Method for extracting exosome of milk product

Abstract

The invention relates to the technical field of biology, in particular to a method for extracting exosomes of dairy products. The invention provides a method for extracting an exosome of a cow milk product, which comprises the following steps: carrying out first centrifugation on the cow milk product to obtain a first supernatant; mixing the first supernate with chymosin and then incubating; performing secondary centrifugation on the cow milk product incubated in the step (2) to obtain a second supernatant; filtering the second supernate to obtain filtrate; centrifuging the filtrate for the third time to obtain a first precipitate; purifying the first precipitate; the rotating speed of the second-time centrifugation is higher than that of the first-time centrifugation, and the rotating speed of the third-time centrifugation is higher than that of the second-time centrifugation; the adding amount of the chymosin is that the final concentration of the chymosin is 2.5 to 12.5 mg / mL. According to the method for extracting the cow milk exosome, the extraction efficiency and the extraction purity are high, the extraction time is shortened, and a high-quality raw material is provided for subsequent deep research in the biomedicine field and development of a drug delivery system.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for extracting exosomes from dairy products. Background Art

[0002] Exosomes are a type of extracellular vesicles with a diameter of about 30 to 150 nanometers, which are widely present in a variety of biological fluids, including blood, urine, and breast milk. They contain proteins, nucleic acids, and other bioactive molecules, participate in intercellular communication, and play an important role in the occurrence and development of a variety of diseases.

[0003] Exosomes in bovine milk are believed to have multiple biological activities, including anti-inflammatory, antioxidant and immunomodulatory functions, and therefore have become one of the research hotspots. At present, the main methods for extracting exosomes from bovine milk include ultracentrifugation, ultrafiltration, size exclusion chromatography, etc. However, these methods are usually complicated, time-consuming and costly. Therefore, the development of a simple, fast and efficient extraction method is of great significance for promoting the research and application of bovine milk exosomes.

[0004] At present, there are many methods for extracting exosomes, including ultracentrifugation (high yield and high purity), filtration centrifugation (simple and time-saving but insufficient purity), immunomagnetic bead method (high specificity but low efficiency), chromatography (high purity but low yield), precipitation method (simple and high yield but low purity), and affinity chromatography (low purity but low yield).

[0005] As a biological fluid with complex and heterogeneous composition, milk contains high abundance of milk protein (>30g / L) and exists in a variety of aggregation states (free protein molecules and soluble colloids or micelles). Casein is also in a highly phosphorylated state. The surface charge state of casein is highly similar to the phospholipid surface of exosomes, making it difficult to separate using ion exchange chromatography. In addition, bovine milk also contains other colloidal structures of comparable size to bovine milk exosomes, such as milk fat globules and casein micelles. Casein accounts for 80% of the total amount of bovine milk protein, and the average density of casein micelles is 1.0632g / mL, which is similar to the density of exosomes (1.1-1.2g / mL), and this value is likely to change with changes in the storage and processing conditions of milk. These micelle structures are similar in size and density to bovine milk exosomes, so they will be co-separated with bovine milk exosomes during the extraction of bovine milk exosomes, resulting in contamination of bovine milk exosomes.

[0006] Patent CN109468265B discloses a method for preparing bovine milk exosomes, which involves a centrifugation step to remove the milk fat layer, followed by adjusting the acidity of the supernatant and treating it with rennet to remove casein, and finally collecting the bovine milk exosomes by microfiltration. It is worth noting that the implementation of this method obtains a crude extract of exosomes with low extraction purity. For the effect of the separated exosomes, this method has not yet been characterized and verified by scientific and rigorous means, thus limiting its accuracy and reliability evaluation in application.

[0007] Patent CN113061571A discloses a method for isolating and identifying Holstein milk exosomes. After obtaining a crude extract by removing milk fat and milk protein through centrifugation, the crude extract is purified by a combination of methods such as density gradient centrifugation. Although this method can obtain exosomes, it does not remove the interference of milk protein, and the crude extract of exosomes is obtained. The extraction purity is low, and the operation is very cumbersome, and the flux and yield are low. In addition, the raw material separated by this method is raw milk, which has certain safety risks and cannot be extended to other commercial dairy products.

[0008] Patent CN114891723B discloses a method for isolating and identifying Holstein cow milk exosomes, which uses a kit for extraction. Although it is simple, it is costly and has low purity. In addition, the reliability and accessibility of the kit are uncontrollable and the price is relatively high, which limits its widespread use.

[0009] Patent CN115340978B discloses a method for preparing bovine milk exosomes, which involves very complicated steps, mainly including: involving a centrifugation step to remove the milk fat layer, adjusting the acidity of the supernatant to remove milk protein, and then adjusting the acidic concentrated whey with alkaline substances to obtain neutral whey, and finally the neutral whey is first subjected to molecular exclusion chromatography and density gradient centrifugation to obtain an exosome gradient liquid, and finally the bovine milk exosomes are collected by ultracentrifugation. Although this method can obtain a good purity, the operation is very cumbersome, the acidification and alkalization process of the supernatant pretreatment will damage the exosomes, it is time-consuming and has a low yield, and the use of high-value reagents such as hollow fiber columns and iodixanol leads to high costs.

[0010] Looking at the existing technologies, there is still a lack of a preparation method for milk-derived exosomes that can take into account fast exosome extraction speed, high purity and reasonable cost. Summary of the invention

[0011] Based on the above-mentioned defects of the prior art, the present application provides a method for extracting exosomes from bovine dairy products, which simplifies the method steps, optimizes the dosage of rennet and the differential ultracentrifugation conditions, and realizes the efficient extraction and purification of exosomes. It has the advantages of simple operation, short time consumption, high purity and good stability.

[0012] The first aspect of the present application provides a method for extracting exosomes from dairy products, comprising the following steps:

[0013] 1) performing a first centrifugation on the dairy product to obtain a first supernatant;

[0014] 2) mixing the first supernatant with chymosin and then incubating;

[0015] 3) centrifuging the dairy product after incubation in 2) for the second time to obtain a second supernatant;

[0016] 4) filtering the second supernatant to obtain a filtrate;

[0017] 5) centrifuging the filtrate for a third time to obtain a first precipitate;

[0018] 6) Purifying the first precipitate to obtain exosomes.

[0019] Specifically, the rotation speed of the second centrifugation is higher than that of the first centrifugation, and the rotation speed of the third centrifugation is higher than that of the second centrifugation.

[0020] Specifically, the speed of the first centrifugation is 956-2655 g, and the time of the first centrifugation is 20-30 minutes.

[0021] Specifically, the amount of chymosin added is such that the final concentration of chymosin is 2.5 to 12.5 mg / mL.

[0022] Specifically, the rotation speed of the second centrifugation is 10621-20817 g, the time of the second centrifugation is 25-35 minutes, and the temperature of the second centrifugation is 2-8°C.

[0023] Specifically, the rotation speed of the third centrifugation is 117524-183632g, the time of the third centrifugation is 60-80 minutes, and the temperature of the third centrifugation is 2-8°C.

[0024] Specifically, the purification comprises the following steps:

[0025] a) centrifuging for the fourth time to obtain the second precipitate;

[0026] b) The second precipitate is resuspended and then ultrafiltered to obtain the supernatant, which is the exosome.

[0027] Specifically, the rotation speed of the fourth centrifugation is 117524-183632g, the time of the fourth centrifugation is 60-80 minutes, and the temperature of the fourth centrifugation is 2-8°C.

[0028] The second aspect of the present application provides an exosome prepared by the method of the first aspect.

[0029] The third aspect of the present application provides the application of the method of the first aspect mentioned above in extracting exosomes from dairy products, wherein the dairy products are selected from any one or more of raw milk / raw milk, low-temperature pasteurized milk, ultra-high temperature sterilized milk, and whole milk powder.

[0030] The beneficial effects of this application are:

[0031] 1. Gentle extraction to protect the integrity of exosomes

[0032] This application innovatively uses chymosin (chymosin) as a treatment method, which effectively precipitates milk protein while avoiding the step of using acid and alkali to adjust pH in traditional methods, thereby completely eliminating the risk of exosome structural damage and deformation caused by acid and alkali treatment. This gentle treatment method ensures the structural integrity and biological activity of the exosome vesicle membrane, retains a high-quality foundation for subsequent applications, and has a good coagulation effect at a low cost.

[0033] 2. Simplify the process and reduce costs

[0034] Compared with the complex multi-step combined treatment of tangential flow concentration, size exclusion and density gradient centrifugation in traditional methods, this application only needs two main steps, rennet treatment combined with differential ultracentrifugation, to achieve effective separation and preliminary purification of exosomes. This significant simplification of the process not only reduces equipment requirements and operational complexity, but also significantly reduces production costs, improves production efficiency, and is more conducive to large-scale production.

[0035] 3. Efficient extraction and high purity output

[0036] Although the steps are simplified, the method of the present application ensures efficient separation and purification of exosomes by screening and optimizing, accurately controlling the chymosin action conditions and differential ultracentrifugation parameters. The exosomes finally obtained are not only of high purity, but also have a purity of 1×10 8 It has a high extraction rate of 100 particles / μg protein and maintains good biological activity and functional integrity, significantly improving the extraction efficiency and purity of exosomes and shortening the extraction time to 4-5 hours, providing high-quality raw materials for subsequent in-depth research in the biomedical field and the development of drug delivery systems.

[0037] 4. Huge potential for commercial application

[0038] Since the method of the present application is directly applicable to commercial dairy products, the method of the present application can successfully extract milk exosomes from a variety of commercial dairy products, especially for ultra-high temperature sterilized milk and whole milk powder, which are difficult to extract exosomes. It has a good extraction effect, which means that the source of raw materials is wide and stable, providing a solid guarantee for the commercial production of exosomes. In addition, the high extraction efficiency and high-quality product characteristics make this method have broad application prospects in many fields such as health products, medicines and biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is an electron micrograph of exosomes extracted from different bovine dairy products in Example 1 of the present application.

[0040] Figure 2 This is a statistical diagram of the particle size distribution of exosomes extracted from different bovine dairy products in Example 1 of the present application.

[0041] Figure 3 These are the BCA protein concentration values ​​of exosomes from different bovine dairy products extracted in Example 1 of the present application.

[0042] Figure 4 The purity values ​​of exosomes from different bovine dairy products extracted in Example 1 of the present application.

[0043] Figure 5 This is the expression diagram of specific proteins of exosomes extracted from different bovine dairy products in Example 1 of the present application.

[0044] Figure 6 This is the screening of chymosin in Comparative Example 1 of the present application.

[0045] Figure 7 This is a graph showing the specific protein expression of exosomes from different bovine dairy products extracted by the kit method in Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0046] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0047] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0048] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0049] The present application provides a method for extracting exosomes from dairy products, comprising the following steps:

[0050] 1) performing a first centrifugation on the dairy product to obtain a first supernatant;

[0051] 2) mixing the first supernatant with chymosin and then incubating;

[0052] 3) centrifuging the dairy product after incubation in 2) for the second time to obtain a second supernatant;

[0053] 4) filtering the second supernatant to obtain a filtrate;

[0054] 5) centrifuging the filtrate for a third time to obtain a first precipitate;

[0055] 6) Purifying the first precipitate to obtain exosomes.

[0056] In the present application, the dairy products refer to various foods extracted or processed from milk, including raw milk, raw milk, fresh milk, pasteurized milk, high-temperature sterilized milk, long-shelf life milk, milk powder, and whey powder.

[0057] In a specific embodiment of the present application, the dairy products include but are not limited to raw milk / raw milk, low-temperature pasteurized milk, ultra-high temperature sterilized milk, and whole milk powder. The raw milk / raw milk refers to the original milk squeezed directly from the cow's body without any sterilization, homogenization, filtration or other processing; the low-temperature pasteurized milk refers to the milk that has been low-temperature pasteurized by heating at 72-85°C for 10-15 seconds; the ultra-high temperature sterilized milk refers to the milk that has been ultra-high temperature instantaneous sterilization by heating at 135-150°C for 4-15 seconds to completely destroy the microorganisms and spores that can grow therein. The high-temperature treatment at 135-150°C causes the structures of some biological molecules in the milk to change, including exosomes Integrity and stability, while high temperature damages the membrane structure of exosomes, thus affecting their subsequent separation and purification, making it difficult to extract exosomes therefrom; the whole milk powder refers to a powdered product made by sterilizing fresh milk and then removing water from the milk through processes such as spray drying or drum drying. The spray drying process will damage the structure and function of exosomes. At the same time, whole milk powder contains a high fat content. These fat molecules may interact with exosomes and affect the separation efficiency of exosomes, making it difficult to extract exosomes therefrom.

[0058] The method for extracting exosomes from dairy products of the present application can successfully extract exosomes from a wide range of dairy products, especially when the dairy product is ultra-high temperature sterilized milk or whole milk powder, it still has a good extraction effect.

[0059] In a specific embodiment of the present application, differential ultracentrifugation is used, which means that particles of different sizes and densities are precipitated in sequence by gradually increasing the centrifugal force, thereby achieving separation. Specifically, the rotation speed of the second centrifugation is higher than the rotation speed of the first centrifugation, and the rotation speed of the third centrifugation is higher than the rotation speed of the second centrifugation.

[0060] The centrifugal speed can be expressed in g or rpm, and the two can be converted to each other according to existing formulas.

[0061] In a specific embodiment of the present application, in step 1), the first centrifugation is a low-speed centrifugation for removing large particle impurities and milk fat, and a first supernatant is obtained after the first centrifugation.

[0062] In a specific embodiment of the present application, in step 1), the rotation speed of the first centrifugation can be 956~2655g. When expressed in rpm, the rotation speed of the first centrifugation is 3000~5000rpm, for example, it can be 3000rpm, 3200rpm, 3400rpm, 3600rpm, 3800rpm, 4000rpm, 4200rpm, 4400rpm, 4600rpm, 4800rpm, 5000rpm.

[0063] In a specific embodiment of the present application, in step 1), the time of the first centrifugation can be 10 to 30 minutes, for example, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, or 30 minutes.

[0064] In a specific embodiment of the present application, in step 2), the chymosin can be Rennin derived from calf stomach, Rennin extracted from plants, or chymosin derived from bovine pancreas, and can specifically be products of Sigma, MCE, Sangon Biotech and other companies; preferably, the chymosin can be chymosin from Sangon Biotech, which has low cost and good coagulation effect, and can achieve good coagulation effect at low cost and low dose. The present application adds chymosin to cause milk protein to coagulate, thereby precipitating the milk protein, thereby avoiding the step of using acid and alkali to adjust pH in the traditional method.

[0065] In a specific embodiment of the present application, in step 2), the final concentration of the chymosin can be 2.5 to 12.5 mg / mL, for example, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 10 mg / mL, 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL, 12 mg / mL, 12.5 mg / mL. The final concentration refers to the concentration of a component or substance in the final mixed solution, specifically refers to the concentration of chymosin in the mixture of chymosin and the first supernatant after the addition of chymosin. At this dosage of chymosin, it is beneficial to improve the efficiency of extraction and the purity of the extracted exosomes. When the rennet concentration is too low, the whey after coagulation is less or the coagulation cannot be performed. When the rennet concentration is too high, the cost is high and waste is caused.

[0066] In a specific embodiment of the present application, in step 2), the first supernatant needs to be incubated after being mixed with rennet, and the incubation time can be 30 to 50 minutes, for example, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 40 minutes, 42 minutes, 44 minutes, 46 minutes, 48 ​​minutes, or 50 minutes.

[0067] In a specific embodiment of the present application, in step 2), the incubation temperature can be 36.5-37.5°C, for example, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37.0°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C.

[0068] In a specific embodiment of the present application, in step 3), the second centrifugation is a low-temperature high-speed centrifugation, which is used to separate and obtain whey liquid after casein is precipitated by rennet treatment, and the second supernatant obtained after the second centrifugation is the whey liquid.

[0069] In a specific embodiment of the present application, in step 3), the rotation speed of the second centrifugation can be 10621g~20817g. When expressed in rpm, the rotation speed of the second centrifugation can be 10000~14000rpm, for example, it can be 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm.

[0070] In a specific embodiment of the present application, in step 3), the time for the second centrifugation can be 25 to 35 minutes, for example, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, or 35 minutes.

[0071] In a specific embodiment of the present application, in step 3), the temperature of the second centrifugation can be 2-8°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C.

[0072] In a specific embodiment of the present application, in step 4), impurities and bacteria are removed by filtration to obtain a clean, sterile filtrate.

[0073] In a specific embodiment of the present application, in step 4), the filtration may be performed by using two layers of micron-grade filter membranes for simultaneous filtration, which are 0.45 μm and 0.22 μm filter membranes respectively, or by using 0.45 μm and 0.22 μm filter membranes for separate filtration.

[0074] In a specific embodiment of the present application, in step 5), the third centrifugation is ultracentrifugation, which is used to obtain preliminarily separated exosomes from the impurity-free and sterile filtrate obtained after filtration.

[0075] In a specific embodiment of the present application, in step 5), the rotation speed of the third centrifugation can be 117524~183632g. When expressed in rpm, the rotation speed of the third centrifugation can be 40000~50000rpm, for example, it can be 40000rpm, 41000rpm, 42000rpm, 43000rpm, 44000rpm, 45000rpm, 46000rpm, 47000rpm, 48000rpm, 49000rpm, 50000rpm.

[0076] In a specific embodiment of the present application, in step 5), the time for the third centrifugation can be 60 to 80 minutes, for example, 60 minutes, 62 minutes, 64 minutes, 66 minutes, 68 minutes, 70 minutes, 72 minutes, 74 minutes, 76 minutes, 78 minutes, or 80 minutes.

[0077] In a specific embodiment of the present application, in step 5), the temperature of the third centrifugation can be 2-8°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C.

[0078] If the speed is too low or the time is too short, the following problems will occur: First, the exosomes cannot be effectively separated from other cellular components (such as proteins, cell fragments, etc.) by centrifugal force, thus affecting the purity and yield of the exosomes. Second, the increase in residual impurities: In the case of insufficient centrifugation conditions, more impurities may be retained with the exosomes, which increases the difficulty of subsequent purification and may also affect the biological activity of the exosomes and their performance in analytical or therapeutic applications. Third, exosome loss: Too low a speed or too short a time may also cause some exosomes to fail to be effectively collected, resulting in the loss of exosomes, especially when dealing with samples containing a large number of other cellular components.

[0079] On the contrary, if the speed is too high or the time is too long, it may also bring some adverse effects: First, the destruction of the exosome structure: Too high a speed or long-term centrifugation may cause physical damage to the membrane structure of the exosomes, causing the exosomes to rupture and leak their contents, thereby affecting the integrity and function of the exosomes. Second, increased nonspecific binding: Long-term high-speed centrifugation may cause nonspecific binding of exosomes to other molecules or particles. These additional binding substances may interfere with the subsequent analysis or application of the exosomes. Third, increased energy consumption and costs: High speed and long-term centrifugation not only increase the energy consumption of the equipment, but may also shorten the service life of the centrifuge, thereby increasing the cost of the experiment.

[0080] After optimization of the present application, under the above-mentioned third centrifugation condition, the method has the best effect of extracting exosomes.

[0081] In a specific embodiment of the present application, in step 6), the purification specifically comprises the following steps:

[0082] a) centrifuging the first precipitate for a fourth time to obtain a second precipitate;

[0083] b) The second precipitate is resuspended and then ultrafiltered to obtain the supernatant, which is the exosome.

[0084] In a specific embodiment of the present application, in step a), the fourth centrifugation is low-temperature ultracentrifugation, which is used for preliminary purification of exosomes.

[0085] In a specific embodiment of the present application, in step a), the rotation speed of the fourth centrifugation can be 117524~183632g. When expressed in rpm, the rotation speed of the fourth centrifugation can be 40000~50000rpm, for example, it can be 40000rpm, 41000rpm, 42000rpm, 43000rpm, 44000rpm, 45000rpm, 46000rpm, 47000rpm, 48000rpm, 49000rpm, 50000rpm.

[0086] In a specific embodiment of the present application, in step a), the time of the fourth centrifugation can be 60 to 80 minutes, for example, 60 minutes, 62 minutes, 64 minutes, 66 minutes, 68 minutes, 70 minutes, 72 minutes, 74 minutes, 76 minutes, 78 minutes, or 80 minutes.

[0087] After optimization of the present application, under the above-mentioned fourth centrifugation conditions, the method has the best exosome purification effect. If the speed is too low and the time is too short, the exosome yield and purity will be low. If the speed is too high and the time is too long, the integrity and function of the exosomes will be affected.

[0088] In a specific embodiment of the present application, in step a), the temperature of the fourth centrifugation can be 2-8°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C.

[0089] In a specific embodiment of the present application, in step b), the second precipitate is resuspended with 1×PBS pre-cooled at 4°C.

[0090] In a specific embodiment of the present application, in step b), the molecular weight cutoff of the ultrafiltration tube may be 100Kda, specifically, it means that an ultrafiltration membrane with a molecular weight cutoff of 100kDa is installed inside the ultrafiltration tube.

[0091] In a specific embodiment of the present application, in step b), during ultrafiltration, the liquid is separated by passing through an ultrafiltration membrane in an ultrafiltration tube through low-temperature and low-speed centrifugation.

[0092] In a specific embodiment of the present application, in step b), the centrifugal speed during ultrafiltration can be 956-2655g. When expressed in rpm, the centrifugal speed during ultrafiltration can be 3000-5000rpm, for example, 3000rpm, 3200rpm, 3400rpm, 3600rpm, 3800rpm, 4000rpm, 4200rpm, 4400rpm, 4600rpm, 4800rpm, 5000rpm.

[0093] In a specific embodiment of the present application, in step b), the centrifugation time during ultrafiltration can be 20 to 40 minutes, for example, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, or 40 minutes.

[0094] In a specific embodiment of the present application, in step b), the centrifugal temperature during ultrafiltration can be 2-8°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C.

[0095] The present application also provides an exosome, which is prepared by the above method for extracting exosomes from dairy products.

[0096] In a specific embodiment of the present application, the particle size of the bovine milk exosomes extracted from the bovine milk product by the above-mentioned method for extracting exosomes from bovine milk products is less than 200 nm, the concentration of the exosomes is 300-400 μg / mL, and the number of exosome particles per unit protein is higher than the limit value of 1×10 published in the reference human cell-derived exosome group standard. 8 Particles / μg protein, with high purity.

[0097] The present application also provides the application of the above method in extracting exosomes from dairy products, wherein the dairy products are selected from any one or more of raw milk / raw milk, low-temperature pasteurized milk, ultra-high temperature sterilized milk, and whole milk powder.

[0098] The present application is further described below by examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, and related fields conventional in the art. These technologies have been well described in the existing literature. In the following examples, the instruments, raw materials, and reagents used can all be obtained commercially, and all meet relevant national standards. Unless otherwise specified, the instruments, reagents, and materials used in the examples are all obtained by conventional means.

[0099] Main instrument and equipment models involved

[0100] (1) High-speed centrifuge: Eppendorf 5804R

[0101] (2) Ultracentrifuge and rotor model: Beckman XPN-100; rotor: Type70Ti

[0102] (3)TEM model: Tecnai G2 spirit Biotwin biological transmission electron microscope

[0103] (4) Particle size analyzer: ParticLe Metrix Zetaview TWIN

[0104] (5) WB imager: Tanon 5200

[0105] The main reagents and consumables involved in WB identification in the examples are:

[0106] (1) Protein sample preparation

[0107] Protein lysis buffer (Beyotime P0013K), protease inhibitor complex (Sangon C600387-0001), protein marker (Sangon C610013-0250), BCA kit (Thermo Fisher 23227), loading buffer (Sangon C516031-0005), precast gel (Sangon C651105-0001) PVDF membrane (Merck Millipore ISEQ00005)

[0108] (2) Antibodies

[0109] Primary antibody rabbit anti-antibody: Anti-CD9 (Abcam ab307085), Anti-CaLnexin (Abcam ab133615), Anti-CD63 (MCEHY-P80604), Anti-CD81 (MCEHY-P80608), Anti-TSG101 antibody (MCEHY-P80924). Secondary antibody: HRP-labeled goat anti-rabbit IgG (Sangon D110058-0100)

[0110] Example 1 Method for isolating milk exosomes from different commercial dairy products

[0111] 1. Material Preparation

[0112] (1) Reference material: Raw milk / raw cow milk (Sample 1-RM) was sourced from the fresh milk filling truck of Bright Dairy & Food Co., Ltd. The sample was stored in a 4°C low-temperature refrigerator for future use to ensure that its quality met the relevant standards.

[0113] (2) Experimental samples: Bright Ubiquiti low-temperature pasteurized milk (sample 2-CM), Bright Ubiquiti ultra-high temperature sterilized milk (sample 3-UHT), and Bright whole milk powder (sample 4-WMP) sold on the market. The relevant samples were stored in a 4°C low-temperature refrigerator for future use to ensure that their quality met the relevant standards.

[0114] 2. Pretreatment of dairy product samples and preparation of exosomes

[0115] (1) Homogenization: According to the sample formula table, take samples with the same protein amount by calculation and set aside.

[0116] (2) Low-speed centrifugation to remove milk fat and impurities: The sample from step (1) was subjected to low-speed centrifugation at 3000 rpm (956 g) for 10 minutes to remove large particles of impurities and milk fat, and the upper whey after low-speed centrifugation was selected for the next step of processing.

[0117] (3) Chymosin treatment to precipitate casein: Take the supernatant from step (2), add an appropriate amount of chymosin (5 mg / mL), mix thoroughly, and incubate at 37.5°C for 30 minutes to allow the milk protein to agglutinate.

[0118] (4) Separating the whey by high-speed centrifugation: The rennet-treated dairy product is subjected to low-temperature high-speed centrifugation at 4°C and 10,000 rpm (10,621 g) for 25 minutes, and the supernatant is obtained after low-temperature high-speed centrifugation.

[0119] (5) Filtering through two layers of micron-grade filter membranes to obtain a purified, sterile supernatant: Filter the supernatant obtained in step (4) through 0.45 μm and 0.22 μm filters in turn to obtain a purified, sterile filtrate for later use.

[0120] (6) Ultracentrifugation to obtain a crude extract of bovine milk exosomes: The filtrate after filtration in step (5) is subjected to low-temperature ultracentrifugation, centrifuged at 4°C, 48,000 rpm (169,235 g) for 60 minutes, and the upper layer of liquid is discarded. The lower layer of precipitate obtained is the initially separated bovine milk exosomes.

[0121] (7) Exosome purification I: Add 26 mL of 1× PBS precooled at 4°C to the precipitate obtained in step (6), resuspend the precipitate, and centrifuge again at 4°C and 48,000 rpm (169,235 g) for 60 min to obtain the precipitate after centrifugation.

[0122] (8) Exosome purification II: The precipitate obtained in (7) was resuspended in 1 mL of pre-cooled PBS, purified by a 100KDa ultrafiltration tube, centrifuged at 4°C, 3000 rpm (956 g) for 30 min, the lower filtrate was discarded, and the supernatant was aspirated to obtain purified bovine milk exosomes.

[0123] (9) The exosomes obtained in step (8) are used immediately or frozen in a -80°C ultra-low temperature freezer.

[0124] In this example, milk exosomes were successfully extracted from different dairy products and subsequently characterized and tested.

[0125] Example 2 Method for isolating milk exosomes from different commercial dairy products

[0126] The difference from Example 1 is that the extraction conditions were changed.

[0127] 1. Material Preparation

[0128] (1) Reference material: Raw milk / raw cow milk (Sample 1-RM) was sourced from the fresh milk filling truck of Bright Dairy & Food Co., Ltd. The sample was stored in a 4°C low-temperature refrigerator for future use to ensure that its quality met the relevant standards.

[0129] (2) Experimental samples: Bright Ubiquiti low-temperature pasteurized milk (sample 2-CM), Bright Ubiquiti ultra-high temperature sterilized milk (sample 3-UHT), and Bright whole milk powder (sample 4-WMP) sold on the market. The relevant samples were stored in a 4°C low-temperature refrigerator for future use to ensure that their quality met the relevant standards.

[0130] 2. Pretreatment of dairy product samples and preparation of exosomes

[0131] (1) Homogenization: According to the sample formula table, take samples with the same protein amount by calculation and set aside.

[0132] (2) Low-speed centrifugation to remove milk fat and impurities: The sample from step (1) was subjected to low-speed centrifugation at 5000 rpm (2655 g) for 30 minutes to remove large particles of impurities and milk fat, and the upper whey after low-speed centrifugation was selected for the next step of processing.

[0133] (3) Chymosin treatment to precipitate casein: Take the supernatant from step (2), add an appropriate amount of chymosin (12.5 mg / mL), mix thoroughly, and incubate at 36.5°C for 50 minutes to allow the milk protein to agglutinate.

[0134] (4) Separating the whey by high-speed centrifugation: The rennet-treated dairy product is subjected to low-temperature high-speed centrifugation at 4°C and 14,000 rpm (20,817 g) for 35 minutes, and the supernatant is obtained after low-temperature high-speed centrifugation.

[0135] (5) Filtering through two layers of micron-grade filter membranes to obtain a purified, sterile supernatant: The supernatant obtained in step (4) is filtered through 0.45 μm and 0.22 μm filters in turn and set aside.

[0136] (6) Ultracentrifugation to obtain a crude extract of bovine milk exosomes: The liquid filtered in step (5) was subjected to low-temperature ultracentrifugation, centrifuged at 4°C, 40,000 rpm (117,524 g) for 80 minutes, and the upper layer of liquid was discarded. The lower layer of precipitate obtained was the initially separated bovine milk exosomes.

[0137] (7) Exosome purification I: Add 26 mL of 1× PBS precooled at 4°C to the precipitate obtained in step (6), resuspend the precipitate, and centrifuge again at 4°C, 40,000 rpm (117,524 g) for 60 min to obtain the precipitate after centrifugation.

[0138] (8) Exosome purification II: The precipitate obtained in (7) was resuspended in 1 mL of pre-cooled PBS, purified by a 100KDa ultrafiltration tube, centrifuged at 4°C, 5000 rpm (2655 g) for 40 min, the lower filtrate was discarded, and the supernatant was aspirated to obtain purified bovine milk exosomes.

[0139] (9) The exosomes obtained in step (8) are used immediately or frozen in a -80°C ultra-low temperature freezer.

[0140] This example successfully extracted milk exosomes from different dairy products.

[0141] Example 3 Characterization of exosomes

[0142] In this example, nanoparticle size analysis (NTA), transmission electron microscopy (TEM), BCA protein concentration determination and protein immunoblotting (WB) were used to identify and characterize the exosomes extracted in Example 1 to confirm their morphological structure, particle size distribution, protein concentration and the presence of specific marker proteins to ensure that the extracted exosomes met the quality requirements.

[0143] 1. Transmission electron microscopy (TEM) observation and identification:

[0144] (1) Sample preparation: Take 5 μL of the exosome-PBS mixture and drop it on a copper mesh. After it stands, aspirate the floating liquid.

[0145] (2) Negative staining: add 10 μL of phosphotungstic acid again, let stand, remove the floating liquid, and dry at room temperature.

[0146] (3) Imaging record: Use TEM imaging at 100 kV and save the image.

[0147] 2. Nanoparticle size analysis (NTA) identification:

[0148] (1) Dilute the sample: dilute the exosome-PBS mixture to the working concentration (about 2000-10000 times).

[0149] (2) Real-time detection: Nanoflow particle size analyzer imaging analysis is used, and the software calculates the particle size distribution and concentration of exosomes in the suspension.

[0150] 3. Western blot (WB) analysis and identification:

[0151] (1) Protein extraction: Thaw exosomes, add lysis buffer, place on ice for 30 min, centrifuge at 8000 rpm at 4°C for 20 min, and collect the supernatant.

[0152] (2) Concentration determination: The samples were tested using a BCA assay kit, a standard curve was prepared, and the exosome protein concentration was determined.

[0153] (3) Sample denaturation and electrophoresis: The samples were mixed with the loading buffer and denatured by boiling, cooled to room temperature, and protein separation was performed using 4-20% SDS-PAGE precast gel (80 V, 120 min).

[0154] (4) Transfer and blocking: The protein was transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 60 min.

[0155] (5) Primary antibody incubation: Wash the membrane with TBST for 5 min x 3 times, add rabbit anti-CD9, CD63, and TSG101 monoclonal antibodies (1:1000) respectively, incubate at 4°C overnight, incubate with primary antibody and secondary antibody successively, and wash with TBST.

[0156] (6) Secondary antibody incubation: Wash the membrane with TBST for 10 minutes x 5 times, add horseradish peroxidase-labeled goat anti-rabbit IgG (1:10000) and react at room temperature for 90 minutes

[0157] (7) Exposure and imaging: After the reaction with the ECL mixed solution, the image is exposed and saved in the imager.

[0158] 4. BCA protein concentration determination

[0159] (1) According to the instructions, use buffer to gradient dilute bovine serum albumin (BSA) standard to prepare 25 μg / mL-2000 μg / mL protein standard.

[0160] (2) In the 96-well microplate protocol, each sample requires 200 μL of working solution. Therefore, prepare the BCA working solution first and calculate the total volume of working solution required according to the formula below. Formula: (number of standards + number of protein samples to be tested) x (number of experimental repetitions) x (volume of working solution for each sample). Mix 50 parts of BCA reagent A with 1 part of BCA reagent B (ratio of reagent A to reagent B = 50:1) to prepare the working solution.

[0161] (3) Add 10 μL of sample to each well of the standard sample and the well to be tested. Use a dispenser to add 200 μL of working solution to each well and shake for 30 seconds to mix thoroughly. Seal the 96-well microplate and incubate at 37°C for 30 minutes.

[0162] (4) After the 96-well microplate has cooled to room temperature, the absorbance at 562 nm is measured using an ELISA reader.

[0163] (5) Draw a standard curve and calculate the concentration of the protein sample. Specifically: First, subtract the average absorbance of the blank standard at 562 nm from the absorbance of each standard and the protein sample to be tested at 562 nm; second, plot the average absorbance of the BSA standard at 562 nm after blank correction against its concentration (μg / mL) to draw a standard curve; third, use the standard curve to determine the protein concentration of each protein sample to be tested.

[0164] Experimental results:

[0165] The extracted exosomes were identified by different detection indicators, including transmission electron microscopy (TEM) to observe whether the extracted exosome membrane structure was intact and whether the size was in line with expectations. Figure 1 As shown in the figure, at the same magnification, typical "saucer-shaped" exosomes can be observed in the resuspensions of exosome samples 1-4 from different dairy products, and the size is more in line with expectations.

[0166] The particle size and particle number concentration of exosomes were analyzed by nanoparticle tracking technology (NTA) to evaluate the efficiency of exosome extraction. Figure 2 As shown, the resuspensions of exosome samples 1-4 from different bovine dairy products are mainly distributed in the range of less than 200 nm. Specifically, samples 1, 2, and 4 have a particle size peak at around 120 nm, and sample 3 has a particle size peak at around 150 nm.

[0167] The protein concentration was determined by the BCA method as follows Figure 3 As shown, the concentrations of exosome samples 1-4 from different dairy products are moderate, and the concentrations are: 332μg / mL, 359μg / mL, 367μg / mL, 346μg / mL, ranging from 300-400μg / mL. The purity of exosomes is evaluated based on the number of exosome particles per unit protein (the ratio of exosome particle concentration to protein concentration). The larger the ratio, the higher the purity. Figure 4 As shown in Figure 2, the ratio of the number of exosome samples 1-4 prepared from different dairy products to the amount of protein is: 3.82×10 9 Particles / μg protein, 4.69×10 9 Particles / μg protein, 6.65×10 8Particles / μg protein, 1.79×10 9 The number of particles / μg protein was higher than the limit value of 1×10 8 particles / μg protein, indicating high purity. In addition, Figure 5 As shown, specific proteins CD9, CD63 and TSG101 can be detected in exosome samples 1-4 from different dairy products, but Calnexin protein cannot be detected, which indicates that the exosomes extracted by the method have good purity.

[0168] Comparative Example 1 Effect of different rennet on extraction effect

[0169] Rennet No. 1 is Rennin derived from calf stomach, purchased from Sigma with the item number R4877; Rennet No. 2 is Rennin extracted from plants, purchased from MCE with the item number HY-P2810; Rennet No. 3 is chymosin derived from bovine pancreas, purchased from Sangon Biotech with the item number A600307.

[0170] According to the existing public information, the above three chymosin products were tested in this example. All products were guaranteed to have their enzyme activity standardized to 1000 USP U / mg. The USP unit here clearly defines the standard that the change in absorbance at a wavelength of 237 nanometers caused by enzymatic hydrolysis of ATEE per minute reaches 0.0075 under ideal conditions of 25°C and pH 7.0. The method for detecting the clotting effect refers to the national standard of compound chymosin [WS10001-HD(0856)-2002], such as Figure 6 As shown, enzyme No. 3 has the best cost-effectiveness, with low cost and good coagulation effect. In unit time, a dosage of 2.5-12.5 mg / mL can achieve a coagulation effect that is no worse than that of enzyme No. 1 and enzyme No. 2.

[0171] Comparative Example 2 Effect of Extraction Method on Extraction Effect

[0172] In this example, an exosome extraction kit (precipitation method) was used to extract exosomes, and the extraction effect (purity) was compared with the method of the present application.

[0173] The specific operation process of the kit method is as follows:

[0174] The first four steps (steps 1 to 4) of the sample pretreatment method are the same as those of Example 1, starting from step 5:

[0175] a. Add 190 μL of cell supernatant exosome extraction reagent to every 1 ml of supernatant sample prepared in step 4, mix well by pipetting, and place at 4°C overnight.

[0176] b. Centrifuge at 10,000 × g at 4°C for 30 minutes. Carefully remove the supernatant with a 1 mL pipette tip. Try to remove as much supernatant as possible without touching the precipitate. Collect the precipitate, which is the exosome.

[0177] c. The exosome pellet obtained by centrifugation can be resuspended in an appropriate amount of PBS for subsequent WB experiments.

[0178] d. The obtained exosomes heavy suspension was used for subsequent experiments such as Western blot.

[0179] like Figure 7 As shown, the Western Blot (WB) results clearly show that the leftmost 2-CM sample is a positive control. The 2-CM sample (located on the leftmost) used as the experimental benchmark shows a strong positive control signal. Compared with the positive control, the exosome positive proteins (TSG101, CD81, CD63) cannot be detected in the samples extracted by the kits 3 and 4, indicating that compared with the method of the present application, the kit method has a poor extraction effect on samples 3 and 4, and the kit fails to extract samples 3 and 4, indicating that the traditional method of extracting bovine milk exosomes has strong pertinence and limitations, and has a poor extraction effect on bovine dairy products that are difficult to extract exosomes, and cannot achieve a good extraction effect for a wide range of bovine dairy products. The method of the present application can successfully extract exosomes from a variety of commercial bovine dairy products, indicating the effectiveness of the extraction system of the present application.

[0180] The above embodiments are merely illustrative of the principles and effects of the present application and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed in the present application shall still be covered by the claims of the present application.

Claims

1. A method for extracting exosomes from dairy products, comprising the following steps: 1) performing a first centrifugation on the dairy product to obtain a first supernatant; 2) mixing the first supernatant with chymosin and then incubating; 3) centrifuging the dairy product after incubation in 2) for the second time to obtain a second supernatant; 4) filtering the second supernatant to obtain a filtrate; 5) centrifuging the filtrate for a third time to obtain a first precipitate; 6) purifying the first precipitate to obtain exosomes; The rotation speed of the second centrifugation is higher than that of the first centrifugation, and the rotation speed of the third centrifugation is higher than that of the second centrifugation.

2. The method according to claim 1, characterized in that In step 1), the speed of the first centrifugation is 956-2655 g, and the time of the first centrifugation is 10-30 minutes.

3. The method according to claim 1, characterized in that In step 2), the amount of chymosin added is such that the final concentration of chymosin is 2.5 to 12.5 mg / mL; and / or, the chymosin comprises chymotrypsin; And / or, the incubation time is 30 to 50 minutes, and the incubation temperature is 36.5 to 37.5°C.

4. The method according to claim 1, characterized in that: In step 3), the speed of the second centrifugation is 10621-20817 g, the time of the second centrifugation is 25-35 minutes, and the temperature of the second centrifugation is 2-8°C.

5. The method according to claim 1, characterized in that In step 4), the pore size of the filter membrane used for filtration is 0.22 to 0.45 μm.

6. The method according to claim 1, characterized in that In step 5), the rotation speed of the third centrifugation is 117524-183632g, the time of the third centrifugation is 60-80 minutes, and the temperature of the third centrifugation is 2-8°C.

7. The method according to claim 1, characterized in that In step 6), the purification comprises the following steps: a) centrifuging the first precipitate for a fourth time to obtain a second precipitate; b) The second precipitate is resuspended and then ultrafiltered to obtain a supernatant, which is the exosome; Preferably, in step a), the speed of the fourth centrifugation is 117524-183632 g, the time of the fourth centrifugation is 60-80 minutes, and the temperature of the fourth centrifugation is 2-8° C.; Preferably, in step b), the centrifugal speed during ultrafiltration is 956-2655 g, the centrifugal time during ultrafiltration is 20-40 minutes, and the centrifugal temperature during ultrafiltration is 2-8°C.

8. The method according to claim 1, characterized in that The cow milk product is selected from any one or more of raw milk / raw cow milk, low-temperature pasteurized milk, ultra-high temperature sterilized milk, and whole milk powder.

9. An exosome prepared by the method according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 in extracting exosomes from dairy products, wherein the dairy products are selected from any one or more of raw milk / raw milk, low-temperature pasteurized milk, ultra-high temperature sterilized milk, and whole milk powder.

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