CA3205241A1 - Milk-derived exosome and extraction method therefor - Google Patents

Abstract

The present application relates to a method for extracting milk-derived exosomes. The method sequentially comprises the following steps: (1) subpackaging and centrifuging raw milk, and collecting a clear liquid; (2) centrifuging the clear liquid obtained in step (1), and collecting a clear liquid in the middle layer; (3) centrifuging the clear liquid in the middle layer obtained in step (2), collecting a clear liquid, mixing the collected clear liquid with a PBS buffer solution and an exosome extraction reagent uniformly, and performing incubation; (4) centrifuging the product of step (3), retaining the precipitate, and re-suspending the precipitate with a PBS buffer solution; and (5) centrifuging the product of step (4) and collecting the supernatant to obtain the milk-derived exosome. Compared with milk-derived exosomes prepared by other methods, the milk-derived exosome prepared by the method for extracting milk-derived exosomes provided in the present application is more complete in morphology, requires less sample volume, and contains more types of milk-derived exosome proteins after separation, which is more conducive to analysis and research work.

Description

MILK-DERIVED EXOSOME AND EXTRACTION METHOD THEREFOR
CROSS-REFERENCE
The present application claims priority to Chinese patent application titled "MILK-DERIVED
EXOSOME AND EXTRACTION METHOD THEREFOR" and filed with the China Patent Office, with the application number of 2022107559493 and the application date of June 30, 2022, the entire contents of which are incorporated herein by reference.
Technical Field The present application relates to the technical field of dairy products, in particular to a milk-derived exosome and an extraction method therefor.
Background Art Exosomes are round or oval vesicles with double-membrane structures actively secreted by cells, with a diameter of 30-200 nm and a density of 1.13-1.19 g/mL. In 1983, small vesicles of 50 nm were discovered for the first time in the study of transferrin receptors in reticulocytes. In 1987, the Canadian scholar Rose Johnstone created the word "Exosome" and believed that exosomes are just a tool for the "excretion" of cells into the external environment. In 2007, Valadi et al.
proposed that exosomes contain mRNA and miRNA and can transmit them to another cell. Since then, the functions and mechanisms of exosomes started to be extensively studied. Exosomes exist in various body fluid environments such as serum, urine, saliva, milk and amniotic fluid.
Exosomes in biological fluids indicate the functional states of their origin cells, and can be used as bio-diagnostic markers for liver cancer, prostate cancer and other diseases in medicine. Since the membrane structures of exosomes can also be used as a drug carrier to exhibit high-efficiency drug delivery characteristics, for example, exosomes where unstable curcumin are loaded can improve the therapeutic effect.
Milk-derived exosomes are small vesicles secreted by mammary epithelial cells, and their main components are proteins, lipids and nucleic acids. The membrane surfaces of milk-derived exosomes have specific membrane proteins, such as the most abundant tetraspanins (CD9, CD82, CD81 and CD63), co-stimulatory molecules (CD54) and adhesion molecules (CD11b). Lipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolarnine, sphingomyelin, and phosphatidylinositol) are also commonly found in exosomes. Nucleic acids in milk-derived exosomes mainly comprise non-coding single-stranded RNA molecules (micro RNA, miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), mRNA and tRNA, etc. Milk-derived Date Recue/Date Received 2023-06-30 exosomes play an important role in physiological processes, such as mediating cell communication, promoting cell growth, and participating in immune responses.
At present, the commonly used exosome separation technology is centrifugation, but centrifugation would damage the morphologies of exosomes.
In view of this, the present application is hereby provided.
Summary of the Invention In order to solve the existing technical problems, the present application provides a new method for extracting milk-derived exosomes, and the method has little influence on the morphologies of exosomes.
In order to achieve the above-mentioned object, the technical solution used in the present application is as follows:
In a first aspect, the present application provides a method for extracting milk-derived exosomes.
The method sequentially comprises the following steps:
(1) Subpackaging and centrifuging raw milk, and collecting a clear liquid;

(2) Centrifuging the clear liquid obtained in step (1), and collecting a clear liquid in the middle layer;

(3) Centrifuging the clear liquid in the middle layer obtained in step (2), collecting a clear liquid, mixing the collected clear liquid with a PBS buffer solution and an exosome extraction reagent uniformly, and performing incubation;

(4) Centrifuging the product of step (3), retaining the precipitate, and re-suspending the precipitate with a PBS buffer solution; and

(5) Centrifuging the product of step (4) and collecting the supernatant to obtain the milk-derived exosome.
Preferably or optionally, the centrifuging process in step (1) is performed at room temperature, 1500-2500 X g for 5-15 min.
Preferably or optionally, the centrifuging process in step (2) is performed at room temperature, 8500-10000 X g for 25-35 mm, preferably 30 mm.
Preferably or optionally, the centrifuging process in step (3) is performed at room temperature, 8500-10000 X g for 25-35 mm, preferably 30 mm.

Date Recue/Date Received 2023-06-30 Preferably or optionally, in step (3), the volume ratio of the clear liquid to the PBS buffer solution to the exosome extraction reagent is 1: 1: 1.
Preferably or optionally, the exosome extraction reagent is a total exosome separation reagent produced by the Thenno Fisher company.
Preferably or optionally, in step (3), the incubation time is 25-35 min, preferably 30 min.
Preferably or optionally, the centrifuging process in step (4) is peifonned at room temperature, 8500-10000X g for 9-11 min, preferably 10 min.
Preferably or optionally, the centrifuging process in step (5) is peifonned at room temperature, 8500-10000 X g for 4-6 min, preferably 5 min.
In a second aspect, the present application further provides a milk-derived exosome which is prepared by the above-mentioned extraction method.
BENEFICIAL EFFECTS
Compared with milk-derived exosomes prepared by other methods, the milk-derived exosome prepared by the method for extracting milk-derived exosomes provided in the present application is more complete in morphology, requires less sample volume, and contains more types of milk-derived exosome proteins after separation, which is more conducive to analysis and research work.
Brief Description of the Drawings FIG. 1 is a transmission electron microscope image of the milk-derived exosome prepared in Example 1;
FIG. 2 is a transmission electron microscope image of the milk-derived exosome prepared in Example 2;
FIG. 3 is a transmission electron microscope image of the milk-derived exosome prepared in Comparative Example 1;
FIG. 4 is a transmission electron microscope image of the milk-derived exosome prepared in Comparative Example 2;
FIG. 5 is a transmission electron microscope image of the milk-derived exosome prepared in comparative example 3;
FIG. 6 is a transmission electron microscope image of the milk-derived exosome prepared in Date Recue/Date Received 2023-06-30 Comparative Example 4;
FIG. 7 is a Venn diagram showing the types of exosome proteins obtained by the separation of milk-derived exosomes prepared in Example 1 and Comparative Examples 1 and 3.
Detailed Description of the Invention In order to facilitate understanding of the present application, the present application is more comprehensively described in detail in conjunction with the drawings and preferred experimental examples of the description, but the scope of protection of the present application is not limited to the following specific examples.
Unless otherwise defined, all technical terms used hereinafter have the same meaning as that commonly understood by a person skilled in the art. The technical terms used herein are only for the purpose of describing specific examples, and are not intended to limit the scope of protection of the present application.
Unless otherwise specified, all kinds of raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.
Example 1 This example provides a method for extracting a milk-derived exosome.
The raw milk of the milk-derived exosome is derived from a fresh milk delivery truck of Beijing Sanyuan Foods Co., Ltd.
Raw milk was subpackaged into 1 mL centrifuge tubes, 1 mL of sample per tube, and centrifuged at room temperature, 2000 X g for 10 min, and a clear liquid was collected after centrifugation was completed. Centrifugation was performed again at room temperature, 10000 X
g for 30 min, and a clear liquid in the middle layer was collected.
The clear liquid in the middle layer was centrifuged at room temperature, 10000X g for 30 min, and then 200 1, of clear liquid was collected and mixed with 200 I, of a PBS
buffer solution.
Then, 200 pl of a total exosome separation reagent produced by the Thenno Fisher company was added, the mixture was mixed uniformly and subjected to incubation and reaction for 30 min, then centrifugation was performed at room temperature, 10000 X g for 10 min, and the precipitate was collected.

Date Recue/Date Received 2023-06-30 The precipitate was resuspended with 50 1., of a PBS buffer solution, centrifuged at 10000 X g for 5min at room temperature, and the supematant was collected to obtain an extracted exosome.
Example 2 This example provides a method for extracting a milk-derived exosome.
The raw milk of the milk-derived exosome is derived from ilactou milk powder produced by Beijing Sanyuan Foods Co., Ltd.
The extraction process in Example 2 is the same as that in Example 1.
Comparative Example 1 This comparative example provides a method for extracting a milk-derived exosome.
The raw milk of the milk-derived exosome is derived from a fresh milk delivery truck of Beijing Sanyuan Foods Co., Ltd.
Raw milk was subpackaged into 1 mL centrifuge tubes, 1 mL of sample per tube, and centrifuged at room temperature, 2000 X g for 10 min, and a clear liquid was collected after centrifugation was completed. Centrifugation was performed again at room temperature, 10000 X
g for 30 min, and a clear liquid in the middle layer was collected.
The clear liquid in the middle layer was centrifuged at room temperature, 10000 X g for 30 min, and then 200 1t1, of clear liquid was collected and mixed with 200 tiL of a PBS buffer solution.
Then, 200 1iL of a high-efficiency exosome precipitation reagent produced by Invent Biotechnologies (Beijing), Inc. was added, the resulting mixture was mixed uniformly and subjected to incubation and reaction at room temperature for 30 min, centrifugation was performed at room temperature, 10000 X g for 10 min, and the precipitate was collected.
The precipitate was resuspended with 50 [IL of a PBS buffer solution, centrifuged at room temperature, 10000X g for 5 min, and the supernatant was collected to obtain an extracted exosome.
Comparative Example 2 This comparative example provides a method for extracting a milk-derived exosome.
Date Recue/Date Received 2023-06-30 The raw milk of the milk-derived exosome is derived from ilactou milk powder produced by Beijing Sanyuan Foods Co., Ltd.
The extraction process of Comparative Example 2 was the same as that of Comparative Example 1.
Comparative Example 3 This comparative example provides a method for extracting a milk-derived exosome.
The raw milk of the milk-derived exosome is derived from a fresh milk delivery truck of Beijing Sanyuan Foods Co., Ltd.
50 mL of raw milk was placed in an ultracentrifuge tube and centrifuged at 4 C, 3000 rpm for 60 min, the supernatant was collected and centrifuged at 4 C, 16000 rpm for 20 min, the fat on the surface was removed, a clear liquid was collected and centrifuged at 4 C, 30000 rpm for 60 min, and loose particles were collected.
The collected loose particles were re-suspended with PBS, then dissolved with a 0.3 mol/L
sucrose solution and centrifuged at 4 C, 20000 rpm for 120 min, the precipitate was collected, re-suspended with PBS and then filtered via a 0.22 membrane, and a clear liquid obtained after filtration was collected to obtain an exosome.
Comparative Example 4 This comparative example provides a method for extracting a milk-derived exosome.
The raw milk of the milk-derived exosome is derived from a fresh milk delivery truck of Beijing Sanyuan Foods Co., Ltd.
50 mL of raw milk was placed in an ultracentrifuge tube and centrifuged at 4 C, 3000 rpm for 60 min, the supernatant was collected and centrifuged at 4 C, 16000 rpm for 20 min, the fat on the surface was removed, and a clear liquid was collected.
200 jtL of clear liquid was mixed uniformly with 200 pl of a PBS buffer solution, then 200 111, of a high-efficiency exosome precipitation reagent produced by Invent Biotechnologies (Beijing), Inc. was added, and the resulting mixture was mixed unifointly, subjected to incubation and reaction at room temperature for 30 min, and then centrifuged at room temperature, 10000 X g

6 Date Recue/Date Received 2023-06-30 for 10 mm, and the precipitate was collected.
The precipitate was re-suspended with 50 irt of a PBS buffer solution, centrifuged at room temperature, 10000 X g for 5 min, and the supernatant was collected to obtain an extracted exosome.
Effect Example 1 The morphologies of the milk-derived exosomes obtained in Examples 1-2 and Comparative Examples 1-4 were observed under a transmission electron microscope (TEM), and the results are shown in FIGs. 1-6.
As can be seen from FIGs. 1-6, the milk-derived exosomes obtained in Examples 1-2 and Comparative Examples 1-3 all have a diameter range of 30-150 nm, and all have cup-shaped double-layer membrane structures. The milk-derived exosomes obtained by the methods provided in Comparative Examples 2-3 are incomplete in morphology and have a messy background, whereas the milk-derived exosomes obtained by the methods in Comparative Example 1 and Example 1 are very complete in morphology and show a complete "cup and plate" structure. The milk-derived exosome obtained by the method in Comparative Example 4 is completely broken.
Effect Example 2 The milk-derived exosomes obtained in Example 1 and Comparative Examples 1 and 3 were respectively treated, and the specific treatment method was as follows: 100 trI, of 0.1% RapiGest SF reagent produced by the Waters company was added to the milk-derived exosome, the resulting liquid was mixed uniformly, and the resulting mixture was ultrasonically dissolved for 30 min; then 100 fiL of DTI' reagent was added, and the resulting mixture was placed in a water bath at 56 C for 1 h; after the reaction was completed, the resulting product was cooled down to room temperature naturally, 1001.IL of 0.1 mol/L IAA was added, and reaction was performed in the dark for 40 min; then 50 mt of 1 mg/mL trypsin was added, enzyme digestion was performed overnight, and typsin was added once more halfway; after the enzymatic hydrolysis was completed, an appropriate amount of 0.5 mol/L HCl was added to terminate the reaction for 50 mm, and the RapiGest SF reagent was made to precipitate; and desalting treatment was performed with an HLB column, the precipitate produced by the RapiGest SF
reagent was removed under an acidic condition, the sample was collected, lyophilization was performed, the lyophilized sample was redissolved with a 0.1% formic acid aqueous solution, centrifugation was performed at 10000X g for 10 min, and the supernatant was collected for later use.

7 Date Recue/Date Received 2023-06-30 The treated milk-derived exosomes were detected by using nanoliter liquid chromatography connected in series with Q Exactive Orbitrap mass spectrometer, and a Venn diagram showing the types of proteins was plotted according to the detection results, as shown in FIG. 7.
As can be seen from FIG. 7, the milk-derived exosome prepared in Example 1 can be separated to obtain 315 kinds of bovine milk exosome proteins, the milk-derived exosome prepared in Comparative Example 1 can be separated to obtain 267 kinds of bovine milk exosome proteins, and the milk-derived exosome prepared in Comparative Example 3 can be separated to obtain 307 kinds of bovine milk exosome proteins.
In summary, compared with a traditional high-speed centrifugation method, the method for extracting milk-derived exosomes provided in the present application has the advantages that the prepared milk-derived exosome is more complete in morphology and requires less sample volume by means of the optimization of the process and the selection of reagent. Compared with similar reagents, the prepared milk-derived exosome can obtain more types of exosome proteins after separation and thus is more suitable for subsequent analysis of exosome proteomics.
Finally, it should be noted that: the above-mentioned examples are only used to illustrate the technical solution of the present application, rather than to limit them.
Although the present application is described in detail with reference to the foregoing examples, a person skilled in the art should understand that it is still possible to amend the technical solutions described in the foregoing examples or to make equivalent replacements to some of the technical features thereof;
and these amendments or replacements should not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the examples of the present application.
INDUSTRIAL PRACTICABILITY
The present application relates to a method for extracting milk-derived exosomes. Compared with milk-derived exosomes prepared by other methods, the milk-derived exosome prepared by the method for extracting a milk-derived exosome provided in the present application is more complete in morphology, requires less sample volume, and contains more types of milk-derived exosome proteins after separation, which is more conducive to analysis and research work.

8 Date Recue/Date Received 2023-06-30

Claims (10)

1. A method for extracting milk-derived exosomes, wherein the method sequentially comprises the following steps:
(1) Subpackaging and centrifuging raw milk, and collecting a clear liquid;
(2) Centrifuging the clear liquid obtained in step (1), and collecting a clear liquid in the middle layer;
(3) Centrifuging the clear liquid in the middle layer obtained in step (2), collecting a clear liquid, mixing the clear liquid with a PBS buffer solution and an exosome extraction reagent uniformly, and performing incubation;
(4) Centrifuging the product of step (3), retaining the precipitate, and re-suspending the precipitate with a PBS buffer solution; and (5) Centrifuging the product of step (4) and collecting the supernatant to obtain the milk-derived exosome.

2. The method for extracting milk-derived exosomes according to claim 1, wherein the centrifuging process in step (1) is performed at room temperature, 1500-2500 X
g for 5-15 min.

3. The method for extracting milk-derived exosomes according to claim 1, wherein the centrifuging process in step (2) is performed at room temperature, 8500-10000 X g for 25-35 min, preferably 30 min.

4. The method for extracting milk-derived exosomes according to claim 1, wherein the centrifuging process in step (3) is performed at room temperature, 8500-10000 X g for 25-35 min, preferably 30 min.

5. The method for extracting milk-derived exosomes according to claim 1, wherein in step (3), the volume ratio of the clear liquid to the PBS buffer solution to the exosome extraction reagent is 1 : 1 : 1.

6. The method for extracting milk-derived exosomes according to claim 5, wherein the exosome extraction reagent is a total exosome separation reagent produced by the Thermo Fisher company.

7. The method for extracting milk-derived exosomes according to claim 1, wherein in step (3), the incubation time is 25-35 min, preferably 30 min.

Date Recue/Date Received 2023-06-30

8. The method for extracting milk-derived exosomes according to claim 1, wherein the centrifuging process in step (4) is performed at room temperature, 8500-10000 X g for 9-11 min, preferably 10 min.

9. The method for extracting milk-derived exosomes according to claim 1, wherein the centrifuging process in step (5) is performed at room temperature, 8500-10000 X g for 4-6 min, preferably 5 min.

10. A milk-derived exosome, wherein the milk-derived exosome is prepared by the extraction method according to any one of claims 1-9.
Date Recue/Date Received 2023-06-30