Note: Descriptions are shown in the official language in which they were submitted.
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
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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.
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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
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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.
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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
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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.
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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.
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