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Production method of metallothionein of cordyceps sinensis

A technology of metallothionein and its production method, which is applied in the field of mass production of metallothionein, can solve the problems that animals are easily restricted by natural conditions and scale, infectious diseases caused by biochemical products, and have high safety, so as to solve the problem of the production source of metallothionein , high yield of metallothionein and low production cost

Inactive Publication Date: 2011-10-05
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method of inducing animal production of MT has the following disadvantages: raising animals is easily restricted by natural conditions and scale; animal-derived biochemical products have potential hidden dangers of infectious diseases
Therefore, there has been a lack of a high-yield, low-cost, high-safety technology and method suitable for large-scale production of MT.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Strain: The strain of Cordyceps militaris (L.) Link was obtained from the China Forestry Microorganism Culture Collection and Management Center, numbered cfcc 5974.

[0022] Test tube slant culture (activation):

[0023] Preparation medium: Glucose 25g, peptone 5g, yeast extract 1g, agar powder 15g in every liter of culture solution, adjust the pH value to 7.0, subpackage in test tubes, sterilize, and make inclined plane; Insert the slant of the test tube under bacterial conditions and incubate at 25°C for 7 days.

[0024] Triangular flask liquid seed culture:

[0025] Prepare medium: Glucose 20g, peptone 5g, yeast extract 1g, pH value 7.0 in each liter of culture medium, divide the prepared medium into 500ml Erlenmeyer flasks, each bottle contains 200ml, seal and store at 121℃ Sterilize under high temperature for 20 minutes, take out the culture medium, and cool naturally to 30°C; under aseptic conditions, insert 1-2 blocks of slant cultures the size of soybean grain...

Embodiment 2

[0037] Strains: Cordyceps hawkesii Gray (Cordyceps hawkesii Gray) strains, derived from China Forestry Microorganism Culture Collection and Management Center, number cfcc 5967.

[0038] Test tube slant culture (activation):

[0039] Preparation medium: Glucose 20g, peptone 10g, yeast extract 5g, agar powder 10g in every liter of culture solution, adjust the pH value to 6.0, subpackage in test tubes, sterilize, and make inclined plane; Insert the slant of the test tube under bacterial conditions and incubate at 15°C for 10 days.

[0040] Triangular flask liquid seed culture:

[0041] Prepare medium: Glucose 10g, peptone 10g, yeast extract 5g, pH value 6.0 in each liter of culture medium, divide the prepared medium into 500ml Erlenmeyer flasks, each bottle contains 200ml, seal and store at 120°C Sterilize under high temperature for 30 minutes, take out the culture medium, and cool naturally to 25°C; under aseptic conditions, add 1-2 blocks of slant cultures the size of soybean...

Embodiment 3

[0054] Strain: Kyushu Cordyceps kyusyuensis strain (Cordyceps kyusyuensis) comes from China Forestry Microorganism Culture Collection Management Center, number cfcc 89250.

[0055] Test tube slant culture (activation):

[0056] Preparation medium: 5g of glucose, 8g of peptone, 10g of yeast extract, 20g of agar powder in every liter of culture solution, adjust the pH value to 5, subpackage in test tubes, sterilize, and make inclined planes; Insert the slant of the test tube under bacterial conditions and incubate at 30°C for 5 days.

[0057] Triangular flask liquid seed culture:

[0058] Preparation of medium: 15g of glucose, 8g of peptone, 10g of yeast extract in each liter of culture medium, pH value is 5, divide the prepared medium into 500ml Erlenmeyer flasks, each bottle contains 200ml, seal and store at 130°C Sterilize under high temperature for 18 minutes, take out the culture medium, and cool naturally to 20°C; under aseptic conditions, add 1-2 blocks of slant culture...

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Abstract

The invention discloses a production method of metallothioneins of cordyceps sinensis, particularly comprising the following steps of: taking cordyceps sinensis; carrying out liquid culture (adding zinc sulfate for induction); filtering mycelia to obtain mycelia which are rich in the metallothioneins; adding a buffer solution, and grinding to disrupt cells; adding an organic denatured solution, and stirring to denature foreign proteins; centrifugally removing the foreign proteins; heating a supernatant for denaturing; centrifugally removing denatured proteins; fast cooling to room temperature; carrying out ultrafiltration concentration to obtain a concentrated solution; carrying out chromatography through G-50; monitoring an MT (Metallothionein) eluent through a sulfhydryl reagent method,and collecting the MT eluent; and carrying out vacuum freeze drying on the MT eluent to obtain MT freeze-dried powder. The invention has the advantages of easiness in operation, short production period, high yield of MT contained in the mycelia or sporocarps and factory production.

Description

Technical field: [0001] The invention relates to a method for producing metallothionein from Cordyceps, in particular to a method for inducing a large amount of metallothionein produced by fungi of the genus Cordyceps, and using the mycelium of the fungus of the genus Cordyceps as a raw material to produce a large amount of metallothionein. Background technique: [0002] In 1957, Margoshes and Vallee discovered a low-molecular-weight protein rich in Cd and Zn in horse kidneys, which was then purified by Kagl and Vallee and named Metallothionein (MT). MT widely exists in the biological world. MT in animals is mainly synthesized in the liver and exists in the blood and kidney. Metallothionein is a non-enzymatic protein with a small molecular weight (6000-7000) and rich in cysteine, which contains elements such as Cd, Cu, Zn, Hg, Au, and does not contain aromatic amino acids and histidine Residues. It has the following characteristics: low molecular weight; containing 30% cys...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/825C07K1/36C07K1/34C07K1/16C12R1/645
Inventor 程显好张聪聪马天云李维焕董洪新刘宇
Owner LUDONG UNIVERSITY
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