Fruit and vegetable exosome extraction method based on ligand fishing

Abstract

The invention discloses a fruit and vegetable exosome extraction method based on ligand fishing. The fruit and vegetable exosome extraction method comprises the following steps: suspending amino-functionalized Fe3O4 (at) SiO2 nanoparticles in a glutaraldehyde solution, and adding activated agglutinin to obtain Fe3O4 (at) SiO2-agglutinin nanoparticles; adding a glutaraldehyde solution into agarose gel to obtain a size exclusion chromatographic column filler; the method comprises the following steps: dispersing Fe3O4 (at) SiO2-lectin nanoparticles in a PBS buffer solution to obtain a uniform suspension, adding the uniform suspension into a size exclusion chromatographic column filler to obtain a uniform slurry, filling the size exclusion chromatographic column with the uniform slurry, and flushing with the buffer solution to form a magnetic bead stationary phase; and loading fruit and vegetable supernatant into a size exclusion chromatographic column, passing through the magnetic bead stationary phase, cleaning residual impurities combined on the exosome-magnetic bead compound by using a buffer solution, and dissociating the exosome from the magnetic beads by using a competitive eluent of the exosome to obtain the high-purity fruit and vegetable exosome. The problem that an existing exosome extraction method is difficult to meet the requirements of high efficiency, simplicity, high purity and high activity at the same time is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome separation, and specifically relates to a method for extracting exosomes from fruits and vegetables based on ligand fishing. Background Art

[0002] Exosomes are tiny vesicles with a lipid bilayer membrane, approximately 30-150 nanometers in size, secreted by most cells in the body. They play a vital role in intercellular communication, transport of substances, and information transfer. Fruit and vegetable exosomes contain a variety of bioactive molecules, such as proteins, lipids, DNA, and RNA, and exhibit enormous potential in agriculture, food, and medicine.

[0003] Currently, the methods that can be used to effectively isolate exosomes include ultracentrifugation, density gradient centrifugation, ultrafiltration centrifugation, immunomagnetic beads, chromatography, and polymer precipitation. Ultracentrifugation separates particles of different sizes through multiple centrifugation cycles. This is relatively simple, but time-consuming and requires expensive ultracentrifuges. It can also easily cause exosome aggregation and structural damage, resulting in reduced purity and activity. Density gradient centrifugation can obtain exosomes of higher purity, but the initial preparation is cumbersome and requires specialized density gradient media. This is also time-consuming and results in lower yields. Ultrafiltration centrifugation utilizes the pore size of ultrafiltration membranes for separation. While simple and rapid, it is subject to membrane contamination and exosome loss, and purity needs to be improved. Immunomagnetic bead-based methods, based on the specific binding of exosome surface markers to antibody-modified magnetic beads, offer advantages such as high specificity and relatively simple operation. However, the non-neutral pH and non-physiological salt concentrations can affect the bioactivity of exosomes, hindering subsequent experiments. Chromatography separates exosomes based on the differences in adsorption and elution characteristics between exosomes and other impurities on chromatographic media, resulting in uniform exosomes. However, it requires specialized chromatographic equipment and is relatively expensive. Polymer precipitation, which precipitates exosomes by adding polymers (such as polyethylene glycol), is simple to use but exhibits lower purity and higher levels of impurities.

[0004] In summary, existing exosome extraction methods all have certain limitations and are unable to simultaneously meet the requirements of efficient, simple, high-purity, and high-activity extraction of fruit and vegetable exosomes. Therefore, the development of a new method for extracting fruit and vegetable exosomes is of great practical significance. Summary of the Invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a method for extracting exosomes from fruits and vegetables based on ligand fishing, which solves the problem that existing exosome extraction methods are difficult to simultaneously meet the requirements of high efficiency, simplicity, high purity and high activity.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for extracting exosomes from fruits and vegetables based on ligand fishing, comprising the following steps:

[0008] Step 1: Wash the fruits and vegetables, cut them into pieces, squeeze the juice, and collect the supernatant of the fruits and vegetables by centrifugation;

[0009] Step 2: suspending the amino-functionalized Fe3O4@SiO2 nanoparticles in a glutaraldehyde solution for 0.5-1 hour to activate the suspension, wherein the amino-functionalized Fe3O4@SiO2 nanoparticles: glutaraldehyde = 1 g: (1.73-3.46) mL, by mass volume ratio; adding activated lectin to the suspension, by mass ratio of lectin: amino-functionalized Fe3O4@SiO2 nanoparticles = 1: (1-10), stirring at room temperature for 30-50 minutes, immobilizing the lectin on the amino-functionalized Fe3O4@SiO2 nanoparticles, separating the magnetic beads by a magnetic field, washing, and drying to obtain Fe3O4@SiO2-lectin nanoparticles;

[0010] Step 3, adding glutaraldehyde solution to the agarose gel, wherein the volume ratio of the glutaraldehyde solution to agarose is 2%-5%, stirring at 4° C. for 12-24 hours to obtain hydroxyl-activated agarose gel, and washing with PBS buffer multiple times to obtain a size exclusion chromatography column filler;

[0011] Step 4: Dispersing the Fe3O4@SiO2-lectin nanoparticles obtained in step 2 in PBS buffer at a mass-to-volume ratio of 20-100 mg / mL to obtain a uniform suspension; adding the uniform suspension to the size exclusion chromatography column filler obtained in step 3 at a volume ratio of the uniform suspension to the size exclusion chromatography column filler of 1:(5-10); stirring at 37°C for 6-12 hours, and then washing with PBS buffer and ethanol solution in sequence to obtain a uniform slurry;

[0012] Step 5, loading the uniform slurry described in step 4 into a size exclusion chromatography column, and simultaneously washing the size exclusion chromatography column with PBS buffer to form a magnetic bead stationary phase;

[0013] In step 6, the fruit and vegetable supernatant obtained in step 1 is loaded onto a size exclusion chromatography column and pushed through the magnetic bead stationary phase under gravity or low pressure. The large particle impurity effluent is discarded, and the impurities remaining on the exosome-magnetic bead complex are first washed with PBS buffer, and then the absorbance of the PBS buffer is detected by ultraviolet light. When the absorbance gradually decreases and tends to be stable, the exosome-magnetic bead complex is eluted with an exosome competitive eluent at a certain flow rate to dissociate the exosomes from the magnetic beads, and the eluate is collected to obtain high-purity fruit and vegetable exosomes.

[0014] Furthermore, in step 1, the fruit or vegetable is any one of watermelon, grape or carrot, and the centrifugation condition is 3000×g at 4° C. for 30 min.

[0015] Furthermore, the preparation method of amino-functionalized Fe3O4@SiO2 nanoparticles in step 2 is specifically as follows:

[0016] Fe3O4 nanoparticles were dispersed in an 80% by volume ethanol solution at a mass-to-volume ratio of 1 g of Fe3O4 nanoparticles to (10-20) mL of ethanol solution. Ammonia water was added as a catalyst, and after ultrasonic stirring for 30 minutes, tetraethyl orthosilicate was added at a mass ratio of 1:6-7 of Fe3O4 nanoparticles to tetraethyl orthosilicate. After ultrasonication at room temperature for 6-8 hours, the Fe3O4@SiO2 magnetic nanoparticles were collected using a magnet, washed, and dried.

[0017] The Fe3O4@SiO2 magnetic nanoparticles are dispersed in anhydrous ethanol, and the mass volume ratio of the Fe3O4@SiO2 magnetic nanoparticles: anhydrous ethanol is 1 g: (10-20) mL. 3-aminopropyltriethoxysilane is evenly added by ultrasonic dispersion, and the mass volume ratio of the Fe3O4@SiO2 magnetic nanoparticles: 3-aminopropyltriethoxysilane is 1 g: (0.1-1) mL. After stirring at room temperature for 6-10 hours, the mixture is adsorbed and recovered by a magnet and washed and dried to obtain amino-functionalized Fe3O4@SiO2 nanoparticles.

[0018] Furthermore, in step 2, the washing is performed by washing with ethanol and deionized water for 3-5 times respectively; and the drying is performed by vacuum freeze drying.

[0019] Furthermore, the lectin in step 2 is any one of concanavalin A, wheat germ agglutinin and lentil agglutinin.

[0020] Furthermore, the mass volume concentration of the glutaraldehyde solution in step 2 and step 3 is 25%.

[0021] Furthermore, the pore size of the size exclusion chromatography column in step 5 is 1000A, the inner diameter is 7.8-10 mm, and the length is 200-300 mm. The agarose gel is any one of CL-4B or CL-6B. The flow rate of the PBS buffer is 0.5-2 mL / min.

[0022] Furthermore, in step 6, the sample loading flow rate of the fruit and vegetable supernatant is 0.1-0.5 mL / min.

[0023] Furthermore, the competitive eluent in step 6 includes glucose and PBS buffer, and the mass-to-volume ratio of the glucose:PBS buffer is (18g-180g):1L, the flow rate of the competitive eluent is 0.5-1mL / min, and the concentration is 0.1-1mol / L.

[0024] Furthermore, the PBS buffer is 1×PBS buffer.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention incorporates Fe3O4@SiO2-lectin nanoparticles into a size exclusion chromatography column. The amino-functionalized Fe3O4@SiO2 nanoparticles possess superparamagnetic properties, are chemically stable, and can be rapidly separated under an applied magnetic field. The lectin has highly specific recognition for glycoproteins on the surface of fruit and vegetable exosomes, enabling efficient enrichment of fruit and vegetable exosomes with simple operation and mild conditions. This method utilizes the specific binding characteristics of magnetic nanoparticles and lectins to enhance the enrichment of fruit and vegetable exosomes. The entire enrichment process does not disrupt the structure and activity of the exosomes, effectively removing impurities and thus improving the purity of the exosomes.

[0027] The amino-functionalized Fe3O4@SiO2 magnetic nanoparticles of the present invention can be reused multiple times. The surface of Fe3O4 is easy to modify, and the biocompatibility of the magnetic nanoparticles can be improved after modification with SiO2.

[0028] The present invention uses magnetic beads to fish for exosomes of fruits and vegetables to solve the drawbacks of conventional methods for fruits and vegetables with high moisture and high enzyme activity. Compared with ultracentrifugation, it does not require expensive ultracentrifuges, is time-saving, and causes less damage to exosomes. Compared with filtration centrifugation and polymer precipitation, it has higher purity and the obtained exosomes are purer. Compared with other extraction methods, it has strong specificity and therefore has excellent application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a comparison chart of the quality of exosomes obtained by the method of the present invention and the existing ultracentrifugation method. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is described in detail below with reference to specific embodiments.

[0031] The present invention provides a ligand-fishing-based method for extracting exosomes from fruits and vegetables. This method involves modifying amino-functionalized ferroferric oxide magnetic microspheres with silica, then immobilizing them with a lectin that has a high affinity for fruit and vegetable exosomes. This lectin is then immobilized on a size-exclusion chromatography column to capture the exosomes. This method leverages the properties of magnetic nanoparticles and size exclusion to enrich fruit and vegetable exosomes under mild conditions. Compared to traditional methods, it offers advantages such as high specificity and purity.

[0032] Nano-ferroferric oxide is a type of nanomaterial with the advantages of easy synthesis, superparamagnetism, high saturation magnetization, good biocompatibility and low toxicity.

[0033] Size exclusion chromatography is a chromatographic technique that separates molecules based on their size. It utilizes the pores of the porous packing in a chromatographic column to prevent larger molecules from entering the pores and rapidly flow out of the column, while smaller molecules are retained by the pores, thereby separating molecules of different sizes. This method operates under mild operating conditions, typically at room temperature, without damaging bioactive molecules. It effectively removes impurities and improves the purity of the target molecule.

[0034] Example 1: Extraction of watermelon exosomes

[0035] Step 1: Select ripe watermelon pulp and cut it into small pieces. After squeezing the juice in a juicer, transfer it to a centrifuge tube. Centrifuge it at 4°C and 3000×g for 30 minutes at 4°C to collect the supernatant.

[0036] Step 2: Disperse 1g of Fe3O4 nanoparticles in 10mL of 80% ethanol solution by volume, add an appropriate amount of ammonia as a catalyst, and ultrasonically stir for 30 minutes. Slowly add ethyl orthosilicate at a mass ratio of 1:6 between Fe3O4 nanoparticles and ethyl orthosilicate. After ultrasonication at room temperature for 6 hours, collect the Fe3O4@SiO magnetic nanoparticles with a magnet, wash them three times with ethanol and three times with deionized water, and then freeze-dry them in a vacuum oven for later use.

[0037] 1 g of dried Fe3O4@SiO nanoparticles was dispersed in 10 mL of anhydrous ethanol and ultrasonically treated for 30 min to make it uniformly dispersed. 3-Aminopropyltriethoxysilane was added according to the mass volume ratio of Fe3O4@SiO nanoparticles to 3-aminopropyltriethoxysilane of 1:0.1, and stirred at room temperature for 6 h. After stirring, the mixture was recovered by magnetic adsorption and washed three times with ethanol and deionized water respectively, and then freeze-dried in vacuum to obtain amino-functionalized Fe3O4@SiO nanoparticles.

[0038] Immobilization of lectin: A certain amount of glutaraldehyde solution with a mass volume concentration of 25% was measured, and the amino-functionalized Fe3O4@SiO2 nanoparticles: glutaraldehyde were 1 g: 1.73 mL by mass volume ratio. The amino-functionalized Fe3O4@SiO nanoparticles were suspended in the 25% glutaraldehyde solution, activated for 0.5 h to obtain a suspension, and activated concanavalin A (ConA) was added. The mass ratio of concanavalin A (ConA) to amino-functionalized Fe3O4@SiO nanoparticles was 1:1. After stirring at room temperature for 30 min, the magnetic beads were separated by a magnetic field, washed with deionized water for 3 times, and vacuum freeze-dried to obtain Fe3O4@SiO-concanavalin A nanoparticles. The magnetic property was used to realize efficient separation, that is, to quickly recover the amino-functionalized Fe3O4@SiO2 nanoparticles (combined with lectin) for washing and drying: (remove unreacted glutaraldehyde, lectin and other impurities).

[0039] Step 3: Select agarose gel CL-4B as the filler of the size exclusion chromatography column, add a 2% volume fraction of glutaraldehyde solution, and stir at 4°C for 12 hours to activate the hydroxyl groups of the agarose gel. After activation, wash with 1xPBS buffer multiple times until no glutaraldehyde residue is detected in the washing solution, and obtain the size exclusion chromatography column filler.

[0040] Step 4: Take the Fe3O4@SiO-concanavalin A nanoparticles prepared in step 2, and disperse them in 1xPBS buffer at a mass volume ratio of 20 mg / mL to prepare a uniform suspension. Add the uniform suspension to the size exclusion chromatography column filler obtained in step 3, and the uniform suspension: size exclusion chromatography column filler is 1:5 by volume ratio. Stir at 37°C for 6 hours. After stirring, wash with 1xPBS and ethanol solution in sequence to obtain a uniform slurry.

[0041] Step 5: Load the uniform slurry obtained in step 4 into a size exclusion column with an inner diameter of 7.8 mm, a length of 200 mm, and a pore size of 1000 A. At the same time, flush the chromatography column with 1xPBS buffer at a flow rate of 0.5 mL / min to form a magnetic bead stationary phase.

[0042] Step 6: The pretreated watermelon sample extract was slowly loaded onto the chromatographic column at a flow rate of 0.1 mL / min. Under the action of gravity, the extract was slowly and steadily passed through the magnetic bead stationary phase to obtain an effluent of large particle impurities that needed to be discarded; the impurities remaining on the watermelon exosome-magnetic bead complex were first washed with an eluent (1×PBS buffer), and then the absorbance of the eluent was detected by ultraviolet light. When the absorbance gradually decreased and tended to be stable, the exosomes bound to the magnetic bead stationary phase were eluted with 0.1 mol / L exosome competitive eluent at a flow rate of 0.5 mL / min. The competitive eluent included glucose and 1×PBS buffer, and the mass volume ratio was 18 g:1 L. The eluent was collected to obtain high-purity watermelon exosomes.

[0043] Example 2: Extraction of grape exosomes

[0044] Step 1: Select fresh grapes, squeeze the juice in a juicer, transfer it to a centrifuge tube, centrifuge at 4°C and 3000×g for 30 minutes, and collect the supernatant.

[0045] Step 2: Disperse 1 g of Fe3O4 nanoparticles in 15 mL of 80% ethanol solution by volume, add ammonia water and ultrasonically stir for 30 minutes. Slowly add ethyl orthosilicate at a mass ratio of 1:6.5 between Fe3O4 nanoparticles and ethyl orthosilicate, ultrasonicate at room temperature for 7 hours, collect Fe3O4@SiO magnetic nanoparticles with a magnet, wash them four times with ethanol and four times with deionized water, and freeze-dry them in a vacuum for later use.

[0046] 1 g of dried Fe3O4@SiO2 magnetic nanoparticles was dispersed in 15 mL of anhydrous ethanol and sonicated for 30 min. 3-Aminopropyltriethoxysilane was added at a mass volume ratio of 1:0.5 for Fe3O4@SiO nanoparticles and 3-aminopropyltriethoxysilane. The mixture was stirred at room temperature for 8 h, recovered by magnetic adsorption, washed four times with ethanol and deionized water, and freeze-dried in a vacuum to obtain amino-functionalized Fe3O4@SiO nanoparticles.

[0047] Lectin immobilization: A certain amount of 25% mass-to-volume glutaraldehyde solution was measured, and the amino-functionalized Fe3O4@SiO2 nanoparticles: glutaraldehyde = 1 g: 3.46 mL, based on the mass-to-volume ratio. The amino-functionalized Fe3O4@SiO nanoparticles were suspended in the 25% glutaraldehyde solution and activated for 0.7 h to obtain a suspension. Activated wheat germ agglutinin (WGA) was added at a mass ratio of wheat germ agglutinin (WGA) to amino-functionalized Fe3O4@SiO nanoparticles of 1:5. The suspension was stirred at room temperature for 40 min, and the magnetic beads were separated by a magnetic field. The beads were washed four times with deionized water and freeze-dried in a vacuum to obtain Fe3O4@SiO-wheat germ agglutinin nanoparticles.

[0048] Step 3: Select Sepharose CL-6B as the filler for the size exclusion chromatography column, add a 3% volume fraction of glutaraldehyde solution, and stir at 4°C for 18 hours to activate the hydroxyl groups of the agarose gel. After activation, wash with 1× PBS buffer several times until no glutaraldehyde residue is detected in the washing solution, thereby obtaining the size exclusion chromatography column filler.

[0049] Step 4: Take the Fe3O4@SiO-wheat germ agglutinin nanoparticles prepared in step 2 and disperse them in 1×PBS buffer at a mass volume ratio of 60 mg / mL to prepare a suspension; add the uniform suspension to the size exclusion chromatography column filler obtained in step 3, and add the uniform suspension to the size exclusion chromatography column filler at a volume ratio of 1:8. Stir at 37°C for 9 hours, and wash with 1×PBS and ethanol solution in sequence to obtain a uniform slurry.

[0050] Step 5: The slurry was loaded into a size exclusion column with an inner diameter of 9 mm, a length of 250 mm, and a pore size of 1000 Å, and the column was flushed with 1× PBS buffer at a flow rate of 1.5 mL / min to form a magnetic bead stationary phase.

[0051] Step 6: The pretreated grape sample extract was loaded onto the chromatographic column at a flow rate of 0.3 mL / min, and the extract was pushed through the magnetic bead stationary phase under low pressure to obtain the effluent containing large particle impurities that needed to be discarded; the impurities remaining on the watermelon exosome-magnetic bead complex were first washed with eluent (1×PBS buffer), and then the absorbance of the eluent was detected by ultraviolet light. When the absorbance gradually decreased and tended to be stable, the exosomes bound to the magnetic bead stationary phase were eluted with 0.5 mol / L competitive eluent at a flow rate of 0.7 mL / min. The competitive eluent included glucose and 1×PBS buffer, and the mass volume ratio was glucose: PBS buffer = 100 g: 1 L. The eluent was collected to obtain high-purity exosomes.

[0052] Example 3: Extraction of carrot exosomes

[0053] Step 1: Take fresh carrots, wash and peel them, cut them into small pieces, squeeze the juice in a juicer, transfer it to a centrifuge tube, centrifuge at 4°C and 3000×g for 30 minutes, and collect the supernatant.

[0054] Step 2: Disperse 1 g of Fe3O4 nanoparticles in 20 mL of 80% ethanol solution by volume, add ammonia water and ultrasonically stir for 30 min, add ethyl orthosilicate at a mass ratio of 1:7 between Fe3O4 nanoparticles and ethyl orthosilicate, ultrasonicate at room temperature for 8 h, collect Fe3O4@SiO magnetic nanoparticles with a magnet, wash them 5 times with ethanol and deionized water, and freeze-dry them in a vacuum for later use.

[0055] 1 g of dried Fe3O4@SiO2 magnetic nanoparticles was dispersed in 20 mL of anhydrous ethanol and sonicated for 30 min. 3-Aminopropyltriethoxysilane was added at a mass volume ratio of 1:1 between Fe3O4@SiO nanoparticles and 3-aminopropyltriethoxysilane. The mixture was stirred at room temperature for 10 h, recovered by magnetic adsorption, washed five times with ethanol and deionized water, and freeze-dried in a vacuum chamber to obtain amino-functionalized Fe3O4@SiO nanoparticles.

[0056] Lectin immobilization: A certain amount of 25% mass-volume glutaraldehyde solution was measured, and the amino-functionalized Fe3O4@SiO2 nanoparticles: glutaraldehyde = 1 g: 2.59 mL, based on the mass-volume ratio. The amino-functionalized Fe3O4@SiO2 nanoparticles were suspended in the 25% glutaraldehyde solution and activated for 1 h to obtain a suspension. Activated lentil agglutinin (LCA) was added at a mass ratio of lentil agglutinin (LCA) to amino-functionalized Fe3O4@SiO2 nanoparticles of 1:10. The suspension was stirred at room temperature for 50 min, and the magnetic beads were separated by a magnetic field. The beads were washed five times with deionized water and freeze-dried in a vacuum to obtain Fe3O4@SiO-lentil agglutinin nanoparticles.

[0057] Step 3: Select sepharose CL-4B as the filler for the size exclusion chromatography column, add a 5% volume fraction of glutaraldehyde solution, and stir at 4°C for 24 hours to activate the hydroxyl groups of the sepharose. After activation, wash with 1× PBS buffer several times until no glutaraldehyde residue is detected in the washing solution to obtain the size exclusion chromatography column filler.

[0058] Step 4: Take the Fe3O4@SiO-lentil lectin nanoparticles prepared in step 2 and disperse them in 1×PBS buffer at a mass volume ratio of 100 mg / mL to prepare a uniform suspension; add the uniform suspension to the size exclusion chromatography column filler obtained in step 3, and stir at a volume ratio of the uniform suspension: size exclusion chromatography column filler = 1:10 at 37°C for 12 hours. After stirring, wash with 1×PBS and ethanol solution in sequence to obtain a uniform slurry.

[0059] Step 5: The uniform slurry was loaded into a size exclusion column with an inner diameter of 10 mm, a length of 300 mm, and a pore size of 1000 Å, and the column was flushed with 1×PBS buffer at a flow rate of 2 mL / min to form a magnetic bead stationary phase.

[0060] Step 6: The pretreated carrot sample extract is loaded onto the chromatographic column at a flow rate of 0.5 mL / min, and the extract is pushed through the magnetic bead stationary phase under gravity or low pressure to obtain an effluent containing large particle impurities that need to be discarded; the impurities remaining on the watermelon exosome-magnetic bead complex are first washed with an eluent (1×PBS buffer), and then the absorbance of the eluent is detected by ultraviolet light. When the absorbance gradually decreases and tends to be stable, the exosomes bound to the magnetic bead stationary phase are eluted with a 1 mol / L competitive eluent at a flow rate of 1 mL / min. The competitive eluent includes glucose and 1×PBS buffer, and the mass volume ratio is glucose:PBS buffer = 180 g:1 L. The eluent is collected to obtain high-purity exosomes.

[0061] Experimental part: Comparative experiment of exosome recovery

[0062] Ultracentrifugation, the earliest technique used for exosome isolation and a common technique in existing literature, is considered the "gold standard" for exosome isolation. However, this method is time-consuming and labor-intensive, requiring dedicated ultracentrifuges. Furthermore, fruits and vegetables are characterized by high moisture content and high enzyme activity. Excessively high centrifugation speeds can lead to the loss of a significant amount of exosomes when collected using ultracentrifugation, reducing the yield. The present invention, however, utilizes Fe3O4@SiO2-lectin nanoparticles loaded into a size-exclusion chromatography column, enabling efficient enrichment of fruit and vegetable exosomes. The conditions are mild, effectively removing impurities and improving exosome purity.

[0063] The fruit and vegetable solutions collected in Examples 1-3 were used to extract exosomes using ultracentrifugation and ligand fishing methods. For the ultracentrifugation method, the collected sample supernatant was centrifuged at 100,000 × g for 70 minutes, the supernatant was removed, and an appropriate amount of phosphate buffer solution was added to the centrifuge tube containing the exosome precipitate for washing. The precipitate was then centrifuged again at 100,000 × g for 70 minutes and resuspended in PBS to obtain ultracentrifugation exosomes. For the ligand fishing method, exosomes were collected according to the method described in the examples.

[0064] like Figure 1 As shown, the sample group is the exosomes obtained by ligand fishing method, and the control group is the exosomes obtained by ultracentrifugation method. The quality of the exosomes separated by ligand fishing method is higher than that of the exosomes obtained by ultracentrifugation method.

[0065] Depend on Figure 1It can be seen that the mass (μg / mL) of exosomes collected using the ligand fishing method is higher than that of the ultracentrifugation method, with asterisks indicating significant differences. This indicates that the method of adsorbing glycoproteins on the surface of fruit and vegetable exosomes through lectins is highly specific; while improving exosome quality, it can more effectively remove impurities and obtain exosomes of higher purity. Therefore, the present invention utilizes the properties of magnetic nanoparticles and size exclusion to enrich fruit and vegetable exosomes, which is characterized by strong specificity and high purity, and can achieve efficient collection of fruit and vegetable exosomes.

Claims

1. A method for extracting exosomes from fruits and vegetables based on ligand fishing, characterized in that: The following steps are involved: Step 1: Wash the fruits and vegetables, cut them into pieces, squeeze the juice, and collect the supernatant of the fruits and vegetables by centrifugation; Step 2: suspending the amino-functionalized Fe3O4@SiO2 nanoparticles in a glutaraldehyde solution for 0.5-1 hour to activate the suspension, wherein the amino-functionalized Fe3O4@SiO2 nanoparticles: glutaraldehyde = 1 g: (1.73-3.46) mL, by mass volume ratio; adding activated lectin to the suspension, by mass ratio of lectin: amino-functionalized Fe3O4@SiO2 nanoparticles = 1: (1-10), stirring at room temperature for 30-50 minutes, immobilizing the lectin on the amino-functionalized Fe3O4@SiO2 nanoparticles, separating the magnetic beads by a magnetic field, washing, and drying to obtain Fe3O4@SiO2-lectin nanoparticles; Step 3, adding glutaraldehyde solution to the agarose gel, wherein the volume ratio of the glutaraldehyde solution to agarose is 2%-5%, stirring at 4° C. for 12-24 hours to obtain hydroxyl-activated agarose gel, and washing with PBS buffer multiple times to obtain a size exclusion chromatography column filler; Step 4: Dispersing the Fe3O4@SiO2-lectin nanoparticles obtained in step 2 in PBS buffer at a mass-to-volume ratio of 20-100 mg / mL to obtain a uniform suspension; adding the uniform suspension to the size exclusion chromatography column filler obtained in step 3 at a volume ratio of the uniform suspension to the size exclusion chromatography column filler of 1:(5-10); stirring at 37°C for 6-12 hours, and then washing with PBS buffer and ethanol solution in sequence to obtain a uniform slurry; Step 5, loading the uniform slurry described in step 4 into a size exclusion chromatography column, and simultaneously washing the size exclusion chromatography column with PBS buffer to form a magnetic bead stationary phase; In step 6, the fruit and vegetable supernatant obtained in step 1 is loaded onto a size exclusion chromatography column and pushed through the magnetic bead stationary phase under gravity or low pressure. The impurities remaining on the exosome-magnetic bead complex are first washed with PBS buffer, and then the absorbance of the PBS buffer is detected by ultraviolet light. When the absorbance gradually decreases and tends to be stable, the exosome-magnetic bead complex is eluted with an exosome competitive eluent at a certain flow rate to dissociate the exosomes from the magnetic beads, and the eluate is collected to obtain high-purity fruit and vegetable exosomes.

2. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1, characterized in that: In step 1, the fruit or vegetable is any one of watermelon, grape or carrot, and the centrifugation condition is 3000×g at 4° C. for 30 min.

3. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1, characterized in that: The preparation method of amino-functionalized Fe3O4@SiO2 nanoparticles in step 2 is specifically as follows: Fe3O4 nanoparticles were dispersed in an 80% by volume ethanol solution at a mass-to-volume ratio of 1 g of Fe3O4 nanoparticles to (10-20) mL of ethanol solution. Ammonia water was added as a catalyst, and after ultrasonic stirring for 30 minutes, tetraethyl orthosilicate was added at a mass ratio of 1:6-7 of Fe3O4 nanoparticles to tetraethyl orthosilicate. After ultrasonication at room temperature for 6-8 hours, the Fe3O4@SiO2 magnetic nanoparticles were collected using a magnet, washed, and dried. The Fe3O4@SiO2 magnetic nanoparticles are dispersed in anhydrous ethanol, and the mass volume ratio of the Fe3O4@SiO2 magnetic nanoparticles: anhydrous ethanol is 1 g: (10-20) mL. 3-aminopropyltriethoxysilane is evenly added by ultrasonic dispersion, and the mass volume ratio of the Fe3O4@SiO2 magnetic nanoparticles: 3-aminopropyltriethoxysilane is 1 g: (0.1-1) mL. After stirring at room temperature for 6-10 hours, the mixture is adsorbed and recovered by a magnet and washed and dried to obtain amino-functionalized Fe3O4@SiO2 nanoparticles.

4. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1, characterized in that: In step 2, the washing is performed by washing with ethanol and deionized water for 3-5 times respectively; and the drying is performed by vacuum freeze drying.

5. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1, characterized in that: The lectin in step 2 is specifically any one of concanavalin A, wheat germ agglutinin and lentil agglutinin.

6. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1, characterized in that: The mass volume concentration of the glutaraldehyde solution in step 2 and step 3 is 25%.

7. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1, characterized in that: The size exclusion chromatography column in step 5 has a pore size of 1000A, an inner diameter of 7.8-10 mm, and a length of 200-300 mm. The agarose gel is either CL-4B or CL-6B. The flow rate of the PBS buffer is 0.5-2 mL / min.

8. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1, characterized in that: The sample loading flow rate of the fruit and vegetable supernatant in step 6 is 0.1-0.5 mL / min.

9. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1, characterized in that: The competitive eluent in step 6 includes glucose and PBS buffer, and the mass-to-volume ratio is glucose:PBS buffer = (18g-180g):1L. The flow rate of the competitive eluent is 0.5-1mL / min, and the concentration is 0.1-1mol / L.

10. The method for extracting exosomes from fruits and vegetables based on ligand fishing according to claim 1 or 9, characterized in that: The PBS buffer is 1×PBS buffer.

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