in the extracellular vesicles.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for isolating and / or enriching extracellular vesicles and the resulting extracellular vesicle-rich compositions. Background Technology

[0002] Extracellular vesicles (EVs) are nanoscale phospholipid bilayer vesicles released from cells and serve as key mediators for intercellular communication. EVs contain bioactive molecules such as proteins, lipids, nucleic acids, and amino acids, making them ideal drug carriers for oral and transdermal drug delivery. Based on their size and release mechanism, they are classified into exosomes (30-150 nm in diameter), microvesicles (100-1000 nm in diameter), and apoptotic bodies (>1000 nm in diameter); current research focuses on the isolation and application of exosomes (30-150 nm).

[0003] Taking common exosomes as an example, the existing exosome purification techniques mainly include the following methods:

[0004] 1. Ultracentrifugation (UC)

[0005] Ultracentrifugation is the most commonly used method for purifying exosomes, but it is small-scale and time-consuming. Repeated centrifugation may also damage the vesicles, thereby reducing their quality and yield.

[0006] 2. Density gradient centrifugation method

[0007] Under the action of ultracentrifugation, the sample is distributed in sucrose solution (or iodixanol solution) to form layers of different densities. The target layer solution is collected, and the exosome precipitate is collected by ultracentrifugation. Density gradient centrifugation is a complicated and time-consuming operation. Repeated centrifugation may also damage the vesicles, reducing their quality and yield.

[0008] 3. Ultrafiltration centrifugation method

[0009] When extracting exosomes using ultrafiltration centrifugation, the pores of the ultrafiltration membrane may become clogged, leading to particle aggregation, which in turn shortens the lifespan of the ultrafiltration tube and reduces separation efficiency.

[0010] 4. Size exclusion chromatography

[0011] Size exclusion chromatography separates exosomes based on the pore size of the gel and the diameter of the exosome particles (30-150 nm). However, it is limited in scale and time-consuming. Currently, commercially available exosome extraction kits use this method and can be used for the separation of small sample volumes.

[0012] 5. Magnetic bead immunoaffinity capture method

[0013] By utilizing the unique protein markers on the surface of exosomes, magnetic beads coated with antibodies to the markers can be incubated with characteristic exosome vesicles to adsorb and separate the exosomes. However, this method is costly and not conducive to subsequent applications.

[0014] 6. PEG precipitation separation method

[0015] Precipitated exosomes by polyethylene glycol (PEG) present several problems: PEG is difficult to remove, affecting the purity and recovery rate of exosomes, which is detrimental to subsequent applications.

[0016] All of the above exosome extraction methods suffer from problems such as small operation scale and low efficiency.

[0017] Animal milk contains abundant extracellular vesicles, is widely available, and has a low cost, making it a good raw material for preparing extracellular vesicles. However, there are certain problems in preparing extracellular vesicles from milk. Taking cow's milk as an example, cow's milk contains high abundances of milk protein (>30 g / L). -1 Milk exosomes exist in various aggregate states (free protein molecules and soluble colloids or micelles), with casein being highly phosphorylated. The surface charge state of casein is very similar to that of the phospholipid surface of exosomes. During ultracentrifugation (UC) purification, some milk proteins settle along with the exosomes and cannot be separated. For the separation of exosomes from milk, existing literature reports the removal of casein first, using methods such as citric acid removal, sodium phosphate precipitation, ammonium sulfate precipitation, and EDTA removal. After treatment with these methods, ultracentrifugation or tangential flow ultrafiltration is generally used for the subsequent extraction of exosomes. Summary of the Invention

[0018] When preparing extracellular vesicles (EVs) from milk, casein is usually removed first using precipitation. During precipitation, exosome surface membrane proteins easily aggregate with other proteins, resulting in the removal of numerous microvesicles and apoptotic bodies along with the precipitate. The inventors unexpectedly discovered that adding a dispersant before casein precipitation significantly improves the extraction efficiency of EVs and increases the particle concentration of EVs in the extract.

[0019] This invention provides a method for isolating and / or enriching extracellular vesicles, comprising:

[0020] 1) Provide a milk source, optionally a defatted milk source;

[0021] 2) Contact the emulsion source with the dispersant to obtain a mixture of the emulsion source and the dispersant;

[0022] 3) Contact the mixture obtained in step 2) with the precipitant to form a precipitate; and

[0023] 4) Separate the precipitate to obtain a composition rich in extracellular vesicles.

[0024] As used in this article, the term "milk source" refers to animal milk or its products, which can provide extracellular vesicles. "Animal milk" refers to the secretions from the mammary glands of female mammals, including but not limited to cow's milk, sheep's milk, horse's milk, and camel's milk.

[0025] The term "extracellular vesicles (EVs)" as used in this article refers to vesicle-like bodies with a double-membrane structure that detach from the cell membrane or are secreted by the cell, or vesicle structures with the same structure. Their diameter ranges from 40 nm to 1000 nm, and they are mainly in the form of microvesicles (MVs) and exosomes (Exs). Extracellular vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, and breast milk), carrying a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, etc., and participating in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation.

[0026] The term "dispersant" as used in this article refers to a substance that can uniformly disperse various components in milk, such as extracellular vesicles, protein particles, and minerals, in a continuous phase.

[0027] In one embodiment, the defatting method described in step 1) includes: allowing the dairy product to settle naturally (e.g., at 2-8°C) until a distinct fat layer forms on the surface, and removing the fat by filtration, for example using a filter such as gauze, a metal mesh, or a filter membrane.

[0028] In another embodiment, the degreasing method described in step 1) uses centrifugation to remove fat, for example, using a centrifuge such as a disc separator.

[0029] In one embodiment, the dispersant in step 2) is selected from amphoteric molecules, surfactants, divalent metal salts, chelating agents, and C2-C8 alcohols.

[0030] In one specific embodiment, the amphoteric molecule is an amino acid and / or its salt, for example, the amino acid is selected from arginine, proline, lysine, histidine, threonine, serine, glycine, alanine, cysteine, valine, methionine, isoleucine, phenylalanine, tryptophan, citrulline, and ornithine.

[0031] In another embodiment, the surfactant is an amphoteric surfactant, a nonionic surfactant, an anionic surfactant, and / or a cationic surfactant. For example, the amphoteric surfactant includes amino acid-type amphoteric surfactants and betaine-type amphoteric surfactants; the nonionic surfactant is selected from polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80), polyoxyethylene-polyoxypropylene copolymer, and poloxamer 188; the anionic surfactant includes carboxylate-type anionic surfactants, sulfate-type anionic surfactants, and sulfonate-type anionic surfactants; and / or the cationic surfactant includes quaternary ammonium-type cationic surfactants, imidazoline-type cationic surfactants, and amine-type cationic surfactants.

[0032] In yet another embodiment, the divalent metal salt is a magnesium salt, a zinc salt, and / or a calcium salt, specifically, for example, the magnesium salt is magnesium sulfate, zinc sulfate, calcium chloride, and / or magnesium chloride.

[0033] In another embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA), nitric acid triacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and / or a salt thereof.

[0034] In yet another embodiment, the C2-C8 alcohol is a C2-C8 diol, such as hexanediol.

[0035] In one embodiment, the final concentration of the dispersant is 0.01 g / ml to 1.0 g / ml, preferably 0.04 g / 100ml to 0.67 g / 100ml, more preferably 0.08 g / 100ml to 0.50 g / ml, for example 0.08 g / 100ml, 0.10 g / 100ml, 0.12 g / 100ml, 0.13 g / 100ml, 0.20 g / 100ml, 0.24 g / 100ml, 0.30 g / 100ml, 0.33 g / 100ml, 0.36 g / 100ml, 0.40 g / 100ml, or 0.50 g / 100ml.

[0036] In another embodiment, the final concentration of the dispersant is 0.001 mol / L to 0.13 mol / L, preferably 0.0033 mol / L to 0.12 mol / L, for example 0.0033 mol / L, 0.004 mol / L, 0.03 mol / L, 0.08 mol / L, or 0.12 mol / L.

[0037] In one embodiment, the precipitant in step 3) is selected from salting-out precipitants, isoelectric point precipitants, and rennet precipitants.

[0038] The "salting-out precipitant" mentioned in this article refers to a class of substances that can cause proteins to precipitate by increasing the ionic strength in the solution.

[0039] The "isoelectric point precipitant" mentioned in this article refers to a class of substances that adjust the pH value to reach the isoelectric point of the protein, thereby causing the protein to precipitate.

[0040] The "rennet precipitant" mentioned in this article refers to a class of enzymes that can catalyze protein coagulation and precipitation.

[0041] In one specific embodiment, the salting-out precipitant is such as ammonium sulfate, sodium sulfate, or sodium chloride, and the pH of the mixture and / or composition is adjusted to 4.8-5.8.

[0042] In another embodiment, the isoelectric point precipitant is an acid, preferably selected from hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, lactic acid, and malic acid. Preferably, the isoelectric point precipitant is added to adjust the pH to 4.6-4.8.

[0043] In yet another embodiment, the rennet is selected from animal rennet, plant rennet, and microbial rennet; for example, the animal rennet is selected from porcine pepsin and bovine pepsin, and the plant rennet is selected from papain, bromelain, and figase.

[0044] In one embodiment, the method for separating and / or enriching extracellular vesicles according to the present invention further includes concentrating the extracellular vesicle-rich composition; for example, using ultrafiltration, dialysis, or microfiltration; specifically, the ultrafiltration is tangential flow ultrafiltration, for example, the tangential flow ultrafiltration membrane assembly used in the tangential flow ultrafiltration is a flat sheet ultrafiltration assembly and / or a hollow fiber ultrafiltration assembly; and specifically, the microfiltration employs a microporous membrane with a pore size of 1 μm.

[0045] The present invention also provides an extracellular vesicle-rich composition obtained by the method for isolating and / or enriching extracellular vesicles as described in the present invention.

[0046] The method of the present invention can achieve at least one of the following beneficial effects:

[0047] 1) After defatting the dairy products, add an appropriate amount of dispersant and mix evenly before adding a precipitant to remove the precipitate. This can reduce the encapsulation of EVs by protein precipitates, increase the particle concentration of EVs in the solution, and obtain a milk extract rich in EVs and whey protein and other nutrients.

[0048] 2) The preparation method of the present invention is low in cost and high in efficiency, and can extract extracellular vesicles on a large scale and with high yield. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0050] Figure 1 The distribution of EV particle number in the extract of the same milk sample precipitated with different acids; among which... Figure 1 (A) is a graph showing the particle number distribution in extracts precipitated with different acids; Figure 1 (B) Particle size distribution and particle number detection were performed using a nanoflow cytometer (NanoFCM). The results showed that there was no significant difference in the number of particles in the extract when different acid-adjusted milk pH values ​​were used in the range of 4.6 to 4.8.

[0051] Figure 2 Different rennets were used to precipitate the same milk sample, among which Figure 2 (A) shows the particle number distribution results in the extract of pepsin (porcine), pepsin (bovine), papain, bromelain, figase, and Hualing microbial chymoses. Figure 2 (B) The particle size distribution and particle number were detected using a nanoflow cytometer (NanoFCM). The results showed that there was no significant difference in the number of EVs in the extract when different rennets were used.

[0052] Figure 3 The graph shows the particle concentration changes of milk extract solution after adding different dispersants, using saturated ammonium sulfate as a precipitant. The ratio of milk to saturated ammonium sulfate solution was 3:2. The dispersants included amino acid surfactants: threonine, serine, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, phenylalanine, lysine, arginine hydrochloride, histidine, citrulline, and ornithine; nonionic surfactants: polysorbates 40 (Tween 40), 60 (Tween 60), and 80 (Tween 80); nonionic surfactants: polyoxyethylene esters: poloxamer 188; and other dispersants: magnesium chloride, magnesium sulfate, EDTA, and 1,6-hexanediol. The addition of different volumes of dispersants increased the number of EV particles, as shown in the graphs. Figure 3 (A) Figure 3 (B) Figure 3 (C) Figure 3 (D) Figure 3 (E) Figure 3 (F) Figure 3 (G) Figure 3 (H).

[0053] Figure 4The graph shows the changes in particle concentration of milk extract solution after adding different dispersants, with acetic acid as the precipitant. The dispersants include poloxamer 188, arginine hydrochloride, proline, lysine, histidine, magnesium chloride, magnesium sulfate, and EDTA. Adding different volumes of dispersant increases the number of EV particles; excessive amounts cause turbidity in the supernatant, leading to filtration difficulties. See below for details. Figure 4 (A) Figure 4 (B) Figure 4 (C).

[0054] Figure 5 The graph shows the change in particle concentration of milk extract solution after adding different dispersants, with citric acid as the precipitant. The dispersants include poloxamer 188, arginine hydrochloride, proline, lysine, histidine, magnesium chloride, magnesium sulfate, and EDTA.

[0055] Figure 6 The graph shows the change in particle concentration of milk extract solution after adding different dispersants as a pepsin (porcine) precipitant. The dispersants include poloxamer 188, arginine hydrochloride, proline, lysine, histidine, magnesium chloride, magnesium sulfate, and EDTA.

[0056] Figure 7 The graph shows the change in particle concentration of milk extract solution after adding different dispersants, which are used as bromelain precipitants. The dispersants include poloxamer 188, arginine hydrochloride, proline, lysine, histidine, magnesium chloride, magnesium sulfate, and EDTA.

[0057] Figure 8 Electron micrographs of milk extract (including vesicles of different sizes), in which... Figure 8 (A) is a transmission electron microscope image showing the results of uranium acetate staining; Figure 8 (B) Transmission electron microscopy results of phosphotungstic acid staining, showing that different staining agents were used to produce large and small extracellular vesicles in the transmission electron microscopy images.

[0058] Figure 9 Western blot (WB) analysis of extracellular vesicle characteristic proteins (CD81, Alix, TSG101) from milk extract.

[0059] Figure 10 This is a transmission electron microscope (TEM) image of a goat milk extract stained with uranium acetate. The TEM image shows clearly visible large and small extracellular vesicles. Detailed Implementation

[0060] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0061] This invention discloses a method for preparing milk extracts. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to test and apply the technology of this invention.

[0062] The present invention will be further illustrated below with reference to the embodiments:

[0063] Example 1: Different dispersants added before saturated ammonium sulfate precipitation.

[0064] Dispersant solutions were prepared using purified water as follows: 10% threonine, 10% serine, 10% proline, 10% glycine, 10% alanine, 10% cysteine, 2% valine, 2% methionine, 2% isoleucine, 2% phenylalanine, 10% lysine, 10% arginine hydrochloride, 2% histidine, 10% citrulline, 2% ornithine, 10% Tween 40, 10% Tween 60, 10% Tween 80, 10% poloxamer 188, 2M magnesium chloride, 2M magnesium sulfate, and 0.1M... EDTA and 10% hexanediol aqueous solution were added to 3 mL of skim milk and each of the above dispersant solutions. After mixing thoroughly, the mixture was left to stand at room temperature for 10 min. Then, 2 mL of saturated ammonium sulfate solution was added to each of the precipitant solutions. After mixing thoroughly, the mixture was left to stand at room temperature for 10 min. The mixture was then centrifuged at 4000g for 20 min. The supernatant was collected and the particle number was determined using a nanofluid cytometer (N30E type, Xiamen Fulu Biotechnology Co., Ltd.). The particle change trend after adding different volumes of each dispersant was investigated. The experimental addition amounts are shown in the table below.

[0065] Table 1. Different dispersant dosages in saturated ammonium sulfate precipitation

[0066]

[0067] Table 2. Yield of different dispersants in saturated ammonium sulfate precipitation.

[0068]

[0069]

[0070] Note: Percentage increase in particle count = (Number of particles in test n / Number of particles in test 1 - 1) × 100%, where n is 2, 3, 4, 5, 6 or 7.

[0071] from Figure 3As shown in Table 2, the addition of different dispersants can improve the extraction efficiency of extracellular vesicles (EVs), i.e., increase the percentage of particle count. The following calculation method for the total volume ratio of dispersant to milk and precipitant is: Total volume ratio of dispersant to milk and precipitant (%) = Dispersant volume / (Milk volume + Precipitant volume) * 100%.

[0072] For example, when 10% threonine solution was added, and the total volume ratio of milk and precipitant was 3.6% (i.e., the final concentration of threonine was 0.36 g / 100 ml), the number of particles in the extract solution increased by 76.8% (compared to the number of particles without the addition of dispersant).

[0073] When 10% serine solution was added, the number of particles in the extract solution increased by 67.7% (compared to the number of particles without the addition of dispersant) when the total volume ratio of serine solution to milk and precipitant was 3.6% (i.e., the final serine concentration was 0.36 g / 100 ml).

[0074] When 10% proline solution was added, the number of particles in the extract solution increased by 82% when the total volume ratio of proline solution to milk and precipitant was 3.6% (i.e., the final proline concentration was 0.36 g / 100 ml).

[0075] When 10% glycine solution was added, the number of particles in the extract solution increased by 78.5% (compared to the number of particles without the addition of dispersant) when the total volume ratio of the extract to milk and precipitant was 3.0% (i.e., the final glycine concentration was 0.30 g / 100 ml).

[0076] When 10% alanine solution was added, the number of particles in the extract solution increased by 67.2% (compared to the number of particles without the addition of dispersant) when the total volume ratio of alanine to milk and precipitant was 3.0% (i.e., the final concentration of alanine was 0.30 g / 100 ml).

[0077] When 10% cysteine ​​solution was added, the number of particles in the extract solution increased by 65.7% (compared to the number of particles without the addition of dispersant) when the total volume ratio of milk and precipitant was 3.0% (i.e., the final cysteine ​​concentration was 0.30 g / 100 ml).

[0078] When 2% valine solution was added, the number of particles in the extract solution increased by 98.7% (compared to the number of particles without dispersant) when the total volume ratio of valine solution to milk and precipitant was 5.0% (i.e., the final concentration of valine was 0.10 g / 100 ml).

[0079] When 2% methionine solution was added, the number of particles in the extract solution increased by 107.1% (compared to the number of particles without the addition of dispersant) when the total volume ratio of milk and precipitant was 4.0% (i.e., the final concentration of methionine was 0.08 g / 100 ml).

[0080] When 2% isoleucine solution was added, the number of particles in the extract solution increased by 71.7% (compared to the number of particles without dispersant) when the total volume ratio of milk and precipitant was 4.0% (i.e., the final concentration of isoleucine was 0.08 g / 100 ml).

[0081] When 10% lysine solution was added, the number of particles in the extract solution increased by 178% when the total volume ratio of lysine to milk and precipitant was 4.0% (i.e., the final concentration of lysine was 0.40 g / 100 ml).

[0082] When 10% arginine hydrochloride solution was added, the number of particles in the extract solution increased by 87.8% when the total volume ratio of arginine hydrochloride to milk and precipitant was 2.4% (i.e., the final concentration of arginine hydrochloride was 0.24 g / 100 ml).

[0083] When 10% citrulline hydrochloride solution was added, the number of particles in the extract solution increased by 89.4% when the total volume ratio of milk and precipitant was 3.0% (i.e., the final concentration of citrulline hydrochloride was 0.30 g / 100 ml).

[0084] When 2% phenylalanine solution was added, the number of particles in the extract solution increased by 129.5% (compared to the number of particles without the addition of dispersant) when the total volume ratio of phenylalanine to milk and precipitant was 5.0% (i.e., the final concentration of phenylalanine was 0.10 g / 100 ml).

[0085] When 2% histidine solution was added, the number of particles in the extract solution increased by 378.9% when the total volume ratio of histidine to milk and precipitant was 6.0% (i.e., the final concentration of histidine was 0.12 g / 100 ml).

[0086] When 2% ornithine solution was added, the number of particles in the extract solution increased by 105.5% (compared to the number of particles without the addition of dispersant) when the total volume ratio of ornithine to milk and precipitant was 4.0% (i.e., the final concentration of ornithine was 0.08 g / 100 ml).

[0087] When 10% Tween 40 solution was added, the number of particles in the extract solution increased by 50.1% when the total volume ratio of milk and precipitant was 2.4% (i.e., the final concentration of Tween 40 was 0.24 g / 100 ml).

[0088] When 10% Tween 60 solution was added, the number of particles in the extract solution increased by 45.9% when the total volume ratio of milk and precipitant was 2.4% (i.e., the final concentration of Tween 60 was 0.24 g / 100 ml).

[0089] When 10% Tween 80 solution was added, the number of particles in the extract solution increased by 54.3% when the total volume ratio of milk and precipitant was 2.4% (i.e., the final concentration of Tween 80 was 0.24 g / 100 ml).

[0090] When 10% poloxamer 188 solution was added, the number of particles in the extract solution increased by 24% (compared to the number of particles without the addition of dispersant) when the total volume ratio of milk and precipitant was 2.0% (i.e., the final concentration of poloxamer 188 was 0.20 g / 100 ml).

[0091] When 2 mol / L magnesium chloride solution was added, the number of particles in the extraction solution increased by 96.5% when the total volume ratio of magnesium chloride to milk and precipitant was 4.0% (i.e., the final concentration of magnesium chloride was 0.08 mol / L).

[0092] When 2 mol / L magnesium sulfate solution was added, the number of particles in the extraction solution increased by 106.7% when the total volume ratio of magnesium sulfate to milk and precipitant was 2.4% (i.e., the final concentration of magnesium sulfate was 0.048 mol / L).

[0093] When 0.1 mol / L EDTA solution was added, the number of particles in the extraction solution increased by 49.9% when the total volume ratio of EDTA solution to milk and precipitant was 2.4% (i.e., the final EDTA concentration was 0.00024 mol / L).

[0094] When 10% hexanediol solution was added, the number of particles in the extract solution increased by 58.1% (compared to the number of particles without the addition of dispersant) when the total volume ratio of hexanediol to milk and precipitant was 2.4% (i.e., the final concentration of hexanediol was 0.24 g / 100 ml).

[0095] This indicates that the addition of the above dispersants can significantly increase the number of extracellular vesicles (EVs) in the extract.

[0096] Example 2: Comparison of precipitation from different acids in milk

[0097] Add 5 mL of skim milk to 10 mL centrifuge tubes. Adjust the pH of the skim milk to 4.6–4.8 using different acid solutions, such as hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, lactic acid, and malic acid. Shake to mix thoroughly, let stand at room temperature for 20 minutes, then centrifuge at 10°C for 4000 g for 20 minutes. Collect the supernatant and determine the particle count using a nanoflow cytometer. The results are shown in the table below and appendix. Figure 1 .

[0098] Table 3. Results of particle number and particle size distribution in milk extracts precipitated by different acids.

[0099]

[0100] from Figure 1 As can be seen from Table 3, there was no significant difference in the number of particles and particle size distribution of milk extracts obtained by precipitation with different acids.

[0101] Example 3: Comparison of precipitation by different rennets in milk

[0102] In a 10mL centrifuge tube, take 5mL of skim milk and add different rennets (2% aqueous solution, incubated at 37℃ for 30min before use; prepare fresh before use to ensure enzyme activity), such as pepsin (porcine), pepsin (bovine), papain, bromelain, fig protease, and Hualing microbial rennet. (Due to the different enzyme activities of the reagents, select enzymes with an activity of not less than 100,000 U / g and an addition amount not exceeding 150μL). Shake to mix thoroughly, incubate at 30-45℃ for 30 minutes to allow coagulation, centrifuge at 4000g for 20min, and use a nanofluid cytometer to determine the particle count of the supernatant. The results are shown in the table below and appendix. Figure 2 .

[0103] Table 4. Results of particle number and particle size distribution in milk extracts precipitated by different rennets.

[0104]

[0105] from Figure 2 As can be seen from Table 4, there were no significant differences in the number of particles and particle size distribution among milk extracts obtained by precipitation with different rennets.

[0106] Example 4: Acetic acid precipitation - different dispersants added before precipitation

[0107] Dispersant solutions of 10% poloxamer 188, 10% arginine hydrochloride, 10% proline, 2M magnesium chloride, 2M magnesium sulfate, 0.1M EDTA, 10% lysine, and 2% histidine were prepared using purified water. 3 mL of skim milk was added to each of the above dispersant solutions, mixed thoroughly, and allowed to stand at room temperature for 10 min. A few drops of dilute acetic acid solution (to adjust the pH to 4.6-4.8) were added, mixed thoroughly, allowed to stand at room temperature for 10 min, and then centrifuged at 4000g for 20 min. The supernatant was collected and the particle number was determined using a nanofluid cytometer. The particle size distribution was investigated after adding different volumes of each dispersant. The experimental amounts are shown in the table below.

[0108] Table 5. Different dispersant dosages in acetic acid precipitation

[0109] name Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 milk 3mL 3mL 3mL 3mL 3mL 3mL 3mL 10% Poloxamer 188 0μL 50μL 100μL 120μL 150μL 180μL 200μL 10% Arginine Hydrochloride 0μL 50μL 100μL 120μL 150μL 180μL 200μL 10% proline 0μL 50μL 100μL 120μL 150μL 180μL 200μL 2M magnesium chloride 0μL 50μL 100μL 120μL 150μL 180μL 200μL 2M magnesium sulfate 0μL 50μL 100μL 120μL 150μL 180μL 200μL 0.1M EDTA 0μL 50μL 100μL 120μL 150μL 180μL 200μL 10% Lysine 0μL 50μL 100μL 120μL 150μL 180μL 200μL 2% histidine 0μL 100μL 150μL 200μL 250μL 300μL 400μL

[0110] Table 6. Results of changes in particle yield of different dispersants in acetic acid precipitation.

[0111] name Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 milk 3mL 3mL 3mL 3mL 3mL 3mL 3mL 10% Poloxamer 188 100.0％ 104.3％ 120.5％ 112.4％ 82.0％ 85.7％ 84.5％ 10% Arginine Hydrochloride 100.0％ 95.0％ 101.9％ 117.4％ 126.7％ 97.5％ 83.2％ 10% proline 100.0％ 113.0％ 117.4％ 134.2％ 142.2％ 93.8％ 94.4％ 2M magnesium chloride 100.0％ 111.2％ 105.0％ 114.3％ 112.4％ 131.7％ 109.3％ 2M magnesium sulfate 100.0％ 112.4％ 111.2％ 126.7％ 94.4％ 98.8％ 108.1％ 0.1M EDTA 100.0％ 105.6％ 114.3％ 95.7％ 76.4％ 77.0％ 75.8％ 10% Lysine 100.0％ 110.6％ 128.6％ 108.7％ 114.3％ 96.9％ 100.0％ 2% histidine 100.0％ 95.7％ 116.1％ 151.6％ 134.2％ 124.8％ 119.3％

[0112] Note: Percentage increase in particle count = (Number of particles in test n / Number of particles in test 1 - 1) × 100%, where n is 2, 3, 4 or 5.

[0113] from Figure 4As can be seen from Table 6:

[0114] When 10% poloxamer 188 solution was added, the number of particles in the extract solution increased by 20.5% (compared to the number of particles without the addition of dispersant) when the total volume ratio of milk and precipitant was 3.3% (i.e., the final concentration of poloxamer 188 was 0.33 g / 100 ml).

[0115] When 10% arginine hydrochloride solution was added, the number of particles in the extract solution increased by 26.7% (compared to the number of particles without the addition of dispersant) when the total volume ratio of milk and precipitant was 5.0% (i.e., the final concentration of arginine hydrochloride was 0.50 g / 100 ml).

[0116] When 10% proline solution was added, the number of particles in the extract solution increased by 42.2% when the total volume ratio of proline solution to milk and precipitant was 5.0% (i.e., the final concentration of proline was 0.50 g / 100 ml).

[0117] When 2 mol / L magnesium chloride solution was added, the number of particles in the extraction solution increased by 31.7% when the total volume ratio of magnesium chloride to milk and precipitant was 6.0% (i.e., the final concentration of magnesium chloride was 0.12 mol / L).

[0118] When 2 mol / L magnesium sulfate solution was added, the number of particles in the extraction solution increased by 26.7% when the total volume ratio of magnesium sulfate to milk and precipitant was 4.0% (i.e., the final concentration of magnesium sulfate was 0.08 mol / L).

[0119] When 0.1 mol / L EDTA solution was added, the number of particles in the extraction solution increased by 14.3% when the total volume ratio of EDTA solution to milk and precipitant was 3.3% (i.e., the final EDTA concentration was 0.0033 mol / L).

[0120] When 10% lysine solution was added, the number of particles in the extract solution increased by 28.6% when the total volume ratio of lysine to milk and precipitant was 3.3% (i.e., the final concentration of lysine was 0.33 g / 100 ml).

[0121] When 2% histidine solution was added, the number of particles in the extract solution increased by 51.6% when the total volume ratio of histidine to milk and precipitant was 6.7% (i.e., the final concentration of histidine was 0.13 g / 100 ml).

[0122] This indicates that the addition of the above dispersants can significantly increase the number of extracellular vesicle particles in the extract.

[0123] Example 5: Citric Acid Precipitation - Different Dispersants Added Before Precipitation

[0124] Dispersant solutions of 10% poloxamer 188, 10% arginine hydrochloride, 10% proline, 2M magnesium chloride, 2M magnesium sulfate, 0.1M EDTA, 10% lysine, and 2% histidine were prepared using purified water. 3 mL of skim milk was added to each of the above dispersant solutions, mixed thoroughly, and allowed to stand at room temperature for 10 min. A few drops of 20% citric acid solution (to adjust the final pH to 4.6-4.8) were added, mixed thoroughly, allowed to stand at room temperature for 10 min, and then centrifuged at 4000g for 20 min. The supernatant was collected and the particle number was determined using a nanofluid cytometer. The particle change trend after adding different volumes of each dispersant was investigated. The experimental addition amounts were the same as in Table 5. The yield changes of different dispersant particles in citric acid precipitation are shown in the table below.

[0125] Table 7 Results of changes in particle yield of different dispersants in citric acid precipitation.

[0126]

[0127] Note: Percentage increase in particle count = (Number of particles in test n / Number of particles in test 1 - 1) × 100%, where n is 2, 3, 4 or 5.

[0128] from Figure 5 As can be seen from Table 7:

[0129] When 10% poloxamer 188 solution was added, the number of particles in the extract solution increased by 17.9% (compared to the number of particles without the addition of dispersant) when the total volume ratio of milk and precipitant was 4.0% (i.e., the final concentration of poloxamer 188 was 0.40 g / 100 ml).

[0130] When 10% arginine hydrochloride solution was added, the number of particles in the extract solution increased by 21.6% (compared to the number of particles without the addition of dispersant) when the total volume ratio of arginine hydrochloride to milk and precipitant was 5.0% (i.e., the final concentration of arginine hydrochloride was 0.50 g / 100 ml).

[0131] When 10% proline solution was added, the number of particles in the extract solution increased by 34.0% when the total volume ratio of proline solution to milk and precipitant was 5.0% (i.e., the final concentration of proline was 0.50 g / 100 ml).

[0132] When 2 mol / L magnesium chloride solution was added, the number of particles in the extraction solution increased by 24.1% when the total volume ratio of magnesium chloride to milk and precipitant was 6.0% (i.e., the final concentration of magnesium chloride was 0.12 mol / L).

[0133] When 2 mol / L magnesium sulfate solution was added, the number of particles in the extraction solution increased by 26.5% when the total volume ratio of magnesium sulfate to milk and precipitant was 4.0% (i.e., the final concentration of magnesium sulfate was 0.08 mol / L).

[0134] When 0.1 mol / L EDTA solution was added, the number of particles in the extraction solution increased by 12.3% when the total volume ratio of EDTA solution to milk and precipitant was 3.3% (i.e., the final EDTA concentration was 0.0033 mol / L).

[0135] When 10% lysine solution was added, the number of particles in the extraction solution increased by 31.5% when the total volume ratio of lysine to milk and precipitant was 3.3% (i.e., the final concentration of lysine was 0.33 g / 100 ml).

[0136] When 2% histidine solution was added, the number of particles in the extract solution increased by 36.4% when the total volume ratio of histidine to milk and precipitant was 6.7% (i.e., the final concentration of histidine was 0.13 g / 100 ml).

[0137] This indicates that the addition of the above dispersants can significantly increase the number of extracellular vesicle particles in the extract.

[0138] Example 6: Adding different dispersants before pepsin (porcine-derived) precipitation

[0139] Dispersant solutions of 10% poloxamer 188, 10% arginine hydrochloride, 10% proline, 2M magnesium chloride, 2M magnesium sulfate, 0.1M EDTA, 10% lysine, and 2% histidine were prepared using purified water. 3 mL of skim milk was added to each of the above dispersant solutions, mixed thoroughly, and allowed to stand at room temperature for 10 min. Then, 50 μL of 2% pepsin (porcine-derived) precipitant solution was added, mixed thoroughly, and allowed to coagulate in a 37℃ incubator for 30 min. The mixture was then centrifuged at 4000g for 20 min, and the supernatant was used to determine the number of particles using a nanofluid cytometer. The particle change trend after adding different volumes of each dispersant was investigated. The experimental addition amounts were the same as in Table 5. The yield changes of different dispersant particles in the pepsin (porcine-derived) precipitate are shown in the table below.

[0140] Table 8. Results of yield changes of different dispersants in pepsin (porcine-derived) precipitate.

[0141]

[0142]

[0143] Note: Percentage increase in particle count = (Number of particles in test n / Number of particles in test 1 - 1) × 100%, where n is 2, 3 or 4.

[0144] from Figure 6 As can be seen from Table 8:

[0145] When 10% poloxamer 188 solution was added, the number of particles in the extract solution increased by 15.9% (compared to the number of particles without the addition of dispersant) when the total volume ratio of milk and precipitant was 3.3% (i.e., the final concentration of poloxamer 188 was 0.33 g / 100 ml).

[0146] When 10% arginine hydrochloride solution was added, the number of particles in the extract solution increased by 20.7% (compared to the number of particles without the addition of dispersant) when the total volume ratio of arginine hydrochloride to milk and precipitant was 4.0% (i.e., the final concentration of arginine hydrochloride was 0.40 g / 100 ml).

[0147] When 10% proline solution was added, the number of particles in the extract solution increased by 19.1% when the total volume ratio of proline solution to milk and precipitant was 4.0% (i.e., the final proline concentration was 0.40 g / 100 ml).

[0148] When 2 mol / L magnesium chloride solution was added, the number of particles in the extraction solution increased by 12.6% when the total volume ratio of magnesium chloride to milk and precipitant was 1.7% (i.e., the final concentration of magnesium chloride was 0.03 mol / L).

[0149] When 2 mol / L magnesium sulfate solution was added, the number of particles in the extraction solution increased by 13.4% when the total volume ratio of magnesium sulfate to milk and precipitant was 1.7% (i.e., the final concentration of magnesium sulfate was 0.03 mol / L).

[0150] When 0.1 mol / L EDTA solution was added, the number of particles in the extraction solution increased by 11.0% when the total volume ratio of EDTA solution to milk and precipitant was 4.0% (i.e., the final EDTA concentration was 0.004 mol / L).

[0151] When 10% lysine solution was added, the number of particles in the extract solution increased by 16.7% when the total volume ratio of lysine to milk and precipitant was 3.3% (i.e., the final concentration of lysine was 0.33 g / 100 ml).

[0152] When 2% histidine solution was added, the number of particles in the extract solution increased by 16.3% when the total volume ratio of histidine to milk and precipitant was 5.0% (i.e., the final concentration of histidine was 0.10 g / 100 ml).

[0153] This indicates that the addition of the above dispersants can significantly increase the number of extracellular vesicle particles in the extract.

[0154] Example 7: Adding different dispersants before precipitating bromelain

[0155] Dispersant solutions of 10% poloxamer 188, 10% arginine hydrochloride, 10% proline, 2M magnesium chloride, 2M magnesium sulfate, 0.1M EDTA, 10% lysine, and 2% histidine were prepared using purified water. 3 mL of skim milk was added to each of the above dispersant solutions, mixed thoroughly, and allowed to stand at room temperature for 10 min. Then, 100 μL of 2% bromelain solution was added as a precipitant, mixed thoroughly, and allowed to coagulate in a 37℃ incubator for 30 min. The mixture was then centrifuged at 4000g for 20 min, and the supernatant was used to determine the number of particles using a nanofluid cytometer. The particle change trend after adding different volumes of each dispersant was investigated. The experimental addition amounts were the same as in Table 5. The yield changes of different dispersant particles in the bromelain precipitate are shown in the table below.

[0156] Table 9. Results of changes in particle yield of different dispersants in bromelain precipitation.

[0157]

[0158] Note: Percentage increase in particle count = (Number of particles in test n / Number of particles in test 1 - 1) × 100%, where n is 2, 3, 4 or 5.

[0159] from Figure 7 As can be seen from Table 9, the number of EV particles in the obtained milk extract was significantly increased after the addition of the dispersant.

[0160] When 10% poloxamer 188 solution was added, the number of particles in the extract solution increased by 9.3% (compared to the number of particles without the addition of dispersant) when the total volume ratio of milk and precipitant was 3.3% (i.e., the final concentration of poloxamer 188 was 0.33 g / 100 ml).

[0161] When 10% arginine hydrochloride solution was added, the number of particles in the extract solution increased by 11.7% (compared to the number of particles without the addition of dispersant) when the total volume ratio of arginine hydrochloride to milk and precipitant was 4.0% (i.e., the final concentration of arginine hydrochloride was 0.40 g / 100 ml).

[0162] When 10% proline solution was added, the number of particles in the extract solution increased by 11.7% when the total volume ratio of proline solution to milk and precipitant was 4.0% (i.e., the final concentration of proline was 0.40 g / 100 ml).

[0163] When 2 mol / L magnesium chloride solution was added, the number of particles in the extraction solution increased by 9.7% when the total volume ratio of magnesium chloride to milk and precipitant was 1.7% (i.e., the final concentration of magnesium chloride was 0.03 mol / L).

[0164] When 2 mol / L magnesium sulfate solution was added, the number of particles in the extraction solution increased by 7.4% when the total volume ratio of magnesium sulfate to milk and precipitant was 1.7% (i.e., the final concentration of magnesium sulfate was 0.03 mol / L).

[0165] When 0.1 mol / L EDTA solution was added, the number of particles in the extraction solution increased by 8.6% when the total volume ratio of EDTA solution to milk and precipitant was 4.0% (i.e., the final EDTA concentration was 0.004 mol / L).

[0166] When 10% lysine solution was added, the number of particles in the extract solution increased by 10.1% when the total volume ratio of lysine to milk and precipitant was 3.3% (i.e., the final concentration of lysine was 0.33 g / 100 ml).

[0167] When 2% histidine solution was added, the number of particles in the extract solution increased by 12.5% ​​when the total volume ratio of histidine to milk and precipitant was 5.0% (i.e., the final concentration of histidine was 0.10 g / 100 ml).

[0168] This indicates that the addition of the above dispersants can increase the number of extracellular vesicle particles in the extract.

[0169] Example 8: Characterization of extracellular vesicles in milk extract

[0170] The prominent features of this milk extract are the presence of whey protein and extracellular vesicle (10-1000nm) particles. The structure of the extracellular vesicles was observed using transmission electron microscopy, and the characteristic proteins of the extracellular vesicles in the milk extract were identified using Western blotting (WB).

[0171] Transmission electron microscopy characterization:

[0172] Sample preparation: Prepare milk extract 10g, add ultrapure water 40g, vortex mix, and centrifuge briefly.

[0173] Preparation of the copper mesh for electron microscopy: Remove the staining solution (uranyl acetate) from the brown ampoule and place it in a 1.5 mL centrifuge tube (wrapped in aluminum foil to protect from light), and centrifuge at 12000 rpm for 1 min. Use tweezers to remove the copper mesh for electron microscopy onto a glass culture dish, face up, and place it in a plasma cleaner for 5 min.

[0174] Copper mesh sample preparation: Take a small piece of sealing film, apply 15g of the sample to the sealing film, and carefully pick up the copper mesh with tweezers so that the front side is attached to the sample. Time for 10 minutes. Apply 15g of each dye solution to the bottom of each sample, 3 sets. After time is up, use tweezers to pick up the copper mesh, blot it dry on filter paper, dip it into the first set of dye solutions, and blot it dry with filter paper; repeat the operation for the second set, blotting dry; place the copper mesh on the third set of dye solutions, and time for 10 minutes. Use tweezers to pick up the copper mesh, blot it dry on filter paper, place the copper mesh on a plate, and store at room temperature.

[0175] Photography: The prepared copper mesh is loaded into the electron microscope feed rod and placed into the electron microscope to photograph the same field of view.

[0176] Using uranium acetate staining, the extracellular vesicles photographed showed cup-shaped structures and clearly contained both large and small vesicles (see attached image). Figure 8 (A)

[0177] In the above sample preparation, the sample was mixed with an equal volume of tissue cell fixative (2% PFA / 2.5% Gluta) and refrigerated overnight (5℃±3℃). The next day, the staining agent phosphotungstic acid (2% aqueous solution, replacing uranium acetate) was used in the same procedure as in the "copper mesh sample preparation". Using phosphotungstic acid staining, the outlines of extracellular vesicles against a "black" background were clearly visible, showing both large and small vesicles (see attached image). Figure 8 (B)

[0178] Western blotting (WB) characterization:

[0179] The WB measurement results are attached. Figure 9 Milk extract contains extracellular vesicle characteristic membrane protein CD81, and intracellular proteins Alix and TSG101.

[0180] Example 9: Transmission electron microscopy characterization of extracellular vesicles in goat milk extract

[0181] Fresh goat milk was centrifuged at 4000g for 10 minutes to remove the supernatant fat. Histidine dispersant solution was added to the skimmed goat milk, and the pH was adjusted to 4.6-4.8 with acetic acid solution to remove the precipitate. The goat milk extract solution rich in extracellular vesicles was collected. Following the "Transmission Electron Microscopy Characterization" procedure in Example 8, the sample was stained with uranium acetate. The photographed goat milk extract showed extracellular vesicles with cup-shaped structures, and clearly contained both large and small vesicles (see attached image). Figure 10 .

Claims

1. A method for isolating and / or enriching extracellular vesicles, comprising: 1) Provide a milk source, optionally a defatted milk source; 2) Contact the emulsion source with the dispersant to obtain a mixture of the emulsion source and the dispersant; 3) Contact the mixture obtained in step 2) with the precipitant to form a precipitate; and 4) Separate the precipitate to obtain a composition rich in extracellular vesicles.

2. The method according to claim 1, wherein the degreasing treatment is achieved by filtration, centrifugation and / or placement.

3. The method according to claim 1 or 2, wherein the dispersant is selected from amphoteric molecules, surfactants, divalent metal salts, chelating agents, and C2-C8 alcohols.

4. The method according to claim 3, wherein the amphoteric molecule is an amino acid and / or its salt, for example, the amino acid is selected from arginine, proline, lysine, histidine, threonine, serine, glycine, alanine, cysteine, valine, methionine, isoleucine, phenylalanine, tryptophan, citrulline, and ornithine.

5. The method according to claim 3, wherein the surfactant is an amphoteric surfactant, a nonionic surfactant, an anionic surfactant, and / or a cationic surfactant, for example, the amphoteric surfactant includes amino acid-type amphoteric surfactants and betaine-type amphoteric surfactants; the nonionic surfactant is selected from polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80), polyoxyethylene-polyoxypropylene copolymer, and poloxamer 188; the anionic surfactant includes carboxylate-type anionic surfactants, sulfate-type anionic surfactants, and sulfonate-type anionic surfactants; and / or the cationic surfactant includes quaternary ammonium-type cationic surfactants, imidazoline-type cationic surfactants, and amine-type cationic surfactants.

6. The method according to claim 3, wherein the divalent metal salt is a magnesium salt, a zinc salt, and / or a calcium salt, specifically, for example, the magnesium salt is magnesium sulfate, zinc sulfate, calcium chloride, and / or magnesium chloride.

7. The method according to claim 3, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA), nitric acid triacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and / or a salt thereof.

8. The method according to claim 3, wherein the C2-C8 alcohol is a C2-C8 diol, such as hexanediol.

9. The method according to any one of claims 1 to 8, wherein the final concentration of the dispersant is 0.01 g / ml to 1.0 g / ml, preferably 0.04 g / 100ml to 0.67 g / 100ml, more preferably 0.08 g / 100ml to 0.50 g / ml, for example 0.08 g / 100ml, 0.10 g / 100ml, 0.12 g / 100ml, 0.13 g / 100ml, 0.20 g / 100ml, 0.24 g / 100ml, 0.30 g / 100ml, 0.33 g / 100ml, 0.36 g / 100ml, 0.40 g / 100ml, or 0.50 g / 100ml.

10. The method according to any one of claims 1 to 8, wherein the final concentration of the dispersant is 0.001 mol / L to 0.13 mol / L, preferably 0.0033 mol / L to 0.12 mol / L, for example 0.0033 mol / L, 0.004 mol / L, 0.03 mol / L, 0.08 mol / L, or 0.12 mol / L.

11. The method according to any one of claims 1 to 10, wherein the precipitant is selected from salting-out precipitants, isoelectric point precipitants, and rennet precipitants.

12. The method of claim 11, wherein the salting-out precipitant, such as ammonium sulfate, is used, and the pH of the mixture and / or composition is adjusted to 4.8-5.

8.

13. The method of claim 11, wherein the isoelectric point precipitant, such as an acid, is used, preferably selected from hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, lactic acid, and malic acid.

14. The method of claim 13, wherein the isoelectric point precipitant is added to adjust the pH to 4.6-4.

8.

15. The method according to claim 11, wherein the rennet is selected from animal rennet, plant rennet, and microbial rennet; for example, the animal rennet is selected from porcine pepsin and bovine pepsin, and the plant rennet is selected from papain, bromelain, and figase.

16. The method according to any one of claims 1 to 15, further comprising concentrating the composition rich in extracellular vesicles; for example, using ultrafiltration, dialysis or microfiltration; specifically, the ultrafiltration is tangential flow ultrafiltration, for example, the tangential flow ultrafiltration membrane assembly used in the tangential flow ultrafiltration is a flat sheet ultrafiltration assembly and / or a hollow fiber ultrafiltration assembly; and specifically, the microfiltration employs a microporous membrane with a pore size of 1 μm.

17. A composition rich in extracellular vesicles obtained by the method of any one of claims 1 to 16.

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