Preparation method for extracting and separating anti-tumor active protein from shiitake mushrooms

A technology of anti-tumor activity and active protein, which is applied to the preparation method of peptides, anti-tumor drugs, chemical instruments and methods, etc., to achieve good safety, good anti-tumor effect, and loose tissue structure

Inactive Publication Date: 2021-07-13
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research involving the extraction of isolated protein substances from edible shiitake mushrooms and their anti-tumor activity in vitro

Method used

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  • Preparation method for extracting and separating anti-tumor active protein from shiitake mushrooms
  • Preparation method for extracting and separating anti-tumor active protein from shiitake mushrooms
  • Preparation method for extracting and separating anti-tumor active protein from shiitake mushrooms

Examples

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Effect test

Embodiment 1

[0039] The preparation method of extracting and separating anti-tumor protein extract LEP from Lentinus edodes (flow process sees attached figure 1 ), including the following steps:

[0040] (1) Pretreatment: Clean the mushroom fruiting bodies, absorb the surface moisture with a paper towel, quick-freeze in liquid nitrogen and grind into powder, pass through a 100-mesh sieve for later use;

[0041](2) Composite enzymatic hydrolysis: take 100 g of shiitake mushroom powder, add 1 L of physiological saline to submerge the shiitake mushroom powder, add cellulase (3000IU), hemicellulase (12000IU) and protease inhibitor (cocktail 1g), and stir at 4°C for 14h;

[0042] (3) Extract active protein: Add the above enzymolysis solution into a supercritical extraction kettle equipped with a stirring and heating device, control the temperature at 35°C, and the pressure at 55MPa, seal the top, start stirring, and the stirring speed is 10m / s, Transport CO 2 Gas, the flow rate is 20L / h, and ...

Embodiment 2

[0046] The preparation method of extracting and separating anti-tumor protein extract LEP from Lentinus edodes (flow process sees attached figure 1 ), including the following steps:

[0047] (1) Pretreatment: Clean the mushroom fruiting bodies, absorb the surface moisture with a paper towel, quick-freeze in liquid nitrogen and grind into powder, pass through a 100-mesh sieve for later use;

[0048] (2) Composite enzymatic hydrolysis: Take 100 g of shiitake mushroom powder, add 1 L of physiological saline to submerge the shiitake mushroom powder, add cellulase (5000IU), hemicellulase (13000IU) and protease inhibitor (cocktail 1g), and stir at 4°C for 16h;

[0049] (3) Extract active protein: Add the above enzymolysis solution into a supercritical extraction kettle equipped with a stirring and heating device, control the temperature at 35°C, and the pressure at 55MPa, seal the top, start stirring, and the stirring speed is 10m / s, Transport CO 2 Gas, the flow rate is 20L / h, and...

Embodiment 3

[0053] The preparation method of extracting and separating anti-tumor protein extract LEP from Lentinus edodes (flow process sees attached figure 1 ), including the following steps:

[0054] (1) Pretreatment: Clean the mushroom fruiting bodies, absorb the surface moisture with a paper towel, quick-freeze in liquid nitrogen and grind into powder, pass through a 100-mesh sieve for later use;

[0055] (2) Composite enzymatic hydrolysis: Take 100 g of shiitake mushroom powder, add 1 L of physiological saline to submerge the shiitake mushroom powder, add cellulase (5000IU), hemicellulase (13000IU) and protease inhibitor (cocktail 1g), and stir at 4°C for 16h;

[0056] (3) Extract active protein: Add the above enzymolysis solution into a supercritical extraction kettle equipped with a stirring and heating device, control the temperature at 40°C, and the pressure at 60MPa, seal the top, start stirring, and the stirring speed is 15m / s, Transport CO 2 Gas, the flow rate is 25L / h, and...

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Abstract

The invention provides a preparation method for extracting and separating anti-tumor active protein from shiitake mushrooms. The preparation method comprises the following steps: (1) pretreatment: cleaning fresh shiitake mushrooms, quickly freezing with liquid nitrogen, grinding, crushing and sieving; (2) composite enzymolysis: immersing the shiitake mushroom powder with normal saline, adding cellulase, hemicellulase and a protease inhibitor, and stirring for 12-16 hours; (3) extracting active protein: extracting the mushroom powder subjected to composite enzymolysis by using supercritical CO2 fluid, and separating in a separation kettle to obtain the active protein; (4) active protein separation: carrying out SourceQ anion exchange chromatography separation and polysaccharide removal, then carrying out Superdex200 gel filtration chromatography, detecting the cytotoxicity by using human lung adenocarcinoma A549 cells, and carrying out separation to obtain the mushroom protein extract LEP with high antitumor activity; and (5) freeze-drying and storing. According to the method, the effect of efficiently extracting the mushroom active protein is achieved through the composite enzymolysis and supercritical extraction processes, and the purpose of separating the anti-tumor active protein is improved by adopting an activity tracking separation method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method for extracting and isolating anti-tumor active protein from shiitake mushrooms. Background technique [0002] Lentinus genus belongs to Basidiomycetes, Agaricales, Tricholomatacete, Lentinus, scientific name Lentinus edodes, is the second largest fungus in the world and is widely planted in my country. The meat of shiitake mushrooms is thick and tender, with delicious taste, unique aroma and rich nutrition. It is a kind of food with the same source of food and medicine, and has high economic, nutritional and medicinal value. Mushrooms contain a lot of carbohydrates and cellulose, low fat, high protein, and a variety of minerals and trace elements. At present, shiitake mushrooms have been found to have anti-tumor, anti-carcinogen, liver protection, blood lipid lowering, anti-oxidation, immune regulation, anti-fungal, bacterial, and viral effects. [0003] There ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/18C07K1/16C07K1/14A61K36/07A61P35/00
CPCC12P21/06C07K1/18C07K1/16C07K1/145A61K36/07A61P35/00Y02P20/54
Inventor 辛凤姣刘宇思李真温博婷王钰璐谷天一
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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