A method of extracting milk exosomes

A technology of exosomes and milk, which is applied in the biological field, can solve the problems of high extraction cost, small volume of extracted samples, and high price, and achieve the effect of increasing the volume of sample extraction, saving the amount of sample preparation, and saving the cost of consumables

Active Publication Date: 2021-06-22
广州市创唯曦旺生物科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Known information shows: 1) The kit method, the extraction sample volume is small, the price is expensive, and the extraction cost is high; 2) The density gradient centrifugation method is cumbersome, takes a long time, and has a large loss; 3) The ultra-speed exosome method obtains The rate is higher, and the steps are simpler than the density gradient, but its purity is very controversial
Except for the kit method that does not require an ultra-high-speed centrifuge and can complete the extraction steps in an ordinary molecular laboratory, the other two methods need to rely on this relatively specific instrument, and the amount of single sample extraction is too small ; It is difficult to achieve mass production or large-volume trials

Method used

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  • A method of extracting milk exosomes
  • A method of extracting milk exosomes
  • A method of extracting milk exosomes

Examples

Experimental program
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Effect test

Embodiment 1

[0036] As a method for extracting milk exosomes according to an embodiment of the present invention, the method includes the following steps:

[0037] (1) Thaw milk samples stored in a -80°C refrigerator, then centrifuge at 2000g, 4°C, for 30 minutes, collect supernatant A, and precipitate into milk lipoprotein and mammary gland cell fragments (cream layer);

[0038] (2) Centrifuge the collected supernatant A at 12000 g, 4° C., for 30 min, collect the supernatant B, and precipitate milk fat protein, casein and other cell debris (cell debris layer);

[0039] (3) further adjust the pH of the collected supernatant B to 5.7;

[0040] (4) Add rennet to the supernatant B after pH adjustment under the condition of 36°C water bath, the amount of rennet added is: the volume of rennet with enzyme activity 17-18 and supernatant B The ratio is 1:10, and the milk protein precipitate (rennet precipitation layer) is precipitated in 8-10 minutes to obtain supernatant C;

[0041] (5) The sup...

experiment example 1

[0048] 1. Determination of enzyme activity

[0049] Adopt the Arima method: Take 5 ml of 100 g / L skim milk and incubate at a certain temperature (35°C) for 5 minutes, add 0.5 ml of enzyme solution, mix quickly and evenly, and accurately record the time (seconds) from adding the enzyme solution to the coagulation of the emulsion, The amount of enzyme that coagulates 1 ml of 100 g / L skim milk in 40 minutes is defined as a Soxhlet unit (Gao Weidong et al., 2010, Xu Su, 1996).

[0050] Chymosin activity (U)=(2400 / T)*(5 / 0.5)*D

[0051] T - solidification time

[0052] D—dilution factor

[0053] 2. Milk exosome RNA extraction

[0054] The Trizol one-step method was used to extract total RNA from exosome samples, and the specific steps were as follows:

[0055] (1) Put the milk exosome samples obtained in Example 1 and Comparative Example 1 into a 2mL centrifuge tube with 1mL Trizol reagent and quickly homogenize for 20s, and the homogenization time can be appropriately increased...

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Abstract

The present invention provides a method for extracting milk exosomes, the method comprising the following steps: (1) taking a milk sample, centrifuging at 2000g, 4°C, and 30min, and collecting the supernatant A; (2) collecting the collected Supernatant A was centrifuged at 12000g, 4°C, 30min, and supernatant B was collected; (3) The collected supernatant B was further adjusted to pH 5.5-6.0; (4) Under the condition of 36-37°C water bath, Adding chymosin to the supernatant B after adjusting the pH to precipitate milk protein to obtain supernatant C; (5) freeze-drying the subcomponents of supernatant C that can pass through a 0.22 μM membrane into powder, namely for milk exosomes. Compared with the traditional centrifugal-ultrahigh-speed centrifugation method, the method for extracting milk exosomes of the present invention reduces the steps of the ultrahigh-speed centrifugation method, saves the cost of consumables used in extraction, and greatly increases the sample extraction volume.

Description

technical field [0001] The invention belongs to the field of biotechnology methods, and in particular relates to a method for extracting milk exosomes. Background technique [0002] Exosomes are a type of membrane-bound vesicles formed by endocytosis, which are released into the extracellular environment by fusion of multivesicular bodies (MVBs) and appear round or cup-shaped under a transmission electron microscope. Phospholipid bilayer structure with a diameter of 40-100nm. Studies have shown that different cells in vivo and in vitro can secrete exosomes under physiological or pathological conditions. In addition, exosomes are also found in various body fluids and breast milk of animals or in vivo. And it has also been proved that the materials in the exosome are closely related to the origin and production conditions of cells. Studies have shown that mRNA and miRNA are found in milk exosomes of humans, cows and pigs and can be transported into immune cells, potentially...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/0631C12N2509/00
Inventor 陈婷张永亮习欠云孙加节史合群刘燕吴海雷
Owner 广州市创唯曦旺生物科技有限公司
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