Exosome extraction method based on plant juice characteristics

Abstract

The invention discloses an exosome extraction method based on plant juice characteristics. The method comprises the following steps: firstly, according to the characteristics of plant juice, dividing samples into four types, namely a clear low-viscosity type sample, a medium-thick type sample, a high-viscosity type sample and a high-fiber type sample, and adopting different pretreatment modes: washing the clear type sample, cutting into blocks, and directly juicing; performing enzymolysis after medium-concentration washing and juicing; performing enzymolysis after high-viscosity homogenate juicing; and the high-fiber type is firstly subjected to enzymolysis and softening, and then homogenate and squeeze juice. Removing impurities through two-step differential centrifugation, and finally obtaining the high-purity exosome through ultrafiltration concentration, PEG precipitation, PBS resuspension and filtration. According to the method disclosed by the invention, various means such as physical crushing, enzymolysis, differential centrifugation, ultrafiltration and precipitation are comprehensively utilized, so that the yield and purity of the exosome are greatly improved, the varieties of plants for preparing the exosome are widened, and a technical support is provided for application research of the plant exosome.

Description

Technical Field

[0001] The present invention relates to the technical field of exosomes, and in particular to a method for extracting exosomes based on the characteristics of plant juice. Background Art

[0002] Exosomes are nano-scale membrane vesicles secreted by cells that can carry proteins, RNA, lipids and other molecules to participate in intercellular signaling. In recent years, plant exosomes have become a hot topic of research as an important medium for the interaction between plant cells and the external environment and other plant cells. Plant exosomes play an irreplaceable and important role in plant growth, development and stress response. This tiny but powerful cell communication tool is gradually revealing its deep-seated significance in life activities.

[0003] However, due to the complexity and diversity of plant tissue structure, the separation and purification of exosomes from plants has always faced many technical bottlenecks. The most widely used ultracentrifugation method currently has inherent defects such as cumbersome operation, long time consumption, large sample loss, and low purity. Especially for plant samples with high viscosity and high cellulose content, ultracentrifugation is difficult to effectively break the cell wall, resulting in low exosome recovery rate.

[0004] Therefore, it is of great significance to provide an efficient, targeted plant exosome extraction method that can improve the purity of exosomes. The key to new technological innovation lies in the systematic and refined optimization of each link from sample pretreatment, centrifugation to concentration and purification. By deeply analyzing the physicochemical properties of plant juices, building a differentiated and personalized extraction process, and combining multiple methods, the extraction yield and quality of exosomes can be significantly improved, and their biological integrity and functional characteristics can be protected to the greatest extent. Summary of the invention

[0005] The purpose of the present invention is to provide an exosome extraction method based on the characteristics of plant juice to solve the limitations of existing plant exosome separation and purification methods.

[0006] A method for extracting exosomes based on the characteristics of plant juice, the method comprising the following steps: 1) Pretreatment of plant samples: Plant samples were classified according to the characteristics of the juice: a. clear and low-viscosity plants, b. medium-viscosity plants, c. high-viscosity plants, and d. high-fiber plants; After classification, washing, cutting, enzymolysis, homogenization and juicing pretreatment are carried out respectively; 2) Two-step differential centrifugation to remove impurities, centrifuging the extracted plant juices in sequence; 3) Enzymatic treatment: Enzymatic treatment of medium-viscous plant juice, high-viscous plant juice and high-fiber plant juice; 4) Ultrafiltration and concentration: Filter the enzymatically hydrolyzed plant juice through a membrane filter and use an ultrafiltration membrane to concentrate the plant juice; 5) Polymer precipitation: Add polyethylene glycol (PEG) to the concentrated plant juice, let it stand and then centrifuge it. Use phosphate buffered saline (PBS) to resuspend the precipitate to obtain the exosome sample.

[0007] Preferably, the plant samples are classified into the following four categories: a. Clear and low-viscosity plants: high juice yield, clear and transparent liquid, representative plants include cucumbers, watermelons, and tomatoes; b. Medium viscous plants: moderate juice yield, slightly turbid liquid, representative plants include apples, pears, and grapes; c. High viscosity plants: The amount of juice produced is small and the liquid is viscous. Representative plants include aloe vera, seaweed, and berries; d. High-fiber plants: They are difficult to produce juice and have high fiber content. Representative plants include broccoli, celery, and root vegetables.

[0008] Preferably, for clear and low-viscosity plants, the juice is directly extracted by cold pressing after washing and cutting without enzymatic treatment; for medium-viscosity plants, enzymatic hydrolysis is performed after differential centrifugation; for high-viscosity plants, phosphate buffer is first added for dilution, the juice is extracted by homogenization, and then enzymatic hydrolysis is performed; for high-fiber plants, pre-enzymatic hydrolysis is required to soften the tissue before homogenization.

[0009] Preferably, for high-fiber plants, the tissue needs to be pre-enzymatically softened before homogenization, and one or more of cellulase and xylanase are selected at a concentration of 0.3%-1%, the enzymatic hydrolysis temperature is 37° C., and the time is 1-2 hours.

[0010] Preferably, the low-speed centrifugation is carried out at 4°C, 3000-6000 rpm, for 20-40 minutes, and the supernatant is collected; the high-speed centrifugation is carried out at 4°C, 10,000-12,000 rpm, for 20-40 minutes, and the supernatant is collected; Preferably, during the extraction process of exosomes from medium-viscous plants, high-viscous plants and high-fiber plants, one or more of pectinase, cellulase, xylanase and protease are added at a concentration of 0.3%-1%, the enzymatic hydrolysis temperature is 37°C, and the time is 1-2 hours.

[0011] Preferably, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa, and the centrifugal concentration is performed at 4°C and 3000 rpm, with a concentration multiple of 5-10 times.

[0012] Preferably, the polymer precipitation uses polyethylene glycol (PEG) as a precipitant, the PEG addition volume ratio (exosome crude extract: PEG) is 4:1-5:1, and the precipitation is allowed to stand at 4°C for 8-24 hours.

[0013] This technology classifies plant sample juices according to their physical and chemical properties, and adopts differentiated pretreatment methods, combined with two-step differential centrifugation, ultrafiltration, enzymatic hydrolysis, polymer precipitation and other methods, to significantly improve the extraction efficiency and purity of exosomes.

[0014] The specific steps are as follows: 1. Pretreatment of plant samples According to the characteristics of plant juice, plants are divided into four categories: clear and low-viscosity type, medium-viscosity type, high-viscosity type and high-fiber type. Different sample cleaning, disinfection, cutting and juice extraction treatments are carried out for different types of plants. Double-layer sterile gauze is used to filter out large particles of fiber and impurities to obtain plant juice.

[0015] 2. Two-step differential centrifugation The first step is low-speed centrifugation: centrifuge the plant juice at 3000-6000 rpm for 20-40 minutes at 4°C to remove cell debris and large particle impurities, and collect the supernatant.

[0016] The second step is high-speed centrifugation: centrifuge the supernatant at 10,000-12,000 rpm for 20-40 minutes at 4°C to further remove the precipitate and obtain a clear supernatant.

[0017] 3. Enzymatic treatment For medium-viscosity, high-viscosity and high-fiber plants, add one or more of pectinase, cellulase, xylanase and protease, and perform enzymatic hydrolysis at 37°C for 1-2 hours to decompose polysaccharides and fiber substances and reduce viscosity.

[0018] 4. Filtration and ultrafiltration concentration Filter through 0.45 μm and 0.22 μm filter membranes to remove tiny impurities. Use 100-300 kDa molecular cutoff ultrafiltration membrane to concentrate the supernatant, centrifuge and concentrate at 4°C and 3000 rpm, with a concentration factor of 5-10.

[0019] 5. Polymer precipitation and purification Polyethylene glycol (PEG) was added to the concentrated plant juice at a volume ratio of 4:1-5:1 (exosome crude extract: PEG), and the mixture was allowed to stand at 4°C for 8-24 hours, centrifuged at 10,000 rpm at 4°C for 1 hour, and the supernatant was discarded. The exosome precipitate was resuspended in phosphate buffered saline (PBS) and filtered through a 0.22 μm filter membrane to obtain purified exosomes.

[0020] The exosome purification method used in the present invention divides plants into four categories according to the characteristics of plant juice: 1) clear and low-viscosity type, such as watermelon, cucumber, etc., with large juice yield and clear juice; 2) medium-thick type, such as apple, pear, etc., with medium juice yield and slightly turbid juice; 3) high-viscosity type, such as aloe vera, plantain, etc., with low juice yield and high juice viscosity; 4) high-fiber type, such as stem vegetables, woody plants, etc., with difficulty in juicing and high fiber content.

[0021] According to one embodiment of the present invention, when extracting clear low-viscosity plant or medium-viscosity plant exosomes, the pretreatment method used includes: directly cutting into pieces after washing and disinfection, squeezing juice with a cold juicer, and then filtering According to one embodiment of the present invention, when extracting highly viscous plant exosomes, the pretreatment method used includes: washing, disinfecting, chopping, adding pre-cooled PBS buffer, homogenizing, extracting juice and filtering.

[0022] According to one embodiment of the present invention, when extracting high-fiber plant exosomes, the pretreatment method used includes: washing, disinfecting, chopping, placing in an enzymatic hydrolysis solution (containing one or more of cellulase and xylanase), enzymatic hydrolysis at 37°C for 1-2 hours to soften the fiber structure, and then homogenizing and filtering the juice.

[0023] The purpose of the present invention is to solve the problems existing in the prior art and establish a new technology for extracting plant exosomes with strong pertinence and wide versatility. The present invention has the following innovations and advantages: 1. Adopting a differentiated processing strategy, fully considering the characteristic differences between plant samples, greatly broadening the applicable plant species for exosome extraction; 2. It cleverly combines multiple pretreatment methods such as physical crushing and enzymatic softening, effectively overcoming the barrier of plant cell walls and significantly improving the extraction rate of exosomes; 3. Ultrafiltration and polymer precipitation technologies were introduced to ensure the integrity of exosomes while improving the purity of extraction, thus obtaining high-quality exosome products; 4. The process is simple and flexible, and the required instruments and equipment are common, which is suitable for most laboratories to carry out the separation and purification of plant exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To compare the particle size distribution and morphological characteristics of watermelon exosomes extracted by combined method and ultracentrifugation method.

[0025] Figure 2 To compare the particle size distribution and morphological characteristics of grape exosomes extracted by combined method and ultracentrifugation method.

[0026] Figure 3 To compare the particle size distribution and morphological characteristics of aloe exosomes extracted by combined method and ultracentrifugation method.

[0027] Figure 4 To compare the particle size distribution and morphological characteristics of broccoli exosomes extracted by combined method and ultracentrifugation method. DETAILED DESCRIPTION

[0028] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0029] Example 1: Extraction of exosomes from clear and low-viscosity plants This example uses watermelon as a representative sample to elaborate on the extraction technology route of clear and low-viscosity plant exosomes.

[0030] The specific steps are as follows: 1. Sample preparation: 11 kg of fresh, mature, and juicy Jingyan Sweet King watermelon were selected, the surface of the watermelon was cleaned and disinfected, and the surface was wiped with sterile gauze to ensure the sample was clean. The skin was peeled and the pulp was obtained to obtain 9 kg of pulp.

[0031] 2. Juice Extraction: 2.1 Chop the watermelon pulp and use a cold press juicer to squeeze the juice at 4℃.

[0032] 2.2 Use double-layer sterile gauze to filter and remove pulp fibers and impurities.

[0033] 2.3 Collect 1200 mL of clear and uniform juice for subsequent processing.

[0034] 3 Two-step differential centrifugation: 3.1 The first step is low-speed centrifugation: centrifuge the watermelon juice at 4°C and 3000 rpm for 30 minutes and collect the supernatant.

[0035] 3.2 Second step: high-speed centrifugation: centrifuge the supernatant at 4°C, 10,000 rpm for 30 minutes and collect 1,000 mL of supernatant.

[0036] 4Ultrafiltration concentration: The obtained supernatant was centrifuged using a 100 kDa molecular cutoff ultrafiltration membrane at 4°C, 3000 rpm, for 10 min, and the operation was repeated until the supernatant was concentrated to 100 mL.

[0037] 5. Polymer precipitation: 5.1 Add 25 mL of polyethylene glycol to 100 mL of watermelon supernatant concentrate and let stand at 4°C for 12 hours.

[0038] 5.2 After standing, centrifuge at 10,000 rpm at 4°C for 1 hour.

[0039] 5.3 Discard the supernatant and resuspend the pellet in 10 mL phosphate buffered saline (PBS).

[0040] 5.4 Centrifuge the resuspension at 10,000 rpm at 4°C for 2 minutes and take the supernatant.

[0041] 5.5 Filter the supernatant using 0.45 μm and 0.22 μm filter membranes in turn to obtain watermelon juice exosomes.

[0042] 6. Exosome identification and preservation: 6.1 Observe the morphology of exosomes by transmission electron microscopy (TEM).

[0043] 6.2 Detection of exosome particle number concentration using nanoparticle tracking analysis (NTA).

[0044] 6.3 Store at -80℃ after aliquoting.

[0045] 7. Data comparison and analysis: Table 1 Comparison of the effects of combined method and ultracentrifugation method in extracting exosomes from watermelon juice.

[0046]

[0047] 8Result analysis: As shown in Table 1, the concentration of watermelon exosome particles extracted by the combined method was about 2.26 times higher than that by the ultracentrifugation method. Figure 1 The results showed that the diameter of watermelon exosome particles extracted by the combined method was concentrated in the range of 50-200nm, and the particle size distribution was uniform. Under the electron microscope, the exosomes were uniformly dispersed, with clear boundaries and complete double-layer membrane structure. Compared with the ultracentrifugation method, the exosome membrane structure obtained by this method was more intact, and the denatured and damaged particles were significantly reduced.

[0048] Example 2: Extraction of exosomes from medium-viscous plants This example uses grapes as a representative sample to elaborate on the extraction technology route of medium-viscous plant exosomes.

[0049] The specific steps are as follows: 1. Sample preparation: Select 5 kg of Karen seedless red grapes, prepare disinfectant according to the recommended dilution ratio for fruits and vegetables, soak for 20 minutes, wash with purified water, and wipe the surface with sterile gauze to ensure the cleanliness of the sample.

[0050] 2. Juice Extraction: 2.1 Put the washed grapes into the cold press juicer and squeeze the juice at 4℃.

[0051] 2.2 Use double-layer sterile gauze to filter and remove pulp fibers and impurities.

[0052] 2.3 Collect 1200 mL of clear and uniform juice for subsequent processing.

[0053] 3 Two-step differential centrifugation: 3.1 The first step is low-speed centrifugation: centrifuge the watermelon juice at 4°C and 3000 rpm for 30 minutes and collect the supernatant.

[0054] 3.2 Second step: high-speed centrifugation: centrifuge the supernatant at 4°C, 10,000 rpm for 30 minutes to obtain 1000 mL of supernatant.

[0055] 4 Enzymatic hydrolysis The pH value of the supernatant was adjusted to 7.4, and 0.5% pectinase, 0.3% cellulase, 0.2% xylanase and 0.1% protease were added. The mixture was gently shaken and enzymolysis was carried out at 37°C for 60 minutes.

[0056] 5 Centrifugation and Filtration The enzymatic hydrolysate was centrifuged at 4°C and 10,000 rpm for 30 minutes, and the supernatant was collected. The supernatant was filtered using 0.45 μm and 0.22 μm filter membranes in turn to obtain 700 mL of filtrate.

[0057] 6Ultrafiltration concentration: Select a 100 kD molecular cutoff ultrafiltration membrane, centrifuge the filtrate at 4°C, 3000 rpm, for 10 minutes, repeat the operation to obtain 70 mL of concentrated solution.

[0058] 7 Polymer precipitation: 7.1 Add 17.5 mL of polyethylene glycol to 70 mL of grape supernatant concentrate and let stand at 4°C for 12 hours.

[0059] 7.2 After standing, centrifuge at 10,000 rpm at 4°C for 1 hour.

[0060] 7.3 Discard the supernatant and resuspend the pellet in 7 mL of phosphate buffered saline (PBS).

[0061] 7.4 Centrifuge the resuspension at 10,000 rpm at 4°C for 2 minutes and collect the supernatant.

[0062] 7.5 Filter the supernatant using a 0.22 μm filter membrane to obtain grape juice exosomes.

[0063] 8 Exosome identification and preservation: 8.1 Observe the morphology of exosomes by transmission electron microscopy (TEM).

[0064] 8.2 Detection of exosome particle number concentration using nanoparticle tracking analysis (NTA).

[0065] 8.3 Store at -80℃ after aliquoting.

[0066] 9. Data comparison and analysis: Table 2 Comparison of the effects of combined method and ultracentrifugation method in extracting exosomes from grape juice.

[0067]

[0068] Result analysis: As shown in Table 2, the number concentration of exosomes extracted by the combined method is about 2.77 times that of the sample obtained by ultracentrifugation. Figure 2 The results showed that the particle size of exosomes extracted by the combined method was mainly distributed in the range of 40-180nm, which is consistent with the characteristics of exosomes. This shows that enzymatic hydrolysis reduces the sample viscosity and improves the release efficiency of exosomes. The exosomes have regular morphology, good dispersion, and a clearly visible double-layer membrane structure. No obvious agglomeration or membrane fragments were observed in the sample, indicating that the optimized extraction process effectively avoided the aggregation and damage of exosomes.

[0069] Example 3: Extraction of exosomes from highly viscous plants This example uses aloe vera as a representative sample to elaborate on the extraction technology route of highly viscous plant exosomes.

[0070] The specific steps are as follows: 1. Sample preparation: Use 1kg of aloe vera and prepare disinfectant according to the recommended dilution ratio for fruits and vegetables. Soak for 20 minutes, wash with purified water, and wipe the surface with sterile gauze to ensure the sample is clean.

[0071] 2. Juice Extraction: 2.1 Chop the aloe vera, add 1000 mL of phosphate buffered saline (PBS) precooled to 4°C and mix, use a cold press juicer to extract the aloe vera juice, and squeeze the juice at 4°C.

[0072] 2.2 Use double-layer sterile gauze to filter and remove pulp fibers and impurities.

[0073] 2.3 Collect 900 mL of clear and uniform juice for subsequent processing.

[0074] 3 Two-step differential centrifugation: 3.1 First step: low-speed centrifugation: centrifuge the aloe vera juice at 4°C, 3000 rpm for 30 minutes and collect the supernatant.

[0075] 3.2 Second step: high-speed centrifugation: centrifuge the supernatant at 4°C, 10,000 rpm for 30 minutes to obtain 800 mL of supernatant.

[0076] 4 Enzymatic hydrolysis The pH value of the supernatant was adjusted to 7.4, and 0.5% pectinase, 0.3% cellulase, 0.2% xylanase and 0.1% protease were added. The mixture was gently shaken and enzymolysis was carried out at 37°C for 60 minutes.

[0077] 5 Centrifugation and Filtration The enzymatic hydrolysate was centrifuged at 4°C and 10,000 rpm for 30 minutes, and the supernatant was collected. The supernatant was filtered using 0.45 μm and 0.22 μm filter membranes in turn to obtain 700 mL of filtrate.

[0078] 6Ultrafiltration concentration: Select an ultrafiltration membrane with a molecular cutoff of 100 kDa, centrifuge the filtrate at 4°C, 3000 rpm, for 10 min, and repeat the operation until the supernatant is concentrated to 70 mL.

[0079] 7 Polymer precipitation: 7.1 Add 17.5 mL of polyethylene glycol to 70 mL of aloe vera concentrate and let stand at 4°C for 12 hours.

[0080] 7.2 After standing, centrifuge at 10,000 rpm at 4°C for 1 hour.

[0081] 7.3 Discard the supernatant and resuspend the pellet in 7 mL of phosphate buffered saline (PBS).

[0082] 7.4 Centrifuge the resuspension at 10,000 rpm at 4°C for 2 minutes and collect the supernatant.

[0083] 7.5 Filter the supernatant using a 0.22 μm filter membrane to obtain aloe exosomes.

[0084] 8 Exosome identification and preservation: 8.1 Observe the morphology of exosomes by transmission electron microscopy (TEM).

[0085] 8.2 Detection of exosome particle number concentration using nanoparticle tracking analysis (NTA).

[0086] 8.3 Store at -80℃ after aliquoting.

[0087] 9. Data comparison and analysis: Table 3 Comparison of the effects of combined method and ultracentrifugation method in extracting aloe exosomes.

[0088]

[0089] Result analysis: The data in Table 3 show that the particle concentration of the combined method is about 2.37 times that of the sample obtained by the ultracentrifugation method. Figure 3 The results showed that the diameter of exosome particles extracted by the combined method ranged from 50 to 200 nm. Electron microscopy results showed that the influence of high sample viscosity was effectively overcome by optimizing the number of enzymatic treatments. Under the electron microscope, it was observed that the exosomes were complete in morphology, evenly distributed, with clear boundaries and a typical central concave structure.

[0090] Example 4: Extraction of exosomes from high-fiber plants This example uses broccoli as a representative sample to elaborate on the extraction technology route of high-fiber plant exosomes.

[0091] The specific steps are as follows: 1. Sample preparation: Select 1.5 kg of fresh broccoli, prepare disinfectant according to the recommended dilution ratio for fruits and vegetables, soak for 20 minutes, wash with purified water, and wipe the surface with sterile gauze to ensure the sample is clean.

[0092] 2. Juice Extraction: 2.1 The first enzymatic hydrolysis treatment: Chop the broccoli, add 1000 mL of enzymatic hydrolysis solution containing 0.5% cellulase and 1% xylanase, and perform enzymatic hydrolysis at 37°C for 2 hours to soften the high-fiber structure and partially decompose the cellulose and hemicellulose in the cell wall.

[0093] 2.2 Homogenization: The enzymatically hydrolyzed sample was placed in an ice bath and homogenized three times using a high shear homogenizer (15,000 rpm, 3 min each time) to fully disrupt the cells and release the juice.

[0094] 2.3 Coarse filtration: Use double-layer sterile gauze to filter to remove plant fibers and impurities, and collect 900 mL of juice for subsequent processing.

[0095] 3 Two-step differential centrifugation: 3.1 The first step is low-speed centrifugation: centrifuge the broccoli juice at 4°C, 3000 rpm for 30 minutes and collect the supernatant.

[0096] 3.2 Second step: high-speed centrifugation: centrifuge the supernatant at 4°C, 10,000 rpm for 30 minutes to obtain 800 mL of supernatant.

[0097] 4 Second enzymatic hydrolysis The pH value of the supernatant was adjusted to 7.4, and 0.5% pectinase, 0.3% cellulase, 0.2% xylanase and 0.1% protease were added. The mixture was gently shaken and enzymolysis was carried out at 37°C for 60 minutes.

[0098] 5 Centrifugation and Filtration The enzymatic hydrolysate was centrifuged at 4°C and 10,000 rpm for 30 minutes, and the supernatant was collected. The supernatant was filtered using 0.45 μm and 0.22 μm filter membranes in turn to obtain 700 mL of filtrate.

[0099] 6Ultrafiltration concentration: Select an ultrafiltration membrane with a molecular cutoff of 100 kDa, centrifuge the filtrate at 4°C, 3000 rpm, for 10 min, and repeat the operation until the supernatant is concentrated to 70 mL.

[0100] 7 Polymer precipitation: 7.1 Add 17.5 mL of polyethylene glycol to 70 mL of broccoli supernatant concentrate and let stand at 4°C for 12 hours.

[0101] 7.2 After standing, centrifuge at 10,000 rpm at 4°C for 1 hour.

[0102] 7.3 Discard the supernatant and resuspend the pellet in 7 mL of phosphate buffered saline (PBS).

[0103] 7.4 Centrifuge the resuspension at 10,000 rpm at 4°C for 2 minutes and collect the supernatant.

[0104] 7.5 Filter the supernatant using a 0.22 μm filter membrane to obtain broccoli exosomes.

[0105] 8 Exosome identification and preservation: 8.1 Observe the morphology of exosomes by transmission electron microscopy (TEM).

[0106] 8.2 Detection of exosome particle number concentration using nanoparticle tracking analysis (NTA).

[0107] 8.3 Store at -80℃ after aliquoting.

[0108] 9. Data comparison and analysis: Table 4 Comparison of the effects of combined method and ultracentrifugation method in extracting broccoli exosomes.

[0109]

[0110] Result analysis: The data in Table 4 show that the particle concentration of the combined method is about 3.62 times that of the sample obtained by the ultracentrifugation method. Figure 4 The results showed that the diameter of exosome particles extracted by the combined method ranged from 50 to 200 nm, and the particle size distribution was relatively uniform. Electron microscopy showed that the exosomes had a regular morphology, a well-preserved double-layer membrane structure, and good dispersion. The above results indicate that the two-step enzymatic hydrolysis strategy significantly improved the release efficiency and morphological integrity of exosomes.

[0111] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for extracting exosomes based on the characteristics of plant juice, characterized in that: The method comprises the following steps: 1) Pretreatment of plant samples: Plant samples were classified according to the characteristics of the juice: a. clear and low-viscosity plants, b. medium-viscosity plants, c. high-viscosity plants, and d. high-fiber plants; After classification, washing, cutting, enzymolysis, homogenization and juicing pretreatment are carried out respectively; 2) Two-step differential centrifugation to remove impurities, centrifuging the extracted plant juices in sequence; 3) Enzymatic treatment: Enzymatic treatment of medium-viscous plant juice, high-viscous plant juice and high-fiber plant juice; 4) Ultrafiltration and concentration: Filter the enzymatically hydrolyzed plant juice through a membrane filter and use an ultrafiltration membrane to concentrate the plant juice; 5) Polymer precipitation: Add polyethylene glycol (PEG) to the concentrated plant juice, let it stand and then centrifuge it. Use phosphate buffered saline (PBS) to resuspend the precipitate to obtain the exosome sample.

2. The method for extracting exosomes based on the characteristics of plant juice according to claim 1, characterized in that: The plant samples were classified into the following four categories: a. Clear and low-viscosity plants: high juice yield, clear and transparent liquid, representative plants include cucumbers, watermelons, and tomatoes; b. Medium viscous plants: moderate juice yield, slightly turbid liquid, representative plants include apples, pears, and grapes; c. High viscosity plants: The amount of juice produced is small and the liquid is viscous. Representative plants include aloe vera, seaweed, and berries; d. High-fiber plants: They are difficult to produce juice and have high fiber content. Representative plants include broccoli, celery, and root vegetables.

3. The method for extracting exosomes based on the characteristics of plant juice according to claim 2, characterized in that: For clear and low-viscosity plants, wash and cut them into pieces and then directly cold-press the juice without enzymatic treatment; for medium-viscosity plants, enzymatic hydrolysis is performed after differential centrifugation; for high-viscosity plants, phosphate buffer is first added to dilute them, and then the juice is extracted by homogenization and then enzymatic hydrolysis is performed; for high-fiber plants, pre-enzymatic hydrolysis is required to soften the tissue before homogenization.

4. The method for extracting exosomes based on the characteristics of plant juice according to claim 3, characterized in that: For high-fiber plants, the tissue needs to be pre-enzymatically softened before homogenization. One or more of cellulase and xylanase should be selected at a concentration of 0.3%-1%, the enzymatic hydrolysis temperature is 37°C, and the time is 1-2 hours.

5. The method for extracting exosomes based on the characteristics of plant juice according to claim 1, characterized in that: Two-step differential centrifugation: low-speed centrifugation at 4°C, 3000-6000 rpm, centrifugation for 20-40 minutes, and collection of the supernatant; high-speed centrifugation at 4°C, 10,000-12,000 rpm, centrifugation for 20-40 minutes, and collection of the supernatant.

6. The method for extracting exosomes based on the characteristics of plant juice according to claim 1, characterized in that: Enzymatic hydrolysis: during the extraction of exosomes from medium-viscous plants, high-viscous plants and high-fiber plants, one or more of pectinase, cellulase, xylanase and protease were added at a concentration of 0.3%-1%, the enzymatic hydrolysis temperature was 37°C, and the time was 1-2 hours.

7. The method for extracting exosomes based on the characteristics of plant juice according to claim 1, characterized in that: Ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa, and centrifuges at 4°C and 3000 rpm to concentrate the product 5-10 times.

8. The method for extracting exosomes based on the characteristics of plant juice according to claim 1, characterized in that: Polyethylene glycol (PEG) was used as the precipitant for polymer precipitation. The volume ratio of PEG addition was 4:1-5:1 for crude exosome extract and PEG, and the precipitation was allowed to stand at 4°C for 8-24 hours.

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