Exosome separation and concentration method using marine animal source tissue as raw material

Abstract

The invention relates to the technical field of bioengineering, in particular to an exosome separation and concentration method with marine animal source tissue as a raw material. Comprising the following steps: freeze-drying and crushing a marine animal source tissue sample; performing sample enzymolysis; and removing impurities and concentrating. According to the method disclosed by the invention, the exosome in the cells is fully dissociated through freeze-dried powder crushing and composite enzymolysis processes, and the filtering speed of the sample is accelerated by virtue of ultrasonic oscillation, so that the rapid and high-purity separation and concentration of the exosome in the marine animal source tissue are realized.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for separating and concentrating exosomes using marine animal tissues as raw materials. Background Technology

[0002] Exosomes are small membrane vesicles containing complex RNA and proteins, with a diameter of approximately 30–150 nm, secreted by living cells and bacteria. They primarily originate from multivesicles formed by the invagination of lysosomal microparticles within cells, and are released into the extracellular matrix after the outer membrane of the multivesicle fuses with the cell membrane. Exosomes were first discovered in sheep reticulocytes in 1983, and Johnstone named them "exosomes" in 1987. As important mediators of long-range cell communication, exosomes are closely related to the occurrence and development of many diseases, including tumors and inflammation.

[0003] Recent studies have revealed that plant and animal-based foods are rich in exosome-like nanovesicles, which can carry various bioactive substances such as functional proteins, flavonoids, and polyphenols. Notably, oral ingestion of food-derived exosome-like nanovesicles is unaffected by the digestive tract environment, including enzymes and pH, allowing the bioactive components they carry to be directly absorbed into the bloodstream without degradation. These nanovesicles exhibit a variety of biological effects, including improving enteritis, anti-cancer activity, promoting bone formation, protecting the liver, and improving memory.

[0004] Currently, exosome isolation techniques include ultracentrifugation, size exclusion, immunocapture, and PEG precipitation. Ultracentrifugation is time-consuming, labor-intensive, and requires expensive equipment, making it difficult to obtain sufficient quantities of exosomes for subsequent experimental analysis. Size exclusion methods collect small amounts of exosomes. While PEG precipitation can isolate exosomes on a large scale, it often introduces a large amount of contaminating proteins into the product, resulting in low purity of the isolated exosomes. At present, exosome research in my country still largely relies on the cumbersome process of ultracentrifugation and imported extraction kits, which seriously hinders the development of exosome research and application in my country. Finding a method that can efficiently isolate exosomes while ensuring their purity is an urgent problem to be solved in this field. Chinese patent CN118546861A discloses an exosome for treating lung injury, its preparation method, and its application. The method involves isolating embryonic cells from gastrulation-stage aquatic animals using trypsin digestion, followed by purification through centrifugation and anion-exchange column chromatography to obtain exosomes. However, relying solely on this combination of centrifugation and anion-exchange column chromatography has limited effectiveness in removing solids and cell debris from the sample, and it struggles to effectively separate impurities such as bacteria and small proteins, resulting in low purity exosomes with significant protein residue. Furthermore, the anion-exchange column chromatography procedure is relatively cumbersome and has a long purification cycle, hindering rapid exosome preparation. The column chromatography process may also result in exosome adsorption loss, further affecting the recovery rate and concentration. Therefore, further research is needed on methods for the isolation and concentration of exosomes from marine animal tissues. Summary of the Invention

[0005] The technical problem to be solved by this invention is that current exosome separation techniques include ultracentrifugation, molecular exclusion, immunocapture, and PEG precipitation. These methods mostly rely on the physical properties of exosomes, such as density, size, or solubility, and are time-consuming, resulting in many impurities in the product. They are also difficult to effectively distinguish interfering particles such as heterogeneous protein aggregates in marine animal tissues, which hinders the development of exosome research and application in my country.

[0006] To address the aforementioned issues, this invention employs a freeze-drying and pulverization process combined with enzymatic hydrolysis to fully release exosomes from cells. Furthermore, ultrasonic oscillation is used to accelerate the filtration process, thereby achieving rapid, high-purity separation and concentration of exosomes from marine animal-derived tissues.

[0007] To achieve the above objectives, the technical solution used in this invention is: a method for separating and concentrating exosomes from marine animal-derived tissues, comprising the following steps: (1) Freeze-drying and pulverizing of marine animal tissue samples: Fresh marine animal tissue samples are cut into pieces, freeze-dried, pulverized, and sieved. Freeze-drying can fully pulverize and disperse the animal tissue samples in a small volume of physiological saline. Direct homogenization of undried tissue samples results in incomplete pulverization and consumes a large amount of physiological saline, which leads to uncontrolled sample volume during subsequent enzymatic digestion, thus hindering the extraction of exosomes.

[0008] (2) Sample enzymatic digestion: After the sample is dispersed in water, 0.2%~2% of papain and 0.2%~1% of trypsin by wet weight of the sample are added. After stirring at 37-55℃ for 15-30 min, the sample is rapidly cooled to 0-10℃ and protease inhibitor and 0.5%~2% of sodium ascorbate by wet weight of the sample are added. After stirring, the sample is temporarily stored at 4℃. Papain catalyzes the cysteine ​​residue (Cys25) at the active site, which forms a covalent intermediate complex with the substrate through the thiol group (-SH), breaking the peptide bond. It has a broad substrate specificity, preferentially hydrolyzing the carboxyl-terminal peptide bond of aromatic amino acids (phenylalanine, tyrosine, tryptophan) or hydrophobic amino acids. It has a stronger ability to degrade proteins and can hydrolyze large protein molecules into small peptide molecules or even amino acids. Trypsin catalyzes the serine residue (Ser195) at the active site, which completes the hydrolysis by nucleophilic attack on the carbonyl carbon of the substrate peptide bond. It exhibits high substrate specificity, specifically hydrolyzing the carboxyl-terminal peptide bonds of lysine and arginine, producing mostly polypeptide fragments of specific lengths and generating fewer free amino acids.

[0009] Their synergistic effect is reflected in substrate-specific complementarity, enabling broad-spectrum protein degradation. Trypsin specifically hydrolyzes the C-terminal peptide bonds of lysine and arginine, preferentially acting on polar protein structures (such as integrins and fibronectin) in the extracellular matrix and cell debris, rapidly breaking the binding sites of exosomes to the cell / matrix and initially releasing the encapsulated exosomes. Papain, on the other hand, prefers to hydrolyze the C-terminal peptide bonds of aromatic amino acids (phenylalanine, tyrosine, tryptophan) and hydrophobic amino acids, further degrading the hydrophobic regions of proteins exposed after trypsin action (such as the hydrophobic domains of collagen and hydrophobic fragments of albumin in body fluids). The combined use of the two can cover both polar and hydrophobic peptide bond sites, improving the degradation efficiency of miscellaneous proteins by 30%-50% compared to single enzyme treatment, and avoiding the co-precipitation of exosomes and miscellaneous proteins due to insufficient action of a single enzyme.

[0010] Advantages of using papain at concentrations of 0.2%~2%: 1. Wide concentration range suitable for different sample types. At low concentrations (0.2%~0.5%), it is suitable for exosomes derived from body fluids (plasma, serum, saliva) or cell culture supernatants. These samples have relatively low levels of impurities, and low concentrations of papain are sufficient to degrade free albumin, immunoglobulins, and other hydrophobic proteins, while avoiding excessive degradation of exosome membrane proteins. At high concentrations (1%~2%), it is suitable for tissue homogenate samples (muscle, viscera) or samples rich in matrix. These samples contain a large amount of recalcitrant hydrophobic proteins such as collagen and fibrin. High concentrations of papain can deeply disintegrate the extracellular matrix network, releasing encapsulated exosomes and increasing yield. 2. Higher concentration than trypsin, enhancing synergistic impurity removal effect. The upper limit of the final concentration of papain (2%) is twice that of trypsin (1%). This setting aligns with the division of labor between the two enzymes: trypsin is responsible for "dissociation" (efficiently breaking the polar binding sites of cell-exosomes at low concentrations), while papain is responsible for "deep decontamination" (requiring higher concentrations to cover more hydrophobic peptide bond sites). The combination of the two forms a synergistic closed loop of "dissociation + degradation". 3. Maintains high efficiency activity in the 37-55℃ range. The reaction temperature (37-55℃) is the high efficiency activity range of papain. Within this concentration range, the enzyme amount can be rapidly degraded within 15-30 minutes, without extending the reaction time, thus reducing the risk of exosome membrane damage.

[0011] Advantages of using trypsin at concentrations of 0.2%–1%: 1. Low concentration range avoids the risk of membrane protein degradation. Trypsin has the potential to degrade exosome membrane surface markers (CD63, CD81, TSG101), which are rich in lysine / arginine sites. If the trypsin concentration is too high (>1%), it will directly damage membrane proteins, leading to the failure of downstream exosome identification. The concentration range of 0.2%–1% ensures that trypsin efficiently hydrolyzes cell surface integrins and matrix polar proteins (achieving exosome dissociation) while avoiding excessive attack on membrane proteins, balancing "dissociation efficiency" and "exosome integrity". 2. Forms a concentration gradient with papain, avoiding substrate competition. The upper limit of trypsin concentration is lower than that of papain, which avoids competitive binding of the two enzymes to the substrate: trypsin preferentially binds to polar amino acid sites, quickly opening the protein conformation, and then papain binds to the exposed hydrophobic sites. The two act in an orderly manner, rather than competing for the substrate, thus improving the overall degradation efficiency. 3. Low concentration facilitates rapid termination of the reaction. Trypsin activity is highly sensitive to inhibitors (soybean trypsin inhibitor) and low temperature (0-10℃). At low concentrations, only a small amount of inhibitor needs to be added to completely terminate the reaction, reducing the interference of inhibitor residues on subsequent exosome purification.

[0012] The core synergistic advantage of the dual-enzyme ratio (papain:trypsin = 1:1~2:1) lies in its concentration ratio, ranging from 1:1 (0.2%:0.2%) to 2:1 (2%:1%). This ratio is based on optimal substrate-specific complementarity: when the sample contains few contaminating proteins and exosomes are easily released, a 1:1 ratio is used, ensuring gentle action of both enzymes and maximizing protection of exosome structure; when the sample matrix is ​​complex and exosomes are tightly encapsulated, a 2:1 ratio is used to enhance the impurity removal ability of papain while keeping the trypsin concentration within limits to avoid membrane damage. Compared to "equal proportion high concentration" or "trypsin excess" methods, this ratio can increase exosome recovery by 20%~30% and reduce contaminating protein residue by 15%~25%.

[0013] Sodium ascorbate mainly plays four core roles: antioxidant protection, assisting in terminating enzymatic hydrolysis, maintaining the structural integrity of exosomes, and preventing oxidative cross-linking of impurities. The specific mechanisms are broken down below in conjunction with experimental conditions: 1. Inhibits oxidative damage during enzymatic hydrolysis and protects exosome membrane structure. Enzymatic hydrolysis is carried out at 37-55℃ with stirring, which easily generates reactive oxygen species (ROS) in the system. These free radicals attack the phospholipid bilayer of the exosome membrane and the amino acid side chains of membrane proteins (such as tyrosine and tryptophan residues), leading to vesicle rupture and degradation of membrane markers (CD63, CD81), affecting the recovery rate and subsequent identification of exosomes. Sodium ascorbate is a highly efficient water-soluble antioxidant that can scavenge ROS in the system by providing hydrogen atoms, while reducing oxidized thiol groups (-SH) of membrane proteins, maintaining the stability of the exosome membrane and avoiding vesicle rupture caused by high-temperature stirring.

[0014] 2. Rapid Termination of Protease Activity. The experiment employed a combined termination strategy of rapid cooling (0-10℃) + protease inhibitor + sodium ascorbate. Sodium ascorbate can assist in terminating enzyme activity in two ways: ① by lowering the redox potential of the system, inhibiting the oxidation of cysteine ​​residues (-SH) at the active site of papain, indirectly weakening its catalytic ability; ② by forming a synergistic effect with low temperature and protease inhibitor, accelerating the loss of both enzyme activities and avoiding excessive exosome damage caused by the "tailing" of the enzymatic reaction.

[0015] 3. Maintaining a weakly acidic environment in the system reduces non-specific aggregation of exosomes. Sodium ascorbate, when dissolved in water, forms a weak acid, which can maintain the pH of the system after enzymatic hydrolysis in the range of 5.5-6.5. This pH is close to the isoelectric point of exosomes, which can reduce the electrostatic repulsion between exosome particles, prevent non-specific aggregation of vesicles due to pH fluctuations, and ensure the homogeneity of exosomes in subsequent temporary storage and purification steps.

[0016] 4. Prevent oxidative cross-linking of contaminating proteins and improve subsequent purification efficiency. If contaminating protein fragments generated from enzymatic hydrolysis undergo oxidative cross-linking, they easily form complexes with exosomes, increasing the difficulty of subsequent ultracentrifugation and ultrafiltration separation. Sodium ascorbate can inhibit the oxidative cross-linking reaction of contaminating proteins, allowing them to exist in the form of soluble peptides, which are easier to remove effectively in subsequent steps, thus improving the final purity of exosomes.

[0017] The dosage range of 0.5%-2% of the sample wet weight ensures sufficient antioxidant effect while avoiding interference from high concentrations of sodium ascorbate. Low concentration (0.5%-1%): suitable for samples with low levels of impurities such as body fluids, sufficient to remove ROS and help terminate enzyme activity; High concentration (1%-2%): suitable for samples with high matrix, such as tissue homogenates, capable of handling stronger oxidative stress while inhibiting cross-linking of impurities.

[0018] (3) Impurity removal and concentration: After centrifugation, the supernatant of the sample in step (2) is taken and filtered through 0.45, 0.22 and 0.15 μm filter membranes in sequence; the effluent is filtered through a 30 nm filter membrane under ultrasonic conditions, the stagnant liquid is collected and filtered through a 0.22 μm filter membrane, and the effluent is sterile and concentrated exosomes.

[0019] In the exosome impurity removal and concentration steps, the core role of ultrasonic treatment is to optimize the filtration efficiency of the 30 nm filter membrane, prevent membrane clogging, and ensure the homogeneity and recovery rate of exosomes. The specific mechanism is broken down in conjunction with the experimental procedure as follows: 1. Dispersing exosome aggregates to improve filtration flux. After enzymatic hydrolysis, centrifugation, and multi-stage filtration (0.45, 0.22, 0.15 μm) pretreatment, exosomes in the supernatant may still form nanoscale aggregates due to electrostatic and hydrophobic interactions. If directly passed through a 30 nm filter membrane, these aggregates will clog the membrane's pore channels, leading to a sharp decrease in filtration rate or even complete membrane blockage. High-frequency mechanical vibrations generated by ultrasound can disrupt the intermolecular forces of the aggregates through cavitation, dispersing them into monodisperse exosome vesicles. This allows the vesicles to more easily approach and pass through the pores of the 30 nm filter membrane, significantly improving filtration flux and treatment efficiency.

[0020] 2. Reduce non-specific adsorption of exosomes on the filter membrane surface. Non-specific adsorption easily occurs between the filter membrane (such as polycarbonate or cellulose ester materials) and the exosome membrane, causing irreversible loss of exosomes. The mechanical vibration of ultrasound can generate continuous shear force, weakening the adsorption force between exosomes and the filter membrane surface, reducing the retention of exosomes on the filter membrane surface, and thus improving the exosome recovery rate.

[0021] 3. Assists in the removal of impurities and maintains membrane permeability. During filtration, trace amounts of impurities such as proteins and peptide fragments that were not removed in previous steps may adsorb onto the surface of the 30 nm filter membrane or become embedded in its pores. The cavitation effect and shear force of ultrasound can remove these adsorbed impurities, maintain the permeability of the filter membrane pores, avoid increased filtration pressure due to impurity accumulation, and ensure the stability of the concentration process.

[0022] 4. Gentle processing, preserving the structural integrity of exosomes. The vesicle structure of exosomes is sensitive to high-intensity mechanical forces. In this process, ultrasound is performed simultaneously with filtration through a 30 nm filter membrane, employing low-intensity, short-duration ultrasound (typically 50-150 W power, 3-5 min). Under these conditions, ultrasound acts only on the aggregates and adsorption layer, without damaging the phospholipid bilayer structure of the exosomes or degrading membrane surface markers (CD63, CD81), thus ensuring the integrity and activity of the final product.

[0023] This invention develops a novel exosome purification system using a "compound enzymatic hydrolysis + ultrasonic filtration" system. First, freeze-drying breaks down large animal tissues into smaller particles, preventing internal digestion difficulties and external over-digestion during subsequent enzymatic hydrolysis. Second, the compound enzymatic hydrolysis process utilizes proteases with different components to efficiently and rapidly hydrolyze animal tissues in a short time, saving time. Third, short-duration, high-speed centrifugation quickly removes solid impurities from the sample. Fourth, leveraging the relatively fixed particle size range of exosomes, a large-size filter membrane removes large particles such as cell debris, bacteria, and polysaccharides, while a small-size filter membrane removes small molecules such as proteins and water. Ultrasonic oscillation further accelerates the filtration process. This achieves rapid, high-purity separation and concentration of exosomes from marine animal tissues.

[0024] Furthermore, in step (1), after the sample is cut into pieces, it is placed at -80℃ for 40 min to 1 h, and then the sample is taken out and placed in a freeze dryer with the partition temperature set to -10 to 20℃ for continuous freeze drying for 15 to 20 h.

[0025] Further, in step (1), the fresh marine animal tissue was washed with physiological saline prepared with ultrapure water and cut into 2 cm*2 cm*2 cm pieces with a sterile scalpel in a sterile environment.

[0026] Further, in step (1), the samples are placed at -80℃ for 40 min to 1 h without contacting each other, and then taken out for freeze-drying. The core advantage of pre-freezing at -80℃ is that it can form small ice crystals, protecting the exosome vesicle structure. Marine animal tissues are rich in water and collagen. If freeze-dried directly, large ice crystals will slowly form inside the tissue. The mechanical compression of the ice crystals will puncture the phospholipid bilayer of the exosomes, causing the vesicles to rupture and lose their contents. The low temperature environment of -80℃ can make the water in the tissue freeze rapidly, forming small and evenly distributed ice crystals. These small ice crystals cause minimal mechanical damage to cells and exosomes, and can preserve the integrity of exosomes to the greatest extent. The pre-freezing time of 40 min to 1 h can ensure that the tissue is completely frozen from the surface to the inside, avoiding the phenomenon of "dry outside and wet inside" during freeze-drying.

[0027] Further, after the freeze-drying in step (1), the sample is taken out and pulverized using a food processor. After pulverization, it is passed through a 100-mesh sieve, and the retained portion is further pulverized and sieved. The advantage of this step is that it precisely controls the powder particle size, ensuring sufficient contact between the substrate and the enzyme. The sieve aperture corresponding to a 100-mesh sieve is approximately 150 μm. After sieving, coarse particles with a particle size >150 μm can be retained, leaving only fine powder with a uniform particle size. The specific surface area of ​​the fine powder from the freeze-dried tissue is greatly increased. In the subsequent enzymatic hydrolysis steps, papain and trypsin can maximize the contact area with the tissue matrix, quickly penetrate into the tissue interior, and efficiently hydrolyze extracellular matrix proteins (such as collagen and fibrin), promoting the full release of exosomes. If the powder particle size is too large or uneven, it will lead to insufficient contact between the enzyme and the substrate, and some tissue matrix cannot be degraded, resulting in low exosome release efficiency and poor reproducibility.

[0028] Further, in step (2), the sieved sample powder is collected and dispersed in physiological saline prepared with ultrapure water. After sonication for 5 minutes, the sample is heated to 37-55°C and then protease is added.

[0029] Further, in step (2), add 0.1%~1% collagenase or 0.1%~1% bone collagenase by wet weight of the sample. Collagenase needs to be added to samples containing skin tissue, and bone collagenase needs to be added to samples containing cartilage tissue.

[0030] Furthermore, the centrifugation conditions in step (3) are 4℃ and 10000 rpm for 8~10 min.

[0031] The centrifugation conditions have the following effects: 1. Low temperature of 4℃: protects the structure and activity of exosomes and inhibits residual enzyme activity.

[0032] ① Avoid thermal damage to exosomes: The phospholipid bilayer membrane of exosomes is sensitive to temperature. Centrifugation at room temperature will cause increased vesicle membrane fluidity, structural rupture, and even degradation of membrane markers (CD63, CD81) due to frictional heat and system temperature rise. Low temperature of 4℃ can maintain the stability of the membrane structure and preserve the integrity of exosomes to the greatest extent.

[0033] ② Inhibit residual protease activity: After the enzymatic hydrolysis step, a small amount of papain and trypsin remain in the system. Low temperature can significantly reduce the catalytic activity of these enzymes, prevent them from continuously degrading exosome membrane proteins during centrifugation, and block the "tailing effect" of the enzymatic hydrolysis reaction.

[0034] ③ Reduce exosome aggregation: Low temperature can reduce the intensity of molecular thermal motion, weaken the hydrophobic interaction and electrostatic attraction between exosomes, avoid non-specific aggregation of exosomes during centrifugation, and ensure the monodispersity of exosomes in the supernatant.

[0035] 2. 10,000 rpm rotation speed: Precisely traps large particulate impurities, achieving efficient separation of exosomes. The magnitude of centrifugal force directly determines the impurity trapping efficiency. 10,000 rpm corresponds to a relative centrifugal force of approximately 10,000 × g, which has a clear separation target.

[0036] Highly efficient sedimentation of large particles: It can completely settle large particles with a diameter >200 nm, such as undigested tissue fragments, cell debris, and fibrin clots. If these impurities enter the subsequent filtration steps, they will quickly clog the 0.45 μm and 0.22 μm filter membranes, reducing the filtration flux.

[0037] Ensure exosomes remain in the supernatant: Exosomes typically have a particle size of 30~150 nm. Under a centrifugal force of 10000×g, they cannot be effectively precipitated and will remain completely in the supernatant. This avoids exosomes being carried away and precipitated due to excessive centrifugal force, resulting in product loss.

[0038] 3. Centrifugation time of 8-10 min: This balances sedimentation efficiency and experimental efficiency. 8 min is sufficient for large particles of impurities at a centrifugal force of 10000×g to completely settle to the bottom of the centrifuge tube, forming a dense precipitate and preventing turbidity in the supernatant. Extending the centrifugation time can further ensure the sedimentation of stubborn impurities (such as dense connective tissue fragments), but after 10 min, the sedimentation efficiency tends to saturate, with no additional gain.

[0039] Furthermore, in step (3), when the effluent is filtered sequentially through a 30 nm filter membrane under ultrasonic conditions, continuous stirring is performed, and physiological saline solution prepared from ultrapure water is continuously added until 100 mL of sample 1 remains. Filtration is then stopped to obtain stagnant liquid, at which point impurities with a particle size of less than 30 nm have been removed.

[0040] An exosome prepared by the above method using marine animal tissue as raw material.

[0041] An ultrasonic filtration device includes a storage bottle, a filter membrane placement area, a vacuum valve, and a collection bottle. The storage bottle is funnel-shaped and has a cap on top. A stirrer is located in the center of the cap, and an ultrasonic vibrator is located on one side. The storage bottle is connected to the collection bottle in sequence through the filter membrane placement area and the vacuum valve.

[0042] Furthermore, rubber gaskets are installed above the filter membrane placement area and below the vacuum extraction valve.

[0043] The application of the above-mentioned ultrasonic filtration device in the separation of exosomes.

[0044] The beneficial effects of this invention are as follows: (1) Low separation cost and easy setup. The equipment used in this invention are all common laboratory instruments and equipment, which are easy to operate. The consumable cost is low, and a high-performance exosome separation device can be quickly and independently set up.

[0045] (2) Rapid and convenient separation with short time consumption. This invention breaks down the process into smaller steps, including freeze-drying, pulverization, enzymatic hydrolysis, centrifugation, membrane filtration, and concentration, to rapidly separate and purify exosomes from samples. Each step is time-efficient, maximizing the extraction of exosomes while ensuring that their structure and function remain unaffected. During membrane filtration and concentration, the use of ultrasonic technology and rotary stirring effectively solves the problem of impurities or exosomes clogging the filter membrane, thereby improving purification efficiency.

[0046] (3) High purity and few impurities. In this invention, exosomes in cells are fully released through freeze-drying, pulverization and compound enzymatic hydrolysis. Then, through centrifugation, membrane filtration and concentration, impurities such as solids, cell debris, bacteria and small molecules in the sample are quickly removed, and finally a high-purity and high-concentration exosome sample is obtained. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0048] Figure 1 The effects of freeze-drying, pulverization, and homogenization on the processing time of large yellow croaker muscle tissue and the concentration of exosomes in the concentrate were investigated.

[0049] Figure 2The effect of ultrasonic treatment on the processing time of large yellow croaker muscle tissue and the concentration of exosomes in the concentrate.

[0050] Figure 3 This is a product design drawing of the ultrasonic filtration device of the present invention. In the drawing, 1 is a liquid storage bottle, 2 is a filter membrane placement area, 3 is a vacuum extraction valve, 4 is a collection bottle, 5 is a stirrer, 6 is an ultrasonic vibrator, and 7 is a rubber gasket.

[0051] Figure 4 This is a TEM characterization result of purified exosomes from the adductor muscle of scallops.

[0052] Figure 5 This is a figure showing the NTA characterization results of purified exosomes from the adductor muscle of scallops.

[0053] Figure 6 This is a TEM characterization result of purified exosomes from the hippocampus.

[0054] Figure 7 This is a figure showing the NTA characterization results of purified exosomes from the hippocampus.

[0055] Figure 8 This is a TEM characterization result of exosomes purified from large yellow croaker muscle cell culture medium.

[0056] Figure 9 This is a figure showing the NTA characterization results of exosomes purified from large yellow croaker muscle cell culture medium.

[0057] Figure 10 These are images showing the morphology of sea bass muscle hydrolysate under different enzymatic treatments and the TEM characterization results of purified exosomes.

[0058] Figure 11 The effects of different enzyme components on the processing time of large yellow croaker muscle tissue and the concentration of exosomes in the concentrate were investigated.

[0059] Figure 12 This study investigates the effects of different enzyme components on the concentration and purity of exosomes in clams.

[0060] Figure 13 The effect of different enzyme components on the concentration and purity of exosomes in squid.

[0061] Figure 14 The effects of freeze-drying, pulverization, and homogenization on the processing time of scallop adductor muscle tissue and the concentration of exosomes in the concentrate were investigated. Detailed Implementation

[0062] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0063] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0064] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0065] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0066] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available or can be prepared by existing methods.

[0068] Example 1: like Figure 3 As shown, an ultrasonic filtration device includes a storage bottle 1, a filter membrane placement area 2, a vacuum valve 3, and a collection bottle 4. The storage bottle 1 is a funnel-shaped cylinder with a cap on top. A stirrer 5 is located in the center of the cap, and an ultrasonic vibrator 6 is located on one side. The storage bottle 1 is connected to the collection bottle 4 in sequence through the filter membrane placement area 2 and the vacuum valve 3.

[0069] Furthermore, a rubber gasket 7 is provided above the filter membrane placement area 2 and below the vacuum extraction valve 3.

[0070] This product consists of three parts: upper, middle, and lower. The upper part is a storage bottle, mainly used for sample filling, filtration, and concentration. A stirring paddle is installed at the top center of the storage bottle to agitate the sample during filtration, preventing large particles from precipitating and clogging the filter membrane. An ultrasonic vibrator is also installed at the top of the storage bottle to prevent nanoscale exosomes from clogging the filter membrane during purification and concentration, thus preventing excess aqueous phase from being trapped. The middle part connects to the storage bottle at the top, allowing for the placement of filter membranes with different pore sizes for impurity removal or concentration; the lower part is a collection bottle for collecting waste liquid or filtrate containing exosomes. A vacuum valve extends from the side of the middle part, connected to a vacuum pump via a rubber hose. The lower part is a collection bottle for collecting the exosome filtrate from impurity removal and the waste liquid from concentration.

[0071] The ultrasonic filtration device of this embodiment is used in the following examples.

[0072] Example 2: Sample preparation: 200 g of scallop adductor muscle was taken, cut into quarters, rinsed with physiological saline prepared with ultrapure water, spread evenly on aluminum foil, and frozen at -80℃ for 1 h. The sample was then freeze-dried in a freeze dryer. After freeze-drying, the sample was removed and ground into powder using a grinder. After passing through a 100-mesh sieve, the sample was dispersed in 1 L of physiological saline prepared with ultrapure water and sonicated for 5 min; this was designated as the vacuum freeze-drying group. Another 200 g of scallop adductor muscle was taken, added to 1 L of physiological saline prepared with ultrapure water, and directly homogenized; this was designated as the homogenization group. The sample was then placed in a water bath and heated to 37℃. Papain (1% by wet weight) and trypsin (0.5% by wet weight) were added, and the mixture was stirred at 200 rpm. After 20 min, the sample was rapidly cooled to 0-10℃, and 100 mg of protease inhibitor and 0.5% sodium ascorbate (wet weight of the sample) were added. After stirring for another 30 s, the sample was temporarily stored at 4℃. The sample was centrifuged at 10,000 rpm for 10 min at 4℃, and the supernatant was retained and recorded as the scallop hydrolysate. Using a vacuum filtration device, the scallop hydrolysate was filtered sequentially through 0.45, 0.22, and 0.15 μm filter membranes. Impurities larger than 150 nm in the effluent at this point had been removed, and this effluent was recorded as the scallop effluent. The scallop effluent was added to the vacuum filtration device, and a 30 nm filter membrane was used for filtration. During filtration, the ultrasonic vibrator was turned on, and a stirring paddle was placed above the funnel to continuously stir the scallop effluent at a speed of 100 rpm. When filtering the scallop eluent, add 400 mL of physiological saline for every 100 mL of scallop eluent added, until only 100 mL of scallop eluent remains in the funnel. Stop filtration, collect the remaining scallop eluent, and filter it through a 0.22 μm filter membrane into a pyrogen-free centrifuge tube. Record this as scallop concentrate. At this point, the scallop concentrate contains concentrated scallop adductor muscle exosomes. Send the scallop concentrate for TEM analysis to determine if it contains exosome particles.

[0073] result: like Figure 14 As shown, the processing time of the scallop adductor muscle samples in the vacuum freeze-drying group was significantly shorter than that in the homogenization group. This indicates that processing animal tissue samples by "freeze-drying and pulverizing" saves processing time compared to processing animal tissue samples by "homogenization".

[0074] like Figure 4 As shown, the scallop concentrate contains abundant exosome particles. NTA analysis revealed a concentration of 4.1 × 10⁻⁶. 12 Particles / mL, with exosomes having a diameter of 108.8 nm accounting for 98.8% ( Figure 5 This demonstrates that the exosome separation and concentration method can effectively separate and purify exosomes in actual shellfish samples.

[0075] Example 3: 400 g of large yellow croaker muscle tissue was cut into 2 cm*2 cm*1 cm cubes and divided into two equal portions, named the vacuum freeze-drying group and the homogenization group. The vacuum freeze-drying group samples were frozen at -80℃ for 2 h and then vacuum dried. The dried samples were then crushed using a grinder, passed through a 70-mesh sieve, and dissolved in 1 L of physiological saline. The homogenization group samples were directly added to 1 L of physiological saline, then homogenized thoroughly using a homogenizer and sonicated for 5 min. The samples were then heated to 37℃ in a water bath, and 1% papain and 0.5% trypsin (wet weight of the sample) were added, followed by stirring at 200 rpm. After 20 min, the samples were rapidly cooled to 0-10℃, and 100 mg of protease inhibitor and 0.5% sodium ascorbate (wet weight of the sample) were added. Stirring continued for 30 s, and the samples were temporarily stored at 4℃. The samples were centrifuged at 4℃ and 10000 rpm for 10 min, and the supernatant was retained and recorded as the large yellow croaker meat enzymatic hydrolysate. Using a vacuum filtration apparatus, the enzymatic hydrolysate of large yellow croaker meat was filtered sequentially through 0.45, 0.22, and 0.15 μm filter membranes. Impurities larger than 150 nm in the effluent at this point had been removed; this effluent was recorded as the large yellow croaker meat effluent. The large yellow croaker meat effluent was added to the vacuum filtration apparatus, and a 30 nm filter membrane was selected for filtration. During filtration, the ultrasonic vibrator was turned on, and a stirring paddle was placed above the funnel to continuously stir the large yellow croaker meat effluent at a speed of 100 rpm. During filtration, 400 mL of physiological saline was added simultaneously for every 100 mL of large yellow croaker meat effluent added, until only 100 mL of large yellow croaker meat effluent remained in the funnel. Filtration was then stopped, and the remaining large yellow croaker meat effluent was collected and filtered through a 0.22 μm filter membrane into a pyrogen-free centrifuge tube, and recorded as the large yellow croaker meat concentrate. At this point, the concentrated croaker meat liquid contains concentrated croaker muscle exosomes.

[0076] The results are as follows Figure 1 As shown, the processing time for the large yellow croaker flesh samples in the vacuum freeze-drying group was significantly shorter than that in the homogenization group. This indicates that processing animal tissue samples via freeze-drying and pulverization saves processing time compared to homogenization. This demonstrates that freeze-drying allows for thorough pulverization and dispersion of animal tissue samples in a small volume of physiological saline. Direct homogenization of undried tissue samples results in incomplete pulverization and consumes a large amount of physiological saline, leading to uncontrolled sample volume during subsequent enzymatic digestion, which hinders exosome extraction.

[0077] Example 4: 800 g of large yellow croaker muscle tissue was cut into 2 cm*2 cm*1 cm cubes, rinsed with physiological saline prepared with ultrapure water, spread evenly on aluminum foil, and frozen at -80℃ for 1 h. The sample was then freeze-dried in a freeze dryer. After freeze-drying, the sample was removed and ground into powder using a grinder. After passing through a 100-mesh sieve, the sample was dispersed in 6 L of physiological saline prepared with ultrapure water and sonicated for 5 min. The sample was then heated to 37℃ in a water bath, and 1% papain and 0.5% trypsin (wet weight) of the sample were added, with stirring at 200 rpm. After 20 min, the sample was rapidly cooled to 0-10℃, and 100 mg of protease inhibitor and 0.5% sodium ascorbate (wet weight) of the sample were added. Stirring continued for 30 s, and the sample was temporarily stored at 4℃. The sample was centrifuged at 10000 rpm for 10 min at 4℃, and the supernatant was retained and recorded as the large yellow croaker muscle enzymatic hydrolysate.

[0078] To verify the effectiveness of ultrasonic treatment, a control experiment was set up. Ultrasonic treatment group: 3 L of supernatant from the enzymatic hydrolysate of large yellow croaker muscle after centrifugation was filtered sequentially through 0.45, 0.22, and 0.15 μm filters; the effluent was filtered through a 30 nm filter under ultrasonic conditions, and the retentate was collected and filtered through a 0.22 μm filter; the retentate was the sterile exosome concentrate. Control group: 3 L of supernatant from the enzymatic hydrolysate of large yellow croaker muscle after centrifugation was filtered sequentially through 0.45, 0.22, and 0.15 μm filters; the effluent was filtered through a 30 nm filter without ultrasonic conditions, and the retentate was collected and filtered through a 0.22 μm filter; the retentate was the sterile exosome concentrate. The large yellow croaker muscle exosome concentrate was sent to an NTA analyzer to determine the exosome concentration in the sample.

[0079] The results are as follows Figure 2 As shown, the processing time of the large yellow croaker meat sample in the ultrasonic treatment group was significantly shorter than that in the control group, and the concentration of exosomes obtained was also significantly higher in the ultrasonic treatment group. During the purification process, the filtration rate of the control group gradually decreased, while the ultrasonic treatment group maintained a relatively stable filtration rate. This indicates that ultrasonic treatment can effectively prevent the filter membrane from being clogged by macromolecules in the sample. Clogged filter membranes not only require frequent replacement, delaying purification time and increasing purification costs, but the clogged filter membrane itself also blocks the filtration of some exosomes, resulting in a decrease in the concentration of exosomes in the purified sample.

[0080] Example 5: Sample preparation: 200 g of seahorse was broken down into soybean-sized pieces, rinsed with physiological saline prepared with ultrapure water, spread evenly on aluminum foil, and frozen at -80℃ for 40 min. The sample was then freeze-dried in a lyophilizer. After freeze-drying, the sample was removed and ground into powder using a grinder. The powder was then passed through a 100-mesh sieve and dispersed in 1 L of physiological saline prepared with ultrapure water, followed by sonication for 5 min. Larger particles that failed to pass through the sieve were dispersed in 500 mL of physiological saline prepared with ultrapure water (4℃) and homogenized under ice bath conditions. After thorough homogenization, the two sample portions were mixed together. The sample was then heated to 37℃ in a water bath, and 1% (wet weight) of papain, 0.5% (trypsin), 0.5% (collagenase), and 0.1% (bone collagenase) of the sample were added, followed by stirring at 200 rpm. After 20 min, the sample was rapidly cooled to 0-10℃, and 100 mg of protease inhibitor and 0.5% sodium ascorbate (wet weight of the sample) were added. Stirring was continued for 30 s, and the sample was temporarily stored at 4℃. The sample was centrifuged at 10,000 rpm for 8 min at 4℃, and the supernatant was retained and recorded as the hippocampal hydrolysate. A vacuum filtration apparatus was used to filter the hippocampal hydrolysate sequentially through 0.45, 0.22, and 0.15 μm filter membranes. Impurities larger than 150 nm in the effluent were removed at this point, and this effluent was recorded as the hippocampal effluent. The hippocampal effluent was added to the vacuum filtration apparatus, and a 30 nm filter membrane was selected for filtration. During filtration, the ultrasonic vibrator was turned on, and a stirring paddle was placed above the funnel to continuously stir the hippocampal effluent at a speed of 100 rpm. During the filtration of the hippocampal effluent, 400 mL of physiological saline was added for every 100 mL of hippocampal effluent added, until only 100 mL of effluent remained in the funnel. Filtration was then stopped, and the remaining effluent was collected and filtered through a 0.22 μm filter membrane into a pyrogen-free centrifuge tube, which was recorded as the hippocampal concentrate. At this point, the hippocampal concentrate contained concentrated hippocampal exosomes. The hippocampal concentrate was sent for TEM analysis to determine the presence of exosome particles. Results are as follows... Figure 6 As shown, the hippocampal concentrate contains abundant exosome particles. NTA analysis revealed a concentration of 6.4 × 10⁻⁶. 10 Particles / mL, with exosomes having a diameter of 141.7 nm accounting for 97.9% ( Figure 7 Compared to the adductor muscle of scallops, seahorses of the same weight not only contain skeletons but are also more difficult to grind and homogenize. This indicates that the exosome separation and concentration method can effectively separate and purify exosomes in actual fish samples.

[0081] Comparative Example 1: Sample preparation: Take 500 mL of waste culture medium used to culture large yellow croaker muscle cells, centrifuge the medium at 4℃ and 10000 rpm for 10 min, and retain the supernatant. Use a vacuum filtration device to filter the centrifuged medium through 0.45, 0.22, and 0.15 μm filter membranes sequentially. Impurities with a particle size greater than 150 nm have been removed from the effluent at this point, and the effluent is recorded as effluent A. Add 0.5% sodium ascorbate by wet weight of the sample to sample A to prevent oxidation. Add effluent A to the vacuum filtration device, and select a 30 nm filter membrane to filter effluent A. During filtration, turn on the ultrasonic vibrator and place a stirring paddle above the funnel to continuously stir effluent A at a speed of 100 rpm. When filtering effluent A, add 600 mL of physiological saline for every 100 mL of effluent A added, until only 100 mL of effluent A remains in the funnel. Stop filtration, collect the remaining effluent A, and filter it through a 0.22 μm filter membrane into a pyrogen-free centrifuge tube. Record this as the concentrated yellow croaker culture medium. At this point, the concentrated yellow croaker culture medium contains concentrated exosomes from in vitro cultured muscle cells of large yellow croaker. Send the concentrated yellow croaker culture medium for TEM analysis to determine the presence of exosome particles. Results are as follows... Figure 8 As shown, the concentrated culture medium for yellow croaker contained abundant exosome particles. NTA analysis revealed a concentration of 8.6 × 10⁻⁶. 11 Particles / mL, with exosomes having a diameter of 136.8 nm accounting for 99.4% ( Figure 9 This indicates that the exosome isolation and concentration method can effectively remove impurities and achieve exosome purification in actual in vitro cultured cell samples.

[0082] Comparative Example 2: To ensure complete separation of exosomes from animal tissues, thorough enzymatic digestion of the samples is necessary. Different enzymatic digestion treatments were performed on the dorsal muscle tissue of a perch. First, 600 g of the sample was divided into three equal portions and broken down into 1 cm * 1 cm * 1 cm pieces. After rinsing with physiological saline prepared with ultrapure water, the samples were spread evenly on aluminum foil and frozen at -80°C for 40 min. The samples were then lyophilized. After lyophilization, the samples were removed and ground into powder using a grinder. The powder was then passed through a 100-mesh sieve and dispersed in 1 L of physiological saline prepared with ultrapure water, followed by sonication for 5 min. Larger particles that failed to pass through the sieve were dispersed in 500 mL of physiological saline prepared with ultrapure water (4°C) and homogenized under suitable conditions. After thorough homogenization, the two sample portions were combined. The samples were then heated to 37°C in a water bath. The control group received no added enzymes and was stirred at 200 rpm. Experimental group 1 received 0.2% papain and 0.2% trypsin (wet weight of the sample), and was stirred at 200 rpm. Experimental group 2 received 1% papain and 1% trypsin (wet weight of the sample), and was stirred at 200 rpm. After 20 min, 100 mg of protease inhibitor and 0.5% sodium ascorbate (wet weight of the sample) were added to each of the three treatment groups, and stirring was continued for 30 s. The samples were then temporarily stored at 4°C. The samples were centrifuged at 10,000 rpm for 8 min at 4°C, and the supernatant was retained and recorded as the sea bass enzymatic hydrolysate. The sea bass enzymatic hydrolysate was filtered sequentially through 0.45, 0.22, and 0.15 μm filter membranes. Impurities larger than 150 nm were removed from the effluent at this point, and this effluent was recorded as the sea bass effluent. The sea bass effluent was added to a vacuum filtration apparatus and filtered through a 30 nm filter membrane. During filtration, an ultrasonic vibrator was turned on, and a stirring paddle was placed above the funnel to continuously stir the effluent at 100 rpm. For every 100 mL of sea bass effluent added, 400 mL of physiological saline was added simultaneously until only 100 mL of effluent remained in the funnel. Filtration was then stopped, and the effluent was collected and filtered through a 0.22 μm filter membrane into a pyrogen-free centrifuge tube, which was then labeled as sea bass concentrate. At this point, the sea bass concentrate contained concentrated sea bass exosomes. The sea bass concentrate was sent for TEM analysis to determine the presence of exosome particles. The results are as follows... Figure 10 As shown, the control group enzyme hydrolysate ( Figure 10 The image (A) contains obvious impurities such as foam and flocculent material, and the TEM image is also filled with granular and flocculent impurities, making it difficult to observe exosome vesicles. Figure 10 (D). In experimental group 1, 0.2% papain and 0.2% trypsin were added to the sample wet weight, and the enzymatic hydrolysate ( Figure 10(B) The solution was clear and transparent, without foam or visible impurities. This indicates that the addition of protease effectively reduced the amount of impurities in the hydrolysate. Insoluble protein impurities were broken down into peptides or small molecules, which were still clearly identifiable in TEM. Figure 10 (E), but no flocculent impurities were visible in the field of view, and exosome vesicles could be observed. Experimental group 2 added 1% papain and 1% trypsin by wet weight of the sample, and its enzymatic hydrolysate ( Figure 10 Group C was clear and transparent, with no foam or visible impurities, just like Group 1. This indicates that the addition of high-concentration protease can still effectively reduce the number of impurities in the enzymatic hydrolysate. Insoluble protein impurities were almost completely decomposed, and particulate impurities were difficult to observe in TEM. Figure 10 In the middle section (F), almost no impurities were observed in the field of view, and a considerable number of exosome vesicles were visible. The results indicate that 1% papain and 1% trypsin by wet weight of the sample can effectively enzymatically hydrolyze the dorsal muscle of the perch, eliminating impurities and obtaining a highly pure exosome concentrate. It is worth noting that the type and concentration of proteases need to be flexibly adjusted according to different biological tissue samples. For samples containing skin tissue, additional collagenase is required, and for samples containing cartilage tissue, additional bone collagenase is required.

[0083] Comparative Example 3: To ensure complete separation of exosomes from animal tissues, thorough enzymatic digestion of the samples is necessary. Different enzyme components were used to digest the dorsal muscle tissue of a sea bass. First, 1400 g of the sample was divided into seven equal portions, each 1 cm × 1 cm × 1 cm in size. After rinsing with physiological saline prepared with ultrapure water, the samples were spread evenly on aluminum foil and frozen at -80°C for 40 min. The samples were then lyophilized. After lyophilization, the samples were removed and ground into powder using a grinder. The powder was then passed through a 100-mesh sieve and dispersed in 1 L of physiological saline prepared with ultrapure water, followed by sonication for 5 min. Larger particles that failed to pass through the sieve were dispersed in 500 mL of physiological saline prepared with ultrapure water (4°C) and homogenized under ice bath conditions. After thorough homogenization, the two sample portions were combined. The samples were then placed in a water bath and heated to 37°C. For experimental group 1, 1% of the sample's wet weight of papain was added, and the mixture was stirred at 200 rpm. For experimental group 2, 1% of the sample's wet weight of trypsin was added, and the mixture was stirred at 200 rpm. For experimental group 3, 1% of the sample's wet weight of collagenase was added, and the mixture was stirred at 200 rpm. For experimental group 4, 1% of the sample's wet weight of papain and 1% of trypsin were added, and the mixture was stirred at 200 rpm. For experimental group 5, 1% of the sample's wet weight of papain and 1% of collagenase were added, and the mixture was stirred at 200 rpm. For experimental group 6, 1% of the sample's wet weight of trypsin and 1% of collagenase were added, and the mixture was stirred at 200 rpm. For experimental group 7, 1% of the sample's wet weight of papain, 1% of trypsin, and 1% of collagenase were added, and the mixture was stirred at 200 rpm. After 20 min, 100 mg of protease inhibitor and 0.5% sodium ascorbate (wet weight of the sample) were added to each of the seven treatment groups. After stirring for another 30 s, the samples were temporarily stored at 4℃. The samples were centrifuged at 10,000 rpm for 8 min at 4℃, and the supernatant was retained and recorded as the sea bass enzymatic hydrolysate. The sea bass enzymatic hydrolysate was filtered sequentially through 0.45, 0.22, and 0.15 μm filter membranes using a vacuum filtration device. Impurities larger than 150 nm in the effluent were removed at this point, and this effluent was recorded as the sea bass effluent. The sea bass effluent was added to the vacuum filtration device, and a 30 nm filter membrane was used for filtration. During filtration, the ultrasonic vibrator was turned on, and a stirring paddle was placed above the funnel to continuously stir the sea bass effluent at a speed of 100 rpm. When filtering the sea bass effluent, add 400 mL of physiological saline for every 100 mL of sea bass effluent added, until only 100 mL of sea bass effluent remains in the funnel. Stop filtration, collect the sea bass effluent, and filter it through a 0.22 μm filter membrane into a pyrogen-free centrifuge tube, labeling it as sea bass concentrate. At this point, the sea bass concentrate contains concentrated sea bass exosomes. Send the sea bass concentrate to an NTA analyzer to determine the concentration of exosomes.

[0084] like Figure 11 As shown, "1" represents papain, "2" represents trypsin, and "3" represents collagenase. Compared with experimental groups 1 and 2, experimental group 3 had a significantly longer processing time and a significantly lower concentration of exosomes after treatment, indicating that collagenase is not suitable for the enzymatic hydrolysis of sea bass dorsal muscle. Compared with experimental group 4, experimental groups 1 and 2 had longer processing times and lower concentrations of exosomes in the concentrated solution, indicating that using a composite enzyme is more effective than using a single type of enzyme. There were little difference in processing time and exosome concentration in the concentrated solution between experimental groups 1 and 5, 2 and 6, and 4 and 7, showing that the addition of collagenase had no effect on the sample processing time or exosome concentration. These results indicate that appropriate enzymes should be selected for enzymatic hydrolysis based on different biological samples.

[0085] Comparative Example 4: When processing biological samples with low protein content, such as clams, enzymatic hydrolysis was performed using papain:trypsin ratios of 1:1, 2:1, and 1:2, respectively, with other conditions remaining constant. The results are as follows: Figure 12 As shown, the 1:1 and 2:1 enzyme fraction treatment groups did not differ significantly in exosome concentration and purity, but the 1:1 enzyme fraction treatment group was significantly better than the 1:2 enzyme fraction treatment group. Therefore, to save costs, the 1:1 enzyme fraction can be selected for enzymatic digestion of marine animal tissue samples with low protein content.

[0086] When processing biological samples with high protein content, such as squid, enzymatic hydrolysis was performed using papain:trypsin ratios of 1:1, 2:1, and 1:2, respectively, with other conditions remaining constant. The results are as follows: Figure 13 As shown, the 1:1 and 1:2 enzyme fraction treatment groups showed little difference in exosome concentration and purity, but were lower than the 2:1 enzyme fraction treatment group. This indicates that papain's ability to remove impurities is effective in removing extraneous proteins from the sample, and the 2:1 enzyme fraction can be selected for enzymatic hydrolysis of marine animal tissue samples with high protein content.

[0087] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for isolating and concentrating exosomes from marine animal tissues, characterized in that... Includes the following steps: (1) Freeze-drying and pulverizing of marine animal tissue samples: Cut fresh marine animal tissue samples into pieces, freeze-dry, pulverize and sieve after freeze-drying; (2) Sample enzymatic digestion: After the sample is dispersed in water, add 0.2%~2% of papain and 0.2%~1% of trypsin by wet weight of the sample. Stir at 37-55℃ for 15-30 min. Then, quickly cool the sample to 0-10℃ and add protease inhibitor and 0.5%-2% of sodium ascorbate by wet weight of the sample. Stir well and store temporarily at 4℃. (3) Impurity removal and concentration: After centrifugation, the supernatant of the sample in step (2) is taken and filtered through 0.45, 0.22 and 0.15 μm filter membranes in sequence; the effluent is filtered through a 30 nm filter membrane under ultrasonic conditions, the stagnant liquid is collected and filtered through a 0.22 μm filter membrane, and the effluent is sterile and concentrated exosomes.

2. The method for separating and concentrating exosomes as described in claim 1, characterized in that: After cutting the sample into pieces, place it at -80℃ for 40 min to 1 h, then remove the sample and place it in a freeze dryer with the partition temperature set to -10 to 20℃ for continuous freeze drying for 15 to 20 h.

3. The method for separating and concentrating exosomes as described in claim 1, characterized in that: After the freeze-drying process in step (1), the sample is taken out and crushed using a food processor. After crushing, it is passed through a 100-mesh sieve. The retained portion is then crushed and sieved again.

4. The method for separating and concentrating exosomes as described in claim 1, characterized in that: Step (2) Collect the sieved sample powder and disperse it in physiological saline prepared with ultrapure water. After sonication for 5 minutes, heat the sample to 37-55℃ and then add protease.

5. The method for separating and concentrating exosomes as described in claim 1, characterized in that: Step (2) Add 0.1%~1% collagenase or 0.1%~1% bone collagenase of the sample wet weight.

6. The method for separating and concentrating exosomes as described in claim 1, characterized in that: The centrifugation conditions in step (3) are 4℃ and 10000 rpm for 8~10 min.

7. The method for separating and concentrating exosomes as described in claim 1, characterized in that: In step (3), the effluent is filtered sequentially through a 30 nm filter membrane under ultrasonic conditions while being continuously stirred and physiological saline solution prepared from ultrapure water is continuously added until 100 mL of sample 1 remains. Filtration is then stopped to obtain the retentate.

8. An exosome prepared from marine animal tissues by the method described in any one of claims 1-7.

9. An ultrasonic filtration device, characterized in that: The device is used for 30 nm filter membrane filtration in step (3) of claim (1). The device includes a storage bottle, a filter membrane placement area, a vacuum valve and a collection bottle. The storage bottle is funnel-shaped and has a cap on top. A stirrer is provided in the center of the cap and an ultrasonic vibrator is provided on one side. The storage bottle is connected to the collection bottle in sequence through the filter membrane placement area and the vacuum valve.

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