AU2020103733A4

AU2020103733A4 - Method for Separating Exosome from Cooked Meat Samples

Info

Publication number: AU2020103733A4
Application number: AU2020103733A
Authority: AU
Inventors: Lei Chen; Mailin Gan; Bohong Li; Xuewei Li; Linyuan Shen; Yuhao Xie; Yiting Yang; Jingjun Zhang; Shunhua Zhang; Li Zhu
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2020-11-27
Filing date: 2020-11-27
Publication date: 2021-02-11
Anticipated expiration: 2028-11-27

Abstract

The invention discloses a method for separating exosome from cooked meat samples, comprising the following steps: removing fascia, blood stains, fat and abnormal meat samples of fresh meat and then mashing it into mince. Then wrapping the mince with medical gauze and boiling it in double distilled water at 100 C for 40 minutes. Centrifuging the obtained broth to remove cell debris and impurities and then precipitating exosome by ultra-high-speed centrifugation. Dissolving it in PBS solution, and then using exosome extraction indicator to separate and purify exosome. The method can effectively separate exosome from cooked meat samples and ensure the purity of exosome. Meanwhile, the operation of the method is simple.

Description

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTIONENTITLED:

Method for Separating Exosome from Cooked Meat Samples

The invention is described in the following statement:-

Method for Separating Exosome from Cooked Meat Samples

TECHNICAL FIELD

The invention belongs to the field of cell biology, and particularly relates to a method for

separating exosome from cooked meat samples.

BACKGROUND

Secreted by living cells, exosomes are vesicles containing DNA, RNA, protein and lipid

with the size of 30-200nm in late endosomes (or multivesicular bodies). Besides, they

widely exist in various body fluids such as serum, plasma and urine.

It has been found that exosome and its contents can be obtained from diet, for example,

milk that crosses the boundary of species. Human intestinal, vascular endothelial cells

and rat intestinal cells can transport milk exosome by endocytosis and secrete miRNA

and other substances through the lateral basement membrane. In milk, the miRNA

encapsulated in exosome has permeability to intestinal barrier. Human beings absorb

miRNA from milk, and these miRNAs are transmitted to circulating immune cells, thus

affecting gene expression. Then, in the study of the cross-border regulation mediated by

exogenous plant miRNA and the plant miRNA absorption and transfer mechanism, it has

been found that atypical miRNA-2911 in honeysuckle decoction can directly target

influenza A virus and treat colds, because the extract of honeysuckle decoction can inhibit

the replication and pathogenesis of dengue virus 2. There are studies reporting that

miRNA coated by exosome-like vesicle has been identified in 11 edible fruits and

vegetables. These edible plant-derived exosome-like nanoparticles (EPDELNs) are

natural plant ultrastructure similar to exosome. miRNA plays an important regulation role in physiology and pathology of animals and plants; these plant miRNAs can be selectively coated in exosome to make them exist stably.

Exosomes are involved in the regulation of important cellular physiological activities,

and their roles in immune response, apoptosis, angiogenesis, inflammation and

coagulation have been reported. Exosomes have become potential targets, early

diagnostic markers and targeted drug carriers for the treatment of various diseases. In this

way, exosomes become an important research hotspot in the field of life science and

medicine in recent years.

As we all know, exosome can be isolated from cell culture supernatant, various body

fluids such as blood and urine, and even various lavage fluids. Because the tissues are

rich in cells and capillaries, previous researchers have obtained exosome from heart and

nerve tissues. Meanwhile, muscle quality is an important economic trait. Recent studies

have found that exosome secreted by differentiated human skeletal muscle cells can

trigger myogenesis of stem cells and induce muscle regeneration or affect the function of

receptor cells in other tissues. The myotubes formed by mouse C2C12 differentiation can

also secrete exosome to regulate the differentiation of skeletal muscle cells, but at present,

there is no report on exosomes directly secreted by tissue skeletal muscle.

Nowadays, meat has become an important source of essential nutrients for human beings.

These nutrients are very important to human health. Meat is especially rich in protein and

has high biological value. Cooked meat refers to the meat food after cooking and high

temperature treatment. High-temperature treatment can change the properties of raw meat,

loosen the structure of protein, and facilitate digestion by acid and enzyme in digestive

tract. While the quality of muscle is directly related to the growth and development of muscle. Skeletal muscle is mainly involved in maintaining body movement and energy metabolism. Mammalian skeletal muscle accounts for about 40% ~ 60% of body weight, which has long been the main object of animal production and meat quality research.

In order to solve the problems related to the existence and extraction of exosomes in

cooked meat products, the invention adopts simple and easy-to-operate method to extract

exosomes from cooked meat, so as to finally achieve the purpose of separating and

purifying exosome from muscle tissue and proving the existence of exosome in cooked

meat.

SUMMARY

In order to solve the above problems, the present invention provides a method for

separating and purifying exosomes from cooked meat, which can effectively separate and

purify exosomes from cooked meat and obtain exosome with high concentration.

In order to achieve the above purpose, the technical scheme adopted by the invention is

as follows.

The method for separating exosomes from cooked meat samples comprises the following

steps.

(1) Removing fascia, blood stains, fat and abnormal meat samples from raw meat and

stirring it into mince. Then wrapping the mince with medical gauze and boiling it in

double distilled water at 100 C for 40 to 60 minutes. Then filter out meat foam through

gauze to obtain broth.

(2) After cooling the broth, centrifuging the broth with 2,000xg centrifugal force to

separate meat foam and impurities, and then centrifuging the supernatant at 4C with

12,000xg centrifugal force.

(3) After centrifugation, keeping the supernatant and filtering it through 0.45tm and

0.2tm filters to remove cell debris from broth. Precipitating exosome in the filtrate by

ultra-high-speed centrifugation and adding PBS solution to suspend exosome.

(4) Adding purification reagent to the obtained solution for purification.

Further, in the step (1), fascia, fat and abnormal meat samples should be removed as

much as possible from the raw meat samples. Wherein, the abnormal meat samples refer

to meat with bloodstains, pustules, abnormal colour, fester, malformation, pathological

changes and so on. The used instruments, gauze and medical gauze are all washed with

double distilled water.

Further, the specific operation of step (2) is as follows.

(1) Split charging the broth in 15 ml centrifuge tubes;

(2) Centrifuging with a conventional centrifuge 2000xg for 30 minutes and discarding the

precipitate while keeping the supernatant.

(3) Putting the supernatant into a 2ml centrifuge tube.

(4) Using a high-speed centrifuge to centrifuge at 4C with 12,000xg for 45 minutes and

discarding the sediment and the upper lipid layer and keep the intermediate supernatant.

Further, the specific operation of step (3) is as follows.

(1) Let the supernatant obtained by centrifugation continuously pass through filters of

0.45am and 0.2am.

(2) Split charging the obtained filtrate into centrifuge tubes of an ultra-high-speed

centrifuge and trimming them to make the weight difference of centrifuge tubes does not

exceed 0.0050g.

(3) Centrifuging the filtrate with an ultra-high-speed centrifuge at 15°C, 160,000xg for

120 minutes.

(4) After centrifugation, pouring the supernatant out and dissolving and suspending

exosome again with 100L PBS solution.

Further, the specific operation of step (4) is as follows.

Exosome purification. Transferring 1.5ml of exosome suspension obtained in step (3) to a

2ml centrifuge tube and adding 0.5ml of Ribo TM Exosome Isolation Reagent (for other

body fluids). Then blowing it evenly with a pipettor (liquid turbidity can be seen in the

mixed state) and standing it in a refrigerator at 4C overnight. Then centrifuging at

1500xg, 4C for 30 minutes. Discarding the supernatant to obtain exosome.

Compared with extracting exosome from serum, plasma and urine, the method has the

following beneficial technical effects. The operation of extracting exosomes from

cooked meat muscle tissue is simple. Moreover, this method removes muscle cell debris

and other impurities which effectively separates exosome and improve its purity. Ultra

high-speed centrifugation can separate exosome from large cooked meat samples, and the

exosome has higher biological activity, which will not affect the subsequent experiments.

BRIEF DESCRIPTION OF THE FIGURES

The attached figures are used to further illustrate the present invention in combination

with specific embodiments.

Figure. 1 is the particle size distribution of exosome in cooked pork.

Figure. 2 is the flow identification of exosome in cooked pork.

Figure. 3 is the statistics of flow identification results of exosome in cooked pork.

DESCRIPTION OF THE INVENTION

The technical scheme in the embodiments of the present invention will be described

clearly and completely with reference to the figures in the embodiments of the present

invention. Obviously, the described embodiments are only parts of the embodiments of

the present invention, not all of them. Based on the embodiments of the present invention,

all other embodiments obtained by ordinary technicians in the field without creative

labour should belong to the protection scope of the present invention.

Embodiment Extracting exosome from cooked pork

1. Removing fascia, blood stains, fat and abnormal meat samples from pig psoas major

muscle as much clear as possible and remove lateral internal septum and related muscle

tissue in chicken breast. Besides, the abnormal meat samples refer to meat with

bloodstains, pustules, abnormal colour, fester, malformation, pathological changes and so

on. Stirring the meat into mince and then wrapping the mince with medical gauze and

boiling it in double distilled water at 100°C for 40 to 60 minutes. It should be noted that

the used instruments, gauze and medical gauze are all washed with double distilled water.

Then filter out meat foam through gauze to obtain broth.

2. After cooling the broth, split charging it in 15 ml centrifuge tubes and centrifuging it

with 2,000xg centrifugal force to separate meat foam and impurities. Then transferring

the supernatant into a 2ml centrifuge tube and using a high-speed centrifuge to centrifuge

at 4C with 12,000xg. Discarding the sediment and the upper lipid layer and keeping the

intermediate supernatant.

3. Let the supernatant obtained by centrifugation continuously pass through filters of

0.45um and 0.2um. Split charging the obtained filtrate into centrifuge tubes of an ultra- high-speed centrifuge and trimming them to make the weight difference of centrifuge tubes does not exceed 0.0050g. Centrifuging the filtrate with an ultra-high-speed centrifuge at 15'C, 160,000xg for 120 min. After centrifugation, pouring the supernatant out and dissolving and suspending exosome again with 100L PBS solution.

4. Adding 1/3 exosome extraction indicator to exosome, mixing them upside down until

they are completely mixed. Standing them in a refrigerator at 4C overnight. Transferring

them to a new 2mL centrifuge tube for centrifuging at 1500xg, 4C for 30 minutes.

Discarding the supernatant and dissolving and suspending exosome again with PBS

solution to obtain exosome with high certification.

5. Detection of surface markers by flow cytometer. The specific steps of it are as

follows.

(1) Taking 100ul exosomes filtered and homogenized by PBS and sealing it and storing it

on ice.

(2) Labelling the unstained exosome as negative control and NC is labelled.

(3) Staining two antibodies in the sample and labelling them as CD63 and CD81.

(4) Performing test according to the operating procedures of the instrument BD-accuri

C6-flow cytometer.

Staining two groups of antibodies-CD63 and CD81, and then subjecting them to flow

antibody surface antigen detection. Using BD-accuri-C6-flow cytometer to analyse the

expression of surface proteins CD63 and CD81 in exosomes from plain boiled pork and

exosomes from red meat, respectively. The detection results show that the two proteins

both have positive signals, wherein the positive rates of CD63 and CD81 in exosome from cooked meat are 84.5% and 95.9%, respectively (shown in Fig. 1 and Table 1). The above results confirm that there are exosomes in cooked meat.

Table 1 The proportion of exosomes labelled by flow cytometer

Antibody Type Positive Rate (%)

CD63 84.5 CD81 95.9 6. Using Nanosight to measure the size of exosome and getting the average particle size

and the main peak which are both within the particle size range of exosome. The particle

distribution index (PDI) is within 0.08-0.7, which indicating that the intensity is

moderate, and test result confidence is high. Through separation, the exosomes with

particle size of 40nm-100nm in cooked meat sample account for 98% (shown in Fig.2).

7. Scanning exosome by electron microscope (EM) to observe its morphology.

After a series of treatments, centrifugation and ultra-centrifugation, exosome is obtained.

Negative staining it and observing its morphology under transmission electron

microscope (TEM). Dropping 25d1exosome solution on the copper mesh and standing it

for 1 minute at room temperature. Using clean filter paper to absorb the liquid from the

side, dropping 30[1 3% phosphotungstic acid solution (pH = 6.8) on the copper mesh, and

performing negative stain for 3 minutes at room temperature. Absorbing the negative

stain solution with filter paper and drying it with incandescent lamp for about 10 minutes.

Observing it under TEM and taking pictures.

Utilizing EM to detect the existence and morphology of exosome. It can be observed that

there are a large number of isolated exosome-like microcapsules (shown in Fig. 3) in

cooked psoas major muscle. Besides, the density in the middle is lower than that at the

edge which is lipid bilayer membrane structure. The diameter of the whole exosome is

-100nm, and the membrane structure is complete. Its surface morphology showed a flat

circular structure similar to exosomes in human saliva and milk reported by predecessors.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for separating exosome from cooked meat samples, characterized by

comprising the following steps.

gauze to obtain broth.

12,000xg centrifugal force.

ultra-high-speed centrifugation and adding PBS solution to suspend exosome.

(4) Adding purification reagent to the obtained solution for purification.

2. The method for separating exosome from cooked meat samples according to claims 1,

characterized in that in step (1), fascia, fat and abnormal meat samples should be

removed as much as possible from the raw meat samples. Wherein, the abnormal meat

samples refer to meat with bloodstains, pustules, abnormal colour, fester, malformation,

pathological changes and so on. The used instruments, gauze and medical gauze are all

washed with double distilled water.

3. The method for separating exosome from cooked meat samples according to claims 1,

characterized by the specific operation of step (2).

(1) Split charging the broth in 15 ml centrifuge tubes;

precipitate while keeping the supernatant.

(3) Putting the supernatant into a 2ml centrifuge tube.

discarding the sediment and the upper lipid layer and keeping the intermediate

supernatant.

4. The method for separating exosome from cooked meat samples according to claims 1,

characterized by the specific operation of step (3).

0.45tm and 0.2[tm.

exceed 0.0050g.

120 minutes.

exosome again with 100tL PBS solution.

5. The method for separating exosome from cooked meat samples according to claim 1,

characterized by the specific operation of step (4).

Transferring 1.5ml of exosome suspension obtained in step (3) to a 2ml centrifuge tube

and adding 0.5ml of Ribo TM Exosome Isolation Reagent (for other body fluids). Then

blowing it evenly with a pipettor (liquid turbidity can be seen in the mixed state) and standing it in a refrigerator at 4C overnight. Then centrifuging at 1500xg, 4C for 30 minutes. Discarding the supernatant to obtain exosome.