CN102206693A - Production method of metallothionein of cordyceps sinensis - Google Patents
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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:
The present invention relates to a kind of production method of Chinese caterpillar fungus metallothionein(MT), relate to a kind of Cordyceps sinensis fungus of inducing specifically and produce metallothionein(MT) in a large number, is the method for raw material greatly preparing metallothionein with the Cordyceps sinensis fungus mycelium.
Background technology:
Nineteen fifty-seven Margoshes and Vallee find a kind of low molecular weight protein that is rich in Cd, Zn in the kidney of horse, Kagl and Vallee purify it subsequently, and the called after metallothionein(MT) (Metallothionein, MT).MT extensively is present in the organic sphere, and the intravital MT of animal is mainly synthetic in liver, exists in blood, kidney.Metallothionein(MT) is a kind of molecular weight less (6000～7000), is rich in the non-enzymatic protein of halfcystine that it contains elements such as Cd, Cu, Zn, Hg, Au, does not contain die aromatischen Aminosaeuren and histidine residues.Have following feature: molecular weight is low; Contain 30% halfcystine and do not have die aromatischen Aminosaeuren; Sulfydryl and metal ratio are 3: 1 or 2: 1,4: 1; Metal content height, every mol contain 6 atoms metals.
Recent study finds that metallothionein(MT) has many biological functions, and as removing interior free yl, its ability of removing free radical is SOD and gsh (GSH) 10000 times; Can separate the removing heavy metals toxin, can combine closely with toxic metal such as lead, arsenic, mercury, separate the removing heavy metals toxin, be clinical optimal biologic detoxication agent at present; Energy radioprotective and uviolizing, the cell tissue that MT content is abundant can be resisted from radiation or the damage of UV-induced cell tissue; Can participate in metabolism of trace elements in the body, can discharge metal ion automatically according to trace element status in the body, micro-balance in the scarce element in the added body, control agent is built up health; Can also prevent cell carcinogenesis, can eliminate free radical, heavy metal, thereby prevent their carcinogenic, mutagenesis, can help to keep cell eubolism and division, the exciting human immunologic function strengthens the anti-cancer and kill cancer action of human body etc.
Along with to the going deep into of researchs such as metallothionein(MT) structure, function, its purposes is increasingly extensive, and is increasing to its demand.The report of at present existing many metallothionein(MT) production methods, most of production method is to induce certain general domesticated animal (as rabbit, pig etc.) to produce MT by certain heavy metal species, adopts the separation and purification from this animal viscera of corresponding physics and chemistry technology to obtain the MT goods then.The method that this induced animal is produced MT has following shortcoming: feeding animals is subjected to the restriction of natural condition and scale easily; The biochemical product of animal-origin has the potential risk of communicate illness.So lack a kind of high yield, low cost, safe, the technology and the method that are suitable for large-scale production MT up to now always.
Summary of the invention:
The objective of the invention is to overcome the deficiency of above-mentioned prior art and provide a kind of simple to operate, with short production cycle, MT productive rate height in mycelium or the sporophore can carry out the production method of the Chinese caterpillar fungus metallothionein(MT) that batch production produces.
The present invention can induce Cordyceps to produce metallothionein(MT) in a large number by adding a certain amount of zinc sulfate in the Cordyceps sinensis fungus artificial culture process.Technological process is:
Cordyceps → liquid culture (add zinc sulfate induce) → mycelium filters → is rich in the mycelium of metallothionein(MT) → with damping fluid, grinds smudge cells → add organic sex change, stirring, foreign protein sex change → centrifugal removal foreign protein → stillness of night heat denatured → centrifugal removal metaprotein → be cooled to room temperature → ultrafiltration and concentration → concentrated solution → G-50 chromatography → sulfhydryl reagent method monitoring rapidly to collect MT elutriant → MT elutriant vacuum lyophilization → MT lyophilized powder.
Purpose of the present invention can reach by following measure: a kind of production method of Chinese caterpillar fungus metallothionein(MT) is characterized in that its concrete steps are:
(1) cultivates the Cordyceps sinensis fungus mycelium: Cordyceps sinensis fungus is carried out test tube slant spawn culture (activation), the cultivation of triangular flask liquid seeds, seed tank culture and fermentor cultivation, the substratum of fermentor cultivation step is: glucose 10-50g in every liter of nutrient solution, peptone 5-10g, yeast extract 1-10g, zinc sulfate 5-20g, the pH value is 5.0-7.0; The fermentor tank volume 60%-80% that feeds intake, inoculation weight 5-10%, 20～30 ℃ of temperature, ventilate 1: 0.3～0.8, mixing speed 80～150rpm is cultured to the mycelium recovery rate more than or equal to 1.2%, the substratum reducing sugar content was put a jar results mycelium smaller or equal to 0.2% o'clock;
(2) Plate Filtration: cultured Cordyceps sinensis fungus mycelium is filtered with flame filter press, take out mycelium through behind the Plate Filtration, the mycelium that obtains can be used for the extraction of metallothionein(MT);
(3) grinding makes cytoclasis, foreign protein sex change:
0.05mol/LTris (the Tutofusin tris)-HCl that is equivalent to 1.5～3 times of mycelium weights will be added in the above-mentioned mycelium, pH 8.6, with shredder grinding, homogenate, to cytoclasis, the chloroform and the ethanol of adding and mycelium and Tris (Tutofusin tris)-weight such as HCl, chloroform and alcoholic acid volume ratio are 0.08: 1, and sex change removes foreign protein, stir about 5～10min;
Cytoclasis liquid is centrifugal in 5000～10000r/min, abandon precipitation, get supernatant liquor; 80～90 ℃ of thermally denature 5-10min of supernatant liquor, the precipitation foreign protein leaves standstill more than the 0.5h, and the above centrifugal precipitation of abandoning of 8000r/min gets supernatant liquor, is the crude extract of MT; Crude extract is concentrated less than 2000 membrane ultrafiltration with molecular weight cut-off, gets concentrated solution;
(4) column chromatography: adopt sephadex G-50 to carry out column chromatography for separation the concentrated solution of the extracting solution of above-mentioned MT, with the deionized water wash-out, detect the absorption peak of elutriant at 254nm, absorption peak is partly auxiliary monitors with the sulfhydryl reagent method, collect the maximum absorption band that the sulfhydryl reagent method monitors, be the metallothionein(MT) part; The MT collection liquid cooling freeze-drying that column chromatography obtains is dry, obtain Chinese caterpillar fungus metallothionein(MT) lyophilized powder.
In order further to realize purpose of the present invention, described test tube slant spawn culture (activation) step: preparation substratum: glucose 5-25g in every liter of nutrient solution, peptone 5-10g, yeast extract 1-10g, agar powder 10-20g, adjust pH is 5.0-7.0, is sub-packed in the test tube, sterilization, bevel; Cordyceps species is inserted the test tube slant under aseptic condition, cultivated 5-10 days for 15～30 ℃.
In order further to realize purpose of the present invention, described triangular flask liquid seeds culturing step: preparation substratum: glucose 10-20g in every liter of nutrient solution, peptone 5-10g, yeast extract 1-10g, the pH value is 5.0-7.0, and the prepared culture medium branch is packed in the 500ml Erlenmeyer flask, every bottled 200ml, seal the back and under 120～130 ℃ of conditions, sterilized 18～30 minutes, take out substratum, naturally cool to 20～30 ℃; Every bottle graft is gone into the slant culture 1-2 piece of soybean grain size under aseptic condition, and 15～30 ℃, shaking speed 120rpm cultivated 5 days～10 days.
In order further to realize purpose of the present invention, described seed tank culture step: preparation substratum: glucose 10-20g in every liter of nutrient solution, peptone 5-10g, yeast extract 1-10g, the pH value is 5.0-7.0, volume 60%-80% feeds intake, sterilization in batches, insert cultured triangular flask liquid spawn, inoculum size 2-10%, 15～30 ℃ of culture temperature, ventilated 1: 0.5, mixing speed 120rpm cultivated 2-5 days for 15～30 ℃ in seeding tank, to mycelium recovery rate 0.4-0.8% (doing meter).
The present invention can produce following positively effect and obvious improvement compared with the prior art: the applicant passes through Cordyceps sinensis fungus fundamental research for many years, found that Cordyceps sinensis fungus produces the high ability of metallothionein(MT), can solve the problem that metallothionein(MT) produces the source.The present invention is with short production cycle, metallothionein(MT) productive rate height, the advantage that production cost is low.
Embodiment:
Below the specific embodiment of the present invention is elaborated:
Embodiment 1:
Bacterial classification: Cordyceps militaris (L.) Link. (Cordyceps militaris (L.) Link) bacterial classification derives from China Forest microbial strains preservation administrative center, numbering cfcc 5974.
Test tube slant spawn culture (activation):
The preparation substratum: glucose 25g in every liter of nutrient solution, peptone 5g, yeast extract 1g, agar powder 15g, adjust pH is 7.0, is sub-packed in the test tube sterilization, bevel; Cordyceps militaris spawn is inserted the test tube slant under aseptic condition, cultivated 7 days for 25 ℃.
The triangular flask liquid seeds is cultivated:
Preparation substratum: glucose 20g in every liter of nutrient solution, peptone 5g, yeast extract 1g, the pH value is 7.0, the prepared culture medium branch is packed in the 500ml Erlenmeyer flask into every bottled 200ml, seal the back and under 121 ℃ of conditions, sterilized 20 minutes, take out substratum, naturally cool to 30 ℃; Every bottle graft is gone into the slant culture 1-2 piece of soybean grain size under aseptic condition, and 30 ℃, shaking speed 120rpm cultivated 5 days.
Seed tank culture:
Preparation substratum: glucose 20g in every liter of nutrient solution, peptone 5g, yeast extract 1g, the pH value is 7.0, the volume 80% that feeds intake, sterilization inserts cultured triangular flask liquid spawn in batches, inoculation weight 5%, 25 ℃ of culture temperature were ventilated mixing speed 120rpm 1: 0.5, in seeding tank, cultivated 2 days for 15～30 ℃, to mycelium recovery rate 0.4% (doing meter);
Fermentor cultivation:
The preparation substratum: glucose 30g in every liter of nutrient solution, peptone 8g, yeast extract 5g, zinc sulfate 15g, the pH value is 6.5; The fermentor tank volume 70% that feeds intake, inoculation weight 6%, 20～30 ℃ of temperature, ventilate 1: 0.3～0.8, mixing speed 120rpm is cultured to mycelium recovery rate (doing meter) more than or equal to 1.2%, the substratum reducing sugar content was put a jar results mycelium smaller or equal to 0.2% o'clock.
Plate Filtration:
Cultured Cordyceps militaris (L.) Link. fungus filament is filtered with flame filter press, take out mycelium through behind the Plate Filtration, 5 tons of fermentor tanks obtain mycelium weight in wet base 380Kg.
Grinding makes cytoclasis, the foreign protein sex change:
0.05mol/L Tris (the Tutofusin tris)-HCl (pH 8.6) that adds 760Kg in the wet mycelium is with shredder grinding, homogenate, to cytoclasis, the chloroform and the ethanol that add 1040Kg, chloroform and alcoholic acid volume ratio are 0.08: 1, and sex change removes foreign protein, stir about 7min.
Cytoclasis liquid is centrifugal in 8000r/min, abandon precipitation, get supernatant liquor; 85 ℃ of thermally denature 8min of supernatant liquor, the precipitation foreign protein, leave standstill 0.5h after, the above centrifugal precipitation of abandoning of 8000r/min, supernatant liquor, be the crude extract of MT; Crude extract is concentrated less than 2000 membrane ultrafiltration with molecular weight cut-off, gets concentrated solution 20Kg.
The concentrated solution of the extracting solution of column chromatography: MT adopts sephadex G-50 to carry out column chromatography for separation, with the deionized water wash-out, detect the absorption peak of elutriant at 254nm, absorption peak is partly auxiliary monitors with the sulfhydryl reagent method, collect the maximum absorption band that the sulfhydryl reagent method monitors, be the metallothionein(MT) part; It is dry that the MT that column chromatography obtains collects the liquid cooling freeze-drying, obtains Chinese caterpillar fungus metallothionein(MT) lyophilized powder 4.25Kg.
Embodiment 2:
Bacterial classification: Cordyceps hawkesii Gary (Cordyceps hawkesii Gray) bacterial classification derives from China Forest microbial strains preservation administrative center, numbering cfcc 5967.
Test tube slant spawn culture (activation):
The preparation substratum: glucose 20g in every liter of nutrient solution, peptone 10g, yeast extract 5g, agar powder 10g, adjust pH is 6.0, is sub-packed in the test tube sterilization, bevel; Cordyceps militaris spawn is inserted the test tube slant under aseptic condition, cultivated 10 days for 15 ℃.
The triangular flask liquid seeds is cultivated:
Preparation substratum: glucose 10g in every liter of nutrient solution, peptone 10g, yeast extract 5g, the pH value is 6.0, the prepared culture medium branch is packed in the 500ml Erlenmeyer flask into every bottled 200ml, seal the back and under 120 ℃ of conditions, sterilized 30 minutes, take out substratum, naturally cool to 25 ℃; Every bottle graft is gone into the slant culture 1-2 piece of soybean grain size under aseptic condition, and 20 ℃, shaking speed 120rpm cultivated 7 days.
Seed tank culture:
Preparation substratum: glucose 10g in every liter of nutrient solution, peptone 10g, yeast extract 10g, the pH value is 6.0, the volume 70% that feeds intake, sterilization inserts cultured triangular flask liquid spawn, inoculum size 10%, 30 ℃ of culture temperature were ventilated mixing speed 120rpm 1: 0.5, in seeding tank, cultivated 3 days for 15 ℃～30 ℃, to mycelium recovery rate 0.5% (doing meter);
Fermentor cultivation:
The preparation substratum: glucose 50g in every liter of nutrient solution, peptone 10g, yeast extract 10g, zinc sulfate 20g, the pH value is 7.0; The fermentor tank volume 80% that feeds intake, inoculation weight 10%, 20～30 ℃ of temperature, ventilate 1: 0.3～0.8, mixing speed 150rpm is cultured to mycelium recovery rate (doing meter) more than or equal to 1.2%, the substratum reducing sugar content was put a jar results mycelium smaller or equal to 0.2% o'clock.
Plate Filtration:
Cultured Cordyceps hawkesii Gary mycelium is filtered with flame filter press, take out mycelium through behind the Plate Filtration, 5 tons of fermentor tanks obtain mycelium weight in wet base 400Kg.
Grinding makes cytoclasis, the foreign protein sex change:
0.05mol/L Tris (the Tutofusin tris)-HCl (pH 8.6) that adds 1200Kg in the wet mycelium is with shredder grinding, homogenate, to cytoclasis, the chloroform and the ethanol that add 1600Kg, chloroform and alcoholic acid volume ratio are 0.08: 1, and sex change removes foreign protein, stir 10min.
Cytoclasis liquid is centrifugal in 5000r/min, abandon precipitation, get supernatant liquor.90 ℃ of thermally denature 5min of supernatant liquor, the precipitation foreign protein, leave standstill 0.5h after, the above centrifugal precipitation of abandoning of 8000r/min, supernatant liquor, be the crude extract of MT; Crude extract is concentrated less than 2000 membrane ultrafiltration with molecular weight cut-off, gets concentrated solution 21Kg.
The concentrated solution of the extracting solution of column chromatography: MT adopts sephadex G-50 to carry out column chromatography for separation, with the deionized water wash-out, detect the absorption peak of elutriant at 254nm, absorption peak is partly auxiliary monitors with the sulfhydryl reagent method, collect the maximum absorption band that the sulfhydryl reagent method monitors, be the metallothionein(MT) part.
It is dry that the MT that column chromatography obtains collects the liquid cooling freeze-drying, obtains Chinese caterpillar fungus metallothionein(MT) lyophilized powder 4.85Kg.
Embodiment 3:
Bacterial classification: kyushu aweto bacterial classification (Cordyceps kyusyuensis) derives from China Forest microbial strains preservation administrative center, numbering cfcc 89250.
Test tube slant spawn culture (activation):
The preparation substratum: glucose 5g in every liter of nutrient solution, peptone 8g, yeast extract 10g, agar powder 20g, adjust pH is 5, is sub-packed in the test tube sterilization, bevel; Cordyceps militaris spawn is inserted the test tube slant under aseptic condition, cultivated 5 days for 30 ℃.
The triangular flask liquid seeds is cultivated:
Preparation substratum: glucose 15g in every liter of nutrient solution, peptone 8g, yeast extract 10g, the pH value is 5, the prepared culture medium branch is packed in the 500ml Erlenmeyer flask into every bottled 200ml, seal the back and under 130 ℃ of conditions, sterilized 18 minutes, take out substratum, naturally cool to 20 ℃; Every bottle graft is gone into the slant culture 1-2 piece of soybean grain size under aseptic condition, and 25 ℃, shaking speed 120rpm cultivated 7 days.
Seed tank culture:
Preparation substratum: glucose 10g in every liter of nutrient solution, peptone 8g, yeast extract 5g, the pH value is 5, the volume 60% that feeds intake, sterilization inserts cultured triangular flask liquid spawn, inoculation weight 2%, 15 ℃ of culture temperature were ventilated mixing speed 120rpm 1: 0.5, in seeding tank, cultivated 5 days for 15 ℃～30 ℃, to mycelium recovery rate 0.8% (doing meter);
Fermentor cultivation:
The preparation substratum: glucose 10g in every liter of nutrient solution, peptone 5g, yeast extract 1g, zinc sulfate 5g, the pH value is 5.0; The fermentor tank volume 60% that feeds intake, inoculation weight 5%, 20～30 ℃ of temperature, ventilate 1: 0.3～0.8, mixing speed 80rpm is cultured to mycelium recovery rate (doing meter) more than or equal to 1.2%, the substratum reducing sugar content was put a jar results mycelium smaller or equal to 0.2% o'clock.
Plate Filtration:
Cultured kyushu aweto mycelium is filtered with flame filter press, take out mycelium through behind the Plate Filtration, 5 tons of fermentor tanks obtain mycelium weight in wet base 300Kg.
Grinding makes cytoclasis, the foreign protein sex change:
0.05mol/LTris (the Tutofusin tris)-HCl (pH 8.6) that adds 450Kg in the wet mycelium is with shredder grinding, homogenate, to cytoclasis, the chloroform and the ethanol that add 750Kg, chloroform and alcoholic acid volume ratio are 0.08: 1, and sex change removes foreign protein, stir about 5min.
Cytoclasis liquid is centrifugal in 10000r/min, abandon precipitation, get supernatant liquor.80 ℃ of thermally denature 10min of supernatant liquor, the precipitation foreign protein, leave standstill 0.5h after, the above centrifugal precipitation of abandoning of 8000r/min, supernatant liquor, be the crude extract of MT; Crude extract is concentrated less than 2000 membrane ultrafiltration with molecular weight cut-off, gets concentrated solution 15Kg.
The concentrated solution of the extracting solution of column chromatography: MT adopts sephadex G-50 to carry out column chromatography for separation, with the deionized water wash-out, detect the absorption peak of elutriant at 254nm, absorption peak is partly auxiliary monitors with the sulfhydryl reagent method, collect the maximum absorption band that the sulfhydryl reagent method monitors, be the metallothionein(MT) part.
It is dry that the MT that column chromatography obtains collects the liquid cooling freeze-drying, obtains Chinese caterpillar fungus metallothionein(MT) lyophilized powder 3.55Kg.
Claims (4)
1. the production method of a Chinese caterpillar fungus metallothionein(MT) is characterized in that its concrete steps are:
(1) cultivates the Cordyceps sinensis fungus mycelium: Cordyceps sinensis fungus is carried out test tube slant spawn culture (activation), the cultivation of triangular flask liquid seeds, seed tank culture and fermentor cultivation, the substratum of fermentor cultivation step is: glucose 10-50g in every liter of nutrient solution, peptone 5-10g, yeast extract 1-10g, zinc sulfate 5-20g, the pH value is 5.0-7.0; The fermentor tank volume 60%-80% that feeds intake, inoculation weight 5-10%, 20～30 ℃ of temperature, ventilate 1: 0.3～0.8, mixing speed 80～150rpm is cultured to the mycelium recovery rate more than or equal to 1.2%, the substratum reducing sugar content was put a jar results mycelium smaller or equal to 0.2% o'clock;
(2) Plate Filtration: cultured Cordyceps sinensis fungus mycelium is filtered with flame filter press, take out mycelium through behind the Plate Filtration, the mycelium that obtains can be used for the extraction of metallothionein(MT);
(3) grinding makes cytoclasis, foreign protein sex change:
0.05mol/LTris (the Tutofusin tris)-HCl that is equivalent to 1.5～3 times of mycelium weights will be added in the above-mentioned mycelium, pH 8.6, with shredder grinding, homogenate, to cytoclasis, the chloroform and the ethanol of adding and mycelium and Tris (Tutofusin tris)-weight such as HCl, chloroform and alcoholic acid volume ratio are 0.08: 1, and sex change removes foreign protein, stir about 5～10min;
Cytoclasis liquid is centrifugal in 5000～10000r/min, abandon precipitation, get supernatant liquor; 80～90 ℃ of thermally denature 5-10min of supernatant liquor, the precipitation foreign protein leaves standstill more than the 0.5h, and the above centrifugal precipitation of abandoning of 8000r/min gets supernatant liquor, is the crude extract of MT; Crude extract is concentrated less than 2000 membrane ultrafiltration with molecular weight cut-off, gets concentrated solution;
(4) column chromatography: adopt sephadex G-50 to carry out column chromatography for separation the concentrated solution of the extracting solution of above-mentioned MT, with the deionized water wash-out, detect the absorption peak of elutriant at 254nm, absorption peak is partly auxiliary monitors with the sulfhydryl reagent method, collect the maximum absorption band that the sulfhydryl reagent method monitors, be the metallothionein(MT) part; The MT collection liquid cooling freeze-drying that column chromatography obtains is dry, obtain Chinese caterpillar fungus metallothionein(MT) lyophilized powder.
2. the production method of a kind of Chinese caterpillar fungus metallothionein(MT) according to claim 1, it is characterized in that described test tube slant spawn culture (activation) step: preparation substratum: glucose 5-25g in every liter of nutrient solution, peptone 5-10g, yeast extract 1-10g, agar powder 10-20g, adjust pH is 5.0-7.0, is sub-packed in the test tube, sterilization, bevel; Cordyceps species is inserted the test tube slant under aseptic condition, cultivated 5-10 days for 15～30 ℃.
3. the production method of a kind of Chinese caterpillar fungus metallothionein(MT) according to claim 1, it is characterized in that described triangular flask liquid seeds culturing step: preparation substratum: glucose 10-20g in every liter of nutrient solution, peptone 5-10g, yeast extract 1-10g, the pH value is 5.0-7.0, and the prepared culture medium branch is packed in the 500ml Erlenmeyer flask, every bottled 200ml, seal the back and under 120～130 ℃ of conditions, sterilized 18～30 minutes, take out substratum, naturally cool to 20～30 ℃; Every bottle graft is gone into the slant culture 1-2 piece of soybean grain size under aseptic condition, and 15～30 ℃, shaking speed 120rpm cultivated 5 days～10 days.
4. the production method of a kind of Chinese caterpillar fungus metallothionein(MT) according to claim 1, it is characterized in that described seed tank culture step: preparation substratum: glucose 10-20g in every liter of nutrient solution, peptone 5-10g, yeast extract 1-10g, the pH value is 5.0-7.0, volume 60%-80% feeds intake, sterilization in batches, insert cultured triangular flask liquid spawn, inoculum size 2-10%, 15～30 ℃ of culture temperature, ventilated 1: 0.5, mixing speed 120rpm cultivated 2-5 days for 15～30 ℃ in seeding tank, to mycelium recovery rate 0.4-0.8% (doing meter).
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586271A (en) * | 2015-12-25 | 2016-05-18 | 江苏大学 | Method for producing cordyceps sinensis mycelium raw material from mutant strain |
| CN107056935A (en) * | 2016-09-27 | 2017-08-18 | 济南米铎碳新能源科技有限公司 | The preparation method of metallothionein |
| CN107049844A (en) * | 2017-05-11 | 2017-08-18 | 炎黄（固安）生物科技有限公司 | A kind of metallothionein facial mask |
| CN107384996A (en) * | 2017-06-26 | 2017-11-24 | 盘古基因生物工程(南京)股份有限公司 | A kind of method and its application for inducing saccharomyces cerevisiae high yield metallothionein |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1130687A (en) * | 1995-03-07 | 1996-09-11 | 戴根春 | Process for preparing metallothionein |
| CN1138098A (en) * | 1996-02-06 | 1996-12-18 | 北京大学 | Selection fermentation and extraction technology of brewing yeast metallothionein producing strain |
| CN1583790A (en) * | 2004-05-26 | 2005-02-23 | 中国科学院亚热带农业生态研究所 | Extracting method for pork liver metal sulfur protein |
| CN101085814A (en) * | 2006-06-09 | 2007-12-12 | 湖南麓谷生物技术有限公司 | Industrial extraction and purification method for rabbit liver zinc metallothionein |
| CN101397542A (en) * | 2008-09-28 | 2009-04-01 | 鲁东大学 | Artificial culture method of zinc-enriched Chinese caterpillar fungus mycelia and culture medium thereof |
| CN101580532A (en) * | 2008-05-12 | 2009-11-18 | 湖南农业大学 | Extracting method of beef liver metallothionein |
-
2011
- 2011-03-23 CN CN 201110076199 patent/CN102206693B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1130687A (en) * | 1995-03-07 | 1996-09-11 | 戴根春 | Process for preparing metallothionein |
| CN1138098A (en) * | 1996-02-06 | 1996-12-18 | 北京大学 | Selection fermentation and extraction technology of brewing yeast metallothionein producing strain |
| CN1583790A (en) * | 2004-05-26 | 2005-02-23 | 中国科学院亚热带农业生态研究所 | Extracting method for pork liver metal sulfur protein |
| CN101085814A (en) * | 2006-06-09 | 2007-12-12 | 湖南麓谷生物技术有限公司 | Industrial extraction and purification method for rabbit liver zinc metallothionein |
| CN101580532A (en) * | 2008-05-12 | 2009-11-18 | 湖南农业大学 | Extracting method of beef liver metallothionein |
| CN101397542A (en) * | 2008-09-28 | 2009-04-01 | 鲁东大学 | Artificial culture method of zinc-enriched Chinese caterpillar fungus mycelia and culture medium thereof |
Non-Patent Citations (7)
| Title |
|---|
| 《Biochemistry》 19851231 Karl Munger et al Copper metallothionein from the fungus Agricus bisporus chemical and spectroscopic properties 6571-6576页 1-4 第24卷, * |
| KARL MUNGER ET AL: "Copper metallothionein from the fungus Agricus bisporus chemical and spectroscopic properties", 《BIOCHEMISTRY》 * |
| SATOMI ONOSAKA ET AL: "The inducedsynthesis of metallothionein in various tissues of rats in response to metals. II. Influence of zinc status and specific effect on pancreatic metallothionein", 《TOXICOLOBY》 * |
| 刘安玲 等: "柱状田头菇(茶薪菇)金属硫蛋白的分离纯化与特性研究", 《菌物系统》 * |
| 李连平 等: "小球藻锌结合类金属硫蛋白的提取和分离", 《食品与发酵工业》 * |
| 王能飞: "一株红酵母Y11吸附镉的特性研究及其金属硫蛋白的分离纯化", 《中国优秀硕士学位论文全文数据库》 * |
| 程显好 等: "蛹虫草对锌的耐性与富集特征", 《生态学报》 * |
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