Method for extracting micromolecular protein from walnut kernel skin and application thereof

A technology of small molecular protein and walnut kernel, which is applied in the field of walnut protein to achieve the effect of simple and easy implementation.

Active Publication Date: 2019-03-01
SOUTHWEST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the existing methods for obtaining small molecular proteins are chemical hydrolysis and enzymatic hydrolysis, and the obtained small peptides are obtained by hydrolyzing large molecular proteins. The method of directly separating and purifying small molecular proteins needs to be developed urgently

Method used

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  • Method for extracting micromolecular protein from walnut kernel skin and application thereof
  • Method for extracting micromolecular protein from walnut kernel skin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Extraction and separation of walnut kernel skin small molecular protein

[0029] 1. Obtaining walnut kernel skin

[0030] Buy fresh walnuts, tear off the wet walnut skins, and rinse them with clean water.

[0031] 2. Obtain water-soluble protein from walnut kernel skin

[0032] Cut the walnut kernel skin into pieces, add 0.1M PB solution (Na 2 HPO 4 -NaH 2 PO4, pH6.0) and protease inhibitors were ground in a grinding cymbal, and the grinding process was kept at a low temperature on ice, and 300 μL PB solution was added to every 1 mg of walnut kernel skin. Grind the walnut kernels into a uniform paste and dilute it with PB solution. The obtained walnut kernel skin grinding liquid is ultrasonically broken with an ultrasonic cell breaker (ultrasonic 3sec, pause 3sec, every 10min is a cycle, each cycle interval is 20min, a total of three cycles), before ultrasonication, add glass sand to the ultrasonic solution to The cell wall was fully ruptured. During th...

Embodiment 2

[0036] Embodiment two: the antibacterial action of walnut kernel bark small molecule protein

[0037] 1 Dialysis treatment

[0038] Carry out dialysis to the small molecule protein solution of walnut kernel skin extracted and separated obtained in Example 1 to reduce the salt concentration therein. The dialysis bag has a certain molecular weight cut-off. Macromolecular substances such as proteins above this molecular weight cannot pass through, while some small molecular substances such as inorganic salts and monosaccharides can pass through the semi-permeable membrane, which can separate protein molecules from small molecular substances. During dialysis, put walnut kernel skin small molecular protein solution in a semipermeable membrane dialysis bag, put it into the distilled water of the dialysate, and the dialysate can be replaced until the small molecular substances such as inorganic salts in the dialysis bag are gradually reduced to the minimum. . After the dialysis is ...

Embodiment 3

[0047] Example 3: Anti-cancer effect of walnut kernel skin small molecule protein

[0048] 1 Experimental materials

[0049] SGC (human gastric adenocarcinoma cells), HEK293 (human embryonic kidney cells), Huh7 (human liver cancer cells), rat splenocytes and rat macrophages, the above cells were purchased from Beina Chuanglian Biotechnology Co., Ltd.; thiazole blue (MTT); Dimethylsulfoxide (DMSO).

[0050] 2 Experimental methods

[0051] In aseptic operation, the cells were inoculated into 96-well plates, and the inoculum volume of each well was 5×10 3 After 12 hours of cell culture, walnut kernel skin small molecular protein with different concentration gradients was added; after 26 hours of co-cultivation, 0.5 mg / mL MTT was added, and the culture was continued for 4 hours; MTT reacted with mitochondria in living cells to form blue-purple After the reaction, carefully suck out the cell supernatant, add DMSO to dissolve the formazan at the bottom of the well, shake for 10 m...

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Abstract

The invention relates to the technical field of walnut protein, and particularly relates to a method for extracting micromolecular protein from walnut kernel skin and application thereof. The micromolecular protein extraction method is characterized in that the walnut kernel skin protein is preliminarily separated according to the molecular weight by utilizing the dextran gel chromatography principle, and the protein component with the smallest molecular weight is selected for activity research. The micromolecular protein of the walnut kernel skin has bacteriostatic and anticancer effects through bacteriostatic tests and cell proliferation tests; the micromolecular protein has the best inhibitory activity on pseudomonas aeruginosa and enterococcus faecalis; the MIC (minimum bacteriostaticconcentration) values are 14.5 and 7.5Mug / mL respectively; at the concentration of 120Mug / mL, the survival rate of Huh7 (human hepatocellular carcinoma cell) is 50%, and the survival rate of SGC (human gastric carcinoma cells) is only 10%. The walnut kernel skin micromolecular protein can be applied to antibacterial drugs and anticancer drugs.

Description

technical field [0001] The invention relates to the technical field of walnut protein, in particular to a small molecular protein extraction method and application of walnut kernel skin. Background technique [0002] The skin of the walnut kernel is rich in nutrients and rich in minerals and vitamins. Contains carbohydrates, calcium, phosphorus, iron, thiamine, riboflavin, niacin, these nutrients are high, and these are difficult to supplement in common foods. The skin of the walnut is rich in vitamin B and E, which can prevent cell aging, and has the effects of strengthening the brain, enhancing memory and delaying aging. Also contain special vitamin composition in the walnut, not only do not raise cholesterol, can also reduce intestinal absorption of cholesterol, suitable for patients with arteriosclerosis, hypertension and coronary heart disease. The skin of walnut kernel contains linolenic acid, calcium, phosphorus and iron, which is an ideal skin beauty agent for the ...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K1/16A61K38/16A61P31/04A61P35/00
CPCA61K38/16A61P31/04A61P35/00C07K1/145C07K1/16
Inventor 阚欢刘云范方宇郭磊张雪春郭安
Owner SOUTHWEST FORESTRY UNIVERSITY
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